Distinct niche structures and intrinsic programs of fallopian tube and ovarian surface epithelial cells.

Epithelial ovarian cancer (EOC) can originate from either fallopian tube epithelial (FTE) or ovarian surface epithelial (OSE) cells, but with different latencies and disease outcomes. To address the basis of these differences, we performed single cell RNA-sequencing of mouse cells isolated from the distal half of fallopian tube (FT) and surface layer of ovary. We find at the molecular level, FTE secretory stem/progenitor cells and OSE cells resemble mammary luminal progenitors and basal cells, respectively. An FT stromal subpopulation, enriched with Pdgfra + and Esr1 + cells, expresses multiple secreted factor (e.g., IGF1) and Hedgehog pathway genes and may serve as a niche for FTE cells. In contrast, Lgr5 + OSE cells express similar genes largely by themselves, raising a possibility that they serve as their own niche. The differences in intrinsic expression programs and niche organizations of FTE and OSE cells may contribute to their different courses toward the development of EOCs.

Selective inhibition of integrin αvß6 leads to rapid induction of urinary bladder tumors in cynomolgus macaques.

Administration of a novel and selective small molecule integrin αvß6 inhibitor, MORF-627, to young cynomolgus monkeys for 28 days resulted in the rapid induction of epithelial proliferative changes in the urinary bladder of 2 animals, in the absence of test agent genotoxicity. Microscopic findings included suburothelial infiltration by irregular nests and/or trabeculae of epithelial cells, variable cytologic atypia, and high mitotic rate, without invasion into the tunica muscularis. Morphologic features and patterns of tumor growth were consistent with a diagnosis of early-stage invasive urothelial carcinoma. Ki67 immunohistochemistry demonstrated diffusely increased epithelial proliferation in the urinary bladder of several monkeys, including those with tumors, and αvß6 was expressed in some epithelial tissues, including urinary bladder, in monkeys and humans. Spontaneous urothelial carcinomas are extremely unusual in young healthy monkeys, suggesting a direct link of the finding to the test agent. Inhibition of integrin αvß6 is intended to locally and selectively block transforming growth factor beta (TGF-ß) signaling, which is implicated in epithelial proliferative disorders. Subsequent in vitro studies using a panel of integrin αvß6 inhibitors in human bladder epithelial cells replicated the increased urothelial proliferation observed in monkeys and was reversed through exogenous application of TGF-ß. Moreover, analysis of in vivo models of liver and lung fibrosis revealed evidence of epithelial hyperplasia and cell cycle dysregulation in mice treated with integrin αvß6 or TGF-ß receptor I inhibitors. The cumulative evidence suggests a direct link between integrin αvß6 inhibition and decreased TGF-ß signaling in the local bladder environment, with implications for epithelial proliferation and carcinogenesis.

LINC00665 interacts with BACH1 to activate Wnt1 and mediates the M2 polarization of tumor-associated macrophages in GC.

Gastric cancer (GC) remains one of the prevalent causes of cancer-related deaths globally. Long non-coding RNAs (lncRNAs) have been associated with different cancers. The polarization of macrophages towards the M2 (alternatively activated) phenotype promotes immunologic tolerance and can induce gastric tumorigenesis. Thus far, lncRNAs have been shown to modulate the differentiation of immune cells. Here, we investigated the biological effects of LINC00665 on the progression of GC and explored the mechanisms underlying its ability to mediate the polarization of macrophages towards the M2 phenotype. We report that the levels of LINC00665 were increased in GC tissues. Furthermore, this increase in LINC00665 expression could be associated with decreased overall survival (OS), progression-free survival (PFS), and post-progression survival (PPS). Using cell-based macrophage polarization models, we demonstrated that LINC00665 upregulation in GC cells facilitated the polarization of macrophages towards the M2 but not M1 (classically activated) phenotype. Furthermore, the loss of LINC00665 prevented the M2 polarization of macrophages. Mechanically, we identified that Wnt1 was the downstream target of LINC00665. Additionally, LINC00665 could directly interact with the transcription factor BTB domain and CNC homology 1 (BACH1). The interaction between LINC00665 and BACH1 resulted in the activation and binding of BACH1 to the Wnt1 promoters. Furthermore, BACH1 silencing could inhibit GC progression, which highlighted a crucial role for BACH1 in LINC00665-mediated Wnt1 activation. In addition, genetic Wnt1 overexpression effectively abolished the repression of Wnt signaling after BACH1 depletion and mediated GC development by supporting M2 macrophage polarization. In conclusion, we report that LINC00665 modulates M2 macrophage polarization and suggest that it may facilitate macrophage-dependent GC progression.

