The influence of P2Y12 gene polymorphisms on clopidogrel therapy in patients after percutaneous coronary intervention.

Aim: We aimed to define the influence of P2Y12 polymorphisms (rs6801273, rs2046934, and rs6809699), diabetes, hypertension, obesity, hypercholesterolemia, statins intake, and smoking habit on clopidogrel therapy in patients undergoing percutaneous coronary intervention. Materials & methods: We used PCR-RFLP and PCR-ASO for P2Y12 genotype analysis. The effectiveness of the therapy was measured with the VerifyNow method and defined in platelet reactivity units. Results: Studied polymorphisms had no statistically significant influence on PRU before (PRU0) and 6 months (PRU6) after the procedure. H1/H1 diabetic carriers had significantly higher PRU6 values than patients without diabetes. Obese H1/H2 subjects had significantly lower PRU6 values than H1/H2 non-obese carriers. Conclusion: We found that obesity and diabetes may influence the long-term outcome of antiplatelet therapy.

Clopidogrel is a medicine that prevents platelets in the blood from clumping and blocking arteries. When the structure of the protein (e.g., P2Y12), responsible for response to clopidogrel is changed, we can observe less efficient therapy. Said changes can be caused for example by genetic polymorphisms, which are two or more variants of the same gene. This is why we wanted to check the impact of P2Y12 polymorphisms. We also wanted to check the impact of diabetes, high blood pressure, being overweight, high cholesterol blood level, cholesterol-reducing drugs, and smoking habits on clopidogrel treatment in patients after a procedure that unblocks blood vessels of the heart to restore its blood supply (percutaneous coronary intervention). We measured the efficacy of the treatment with platelet reactivity units (PRU). Studying polymorphisms had no impact on treatment efficacy before (PRU0) and 6 months (PRU6) after the medical procedure. We found that diabetes can cause higher platelet reactivity after 6 months of therapy. We noticed that being overweight may also be important, as obese patients had lower platelet reactivity values.

Genetic Variability in Leishmaniasis-Causing Leishmania infantum in Humans and Dogs from North-East Spain.

Leishmania infantum is the primary cause of visceral and cutaneous leishmaniasis in the European Mediterranean region. Subspecies-level characterization of L. infantum aids epidemiological studies by offering insights into the evolution and geographical distribution of the parasite and reservoir identity. In this study, conducted in north-east Spain, 26 DNA samples of L. infantum were analyzed, comprising 21 from 10 humans and 5 from 5 dogs. Minicircle kinetoplast DNA (kDNA) polymerase chain reaction assays using primers MC1 and MC2, followed by sequencing, were employed to assess intraspecific genetic variability. Single-nucleotide polymorphism (SNP) analysis detected seven genotypes (G1, G2, G12*-G15*, and G17*), with five being reported for the first time (*). The most prevalent was the newly described G13 (54%), while the other currently identified genotypes were predominantly found in single samples. The in silico restriction fragment length polymorphism (RFLP) method revealed five genotypes (B, F, N, P, and W), one of them previously unreported (W). Genotype B was the most prevalent (85%), comprising three SNP genotypes (G1, G2, and G13), whereas the other RFLP genotypes were associated with single SNP genotypes. These kDNA genotyping methods revealed significant intraspecific genetic diversity in L. infantum, demonstrating their suitability for fingerprinting and strain monitoring.

Prevalence and effect of PIK3CA H1047R somatic mutation among Indian head and neck cancer patients.

PIK3CA is one among the several mutated genes in cancer, including head and neck squamous cell carcinoma (HNSCC). H1047R is a hotspot somatic mutation in PIK3CA that occurs most frequently in several forms of cancers. Distribution of PIK3CA H1047R mutation in Indian HNSCC patients was screened and its effect on disease progression and response to treatment was analysed in this study. Genomic DNA was extracted from tumour biopsies of HNSCC patients (n = 48) and polymerase chain reaction coupled restriction fragment length polymorphism (PCR-RFLP) technique was used to screen for the mutation. Overall survival (OS) and Progression-free survival (PFS) of the patients were calculated in order to study effect of this mutation on survival and response to treatment respectively. Results showed that irrespective of patients' criteria, twenty-five patients (52 %) carried a heterozygous form of mutation (His/Arg) and the rest (48 %) were wild type (His/His). The mean OS of the cohort with the mutation was 20.451 months (SE ± 1.710 months) while 26.31 months (SE ± 2.431) was in wild type population. PFS of the patients with the mutation was 18.612 months (SE ± 2.072), and for the wild type population, it was 26.31 months (SE ± 2.431). These observations suggest that Indian HNSCC patients with PIK3CA H1047R mutation have poor prognosis.

