Single-cell analyses reveal evolution mimicry during the specification of breast cancer subtype.

Background: The stem or progenitor antecedents confer developmental plasticity and unique cell identities to cancer cells via genetic and epigenetic programs. A comprehensive characterization and mapping of the cell-of-origin of breast cancer using novel technologies to unveil novel subtype-specific therapeutic targets is still absent. Methods: We integrated 195,144 high-quality cells from normal breast tissues and 406,501 high-quality cells from primary breast cancer samples to create a large-scale single-cell atlas of human normal and cancerous breasts. Potential heterogeneous origin of malignant cells was explored by contrasting cancer cells against reference normal epithelial cells. Multi-omics analyses and both in vitro and in vivo experiments were performed to screen and validate potential subtype-specific treatment targets. Novel biomarkers of identified immune and stromal cell subpopulations were validated by immunohistochemistry in our cohort. Results: Tumor stratification based on cancer cell-of-origin patterns correlated with clinical outcomes, genomic aberrations and diverse microenvironment constitutions. We found that the luminal progenitor (LP) subtype was robustly associated with poor prognosis, genomic instability and dysfunctional immune microenvironment. However, the LP subtype patients were sensitive to neoadjuvant chemotherapy (NAC), PARP inhibitors (PARPi) and immunotherapy. The LP subtype-specific target PLK1 was investigated by both in vitro and in vivo experiments. Besides, large-scale single-cell profiling of breast cancer inspired us to identify a range of clinically relevant immune and stromal cell subpopulations, including subsets of innate lymphoid cells (ILCs), macrophages and endothelial cells. Conclusion: The present single-cell study revealed the cellular repertoire and cell-of-origin patterns of breast cancer. Combining single-cell and bulk transcriptome data, we elucidated the evolution mimicry from normal to malignant subtypes and expounded the LP subtype with vital clinical implications. Novel immune and stromal cell subpopulations of breast cancer identified in our study could be potential therapeutic targets. Taken together, Our findings lay the foundation for the precise prognostic and therapeutic stratification of breast cancer.

Production of a Bacteriocin Like Protein PEG 446 from Clostridium tyrobutyricum NRRL B-67062.

Clostridium tyrobutyricum strain NRRL B-67062 was previously isolated from an ethanol production facility and shown to produce high yields of butyric acid. In addition, the cell-free supernatant of the fermentation broth from NRRL B-67062 contained antibacterial activity against certain Gram-positive bacteria. To determine the source of this antibacterial activity, we report the genome and genome mining of this strain. The complete genome of NRRL B-67062 showed one circular chromosome of 3,242,608 nucleotides, 3114 predicted coding sequences, 79 RNA genes, and a G+C content of 31.0%. Analyses of the genome data for genes potentially associated with antimicrobial features were sought after by using BAGEL-4 and anti-SMASH databases. Among the leads, a polypeptide of 66 amino acids (PEG 446) contains the DUF4177 domain, which is an uncharacterized highly conserved domain (pfam13783). The cloning and expression of the peg446 gene in Escherichia coli and Bacillus subtilis confirmed the antibacterial property against Lactococcus lactis LM 0230, Limosilactobacillus fermentum 0315-25, and Listeria innocua NRRL B-33088 by gel overlay and well diffusion assays. Molecular modeling suggested that PEG 446 contains one alpha-helix and three anti-parallel short beta-sheets. These results will aid further functional studies and facilitate simultaneously fermentative production of both butyric acid and a putative bacteriocin from agricultural waste and lignocellulosic biomass materials.

Novel endolysin LysMP for control of Limosilactobacillus fermentum contamination in small-scale corn mash fermentation.

