Category Archives: Cell Biology

Cell Biology

Multiplexed whole-animal imaging with reversibly switchable optoacoustic proteins – Science Advances

INTRODUCTION

Photo- or optoacoustic (OA) imaging combines optical contrast with ultrasound resolution, enabling high-resolution, real-time in vivo imaging well beyond the 1-mm penetration depth typical of microscopy methods (1, 2). OA has already provided intriguing insights into tumor heterogeneity (3), neuronal dynamics (4), psoriasis (5), and brown fat metabolism (6) based on endogenous contrast from hemoglobin and lipids (7, 8). This is complemented by theranostic research (9, 10) and clinical application (11), e.g., imaging of Crohns disease (12). However, OA imaging has not yet become a routine tool in life sciences because of the lack of strong OA contrast agents that can be expressed in desired cell types (13). The few transgenic labels used in OA so far (8) give weak signals that cannot rise above the strong background due to hemoglobin. Photochromic proteins that can be reversibly switched between two states by light can overcome this limitation by entirely separating the label signal, which modulates in accordance with the illumination, from the background, which remains constant (14). This concept, despite being validated in several studies (1520), has not been implemented widely because it requires complex instrumentation and data analysis tools. Here, we introduce two reversibly switchable OA proteins (rsOAPs) and demonstrate their use with widely accessible off-the-shelf commercial imaging systems as well as our open-access machine learning (ML)based software code for analysis. One of our new rsOAPs shows high switching speeds and dynamic range of photomodulation that allow us to resolve the signals of different cell populations labeled with differentiable rsOAPs in close proximity in the same animal, demonstrating the potential for simultaneous tracking of different cellular processes through temporal multiplexing.

(A) Homology model (iTasser, based on 6g1y) of ReBphP-PCM. Truncation sides indicated. (B) Schematic representation of truncations. (C) Photoinduced differential spectra for truncations. (D and E) Similar representations for RpBphP1. (F) Stabilization of the BV D-ring in RpBphP1 and DrBphP. (G) Similar representation for PaBphP, which shows an arginine similar to ReBphP, presumably abstracting D194 and destabilizing the Pfr state, yielding a faster photoswitching.

Bacterial photoreceptors called bacteriophytochromes (BphPs) (21) have emerged as most suitable for rsOAP development due to their strong absorption in the near-infrared range and low photofatigue (22). To identify the most promising candidate for further development, we screened eight native BphPs (table S1) and selected the one from Rhizobium etli. A set of truncations enabled us to minimize its size and optimize its photoswitching characteristics. In brief, on the basis of existing structural data as well as homology models, we created truncations containing the minimum PAS-GAF-PHY photosensory core domains [photosensory core module (PCM)] together with extra amino acids from the annotated linkers between PHY and histidine kinase domains and tested their characteristics in regard to signal generation and photoswitching (Fig. 1, A to E, and note S1). The final variant ReBphP-PCM shows twofold larger change in OA signal (Fig. 2G), more than fivefold faster switching (Fig. 2, C and D), and greater resistance to photofatigue than other rsOAPs (Fig. 2E), while its high molar absorbance is on par with the recently described Deinococcus radiodurans DrBphP-PCM (92,000 M1 cm1; Fig. 2, B and G) (19). Those characteristics enable higher numbers of switching cycles per second, which improves sensitivity and allows imaging over longer timeframes. On the molecular level, this acceleration of switching speed is the result of a less stabilized Pfr state favoring the photoinduced transition to Pr. The destabilization is likely caused by an arginine present in ReBphP but not in RpBphP1 and DrBphP. This arginine, by interacting with a conserved aspartate, which, in turn, interacts with the D-ring of the Pfr state chromophore, weakens Pfr stabilization (Fig. 1, F and G, and note S1).

(A) Principle of photoswitching in BphPs (top) and concept of temporal unmixing of two labels (green ball and blue star; bottom). Illumination shown in dark red (780 nm) and red (630 nm). Pr refers to the red state, while Pfr refers to the far-red state. The bottom part of the panel was adapted with permission from (14). (B) Absorbance spectra of Pr and Pfr states of the three rsOAPs in comparison to hemoglobin (HbO2 and Hb, 1999, S. Prahl, omlc.org). (C) Switching cycles of the rsOAPs. Only OA signal at 770 nm is shown. a.u., arbitrary units. (D) Single switching cycle from (C), shown with an exponential fit. (E) Photofatigue of the proteins per cycle. (F) Absorbance ratio between the Pfr and Pr state for different wavelengths. (G) Absorbance (filled bars) and OA signal intensity (hollow) ratio between the Pfr and Pr state for the three rsOAPs at 770 nm. (H) Matthews coefficient shown as a function of number of cycles and pulses. Shown is the analysis of a 4T1 tumor expressing ReBphP-PCM; histology was used as ground truth. All proteins have been adjusted to equal Soret peak absorption.

Our truncation strategy also proved successful in obtaining a switchable RpBphP1-PCM from Rhodopseudomonas palustris, in contrast to a previous report that truncated forms of this protein do not undergo reversible switching (19). Our engineered RpBphP1-PCM maintains the far-red state (Pfr) extinction coefficient and photochromic behavior of the parental RpBphP1 (Figs. 1E and 2B and fig. S1), and the change in its OA signal following illumination at 770 nm is similar to that of the previously described DrBphP-PCM (Fig. 2G). (Plasmid for expressing ReBphP-PCM in bacteria and eukaryotic cells or for introduction into viral vectors can be obtained from Addgene.) Both new rsOAPs are monomeric (fig. S2) and show higher expression in mammalian cells than the full-length parental proteins (fig. S3). The two developed rsOAPs and DrBphP-PCM show distinctive switching speeds, which is the reason for our ability to discriminate the proteins in vivo successfully. As a result, probes expressed in different cells in close proximity in the animal can be distinguished during high-resolution OA imaging.

We performed all OA imaging using an off-the-shelf, commercially available multispectral OA tomography device with a 10-Hz pulsed tunable laser and a 256-element transducer array (MSOT, iThera Medical). Off-switching of rsOAPs was achieved with light at 770 nm, which gave the highest difference in OA signal intensity between the on and off states (fig. S5A), while on switching was achieved using light at 680 nm. Lower wavelengths did not substantially improve the transition to the on state (fig. S5B). The number of laser pulses per wavelength was chosen to cover the full switching kinetics, but it can be significantly reduced using information-content analysis, which allows an estimate of the minimal number of cycles and pulses per cycle required to discern the labeled structure, thus effectively limiting imaging dwell time, which is essential for, e.g., time-resolved studies (Fig. 2H, fig. S6, and note S2). All temporal unmixing was conducted with in-house code developed to analyze time-varying patterns in the reconstructed data in the frequency and time domains using classic ML approaches (Fig. 3, Materials and Methods, and notes S3 and S4). In brief, after running fluence and motion correction on the data, a range of distinctive features was extracted from the photomodulated signal for each voxel of the tomography images. On the basis of a set of these data and corresponding histology as ground truth, a bagged random forest algorithm (23) was trained and validated on independent datasets of a different type to prevent overfitting. The ensuing model was then used to analyze all data in this study. The code for data preparation, for analysis with the model used in this work, and for generation of new models is available to the community along with graphical user interfaces.

The time-varying patterns in the OA raw data are extracted in the Feature calculation (blue) and analyzed using a classification model in the Data analysis step (yellow). In Model building (green), a classification model is trained based on imaging data with associated histology ground truth. In the script, two algorithms can be selected: bagged tree or support vector machine. For uniformity, the images shown in this work exclusively use the bagged tree approach, although the support vector machine has some virtues (note S4).

The OA imaging scheme is shown in Fig. 4A. First, we used rsOAPs for superficial in vivo imaging. We imaged the development of 4T1 mouse mammary gland tumors coexpressing ReBphP-PCM and green fluorescent protein (GFP) after they were grafted onto the backs of FoxN1 nude mice (n = 3). The initial population of 0.8 106 injected cells was readily visualized separate from all background absorbers (Fig. 4B), as was the growing tumor mass at all days after injection (fig. S7, A to D). To test whether this imaging is also possible in brain tissue after light passes through the skull, we implanted 0.7 106 4T1 cells coexpressing ReBphP-PCM and GFP at a depth of 3.6 mm and imaged them immediately thereafter. Comparison of the OA images with fluorescence images obtained after sacrificing mice revealed perfect overlap of the labeling, confirming background-free identification of 1.4 105 cells deep in the mouse brain (Fig. 4C). Next, we used the same rsOAP to image deep-seated tumors of HCT116 human colon carcinoma cells implanted intraperitoneally (n = 2). From day 3 onward, we were able to visualize the growth of several individual tumor sites to a depth of ~1 cm (fig. S7, E and F). Comparison of OA images and histology obtained after sacrifice confirmed identification of all malignant tissue (Fig. 4, D to F, and fig. S7, E and G), including small tumors or metastatic patches containing less than 10,000 cells (fig. S7, I and J).

In certain experiments, GFP was coexpressed to allow fluorescence imaging of histology slices. (A) Schematic of OA tomography used in this work. (B) 4T1 cells (0.8 106 injected subcutaneously) stably expressing ReBphP-PCM and imaged on day 9. (C) 4T1 cells (0.7 106 injected intracranially) stably expressing ReBphP-PCM imaged at a depth of 3.6 mm in the brain (arrow I) immediately after injection. (D) Volume representation of HCT116 cells (1.5 106 injected intraperitoneally) stably expressing ReBphP-PCM at consecutive time points. (E) Histology of the same mouse at day 14. (D and E) Arrows indicate distinctive tumor masses. (F) Certainty of prediction (weighted sum of tree scores) indicating quality of discerning label signal or background of regions of interest shown in (E) (right). (G) Imaging of the indicated concentrations of Jurkat T cells in Matrigel expressing ReBphP-PCM immediately after subcutaneous implantation; because of the polymerization process, no homogeneity is expected. (H) Imaging of the indicated concentrations of E. coli expressing ReBphP-PCM in Matrigel immediately after subcutaneous implantation. In (B), (C), (G), and (H), color maps refer to R2 (detection quality). All slices are single representative slices. All scale bars, 1 mm. Earlier time points and data from additional mice can be found in fig. S7.

To assess the sensitivity of imaging with our rsOAPs, we imaged dorsal implants of Matrigel containing different numbers of Jurkat T lymphocytes stably coexpressing ReBphP-PCM and GFP in mice (Fig. 4G). We detected populations as small as 500 cells/l, suggesting the potential for sensitive tracking of immune processes. Similarly, imaging of dorsal implants of Matrigel containing bacteria expressing ReBphP-PCM detected populations as small as 14,000 bacteria/l (Fig. 4H). This sensitivity may be useful for studying and optimizing bacteria-based tumor therapies (24).

