Restriction Fragment Research Articles

Diversity and Structure of Soil Bacterial Communities in the Area of Non-ferrous Metal Plant Revealed by 16S rRNA Gene Retrieval

The goals of the study were to assess the diversity and structure of the bacterial communities within the soil depth along a gradient of heavy metal (HM) contamination and to identify indigenous bacterial species in the agricultural area of a non-ferrous metal plant KCM 2000 Group (Plovdiv) by using 16S rRNA gene retrieval. 16S rRNA gene clone libraries were constructed for nine samples, which were collected from two soil depths in June 2020. From each library, up to 100 clones were analysed and grouped into operational taxonomic units (OTUs) by restriction fragment length polymorphism (RFLP). The representatives of the OTUs were sequenced, followed by phylogenetic analysis. The results revealed that phyla Proteobacteria (11.11–71.43%) and Actinomycetota (3.57–33%) were the most abundant. Surface soils (12 phyla and 15 classes) were more diverse than subsurface ones (7 phyla and 12 classes). The lowest diversity at both phylum and class levels was calculated for the moderately contaminated soils from the two studied depths. Thirteen 16S rDNA sequences were identified to a species level, and they belonged to Proteobacteria, Actinomycetota and Firmicutes. This study highlighted that both HM contamination and soil depth caused shifts in diversity and structure of soil bacterial communities.

First Report of a Phytoplasma Strain in the Elm Yellows Group (16SrV) associated with Virginia Creeper in Maryland, USA.

Virginia creeper (Parthenocissus quinquefolia [L.] Planch.) is a deciduous flowering vine in the Vitaceae family. Native to eastern North America, it is often used ornamentally as a climbing vine or as ground cover due to its rapid growth and foliage color in the fall. In July of 2022, along exterior walls of a private property in Lanham, MD, two Virginia creeper (VC) vines were observed displaying symptoms of yellow mottling and premature reddening of leaves. To investigate the cause of these symptoms, two symptomatic leaf samples and one asymptomatic leaf samples from a third vine in the same vicinity were collected for further analysis. A Qiagen DNeasy Plant Mini Kit was used to extract total DNA from leaf tissues according to the manufacturer's instructions. A phytoplasma-specific real-time PCR (Hodgetts et al. 2009) was used to test the DNA extracts, which detected the presence of phytoplasmas in the two DNA samples derived from symptomatic vines. The near full-length of the 16S ribosomal RNA gene was then amplified by seminested PCR from these samples with primers P1/16S-SR followed by P1A/16S-SR (Deng, and Hiruki 1991; Lee et al. 2004) and Sanger sequenced using primers P1A and 16S-SR. Analysis of the obtained 16S rDNA sequences revealed no variation between the two plant samples, and one sequence was deposited in GenBank representing the phytoplasma strain named VC-MD1 (GenBank PP746981). A BLASTn search of the 16S rRNA gene sequence in the NCBI nucleotide database, showed 99.93% sequence identity with the phytoplasma strain AldY-WA1 (GenBank MZ557341) from red alder in Washington, a phytoplasma associated with VC plants in southern Florida (GenBank AF305198) (Harrison et al. 2001), and other strains detected in grapevines in Europe described as "flavescence dorée" phytoplasma (GenBank AF176319) (Davis, and Dally 2001). The virtual restriction fragment length polymorphism pattern derived from iPhyClassifier (Zhao et al. 2009), indicated that VC-MD1 is indeed a member of the 16SrV-C phytoplasma subgroup. To confirm the identification, the partial spc operon and the partial tuf gene were amplified as previously described (Lee et al. 2010; Makarova et al. 2012). Specifically, the spc operon region was amplified using a nested PCR approach with primer set L15F1A-a/MapR1 followed by L15F1A-b/MapR1A-b. Sequence data obtained from the two loci were deposited to GenBank with accession numbers PP746982 (spc) and PP746983 (tuf). BLAST searches querying the nucleotide sequences of the spc operon and tuf gene showed 95.39% and 99.05% identity, respectively, to the corresponding loci of 'Candidatus Phytoplasma rubi' strain RS (GenBank CP114006) and hemp dogbane yellows phytoplasma strain HD1 (GenBank FR686506). Phylogenetic analysis based on secY and tuf gene sequences suggest that the VC-MD1 strain is evolutionary closest to 16SrV-C phytoplasma strains detected in various hosts in the United States, including HD1 and AldY-WA1. These North American strains cluster together on a distinct branch within the elm yellows group phytoplasmas. For the State of Maryland, this detection represents the first report of a phytoplasma strain member of the16SrV-C subgroup infecting VC plants. A phytoplasma of the same subgroup was previously detected in Florida in asymptomatic VC vines (Harrison et al. 2001). The 16S rRNA gene sequences of the two VC phytoplasma strains are nearly identical, differing by just a single nucleotide. The disease transmission vectors of the VC-MD1 strain and the prevalence of the disease in the region remains undetermined.

