Single-molecule optical mapping enables quantitative measurement of D4Z4 repeats in facioscapulohumeral muscular dystrophy (FSHD).

PURPOSE: Facioscapulohumeral muscular dystrophy (FSHD) is a common adult muscular dystrophy. Over 95% of FSHD cases are associated with contraction of the D4Z4 tandem repeat (~3.3 kb per unit) at 4q35 with a specific genomic configuration (haplotype) called 4qA. Molecular diagnosis of FSHD typically requires pulsed-field gel electrophoresis with Southern blotting. We aim to develop novel genomic and computational methods for characterising D4Z4 repeat numbers in FSHD. METHODS: We leveraged a single-molecule optical mapping platform that maps locations of restriction enzyme sites on high molecular weight (>150 kb) DNA molecules. We developed bioinformatics methods to address several challenges, including the differentiation of 4qA with 4qB alleles, the differentiation of 4q35 and 10q26 segmental duplications, the quantification of repeat numbers with different enzymes that may or may not have recognition sites within D4Z4 repeats. We evaluated the method on 25 human subjects (13 patients, 3 individual control subjects, 9 control subjects from 3 families) labelled by the Nb.BssSI and/or Nt.BspQI enzymes. RESULTS: We demonstrated that the method gave a direct quantitative measurement of repeat numbers on D4Z4 repeats with 4qA allelic configuration and the levels of postzygotic mosaicism. Our method had high concordance with Southern blots from several cohorts on two platforms (Bionano Saphyr and Bionano Irys), but with improved quantification of repeat numbers. CONCLUSION: While the study is limited by small sample size, our results demonstrated that single-molecule optical mapping is a viable approach for more refined analysis on genotype-phenotype relationships in FSHD, especially when postzygotic mosaicism is present.

Using Secure Multi-Party Computation to Protect Privacy on a Permissioned Blockchain.

The development of information technology has brought great convenience to our lives, but at the same time, the unfairness and privacy issues brought about by traditional centralized systems cannot be ignored. Blockchain is a peer-to-peer and decentralized ledger technology that has the characteristics of transparency, consistency, traceability and fairness, but it reveals private information in some scenarios. Secure multi-party computation (MPC) guarantees enhanced privacy and correctness, so many researchers have been trying to combine secure MPC with blockchain to deal with privacy and trust issues. In this paper, we used homomorphic encryption, secret sharing and zero-knowledge proofs to construct a publicly verifiable secure MPC protocol consisting of two parts-an on-chain computation phase and an off-chain preprocessing phase-and we integrated the protocol as part of the chaincode in Hyperledger Fabric to protect the privacy of transaction data. Experiments showed that our solution performed well on a permissioned blockchain. Most of the time taken to complete the protocol was spent on communication, so the performance has a great deal of room to grow.

Somatic variants in new candidate genes identified in focal cortical dysplasia type II.

OBJECTIVE: Focal cortical dysplasia type II (FCDII) is a malformation of cortex development commonly found in children with drug-resistant epilepsy. FCDII has been associated with somatic mutations in mammalian target of rapamycin (mTOR)-related pathway genes and an upregulation of mTOR. Somatic mutations were found in 10%-63% of FCDII samples; the frequency of the mutant allele was 0.93%-33.5%. This study aimed to find new candidate genes involved in FCDII. METHODS: We collected resected FCD lesions, perilesional brain tissues, and peripheral blood from 17 children with pathologically confirmed FCDII. We performed whole exome sequencing and followed a set of screening and analysis strategies to identify potentially deleterious somatic variants (PDSVs) in brain-expressed genes. We performed site-specific amplicon sequencing to validate the results. We also performed an in vitro functional study on an IRS1 variant. RESULTS: In six of 17 samples, we identified seven PDSVs in seven genes, including two frameshift variants and five missense variants. The frequencies of the variant allele were 1.29%-5.50%. The genes were MTOR, TSC2, IRS1, RAB6B, RALA, HTR6, and ZNF337. PDSVs in IRS1, RAB6B, ZNF337, RALA, and HTR6 had not been previously associated with FCD. In one lesion, two PDSVs were found in two genes. In a transfected cell line, we demonstrated that the c.1791dupG (identified in FCDII from Patient 1) led to a truncated IRS1 and significant mTOR hyperactivation compared to cells that carried wild-type IRS1. mTOR was also activated in FCDII tissue from Patient 1. SIGNIFICANCE: Seven PDSVs were identified in FCDII lesions in six of 17 children. Five variant genes had not been previously associated with cortical malformations. We demonstrated that the IRS1 variant led to mTOR hyperactivation in vitro. Although functional experiments are needed, the results provide evidence for novel candidate genes in the pathogenesis of FCDII.