A strong advantage of photocontrollable labels is the possibility to delineate multiple labels based on their individual switching kinetics. To demonstrate this, we imaged 1-mm alginate beads filled with Escherichia coli expressing ReBphP-PCM, RpBphP1-PCM, or DrBphP-PCM. All beads were unambiguously identified on the basis of their switching kinetics (Fig. 5A). The same differentiation was achieved in vivo after implanting Jurkat T lymphocytes expressing ReBphP-PCM or DrBphP1-PCM and E. coli expressing RpBphP1-PCM into the back of mice (Fig. 5B).

(A) Imaging of an alginate bead phantom containing E. coli expressing rsOAPs ReBphP-PCM, RpBphP1-PCM, and DrBphP-PCM. (B) Imaging of Jurkat T cells and E. coli (1.4 106) expressing each of the three rsOAPs imaged immediately after implantation into a 4T1 tumor. (C) Imaging of a 4T1 tumor with implants of two Jurkat T cells expressing rsOAPs. Zones of mixture of the two populations with distinct kinetics are colored yellow. In (A) to (E), color maps indicate clusters showing distinguishable kinetics. (D) Imaging of a 4T1 tumor stably expressing ReBphP-PCM at day 9 (arrows II and III) imaged immediately after E. coli (108 cells) expressing DrBphP-PCM have been injected into the tumor (arrow I). Histology confirmation is inferred from fluorescence in DrBphP-PCM (Cy5 only) and ReBphP-PCM (GFP primarily). (E) Volume representation of k. All slices are single representative slices. All scale bars, 1 mm.

Because the kinetics of photoswitching are energy dependent, fluence changes due to light attenuation by surrounding absorbersphotochromic or staticcomplicates temporal multiplexing (note S5). Thus, one aim of our development of the fast-switching ReBphP-PCM was to achieve a switching time constant clearly separate from other rsOAPs. We show that 4T1 tumor expressing ReBphP-PCM and GFP are readily distinguished from infiltrating DrBphP-PCMexpressing E. coli cells (intratumorally injected 108 bacteria; Fig. 5, D and E). This means that multiplexing is possible for co-registration studies and that the concentrations of the labels can be estimated based on the convoluted kinetics (fig. S8). Similarly, we show this for two populations of rsOAP labeled Jurkat T lymphocytes in a 4T1 tumor (intratumorally injected 5 105 cells; Fig. 5C). Hence, temporally unmixed multiplexed OA imaging of cells of the immune system enables following their function and involvement in disease mechanism in vivo, longitudinal on the organism level.

The combination of OA and transgenic rsOAP labels allows the tracking of specific cell populations in vivo, which can open up possibilities for longitudinal studies of intact animals in diverse fields such as immunology, developmental biology, neurology, and cancer research. To support these studies, we describe next-generation rsOAPs that provide faster switching and greater resistance to photofatigue than existing rsOAPs, allowing highly sensitive detection, and importantly true multiplexing, without interference from hemoglobin or other abundant absorbers in vivo. These rsOAPs can be used with off-the-shelf equipment and our ML-based open-access image processing code to detect populations of fewer than 500 cells in vivo. The approach relies entirely on a time series of images, thus making the concept translatable between different OA imaging devices. These tools will facilitate the wider use of OA imaging in life sciences, particularly for the study of cellular dynamics and interactions on the level of whole organisms.

RpBphP1 (16) was obtained from Addgene (V. Verkhusha, plasmid no. 79845). Mammalian optimized ReBphP was synthesized as gene strings (GeneArt, Life Technologies, Regensburg, Germany). All other BphPs used in the study have been a gift from A. Mglich (University of Bayreuth, Germany).

For bacterial protein expression, the coding sequences of all BphPs used in the study except RpBphP1 were polymerase chain reaction (PCR)amplified as a Nde I/Xho I fragment and cloned into the second multiple cloning site of the pET-Duet1 vector (Novagen, Merck Millipore). RpBphP1 was PCR-amplified as a Nde I/Pac I fragment and cloned into the second multiple cloning site of the pET-Duet1 vector. In addition, for biliverdin synthesis, the heme oxygenase (HO) of Nostoc sp. was cloned using Nco I/Hind III into the first multiple cloning site of pET-Duet1.

For equimolar mammalian expression, first, ReBphP_P2A and mCherry were PCR-amplified and then stitched using overlap PCR as an Eco RI/Xba I fragment and cloned in a pcDNA3.0 vector (Thermo Fisher Scientific). Later similar constructs for other BphPs were made by amplifying them as Eco RI/Not I fragment and inserted in place of ReBphP1-PCM in the above construct. The resulting plasmids allowed the equimolar coexpression of RpBphP1, RpBphP1-PCM, ReBphP-PCM, ReBphP-PCM, or DrBphP-PCM and mCherry proteins.

Proteins have been expressed in E. coli strain BL21 (DE3) (New England Biolabs, #C2527). In brief, plasmids expressing BphPs and HO were transformed into the BL21 host cells. Bacterial cells were grown in LB media supplemented with ampicillin at 37C until the culture reached OD (optical density) 0.6, followed by induction of protein expression by addition of IPTG (isopropyl--d-thiogalactopyranoside) and further incubation for 16 to 18 hours at 22C. The next day, the bacterial pellet was collected by centrifugation and pellet was resuspended in phosphate-buffered saline (PBS). After cell lysis, proteins were purified by immobilized metal affinity chromatography in PBS, followed by gel filtration on a HiLoad 26/600 Superdex 75 pg (GE Healthcare Life Sciences, Freiburg, Germany).

For absorption spectra, the purification buffer was exchanged against PBS and the proteins were measured with a Shimadzu UV-1800 spectrophotometer (Shimadzu Inc., Kyoto, Japan) using a 100-l quartz cuvette. To measure the ON (Pfr) and OFF (Pr) spectra of respective proteins, photoswitching was carried out using 650/20-nm or 780/20-nm light-emitting diodes (Thorlabs) placed above the quartz cuvette in the spectrophotometer.

Fluorescence measurements for all BphPs were performed with a Cary Eclipse Fluorescence spectrophotometer (Varian Inc., Australia). Photoswitching was carried out as above. Fluorescence measurement was done by fixing excitation wavelength at 700 nm and emission wavelength at 720 nm. Excitation wavelength and emission slit were set to 5 nm, and the absorbance at the excitation wavelength was always equal to 0.1 to avoid inner filter effects.

4T1 and Jurkat cells were maintained in RPMI 1640. HeLa and HCT116 cells were maintained in Dulbeccos modified Eagles medium (DMEM) and McCoy 5A medium, respectively. All media were supplemented with 10% fetal bovine serum (Invitrogen) and antibiotics [penicillin (100 U/ml) and streptomycin (100 mg/ml)]. Cells were cultivated at 37C and 5% CO2.

Tissue culture. The Platinum-E and RD114 packaging cell lines were cultivated in cDMEM (Complete Dulbeccos modified Eagle medium), HCT116 cell line was grown in McCoy 5A medium (Life Technologies), and 4T1 and Jurkat cells were cultured in cRPMI (Complete Roswell Park Memorial Institute)1640 Medium. All media were supplemented with 10% fetal calf serum, 0.025% l-glutamine, 0.1% Hepes, 0.001% gentamicin, and 0.002% streptomycin.

Generation of constructs. ReBphP-PCM-IRES-GFP was amplified using specific primers (5-ATTAGCGGCCGCGCCACCATGAGCGGCACCAGAG-3 and 5-ATTAGAATTCTCACTTGTACAGCTCGTCCATGCCGTGAGTG-3) and cloned into the mP71 using Not I and Eco RI restriction sites. The mP71 vector was a gift from W. Uckert.

Generation of cell lines. For retrovirus production, Platinum-E or RD114 packaging cells were transfected with the retroviral vector mP71-ReBphP-PCM-IRES-GFP using calcium phosphate precipitation. The supernatant of the packing cells was collected at 48 and 72 hours after transfection and purified from the remaining cells by centrifugation at 1500 rpm at 4C for 7 min. One day before transduction, nontissue culturetreated 48-well plates were coated with RetroNectin (Clontech) according to the manufacturers recommendations overnight at 4C. After washing once with PBS, virus supernatant was added and centrifuged at 3000g and 32C for 2 hours. Virus supernatant was removed, and cell lines (4T1, HCT116, and Jurkat) were added in 400 l of the respective medium supplemented with 1:100 LentiBOOST Solution A and 1:100 LentiBOOST Solution B (Sirion Biotech). Cells were then spinoculated at 800g at 32C for 1.5 hours. After 5 days of culture, cells were sorted for high expression of GFP using flow cytometry.

All animal experiments were approved by the government of Upper Bavaria and were carried out in accordance with the approved guidelines. For 4T1 xenografts of stably expressing ReBphp-PCM and GFP, 0.8 106 cells in PBS have been implanted in the back of FoxN1 nude mice (Charles River Laboratories, Boston, USA) and maintained for 9 days. For HCT116 cells expressing ReBphP-PCM and GFP, 1.5 106 cells in 200 l PBS have been injected intraperitoneally in FoxN1 nude mice and were maintained for 14 days. For intracranial injections of stably expressing ReBphP-PCM and GFP 4T1 cells, mice were first anesthetized according to the animal protocol. The head of the mouse was fixed in a Stereotaxic frame (David Kopf Instruments, model 940), an incision in the skin was made using a scalpel, and a small hole was drilled into the skull. Later, 5-l cells (0.14 106 cells/l) were injected slowly with a 10-l Hamilton syringe (26Gs). The incision in the skin was closed using Histoacryl (B. Braun Melsungen AG). The mice were scanned in MSOT and sacrificed immediately after scanning. For Matrigel implants of Jurkat cells expressing ReBphP-PCM, different concentrations of cells ranging from 6400 to 500 cells/l were implanted subcutaneously in the back of the mice. Similarly, bacterial cells expressing ReBphP-PCM in different concentrations (1.4 105 to 1.4 104 cells/l) were also implanted in the back of the mice. For multiplexing experiment, bacterial cells expressing rsOAPs individually with the concentration of 1.4 106 cells/l were implanted on the back of the mice in the same plane. For multiplexing experiment in vivo, intratumoral injections, bacterial cells expressing DrBphP-PCM resuspended in PBS have been injected into the 4T1 tumor expressing ReBphP-PCM and GFP using an insulin syringe with a 30-gauge needle.

For all MSOT imaging, mice have been anesthetized using 2% isoflurane in O2. Anesthetized mice were placed in the MSOT holder using ultrasound gel and water as coupling media. After termination of the experiments, all mice have been sacrificed and stored at 80C for cryosectioning.