Development of PCR-based genetic test for detection of TTF1 mutation causing abortion in Holstein Friesian cattle.

A stop-gain mutation (rs715966442; BTA11: 1,02,463,944 nucleotide position) in transcription termination factor, RNA polymerase I (TTF1) gene causes abortion in Holstein Friesian (HF) cattle. A PCR-restriction fragment length polymorphism (PCR-RFLP)-based genetic test has been developed and validated to screen the TTF1 mutation locus in HF cattle. The mutation locus was screened in 80 HF and HF crossbreds using the protocol, which revealed two animals as carriers of the mutant TTF1 allele. The test employed is cost-effective, rapid and precise and can be utilized as an effective tool for the screening of TTF1 mutation carriers in HF cattle population.

Potential risk factors for hypospadias and negative correlation with DICER1 (rs3742330) A>G variant in Algerian population: A case-control study.

BACKGROUND: Hypospadias continues to be a prevalent congenital anomaly affecting the male external genitalia, characterized by an unclear origin and complex treatment approaches. This study aimed to investigate the risk factors associated with hypospadias and explore its genetic link with the DICER1 rs3742330 variant. METHODS: The study involved two groups: 105 male children with hypospadias and 111 healthy male children as matched controls. Detailed history and physical examinations were conducted for all patients and controls. PCR-restriction fragment length polymorphism was utilized to identify the DICER1 rs3742330 variant, analyzing genotype distribution and allele frequency. Logistic regression analysis estimated the risk factors for hypospadias. RESULTS: The mean age in the hypospadias group was 4.56 ± 2.50 years. The most prevalent type of hypospadias observed was the anterior type in 60 children (57.14%). Intrauterine growth restriction, advanced maternal age, and gestational hypertension were identified as significant risk factors for hypospadias (p = .011, p = .016, and p = .041, respectively). Regarding the genetic study, no significant difference was found in both genotype and allele frequencies of the DICER1 rs3742330 variant between case and control groups. CONCLUSIONS: The rs3742330 variant in the DICER1 gene showed no association with hypospadias cases in the Algerian population. However, multivariate logistic regression analysis identified preterm birth, low birth weight, intrauterine growth restriction, advanced maternal age, gestational diabetes, and rural residence as the most significant independent predictors for hypospadias.

Finger printing-RFLP analysis of chromosomal IS6110 insertion sequence and PCR diagnosis of pulmonary tuberculosis, isolated from patients in El Hajeb region of Morocco.

Tuberculosis (TB) is one of the contagious diseases caused by M. tuberculosis (MTB) bacteria. Prompt diagnosis is one of the active solutions to control the spread of this infection. Besides, a targeted, specific and non-complex diagnosis can prove promising in this type of epidemic. This study was designed to compare the efficiencies of a diagnosis by Ziehl-Neelsen staining (ZN) and by the polymerase chain reaction (PCR) technique. Samples presented smear-positive pulmonary TB were subjected to Chromosomal restriction fragment length polymorphism of IS6110 (IS6110-RFLP) for fingerprinting profile determination. The results showed that out of 100 sputum samples of suspected case, 53 were positive. Numbers of positive individuals for tuberculosis obtained by the different diagnostic techniques, to know, (ZN staining; culture and PCR) were respectively: 6, 25 and 22. Chromosomal RFLP fingerprinting profile revealed the presence of five different genotypes obtained from seven tested isolates. These results suggest that molecular techniques are alternative tool for fast and specific diagnosis of pulmonary MTB from sputum.

[Identification of Cervi Cornu (Cervus elaphus) and its formula granules based on PCR-RFLP].

