DNA PCR Research Articles

Detection of Leishmania donovani DNA from Oral Swab in Visceral Leishmaniasis

Visceral leishmaniasis (VL) is the most severe form of leishmaniasis and is fatal if left untreated in over 95% of cases. Leishmaniasis is one of the neglected tropical diseases that tend to thrive in developing regions of the world where inadequate access to healthcare makes it difficult for some people to even receive a diagnosis. This study examined the usefulness of oral swabs as specimens for VL diagnosis, by detecting Leishmania donovani DNA in oral swabs from both VL patients and L. donovani-infected mice. Eighty oral swab (OS) and blood buffy coat (BC) samples were collected from suspected VL cases in Bangladesh. These samples were evaluated using Leishmania kinetoplast minicircle DNA (kDNA) in real-time PCR, and the results showed that 62.5% (50/80) and 67.5% (54/80) of the cases tested positive for the BC specimen and OS, respectively. The OS positivity was statistically comparable to the BC. L. donovani DNA was also detected in an oral swab of all infected BALB/c mice by conventional PCR targeting the large subunit ribosomal RNA gene (LSUrRNA), while it was negative in uninfected mice. This study highlights the potential of practical methods for the molecular diagnosis of VL using oral swabs as a non-invasive, simple, and accurate approach.

Molecular Characterization of Trypanosoma cruzi from Triatomine Species in São Paulo State, an Area Free of Vector-Borne Chagas Disease

Chagas disease (ChD) is a neglected tropical disease caused by Trypanosoma cruzi, endemic in 21 countries across the Americas, with increasing cases globally. In São Paulo, Brazil, vector control has focused on Triatoma infestans, but secondary triatomine species continue to pose transmission risks. This study aimed to investigate the prevalence of T. cruzi in triatomine feces and characterize its genetic diversity using molecular techniques. Fecal samples were collected from 570 triatomines across 25 municipalities in São Paulo, followed by DNA extraction and PCR amplification targeting the mitochondrial cytochrome b gene and the V7V8 region of the 18S rRNA gene. The results revealed a low overall infection rate (3.2%). However, excluding the triatomines collected in palm trees, all of which were negative, we found mainly Panstrongylus megistus in residences and peridomiciles, showing the highest infection rate (65%) for T. cruzi, followed by Triatoma sordida and Rhodnius neglectus. Phylogenetic analysis confirmed that DTU TcI was the most prevalent genotype, consistent with previous findings in the region. This study highlights the importance of continued vector surveillance, as these secondary species are capable of maintaining T. cruzi transmission in both urban and rural environments, underscoring the ongoing risk of ChD resurgence in São Paulo.

P-772. Whole Genome Analysis of Mycobacterium intracellulare isolates from Colonic Biopsy In Inflammatory Bowel Disease Patients

Abstract Background A long debate has prevailed over cause of Crohn's disease with published evidence for Mycobacterium paratuberculosis (MAP).Our study was designed to look for mycobacterial pathogens by culture and DNA PCR in patients with inflammatory bowel disease(IBD). Phylogenetic Analysis of M. intracellulare isolate genomes compared with reference M. intracellular strain ATCC 13950T Circular representation of four M. intracellulare isolate genomes compared with reference M. intracellular strain ATCC 13950T. Inner to outer four concentric rings show isolates from this study. The information is read from outer circle to inner as follows: genome size, genes on the forward strand, genes on the reverse strand, t-RNA, and r-RNA of reference strain.The percentage similarity is represented by different color codes. Zoomed portion represent absent genetic region in four isolates. Methods Colonic biopsy and Blood Buffy coat samples were processed for cultures and DNA detection for mycobacterial pathogens (including MAP) in 889 patients with inflammatory bowel disease; Crohn’s disease (CD)( clinical manifestations, endoscopic,radiological and histological features as per ECCO consensus guidelines), Intestinal tuberculosis,Controls (Adult patients with hemorrhoidal bleeding and no other colonic disease undergoing sigmoidoscopy). Results Four cultures from CD patients grew Mycobacterium intracellulare. Whole Genome Sequencing identified deletion regions in the studied isolates, with 156 protein-coding genes absent in all four isolates compared to the reference genome (M. intracellulare Strain ATCC 13950T). Antibiotic resistance and virulence factor analyses indicated resistance to rifampicin and isoniazid and an abundance of virulence factor genes in M. Intracellulare species. Pan and core gene analyses suggested a closed pan-genome in M.intracellulare, with limited distribution of antibiotic resistance genes. Conclusion M. Intracellulare is reported in immune-compromised patientsin several studies. The results of the core genome phylogenetic analysis suggest that factors beyond the isolation sources of M.intracellulare strains may contribute to shape their evolutionary trajectory. Functional analysis indicated a gradual decrease in metabolic capacity across clusters, suggesting an ongoing genome streamlining process in M. Intracellulare species. Essential gene prediction identified strain specific essential genes, emphasizing the importance of energy-related processes. Homologous recombination rate analysis suggested recombination in M. intracellulare strains, with specific parameters showing variation compared to other pathogenic species. This comprehensive study contributes valuable information about the genomic characteristics, evolution, and functional profiles of M. intracellulare strains. Disclosures All Authors: No reported disclosures

