Isolation and Characterization of a Novel Virulent Phage ASG01 of Aeromonas salmonicida and Its Cell Wall Hydrolase Activity.

Aeromonas salmonicida is an important pathogen that causes furunculosis in trout and salmon with high morbidity and mortality, resulting in significant economic losses in aquaculture. Overuse of antibiotics has led to the continuous emergence of drug-resistant strains. Hence, there is an urgent need to find an alternative environmentally friendly antimicrobial agent. In this study, we isolated a virulent phage of A. salmonicida, named ASG01, which belongs to the Myoviridae family and maintains lytic activity at a pH value range from 4 to 12 and in the temperature range from 30 °C to 60 °C. The whole genomic sequence of ASG01 showed 82% similarity to Aeromonas phage pAh6-C. The cell wall hydrolase (Cwh)-encoding gene from the genome of ASG01 was predicted and heterologously expressed. Notably, in the absence of additional phage genes, endogenous expression of Cwh could lyse E. coli cells and greatly inhibit the growth of tested fish pathogenic bacteria. The lytic activity of Cwh was eliminated when the predicted active site was mutated. These results indicate that Cwh of ASG01 possessed excellent lytic activity and a wide antibacterial spectrum, suggesting its potential as an effective enzybiotic.

Targeting oxidative phosphorylation to increase the efficacy of immune-combination therapy in renal cell carcinoma.

BACKGROUND: Immune checkpoint inhibitors (ICIs) are the standard of care for metastatic renal cell carcinoma (RCC); however, most patients develop de novo or acquired resistance to ICIs. Oxidative phosphorylation (OXPHOS) has been rarely explored as a potential target for correcting ICI resistance. METHODS: We systematically analyzed RNA sequencing and clinical data from CheckMate, JAVELIN Renal 101, and NCT01358721 clinical trials, and clinicopathological data of 25 patients from Tongji Hospital to investigate the relationship between OXPHOS and ICI resistance. The Ndufb8-knockdown Renca cell line was derived to determine the effect of OXPHOS on RCC immunotherapy in vivo. RESULTS: An analysis of the CheckMate series data revealed that high OXPHOS levels are risk factors for ICI in patients with RCC, but are affected by thevon Hippel-Lindau protein (VHL) and hypoxia-inducible factor-1α status. This result is consistent with correlation between clinicopathological characteristics and prognostic observations at our institute. Knockdown of the mitochondrial complex I subunit Ndufb8 of the Renca cell line had no effect on cell growth and migration in vitro, but slowed down cell growth in vivo. Among anti-programmed death ligand 1 (PD-L1)-treated BALB/c mice, shNdufb8 Renca tumors grew slower than shControl Renca tumors and the corresponding mice survived longer. Flow cytometry revealed that CD8+ T cells in shNdufb8 Renca tumors, which were exposed to a lower degree of hypoxia and expressed less programmed death-1 (PD-1) and T-cell immunoglobulin domain and mucin domain 3 (TIM-3), secreted more interferon-γ after stimulation. Immunofluorescence demonstrated that the shNdufb8 Renca tumors had a higher proportion of CD8+ T cells and the proportion of these cells was lower in the hypoxic area. CONCLUSIONS: OXPHOS is a reliable predictor of immunotherapy response in RCC and is more pronounced in metastatic lesions. RCC cells generate a hypoxic tumor microenvironment and inhibit T-cell function through oxidative metabolism, thereby leading to immunotherapy resistance.

The splicing factor Prpf31 is required for hematopoietic stem and progenitor cell expansion during zebrafish embryogenesis.

