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Background: Esophageal carcinoma (EC) and gastric cardiac adenocarcinoma (GCA) have high incidence rates in the Chaoshan region of South China. Multifocal esophageal and cardiac cancer (MECC) is commonly observed in this region in clinical practice. However, the genomic characteristics of MECC remains unclear. Materials and methods: In this study, a total of 2123 clinical samples of EC and GCA were analyzed to determine the frequency of multifocal tumors, as well as their occurrence sites and pathological types. Cox proportional hazards regression was used to model the relationship between age, sex, and tumor state concerning survival in our analysis of the cohort of 541 patients with available follow-up data. We performed whole-genome sequencing on 20 tumor foci and 10 normal samples from 10 MECC patients to infer clonal structure on 6 MECC patients to explore genome characteristics. Result: The MECC rate of EC and GCA was 5.65% (121 of 2123). Age and sex were potential factors that may influence the risk of MECC (p < 0.001). Furthermore, MECC patients showed worse survival compared with single tumor patients. We found that 12 foci from 6 patients were multicentric origin model (MC), which exhibited significant heterogeneity of variations in paired foci and had an increased number of germline mutations in immune genes compared to metastatic model. In MC cases, different lesions in the same patient were driven by distinct mutation and copy number variation (CNV) events. Although TP53 and other driver mutation genes have a high frequency in the samples, their mutation sites show significant heterogeneity in paired tumor specimens. On the other hand, CNV genes exhibited higher concordance in paired samples, especially in the amplification of oncogenes and the deletion of tumor suppressor genes. Conclusions: The extent of inter-tumor heterogeneity suggests both monoclonal and polyclonal origins of MECC, which could provide insight into the genome diversity of MECC and guide clinical implementation. [ABSTRACT FROM AUTHOR]

Background: Heterakis gallinarum (H. gallinarum) is a common poultry parasite that can be found in the ceca of many gallinaceous bird species, causing minor pathology and reduced weight gain. Most infections go unnoticed in commercial flocks due to the dependence on fecal egg counts, which are prone to false-negative diagnoses. Furthermore, there is a lack of research on gastrointestinal nematodes that use molecular identification methods, which could be essential for rapid diagnosis and developing efficient control approaches. As a result, the study aimed to look at the cause of mortality in layer chickens induced by H. gallinarum in Egyptian poultry farms using morphological, ultrastructural, and molecular characterization. Histopathological, immunohistochemical, and cell-mediated immune responses from damaged cecal tissues were also examined. Results: Seventy bird samples from ten-layer flocks of different breeds (Native, white, and brown layers) suffering from diarrhea, decreased egg output, and emaciation were collected. Cecal samples were collected from affected and non-affected birds and were examined for parasitic diseases using light and a scanning electron microscope. The mitochondrial cytochrome oxidase 1 (COX1) gene was used to characterize H. gallinarum. Our results showed that the collected nematodal worms were identified as H. gallinarum (male and female), further confirmed by COX1 gene amplification and sequence alignment. Gene expression analysis of the inflammatory markers in infected tissues showed a significant up-regulation of IL-2, IFN-γ, TLR-4, and IL-1β and a significant down-regulation of the anti-inflammatory IL-10. The mRNA level of the apoptotic cas-3 revealed apoptotic activity among the H. gallinarum samples compared to the control group. Conclusions: Our results implemented the use of molecular methods for the diagnosis of Heterakis, and this is the first report showing the tissue immune response following infection in layers: upregulation of IL-1β, IFN-γ, Il-2, and TLR-4, while down-regulation of anti-inflammatory IL-10 in cecal tissue, Cas-3 apoptotic activity and Nuclear factor-κB (NF-κB)activity with immunophenotyping of T-cells in Heterakis infected tissue. [ABSTRACT FROM AUTHOR]

[Objectives] A recombinase polymerase amplification(RPA)system was constructed for the rapid detection of Fusarium oxysporum f. sp. niveum (FON) in order to provide technical support for the rapid detection of watermelon wilt in the field. [Methods] The genome sequences of 11 pathogenic fungi, including FON, were downloaded from the NCBI website for bioinformatics analysis using Seq Hunter 2 software. Four specific gene fragments of FON were obtained by comparison and primers were designed and screened for them. The specificity and sensitivity of the primers were verified using polymerase chain reaction(PCR), and in addition, the specificity and sensitivity of the primers were verified using the established RPA detection system, and finally, the RPA detection evaluation of artificially inoculated watermelon plants was performed. [Results] The RPA detection system for FON was established, and the optimal reaction time(40 min)and optimal reaction temperature (30 ℃) of the amplification system of DNA constant temperature rapid amplification kit were explored, and the detection limit of the optimal reaction system was 100 pg·μL-1. When using the DNA constant temperature rapid amplification kit(colloidal gold test strip type)for amplification, it only needed to react at 38 ℃ for 12 min, and 50 μL of the amplified product diluted 20 times was detected with the colloidal gold test strip. After 5 min, the results could be judged, and the detection limit was 100 pg·μL-1. [Conclusion] The RPA detection system of FON was successfully constructed. The established RPA detection system could detect FON from the genomic DNA of infected watermelon plant tissues, which could provide technical support for the rapid detection of watermelon Fusarium wilt in the field. [ABSTRACT FROM AUTHOR]

Breast cancer is a heterogeneous disease, and its subtypes are characterized by hormone receptor and human epidermal growth factor receptor 2 (HER2) expression status. "HER2-low" tumors, which exhibit a low level of HER2 expression (immunohistochemistry 1+ or 2+ without gene amplification), were conventionally considered not amenable to anti-HER2 targeting agents based on the results of a phase III trial of trastuzumab. However, this perspective is being challenged by the emergence of novel anti-HER2 antibody-drug conjugates, such as trastuzumab-deruxtecan. These innovative therapies have demonstrated remarkable efficacy against HER2-low breast cancer, shedding new light on a previously overlooked category of breast cancer. Such promising results highlight the need for in-depth investigations of the biology and prognostic implications of HER2-low tumors. In this review, we comprehensively summarize the current evidence surrounding this topic and highlight areas that warrant further exploration and research in the future. [ABSTRACT FROM AUTHOR]

Recombinase polymerase amplification (RPA) is an isothermal DNA amplification reaction at around 41 °C using recombinase (Rec), single-stranded DNA-binding protein (SSB), strand-displacing DNA polymerase (Pol), and an ATP-regenerating enzyme. Considering the onsite use of RPA reagents, lyophilized RPA reagents with long storage stability are highly desired. In this study, as one of the approaches to solve this problem, we attempted to use a thermostable pyruvate kinase (PK). PK gene was isolated from a thermophilic bacterium Thermotoga maritima (Tma -PK). Tma -PK was expressed in Escherichia coli and purified from the cells. Tma -PK exhibited higher thermostability than human PK. The purified Tma -PK preparation was applied to RPA as an ATP-regenerating enzyme. Liquid RPA reagent with Tma -PK exhibited the same performance as that with human PK. Lyophilized RPA reagent with Tma -PK exhibited higher performance than that with human PK. Combined with our previous results of RPA reagents of thermostable Pol from a thermophilic bacterium, Aeribacillus pallidus , the results in this study suggest that thermostable enzymes are preferable to mesophilic ones as a component in lyophilized RPA reagents. [ABSTRACT FROM AUTHOR]

Antibody drugs play a vital role in diagnostics and therapy. However, producing antibodies from mammalian cells is challenging owing to cellular heterogeneity, which can be addressed by applying droplet-based microfluidic platforms for high-throughput screening (HTS). Here, we designed an integrated system based on disulfide-bonded redox-responsive hydrogel beads (redox-HBs), which were prepared through enzymatic hydrogelation, to compartmentalize, screen, select, retrieve, and recover selected Chinese hamster ovary (CHO) cells secreting high levels of antibodies. Moreover, redox-HBs were functionalized with protein G as an antibody-binding module to capture antibodies secreted from encapsulated cells. As proof-of-concept, cells co-producing immunoglobulin G (IgG) as the antibody and green fluorescent protein (GFP) as the reporter molecule, denoted as CHO(IgG/GFP), were encapsulated into functionalized redox-HBs. Additionally, antibody-secreting cells were labeled with protein L-conjugated horseradish peroxidase using a tyramide amplification system, enabling fluorescence staining of the antibody captured inside the beads. Redox-HBs were then applied to fluorescence-activated droplet sorting, and selected redox-HBs were degraded by reducing the disulfide bonds to recover the target cells. The results indicated the potential of the developed HTS platform for selecting a single cell viable for biopharmaceutical production. [ABSTRACT FROM AUTHOR]

