Gene Amplification Research Articles

Pan-cancer analysis reveals copper transporters as promising potential targets

BackgroundCopper transport proteins (SLC31A1, ATP7A, ATP7B) regulate copper levels in the body and may be involved in tumor development. However, their comprehensive expression and function across various cancers remain unclear. MethodsThe expressions of copper transporters in 33 tumors and normal tissues were analyzed using TCGA, GTEx, CCLE, ULCAN, and HPA databases. Cox regression assessed their impact on patient survival. Gene alterations were explored using cBioPortal. Spearman correlation tests were performed to investigate the associations between copper transporters and tumor mutation burden (TMB), microsatellite instability (MSI), and infiltration of immune cells. Gene functions were analyzed using STRING and GeneMANIA databases. Drug sensitivity was assessed using GSCALite database. ATP7B expression in lung squamous cell carcinoma (LUSC) was validated by immunohistochemical staining. ResultsCopper transporters exhibited variable expression patterns across various cancer types, indicating their potential dual role as either oncogenes or tumor suppressor genes, depending on the cancer type. Significant associations were found between these transporters and tumor stage, as well as prognosis in most tumors studied. Pathway analysis identified links between copper transporters and tumor-related pathways like apoptosis and RAS/MAPK. Copy number variation (CNV) analysis revealed varying degrees of gene amplification and deletion of copper transporters in most tumors. Copper transporters exhibited strong correlations with immune features, including TMB, MSI, and immune-infiltrating cells, suggesting their potential role in guiding immunotherapy. They were also associated with sensitivity to various chemotherapeutic and immunotherapeutic drugs. Immunohistochemical tests validated the correlation between elevated ATP7B level and worse progression-free survival (PFS) in LUSC. ConclusionCopper transporters may serve as potential tumor markers and therapeutic targets.

Methicillin-Resistant Staphylococcus aureus (MRSA) in Different Food Groups and Drinking Water.

Methicillin-resistant Staphylococcus aureus (MRSA) has been included by the World Health Organization in its list of "priority pathogens" because of its widespread prevalence and the severity of the infections it causes. The role of food in infections caused by MRSA is unknown, although strains of this microorganism have been detected in various items for human consumption. In order to gain an overview of any possible role of food in MRSA infections, a review was undertaken of studies published between January 2001 and February 2024 relating to MRSA. These comprised research that focused on fish and shellfish, eggs and egg products, foods of vegetable origin, other foodstuffs (e.g., honey or edible insects), and drinking water. In most of these investigations, no prior enrichment was carried out when isolating strains. Three principal methods were used to confirm the presence of MRSA, namely amplification of the mecA gene by PCR, amplification of the mecA and the mecC genes by PCR, and disc diffusion techniques testing susceptibility to cefoxitin (30 μg) and oxacillin (1 μg). The great diversity of methods used for the determination of MRSA in foods and water makes comparison between these research works difficult. The prevalence of MRSA varied according to the food type considered, ranging between 0.0% and 100% (average 11.7 ± 20.3%) for fish and shellfish samples, between 0.0% and 11.0% (average 1.2 ± 3.5%) for egg and egg products, between 0.0% and 20.8% (average 2.5 ± 6.8%) for foods of vegetable origin, between 0.6% and 29.5% (average 28.2 ± 30.3%) for other foodstuffs, and between 0.0% and 36.7% (average 17.0 ± 14.0%) for drinking water.

PCR identification of Enterococcus Species as a Primary Pathogen in Neonatal Bloodstream Infections at Damascus University Paediatric Hospital

