Abstract

The human bronchial epithelium is composed of multiple distinct cell types that cooperate to defend against environmental insults. While studies have shown that smoking alters bronchial epithelial function and morphology, its precise effects on specific cell types and overall tissue composition are unclear. We used single-cell RNA sequencing to profile bronchial epithelial cells from six never and six current smokers. Unsupervised analyses led to the characterization of a set of toxin metabolism genes that localized to smoker ciliated cells, tissue remodeling associated with a loss of club cells and extensive goblet cell hyperplasia, and a previously unidentified peri-goblet epithelial subpopulation in smokers who expressed a marker of bronchial premalignant lesions. Our data demonstrate that smoke exposure drives a complex landscape of cellular alterations that may prime the human bronchial epithelium for disease.

The human bronchus is lined with a pseudostratified epithelium that acts as a physical barrier against exposure to harmful environmental insults such as inhaled toxins, allergens, and pathogens (1, 2). The bronchial epithelium is a complex tissue, predominantly composed of ciliated, goblet, club, and basal epithelial cells. These cell types cooperate to perform mucociliary clearance, which is the process that mediates the capture and removal of inhaled substances (1, 2). Goblet cells secrete components of a mucosal lining that entraps inhaled particulate matter, which is propelled out of the airways by mechanical beating of ciliated cells (1, 2). Club cells have both secretory (3) and progenitor (4) functions, and basal cells are multipotent progenitors responsible for normal tissue homeostasis (57). Interplay among these cells is required for proper function and long-term maintenance of the bronchial epithelium, but exposure to substances, such as tobacco smoke, might alter or injure specific cell types and lead to tissue-wide dysfunction.

Inhalation of tobacco smoke exposes the bronchial epithelium to toxins, carcinogens, and free radicals (811), but cellular injuries and abnormalities associated with this exposure are complex and have not been fully characterized. Previous studies have described smoking-induced epithelial changes, such as increased goblet cell numbers (1214) and reduced ciliary length (15, 16). Robust transcriptomic alterations have also been observed in the bronchial epithelium of smokers, involving the up-regulation of genes linked to xenobiotic metabolism and the oxidative stress response (17, 18). Furthermore, it has been reported that a subset of gene expression alterations detected in smokers persists years after smoking cessation (18). However, the aforementioned transcriptomic studies profiled bronchial tissue in bulk, masking cell typespecific contributions to the smoking-associated gene expression signature.

To overcome the limitations of bulk tissue analyses, we used single-cell RNA sequencing (scRNA-Seq) to profile the transcriptomes of individual bronchial cells from healthy never and current smokers. We identified bronchial subpopulations using an unsupervised machine learning algorithm and immunostained bronchial tissue from independent cohorts of never and current smokers to validate robust smoking-associated findings. In the airways of smokers, we described a metabolic response specific to ciliated cells, a shift in the presence of club and goblet cells, and the emergence of a previously uncharacterized epithelial subpopulation.

Bronchial brushings were procured by bronchoscopy from the right mainstem bronchus of six healthy current smokers and six healthy never smokers (table S1), and single ALCAM+ epithelial cells (19) and CD45+ white blood cells (WBCs) were isolated from each donor (Fig. 1A and fig. S1). The CEL-Seq scRNA-Seq protocol (20) was used to profile the transcriptomes of 84 epithelial cells and 11 WBCs from each of the 12 donors (1140 total cells: 1008 epithelial cells and 132 WBCs). Low-quality cells were excluded from downstream analyses, leaving 796 cells (753 epithelial cells and 43 WBCs) (figs. S2 and S3) expressing an average of 1817 genes per cell. Expression of known marker genes for bronchial cell types was detected in largely nonoverlapping cells, including KRT5 for basal cells, FOXJ1 for ciliated cells, SCGB1A1 for club cells, MUC5AC for goblet cells, and CD45 for WBCs (Fig. 1B). Given the relatively small number of subjects, we sought to determine whether smoking-associated gene expression changes identified in these donors reflected those observed in a larger, independent cohort of never and current smokers. Data from all cells procured from each donor were combined to generate in silico bulk bronchial brushings. Analysis of differential expression between never and current smoker in silico bulk samples revealed associations that were highly correlated (Spearmans r = 0.45) with those observed in a previously published bulk bronchial brushing dataset generated by microarray (fig. S4) (18).

(A) Bronchial brushings were procured from the right mainstem bronchus of six never smokers and six current smokers. Bronchial tissue was dissociated, single cells were isolated by fluorescence-activated cell sorting (FACS), and single-cell RNA libraries were prepared and sequenced. (B) t-distributed stochastic neighbor embedding (t-SNE) was performed to illustrate transcriptomic relationships among cells. Donor smoking status (NS, never smoker; CS, current smoker) was visualized for each cell as well as expression of bronchial cell type marker genes [z-normalized transcripts per million (TPM) values] across all cells: KRT5 (basal), FOXJ1 (ciliated), SCGB1A1 (club), MUC5AC (goblet), and CD45 (WBC). (C) An unsupervised analytical approach (LDA) was used to identify distinct cell clusters and sets of coexpressed genes. Cell clusters were defined by unique gene set expression patterns, and never or current smoker cell enrichment was assessed.

To characterize cellular subpopulations beyond known cell type markers, we used latent Dirichlet allocation (LDA) as an unsupervised framework to assign cells to clusters and identify distinct sets of coexpressed genes across all cells (Fig. 1C). LDA divided the dataset into 13 distinct cell clusters and 19 sets of coexpressed genes (Fig. 2, A and B, and figs. S5 to S8). Each cell cluster was defined by the expression of a unique combination of gene sets, and each gene set was defined by a unique expression pattern among clusters (Fig. 2, A and B, and fig. S9). Cell types were defined for 8 of the 13 clusters based on medium to high marker gene expression: Cell clusters C-2 and C-4 expressed KRT5, C-5 and C-11 expressed FOXJ1, C-1 and C-8 expressed SCGB1A1, and C-3 expressed MUC5AC (Fig. 2C). Cluster C-7 expressed WBC marker CD45 (Fig. 2C), and Fishers exact test was used to show that C-7 was enriched with sorted CD45+ cells (P = 9.6 1047, Fishers exact test). C-7 cells also expressed several T cell receptor genes (e.g., TRBC2 and TRGC1), indicating a T cell lineage (fig. S10). Low levels of SCGB1A1 transcripts were detected in cluster C-10 (SCGB1A1low), and CFTR was expressed by cluster C-13, which suggests that these cells may be ionocytes (fig. S11) (21). Marker gene expression was not detected in clusters C-6, C-9, and C-12 (Fig. 2C). Enrichment [false discovery rate (FDR) q < 0.05] of current smoker cells was observed in goblet cell cluster C-3, as well as C-9 and C-12, whereas that of never smoker cells was observed in club cell cluster C-1 and basal cluster C-4 (Fig. 2D). Donor-specific contributions of cells to each cluster were variable; however, most of the never and current smokers contributed to each never and current smokerassociated cell cluster, respectively (fig. S12). Furthermore, a subset of gene sets expressed by specific clusters of ciliated, club, goblet, and basal cells, as well as those without a cell type designation, was differentially expressed between never and current smokers in transcriptomic data generated from bulk bronchial tissue (Fig. 2, A and B, and fig. S13) (18). Therefore, smoking-induced gene expression changes reported in bulk tissue are likely driven by alterations to multiple bronchial cell types.

(A) Global transcriptomic profiles of 13 bronchial cell clusters were defined by expression of unique combinations of 19 gene sets and visualized by heatmap (z-normalized TPM values). (B) A MetaGene was generated for each gene set (GS-1 to GS-19), and mean cluster-specific expression was designated: high (pink), medium (white), low (light gray), or not expressed (dark gray). (C) Mean expression of marker genes was summarized for each cluster designated: high (pink), medium (white), low (light gray), or not expressed (dark gray). (D) Per-cluster percentage of total cells and the ratio of never and current smoker cells were calculated, and per-cluster statistical enrichment (FDR q < 0.05, indicated in blue) of NS or CS cells was assessed.

We characterized transcriptomic similarities and differences among FOXJ1+ clusters C-5 and C-11 to define ciliated cell subpopulations detected in never and current smokers. Our data revealed that both clusters of ciliated cells expressed gene set GS-2 but could be differentiated based on expression of gene set GS-3 by cluster C-5 and gene set GS-7 by cluster C-11 (Fig. 3A). GS-2 contains FOXJ1, in addition to genes involved with ciliary assembly, maintenance, and function, such as motor protein genes (e.g., DYNLL1 and DNAH9) and intraflagellar transport genes (e.g., IFT57 and IFT172) (Fig. 3A and extended table S3). GS-2 also includes antioxidant genes (e.g., PRDX5, GPX4, and GSTA2), known transcriptional regulators of ciliogenesis [e.g., RFX2 (22, 23) and RFX3 (24, 25)], and surface proteins not previously attributed to ciliated cells (e.g., CDHR3 and CD59). GS-3 contains genes with known roles in airway ciliary biology, such as IFT88 (required for ciliary formation) (2628) and DNAH5 (required for ciliary motility) (2931). By contrast, gene set GS-7 is enriched with cell cycleassociated genes (extended table S3), such as CDK1 and CCNB1 (G1-S transition) and TOP2A (S-phase DNA replication), as well as the transcription factor HES6. Therefore, clusters C-5 and C-11 likely represent functionally distinct subpopulations of FOXJ1+ ciliated cells.

(A) Expression of gene sets GS-2, GS-3, and GS-7 in clusters C-5 and C-11 was visualized by heatmap (z-normalized TPM values). (B) Cluster C-5 was split into never and current smoker subsets, and expression of GS-8 genes was visualized by heatmap. (C) Bronchial tissue procured from an independent cohort of never and current smokers (UMCG cohort, table S2) was immunostained for AKR1B10, Ac--Tub, and KRT8. Representative images of never smoker (left) and current smoker (right) tissue were displayed. Arrows specify examples of AKR1B10+ ciliated cells (Ac--Tub+). (D) An increase in tissue length (m)normalized numbers of AKR1B10+ Ac--Tub+ cells was observed in current smokers relative to never smokers [P = 7.4 107, Wilcoxon rank-sum (WRS) test].

