Polymerase Chain Reaction Sequencing Research Articles

Clinical and molecular analysis of JCPyV and BKPyV infection and associated risk of urothelial carcinoma development in the upper tract

BackgroundHuman polyomaviruses (HPyVs), JC polyomavirus (JCPyV) and BK polyomavirus (BKPyV), have been found in upper tract urothelial carcinoma UTUC; however, the association of the viral oncogenic factors and clinical characteristics of UTUC remains unclear. This study aimed to investigate the prevalence of JCPyV and BKPyV in UTUC and their correlation with cancer progression among the southwest Taiwanese population from 2020 to 2022.MethodsA total of 72 paraffin-embedded UTUC tissue samples and 41 adjacent tissue samples were collected from 72 patients. Nested polymerase chain reaction and DNA sequencing were used to detect viral DNA and genotypes. Immunohistochemistry was performed using anti- large T (LT) and anti-p53 monoclonal antibodies to detect the expression of viral early LT protein and cellular p53 protein, respectively.ResultsThe overall prevalence of JCPyV and BKPyV were higher in UTUC than in adjacent tissue samples (65.3% [47/72] vs. 17.1% [7/41]). JCPyV and BKPyV were detected in 95.7% (45/47) and 4.3% (2/47) of the HPyVs-positive UTUC samples, respectively. JCPyV-TW-3 was the predominant strain of JCPyV infection. In UTUC samples, the LT protein of JCPyV and BKPyV positivity rate was 65.3%, while that of mutant p53 protein was 52.7%. JCPyV infection and LT protein expression increased the odds ratio (OR) of UTUC by 9.13-fold. The OR of UTUC was higher by 10.34-fold in patients with mutant p53 and by 10.37-fold in those with simultaneous LT and mutant p53 expression. The presence of LT protein in UTUC patients may increase the OR of mutant p53 protein expression by 2.93-fold compared to its absence. Women had a 5.19-fold higher superiority of JCPyV infection and LT expression than men. Patients with chronic kidney disease (CKD) had a 3.15-fold higher OR for mutant p53 protein expression than those without it. In the UTUC advanced stages, the OR of virus and LT expression was 3.18-fold higher compared to those who do not require chemotherapy.ConclusionsJCPyV infection is highly prevalent in UTUC, and the presence of CKD concurrent with high expressions of LT and mutant p53 proteins in patients may be a useful indicator for chemotherapy and poor prognosis.

Establishment and characterization of liver-specific Apoa4-Cre and Cyp2c11-Cre rat models in juvenile and adult stages.

Liver diseases are a major contributor to both morbidity and mortality. Conditional knockout animals are always produced through crossing floxed animals with a tissue-specific Cre animal. The use of floxed rat resource has rapidly increased, but the liver-specific Cre rat lines for studying liver diseases and interested genes are limited, especially in a spatially and temporally restricted manner. RNA sequencing and real-time polymerase chain reaction (PCR) were used to screen and confirm the presence of liver-specific genes. Apoa4-Cre rats and Cyp2c11-Cre rats were produced by CRISPR/Cas9 knockin. Rosa26-imCherry rats were employed to hybridize with the Cre rats to obtain the Apoa4-Cre/Rosa26-imCherry and Cyp2c11-Cre/Rosa26-imCherry rats. The temporal and spatial patterns of Cre expression were determined by the observation of red fluorescence on tissue sections. Hematoxylin-eosin stain was used to evaluate the liver histopathologic changes. The blood biochemical analysis of several liver enzymes and liver lipid profile was performed to evaluate the liver function of Cre rats. Apoa4 and Cyp2c11 were identified as two liver-specific genes. Apoa4-Cre and Cyp2c11-Cre rats were produced and hybridized with Rosa26-imCherry rats. The red fluorescence indicated that the Cre recombinases were specially expressed in the juvenile and adult liver and not in other organs of two hybridized rats. All the blood biochemical parameters except low-density lipoprotein (LDL) did not change significantly in the Cre rats. No histological alterations were detected in the livers of the Cre rats. Liver-specific Apoa4-Cre and Cyp2c11-Cre rats have been established successfully and could be used to study gene knockout, specifically in juvenile and adult liver.