Analysis of Community Composition of Bacterioplankton in Changle Seawater in China by Illumina Sequencing Combined with Bacteria Culture.

OBJECTIVES: To characterize the abundance and relative composition of seawater bacterioplankton communities in Changle city using Illumina MiSeq sequencing and bacterial culture techniques. METHODS: Seawater samples and physicochemical factors were collected from the coastal zone of Changle city on 8 September 2019. Nineteen filter membranes were obtained after using a suction filtration system. We randomly selected eight samples for total seawater bacteria (SWDNA group) sequencing and three samples for active seawater bacteria (SWRNA group) sequencing by Illumina MiSeq. The remaining eight samples were used for bacterial culture and identification. Alpha diversity including species coverage (Coverage), species diversity (Shannon-Wiener and Simpson index), richness estimators (Chao1), and abundance-based richness estimation (ACE) were calculated to assess biodiversity of seawater bacterioplankton. Beta diversity was used to evaluate the differences between samples. The species abundance differences were determined using the Wilcoxon rank-sum test. Statistical analyses were performed in R environment. RESULTS: The Alpha diversity in the SWDNA group in each index was ACE 3206.99, Chao1 2615.12, Shannon 4.64, Simpson 0.05, and coverage 0.97; the corresponding index was ACE 1199.55, Chao1 934.75, Shannon 3.49, Simpson 0.09, and coverage 0.99. The sequencing results of seawater bacterial genes in the coastal waters of Changle city showed that the phyla of high-abundance bacteria of both the SWDNA and SWRNA groups included Cyanobacteria, Proteobacteria, and Bacteroidetes. The main classes included Oxyphotobacteria, Alphaproteobacteria, and Gammaproteobacteria. The main genera included Synechococcus CC9902, Chloroplast, and Cyanobium_PCC-6307. Beta diversity analysis showed a significant difference between the SWDNA and SWRNA groups (P < 0.05). The species abundance differences between SWDNA and SWRNA groups after Wilcoxon rank-sum test showed that, at the phylum level, Actinomycetes was more abundant in SWDNA group (9.17 vs 1.02%, P < 0.05); at the class level, Actinomycetes (δ- Proteus) was more abundant in SWDNA group (9.47% vs 1.01%, P < 0.05); and at the genus level, Chloroplast was more abundant in SWRNA group (13.07% vs 44.57%, P < 0.05). Nine species and 53 colonies were found by bacterial culture: 20 strains of Vibrio (37.74%), 22 strains of Enterobacter (41.51%), and 11 strains of non-fermentative bacteria (20.75%). CONCLUSION: Illumi MiSeq sequencing of seawater bacteria revealed that the total bacterial community groups and the active bacterial community groups mainly comprised Cyanobacteria, Proteobacteria, and Bacteroides at the phylum level; Oxyphotobacteria, α-Proteobacteria, and γ-Proteobacteria at the class level; with Synechococcus_CC9902, Chloroplast, and Cyanobium_PCC-6307 comprising the predominant genera. Exploring the composition and differences of seawater bacteria assists understanding regarding the biodiversity and the infections related to seawater bacteria along the coast of the Changle, provides information that will aid in the diagnosis and treatment of such infections.

Three subtypes of lung cancer fibroblasts define distinct therapeutic paradigms.

Cancer-associated fibroblasts (CAFs) are highly heterogeneous. With the lack of a comprehensive understanding of CAFs' functional distinctions, it remains unclear how cancer treatments could be personalized based on CAFs in a patient's tumor. We have established a living biobank of CAFs derived from biopsies of patients' non-small lung cancer (NSCLC) that encompasses a broad molecular spectrum of CAFs in clinical NSCLC. By functionally interrogating CAF heterogeneity using the same therapeutics received by patients, we identify three functional subtypes: (1) robustly protective of cancers and highly expressing HGF and FGF7; (2) moderately protective of cancers and highly expressing FGF7; and (3) those providing minimal protection. These functional differences among CAFs are governed by their intrinsic TGF-ß signaling, which suppresses HGF and FGF7 expression. This CAF functional classification correlates with patients' clinical response to targeted therapies and also associates with the tumor immune microenvironment, therefore providing an avenue to guide personalized treatment.

Assessment of seawater bacterial infection in rabbit tibia by Illumina MiSeq sequencing and bacterial culture.