Pre-mRNA splicing is a precise regulated process and is crucial for system development and homeostasis maintenance. Mutations in spliceosomal components have been found in various hematopoietic malignancies (HMs) and have been considered as oncogenic derivers of HMs. However, the role of spliceosomal components in normal and malignant hematopoiesis remains largely unknown. Pre-mRNA processing factor 31 (PRPF31) is a constitutive spliceosomal component, which mutations are associated with autosomal dominant retinitis pigmentosa. PRPF31 was found to be mutated in several HMs, but the function of PRPF31 in normal hematopoiesis has not been explored. In our previous study, we generated a prpf31 knockout (KO) zebrafish line and reported that Prpf31 regulates the survival and differentiation of retinal progenitor cells by modulating the alternative splicing of genes involved in mitosis and DNA repair. In this study, by using the prpf31 KO zebrafish line, we discovered that prpf31 KO zebrafish exhibited severe defects in hematopoietic stem and progenitor cell (HSPC) expansion and its sequentially differentiated lineages. Immunofluorescence results showed that Prpf31-deficient HSPCs underwent malformed mitosis and M phase arrest during HSPC expansion. Transcriptome analysis and experimental validations revealed that Prpf31 deficiency extensively perturbed the alternative splicing of mitosis-related genes. Collectively, our findings elucidate a previously undescribed role for Prpf31 in HSPC expansion, through regulating the alternative splicing of mitosis-related genes.

The splicing factor DHX38 enables retinal development through safeguarding genome integrity.

DEAH-Box Helicase 38 (DHX38) is a pre-mRNA splicing factor and also a disease-causing gene of autosomal recessive retinitis pigmentosa (arRP). The role of DHX38 in the development and maintenance of the retina remains largely unknown. In this study, by using the dhx38 knockout zebrafish model, we demonstrated that Dhx38 deficiency causes severe differentiation defects and apoptosis of retinal progenitor cells (RPCs) through disrupted mitosis and increased DNA damage. Furthermore, we found a significant accumulation of R-loops in the dhx38-deficient RPCs and human cell lines. Finally, we found that DNA replication stress is the prerequisite for R-loop-induced DNA damage in the DHX38 knockdown cells. Taken together, our study demonstrates a necessary role of DHX38 in the development of retina and reveals a DHX38/R-loop/replication stress/DNA damage regulatory axis that is relatively independent of the known functions of DHX38 in mitosis control.

Identification of alternative splicing associated with clinical features: from pan-cancers to genitourinary tumors.

Background: Alternative splicing events (ASEs) are vital causes of tumor heterogeneity in genitourinary tumors and many other cancers. However, the clinicopathological relevance of ASEs in cancers has not yet been comprehensively characterized. Methods: By analyzing splicing data from the TCGA SpliceSeq database and phenotype data for all TCGA samples from the UCSC Xena database, we identified differential clinical feature-related ASEs in 33 tumors. CIBERSORT immune cell infiltration data from the TIMER2.0 database were used for differential clinical feature-related immune cell infiltration analysis. Gene function enrichment analysis was used to analyze the gene function of ASEs related to different clinical features in tumors. To reveal the regulatory mechanisms of ASEs, we integrated race-related ASEs and splicing quantitative trait loci (sQTLs) data in kidney renal clear cell carcinoma (KIRC) to comprehensively assess the impact of SNPs on ASEs. In addition, we predicted regulatory RNA binding proteins in bladder urothelial carcinoma (BLCA) based on the enrichment of motifs around alternative exons for ASEs. Results: Alternative splicing differences were systematically analyzed between different groups of 58 clinical features in 33 cancers, and 30 clinical features in 24 cancer types were identified to be associated with more than 50 ASEs individually. The types of immune cell infiltration were found to be significantly different between subgroups of primary diagnosis and disease type. After integrating ASEs with sQTLs data, we found that 63 (58.9%) of the race-related ASEs were significantly SNP-correlated ASEs in KIRC. Gene function enrichment analyses showed that metastasis-related ASEs in KIRC mainly enriched Rho GTPase signaling pathways. Among those ASEs associated with metastasis, alternative splicing of GIT2 and TUBB3 might play key roles in tumor metastasis in KIRC patients. Finally, we identified several RNA binding proteins such as PCBP2, SNRNP70, and HuR, which might contribute to splicing differences between different groups of neoplasm grade in BLCA. Conclusion: We demonstrated the significant clinical relevance of ASEs in multiple cancer types. Furthermore, we identified and validated alternative splicing of TUBB3 and RNA binding proteins such as PCBP2 as critical regulators in the progression of urogenital cancers.

SPPUSM: An MS/MS spectra merging strategy for improved low-input and single-cell proteome identification.