BACKGROUND: Traditional bioethanol fermentation industries are not operated under strict sterile conditions and are prone to microbial contamination. Lactic acid bacteria (LAB) are often pervasive in fermentation tanks, competing for nutrients and producing inhibitory acids that have a negative impact on ethanol-producing yeast, resulting in decreased yields and stuck fermentations. Antibiotics are frequently used to combat contamination, but antibiotic stewardship has resulted in a shift to alternative antimicrobials. RESULTS: We demonstrate that endolysin LysMP, a bacteriophage-encoded peptidoglycan hydrolase, is an effective method for controlling growth of LAB. The LysMP gene was synthesized based on the prophage sequence in the genome of Limosilactobacillus fermentum KGL7. Analysis of the recombinant enzyme expressed in E. coli and purified by immobilized metal chelate affinity chromatography (IMAC) showed an optimal lysis activity against various LAB species at pH 6, with stability from pH 4 to 8 and from 20 to 40 °C up to 48 h. Moreover, it retains more than 80% of its activity at 10% ethanol (v/v) for up to 48 h. When LysMP was added at 250 µg/mL to yeast corn mash fermentations containing L. fermentum, it reduced bacterial load by at least 4-log fold compared to the untreated controls and prevented stuck fermentation. In comparison, untreated controls with contamination increased from an initial bacterial load of 1.50 × 107 CFU/mL to 2.25 × 109 CFU/mL and 1.89 × 109 CFU/mL after 24 h and 48 h, respectively. Glucose in the treated samples was fully utilized, while untreated controls with contamination had more than 4% (w/v) remaining at 48 h. Furthermore, there was at least a fivefold reduction in lactic acid (0.085 M untreated contamination controls compared to 0.016 M treated), and a fourfold reduction in acetic acid (0.027 M untreated contamination controls vs. 0.007 M treated), when LysMP was used to treat contaminated corn mash fermentations. Most importantly, final ethanol yields increased from 6.3% (w/v) in untreated contamination samples to 9.3% (w/v) in treated contamination samples, an approximate 50% increase to levels comparable to uncontaminated controls 9.3% (w/v). CONCLUSION: LysMP could be a good alternative to replace antibiotics for mitigation of LAB contamination in biofuel refineries.

Single-cell analysis of white adipose tissue reveals the tumor-promoting adipocyte subtypes.

BACKGROUND: The tumor-adipose microenvironment (TAME) is characterized by the enrichment of adipocytes, and is considered a special ecosystem that supports cancer progression. However, the heterogeneity and diversity of adipocytes in TAME remains poorly understood. METHODS: We conducted a single-cell RNA sequencing analysis of adipocytes in mouse and human white adipose tissue (WAT). We analyzed several adipocyte subtypes to evaluate their relationship and potential as prognostic factors for overall survival (OS). The potential drugs are screened by using bioinformatics methods. The tumor-promoting effects of a typical adipocyte subtype in breast cancer are validated by performing in vitro functional assays and immunohistochemistry (IHC) in clinical samples. RESULTS: We profiled a comprehensive single-cell atlas of adipocyte in mouse and human WAT and described their characteristics, origins, development, functions and interactions with immune cells. Several cancer-associated adipocyte subtypes, namely DPP4+ adipocytes in visceral adipose and ADIPOQ+ adipocytes in subcutaneous adipose, are identified. We found that high levels of these subtypes are associated with unfavorable outcomes in four typical adipose-associated cancers. Some potential drugs including Trametinib, Selumetinib and Ulixertinib are discovered. Emphatically, knockdown of adiponectin receptor 1 (AdipoR1) and AdipoR2 impaired the proliferation and invasion of breast cancer cells. Patients with AdipoR2-high breast cancer display significantly shorter relapse-free survival (RFS) than those with AdipoR2-low breast cancer. CONCLUSION: Our results provide a novel understanding of TAME at the single-cell level. Based on our findings, several adipocyte subtypes have negative impact on prognosis. These cancer-associated adipocytes may serve as key prognostic predictor and potential targets for treatment in the future.

First report of Fusarium falciforme causing root rot of pomegranate (Punica granatum L.) in China.

