The Trimeric Autotransporter Adhesin SadA from Salmonella spp. as a Novel Bacterial Surface Display System.

Bacterial surface display platforms have been developed for applications such as vaccine delivery and peptide library screening. The type V secretion system is an attractive anchoring motif for the surface expression of foreign proteins in gram-negative bacteria. SadA belongs to subtype C of the type V secretion system derived from Salmonella spp. and promotes biofilm formation and host cell adherence. The inner membrane lipoprotein SadB is important for SadA translocation. In this study, SadA was used as an anchoring motif to expose heterologous proteins in Salmonella typhimurium using SadB. The ability of SadA to display heterologous proteins on the S. typhimurium surface in the presence of SadB was approximately three-fold higher than that in its absence of SadB. Compared to full-length SadA, truncated SadAs (SadA877 and SadA269) showed similar display capacities when exposing the B-cell epitopes of urease B from Helicobacter pylori (UreB158-172aa and UreB349-363aa). We grafted different protein domains, including mScarlet (red fluorescent protein), the urease B fragment (UreBm) from H. pylori SS1, and/or protective antigen domain 4 from Bacillus anthracis A16R (PAD4), onto SadA877 or SadA1292. Whole-cell dot blotting, immunofluorescence, and flow cytometric analyses confirmed the localization of Flag×3-mScarlet (~30 kDa) and Flag×3-UreBm-mScarlet (~58 kDa) to the S. typhimurium surface using truncated SadA877 or SadA1292 as an anchoring motif. However, Flag×3-UreBm-PAD4-mScarlet (~75 kDa) was displayed on S. typhimurium using SadA1292. The oral administrated pSadBA1292-FUM/StmΔygeAΔmurI and pSadBA877-FUM/StmΔygeAΔmurI could elicit a significant mucosal and humoral immunity response. SadA could thus be used as an anchoring motif for the surface expression of large heterologous proteins as a potential strategy for attenuated bacterial vaccine development.

Skin characterization of diabetes mellitus revealed by polarization-sensitive optical coherence tomography imaging.

Significance: Diabetes can lead to the glycation of proteins and dysfunction of skin collagen. Skin lesions are a prevalent clinical symptom of diabetes mellitus (DM). Early diagnosis and assessing the efficacy of treatment for DM are crucial for patient health management. However, performing a non-invasive skin assessment in the early stages of DM is challenging. Aim: By using the polarization-sensitive optical coherent tomography (PS-OCT) imaging technique, it is possible to noninvasively assess the skin changes caused by diabetes. Approach: The PS-OCT was used to monitor the polarization characteristics of mouse skin at different stages of diabetes. Results: Based on a multi-layered adhesive tape model, we found that the polarization characteristics (retardation, optic axis, and polarization uniformity) were sensitive to the microstructure changes in the samples. Through this method, we observed significant changes in the polarization states of the skin as diabetes progressed. This was in line with the detected microstructure changes in skin collagen fibers using scanning electron microscopy. Conclusions: This study presents a highly useful approach for non-invasive skin assessment of diabetes.

Potential of dietary hemp and cannabinoids to modulate immune response to enhance health and performance in animals: opportunities and challenges.