Bacterial septicaemia is one of the most common causes of death in newborns throughout the globe. Recently, gram positive bacteria have been detected as a main responsible of hospital acquired blood infection in developed country especially Enterococcus sp. and Staphylococcus sp. This preliminary study aimed to identify Enterococcus species and Staphylococcus species isolated form acquired blood infection in the department of premature and neonates at paediatric university hospital in Damascus, based on PCR technology. the antibiotic sensitivity these pathogens was also investigated against common antibiotics usually used to treat septicaemia in this hospital. Bacterial gDNA was extracted directly from blood samples of children with acquired Septicemia. The bacterial infection was confirmed in 50 samples based on the amplification of 16srDNA gene. Genus Enterococcus sp. and Staphylococcus species were identified as source of infection in samples using specific primers via PCR technique. The antibiotic sensitivity of isolated bacterial strains was studied using Kirby-Bauer disc method. Our study showed that 47 of the 50 samples of the infection were bacterial. The rate of blood infection caused by Enterococcus species was 24%, and 4% for Staphylococcus sp. the isolated bacteria showed a high sensitivity to Amikacin, medium sensitivity to Vancomycin, and less sensitivity to Ampicillin and Gentamycin. Enterococcus species Showed good sensitivity to Amikacin and Vancomycin and low sensitivity to Gentamycin, while was resistant to Ampicillin. Furthermore, the two isolated stains Enterococcus sp and Staphylococcus species showed a good sensitivity against chlorhexidine. This study confirmed the importance of molecular technologies in diagnosing the acquired septicaemia and identifying the specific infectious agent. The molecular test showed that Enterococcus sp. was an important cause of acquired blood infection in neonates respect to ampicillin resistant staphylococcus species.

DNA Extraction and PCR Optimization of Coffea arabica L. and Coffea canephora Pierre ex A. Froehner

DNA isolation is an important step for further molecular analysis. Coffee plants contain polysaccharides, polyphenols, and secondary metabolites which can contaminate the results of DNA isolation. This study was conducted to isolate DNA from arabica and robusta coffee leaves using a modified CTAB method and PCR optimization for amplification of matK and rbcL genes. DNA was isolated using buffers (CTAB 10%, PVP 1%, β-mercaptoethanol 1%, Tris-HCl 1M, NaCl 5M, and EDTA 0.5%), and eluted with TE-RNase. Genomic DNA of ten coffee plants was successfully isolated with concentrations ranging from 33-146 nm/µL with purity (A260/A280) 1.7-1.9. Based on the genomic DNA isolated, matK and rbcL genes were amplified with initial denaturation conditions of 94°C (for 1 minute of matK and 4 minutes of rbcL) followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing (adjusted to each primer), and extension at 72°C for 1 minute. The optimal PCR conditions were effective to amplify matK (900 bp) and rbcL (600 bp) genes. Thus, the modified DNA isolation method and PCR optimization can be used as an efficient tool for further molecular analysis of Coffea arabica L. and Coffea canephora Pierre ex A. Froehner.

Multiple-Locus Variable-Number Tandem Repeat Analysis of Helicobacter Pylori Strains Isolated from Biopsy Samples

Background: Multiple-locus variable-number tandem-repeat analysis (MLVA) is a reliable, fast, and useful method for conducting phylogenetic and genotyping analyses. However, its application in Helicobacter pylori typing is limited. Objectives: This study aimed to determine the genetic diversity of H. pylori strains isolated from biopsy samples using six variable loci in MLVA typing. Methods: Over one year, we collected 70 non-duplicative biopsy samples from patients at Rohani Hospital, Babol, in northern Iran. After DNA extraction, H. pylori strains were confirmed through the amplification of the glmM gene in the PCR test. Multiple-locus variable-number tandem-repeat analysis typing utilized six VNTR markers: VNTR-180, VNTR-263, VNTR-614, VNTR-607, VNTR-2181, and VNTR-2457. The PHYLOViZE software was employed to construct the phylogenetic tree. Results: Out of the 70 specimens, 43 tested positive for H. pylori. The phylogenetic tree revealed some strains with completely similar MLVA patterns. Our findings indicated that type 2 is the dominant type in this region, with most patients infected with this type exhibiting gastritis. Conclusions: The high allelic variability observed at VNTR-614, -607, and -2457 indicates high intraspecific variability for these markers in all isolates from our region. Consequently, these VNTRs can be considered ideal sites for typing.