Pomegranate (Punica granatum L.) is a deciduous shrub or small tree that is native to Iran and Afghanistan. It is also a commercially important fruit tree in China and worldwide. In the summer of 2022, a serious root rot disease occurred in some pomegranate orchards in Xichuan County(32º42´ N, 111º48´ E), Henan Province, China, with an incidence of ~30%. Symptoms included leaf yellowing and wilting, root browning and rotting, and stem-base cracking, eventually leading to defoliation and death. To isolate the causal agent, small pieces (5×5 mm) of diseased root from six trees were surface-sterilized by dipping in 2% NaClO for 8 min followed by 70% ethanol for 15 s, rinsed five times with sterile water, and plated on potato dextrose agar (PDA), then incubated at 28°C in the dark for 5 days. Fifteen pure fungal isolates with the same morphological characteristics were obtained from 24 pieces of roots. All isolates produced white fluffy mycelia. Microconidia were hyaline, oval or reniform, with zero to one septa and dimensions of 7.1 to 19.9 (average 14.5 )× 3.8 to 8.0 (average 5.6) µm (n = 100). Macroconidia were sickle-shaped, one to four septate, and 20.1 to 40.8 (average 26.5) × 4.8 to 8.6 (average 6.5) µm (n = 100). Chlamydospores were spherical, single, in pairs or chains, and 5.6 to 9.8 (average 6.8) µm in diameter (n = 100). Based on the above characteristics, the pathogens were identified as Fusarium sp. (Leslie and Summerell 2006). Genomic DNA was extracted from mycelia of two representative isolates Fs1 and Fs3. The internal transcribed spacer (ITS), translation elongation factor 1-alpha (TEF-1α) and RNA polymerase II second largest subunit (RPB2) sequences were PCR amplified using primer pairs of ITS1/ITS4, EF1/EF2, and RPB2-5f2/RPB2-7cr, RPB2-7cf/RPB2-11ar (O'Donnell et al., 2022), respectively. BLAST analysis showed that the ITS, TEF-1α and RPB2 sequences of isolates Fs1(GenBank accession nos. OK001765, OQ921726 and OQ928396) and Fs3 (GenBank accession nos. OK001771, OQ921727 and OQ928397) showed 99%-100% identity with multiple GenBank sequences of Fusarium falciforme (KY617066, MN064683, KF255514, OQ933361, KY556711 and ON331935). A phylogenetic tree based on concatenated sequences of ITS, TEF-1α and RPB2 using maximum-likelihood analysis revealed that both isolates Fs1 and Fs3 were in the same clade with F. falciforme strains. Based on the morphological and molecular characteristics, the isolates were identified as members of F. falciforme. For pathogenicity testing, conidial suspensions (1×108 spores /mL) of isolates Fs1 and Fs3 were poured onto the roots of healthy pomegranate that had been planted in pots two months previously. Ten plants were inoculated for each isolate. Control plants were drenched with sterile water. After 3 months, inoculated plants developed leaf yellowing and wilting accompanied by root browning and rotting, much like symptoms observed in field plants. The same fungi re-isolated from the experimental plants were confirmed to be F. falciforme by morphology and sequence analysis. This is the first report of F. falciforme causing root rot on pomegranate. F. falciforme is a ubiquitous soil-borne pathogen that causes root rot on multiple plants around the world (Xu F., et al. 2022; Qiu R., et al. 2023). The results of pathogen identification are essential precursors to development of effective control of the disease.

The ALOX5 inhibitor Zileuton regulates tumor-associated macrophage M2 polarization by JAK/STAT and inhibits pancreatic cancer invasion and metastasis.

5-lipoxygenase (encoded by ALOX5) plays an important role in immune regulation. Zileuton is currently the only approved ALOX5 inhibitor. However, the mechanisms of ALOX5 and Zileuton in progression of pancreatic cancer remain unclear. Therefore, we investigated the effects of Zileuton on tumor-associated macrophage M2 polarization and pancreatic cancer invasion and metastasis, both in vivo and in vitro. In bulk RNA sequencing (RNA-seq) and single-cell RNA sequencing (scRNA-seq) analyses, we found a significant association between elevated levels of ALOX5 and poor survival, adverse stages, M2 macrophage infiltration, and the activation of JAK/STAT pathways in macrophages. In clinical samples, immunofluorescence, quantitative real-time PCR and immunohistochemical results verified the high expression of ALOX5 in pancreatic cancer, primarily in macrophages. We constructed PANC-1 human pancreatic cancer cells and macrophages overexpressing ALOX5 using lentivirus. In PANC-1 pancreatic cancer cells, low-dose Zileuton inhibited PANC-1 cell invasion and migration by blocking ALOX5. In macrophages, ALOX5 induced the M2-like phenotype through the JAK/STAT pathway and promoted the chemotaxis of macrophages towards PANC-1 cells, while Zileuton can inhibit these effects. We constructed the nude mouse model of in situ transplantation tumor of pancreatic cancer. After treatment with Zileuton, the mice showed increased survival rates and reduced liver metastasis. These findings indicate that ALOX5 regulates tumor-associated macrophage M2 polarization via the JAK/STAT pathway and promotes invasion and metastasis in pancreatic cancer. Zileuton can inhibit these effects by inhibiting ALOX5. These results provide a theoretical basis for the potential use of Zileuton in the treatment of pancreatic cancer.