Cannabinoids are a group of bioactive compounds abundantly present in Cannabis sativa plant. The active components of cannabis with therapeutic potential are known as cannabinoids. Cannabinoids are divided into three groups: plant-derived cannabinoids (phytocannabinoids), endogenous cannabinoids (endocannabinoids), and synthetic cannabinoids. These compounds play a crucial role in the regulation various physiological processes including the immune modulation by interacting with the endocannabinoid system (A complex cell-signaling system). Cannabinoid receptor type 1 (CB1) stimulates the binding of orexigenic peptides and inhibits the attachment of anorexigenic proteins to hypothalamic neurons in mammals, increasing food intake. Digestibility is unaffected by the presence of any cannabinoids in hemp stubble. Endogenous cannabinoids are also important for the peripheral control of lipid processing in adipose tissue, in addition to their role in the hypothalamus regulation of food intake. Regardless of the kind of synaptic connection or the length of the transmission, endocannabinoids play a crucial role in inhibiting synaptic transmission through a number of mechanisms. Cannabidiol (CBD) mainly influences redox equilibrium through intrinsic mechanisms. Useful effects of cannabinoids in animals have been mentioned e.g., for disorders of the cardiovascular system, pain treatment, disorders of the respiratory system or metabolic disorders. Dietary supplementation of cannabinoids has shown positive effects on health, growth and production performance of small and large animals. Animal fed diet supplemented with hemp seeds (180 g/day) or hemp seed cake (143 g/kg DM) had achieved batter performance without any detrimental effects. But the higher level of hemp or cannabinoid supplementation suppress immune functions and reduce productive performance. With an emphasis on the poultry and ruminants, this review aims to highlight the properties of cannabinoids and their derivatives as well as their significance as a potential feed additive in their diets to improve the immune status and health performance of animals.

Multilayer amnion-PCL nanofibrous membrane loaded with celecoxib exerts a therapeutic effect against tendon adhesion by improving the inflammatory microenvironment.

Tendon adhesion is a common complication after tendon surgery. The inflammatory phase of tendon healing is characterized by the release of a large number of inflammatory factors, whose mediated excessive inflammatory response is an important cause of tendon adhesion formation. Nonsteroidal anti-inflammatory drugs(NSAIDs) were used to prevent tendon adhesions by reducing the inflammatory response. However, recent studies have shown that the NSAIDs partially impairs tendon healing. Therefore, optimizing the anti-adhesive membrane loaded with NSAIDs to mitigate the effects on tendon healing requires further in-depth study. Amniotic membranes(AM) are natural polymeric semi-permeable membranes from living organisms that are rich in matrix, growth factors, and other active ingredients. In this study, we used electrostatic spinning technology to construct multifunctional nanofiber membranes of the PCL membrane loaded with celecoxib and AM. In vitro cellular assays revealed that celecoxib-loaded PCL membranes significantly inhibited the adhesion and proliferation of fibroblasts with increasing concentrations of celecoxib. In a rabbit tendon repair model, biomechanical tests further confirmed that the PCL membrane loaded with celecoxib had better anti-adhesion effects. Further experimental studies revealed that the PCL/AM membrane improved the inflammatory microenvironment by downregulating the expression of pro-inflammatory factors such as COX-2, IL-1ß, and TNF-α proteins; and inhibiting the synthesis of COL I and COL Ⅲ. The PCL/AM membrane can continuously release celecoxib to reduce the inflammatory response and deliver growth factors to the damaged area to build a suitable microenvironment for tendon repair, which provides a new direction to improve the repair efficiency of tendon.

Regulatory Mechanism of Peroxisome Number Reduction Caused by FgPex4 and FgPex22-like Deletion in Fusarium graminearum.

Peroxisomes are single-membrane-bound organelles that play critical roles in eukaryotic cellular functions. Peroxisome quantity is a key factor influencing the homeostasis and pathogenic processes of pathogenic fungi. The aim of the present study was to investigate the underlying mechanisms of the reduction in number of peroxisomes in Fusarium graminearum consequent to FgPex4 and FgPex22-like deletion. The number of peroxisomes decreased by 40.55% and 39.70% when FgPex4 and FgPex22-like, respectively, were absent. Peroxisome biogenesis-related proteins, as well as inheritance- and division-related dynamin-like proteins were reduced at the transcriptional level in the mutant strains. In addition, the degree of pexophagy was intensified and the accumulation of ubiquitinated FgPex5 was also increased in F. graminearum when FgPex4 or FgPex22-like was absent. The findings suggest that FgPex4 and FgPex22-like influence the number of peroxisomes by influencing peroxisome biogenesis and pexophagy.