Molecular diagnosis and phylogenetic insights of Eimeria species infecting buffaloes (Bubalus bubalis) in Meghalaya's subtropical hilly region, India

Coccidiosis is an intestinal infection caused by Eimeria spp. that results in economic losses owing to morbidity and mortality in young buffalo calves. This study aimed for molecular diagnosis and phylogenetic analysis of Eimeria spp. in buffaloes of Meghalaya's sub-tropical mountainous terrain. Fresh buffaloes' fecal samples were collected from buffalo farms of Umling, Umsning and Bhoirymbong blocks, Ri Bhoi, Meghalaya and screened for Eimeria oocysts using flotation and modified McMaster methods. Fecal sample examination revealed 27.44 % (87/317) infection in buffaloes. Age wise, 64.44 % (29/45), 25.35 % (36/142) and 16.92 % (22/130) infections were recorded in <6 months, 6 months to 1 year and 1–2 year old buffaloes, respectively. Morphological characterization of Eimeria spp. revealed E. bovis (21.83 %), E. bareillyi (18.39 %), E. zuernii (11.49 %), E. ellipsoidalis (3.44 %) and mixed infection (44.82 %). Amplification of ITS-1 gene confirmed Eimeria spp. (410 bp), E. bovis (238 bp) and E. zuernii (344 bp). Phylogenetic analysis of E. bovis Umiam isolate revealed that these were closely related to the E. bovis isolate from South Korea (MH245198.1), and Turkey (KU351711.1) and distantly related to the isolates from Jammu and Kashmir (OQ103422.1) and Uttar Pradesh, Mathura (OK486542.1). E. zuernii isolate from Umiam, Meghalaya was observed to be phylogenetically close to the isolates from South Korea (MH245202.1), Japan (LC171339.1) and Turkey (KU351715.1), whereas phylogenetic divergence was observed between, E. zuernii isolate from Umiam, Meghalaya with isolates of Andhra Pradesh, Tirupati (MN601278.1) and Jammu and Kashmir (OQ103424.1). Therefore, treatment and effective control strategies should be implemented immediately to prevent spread of infection in the buffaloes.

EGFR mutations and abnormal trafficking in cancers.

Epidermal growth factor receptor (EGFR) is a transmembrane tyrosine kinase receptor and a member of the ErbB receptor family. As a significant cancer driver, EGFR undergoes mutations such as gene amplification or overexpression in a wide range of malignant tumors and is closely associated with tumorigenesis. This review examines the aberrant expression of EGFR in several common cancers and summarizes the current therapeutic strategies developed for this receptor. Additionally, this review compares the differences in EGFR activation, internalization, endocytosis, and sorting in normal and cancer cells, and highlights some regulatory factors that influence its trafficking process.Kindly check and confirm the edit made in the title.Yes, correctAs per journal instructions structured abstract is mandatory kindly provideThe abstract format does not apply to Review articles.

The DNA repair pathway as a therapeutic target to synergize with trastuzumab deruxtecan in HER2-targeted antibody–drug conjugate–resistant HER2-overexpressing breast cancer