A Prediction Model of Essential Hypertension Based on Genetic and Environmental Risk Factors in Northern Han Chinese.

Background: Essential hypertension (EH) is a chronic disease of universal high prevalence and a well-established independent risk factor for cardiovascular and cerebrovascular events. The regulation of blood pressure is crucial for improving life quality and prognoses in patients with EH. Therefore, it is of important clinical significance to develop prediction models to recognize individuals with high risk for EH. Methods: In total, 965 subjects were recruited. Clinical parameters and genetic information, namely EH related SNPs were collected for each individual. Traditional statistic methods such as t-test, chi-square test and multi-variable logistic regression were applied to analyze baseline information. A machine learning method, mainly support vector machine (SVM), was adopted for the development of the present prediction models for EH. Results: Two models were constructed for prediction of systolic blood pressure (SBP) and diastolic blood pressure (DBP), respectively. The model for SBP consists of 6 environmental factors (age, BMI, waist circumference, exercise [times per week], parental history of hypertension [either or both]) and 1 SNP (rs7305099); model for DBP consists of 6 environmental factors (weight, drinking, exercise [times per week], TG, parental history of hypertension [either and both]) and 3 SNPs (rs5193, rs7305099, rs3889728). AUC are 0.673 and 0.817 for SBP and DBP model, respectively. Conclusions: The present study identified environmental and genetic risk factors for EH in northern Han Chinese population and constructed prediction models for SBP and DBP.

Fast Radio Map Construction by using Adaptive Path Loss Model Interpolation in Large-Scale Building.

The radio map construction is usually time-consuming and labor-sensitive in indoor fingerprinting localization. We propose a fast construction method by using an adaptive path loss model interpolation. Received signal strength (RSS) fingerprints are collected at sparse reference points by using multiple smartphones based on crowdsourcing. Then, the path loss model of an access point (AP) can be built with several reference points by the least squares method in a small area. Afterwards, the RSS value can be calculated based on the constructed model and corresponding AP's location. In the small area, all models of detectable APs can be built. The corresponding RSS values can be estimated at each interpolated point for forming the interpolated fingerprints considering RSS loss, RSS noise and RSS threshold. Through combining all interpolated and sparse reference fingerprints, the radio map of the whole area can be obtained. Experiments are conducted in corridors with a length of 211 m. To evaluate the performance of RSS estimation and positioning accuracy, inverse distance weighted and Kriging interpolation methods are introduced for comparing with the proposed method. Experimental results show that our proposed method can achieve the same positioning accuracy as complete manual radio map even with the interval of 9.6 m, reducing 85% efforts and time of construction.

Long-read sequencing identified a causal structural variant in an exome-negative case and enabled preimplantation genetic diagnosis.

BACKGROUND: For a proportion of individuals judged clinically to have a recessive Mendelian disease, only one heterozygous pathogenic variant can be found from clinical whole exome sequencing (WES), posing a challenge to genetic diagnosis and genetic counseling. One possible reason is the limited ability to detect disease causal structural variants (SVs) from short reads sequencing technologies. Long reads sequencing can produce longer reads (typically 1000 bp or longer), therefore offering greatly improved ability to detect SVs that may be missed by short-read sequencing. RESULTS: Here we describe a case study, where WES identified only one heterozygous pathogenic variant for an individual suspected to have glycogen storage disease type Ia (GSD-Ia), which is an autosomal recessive disease caused by bi-allelic mutations in the G6PC gene. Through Nanopore long-read whole-genome sequencing, we identified a 7.1 kb deletion covering two exons on the other allele, suggesting that complex structural variants (SVs) may explain a fraction of cases when the second pathogenic allele is missing from WES on recessive diseases. Both breakpoints of the deletion are within Alu elements, and we designed Sanger sequencing and quantitative PCR assays based on the breakpoints for preimplantation genetic diagnosis (PGD) for the family planning on another child. Four embryos were obtained after in vitro fertilization (IVF), and an embryo without deletion in G6PC was transplanted after PGD and was confirmed by prenatal diagnosis, postnatal diagnosis, and subsequent lack of disease symptoms after birth. CONCLUSIONS: In summary, we present one of the first examples of using long-read sequencing to identify causal yet complex SVs in exome-negative patients, which subsequently enabled successful personalized PGD.