The complete mitochondrial genome of the Anas platyrhynchos Linnaeus, 1758 breed Longshengcui and its phylogenetic analyses.

Duck breed Longshengcui (Anas platyrhynchos Linnaeus, 1758 breed Longshengcui, LSC) is one of the famous native breed of the Guangxi Zhuang Nationality Autonomous Region in China. In this study, we report the complete mitochondrial genome of LSC. The mitogenome (GenBank accession no. MZ895120) has 16,602 bp in length and consisted of the well-known 13 protein-coding genes, 22 tRNA genes, two rRNA genes, and the control region. The phylogenetic analysis showed that LSC and Zhijiang duck have highly similar genetic relationship. These results are helpful for the conservation of genetic resources and phylogeny of this species.

Nanofibrous polycaprolactone/amniotic membrane facilitates peripheral nerve regeneration by promoting macrophage polarization and regulating inflammatory microenvironment.

Appropriate levels of inflammation are an important part of functional repair of nerve damage. However, excessive inflammation can cause the continuous activation of immune inflammatory cells and degeneration of nerve cells. Regulating the temporal and spatial changes in M1/M2 macrophages can regulate the local inflammatory immune environment of the tissue to promote its transformation to a direction conducive to tissue repair.In the present study, a multi-layer multifunctional nanofiber composite membrane of polycaprolactone(PCL) and amniotic membrane (AM) was constructed using electrospinning. In vitro studies have shown that the PCL/AM composite promoted the axon growth of SH-SY5Y cells and induced their differentiation into neurons. The PCL/AM composite wrapped the nerve stump to form a microenvironment that was conducive to nerve regeneration, blocked the invasion of scar tissue, promoted the recruitment of macrophages and moderate polarization to M2, enhanced the expression of anti-inflammatory factors IL-10 and IL-13, inhibited the expression of pro-inflammatory factors IL-6 and TNF-α, and induced myelin sheath and axon regeneration. By releasing various bioactive substances to regulate the polarization of M2 macrophages and formation of anti-inflammatory factors, the PCL/AM composite can enhance axonal regeneration and improve nerve repair.

A dual functional Ti-Ga alloy: inhibiting biofilm formation and osteoclastogenesis differentiation via disturbing iron metabolism.

BACKGROUND: Although biomedical implants have been widely used in orthopedic treatments, two major clinical challenges remain to be solved, one is the bacterial infection resulting in biofilm formation, and the other is aseptic loosening during implantation due to over-activated osteoclastogenesis. These factors can cause many clinical issues and even lead to implant failure. Thus, it is necessary to endow implants with antibiofilm and aseptic loosening-prevention properties, to facilitate the integration between implants and bone tissues for successful implantation. To achieve this goal, this study aimed to develop a biocompatible titanium alloy with antibiofilm and anti-aseptic loosening dual function by utilizing gallium (Ga) as a component. METHODS: A series of Ti-Ga alloys were prepared. We examined the Ga content, Ga distribution, hardness, tensile strength, biocompatibility, and anti-biofilm performance in vitro and in vivo. We also explored how Ga3+ ions inhibited the biofilm formation of Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) and osteoclast differentiation. RESULTS: The alloy exhibited outstanding antibiofilm properties against both S. aureus and E. coli in vitro and decent antibiofilm performance against S. aureus in vivo. The proteomics results demonstrated that Ga3+ ions could disturb the bacterial Fe metabolism of both S. aureus and E. coli, inhibiting bacterial biofilm formation. In addition, Ti-Ga alloys could inhibit receptor activator of nuclear factor-κB ligand (RANKL)-dependent osteoclast differentiation and function by targeting iron metabolism, then suppressing the activation of the NF-κB signaling pathway, thus, showing their potential to prevent aseptic loosening. CONCLUSION: This study provides an advanced Ti-Ga alloy that can be used as a promising orthopedic implant raw material for various clinical scenarios. This work also revealed that iron metabolism is the common target of Ga3+ ions to inhibit biofilm formation and osteoclast differentiation.