Microbial fuel cells (MFCs) represent a promising technology to generate bio-electricity and synchronously reduce wastewater pollutants. The presence of exoelectrogens in wastewater is critical for bio-electricity and pollutant reduction, but the performance of exoelectrogens at different pH levels remains unknown. This study aims to bridge this gap by offering an integrated approach to understanding the performance of exoelectrogens under varying substrate pH, particularly in bio-electricity generation and pollutant reduction in sugarbeet processing wastewater (SBWW). Three pH levels (ranging from acidic to alkaline) were studied and MFC’s electricity output was measured. Later, current density, power density, and coulombic efficiency (CE) were calculated. Both pre- and post-experiment substrate samples were analysed with inductively coupled plasma (ICP). Furthermore, 16S rRNA gene analysis, DNA amplification, sequencing library preparation, and bioinformatics workflows on post-experiment samples of the substrate and anode samples were conducted. A diverse community of microorganisms was identified, especially Alphaproteobacteria, Gammaproteobacteria, and Deltaproteobacteria (Geobacter). Bacteroidetes and Desulfovibrio were the major exoelectrogens responsible for electricity generation. Among the three pH levels tested, the most alkaline pH level (9.5±0.1) outperformed the others, achieving a 54% higher power density, 21% greater current density, and a 40% higher CE compared to the acidic pH level (6.5±0.1). Around 50–99% of pollutants were removed from the SBWW. The study revealed that Gammaproteobacteria thrive and perform better in alkaline environment. [ABSTRACT FROM AUTHOR]

[Display omitted] The cancer therapeutic efficacy of the peroxidase (POD)-mimicking nanozyme-based monotherapy is significantly hindered due to insufficient intratumoral hydrogen peroxide (H 2 O 2) and glutathione (GSH) consumption effect on reactive oxygen species (ROS). In this study, we present the development of poly (o -phenylenediamine)@gold nanoparticles (AuNPs) (PoPD@Au) nanocomposites for multifunctional catalytic-photothermal therapy. These nanocomposites exhibit triple distinct nanozymatic activities, i.e. , POD-like activity that catalyzes H 2 O 2 to ROS, glucose oxidase (GOx)-like activity that supplements endogenous H 2 O 2 , and GSH depleting activity that decreases the ROS consumption efficiency. This open source and reduce expenditure strategy for ROS generation allows for the amplification of tumor oxidative stress, thereby enhancing anti-tumor efficiency. Additionally, the PoPD@Au nanocomposites demonstrate outstanding photothermal conversion efficiency, contributing to the synergistic effect between PoPD and AuNPs. Moreover, we reveal the improved photothermal performance of PoPD@Au triggered by the tumor microenvironment pH, which provides additional benefits for targeted catalytic-photothermal therapy. This "four-in-one" design of PoPD@Au enables efficient anti-tumor effects both in vitro and in vivo , making it a universal strategy for engineering catalytic-photothermal therapeutic nanoagents. [ABSTRACT FROM AUTHOR]

Background: Chordoma, a rare malignant tumor arising from notochordal tissue, usually occurs along the spinal axis. Only a few published reports of primary lung chordomas exist. Herein, we present a case of primary lung chordoma and discuss important considerations for diagnosing rare chordomas. Case presentation: We report a case of primary lung chordoma in a 39-year-old male with a history of testicular mixed germ-cell tumor of yolk sac and teratoma. Computed tomography revealed slow-growing solid lesions in the left lower lobe. We performed wedge resection for suspected germ-cell tumor lung metastasis. Histologically, large round or oval cells with eosinophilic cytoplasm were surrounded by large cells with granular, lightly eosinophilic cytoplasm. Tumor cells were physaliphorous. Immunohistochemistry was positive for brachyury, S-100 protein, epithelial membrane antigen, vimentin, and cytokeratin AE1/AE3, suggesting pulmonary chordoma. Re-examination of the testicular mixed germ-cell tumor revealed no notochordal elements. Although some areas were positive for brachyury staining, hematoxylin and eosin (HE) staining did not show morphological features typical of chordoma. Complementary fluorescence in situ hybridization (FISH) of the lung tumor confirmed the absence of isochromosome 12p and 12p amplification. Thus, a final diagnosis of primary lung chordoma was established. Conclusions: In patients with a history of testicular mixed germ cell tumors, comparison of histomorphology using HE and Brachyury staining of lung and testicular tumors, and analyzing isochromosome 12p and 12p amplification in lung tumors using FISH is pivotal for the diagnosis of rare lung chordomas. [ABSTRACT FROM AUTHOR]

The emergence of extensively-resistant strains of Klebsiella pneumoniae (K. pneumoniae) in healthcare settings is linked to prolonged hospitalization and uncontrolled use of antibiotics. There is a paucity of data regarding the prevalence and mechanisms of colistin and fosfomycin resistance encoding genes rate and mechanisms in Iran. The objective of this study was to determine the prevalence of biofilm formation and fosfomycin and colistin resistance among K. pneumoniae strains producing ESBL and carbapenemases by detecting the mcr-1, mcr-2, and fosA genes in Tehran, Iran, during the 2020-2021 period. After collecting 73 samples, the isolates were identified using biochemical tests. Antibiotic susceptibility test was performed using the disk diffusion method. The phenotypic determination of extended-spectrum beta-lactamases (ESBLs) and carbapenemase enzymes was conducted using combined disk and CARBA-NP tests, respectively. The biofilm formation was conducted using a microtiter tissue plate assay. Polymerase chain reaction (PCR) was employed to detect the mcr-1, mcr-2 and fosA genes, which are associated with colistin and fosfomycin resistance, respectively. The highest resistance rate was observed against ampicillin (97%), chloramphenicol (90%), and ciprofloxacin (87%), respectively. In contrast, the lowest resistance rate was noted against gentamicin (4%), amikacin (10%), and cotrimoxazole (18%). Moreover, 44 and 23 isolates were identified as ESBL and carbapenemase -producing K. pneumonia), respectively. Of the fortyeight isolates that formed strong biofilms, one was a non-biofilm producer. The PCR test revealed the amplification of the fosA2 gene in four isolates and the mcr-2 genes in one isolate. However, no amplification of the fosA3 or mcr-1 genes was observed. The present study demonstrated that the frequency of K. pneumoniae isolates producing ESBL and carbapenemase, as well as mcr-1, mcr-2 and fosA genes, was relatively low. However, given the potential for these genes to be disseminated more widely, it is imperative to implement effective isolation and control measures. Moreover, these strains demonstrated the capacity to form biofilms in vitro, which can lead to persistent infections in the hospital settings. [ABSTRACT FROM AUTHOR]

Stress-associated proteins (SAPs) are known to play an important role in plant responses to abiotic stresses. This study systematically identified members of the sunflower SAP gene family using sunflower genome data. The genes of the sunflower SAP gene family were analyzed using bioinformatic methods, and gene expression was assessed through fluorescence quantification (qRT-PCR) under salt and drought stress. A comprehensive analysis was also performed on the number, structure, collinearity, and phylogeny of seven Compositae species and eight other plant SAP gene families. The sunflower genome was found to have 27 SAP genes, distributed across 14 chromosomes. The evolutionary analysis revealed that the SAP family genes could be divided into three subgroups. Notably, the annuus variety exhibited amplification of the SAP gene for Group 3. Among the Compositae species, C. morifolium demonstrated the highest number of collinearity gene pairs and the closest distance on the phylogenetic tree, suggesting relative conservation in the evolutionary process. An analysis of gene structure revealed that Group 1 exhibited the most complex gene structure, while the majority of HaSAP genes in Group 2 and Group 3 lacked introns. The promoter analysis revealed the presence of cis-acting elements related to ABA, indicating their involvement in stress responses. The expression analysis indicated the potential involvement of 10 genes (HaSAP1, HaSAP3, HaSAP8, HaSAP10, HaSAP15, HaSAP16, HaSAP21, HaSAP22, HaSAP23, and HaSAP26) in sunflower salt tolerance. The expression of these 10 genes were then examined under salt and drought stress using qRT-PCR, and the tissue-specific expression patterns of these 10 genes were also analyzed. HaSAP1, HaSAP21, and HaSAP23 exhibited consistent expression patterns under both salt and drought stress, indicating these genes play a role in both salt tolerance and drought resistance in sunflower. The findings of this study highlight the significant contribution of the SAP gene family to salt tolerance and drought resistance in sunflower. [ABSTRACT FROM AUTHOR]

Chimeric antigen receptor gene-modified T (CAR-T) cell immunotherapy is considered as one of the most promising tumor treatments. The number of effector CAR-T cells is a key factor in determining the therapeutic effect of CAR-T cell therapy. The expansion of CAR-T cells in vitro is time and energy-consuming. After transfusion, it's difficult for CAR-T cells to infiltrate into solid tumors, resulting in a significant decrease in the number of CAR-T cells that can effectively inhibit solid tumors. Currently, the amplification of CAR-T cells has problems in enhancing amplification specificity and treatment safety, which hinders the clinical transformation of CAR-T cell therapy. In recent years, achievements in new immunity agonist and their downstream signals have provided more options for CAR-T amplification, and the novel administration methods of immunity agonist have further improved the safety of CAR-T amplification in vivo. This review analyzes the current challenges in CAR-T cell amplification, and systematically expounds the new strategies of CAR-T cell amplification in vitro and in vivo in recent years, providing new thoughts for the efficacy and capacity optimization of CAR-T cell therapy. [ABSTRACT FROM AUTHOR]