DMDtoolkit: a tool for visualizing the mutated dystrophin protein and predicting the clinical severity in DMD.

BACKGROUND: Dystrophinopathy is one of the most common human monogenic diseases which results in Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). Mutations in the dystrophin gene are responsible for both DMD and BMD. However, the clinical phenotypes and treatments are quite different in these two muscular dystrophies. Since early diagnosis and treatment results in better clinical outcome in DMD it is essential to establish accurate early diagnosis of DMD to allow efficient management. Previously, the reading-frame rule was used to predict DMD versus BMD. However, there are limitations using this traditional tool. Here, we report a novel molecular method to improve the accuracy of predicting clinical phenotypes in dystrophinopathy. We utilized several additional molecular genetic rules or patterns such as "ambush hypothesis", "hidden stop codons" and "exonic splicing enhancer (ESE)" to predict the expressed clinical phenotypes as DMD versus BMD. RESULTS: A computer software "DMDtoolkit" was developed to visualize the structure and to predict the functional changes of mutated dystrophin protein. It also assists statistical prediction for clinical phenotypes. Using the DMDtoolkit we showed that the accuracy of predicting DMD versus BMD raised about 3% in all types of dystrophin mutations when compared with previous methods. We performed statistical analyses using correlation coefficients, regression coefficients, pedigree graphs, histograms, scatter plots with trend lines, and stem and leaf plots. CONCLUSIONS: We present a novel DMDtoolkit, to improve the accuracy of clinical diagnosis for DMD/BMD. This computer program allows automatic and comprehensive identification of clinical risk and allowing them the benefit of early medication treatments. DMDtoolkit is implemented in Perl and R under the GNU license. This resource is freely available at http://github.com/zhoujp111/DMDtoolkit , and http://www.dmd-registry.com .

Transcriptome sequencing of HER2-positive breast cancer stem cells identifies potential prognostic marker.

In cancer stem cell theory, breast cancer stem cells (BCSCs) are postulated to be the root cause of recurrence and metastasis in breast cancer. Discovery of new biomarkers and development of BCSC-targeted therapy are practical issues that urgently need to be addressed in the clinic. However, few breast cancer stem cell targets are known. Given that there are few BCSCs, performing transcriptome sequencing on them thus far has not been possible. With the emergence of single-cell sequencing technology, we have now undertaken such a study. We prepared single-cell suspensions, which were sorted using flow cytometry from breast tumor tissue and adjacent normal breast tissue from two HER2-positive patients. We obtained BCSCs, breast cancer cells, mammary cells, and CD44+ mammary cells. Transcriptome sequencing was then performed on these four cell types. Using bioinformatics, we identified 404 differentially expressed BCSC genes from the HER2-positive tumors and preliminary explored transcriptome characteristics of BCSCs. Finally, by querying a public database, we found that CA12 was a novel prognostic biomarker in HER2-positive breast cancer, which also had prognostic value in all breast cancer types. In conclusion, our results suggest that CA12 may be associated with BCSCs, especially HER2-positive BCSCs, and is a potential novel therapeutic target and biomarker.

Knockdown of p62/sequestosome 1 attenuates autophagy and inhibits colorectal cancer cell growth.

p62/sequestosome-1 is a multifunctional adapter protein implicated in selective autophagy, cell signaling pathways, and tumorigenesis, and plays an important role at the crossroad between autophagy and cancer. But, the connection between autophagy and cancer is complex and in some cases contradictory. Human colorectal cancer tissues from patients were analyzed for expression of p62 and Microtubule-associated protein light chain 3 (LC3, an autophagosome marker) using immunostaining, western blotting, real-time PCR, and confocal microscopy. To study the effects of p62 on autophagy and cell growth, shRNA for p62 was applied and cell growth curve was monitored in human colorectal cancer cell. In vivo experiments were done using the mouse xenograft model. We showed that up-regulated expression of p62 and LC3 in colorectal cancer tissues. We also demonstrated that specifically knockdown the expression of p62 showed significantly inhibitory effects not only on autophagy activation, but also on tumor growth both in vitro and xenograft tumors model. The ectopic overexpression of p62 and autophagy activation contributes to colorectal tumorigenesis. p62 and autophagy will be therapy targets for the treatment of colorectal cancer.

