Sanger Sequencing Technique Research Articles

Microsatellite Markers & Mitochondrial D-Loop Based Phylogenetic And Diversity Analysis In Gabrali Cattle.

The Gabrali cattle is a multipurpose breed, native to Khyber Pakhtunkhwa, Pakistan. Despite its economic importance, scientific data about its phylogeny and genetic diversity is scarce. To address this issue, the present study was conducted on thirty (30) unrelated Gabrali male and female animals, from which blood samples were collected and DNA was extracted. Twelve (12) microsatellite loci and 1159bp of D-loop region were amplified via PCR and visualized on a 2% agarose gel. Sanger sequencing technique was used to sequence the D-loop amplicons. The microsatellite loci revealed 83 alleles with MNA (mean no of alleles per locus) = 8.8, the Ho (observed heterozygosity) and He (expected heterozygosity) of all loci were 0.58 and 0.50 respectively, mean PIC (Polymorphic Information content) = 0.59 and FIS (Inbreeding coefficient) = 0.056 using GeneAlEx® software. Microsatellite analysis revealed a normal allelic distribution across the breed, consistent with Hardy-Weinberg equilibrium. D-loop sequencing revealed eight haplotypes with 30 SNPs using DNA SP 6.0 software. High AT content was observed than GC content i.e. 55.9% and 44.1% respectively, while the transition to transversion ratio was R = 10:1. The value of Haplotype and Nucleotides diversity was Hd = 0.8601 ± SD = 0.0895 and Π = 0.0136 ± SD = 0.00197 respectively. Phylogenetic analysis done using Neighbor-joining method with bootstraps value of 1000, revealed the monophyletic clade of both Gabrali and Bos indicus haplotypes. Furthermore, the introgression of genes from national cattle breeds into Gabrali cattle was observed. It is concluded that Gabrali cattle have common ancestry with Bos indicus having no threats of genetic bottleneck having substantial genetic diversity.

Microbial and Sensory Characteristics of Traditional Watery Kimchi (Dongchimi) Fortified with Probiotics

Dongchimi, a traditional Korean watery kimchi, relies on complex interactions among diverse lactic acid bacteria (LAB) to maintain its freshness and quality. Recently, dongchimi has gained attention as a health-promoting food due to its content of probiotics and prebiotics. In this study, six probiotic strains were employed into dongchimi fermentation, and its sensory and microbial characteristics were evaluated. The LAB-enriched dongchimi demonstrated superior sensory preference (63%) and significantly higher LAB counts (average 5.2 × 107 CFU/ml) compared to traditional dongchimi. Furthermore, microbial diversity between the LAB-enriched and traditional dongchimi was analyzed during the fermentation process using both culture-dependent Sanger sequencing and culture-independent metabarcoding techniques, employing 16S ribosomal RNA gene sequences. Lactiplantibacillus plantarum was identified as the dominant probiotic strain in both types of dongchimi, while other probiotics, including Bifidobacterium bifidum, B. animalis, Limosilactobacillus Fermentum, and Heyndrickxia coagulans, were exclusively detected in the LAB-enriched dongchimi. In conclusion, Lactiplanti. plantarum and Limosi. Fermentum were identified as the most effective probiotics for dongchimi fermentation. These results offer critical insights into the microbial ecology and probiotic strains essential for optimizing synbiotic dongchimi, thereby reinforcing health claims related to probiotics and prebiotics.

Students’ Habits and Identification of Bacteria on Inanimate Surface of Educational Setting

Introduction: Diponegoro Clinic reported 29.1% of students seek infectious diseases treatment. Student habits in the classroom were thought to play roles in the presence of bacteria. Surfaces of inanimate objects in the classroom were potential source for bacteria. The research objectives were analyzing student’s habits and identifying bacteria on the surface of inanimate objects in the classroom. Methods: Four types of samples, including the surfaces of tables, chairs, flips of air conditioners, and floors were taken from 13 faculties at Universitas Diponegoro. Plate count agar media were used to isolate bacterial colonies, and PCR analysis was performed for DNA extraction and amplification. DNA Sanger sequencing techniques were used for genetic bacterial identification. The online questionnaire was used to assess student habits in the classroom. Two hundred students responded. Results and Discussion: Acinetobacter baumannii and Staphylococcus aureus were found in classrooms. These bacteria were associated with respiratory tract infections. This study revealed that 86.5% were between the ages of 17-21, 60.81% were from outside Semarang City, and 88.33% lived in Semarang City. About 60.81% of respondents studied in health sciences. Furthermore, it was reported that 66.67% of respondents were sick in the last few weeks, attended class despite being sick (72.52%), and coughed and sneezed in class (40.99%). Conclusion: Bacteria associated with respiratory tract infection were found. Students' habits in the classroom were potentially caused by the presence of these bacteria. The use of antibacterial agents could reduce the presence of bacteria on inanimate object surfaces.