Association of KCNJ11 gene (rs5219) polymorphism with HOMA-IR & HOMA B values in type 2 diabetes mellitus in India: A case-control study

Objectives Type 2 diabetes mellitus (T2DM) is a complex illness that results from either insulin resistance or insufficient insulin, which raises blood sugar levels. Numerous genes interact to influence the secretion of insulin. A gene of great interest is KCNJ11 of subfamily-J, member 11, which functions as an inwardly rectifying ATP-sensitive potassium (KATP) channel in pancreatic beta cells and is involved in glucose-stimulated insulin release. Material & Methods The present case-control study attempts to delineate the genetic impact of KCNJ11 (rs5219) gene polymorphism on the risk of T2DM in the Indian population. It involves 55 patients with type 2 diabetes (fasting plasma glucose of >126 mg/dl, 2-h glucose of >200 mg/dl, or HbA1c level of >6.4%) and 55 healthy controls (fasting plasma glucose of <100 mg/dl, 2-h glucose of <140 mg/dl, or HbA1c level of <6.4%). polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) was used to study KCNJ11 polymorphism through a standard protocol. Enzyme Linked Immunosorbent Assay (ELISA) was used to estimate serum Insulin levels. HOMA-IR & HOMA-β values were calculated. Statistical analysis was done using t-test, Chi-Square test, and One-way analysis of variance (ANOVA) test. Results Serum insulin levels and HOMA-IR values were significantly decreased in cases than in the control group. Logistic regression analysis showed that the frequency of KK genotype in T2DM individuals (21.8%) was higher than the control group (9%) (p = 0.01). Frequency of K allele (38%) in patients was higher than the control group (18%) (p = 0.001). The K allele risk in diabetic patients was 9.9 times higher as compared to controls (p = 0.001, OR 9.9, 95%Cl 0.036–0.36). Homeostatic model assessment β (HOMA-β) values of KK genotype (59.9±27.8315) were lower than that of EK (76.8±33.23) and EE (127.9±44.59) genotypes (p < 0.001). Conclusion The presence of KCNJ11 (rs 5219) gene polymorphism shows a noteworthy correlation with the likelihood of developing T2DM among the North Indian population. K allele is more likely to be present in individuals with T2DM than the control group. Moreover, HOMA-β values of those with the KK genotype were found to be lower than the individuals having EK and EE genotypes.

Synergistic potential of essential oil combinations against Microsporum, Trichophyton, and Epidermophyton.