Cervi Cornu is the ossified antler, or the base antler that falls off in the spring of the following year after the pilose antler is sawn off from Cervus elaphus or C. nippon, as a precious traditional Chinese medicine, has been recognized for its medicinal value and widely used in clinical practice. However, the origins of Cervi Cornu are miscellaneous, and Cervi Cornu is even mixed with adulterants in the market. Currently, there is a shortage of ways to identify Cervi Cornu and no standard to control the quality of Cervi Cornu. So it is valuable to develop a way to effectively identify Cervi Cornu from the adulterants. In this study, the differences in the mitochondrial barcode cytochrome b(Cytb) gene sequences of C. elaphus, C. nippon and their related species were compared and the specific single nucleotide polymorphism(SNP) sites on the Cytb sequences of Cervi Cornu were screened out. According to the screened SNPs, Cervi Cornu-specific primers dishmy-F and dishmy-R were designed. The PCR system was established and optimized, and the tolerance and feasibility of Taq polymerases and PCR systems affecting the repeatability of the PCR method were investigated. The amplification products of C. elaphus and C. nippon were digested using the restriction enzyme MseⅠ. The results showed that after electrophoresis of the product from PCR with the annealing temperature of 56 ℃ and 35 cycles, a single specific band at about 100 bp was observed for C. elaphus samples, and the product of C. elaphus samples was 60 bp shorter than that of C. nippon samples. There was no band for adulterants from other similar species such as Alces alces, Rangifer tarandus, Odocoileus virginianus, O. hemionus, Cap-reolus pygargus, Przewalskium albirostis and negative controls. The polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) method established in this study can quickly and accurately identify Cervi Cornu originated from C. elaphus in crude drugs, standard decoctions, and formula granules, and distinguish the origins of Cervi Cornu products, i.e., C. nippon and similar species. This study can be a reference for other studies on the quality standard of other formula granules of traditional Chinese medicines.

Molecular identification of Sarcocystis species in wild boar (Sus scrofa) and pigs (Sus scrofa domesticus) in Brazil.

Sarcocystis spp. are protozoan parasites that form cysts in the organs and musculature of various animal species. The species Sarcocystis miescheriana and Sarcocystis suihominis are pathogenic to pigs and wild boars (Sus scrofa), acting as intermediate hosts, while humans are the definitive host for S. suihominis. To date, there have been no reports of the identification of these coccidian species in Sus scrofa in Brazil. Therefore, in this study, we conducted the first molecular identification of Sarcocystis species using PCR-RFLP and sequencing. A total of 210 samples were analyzed, of this total, 67 tested positive for Sarcocystis spp., representing 31.9% of the total samples assessed. Out of the total positive samples, 55 (82.1%) were identified as S. miescheriana and 8 (11.9%) as S. suihominis, a zoonotic species. Additionally, other species related to bovines, such as S. cruzi and zoonotic S. hominis, were detected in 3.0% of the samples, serving as contaminants in the pork products. The presence of S. suihominis in swine and wild boar samples is concerning due to the zoonotic risk and potential environmental contamination, as humans act as definitive hosts, also for the presence of S. hominis as a bovine contaminant in pork sausages. Furthermore, we confirmed the efficacy of the PCR-RFLP technique as a reliable tool for the identification of Sarcocystis species, demonstrating its potential use in laboratories for molecular diagnosis and rapid identification of these parasites, aiming to protect public health and ensure food safety.

An artificial-restriction fragment length polymorphism (A-RFLP) method for genotyping intronic SNP rs7587633 C/T of ATG16L1.

SNPs could either cause a disorder or directly alter the efficacy of a particular treatment and act as biological markers. The SNP rs7587633 C/T present in the intronic region of the ATG16L1 gene has been studied for its role in psoriasis vulgaris and Palmoplantar pustulosis. To genotype rs7587633 C/T using PCR-RFLP no restriction site is present for any of the restriction enzymes at the SNP position. To develop an artificial-RFLP method for genotyping rs7587633 C/T, the forward primer was designed in such a way that it resulted in the creation of an EcoRI restriction site in the amplified product which could further be digested with EcoRI to find the genotype of the individual. The newly developed A-RFLP method was applied to genotype the SNP rs7587633 C/T in DNA samples of 100 healthy control individuals. The allelic and genotypic frequencies of the SNPs were 0.80(C), 0.20(T) and 65%(CC), 31%(CT) and 4%(TT), respectively. In conclusion, we developed an A-RFLP method to genotype the SNP rs7587633 C/T which is not present in any of the natural restriction sites and this method could be applied to genotype this SNP in various populations/diseases to find its role.