P-2172. Development of a Point-of-Care Diagnostic for Congenital Cytomegalovirus

Abstract Background Cytomegalovirus (CMV), is the most common congenital infection in the United States. Congenital CMV (cCMV) affects 3-6 per 1000 live born infants yearly. Up to 80% of cCMV cases are asymptomatic but can result in long term sequelae, including sensorineural hearing loss. Despite improved diagnostic strategies and therapeutics, universal screening programs are not standard care. Our laboratory is addressing this gap by designing a novel isothermal CRISPR-based point-of-care assay for cCMV. Preliminary studies using laboratory samples and conditions have demonstrated the feasibility of our approach. Methods A CRISPR-Cas12a CMV specific detection system was designed against the CMV gene UL123 (immediate-early gene 1; IE1). Briefly, CMV was spiked into nuclease-free water samples and DNA was isolated using commercial kits. DNA was then amplified using standard PCR techniques. Amplified viral DNA was added to a reaction containing CRISPR-Cas12a, a fluorescent detection probe, and CMV UL123 guide RNA. The reaction was incubated at 37°C for 20 minutes. Fluorescent intensity of CMV spiked samples was compared to a no template control. The positive cutoff was calculated using the mean of the no template control + 3*SD. Results Using this strategy, we observed a limit of detection at 10 infectious units/mL of CMV. There was no cross reactivity to other alpha- or betaherpesviruses. This was repeated using CMV-spiked human serum, saliva, and urine samples, which resulted in a similar limit of detection. To further improve our assay, we replaced the DNA isolation and PCR steps with an isothermal one-pot reaction, using a UL123 padlock probe based rolling circle amplification system. Employing this strategy, we can detect CMV in samples in 2 hours at 37°C, without the need for DNA isolation, prior amplification, or the use of PCR equipment. This approach was validated by designing targets to detect Epstein Barr Virus (EBV). Conclusion We continue efforts to validate our isothermal point-of-care diagnostic to encourage cCMV universal screening implementation. Disclosures All Authors: No reported disclosures

Comparison of Lysis and Amplification Methodologies for Optimal 16S rRNA Gene Profiling for Human and Mouse Microbiome Studies

When conducting sequence-based analysis of microbiome samples, it is important to accurately represent the bacterial communities present. The aim of this study was to compare two commercially available DNA isolation and PCR amplification approaches to determine their impact on the taxonomic composition of microbiome samples following 16S rRNA gene sequencing. A well-established 16S rRNA gene profiling approach, which was widely used in the Human Microbiome Project (HMP), was compared with a novel alkaline degenerative technique that utilizes alkaline cell lysis in combination with a degenerate pool of primers for nucleic acid extraction and PCR amplification. When comparing these different approaches for the microbiome profiling of human and mouse fecal samples, we found that the alkaline-based method was able to detect greater taxonomic diversity. An in silico analysis of predicted primer binding against a curated 16S rRNA gene reference database further suggested that this novel approach had the potential to reduce population bias found with traditional methods, thereby offering opportunities for improved microbial community profiling.

