Identification of prognostic biomarkers for cholangiocarcinoma by combined analysis of molecular characteristics of clinical MVI subtypes and molecular subtypes.

Cholangiocarcinoma (CCA) is widely noted for its high degree of malignancy, rapid progression, and limited therapeutic options. This study was carried out on transcriptome data of 417 CCA samples from different anatomical locations. The effects of lipid metabolism related genes and immune related genes as CCA classifiers were compared. Key genes were derived from MVI subtypes and better molecular subtypes. Pathways such as epithelial mesenchymal transition (EMT) and cell cycle were significantly activated in MVI-positive group. CCA patients were classified into three (four) subtypes based on lipid metabolism (immune) related genes, with better prognosis observed in lipid metabolism-C1, immune-C2, and immune-C4. IPTW analysis found that the prognosis of lipid metabolism-C1 was significantly better than that of lipid metabolism-C2 + C3 before and after correction. KRT16 was finally selected as the key gene. And knockdown of KRT16 inhibited proliferation, migration and invasion of CCA cells.

Rice false smut virulence protein subverts host chitin perception and signaling at lemma and palea for floral infection.

The flower-infecting fungus Ustilaginoidea virens causes rice false smut, which is a severe emerging disease threatening rice (Oryza sativa) production worldwide. False smut not only reduces yield, but more importantly produces toxins on grains, posing a great threat to food safety. U. virens invades spikelets via the gap between the 2 bracts (lemma and palea) enclosing the floret and specifically infects the stamen and pistil. Molecular mechanisms for the U. virens-rice interaction are largely unknown. Here, we demonstrate that rice flowers predominantly employ chitin-triggered immunity against U. virens in the lemma and palea, rather than in the stamen and pistil. We identify a crucial U. virens virulence factor, named UvGH18.1, which carries glycoside hydrolase activity. Mechanistically, UvGH18.1 functions by binding to and hydrolyzing immune elicitor chitin and interacting with the chitin receptor CHITIN ELICITOR BINDING PROTEIN (OsCEBiP) and co-receptor CHITIN ELICITOR RECEPTOR KINASE1 (OsCERK1) to impair their chitin-induced dimerization, suppressing host immunity exerted at the lemma and palea for gaining access to the stamen and pistil. Conversely, pretreatment on spikelets with chitin induces a defense response in the lemma and palea, promoting resistance against U. virens. Collectively, our data uncover a mechanism for a U. virens virulence factor and the critical location of the host-pathogen interaction in flowers and provide a potential strategy to control rice false smut disease.

Molecular profiles of different PD-L1 expression in patients with esophageal squamous cell carcinoma.

BACKGROUND: PD-1/PD-L1 inhibitors are approved treatments for patients with esophageal squamous cell carcinoma (ESCC). The present investigation aspired to explore the interrelation between molecular phenotype and PD-L1 expression in ESCC. METHODS: PD-L1 testing and targeted next-generation sequencing (NGS) were performed on tumoral tissues from 139 ESCC patients. Tumor-infiltrating lymphocytes (TILs) were scrutinized using a tyramide signal amplification system combined with immunohistochemistry. RESULTS: Among enrolled patients, 36.7% displayed high PD-L1 expression (combined positive score [CPS] ≥10). BRCA1 and NF1 gene mutations were significantly associated with high PD-L1 expression (p < .05) while TGFß pathway alterations were linked to low PD-L1 expression (p = .02). High copy number instability (CNI) and copy number alterations (CNA) were correlated with low PD-L1 expression. Patients with CDKN2A deletion exhibited higher PD-L1 expression. Varying types of TILs were observed across different PD-L1 expression groups. The ratio of CD8+PD-L1+ T cells and CD8+PD-1+ T cells to CD8+ T cells remained comparable in both tumoral and stromal regions, but the ratio of CD68+PD-L1+ macrophages to CD68+ macrophages was higher than the ratio of CD68+PD-1+ macrophages to CD68+ macrophages. CPS was significantly correlated with PD-L1+ lymphocytes and CD68+ macrophages in the tumoral region. CD8+ T cell infiltration was positively correlated with PD-1+ cells in both tumoral and stromal regions. CONCLUSION: In this study, we presented the prevalence rates of PD-L1 expression in Chinese ESCC patients. The association of genetic profiles with PD-L1 expression levels also provide the clue that genomic phenotype may interact with the immunologic phenotype in ESCC.