Molecular characterization of clinical and environmental isolates from the Cryptococcus neoformans/C. Gattii species complexes of Maceió, Alagoas, Brazil.

Cryptococcosis is one of the major life-threatening opportunistic/systemic fungal diseases of worldwide occurrence, which can be asymptomatic or establish pneumonia and meningoencephalitis mainly in immunosuppressed patients, caused by the Cryptococcus neoformans and C. gattii species complexes. Acquisition is by inhaling fungal propagules from avian droppings, tree hollows and decaying wood, and the association of the molecular types with geographic origin, virulence and antifungal resistance have epidemiological importance. Since data on cryptococcosis in Alagoas are limited, we sought to determine the molecular types of etiological agents collected from clinical and environmental sources. We evaluated 21 isolates previously collected from cerebrospinal fluid and from environment sources (pigeon droppings and tree hollows) in Maceió-Alagoas (Brazil). Restriction fragment length polymorphism of URA5 gene was performed to characterize among the eight standard molecular types (VNI-VNIV and VGI-VGIV). Among isolates, 66.67% (14) were assigned to C. neoformans VNI - 12 of them (12/14) recovered from liquor and 2 from a tree hollow (2/14). One isolate from pigeon droppings (4.76%) corresponded to C. neoformans VNIV, while five strains from tree hollows and one from pigeon droppings (6, 28.57%) to C. gattii VGII. VNI-type was present in clinical and environmental samples and most C. neoformans infections were observed in HIV-positive patients, while types VNIV and VGII were prevalent in environmental sources in Alagoas. This is the first molecular characterization of Cryptococcus spp. in Alagoas, our study provides additional information on the ecoepidemiology of Cryptococcus spp. in Brazil, contributing to a closer view of the endemic species.

First report of Meloidogyne hapla on hemp (Cannabis sativa) in Oregon.

Hemp is a crop that has gained interest in Washington and Oregon. As with other crops, hemp production faces challenges due to biotic factors, including plant-parasitic nematodes. During a survey for plant-parasitic nematodes associated with hemp, Meloidogyne sp. was found in a composite root sample collected in Oregon. Morphological characterization of second-stage juveniles identified the nematode as Meloidogyne hapla. Molecular identification confirmed the population as M. hapla. To our knowledge, this is the first report of M. hapla on hemp in the Pacific Northwest of the United States.

The Association of miRNA-146a Gene Variation and Multiple Sclerosis in The Iranian Population.

Background: Multiple sclerosis (MS) is a complex human autoimmune-type inflammatory disease of the central nervous system (CNS). MicroRNA-146a (miR-146a) belongs to an endogenous and non-coding RNA family with 18-22 nucleotides long, which modulates the innate and adaptive immune response. Methods: Our study aimed to investigate a possible association between rs2910164 and rs2431697 polymorphisms of the miR-146a gene and multiple sclerosis in the Iranian population. A total of 60 MS cases and 100 controls were recruited. Single nucleotide polymorphism (SNP) rs2431697 was genotyped by utilizing polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), and SNP rs2910164 was genotyped by using Tetra-primer ARMS-PCR. Statistical Analysis conducted by the chi-squared test utilizing SPSS version 21.0 Software. The Hardy-Weinberg equilibrium assumption was evaluated. Results: The results of the present study suggest the miR-146a gene rs2431697 polymorphism is not associated with multiple sclerosis. However, there is a significant relationship between polymorphism rs2910164 of the miR-146a gene and multiple sclerosis in the population studied (P = 0.012). Conclusion: Our data provide evidence that the miR-146a gene may be involved in creating the susceptibility to MS in the Iranian population.

Association between MUC1 rs4072037 polymorphism and Helicobacter pylori in patients with gastric cancer.

