Nucleotide Position Research Articles

Molecular and in silico investigation of a novel ECHS1 gene mutation in a consanguine family with short-chain enoyl-CoA hydratase deficiency and Mt-DNA depletion: effect on trimer assembly and catalytic activity.

Short-chain enoyl-CoA hydratase deficiency (ECHS1D) is a rare congenital metabolic disorder that follows an autosomal recessive inheritance pattern. It is caused by mutations in the ECHS1 gene, which encodes a mitochondrial enzyme involved in the second step of mitochondrial β-oxidation of fatty acids. The main characteristics of the disease are severe developmental delay, regression, seizures, neurodegeneration, high blood lactate, and a brain MRI pattern consistent with Leigh syndrome. Here, we report three patients belonging to a consanguineous family who presented with mitochondrial encephalomyopathy. Whole-exome sequencing revealed a new homozygous mutation c.619G > A (p.Gly207Ser) at the last nucleotide position in exon 5 of the ECHS1 gene. Experimental analysis showed that normal ECHS1 pre-mRNA splicing occurred in all patients compared to controls.Furthermore, three-dimensional models of wild-type and mutant echs1 proteins revealed changes in catalytic site interactions, conformational changes, and intramolecular interactions, potentially disrupting echs1 protein trimerization and affecting its function. Additionally, the quantification of mtDNA copy number variation in blood leukocytes showed severe mtDNA depletion in all probands.

In Silico Identification of Sugarcane Genome-Encoded MicroRNAs Targeting Sugarcane Mosaic Virus

Sugarcane mosaic virus (SCMV) (genus, Potyvirus; family, Potyviridae) is widespread, deleterious, and the most damaging pathogen of sugarcane (Saccharum officinarum L. and Saccharum spp.) that causes a substantial barrier to producing high sugarcane earnings. Sugarcane mosaic disease (SCMD) is caused by a single or compound infection of SCMV disseminated by several aphid vectors in a non-persistent manner. SCMV has flexuous filamentous particle of 700–750 nm long, which encapsidated in a positive-sense, single-stranded RNA molecule of 9575 nucleotides. RNA interference (RNAi)-mediated antiviral innate immunity is an evolutionarily conserved key biological process in eukaryotes and has evolved as an antiviral defense system to interfere with viral genomes for controlling infections in plants. The current study aims to analyze sugarcane (Saccharum officinarum L. and Saccharum spp.) locus-derived microRNAs (sof-miRNAs/ssp-miRNAs) with predicted potential for targeting the SCMV +ssRNA-encoded mRNAs, using a predictive approach that involves five algorithms. The ultimate goal of this research is to mobilize the in silico- predicted endogenous sof-miRNAs/ssp-miRNAs to experimentally trigger the catalytic RNAi pathway and generate sugarcane cultivars to evaluate the potential antiviral resistance surveillance ability and capacity for SCMV. Experimentally validated mature sugarcane (S. officinarum, 2n = 8X = 80) and (S. spp., 2n = 100–120) sof-miRNA/ssp-miRNA sequences (n = 28) were downloaded from the miRBase database and aligned with the SCMV genome (KY548506). Among the 28 targeted mature locus-derived sof-miRNAs/ssp-miRNAs evaluated, one sugarcane miRNA homolog, sof-miR159c, was identified to have a predicted miRNA binding site, at nucleotide position 3847 of the SCMV genome targeting CI ORF. To verify the accuracy of the target prediction accuracy and to determine whether the sugarcane sof-miRNA/ssp-miRNA could bind the predicted SCMV mRNA target(s), we constructed an integrated Circos plot. A genome-wide in silico-predicted miRNA-mediated target gene regulatory network was implicated to validate interactions necessary to warrant in vivo analysis. The current work provides valuable computational evidence for the generation of SCMV-resistant sugarcane cultivars.

Botanical Biometrics: Exploring Morphological, Palynological, and DNA Barcoding Variations in White Kwao Krua (Pueraria candollei Grah. ex Benth. and P. mirifica Airy Shaw & Suvat.)

White Kwao Krua, a crucial Thai medicinal plant, contains various phytoestrogen substances used to alleviate menopausal symptoms in estrogen-deficient women. It originates from two species, namely Pueraria candollei Wall. ex Benth. and P. mirifica Airy Shaw & Suvat. However, there exists morphological variation, and the taxonomic status between both species is ambiguous, making discrimination challenging. In this study, we aim to clarify and differentiate the morphological characteristics, palynology, and DNA barcoding of both species. The morphological results showed the stipule size is a phenotypic marker for the differentiation of both species during the vegetative stage. The palynological results, however, exhibited similarity. Through an examination of nucleotide sequences and neighbor-joining tree analysis, it was determined that the DNA barcoding of the matK region has the capability to distinguish between P. candollei and P. mirifica at nucleotide position 702. Specifically, P. candollei manifested a G base, contrasting with the C base observed in P. mirifica. This study concludes that stipule size and the matK gene in DNA barcoding serve as a distinctive characteristic for distinguishing between P. candollei and P. mirifica. These methodologies prove valuable for ensuring the accurate identification of white Kwao Krua for horticulturists.