BackgroundAnti-HER2 therapies, including the HER2 antibody–drug conjugates (ADCs) trastuzumab emtansine (T-DM1) and trastuzumab deruxtecan (T-DXd), have led to improved survival outcomes in patients with HER2-overexpressing (HER2+) metastatic breast cancer. However, intrinsic or acquired resistance to anti-HER2–based therapies remains a clinical challenge in these patients, as there is no standard of care following disease progression. The purpose of this study was to elucidate the mechanisms of resistance to T-DM1 and T-DXd in HER2+ BC patients and preclinical models and identify targets whose inhibition enhances the antitumor activity of T-DXd in HER2-directed ADC-resistant HER2+ breast cancer in vitro and in vivo.MethodsTargeted DNA and whole transcriptome sequencing were performed in breast cancer patient tissue samples to investigate genetic aberrations that arose after anti-HER2 therapy. We generated T-DM1 and T-DXd–resistant HER2+ breast cancer cell lines. To elucidate their resistance mechanisms and to identify potential synergistic kinase targets for enhancing the efficacy of T-DXd, we used fluorescence in situ hybridization, droplet digital PCR, Western blotting, whole-genome sequencing, cDNA microarray, and synthetic lethal kinome RNA interference screening. In addition, cell viability, colony formation, and xenograft assays were used to determine the synergistic antitumor effect of T-DXd combinations.ResultsWe found reduced HER2 expression in patients and amplified DNA repair–related genes in patients after anti-HER2 therapy. Reduced ERBB2 gene amplification in HER2-directed ADC–resistant HER2+ breast cancer cell lines was through DNA damage and epigenetic mechanisms. In HER2-directed ADC–resistant HER2+ breast cancer cell lines, our non-biased RNA interference screening identified the DNA repair pathway as a potential target within the canonical pathways to enhance the efficacy of T-DXd. We validated that the combination of T-DXd with ataxia telangiectasia and Rad3-related inhibitor, elimusertib, led to significant breast cancer cell death in vitro (P < 0.01) and in vivo (P < 0.01) compared to single agents.ConclusionsThe DNA repair pathways contribute to HER2-directed ADC resistance. Our data justify exploring the combination treatment of T-DXd with DNA repair–targeting drugs to treat HER2-directed ADC–resistant HER2+ breast cancer in clinical trials.

Chicken Immunization followed by RNA Extraction and cDNA Synthesis for Antibody Library Preparation.

Effective isolation of specific antibodies from immunological repertoires requires the generation of a diverse library against a specific antigen of interest, as well as efficient selection procedures, such as bio-panning and phage ELISA. Key to this is the generation of a good immune response in the host, followed by preparation of high-quality RNA and cDNA from which a library can be constructed by the amplification and cloning of immunoglobulin heavy and light chain genes. The first step in the construction of such an "immune library" is a successful course of immunization. Detection of a strong serum antibody titer will theoretically then result in a pool of extracted RNA that is enriched for transcripts of genes encoding the antibody of interest. Chicken antibodies have been widely used for research and diagnostic purposes, largely because of both their cross-reactivity to epitopes shared by humans, mice, primates, and other mammals, and their simple characteristics, with chickens featuring single functional copies of V H /J H and V λ /J λ gene pairs. In chickens, antibodies against an antigen of interest can be detected in the serum as soon as 5-7 d after immunization. Once the antibody titer reaches an appropriate level in the serum, the spleen, bursa of Fabricius, and bone marrow are then harvested, and antibody libraries can be prepared from extracted RNA. Here, we describe a protocol for chicken immunization with an antigen of interest, followed by RNA extraction from the relevant tissues and cDNA synthesis, which users can use for antibody library construction.

Molecular characterization and validation of zinc solubilization potential of bacteria isolated from onion (Allium cepa L.) rhizosphere

Zinc (Zn) is essential for optimal plant growth and nutrition, while zinc-solubilizing bacteria (ZSB) enhance plant accessibility to zinc by converting its insoluble forms into usable ones. The primary objective of this research was to isolate and identify Zn-solubilizing strains from the onion rhizosphere and evaluate their ability to solubilize different insoluble Zn compounds, including ZnO, ZnCO3, and Zn3(PO4)2. Of the nineteen bacterial isolates retrieved, fifteen demonstrated the ability to solubilize inorganic Zn compounds based on plate assay techniques. The Zn-solubilizing bacterial strains, selected for their qualitative properties, underwent quantitative testing in broth culture using atomic absorption spectrophotometry (AAS) and Field Emission-Scanning Electron Microscopy with Energy Dispersive X-ray Spectroscopy (FE-SEM-EDS). Seven bacterial isolates with a high capacity to solubilize Zn were identified based on 16S rRNA gene amplification and sequence analysis. These isolates were affiliated with Pantoea eucrina, Pantoea dispersa, Pseudomonas aeruginosa, Bacillus velezensis, and Pseudomonas fluorescens. Notably, this study is the first to report the potential of Pantoea eucrina as a ZSB. The strain Pantoea eucrina ZSC9 exhibited the maximum Zn solubilization index (8.85) and the highest soluble Zn content (624 mg L−1) among the three insoluble Zn compounds, with ZnO being the most effectively solubilized on the 10th day of incubation in ZnO-enriched basal medium. All bacterial isolates were more effective at solubilizing ZnO than ZnCO3 and Zn3(PO4)2, and this solubilization led to a significant drop in the pH of the broth. Pantoea eucrina ZSC9 showed the maximum pH reduction (3.82) in a ZnO-supplemented medium, indicating a negative correlation between broth pH and Zn solubilization. Seven selected ZSB strains were assessed for their potential to enhance onion growth under pot house conditions. The results showed that inoculating onion seedlings with these ZSB strains significantly improved their growth. Among the strains tested, ZSC9, ZSC10, and ZSC17 were the most effective, enhancing growth attributes such as plant height, bulb diameter, fresh and dry weight of bulbs, Zn uptake by the plant, and overall bulb yield compared to other strains. These findings suggest that these ZSB isolates have great potential as biofertilizers to enhance plant growth and development.