BACKGROUND: Bactrocera correcta is a quarantine pest that negatively impacts the fruit and vegetable industry. Differentiating B. correcta from similar species, especially in non‐adult stages, remains challenging. Rapid molecular identification techniques, such as recombinase polymerase amplification (RPA) combined with CRISPR/Cas12a and multienzyme isothermal rapid amplification with lateral flow dipstick (MIRA‐LFD), play a crucial role in early monitoring and safeguarding agricultural production. Our study introduces two methods for the rapid visual identification of B. correcta. RESULTS: Bactrocera correcta specific RPA primers, CRISPR RNA (crRNA), and the LFD probe were designed based on the cox1 genes. The RPA reaction conditions were optimized (at 37 °C for 8 min) for effective template DNA amplification. Two nucleic acid detection methods were established to visualize RPA. In the RPA‐CRISPR/Cas12a system, the optimal LbCas12a/crRNA concentration ratio was 200:400 nmol L−1. Successful amplification was determined by the presence or absence of green fluorescence following 15 min incubation at 37 °C. The MIRA‐LFD system achieved precise identification of the target species within 4 min at 37 °C. Both methods exhibited high specificity and sensitivity, allowing for detection from 1.0 × 10−1 ng μL−1 of DNA. Combined with rapid DNA extraction, rapid identification of individual B. correcta at different developmental stages was achieved, enhancing the practicality and convenience of the established methods. CONCLUSION: Our research findings demonstrate that both the RPA‐CRISPR/Cas12a and MIRA‐LFD methods for B. correcta detection was accurate and rapid (within 30 min and 10 min, respectively), at 37 °C. Our methods do not rely on expensive equipment, thus possess high practical value, providing improved identification solutions for port quarantine pests and field applications. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

The study investigated the insecticide tolerance and dissipation potential of five Trichoderma isolates, namely T. asperellum (4 isolates) and T. harzianum (single isolate), to thiamethoxam and chlorantraniliprole in treated liquid medium and tomato fruit. The isolates were identified using ITS gene amplification and sequencing regions. In vitro tolerance was assessed using a poisoned medium technique, with mycelial placed onto Potato Dextrose Agar (PDA.) media supplemented with commercial insecticides at recommended doses. Dissipation potential was determined using the isolates after independently inoculating them into PDB liquid media with Actara and Voliam Flexi. The results showed that the inhibitory effect of thiamethoxam and chlorantraniliprole was almost zero, and the five isolates were competent to reach 9 cm growth like the control after 96 h of incubation at 25°C ± 2°C. Mycelial fresh weights of T. harzianum EGY-T4 and T. asperellum EGY-T5 were significantly equal to their respective control without insecticide. T. asperellum EGY-T1 had the highest dissipation rate of 36% and 30% for thiamethoxam in Actara and Voliam Flexi when applied at the higher dose of 100 mg L-1. The dissipation rate of thiamethoxam was recorded at 92.2% when applied in combination with T. asperellum EGY-T1, reducing the PHI from 7 to 4 days. Chlorantraniliprole was dissipated to the extent of 51.3 and 99.5% when applied in combination with T. asperellum EGY-T1 and T. harzianum EGY-T4, respectively, with residues reaching up to 0.12 and 0.21 mg kg-1 after one day. [ABSTRACT FROM AUTHOR]

Seafood is a prime target for fraudulent activities due to the complexity of its supply chain, high demand, and difficult discrimination among species once morphological characteristics are removed. Instances of seafood fraud are expected to increase due to growing demand. This manuscript reviews the application of DNA‐based methods for commercial fish authentication and identification from 2000 to 2023. It explores (1) the most common types of commercial fish used in assay development, (2) the type of method used, (3) the gene region most often targeted, (4) provides a case study of currently published assays or primer‐probe pairs used for DNA amplification, for specificity, and (5) makes recommendations for ensuring standardized assay‐based reporting for future studies. A total of 313 original assays for the detection and authentication of commercial fish species from 191 primary articles published over the last 23 years were examined. The most explored DNA‐based method was real‐time polymerase chain reaction (qPCR), followed by DNA sequencing. The most targeted gene regions were cytb (cytochrome b) and COI (cytochrome c oxidase 1). Tuna was the most targeted commercial fish species. A case study of published tuna assays (n = 19) targeting the cytb region found that most assays were not species‐specific through in silico testing. This was conducted by examining the primer mismatch for each assay using multiple sequence alignment. Therefore, there is need for more standardized DNA‐based assay reporting in the literature to ensure specificity, reproducibility, and reliability of results. Factors, such as cost, sensitivity, quality of the DNA, and species, should be considered when designing assays. [ABSTRACT FROM AUTHOR]

The emergence of acquired resistance to EGFR-tyrosine kinase inhibitors (TKIs) is almost inevitable even after a remarkable clinical response. Secondary mutations such as T790M and C797S are responsible for the resistance to 1st/2nd-generation (1/2G) TKIs and 3G TKIs, respectively. To overcome both the T790M and C797S mutations, novel 4G EGFR-TKIs are now under early clinical development. In this study, we evaluated the efficacy of a 4G EGFR-TKI in the treatment of lung cancer with EGFR mutation as well as explored resistance mechanisms to a 4G TKI. First, we compared the efficacies of seven TKIs including a 4G TKI, BI4020, against Ba/F3 cell models that simulate resistant tumors after front-line osimertinib treatment failure because of a secondary mutation. We also established acquired resistant cells to BI4020 by chronic drug exposure. Ba/F3 cells with an osimertinib-resistant secondary mutation were refractory to all 3G TKIs tested (alflutinib, lazertinib, rezivertinib, almonertinib, and befotertinib). BI4020 inhibited the growth of C797S-positive cells; however, it was not effective against L718Q-positive cells. Erlotinib was active against all Ba/F3 cells tested. In the analysis of resistance mechanisms of BI4020-resistant (BIR) cells, none harbored secondary EGFR mutations. HCC827BIR cells had MET gene amplification and were sensitive to a combination of capmatinib (MET-TKI) and BI4020. HCC4006BIR and H1975BIR cells exhibited epithelial-to-mesenchymal transition. This study suggests that erlotinib may be more suitable than 4G TKIs to overcome secondary mutations after front-line osimertinib. We found that off-target mechanisms that cause resistance to earlier-generation TKIs will also cause resistance to 4G TKIs. [ABSTRACT FROM AUTHOR]

The aim of the study was to establish a live/dead qPCR with propidium monoazide (PMA) that can quantitatively differentiate between viable/non-viable microorganisms in dental hard tissues. Human premolars (n = 88) were prepared with nickel–titanium instruments and incubated with E. faecalis (21 d). Subsequently, the bacteria in half of the teeth were devitalized by heat inactivation (100 °C, 2 h). The following parameters were tested: PMA concentrations at 0 µmol (control), 50 µmol, 100 µmol, and 200 µmol; PMA incubation times of 30 min and 60 min, and blue light treatment for 30 min and 60 min. The teeth were ground using a cryomill and the bacterial DNA was quantified using qPCR, ANOVA, and p = 0.05. The qPCR of the control group detected a similar number of avital 9.94 × 106 and vital 1.61 × 107 bacterial cells. The use of PMA inhibited the amplification of DNA from non-viable cells during qPCR. As a result, the best detection of avital bacteria was achieved with the following PMA parameters: (concentration, incubation time, blue light treatment) 200-30-30; 5.53 × 104 (avital) and 1.21 × 100.7 (vital). The live/dead qPCR method using PMA treatment is suitable for the differentiation and quantification of viable/non-viable microorganisms in dentin, as well as to evaluate the effectiveness of different preparation procedures and antimicrobial irrigants in other biological hard substances. [ABSTRACT FROM AUTHOR]

Abstract Random mutagenesis, such as error-prone PCR (epPCR), is a technique capable of generating a wide variety of a single gene. However, epPCR can produce a large number of mutated gene variants, posing a challenge in ligating these mutated PCR products into plasmid vectors. Typically, the primers for mutagenic PCRs incorporate artificial restriction enzyme sites compatible with chosen plasmids. Products are cleaved and ligated to linearized plasmids, then recircularized by DNA ligase. However, this cut-and-paste method known as ligation-dependent process cloning (LDCP), has limited efficiency, as the loss of potential mutants is inevitable leading to a significant reduction in the library’s breadth. An alternative to LDCP is the circular polymerase extension cloning (CPEC) method. This technique involves a reaction where a high-fidelity DNA polymerase extends the overlapping regions between the insert and vector, forming a circular molecule. In this study, our objective was to compare the traditional cut-and-paste enzymatic method with CPEC in producing a variant library from the gene encoding the red fluorescent protein (DsRed2) obtained by epPCR. Our findings suggest that CPEC can accelerate the cloning process in gene library generation, enabling the acquisition of a greater number of gene variants compared to methods reliant on restriction enzymes.