Dermatophyte infections globally account for 20 to 25% of fungal infections. Dermatophytes have begun exhibiting antifungal drug resistance, making it challenging to treat this particular infection. Essential oils could be used as alternative solutions as they have been used for a long period to treat different infections. The research has demonstrated the antifungal efficacy of cinnamon, clove, lemongrass, tea tree, thyme, and garlic essential oils, and the impact of their combinations was assayed against Microsporum canis, Trichophyton tonsurans, T. violaceum, T. verrucosum, and Epidermophyton floccosum. Polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) was used to identify the most prevalent M. canis. The accession number of M. canis was obtained as ON007275. All tested essential oils exhibited antidermatophytic action except garlic. A synergistic effect was attained by cinnamon + clove, cinnamon + lemongrass, clove + lemongrass, clove + tea tree, and thyme + tea tree combinations. Concerning antifungal activity, M. canis was the most susceptible dermatophytic species, except in the case of thyme T. violaceum, which was the most susceptible dermatophytic species. The maximum inhibition was recorded in the cases of cinnamon and cinnamon + lemongrass combination against M. canis. The least minimum inhibitory concentrations were attained by cinnamon and clove against M. canis, cinnamon + clove against M. canis and T. violaceum, and cinnamon + lemongrass against M. canis, T. violaceum, T. verrucosum, and E. floccosum. The least minimum fungicidal concentration showed by cinnamon against M. canis, cinnamon + clove against M. canis and T. violaceum, cinnamon + lemongrass against M. canis, T. violaceum, T. verrucosum, and E. floccosum, and clove + lemongrass against M. canis.

Identification of Fish Species Involved with Ciguatera Food Poisoning in Okinawan Waters by Using PCR-RFLP analysis

Ciguatera fish poisoning (CFP), known as a seafood-borne disease, is caused by consumption of fish contaminated with ciguatoxins in tropical and subtropical sea. The ciguatera fishes, Variola louti, Lutjanus monostigma and L. bohar have an absolute majority in the Ryukyu Archipelago, southwestern Japan. We developed the cluster analysis of phylogenetic tree by using mitochondrial (mt) DNA 16S rRNA sequences of V. louti, L. monostigma and L. bohar and differentiate them from morphologically similar species (L. fulviflamma, L. russellii, L. argentimaculatus, Plectropomus leopardus and V. albimarginata) in our previous study. The fish were acquired from the coastal waters of the Ryukyu Archipelago, and a polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) marker of the mtDNA 16S rRNA region was used, employing the restriction enzymes BmgT120 I, Dde I, and SnaB I, to identify the fish species responsible for CFP. These results showed that a PCR-RFLP marker can be obtained more easily than a nucleotide sequence.

Study of the population genetic structure of Opisthorchis-like eggs in northern Thailand using mitochondrial genes.

Opisthorchis-like eggs are a public health problem in northern and northeastern Thailand. However, the genetic epidemiology and structure of these parasites in northern Thailand are unknown. Thus, this study investigated their population genetic structure using cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 1 (nad1) nucleotide sequences. A study was conducted in the hill tribe regions of Chiang Mai Province, northern Thailand. Internal transcribed spacer 2 polymerase chain reaction and restriction fragment length polymorphism were used to distinguish 205 positive feces samples for Opisthorchis-like eggs. The results showed that the prevalence of O. viverrini and Haplorchis taichui was 10.5% and 38.2%, respectively, and the co-infection rate was 37.2%. To determine the genetic structure of O. viverrini and H. taichui using cox1 and nad1 genes, genetic analysis was performed using 30 randomly chosen fecal samples for Opisthorchis-like eggs. Pairwise FST analysis indicated that O. viverrini and H. taichui displayed nonsignificant genetic differentiation within Chiang Mai Province and between interpopulations from different geographic areas. Moreover, within the intrapopulation in Chiang Mai Province,cox1 presented higher gene flow than nad1 in O. viverrini, while nad1 demonstrated higher gene flow than cox1 in H. taichui. The neutrality tests based on Fu's Fs indicated population expansion and selective sweep from bottleneck or hitchhiking in O. viverrini and H. taichui populations, supported by haplotype network patterns. Phylogenetic tree analysis based on cox1 and nad1 revealed the monophyly of O. viverrini and H. taichui and genetic relationships with other isolates collected from Thailand, Lao People's Democratic Republic (PDR), and Vietnam. This study investigated the molecular discrimination and genetic structure of Opisthorchis-like eggs in northern Thailand. The genetic information derived from this study could be associated with the background, molecular epidemiology, and disease severity of these parasites.