Saccharomyces cerevisiae surface display of endolysin LysKB317 for control of bacterial contamination in corn ethanol fermentations.

Control of bacterial contamination in bioethanol fermentation facilities has traditionally relied on chemical-based products such as hop acids and use of antibiotics. Recent emphasis on antibiotic stewardship has prompted new research into the development of alternative approaches to microbial remediation strategies. We recently described a recombinant peptidoglycan hydrolase, endolysin LysKB317, which inhibited Limosilactobacillus fermentum strains in corn mash fermentation. Here, Saccharomyces cerevisiae EBY100 was used to anchor recombinant LysKB317 using cell surface display with the a-agglutinin proteins Aga1p-Aga2p. Immunostaining and confocal fluorescence were used for localization of the extracellular interface of the cells. Yeast surface-expressed endolysin demonstrated an 83.8% decrease in bacterial cell counts compared to a 9.5% decrease in control yeast. Recombinant S. cerevisiae expressing LysKB317 used for small-scale corn mash fermentation, when infected with L. fermentum, could proactively control bacterial infection for 72 h with at least 1-log fold reduction. Analysis of fermentation products showed improved ethanol concentrations from 3.4% to at least 5.9% compared to the infection-only control and reduced levels of lactic and acetic acid from 34.7 mM to 13.8 mM and 25.5 mM to 18.1 mM, respectively. In an optimized yeast surface display system, proactive treatment of bacterial contaminants by endolysin LysKB317 can improve fermentation efficiency in the presence of L. fermentum contamination.

Spatial heterogeneity and Immune infiltration of cellular lysosomal pathways reveals a new blueprint for tumor heterogeneity in esophageal cancer.

Background: Esophageal squamous cell carcinoma (ESCC) is a common Malignant tumor of digestive tract which have a potential association with lysosomal pathway. The purpose of this study was to explore the correlation between lysosome pathway and immune infiltration of ESCC. Methods: The cell type annotation of ESCC patients and the distribution of their gene markers were analyzed by single cell data. They were also grouped according to the expression of lysosomal pathways. Gene set variation analysis (GSVA) enriched pathway scoring, Cellchat cell communication was performed to demonstrate the tumour-associated pathway scores and interactions of different cell populations. Relevant differential genes were screened, prognostic risk markers were constructed and direct associations of lysosomal pathway-related gene risk scores with immune infiltration and tumour treatment drug sensitivity were assessed by algorithms. In cellular experiments, qPCR and flow cytometry were used to assess the role of the lysosomal pathway gene-MT1X on tumour cell development. Results: ESCC single cell data were annotated into 7 Cluster clusters by t-sne downscaling analysis. Cellchat analysis revealed that the "MIF" cellular communication network is the main communication mode of the lysosomal pathway in ESCC cells. The lysosomal pathway genetic risk model was found to be significantly different from ESCC prognosis in both the training and validation groups. The lysosome pathway gene risk model was associated with treatment resistance in ESCC patients using oncopredict R package. The correlation between the expression of lysosomal-DEG and tumour immune infiltration and immune cell types by the MCPcounter method. Cellular assays showed that the lysosomal pathway gene MT1X was less expressed in oesophageal cancer cells than in normal oesophageal epithelial cells. Knockdown of MT1X significantly promoted the growth rate of oesophageal cancer cells. Conclusion: Based on the single cell sequencing technology and transcriptomic analysis, we confirmed that there is a close association between the lysosomal pathway and the immune infiltration and treatment sensitivity of ESCC, which may be a potential target for a new direction of ESCC therapy.

Research Progress on Factors Affecting Oil-Absorption Performance of Cement-Based Materials.