Purpose: To determine the prevalent microbiological profile of biliary atresia (BA) patients at the time of its occurrence by studying their intestinal flora. Methods: A total of 118 gut microbiota samples from three groups of 43 BA patients, 33 disease controls (DC) with other cholestatic diseases and 42 healthy controls (HC), were analyzed by deep mining of public data. Subsequently, a total of 23 fecal samples from three groups of clinically collected patients (11 BA, 6 DC and 6 HC) were sequenced for 16S rRNA gene amplification and analyzed for serum butyrate (BU) level by liquid chromatography. Results: Taxonomic analysis revealed significant differences in the composition of the intestinal microbiota between BA patients and controls, with a reduction in diversity and a higher abundance of Proteobacteria, Streptococcus and Lactobacillus in the BA group. Database and clinical data analyses concluded that Streptococcus/Bacteroides (AUC = 0.9035, 95% CI 0.8347–0.9722, P < 0.0001) or Streptococcus/Eggerthella (AUC = 0.8333, 95% CI 0.6340–1.000, P = 0.027) was the best microbiota to differentiate between BA and DC. Serum butyrate levels were low in the BA and DC groups and differed from the HC group (P = 0.01, P = 0.04). Butyrate levels in BA were negatively correlated with jaundice clearance and cholangitis, but not statistically significant. Conclusions: Our study reveals changes in the composition of the gut microbiota in BA, especially the butyrate-producing microbiota, and suggests the potential for using gut microbiota as a noninvasive diagnostic benefit for BA. Low levels of serum butyrate in BA may indicate a poor prognosis. [ABSTRACT FROM AUTHOR]

Targeted therapy is crucial for advanced colorectal cancer (CRC) positive for genetic drivers. With advances in deep sequencing technology and new targeted drugs, existing standard molecular pathological detection systems and therapeutic strategies can no longer meet the requirements for careful management of patients with advanced CRC. Thus, rare genetic variations require diagnosis and targeted therapy in clinical practice. Rare gene mutations, amplifications, and rearrangements are usually associated with poor prognosis and poor response to conventional therapy. This review summarizes the clinical diagnosis and treatment of rare genetic variations, in genes including erb-b2 receptor tyrosine kinase 2 (ERBB2), B-Raf proto-oncogene, serine/threonine kinase (BRAF), ALK receptor tyrosine kinase/ROS proto-oncogene 1, receptor tyrosine kinase (ALK/ROS1), neurotrophic receptor tyrosine kinases (NTRKs), ret proto-oncogene (RET), fibroblast growth factor receptor 2 (FGFR2), and epidermal growth factor receptor (EGFR), to enhance understanding and identify more accurate personalized treatments for patients with rare genetic variations.

Antifreeze proteins (AFPs) are found in a variety of marine cold‐water fishes where they prevent freezing by binding to nascent ice crystals. Their diversity (types I, II, III and antifreeze glycoproteins), as well as their scattered taxonomic distribution hint at their complex evolutionary history. In particular, type I AFPs appear to have arisen in response to the Late Cenozoic Ice Age that began ~ 34 million years ago via convergence in four different groups of fish that diverged from lineages lacking this AFP. The progenitor of the alanine‐rich α‐helical type I AFPs of sculpins has now been identified as lunapark, an integral membrane protein of the endoplasmic reticulum. Following gene duplication and loss of all but three of the 15 exons, the final exon, which encoded a glutamate‐ and glutamine‐rich segment, was converted to an alanine‐rich sequence by a combination of frameshifting and mutation. Subsequent gene duplications produced numerous isoforms falling into four distinct groups. The origin of the flounder type I AFP is quite different. Here, a small segment from the original antiviral protein gene was amplified and the rest of the coding sequence was lost, while the gene structure was largely retained. The independent origins of type I AFPs with up to 83% sequence identity in flounder and sculpin demonstrate strong convergent selection at the level of protein sequence for alanine‐rich single alpha helices that bind to ice. Recent acquisition of these AFPs has allowed sculpins to occupy icy seawater niches with reduced competition and predation from other teleost species. [ABSTRACT FROM AUTHOR]

Testicular germ cell tumours (GCT) are divided into three different subtypes (types I–III) regarding to their developmental origin, histological differences and molecular features. Type I GCT develop from disturbed primordial germ cells and most commonly occur in children and young adolescents, which is why they are referred to as prepubertal GCT. Type II GCT develop from a non‐invasive germ cell neoplasia in situ (GCNIS) and show an isochromosome 12p (i12p) or gain of 12p material as a common and characteristic molecular alteration. Type III GCT originate from distorted postpubertal germ cells (e.g. spermatogonia) in adult patients and have changes on chromosome 9 with amplification of the DMRT1 gene. Type I GCT encompass prepubertal‐type teratomas and yolk‐sac tumours (YST). Type II GCT include seminoma, embryonal carcinoma, choriocarcinoma, postpubertal‐type teratoma and postpubertal‐type YST. Types I and II GCT both show similar morphology, but are separated from each other by the detection of a GCNIS and an i12p in type II GCT. For type II GCT it is especially important to detect non‐seminomatous elements, as these tumours have a worse biological behaviour and need a different treatment to seminomas. In contrast to types I and II GCT, type III tumours are equivalent to spermatocytic tumours and usually occur in elderly men, with few exceptions in young adults. The development of types I and II GCT seems to depend not upon driver mutations, but rather on changes in the epigenetic landscape. Furthermore, different pluripotency associated factors (e.g. OCT3/4, SOX2, SOX17) play a crucial role in GCT development and can be used as immunohistochemical markers allowing to distinguish the different subtypes from each other in morphologically challenging tissue specimens. Especially in metastatic sites, a morphological and immunohistochemical diagnostic algorithm is important to detect small subpopulations of each non‐seminomatous GCT subtype, which are associated with a poorer prognosis and need a different treatment. Furthermore, primary extragonadal GCT of the retroperitoneum or mediastinum develop from misguided germ cells during embryonic development, and might be challenging to detect in small tissue biopsies due to their rarity at corresponding sites. This review article summarises the pathobiological and developmental aspects of the three different types of testicular GCT that can be helpful in the histopathological examination of tumour specimens by pathologists. [ABSTRACT FROM AUTHOR]

The incidence of beta-lactam resistance among clinical isolates is a major health concern. A key method to study the emergence of antibiotic resistance is adaptive laboratory evolution. However, in the case of the beta-lactam ampicillin, bacteria evolved in laboratory settings do not recapitulate clinical-like resistance levels, hindering efforts to identify major evolutionary paths and their dependency on genetic background. Here, we used the Microbial Evolution and Growth Arena (MEGA) plate to select ampicillin-resistant Escherichia coli mutants with varying degrees of resistance. Whole-genome sequencing of resistant isolates revealed that ampicillin resistance was acquired via a combination of single-point mutations and amplification of the gene encoding beta-lactamase AmpC. However, blocking AmpC-mediated resistance revealed latent adaptive pathways: strains deleted for ampC were able to adapt through combinations of changes in genes involved in multidrug resistance encoding efflux pumps, transcriptional regulators, and porins. Our results reveal that combinations of distinct genetic mutations, accessible at large population sizes, can drive high-level resistance to ampicillin even independently of beta-lactamases. Bacterial resistance to beta-lactam antibiotics is on the rise, but laboratory evolution studies do not always recapitulate clinical resistance levels. Here, the authors select Escherichia coli mutants with varying degrees of beta-lactam resistance, showing that combinations of distinct genetic mutations, accessible at large population sizes, can drive high-level resistance independently of beta-lactamases. [ABSTRACT FROM AUTHOR]