Platelet RNA enables accurate detection of ovarian cancer: an intercontinental, biomarker identification study.

Platelets are reprogrammed by cancer via a process called education, which favors cancer development. The transcriptional profile of tumor-educated platelets (TEPs) is skewed and therefore practicable for cancer detection. This intercontinental, hospital-based, diagnostic study included 761 treatment-naïve inpatients with histologically confirmed adnexal masses and 167 healthy controls from nine medical centers (China, n = 3; Netherlands, n = 5; Poland, n = 1) between September 2016 and May 2019. The main outcomes were the performance of TEPs and their combination with CA125 in two Chinese (VC1 and VC2) and the European (VC3) validation cohorts collectively and independently. Exploratory outcome was the value of TEPs in public pan-cancer platelet transcriptome datasets. The AUCs for TEPs in the combined validation cohort, VC1, VC2, and VC3 were 0.918 (95% CI 0.889-0.948), 0.923 (0.855-0.990), 0.918 (0.872-0.963), and 0.887 (0.813-0.960), respectively. Combination of TEPs and CA125 demonstrated an AUC of 0.922 (0.889-0.955) in the combined validation cohort; 0.955 (0.912-0.997) in VC1; 0.939 (0.901-0.977) in VC2; 0.917 (0.824-1.000) in VC3. For subgroup analysis, TEPs exhibited an AUC of 0.858, 0.859, and 0.920 to detect early-stage, borderline, non-epithelial diseases and 0.899 to discriminate ovarian cancer from endometriosis. TEPs had robustness, compatibility, and universality for preoperative diagnosis of ovarian cancer since it withstood validations in populations of different ethnicities, heterogeneous histological subtypes, and early-stage ovarian cancer. However, these observations warrant prospective validations in a larger population before clinical utilities.

Effect of muscle activation on dynamic responses of neck of pilot during emergency ejection: a finite element study.

To determine the effect of muscle activation on the dynamic responses of the neck of a pilot during simulated emergency ejections. A complete finite element model of the pilot's head and neck was developed and dynamically validated. Three muscle activation curves were designed to simulate different activation times and levels of muscles during pilot ejection: A is the unconscious activation curve of the neck muscles, B is the pre-activation curve, and C is the continuous activation curve. The acceleration-time curves obtained during ejection were applied to the model, and the influence of the muscles on the dynamic responses of the neck was investigated by analyzing both angles of rotation of the neck segments and disc stresses. Muscle pre-activation reduced fluctuations in the angle of rotation in each phase of the neck. Continuous muscle activation caused a 20% increase in the angle of rotation compared to pre-activation. Moreover, it resulted in a 35% increase in the load on the intervertebral disc. The maximum stress on the disc occurred in the C4-C5 phase. Continuous muscle activation increased both the axial load on the neck and the posterior extension angle of rotation of the neck. Muscle pre-activation during emergency ejection has a protective effect on the neck. However, continuous muscle activation increases the axial load and rotation angle of the neck. A complete finite element model of the pilot's head and neck was established and three neck muscle activation curves were designed to investigate the effects of muscle activation time and level on the dynamic response of the pilot's neck during ejection. This increased insights into the protection mechanism of neck muscles on the axial impact injury of the pilot's head and neck.

An optical clearing imaging window: Realization of mouse brain imaging and manipulation through scalp and skull.

Cortical visualization is essential to understand the dynamic changes in brain microenvironment under physiopathological conditions. However, the turbid scalp and skull severely limit the imaging depth and resolution. Existing cranial windows require invasive scalp excision and various subsequent skull treatments. Non-invasive in vivo imaging of skull bone marrow, meninges, and cortex through scalp and skull with high resolution yet remains a challenge. In this work, a non-invasive trans-scalp/skull optical clearing imaging window is proposed for cortical and calvarial imaging, which is achieved by applying a novel skin optical clearing reagent. The imaging depth and resolution are greatly enhanced in near infrared imaging and optical coherence tomography imaging. Combining this imaging window with adaptive optics, we achieve the visualization and manipulation of the calvarial and cortical microenvironment through the scalp and skull using two-photon imaging for the first time. Our method provides a well-performed imaging window and paves the way for intravital brain studies with the advantages of easy-operation, convenience and non-invasiveness.