We found that ciliated cells from current smokers expressed a distinct transcriptional signature. Specifically, the current smoker subset of cluster C-5 FOXJ1+ cells expressed gene set GS-8, which was enriched with genes encoding enzymes implicated in aldehyde and ketone metabolism, such as ALDH3A1, AKR1C1, and AKR1B10 (Fig. 3B). This finding suggested that the gene expression response to toxic aldehydes and ketones present in tobacco smoke (8, 9) might be restricted to ciliated epithelial cells. To confirm that this set of enzymes localized to ciliated cells, we immunostained bronchial tissue procured from an independent cohort of never and current smokers [University Medical Center Groningen (UMCG) cohort, table S2] for the aldo-keto reductase AKR1B10, as well as cilia-specific acetylated -tubulin (Ac--Tub) and the luminal cytokeratin KRT8, which is expressed by all nonbasal cells (Fig. 3C). We found that AKR1B10 was robustly expressed in the airways of current smokers, and numbers of AKR1B10+ ciliated cells were significantly higher than those observed in never smokers (P = 7.4 107; Fig. 3, C and D). AKR1B10 was detected throughout the cytoplasm of smoker ciliated cells, as well as at the base of the cilia (Fig. 3C). AKR1B10+ ciliated cells were uncommon in never smokers, and overall low magnitude of AKR1B10 expression was observed in these cells (Fig. 3C). We detected rare instances of nonciliated AKR1B10+ KRT8+ cells (fig. S14A), but AKR1B10+ KRT8 cells were not observed. We also confirmed that AKR1B10 was not expressed by current smoker MUC5AC+ goblet cells (fig. S14B). Overall, these results demonstrate that ciliated cells express a specific set of detoxification genes in response to smoke exposure.

Our data revealed that the largest cluster of SCGB1A1+ cells, C-1, was enriched with never smoker cells (Fig. 2D), indicating that this subpopulation of club cells was depleted from the airways of smokers. C-1 cells distinctly expressed high levels of gene set GS-19, which contains MUC5B, in addition to SCGB3A1 and transcription factors TCF7, FOS, and JUN (Fig. 4A). However, SCGB1A1 (included in gene set GS-17) was also highly expressed by cluster C-8, which was not affected by smoking status (Fig. 2D). Therefore, these results indicate that smoking is associated with a decrease in MUC5B+ SCGB1A1+ (C-1) club cell content. Furthermore, gene set GS-13, which contains immunologically relevant genes BPIFB1 (32) and PIGR (33) (Fig. 4A), was expressed by SCGB1A1+ cells (C-1 and C-8) as well as MUC5AC+ cluster C-3, indicating that there may be functional overlap among club and goblet cells.

(A) Expression of gene sets GS-19, GS-17, GS-13, and GS-1 in clusters C-1, C-8, and C-3 was visualized by heatmap (z-normalized TPM values). Bronchial tissue procured from an independent cohort of never and current smokers (UMCG cohort, table S2) was immunostained for MUC5B and MUC5AC. (B) Representative images of never smoker tissue, MN current smoker tissue, and current smoker GCH were displayed. Arrows specify examples of MUC5B+, MUC5B+ MUC5AC+, and MUC5AC+ cells. Changes in tissue length (m)normalized numbers of (C) MUC5B+ cells (MN decrease, P = 0.02; GCH decrease, P = 1.8 105), (D) MUC5B+ MUC5AC+ cells (GCH decrease, P = 0.02), and (E) MUC5AC+ cells (MN increase, P = 1.5 106; GCH increase, P = 7.4 107) were observed (WRS test) in current smoker MN and GCH tissue relative to never smokers (WRS test). (F) Average proportions of MUC5B+, MUC5B+ MUC5AC+, and MUC5AC+ cells observed in never smokers, as well as MN and GCH current smoker tissue are displayed.

The MUC5AC+ goblet cell cluster C-3 was significantly enriched with current smoker cells (Fig. 2D), which is consistent with previous studies showing that smoking is associated with increased bronchial goblet cell abundance (1214). Cluster C-3 expressed gene set GS-1, which contains the goblet cell marker gene MUC5AC as well as several genes with known roles in goblet cell biology, such as SPDEF (34), AGR2 (35), and TFF3 (36) (Fig. 4A). Genes associated with the unfolded protein response are present in GS-1 (e.g., KDLER3 and DNAJC10) (extended table S3). We also identified several unique goblet cell surface markers (e.g., CLDN10, TSPAN8, and TSPAN13), as well as a transcription factor (NKX3-1) whose role in the goblet cell transcriptional program is unknown (Fig. 4A). Therefore, these data indicate that smoking is associated with increased numbers of MUC5AC+ goblet cells.

To confirm smoking-associated shifts in club and goblet cell numbers, we immunostained bronchial tissue procured from an independent cohort of never and current smokers (UMCG cohort, table S2) for markers of club (MUC5B) and goblet (MUC5AC) cells (Fig. 4B). Imaging data revealed cell subpopulations that exclusively express MUC5B or MUC5AC, as well as those that coexpress both MUC5B and MUC5AC (Fig. 4B). The airways of never smokers contained similar numbers of MUC5B+, MUC5B+, MUC5AC+, and MUC5AC+ cells (Fig. 4, B and F). The bronchial epithelium of current smokers, however, took on two distinct phenotypes: tissue regions described as morphologically normal (MN), which were similar to never smokers, and regions characterized by high MUC5AC+ cell density, referred to as goblet cell hyperplasia (GCH) (Fig. 4B and fig. S15). In the MN smoker tissue, we observed a significant decrease in MUC5B+ cells (P = 0.02) (Fig. 4C) and a significant increase in MUC5AC+ cells (P = 1.5 106) (Fig. 4E), relative to never smokers, but no change in MUC5B+ MUC5AC+ content was observed (Fig. 4D). Differences between smoker GCH and never smoker epithelium, however, were more pronounced. Near-complete loss of MUC5B+ cells was observed in smoker GCH (P = 1.8 105; Fig. 4C), along with a significant loss of MUC5B+ MUC5AC+ cells (P = 0.02; Fig. 4D), relative to never smokers. GCH-associated alterations were accompanied by a 13-fold increase in MUC5AC+ cells (P = 7.4 107; Fig. 4, E and F). Additional immunostaining for KRT5 expression in the same bronchial tissue revealed that basal cell content was not affected by smoking status and did not vary between MN and GCH regions (fig. S16). Overall, these findings indicate that smoking is associated with a loss of club cells, increased numbers of goblet cells, and substantial GCH airway remodeling.

We sought to establish the identity of cluster C-9, which was strongly enriched with current smoker cells and did not express established cell type marker genes (e.g., KRT5, FOXJ1, SCGB1A1, and MUC5AC) (Fig. 2C). C-9 cells expressed high levels of gene set GS-12, which contains the luminal cytokeratin KRT8 (Fig. 5A). Additional cytokeratin genes were also present in GS-12, such as KRT13 and KRT19, as well as antioxidant genes, such as TXN and GPX1 (Fig. 5A). Cluster C-9 also expressed gene set GS-16, which was detected at low levels in MUC5AC+ cells (C-3) and contained the xenobiotic metabolism gene CYP1B1 (Fig. 5A). Furthermore, high expression of gene set GS-15 was detected in both C-9 and MUC5AC+ cells (C-3) (Fig. 5, A to C), suggesting that this cluster may have a functional relationship with goblet cells. GS-15 contains several genes previously reported to be persistently up-regulated after smoking cessation (e.g., CEACAM5, CEACAM6, and UPK1B) (18), one of which has been explicitly linked to lung squamous cell carcinoma (SCC) and premalignancy (CEACAM5) (37).

(A) Expression of gene sets GS-12, GS-16, GS-15, and MUC5AC in clusters C-3 and C-9 was visualized by heatmap (z-normalized TPM values). (B) t-SNE was used to visualize cluster C-3 and C-9 cells as well as (C) CEACAM5 expression (z-normalized TPM values) across all cells. (D) Bronchial tissue procured from an independent cohort of never and current smokers (UCL cohort, table S3) was immunostained for CEACAM5, KRT8, and MUC5AC. Representative images of never smoker tissue and current smoker GCH were displayed. Arrows specify examples of CEACAM5+ KRT8+ MUC5AC PG cells. (E) A significant increase in tissue length (m)normalized numbers of CEACAM5+ KRT8+ MUC5AC cells in current smoker GCH tissue, relative to never smokers, was observed (P = 0.004, WRS test).

To validate the presence of cluster C-9 cells in the airways of current smokers, we immunostained bronchial tissue procured from a second independent cohort of never and current smokers [University College London (UCL) cohort, table S3] for KRT8, MUC5AC (goblet cells), and Ac--Tub (ciliated cells). KRT8+ MUC5AC Ac--Tub cells that were morphologically distinct from goblet and ciliated cells were detected in significantly higher numbers in GCH regions of current smokers relative to never smokers (fig. S17). To confirm that there was functional overlap between goblet cells and this subpopulation of KRT8+ MUC5AC Ac--Tub cells, we immunostained bronchial tissue (UCL cohort, table S3) for CEACAM5, in addition to KRT8 and MUC5AC. Increased numbers of CEACAM5+ KRT8+ MUC5AC cells were detected in GCH regions of current smokers relative to never smokers (P = 0.004) (Fig. 5, D and E), although variable content among donors was observed. Within current smoker GCH tissue regions, CEACAM5+ KRT8+ MUC5AC cells were typically found in close proximity to goblet cells (CEACAM5+ KRT8+ MUC5AC+) and were therefore named peri-goblet (PG) cells (UCL cohort, Fig. 5D; UMCG cohort, fig. S18). CEACAM5 expression in goblet cells was phenotypically punctate and colocalized with MUC5AC in both never and current smokers (Fig. 5D and fig. S18). In PG cells, however, CEACAM5 localized to the plasma membrane and cytoplasm (Fig. 5D and fig. S18). Overall, these data indicate that PG cells are a previously unidentified, bronchial epithelial subpopulation associated with smoking-induced GCH.

Previous transcriptomic studies have shown that smoking is associated with a robust bronchial gene expression signature (17, 18). Interrogation of bronchial tissue at single-cell resolution revealed that elements of this signature were derived from different cell subpopulations. Overall, we found smoking-associated phenotypes that included a metabolic response that localized to ciliated cells, a cell type shift that involved club cell loss and goblet cell expansion, and a previously uncharacterized subpopulation of PG epithelial cells present within regions of GCH (fig. S19).

We identified a gene set (GS-8) specifically expressed by smoker ciliated cells (C-5) that contains genes encoding families of enzymes, such as aldehyde dehydrogenases (e.g., ALDH3A1 and ALDH1A3) and aldo-keto reductases (e.g., AKR1B10 and AKR1C1), capable of breaking down tobacco smokederived chemical compounds, such as toxic aldehydes (e.g., formaldehyde and acrolein) and ketones (e.g., acetone and methyl vinyl ketone) (8, 9). This finding suggests that ciliated cells exhibit a cell typespecific coping mechanism that may convey resistance to certain forms of smoking-induced toxicity. Links between this mechanism and previously reported smoking phenotypes, such as reduced ciliary length (15), however, are unclear. This finding might also highlight a protective function with tissue-wide significance, in which the bronchial epitheliums capacity for detoxification may be compromised if ciliated cells are lost because of injury or disease.