Background: Adult skeletal muscle contains resident muscle stem cells (MuSC) with high myogenic and engraftment potentials, making them suitable for cell therapy and regenerative medicine approaches. However, purification process of MuSC remains a major hurdle to their use in the clinic. Indeed, muscle tissue enzymatic dissociation triggers a massive activation of stress signaling pathways, among which P38 and JNK MAPK, associated with a premature loss of MuSC quiescence. While the role of these pathways in the myogenic progression of MuSC is well established, the extent to which their dissociation-induced activation affects the functionality of these cells remains unexplored. Methods: We assessed the effect of P38 and JNK MAPK induction on stemness marker expression and MuSC activation state during isolation by pharmacological approaches. MuSC functionality was evaluated by in vitro assays and in vivo transplantation experiments. We performed a comparative analysis of the transcriptome of human MuSC purified with pharmacological inhibitors of P38 and JNK MAPK (SB202190 and SP600125, respectively) versus available RNAseq resources. Results: We monitored PAX7 protein levels in murine MuSC during muscle dissociation and demonstrated a two-step decline partly dependent on P38 and JNK MAPK activities. We showed that simultaneous inhibition of these pathways throughout the MuSC isolation process preserves the expression of stemness markers and limits their premature activation, leading to improved survival and amplification in vitro as well as increased engraftment in vivo. Through a comparative RNAseq analysis of freshly isolated human MuSC, we provide evidence that our findings in murine MuSC could be relevant to human MuSC. Based on these findings, we implemented a purification strategy, significantly improving the recovery yields of human MuSC. Conclusion: Our study highlights the pharmacological limitation of P38 and JNK MAPK activities as a suitable strategy to qualitatively and quantitatively ameliorate human MuSC purification process, which could be of great interest for cell-based therapies. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis, the causative agent of tuberculosis, is a pathogenic bacterium that has claimed millions of lives since the Middle Ages. According to the World Health Organization's report, tuberculosis ranks among the ten deadliest diseases worldwide. The presence of an extensive array of genes and diverse proteins within the cellular structure of this bacterium has provided us with a potent tool for diagnosis. While the culture method remains the gold standard for tuberculosis diagnosis, it is possible that molecular diagnostic methods, emphasis on the identification of mutation genes (e.g., rpoB and gyrA) and single nucleotide polymorphisms, could offer a safe and reliable alternative. Over the past few decades, as our understanding of molecular genetics has expanded, methods have been developed based on gene expansion and detection. These methods typically commence with DNA amplification through nucleic acid targeted techniques such as polymerase chain reaction. Various molecular compounds and diverse approaches have been employed in molecular assays. In this review, we endeavor to provide an overview of molecular assays for the diagnosis of tuberculosis with their properties (utilization, challenges, and functions). The ultimate goal is to explore the potential of replacing traditional bacterial methods with these advanced molecular diagnostic techniques. [ABSTRACT FROM AUTHOR]

In this study, a microfluidic-based system utilizing colorimetric loop-mediated isothermal amplification (LAMP) is introduced for the quantitative analysis of nucleic acid targets. This system offers a user-friendly and cost-effective platform for the multiplexed genetic diagnosis of various infectious diseases across multiple samples. It includes time-lapse imaging equipment for capturing images of the microfluidic device during the LAMP assay and a hue-based quantitative analysis software to analyze the LAMP reaction, streamlining diagnostic procedures. An electric pipette was used to simplify the loading of samples and LAMP reagents into the device, allowing easy operation even by untrained individuals. The hue-based analysis software employs efficient image processing and post-processing techniques to calculate DNA amplification curves based on color changes in multiple reaction chambers. This software automates several tasks, such as identifying reaction chamber areas from time-lapse images, quantifying color information within each chamber, correcting baselines of DNA amplification curves, fitting experimental data to theoretical curves, and determining the threshold time for each curve. To validate the developed system, conventional off-chip LAMP assays were conducted with a 25 μL reaction mixture in 0.2 mL polymerase chain reaction (PCR) tubes using a real-time turbidimeter. The results indicated that the threshold time obtained using the colorimetric LAMP assay in the developed system is comparable to that obtained with real-time turbidity measurements in PCR tubes, demonstrating the system's capability for quantitative analysis of target nucleic acids, including those from human herpesviruses. [ABSTRACT FROM AUTHOR]

The DNA damage response is critical for maintaining genome integrity and is commonly disrupted in the development of cancer. PPM1D (protein phosphatase Mg2+/Mn2+-dependent 1D) is a master negative regulator of the response; gain-of- function mutations and amplifications of PPM1D are found across several human cancers making it a relevant pharmacological target. Here, we used CRISPR/Cas9 screening to identify synthetic-lethal dependencies of PPM1D, uncovering superoxide dismutase-1 (SOD1) as a potential target for PPM1D-mutant cells. We revealed a dysregulated redox landscape characterized by elevated levels of reactive oxygen species and a compromised response to oxidative stress in PPM1D-mutant cells. Altogether, our results demonstrate a role for SOD1 in the survival of PPM1D-mutant leukemia cells and highlight a new potential therapeutic strategy against PPM1D-mutant cancers. [ABSTRACT FROM AUTHOR]

The study aimed to develop a quantitative colorimetric loop-mediated isothermal amplification technique using the phenol red indicator (QLAMP-PhR) for detecting Fusobacterium nucleatum (Fn) levels in colorectal cancer (CRC) patients and healthy individuals. QLAMP-PhR assays were conducted on 251 stool samples specific for the Fn FadA gene. Six primers were synthesized and utilized with master mix reagents, and a phenol red indicator was employed to enhance the QLAMP-PhR technique. A standard quantitative analysis curve was generated using a logarithmic function (absorbance vs. concentration) by serially diluting the copy number of genomic DNA templates (Fn ATCC25586). The CRC group exhibited a significantly higher abundance of Fn compared to the healthy control group (P < 0.001). These findings suggest that the QLAMP-PhR technique effectively identifies Fn specifically by its gene for the key virulence factor FadA. Additionally, ideas for developing a real-time QLAMP-PhR test were presented. Compared to the traditional polymerase chain reaction (PCR) technique, QLAMP-PhR offers several advantages including rapidity, simplicity, specificity, sensitivity, and cost-effectiveness method that can quantitatively screen for Fn presence in normal populations. The QLAMP-PhR method represents a sensitive and specific amplification assay for the rapid detection of the Fn pathogen. To the best of our knowledge, this study is the first to report the application of QLAMP-PhR for detecting FadA in Fn. [ABSTRACT FROM AUTHOR]

Background: There is a growing demand for fast and reliable plant biomolecular analyses. DNA extraction is the major bottleneck in plant nucleic acid-based applications especially due to the complexity of tissues in different plant species. Conventional methods for plant cell lysis and DNA extraction typically require extensive sample preparation processes and large quantities of sample and chemicals, elevated temperatures, and multiple sample transfer steps which pose challenges for high throughput applications. Results: In a prior investigation, an ionic liquid (IL)-based modified vortex-assisted matrix solid phase dispersion approach was developed using the model plant, Arabidopsis thaliana (L.) Heynh. Building upon this foundational study, the present study established a simple, rapid and efficient protocol for DNA extraction from milligram fragments of plant tissue representing a diverse range of taxa from the plant Tree of Life including 13 dicots and 4 monocots. Notably, the approach was successful in extracting DNA from a century old herbarium sample. The isolated DNA was of sufficient quality and quantity for sensitive molecular analyses such as qPCR. Two plant DNA barcoding markers, the plastid rbcL and nuclear ribosomal internal transcribed spacer (nrITS) regions were selected for DNA amplification and Sanger sequencing was conducted on PCR products of a representative dicot and monocot species. Successful qPCR amplification of the extracted DNA up to 3 weeks demonstrated that the DNA extracted using this approach remains stable at room temperature for an extended time period prior to downstream analysis. Conclusions: The method presented here is a rapid and simple approach enabling cell lysis and DNA extraction from 1.5 mg of plant tissue across a broad range of plant taxa. Additional purification prior to DNA amplification is not required due to the compatibility of the extraction solvents with qPCR. The method has tremendous potential for applications in plant biology that require DNA, including barcoding methods for agriculture, conservation, ecology, evolution, and forensics. [ABSTRACT FROM AUTHOR]

Recent focus has been given to nanoparticles as an alternative fungicidal compound instead of chemical ones. More environmentally friendly ways of synthesis are the highest priority regarding the antifungal agents in the agriculture sector. Therefore, in this research, hyssop (H. officinalis) and sage (S. officinalis) aqueous extracts were prepared and used as a reducing source in the green synthesis of silver nanoparticles (AgNPs). Aqueous extracts and green synthesized AgNPs were examined for phytochemical composition and antioxidant capacity. Hyssop and sage extracts based AgNPs were analyzed using UV-vis spectrometry, SEM-EDS, and TEM-EDS. Antifungal activity against Fusarium spp. isolates collected from different infected crops was determined. Fusarium spp. isolates from strawberry, asparagus, pea, carrot, wheat, and rapeseed samples identified at the molecular level by translation elongation factor 1-alpha (TEF1α) gene amplification and sequencing. Green synthesized AgNPs had lower phytochemical content, however higher antioxidant activity compared to pure extracts. Both hyssop and sage extracts are suitable reducing agents for AgNPs formation, and sage extract results in larger particle size. Aqueous hyssop extract had higher antifungal activity than aqueous sage extract. However, a 10% concentration of whole sage extract based AgNPs solution, added to the PDA medium, and a 5% concentration of hyssop extract based AgNPs inhibited Fusarium spp. the most. F. proliferatum was the most sensitive to all treatments among the other fungi. [ABSTRACT FROM AUTHOR]