Introduction of Panax notoginseng into pine forests significantly enhances the diversity, stochastic processes, and network complexity of nitrogen-fixing bacteria in the soil

IntroductionNitrogen-fixing bacteria (NFB) have a pivotal impact on the nitrogen cycle within agroforestry systems. The organic management of the Panax notoginseng (sanqi)-Pinus armandii agroforestry (SPA) system resulted in nitrogen deficiency because of the lack of application of chemical fertilizers. Therefore, assessing the variability in NFB due to the cultivation of sanqi in the SPA system becomes crucial.MethodsThe seasonal dynamics in the abundance, diversity, and community structure of NFB in the soil of monocropping pine (MP) and SPA systems were assessed using real-time quantitative polymerase chain reaction and high-throughput sequencing technology.Results and discussionSanqi cultivation triggered a decrease in the abundance of NFB but increased α diversity. Additionally, significant differences in the community structure of NFB were noted between the MP and SPA systems. Moreover, the abundance of Bradyrhizobium and Azospirillum increased in the soil after sanqi was cultivated. Furthermore, the cultivation of sanqi broadened the ecological niche breadth of NFB and increased the stochasticity in its community structure assembly (i.e., dispersal limitation). Additionally, the SPA system increased the network complexity but not the stability of NFB. The structural equation model (SEM) revealed that pH directly impacted the network complexity and stability of NFB in the SPA system. Sanqi cultivation positively influences the community characteristics of NFB in the soil in the SPA system. Our study provides new insights into nitrogen cycling and utilization in the SPA system.

Association study of the HLA class I system with psoriatic arthritis in Southern Tunisia: a case-control study.

Psoriatic arthritis (PsA) is a chronic inflammatory rheumatism belonging to the spondyloarthritis family. It is a multifactorial disease whose genetic determinism is still poorly understood. It is favored by environmental factors in genetically predisposed individuals. This study aims to investigate the association of HLA-A, HLA-B, and HLA-C alleles with PsA and its various manifestations in patients from Southern Tunisia. A case-control association study was conducted involving 48 PsA patients and 123 controls. HLA-A and HLA-B typing was performed using microlymphocytotoxicity complement-dependent assays, and HLA-C typing was done using polymerase chain reaction-sequence specific primer. Positive associations were confirmed for HLA-B27 and -C*06 alleles with PsA in our Southern Tunisian population with a more pronounced association in cases of -C*06 homozygosity. The HLA-B*27-C*02 and HLA-B*50-C*06 haplotypes were significantly more frequent in PsA patients, whereas the HLA-B*35-C*04 haplotype was negatively associated with PsA. Specific HLA alleles were linked to disease manifestations: -C04 with female sex, -B27 with familial spondyloarthritis, and -B17 with sporadic PsA. Cervical spine involvement was associated with -C02, and hip involvement with -B35 and -C02. Uveitis was linked to -C02 and -B27. A negative association was observed between a BASFI score > 4 and the presence of -B45 and -C07. The HLA class I system contributes to PsA susceptibility in the Southern Tunisian population. Key Points • This is the first study exploring the association between HLA class I alleles and psoriatic arthritis in Southern Tunisia. • HLA-B27 and -C06 alleles are strongly associated with PsA, with a more pronounced effect in -C06 homozygosity. • Specific HLA alleles are linked to clinical manifestations: -B27 is associated with familial PsA, -B17 with sporadic PsA, and -B35 with hip involvement. • These findings provide insights into the genetic contribution to PsA pathophysiology in Southern Tunisia and highlight the role of HLA alleles in specific disease features.

Tracing Emergence of SARS-CoV-2 Variants: Insights from Comprehensive Assessment Using Reverse Transcription Polymerase Chain Reaction and Whole Genome Sequencing

Tracing Emergence of SARS-CoV-2 Variants: Insights from Comprehensive Assessment Using Reverse Transcription Polymerase Chain Reaction and Whole Genome Sequencing

P-885. Clinical Characteristics and Molecular Epidemiology of Invasive Nontypeable Haemophilus influenzae Disease at a Tertiary Medical Center in Washington, DC