Phantom and mice data were acquired using a commercially available MSOT scanner (MSOT In Vision 256-TF, iThera Medical GmbH, Munich, Germany). In brief, nanosecond pulsed light was generated from a tunable optical parametric oscillator (OPO) laser and delivered to the sample through a ring-type fiber bundle. The wavelengths, 680 and 770 nm, were used for photoswitching and imaging in phantoms and in mice. Light absorbed by the sample generates an acoustic signal that propagates through the sample and is detected outside the sample by a cylindrically focused 256-element transducer. The transducer array had a central frequency of 5 MHz (6 dB was approximately 90%) with a radius of curvature of 40 mm and an angular coverage of 270. Acoustic signals were detected as time series pressure readouts at 2030 discrete time points at 40 MS/s (Mega-samples per second). The acquired acoustic data were reconstructed using the ViewMSOT version 3.8.1.04 (iThera Medical GmbH, Munich, Germany) software with the following settings: 50 kHz to 6.5 MHz; trim speed of 7.

All data analysis was conducted using MATLAB2018b. The data reconstructed with ViewMSOT were loaded into MATLAB by iThera MATLAB code (iThera MATLAB, version: msotlib_beta_rev75). All analyses were carried out with the code provided along with this manuscript (note S3). In brief, movement correction was done by phase correlation preliminary to optimization-based image co-registration with the intensity and nonrigid co-registration of frames of the first cycle being used as reference. For further processing, different features of the time series have been computed and are used for classification/switching label detection using an ML model. For fast Fourier transform, repetitive frequency of the whole concatenated signal for each image point is computed to identify signals corresponding to the illumination schedule. For exponential fitting, the normalized mean kinetic of all cycles is used. Then, the coefficients compared to an expected exponential kinetic are calculated and used as a quality measure. Here, positive and negative exponential are considered. Using fit coefficients and quality of fit (R2) as measures, only 77% accuracy compared to a ground truth is achieved. Thus, additional features are invoked. Overall, all analyzed features are (i and ii) the coefficient for the exponential fit (exp(b(x + 1)) and exp(b(x 1) + 1) of the mean kinetic (mean of all cycles); (iii) R2 of the fit; (iv) the mean intensity over the concatenated signal; (v) max-min of all the data at the pixel; (vi to ix) median maximums and minimums of cycles along with SD; (x) number of cycles with positive or negative trend; (xi) the length of the part of the cycle that shows a trend, i.e., at what point the signal vanishes in the noise; and (xii) Fourier coefficient for the expected frequency defined by the photocontrol schedule. All those are used as predictor values for an unmixing model based on random forest approaches (23, 25)for overall model, trained on 4T1 day 9 as well as highest concentration of Jurkat T lymphocytes. We used 50 trees in the ensemble, as further increase of number did not lead to out-of-bag error decrease. This approach resulted in model performance increase up to 96% of positive predictive value for ground truth (see note S4 for more details on the use of ML in this work).

For visualization, data were not further processed and are shown against the respective slice at 680 nm as anatomy information, except in the case of 4T1 injected in brain where the anatomy is shown at 900 nm. Representative slices are shown. For clustering, appropriate ranges of the kinetic parameter were chosen on the unmixed data to distinguish different labels.

After sacrificing, the mice were cryopreserved at 80C. To detect the fluorescence in tumors, the respective part of the mouse was embedded in Tissue-Tek O.C.T. (Sakura Finetek Europe B.V., Zoeterwonde, The Netherlands). Sections (10 m) were cut (Leica CM1950, Leica Microsystems, Wetzlar, Germany) for brain, 4T1, and HCT116 mice at the interval of 150, 250, and 500 m, respectively, and imaged using a 482/35-nm bandpass for excitation and 535/38-nm bandpass filter for detection of GFP fluorescence. Images were taken using an Andor LucaR charge-coupled device camera (DL-604M, Andor Technology, Belfast, UK) with 10-s exposure and a gain of 10. On the basis of the histology, ground truth on co-registered images was created using a semiautomatic procedure based on anatomical markers and intensity-based multimodal co-registration together with a nonrigid spline-based method and human (two independent)based selection of signals in the fluorescence images.

A 2 to 4% (w/v) aqueous solution of sodium alginate was prepared in PBS. E. coli strain BL21 cells expressing rsOAPs were harvested by centrifugation (4000 rpm, 20 min) and resuspended in PBS. The cell suspensions were then mixed with sterile alginate. Beads were formed by filling the alginate cell mixtures in the syringe with 30-gauge needle, followed by centrifugation at 300 rpm, which allowed the addition of the mixtures into sterile CaCl2 (200 mM). The cell-containing beads, 1 mm in diameter, were allowed to solidify for 10 min before CaCl2 was replaced by fresh distilled water. The cell beads were then randomly distributed in the agar phantom with 1.5% (w/w) agar and 3.5% (v/v) intralipid emulsion and imaged in MSOT as described elsewhere.

For OA characterization of rsOAPs, custom-made experimental setup was used as described earlier (22). Briefly, nanosecond excitation pulses were generated by an OPO laser (SpitLight DPSS 250 ZHGOPO, InnoLas) running at a repetition rate of 50 Hz. Constant pulse energy was ensured using a half-wave plate in a motorized rotation stage (PRM1Z8, Thorlabs) and a polarizing beam splitter; using a lookup table and adapting the polarization with the half-wave plate, we kept the power constant at 1.3 mJ (otherwise mentioned) over the whole illumination schedule. Samples were injected into an acoustically coupled flow chip (-Slide I 0.2 Luer, hydrophobic, uncoated, ibidi) and illuminated from one side using a fiber bundle (CeramOptec) at a constant pulse energy of 1.3 mJ at the fiber output. Photoswitching was carried out by illuminating the sample alternatively with 650- and 780-nm light. OA signals were detected with a cylindrically focused single-element transducer (V382-SU, 3.5 MHz, Olympus) followed by signal amplification by 60 dB with a wide-band voltage amplifier (DHPVA-100, Femto) and digitized at 100 MS/s with a data acquisition card (RZE-002 400, GaGe). Dependency of Pfr Pr conversion on 770-nm pulse energy was measured with different pulse energies (0.4, 0.7, 1.0, and 1.3 mJ). Dependency of Pfr Pr conversion on repetition rate of laser was measured with three different laser repetition rates (10, 25, and 50 Hz). Effect of different switching ON wavelength and resulting dynamic range at 770 nm was measured using different switching ON wavelength ranging from 630 to 680 nm.

Read more:
Multiplexed whole-animal imaging with reversibly switchable optoacoustic proteins - Science Advances

Cell Biology

Growth of the Life Science Tools Market has been on the back of all-encompassing Demand for High throughput Measurement Techniques – BioSpace

The drive for application in life sciences tools between life science researcher stems from the need for bioinformatics for unearthing a diverse array of scientific data. The research fraternity and industry alike leverage life science tools for gaining insights into various elements: DNA and RNA sequences, protein structures, biological pathways in drug making, and biological signals useful for disease prognosis. The major application areas in life sciences market include genetics and cell biology. Life sciences tools hold potential in biological data acquisition, data mining, and analysis. The life sciences industry affinity to leverage the potential of computation tools in synthetic and systems biology has led to the evolution of the life sciences tools market.

The report on the life sciences tools market provides a scrutiny of key growth dynamics, insights into emerging regulatory landscape, and elements of the competitive dynamics. The study offers a qualitative and quantitative analysis of new technological avenues. In addition, it offers a measure of potential of these developments for vaccine development and drug making.

Get Brochure of the Report @ https://www.tmrresearch.com/sample/sample?flag=B&rep_id=6591

Global Life Science Tools Market: Growth Dynamics

The growth of the life science tools market has been on the back of all-encompassing demand for high throughput measurement techniques across all domains of life sciences. Advances in omics, emerging avenues in synthetic and systems biology have unlocked new potential for market players. Perhaps, the most significant impetus to the expansion of life sciences market comes from the growing application of bioinformatics tools. The veritable explosion of sequencing data, notably from cancer research, has spurred demand for life sciences tools. Over the years, such research initiatives have formed a part of large-scale genomic research for precision medicine. A wide array of tools have been unveiled that will make collaborative approaches easy to adopt for life science researchers, such as cloud technology for cancer medicines.

Global Life Science Tools Market: Notable Developments

The life sciences tools market has in recent years witnessed a number of large-scale acquisition and strategic collaborations. Technology players has expanded their portfolio by forging collaborations with research institutes. A notable instance is Intel collaborating with Oregon Health & Science University to develop next-generation life sciences tools for advancing industry efforts in developing precision medicine for cancer. The project, concedes the partners, is one of its kind as it will enable life sciences researchers to offer clinicians tools for diagnosis disease based just on genome of a personprobably by the end of 2020. Several research institutes are expected to make a foray to make such initiatives reach fruition faster, thereby opening vast revenues streams in the life science tools market.

Buy this Premium Report @ https://www.tmrresearch.com/checkout?rep_id=6591&ltype=S

Some of the well-entrenched players in the life sciences tools market are Merck KGaA, Hitachi, Ltd., Danaher Corporation, Bio-Rad Laboratories, Inc., F. Hoffmann-La Roche Ltd., and Agilent Technologies.

Global Life Science Tools Market: Regional Assessment

Developed regions are seeing the growing trend of technology players unveiling scalable solutions for various omics projects. Markets such as North America has thus been hotbed of opportunities for the past several years. Countries such as the U.S. is seeing the rapid uptake of advances data analytics solutions by life sciences researchers, resulting in incredible scope in these markets.

On the other hand, the growing research in precision medicine has spurred the demand for life sciences tools in emerging markets. A notable emerging market is that of Asia Pacific. Life sciences organization in countries such as China have ramped up their efforts to unlock the potential of computation tools for systems biology. Other key regions in the life science tools market are Europe, the Middle East, and Latin America. These regional markets are seeing vast potential due to growing focus on governments on omics research that increasingly need technologically advanced life sciences tools.

Get Table of Content of the Report @ https://www.tmrresearch.com/sample/sample?flag=T&rep_id=6591

About TMR Research

TMR Research is a premier provider of customized market research and consulting services to business entities keen on succeeding in todays supercharged economic climate. Armed with an experienced, dedicated, and dynamic team of analysts, we are redefining the way our clients conduct business by providing them with authoritative and trusted research studies in tune with the latest methodologies and market trends.

Contact:

TMR Research,

3739 Balboa St # 1097,

San Francisco, CA 94121

United States

Tel: +1-415-520-1050

Visit Site: https://www.tmrresearch.com/

See the original post:
Growth of the Life Science Tools Market has been on the back of all-encompassing Demand for High throughput Measurement Techniques - BioSpace

Cell Biology

Linking glutamate receptor movements and synapse function – Science Magazine

Receptors moving in and out of the synapse

The number of neurotransmitter receptors and their spatial organization on the postsynaptic site is a central determinant of synaptic efficacy. Sophisticated techniques to visualize and track the movement of single molecules have provided us with profound new insights into these dynamics. We now know that neurotransmitter receptors undergo movements on different scales. Groc and Choquet review our present understanding of the mechanisms that regulate glutamate receptor localization and clustering. Receptor movements are fundamental to basic synaptic function and participate in many forms of synaptic plasticity.