Analysis of Single Nucleotide Polymorphisms of Liver X Receptor Alpha (LXR-α) Gene in Diabetic Kidney Disease.

Introduction Genetic factors significantly contribute to the progression of diabetic kidney disease (DKD), which is one of the world's most common causes of chronic kidney disease (CKD). Inflammation, glucose homeostasis, and lipid metabolism are all significantly influenced by the liver X receptor alpha (LXR-α) gene. Single nucleotide polymorphisms in the liver X receptor alpha gene may result in the altered function of the gene, making the individual susceptible to DKD. The purpose of this article is to examine the frequency of expression of single nucleotide polymorphisms (SNPs) in exon 7 of the LXR-α gene in people with type 2 diabetes (T2DM) and DKD. Gaining an understanding of these genetic differences could improve risk assessmentand therapy approaches while providing fresh perspectives on the molecular mechanisms underlying DKD. Aims and objectives To identify specific SNPs in the LXR-αgene in T2DMand DKD patients. Methods The study was conducted between May 2022 and May 2023 and it involved 120 T2DM and 120 DKD patients. All the clinical history of the study participants were recorded. Blood samples were collected from the participants and genomic DNA was isolated and was followed by polymerase chain reaction (PCR) using exon 7 specified primers of LXR-α gene. To find mutations in the LXR-α gene, the PCR products were subsequently analyzed using the capillary-based Sanger sequencing technique. Results A total of seven SNPs were detected in T2DM patients, and 20 SNPs were detected in DKD patients. SNP rs7120118 was found with the highest frequency of 100 (83.33%) in T2DM patients and 80 (66.66%) in DKD patients. SNP rs1956299169 was found to have the second highest frequency of 60 (50%) among T2DM patients and SNP rs1252683195 was found to have a frequency of 20 (16.7%). Othercommon SNPs were rs1956299169, with 40 (33.33%) in DKD and 60 (50%) in T2DM, and rs12574081, with 20 (16.71%) in T2DM and 10 (8.33%) in T2DM. Conclusion The study detects the highest and the most common SNP rs7120118, which may be associated with T2DM and DKD. Thereby, ithelps to conduct furtherassociation studies with larger populations and to diagnose the disease.

Clinical whole Exome Sequencing Reveals Novel Homozygous Missense Variant in the PMPCA Gene causing Autosomal Recessive Spinocerebellar Ataxia.

Autosomal recessive cerebellar ataxias (ARCA) are rare heterogenous neurodegenerative disorders characterized by degeneration of the cerebellum and spinal cord with an early onset before the age of 20 years. PMPCA (MIM: 613036), is a key enzyme in mitochondrial protein processing which is critical for cell survival and growth. Our objective was to investigate Peptidase, Mitochondrial Processing Subunit Alpha (PMPCA) mutations linked with Spinocerebellar ataxia, autosomal recessive 2 (SCAR2). In the current study, Whole Exome Sequencing (WES) was done followed by Sanger sequencing for the validation of the WES results. WES results identified a novel homozygous variant, NM_015160.2: c.802C>T p.(Arg268Trp) in PMPCA gene. Mutation in this gene leads to progressive cerebellar ataxia with fine motor skills difficulties, intentional tremors, slow slurred speech and learning difficulties in a 12-year-old Saudi patient. WES results were further validated by Sanger sequencing technique. Identified phenotype in our case was similar as previously described for SCAR2 related conditions. To our knowledge, this is the first reported mutation in PMPCA gene leading to SCAR2 in Saudi Arabia. These findings will enrich the scarce literature, further provide a new insight on the role of PMPCA gene-related disorders leading to SCAR2 and expand the disease concept. In addition, this will help to establish a database for the disease and its causative factors will further help in controlling diseases resulting from consanguinity in Saudi population.

Characterization of the complete mitochondrial genome of the Sunda stink-badger (Mydaus javanensis) from the island of Borneo.