Abstract Background The incidence of invasive nontypeable H. influenzae (NTHi) is on the rise, and NTHi now causes the majority of invasive H. influenzae infections across all age groups. While these presentations are usually among people at the extremes of age, we observed a recent increase in invasive NTHi cases in adults at our institution compared to previous years. Here, we report their clinical presentation and the subsequent molecular epidemiology investigation. Characteristics of Patients With Invasive Nontypeable Haemophilus influenzae (NTHi) Infection, GW Hospital, December 2022-August 2023 Abbreviations: HIV, human immunodeficiency virus; MSM, men who have sex with men; MSK, musculoskeletal; LOS, length of hospital stay; N/A, information not available; HBV, hepatitis B virus; C1, clonal cluster 1; C2 clonal cluster 2; NC, non-cluster * in cells/mm3 Methods We describe the demographics, clinical characteristics, and outcomes of individuals hospitalized at our institution with invasive NTHi disease between December 2022 and August 2023. Invasive NTHi disease was defined as infections beyond the respiratory tract or skin. Data was obtained from retrospective chart abstraction. Available bacterial isolates were sent to the Georgia Emerging Infections Program laboratory for polymerase chain reaction (PCR) and whole genome sequencing (WGS), and were categorized as belonging to clusters 1 or 2 (C1, C2) or as non-cluster, as described previously. Results Nine cases [median age 55 (32-76) years, 100% male, 89% Black] were identified. Five individuals were living with HIV (PWH) on antiretroviral therapy, with median CD4 count 348 (range 32-984) cells/mm3, and all except one were virologically suppressed. Six patients identified as men who have sex with men (MSM). Five patients presented with musculoskeletal manifestations. Seven isolates were available for PCR/WGS: one isolate belonged to C1, three to C2, and three as non-cluster. The mean hospital stay was 11 (range 3-28) days. Three patients required admission to the intensive care unit, all of which died. Conclusion This case series identified nine patients within a nine-month span presenting with invasive NTHi disease, an abrupt rise from our local historical data. We noted an overrepresentation of PWH and MSM, with over half of isolates belonging to two previously identified clonal clusters associated with similar demographics. Future research is needed to better understand novel transmission routes, clinical characteristics, genotypic relatedness, and geographic spread of invasive NTHi disease. Disclosures Rebecca Yee, Ph.D, Biomerieux: Grant/Research Support

Building the Future of Clinical Diagnostics: An Analysis of Potential Benefits and Current Barriers in CRISPR/Cas Diagnostics.

Advancements in molecular diagnostics, such as polymerase chain reaction and next-generation sequencing, have revolutionized disease management and prognosis. Despite these advancements in molecular diagnostics, the field faces challenges due to high operational costs and the need for sophisticated equipment and highly trained personnel besides having several technical limitations. The emergent field of CRISPR/Cas sensing technology is showing promise as a new paradigm in clinical diagnostics, although widespread clinical adoption remains limited. This perspective paper discusses specific cases where CRISPR/Cas technology can surmount the challenges of existing diagnostic methods by stressing the significant role that CRISPR/Cas technology can play in revolutionizing clinical diagnostics. It underscores the urgency and importance of addressing the technological and regulatory hurdles that must be overcome to harness this technology effectively in clinical laboratories.

Trichosporon asahii: emerging challenges in pathogenesis and drug resistance.

Trichosporon asahii (T. asahii) is an opportunistic pathogenic fungus that often causes severe infections in immunosuppressed patients. Among Trichosporon species, T. asahii is the most pathogenic and lethal species. Current research faces challenges related to unknown pathogenic mechanisms, complex resistance mechanisms, insufficiently rapid and accurate diagnostic methods, and insufficient research on susceptibility to infection. These issues need to be explored in depth. This review summarizes research progress on the origin and classification of T. asahii, its virulence factors and pathogenic mechanisms, epidemiological characteristics, infection modes, diagnostic methods, drug treatment options, and drug resistance mechanisms. Traditional culture combined with molecular biology techniques, such as polymerase chain reaction and gene sequencing, has improved the accuracy and speed of detection. Treatment relies mainly on azole antifungal drugs and amphotericin B; however, patients are facing the problem of drug resistance. New techniques, such as gene knockout and gene sequencing, have identified resistance mechanisms, thus supporting the development of novel antifungal drugs. In summary, an in-depth study of T. asahii will aid in developing more effective diagnostic and therapeutic methods and improve patient prognosis.