Simultaneous Extraction of Bone Marrow RNA and DNA from Patients with Hematologic Diseases Using a Combined Magnetic Bead Method within 1 Hour.

TRIzolTM is widely used for RNA and DNA extraction. However, this method is laborious and time-consuming. The objective of this study was to validate a time-effective and labor-saving protocol. The TRIzol method was used to separate the aqueous phase, protein, and phenol layer of bone marrow samples from 12 patients with hematological diseases. Subsequently, RNA and DNA were extracted from the aqueous layer containing RNA and phenol layer containing DNA, respectively, using magnetic bead extraction kits. The quantity and purity of extracted RNA and DNA were examined using a NanoDrop spectrophotometer. Quantitative fluorescence PCR amplification of the ABL1 gene was performed to verify the effectiveness of the extracted RNA and DNA for downstream experiments. RNA and DNA from another 16 bone marrow samples were extracted to compare the performance of the two methods. Co-extraction of RNA and DNA was completed within 1 h. The data showed that RNA and DNA yield ranged from 13.1 to 204.5 ng/µL and 33.1 to 228.8 ng/µL, respectively. The A260/A280 ratios of RNA and DNA samples ranged from 1.82 to 2.01 and 1.73 to 1.91, respectively. RNA and DNA extracted using this scheme exhibited ideal performance in quantitative fluorescence PCR. The present protocol showed better quality and effectiveness in extracting RNA and DNA compared to the TRIzol method. This protocol for RNA and DNA co-extraction is fast, labor-saving, and high throughput. It can be adopted for routine molecular biology analyses, particularly for non-reproducible specimens.

Laundry Isolate Delftia sp. UBM14 Capable of Biodegrading Industrially Relevant Aminophosphonates.

Phosphonates such as ethylenediaminetetra (methylenephosphonic acid) (EDTMP) and aminotris (methylenephosphonic acid) (ATMP) are used every day in water treatment processes or in household products. Their consumption is still increasing, regardless of the debates on their environmental impact. Here, the microbial characterisation and determination of the biodegradation potential of selected industrially relevant phosphonates for the isolate Delftia sp. UMB14 is reported. The opportunistic strain was isolated from a biofilm that was derived from a conventional washing machine using conventional detergents containing phosphonates. In antimicrobial susceptibility testing, the strain was only susceptible to sulfonamide, tetracycline, and chloramphenicol. Physiological and biochemical characteristics were determined using the BIOLOG EcoPlate assay. Most importantly, the strain was shown to convert D-malic acid and D-mannitol, as confirmed for strains of Delftia lacustris, and thus the new isolate could be closely related. Biodegradation tests with different phosphonates showed that the strain preferentially degrades ATMP and EDTMP but does not degrade glyphosate (GS) and amino (methylphosphonic acid) (AMPA). A specific gene amplification confirmed the presence of phnX (phosphonoacetaldehyde hydrolase) and the absence of PhnJ (the gene for the core component of C-P lyase). The presence of PhnCDE is strongly suggested for the strain, as it is common in Delftia lacustris species.