Performance analysis of Leica Biosystems p16 monoclonal antibody in oropharyngeal squamous cell carcinoma

BackgroundHead and neck squamous cell carcinoma (HNSCC) is the sixth leading cause of cancer death globally, with newly diagnosed oropharyngeal squamous cell carcinoma (OPSCC) cases rising to 54,000 in the US alone in the year 2022. Recently, human papilloma virus (HPV) infection was more prevalent in OPSCC patients than the traditionally known carcinogens such as tobacco or alcohol. HPV 16 is the most common causative HPV strain, which is found in 5–10% of HNSCC patients. HPV 16’s E6 and E7 oncoproteins bind and inactivate p53 and retinoblastoma (Rb) tumor-suppressing genes. This causes aberrant over-expression of the cell cycle inhibitor gene, p16, leading to tumorigenesis. Leica Biosystems (LBS) has developed a p16 antibody (6H12 clone) for qualitatively identifying the p16 protein in formalin-fixed paraffin-embedded (FFPE) tissue by immunohistochemical staining. This method comparison study tested the concordance rates between ready-to-use (RTU) LBS p16/LBS RTU p16 antibody and Roche Tissue Diagnostics (RTD) CINtec p16 Histology immunohistochemical (IHC) assays by measuring overall agreement (OA), average positive agreement (APA), and average negative agreement (ANA) rates in 170 OPSCC FFPE cases. Interobserver agreement of the 2 assays and LBS RTU p16 comparison with the standard HPV molecular assays (DNA ISH and PCR) were also assessed.MethodsOne hundred and seventy (170) unique oropharyngeal cancer cases were stained for qualitative analysis by the LBS p16 antibody on BOND III. This assay was compared to Ventana’s RTD E6H4 (CINtec) clone on Benchmark XT. A stained core was considered p16 positive if the Histoscore (H score) was ≥ 140 and negative if H < 140.ResultsAcross the pathologists, the agreement rate between the 2 assays ranged from OA, 98.7 – 98.8%, ANA, 98.8 -98.9%, and APA, 98.6%. For LBS RTU p16, the interobserver agreement was OA, 98.7%, ANA, 98.8%, and APA, 98.6%; while for RTD CINtec p16 assay, the concordance was OA, 98.7%, ANA, 98.8% and APA, 98.6%. In comparison to the HPV molecular testing, DNA ISH, and PCR, across pathologists, LBS p16 clone (LBS RTU p16) showed a concordance rate of 85.8-86.9% and 87.6-88.8%, respectively.ConclusionLBS p16 monoclonal antibody demonstrated high concordance with CINtec p16 IHC assay across all the endpoints, suggesting a potential use of LBS RTU p16 clone in detecting p16 protein in oropharyngeal cancer cases.

Alcohol-induced liver injury is mediated via α4-containing nicotinic acetylcholine receptors expressed in hepatocytes.

Our previous study demonstrated that alcohol induced the expression of the α4 subunit of nicotinic acetylcholine receptors (nAChRs) in the livers of wild type mice (WT), and that whole-body α4 nAChR knockout mice (α4KO) showed protection against alcohol-induced steatosis, inflammation, and injury. Based on these findings, we hypothesized that hepatocyte-specific α4 nAChRs may directly contribute to the detrimental effects of alcohol on the liver. Hepatocyte-specific α4 knockout mice (α4HepKO) were generated, and the absence of α4 nAChR was confirmed through PCR of genomic DNA. Female WT and α4HepKO mice were exposed to alcohol in the NIAAA chronic + binge model. After 10 days on the Lieber-DeCarli liquid diet containing 5% (vol/vol) alcohol or isocaloric maltose-dextrin, the mice were gavaged with a single dose of alcohol or isocaloric maltose-dextrin. The mice were euthanized 9 h later and their organs harvested. Additionally, hepatocytes were isolated from WT, α4HepKO, α4floxed, and α4KO mice and exposed to 80 mM alcohol invitro for 24 h. Steatosis, inflammation, and cell injury were assessed in both liver and isolated hepatocytes. In WT mice, alcohol exposure resulted in hepatic steatosis, inflammation, and injury as evidenced by increased liver triglycerides, neutrophil infiltration, and serum concentrations of liver enzymes. All of these responses were markedly lower in α4HepKO mice. mRNA expression of genes involved in lipogenesis (Srebf1, Fasn, and Dgat2) and inflammation (TNFα, Cxcl5, Cxcl1, and Serpine1) were increased in the livers of WT mice exposed to alcohol invivo and in WT hepatocytes exposed to alcohol invitro. These changes were not observed in liver or hepatocytes from mice lacking α4 nAChRs. α4 nAChRs expressed in hepatocytes mediate alcohol-associated hepatoxicity. Therefore, the development of therapeutic strategies targeting hepatocyte α4-containing nAChRs could help reduce the burden of ALD.