OBJECTIVES: We aimed to explore the bacterial community composition following ocean bacterial infection using an animal model. METHODS: This animal-based experiment was conducted from September 2019 to November 2019. Eighteen seawater filter membranes were collected from Changle City, Fujiian Province, China, on September 8, 2019. Ten filter membranes were used for implantation. Eight filter membranes that were used in the bacterial culture for the exploration of seawater bacteria were assigned to the seawater group (SG). Fourteen healthy adult New Zealand rabbits were randomly divided into the experimental group (EG) and control group (CG). Seawater filter membranes and asepsis membranes were implanted into the tibia in the EG and CG, respectively. One week after surgery, tibial bone pathology tissues were collected and assessed using light microscopy and scanning electron microscopy (SEM). Medullary cavity tissues were collected for the performance of Illumina MiSeq sequencing and bacterial culture. The differences between EG and CG were assessed by pathological observation under light microscopy and SEM, high-throughput bacterial sequencing, and bacterial culture. RESULTS: Compared with the CG, the infection rate was 100%, and the mortality value was 20% after the implantation of the filter membranes in the EG. Both light microscopy and SEM showed that a large number of bacteria were distributed in the bone marrow cavity after ocean bacterial infection. No bacterial growth was found in the CG. Illumina MiSeq sequencing found that Firmicutes, Proteobacteria, Thermotogae, Fusobacteria, Bacteroidetes, and Actinobacteria were the dominant bacteria at the phylum level and Clostridium_sensu_stricto_7, Haloimpatiens, Clostridium_sensu_stricto_15, Clostridiaceae_1, Clostridium_sensu_stricto_18, and Oceanotoga were the dominant bacteria in genus level among the EG. In the bacterial culture of the medullary cavity tissues, Klebsiella pneumoniae, Shewanella algae, Staphylococcus aureus, Escherichia coli, Enterobacter cloacae, and Vibrio vulnificus were the predominant infective species. Moreover, compared with the SG, the EG showed a higher detection rate of E. coli and S. aureus (P = 0.008 and P = 0.001, respectively). The detection rates of V. alginolyticus, V. parahaemolyticus, and V. fluvialis were higher in the SG than the EG (P = 0.007, P = 0.03, and P = 0.03, respectively). CONCLUSIONS: Our model, which was comprehensively evaluated using four techniques: histopathology and SEM observation, gene detection, and bacteria culture, provides a scientific basis for the clinical diagnosis and treatment of patients in such settings.

Aging-Associated Alterations in Mammary Epithelia and Stroma Revealed by Single-Cell RNA Sequencing.

Aging is closely associated with increased susceptibility to breast cancer, yet there have been limited systematic studies of aging-induced alterations in the mammary gland. Here, we leverage high-throughput single-cell RNA sequencing to generate a detailed transcriptomic atlas of young and aged murine mammary tissues. By analyzing epithelial, stromal, and immune cells, we identify age-dependent alterations in cell proportions and gene expression, providing evidence that suggests alveolar maturation and physiological decline. The analysis also uncovers potential pro-tumorigenic mechanisms coupled to the age-associated loss of tumor suppressor function and change in microenvironment. In addition, we identify a rare, age-dependent luminal population co-expressing hormone-sensing and secretory-alveolar lineage markers, as well as two macrophage populations expressing distinct gene signatures, underscoring the complex heterogeneity of the mammary epithelia and stroma. Collectively, this rich single-cell atlas reveals the effects of aging on mammary physiology and can serve as a useful resource for understanding aging-associated cancer risk.

Prevention of nosocomial COVID-19 infections in otorhinolaryngology-head and neck surgery.

Coronavirus disease 2019 (COVID-19) has rapidly evolved into a pandemic, causing a global public health crisis. Many frontline healthcare workers providing ear, nose and throat services have been reported to contract COVID-19 at work. Early during the COVID-19 outbreak, several medical professionals in Otolaryngology-Head and Neck Surgery were infected in Wuhan, China. A series of measures were then taken immediately, which successfully halted the spread of the disease. Here we would like to share the lessons we have learned, and our experience to protect our health care workers during the COVID-19 pandemic.

LINC00665 induces gastric cancer progression through activating Wnt signaling pathway.