Effect of VDR and TLR2 gene variants on the clinical course of patients with COVID-19 disease.

The coronavirus disease 2019 (COVID-19) pandemic, which has caused a major global health crisis, primarily targets the upper and lower respiratory tract. But infected individuals may experience different clinical symptoms, ranging from asymptomatic to critical. The vitamin D receptor (VDR) and Toll-like receptor 2 (TLR2) polymorphisms play a role in the immune response. This study aimed to evaluate the effect of VDR Bsml (rs1544410) and TLR2 23bp indel variants on the clinical status of Turkish patients with COVID-19 disease. A total of 312 people, including 106 intensive care unit (ICU) patients, 103 symptomatic hospitalized patients, and 103 healthy controls, were included in the study. The VDR BsmI and TLR2 23bp indel were genotyped using polymerase chain reaction and/or restriction fragment length fraction methods. The VDR BsmI b/b genotype and b allele were higher in symptomatic patients compared to the healthy control group (p = 0.035). The VDR BsmI B/B and B/b genotype distribution did not differ between ICU patients and both symptomatic patients and controls (p > 0.05). We found that B/B:B/b+b/b and B/B+B/b:b/b were significantly different in symptomatic patients compared to controls (p = 0.033 and p = 0.041, respectively). The VDR BsmI b/b genotype distribution was found to be lower in deceased patients than in living patients (p = 0.023). There was no significant difference between the groups in terms of TLR2 23bp indel genotype and allele distribution (p > 0.05). Our study results suggest that the VDR BsmI b allele may have a role in COVID-19 patients with symptomatic findings. These data need to be repeated in different ethnic and larger sample groups.

First Report on the Molecular Detection and Genetic Characterization of Toxoplasma gondii From Donkeys in Kenya.

The present study was conducted to determine the presence of Toxoplasma gondii in donkeys by molecular tests and genetic diversity analysis of the obtained DNA samples from central Kenya. A total of 363 blood samples were collected from donkeys in Meru and Kirinyaga Counties, and 96 samples that were previously seropositive for T. gondii using indirect ELISA were subjected to nested PCR based on the amplification of the internal transcribed spacer 1 (ITS-1) gene followed by DNA sequencing and phylogenetic analysis. Genotyping was performed on 15 selected positive samples using multilocus nested polymerase chain reaction restriction fragment length polymorphism (Mn-PCR-RFLP) with eight genetic markers ('SAG 2, 5'SAG 2, Alt. SAG 2, SAG 3, GRA 6, C29-2, BTUB and L358). Toxoplasma gondii DNA was detected in 36.5% (35/96) of the blood samples. The sequences obtained exhibited 98.2-99.5% homology with those deposited in GenBank. Phylogenetic analysis demonstrated that the obtained sequences are conserved and clustered with those of infecting animals from other regions of the world. Eighteen distinct T. gondii haplotypes were identified to be circulating in donkeys from central Kenya. The T. gondii DNA samples exhibited high haplotype diversity (Hd: 0.915) and limited genetic diversity (π = 0.01027). PCR-RFLP of T. gondii DNA-positive samples revealed three different genetic combinations that consisted of alleles I, II and III, indicating the dissemination of atypical genotypes. This study demonstrated that T. gondii is widespread in donkeys from Kenya and could be a possible source of infection in humans. These findings are important for designing control strategies for this parasite to improve the livestock sector, which is one of the main sources of livelihood for farmers in Kenya.

Identification of <em>Candida</em> species isolated from cancer patients by Polymerase Chain Reaction – Restriction Fragment Length Polymorphism in Apeksha Hospital, Maharagama, Sri Lanka

Identification of <em>Candida</em> species isolated from cancer patients by Polymerase Chain Reaction – Restriction Fragment Length Polymorphism in Apeksha Hospital, Maharagama, Sri Lanka

Single Nucleotide Polymorphism-based Identification of Bacterial Artificial Chromosome-mediated Homologous Recombination.