Hematogenous Dissemination of Scedosporiosis in an Immunocompetent Patient

Abstract Scedosporiosis is becoming an increasingly recognized cause of treatment-resistant opportunistic infections in immunocompromised patients. Fungal infections with Scedosporium spp. have also been seen in immunocompetent patients with previously damaged bronchopulmonary trees. Presentation may be associated with mycetoma, sinopulmonary involvement, extrapulmonary localized infections, and, in rare cases, disseminated infections. We present a case of an immunocompetent 57-year-old woman with prior pulmonary tuberculosis who was found to have Scedosporium boydii/apiospermum pneumonia and Scedosporium osteomyelitis. Multiple bone cultures were negative prior to diagnostic confirmation by 28S ribonucleic acid polymerase chain reaction sequencing. This case illustrates the rare occurrence of hematogenous spread in an immunocompetent patient and the diagnostic and therapeutic challenges associated with Scedosporium spp. infections.

Multi-tiered strategy for large-scale wastewater detection of SARS-CoV-2 in low-case settings provides confidence for public health actions

ABSTRACT Wastewater surveillance has played a pivotal role in monitoring SARS-CoV-2 transmission worldwide. However, developing and implementing the methods underpinning these programmes in regions with prolonged periods of low community transmission has proven challenging. In Victoria, Australia, wastewater surveillance provided early warning of unknown community infections and informed timely public health decisions to limit their spread when case numbers were low. To achieve this, we developed a methodological approach sensitive to extremely low viral loads and could readily identify false positives within short turnaround times. Here, we describe the successful development, implementation, and evaluation of analytic methods using Reverse Transcriptase Quantitative Polymerase Chain Reaction (RT-qPCR) and amplicon sequencing in tandem with CRISPR DETECTR in an ongoing, large-scale surveillance programme to detect SARS-CoV-2 in wastewater in Victoria, Australia. Our study covers ten months, from July 2020 to April 2021, and includes all state-wide health districts and prolonged periods with no known, active community cases among the ∼6.7 million population.

Construction of a High-Density Genetic Linkage Map and QTL Mapping for Stem Rot Resistance in Passion Fruit (Passiflora edulis Sims).

The passion fruit (Passiflora edulis Sims) is a diploid plant (2n = 2x = 18) and is a perennial scrambling vine in Southern China. However, the occurrence and spread of stem rot in passion fruit severely impact its yield and quality. In this study, we re-sequenced a BC1F1 population consisting of 158 individuals using whole-genome resequencing. We constructed a high-density genetic linkage map and identified the quantitative trait locus (QTL), and analyzed candidate genes associated with stem rot resistance in passion fruit. Based on the passion fruit reference genome (MER), a high-density genetic linkage map was constructed with 1,180,406 single nucleotide polymorphisms (SNPs). The map contains nine linkage groups, covering a total genetic distance of 1559.03 cM, with an average genetic distance of 311.81 cM. The average genetic distance between 4206 bins was 0.404 cM, and the average gap length was 10.565 cM. The collinearity correlation coefficient between the genetic map and the passion fruit genome was 0.9994. Fusarium solani was used to infect the BC1F1 population, and the resistance to stem rot showed a continuous distribution. A QTL, qPSR5, was mapped to the 113,377,860 bp-114,811,870 bp genomic region on chromosome 5. We performed RNA sequencing (RNA-seq) and real-time quantitative polymerase chain reaction (RT-qPCR) to analyze the expression levels of predicted genes in the candidate region and identified ZX.05G0020740 and ZX.05G0020810 as ideal candidate genes for stem rot resistance in passion fruit. The findings in this study not only lay the foundation for cloning the qPSR5 responsible for stem rot resistance but also provide genetic resources for the genetic improvement of passion fruit.

Identification of the Highly Polymorphic Prion Protein Gene (PRNP) in Frogs (Rana dybowskii).

Prion diseases are fatal neurodegenerative diseases that can be transmitted by infectious protein particles, PrPScs, encoded by the endogenous prion protein gene (PRNP). The origin of prion seeds is unclear, especially in non-human hosts, and this identification is pivotal to preventing the spread of prion diseases from host animals. Recently, an abnormally high amyloid propensity in prion proteins (PrPs) was found in a frog, of which the genetic variations in the PRNP gene have not been investigated. In this study, genetic polymorphisms in the PRNP gene were investigated in 194 Dybowski's frogs using polymerase chain reaction (PCR) and amplicon sequencing. We carried out in silico analyses to predict functional alterations according to non-synonymous single nucleotide polymorphisms (SNPs) using PolyPhen-2, PANTHER, SIFT, and MutPred2. We used ClustalW2 and MEGA X to compare frog PRNP and PrP sequences with those of prion-related animals. To evaluate the impact of the SNPs on protein aggregation propensity and 3D structure, we utilized AMYCO and ColabFold. We identified 34 novel genetic polymorphisms including 6 non-synonymous SNPs in the frog PRNP gene. The hydrogen bond length varied at codons 143 and 207 according to non-synonymous SNPs, even if the electrostatic potential was not changed. In silico analysis predicted S143N to increase the aggregation propensity, and W6L, C8Y, R211W, and L241F had damaging effects on frog PrPs. The PRNP and PrP sequences of frogs showed low homology with those of prion-related mammals. To the best of our knowledge, this study was the first to discover genetic polymorphisms in the PRNP gene in amphibians.