Several studies have reported that smoking is associated with increased mucous production and GCH in the bronchus (1214, 3840). Loss of club cells (SCGB1A1+) has been reported in smoker bronchioles (11, 12), but this is the first instance in which a similar observation has been made in the mainstem bronchus. We confirmed that GCH is a regional phenomenon interspersed among MN tissue areas. The determinants of GCH prevalence are unclear, but it has been shown that cytokines [e.g., interleukin-13 (IL-13) and (IL-4)] (4143) and viral infection (e.g., Rhino virus and polyinosinic:polycytidylic acid) (44, 45) can increase MUC5AC expression and goblet cell abundance. The specific catalyst for GCH in response to smoke exposure is unknown, but reports of its co-occurrence with airway inflammation suggest that immunological interplay may be a factor (14). Furthermore, there is evidence that both basal and club cells are capable of goblet cell differentiation (32, 46). However, the origins of newly formed goblet cells in the airways of smokers have not been explicitly described. Functional implications for goblet cell expansion and club cell loss are unclear, but a similar phenotype has been described in the airways of asthmatics, in which diminished mucosal fluidity, the formation of mucosal plugs, and impaired mucociliary clearance were observed (47, 48). Murine models have also shown that MUC5B loss is associated with impaired mucociliary clearance, airflow obstruction, and respiratory infection (49).

Smoking-induced GCH was associated with the presence of a previously uncharacterized subpopulation of CEACAM5+ KRT8+ MUC5AC PG epithelial cells. The origins of PG cells are unclear, but a KRT8+ undifferentiated epithelial subpopulation derived from basal cells, referred to as suprabasal, has been described in murine models (46, 50). Suprabasal cells act as intermediate precursors to ciliated and secretory cells during basal cell differentiation under normal conditions (46) and, after injury, as a repair mechanism (50). However, the suprabasal phenomenon has not been characterized in the human bronchus, and little is known regarding human intermediate epithelial subpopulations. Furthermore, the involvement of a KRT8+ intermediate state in club cell transdifferentiation (4, 34) has not been explored. Goblet cell differentiation required for the onset and maintenance of smoking-associated GCH might involve a pro-goblet precursor subpopulation, but the explicit role of PG cells in this context requires further investigation.

It has been reported that CEACAM5 expression is persistently up-regulated in the airways of former smokers, whereas genes specifically expressed by goblet cells, such as MUC5AC, SPDEF, and AGR2, return to normal, never smoker levels post-smoking cessation (18). These findings suggest that goblet cell expansion in the airways of smokers is reversible, whereas the emergence of CEACAM5+ PG cells might have long-term implications. The functional consequences of the presence of PG cells are unclear, but irreversible alterations to bronchial epithelial composition might underlie chronic disease states. Although PG cells were identified in this study in the absence of established disease phenotypes, CEACAM5+ KRT5+ cells have been detected in bronchial premalignant lesions and lung SCC (37). CEACAM5 has also been detected in numerous additional cancer types (51, 52), and several genes that are coexpressed with CEACAM5 (i.e., detected in GS-15) have been implicated in carcinogenesis, such as UPK1B (53), MSLN (54, 55), and PSCA (56, 57). Therefore, investigation of mechanisms linking the presence and variable abundance of GCH-associated CEACAM5+ PG cells and premalignant lesion-associated CEACAM5+ KRT5+ cells might provide insight into smoking-induced conditions that promote lung carcinogenesis.

These data demonstrate that human bronchial epithelial exposure to tobacco smoke drives ciliated cellspecific toxin metabolism and leads to both club cell depletion and goblet cell expansion. A novel subpopulation of PG cells was also detected in the bronchial airways of smokers in association with GCH. These results will enable us to more precisely define the landscape of smoking-induced epithelial abnormalities. Future work will use experimental systems to define the consequences of specific, smoke-derived chemical compounds and investigate the recapitulation and reversal of cell and molecular phenotypes observed in this study. Furthermore, these findings may be leveraged to improve diagnostics and develop preventative strategies for smoking-associated lung diseases.

At Boston University Medical Center, healthy volunteer never smokers (n = 6) and current smokers (n = 6) underwent a bronchoscopy to obtain brushings from the right mainstem bronchus, as described previously (17, 18). Eligible volunteers included subjects who (i) were between the ages of 19 and 55; (ii) did not use inhaled or intranasal medications; (iii) did not have a history of chronic obstructive pulmonary disease, asthma, pulmonary fibrosis, sarcoid, or head and neck/lung cancer; (iv) did not use marijuana; (v) did not have a respiratory infection within the past 6 weeks; and (vi) did not use other tobacco products (i.e., pipe, cigar, and chewing). Spirometry was performed to assess lung function (e.g., FEV1/FVC). Exhaled carbon monoxide (Smokerlyzer Carbon Monoxide Monitor, model EC-50; Bedfont Scientific Ltd.) and urine cotinine (NicAlert; Confirm BioSciences) levels were measured to confirm smoking status. The Institutional Review Board approved the study, and all subjects provided written informed consent.

Bronchial brushings were treated with 0.25% trypsin/EDTA for 20 min and stained for sorting using a BD FACSAria II. Gating based on forward scatter height (FSC-H) versus forward scatter area (FSC-A) was applied to sort only singlet events (fig. S1A). Dead cells (LIVE/DEAD Fixable Aqua Dead Cell Stain, Thermo Fisher; L34957) and red blood cells expressing GYPA/B (fig. S1B) on their surface [allophycocyanin (APC) anti-CD235ab; BioLegend, 306607] were stained and excluded. ALCAM+ epithelial cells [phycoerythrin (PE) anti-CD166; BioLegend, 343903] and CD45+ WBCs (APC-Cy7 anti-CD45; BD, 561863) were stained (fig. S1C) and sorted into 96-well polymerase chain reaction (PCR) plates containing lysis buffer [0.2% Triton X-100, 2.5% RNaseOUT (Thermo Fisher; 10777019)] compatible with downstream RNA library preparation. In each 96-well PCR plate for each subject, we sorted 84 ALCAM+ cells and 11 CD45+ cells and maintained one empty well as a negative control. The plates were frozen on dry ice and stored at 80C until preparation for sequencing.

Massively parallel scRNA-Seq of human bronchial airway cells was performed using a modified version of the CEL-Seq RNA library preparation protocol (20). For each of the 12 recruited donors, one frozen 96-well PCR plate containing sorted cells was thawed on ice, and RNA was directly reverse-transcribed (Thermo Fisher, AM1751) from whole-cell lysate using primers composed of an anchored poly(dT), the 5 Illumina adaptor sequence, a six-nucleotide well-specific barcode, a five-nucleotide unique molecular identifier (UMI), and a T7 RNA polymerase promoter. All primer sequences were listed in extended table S1. Samples were additionally supplemented with ERCC RNA Spike-In Mix (1:1,000,000 dilution; Thermo Fisher, 4456740) for quality control. Complementary DNA generated from each of the 96 wells per plate was pooled, subjected to second-strand synthesis (Thermo Fisher, AM1751), and amplified by in vitro transcription (Thermo Fisher, AM1751). Amplified RNA was chemically fragmented (New England BioLabs, E6150) and ligated to an Illumina RNA 3 adapter (Illumina, RS-200-0012). Samples were again reverse-transcribed using a 3 adaptor-specific primer and amplified using indexed Illumina RNA PCR primers (Illumina, RS-200-0012). In total, 1152 samples (1008 epithelial cells, 132 WBCs, and 12 negative controls) were sequenced on an Illumina HiSeq 2500 in rapid mode, generating paired-end reads (15 nucleotides for read 1, 7 nucleotides for index, and 52 nucleotides for read 2).

Illuminas bcl2fastq2 software (v2.19.1) was used to demultiplex the sequencing output to 12 plate-level FASTQ files (1 per 96-well plate). A python-based pipeline (https://github.com/yanailab/CEL-Seq-pipeline) was used to (i) demultiplex each plate-level FASTQ file to 96 cell-level FASTQ files, trim 52 nucleotide reads to 35 nucleotides, and append UMI information from read 1 (R1) to the header of read 2 (R2); (ii) perform genomic alignment of R2 with Bowtie2 (v2.2.2) using a concatenated hg19/External RNA Controls Consortium (ERCC) reference assembly; and (iii) convert aligned reads to gene-level counts using a modified version of the HTSeq (v0.5.4p1) python library that identifies reads aligning to the same location with identical UMIs and reduces them to a single count. One UMI-corrected count was then referred to as a transcript. The pipeline was configured with the following settings: alignment quality (min_bc_quality) = 10, barcode length (bc_length) = 6, UMI length (umi_length) = 5, cut_length = 35.

The quality of each cell was assessed by examining the total number of reads, total reads aligned to hg19, total reads aligning to genes (pre-UMI correction), total transcript counts, and total genes with at least one detected transcript. Cells were excluded from downstream analyses if the total number of transcripts was not twofold greater than the total background-level transcripts detected in the empty well negative control on each plate (fig. S3). Cells were also excluded from downstream analyses if there was a weak Pearson correlation (r < 0.7) between detected ERCC RNA Spike-In transcript counts (log10) and ERCC input concentration (log10) (amol/ml) (fig. S3). All nonprotein-coding genes and genes with less than two transcript counts in five cells were removed from the dataset. The remaining 7680 genes measured across 796 cells were used for subsequent analyses.

LDA from the topicmodels R package (v0.2-6) was used to generate probabilistic representations of cell clusters and gene sets present in the dataset, referred to as Cell-States and Gene-States. The input for the Cell-State model required a counts data matrix where cells were columns and genes were rows, whereas for the Gene-State model, the same matrix was transposed (i.e., genes were columns and cells were rows). Models were fit using the variational expectationmaximization (VEM) algorithm with the following parameters: nstart = 5, seed = 12345, estimate.alpha = TRUE, estimate.beta = TRUE. The given parameter k determined the number of Cell-States and Gene-States to be estimated by the model. The optimal value of k was determined by fivefold cross-validation and evaluation of model perplexity. For the Gene-State model, cells were randomly partitioned into training (80%) and test (20%) sets, whereas for the Cell-State model, genes were randomly partitioned into training (80%) and test (20%) sets. Models were then fit to the training set, and perplexity was estimated to evaluate model fit for the held-out test set. Fifty iterations of this process were performed for k = 2 to 50, mean perplexity was calculated at each k, and the minimum mean perplexity was selected as the optimal value of k (i.e., k.opt), which was k = 13 for the Cell-State model and k = 19 for the Gene-State model (fig. S6).