Science, this issue p. eaay4631

Since it was established that the cognitive brain is formed mostly by an interconnected network of neurons that communicate at contact sites termed synapses, intense research has aimed at identifying their molecular composition and physiological roles. The discovery that the efficacy of synaptic transmission can be modified by neuronal activity has undoubtedly been a major step in understanding brain function. The various forms of activity-dependent synaptic plasticity were early on proposed to play central roles in brain adaptation, learning, and memory. This motivated neurophysiologists to understand the mechanisms of synaptic plasticity, initially within the sole framework of the quantal properties of transmitter release, largely ignoring the cell biology revolution that was occurring in parallel. In the 1970s, at the same time that synaptic plasticity was discovered, the fluidity of cell membranes was established. Surprisingly, these contemporary findings seldom crossed paths. As cell biologists established the major roles of receptor trafficking in cell function, neurophysiologists still largely viewed synapse function as based on unitary receptor properties and control of transmitter release. It has been only about 20 years since the two fields cross-fertilized and the regulation of receptor movements into and out of synapses emerged as a fundamental mechanism for synaptic plasticity.

Largely based on the development of imaging approaches, including single-molecule tracking, receptors have been demonstrated to undergo a variety of movements, from long-range rapid motor-based intracellular transport, to short-range Brownian surface diffusion, and intercompartment exchange by membrane trafficking. For efficient synaptic transmission, receptors must accumulate in front of neurotransmitter release sites. This is accomplished through a set of interactions with intracellular scaffold proteins, transmembrane auxiliary subunits, or adhesion proteins and other extracellular elements. This duality of receptor movements and stabilization has led to the important concept that the number of functionally responsive receptors at synapses results from the interplay between reversible receptor stabilization and dynamic equilibrium between pools of receptors in the synaptic, extrasynaptic, and intracellular compartments. Coarse receptor distribution along dendrites is largely achieved by intracellular transport. Because exchange of receptors between surface and intracellular compartments seems to occur largely at extrasynaptic sites, reversible surface receptor diffusion trapping at synapses has emerged as a central mechanism to control their availability for synaptic activation. Receptor stabilization and movements are all profoundly regulated by short- and long-term neuronal activity patterns. Reciprocally, evidence has accumulated that receptor movements participate in many forms of synaptic plasticity. Notably, altered receptor movements are observed in many neurodevelopmental, psychiatric, or neurodegenerative pathological models as indicated in the figure [the + and signs indicate the reported positive and negative modulation of the indicated trafficking and stabilization processes during either normal (blue) or pathological (red) synaptic function]. Whether altered receptor trafficking represents the primum movens of some neurological diseases remains to be established, but is certainly an attractive hypothesis.

Most receptor trafficking studies have been performed in reduced experimental systems such as neuronal cultures. This has limited our understanding of the physiological impact of these processes. The development of brighter and smaller probes together with new imaging modalities are on the verge of allowing routine measurement of receptor movements in more physiological settings such as brain slices and in vivo. There is little doubt that qualitatively comparable trafficking modalities will be identified. Reciprocally, tools are being developed to control the various types of receptor movements, from blocking surface diffusion by receptor cross-linking to stopping receptor exocytosis with light-activated toxins. Often, these trafficking tools do not impair basic synaptic function, because resilience of the synapse to trafficking alterations is high owing to the amount of available receptors, as well as the trapping capacities and nanoscale organization of the synapse. Combining measurement and control of receptor movements will not only allow better understanding of their contribution to synaptic and neuronal function but also provide valuable tools for identifying the role of synaptic plasticity in higher brain functions. Controlling receptor movements or stabilization may eventually represent an alternative therapeutic strategy to receptor activity modulation approaches in a variety of synaptic and network-based brain diseases.

Movements of large amplitude constantly reshuffle the receptor distribution (e.g., surface diffusion and intracellular transport). Movements at interfaces (e.g., between synaptic and extrasynaptic sites, between intracellular and surface compartments) are of small amplitude but have huge functional impacts. Each of these movements is highly regulated and finely tuned in physiological and pathological conditions.

Regulation of neurotransmitter receptor content at synapses is achieved through a dynamic equilibrium between biogenesis and degradation pathways, receptor stabilization at synaptic sites, and receptor trafficking in and out synapses. In the past 20 years, the movements of receptors to and from synapses have emerged as a series of highly regulated processes that mediate postsynaptic plasticity. Our understanding of the properties and roles of receptor movements has benefited from technological advances in receptor labeling and tracking capacities, as well as from new methods to interfere with their movements. Focusing on two key glutamatergic receptors, we review here our latest understanding of the characteristics of receptor movements and their role in tuning the efficacy of synaptic transmission in health and brain disease.

Link:
Linking glutamate receptor movements and synapse function - Science Magazine

Cell Biology

COVID 19 Impact on Cell Culture Market: 2020 Global Industry Size, Segments, Future Trends, Growth Factors, Company Profiles and Forecast till 2026 -…

The Cell Culture Market is expected to grow during the forecast period owing to the increase in awareness regarding the potential benefits regarding cell culture based vaccines. Additionally, the rising demand for monoclonal antibodies can also help in augmenting the market growth. However, high cost of cell biology research might impede the growth of the market.

Get Sample Copy of this Report @https://www.orianresearch.com/request-sample/1154326

Report Covers Market Segment by Manufacturers: Becton, Dickinson and Company Cellgenix Corning Danaher Eppendorf Fujifilm Irvine Scientific (Acquired By Fujifilm Corporation).

Cell culture is the process by which cells are grown under controlled conditions, generally outside of their natural environment. Cell culture is one of the major tools used in cellular and molecular biology, since it provides excellent model systems for studying the normal physiology and biochemistry of cells and the effects of drugs and toxic compounds on the cells. It is also used in the development of biological compounds.

Cell Culture Industry report offers a comprehensive insight into the development policies and plans in addition to manufacturing processes and cost structures. On the basis of product, this report displays the cost structure, sales revenue, sales volume, gross margin, market share and growth rate.

Pharmaceutical & Biotechnology Companies Hospitals and Diagnostic Laboratories Research Institutes Cell Banks

Key Benefits of the Report:

Global, and regional, product type & application market size and their forecast from 2019-2026 Identification and detailed analysis on key market dynamics, such as, drivers, restraints, opportunities, and challenges influencing the growth of the market

Detailed analysis on industry outlook with market specific PEST analysis, and Supply Chain to better understand the market and build expansion strategies Identification of key market players and comprehensively analyze their market share and core competencies, detailed financial positions, key products, and unique selling points Analysis on key players strategic initiatives and competitive developments, such as agreements & joint ventures, mergers & acquisitions, expansion, and new product launches in the market Expert interviews and their insights on market trends, market shift, current and future outlook, and factors impacting vendors short term & long term strategies Detailed insights on emerging regions, product type and application with qualitative and quantitative information and facts Identification of the key patents filed in the field of Cell Culture equipment and other related technologies.

Target Audience: Cell Culture providers Traders, Importer and Exporter Raw material suppliers and distributors Research and consulting firms Government and research organizations Associations and industry bodies

We have assigned weights to these parameters and quantified their market impacts using the weighted average analysis to derive the expected market growth rate.The market estimates and forecasts have been verified through exhaustive primary research with theKey Industry Participants (KIPs) which typically include: Original Equipment Manufacturer Component Supplier Distributors Government Body & Associations Research Institute

Orian Research is one of the most comprehensive collections of market intelligence reports on the World Wide Web. Our reports repository boasts of over 500000+ industry and country research reports from over 100 top publishers. We continuously update our repository so as to provide our clients easy access to the worlds most complete and current database of expert insights on global industries, companies, and products. We also specialize in custom research in situations where our syndicate research offerings do not meet the specific requirements of our esteemed clients.

See the original post:
COVID 19 Impact on Cell Culture Market: 2020 Global Industry Size, Segments, Future Trends, Growth Factors, Company Profiles and Forecast till 2026 -...

Cell Biology

Majority of Evaluable Patients Across Genotypes Achieve Transfusion Independence and Maintain It with Near-Normal Hemoglobin Levels in Phase 3 Studies…

89% of evaluable patients (17/19) with transfusion-dependent -thalassemia who do not have a 0/0 genotype achieved transfusion independence with 11.9 g/dL median weighted average total hemoglobin (Hb) level in HGB-207

Data from exploratory analyses of HGB-207 show improved markers of blood cell production and bone marrow function in patients who achieved transfusion independence

85% of patients (11/13) with a 0/0 genotype or IVS-I-110 mutation in HGB-212 have been transfusion-free for at least 7 months

bluebird bio, Inc. (Nasdaq: BLUE) today announced that new data from ongoing Phase 3 studies of betibeglogene autotemcel (beti-cel; formerly LentiGlobin for -thalassemia gene therapy) show pediatric, adolescent and adult patients with a range of genotypes of transfusion-dependent -thalassemia (TDT) achieve and maintain transfusion independence with hemoglobin (Hb) levels that are near-normal (10.5 g/dL). These data are being presented at the Virtual Edition of the 25th European Hematology Association (EHA25) Annual Congress.

"With more than a decade of clinical experience evaluating gene therapy in patients with transfusion dependent -thalassemia across a wide range of ages and genotypes, we have built the most comprehensive understanding of treatment outcomes in the field," said David Davidson, M.D., chief medical officer, bluebird bio. "Seeing patients achieve transfusion independence and maintain that positive clinical benefit over time with robust hemoglobin levels reflects our initial vision of the potential of beti-cel. The accumulating long-term data demonstrating improvements in bone marrow histology, iron balance and red cell biology support the potential of beti-cel to correct the underlying pathophysiology of transfusion-dependent -thalassemia."

A total of 60 pediatric, adolescent and adult patients across genotypes of TDT have been treated with beti-cel in the Phase 1/2 Northstar (HGB-204) and HGB-205 studies, and the Phase 3 Northstar-2 (HGB-207) and Northstar-3 (HGB-212) studies as of March 3, 2020. In studies of beti-cel, transfusion independence is defined as no longer needing red blood cell transfusions for at least 12 months while maintaining a weighted average Hb of at least 9 g/dL.

TDT is a severe genetic disease caused by mutations in the -globin gene that results in significantly reduced or absent adult hemoglobin (HbA). In order to survive, people with TDT maintain Hb levels through lifelong, chronic blood transfusions. These transfusions carry the risk of progressive multi-organ damage due to unavoidable iron overload.