A natural allele of proteasome maturation factor improves rice resistance to multiple pathogens.

Crops with broad-spectrum resistance loci are highly desirable in agricultural production because these loci often confer resistance to most races of a pathogen or multiple pathogen species. Here we discover a natural allele of proteasome maturation factor in rice, UMP1R2115, that confers broad-spectrum resistance to Magnaporthe oryzae, Rhizoctonia solani, Ustilaginoidea virens and Xanthomonas oryzae pv. oryzae. Mechanistically, this allele increases proteasome abundance and activity to promote the degradation of reactive oxygen species-scavenging enzymes including peroxidase and catalase upon pathogen infection, leading to elevation of H2O2 accumulation for defence. In contrast, inhibition of proteasome function or overexpression of peroxidase/catalase-encoding genes compromises UMP1R2115-mediated resistance. More importantly, introduction of UMP1R2115 into a disease-susceptible rice variety does not penalize grain yield while promoting disease resistance. Our work thus uncovers a broad-spectrum resistance pathway integrating de-repression of plant immunity and provides a valuable genetic resource for breeding high-yield rice with multi-disease resistance.

Virsearcher: Identifying Bacteriophages from Metagenomes by Combining Convolutional Neural Network and Gene Information.

Metagenome sequencing provides an unprecedented opportunity for the discovery of unknown microbes and viruses. A large number of phages and prokaryotes are mixed together in metagenomes. To study the influence of phages on human bodies and environments, it is of great significance to isolate phages from metagenomes. However, it is difficult to identify novel phages because of the diversity of their sequences and the frequent presence of short contigs in metagenomes. Here, virSearcher is developed to identify phages from metagenomes by combining the convolutional neural network (CNN) and the gene information of input sequences. Firstly, an input sequence is encoded in accordance with the different functions of its coding and the non-coding regions and then is converted into word embedding code through a word embedding layer before a convolutional layer. Meanwhile, the hit ratio of the virus genes is combined with the output of the CNN to further improve the performance of the network. The genes used by virSearcher consist of complete and incomplete genes. Experiments on several metagenomes have showed that, compared with others, virSearcher can significantly improve the performance for the identification of short sequences, while maintaining the performance for long ones. The source code of virSearcher is freely available from http://github.com/DrJackson18/virSearcher.

Overproduction of OsRACK1A, an effector-targeted scaffold protein promoting OsRBOHB-mediated ROS production, confers rice floral resistance to false smut disease without yield penalty.

Grain formation is fundamental for crop yield but is vulnerable to abiotic and biotic stresses. Rice grain production is threatened by the false smut fungus Ustilaginoidea virens, which specifically infects rice floral organs, disrupting fertilization and seed formation. However, little is known about the molecular mechanisms of the U. virens-rice interaction and the genetic basis of floral resistance. Here, we report that U. virens secretes a cytoplasmic effector, UvCBP1, to facilitate infection of rice flowers. Mechanistically, UvCBP1 interacts with the rice scaffold protein OsRACK1A and competes its interaction with the reduced nicotinamide adenine dinucleotide phosphate oxidase OsRBOHB, leading to inhibition of reactive oxygen species (ROS) production. Although the analysis of natural variation revealed no OsRACK1A variants that could avoid being targeted by UvCBP1, expression levels of OsRACK1A are correlated with field resistance against U. virens in rice germplasm. Overproduction of OsRACK1A restores the OsRACK1A-OsRBOHB association and promotes OsRBOHB phosphorylation to enhance ROS production, conferring rice floral resistance to U. virens without yield penalty. Taken together, our findings reveal a new pathogenic mechanism mediated by an essential effector from a flower-specific pathogen and provide a valuable genetic resource for balancing disease resistance and crop yield.