Background: The MUC1 gene encodes glycoproteins attached to cell membrane that play a protective role in gastric cancer and protect epithelial surfaces against external factors such as Helicobacter pylori. H. pylori infection can induce a cascade of innate and acquired immune responses in gastric mucosa. Relationship between rs4072037G>A polymorphism of MUC1 gene and increased susceptibility to H. pylori infection aimed to investigate in patients with gastric cancer in Mazandaran, northern Iran. Methods: A case-control study was conducted on 99 patients with gastric cancer (H. pylori positive and negative) and 98 controls (H. pylori positive and negative) without gastric cancer (confirmed by pathological biopsy samples obtained during endoscopy). H. pylori infection was diagnosed by histological examination using Giemsa staining. Genomic DNA extracted from peripheral blood was analyzed by PCR-RFLP technique. Results: Analysis of all genetic models showed no significant relationship between rs4072037G>A polymorphism and risk of gastric cancer (GC). The relationship between H. pylori infection and rs4072037G>A polymorphism showed an increased susceptibility to gastric cancer in both positive and negative H. pylori groups (including case and control groups). The genetic model of GA/GG and H. pylori- positive versus GA/GG and H. pylori-negative showed a significantly increased susceptibility to gastric cancer (OR=0.251, CI: 0.128-0.493, P=0.000). Conclusion: These findings indicate that rs4072037G>A polymorphism may interact with H. pylori infection to increase the risk of GC.

Detection of Booroola Polymorphism of Bone Morphogenetic Protein Receptor 1b and Embrapa Polymorphism of Growth Differentiation Factor 9 in Sheep in Thailand.

This study aimed to investigate the appearance and frequencies of the Booroola polymorphism of the bone morphogenetic protein receptor 1b (BMPR1B) gene (FecB) and the Embrapa polymorphism of the growth differentiation factor 9 (GDF9) gene (FecGE) in sheep in Thailand. A total of 454 crossbred sheep blood samples were collected from four provinces in Thailand during August 2022 to July 2023. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was used to identify the FecB and FecGE genotypes. The history of ewe birth types was collected from the owners to analyze the association between fecundity (Fec) genotypes and the history of birth types. The genotypic frequencies of FecB for homozygous genotype (B/B), heterozygous genotype (+/B), and wildtype (+/+) were 0.22%, 1.54%, and 98.24%, respectively. Meanwhile, the genotypic frequencies of FecGE for homozygous genotype (E/E), heterozygous genotype (+/E), and wildtype (+/+) were 0.00%, 2.42%, and 97.58%, respectively. Furthermore, three ewes exhibited both FecB and FecGE genotypes. Fisher's exact test revealed that possession of the FecB genotype was associated with multiple births (p < 0.01). Both FecB and FecGE mutations were identified in crossbred sheep in Thailand. Sheep containing FecB allele could be alternative candidates to be selected to improve the prolificacy of crossbred sheep in Thailand.

Mutagenic primer-based novel multiplex PCR-RFLP technique to genotype BECN1 SNPs rs10512488 and rs11552192.

BACKGROUND: Single Nucleotide Polymorphisms (SNPs) in candidate autophagy gene BECN1 could influence its functions thereby autophagy process. BECN1 noncoding SNPs were found to be significantly associated with neurodegenerative disease and type 2 diabetes mellitus. This study aimed to develop a simultaneous genotyping technique for two BECN1 SNPs (rs10512488 and rs11552192). METHODS: A mutagenic primer-based approach was used to introduce a NdeI restriction site to genotype rs10512488 by Artificial-Restriction Fragment Length Polymorphism (A-RFLP) along with rs11552192 by Polymerase Chain Reaction (PCR)-RFLP. Multiplexing PCR and restriction digestion reactions were set up for simultaneous genotyping of both SNPs in 100 healthy individuals. Genotypic and allele frequencies were manually calculated, and the Hardy-Weinberg Equilibrium was assessed using the chi-square test. RESULTS: We successfully developed PCR and RFLP conditions for the amplification and restriction digestion of both SNPs within the same tube for genotyping. The results of genotyping by newly developed multiplexing PCR-RFLP technique were concordant with the genotypes obtained by Sanger sequencing of samples. Allelic frequencies of rs10512488 obtained were 0.15 (A) and 0.85 (G), whereas allelic frequencies of rs11552192 were 0.16 (T) and 0.84 (A). CONCLUSION: The newly developed technique is rapid, cost-effective and time-saving for large-scale applications compared to sequencing methods and would play an important role in low-income settings. For the first time, allelic frequencies of rs10512488 and rs11552192 were reported among the North Indian population.