Negative binomial generalized linear models were built using the MASS R package (v7.3-45) for each Gene-State (n = 19) and each Cell-State (n = 13), in which States were treated as inferred, independent variables and genes or cells, respectively, were treated as dependent variables. A cell was assigned to a Cell-State if a significant association (FDR q < 0.05) was observed with positive directionality (regression coefficient > 1). Similarly, a gene was assigned to a Gene-State if a significant, positive association was observed (FDR q < 1 105, regression coefficient > 1). If multiple State associations were observed for a given gene or cell, assignment was determined on the basis of the strongest State association (i.e., minimum FDR q). Additional metrics for gene set and cluster assignment include State Specificity and State Similarity. LDA (see the previous section) also assigned a probability to each gene (or cell) for each Gene-State (or Cell-State), and State Specificity was calculated by dividing that probability by the sum of probabilities across all Gene-States (or Cell-States). A minimum State Specificity of 0.1 was required for gene or cell assignment. State Similarity was calculated by assessing the cosine (q) similarity between each Gene-State and relative expression of each gene (gene counts divided by total counts for each cell). A minimum State Similarity of 0.4 was required for gene assignment. All downstream analyses used the 785 cells that fit the criteria for Cell-State assignment and 676 genes that fit the criteria for Gene-State assignment. Statistical modeling results, State Specificity, and State Similarity values for all genes, regardless of assignment status, were included in extended table S2.

Before heatmap visualization, transcript counts were transformed to z-normalized transcripts per million (TPM). Genes (top to bottom) and cells (left to right) were ordered according to the strength of statistical association (FDR q) with respective assigned Gene-States and Cell-States. The tsne R package v0.1-3 was used for dimensionality reduction by t-distributed stochastic neighbor embedding (t-SNE). Modified parameters include k = 2 and seed = 1234. Input for t-SNE was z-normalized TPM values across genes with at least three transcript counts in three cells (n = 4914 genes). Gene expression overlay onto t-SNE visualization was also performed using z-normalized TPM values.

The enrichR R package (v0.0.0.9000) was used as an interface for the web-based functional annotation tool, Enrichr, to identify Gene Ontology (GO) terms from the GO Biological Process 2015 library significantly associated with each gene set (58, 59). Functional annotation results were listed in extended table S3.

Raw CEL files obtained from the Gene Expression Omnibus (GEO) for series GSE7895 were normalized to produce gene-level expression values using the implementation of the Robust Multiarray Average (RMA) in the affy R package (v1.36.1) and an Entrez Gene-specific probeset mapping (17.0.0) from the Molecular and Behavioral Neuroscience Institute (Brainarray) at the University of Michigan (http://brainarray.mbni.med.umich.edu/).

Bronchial brushings were reconstructed in silico from the single-cell data by taking the sum of all transcript counts for each gene across all cells procured from each donor. Negative binomial generalized linear models were built using the MASS R package (v7.3-45), modeling transcript counts as a function of smoking status (FDR q < 0.05: n = 593 genes). In parallel, using never and current smoker bulk bronchial brushing microarray data (GEO series GSE7895), linear models were built using the stats R package (R v3.2.0), modeling gene-level expression values as a function of smoking status (FDR q < 0.05: n = 689 genes). The correlation between test statistics generated from both models was then measured to compare differential expression results (fig. S4A). Using the overlap among smoking-associated genes identified in both models (n = 155 genes), correlations (Spearman) among in silico bronchial brushings and bulk bronchial brushings were examined (fig. S4B).

Using published microarray data generated from bulk bronchial brushings procured from never and current smokers (GEO series GSE7895), RMA-transformed values for each gene were z-normalized. MetaGene values were then generated by computing the mean z score across all genes in each gene set (GS-1 to GS-19) for each sample. Linear models were built using the stats R package (R v3.2.0), modeling MetaGene expression as a function of donor smoking status and age. For metagenes that were associated with smoking status (FDR q < 0.05), but not age, if the mean current smoker value was greater than or less than the mean never smoker value, the gene set was considered to be up- or down-regulated in current smokers, respectively.

TPM values for cell type marker genes (KRT5, FOXJ1, SCGB1A1, MUC5AC, and CD45) were z-normalized across all cells. Cluster-specific mean expression was designated high (pink) if expression exceeded 1 SD above the mean value across all cells, medium (white) if expression exceeded one-half of an SD above the mean value across all cells, and low (light gray) if expression exceeded the mean value across all cells. If cluster-specific mean expression was designated high, medium, or low for KRT5, FOXJ1, SCGB1A1, MUC5AC, or CD45 (PTPRC), that cluster was assigned the cell type of basal, ciliated, club, goblet, or WBC, respectively. Cluster-specific mean expression below the mean value across all cells indicated that a given cluster did not express a given marker gene (dark gray).

To assess smoking statusspecific cell enrichment for each cluster, logistic regression was performed using the stats R package (R v3.2.0), modeling each cluster assignment as a function of donor smoking status and the number of cells contributed by each donor. For clusters that were associated with smoking status (FDR q < 0.05), but not the number of cells contributed by each donor, the directionality of the regression coefficient was leveraged to assign never or current smoker status.

Transcript counts were transformed to z-normalized TPM. MetaGene values were then generated by computing the mean z score across all genes in each gene set (GS-1 to GS-19) for each cell. Cluster-specific MetaGene expression was designated high (pink) if mean expression exceeded 1 SD above the mean value across all cells, medium (white) if mean expression exceeded one-half of an SD above the mean value across all cells, and low (light gray) if mean expression exceeded the mean value across all cells. Cluster-specific mean expression below the mean value across all cells indicated that a given cluster did not express a given gene set (dark gray).

Bronchial tissue was collected from patients undergoing lung resection. All specimens were procured at least 5 cm from bronchial sites affected by disease diagnoses, and analyses indicated that tissue was histologically normal. The UMCG cohort (table S2) included specimens analyzed in collaboration with the UMCG collected from four never smokers and four current smokers. Specimens were obtained from the tissue bank in the UMCG Department of Pathology. The study protocol was consistent with the Research Code of the UMCG and Dutch national ethical and professional guidelines (Code of conduct; Dutch federation of biomedical scientific societies; http://www.federa.org). The UCL cohort (table S3) included specimens analyzed in collaboration with the UCL collected from five never smokers and five current smokers. Ethical approval was sought and obtained from the UCL Hospital Research Ethics Committee (REC reference 06/Q0505/12). This study was carried out in accordance with the Declaration of Helsinki (2000) of the World Medical Association.

Formalin-fixed paraffin-embedded lung sections were cut at 4 mm, tissue was probed with primary antibodies (listed below) and secondary antibodies with fluorescent conjugates (Invitrogen Alexa Fluor 488, 594, 647), and nuclear staining was performed with 4,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher, R37606). Immunostaining was performed using the following primary antibodies: mouse antiAc--Tub (Sigma, T6793), rabbit antiAc--Tub (Enzo Life Sciences, BML SA4592), rabbit anti-AKR1B10 (Sigma, HPA020280), rabbit anti-CEACAM5 (Abcam, ab131070), chicken anti-KRT5 (BioLegend, 905-901), rat anti-KRT8 (Developmental Studies Hybridoma Bank, University of Iowa; TROMA-I), mouse anti-MUC5AC (Abcam, ab3649), and rabbit anti-MUC5B (Sigma, HPA008246). Imaging of staining panels analyzed in collaboration with investigators at the UMCG (table S2) (e.g., AKR1B10/Ac--Tub/KRT8: Fig. 3C; AKR1B10/MUC5AC/KRT8: fig. S13; MUC5B/MUC5AC: Fig. 4B; MUC5B/MUC5AC/KRT5: fig. S14; CEACAM5/KRT8/MUC5AC: fig. S18) was performed using a Carl Zeiss LSM 710 NLO confocal microscope at 63 objective magnification at the Boston University School of Medicine Multiphoton Microscope Core Facility. Imaging of staining panels analyzed in collaboration with investigators at the UCL (table S3) (e.g., CEACAM5/KRT8/MUC5AC: Fig. 5D; KRT8/MUC5AC/Ac--Tub: fig. S15) was performed using a Leica TCS Tandem confocal microscope at 63 objective magnification.

All imaging data were analyzed using ImageJ Fiji software. For each image, cells were counted relative to the measured length of the epithelium in micrometers (cells per micrometer). Mean cell counts per micrometer (cells per millimeter) were then calculated for never smokers (treated as the control), and individual values for each image from never and current smokers were calculated relative to the never smoker mean (i.e., relative cells per millimeter). We analyzed three images for each donor and assessed smoking-associated changes using the Wilcoxon rank-sum test. For panels in which MUC5AC was stained, current smoker tissue was assigned the phenotypic status of either MN or GCH based on qualitative assessment of goblet cell density and stratification. For each current smoker, three images of each status were analyzed.

Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/12/eaaw3413/DC1

Table S1. Bronchial brushings were procured from six never smokers and six current smokers.

Table S2. Bronchial tissue was obtained by lung resection from four never smokers and four current smokers at the UMCG.

Table S3. Bronchial tissue was obtained by lung resection from five never smokers and five current smokers at the UCL Hospital.

Fig. S1. Single bronchial cells were isolated by FACS.

Fig. S2. scRNA-Seq data quality were evaluated for each donor.

Fig. S3. Low-quality cells were excluded from downstream analyses.

Fig. S4. Bronchial brushings reconstructed in silico from single-cell data resemble data generated from bulk bronchial brushings.

Fig. S5. LDA was used to identify Cell-States and Gene-States.

Fig. S6. Gene-State and Cell-State model optimization.

Fig. S7. LDA was used to identify 13 cell clusters.

Fig. S8. LDA was used to identify 19 gene sets.

Fig. S9. Gene set expression across cell clusters.

Fig. S10. T cell receptor genes were detected in CD45+ cell cluster.

Fig. S11. Cluster 13 cells expressed CFTR.

Fig. S12. Distributions of cell clusters within each subject.

Fig. S13. Smoking-associated differential expression of each gene set was analyzed in published bulk bronchial brushing data.

Fig. S14. Nonciliated cell AKR1B10 expression was uncommon.

Fig. S15. MN and GCH tissue regions were distributed throughout the bronchial airways of current smokers.

Fig. S16. Basal cell numbers were not altered in smokers.

Fig. S17. Increased numbers of indeterminate KRT8+ cells were observed in GCH smoker tissue.

Fig. S18. PG cells were enriched in regions of GCH within the airways of smokers.

Fig. S19. Smoking-induced heterogeneity was observed in the human bronchial epithelium.

Extended table S1. Primer sequences for scRNA-Seq.

Extended table S2. Statistical modeling results, State Specificity, and State Similarity values for all genes.

Extended table S3. Functional annotation results for each gene set.

This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license, which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited.