The Mephitidae is a family of skunks and stink-badgers that includes 12 extant species in four genera, namely, Mydaus, Conepatus, Mephitis and Spilogale. Mydaus is the only genus within Mephitidae found outside the American continent, with its distribution limited to the islands of Borneo, Indonesia and Philippines. There are two extant species of Mydaus i.e., javanensis and marchei. Currently, complete mitogenomes are unavailable for either species. Here, we present the characterization of the first complete mitogenome for the Sunda stink-badger (Mydaus javanensis) from the island of Borneo. Muscle tissue was obtained and the DNA was sequenced using a combination of Illumina Barcode Tagged Sequence (BTSeq) and Sanger sequencing techniques. The genome was annotated with MITOS and manually checked for accuracy. A circular map of the mitogenome was constructed with Proksee. Relative synonymous codon usage (RSCU) and codon frequency were calculated using MEGA-X. The protein coding genes (PCGs) were aligned with reference sequences from GenBank and used for the construction of phylogenetic trees (maximum liklihood (ML) and Bayesian inference (BI)). Additionally, due to the lack of available complete genomes in public databases, we constructed another tree with the cyt b gene. The complete circular mitogenome was 16,391 base pairs in length. It comprises the typical 13 protein-coding genes, 22 tRNAs, two ribosomal RNA genes, one control region (CR) and an L-strand replication origin (OL). The G+C content was 38.1% with a clear bias towards A and T nucleotides. Of the 13 PGCs, only ND6 was positioned in the reverse direction, along with five other tRNAs. Five PCGs had incomplete stop codons and rely on post-transcriptional polyadenylation (TAA) for termination. Based on the codon count, Leucine was the most common amino acid (589), followed by Threonine (332) and Isoleucine (325). The ML and BI phylogenetic trees, based on concatenated PCGs and the cyt b gene, respectively, correctly clustered the species with other members of the Mephitidae family but were unique enough to set it apart from Conepatus, Mephitis and Spilogale. The results confirm Mydaus as a member of the mephitids and the mitogenome will be useful for evolutionary analysis and conservation of the species.

CircSEC24B activates autophagy and induces chemoresistance of colorectal cancer via OTUB1-mediated deubiquitination of SRPX2

Circular RNAs (circRNAs) are a type of regulatory RNA that feature covalently closed single-stranded loops. Evidence suggested that circRNAs play important roles in the progression and development of various cancers. However, the impact of circRNA on autophagy-mediated progression of colorectal cancer (CRC) remains unclear. The objective of this project was to investigate the influence of circSEC24B on autophagy and its underlying mechanisms in CRC. To validate the presence and circular structure of circSEC24B in CRC cells and tissues, PCR and Sanger sequencing techniques were employed. Drug resistance and invasive phenotype of CRC cells were evaluated using CCK8, transwell, and Edu assays. Gain- and loss-of-function experiments were conducted to assess the effects of circSEC24B and its protein partner on the growth, invasion, and metastasis of CRC cells in vitro and in vivo. Interactions between circSEC24B, OTUB1, and SRPX2 were analyzed through immunofluorescence, RNA-pulldown, and RIP assays. Mass spectrometry analysis was used to identify potential binding proteins of circRNA in CRC cells. Vectors were constructed to investigate the specific structural domain of the deubiquitinating enzyme OTUB1 that binds to circSEC24B. Results showed that circSEC24B expression was increased in CRC tissues and cell lines, and it enhanced CRC cell proliferation and autophagy levels. Mechanistically, circSEC24B promoted CRC cell proliferation by regulating the protein stability of SRPX2. Specifically, circSEC24B acted as a scaffold, facilitating the binding of OTUB1 to SRPX2 and thereby enhancing its protein stability. Additionally, evidence suggested that OTUB1 regulated SRPX2 expression through an acetylation-dependent mechanism. In conclusion, this study demonstrated that circSEC24B activated autophagy and induced chemoresistance in CRC by promoting the deubiquitination of SRPX2, mediated by the deubiquitinating enzyme OTUB1.

Clinical and molecular characterization of myotonia congenita using whole-exome sequencing in Egyptian patients.

Myotonia Congenita (MC) is a rare disease classified into two major forms; Thomsen and Becker disease caused by mutations in the CLCN1 gene, which affects muscle excitability and encodes voltage-gated chloride channels (CLC-1). While, there are no data regarding the clinical and molecular characterization of myotonia in Egyptian patients. Herein, we report seven Egyptian MC patients from six unrelated families. Following the clinical diagnosis, whole-exome sequencing (WES) was performed for genetic diagnosis. Various in silico prediction tools were utilized to interpret variant pathogenicity. The candidate variants were then validated using Sanger sequencing technique. In total, seven cases were recruited. The ages at the examination were ranged from eight months to nineteen years. Clinical manifestations included warm-up phenomenon, hand grip, and percussion myotonia. Electromyography was performed in all patients and revealed myotonic discharges. Molecular genetic analysis revealed five different variants. Of them, we identified two novel variants in the CLCN1 gene ( c.1583G > C; p.Gly528Ala and c.2203_2216del;p.Thr735ValfsTer57) and three known variants in the CLCN1 and SCN4A gene. According to in silico tools, the identified novel variants were predicted to have deleterious effects. As the first study to apply WES among Egyptian MC patients, our findings reported two novel heterozygous variants that expand the CLCN1 mutationalspectrum for MC diagnosis. These results further confirm that genetic testing is essential for early diagnosis of MC, which affects follow-up treatment and prognostic assessment in clinical practice.