"Patients with transfusion-dependent -thalassemia do not make enough healthy red blood cells and cannot live without chronic transfusions; for patients that means a lifetime of necessary visits to a hospital or clinic and reliance on an often unreliable blood supply, which compounds the challenges of managing this disease," said presenting study author Professor John B. Porter, MA, M.D., FRCP, FRCPath, University College London Hospital, London, UK. "These results showing patients free from transfusions and maintaining near-normal hemoglobin levels after treatment with beti-cel is a positive outcome for people living with transfusion-dependent -thalassemia. In addition, we now have more data that provide further evidence that most of these patients have a measurable improvement in markers of healthy red blood cell production."

Beti-cel is a one-time gene therapy designed to address the underlying genetic cause of TDT by adding functional copies of a modified form of the -globin gene (A-T87Q-globin gene) into a patients own hematopoietic (blood) stem cells (HSCs). This means there is no need for donor HSCs from another person, as is required for allogeneic HSC transplantation (allo-HSCT). Once a patient has the A-T87Q-globin gene, they have the potential to produce HbAT87Q, which is gene therapy-derived Hb, at levels that eliminate or significantly reduce the need for transfusions.

As of March 3, 2020, all 23 patients in HGB-207 were treated and have been followed for a median of 19.4 months. These patients ranged in age from four to 34 years, including eight pediatric (<12 years of age) and 15 adolescent/adult (>12 years of age) patients. Only 19 patients were evaluable for transfusion independence; four additional patients do not yet have sufficient follow-up to be assessed for transfusion independence.

Eighty-nine percent of evaluable patients (17/19) achieved transfusion independence, with median weighted average total Hb levels of 11.9 g/dL (min-max: 9.4 12.9 g/dL) over a median of 19.4 months of follow-up to date (min-max: 12.3 31.4 months). These 17 patients previously required a median of 17.5 transfusions per year (min-max: 11.5 37 transfusions per year).

Improved iron levels, as measured by serum ferritin and hepcidin levels (proteins involved in iron storage and homeostasis), were observed and trends toward improved iron management were seen. Over half of patients stopped chelation therapy, which is needed to reduce excess iron caused by chronic blood transfusions. Seven out of 23 patients began using phlebotomy for iron reduction.

Analysis of Healthy Red Blood Cell Production

In exploratory analyses, biomarkers of ineffective erythropoiesis (red blood cell production) were evaluated in patients who achieved transfusion independence in HGB-207.

The myeloid to erythroid (M:E) ratio in bone marrow from patients who achieved transfusion independence increased from a median of 1:3 (n=17) at baseline to 1:1.2 (n=16) at Month 12. Improvement of the M:E ratio, the ratio of white blood cell and red blood cell precursors in the bone marrow, suggests an improvement in mature red blood cell production. Images illustrating the bone marrow cellularity at baseline, Month 12 and Month 24 are available in the EHA25 presentation (abstract #S296): "Improvement in erythropoiesis in patients with transfusion-dependent -thalassemia following treatment with betibeglogene autotemcel (LentiGlobin for -thalassemia) in the Phase 3 HGB-207 study".

Additionally, biomarkers of erythropoiesis continue to demonstrate a trend toward normalization in patients who achieved transfusion independence, including improved levels over time of erythropoietin, a hormone involved in red blood cell production; reticulocytes, immature red blood cells; and soluble transferrin receptor, a protein measured to help evaluate iron status. The continued normalization of red blood cell production over time among some patients who achieved transfusion independence supports the disease-modifying potential of beti-cel in patients with TDT.

Northstar-3 (HGB-212) Efficacy

As of March 3, 2020, 15 patients (genotypes: 9 0/0, 3 0/ +IVS1-110, 3 homozygous IVS-1-110 mutation) were treated and had a median follow-up of 14.4 months (min-max: 1.124.0 months). Median age at enrollment was 15 (min-max: 4 33 years).

Six of eight evaluable patients achieved transfusion independence, with median weighted average total Hb levels of 11.5 g/dL (min-max: 9.5 13.5 g/dL), and continued to maintain transfusion independence for a median duration of 13.6 months (min-max: 12.2 21.2 months) as of the data cutoff.

Eighty-five percent of patients (11/13) with at least seven months of follow-up had not received a transfusion in more than seven months at time of data cutoff. These 11 patients previously required a median of 18.5 transfusions per year (min-max: 11.0 39.5 transfusions per year). In these patients, gene therapy-derived HbAT87Q supported total Hb levels ranging from 8.814.0 g/dL at last visit.

Betibeglogene autotemcel Safety

Non-serious adverse events (AEs) observed during the HGB-207 and HGB-212 trials that were considered related or possibly related to beti-cel were tachycardia, abdominal pain, pain in extremities, leukopenia, neutropenia and thrombocytopenia. One serious event of thrombocytopenia was considered possibly related to beti-cel.

In HGB-207, serious events post-infusion in two patients included three events of veno-occlusive liver disease and two events of thrombocytopenia. In HGB-212, serious events post-infusion in two patients included two events of pyrexia.

Additional AEs observed in clinical studies were consistent with the known side effects of HSC collection and bone marrow ablation with busulfan, including SAEs of veno-occlusive disease.

In both Phase 3 studies, there have been no deaths, no graft failure, no cases of vector-mediated replication competent lentivirus or clonal dominance, no leukemia and no lymphoma.

The presentations are now available on demand on the EHA25 website:

About betibeglogene autotemcel

The European Commission granted conditional marketing authorization (CMA) for betibeglogene autotemcel (beti-cel; formerly LentiGlobin gene therapy for -thalassemia), marketed as ZYNTEGLO gene therapy, for patients 12 years and older with transfusion-dependent -thalassemia (TDT) who do not have a 0/0 genotype, for whom hematopoietic stem cell (HSC) transplantation is appropriate, but a human leukocyte antigen (HLA)-matched related HSC donor is not available. On April 28, 2020, the European Medicines Agency (EMA) renewed the CMA for ZYNTEGLO, supported by data from 32 patients treated with ZYNTEGLO, including three patients with up to five years of follow-up.

TDT is a severe genetic disease caused by mutations in the -globin gene that result in reduced or significantly reduced hemoglobin (Hb). In order to survive, people with TDT maintain Hb levels through lifelong chronic blood transfusions. These transfusions carry the risk of progressive multi-organ damage due to unavoidable iron overload.

Beti-cel adds functional copies of a modified form of the -globin gene (A-T87Q-globin gene) into a patients own hematopoietic (blood) stem cells (HSCs). Once a patient has the A-T87Q-globin gene, they have the potential to produce HbAT87Q, which is gene therapy-derived hemoglobin, at levels that may eliminate or significantly reduce the need for transfusions.

Non-serious adverse events (AEs) observed during clinical studies that were attributed to beti-cel included abdominal pain, thrombocytopenia, leukopenia, neutropenia, hot flush, dyspnea, pain in extremity and non-cardiac chest pain. Two serious adverse events (SAE) of thrombocytopenia was considered possibly related to beti-cel.

Additional AEs observed in clinical studies were consistent with the known side effects of HSC collection and bone marrow ablation with busulfan, including SAEs of veno-occlusive disease.

The CMA for beti-cel is valid in the 27 member states of the EU as well as UK, Iceland, Liechtenstein and Norway. For details, please see the Summary of Product Characteristics (SmPC).

The U.S. Food and Drug Administration (FDA) granted beti-cel orphan drug designation and Breakthrough Therapy designation for the treatment of transfusion-dependent -thalassemia. Beti-cel is not approved in the U.S.

Beti-cel continues to be evaluated in the ongoing Phase 3 Northstar-2 and Northstar-3 studies. For more information about the ongoing clinical studies, visit http://www.northstarclinicalstudies.com or clinicaltrials.gov and use identifier NCT02906202 for Northstar-2 (HGB-207) and NCT03207009 for Northstar-3 (HGB-212).

bluebird bio is conducting a long-term safety and efficacy follow-up study (LTF-303) for people who have participated in bluebird bio-sponsored clinical studies of betibeglogene autotemcel or LentiGlobin for SCD. For more information visit: https://www.bluebirdbio.com/our-science/clinical-trials or clinicaltrials.gov and use identifier NCT02633943 for LTF-303.

About bluebird bio, Inc.

bluebird bio is pioneering gene therapy with purpose. From our Cambridge, Mass., headquarters, were developing gene therapies for severe genetic diseases and cancer, with the goal that people facing potentially fatal conditions with limited treatment options can live their lives fully. Beyond our labs, were working to positively disrupt the healthcare system to create access, transparency and education so that gene therapy can become available to all those who can benefit.

bluebird bio is a human company powered by human stories. Were putting our care and expertise to work across a spectrum of disorders including cerebral adrenoleukodystrophy, sickle cell disease, -thalassemia and multiple myeloma using three gene therapy technologies: gene addition, cell therapy and (megaTAL-enabled) gene editing.

bluebird bio has additional nests in Seattle, Wash; Durham, N.C.; and Zug, Switzerland. For more information, visit bluebirdbio.com.

Follow bluebird bio on social media: @bluebirdbio, LinkedIn, Instagram and YouTube.

ZYNTEGLO, LentiGlobin, and bluebird bio are trademarks of bluebird bio, Inc.

bluebird bio Forward-Looking Statements

This release contains "forward-looking statements" within the meaning of the Private Securities Litigation Reform Act of 1995. Any forward-looking statements are based on managements current expectations of future events and are subject to a number of risks and uncertainties that could cause actual results to differ materially and adversely from those set forth in or implied by such forward-looking statements. These risks and uncertainties include, but are not limited to: the risk that the COVID-19 pandemic and resulting impact on our operations and healthcare systems will affect the execution of our development plans or the conduct of our clinical studies; the risk that the efficacy and safety results observed in the patients treated in our prior and ongoing clinical trials of beti-cel may not persist; and the risk that the efficacy and safety results from our prior and ongoing clinical trials will not continue or be repeated with additional patients in our ongoing or planned clinical trials or in the commercial context; the risk that the FDA will require additional information regarding beti-cel, resulting in a delay to our anticipated timelines for regulatory submissions, including submission of our BLA. For a discussion of other risks and uncertainties, and other important factors, any of which could cause our actual results to differ from those contained in the forward-looking statements, see the section entitled "Risk Factors" in our most recent Form 10-Q, as well as discussions of potential risks, uncertainties, and other important factors in our subsequent filings with the Securities and Exchange Commission. All information in this press release is as of the date of the release, and bluebird bio undertakes no duty to update this information unless required by law.

View source version on businesswire.com: https://www.businesswire.com/news/home/20200612005084/en/

Contacts

Media:Catherine Falcetti, 339-499-9436cfalcetti@bluebirdbio.com

Investors:Ingrid Goldberg, 410-960-5022igoldberg@bluebirdbio.com

Elizabeth Pingpank, 617-914-8736epingpank@bluebirdbio.com

See the article here:
Majority of Evaluable Patients Across Genotypes Achieve Transfusion Independence and Maintain It with Near-Normal Hemoglobin Levels in Phase 3 Studies...