Genomic alterations associated with resistance and circulating tumor DNA dynamics for early detection of progression on CDK4/6 inhibitor in advanced breast cancer.

Combined CDK4/6 inhibitor (CDK4/6i) and endocrine therapy significantly improves outcome for patients with estrogen receptor-positive (ER+) metastatic breast cancer, but drug resistance and thus disease progression inevitably occur. Herein, we aimed to identify genomic alterations associated with combined CDK4/6i and endocrine therapy resistance, and follow the levels of specific mutations in longitudinal circulating tumor DNA (ctDNA) for early detection of progression. From a cohort of 86 patients with ER+ metastatic breast cancer we performed whole exome sequencing or targeted sequencing of paired tumor (N = 8) or blood samples (N = 5) obtained before initiation of combined CDK4/6i and endocrine therapy and at disease progression. Mutations in oncogenic genes at progression were rare, while amplifications of growth-regulating genes were more frequent. The most frequently acquired alterations observed were PIK3CA and TP53 mutations and PDK1 amplification. Longitudinal ctDNA dynamics of mutant PIK3CA or private mutations revealed increased mutation levels at progression in 8 of 10 patients (80%). Impressively, rising levels of PIK3CA-mutated ctDNA were detected 4-17 months before imaging. Our data add to the growing evidence supporting longitudinal ctDNA analysis for real-time monitoring of CDK4/6i response and early detection of progression in advanced breast cancer. Further, our analysis suggests that amplification of growth-related genes may contribute to combined CDK4/6i and endocrine therapy resistance.

Defining the bacterial microbiome of ticks in Chongwe and Chisamba Districts of Zambia

BackgroundThe microbiome composition of an arthropod vector may impede the growth of some pathogens, aid colonisation by pathogens or affect vector behaviour in ways that impact the transmission of pathogens. In Zambia, little is known of the microbial communities hosted by ticks and how pathogens like Rickettsia play a role in the microbiome composition. ObjectiveThis study sought to determine the microbiome of Rickettsia-negative and Rickettsia-positive ticks in selected districts of Zambia. MethodsThis was a cross-sectional study carried out on 94 ticks collected from cattle in Chongwe and Chisamba districts. The overall prevalence of Rickettsia spp. was detected using PCR amplification of the ompB gene. Thereafter, both Rickettsia-negative and positive ticks underwent 16S rRNA gene amplification and Illumina high-throughput sequencing. Data was analysed using QIIME2 analysis pipeline. ResultsThe prevalence of Rickettsia was found to be 47.9% (45/94) with prevalence in Amblyomma at 78.5% (22/28), Hyalomma at 68.9% (20/29) and Rhipicephalus having the lowest at 8.1% (3/37). Proteobacteria, Firmicutes, Actinobacteriota and Euryachaeota were the most common phyla, while endosymbionts were uncommonly detected in the ticks. Further analysis showed significant differences in microbiome composition based on Rickettsia detection status (p=0.001) and location (p=0.001), based on the alpha diversity Shannon index, Bray Curtis beta diversity and PERMANOVA, whilst differences according to life stage, tick species and genus was only shown based on the Bray Curtis beta diversity and PERMANOVA analysis. ConclusionUltimately, this study provides valuable insights into the structure of the tick microbiome in parts of Zambia and how it is affected by the presence of Rickettsia.

Sorcin in Cancer Development and Chemotherapeutic Drug Resistance.