Screening for antifolate and artemisinin resistance in Plasmodium falciparum dried-blood spots from three hospitals of Eritrea

Background Antimalarial drug resistance is a major challenge hampering malaria control and elimination. About three-quarters of Eritrea’s population resides in the malaria-endemic western lowlands of the country. Plasmodium falciparum, the leading causative parasite species, has developed resistance to basically all antimalarials. Continued surveillance of drug resistance using genetic markers provides important molecular data for treatment policies which complements clinical studies, and strengthens control efforts. This study sought to genotype point mutations associated with P. falciparum resistance to sulfadoxine-pyrimethamine and artemisinin, in dried-blood spots from three hospitals in the western lowlands of Eritrea. Methods Dried-blood spot samples were collected from patients visiting Adi Quala, Keren and Gash Barka Hospitals, between July and October, 2014. The patients were followed up after treatment with first line artesunate-amodiaquine, and dried-blood spots were collected on day three after treatment. Nested polymerase chain reaction and Sanger sequencing techniques were employed to genotype point mutations in the Pfdhfr (PF3D7_0417200), Pfdhps (PF3D7_0810800) and PfK13 (PF3D7_1343700) partial gene regions. Results Sequence data analyses of PCR-positive isolates found wild-type artemisinin haplotypes associated with resistance (Y493Y, R539R, I543I) in three isolates, whereas four mutant antifolate haplotypes associated with resistance were observed in six isolates. These included the triple-mutant Pfdhfr (S108N, C59R, N51I) haplotype, the double-mutant Pfdhfr (N51I, S108N) haplotype, the single-mutant Pfdhfr (K540E) haplotype, and the mixed-mutant Pfdhfr-Pfdhps (S108N, N51I + K540E) haplotype. Other findings observed were, a rare non-synonymous Pfdhfr V45A mutation in four isolates, and a synonymous Pfdhps R449R in one isolate. Conclusions The mutant antifolate haplotypes observed indicate a likely existence of full SP resistance. Further studies can be carried out to estimate the prevalence of SP resistance. The wild-type artemisinin haplotypes observed suggest artemisinin is still an effective treatment. Continuous monitoring of point mutations associated with delayed parasite clearance in ART clinical studies is recommended.

Genetic landscape for majority and minority HIV-1 drug resistance mutations in antiretroviral therapy naive patients in Accra, Ghana

BackgroundThe successful detection of drug-resistance mutations (DRMs) in HIV-1 infected patients has improved the management of HIV infection. Next-generation sequencing (NGS) to detect low-frequency mutations is predicted to be useful for efficiently testing minority drug resistance mutations, which could contribute to virological failure. This study employed Sanger sequencing and NGS to detect and compare minority and majority drug resistance mutations in HIV-1 strains in treatment-naive patients from Ghana. MethodFrom a previous study, 20 antiretroviral therapy (ART)-naive participants were selected for a cross-sectional study. Sanger sequencing and NGS techniques were used to detect the majority and minority HIV drug resistance (HIVDR) mutations, respectively, in the protease (PR) and partial reverse transcriptase (RT) genes. NGS detected mutations at 1 % and 5 % frequencies and Sanger sequencing at ≥20 % frequencies. The sequences obtained from NGS and Sanger sequencing platforms were submitted to the Stanford HIV drug resistance database for subtyping, mutation identification, and interpretations. ResultsSequences from the twenty participants where the CRF02_AG was the predominant strain (16, 80 %) were analyzed. NGS detected 25 mutations in the RT and PR genes, compared to 21 mutations by Sanger sequencing. Minority DRMs were detected at the prevalence of 55.0 % with NGS against 35 % DRMs by Sanger sequencing. One of the patients had eight different HIVDR variants, with two minority variants. These mutations were directed against PI (K20I and D30DN), NNRTI (Y181C, M23LM and V108I) and NRTI (K65R, M184I, and D67N). ConclusionThe study affirms the usefulness of genomic sequencing for drug resistance testing in HIV. It further shows that Sanger sequencing alone may not be adequate to detect mutations and that NGS capacity should be developed and deployed in the Ghanaian clinical settings for patients living with HIV.