With the wide application of petroleum resources, oil substances have polluted the environment in every link from crude oil extraction to utilization. Cement-based materials are the main materials in civil engineering, and the study of their adsorption capacity for oil pollutants can expand the scope of functional engineering applications of cement-based materials. Based on the research status of the oil-wet mechanism of different kinds of oil-absorbing materials, this paper lists the types of conventional oil-absorbing materials and introduces their application in cement-based materials while outlining the influence of different oil-absorbing materials on the oil-absorbing properties of cement-based composites. The analysis found that 10% Acronal S400F emulsion can reduce the water absorption rate of cement stone by 75% and enhance the oil-absorption rate by 62%. Adding 5% polyethylene glycol can increase the oil-water relative permeability of cement stone to 1.2. The oil-adsorption process is described by kinetic and thermodynamic equations. Two isotherm adsorption models and three adsorption kinetic models are explained, and oil-absorbing materials and adsorption models are matched. The effects of specific surface area, porosity, pore interface, material outer surface, oil-absorption strain, and pore network on the oil-absorption performance of materials are reviewed. It was found that the porosity has the greatest influence on the oil-absorbing performance. When the porosity of the oil-absorbing material increases from 72% to 91%, the oil absorption can increase to 236%. In this paper, by analyzing the research progress of factors affecting oil-absorption performance, ideas for multi-angle design of functional cement-based oil-absorbing materials can be obtained.

Macrolide antibiotics activate the integrated stress response and promote tumor proliferation.

Macrolide antibiotics are widely used antibacterial agents that are associated with autophagy inhibition. This study aimed to investigate the association between macrolide antibiotics and malignant tumors, as well as the effect on autophagy, reactive oxygen species (ROS) accumulation and integrated stress response (ISR). The meta-analysis indicated a modestly higher risk of cancer in macrolide antibiotic ever-users compared to non-users. Further experiments showed that macrolides block autophagic flux by inhibiting lysosomal acidification. Additionally, azithromycin, a representative macrolide antibiotic, induced the accumulation of ROS, and stimulated the ISR and the activation of transcription factor EB (TFEB) and TFE3 in a ROS-dependent manner. Finally, animal experiments confirmed that azithromycin promoted tumor progression in vivo, which could be receded by N-acetylcysteine, an inhibitor of ROS and ISR. Overall, this study reveals the potential role of macrolide antibiotics in malignant progression and highlights the need for further investigation into their effects.

Elevated Angiotensin-II Levels Contribute to the Pathogenesis of Open-Angle Glaucoma Via Inducing the Expression of Fibrosis-Related Genes in Trabecular Meshwork Cells Through a ROS/NOX4/SMAD3 Axis.

Glaucoma including primary open-angle glaucoma (POAG) results from elevations in intraocular pressure (IOP). An eye-localized renin-angiotensin system (RAS) has been implicated in IOP regulation, although its mechanism of action and contribution to glaucoma is poorly understood. Here, we detected significant increases in the levels of angiotensin II (ANGII) in aqueous humor samples from POAG patients. Moreover, we determined that the concentrations of ANGII were positively correlated with IOP, suggesting a role for elevated ANGII levels in eye pathogenesis. Functional investigations demonstrated that ANGII induces the expression of fibrosis-related genes of transformed and primary human trabecular meshwork cells (HTMCs) through the transcriptional upregulation of key fibrotic genes. Parallel experiments using a murine periocular conjunctival fornix injection model confirmed that ANGII induces the expression of fibrosis-related genes in trabecular meshwork (TM) cells in vivo along with increasing IOP. ANGII was revealed to function through increasing the levels of reactive oxygen species (ROS) via selectively upregulating NOX4, with NOX4 knockdown or inhibition with GLX351322 alleviating fibrotic changes induced by ANGII. We further show that ANGII activates Smad3, with both GLX351322 and an inhibitor of Smad3 (SIS3) decreasing the phosphorylation of Smad3 and dampening the ANGII-induced increases in fibrotic proteins. Moreover, NOX4 and Smad3 inhibitors also partially rescued the elevated IOP levels induced by ANGII. Our collective results therefore highlight ANGII as a biomarker and treatment target in POAG together with establishing a causal relationship between ANGII and up-regulation of the expression of fibrosis-related genes of TM cells via a NOX4/ROS axis in cooperation with TGFß/Smad3 signaling.

Human cytomegalovirus infection perturbs neural progenitor cell fate via the expression of viral microRNAs.