Establishment of a CRISPR-Cas9-Mediated Genome Editing System in Flax.

Flax is an important crop used for oil and fiber production. Although genetic engineering has been possible in flax, it is not commonly used to produce cultivars. However, the use of genome editing technology, which can produce site-specific mutations without introducing foreign genes, may be a valuable tool for creating elite cultivars that can be easily cultivated. The purpose of this study was to investigate the potential of genome editing in flax by establishing the clustered regularly interspaced short palindromic repeats (CR ISPR)-CRISPR-associated protein 9 (CRISPR-Cas9) genome editing system using the phytoene desaturase (PDS) gene, which produces albino mutants that are easily identifiable. Four sgRNAs were designed from two PDS genes of Flax (LuPDS1 and LuPDS2), and CRISPR-Cas9 genome editing vectors were constructed. After gene transformation, albino phenotypes were observed in transformed callus and regenerated plantlets on selection media. Polymerase chain reaction (PCR) amplification and sequencing of the PDS genes revealed deletions and insertions in the albino tissues, indicating successful editing of the PDS genes. Potential off-target sites were analyzed, but no off-target mutations were found, indicating the specificity of the CRISPR-Cas9 system. The establishment of a flax genome editing system using the CRISPR-Cas9 technology opens up new possibilities for the genetic engineering of flax. This study demonstrates the potential of genome editing in creating elite cultivars that can be easily cultivated, which can have significant implications for the flax industry.

Liquid Biopsy and Multidisciplinary Treatment for Esophageal Cancer.

Esophageal cancer (EC) is one of the leading causes of cancer-related deaths globally. Surgery is the standard treatment for resectable EC after preoperative chemoradiotherapy or chemotherapy, followed by postoperative adjuvant chemotherapy in certain cases. Upper gastrointestinal endoscopy and computed tomography (CT) are predominantly performed to evaluate the efficacy of these treatments, but their sensitivity and accuracy for evaluating minimal residual disease remain unsatisfactory, thereby requiring the development of alternative methods. In recent years, interest has been increasing in using liquid biopsy to assess treatment responses. Liquid biopsy is a noninvasive technology for detecting cell components in the blood and other body fluids. It involves collecting a small sample of body fluid, which is then analyzed for the presence of components, including circulating tumor DNA (ctDNA), microRNA (miRNA), or circulating tumor cells (CTCs). Further, ctDNA and miRNA are analyzed with various techniques, including digital polymerase chain reaction (dPCR) and next-generation sequencing (NGS). CTCs are isolated by determining surface antigens using immunomagnetic techniques or by filtering the blood according to cell size and rigidity. Several studies indicate that investigating these materials helps predict EC prognosis and recurrence and possibly stratifies high-risk groups. Liquid biopsy may also apply to the selection of cases that have achieved a complete response through preoperative treatment to prevent surgery and preserve the esophagus, as well as identifying the suitability of postoperative chemotherapy and the timing of conversion surgery for unresectable EC. The potential of liquid biopsy to enhance treatment decisions will further advance EC treatment.

Enhancing Neuron Activity Promotes Functional Recovery by Inhibiting Microglia-Mediated Synapse Elimination After Stroke.