Introduction: Muscle invasive bladder cancer (MIBC) remains a prevalent cancer with limited therapeutic options, obviating the need for innovative therapies. The epidermal growth factor receptor (EGFR) is a linchpin in tumor progression and presents a potential therapeutic target in MIBC. Additionally, the EGFR ligands AREG and EREG have shown associations with response to anti-EGFR therapy and improved progression-free survival in colorectal carcinoma. Materials and methods: We investigated the prognostic significance of EGFR, AREG, and EREG in MIBC. Gene expression and copy number analyses were performed via qRT-PCR on tissue samples from 100 patients with MIBC who underwent radical cystectomy at the University Hospital Mannheim (MA; median age 72, interquartile range [IQR] 64–78 years, 25% female). Results were validated in 361 patients from the 2017 TCGA MIBC cohort (median age 69, IQR 60–77 years, 27% female), in the Chungbuk and MDACC cohort. Gene expressions were correlated with clinicopathologic parameters using the Mann-Whitney test, Kruskal-Wallis- test and Spearman correlation. For overall survival (OS), cancer-specific survival (CSS) and disease-free survival (DFS) gene expression was analyzed with Kaplan-Meier and Cox-proportional hazard models. Results: Significant gene expression differences in EGFR, AREG, and EREG could be detected in all cohorts. In the TCGA cohort, EGFR expression was significantly elevated in patients with EGFR amplification and KRAS wildtype. High AREG expression independently predicted longer OS (HR = 0.35, CI 0.19 - 0.63, p = 0.0004) and CSS (HR = 0.42, CI 0.18 – 0.95, p = 0.0378) in the MA cohort. In the TCGA cohort, high EGFR, AREG, and EREG expression correlated with shorter OS (AREG: HR = 1.57, CI 1.12 – 2.20, p = 0.0090) and DFS (EGFR: HR = 1.91, CI 1.31 – 2.8, p = 0.0008). EGFR amplification was also associated with reduced DFS. Discussion: High EGFR and EREG indicate worse survival in patients with MIBC. The prognostic role of AREG should further be investigated in large, prospective series. Divergent survival outcomes between the four cohorts should be interpreted cautiously, considering differences in analysis methods and demographics. Further in vitro investigations are necessary to elucidate the functional mechanisms underlying the associations observed in this study. [ABSTRACT FROM AUTHOR]

Flow cytometry can provide detailed information about protein expression on cell surface and is, therefore, widely used in clinical testing. However, owing to the limited sensitivity of fluorescence signals, detection of low‐expression cell surface markers is challenging. The present report describes a DNA‐mediated, on‐membrane assembly of conjugated polymer nanoparticles (Pdots) that amplifies fluorescence signal from surface markers for sensitive detection via flow cytometry. Single‐stranded DNA (ssDNA)‐conjugated antibodies are first bound to cell surface markers, from which ssDNA‐modified Pdots are sequentially assembled using DNA hybridization. The use of DNA as a linker enables the distance‐controlled assembly of Pdots to prevent fluorescence quenching, whereas their on‐membrane sequential assembly allows amplification of the fluorescence signal without reducing binding ability of antibodies. Thus, two rounds of Pdot assembly achieve 31‐fold amplification of the fluorescence signal from CD19 on Nalm‐6 cells, which is 125‐fold brighter than that obtained using the conventional fluorescent dye‐based method. Moreover, the sequential assembly of 22 nm Pdots shows 24‐fold higher fluorescence than one‐step labeling with 81 nm Pdots, suggesting the advantage of the sequential assembly strategy in avoiding steric hindrance. The proposed method is expected to contribute to the sensitive detection of low‐expression surface markers for early and accurate diagnosis. [ABSTRACT FROM AUTHOR]

It is now understood that the flatfish farming industry faces significant threats from Chinook salmon bafinivirus (CSBV) induced hemorrhagic disease. Developing a prompt and efficient diagnostic method is crucial for effective disease control and minimizing losses in aquaculture production. This research assessed the loop-mediated isothermal amplification (LAMP) technique, using a specific primer set, for rapid and precise detection of CSBV. The primer set for LAMP detection was selected based on its demonstrated high specificity and sensitivity, as confirmed through visualization and gel electrophoresis. The CSBV-LAMP reaction system operated at an effective temperature of 63–67°C for 60 min. Importantly, the LAMP assay could identify viral genomic cDNA at an extremely low concentration of 10 pg/μL within 60 min at 65°C. Moreover, this reaction system accurately detected CSBV in naturally infected flatfish samples, yielding a 100% positive detection rate. [ABSTRACT FROM AUTHOR]

Collective behaviors efficiently impart benefits to a diversity of species ranging from bacteria to humans. Fly larvae tend to cluster and form coordinated digging groups under crowded conditions, yet understanding the rules governing this behavior is in its infancy. We primarily took advantage of the Drosophila model to investigate cooperative foraging behavior. Here, we report that Drosophila‐related species and the black soldier fly have evolved a conserved strategy of cluster digging in food foraging. Subsequently, we investigated relative factors, including larval stage, population density, and food stiffness and quality, that affect the cluster digging behavior. Remarkably, oxygen supply through the posterior breathing spiracles is necessary for the organization of digging clusters. More importantly, we theoretically devise a mathematical model to accurately calculate how the cluster digging behavior expands food resources by diving depth, cross‐section area, and food volume. We found that cluster digging behavior approximately increases 2.2 fold depth, 1.7‐fold cross‐section area, and 1.9 fold volume than control groups, respectively. Amplification of food sources significantly facilitates survival, larval development, and reproductive success of Drosophila challenged with competition for limited food resources, thereby conferring trophic benefits to fitness in insects. Overall, our findings highlight that the cluster digging behavior is a pivotal behavior for their adaptation to food scarcity, advancing a better understanding of how this cooperative behavior confers fitness benefits in the animal kingdom. [ABSTRACT FROM AUTHOR]

Key message: In our study, we discovered a fragment duplication autoregulation mechanism in 'ZS-HY', which may be the reason for the phenotype of red foliage and red flesh in grapes. In grapes, MYBA1 and MYBA2 are the main genetic factors responsible for skin coloration which are located at the color loci on chromosome 2, but the exact genes responsible for color have not been identified in the flesh. We used a new teinturier grape germplasm 'ZhongShan-HongYu' (ZS-HY) which accumulate anthocyanin both in skin and flesh as experimental materials. All tissues of 'ZS-HY' contained cyanidin 3-O-(6″-p-coumaroyl glucoside), and pelargonidins were detected in skin, flesh, and tendril. Through gene expression analysis at different stage of flesh, significant differences in the expression levels of VvMYBA1 were found. Gene amplification analysis showed that the VvMYBA1 promoter is composed of two alleles, VvMYBA1a and 'VvMYBA1c-like'. An insertion of a 408 bp repetitive fragment was detected in the allele 'VvMYBA1c-like'. In this process, we found the 408 bp repetitive fragment was co-segregated with red flesh and foliage phenotype. Our results revealed that the 408 bp fragment replication insertion in promoter of 'VvMYBA1c-like' was the target of its protein, and the number of repeat fragments was related to the increase of trans-activation of VvMYBA1 protein. The activation of promoter by VvMYBA1 was enhanced by the addition of VvMYC1. In addition, VvMYBA1 interacted with VvMYC1 to promote the expression of VvGT1 and VvGST4 genes in 'ZS-HY'. The discovery of this mutation event provides new insights into the regulation of VvMYBA1 on anthocyanin accumulation in red-fleshed grape, which is of great significance for molecular breeding of red-fleshed table grapes. [ABSTRACT FROM AUTHOR]

Anaplasma and Ehrlichia are tick-borne bacterial pathogens that cause anaplasmoses and ehrlichioses in humans and animals. In this study, we examined the prevalence of Anaplasma and Ehrlichia species in ticks and domesticated animals in Suizhou County, Hubei Province in the central China. We used PCR amplification and DNA sequencing of the 16S rRNA, groEL, and gltA genes to analyze. We collected 1900 ticks, including 1981 Haemaphysalis longicornis and 9 Rhipicephalus microplus, 159 blood samples of goats (n = 152), cattle (n = 4), and dogs (n = 3) from May to August of 2023. PCR products demonstrated that Anaplasma bovis, Anaplasma capra, and an Ehrlichia species were detected in the H. longicornis with the minimum infection rates (MIR) of 1.11%, 1.32%, and 0.05%, respectively; A. bovis, A. capra, and unnamed Anaplasma sp. were detected in goats with an infection rate of 26.31%, 1.31% and 1.97%, respectively. Anaplasma and Ehrlichia species were not detected from cattle, dogs and R. microplus ticks. The genetic differences in the groEL gene sequences of the Anaplasma in the current study were large, whereas the 16S rRNA and gltA gene sequences were less disparate. This study shows that ticks and goats in Suizhou County, Hubei Province carry multiple Anaplasma species and an Ehrlichia species, with relatively higher infection rate of A. bovis in goats. Our study indicates that multiple Anaplasma and Ehrlichia species exist in ticks and goats in the central China with potential to cause human infection. [ABSTRACT FROM AUTHOR]