A novel genotype of Babesia microti-like group in Ixodes montoyanus ticks parasitizing the Andean bear (Tremarctos ornatus) in Ecuador.

Babesia species (Piroplasmida) are hemoparasites that infect erythrocytes of mammals and birds and are mainly transmitted by hard ticks (Acari: Ixodidae). These hemoparasites are known to be the second most common parasites infecting mammals, after trypanosomes, and some species may cause malaria-like disease in humans. Diagnosis and understanding of Babesia diversity increasingly rely on genetic data obtained through molecular techniques. Among hard ticks, several Ixodes species are known vectors of Babesia microti-like species in the Northern Hemisphere. Recently, Ixodes and Amblyomma ticks have been recorded parasitizing the Andean bear (Tremarctos ornatus) in Ecuador. Previous reports have suggested babesiosis in a fatal case of this threatened bear species in that country. This study aimed to detect Piroplasmida DNA in hard ticks collected from Andean bears at two sites in Ecuador. This species plays a critical role as an ecological engineer and a seed disperses, contributing significantly to the maintenance and health of Andean ecosystems. Twelve ticks screened with conventional PCR and Piroplasmida DNA was amplified from one Ixodes montoyanus tick collected from a free-living female Andean bear at Llanganates National Park. Two Babesia sequences were characterized: one for the 18S ribosomal rRNA gene and another for the cytochrome c oxidase 1 gene. Phylogenetic analyses for both loci placed these sequences within the B. microti-like clade. This study reports a novel B. microti-like genotype identified in an I. montoyanus parasitizing a female Andean bear, contributing to the knowledge of the diversity of this group in South America. Given their conservation status, future epidemiological surveillance of Babesia and other tick-borne infectious agents in Andean bears is needed.

Concordance of non-invasive plasma cell-free DNA with invasive diagnostics for diagnosis of invasive fungal disease.

Mold plasma cell-free DNA (cfDNA) PCR is a promising non-invasive diagnostic modality for early diagnosis of invasive mold disease (IMD) in immunocompromised patients. Although mold cfDNA PCR has been shown to be highly accurate, the value of invasive procedures to collect specimens for conventional fungal diagnostics following plasma cfDNA testing remains unclear. This retrospective single-center cohort study included patients with mold plasma cfDNA PCR performed 7 days before or 2 days after invasive specimen collection. Mold PCR detected Aspergillus species, Mucorales agents, Fusarium species, and Scedosporium species. Invasive procedures included tissue biopsy and bronchoscopy. The primary endpoint was the concordance of mold plasma cfDNA PCR results with results of conventional fungal tests performed on tissue and bronchoalveolar lavage fluid (BAL). Five hundred and six patients with mold plasma cfDNA PCR resulting ahead of invasive specimen (123 tissue and 426 BAL) results were included, and 437 (86.4%) were immunocompromised. After adjudicating discordant results based on the EORTC/MSGERC definitions for IMD, mold plasma cfDNA PCR and invasive test results were 88.5% (448/506) concordant. In proven cases, 64.7% (11/17) of negative mold plasma cfDNA PCR results occurred in patients with fungal sinusitis (8) and limb infection (3). Non-hematologic malignancy and non-neutropenic states were statistically associated with negative mold plasma cfDNA PCR in patients with proven or probable IMD. Non-invasive mold plasma cfDNA PCR results were highly concordant with invasive specimen fungal test results, thus indicating risk-prone invasive specimen collection can be safely curtailed in immunocompromised patients with suspected IMD.