Exploring the growth trait molecular markers in two sheep breeds based on Genome-wide association analysis.

Growth traits are quantitative traits controlled by multiple micro-effect genes. we identified molecular markers related to sheep growth traits, which formed the basis of molecular breeding. In this study, we randomly selected 100 Qira Black sheep and 84 German Merino sheep for the blood collection the jugular vein to genotype by using the Illumina Ovine SNP 50K Bead Chip. quality control criteria for statistical analysis were: rejection detection rate < 90% and minimum allele frequency (MAF) < 5%. Then, we performed Genome-wide association studies (GWAS) on sheep body weight, body height, body length, and chest circumference using mixed linear models. After getting 55 SNPs with significant correlation, they were annotated by reference genome of Ovis aries genome (Oar_v4.0) and We obtained a total of 84 candidate genes associated with production traits (BMPR1B, HSD17B3, TMEM63C, etc.). We selected BMPR1B for population validation and found a correlation between the FecB locus and body weight traits. Therefore, this study not only supplements the existing knowledge of molecular markers of sheep growth traits, but also has important theoretical significance and reference value for the mining of functional genes of sheep growth traits.

Introduction of the FecGF mutation in GDF9 gene via CRISPR/Cas9 system with single-stranded oligodeoxynucleotide.

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) 9 system has been a recent focus of breeders owing to its potential to improve economically significant traits of livestock. The introduction of defined point mutations into the ovine genome via CRISPR/Cas9-mediated homology-directed repair has been reported; however, indel and mosaic events observed in genetically modified animals limit the practical application of this system in sheep breeding. The FecGF mutation (g. G1111A, p. V371 M) in the growth differentiation factor 9 (GDF9) gene is strongly associated with litter size in Belclare and Norwegian White Sheep. In the present study, we introduced the FecGF mutation in GDF9 by co-injecting the CRISPR/Cas9 system, single-stranded oligodeoxynucleotide (ssODN), and Scr7 into ovine zygotes. Scr7 at various concentrations (0 µM, 1 µM, and 2 µM) had no adverse effects on embryonic development in vitro. No significant differences in total mutation, point mutation, and indel rates in embryos were observed among groups treated with different concentrations of Scr7. However, the mosaicism rates of embryos from zygotes microinjected with 1 and 2 µM Scr7 were significantly lower than that for 0 µM Scr7 (7.7% and 7.5% vs. 19.7%). We successfully obtained lambs with defined nucleotide substitutions by the coinjection of Cas9 mRNA, sgRNA, ssODN, and 1 µM Scr7 into Altay sheep zygotes. The single nucleotide mutation efficiency was 7.69% (3/39) in newborn lambs, with one mosaic. Our findings provide evidence that Scr7 could improve the specificity of the CRISPR/Cas9 system for the introduction of a defined point mutation in livestock to some extent.

GSCA: an integrated platform for gene set cancer analysis at genomic, pharmacogenomic and immunogenomic levels.