SOluble Resistance-related Calcium-binding proteIN (sorcin) earned its name due to its co-amplification with ABCB1 in multidrug-resistant cells. Initially thought to be an accidental consequence of this co-amplification, recent research indicates that sorcin plays a more active role as an oncoprotein, significantly impacting multidrug resistance (MDR). Sorcin is a highly expressed calcium-binding protein, often overproduced in human tumors and multidrug-resistant cancers, and is a promising novel MDR marker. In tumors, sorcin levels inversely correlate with both patient response to chemotherapy and overall prognosis. Multidrug-resistant cell lines consistently exhibit higher sorcin expression compared to their parental counterparts. Furthermore, sorcin overexpression via gene transfection enhances drug resistance to various chemotherapeutic drugs across numerous cancer lines. Conversely, silencing sorcin expression reverses drug resistance in many cell lines. Sorcin participates in several mechanisms of MDR, including drug efflux, drug sequestering, cell death inhibition, gene amplification, epithelial-to-mesenchymal transition, angiogenesis, and metastasis. The present review focuses on the structure and function of sorcin, on sorcin's role in cancer and drug resistance, and on the approaches aimed at targeting sorcin.

Phosphorus-Use-Efficiency Gene Identification in Fabaceae and RSL2 Expansion in Lupinus albus Is Associated with Low-Phosphorus Adaptation.

Phosphorus is critical for plant growth but often becomes less accessible due to its precipitation with cations in soil. Fabaceae, a diverse plant family, exhibits robust adaptability and includes species like Lupinus albus, known for its efficient phosphorus utilization via cluster roots. Here, we systematically identified phosphorus-utilization-efficiency (PUE) gene families across 35 Fabaceae species, highlighting significant gene amplification in PUE pathways in Fabaceae. Different PUE pathways exhibited variable amplification, evolution, and retention patterns among various Fabaceae crops. Additionally, the number of homologous genes of the root hair development gene RSL2 in L. albus was far more than that in other Fabaceae species. Multiple copies of the RSL2 gene were amplified and retained in L. albus after whole genome triplication. The gene structure and motifs specifically retained in L. albus were different from homologous genes in other plants. Combining transcriptome analysis under low-phosphorus treatment, it was found that most of the homologous genes of RSL2 in L. albus showed high expression in the cluster roots, suggesting that the RSL2 gene family plays an important role in the adaptation process of L. albus to low-phosphorus environments and the formation of cluster roots.

Global Study on the Accuracy of Human Epidermal Growth Factor Receptor 2-Low Diagnosis in Breast Cancer.

Recently, a new type of antibody-drug conjugate, trastuzumab-deruxtecan (T-DXd), has been approved for the treatment of metastatic breast cancer with low level of human epidermal growth factor receptor 2 (HER2) gene expression. Thereby, eligibility relies on an accurate diagnosis of HER2-low status defined by immunohistochemistry IHC 1+/2+ with no gene amplification. To assess pathologists' accuracy and training efficacy in the diagnosis of HER2-low. Agreement rates of HER2-low scoring in breast cancer tissue were assessed between expert consensus and real-world pathologists (n = 77 from 14 countries) before and after a specific 4-hour training for HER2-low detection. Two assays were evaluated, the Ventana Pathway 4B5 CDx and the Dako HercepTest (polyclonal). Concordance of the pathologists with consensus score and efficacy of training were measured by Cohen κ, overall rater agreement, and receiver operating characteristic (ROC) curve statistics. In the Ventana 4B5 HER2-low category, baseline agreement rates were >80% but <90%. Negative percentage agreement was improved from 80.6% to 91.1% by training. In the HER2-0 category, positive percentage agreement (74.6%) was the only parameter below the 80% benchmark but was significantly improved to 89.2% after training. Training efficacy was confirmed by ROC curve analysis, which shows improvement for the identification of HER2-0 and HER2-low cases. Finally, in-depth examination of cases with discordant HER2 status disclosed specific issues of HER2-low underscoring and overscoring. The ability of pathologists to achieve acceptable diagnostic accuracy in identifying patients with HER2-low breast cancer could be enhanced by short-term training. Potential routes to improve the quality of HER2-low scoring in clinical practice have been identified.