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Characterizing smoking-induced transcriptional heterogeneity in the human bronchial epithelium at single-cell resolution - Science Advances

MELVILLE, N.Y., Dec. 16, 2019 (GLOBE NEWSWIRE) -- BioRestorative Therapies, Inc. (BioRestorative or the Company) (OTC: BRTX), a life sciences company focused on stem cell-based therapies, announced today feature coverage in the news outlet, IEEE Pulse, a magazine of the IEEE Engineering in Medicine and Biology Society. According to IEEE, it is the worlds largest technical professional organization for the advancement of technology.

To view the IEEE Pulse Magazines article featuring BioRestorative, click here.

The published cover-story article features commentary from Francisco Silva, Chief Scientist and Vice President of Research and Development for BioRestorative, regarding BRTX-100, the Companys lead therapeutic candidate for chronic lumbar disc disease. Once the U.S. Food and Drug Administration (FDA) authorizes the sale of BRTX-100, we would ship it to your doctor, and with a 30-minute procedure the material would be injected into your disc in a 1.5 ml solution, explains Silva. He elaborates on the product, discussing growing and expanding stem cells from the patients bone marrow under hypoxic conditions that mimic those in the normal intervertebral space. We are enriching the cells to be able to survive in this harsh environment, says Silva.

In addition to BRTX-100, the magazine article also highlights BioRestoratives other research pursuit, its ThermoStem program, utilizing brown adipose (fat) derived stem cells to target treatment of metabolic diseases and disorders, like diabetes, obesity and hypertension.

About BioRestorative Therapies, Inc.

BioRestorative Therapies, Inc. (www.biorestorative.com) develops therapeutic products using cell and tissue protocols, primarily involving adult stem cells. Our two core programs, as described below, relate to the treatment of disc/spine disease and metabolic disorders:

Disc/Spine Program (brtxDISC): Our lead cell therapy candidate, BRTX-100, is a product formulated from autologous (or a persons own) cultured mesenchymal stem cells collected from the patients bone marrow. We intend that the product will be used for the non-surgical treatment of painful lumbosacral disc disorders. The BRTX-100 production process utilizes proprietary technology and involves collecting a patients bone marrow, isolating and culturing stem cells from the bone marrow and cryopreserving the cells. In an outpatient procedure, BRTX-100 is to be injected by a physician into the patients damaged disc. The treatment is intended for patients whose pain has not been alleviated by non-invasive procedures and who potentially face the prospect of surgery. We have received authorization from the Food and Drug Administration to commence a Phase 2 clinical trial using BRTX-100 to treat persistent lower back pain due to painful degenerative discs.

Metabolic Program (ThermoStem): We are developing a cell-based therapy to target obesity and metabolic disorders using brown adipose (fat) derived stem cells to generate brown adipose tissue (BAT). BAT is intended to mimic naturally occurring brown adipose depots that regulate metabolic homeostasis in humans. Initial preclinical research indicates that increased amounts of brown fat in the body may be responsible for additional caloric burning as well as reduced glucose and lipid levels. Researchers have found that people with higher levels of brown fat may have a reduced risk for obesity and diabetes.

Forward-Looking Statements

This press release contains "forward-looking statements" within the meaning of Section 27A of the Securities Act of 1933, as amended, and Section 21E of the Securities Exchange Act of 1934, as amended, and such forward-looking statements are made pursuant to the safe harbor provisions of the Private Securities Litigation Reform Act of 1995. You are cautioned that such statements are subject to a multitude of risks and uncertainties that could cause future circumstances, events or results to differ materially from those projected in the forward-looking statements as a result of various factors and other risks, including, without limitation, whether the Company will be able to consummate the private placement and the satisfaction of closing conditions related to the private placement and those set forth in the Company's Form 10-K filed with the Securities and Exchange Commission. You should consider these factors in evaluating the forward-looking statements included herein, and not place undue reliance on such statements. The forward-looking statements in this release are made as of the date hereof and the Company undertakes no obligation to update such statements.

CONTACT:Email: ir@biorestorative.com

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BioRestorative Therapies Featured in IEEE Pulse Magazine's Cover Story About Stem Cell Therapies for Low Back Pain - GlobeNewswire

A team of scientists from Peter the Great St.Petersburg Polytechnic University (SPbPU) studied the evolutionary ages of human genes and identified a new class of them expressed in tumors -- tumor specifically expressed, evolutionarily novel (TSEEN) genes. This confirms the team's earlier theory about the evolutionary role of neoplasms.

A report about the study was published in Scientific Reports.

A tumor is a pathological new growth of tissues. Due to genetic changes, it has impaired cellular regulation and therefore defective functionality. Tumors can be benign or malignant. Unlike the latter, the former grow slowly, don't metastasize, and are easy to remove. Malignant tumors (cancer) are one of the primary mortality factors in the world.

A team of scientists from Saint Petersburg discovered a new class of evolutionarily novel genes present in all tumors -- the so-called TSEEN (Tumor Specifically Expressed Evolutionarily Novel) genes.

The evolutionary role of these genes is to provide genetic material for the origin of new progressive characteristics. TSEEN genes are expressed in many neoplasms and therefore can be excellent tumor markers."

Prof. Andrei Kozlov, a PhD in Biology, the head of Laboratory "Molecular Virology and Oncology" at Peter the Great St. Petersburg Polytechnic University

The new research confirms a theory that has been proposed by the A. Kozlov earlier. According to it, the number of oncogenes in a human body should correspond to the number of differential cell types. The theory also suggested that the evolution of oncogenes, tumor suppressor genes, and the genes that determine cell differentiation goes on concurrently. The theory is based on the hypothesis of evolution through tumor neofunctionalization, according to which hereditary neoplasms might have played an important role during the early stages of metazoan evolution by providing additional cell masses for the origin of new cell types, tissues, and organs. Evolutionarily novel genes that originate in the DNA of germ cells are expressed in these extra cells.

Prof. Kozlov also made a reference to the article 'Evolutionarily Novel Genes Are Involved in Development of Progressive Traits in Humans' (2019) that has recently been published by his laboratory. In this article the team confirmed their hypothesis using transgenic fish tumors and fish evolutionarily novel genes. The orthologs of such genes are found in the human genome, but in humans they play a role in the development of progressive characteristics not encountered in fish (e.g. lungs, breasts, placenta, ventricular septum in the heart, etc). This confirms the hypothesis about the evolutionary role of tumors. The studies referred to in the article lasted for several years, and their participants used a wide range of methods from the fields of bioinformatics and molecular biology.

"Our work is of great social importance, as the cancer problem hasn't been solved yet. Our theory suggests new prevention and therapy strategies," said Prof. Kozlov. According to him, to fight cancer, a new paradigm should be developed in oncology. TSEEN genes may be used to create new cancer test systems and antitumor vaccines.

Source:

Journal reference:

Makashov, A.A., et al. (2019) Oncogenes, tumor suppressor and differentiation genes represent the oldest human gene classes and evolve concurrently. Scientific Reports. doi.org/10.1038/s41598-019-52835-w.

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Evolutionarily novel genes found to be expressed in tumors - News-Medical.net

From arthritis and heart failure to diabetes and menopause, many conditions are associated with muscle weakness and increased fat deposits.

Now a multidisciplinary team of researchers at the University of Massachusetts Amherst is applying a unique approach to examine the effects of fat tissue on skeletal muscle structure and function in young and older men and women.

Armed with a two-year, $374,188 grant from the National Institute on Aging, lead investigator Jane Kent, professor and chair of kinesiology in the School of Public Health and Health Sciences, and colleagues will combine state-of-the-art, noninvasive magnetic resonance imaging and spectroscopy techniques with whole-body, single-cell and molecular measures of muscle function.

As muscle typically contributes 30-40% of total body mass, this metabolically active tissue plays a direct role in maintaining good health. Currently, we do not know the mechanical consequences of fat infiltration on muscle. Our hypothesis is that fat physically limits muscle strength by interfering with the way the muscle was designed to work."

Jane Kent, lead investigator

The collaborative research is being performed in the Human Magnetic Resonance and Human Health and Performance centers at the Institute for Applied Life Sciences (IALS), where scientists strive to translate fundamental research into innovations that benefit humankind. Advanced data analysis will be carried out in the Muscle Physiology and Muscle Biology laboratories in the Totman building.

Kent is working with kinesiology assistant professor Mark Miller, endocrinologist and research professor of kinesiology Dr. Stuart Chipkin, math and statistics professor emeritus John Buonaccorsi and professor Bruce Damon from the Vanderbilt University Institute of Imaging Science. Graduate students Joseph Gordon III and Christopher Hayden, along with project coordinator Nicholas Remillard, round out the research team.

Kent says the innovative research may yield new knowledge about the effects of fat on muscle activity, information that has potential health benefits.

"Understanding the impact adipose tissue has on skeletal muscle has the potential to markedly alter our approach to mitigating and reversing muscle dysfunction in aging and the large number of conditions associated with increased fat content in muscle," Kent says.

The research team is recruiting volunteers to round out the study group of overweight and obese young adults, age 25-45, and healthy older adults, age 65-75. Participants would be required to visit the campus up to three times and would receive financial compensation for their time.

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Study examines the effects of fat tissue on skeletal muscle structure and function - News-Medical.net

The 3-D view of the nuclear lamina shows a section of the architecture of the delicate meshwork made of lamin filaments (filament rod in dark grey and its globular domains in red) beneath the cell nuclear membrane (transparent grey) and the nuclear pore complexes (blue). Credit: Yagmur Turgay, University of Zurich

Using 3-D electron microscopy, structural biologists from the University of Zurich succeeded in elucidating the architecture of the lamina of the cell nucleus at molecular resolution for the first time. This scaffold stabilizes the cell nucleus in higher eukaryotes and is involved in organizing, activating and duplicating the genetic material. Diseases such as muscular dystrophy and premature aging, caused by mutations in the lamin gene, the major constituent of the lamina, can now be studied more effectively.

Compared to bacteria, in eukaryotes the is located in the cell nucleus. Its outer shell consists of the nuclear membrane with numerous nuclear pores. Molecules are transported into or out of the cell nucleus via these pores. Beneath the membrane lies the nuclear lamina, a threadlike meshwork merely a few millionths of a millimeter thick. This stabilizes the cell nucleus and protects the DNA underneath from external influences. Moreover, the lamina plays a key role in essential processes in the cell nucleus such as the organization of the chromosomes, gene activity and the duplication of genetic material before cell division.

Detailed 3D image of the nuclear lamina in its native environment

Now, for the first time, a team of researchers headed by cell biology professor Ohad Medalia from the Department of Biochemistry at UZH has succeeded in elucidating the molecular architecture of the nuclear lamina in mammalian cells in detail. The scientists studied fibroblast cells of mice using . This technique combines and tomography, and enables cell structures to be displayed in 3D in a quasi-natural state, explains Yagmur Turgay, the first author of the study. The cells are shock-frozen in liquid ethane at minus 190 degrees without being pretreated with harmful chemicals, thereby preserving the cell structures in their original state.