[Strategies of genome-wide association study based on high-throughput sequencing].

Genome-wide association studies (GWASs) have been playing an important role on human complex diseases. Generally speaking, GWAS tries to detect the relationship between genome-wide genetic variants and measurable traits in the population level. Although fruitful, array-based GWASs still exist some problems, for example, the so-called missing heritability--significantly associated SNPs can only explain a small part of phenotypic variation. Other problems include that, in some traits, significantly associated SNPs in one study are hard to be repeated by other studies; and that the functions of significantly associated SNPs are often difficult to interpret. High-throughput sequencing, also known as next-generation sequencing (NGS), could be one of the most promising technologies to solve those problems by quickly producing accurate variations in a high-throughput way. NGS-based GWASs (NGS-GWAS), to some extent, provide a better solution compared with traditional array-based GWASs. We systematically review the strategies and methods for NGS-GWASs, pick out the most feasible and efficient strategies and methods for NGS-GWASs, and discuss their applications in personalized medicine.

Factors influencing recurrence and model development for recurrence of minimally invasive percutaneous transhepatic lithotripsy: a single-center retrospective study.

OBJECTIVE: To identify factors influencing recurrence after percutaneous transhepatic choledochoscopic lithotripsy (PTCSL) and to develop a predictive model. METHODS: We retrospectively analyzed clinical data from 354 patients with intrahepatic and extrahepatic bile duct stones treated with PTCSL at Qinzhou First People's Hospital between February 2018 and January 2020. Patients were followed for three years and categorized into non-recurrence and recurrence groups based on postoperative outcome. Univariate analysis identified possible predictors of stone recurrence. Data were split using the gradient boosting machine (GBM) algorithm, assigning 70% as the training set and 30% as the test set. The predictive performance of the GBM model was assessed using the receiver operating characteristic (ROC) curve and calibration curve, and compared with a logistic regression model. RESULTS: Six factors were identified as significant predictors of recurrence: age, diabetes, total bilirubin, biliary stricture, number of stones, and stone diameter. The GBM model, developed based on these factors, showed high predictive accuracy. The area under the ROC curve (AUC) was 0.763 (95% CI: 0.695-0.830) for the training set and 0.709 (95% CI: 0.596-0.822) for the test set. Optimal cutoff values were 0.286 and 0.264, with sensitivities of 62.30% and 66.70%, and specificities of 77.20% and 68.50%, respectively. Calibration curves indicated good agreement between predicted probabilities and observed recurrence rates in both sets. DeLong's test revealed no significant differences between the GBM and logistic regression models in predictive performance (training set: D = 0.003, P = 0.997 > 0.05; test set: D = 0.075, P = 0.940 > 0.05). CONCLUSION: Biliary stricture, stone diameter, diabetes, stone number, age, and total bilirubin significantly influence stone recurrence after PTCSL. The GBM model, based on these factors, demonstrates robust accuracy and discrimination. Both GBM and logistic regression models effectively predicted stone recurrence post-PTCSL.

Restoration of klotho gene expression induces apoptosis and autophagy in gastric cancer cells: tumor suppressive role of klotho in gastric cancer.

BACKGROUND: The loss of tumor suppressor gene expression is involved in the carcinogenesis of gastric cancer (GC). Klotho is a recently identified tumor suppressor gene that epigenetically inactivated in gastric cancer. However, the signaling pathways involved in the suppressive role of klotho have rarely been reported in gastric cancer. In this study, we investigated the involvement of klotho in gastric cancer cell proliferation, apoptosis, and autophagy as well as the associated signaling. METHODS: Methylation of klotho gene promoter in GC-7901, MNK-45 and AGS gastric cancer cells as well as GES-1 normal gastric epithelial cells was detected by bisulfate-based PCR. Restoration of klotho gene expression was established by applying a demethylating agent and delivering aklotho gene expression vector into GC-7901 cells. Cell viability was measured by CCK-8 assay. Cell apoptosis and cycling were analyzed by flow cytometry. Autophagy was measured by detecting LC3-I and LC3-II expression. Protein levels and phosphorylation were measured by Western blot assay. RESULTS: Methylation of klotho gene promoter and expression of the klotho gene were detected in GC cells. Restoration of klotho gene expression significantly inhibited cell proliferation, induced cell apoptosis, and increased LC3-I/LC3-II expression in GC cells. Restoration of klotho gene expression downregulated the phosphorylation levels of IGF-1 receptor, IRS-1, PI3K, Akt, and mTOR proteins. Both apoptosis and autophagy inhibitors blocked klotho-induced apoptosis and autophagy. CONCLUSION: Klotho is a tumor suppressor in gastric cancer, which regulates IGF-1R phosphorylation and the subsequent activation of IRS-1/PI3K/Akt/mTOR signaling, tumor cell proliferation, apoptosis, and autophagy.