Double-layer focal plane microscopy for high throughput DNA sequencing.

Throughput is one of the most important properties in DNA sequencing. We propose a novel double-layer focal plane microscopy that doubles the DNA sequencing throughput. Each fluorescence channel is divided into two tube lens channels by energy splitting, and the camera is adjusted to take images corresponding to different defocus positions of the objective, thus doubling the information capacity of the microscopy. The microscopy is applied to gene chip, which has high spatial frequency and good uniformity, so the simultaneous imaging of the two tubes has little influence on each other due to the spatial averaging effect. Experimental results show that the image signal to noise ratio (SNR) is reduced by 1%, while the sequencing throughput is doubled.

Characteristics of Genomic Alterations in Pericardial Effusion of Advanced Non-small Cell Lung Cancer.

Background: The feasibility and value of pericardial effusion as a liquid biopsy sample for actionable alteration detection in patients with non-small cell lung cancer (NSCLC) has not been adequately investigated. Here, we aim to reveal genomic alterations between pericardial effusion and paired tumor tissue, plasma (plasma cfDNA), and pleural effusion supernatant (PE-cfDNA) based on second-generation sequencing technology. Material and methods: A total of 26 advanced NSCLC patients were retrospectively studied. The following samples were collected and sequenced using two targeted next-generation sequencing panels: pericardial effusion (n = 26), matched tumor tissue (n = 6), plasma (n = 16), and pleural effusion supernatant (n = 5). Results: A total of 10 actionable alterations were identified in pericardial effusion of the NSCLC patients, including MET amplification, EGFR L858R, EGFR T790M, EGFR exon 19 deletion, EGFR L861Q, KRAS G12C, EML4-ALK (exon 18: exon 20) fusion, EML4-ALK (exon 20: exon 20) fusion, EML4-ALK (exon 6: exon 20) fusion, and ERBB2 exon 20 insertion. All these actionable alterations harbored multiple drug-sensitive targets as well as several drug-resistant targets, such as EGFR T790M. Compared to plasma cfDNA of 16 patients, paired pericardial effusion had higher number of actionable alterations (p = 0.08) as well as higher percentage of the population with actionable alterations (p = 0.16). Moreover, 8 out of 10 actionable alterations with single nucleotide variations (SNVs) or insertions/deletions (indels) had a higher variant allele frequency (VAF) in pericardial effusion than plasma cfDNA. In addition, we identified two actionable alterations in paired pericardial effusion, which were absence in PE-cfDNA. Clearly, 2 out of 3 actionable alterations with SNVs/indels in pericardial effusion had a higher VAF than those in PE-cfDNA. Our finding suggested the importance of pericardial effusion in the optimal selection of patients for targeted therapy. Conclusion: Among liquid biopsy specimens from the advanced NSCLC patients, pericardial effusion may be a better candidate for genomic profiling than plasma cfDNA, while it could serve as a supplement to PE-cfDNA in detecting actionable alterations. Therefore, pericardial effusion might provide a new alternative for selection of patients for better treatment management.

The Flower-Infecting Fungus Ustilaginoidea virens Subverts Plant Immunity by Secreting a Chitin-Binding Protein.

Ustilaginoidea virens is a biotrophic fungal pathogen specifically colonizing rice floral organ and causes false smut disease of rice. This disease has emerged as a serious problem that hinders the application of high-yield rice cultivars, by reducing grain yield and quality as well as introducing mycotoxins. However, the pathogenic mechanisms of U. virens are still enigmatic. Here we demonstrate that U. virens employs a secreted protein UvCBP1 to manipulate plant immunity. In planta expression of UvCBP1 led to compromised chitin-induced defense responses in Arabidopsis and rice, including burst of reactive oxygen species (ROS), callose deposition, and expression of defense-related genes. In vitro-purified UvCBP1 protein competes with rice chitin receptor OsCEBiP to bind to free chitin, thus impairing chitin-triggered rice immunity. Moreover, UvCBP1 could significantly promote infection of U. virens in rice flowers. Our results uncover a mechanism of a floral fungus suppressing plant immunity and pinpoint a universal role of chitin-battlefield during plant-fungi interactions.