Effect of MAOA rs1465108 polymorphism on susceptibility to substance/alcohol use disorder: a novel PCR-RFLP assay for the detection of MAOA rs1465108.

BACKGROUND: The health and social consequences of substance/alcohol use disorders are harmful. Most of the individuals cannot stop using them due to more likely their genetic background. The current study aimed both to develop a novel PCR-RFLP method for genotyping of MAOA rs1465108 and to analyze the effect of MAOA rs1465108 on the risk of alcohol (AUD), opioid (OUD) or methamphetamine (MUD) use disorders and on the depressive and anxiety symptoms in a Turkish population. METHODS AND RESULTS: A total of 353 individual with AUD (n = 154), OUD (n = 160) or MUD (n = 39) and 109 healthy subjects were included. The intensity of anxiety and depressive symptoms and craving and opioid withdrawal were measured by appropriate scales. Logistic regression analysis revealed no association between MAOA rs1465108 polymorphism and substance/alcohol use disorder (p > 0.05). Healthy subjects (3.0) had significantly lower levels of depressive symptoms than individuals with OUD (27.0), AUD (21.0) and MUD (25.5) groups. The severity of depressive symptoms was significantly higher in OUD as compared to AUD. There was a statistically significant difference between individuals with AUD, OUD and MUD in view of the average ages of first use (17, 19 and 20 years, respectively) (p < 0.05). CONCLUSIONS: The results presented here do not support the hypothesis that MAOA rs1465108 is associated with substance/alcohol use disorders. The intensity of depressive symptoms could be changed according to the abused substance type. A novel PCR-RFLP was developed for genotyping of MAOA rs1465108 polymorphism, which could be a better option for laboratories without high technology equipment.

Association of combined -344T/C and K173R polymorphisms in aldosterone synthase gene with type 2 diabetes mellitus in the Moroccan population.

Background: Aldosterone synthase (CYP11B2) is crucial for aldosterone production, and variations in its gene may influence type 2 diabetes mellitus (T2DM) development. This study explores the link between two single nucleotide polymorphisms (SNPs) in the CYP11B2 gene - -344T/C and K173R and T2DM in the Moroccan population . Methods: The research involved 86 individuals with T2DM and 75 control subjects. Genotyping for the -344T/C and K173R SNPs was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis . Result: Results indicated significant differences in the genotype and allelic distribution of the CYP11B2 K173R polymorphism between T2DM patients and control subjects, with P-values of 0.02 and 0.04, respectively. The -344T/C polymorphism showed no significant genomic level differences, but its allelic variations were statistically significant (P=0.01), indicating a notable association between the C allele and T2DM. Furthermore, the K173R polymorphism was found to significantly increase T2DM risk, with a 2.34-fold higher risk in individuals carrying the KR genotype. The study also examined the combined effect of these SNPs. The dominant model analysis (TT vs. TC+CC and KK vs. KR+RR) showed significant differences between T2DM patients and controls for both SNPs. Additionally, a haplotype-based analysis revealed that the C-R haplotype was associated with an increased risk of T2DM. Conclusion: Our study suggests a significant association between the CYP11B2-K173R polymorphism and T2DM in the Moroccan population. Conversely, while the CYP11B2 -344T/C polymorphism exhibits a significant difference in allelic distribution, no significant difference is observed at the genomic level.

Molecular investigation of Anaplasma phagocytophilum and related strains among sheep flocks from different parts of Türkiye; with a note of phylogenetic analyses of Anaplasma phagocytophilum- like 1.