The human hepatitis delta virus (HDV) is a satellite RNA virus that depends on hepatitis B virus (HBV) surface proteins (HBsAg) to assemble into infectious virions targeting the same organ (liver) as HBV. Until recently, the evolutionary origin of HDV remained largely unknown. The application of bioinformatics on whole sequence databases lead to discoveries of HDV-like agents (DLA) and shed light on HDV's evolution, expanding our understanding of HDV biology. DLA were identified in heterogeneous groups of vertebrates and invertebrates, highlighting that the evolution of HDV, represented by eight distinct genotypes, is broader and more complex than previously foreseen. In this study, we focused on the characterization of three mammalian DLA discovered in woodchuck (Marmota monax), white-tailed deer (Odocoileus virginianus), and lesser dog-like bat (Peropteryx macrotis) in terms of replication, cell-type permissiveness, and spreading pathways. We generated replication-competent constructs expressing 1.1-fold over-length antigenomic RNA of each DLA. Replication was initiated by transfecting the cDNAs into human (HuH7, HeLa, HEK293T, A549) and non-human (Vero E6, CHO, PaKi, LMH) cell lines. Upon transfection and replication establishment, none of the DLA expressed a large delta antigen. A cell division-mediated viral amplification assay demonstrated the capability of non-human DLA to replicate and propagate in hepatic and non-hepatic tissues, without the requirement of envelope proteins from a helper virus. Remarkably L-HDAg but not S-HDAg from HDV can artificially mediate envelopment of WoDV and DeDV ribonucleoproteins (RNPs) by HBsAg to form infectious particles, as demonstrated by co-transfection of HuH7 cells with the respective DLA expression constructs and a plasmid encoding HBV envelope proteins. These chimeric viruses are sensitive to HDV entry inhibitors and allow synchronized infections for comparative replication studies. Our results provide a more detailed understanding of the molecular biology, evolution, and virus–host interaction of this unique group of animal viroid-like agents in relation to HDV. [ABSTRACT FROM AUTHOR]

We report on single case of intraplacental choriocarcinoma (IC) coexisting with feto-maternal hemorrhage from our hospital, a rare malignant tumor that occurs in the chorionic villous trophoblast. To investigate genetic and epigenetic changes to the carcinogenesis of IC, we employed cancer gene panel analysis and whole methylation analysis from a recent case of IC. By Short Tandem Repeats analysis, we confirmed that the tumor of present IC was derived from concurrent normal chorionic villous trophoblast cells. No mutation was found in 145 cancer-related genes. Meanwhile, amplification in MDM2 gene was observed. Furthermore, we observed deferentially methylated CpG sites between tumor and surrounding normal placenta in present IC case. These observations suggest that IC might be arisen as a result of aberrations of methylation rather than of DNA mutations. Further studies are needed to clarify association between aberrant methylation and choriocarcinogenesis. [ABSTRACT FROM AUTHOR]

Introduction: Our goal was to investigate the antimicrobial resistance due to beta-lactamase genes and virulent determinants (biofilm-forming ability) expressed by Acinetobacter collected from health settings in Pakistan. A cross-sectional study was conducted for the molecular characterization of carbapenemases and biofilm-producing strains of Acinetobacter spp. Methodology: Two twenty-three imipenem-resistant Acinetobacter isolates were analyzed from 2020 to 2023.The combination disk test and modified hodge test were performed. Biofilm forming ability was determined by polystyrene tube assay. Multiplex polymerase chain reaction (PCR) for virulent and biofilm-forming genes, and 16S rRNA sequencing were performed. Results: 118 (52.9%) carbapenem-resistant Acinetobacter (CR-AB) were isolated from wounds and pus, 121 (54.2%) from males, and 92 (41.2%) from 26-50-years-olds. More than 80% of strains produced β-lactamases and carbapenemases. Based on the PCR amplification of the ITS gene, 174 (78.0%) CR-AB strains were identified from CR-Acinetobacter non-baumannii (ANB). Most CR-AB were strong and moderate biofilm producers. Genetic analysis revealed the blaOXA-23, blaTEM, blaCTX-M blaNDM-1 and blaVIM were prevalent in CR-AB with frequencies 91 (94.8%), 68 (70.8%), 19 (19.7%), 53 (55.2%), 2 (2.0%) respectively. Among virulence genes, OmpA was dominant in CR-AB isolates from wound (83, 86.4%), csuE 63 (80.7%) from non-wound specimens and significantly correlated with blaNDM and blaOXA genes. Phylogenetic analysis revealed three different clades for strains based on specimens. Conclusions: CR-AB was highly prevalent in Pakistan and associated with wound infections. The genes, blaOXA-23, blaTEM, blaCTX-M, and blaNDM1 were detected in CR-AB. Most CR-AB were strong biofilm producers with virulent genes OmpA and csuE. [ABSTRACT FROM AUTHOR]

Introduction: Mycetoma is a chronic granulomatous inflammatory disease of the subcutaneous tissue, which affects deep structures and bone. Most cases of actinomycetoma are caused by members of the genus Nocardia. Case presentation: Here we report the case of a 43-year-old male who presented a disseminated mycetoma on the forearm, chest and neck, characterized by enlarged and erythematous lesions through which seropurulent material drains, and numerous atrophic scars. Molecular identification was performed by 16S gene amplification and sequencing. Nocardia mexicana was identified with 100% identity. Trimethoprimsulfamethoxazole, diaminodiphenyl sulfone and amikacin was a successful treatment after 6 months. Conclusions: Nocardia mexicana is a rare organism that causes mycetoma. We report a case of extensive mycetoma on the forearm with spread to the neck and thorax associated with manipulation of the mouth of a calf. [ABSTRACT FROM AUTHOR]

To study the expression of HER2 in high-grade FIGO3 endometrial endometroid carcinoma (EEC) and to correlate our findings with the clinicopathologic characteristics of this tumor. HER2 expression by immunohistochemistry (IHC) was performed on 10% formalin-fixed paraffin embedded tissue on cases diagnosed as FIGO3 EEC. HER2 expression was interpreted as negative (0), low (1+ and 2+) or positive (3+) using similar criteria as in the breast. HER2 amplification by Fluorescence in situ hybridization (FISH) was performed on cases interpreted as 2+ and 3+ by IHC. One hundred and forty-three FIGO3 EEC were identified. Of these, 70 (49%) cases were HER2 negative (IHC 0), and 73 (51%) cases expressed/amplified HER2 by IHC and/or FISH. Of the 73 cases expressing or amplifying HER2, 59 cases were IHC 1+, 12 cases were IHC 2+, and 2 cases were IHC 3+. FISH testing was performed in 12 cases. Only one of the two HER2 IHC 3+ cases showed HER2 gene amplification by FISH and the other 11 cases were not amplified. The 5-year overall survival (OS) rate for HER2 IHC 1+ cases was 92.20% (95% CI: 83.97–100.00), and the 5-year OS rate for HER2 IHC 2+/3+ cases was 89.50% (95% CI: 56.41–100.00). Our findings indicate that about one half of FIGO3 EEC variably expresses HER2 and with the emerging concept of "HER2 low", anti-HER2 agents may be explored as potential therapeutic options in these patients, for possible survival benefits. [Display omitted] • About 50% of FIGO3 endometrial endometrioid carcinomas (EEC) express the HER2 protein with varying intensities. • Amplification or overexpression of HER2 in FIGO3 EEC is very low. • The therapeutic implication of low-level expression of HER2 in FIGO3 EEC is largely unknown. • Anti-HER2 therapy might be explored as a treatment option in low-level HER2 expression in FIGO3 EEC with potential benefits. [ABSTRACT FROM AUTHOR]

The identification process at the crime scene (TKP) is one of the essential tasks carried out by police investigators. One critical aspect is the collection and analysis of evidence found at the crime scene, referred to as biological evidence (BB). This evidence serves as a crucial source for determining whether an incident occurred and, if so, whether it was a suicide or another criminal act (such as murder, etc.). The biological evidence found can be analyzed using molecular serobiology approaches, which are rapidly advancing in the fields of forensic medicine and medicolegal practice. One such approach involves utilizing DNA traces, although a common challenge is that often the DNA recovered at crime scenes is degraded. Therefore, a DNA amplification method utilizing the Polymerase Chain Reaction (PCR) technique, along with the short tandem repeat (STR) – CODIS identification approach, is necessary. The analysis of DNA traces using the STR-CODIS approach enables investigators to determine whether the incident was purely criminal, a suicide, or another type of accident involving the victim. Furthermore, the DNA analysis using the STR-CODIS method can also aid in identifying the victims discovered at the crime scene. [ABSTRACT FROM AUTHOR]