Bacterial Artificial chromosome (BAC) recombineering is a powerful genetic manipulation tool for the efficient development of recombinant genetic resources. Long homology arms of more than 150 kb composed of BAC constructs not only substantially enhance genetic recombination events, but also provide a variety of single nucleotide polymorphisms (SNPs) that are useful markers for accurately docking BAC constructs at target sites. Even if the BAC construct is homologous to the sequences of the target region, different variations may be distributed between various SNPs within the region and those within the BAC construct. Once the BAC construct carrying these variations was precisely replaced in the target region, the SNP profiles within the target genomic locus were directly replaced with those in the BAC. This alteration in SNP profiles ensured that the BAC construct accurately targeted the designated site. In this study, we introduced restriction fragment length polymorphism or single-strand conformation polymorphism analyses to validate and evaluate BAC recombination based on changes in SNP patterns. These methods provide a simple and economical solution to validation steps that can be cumbersome with large homologous sequences, facilitating access to the production of therapeutic resources or disease models based on BAC-mediated homologous recombination.

LEP rs7799039 and LEPR rs1137101 gene variants are not associated with clinical features in patients with metabolic syndrome in the Turkish population.

Genetic predisposition plays a role in the etiology of metabolic syndrome (MetS), an important health problem worldwide. Leptin (LEP), produced by adipose tissue, plays a crucial role in the development of MetS. In this study, we evaluated the effects of LEP and LEP receptor (LEPR) variants on clinical findings and risk of developing MetS in the Turkish population. A total of 320 patients were included in the study, of whom 150 were patients with MetS and 170 were healthy controls. DNA was extracted from blood samples. LEP rs7799039 and LEPR rs1137101 variants were genotyped using the polymerase chain reaction-based restriction fragment length polymorphism method. The genotype distributions of these variants and clinical and laboratory findings were compared. The LEP rs7799039 GA and AA genotypes and A allele frequencies were higher in participants with MetS than in the control group. For LEP rs7799039, the genotype AA-GA was higher in males, and the GG genotype was higher in females. On analyzing the clinical outcomes associated with these variants, it was observed that individuals possessing LEP rs7799039 GA and AA genotypes displayed elevated levels of triglycerides. In addition, those with the AG-GG genotype of LEPR rs1137101 had lower mean hemoglobin levels. Our results showed that the LEP rs7799039 and LEPR rs1137101 variants may be associated with both the risk of MetS development and clinical findings. Among the various contributors to MetS, a genetic predisposition is commonly recognized as the primary cause.

Exploring the Microbial Diversity of Botswana’s Traditional Sourdoughs

Sourdough is one of the oldest technologies employed by humans to leaven bread because of its ability to enhance the flavour and structure of bread using micro-organisms. However, there is a lack of comprehensive information in Botswana regarding the diversity of sourdough starters and the fermentative micro-organisms responsible for spontaneous fermentation. The present study aimed to explore the microbial species diversity of sourdoughs in Botswana and gain insight into the unique microbial communities involved in sourdough production. A total of nine samples were collected from different areas in Botswana. The microbial diversity in sourdoughs was characterized through the sequencing of amplicons of the 16S ribosomal DNA and internal transcribed spacer regions. In silico polymerase chain reaction–restriction fragment length polymorphism and phylogenetics were utilized to determine the genetic diversity among the isolates. The dominant yeast species identified were Saccharomyces cerevisiae, Wickerhamomyces anomamlus, Pichia kudriazverii and kazachstania humilis. Additionally, the presence of Lactiplantibacillus plantarum, Lacticaseibacillus paracasei, Liquorilactobacillus nageli and Bacillus cereus was also detected. It is worth noting that two species of acetic acid bacteria (AAB), namely Acetobacter pasteurianus and A. indonesiensis, were isolated, though in low levels, but the finding is significant in sourdough fermentation. The low occurrence of AAB (acetic acid bacteria) species observed in this study could be an important finding, as these bacteria are considered understudied, yet they are known to contribute significantly to the final product.

Incidence and Molecular Detection of Genotypes for Giardia lamblia Isolated From Children in Zakho District, Duhok Province, Kurdistan Region, Iraq.