Complex genotype–phenotype correlation of MYH11: new insights from monozygotic twins with highly variable expressivity and outcomes

BackgroundThoracic aortic aneurysm/dissection (TAAD) and patent ductus arteriosus (PDA) are serious autosomal-dominant diseases affecting the cardiovascular system. They are mainly caused by variants in the MYH11 gene, which encodes the heavy chain of myosin 11. The aim of this study was to evaluate the genotype–phenotype correlation of MYH11 from a distinctive perspective based on a pair of monozygotic twins.MethodsThe detailed phenotypic characteristics of the monozygotic twins from the early fetal stage to the infancy stage were traced and compared with each other and with those of previously documented cases. Whole-exome and Sanger sequencing techniques were used to identify and validate the candidate variants, facilitating the analysis of the genotype–phenotype correlation of MYH11.ResultsThe monozygotic twins were premature and presented with PDA, pulmonary hypoplasia, and pulmonary hypertension. The proband developed heart and brain abnormalities during the fetal stage and died at 18 days after birth, whereas his sibling was discharged after being cured and developed normally post follow-up. A novel variant c.766 A > G p. (Ile256Val) in MYH11 (NM_002474.2) was identified in the monozygotic twins and classified as a likely pathogenic variant according to the American College of Medical Genetics/Association for Molecular Pathology guidelines. Reviewing the reported cases (n = 102) showed that the penetrance of MYH11 was 82.35%, and the most common feature was TAAD (41.18%), followed by PDA (22.55%), compound TAAD and PDA (9.80%), and other vascular abnormalities (8.82%). The constituent ratios of null variants among the cases with TAAD (8.60%), PDA (43.8%), or compound TAAD and PDA (28.6%) were significantly different (P = 0.01). Further pairwise comparison of the ratios among these groups showed that there were significant differences between the TAAD and PDA groups (P = 0.006).ConclusionThis study expands the mutational spectrum of MYH11 and provides new insights into the genotype–phenotype correlation of MYH11 based on the monozygotic twins with variable clinical features and outcomes, indicating that cryptic modifiers and complex mechanisms beside the genetic variants may be involved in the condition.

Molecular Detection and Phylogenetic Analysis of the alkB Gene in Klebsiella oxytoca Strains Isolated from the Gut of Tenebrio molitor.

The challenge in polystyrene disposal has caused researchers to look for urgent innovative and ecofriendly solutions for plastic degradation. Some insects have been reported to use polystyrene as their sole carbon source, and this has been linked to the presence of microbes in their guts that aid in plastic digestion. Thus, this study focuses on the molecular detection and phylogenetic analysis of the alkane-1-monooxygenase (alkB) gene in Klebsiella oxytoca strains isolated from the gut of Tenebrio molitor. The alkB gene encodes for alkane-1-monooxygenase, an enzyme involved in the oxidation of inactivated alkanes. This gene can be used as a marker to assess bacteria's ability to biodegrade polystyrene. Three bacterial strains were isolated from the guts of T. molitor mealworms and were confirmed using polymerase chain reaction (PCR) of the 16S ribosomal RNA gene. The primers used in the amplification of the 16S ribosomal RNA region were designed using NCBI, a bioinformatics tool. To detect the presence of the alkB gene in the isolated bacterial strains, a set of primers used in the amplification of this gene was manually designed from the conserved regions of the alkB nucleotide sequences of eleven bacterial species from GenBank. TCOFFE online tool was used to align the alkB sequences of the bacteria, while Jalview and ConSurf were used to view the alignment. The amplified alkB gene was then sequenced using the Sanger sequencing technique, blasted on NCBI to look for similar sequences, and a phylogenetic tree was constructed. Based on the 16S ribosomal RNA gene sequences, the isolated bacterial strains were confirmed to be Klebsiella oxytoca NBRC 102593, Klebsiella oxytoca JCM 1665, and Klebsiella oxytoca ATCC 13182. The alkB gene sequence identical to fourteen alkB gene sequences derived from Actinobacteria whole genome was detected in Klebsiella oxytoca for the first time to the best of our knowledge. The novel nucleotide sequence was published in the NCBI database under accession number OP959069. This gene sequence was found to be for the enzyme alkane-1-monooxygenase and may be one of the enzymes responsible for polystyrene degradation by the putative Klebsiella oxytoca ATCC 13182 in T. molitor.