Anaplasma phagocytophilum is a vector-borne zoonotic pathogen and can infect various vertebrate hosts, especially cattle, sheep, goats, horses, and dogs. Molecular-based studies have revealed that the agent has a high genetic diversity and closely related strains circulate in hosts. In this study, 618 sheep blood samples obtained from different geographic regions of Türkiye were researched for A.phagocytophilum and related strains with PCR, RFLP, and DNA sequence analyses. The DNA of these pathogens was detected in 110 (17.79%) samples. RFLP assay showed that all positive samples were infected with A.phagocytophilum-like 1, whereas A.phagocytophilum-like 2 and A.phagocytophilum were not detected. Partial parts of 16 S rRNA gene of seven randomly selected positive samples were sequenced. The phylogenetic analyses of these isolates revealed that at least two A.phagocytophilum-like 1 isolates circulate among hosts in Türkiye and around the world. A.phagocytophilum-related strains have been reported in molecular-based studies over the last few years, but there is a lack of data on the vector competence, epidemiology, clinical symptoms, and genetic diversity of these pathogens. Therefore, large-scale molecular studies are still needed to obtain detailed data on the above-mentioned topics.

Determination of ITS1 haplotypes of Fritillariae Cirrhosae Bulbus by amplicon sequencing.

BACKGROUND: Fritillariae Cirrhosae Bulbus is an antitussive and expectorant Chinese medicinal material derived from the dried bulbs of six Fritillaria species. In the 2015 edition of the Chinese Pharmacopoeia, the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) is the officially listed method for their authenfication. Specifically, the ~ 300-bp ITS1 amplicon of only Fritillariae Cirrhosae Bulbus but not other Fritillaria species can be cleaved into two smaller fragments with restriction enzyme SmaI. Considering repeated reported cases of incomplete digestion of ITS1 amplicon, this study aims to investigate the possibility of heterogeneous ITS1 sequences contained in the Fritillariae Cirrhosae Bulbus. METHODS: In this study, ITS1 amplicons of Fritillaria Cirrhosae Bulbus and four other Fritillaria species were sequenced on Illumina platform. We utilised high-throughout amplicon sequencing to determine ITS1 haplotypes and their frequencies in Fritillaria genomes. RESULTS: Our results showed that all six botanical sources of Fritillariae Cirrhosae Bulbus indeed possess ITS1 haplotypes with no SmaI restriction site, and the average percentages of ITS1 reads containing SmaI restriction site ranged from 63.60% to 91.81%. CONCLUSION: Our findings suggest that the incomplete digestion in PCR-RFLP analysis of Fritillariae Cirrhosae Bulbus is caused by the presence of ITS1 haplotypes without SmaI restriction site due to intragenomic heterogeneity.

Molecular Analysis of Pseudomonas aeruginosa Isolates with Mutant gyrA Gene and Development of a New Ciprofloxacin Derivative for Antimicrobial Therapy.

This study focuses on the prevalence of Pseudomonas aeruginosa in various medical specimens. In addition, the investigates of this research shows the genetic analysis of pathogen-resistant isolates and chemical modifications to ciprofloxacin. A total of 225 specimens from men and women aged 30 to 60 were carefully collected and examined, including samples from wound, burn, urine, sputum, and ear samples. The data were obtained from AL Muthanna hospitals. PCR-RFLP and gene expression analysis were used to identify resistant strains and explore the genetic basis of antibiotic resistance. A ciprofloxacin derivative was synthesized and confirmed through FT-IR, 1H-NMR, and mass spectroscopy techniques then it was tested as antibacterial agent. Also, molecular docking study was conducted to predict the mechanism of action for the synthesized derivative. The results demonstrated that wound samples had the highest positive rate (33.7%) of P. aeruginosa isolates. The PCR-RFLP testing correlated ciprofloxacin resistance with gyrA gene mutation. Gene expression analysis revealed significant changes in the gyrA gene expression in comparison to the reference rpsL gene subsequent to exposure to the synthesized derivative. Furthermore, the molecular docking investigation illustrated the strategic positioning of the ciprofloxacin derivative within the DNA-binding site of the gyrA enzyme. The examination of genetic expression patterns manifested diverse effects attributed to the CIP derivative on P. aeruginosa, thus portraying it as a viable candidate in the quest for the development of novel antimicrobial agents. Ciprofloxacin derivative may offer new antimicrobial therapeutic options for treating Pseudomonas aeruginosa infections in wound specimens, addressing resistance and gyrA gene mutations.