Single and rare cell analysis provides unique insights into the investigation of biological processes and disease progress by resolving the cellular heterogeneity that is masked by bulk measurements. Although many efforts have been made, the techniques used to measure the proteome in trace amounts of samples or in single cells still lag behind those for DNA and RNA due to the inherent non-amplifiable nature of proteins and the sensitivity limitation of current mass spectrometry. Here, we report an MS/MS spectra merging strategy termed SPPUSM (same precursor-produced unidentified spectra merging) for improved low-input and single-cell proteome data analysis. In this method, all the unidentified MS/MS spectra from multiple test files are first extracted. Then, the corresponding MS/MS spectra produced by the same precursor ion from different files are matched according to their precursor mass and retention time (RT) and are merged into one new spectrum. The newly merged spectra with more fragment ions are next searched against the database to increase the MS/MS spectra identification and proteome coverage. Further improvement can be achieved by increasing the number of test files and spectra to be merged. Up to 18.2% improvement in protein identification was achieved for 1 ng HeLa peptides by SPPUSM. Reliability evaluation by the "entrapment database" strategy using merged spectra from human and E. coli revealed a marginal error rate for the proposed method. For application in single cell proteome (SCP) study, identification enhancement of 28%-61% was achieved for proteins for different SCP data. Furthermore, a lower abundance was found for the SPPUSM-identified peptides, indicating its potential for more sensitive low sample input and SCP studies.

Role of human papillomavirus and associated viruses in bladder cancer: An updated review.

Bladder cancer (BC) is a complex disease affecting the urinary system and is regulated by several carcinogenic factors. Viral infection is one such factor that has attracted extensive attention in BC. Human papillomavirus (HPV) is the most common sexually transmitted infection, and although multiple researchers have explored the role of HPV in BC, a consensus has not yet been reached. In addition, HPV-associated viruses (e.g., human immunodeficiency virus, herpes simplex virus, BK virus, and JC virus) appear to be responsible for the occurrence and progression of BC. This study systematically reviews the relationship between HPV-associated viruses and BC to elucidate the role of these viruses in the onset and progression of BC. In addition, the study aims to provide a greater insight into the biology of HPV-associated viruses, and assess potential strategies for treating virus-induced BC. The study additionally focuses on the rapid development of oncolytic viruses that provide a potentially novel option for the treatment of BC.

Absolute Quantification of Dynamic Cellular Uptake of Small Extracellular Vesicles via Lanthanide Element Labeling and ICP-MS.

Small extracellular vesicles (sEVs) are increasingly reported to play important roles in numerous physiological and pathological processes. Cellular uptake of sEVs is of great significance for functional regulation in recipient cells. Although various sEV quantification, labeling, and tracking methods have been reported, it is still highly challenging to quantify the absolute amount of cellular uptake of sEVs and correlate this information with phenotypic variations in the recipient cell. Therefore, we developed a novel strategy using lanthanide element labeling and inductively coupled plasma-mass spectrometry (ICP-MS) for the absolute and sensitive quantification of sEVs. This strategy utilizes the chelation interaction between Eu3+ and the phosphate groups on the sEV membrane for specific labeling. sEVs internalized by cells can then be quantified by ICP-MS using a previously established linear relationship between the europium content and the particle numbers. High Eu labeling efficiency and stability were demonstrated by various evaluations, and no structural or functional alterations in the sEVs were discovered after Eu labeling. Application of this method revealed that 4020 ± 171 sEV particles/cell were internalized by HeLa cells at 37 °C and 61% uptake inhibition at 4 °C. Further investigation led to the quantitative differential analysis of sEV cellular uptake under the treatment of several chemical endocytosis inhibitors. A 23% strong inhibition indicated that HeLa cells uptake sEVs mainly through the macropinocytosis pathway. This facile labeling and absolute quantification strategy of sEVs with ppb-level high sensitivity is expected to become a potential tool for studying the functions of sEVs in intracellular communication and cargo transportation.

Sensitive N-Glycopeptide Profiling of Single and Rare Cells Using an Isobaric Labeling Strategy without Enrichment.