Use of molecular markers associated with resistance to biotic and abiotic environmental factors in developing breeding material for tomato and pepper in Belarus

Relevance. The development of a system of molecular markers that allows identifying the genetic determinants of resistance to pathogens, as well as the typing of alleles involved in the regulation of anthocyanin accumulation, is the most important condition for increasing the efficiency of the breeding process aimed at enhancing the resistance of cultivated crops to biotic and abiotic stresses.Methodology. The work involved molecular genetic methods of DNA isolation, PCR analysis, restriction, and evaluation of amplification and restriction products in agarose or polyacrylamide gels. The material used included the diverse collections of Solanum lycopersicum and Capsicum annuum, as well as the specimens of closely related wild species.Results. The paper evaluates the effectiveness of 25 molecular markers presented in the literature associated with resistance to tomato and pepper diseases caused by fungal, bacterial, viral pathogens, as well as nematodes. Markers to the alleles of MYB transcription factor genes associated with the regulation of anthocyanin accumulation in tomato (SlMyb12, Anthocyanin1, Anthocyanin2, An-2-like and Atroviolacium) and pepper (Myb113-like1, Myb113-like2 and ETC3-2), recommended for the breeding process aimed at increasing resistance to stressful biotic and abiotic environmental factors, are presented.

Metagenomic estimation of absolute bacterial biomass in the mammalian gut through host-derived read normalization.

Absolute bacterial biomass estimation in the human gut is crucial for understanding microbiome dynamics and host-microbe interactions. Current methods for quantifying bacterial biomass in stool, such as flow cytometry, qPCR, or spike-ins (i.e., adding cells or DNA from an organism not normally found in a sample), can be labor-intensive, costly, and confounded by factors like water content, DNA extraction efficiency, PCR inhibitors, and other technical challenges that add bias and noise. We propose a simple, cost-effective approach that circumvents some of these technical challenges: directly estimating bacterial biomass from metagenomes using bacterial-to-host (B:H) read ratios. We compare B:H ratios to the standard methods outlined above, demonstrating that B:H ratios are useful proxies for bacterial biomass in stool and possibly in other host-associated substrates. We show how B:H ratios can be used to track antibiotic treatment response and recovery in both mice and humans, which showed 403-fold and 45-fold reductions in bacterial biomass during antibiotic treatment, respectively. Our results indicate that host and bacterial metagenomic DNA fractions in human stool fluctuate longitudinally around a stable mean in healthy individuals, and the average host read fraction varies across healthy individuals by < 8-9 fold. B:H ratios offer a convenient alternative to other absolute biomass quantification methods, without the need for additional measurements, experimental design considerations, or machine learning algorithms, enabling retrospective absolute biomass estimates from existing stool metagenomic data.

DNA-compatible one-pot synthesis of multi-substituted dihydrofuran via pyridinium ylide-mediated cyclization.

Synthesis of chemically diverse heterocyclic scaffolds in DNA-encoded libraries is highly demanded. We herein reported a convenient one-pot multi-component on-DNA synthetic strategy to afford multi-substituted 2,3-dihydrofuran scaffolds via pyridinium ylide-mediated cyclization. This reaction exhibited modest to excellent conversions for a broad range of DNA-conjugated aldehydes, β-ketonitriles and pyridinium salts under mild reaction conditions. Furthermore, the compatibility of this strategy with DEL construction was verified by enzymatic DNA ligation, PCR amplification and mock library synthesis.

Nebulization of 2% lidocaine has no detectable impact on the healthy equine respiratory microbiota.