Giardia lamblia is a significant intestinal protozoan in humans worldwide, with a high incidence of infection in developing countries, particularly among children. Molecular analysis has identified eight assemblages (A to H), with A and B more frequently associated with human infections. Regardless of its importance, to the best of our knowledge, this is the first molecular study on assemblages in Giardia lamblia adopted in the Zakho district, province of Duhok, Iraq. The present study aimed to determine the giardiasis infection rate and identify the assemblages of Giardia lamblia in children from four areas in the Zakho district. We collected fecal samples and conducted a microscopic examination. Genomic DNA was extracted, and assemblage identification was done via amplification of the gdhgene using a semi-nested polymerase chain reaction and restriction fragment length polymorphism (RFLP). Out of 31 Giardia-positive samples, 23 were successfully amplified through semi-nested PCR. Nineteen isolates (82.60%) were assigned to assemblage B, and four (17.40%) to assemblage A. Assemblage B was identified as belonging to sub-assemblages B11 and B1V, while assemblage A was identified as sub-assemblages A1 and A11. This study provides insights about Giardia lamblia assemblages in the Zakho district, Duhok province, Iraq, and may serve as a beginning step toward understanding the molecular characterization of Giardia in the studied area.

Diversity in clinical isolates of Ictalurid herpesvirus 1 (IcHV1) from U.S. farm-raised catfish and virulence assessment in channel and channel × blue catfish hybrids.

Ictalurid herpesvirus 1 (IcHV1) is the most significant viral agent in U.S. catfish aquaculture. Little is known regarding the genetic stability and antigenic variability of IcHV1. Herein, the genetic and antigenic diversity of IcHV1 field isolates was assessed by restriction fragment length polymorphism (RFLP) analysis and serum neutralization assays. RFLP analysis identified two distinct genotypes (IcHV1A and IcHV1B), both discrete from blue catfish alloherpesvirus (BCAHV). Neutralization assays with anti-IcHV1 monoclonal antibody Mab-95 indicate shared antigenic determinants for IcHV1A and IcHV1B that are absent from BCAHV, which Mab-95 did not neutralize. Virulence assessments with representative isolates demonstrate significant differences between isolates within RFLP groups and pooled RFLP group data suggest IcHV1B (pooled survival [mean ± SE]: 58.3% ± 2.6) may be more virulent than IcHV1A (survival: 68.6% ± 2.4). Rechallenges with representative IcHV1A and IcHV1B isolates indicate a cross-protective effect, with fish surviving initial exposure to IcHV1A or IcHV1B showing robust protection when subsequently re-exposed to IcHV1A or IcHV1B. This work demonstrated significant differences in virulence between case isolates, identifying two discrete IcHV1 lineages, distinct from BCAHV, with similar virulence in channel and channel × blue catfish hybrids and a cross-protective effect in catfish surviving exposure to either lineage.

Investigation of Vaspin and Visfatin -4689G/T Gene Polymorphisms in Alopecia Areata Patients

Alopecia Areata (AA) is a chronic autoimmune condition that causes recurrent hair bereavement. Genetic and immunological factors act a part in the pathogenesis of AA. The aim of this study was to look into relationship between the vaspin and visfatin -4689G/T gene polymorphisms and AA sensibility in the Turkish population. This study included 80 AA patients and 80 healthy controls. Genomic DNA was extracted of blood samples Vaspin and visfatin -4689G/T gene polymorphisms were determined using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) methods. The observed disparity in vaspin genotypes and allele distribution amid AA patients and healthy controls did not reach statistical significance (χ2 = 2.51, df = 1, p = 0.11 and χ2 = 1.75, df = 1, p = 0.18, respectively). Although visfatin GT genotype was higher in AA patients compared to control, it was not statistically significant. People with the visfatin GT genotype were more likely to be AA than people with the GG genotype [OR (95% CI) = 2.11 (1.04-4.27), p = 0.03]. This study shows that there is no affair amid vaspin and visfatin -4689G/T polymorphism and AA in the Turkish population. However, the TT genotype for the vaspin gene and the GT genotype for the visfatin -4689G/T gene are risk factors for people with AA disease.