The lamin meshwork is a layer thats around 14 nanometers thick, located directly beneath the pore complexes of the nuclear membrane and consists of regions that are packed more or less densely, says Yagmur Turgay, describing the architecture of the nucleoskeleton. The scaffold is made of thin, threadlike structures that differ in length the lamin filaments. Only 3.5 nanometers thick, the lamin filaments are much thinner and more delicate than the structures forming the cytoskeleton outside the in higher organisms.

New approach for research on progeria and muscular dystrophy

The building blocks of the filaments are two proteins type A and B lamin proteins which assemble into polymers. They consist of a long stem and a globular domain, much like a pin with a head. Individual mutations in the lamin gene elicit severe diseases with symptoms such as premature aging (progeria), muscle wasting (), lipodystrophy and damage of the nervous system (neuropathies). Cryo-electron tomography will enable us to study the structural differences in the nuclear lamina in healthy people and in patients with mutations in the lamin gene in detail in the future, concludes Ohad Medalia. According to the structural biologist, this method permits the development of new disease models at molecular level, which paves the way for new therapeutic interventions.

The study is published in Nature.

More information: Yagmur Turgay et al, The molecular architecture of lamins in somatic cells, Nature (2017).

Originally posted here:
Molecular structure of the cell nucleoskeleton revealed for the first time - Spring Hill Insider

The Poor Poet Carl Spitzweg (1838)

Were back! The seventh annual McGill Reads holiday reading list has been carefully compiled and lovingly laid out, highlighting the thought-provoking selection of great reads as suggested by McGill students, staff, faculty and administrators.

Once again, eclectic is our watchword as our list features everything from literary classics to fresh new releases; sci-fi to self-help; best-sellers to biographies; and poetry to graphic novels. And, as always, just as fascinating as the diverse list itself are the backstories behind each selected title that give us a little glimpse into each of our contributors.

Thanks to everyone who participated and may all your books be page-tuners!

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We will kick things off by giving a big McGill Reads welcome to Manal Abou-Ghaida, one of the Universitys newest employees. Im new to McGill (started December 9th!), and I just stumbled on the McGill Reads book list call for entries, writes Abou-Ghaida, Records Administrator, Enrolment Services. I love to read! Im from Edmonton, Alberta, and Im known for starting book clubs in my place of work.

Abou-Ghaida suggests Agatha Christies And Then There Were None because I love a good classic mystery, something thats a page turner, and importantly over the holiday season something that excites me and stirs my imagination.

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From a first-timer, we go to a grizzled McGill Reads vet. One of our lists most enthusiastic supporters, Victor Chisholm has been contributing since the inaugural McGill Reads in 2013.

I am grateful to the Cundill History Prize for providing such interesting options in their annual shortlists and longlists, says the Student Affairs Administrator, Faculty of Science. From the 2019 shortlist, I would be delighted to read Victoria Johnsons American Eden: David Hosack, Botony, and Medicine in the Garden of the Early Republic, because anything that makes us think about plants, without whom our lives would be impossible, must be worth reading!

Ideas on CBC Radio just aired an interview with a past laureate, Maya Jasanoff, who won the 2018 Cundill Prize for her book The Dawn Watch: Joseph Conrad in a Global World. She gave a fascinating account of how to read Conrads writings about colonialism both critically and appreciatively, says the avid cyclist and ice cream connoisseur. Ive never read Conrad, and the prose excerpts that were aired on the radio were astonishing, so I may give Heart of Darkness a try.

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Sean Goldfarb, a graduate student in Cell Biology, has his eyes on a number of books including:

Les racines du ciel, by Romain Gary, whose books La vie deavant soiIread(one of two books Ireadthat ever made me cry), andLes cerfs-volantswhichIm finishing up. Its funny, it took me till Iread a non-English book to be able to say Ive found my favourite author!

Of Human Bondage, by W. Somerset Maugham, which is my mothers favourite book by her favourite author. Just need to find it among all the books at home

The Painted Veil, by W. Somerset Maugham, because Im drawn to it, having found it while looking for Of Human Bondage.

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Domenic Aversano, Customer Service Coordinator, Printing Services is succinct and to the point when it comes to his pair of suggestions for people looking for books to tackle during the break. Aversano recommends The View from Flyover Country, by Sarah Kendzior and How to be Less Stupid about Race, by Crystal M. Fleming. Two books that are essential to understand and challenge who we are and why, he writes.

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Anita Kar admits to having already read most of The Four Agreements by Don Miguel Ruiz, but I want to finish it over the holidays and try to assimilate it into my brain permanently, says the Montreal Neurological Institute and Hospital Communications Officer. The simple reason is because I think the messages in this book will help me be more peaceful in my everyday life, in everyday situations.

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Over the holidays, Ill bereading Annie ProulxsBarkskins, an epic tale that follows the lives of three main characters over the course of 300 years: the first two being French colonists (and their respective progeny) as they eke out a life in North America, and the third being the forests and wild spaces of the new world, perceived at first as vast and endless but gradually recognized as finite and fragile, writes Andra Syvnen, Assistant Dean, Admissions & Recruitment.

The book is beautifully written an amazingly detailed and captivating recounting of hardship, success, adventure, cultural genocide and environmental destruction that is heartbreaking in its vividness. Its impossible to put down.

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Jim Nicell is another longtime contributor to McGill Reads and is renowned for his voracious literary appetite. My wife knows very well that I sneak books into the house when she isnt looking, jokes the Dean of the Faculty of Engineering.

My to read this year is a little eclectic. I plan to get a head start on the holidays next week by starting the new biography of Thomas Edison called Edison by Edmund Morris, says Nicell.

After that, I plan to dive into Maoism: A Global History, which was on my bookshelf well before its author, Julia Lovell, was nominated and then awarded the 2019 Cundill Prize. I often tell my wife that I take great pride in picking winning books each year, but she seems to think that this may simply be due to the fact that I buy too many books. Shes probably right, but dont tell her, says Nicell. And then, to lighten the reading a bit, I am hoping to reread Douglas Adams complete Hitchhikers Guide to the Galaxy series again and, in the process, try to get my kids addicted to it. Hope springs eternal.

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Gwen Wren, U3 Environment, says she will be finishing Margaret Atwoods Testaments. Im a big Atwood fan and I think the issues she engages with through fiction are very prevalent in the world today, writes Wren.

As well, Ill be re-reading Marina Keegans The Opposite of Loneliness, a book of short essays that reminds me to slow down and relish in the moment. Something I often forget in the fast pace life of being a McGill student.

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Cynthia Leehas a pair of books lined up for the break. First, she will tackle We Are the Weather: Saving the Planet Begins at Breakfast, by Jonathan Safran Foer. Recently I watched the documentary, Eating Animals based on Safran Foers book and it was very powerful, I encourage everyone to watch it, says everyones favourite Associate Director, Media Relations. This subject of this book is again global warming and its direct relation to humans consumption of animals for food.

Next, she will revisit When Breath Becomes Air, by Paul Kalanithi. Im rereading this book because its one of my favorites, she says. Its the memoir of Paul Kalanithi, an American neurosurgeon who was diagnosed with stage IV lung cancer at the age of 36. One day he was a doctor treating the dying, and the next he was a patient struggling to live.

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Kim Davies, an Administrator at the Rossy Cancer Network, is nothing if not precise. She says she will read the following three books, in order (with page count);

Malcolm Gladwell, Talking to Strangers: What We Should Know About the People We Dont Know 400 pages

Ryu Murakami, Piercing 192 pages

Ian Williams, Reproduction 464 pages

1,056 pages in 10 days. ~106 pages/day, notes Davies.

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This year I will be less ambitions in my holiday reading, writes regular contributor Kendra Gray. The feature of my holiday reading will beBlowout by Rachel Maddow. Russia, oil, and politics, as told by Rachel Maddow. I can think of no better combination.

Gray also plans on finishingAmin Maaloufs Origins: A Memoir, the story of Maaloufs family and his travels to Cuba to learn about the great-uncle who left Lebanon and made a life in Cuba in the early 1900s. In addition to being a fascinating history of the time, it is addresses issues of belonging, home and success, says Gray.

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Antonia Maioni, Dean of the Faculty of Arts has set three reading goals for the holidays.

The Cundill History Prize (administered by the Faculty of Arts) attracts extraordinary books from around the globe, but only a sliver make it as the three finalists, says Maioni. I plan to go back to two fascinating shortlisted books: A Fistful of Shells by Toby Green and Unruly Waters by Sunil Amrith.

Next, Maioni wants to catch up with Louise Pennys Armand Gamache series. She is so prolific I can barely keep up!

Finally, says Maioni, as a Tudor history buff, I will be re-reading Wolf Hall and Bring Up the Bodies in anticipation of Hilary Mantels new book, the Mirror and the Light, due out in March 2020.

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William Hip Kuen, MSc in Mathematics and Statistics,plans on reading A Message to Garcia by Elbert Hubbard. I have heard that this book is extremely fruitful for university students, and I decided to read it to help myself become a better person in the future, he says.

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Here is my list if my history holds true I will read two of these, and something else, writes McGill Reads regular Kimberley Stephenson, Trade Buyer, Le James McGill University Bookstore. Stephensons list includes:

Spying on the South by Tony Horwitz, because I loved his previous book Confederates in the Attic, and he brilliantly mixes the past with the present.

One Day by Gene Weingarten. Weingarten is the author of Fiddler on the Subway, and the Pulitzer Prize winning feature article Fatal Distraction I am looking forward to a longer piece by him.

Blue Moon by Lee Child, because Reacher.

High Five Joe Ide this is Joe Ides fourth book. If Sherlock Holmes lived in South Los Angeles and was a scrawny kid, then you would have IQ, the detective in this series.

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Melissa-Anne Cobbler, Wellness Advisor, Faculty of Science has two books lined up for the break; Magnetic Equator, by Kaie Kellough; and Here Comes the Sun, by Nicole Y. Dennis-Benn

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If we had a Best Backstory Award, it might very well go to Juliana Rigante, who will be graduating in May 2020 with a Bachelors degree in Pharmacology.

Being a student in science and working part-time can be quite overwhelming at times, so to stay sane, I sing opera at my high school with the same teacher whos been training me since I was 12 years old, she writes. Thats why this year, A Night at the Opera: An Irreverent Guide to The Plots, The Singers, The Composers, The Recordings, by Sir Denis Forman is on my list. I cant wait to educate myself on the songs Ive already sung and perhaps get some inspiration for what I can sing in the New Year!

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What fun! writes Carola Weil, Dean of the School of Continuing Studies. This actually reminds me of a project I initiated at my previous university where the dean of Libraries and I hosted a type of book club for the entire university community based on a list of 80 most influential books in the U.S. Members of the university community could volunteer to read, present and lead a discussion on their book of choice. Very illuminating and a lot of fun.