Adenosine triphosphate can act as a determinant of lysine acetylation of non-native and native substrates.

The protein lysine acetylation includes acetyl-CoA (AcCoA) or acetyl phosphate (AcP)-mediated nonenzymatic acetylation, and enzymatic acetylation. It is widespread in the proteomes but the acetylation levels of most sites are very low. A thorough understanding of the determinants of low acetylation levels is highly important for elucidating the physiological relevance of lysine acetylation. In this study, we constructed a non-native substrate library containing 24 synthesized polypeptides, and we showed that ATP could inhibit the AcCoA-mediated nonenzymatic acetylation of these polypeptides through LC-MS/MS analysis. The acetyltransferase PatZ could acetylated these non-native substrates, and the PatZ-catalyzed acetylation of the polypeptides was also inhibited by ATP. Furthermore, the Western blot showed that ATP also inhibited the nonenzymatic (AcCoA or AcP-mediated) and enzymatic (PatZ-catalyzed) acetylation of acetyl-CoA synthetase Acs, which is a native substrate for acetylation. ATP can also inhibit the autoacetylation of acetyltransferase PatZ. Besides, both ADP and AMP could enhance the AcP-mediated acetylation of Acs, but ADP slightly inhibited the AcCoA-mediated acetylation of Acs. However, both ADP and AMP had no evident inhibition on the PatZ-catalyzed acetylation of Acs. Based on these results, we proposed that ATP can act as an inhibitor of acetylation, and it may regulate the function of PatZ by inhibiting its autoacetylation and compensate for the function of deacetylase CobB.

Genome-wide association study for T lymphocyte subpopulations in swine.

BACKGROUND: Lymphocytes act as a major component of the adaptive immune system, taking very crucial responsibility for immunity. Differences in proportions of T-cell subpopulations in peripheral blood among individuals under same conditions provide evidence of genetic control on these traits, but little is known about the genetic mechanism of them, especially in swine. Identification of the genetic control on these variants may help the genetic improvement of immune capacity through selection. RESULTS: To identify genomic regions responsible for these immune traits in swine, a genome-wide association study was conducted. A total of 675 pigs of three breeds were involved in the study. At 21 days of age, all individuals were vaccinated with modified live classical swine fever vaccine. Blood samples were collected when the piglets were 20 and 35 days of age, respectively. Seven traits, including the proportions of CD4+, CD8+, CD4+CD8+, CD4+CD8-, CD4-CD8+, CD4-CD8- and the ratio of CD4+ to CD8+ T cells were measured at the two ages. All the samples were genotyped for 62,163 single nucleotide polymorphisms (SNP) using the Illumina porcineSNP60k BeadChip. 40833 SNPs were selected after quality control for association tests between SNPs and each immune trait considered based on a single-locus regression model. To tackle the issue of multiple testing in GWAS, 10,000 permutations were performed to determine the chromosome-wise and genome-wise significance levels of association tests. In total, 61 SNPs with chromosome-wise significance level and 3 SNPs with genome-wise significance level were identified. 27 significant SNPs were located within the immune-related QTL regions reported in previous studies. Furthermore, several significant SNPs fell into the regions harboring known immunity-related genes, 14 of them fell into the regions which harbor some known T cell-related genes. CONCLUSIONS: Our study demonstrated that genome-wide association studies would be a feasible way for revealing the potential genetics variants affecting T-cell subpopulations. Results herein lay a preliminary foundation for further identifying the causal mutations underlying swine immune capacity in follow-up studies.

Novel Variant Expands the Clinical Spectrum of CUX2-Associated Developmental and Epileptic Encephalopathies.