Digital PCR is a sensitive new technique for SARS-CoV-2 detection in clinical applications.

The global coronavirus disease 2019 (COVID-19) pandemic has posed great challenges in people's daily lives. Highly sensitive laboratory techniques played a critical role in clinical COVID-19 diagnosis and management. In this study the feasibility of using a new digital PCR-based detection assay for clinical COVID-19 diagnosis was investigated by comparing its performance with that of RT-PCR. Clinical patient samples and samples obtained from potentially contaminated environments were analyzed. The study included 10 patients with confirmed COVID-19 diagnoses, 32 validated samples of various types derived from different clinical timepoints and sites, and 148 environmentally derived samples. SARS-CoV-2 nucleic acids were more readily detected in respiratory tract samples (35.0%). In analyses of environmentally derived samples, the positivity rate of air samples was higher than that of surface samples, probably due to differences in virus concentrations. Digital PCR detected SARS-CoV-2 in several samples that had previously been deemed negative, including 3 patient-derived samples and 5 environmentally derived samples. In this study digital PCR exhibited higher sensitivity than conventional RT-PCR, suggesting that it may be a useful new method for clinical SARS-CoV-2 detection. Improvement of SARS-CoV-2 detection would substantially reduce the rates of false-negative COVID-19 test results, in particular those pertaining to asymptomatic carriers.

HPV genotypic spectrum in Jilin province, China, where non-vaccine-covered HPV53 and 51 are prevalent, exhibits a bimodal age-specific pattern.

BACKGROUND: Human papillomavirus (HPV), the most common sexually transmitted disease, is involved in a series of other diseases. The persistent infection of high-risk HPVs (HR-HPVs) is considered to be the causative agent of cervical cancer, and it is related to noncervical cancers. The present study aims to estimate the HPV prevalence and genotype distribution in Jilin province, China, to guide HPV-related cervical cancer screening and HPV vaccination. METHODS: From October 2017 to September 2019, 21,282 samples (634 male and 20,648 female) were collected for HPV infection detection using an HPV genotyping panel. The age-related HPV prevalence and morbidity of HPV-based disease and HPV prevalence associated with specific diseases were analyzed. RESULTS: A total of 7095 (34.4%) positive for HPV infection of 20648 women, and 164 (25.8%) positive of 634 men. The HPV prevalence among women exhibited a bimodal pattern, with a peak in young group and a second peak in old group, with increased severity of cervical lesions. HPV16 (7.8%), HPV52 (5.8%), HPV58 (5.0%), HPV53 (3.4%), and HPV51 (3.0%) were the most prevalent genotypes among women, and HPV6 (6.0%), HPV11 (5.7%), HPV16 (3.6%), HPV18 (2.7%), and HPV51 (3.0%) were prevalent among men. Non-vaccine-covered HPV53 and 51 were found in 6.3% of HPV infection and 8.9% of cervical cancer in Jilin province. Furthermore, 45.5% of females and 28.6% of males with genital warts were infected with HR-HPV genotypes. CONCLUSION: The HPV genotypic spectrum in Jilin province, where non-vaccine-covered HPV53 and 51 were prevalent, exhibited an age- and cervical lesion-specific pattern, which provides guidance for HPV vaccination and cervical cancer screening. HPV infection in men and benign hyper-proliferative lesions should not be neglected.

Combining Genetic Mutation and Expression Profiles Identifies Novel Prognostic Biomarkers of Lung Adenocarcinoma.