Activating glutamatergic neurons in the ipsilesional motor cortex can promote functional recovery after stroke. However, the underlying molecular mechanisms remain unclear. Clarifying key molecular mechanisms involved in recovery could help understand the development of neuromodulation strategies after stroke. Adeno-associated virus 2/9-CamKIIa-hM3Dq-mCherry was injected into ipsilesional motor cortex by stereotaxic in the photothrombotic stroke model. Starting from the third day after the stroke, male mice were injected intraperitoneally with clozapine-N-oxide every day to activate excitatory neurons. C1q-blocking antibody and annexin V were used to inhibit C1q and exposed phosphatidylserine (EPS), respectively. The cylinder test and grid-walking test were performed to evaluate functional recovery. The potential molecular mechanisms of excitatory neuronal activation on microglia-mediated synaptic pruning after stroke by immunofluorescence, real-time polymerase chain reaction, Western blotting, and RNA sequencing. Activating excitatory neurons significantly promoted functional recovery and inhibited microglia-mediated synaptic pruning after stroke. Furthermore, it decreased EPS and C1q levels in synapses. On the contrary, inhibiting excitatory neurons aggravated functional defects, promoted microglia-mediated synaptic pruning, and increased EPS and C1q levels in synapses. Selective blocking of EPS repressed C1q tagging of synapses and microglia-mediated synaptic pruning and improved functional recovery. Meanwhile, blocking EPS markedly rescued synaptic density, and motor function deteriorated by chemogenetic inhibition. In addition, C1q-blocking antibody prevented phosphatidylserine engulfment by microglia. Together, these data provide mechanistic insight into microglia-mediated synapse pruning after neuronal activation after stroke and identify the role of C1q binding to EPS in stroke treatment during the repair phase.

Managing Helicobacter pylori as an Infectious Disease: Implementation of Antimicrobial Stewardship.

Helicobacter pylori is prevalent globally and implicated in various gastric diseases and malignancies. Rising antibiotic resistance has increasingly compromised the effectiveness of standard H. pylori eradication therapies. This review explores the role of antimicrobial stewardship (AMS) as a structured approach to optimizing H. pylori management through the "5D" strategy: Diagnosis-utilizing advanced diagnostic tools to accurately detect bacterial resistance; Drug-selecting antibiotics tailored to resistance profiles and patient-specific factors; Dosage-optimizing dosing and frequency based on pharmacokinetic properties to maximize efficacy; Duration-employing shorter treatment courses where supported by evidence; and Discontinuation-balancing the benefits and risks of repeated antibiotic treatments. We discuss recent advances in diagnostic technologies, such as polymerase chain reaction and next-generation sequencing, and their impact on therapeutic decision-making. Additionally, we evaluate treatment regimens, with a particular focus on emerging alternatives such as regimens containing potassium-competitive acid blockers. Given the growing global resistance and limited pipeline for new antibiotics, we advocate for a more strategic and resource-conscious approach to H. pylori management, integrating AMS principles within the "One Health" framework to address the pathogen's transmission across humans, animals, and the environment. With advancements in resistance testing and diagnostics, H. pylori therapies are likely to become increasingly personalized and precise. To achieve this, effective AMS implementation necessitates interdisciplinary collaboration to maximize therapeutic outcomes, minimize adverse effects, combat resistance, and reduce healthcare costs.

Response of Soil Bacteria to Short-Term Nitrogen Addition in Nutrient-Poor Areas.

Increasing nitrogen (N) addition induces soil nutrient imbalances and is recognized as a major regulator of soil microbial communities. However, how soil bacterial abundance, diversity, and community composition respond to exogenous N addition in nutrient-poor and generally N-limited regions remains understudied. In this study, we investigated the effects of short-term exogenous N additions on soil bacterial communities using quantitative polymerase chain reaction (PCR) and Illumina Miseq sequencing in an in situ N addition field experiment. The results showed that a low nitrogen addition increased the observed species (Sobs) of the bacterial community, and with the increased nitrogen addition, the Sobs of bacteria gradually decreased, especially the unique OTUs. The relative abundance of Proteobacteria, Actinobacteria, and Gemmatimonadetes increased with increasing nitrogen addition, whereas the relative abundance of Chloroflexi and Firmicutes decreased. Soil properties play an important role in bacterial community structure at phylum or genus levels. Short-term nitrogen addition increased the proportion of nodes from Actinobacteria and Proteobacteria in the co-occurrence network and enhanced the stability of the microbial network. Actinobacteria may play an important role in constructing the network. Our study aims to explore the effects of nitrogen addition on the diversity, composition, and structure of soil bacterial communities in nutrient-poor areas caused by ecological disturbances.

Imperatorin's Effect on Myocardial Infarction Based on Network Pharmacology and Molecular Docking.