Glucocorticosteroids remain the most common pharmaceutical approach for the treatment of equine asthma but can be associated with significant side effects, including respiratory microbiome alterations. The goal of the study was to assess the impact of 2% lidocaine nebulization, a projected alternative treatment of equine asthma, on the healthy equine respiratory microbiota. A prospective, randomized, controlled, blinded, 2-way crossover study was performed, to assess the effect of 1 mg/kg 2% lidocaine (7 treatments over 4 days) on the equine respiratory microbiota compared to control horses (saline and no treatment). Clinical assessments and respiratory samples, including nasal wash, endoscopic tracheal aspirate and bronchoalveolar lavage fluid, were obtained at each sample collection timepoint. The profile of the respiratory bacterial microbiota was evaluated using 16S amplicon sequencing, and clinical data compared using related samples analyses, based on data normality. The treatment did not affect the clinical data or alter the tracheal and nasal microbiota in healthy horses. However, time explained 12.6% of microbiota variation among samples. A significant difference in bacterial composition was observed between nasal and tracheal samples, showing the greatest relative abundance of Actinobacteria and Firmicutes, respectively. Bacterial DNA from bronchoalveolar lavage fluid did not amplify with generic primers targeting the V4 variable region of the prokaryotic small subunit ribosomal RNA gene, despite attempting multiple DNA extraction methods and PCR protocols, and after excluding PCR inhibition. This observation indicates that bronchoalveolar lavage fluid of healthy horses has a low bacterial load.

DNA Barcoding Based on &lt;i&gt;matK &lt;/i&gt;Gene and Phytochemistry Analyses of Local Balinese Kayu Tangi ( &lt;i&gt;Lagerstroemia&lt;/i&gt; sp.)

Kayu tangi (Lagerstroemia sp.) is one of the medicinal plants that is often used traditionally by Balinese people for treating insomnia, diabetes, and dysentery. This study aims to determine the phylogenetic trees and the active compounds in Lagerstroemia tomentosa extract and its antioxidant activities. The methods used in molecular identification are DNA isolation, PCR, and DNA sequencing. The DNA sample was BLASTed to see its homology with the sequences in GenBank. These sequences were then used for phylogenetic tree analysis. The phytochemical analysis was done using the GC-MS method and the antioxidant activity test used the DPPH (1,1-diphenyl-2-picrylhydrazyl) after being reacted with the tested extract. The results of molecular identification showed that the samples used were closely related to Lagerstroemia tomentosa species (MW044208.1). The antioxidant activity test results showed an IC50 of 2.1 ugml-1 which is included in the very strong category. The phytochemical test results showed that the most dominant compounds contained in the plant stem bark were furfural (AUC 8.54%), beta-sitosterol (AUC 6.00%), and gamma-sitosterol (AUC 3.81%). Based on PubChem data, these compounds have antioxidant and antimicrobial properties as well as insecticide and herbicide. In conclusion, kayu tangi is very close to Lagerstroemia tomentosa and it’s potentially used as a medicine with strong antioxidant activities.

Effectiveness of Nasal Flush Treatments in Mycoplasma PCR or DNA Sequencing Positive Tortoises

Abstract Mycoplasmas can cause severe chronic nasal sinusitis in tortoises and box turtles, both in captive and wild populations. The aim of this study was to compare positive and negative Mycoplasma sp. test results prior to and after a nasal flush protocol combined with systemic enrofloxacin, as well as to compare positive and negative results between DNA sequencing and PCR in tortoises with nasal discharge. Fifteen mycoplasma positive tortoises were included in the study. All were treated with the nasal flush protocol. At the end of the nasal flush protocol, 26.7% of the treated tortoises remained mycoplasma positive. One month after the end of the nasal flush protocol, 20% remained positive. The PCR and DNA sequencing tests had good agreement based on a weighted kappa of 0.64. Based on these results, we conclude that the outlined nasal flush protocol is overall successful at acutely resolving nasal discharge and leading to a PCR or DNA sequencing negative tortoise one month after treatment.