Long noncoding RNAs (lncRNAs) are recently recognized as noteworthy regulators of different tumors, counting gastric cancer (GC). Lately, long intergenic noncoding RNA (LINC) 00665 has been verified to display significant parts in several cancers. Be that as it may, its role and mechanism in GC movement still stay uninvestigated. As of now, we observed LINC00665 was obviously GC cells (MKN28, BGC-823, SGC7-901, AGS, HGC-27) in comparison to GES-1 cells, which was identified as human normal gastric epithelial cells. Then, LINC00665 was obviously downregulated in GC cells including AGS and BGC-823 cells. Loss of LINC00665 greatly repressed AGS and BGC-823 cell survival and cell expansion. Moreover, GC cell apoptosis was significantly induced by the loss of LINC00665. For another, we found that the GC cell cycle was also captured in G1 and G2 phases. The experiments on cell migration and invasion indicated that knockdown of LINC00665 restrained GC cell migration and invasion. Modifications in Wnt signaling are closely associated with the development of cancers. Here, we found that Wnt signaling was significantly inactivated by the silence of LINC00665 in GC cells. ß-catenin and cyclinD1 were restrained whereas GSK-3ß was induced by the inhibition of LINC00665 in GC cells. Furthermore, we confirmed the impact of LINC00665 in vivo using xenograft models. Taken these together, we indicated that LINC00665 could function as a novel biomarker in GC progression.

Single-cell trajectories reconstruction, exploration and mapping of omics data with STREAM.

Single-cell transcriptomic assays have enabled the de novo reconstruction of lineage differentiation trajectories, along with the characterization of cellular heterogeneity and state transitions. Several methods have been developed for reconstructing developmental trajectories from single-cell transcriptomic data, but efforts on analyzing single-cell epigenomic data and on trajectory visualization remain limited. Here we present STREAM, an interactive pipeline capable of disentangling and visualizing complex branching trajectories from both single-cell transcriptomic and epigenomic data. We have tested STREAM on several synthetic and real datasets generated with different single-cell technologies. We further demonstrate its utility for understanding myoblast differentiation and disentangling known heterogeneity in hematopoiesis for different organisms. STREAM is an open-source software package.

The microbial community responsible for dechlorination and benzene ring opening during anaerobic degradation of 2,4,6­trichlorophenol.

This study describes the dechlorination ability of acclimated biomass, the high-throughput sequencing of the 16S ribosomal RNA (rRNA) gene of such microorganisms, and the analysis of their community structure in relation to special functions. Two types of acclimated biomass (AB-1 and AB-2) were obtained via different acclimated treatment processes and were used to degrade 2,4,6­trichlorophenol. The degradation pathway and characteristics of trichlorophenol degradation were different between the two groups. AB-1 degraded trichlorophenol only to 4-chlorophenol. AB-2 completely dechlorinated trichlorophenol and opened the benzene ring. The 16S rRNA high-throughput sequencing method was employed to examine the microbial diversity. It was found that the microbial richness and diversity of AB-1 were higher than those of AB-2. Firmicutes and Bacteroidetes were 2.7-fold and 4.3-fold more abundant, respectively, in AB-1 than in AB-2. Dechlorination bacteria in AB-1 mainly included Desulfobulbus, Desulfovibrio, Dechloromonas, and Geobacter. The above-mentioned bacteria were less abundant in AB-2, but the abundance of Desulfomicrobium was twofold higher in AB-2 than in AB-1. The two types of acclimated biomass contained different hydrogen (H2)-producing bacteria. AB-2 showed higher abundance and diversity of hydrogen-producing bacteria. There was no Ignavibacteriae in AB-1, whereas its abundance in AB-2 was 8.4%. In this biomass, Ignavibacteriae was responsible for opening of the benzene ring. This study indicates that the abundance and diversity of microorganisms are not necessarily beneficial to the formation of a functional dechlorinating community. The H2-producing bacteria (which showed greater abundance and diversity) and Ignavibacterium were assumed to be core functional populations that gave AB-2 stronger dechlorination and phenol-degradation abilities. Control of lower oxidation reduction potential (Eh) and higher temperatures by means of fresh aerobic activated sludge as the starting microbial group, caused rapid complete dechlorination of 2,4,6­trichlorophenol and benzene ring opening.

Integrated in situ gas stripping-salting-out process for high-titer acetone-butanol-ethanol production from sweet sorghum bagasse.

BACKGROUND: The production of biobutanol from renewable biomass resources is attractive. The energy-intensive separation process and low-titer solvents production are the key constraints on the economy-feasible acetone-butanol-ethanol (ABE) production by fermentation. To decrease energy consumption and increase the solvents concentration, a novel two-stage gas stripping-salting-out system was established for effective ABE separation from the fermentation broth using sweet sorghum bagasse as feedstock. RESULTS: The ABE condensate (143.6 g/L) after gas stripping, the first-stage separation, was recovered and introduced to salting-out process as the second-stage. K4P2O7 and K2HPO4 were used, respectively. The effect of saturated salt solution temperature on final ABE concentration was also investigated. The results showed high ABE recovery (99.32%) and ABE concentration (747.58 g/L) when adding saturated K4P2O7 solution at 323.15 K and 3.0 of salting-out factor. On this condition, the energy requirement of the downstream distillation process was 3.72 MJ/kg of ABE. CONCLUSIONS: High-titer cellulosic ABE production was separated from the fermentation broth by the novel two-stage gas stripping-salting-out process. The process was effective, which reduced the downstream process energy requirement significantly.