With those kind of literary chops, it comes as no surprise that Weils nightstand has the following stack of books waiting for her:

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A self-proclaimed huge Sci-Fi and historical fantasy fan, Bojan Vastagsays hisreading list is pretty long. However, recently I stumbled across Hugh Howeys Wool (first book of a Silo trilogy) and it blew my mind. It gives a good portrait of how our society is intertwined and how beliefs based on an edited truth impact our lives, says the Solutions Architect, IT Services This means that my holidayreads will be Shift (part 2) and Dust (part 3).

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Ive been enjoying the McGill Reads series for the past few years and I think its time I participated, writes Sarah Delisle, Emergency Planning Officer, Campus Public Safety.

Delisle will readGeorge Takeis They Called Us Enemy, a graphic novel about the actors childhood experiences in Japanese internment camps in the US during World War II. She says the book caught my eye as I was walking through Indigo recently and reminded me of Mark Sakamotos bookForgiveness: A Gift From My Grandparents, which Ireadas part of CanadaReads 2018. Sakamoto tells about his paternal grandparents experiences in WWII internment camps in Canada so I was drawn to They Called Us Enemy as a way to learn about what was going on south of the border. Plus, graphic novels are a format I love for autobiographies!

Delisle also plans to read Daniel Aldrichs Black Wave: How Networks and Governance Shaped Japans 3/11 Disasters.

Black Wave is part of my ever-expanding library of disaster-related books (professional hazard!), she writes. Working in emergency management, Im always interested in how communities respond to, deal with, and recover from large-scale disaster events, so Im intrigued to read about the response to the magnitude 9.0 earthquake (and resulting 60-foot tsunami) that impacted Japan in 2011.

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While Im a pretty avid reader all the time, I do find the holidays an especially satisfying time for getting into some great books, says Chris Buddle, out-going Dean of Students and in-coming Associate Provost, Teaching and Academic Programs.

On the fiction front, our favourite spider-hunting entomologist has lined up three selections.

Of course, Buddle also likes non-fiction and is planning to tackle a trio of tomes.

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Dorothy Redhead, Assistant to the Dean, Faculty of Arts, says she wants to finishreading Ducks and Newbury Port, by Lucy Ellmann and is looking forward to reading Save me the Plums, Ruth Reichls memoir of her time as editor of the magazine Gourmet. It was my favourite magazine and, sadly, is no more having published its last issue in November 2009, says Redhead.

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Sweet-talker Erin McDonagh writes that the annual McGill Reads is the highlight of the McGill holiday season!

Thank you, Erin. Its one of our favourite projects too!

The Teaching and Learning Planner from Teaching and Learning Services has two books lined up for the holidays.

First, theres The Secret History of Jane Eyre by John Pfordresher. The author reconsiders Charlotte Bronts life and experiences, and links them to the development of her novelJane Eyre, says McDonagh. Pfordresher challenges the idea that this small, short-sighted woman from the Yorkshire Moors had a uninteresting, sheltered life by showing how experiences with her brother Branwells addiction, her travels to Brussels and London, and her own thoughts and dreams provided her with ample grist for the mill of her literary imagination.

Next, McDonagh has chosen an all-time classic with a fantastical twist; Alice in Wonderland by Lewis Carroll, with illustrations by Salvador Dali. This book needs no introduction, and rereading it is a yearly holiday treat for me, she says. Last year, however, I discovered this fantastic illustrated version of the tale with works by Salvador Dali. It seems like a completely natural pairing, and adding weird, surrealist art to a weird, surrealist book has certainly yielded a fantastic product.

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Albert Le, a DMD candidate in the Faculty of Dentistry, thinks everyone should read aul Kalanithis When Breath Becomes Air. It is a poignant memoir from a man whose story that can inspire so many of us on the value of life, says Le. I cant do this book justice in describing it myself.

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Having just finished Claremont by local Montreal author, Wiebke von Carolsfeld, Dino Dutz, is singing its praises.

As a film director, I was already a fan of her award-winning films, so naturally I was intrigued toread her first novel, writes the Administrative Coordinator to the Associate Deans at the Schulich School of Music. Its a beautifully and sensitively written story that begins with a horrifying family trauma whose survivors must navigate through an uncertain, unexpected, often awkward aftermath. The characters imperfections are also their strengths, and their relationships to each other are a testament to the strange and unexpected ways that love can guide you through the unthinkable to a new version of family.

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Daniel McCabe wins the 13th Hour Award for the last submission of the year. Truthfully, if we didnt owe him money, he might not have made the cut.

I made a happy discovery recently when I stumbled upon a deeply discounted copy of Nextwave: Agents of H.A.T.E. in a downtown comic book shop, writes the much-admired editor of McGill News. A collaboration between Warren Ellis, one of the comic book worlds most influential writers (the second Iron Man film borrows heavily from one of his works) and the wonderfully versatile Canadian artist Stuart Immonen, Nextwave pokes gentle fun at the absurdity of superheroes, while simultaneously delighting in some of the over-the-top pleasures that super hero comics offer.

McCabe says his Christmas present to himself will be another graphic novel: the years-in-the-making Clyde Fans collection. The book, about a failing family business and the long, troubled relationship between two brothers, is by Seth, another Canadian, who is known for his retro cartoony style. The tone is nuanced and full of melancholy, and Seth, whose artwork has been featured in more than one museum, is one of his mediums most skilled cartoonists exemplified in the careful way he constructs a narrative, panel by panel by panel.

A fan of Emily Nussbaum, McCabe is looking forward to reading her new book I Like to Watch: Arguing My Way Through the TV Revolution. She is a Pulitzer Prize-winning TV critic for The New Yorkerand her collection of essays explores the legacy of Norman Lear, looks at some of the questions raised by the #MeToo movement, and makes the case for whyBuffy the Vampire Slayeris one of the greatest shows in TV history.

One of her favourite shows and one of mine too is Bojack Horseman.Its among the funniest shows around, but its also pointed, wrenching and moving at times, says McCabe. The mastermind behind the show, Raphael Bob-Waksberg, has a new short story collection out, Someone Who Will Love You in All Your Damaged Glory. Given how much I loveBojack,I was thinking of picking up a copy.

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David Syncox says Bren Browns Dare to Lead has been on my desk for the entire fall semester and the hes determined to read it this holiday season. I am excited to read it as it speaks to being courageous and vulnerable as a leader, says the perpetually upbeat Director, Alumni Communities. As a staunch believer in lifelong learning and skills development, Im excited to learn a few strategies to add to my ever growing tool box.

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When Caitlin MacDougall last checked in with us earlier this year for the McGill Reads summer edition, she had already read or listened to 39 of the 75 books she was planning to tackle this year. Take that Jim Nicell!

We are happy to report that the Liaison Officer for the Farm Management and Technology Program has finished her 69th book and is on track to finish by December 31 (woot woot!), she says. Once again, it wouldnt be possible without the McGill Librarys Overdrive app and selection of audiobooks.

Over the holidays, MacDougall plans to read;

Continued here:
McGill Reads: 2019 holiday edition - McGill - McGill Reporter

The stock of ABCAM PLC ORDINARY SHARES UNITED KINGDO (OTCMKTS:ABCZF) registered a decrease of 3.12% in short interest. ABCZFs total short interest was 9,300 shares in December as published by FINRA. Its down 3.12% from 9,600 shares, reported previously. With 4,000 shares average volume, it will take short sellers 2 days to cover their ABCZFs short positions.

The stock increased 0.21% or $0.04 during the last trading session, reaching $18.18. About 296 shares traded. Abcam plc (OTCMKTS:ABCZF) has 0.00% since December 14, 2018 and is . It has by 0.00% the S&P500.

Abcam plc, together with its subsidiaries, produces and distributes research-grade antibodies and associated protein research tools worldwide. The company has market cap of $3.75 billion. The firm primarily offers primary and secondary antibodies, biochemicals, isotype controls, flow cytometry multi-color selectors, kits, loading controls, lysates, peptides, proteins, slides, tags and cell markers, and tools and reagents. It has a 45.56 P/E ratio. The Companys products are used in the areas of cancer, cardiovascular, cell biology, developmental biology, epigenetics and nuclear signaling, immunology, metabolism, microbiology, neuroscience, signal transduction, and stem cells, as well as in drug discovery services and products.

Another recent and important Abcam plc (OTCMKTS:ABCZF) news was published by Investorplace.com which published an article titled: The 3 Best Ways to Buy International Stocks Investorplace.com on July 25, 2016.

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Will ABCAM PLC ORDINARY SHARES UNITED KINGDO (OTCMKTS:ABCZF) Run Out of Steam Soon? Short Interest Is Down - FinanceRecorder

Getting to know cells well helps understand how organisms function. This is one of the aspects that drive scientists, researchers, and physicians to create bioprinting technology to generate living structures that can mimic the actual environment of human tissues. Bioprinters today usually involve a syringe-like mechanism to deposit cell material within a gel or scaffold structure, which helps keep the desired 3D shape while printing and is then washed away or dissolved. A Swedish company called Fluicell is out to change the reigning trend and has just released a new system for cell 3D printing. Based on innovative open-volume microfluidics technology, their brand new bioprinting system, Biopixlar, is capable of generating detailed, multi-cellular biological tissues without the need for a gel matrix.

The Biopixlar bioprinter

Biopixlar is designed for handling scarce and valuable cell sources such as stem cells, primary cells, and patient biopsies. The company has actually begun working at their own labs building full tissue and cancer models, which usually takes them just 24 hours to print thanks to their technology. The system is an all-in-one discovery platform that allows the printing of multiple types of different cells at once with high precision and resolution. One of the fun features is the gamepad interface, used to manually control the position of the print head and deposit the cells. Also, an integrated multi-color fluorescence imaging configuration enables real-time monitoring of the printing process and post-print analysis.

Gavin Jeffries

Fluicell, a spin-off company out of Chalmers University of Technology, in Sweden, has been around since 2012, developing biotech hardware devicesincluding the BioPen and Dynaflow Resolve systemsbut their research has taken them to explore changes in the bioprinting market, namely producing human-like tissue replicas. 3DPrint.com spoke to Gavin Jeffries, co-founder and Chief Technology Officer at Fluicell, to understand the process behind Biopixlar.

How did Fluicell become a pioneer in open-volume microfluidics?

Microfluidics is essentially the control of liquids on a smaller scale and is very useful when scientists need to handle the smallest amounts of liquid or have very rare samples and need fast responses. Over the last 20 years, microfluidics has been advancing quite a lot but has largely focused on chip-based devices, which means the whole field is centered around putting cells or liquids inside another device. When we first started the company we noticed that having something inside a device was restrictive, because within biology you normally want to have your cells in a petri dish or on your microscope, not inside a chip. But at the same time, we wanted to harness the power of microfluidics to use small sample amounts and have those very fast response times, so essentially we came up with a way of very precisely controlling both positive and negative pressures to allow control of liquids outside of our microfluidic chip. Meaning we can still have the function of microfluidics but in an open volume (basically in any kind of biological platform.) Since 2011, this technology has been picked up by different fields for research.