MOTIVATION: Although several prognostic signatures for lung adenocarcinoma (LUAD) have been developed, they are mainly based on a single-omics data set. This article aims to develop a novel set of prognostic signatures by combining genetic mutation and expression profiles of LUAD patients. METHODS: The genetic mutation and expression profiles, together with the clinical profiles of a cohort of LUAD patients from The Cancer Genome Atlas (TCGA), were downloaded. Patients were separated into 2 groups, namely, the high-risk and low-risk groups, according to their overall survivals. Then, differential analysis was performed to determine differentially expressed genes (DEGs) and mutated genes (DMGs) in the expression and mutation profiles, respectively, between the 2 groups. Finally, a prognostic model based on the support vector machine (SVM) algorithm was developed by combining the expression values of the DEGs and the mutation times of the DMGs. RESULTS: A total of 13 DEGs and 7 DMGs were recognized between the 2 groups. Their prognostic values were validated using independent cohorts. Compared with several existing signatures, the proposed prognostic signatures exhibited better prediction performance in the testing set. In addition, it is found that 1 of the 7 DMGs, GRIN2B, is mutated much more frequently in the high-risk group, showing a potential value as a therapy target. CONCLUSIONS: Combining multi-omics data sets is an applicable manner to identify novel prognostic signatures and to improve the prognostic prediction for LUAD, which will be heuristic to other types of cancers.

Characteristics of primary side population cervical cancer cells.

The aim of the present study was to identify and characterize side population (SP) cells in primary cervical cancer. A primary culture was successfully established, and the SP cells were isolated via fluorescence-activated cell sorting. Subsequently, in vitro analysis of clonogenic capacity by soft agar assay and in vivo analysis of tumorigenicity were performed. The isolated SP cells accounted for ~4.73% of the total primary culture cells. The SP cells had a decreased proliferation rate and an increased distribution in G0/G1 compared with non-SP (NSP) cells. Following isolation, SP cells exhibited increased proliferative and self-renewal potency compared with NSP cells. Furthermore, significant ATP binding cassette subfamily G member 2 (ABCG2) expression was detected in SP cells but not in NSP cells. The tumor formation rate of SP cells was longer, and the tumor size and tumor formation rate of SP cells were increased in non-obese diabetic/severe combined immunodeficiency mice. In conclusion, the present study demonstrated that SP cells can be isolated from primary cervical cancer cell culture, and SP cells are enriched with stem cell-like cells that have a high capacity for colony formation and tumorigenesis.

Nicotinamide phosphoribosyltransferase (Nampt) may serve as the marker for osteoblast differentiation of bone marrow-derived mesenchymal stem cells.

Decreased bone volume and strength with aging and enhanced risk of fractures are in part due to reduced number of bone-forming mesenchymal stem cells (MSCs) and cellular dysfunction. In a previous study, we found that osteogenic differentiation of the multipotent and omnipotent preosteoblasts are accompanied by the alterations of intracellular NAD metabolism in which nicotinamide phosphoribosyltransferase (Nampt) plays a regulatory role. The increased Nampt during osteoblast differentiation, the enzyme catalyzing NAD resynthesis from nicotinamide was noted. However, whether Nampt will also be able to affect osteogenic differentiation of primary bone marrow-derived mesenchymal stem cells (BM-MSCs), it is still uncertain. Here we report the role of Nampt in regulating osteoblast differentiation in primary mouse BM-MSCs. We found that Nampt expression was progressively elevated during BM-MSCs osteogenic differentiation. The Nampt inhibitor FK866 or knock-down of Nampt in BM-MSCs led to declined osteoblastogenesis, including attenuated ALP activity, diminished matrix mineralization and down-regulated osteoblast specific marker genes. In addition, declined osteoblastogenesis by Nampt deficiency or addition of FK866 was related to lower intracellular NAD concentration and decreased Sirt1 activity. The present findings demonstrate that osteogenic differentiation in MSCs can be modulated by intracellular NAD metabolism, in which Nampt may serve as an applicable marker for the osteoblast determination.

Increased CD4+CXCR5+T follicular helper cells in diabetic nephropathy.