Simultaneous acetone-butanol-ethanol fermentation, gas stripping, and full-cell-catalyzed esterification for effective production of butyl oleate.

In this study, aiming to improve the economic feasibility of acetone-butanol-ethanol (ABE) fermentation process, generate valuable products and extend the product chain, esterification catalyzed by Candida sp. 99-125 cells was hybrid with the ABE fermentation-gas-stripping integration system. The gas-stripping condensate that contained concentrated ABE products was directly used for esterification without the participation of toxic organic solvents. Full-cell catalysis temperature and the cell dosage rate on oleate production were evaluated and optimized in the esterification process. Under the optimized conditions (35 °C, 8% of cells), ~ 68% of butyl oleate and ~ 12% of ethyl oleate were obtained after 4 h of esterification. The Candida sp. 99-125 cells were able to be reused for at least four cycles. The novel cascade process showed environmental benefits, which also showed promising in improving the economic feasibility of the conventional ABE fermentation process.

Mapping human pluripotent stem cell differentiation pathways using high throughput single-cell RNA-sequencing.

BACKGROUND: Human pluripotent stem cells (hPSCs) provide powerful models for studying cellular differentiations and unlimited sources of cells for regenerative medicine. However, a comprehensive single-cell level differentiation roadmap for hPSCs has not been achieved. RESULTS: We use high throughput single-cell RNA-sequencing (scRNA-seq), based on optimized microfluidic circuits, to profile early differentiation lineages in the human embryoid body system. We present a cellular-state landscape for hPSC early differentiation that covers multiple cellular lineages, including neural, muscle, endothelial, stromal, liver, and epithelial cells. Through pseudotime analysis, we construct the developmental trajectories of these progenitor cells and reveal the gene expression dynamics in the process of cell differentiation. We further reprogram primed H9 cells into naïve-like H9 cells to study the cellular-state transition process. We find that genes related to hemogenic endothelium development are enriched in naïve-like H9. Functionally, naïve-like H9 show higher potency for differentiation into hematopoietic lineages than primed cells. CONCLUSIONS: Our single-cell analysis reveals the cellular-state landscape of hPSC early differentiation, offering new insights that can be harnessed for optimization of differentiation protocols.

Dissecting hematopoietic and renal cell heterogeneity in adult zebrafish at single-cell resolution using RNA sequencing.

Recent advances in single-cell, transcriptomic profiling have provided unprecedented access to investigate cell heterogeneity during tissue and organ development. In this study, we used massively parallel, single-cell RNA sequencing to define cell heterogeneity within the zebrafish kidney marrow, constructing a comprehensive molecular atlas of definitive hematopoiesis and functionally distinct renal cells found in adult zebrafish. Because our method analyzed blood and kidney cells in an unbiased manner, our approach was useful in characterizing immune-cell deficiencies within DNA-protein kinase catalytic subunit (prkdc), interleukin-2 receptor γ a (il2rga), and double-homozygous-mutant fish, identifying blood cell losses in T, B, and natural killer cells within specific genetic mutants. Our analysis also uncovered novel cell types, including two classes of natural killer immune cells, classically defined and erythroid-primed hematopoietic stem and progenitor cells, mucin-secreting kidney cells, and kidney stem/progenitor cells. In total, our work provides the first, comprehensive, single-cell, transcriptomic analysis of kidney and marrow cells in the adult zebrafish.

GiniClust: detecting rare cell types from single-cell gene expression data with Gini index.

High-throughput single-cell technologies have great potential to discover new cell types; however, it remains challenging to detect rare cell types that are distinct from a large population. We present a novel computational method, called GiniClust, to overcome this challenge. Validation against a benchmark dataset indicates that GiniClust achieves high sensitivity and specificity. Application of GiniClust to public single-cell RNA-seq datasets uncovers previously unrecognized rare cell types, including Zscan4-expressing cells within mouse embryonic stem cells and hemoglobin-expressing cells in the mouse cortex and hippocampus. GiniClust also correctly detects a small number of normal cells that are mixed in a cancer cell population.