Human cytomegalovirus (HCMV) preferentially targets neural progenitor cells (NPCs) in congenitally infected fetal brains, inducing neurodevelopmental disorders. While HCMV expresses several microRNAs (miRNAs) during infection, their roles in NPC infection are unclear. Here, we characterized expression of cellular and viral miRNAs in HCMV-infected NPCs during early infection by microarray and identified seven differentially expressed cellular miRNAs and six significantly upregulated HCMV miRNAs. Deep learning approaches were used to identify potential targets of significantly upregulated HCMV miRNAs against differentially expressed cellular messenger RNA (mRNAs), and the associations with miRNA-mRNA expression changes were observed. Gene ontology enrichment analysis indicated cellular gene targets were significantly enriched in pathways involved in neurodevelopment and cell-cycle processes. Viral modulation of selected miRNAs and cellular gene targets involved in neurodevelopmental processes were further validated by real-time quantitative reverse transcription polymerase chain reaction. Finally, a predicted 3' untranslated region target site of hcmv-miR-US25-1 in Jag1, a factor important for neurogenesis, was confirmed by mutagenesis. Reduction of Jag1 RNA and protein levels in NPCs was observed in response to transient expression of hcmv-miR-US25-1. A hcmv-miR-US25-1 mutant virus (ΔmiR-US25) displayed limited ability to downregulate Jag1 mRNA levels and protein levels during the early infection stage compared with the wild type virus. Our collective experimental and computational investigation of miRNAs and cellular mRNAs expression in HCMV-infected NPCs yields new insights into the roles of viral miRNAs in regulating NPC fate and their contributions to HCMV neuropathogenesis.

Macrophages in Tumor-Associated Adipose Microenvironment Accelerate Tumor Progression.

Adipose-tissue macrophages (ATMs), a complex ensemble of diverse macrophage subtypes, are prevalent in the tumor-adipose microenvironment (TAME) and facilitate tumor growth. However, the mechanisms in which the tumor-adipocyte crosstalk may enable the properties and plasticity of macrophages remain unclear. The single-cell RNA-sequence profiling reveals that a subset of macrophages expressed CD163, CCL2, and CCL5 in TAME, exhibiting an immunosuppressive subtype. It is demonstrated that CD163+ macrophages aggregate to surround adipocytes in breast cancer tissues. The expressions of CCL2 and CCL5 are also elevated in TAME and enable the recruitment and polarize macrophages. Mechanically, the level of exosomal miRNA-155 increased in the coculture of tumor cells and adipocytes, and then it promoted the generation and release of CCL2 and CCL5 from adipocytes by targeting the SOCS6/STAT3 pathway. Inhibition of exosomal miRNA-155 in tumor cells reduced the CCL2 and CCL5 levels in tumor-adipocytes coculture and further retarded tumor growth. Finally, the deletion of macrophages partially inhibited adipocyte-induced tumor proliferation. Likewise, inhibiting chemokines and their receptors or suppressing the phosphorylation of STAT3 decreased tumor burden in preclinical models. These results demonstrate that the niche factors in TAME, such as exosomal miRNA-155, regulate the function and polarity of macrophages to facilitate tumor progression.

First Report of Leaf Black Spot Caused by Alternaria alternata on Rosemary (Rosmarinus officinalis L.) in China.