Investigation of Hyalomma turanicum and Hyalomma asiaticum in Pakistan, with notes on the detection of tickborne Rickettsiales

There is limited information on the occurrence of Hyalomma turanicum and Hyalomma asiaticum ticks, as well as associated Rickettsia, Anaplasma, and Ehrlichia species in Pakistan. Addressing this knowledge gap, the current study aimed at morphomolecular confirmation of these ticks and molecular assessment of associated Rickettsiales bacteria (Rickettsia, Anaplasma, and Ehrlichia spp.) in Balochistan, Pakistan. A total of 314 ticks were collected from 74 of 117 (63.2%) hosts, including 41 of 74 (55.4%) goats and 33 of 74 (44.5%) sheep. Subsequently, a subset of microscopically identified ticks was subjected to DNA extraction and PCR to amplify 16S rDNA and cox1 fragments. Additionally, gltA, ompA, and ompB fragments were targeted for Rickettsia spp. and 16S rDNA fragments for both Anaplasma and Ehrlichia spp. The 16S rDNA and cox1 sequences of Hy. turanicum demonstrated 100% identity with those of the same species previously reported from Pakistan. The 16S rDNA and cox1 sequences of Hy. asiaticum exhibited 99.52 and 100% identities, respectively, with corresponding species reported from China, Kazakhstan, and Turkey. The gltA, ompA, and ompB fragments associated with Hy. turanicum showed 100% identities with Rickettsia aeschlimannii reported from Egypt, Italy, Kazakhstan, Kenya, Pakistan, and Senegal. The 16S rDNA sequences of Anaplasma sp. and Ehrlichia sp. associated with both Hy. asiaticum and Hy. turanicum exhibited 99.67 and 100% identities with unknown Anaplasma sp. and Ehrlichia sp. reported from Morocco and Pakistan, respectively. In the 16S rDNA and cox1 phylogenetic trees of ticks, Hy. turanicum and Hy. asiaticum from the current study clustered with their respective species. Similarly, in gltA, ompA, and ompB phylogenetic trees of Rickettsia, R. aeschlimannii of the present study clustered with the same species, whereas Anaplasma sp. and Ehrlichia sp. of this study clustered with undetermined Anaplasma spp. and Ehrlichia spp. in the 16S rDNA phylogenetic tree of Anaplasmataceae. Among the DNA samples from the screened ticks, a coinfection rate of R. aeschlimannii, Anaplasma sp., and Ehrlichia sp. (2 of 80, 2.5%) was observed in Hy. turanicum, whereas individual infection rates were noted as follows: R. aeschlimannii (8 of 80, 10%), Anaplasma sp. (5 of 80, 6.3%), and Ehrlichia sp. (5 of 80, 6.3%). This study marks the first record of molecular characterization of Hy. turanicum and Hy. asiaticum as well as the detection of associated R. aeschlimannii, Anaplasma sp., and Ehrlichia sp. in Balochistan, Pakistan.

Foxa1 disruption enhances human cell integration in human-mouse interspecies chimeras.

Blastocyst complementation can potentially generate a rodent model with humanized nasopharyngeal epithelium (NE) that supports sustained Epstein-Barr virus (EBV) infection, enabling comprehensive studies of EBV biology in nasopharyngeal carcinoma. However, during this process, the specific gene knockouts required to establish a developmental niche for NE remain unclear. We performed bioinformatics analyses and generated Foxa1 mutant mice to confirm that Foxa1 disruption could potentially create a developmental niche for NE. Subsequently, MYD88-inactivated human pluripotent stem cells (hPSCs) were constructed and complemented with Foxa1-deficient mouse blastocysts, withNosip-deficient mouse blastocysts as a control. The chimerism of human cells in mouse embryos was evaluated from E8.5 to E12.5 using genomic DNA PCR and immunohistochemistry. Our bioinformatics analysis indicated that the expression patterns of Foxa1 in E8.5 to E16.5 mouse embryos underscore its critical role in NE development. The generated mice with Foxa1 disordered region mutations displayed morphological abnormality in NE, suggesting Foxa1-knockouts could potentially establish a developmental niche for NE. In chimeric assays, human cells integrated into 80.00% of Foxa1-deficient embryos, compared with the 4.17% in controls. Immunohistochemistry results revealed robust proliferation of human cells in Foxa1-deficient mouse embryos. However, chimeras from Foxa1-deficient mouse embryos did not survive beyond E10.5, hindering the evaluation of human cell integration in mouse NE. Foxa1 disruption in mouse embryos significantly enhances the integration of human cells in human-mouse interspecies chimeras, thereby facilitating the generation of endoderm-derived organs through blastocyst complementation. Overcoming chimeras' embryonic lethality is crucial for successfully generating humanized NE in Foxa1-deficient mouse embryos.