Cancer initiation and progression are likely caused by the dysregulation of biological pathways. Gene set analysis (GSA) could improve the signal-to-noise ratio and identify potential biological insights on the gene set level. However, platforms exploring cancer multi-omics data using GSA methods are lacking. In this study, we upgraded our GSCALite to GSCA (gene set cancer analysis, http://bioinfo.life.hust.edu.cn/GSCA) for cancer GSA at genomic, pharmacogenomic and immunogenomic levels. In this improved GSCA, we integrated expression, mutation, drug sensitivity and clinical data from four public data sources for 33 cancer types. We introduced useful features to GSCA, including associations between immune infiltration with gene expression and genomic variations, and associations between gene set expression/mutation and clinical outcomes. GSCA has four main functional modules for cancer GSA to explore, analyze and visualize expression, genomic variations, tumor immune infiltration, drug sensitivity and their associations with clinical outcomes. We used case studies of three gene sets: (i) seven cell cycle genes, (ii) tumor suppressor genes of PI3K pathway and (iii) oncogenes of PI3K pathway to prove the advantage of GSCA over single gene analysis. We found novel associations of gene set expression and mutation with clinical outcomes in different cancer types on gene set level, while on single gene analysis level, they are not significant associations. In conclusion, GSCA is a user-friendly web server and a useful resource for conducting hypothesis tests by using GSA methods at genomic, pharmacogenomic and immunogenomic levels.

LOF variants identifying candidate genes of laterality defects patients with congenital heart disease.

Defects in laterality pattern can result in abnormal positioning of the internal organs during the early stages of embryogenesis, as manifested in heterotaxy syndrome and situs inversus, while laterality defects account for 3~7% of all congenital heart defects (CHDs). However, the pathogenic mechanism underlying most laterality defects remains unknown. In this study, we recruited 70 laterality defect patients with CHDs to identify candidate disease genes by exome sequencing. We then evaluated rare, loss-of-function (LOF) variants, identifying candidates by referring to previous literature. We chose TRIP11, DNHD1, CFAP74, and EGR4 as candidates from 776 LOF variants that met the initial screening criteria. After the variants-to-gene mapping, we performed function research on these candidate genes. The expression patterns and functions of these four candidate genes were studied by whole-mount in situ hybridization, gene knockdown, and gene rescue methods in zebrafish models. Among the four genes, trip11, dnhd1, and cfap74 morphant zebrafish displayed abnormalities in both cardiac looping and expression patterns of early signaling molecules, suggesting that these genes play important roles in the establishment of laterality patterns. Furthermore, we performed immunostaining and high-speed cilia video microscopy to investigate Kupffer's vesicle organogenesis and ciliogenesis of morphant zebrafish. Impairments of Kupffer's vesicle organogenesis or ciliogenesis were found in trip11, dnhd1, and cfap74 morphant zebrafish, which revealed the possible pathogenic mechanism of their LOF variants in laterality defects. These results highlight the importance of rare, LOF variants in identifying disease-related genes and identifying new roles for TRIP11, DNHD1, and CFAP74 in left-right patterning. Additionally, these findings are consistent with the complex genetics of laterality defects.

Multi-omics characterization of autophagy-related molecular features for therapeutic targeting of autophagy.

Autophagy is a major contributor to anti-cancer therapy resistance. Many efforts have been made to understand and overcome autophagy-mediated therapy resistance, but these efforts have been unsuccessful in clinical applications. In this study, we establish an autophagy signature to estimate tumor autophagy status. We then classify approximately 10,000 tumor samples across 33 cancer types from The Cancer Genome Atlas into autophagy score-high and autophagy score-low groups. We characterize the associations between multi-dimensional molecular features and tumor autophagy, and further analyse the effects of autophagy status on drug response. In contrast to the conventional view that the induction of autophagy serves as a key resistance mechanism during cancer therapy, our analysis reveals that autophagy induction may also sensitize cancer cells to anti-cancer drugs. We further experimentally validate this phenomenon for several anti-cancer drugs in vitro and in vivo, and reveal that autophagy inducers potentially sensitizes tumor cells to etoposide through downregulating the expression level of DDIT4. Our study provides a comprehensive landscape of molecular alterations associated with tumor autophagy and highlights an opportunity to leverage multi-omics analysis to utilize multiple drug sensitivity induced by autophagy.

Peroxisome Proliferator FpPEX11 Is Involved in the Development and Pathogenicity in Fusarium pseudograminearum.