Rosemary (Rosmarinus officinalis L.) is an aromatic, evergreen, medicinally important shrub and widely used for cooking, tea, cosmetics as well as medicinal materials. It is grown in many countries including China that had more than 9300 hm2 of commercial cultivation area in 2021. In March 2020, a leaf spot disease sporadic occurred in field rosemarry plants in Nanyang City (32º51´ N, 111º36´ E), Henan Province, China. The disease outbreaked in September with a disease incidence of 57-83%. Symptoms initially appeared as small brown leaf spots that gradually expanded into dark blackbrown irregular lesions. Most of the spots started from the leaf tip or leaf margin, and gradually spread to the leaf base, resulting in heavy defoliation especially on rainy days. Diseased leaf segments (1×3 mm) were surface-sterilized by dipping in 1% sodium hypochlorite for 1 min, rinsed three times with sterile distilled water, and plated on potato dextrose agar, then incubated at 28°C in the dark for 5 days. Twelve fungal isolates with the same morphological characteristics were obtained from nine affected leaves. The fungal colonies were initially white and turned gray brown with flocculent aerial mycelia and a whorled back. Conidia were frequently born in a long chain, with a short beak, brown or light-brown, 13.2 to 48. 7 (average 26.1) × 4.0 to 13.1 (average 8.0) µm in size (n=148) with 0 to 8 transverse and 0 to 3 longitudinal/oblique septa. Phenotypic features of the isolates agreed with those of Alternaria alternata (Simmons et al. 2007). Two isolates Aa1 and Aa2 were randomly selected for molecular and pathogenicity tests. DNA was extracted from mycelia. Partial sequences of internal transcribed spacer (ITS) and translation elongation factor 1-alpha (TEF1-α) were amplified using the primer pairs ITS1/ITS4 and EFI-728F/EFI-986R (Wei et al. 2022), respectively. The GenBank accession nos. were OK036714 and OK036715 for ITS, and ON951980 and ON951981 for TEF1-α of Aa1 and Aa2, respectively, with a maximal identity of greater than 99% to multiple A. alternata strains. In the neighbour joining phylogenetic tree of the amplified ITS and TEF1-α sequences both Aa1 and Aa2 clustered with A. alternata strains, clearly separating them from other Alternaria spp. For pathogenicity test, conidial suspensions (1×106 spores /mL) of Aa1 and Aa2 were separately sprayed on healthy one-year-old rosemary plants (n=3) with their leaves slightly wounded with a sterilized needle. Control plants (n=3) were sprayed with sterile water. Both inoculated and control plants were incubated at 90% RH, 28 °C. After 14 days, all the inoculated leaves showed black brown lesions similar to those on naturally affected field plants, whereas controls remained symptomless. Fungal cultures with the same phenotypic features as the inocula were constantly re-isolated from the infected leaves. A. alternata was reported as pathogen causing foliar necrosis on rosemary in Italy (Perello et al.1995) and leaf spot (or leaf blight) on multiple plant species such as Actaea dahurica (Hai et al. 2022), and Ligustrum japonicum (Wei et al. 2022) in China. This is the first report of A. alternata causing leaf black spot on rosemary in China.

Neuroendocrine regulations in tissue-specific immunity: From mechanism to applications in tumor.

Immune responses in nonlymphoid tissues play a vital role in the maintenance of homeostasis. Lots of evidence supports that tissue-specific immune cells provide defense against tumor through the localization in different tissue throughout the body, and can be regulated by diverse factors. Accordingly, the distribution of nervous tissue is also tissue-specific which is essential in the growth of corresponding organs, and the occurrence and development of tumor. Although there have been many mature perspectives on the neuroendocrine regulation in tumor microenvironment, the neuroendocrine regulation of tissue-specific immune cells has not yet been summarized. In this review, we focus on how tissue immune responses are influenced by autonomic nervous system, sensory nerves, and various neuroendocrine factors and reversely how tissue-specific immune cells communicate with neuroendocrine system through releasing different factors. Furthermore, we pay attention to the potential mechanisms of neuroendocrine-tissue specific immunity axis involved in tumors. This may provide new insights for the immunotherapy of tumors in the future.

Antibacterial Property and Metagenomic Analysis of Milk Kefir.

Milk kefir fermentation has been used in households for generations. Consumption of milk kefir has been associated with various health benefits, presumably from the probiotics of yeast and bacteria that make up the kefir grains. In addition, many of the microbes are known to produce novel antimicrobial compounds that can be used for other applications. The microbes living inside kefir grains differ significantly depending on geographical location and production methods. In this study, we aimed to use metagenomic analysis of fermented milk by using three different kefir grains (kefir 1, kefir 2, and kefir 3) from different US sources. We analyzed the microbial compositions of the three milk fermentation samples. This study revealed that each sample contains unique and distinct groups of microbes, kefir 1 showed the least diversity, and kefir 3 showed the highest diversity. Kefir 3 is rich in Proteobacteria while kefir 2 is dominated by the Firmicutes. Using bacterial indicator growth analyses carried out by continuous readings from microplate-based bioreactor assays suggested that kefir 2 fermentation filtrate has higher antibacterial property. We have screened 30 purified cultures of kefir 2 sample and isolated two lactic acid bacteria strains with higher antibacterial activities; the two strains were identified as Leuconostoc mesenteroides 28-1 and Lentilactobacillus kefiri 25-2 by 16S genomic PCR with confirmed antibacterial activities of fermentation filtrate after growing under both aerobic and anaerobic conditions.