PHYLOGENETIC ANALYSIS OF TWELVE BULGARIAN SEQUENCES BASED ON PARTIAL OPEN READING FRAME 2 GENOME FRAGMENT OF HEPATITIS E VIRUS

Background: Hepatitis E virus (HEV) causes both acute and chronic liver inflammation. HEV is transmitted through the fecal-oral mechanism and infects both animals and humans. The virus belongs to the Hepeviridae family and its genome is a single stranded RNA molecule. Thanks to molecular sequencing methods different genotypes and subgenotypes have been established. The aim of the present study was to identify and characterize Bulgarian HEV sequences by applying Sanger sequencing technique for a genome fragment in Open Reading Frame 2 (ORF2 region). Material and methods: Twelve retrospective samples from patients with serologically confirmed HEV infection (anti-HEV IgM and IgG positive) were sequenced by Sanger sequencing. Sequencing data were analysed by BioEdit, MEGA11 and NCBI Genbank software tools. Results: The results revealed that all isolates assign to species Paslahepevirus balayani. Phylogenetic analysis showed that HEV isolates were characterized with considerable genetic diversity. The sequences were sub-clustered into the following subgenotypes: HEV-3e, 3m, 3f and 3c. Conclusion: We successfully applied the Sanger method for hepatitis E virus RNA sequencing. The established heterogeneity of subgenotypes requires further study in order to determine the circulation of all possible subgenotypes of HEV in the country.

Screening for antifolate and artemisinin resistance in Plasmodium falciparumdried-blood spots from three hospitals of Eritrea.

Antimalarial drug resistance is a major challenge hampering malaria control and elimination. About three-quarters of Eritrea's population resides in the malaria-endemic western lowlands of the country. Plasmodium falciparum, the leading causative parasite species, has developed resistance to basically all antimalarials. Continued surveillance of drug resistance using genetic markers provides important molecular data for treatment policies which complements clinical studies, and strengthens control efforts. This study sought to genotype point mutations associated with P. falciparum resistance to sulfadoxine-pyrimethamine and artemisinin, in dried-blood spots from three hospitals in the western lowlands of Eritrea. Dried-blood spot samples were collected from patients visiting Adi Quala, Keren and Gash Barka Hospitals, between July and October, 2014. The patients were followed up after treatment with first line artesunate-amodiaquine, and dried-blood spots were collected on day three after treatment. Nested polymerase chain reaction and Sanger sequencing techniques were employed to genotype point mutations in the Pfdhfr (PF3D7_0417200), Pfdhps (PF3D7_0810800) and PfK13 (PF3D7_1343700) partial gene regions. Sequence data analyses of PCR-positive isolates found wild-type artemisinin haplotypes associated with resistance (Y493Y, R539R, I543I) in three isolates, whereas four mutant antifolate haplotypes associated with resistance were observed in six isolates. These included the triple-mutant Pfdhfr (S108N, C59R, N51I) haplotype, the double-mutant Pfdhfr (N51I, S108N) haplotype, the single-mutant Pfdhfr (K540E) haplotype, and the mixed-mutant Pfdhfr-Pfdhps (S108N, N51I + K540E) haplotype. Other findings observed were, a rare non-synonymous Pfdhfr V45A mutation in four isolates, and a synonymous Pfdhps R449R in one isolate. The mutant antifolate haplotypes observed indicate a likely existence of full SP resistance. Further studies can be carried out to estimate the prevalence of SP resistance. The wild-type artemisinin haplotypes observed suggest artemisinin is still an effective treatment. Continuous monitoring of point mutations associated with delayed parasite clearance in ART clinical studies is recommended.

Novel pathogenic variants of SLC38A8 gene and literature review.

This study aimed to analyze the clinical and genetic characteristics of 6 Chinese patients with foveal hypoplasia (FH) caused by the variants of solute carrier family 38 member 8 (SLC38A8), and to describe the genotype and phenotype of SLC38A8 variants from previous literature. All subjects underwent comprehensive ophthalmic examinations. Optical coherence tomography (OCT) was performed to evaluate the structural grade of FH. Pathogenic variants of SLC38A8 gene were identified using panel-based next-generation sequencing and direct Sanger sequencing techniques. Further, all previously reported cases of SLC38A8 variants were re-analyzed together with the novel ones identified in this study. Nystagmus and FH were present in 6 patients with variants of SLC38A8 gene, accompanied by a normal anterior segment. Grade 4 FH was identified in 4 patients. A total of 12 variants of SLC38A8 gene were identified, including 9 novel variants. Systematical analysis revealed that half of the variants (30/60) were missense, the majority of which (23/30) were distributed in the transmembrane (TM) domains. Grade 4 FH was detected in the majority of patients (66%, 23/35). There was no statistical difference in the clinical features between the subgroups of patients with 0, 1 and 2 missense variants. Severe arrest of foveal development was identified in patients with variants of SLC38A8. This study provides a brief summary of the clinical and genetic characteristics of the pathogenic SLC38A8 variants, which is helpful in the differentiation diagnosis of FH.