Developmental and epileptic encephalopathies (DEE) caused by heterozygous deleterious variants in Cut Like Homeobox2 (CUX2) is rare. To the best of our knowledge the only variant associated with a phenotype in this gene is the de novo missense variant c.1768G > A, p.Glu590Lys; however, further additional research is needed to characterize the relationship between disease and variants in this gene. In this study, we reported a patient from a non-consanguineous Chinese family presenting with epilepsy, developmental delay, and speech delay. Additionally, the patient responded well to levetiracetam, and at his last follow-up (5.5 years old), he had discontinued antiepileptic drug treatment and remained seizure-free for 6 months. To identify possible causative variants, trio-whole exome sequencing was performed. We identified a novel de novo missense CUX2 c.2834C > T, p. Thr945Met variant in the patient. Based on clinical and genetics information associated with the bioinformatics analyses, we hypothesized that this variant was the cause of the reported phenotype. AlphaFold and SWISS-MODEL homology modeling servers were used to predict the three-dimensional (3D) structure of CUX2 protein. Predictions based on the 3D-structure modeling indicated that the p.Thr945Met substitution was likely to alter the DNA-binding specificities and affect protein function. On the basis of clinical characteristics and genetic analysis, we presented one case diagnosed with DEE67. Our finding expanded the clinical and molecular spectrum of CUX2 variants.

LINC01915 Facilitates the Conversion of Normal Fibroblasts into Cancer-Associated Fibroblasts Induced by Colorectal Cancer-Derived Extracellular Vesicles through the miR-92a-3p/KLF4/CH25H Axis.

Increasing long non-coding RNAs are reported to regulate the cell growth, apoptosis, and metastasis of cancer-associated fibroblasts (CAFs).This study aimed to explore how LINC01915 influences the conversion of normal fibroblasts (NFs) into CAFs in colorectal cancer (CRC). LINC01915 expression was initially measured in clinical tissue samples and in NFs and CAFs. Identification of the interaction between LINC01915, miR-92a-3p, KLF4, and CH25H was done. The effects of LINC01915, miR-92a-3p, and KLF4 on the angiogenesis, extracellular vesicle (EV) uptake by NFs, and activation of stromal cells were assessed using gain- or loss-of-function approaches. Xenograft mouse models were established to validate these in vitro findings in vivo. EVs were shown to stimulate NF proliferation, migration, and angiogenesis, as well as facilitate NF conversion into CAFs. CRC tissues and CAFs showed downregulated expression of LINC01915, which was associated with poor prognosis of patients. Moreover, employed LINC01915 inhibited tumor angiogenesis, CAF activation, and the uptake of tumor-derived EVs by NFs. Mechanistically, LINC01915 could competitively bind to miR-92a-3p and caused upregulation of the miR-92a-3p target KLF4 which, in turn, promoted the transcription of CH25H, leading to the suppressed uptake of EVs by NFs. The in vivo and in vitro experimental results showed that LINC01915 inhibited the uptake of CRC-derived EVs by NFs through the miR-92a-3p/KLF4/CH25H axis, thus arresting the angiogenesis and the conversion of NFs into CAFs and in turn prevent tumor growth. These data together supported the inhibiting role of LINC01915 in the conversion of NFs into CAFs triggered by the CRC-derived EVs and the ensuing tumor growth, which may be related to its regulation on the miR-92a-3p/KLF4/CH25H axis.

Methylglyoxal Has Different Impacts on the Fungistatic Roles of Ammonia and Benzaldehyde, and Lactoylglutathione Lyase Is Necessary for the Resistance of Arthrobotrys oligospora to Soil Fungistasis.

Biocontrol of root-knot nematode has attracted increasing attention over the past two decades. The inconsistent field performance of biocontrol agents, which is caused by soil fungistasis, often restricts their commercial application. There is still a lack of research on the genes involved in biocontrol fungi response to soil fungistasis, which is important for optimizing practical applications of biocontrol fungi. In this study, the lactoylglutathione lyase-encoding AOL_s00004g335 in the nematophagous fungi Arthrobotrys oligospora was knocked out, and three mutant strains were obtained. The hyphal growth of mutants on the three media was almost the same as that of the wild-type strain, but mutants had slightly higher resistance to NaCl, SDS, and H2O2. Methylglyoxal (MG) significantly increased the resistance of A. oligospora to ammonia, but decreased the resistance to benzaldehyde. Furthermore, the resistance of the mutants to soil fungistasis was largely weakened and MG could not increase the resistance of A. oligospora to soil fungistasis. Our results revealed that MG has different effects on the fungistatic roles of ammonia and benzaldehyde and that lactoylglutathione lyase is very important for A. oligospora to resist soil fungistasis.