Construction and Validation of CRISPR/Cas Vectors for Editing the PDS Gene in Banana (Musa spp.).

Bananas and plantains are important staple food crops affected by biotic and abiotic stresses. The gene editing technique via Clustered Regularly Interspaced Short Palindromic Repeats associated with the Cas protein (CRISPR/Cas) has been used as an important tool for development of cultivars with high tolerance to stresses. This study sought to develop a protocol for the construction of vectors for gene knockout. Here we use the phytoene desaturase (PDS) gene as a case study in Prata-Anã banana by the nonhomologous end junction (NHEJ) method. PDS is a key gene in the carotenoid production pathway in plants and its knockout leads to easily visualized phenotypes such as dwarfism and albinism in plants. Agrobacterium-mediated transformation delivered CRISPR/Cas9 constructs containing gRNAs were inserted into embryogenic cell suspension cultures. This is the first study to provide an effective method/protocol for constructing gene knockout vectors, demonstrating gene editing potential in a Brazilian banana variety. The constitutive (CaMV 35S) and root-specific vectors were successfully assembled and confirmed in transformed Agrobacterium by DNA extraction and PCR. The specificity of transformation protocols makes it possible to use the CRISPR-Cas9 technique to develop Prata-Anã banana plants with enhanced tolerance/resistance to major biotic and abiotic factors.

Molecular Detection of Feline Panleukopenia Virus From Clinical Cases in India

This study was planned for molecular detection of Feline panleukopenia virus from the clinical cases in India. A total of 90 faecal samples from the cats at different ages, breed and sex which showed clinical features such as diarrhea, persistent vomiting and hemorrhagic enteritis in clinics, Tamil Nadu, India in 2020 to 2023 were collected. The faecal samples were processed and the filtered samples were used for DNA extraction and PCR. The molecular study done by detection FM gene. After PCR assay, the results showed that out of 90 samples, 62 samples were positive for FM at 695 bp. The current study showed that the prevalence of infection in females was higher than those of males at age less than 1 year. The cats at local breed showed a percentage of 48.4 which exhibit no differences from foreign breed 51.6 %. In conclusion, the prevalence of FPL infection was high at all breeds and female cats at age less than 1 year.

Prevalence of SPOP and IDH Gene Mutations in Prostate Cancer in a Jordanian Population.

Speckle-type POZ (SPOP) is described as an essential tumor suppressor factor in gastric cancer, colorectal cancer, and prostate cancer (PCa). SPOP gene mutations were reported in primary human PCa. Isocitrate dehydrogenase-1 (IDH1) oncogene mutations were detected in gliomas, acute myeloid leukemia, some benign and malignant cartilaginous tumors, and only 1% of PCa. This study aimed to investigate the prevalence of mutations of SPOP and IDH1 genes in PCa in the Jordanian population. One hundred formalin-fixed paraffin-embedded tissue samples were collected from patients diagnosed with prostate adenocarcinoma. The obtained specimens were subjected to genomic DNA extraction, PCR amplification, and direct sequencing of exons 4, 5, 6, and 7 of the SPOP gene and exon 6 of the IDH1 gene. SPOP gene mutations were found in 17% of PCa cases, while no mutation was detected in the screened exon 6 of the IDH1 gene. Clinicopathological data demonstrated a strong correlation between prostate-specific antigen (PSA) levels and both Gleason score (GS) and the International Society of Urological Pathology (ISUP) grade group (GG). There was no significant correlation between PSA levels and age (p = 0.816) nor there were significant associations for SPOP mutational status with age (p = 0.659), PSA levels (p = 0.395), GS (p = 0.259), and ISUP GG (p = 0.424) in the tested population. The study found a strong correlation between PSA levels and both GS and ISUP GG. It also identified a high frequency (17%) of SPOP gene mutations in Jordanian Arab PCa patients, mainly in exon 7. No IDH1 mutations were detected in exon 6.