Association of VEGFR1, VEGFR2 and VEGFR3 polymorphisms with esophageal cancer risk: a case–control study

BackgroundEsophageal cancer is the eleventh most common cancer and is the seventh leading cause of mortality worldwide. Vascular endothelial growth factor (VEGF) and its receptors pathway are a key regulator of angiogenesis and play an important role in carcinogenesis. The aim of current study was to evaluate the association of five VEGFR polymorphisms with esophageal cancer risk in patients from Punjab, North-west India.MethodsThis case–control study included 310 esophageal cancer patients and 325 age and gender matched healthy controls. VEGFR1-710C/T, VEGFR2-604 T/C (rs2071559), VEGFR2 1192 G/A (rs2305948), VEGFR2 1719A/T (rs1870377) and VEGFR3 (rs72816988) polymorphisms were genotyped by using polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) method. Restriction digestion products were analyzed on 2.4% agarose gel and genotype was assigned to each sample on the basis of fragments obtained after digestion. Randomly 10% samples were repeated by Sanger sequencing to revalidate the results.ResultsThere was a significant association of CT genotype (OR = 0.28; 95%CI, 0.10–0.76; p = 0.01) and T allele (OR = 0.28; 95%CI, 0.10–0.77; p = 0.01) of VEGFR1-710C/T polymorphism with decreased risk of esophageal cancer. TC genotype of VEGFR2-604 T/C (OR = 0.66; 95%CI, 0.44–0.97; p = 0.03) and GA genotype of VEGFR2 1192G/A (OR = 0.54; 95%CI, 0.31–0.95; p = 0.03) polymorphisms were significantly associated with decreased risk of esophageal cancer. There was no significant difference in allele and genotype frequency of VEGFR2 1719A/T and VEGFR3 (rs72816988) polymorphisms between esophageal cancer patients and controls (p > 0.05). Haplotype analysis revealed that haplotype C-604A1719A1192 was significantly associated with the decreased esophageal cancer risk (OR = 0.44; 95%CI, 0.23–0.84; p = 0.01).ConclusionVEGFR1-710C/T, VEGFR2-604 T/C and VEGFR2 1192G/A polymorphisms were associated with the decreased risk of esophageal cancer in patients from Punjab, North-west India.Graphical abstract

Novel Insights into Hb Shaare Zedek Associated with β0-Thalassemia: Molecular Characteristics, Genetic Origin and Diagnostic Approaches.

Hemoglobin Shaare Zedek (Hb SZ) is a rare structural α-Hb variant. Characterizing its genotype-phenotype relationship and genetic origin enhances diagnostic and clinical management insights. We studied a proband and six family members using high-performance liquid chromatography (HPLC), capillary electrophoresis (CE), PCR, and sequencing to analyze α- and β-globin genes and α-globin haplotypes. Pathogenicity predictions and a rapid diagnostic method were developed. The proband, his father, grandfather, and aunt had Hb migrating to the HbH-zone on CE and elevated fetal hemoglobin (HbF) on HPLC. Direct sequencing identified an A to G mutation at codon 56 of the α2-globin gene, characteristic of Hb SZ. Additionally, the proband carried a β-globin gene mutation [HBB.52A>T]. Mild thalassemia-like changes were observed in the proband, whereas individuals with only the Hb SZ variant did not exhibit these changes. Pathogenicity predictions indicated that Hb SZ is benign. The variant can be identified using restriction fragment length polymorphism (RFLP) and allele-specific PCR. The Thai variant of Hb SZ is associated with the haplotype [- - M - - - -]. Hb SZ is a non-pathological variant that minimally affects red blood cell parameters, even when it coexists with β0-thalassemia. HPLC and CE systems cannot distinguish it from other Hbs, necessitating DNA analysis for accurate diagnosis.

Molecular typing methods to characterize Brucella spp. from animals: A review.