Fusarium crown rot (FCR) of wheat, an important soil-borne disease, presents a worsening trend year by year, posing a significant threat to wheat production. Fusarium pseudograminearum cv. b was reported to be the dominant pathogen of FCR in China. Peroxisomes are single-membrane organelles in eukaryotes that are involved in many important biochemical metabolic processes, including fatty acid ß-oxidation. PEX11 is important proteins in peroxisome proliferation, while less is known in the fungus F. pseudograminearum. The functions of FpPEX11a, FpPEX11b, and FpPEX11c in F. pseudograminearum were studied using reverse genetics, and the results showed that FpPEX11a and FpPEX11b are involved in the regulation of vegetative growth and asexual reproduction. After deleting FpPEX11a and FpPEX11b, cell wall integrity was impaired, cellular metabolism processes including active oxygen metabolism and fatty acid ß-oxidation were significantly blocked, and the production ability of deoxynivalenol (DON) decreased. In addition, the deletion of genes of FpPEX11a and FpPEX11b revealed a strongly decreased expression level of peroxisome-proliferation-associated genes and DON-synthesis-related genes. However, deletion of FpPEX11c did not significantly affect these metabolic processes. Deletion of the three protein-coding genes resulted in reduced pathogenicity of F. pseudograminearum. In summary, FpPEX11a and FpPEX11b play crucial roles in the growth and development, asexual reproduction, pathogenicity, active oxygen accumulation, and fatty acid utilization in F. pseudograminearum.

FgLEU1 Is Involved in Leucine Biosynthesis, Sexual Reproduction, and Full Virulence in Fusarium graminearum.

Fusarium head blight (FHB) caused by Fusarium graminearum is a significant disease among cereal crops. In F. graminearum, biosynthesis of leucine, which is a branched chain amino acid, is achieved by converting α-isopropylmalate to ß-isopropylmalate catalyzed by isopropylmalate isomerase encoded by LEU1. Considering the potential for targeting this pathway by fungicides, we characterized the gene FgLEU1 (FGSG-09589) in the Fusarium graminearum genome using bioinformatics methods. For functional characterization, we constructed a deletion mutant of FgLEU1 (ΔLEU1) through homologous recombination. Compared with the wild-type strain PH-1, ΔLEU1 showed slower colony growth and fewer aerial mycelia. Leucine addition was needed to ensure proper mutant growth. Further, ΔLEU1 showed decreased conidial production and germination rates, and could not produce ascospores. Moreover, ΔLEU1 showed complete loss of pathogenicity and reduced ability to produce deoxynivalenol (DON) and aurofusarin. Upstream and downstream genes of FgLEU1 were significantly upregulated in ΔLEU1. Contrary to previous reports, the deletion mutant was more resistant to osmotic stress and cell wall-damaging agents than the wild-type. Taken together, FgLEU1 plays a crucial role in leucine synthesis, aerial mycelial growth, sexual and asexual reproduction, pathogenicity, virulence, and pigmentation in Fusarium graminearum, indicating its potential as a target for novel antifungal agents.

Biological Pathway-Derived TMB Robustly Predicts the Outcome of Immune Checkpoint Blockade Therapy.

Although immune checkpoint blockade (ICB) therapies have achieved great progress, the patient response varies among cancers. In this study, we analyzed the potential genomic indicators contributing to ICB therapy response. The results showed that high tumor mutation burden (TMB) failed to predict response in anti-PD1 treated melanoma. SERPINB3 was the most significant response-related gene in melanoma and mutations in either SERPINB3 or PEG3 can serve as an independent risk factor in melanoma. Some recurrent mutations in CSMD3 were only in responders or non-responders, indicating their diverse impacts on patient response. Enrichment scores (ES) of gene mutations in 12 biological pathways were significantly higher in responders or non-responders. Next, the P-TMB calculated from genes in these pathways was significantly related to patient response with prediction AUC 0.74-0.82 in all collected datasets. In conclusion, our work provides new insights into the application of TMB in predicting patient response, which will benefit to immunotherapy research.