Background: The aim of this study was to determine whether mammographic and sonographic features of malignant breast lesions are correlated with tumor histologic grade, hormonal receptor, human epidermal growth factor receptor 2 (HER2), and Ki-67 status. Materials and Methods: In this retrospective study, imaging and histopathological findings of 187 biopsy-proven breast cancer cases from November 2019 to February 2021 were reviewed. The Chi-square test was used to examine the potential correlation between mammographic and sonographic characteristics with histopathological features such as hormonal receptor, HER2 status, Ki-67 labeling index, and histological grade. Results: We observed that microlobulated margin as well as oval/round morphology in mammograms correlate with triple-negative intrinsic subtype (P = 0.006 and P = 0.004). The presence of calcification in sonography was significantly higher in the luminal-B subtype (P = 0.002). Furthermore, ill-defined margins in mammography were significantly higher in amplified HER2 expression (P = 0.004) in the same manner as an oval/round shape in higher levels of Ki-67 (P = 0.030). Conclusion: Mammography and sonography features may reflect the biological behavior of various subtypes of breast cancer and can detect more aggressive breast cancers that can mimic benign or less malignant appearing lesions. These findings may be an excellent predictor for some subtypes like triple-negative breast cancer. Studying the range of these imaging characteristics may help in better understanding the prognosis, choosing a treatment strategy, and predicting response to treatment. [ABSTRACT FROM AUTHOR]

Six popular and widely cultivated arabica coffee (Coffea arabica L.) varieties of commercial importance namely Selection 5B, Selection 13, Selection 11, Selection 8, Selection 7.3 and Selection 3 were tested for their genetic identity with ISSR markers. Fifteen ISSR primers were tested using genomic DNA of selected coffee varieties. Pooled genomic DNA of all the six varieties was amplified with each ISSR primer with an average of four loci per primer. The size range of locus amplified by all the fifteen primers was ranging from 100 to 1200 bp depending upon on the ISSR primers. Only three out of fifteen primers, namely ISSR4, ISSR6 and ISSR8, were screened based on the number of amplified locus and size range from low to high. The selective ISSR primers distinguished all the six varieties of coffee with unique markers. ISSR 4 amplified two unique markers with a locus size 1300 bp and 950 bp for Sln.5B and 180 bp and 150 bp for Sln.13. ISSR6 had produced five varietal-specific markers with a locus size of 180 bp in Sln.5B, 1250 bp in Sln.11, 350 bp in Sln.3. ISSR8 had amplified seven unique loci across the coffee varieties with 700 bp and 800 bp in Sln.5B, 200 bp and 500 bp in Sln.11 and one locus each in Sln.7.3 and Sln.3 with 300 bp and 150 bp respectively. Repeated amplification of genomic DNA of all the six varieties of coffee with selective ISSR primers produced consistent ISSR genetic fingerprints. Selective ISSR primers were validated with marker parameters resolving power (RP), effective multiplex ratio (EMR), marker index (MI) and polymorphic information content (PIC). Utilisation of these markers in arabica coffee genetic improvement is discussed. [ABSTRACT FROM AUTHOR]

The common deleterious genetic defects in Holstein cattle include haplotypes 1–6 (HH1–HH6), haplotypes for cholesterol deficiency (HCD), bovine leukocyte adhesion deficiency (BLAD), complex vertebral malformation (CVM) and brachyspina syndrome (BS). Recessive inheritance patterns of these genetic defects permit the carriers to function normally, but homozygous recessive genotypes cause embryo loss or neonatal death. Therefore, rapid detection of the carriers is essential to manage these genetic defects. This study was conducted to develop a single‐tube multiplex fluorescent amplification–refractory mutation system (mf‐ARMS) PCR method for efficient genotyping of these 10 genetic defects and to compare its efficiency with the kompetitive allele specific PCR (KASP) genotyping assay. The mf‐ARMS PCR method introduced 10 sets of tri‐primers optimized with additional mismatches in the 3′ end of wild and mutant‐specific primers, size differentiation between wild and mutant‐specific primers, fluorescent labeling of universal primers, adjustment of annealing temperatures and optimization of primer concentrations. The genotyping of 484 Holstein cows resulted in 16.12% carriers with at least one genetic defect, while no homozygous recessive genotype was detected. This study found carrier frequencies ranging from 0.0% (HH6) to 3.72% (HH3) for individual defects. The mf‐ARMS PCR method demonstrated improved detection, time and cost efficiency compared with the KASP method for these defects. Therefore, the application of mf‐ARMS PCR for genotyping Holstein cattle is anticipated to decrease the frequency of lethal alleles and limit the transmission of these genetic defects. [ABSTRACT FROM AUTHOR]

• Comprehensive analysis of prostate cancer samples and cell lines to explore telomeres and associated genes as prognostic biomarkers. • Despite few genetic alterations in localized disease, there is an increase in amplifications in the analyzed genes in metastatic disease. • Upregulation of shelterin and CST genes correlates with a more aggressive form of localized prostate cancer, despite limited genetic alterations. • Longer telomeres are observed in aggressive disease, indicating that increased expression of telomere-related genes could be an early biomarker for prostate cancer aggressiveness. Search for new clinical biomarkers targets in prostate cancer (PC) is urgent. Telomeres might be one of these targets. Telomeres are the extremities of linear chromosomes, essential for genome stability and control of cell divisions. Telomere homeostasis relies on the proper functioning of shelterin and CST complexes. Telomeric dysfunction and abnormal expression of its components are reported in most cancers and are associated with PC. Despite this, there are only a few studies about the expression of the main telomere complexes and their relationship with PC progression. We aimed to evaluate the role of shelterin (POT1, TRF2, TPP1, TIN2, and RAP1) and CST (CTC1, STN1, and TEN1) genes and telomere length in the progression of PC. We evaluated genetic alterations of shelterin and CST by bioinformatics in samples of localized (n = 499) and metastatic castration-resistant PC (n = 444). We also analyzed the expression of the genes using TCGA (localized PC n = 497 and control n = 152) and experimental approaches, with surgical specimens (localized PC n = 81 and BPH n = 10) and metastatic cell lines (LNCaP, DU145, PC3 and PNT2 as control) by real-time PCR. Real-time PCR also determined the telomere length in the same experimental samples. All acquired data were associated with clinical parameters. Genetic alterations are uncommon in PC, but POT1, TIN2, and TEN1 showed significantly more amplifications in the metastatic cancer. Except for CTC1 and TEN1, which are differentially expressed in localized PC samples, we did not detect an expression pattern relative to control and cell lines. Nevertheless, except for TEN1, the upregulation of all genes is associated with a worse prognosis in localized PC. We also found that increased telomere length is associated with disease aggressiveness in localized PC. The upregulation of shelterin and CST genes creates an environment that favors telomere elongation, giving selective advantages for localized PC cells to progress to more aggressive stages of the disease. [ABSTRACT FROM AUTHOR]

Primary cardiac malignancies are rare, with cardiac sarcomas being the main type. Among these, intimal sarcomas are the most common. However, they tend to occur in the great vessels and are rare in the heart, with only a few isolated cases reported. We report a challenging case of a patient with left atrial intimal sarcoma with rhabdomyosarcoma differentiation. The patient was admitted after a physical examination detected left heart occupancy, and initial imaging suspected a left atrial thrombus. The patient then underwent extracorporeal circulation-assisted open cardiac surgery with resection of an atrial mass. The postoperative pathological findings were suggestive of an arterial intimal sarcoma, which included areas of rhabdomyosarcoma differentiation within the tumor tissue. Unfortunately, the patient's tumor recurred 4 months later, and she died due to treatment failure. This case highlights the rarity and risk of misdiagnosis of cardiac intimal sarcoma. Additionally, we aim to improve the understanding of intimal sarcoma through a review of immunohistochemistry and gene amplification techniques. [ABSTRACT FROM AUTHOR]

Vibrio parahaemolyticus (V. parahaemolyticus) is one of the important seafood-borne pathogens that cause a serious gastrointestinal disorder in humans. Recently, biosensors have attracted serious attention for precisely detecting and tracking risk factors in foods. However, a major consideration when fabricating biosensors is to match the low cost of portable devices to broaden its application. In this study, a 3D-printed integrated handheld biosensor (IHB) that combines RPA-CRISPR/Cas12a, a lateral flow strip (LFS), and a handheld device was developed for the ultrasensitive detection of V. parahaemolyticus. Using the preamplification of RPA on tlh gene of V. parahaemolyticus, a specific duplex DNA product was obtained to activate the trans-cleavage activity of CRISPR/Cas12a, which was then utilized to cleave the ssDNA probe. The ssDNA probe was then detected by the LFS, which was negatively correlated with the content of amplified RPA products of the tlh gene. The IHB showed high selectivity and excellent sensitivity for V. parahaemolyticus detection, and the limit of detection was 4.9 CFU/mL. The IHB also demonstrated great promise for the screening of V. parahaemolyticus in samples and had the potential to be applied to the rapid screening of other pathogen risks for seafood and marine environmental safety. [ABSTRACT FROM AUTHOR]