The Molecular Detection, Characterization, and Temperature Dependence of Wolbachia Infections in Field Populations of Aedes albopictus (Diptera: Culicidae) Mosquitoes in Greece

We investigated the prevalence and genetic diversity of Wolbachia pipientis strains in Aedes albopictus populations in Greece. Using a combination of PCR and Sanger sequencing techniques, we genotyped Wolbachia strains in 105 mosquitoes collected across eight different administrative regions in 2021. We found a high prevalence of Wolbachia in both male (90%) and female (97%) mosquitoes. Among the infected samples, 84% had double infections with both wAlbA and wAlbB strains, while 16% had infections with only wAlbB. Our comparison of the Multi-Locus Sequence Typing (MLST) profile, employing gatB–coxA–hcpA–ftsZ–fbpA genotyping, revealed a single MLST profile for each wAlbA and wAlbB strain in Greek populations. The same MLST profiles were also reported in populations from China, Russia, and Argentina, suggesting low levels of global diversity in wAlbA and wAlbB strains. Furthermore, our results indicated a significant association between temperature and the prevalence of single infections (p = 6.498 × 10−7), with higher temperatures correlating with an increased likelihood of single infections. Although male bias showed a tendency towards single infections, the effect was marginally non-significant (p = 0.053). These results were confirmed using a bootstrap-with-replacement analysis approach. Overall, our findings offer novel insights into the distribution and species diversity of Wolbachia strains in Greek Ae. albopictus populations, emphasizing the importance of understanding the short-term plastic and adaptive responses of these organisms to environmental stressors and rapid climate change.

A novel APC mutation associated with Gardner syndrome in a Chinese family

Gardner syndrome (GS) is a specific form of familial adenomatous polyposis (FAP), which manifests as colorectal polyps, multiple osteomas and soft tissue tumors, and in the oral cavity as osteomas of the jaws, odontomas, and abnormal tooth counts. The underlying cause of GS is attributed to mutations in the APC gene. Mutations in this gene disrupt the normal functioning of the protein and lead to the development of GS. To further investigate GS, a family affected by the syndrome was selected from Dongguan, Guangdong Province. The family members underwent a comprehensive survey, which involved collecting clinical data and peripheral venous blood samples. The samples were then used for genetic analysis. Whole exome sequencing (WES) and Sanger sequencing techniques were utilized to screen and identify specific mutation sites in the APC gene. The clinical findings for the GS family included the presence of gastrointestinal polyps and odontomas. After analyzing the genetic sequencing results, a novel mutation site c.4266dupA on the APC gene was found in the patients, which leading to the APC protein truncation. As a result of this study, it is suggested that odontoma may be an early indicator of GS. Additionally, the identification of this novel mutation site in the APC gene expands the known spectrum of genetic mutations associated with the disease. This discovery has significant implications for the early diagnosis of GS, thus enabling timely intervention to reduce the risk of developing colon cancer and other related diseases.

Low Pathogenic Avian Influenza H9N2 Viruses in Morocco: Antigenic and Molecular Evolution from 2021 to 2023.