Single-cell and spatially resolved analysis uncovers cell heterogeneity of breast cancer.

The heterogeneity and the complex cellular architecture have a crucial effect on breast cancer progression and response to treatment. However, deciphering the neoplastic subtypes and their spatial organization is still challenging. Here, we combine single-nucleus RNA sequencing (snRNA-seq) with a microarray-based spatial transcriptomics (ST) to identify cell populations and their spatial distribution in breast cancer tissues. Malignant cells are clustered into distinct subpopulations. These cell clusters not only have diverse features, origins and functions, but also emerge to the crosstalk within subtypes. Furthermore, we find that these subclusters are mapped in distinct tissue regions, where discrepant enrichment of stromal cell types are observed. We also inferred the abundance of these tumorous subpopulations by deconvolution of large breast cancer RNA-seq cohorts, revealing differential association with patient survival and therapeutic response. Our study provides a novel insight for the cellular architecture of breast cancer and potential therapeutic strategies.

Sequence duplication in 3' UTR modulates virus replication and virulence of Japanese encephalitis virus.

Japanese encephalitis virus (JEV), an important neurotropic pathogen, belongs to the genus Flavivirus of the family Flaviviridae and has caused huge threat to public health. It is still obscure regarding the functions of stem-loop (SL) and dumbbell (DB) domains of JEV 3' UTR in viral replication and virulence. In the current study, using the infectious clone of JEV SA14 strain as a backbone, we constructed a series of deletion mutants of 3' UTR to investigate their effects on virus replication. The results showed that partial deletions within SL or DB domain had no apparent effects on virus replication in both mammalian (BHK-21) and mosquito (C6/36) cells, suggesting that they were not involved in viral host-specific replication. However, the entire SL domain deletion (ΔVR) significantly reduced virus replication in both cell lines, indicating the important role of the complete SL domain in virus replication. The revertant of ΔVR mutant virus was obtained by serial passage in BHK-21 cells that acquired a duplication of DB domain (DB-dup) in the 3' UTR, which greatly restored virus replication as well as the capability to produce the subgenomic flavivirus RNAs (sfRNAs). Interestingly, the DB-dup mutant virus was highly attenuated in C57BL/6 mice despite replicating similar to WT JEV. These findings demonstrate the significant roles of the duplicated structures in 3' UTR in JEV replication and provide a novel strategy for the design of live-attenuated vaccines.

Associations between hepatitis B virus infection and risk of colorectal Cancer: a population-based prospective study.

BACKGROUND: Previous studies have observed a close association between hepatitis B virus (HBV) infection and hepatocellular carcinoma (HCC) as well as extrahepatic cancers. However, research concerning the effect of HBV infection on the risk of colorectal cancer (CRC) is rare and inconsistent. This study aims to determine the relationship between HBV infection and new-onset CRC. METHODS: We prospectively examined the relationship between HBV infection and new-onset CRC among 93,390 participants from Kailuan Cohort study. Cox proportional hazards regression models, subgroup analyses and competing risk analyses were used to evaluate the association between HBV infection and the risk of new-onset CRC. RESULTS: During a median follow-up of 11.28 years, 448 incident CRC cases were identified. The adjusted HR (95%confidence interval (CI)) for the association of HBsAg Seropositive with CRC was 1.85(1.15 ~ 2.96) in the Cox regression. Subgroup analyses showed that the HBsAg seropositive group was associated with increased risk of new-onset CRC among male, middle-aged, normal weight, smokers and non-drinker participants, respectively. A positive association of HBV infection with the risk of CRC was observed in the adjusted sub-distribution proportional hazards (SD) models (HRSD = 1.77, 95% CI:1.11-2.84) and cause-specific hazards (CS) models (HRCS = 1.79, 95% CI: 1.13-2.91). CONCLUSIONS: Our results have found a significant association between HBV infection and the risk of incident CRC among Chinese participants. TRIAL REGISTRATION: Kailuan study, ChiCTR-TNRC-11001489. Registered 24 August 2011 - Retrospectively registered, http:// http://www.chictr.org.cn/showprojen.aspx?proj=8050.