Single-cell omics is critical in revealing population heterogeneity, discovering unique features of individual cells, and identifying minority subpopulations of interest. As one of the major post-translational modifications, protein N-glycosylation plays crucial roles in various important biological processes. Elucidation of the variation in N-glycosylation patterns at single-cell resolution may largely facilitate the understanding of their key roles in the tumor microenvironment and immune therapy. However, comprehensive N-glycoproteome profiling for single cells has not been achieved due to the extremely limited sample amount and incompatibility with the available enrichment strategies. Here, we have developed an isobaric labeling-based carrier strategy for highly sensitive intact N-glycopeptide profiling for single cells or a small number of rare cells without enrichment. Isobaric labeling has unique multiplexing properties, by which the "total" signal from all channels triggers MS/MS fragmentation for N-glycopeptide identification, while the reporter ions provide quantitative information. In our strategy, a carrier channel using N-glycopeptides obtained from bulk-cell samples significantly improved the "total" signal of N-glycopeptides and, therefore, promoted the first quantitative analysis of averagely 260 N-glycopeptides from single HeLa cells. We further applied this strategy to study the regional heterogeneity of N-glycosylation of microglia in mouse brain and discovered region-specific N-glycoproteome patterns and cell subtypes. In conclusion, the glycocarrier strategy provides an attractive solution for sensitive and quantitative N-glycopeptide profiling of single/rare cells that cannot be enriched by traditional workflows.

Artificial evolution of OsEPSPS through an improved dual cytosine and adenine base editor generated a novel allele conferring rice glyphosate tolerance.

Exploiting novel endogenous glyphosate-tolerant alleles is highly desirable and has promising potential for weed control in rice breeding. Here, through fusions of different effective cytosine and adenine deaminases with nCas9-NG, we engineered an effective surrogate two-component composite base editing system, STCBE-2, with improved C-to-T and A-to-G base editing efficiency and expanded the editing window. Furthermore, we targeted a rice endogenous OsEPSPS gene for artificial evolution through STCBE-2-mediated near-saturated mutagenesis. After hygromycin and glyphosate selection, we identified a novel OsEPSPS allele with an Asp-213-Asn (D213N) mutation (OsEPSPS-D213N) in the predicted glyphosate-binding domain, which conferred rice plants reliable glyphosate tolerance and had not been reported or applied in rice breeding. Collectively, we developed a novel dual base editor which will be valuable for artificial evolution of important genes in crops. And the novel glyphosate-tolerant rice germplasm generated in this study will benefit weeds management in rice paddy fields.

The emotional facial recognition performance of Chinese patients with schizophrenia: An event-related potentials study.

Background: Patients with schizophrenia have deficits in identifying and recognizing emotional facial expressions. Aim: This study aimed to explore the event-related potential (ERP) responses of patients with schizophrenia (SZ) and healthy controls (HC) using the Chinese Facial Affective Picture System (CFAPS). Methods: This study included 30 SZs and 31 HCs. We asked them to complete the task based on the oddball paradigm, in which three emotional faces (happy, fearful, and neutral) were used as target stimuli. Additionally, the amplitude and latency of the N170 component and the P300 component were recorded synchronously. Results: Compared with HCs, SZs had significantly smaller amplitudes of N170 and P300 to all facial expressions. The pairwise comparison revealed that fearful faces could trigger a significantly larger P300 amplitude in HCs than neutral faces, while the such a difference was not found in SZs. Conclusion: These findings indicated that SZs had a noticeable deficiency in the structural coding of face recognition and available attentional resources.

Tumor heterogeneity in VHL drives metastasis in clear cell renal cell carcinoma.