Fungistatic Mechanism of Ammonia against Nematode-Trapping Fungus Arthrobotrys oligospora, and Strategy for This Fungus To Survive Ammonia.

Soil fungistasis is a phenomenon in which the germination and growth of fungal propagules is widely inhibited in soils. Although fungistatic compounds are known to play important roles in the formation of soil fungistasis, how such compounds act on soil fungi is little studied. In this study, it was found that ammonia (NH3) induced global protein misfolding marked by increased ubiquitination levels of proteins (ubiquitylome data and Western blot verification). The misfolded proteins should trigger the endoplasmic reticulum (ER) stress, which was indicated by electron microscope image and proteome data. Results from the mutants of BiP and proteasome subunit alpha 7 suggested that ER stress played a mechanistic role in inhibiting conidial germination. Results from proteome data indicated that, to survive ammonia fungistasis, conidia first activated the unfolded protein response (UPR) to decrease ER stress and restore ER protein homeostasis, and the function of UPR in surviving ammonia was confirmed by using mutant strains. Second, ammonia toxicity could be reduced by upregulating carbon metabolism-related proteins, which benefited ammonia fixation. The results that metabolites (especially glutamate) could relieve the ammonia fungistasis confirmed this indirectly. Finally, results from gene knockout mutants also suggested that the fungistatic mechanism of ammonia is common for soil fungistasis. This study increased our knowledge regarding the mechanism of soil fungistasis and provided potential new strategies for manipulating soil fungistasis. IMPORTANCE Soil fungistasis is a phenomenon in which the germination and growth of fungal propagules is widely inhibited in soil. Although fungistatic compounds are known to play important roles in the formation of soil fungistasis, how such compounds act on soil fungi remains little studied. This study revealed an endoplasmic reticulum stress-related fungistatic mechanism with which ammonia acts on Arthrobotrys oligospora and a survival strategy of conidia under ammonia inhibition. Our study provides the first mechanistic explanation of how ammonia impacts fungal spore germination, and the mechanism may be common for soil fungistasis. This study increases our knowledge regarding the mechanism of soil fungistasis in fungal spores and provides potential new strategies for manipulating soil fungistasis.

Genome-wide profiling of BK polyomavirus integration in bladder cancer of kidney transplant recipients reveals mechanisms of the integration at the nucleotide level.

Chronic BK polyomavirus (BKPyV) infection is recognized as a potential oncogenic factor of urothelial carcinoma (UC) in renal transplant recipients. Recent studies have reported a positive correlation among BKPyV integration, persistent overexpression of viral large T antigen (TAg), and malignancy, yet little is known about the specific integration mechanisms and the impacts of viral integration. Here, we performed whole-genome sequencing (WGS) and viral capture-based sequencing on high-grade immunohistochemically TAg-positive UCs in two renal transplant recipients. A total of 181 integration sites, including the three found by WGS, were identified by viral capture-based sequencing, indicating its enhanced sensitivity and ability in identifying low-read integration sites in subpopulations of the tumor cells. The microhomologies between human and BKPyV genomes were significantly enriched in the flanking regions of 84.5% the integration sites, with a median length of 7 bp. Notably, 75 human genes formed fusion sequences due to viral insertional integration. Among them, the expression of 15 genes were statistically associated with UC based on GEO2R expression analysis. Our results indicated a multisite and multifragment linear integration pattern and a potential microhomology or nonhomologous end joining integration mechanism at the single-nucleotide level. We put forward a potential selection mechanism driven by immunity and centered on viral integration in the carcinogenesis of BKPyV.

Pedicled ileal flap for duodenal defect after right hemicolectomy.

BACKGROUND/AIMS: To describe a convenient approach for repairing a large duodenal defect after right hemicolectomy for right side colon neoplasm. METHODOLOGY: Six patients with a large duodenal defect after right hemicolectomy with partial duodenectomy for right side colon neoplasm were treated with a pedicled ileal flap to cover the large duodenal defect. RESULTS: Six cases recovered smoothly. There was no perioperative morbidity and mortality in this series, except for one patient who had a duodenal leakage but who got well with medical treatment. Postoperative radiography showed normal duodenal peristalsis. CONCLUSIONS: The pedicled ileal flap procedure for repairing a large duodenal defect after right hemicolectomy is safe and convenient, and is especially indicated for elderly patients or patients with poor general condition.