Cell Biology

An ion channel senses cell swelling and helps cells to choose a response – Washington University in St. Louis Newsroom

After a dry spell, a rainy day can feel rejuvenating. But for plants, a downpour can mean trouble. Faced with water suddenly rushing into its tissues, a plant must control its cells volume or risk them exploding.

New research from Washington University in St. Louis offers clues about how mechanosensitive ion channels in the plants cells respond to swelling by inducing cell death potentially to protect the rest of the plant.

The plants response to cell swelling has been studied for a long time and a lot is known about the signaling events. However, the sensor that detects cell swelling in the first place was not known, said Liz Haswell, professor of biology in Arts & Sciences.

The discovery reported by Haswell and Debarati Basu, postdoctoral research scholar in the Haswell lab, in the June 11 issue of Current Biology provides insight into how plants sense and respond to mechanical signals, such as cell swelling, rather than chemicals signals, such as nutrients or growth factors.

Plant cells are armed with a strong yet flexible outer cell wall that holds back the force of water pushing out from inside the cell. Lacking a skeleton, plants only have the force of water and cellulose to keep them upright. Without that force, they go limp. But as the pressure pushing out turgor pressure becomes too great, the cell swells and an imbalance occurs.

It has been documented in plants that cell swelling leads to a release of calcium into the cell cytoplasm and a buildup of reactive oxygen species, unstable molecules containing oxygen that can lead to cell death. As the cell responds to the swelling, specific genes get turned on or off.

But the player that senses cell swelling has been missing.

Sandwiched between the outer cell wall and the internal contents of the cell is the plasma membrane. Embedded in the plasma membrane are mechanosensitive ion channels or tunnels that release ions in a response to membrane stretch. Mechanosensitive ion channel 10 (MSL10) is one member of the family of mechanosensitive ion channels that is a focus of the Haswell lab.

Basu applied a chemical that would cause the cell wall to lose its strength and become soft. At the same time, she could increase the turgor pressure inside the cell and study the role of MSL10 in the initial steps involved in the cell swelling response.

Plant cells, carrying a mutation that made MSL10 overly active, responded to cell swelling similarly to wildtype plants calcium was released, reactive oxygen species made and gene expression changed. However, the response was more pronounced and missing when the plant cells lacked MSL10.

Basu and Haswell discovered that MSL10 is not only an ion transporter but also a primary responder to cell swelling.

MSL10 is an ion channel, so its tempting to think that it itself is transporting calcium. That may not be true, explained Basu. Our results propose the possibility that MSL10 senses the cell swelling and activates a different channel that then transports the calcium.

As the cell swelled, the cell wall failed to maintain the force of the turgor pressure. But it did not explode. Instead, the cell died. But only plants with functional MSL10 died. In plants lacking MSL10, death was avoided.

This might seem counterintuitive, Haswell said. Why is MSL10 required for cells to die youd expect it to save cells lives during swelling, not the other way around. The key is that cells werent dying a normal kind of death, they were undergoing programmed cell death.

Basu found that MSL10 activates programmed cell death a regulatory mechanism that originates from inside of the cell. Cell damage itself did not cause death; MSL10 triggered a program of cell suicide.

Why the plant triggers cell suicide in response to cell swelling is still a mystery. But Basu and Haswell have some intriguing hypotheses.

The plasma membrane has probably been damaged. So maybe the plant wants to recoup some of that material and incorporate it back into the plant through this regulated process, Basu offered.

Or perhaps these damaged cells are more susceptible to infection, and the plant commits cell suicide as a way to save the plant at the sacrifice of a few cells.

We already know that when a pathogen infects a plant, the plant will kill off a bunch of the cells that are infected to prevent the spread of the infection, Haswell said. This idea of cell suicide in response to mechanical stimuli is intriguing.

Original post:
An ion channel senses cell swelling and helps cells to choose a response - Washington University in St. Louis Newsroom

Cell Biology

bluebird bio : Majority of Evaluable Patients Across Genotypes Achieve Transfusion Independence and Maintain It with Near-Normal Hemoglobin Levels in…

89% of evaluable patients (17/19) with transfusion-dependent -thalassemia who do not have a 0/0 genotype achieved transfusion independence with 11.9 g/dL median weighted average total hemoglobin (Hb) level in HGB-207

Data from exploratory analyses of HGB-207 show improved markers of blood cell production and bone marrow function in patients who achieved transfusion independence

85% of patients (11/13) with a 0/0 genotype or IVS-I-110 mutation in HGB-212 have been transfusion-free for at least 7 months

bluebird bio, Inc. (Nasdaq: BLUE) today announced that new data from ongoing Phase 3 studies of betibeglogene autotemcel (beti-cel; formerly LentiGlobin for -thalassemia gene therapy) show pediatric, adolescent and adult patients with a range of genotypes of transfusion-dependent -thalassemia (TDT) achieve and maintain transfusion independence with hemoglobin (Hb) levels that are near-normal (10.5 g/dL). These data are being presented at the Virtual Edition of the 25th European Hematology Association (EHA25) Annual Congress.

With more than a decade of clinical experience evaluating gene therapy in patients with transfusion dependent -thalassemia across a wide range of ages and genotypes, we have built the most comprehensive understanding of treatment outcomes in the field, said David Davidson, M.D., chief medical officer, bluebird bio. Seeing patients achieve transfusion independence and maintain that positive clinical benefit over time with robust hemoglobin levels reflects our initial vision of the potential of beti-cel. The accumulating long-term data demonstrating improvements in bone marrow histology, iron balance and red cell biology support the potential of beti-cel to correct the underlying pathophysiology of transfusion-dependent -thalassemia.

A total of 60 pediatric, adolescent and adult patients across genotypes of TDT have been treated with beti-cel in the Phase 1/2 Northstar (HGB-204) and HGB-205 studies, and the Phase 3 Northstar-2 (HGB-207) and Northstar-3 (HGB-212) studies as of March 3, 2020. In studies of beti-cel, transfusion independence is defined as no longer needing red blood cell transfusions for at least 12 months while maintaining a weighted average Hb of at least 9 g/dL.

TDT is a severe genetic disease caused by mutations in the -globin gene that results in significantly reduced or absent adult hemoglobin (HbA). In order to survive, people with TDT maintain Hb levels through lifelong, chronic blood transfusions. These transfusions carry the risk of progressive multi-organ damage due to unavoidable iron overload.

Patients with transfusion-dependent -thalassemia do not make enough healthy red blood cells and cannot live without chronic transfusions; for patients that means a lifetime of necessary visits to a hospital or clinic and reliance on an often unreliable blood supply, which compounds the challenges of managing this disease, said presenting study author Professor John B. Porter, MA, M.D., FRCP, FRCPath, University College London Hospital, London, UK. These results showing patients free from transfusions and maintaining near-normal hemoglobin levels after treatment with beti-cel is a positive outcome for people living with transfusion-dependent -thalassemia. In addition, we now have more data that provide further evidence that most of these patients have a measurable improvement in markers of healthy red blood cell production.

Beti-cel is a one-time gene therapy designed to address the underlying genetic cause of TDT by adding functional copies of a modified form of the -globin gene (A-T87Q-globin gene) into a patients own hematopoietic (blood) stem cells (HSCs). This means there is no need for donor HSCs from another person, as is required for allogeneic HSC transplantation (allo-HSCT). Once a patient has the A-T87Q-globin gene, they have the potential to produce HbAT87Q, which is gene therapy-derived Hb, at levels that eliminate or significantly reduce the need for transfusions.

Northstar-2 (HGB-207) Efficacy

As of March 3, 2020, all 23 patients in HGB-207 were treated and have been followed for a median of 19.4 months. These patients ranged in age from four to 34 years, including eight pediatric (<12 years of age) and 15 adolescent/adult (>12 years of age) patients. Only 19 patients were evaluable for transfusion independence; four additional patients do not yet have sufficient follow-up to be assessed for transfusion independence.

Eighty-nine percent of evaluable patients (17/19) achieved transfusion independence, with median weighted average total Hb levels of 11.9 g/dL (min-max: 9.4 12.9 g/dL) over a median of 19.4 months of follow-up to date (min-max: 12.3 31.4 months). These 17 patients previously required a median of 17.5 transfusions per year (min-max: 11.5 37 transfusions per year).

Improved iron levels, as measured by serum ferritin and hepcidin levels (proteins involved in iron storage and homeostasis), were observed and trends toward improved iron management were seen. Over half of patients stopped chelation therapy, which is needed to reduce excess iron caused by chronic blood transfusions. Seven out of 23 patients began using phlebotomy for iron reduction.

Analysis of Healthy Red Blood Cell Production

In exploratory analyses, biomarkers of ineffective erythropoiesis (red blood cell production) were evaluated in patients who achieved transfusion independence in HGB-207.

The myeloid to erythroid (M:E) ratio in bone marrow from patients who achieved transfusion independence increased from a median of 1:3 (n=17) at baseline to 1:1.2 (n=16) at Month 12. Improvement of the M:E ratio, the ratio of white blood cell and red blood cell precursors in the bone marrow, suggests an improvement in mature red blood cell production. Images illustrating the bone marrow cellularity at baseline, Month 12 and Month 24 are available in the EHA25 presentation (abstract #S296): Improvement in erythropoiesis in patients with transfusion-dependent -thalassemia following treatment with betibeglogene autotemcel (LentiGlobin for -thalassemia) in the Phase 3 HGB-207 study.

Additionally, biomarkers of erythropoiesis continue to demonstrate a trend toward normalization in patients who achieved transfusion independence, including improved levels over time of erythropoietin, a hormone involved in red blood cell production; reticulocytes, immature red blood cells; and soluble transferrin receptor, a protein measured to help evaluate iron status. The continued normalization of red blood cell production over time among some patients who achieved transfusion independence supports the disease-modifying potential of beti-cel in patients with TDT.

Northstar-3 (HGB-212) Efficacy

As of March 3, 2020, 15 patients (genotypes: 9 0/0, 3 0/ +IVS1-110, 3 homozygous IVS-1-110 mutation) were treated and had a median follow-up of 14.4 months (min-max: 1.124.0 months). Median age at enrollment was 15 (min-max: 4 33 years).

Six of eight evaluable patients achieved transfusion independence, with median weighted average total Hb levels of 11.5 g/dL (min-max: 9.5 13.5 g/dL), and continued to maintain transfusion independence for a median duration of 13.6 months (min-max: 12.2 21.2 months) as of the data cutoff.

Eighty-five percent of patients (11/13) with at least seven months of follow-up had not received a transfusion in more than seven months at time of data cutoff. These 11 patients previously required a median of 18.5 transfusions per year (min-max: 11.0 39.5 transfusions per year). In these patients, gene therapy-derived HbAT87Q supported total Hb levels ranging from 8.814.0 g/dL at last visit.