Avian influenza viruses pose significant threats to both the poultry industry and public health worldwide. Among them, the H9N2 subtype has gained substantial attention due to its high prevalence, especially in Asia, the Middle East, and Africa; its ability to reassort with other influenza viruses; and its potential to infect humans. This study presents a comprehensive phylogenetic and molecular analysis of H9N2 avian influenza viruses circulating in Morocco from 2021 to 2023. Through an active epidemiological survey, a total of 1140 samples (trachea and lungs) and oropharyngeal swabs pooled into 283 pools, collected from 205 farms located in 7 regions of Morocco known for having a high density of poultry farms, were analyzed. Various poultry farms were investigated (159 broiler farms, 24 layer farms, 10 breeder farms, and 12 turkey breeder farms). A total of 21 AI H9N2 strains were isolated, and in order to understand the molecular evolution of the H9N2 avian influenza virus, their genetic sequences were determined using the Sanger sequencing technique. Phylogenetic analysis was performed using a dataset comprising global H9N2 sequences to determine the genetic relatedness and evolutionary dynamics of the Moroccan strains. The results revealed the continued circulation and diversification of H9N2 avian influenza viruses in Morocco during the study period. Real-time RT-PCR showed a positivity rate of 35.6% (73/205), with cycle threshold values ranging from 19.2 to 34.9. The phylogenetic analysis indicated that all Moroccan strains belonged to a G1-like lineage and regrouped into two distinct clusters. Our newly detected isolates aggregated distinctly from the genotypes previously isolated in Morocco, North and West Africa, and the Middle East. This indicats the potential of virus evolution resulting from both national circulation and cross-border transmission. A high genetic diversity at both nucleotide and amino-acid levels was observed among all the strains isolated in this study, as compared to H9N2 strains isolated in Morocco since 2016, which suggests the co-circulation of genetically diverse H9N2 variants. Newly discovered mutations were detected in hemagglutinin positions 226, 227, and 193 (H3 numbering), which highlights the genetic evolution of the H9N2 AIVs. These findings contribute to our understanding of the evolution and epidemiology of H9N2 in the region and provide valuable insights for the development of effective prevention and control strategies against this emerging avian influenza subtype.

The impact of circulating 25-hydroxyvitamin D and vitamin D receptor variation on leukemia-lymphoma outcome: Molecular and cytogenetic study

Vitamin D (VD) potentially has a crucial function in the development of cancerous cells. This study aims to detect the role of vitamin D concentration and its receptor polymorphisms as possible prognostic biomarkers in patients with leukemia/lymphoma and further will attempt to detect the presence of the Philadelphia chromosome abnormality in chronic myeloid leukemia (CML). Seventy-five patients, in addition to 50 healthy individuals were included. Three single nucleotide polymorphisms of the vitamin D receptor (FokI, Tru91, and ApaI) were identified via Polymerase Chain Reaction- Fragment Length Polymorphism (PCR-RFLP). Sanger sequencing and karyotyping for all patients has been undertaken. Out of 75 patients, 69 (92.0%) were vitamin D deficient. The homozygous genotype TT of FokI is the most commonly found in non-Hodgkin's lymphoma, while the heterozygous CT is observed markedly in CML, chronic lymphoid leukemia, and Hodgkin's lymphoma. The AC and CC genotypes of ApaI are more frequent in patients with CML, while the AC genotype is the most common in HL. In Tru9I, the GG genotype has a wider distribution in individuals diagnosed with leukemia. The PCR-RFLP and Sanger sequencing techniques together confirmed significant genotype respectively. The Philadelphia chromosome, t (9;22) was found in five (17%) cases with CML. There is a marked relationship between FokI, ApaI, and Tru91 polymorphisms and the chance of developing leukemia. In lymphoma, a significant connection between the polymorphisms of FokI and ApaI is frequently detected. Cytogenetic and molecular testing are essential for detection of CML and monitoring therapy response.

Genotypic variants of the tetrahydrobiopterin (BH4) biosynthesis genes in patients with hyperphenylalaninemia from different regions of Iran.

Hyperphenylalaninemia (HPA) is a metabolic disorder classified into phenylalanine-4-hydroxylase (PAH) and non-PAH deficiency. The latter is produced by mutations in genes involved in the tetrahydrobiopterin (BH4) biosynthesis pathway and DNAJC12 pathogenetic variants. The BH4 metabolism, including de novo biosynthesis involved genes (i.e., guanosine 5'-triphosphate cyclohydrolase I (GTPCH/GCH1), sepiapterin reductase (SR/SPR), 6-pyruvoyl-tetrahydropterin synthase (PTPS/PTS)), and two genes that play roles in cofactor regeneration pathway (i.e., dihydropteridine reductase (DHPR/QDPR) and pterin-4α-carbinolamine dehydratase (PCD/PCBD1)). The subsequent systemic hyperphenylalaninemia and monoamine neurotransmitter deficiency lead to neurological consequences. The high rate of consanguineous marriages in Iran substantially increases the incidence of BH4 deficiency. We utilized the Sanger sequencing technique in this study to investigate 14 Iranian patients with non-PAH deficiency. All affected subjects in this study had HPA and no mutation was detected in their PAH gene. We successfully identified six mutant alleles in BH4-deficiency-associated genes, including three novel mutations: one in QDPR, one in PTS, and one in the PCBD1 gene, thus giving a definite diagnosis to these patients. In this light, appropriate patient management may follow. The clinical effect of reported variants is essential for genetic counseling and prenatal diagnosis in the patients' families and significant for the improvement of precision medicine.