Loss of function of the von Hippel-Lindau (VHL) tumor suppressor gene is a hallmark of clear cell renal cell carcinoma (ccRCC). The importance of heterogeneity in the loss of this tumor suppressor has been under reported. To study the impact of intratumoral VHL heterogeneity observed in human ccRCC, we engineered VHL gene deletion in four RCC models, including a new primary tumor cell line derived from an aggressive metastatic case. The VHL gene-deleted (VHL-KO) cells underwent epithelial-to-mesenchymal transition (EMT) and exhibited increased motility but diminished proliferation and tumorigenicity compared to the parental VHL-expressing (VHL+) cells. Renal tumors with either VHL+ or VHL-KO cells alone exhibit minimal metastatic potential. Combined tumors displayed rampant lung metastases, highlighting a novel cooperative metastatic mechanism. The poorly proliferative VHL-KO cells stimulated the proliferation, EMT, and motility of neighboring VHL+ cells. Periostin (POSTN), a soluble protein overexpressed and secreted by VHL non-expressing (VHL-) cells, promoted metastasis by enhancing the motility of VHL-WT cells and facilitating tumor cell vascular escape. Genetic deletion or antibody blockade of POSTN dramatically suppressed lung metastases in our preclinical models. This work supports a new strategy to halt the progression of ccRCC by disrupting the critical metastatic crosstalk between heterogeneous cell populations within a tumor.

A rapid and sensitive single-cell proteomic method based on fast liquid-chromatography separation, retention time prediction and MS1-only acquisition.

Single-cell analysis has received much attention in recent years for elucidating the widely existing cellular heterogeneity in biological systems. However, the ability to measure the proteome in single cells is still far behind that of transcriptomics due to the lack of sensitive and high-throughput mass spectrometry methods. Herein, we report an integrated strategy termed "SCP-MS1" that combines fast liquid chromatography (LC) separation, deep learning-based retention time (RT) prediction and MS1-only acquisition for rapid and sensitive single-cell proteome analysis. In SCP-MS1, the peptides were identified via four-dimensional MS1 feature (m/z, RT, charge and FAIMS CV) matching, therefore relieving MS acquisition from the time consuming and information losing MS2 step and making this method particularly compatible with fast LC separation. By completely omitting the MS2 step, all the MS analysis time was utilized for MS1 acquisition in SCP-MS1 and therefore led to 65%-138% increased MS1 feature collection. Unlike "match between run" methods that still needed MS2 information for RT alignment, SCP-MS1 used deep learning-based RT prediction to transfer the measured RTs in long gradient bulk analyses to short gradient single cell analyses, which was the key step to enhance both identification scale and matching accuracy. Using this strategy, more than 2000 proteins were obtained from 0.2 ng of peptides with a 14-min active gradient at a false discovery rate (FDR) of 0.8%. Comparing with the DDA method, improved quantitative performance was also observed for SCP-MS1 with approximately 50% decreased median coefficient of variation of quantified proteins. For single-cell analysis, 1715 ± 204 and 1604 ± 224 proteins were quantified in single 293T and HeLa cells, respectively. Finally, SCP-MS1 was applied to single-cell proteome analysis of sorafenib resistant and non-resistant HepG2 cells and revealed clear cellular heterogeneity in the resistant population that may be masked in bulk studies.

A three-stage search strategy combining database reduction and retention time filtering to improve the sensitivity of low-input and single-cell proteomic analysis.

When performing proteome profiling of low-input and single-cell samples, achieving deep protein coverage is very challenging due to the sensitivity limitation of current proteomic methods. Herein, we introduce a three-stage search strategy that combines the advantages of database reduction and Δ retention time (ΔRT) filtering. The strategy improves peptide/protein identification and reproducibility by retaining more correct identifications and filtering out incorrect identifications. The raw data were first merged and searched against a Uniprot database with a relaxed false discovery rate (FDR) of 40% to identify the possible detectable proteins. The identified proteins were then used as a new database to search the raw data against with a tighter FDR of 10%. After this, the results were filtered using ΔRT (the difference between the measured and predicted RT) to reduce the incorrect identifications and maintain the FDR below 1%. This strategy resulted in over 30% improvement in proteome coverage for single-cells and samples of similar size. The reproducibility of identification and quantification was also enhanced for the low-input samples. Moreover, the 50% higher number of differential proteins found in the two types of single neurons indicates the application potential of this strategy.

Integrated Analysis of Transcriptome Changes in Osteoarthritis: Gene Expression, Pathways and Alternative Splicing.