Betibeglogene autotemcel Safety

Non-serious adverse events (AEs) observed during the HGB-207 and HGB-212 trials that were considered related or possibly related to beti-cel were tachycardia, abdominal pain, pain in extremities, leukopenia, neutropenia and thrombocytopenia. One serious event of thrombocytopenia was considered possibly related to beti-cel.

In HGB-207, serious events post-infusion in two patients included three events of veno-occlusive liver disease and two events of thrombocytopenia. In HGB-212, serious events post-infusion in two patients included two events of pyrexia.

Additional AEs observed in clinical studies were consistent with the known side effects of HSC collection and bone marrow ablation with busulfan, including SAEs of veno-occlusive disease.

In both Phase 3 studies, there have been no deaths, no graft failure, no cases of vector-mediated replication competent lentivirus or clonal dominance, no leukemia and no lymphoma.

The presentations are now available on demand on the EHA25 website:

About betibeglogene autotemcel

The European Commission granted conditional marketing authorization (CMA) for betibeglogene autotemcel (beti-cel; formerly LentiGlobin gene therapy for -thalassemia), marketed as ZYNTEGLO gene therapy, for patients 12 years and older with transfusion-dependent -thalassemia (TDT) who do not have a 0/0 genotype, for whom hematopoietic stem cell (HSC) transplantation is appropriate, but a human leukocyte antigen (HLA)-matched related HSC donor is not available. On April 28, 2020, the European Medicines Agency (EMA) renewed the CMA for ZYNTEGLO, supported by data from 32 patients treated with ZYNTEGLO, including three patients with up to five years of follow-up.

TDT is a severe genetic disease caused by mutations in the -globin gene that result in reduced or significantly reduced hemoglobin (Hb). In order to survive, people with TDT maintain Hb levels through lifelong chronic blood transfusions. These transfusions carry the risk of progressive multi-organ damage due to unavoidable iron overload.

Beti-cel adds functional copies of a modified form of the -globin gene (A-T87Q-globin gene) into a patients own hematopoietic (blood) stem cells (HSCs). Once a patient has the A-T87Q-globin gene, they have the potential to produce HbAT87Q, which is gene therapy-derived hemoglobin, at levels that may eliminate or significantly reduce the need for transfusions.

Non-serious adverse events (AEs) observed during clinical studies that were attributed to beti-cel included abdominal pain, thrombocytopenia, leukopenia, neutropenia, hot flush, dyspnea, pain in extremity and non-cardiac chest pain. Two serious adverse events (SAE) of thrombocytopenia was considered possibly related to beti-cel.

Additional AEs observed in clinical studies were consistent with the known side effects of HSC collection and bone marrow ablation with busulfan, including SAEs of veno-occlusive disease.

The CMA for beti-cel is valid in the 27 member states of the EU as well as UK, Iceland, Liechtenstein and Norway. For details, please see the Summary of Product Characteristics (SmPC).

The U.S. Food and Drug Administration (FDA) granted beti-cel orphan drug designation and Breakthrough Therapy designation for the treatment of transfusion-dependent -thalassemia. Beti-cel is not approved in the U.S.

Beti-cel continues to be evaluated in the ongoing Phase 3 Northstar-2 and Northstar-3 studies. For more information about the ongoing clinical studies, visit http://www.northstarclinicalstudies.com or clinicaltrials.gov and use identifier NCT02906202 for Northstar-2 (HGB-207) and NCT03207009 for Northstar-3 (HGB-212).

bluebird bio is conducting a long-term safety and efficacy follow-up study (LTF-303) for people who have participated in bluebird bio-sponsored clinical studies of betibeglogene autotemcel or LentiGlobin for SCD. For more information visit: https://www.bluebirdbio.com/our-science/clinical-trials or clinicaltrials.gov and use identifier NCT02633943 for LTF-303.

About bluebird bio, Inc.

bluebird bio is pioneering gene therapy with purpose. From our Cambridge, Mass., headquarters, were developing gene therapies for severe genetic diseases and cancer, with the goal that people facing potentially fatal conditions with limited treatment options can live their lives fully. Beyond our labs, were working to positively disrupt the healthcare system to create access, transparency and education so that gene therapy can become available to all those who can benefit.

bluebird bio is a human company powered by human stories. Were putting our care and expertise to work across a spectrum of disorders including cerebral adrenoleukodystrophy, sickle cell disease, -thalassemia and multiple myeloma using three gene therapy technologies: gene addition, cell therapy and (megaTAL-enabled) gene editing.

bluebird bio has additional nests in Seattle, Wash; Durham, N.C.; and Zug, Switzerland. For more information, visit bluebirdbio.com.

Follow bluebird bio on social media: @bluebirdbio, LinkedIn, Instagram and YouTube.

ZYNTEGLO, LentiGlobin, and bluebird bio are trademarks of bluebird bio, Inc.

bluebird bio Forward-Looking Statements

This release contains forward-looking statements within the meaning of the Private Securities Litigation Reform Act of 1995. Any forward-looking statements are based on managements current expectations of future events and are subject to a number of risks and uncertainties that could cause actual results to differ materially and adversely from those set forth in or implied by such forward-looking statements. These risks and uncertainties include, but are not limited to: the risk that the COVID-19 pandemic and resulting impact on our operations and healthcare systems will affect the execution of our development plans or the conduct of our clinical studies; the risk that the efficacy and safety results observed in the patients treated in our prior and ongoing clinical trials of beti-cel may not persist; and the risk that the efficacy and safety results from our prior and ongoing clinical trials will not continue or be repeated with additional patients in our ongoing or planned clinical trials or in the commercial context; the risk that the FDA will require additional information regarding beti-cel, resulting in a delay to our anticipated timelines for regulatory submissions, including submission of our BLA. For a discussion of other risks and uncertainties, and other important factors, any of which could cause our actual results to differ from those contained in the forward-looking statements, see the section entitled Risk Factors in our most recent Form 10-Q, as well as discussions of potential risks, uncertainties, and other important factors in our subsequent filings with the Securities and Exchange Commission. All information in this press release is as of the date of the release, and bluebird bio undertakes no duty to update this information unless required by law.

View source version on businesswire.com: https://www.businesswire.com/news/home/20200612005084/en/

Read more:
bluebird bio : Majority of Evaluable Patients Across Genotypes Achieve Transfusion Independence and Maintain It with Near-Normal Hemoglobin Levels in...

Cell Biology

Brain Tumor Diagnostics and Therapeutics Market Competitive Environment and Forecast 2017 2025 – 3rd Watch News

Global Brain Tumor Diagnostics and Therapeutics Market: Snapshot

The global market for the diagnostics and therapeutics is gaining traction from a number of factors, such as increasing prevalence of brain cancer, innovations in drug delivery to brain cancer cells, and widespread market for the treatment of brain cancer as well as its associated symptoms. On the other hand, the lack of brain cancer specific drugs, increasing use of generics in chemotherapy, and utterly high cost of brain tumor diagnosis and treatment are a few important challenges faced by the market before attaining its true potential. Nevertheless, in the near future, the vendors of this market are expected to gain new opportunities by improving the technology to reach the cancer tumor at unreachable parts of the brain, although surgery currently remains the most effective mode of treatment.

Based on cancer type, the global brain tumor diagnostics and therapeutics market can be segment into glioma including oligodendroglioma, astrocytoma, choroid plexus papilloma, and ependymoma, medulloblastoma, meningioma, schwannomas, and pituitary adenoma. On the basis of therapeutics, this market can be categorized into brain cancer including radiation therapy, chemotherapy, and targeted therapy. Geographically, the report takes stock of the potential of all important regions such as North America, Asia Pacific, and Europe.

This report on the global brain tumor diagnostics and therapeutics market has been developed by a group of professional market research analysts, with a solitary goal to represent the current scenario as well as the future prospects of the market to the stakeholders connected to the value chain. A number of leading companies operating in this market have also been profiled to highlight the competitive landscape.

Get Sample Copy of the Report @https://www.tmrresearch.com/sample/sample?flag=B&rep_id=1181

Global Brain Tumor Diagnostics and Therapeutics Market: Overview

Increased prevalence of symptoms associated with brain tumor has led to a growing demand for tests detecting the presence of tumors. Some of the common types of tests used for the diagnosis of brain tumor are magnetic resonance imaging (MRI), myelogram, electroencephalography (EEG), tissue sampling or biopsy of surgical removal of a tumor, CT scan, cerebral angiogram or cerebral anteriogram, molecular testing, positron emission tomography (also called PET or PET-CT scan), and neurocognitive assessment.

The growth drivers, opportunities, deterrents, and recent developments in the global market for brain tumor diagnostics and therapeutics have been analyzed in details. The report presents value chain analysis, the supply and demand ratio, market attractiveness, and the past and projected leading market segments. The prominent vendors have been evaluated in depth, considering their market shares, product portfolios, and recent business strategies.

Global Brain Tumor Diagnostics and Therapeutics Market: Drivers and Restraints

Recently, the healthcare sector has been displaying increasing public-private partnerships. These partnerships will prove to be beneficial for the growth of the global brain tumor diagnostics and therapeutics market as they are contributing towards the modernization of radiology and diagnostic imaging services. Research activities in the area of molecular diagnostics aimed at understanding cell biology and recognizing gene mutations related to malignancy are expected to boost growth. Technological advancements, increasing consumer base, advanced healthcare infrastructures, and growing health awareness among people are some of the major growth drivers of the global brain tumor diagnostics and therapeutics market.

On the other hand, the high cost of these tests and lack of trained personnel might restrain the growth of the global brain tumor diagnostics and therapeutics market. Nevertheless, opportunities are likely to materialize from the emergence of new technologies such as chemical exchange saturation transfer (CEST) and sodium magnetic resonance imaging (Na MRI). These technologies not only aid in the diagnosis of tumors, but also in therapies treating them.

Based on types of tumor, the anaplastic astrocytoma, anaplastic oligodendroglioma, low-grade (diffuse) astrocytoma, ependymoma, glioblastoma, and oligodendroglioma can be the major segments.

Read Comprehensive Overview of Report @https://www.tmrresearch.com/brain-tumor-diagnostics-therapeutics-market

Global Brain Tumor Diagnostics and Therapeutics Market: Regional Outlook

Based on region, the global brain tumor diagnostics and therapeutics market can be segmented into the Middle East and Africa, Latin America, North America, Asia Pacific, and Europe. North America, with the U.S at the forefront, is expected to witness significant growth. The increasing demand for brain tumor diagnostics and therapeutics from this region can be attributed to the rising incidence of brain tumors. According to the Central Brain Tumor Registry, the U.S. might witness approximately 79,270 new cases of primary non-malignant and malignant brain and CNS tumors by the end of 2017.

Furthermore, Asia Pacific is slated to expand considerably over the forecast period, as the awareness about the availability and significance of these tests is gradually spreading. The healthcare expenditures and per capita incomes of people in countries across Asia Pacific are also rising, promising further expansion of the brain tumor diagnostics and therapeutics market in the region.