Molecular characterization and identification of African Swine Fever virus isolates from emerging cases of infection in Central Luzon, Philippines

A study that evaluated the molecular characteristics to confirm the identity of the African Swine Fever (ASF) pathogen from emerging cases of infection in Central Luzon, Philippines was initiated. The study involved the collection of 125 blood samples from pigs during the four-month duration of the study when clinical signs of ASF was prevalent in the four out of seven provinces of Central Luzon, Philippines. This work included the amplification of the p72 gene in DNA samples through PCR and confirmation of the identity of the ASF pathogen through DNA sequencing. The DNA sequences of the p72 gene of the ASF pathogen are made up of 412 nucleotides and used as a basis in identifying the Philippine ASF pathogens as ASF virus PH/CL 4/2022, ASF virus PH/CL 3/2022, ASF virus PH/CL 2/2022 and ASF virus PH/CL 1/2022. Philippines ASF virus isolates showed 100% sequence similarity with sequences reported from Malaysia ASF isolate MVZT218/2021, Vietnam ASF isolate D/ASFV/VN/DN-VC/2019, and India ASF isolate ASF/MZ/IND/3 based on BLASTN homology search in the NCBI database. The results of the study revealed close phylogenetic relationships among ASF isolates from Vietnam, Malaysia, India and the Philippines.

Optimizing single-tube nested PCR for enhanced genotyping of single cells and in vitro produced bovine embryos

Single-tube nested PCR (STnPCR) is a technique that improves nested PCR, reducing potential contamination and false-positive results, enhancing the amplification sensitivity. Despite being commonly used for the detection of microorganisms, STnPCR can be a valuable tool for bovine genotyping, encompassing essential targets as ROSA26 and TSPY, pivotal in the fields of animal reproduction, genetic improvement, and transgenic research. The objective of this study was to improve and innovate STnPCR for gene detection in cattle. We aimed to detect the ROSA26 and TSPY genes using low-concentration DNA samples, including single cells, small cell groups (one to five cells), in vitro-produced embryos, and bovine tissue samples. Moreover, we refined STnPCR for gene detection in up to single cells by conducting sensitivity testing with different concentration ratios of internal and external primers. Successful amplification of the ROSA26 and TSPY genes was achieved across all tested primer concentrations, even in single cells, with more consistent results observed at lower primer concentrations. Additionally, simultaneous gene amplification was achieved through STnPCR multiplexing, representing the first study of multiplex STnPCR in cattle. These outcomes not only confirm its effectiveness in detecting genetic markers for animal genetic improvement and transgenic elements but also pave the way for its widespread adoption in reproductive studies in bovines.

Image analysis for bright-field HER2 in situ hybridization: validation for clinical use.

The aim of the present study was to develop and validate a quantitative image analysis (IA) algorithm to aid pathologists in assessing bright-field HER2 in situ hybridization (ISH) tests in solid cancers. A cohort of 80 sequential cases (40 HER2-negative and 40 HER2-positive) were evaluated for HER2 gene amplification with bright-field ISH. We developed an IA algorithm using the ISH Module from HALO software to automatically quantify HER2 and CEP17 copy numbers per cell as well as the HER2/CEP17 ratio. We observed a high correlation of HER2/CEP17 ratio, an average of HER2 and CEP17 copy number per cell between visual and IA quantification (Pearson's correlation coefficient of 0.842, 0.916, and 0.765, respectively). IA was able to count from 124 cells to 47,044 cells (median of 5565 cells). The margin of error for the visual quantification of the HER2/CEP17 ratio and of the average of HER2 copy number per cell decreased from a median of 0.23 to 0.02 and from a median of 0.49 to 0.04, respectively, in IA. Curve estimation regression models showed that a minimum of 469 or 953 invasive cancer cells per case is needed to reach an average margin of error below 0.1 for the HER2/CEP17 ratio or for the average of HER2 copy number per cell, respectively. Lastly, on average, a case took 212.1s to execute the IA, which means that it evaluates about 130 cells/s and requires 6.7s/mm2. The concordance of the IA software with the visual scoring was 95%, with a sensitivity of 90% and a specificity of 100%. All four discordant cases were able to achieve concordant results after the region of interest adjustment. In conclusion, this validation study underscores the usefulness of IA in HER2 ISH testing, displaying excellent concordance with visual scoring and significantly reducing margins of error.