OBJECTIVE: Osteoarthritis (OA) is the most prevalent joint disease characterized by the degeneration of articular cartilage and the remodeling of its underlying bones, resulting in pain and loss of function in the knees and hips. As far as we know, no curative treatments are available except for the joint replacement. The precise molecular mechanisms which are involved in the degradation of cartilage matrix and development of osteoarthritis are still unclear. DESIGN: By analyzing RNA-seq data, we found the molecular changes at the transcriptome level such as alternative splicing, gene expression, and molecular pathways in OA knees cartilage. RESULTS: Expression analysis have identified 457 differential expressed genes including 266 up-regulated genes such as TNFSF15, ST6GALNAC5, TGFBI, ASPM, and TYM, and 191 down-regulated genes such as ADM, JUN, IRE2, PIGA, and MAFF. Gene set enrichment analysis (GSEA) analysis identified down-regulated pathways related to translation, transcription, immunity, PI3K/AKT, and circadian as well as disturbed pathways related to extracellular matrix and collagen. Splicing analysis identified 442 differential alternative splicing events within 284 genes in osteoarthritis, including genes involved in extracellular matrix (ECM) and alternative splicing, and TIA1 was identified as a key regulator of these splicing events. CONCLUSIONS: These findings provide insights into disease etiology, and offer favorable information to support the development of more effective interventions in response to the global clinical challenge of osteoarthritis.

An angled-shape tip-based strategy for highly sensitive proteomic profiling of a low number of cells.

Profiling proteins plays an essential role in understanding the functions and dynamic networks in biological systems. Mass spectrometry-based proteomic analysis commonly requires multistep sample processing, which results in severe sample loss. Although the recently developed microproteomic strategies have substantially reduced sample loss via droplet microfluidic technology, specialized equipment and well-trained personnel are needed, which may limit their wide adoption. Here, we report an angled-shape tip-based strategy for rapid sample preparation and sensitive proteomic profiling of small cell populations (<1000 cells). The angled-shape tip provided a 'reactor' for the entire proteomic sample processing workflow, from cell capture and lysis to protein digestion, eliminating the sample transfer-induced protein loss. The angled-shape tip was surface-treated for anti-protein adsorption which further reduced the sample loss. Using this strategy, 1241 ± 38-4110 ± 37 protein groups and 4010 ± 700-34 879 ± 575 peptides were identified from 10-1000 HeLa cells with high quantification reproducibility in only 4.5 h sample processing time, which was superior to the reported methods and commercial kits, especially for <100 cells. This approach was easily accessible, straightforward to operate, and compatible with flow cytometry-based cell sorting. It showed great potential for in-depth proteomic profiling of rare cells (<1000 cells) in both basic biological research and clinical application.

Insight into thermal regulation of supercapacitors via surface-engineered phase change microcapsules.

Constructing efficient thermal management system to settle the thermal runaway of energy storage devices via employing phase change microcapsules (MEPCMs) is of great significance. However, it is still a challenge that the conventional MEPCMs go against the electrochemical performance and hardly be homogenously fixed in the electrodes. In order to conquer these long-standing critical issues, we designed a novel electrochemically active double-shell phase change microcapsule by introducing polypyrrole on the surface of dense amine resin shell of the conventional inert MEPCM. The active MEPCMs@PPy are uniformly immobilized on the surface of the electrode material using reduced graphene oxide to ensure the stable and efficient operation of the flexible supercapacitor. The assembled all-solid-state supercapacitor containing MEPCMs@PPy (SCs@MEPCMs@PPy) lagged 103 s to 55 °C than the SCs@00 without the added phase change material. At a high temperature of 55 °C and a scan rate of 50 mV s-1, SCs@MEPCMs@PPy exhibits an areal specific capacitance of 110.6 mA cm-2, which is higher than that of the original SCs@MEPCMs. A capacitance retention of 79.8 % and coulombic efficiency of 98.4 % can be reached after 3000 cycles. This study opens a new avenue for developing applicable microencapsulated phase change materials in temperature-regulated electrode systems for supercapacitors and alkaline-ion batteries.

Corrigendum: Transcription factors BARX1 and DLX4 contribute to progression of clear cell renal cell carcinoma via promoting proliferation and epithelial-mesenchymal transition.

[This corrects the article DOI: 10.3389/fmolb.2021.626328.].