BACKGROUND: T follicular helper (Tfh) cells are known to regulate humoral immune response. In this study we examined the correlation of different subsets of peripheral blood Tfh cells in patients with diabetic nephropathy (DN). METHODS: A total of 23 DN patients and 15 healthy controls (HC) were investigated for various subsets of Tfh cells by flow cytometry. The molecules ICOS+, PD-1+, CD28+, CD154+, IL-21+, IFN-γ+, IL-4+, IL-17+ Tfh cells were examined. The subsets of B cells were investigated by flow cytometry. The levels of 24 h urinary protein and estimated glomerular filtration rate (eGFR) were calculated. A potential correlation between the number of different subsets of Tfh cells, B cells and DN, was assessed. RESULTS: The circulating CD4+CXCR5+PD-1+, PD-1+CD154+, PD-1+CD28+, PD-1+IL-21+, PD-1+IL-4+, PD-1+-IL-17+-Tfh cell counts, CD38+CD19+, CD38+CD19+CD40+ B cells and plasma levels of IL-21 were significantly increased in DN patients (p < 0.05), as compared to that in the HC group. Furthermore, the circulating CD4+CXCR5+PD-1+ Tfh cell counts negatively correlated with eGFR; Tfh cell counts positively correlated with 24 h urinary protein concentration in DN patients. Post-treatment, there was a significant reduction in the CD4+CXCR5+PD-1+ Tfh cell counts and its subsets, with a corresponding decrease in plasma levels of IL-6 and IL-17A (p < 0.05) in DN patients, as compared to the HCs. CONCLUSION: An increased number of CD4+CXCR5+PD-1+ Tfh cells were observed in DN patients, which may be new targets for intervention in DN.

Nampt expression increases during osteogenic differentiation of multi- and omnipotent progenitors.

Despite emerging data showing that metabolic changes occur with stem cell differentiation, the cross-talk between factors governing energy metabolism and epigenetic modification is not understood. Nicotinamide adenine dinucleotide (NAD) participates in both energy metabolism and protein modification processes. Changes of the intracellular NAD concentration have been shown to correlate with differentiation of adult and embryonic stem cells. In the present study, we investigated the expression pattern of Nampt, the rate-limiting enzyme in NAD salvaging pathway, during osteogenic differentiation of the multipotent mouse fibroblast C3H10T1/2 and the omnipotent preosteoblast MC3T3-E1 cells. We found that Nampt was increasingly expressed during differentiation in both cell models. The increase of Nampt was associated with higher NAD concentration and Sirt1 activity. Knockdown of Nampt or addition of its specific inhibitor FK866 leads to lower intracellular NAD concentration and decline in osteogenesis. These findings indicate that osteogenic differentiation correlates with intracellular NAD metabolism in which Nampt plays a regulatory role.

Nicotinamide phosphoribosyltransferase (Nampt) affects the lineage fate determination of mesenchymal stem cells: a possible cause for reduced osteogenesis and increased adipogenesis in older individuals.

Human aging is associated with a progressive decline in bone mass and an accumulation of marrow fat. We found that osteoblast differentiation was reduced and adipocyte formation increased in bone marrow stromal cells derived from aged mice compared with young controls. The increased adipogenesis correlated with a relatively lower Sirt1 activity and a lower intracellular NAD(+) concentration. We suppose that these effects were caused by age-related reduction of nicotinamide phosphoribosyltransferase (Nampt), the enzyme catalyzing NAD resynthesis from nicotinamide (NAM). In support of this hypothesis, treatment with Nampt inhibitor FK866 increased adipocyte formation and reduced mineralization in primary cultured bone marrow stromal cells. In addition, knockdown of Nampt in the mouse mesenchymal cell line C3H10T1/2 cells resulted in decreased Sirt1 activity and enhanced adipogenesis. Interestingly, although Nampt deficiency resulted in both decreased intracellular NAD(+) and increased NAM, the cell differentiation could be controlled only by regulation of NAM. These results indicate that the lineage fate determination of mesenchymal stem cells (MSCs) is influenced by cell energy metabolism and points to a possible mechanism for the development of senile osteoporosis. Furthermore, we suggest that side effects on bone should be considered when evaluating the long-term safety of NAD-interfering pharmaceuticals.