Purpose: Myocardial infarction (MI), a severe cardiovascular disease, is the result of insufficient blood supply to the myocardium. Despite the improvements of conventional therapies, new approaches are needed to improve the outcome post-MI. Imperatorin is a natural compound with multiple pharmacological properties and potential cardioprotective effects. Therefore, this work investigated imperatorin's therapeutic effects and its mechanism of action in an MI mouse model. Methods: Network pharmacology, molecular docking, and experimental validation were performed for exploring the pharmacokinetic properties, therapeutic effects, and molecular targets of imperatorin in MI. Permanent ligation of the left anterior descending artery was performed in male C57BL/6 mice to induce MI before treatment with imperatorin once per day for 1 week. Echocardiography, heart histology, RNA sequencing, and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) as well as western blotting were carried out for evaluating cardiac function and structure, as well as gene and protein expression. Results: Imperatorin significantly improved cardiac function, preserved cardiac structure, attenuated cardiac remodeling and fibrosis, and reduced cardiomyocyte apoptosis in MI mice. Eight differentially expressed genes overlapping with key target genes were found, two upregulated and six downregulated. A key target in signaling pathways associated with imperatorin effect in MI was angiotensin-converting enzyme (ACE). Imperatorin inhibited ACE-angiotensin II (Ang II)-angiotensin II Type 1 receptor (AT1R) axis in MI mice. Conclusion: Imperatorin attenuated MI by inhibiting the ACE-Ang II-AT1R axis. Thus, imperatorin might be considered a potential therapeutic agent to cure MI.

Dietary Quercetin Regulates Gut Microbiome Diversity and Abundance in Apis cerana (Hymenoptera Apidae).

Honeybee gut microbiota plays a crucial role in maintaining their health and digestive function. Studies have confirmed that quercetin improves honeybee health by enhancing their pesticide tolerance and survival rates. This study aimed to examine the effects of quercetin on the bee gut microbiome by absolute quantification sequencing. We included 1800 bees from the experimental apiary and exposed them to 151.2, 75.6, and 37.8 mg/L of quercetin. Gut samples were collected on the 5th and 9th days, subjected to a polymerase chain reaction and 16S rRNA sequencing, and analyzed. After 5 days of quercetin treatment, the diversity of the honeybee gut microbiota was altered, and total bacterial copies and Lactobacillus abundance significantly decreased at high quercetin concentrations (151.2 and 75.6 mg/L). On day 9, the gut microbial community had recovered from the adverse effects, and Gilliamella abundance increased in response to 37.8 mg/L quercetin treatment. However, quercetin had no noticeable effects on survival rate, food consumption, and gut structure. Our study confirmed the effect of short-term quercetin intake on the gut microbiota of A. cerana, providing valuable insights into how phytochemicals alter the bee gut microbiome, and their repercussions on host physiology.

A homozygous loss-of-function mutation in CEP250 is associated with acephalic spermatozoa syndrome in humans.

The presence of predominantly headless sperm in semen is a hallmark of acephalic spermatozoa syndrome, which is primarily caused by gene mutations in humans. To identify genetic causes for acephalic spermatozoa syndrome. Polymerase chain reaction and Sanger sequencing were performed to define mutations in SUN5 and PMFBP1. Whole-exome sequencing was performed on the patients to identify pathogenic mutations for infertility. Western blotting and immunofluorescence analysis detected the expression level and localization of CEP250. Co-immunoprecipitation detected the protein-protein interactions. Cep250-KI mice were generated by the CRISPR-Cas9 system. Here, 10 patients diagnosed with acephalic spermatozoa syndrome were recruited, and a homozygous loss-of-function mutation in CEP250 (NM_007186: c. 4710_4723del: p. E1570fs*39) was identified from a consanguineous Han Chinese family. Immunofluorescence experiments revealed a decreased CEP250 signal in the neck region of the patient's sperm compared with the normal. Co-immunoprecipitation results indicated reduced interaction between SUN5/PMFBP1 and mutant CEP250 compared with the wild-type, possibly due to the absence of complete 2272-2442 amino acids. Besides, the patient can be effectively treated with intracytoplasmic sperm injections. Nevertheless, Cep250-KI male mice exhibit non-obstructive azoospermia, which indicates the different functions in CEP250 between human and mouse spermatogenesis. Collectively, CEP250 may represent a novel pathogenic gene for acephalic spermatozoa syndrome in humans, and we provide precise genetic diagnosis and treatment strategies for the patient.