Brucellosis is an infectious disease of animals that can infect humans. The disease causes significant economic losses and threatens human health. A timely and accurate disease diagnosis plays a vital role in the identification of brucellosis. In addition to traditional diagnostic methods, molecular methods allow diagnosis and typing of the causative agent of brucellosis. This review will discuss various methods, such as Bruce-ladder, Suiladder, high-resolution melt analysis, restriction fragment length polymorphism, multilocus sequence typing, multilocus variable-number tandem repeat analysis, and whole-genome sequencing single-nucleotide polymorphism, for the molecular typing of Brucella and discuss their advantages and disadvantages.

Associations of ACE I/D and AGTR1 rs5182 polymorphisms with diabetes and their effects on lipids in an elderly Chinese population

BackgroundDiabetes mellitus is generally accompanied by dyslipidaemia, but inconsistent relationships between lipid profiles and diabetes are noted. Moreover, genetic variations in insertion/deletion (I/D) polymorphisms at angiotensin-converting enzyme gene (ACE) and T/C polymorphisms in the angiotensin type 1 receptor gene (AGTR1) are related to diabetes and lipid levels, but the associations are controversial. Thus, the current research aimed to explore the effects of ACE I/D, AGTR1 rs5182 and diabetes mellitus on serum lipid profiles in 385 Chinese participants with an average age of 75.01 years.MethodsThe ACE I/D variant was identified using the polymerase chain reaction (PCR) method, whereas the AGTR1 rs5182 polymorphism was identified using the PCR-based restriction fragment length polymorphism (PCR-RFLP) method and verified with DNA sequencing. Total cholesterol (TC), triglyceride (TG), apolipoprotein A (ApoA), apolipoprotein B (ApoB), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) levels were measured using routine methods, and the lipid ratios were calculated.ResultsACE I/D, but not AGTR1 rs5182, was a predictor of TG/HDL-C for the whole study population. Both ACE I/D and AGTR1 rs5182 were predictors of HDL-C and LDL-C levels in females but not in males. Moreover, in females, diabetes mellitus and ACE I/D were identified as predictors of TG and TG/HDL-C, whereas AGTR1 rs5182 and diabetes mellitus were predictors of TG/HDL-C. Moreover, diabetes mellitus and the combination of ACE I/D and AGTR1 rs5182 variations were predictors of TG and TG/HDL-C exclusively in females.ConclusionsThe results demonstrated the potential for gender-dependent interactions of ACE I/D, AGTR1 rs5182, and diabetes on lipid profiles. These findings may serve as an additional explanation for the inconsistent changes of blood lipids in individuals with diabetes mellitus, thereby offering a novel perspective for the clinical management of blood lipid levels in diabetic patients.

A rapid quarantine approach for the pathogenic and invasive Phytophthora species associated with imported fruits in China.

Phytophthora spp. represent a pivotal genus of plant pathogens with a global distribution, exerting significant deleterious effects on food safety and forestry ecosystems. Numerous pathogenic and invasive Phytophthora species, introduced through imported fruits, have been frequently detected at Chinese ports. With the rise in global trade activities, the plant quarantine of imported fruits is becoming increasingly important but challenging. Fast, simple, and labor-saving techniques are necessary and anticipated. A polymerase chain reaction restriction fragment length polymorphism capillary electrophoresis (PCR-RFLP-CE) technology-based quarantine approach was developed for 16 Phytophthora species associated with the imported fruits in China. The Ypt1 gene, exhibiting abundant interspecific variations, was selected as the marker gene for PCR. The restriction endonuclease AluI was proven to be capable and compatible in simultaneously separating different Phytophthora species during CE. By combining with a fast and efficient DNA extraction kit, the developed PCR-RFLP-CE technique was successfully applied to identify Phytophthora species in artificially infested fruits. We provide a quick, practical, and high-throughput detection approach for hazardous and invasive Phytophthora species associated with imported fruits in China. This strategy can give good convenience and technological support for carrying out massive quarantine activities at Chinese ports. © 2024 Society of Chemical Industry.