How will the gamepad simplify the user experience?

Biopixlar is a complete discovery platform, with everything embedded in it. Actually, just like a game system, the gamepad interface provides user control over the responsivity of the machine. This control format is ideal for people who are coming into the workforce and who have grown up with advanced interfaces, without the need to use a mouse or a keyboard. We also hoped to focus on the comfort of working with the device, for example, researchers will be able to get a direct response in real-time because it is fully embedded with microscopy, so they will see everything they are doing, every cell they put in, just everything.

Biopixlar is designed to be a complete platform where discovery science is its home and marketplace. Research and development, whether it is looking at disease models or interrogating biological systems, the user has control over building these early-stage models as accurately as possible. These can be found in academia and the pharma industry, so it will be our first bridge between the two market segments.

Closeup of the Biopixlar printhead

What makes Biopixlar so unique?

After one layer of cells is put down, Biopixlar allows them to grow and then pattern them using a molecular cell binder to put the next level of cells, and so on, building up layer by layer and using the extracellular matrix binding agent in between, which would naturally be reproduced by the cells. We chose to use components of the extracellular matrix that are naturally formed with the cells so that the device can pattern them on top of the cells which are printed, allowing for more cells to attach. In this way, researchers will not need to house the cells in any binder to build in three dimensions.

Why is cell viability really high with the Biopixlar system?

That is largely because of the microfluidics within the device. We use a consumable cartridge to load the cells, but inside there is a series of complex circuitry that allows the handling of liquids in a no-sharing regime so the fluids dont rub against each other and the cells are much happier being in this kind of no shared environment. When we patterned the cells at the lab, we noticed that there is no negative impact of printed cells versus putting them in a dish. Moreover, we feel comfortable and very happy that we minimally interfere with the cells when we build them into the structure that we want to create.

Printed skin cancer model

Do you consider Biopixlar will be successful among researchers?

We stand alone within the market of bioprinting because we do not need to use any binding matrix, our goal is to put cells as close as possible to each other so that they begin communicating straight away. Most of the full tissue and cancer models we built at the lab were done within 24 hours, and this is largely due to the fact that we dont have anything in the way of the cells communicating with each other. Additionally, thanks to the gamepad, we can see exactly what we are doing in real-time. The technology sparks interest in the field because people can actually see the bridge between advanced technology and biology and we are now starting to get to a point where we can show results and people are starting to get excited about them.

Is understanding cell behavior at the core of what you do?

The only thing which we are really focusing on is the cells. With Biopixlar, scientists dont have to pattern ink or deposition areas, they will not have to deal with that and instead, focus on the cells. Biopixlar has a unique advantage to see if anything is going wrong because if something were to happen to the cells or the biology during the process, it will be seen directly. Thanks to the high-resolution microscopy, we can interrogate the cells as they are printed or while they are growing. This all-in-one discovery platform approach is necessary to carry out bioprinting while providing advantages over how the biological tissues are actually built.

Printed liver cancer model

How would you describe Biopixlar to a potential buyer?

It is a high-resolution machine that fits in a comfortable lab setting with an easy-to-use experience, built with microscopy for looking at individual cells. Researchers need an accurate micro position to move around all the microcomponents while having a very stable infrastructure because it is moving on the micron size scale, instead of the millimeter size, we wouldnt want it to vibrate and lose calibration in the middle of a print. Overall, it is an accessible, original and optimal resolution device for lab spaces.

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Interview with Gavin Jeffries from Fluicell: Cell Biology is at the Core of our Work - 3DPrint.com

XconomySan Diego

Nearly nine years ago Jeanne Loring and her colleagues at Scripps Research debuted a test that leveraged advances in genomics and data science to determine, without testing in animals, whether human stem cells were pluripotent, or able to become any type of cell in the body.

Being able to prove that has become increasingly important as scientists look to induced pluripotent stem cells (iPSCs)mature, specialized cells that have been reprogrammed as immature cells, regaining the capability of becoming any type of cellas material for new regenerative medicines.

Now Loring and Andres Bratt-Leal, who joined her lab in 2012 as a post-doctoral researcher, have founded a biotech that combines stem cell biology and genomics know-how to advance a potential cell therapy for Parkinsons disease.

The startup announced Thursday it raised a seed round of $6.5 million to support its work. Aspens lead drug candidate, which is in preclinical testing, is intended to replace neurons in the brains of people with the disease, which causes those cells to become damaged or die.

When people with Parkinsons disease lose neurons, they also lose a chemical messenger the cells produce, called dopamine. Without dopamine, communication between nerve cells falters, which leads to the debilitating motor problems that characterize the disease. Existing Parkinsons drugs aim to alter dopamine levels. Aspen, however, wants to fix the upstream problem that leads to those lowered levels by reconstructing patients damaged neural networks.

The cell therapy would involve harvesting patients own living cells through a skin biopsy, reprogramming them to immature cells, or iPSCs, then further engineering them to become predisposed to mature into neurons. Once enough of those cells have been grown in the lab, those neuron precursor cells would be delivered directly to the brain.

Using a patients own cells avoids the dangerous immune system reactions that can occur when donor cells are used in such therapies, and obviates the need for immunosuppression drugs. Two cell therapies that use genetic engineering have been approved by the FDA, both of which take and tweak patients T cells into treatments for cancer. Stem cell transplants have been used to treat some cancers.

Aspen worked to ensure the company could ably manufacture a so-called autologous replacement cell therapy, or one from a patients one cells, by improving the process of differentiating iPSCs into dopamine neurons, Loring says. And the group developed another predictive genomic-based test, similar to the effort Loring spearheaded nearly a decade ago to determine whether cells were pluripotent, that can detect which iPSCs are destined to become neurons.

(Bratt-Leal) put his biological engineering expertise into coming up with a way that was reproducible, that we would get the same cells no matter who we got the original cells from, she says.

The company plans to test the therapy in patients that they determine, through genomic testing, have the most common form of Parkinsons, which is referred to as sporadic and arises without a clear genetic predisposition. It also has a second treatment in the works that it intends to develop for patients with familial forms of the disease, and uses a gene editing toolyet to be selectedto alter their stem cells during the reprogramming process.

Howard Federoff, who was most recently vice chancellor for health affairs and CEO of the UC Irvine Health system, is Aspens CEO. Federoff says he has come to believe that Parkinsons patients need more than just to stabilize their disease They need to turn the clock back.

Many companies are working on drugs to treat Parkinsons, but most are meant to manage symptoms rather than reverse the disease. Levodopa, which supplants missing dopamine, is used widely, but it can cause side effects, including involuntary movement called dyskinesia; and, as the disease progresses, the drug eventually stops working between doses.

Aspen claims it is the only company working toward an autologous neuron replacement. The company, however, will need to raise a Series A round to move its drug candidates through Phase 2 proof-of-concept trials, Loring says.

The company raised its seed round from a group of investors including Domain Associates, Alexandria Venture Investments, Arch Venture Partners, Axon Ventures, OrbiMed, and Section 32. Initially, it was financed through grants from Summit for Stem Cell, a San Diego-based nonprofit.

Sarah de Crescenzo is an Xconomy editor based in San Diego. You can reach her at sdecrescenzo@xconomy.com.

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Aspen Neuro Bags $6.5M to Test Parkinson's Disease Stem Cell Therapy - Xconomy

WASHINGTON One scientists sweet tribute to her father may one day give beekeepers cluesabout their colonies health, as well as help warn others when crop diseases orpollen allergies are about to strike.

Those are all possible applications thatbiochemistry researcher Roco Cornero of George Mason University in Fairfax, Va.,sees for her work on examining proteins in honey. Cornero describedher unpublished work December 9 at the annual joint meeting of the AmericanSociety for Cell Biology and the European Molecular Biology Organization.

Amateur beekeepers often dontunderstand what is stressing bees in their hives, whether lack of water,starvation or infection with pathogens, says Cornero, whose father kept beesbefore his death earlier this year. What we see in the honey can tell us astory about the health of that colony, she says.

Bees are like miniature scientists thatfly and sample a wide variety of environmental conditions, says cell biologist LanceLiotta, Corneros mentor at George Mason. As bees digest pollen, soil and water,bits of proteins from other organisms, including fungi, bacteria and virusesalso end up in the insects stomachs. Honey, in turn, is basically bee vomit,Liotta says, and contains a record of virtually everything the bee came incontact with, as well as proteins from the bees themselves.

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The information archive in honey isunbelievable, Liotta says. But until now, scientists have had a hard timestudying proteins in honey. Its so gooey and sticky and hard to work with,he says. Sugars in honey gum up lab equipment usually used to isolate proteins.

So Cornero developed a method to pullpeptides bits of proteins out of honey using nanoparticles a feat noother researchers have previously managed, Liotta says. Once extracted from thehoney, the peptides are analyzed by mass spectrometry to determine the order ofamino acids that make up each fragment of protein. Those peptides are thencompared with a database of proteins to determine which organisms produced thehoney proteins.

A group of high school students workingat George Mason for the summer collected 13 honey samples from Virginia,Maryland. Two additional samples came from Corneros hometown of Mar del Platain Argentina. The Argentine honey was from the last batches her fathercollected from his bees.

Proteins from bees, microbes and a widevariety of plants were among the components of the honey. Peptides in honeyfrom one sample came from several bacteria, including some that normally livein bees guts and a few disease-causing varieties. Proteins from viruses andparasites that infect bees, including deformed wing virus and Varroa mites,which have been implicatedin colony collapse disorder, were also found in the sample (SN: 1/17/18). Those results could meanbees from that location may have trouble surviving the winter when the insectsimmune systems are less able to fight infections.

Cornero also determined by looking atpollen and plant proteins in the honey that bees had pollinated a variety ofplants, including sunflowers, lilacs, olive trees, red clover, potatoes andtomatoes. By analyzing pollen peptides, scientists may one day be able to learnwhether claims that certain honey is made from wildflowers, clover or orangeblossoms are really true.

Whats more, counting pollen peptides inlocal hives could, for example, give allergy sufferers a better idea of whenhay fever is likely to flare in their area, Cornero says. The researchers alsofound plant virus proteins in the honey, an indication of the types of diseasesthat may be stalking local crops.

Next, Cornero hopes to develop a rapid proteintest that would allow beekeepers to plunge a dipstick into honey and rapidly gaugetheir hives health. Having my dad as a beekeeper, I know how beekeepers work,and it would be a great way to honor his work, she says.

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A biochemists extraction of data from honey honors her beekeeper father - Science News