Companies Mentioned in the Report

Some of the major market players operating in the global brain tumor diagnostics and therapeutics market are Philips Healthcare, Roche Diagnostics, GE Healthcare, Toshiba Corporation, Carestream Health, Bristol Myer Squibb, Hitachi, Ltd., and Siemens Healthineers.

About TMR Research:

TMR Research is a premier provider of customized market research and consulting services to business entities keen on succeeding in todays supercharged economic climate. Armed with an experienced, dedicated, and dynamic team of analysts, we are redefining the way our clients conduct business by providing them with authoritative and trusted research studies in tune with the latest methodologies and market trends.

Contact:

TMR Research,3739 Balboa St # 1097,San Francisco, CA 94121United StatesTel: +1-415-520-1050

Read the rest here:
Brain Tumor Diagnostics and Therapeutics Market Competitive Environment and Forecast 2017 2025 - 3rd Watch News

Cell Biology

A protein that helps treat viruses can later interfere with lung tissue repair – News-Medical.Net

Jun 12 2020

Researchers at the Francis Crick Institute have found that a protein which is initially helpful in the bodys immune response to a virus, can later interfere with the repair of lung tissue. The work, published in Science, highlights the need for careful consideration regarding the use of this protein to treat viruses, including coronavirus.

Tissue sections of mouse lungs, after infection with influenza. The image on the left is the control and the image on the right is from mice without receptors for interferon lambda. The lungs where interferon lambda signalling is blocked (right) shows improved epithelial cell growth and differentiation (in red).

When a virus infects the lungs, the body attempts to defend itself and fight off the infection. One defensive mechanism is the activation of a protein, called interferon lambda, which signals to surrounding lung tissue cells to switch on anti-viral defenses.

Interferon lambda is currently being investigated in clinical trials as a potential treatment for COVID-19, so understanding the biology underlying its anti-viral effects is important.

The research team investigated the effects of this protein in the lab and found that if it is active for an extended period, it inhibits the repair of the lung tissue. This could prolong lung damage and increase the risk of subsequent bacterial infections.

The Crick scientists observed that in mice with influenza, having increased levels of this protein in their lungs meant that their epithelial cells multiplied less. These cells make up the lining of the airspaces in the lung and need to multiply to replace damaged cells and repair damage. This was the case for mice treated with the protein experimentally and also mice that had produced the protein naturally, as a result of their response to the virus.

Furthermore, cultures of human lung epithelial cells treated with this protein were also less able to grow.

This is a really potent protein with many different functions. At the beginning of a viral infection, it is protective, triggering functions that help to fight the virus. However, if it remains in the tissue for too long, it could become harmful.

This means, for any anti-viral treatment that uses this protein, there is a really careful balance that must be made. Clinicians should consider the timing of the treatment, the earlier this better, and the duration of treatment.

Andreas Wack, Author and group leader of the Immunoregulation lab at the Crick

While this research studied mice infected with influenza, the effects of this protein should be similar for other viruses that also cause lung damage, including coronavirus.

The paper has been published alongside research from Harvard Medical School, which found that severe COVID-19 patients showed strong expression of this protein in their lungs.

Understanding how our bodies respond to infection has never been more important. Differences in our immune responses have huge implications for whether a treatment will work and what the side effects might be.

Our results suggest that before pursuing treatment with interferon lambda, doctors should consider at what stage of the disease patients are, as treatment late in infection may increase the risk of prolonged damage.

Jack Major, Lead author and PhD student in the Immunoregulation lab at the Crick

The Crick researchers will continue to study inflammatory pathways in lung infections, including infection with coronavirus.

Source:

Journal reference:

Major, J., et al. (2020) Type I and III interferons disrupt lung epithelial repair during recovery from viral infection. Science. doi.org/10.1126/science.abc2061.

Read the rest here:
A protein that helps treat viruses can later interfere with lung tissue repair - News-Medical.Net

Cell Biology

Neural circuits that control hibernation-like behaviors discovered in mice – News-Medical.Net

Reviewed by Emily Henderson, B.Sc.Jun 11 2020

The dream of suspended animation has long captivated the human imagination, reflected in countless works of mythology and fiction, from King Arthur and Sleeping Beauty to Captain America and Han Solo. By effectively pausing time itself for an individual, a state of stasis promises to enable the repair of lethal injuries, prolong life and allow for travel to distant stars.

While suspended animation may seem a fantasy, a strikingly diverse array of life has already achieved a version of it. Through behaviors like hibernation, animals such as bears, frogs and hummingbirds can survive harsh winters, droughts, food shortages and other extreme conditions by essentially entering into biological stasis, where metabolism, heart rate and breathing slow to a crawl and body temperature drops.

Now, Harvard Medical School neuroscientists have discovered a population of neurons in the hypothalamus that controls hibernation-like behavior, or torpor, in mice, revealing for the first time the neural circuits that regulate this state.

Reporting in Nature on June 11, the team demonstrated that when these neurons are stimulated, mice enter torpor and can be kept in that state for days. When the activity of these neurons is blocked, natural torpor is disrupted.

Another study published simultaneously in Nature by researchers from the University of Tsukuba in Japan also identified a similar population of neurons in the hypothalamus.

By better understanding these processes in mice and other animal models, the authors envision the possibility of one day working toward inducing torpor in humans--an achievement that could have a vast array of applications, such as preventing brain injury during stroke, enabling new treatments for metabolic diseases or even helping NASA send humans to Mars.

The imagination runs wild when we think about the potential of hibernation-like states in humans. Could we really extend lifespan? Is this the way to send people to Mars?"

Sinisa Hrvatin, study co-lead author, instructor in neurobiology in the Blavatnik Institute at HMS

"To answer these questions, we must first study the fundamental biology of torpor and hibernation in animals," Hrvatin said. "We and others are doing this--it is not science fiction."

To reduce energy expenditure in times of scarcity, many animals enter a state of torpor. Hibernation is an extended seasonal form of this. Unlike sleep, torpor is associated with systemic physiological changes, particularly significant drops in body temperature and suppression of metabolic activity. While common in nature, the biological mechanisms that underlie torpor and hibernation are still poorly understood.

The role of the brain, in particular, has remained largely unknown, a question that drove the research efforts of Hrvatin and colleagues, including co-lead author Senmiao Sun, a graduate student in the Harvard Program in Neuroscience, and study senior author Michael Greenberg, the Nathan Marsh Pusey Professor and chair of the Department of Neurobiology in the Blavatnik Institute at HMS.

The researchers studied mice, which do not hibernate but experience bouts of torpor when food is scarce and temperatures are low. When housed at 22 C (72 F), fasting mice exhibited a sharp drop in core body temperature and significant reduction in metabolic rate and movement. In comparison, well-fed mice retained normal body temperatures.

As mice began to enter torpor, the team focused on a gene called Fos--previously shown by the Greenberg lab to be expressed in active neurons. Labeling the protein product of the Fos gene allowed them to identify which neurons are activated during the transition to torpor throughout the entire brain.

This approach revealed widespread neuronal activity, including in brain regions that regulate hunger, feeding, body temperature and many other functions. To see if brain activity was sufficient to trigger torpor, the team combined two techniques--FosTRAP and chemogenetics--to genetically tag neurons that are active during torpor. These neurons could then be re-stimulated later by adding a chemical compound.

The experiments confirmed that torpor could indeed be induced--even in well-fed mice--by re-stimulating neurons in this manner after the mice recovered from their initial bout of inactivity.

However, because the approach labeled neurons throughout the entire brain, the researchers worked to narrow in on the specific area that controls torpor. To do so, they designed a virus-based tool that they used to selectively activate neurons only at the site of injection.

Focusing on the hypothalamus, the region of the brain responsible for regulating body temperature, hunger, thirst, hormone secretion and other functions, the researchers carried out a series of painstaking experiments. They systematically injected 54 animals with minute amounts of the virus covering 226 different regions of the hypothalamus, then activated neurons only in the injected regions and looked for signs of torpor.

Neurons in one specific region of the hypothalamus, known as the avMLPA, triggered torpor when activated. Stimulating neurons in other areas of the hypothalamus had no effect.

"When the initial experiment worked, we knew we had something," Greenberg said. "We gained control over torpor in these mice using FosTRAP, which allowed us to then identify the subset of cells that are involved in the process. It's an elegant demonstration of how Fos can be used to study neuronal activity and behavioral states in the brain."

The team further analyzed the neurons that occupy the region, using single-cell RNA sequencing to look at almost 50,000 individual cells representing 36 different cell types, ultimately pinpointing a subset of torpor-driving neurons, marked by the neurotransmitter transporter gene Vglut2 and the peptide Adcyap1.

Stimulating only these neurons was sufficient to induce rapid drops in body temperature and motor activity, key features of torpor. To confirm that these neurons are critical for torpor, the researchers used a separate virus-based tool to silence the activity of avMLPA-Vglut2 neurons. This prevented fasting mice from entering natural torpor, and in particular disrupted the associated decrease in core body temperature. In contrast, silencing these neurons in well-fed mice had no effect.

"In warm-blooded animals, body temperature is tightly regulated," Sun said. "A drop of a couple of degrees in humans, for example, leads to hypothermia and can be fatal. However, torpor circumvents this regulation and allows body temperatures to fall dramatically. Studying torpor in mice helps us understand how this fascinating feature of warm-blooded animals might be manipulated through neural processes."

The researchers caution that their experiments do not conclusively prove that one specific neuron type controls torpor, a complex behavior that likely involves many different cell types. By identifying the specific brain region and subset of neurons involved in the process, however, scientists now have a point of entry for efforts to better understand and control the state in mice and other animal models, the authors said.

They are now studying the long-term effects of torpor on mice, the roles of other populations of neurons and the underlying mechanisms and pathways that allow avMLPA neurons to regulate torpor.

"Our findings open the door to a new understanding of what torpor and hibernation are, and how they affect cells, the brain and the body," Hrvatin said. "We can now rigorously study how animals enter and exit these states, identify the underlying biology, and think about applications in humans. This study represents one of the key steps of this journey."

The implications of one day being able to induce torpor or hibernation in humans, if ever realized, are profound.

"It's far too soon to say whether we could induce this type of state in a human, but it is a goal that could be worthwhile," Greenberg said. "It could potentially lead to an understanding of suspended animation, metabolic control and possibly extended lifespan. Suspended animation in particular is a common theme in science fiction, and perhaps our ability to traverse the stars will someday depend on it."

Source:

Journal reference:

Hrvatin, S., et al. (2020) Neurons that regulate mouse torpor. Nature. doi.org/10.1038/s41586-020-2387-5.

See the rest here:
Neural circuits that control hibernation-like behaviors discovered in mice - News-Medical.Net