A new HaCV-EBHSV recombinant lagovirus circulating in European brown hares (Lepus europaeus) from Catalonia, Spain

In 2020/2021, several European brown hare syndrome virus (EBHSV) outbreaks were recorded in European hares (Lepus europaeus) from Catalonia, Spain. Recombination analysis combined with phylogenetic reconstruction and estimation of genetic distances of the complete coding sequences revealed that 5 strains were recombinants. The recombination breakpoint is located within the non-structural protein 2C-like RNA helicase (nucleotide position ~ 1889). For the genomic fragment upstream of the breakpoint, a non-pathogenic EBHSV-related strain (hare calicivirus, HaCV; GII.2) was the most closely related sequence; for the rest of the genome, the most similar strains were the European brown hare syndrome virus (EBHSV) strains recovered from the same 2020/2021 outbreaks, suggesting a recent origin. While the functional impact of the atypical recombination breakpoint remains undetermined, the novel recombinant strain was detected in different European brown hare populations from Catalonia, located 20–100 km apart, and seems to have caused a fatal disease both in juvenile and adult animals, confirming its viability and ability to spread and establish infection. This is the first report of a recombination event involving HaCV and EBHSV and, despite the recombination with a non-pathogenic strain, it appears to be associated with mortality in European brown hares, which warrants close monitoring.

Formation of Genetically Determined Resistance against Human Norovirus Infection through Polymorphism of the FUT2 gene: a Review of the Literature

Relevance. Human Noroviruses (HuNoV) are highly contagious pathogens responsible of acute human norovirus infection. HuNoV is the cause of every fifth case of acute non-bacterial gastroenteritis, annually causing about 699 million cases of the disease and more than 200 thousand deaths worldwide. Controlled expression of the HBGA antigens by the FUT2 gene causes resistance to human norovirus. Polymorphisms of the FUT family genes contribute to partial or complete immunity to certain genogroups/ genotypes of norovirus.Aims. To characterize the effect of FUT2 gene polymorphisms on susceptibility to HuNoV.Results. Nonsensemutations of G428A in two homologous alleles contribute to the formation of a secretory-negative phenotype (se), which is a factor determining immunity to noroviruses. Some missense-mutations in the nucleotide positions se385,571 form partial resistance against certain genotypes. People with a secretory-negative phenotype are immune to infection by the GII.4 genotype and its genovariants.Conclusions. The expression of HLA antigens by the functionally inactive FUT2 gene plays a key role in the resistance of the human population to HuNoV. Susceptibility to HuNoV largely depends on the prevalence of HBGA phenotypic diversity among ethnic populations around the world. Targeted screening aimed at identifying polymorphisms of the FUT family will allow identifying risk groups more susceptible to HuNoV.

Phytophthora: taxonomic and phylogenetic revision of the genus-SUPP

Supplementary files:<br />Supplementary File S1. Methods for isolation, maintenance, sporulation, and storage of Phytophthora species.<br />Supplementary File S2. IDphy2 Molecular Protocols for Sanger Sequencing with seven genes. <br />Supplementary File S3. ITS rDNA, COI, MOTUs, 3G HTS and 2G HTS local databases.<br />Supplementary Fig. S1. Order Peronosporales. Maximum likelihood tree inferred with IQ-TREE v. 2 from a three-locus (ITS, COI and β-tub) alignment (Supplementary Table S1). Ultrafast bootstrap support values ≥ 50 % are shown above and posterior probabilities ≥ 0.90 from a Bayesian analysis are below branches.<br />Supplementary Fig. S2. Phytophthora and related obligate biotrophic taxa. Maximum likelihood tree inferred with IQ-TREE v. 2 from a five-locus (COI, β-tub, EF1α, L10 and HSP90) alignment (Supplementary Table S1). Ultrafast bootstrap support values ≥ 50 % are shown above and posterior probabilities ≥ 0.90 from a Bayesian analysis are below branches.<br />Supplementary Fig. S3. Seven-locus Phytophthora phylogeny featuring 213 described species. Bayesian tree inferred with MrBayes v. 3.2.7. Posterior probabilities ≥ 0.90 are shown. Sequence accessions are listed in Table 2.<br />Supplementary Fig. S4. ITS-only maximum likelihood tree. Tree inferred using IQ-TREE v. 2 from a 217 taxon alignment with 1 178 nucleotide positions and 404 binary gap positions. Ultrafast bootstrap support values ≥ 50 % are shown above and posterior probabilities ≥ 0.90 from a Bayesian analysis are below branches.<br /> Supplementary Fig. S5. COI-only maximum likelihood tree. Tree inferred using IQ-TREE v. 2 from a 190 taxon alignment with 744 nucleotide positions. Ultrafast bootstrap support values ≥ 50 % are shown above and posterior probabilities ≥ 0.90 from a Bayesian analysis are below branches.<br /> Supplementary Fig. S6. YPT1-only maximum likelihood tree. Tree inferred using IQ-TREE v. 2 from a 180 taxon alignment with 850 nucleotide positions and 322 binary gap positions. Ultrafast bootstrap support values ≥ 50 % are shown above and posterior probabilities ≥ 0.90 from a Bayesian analysis are below branches.<br /> Supplementary Fig. S7. β-tub-only maximum likelihood tree. Tree inferred using IQ-TREE v. 2 from a 213 taxon alignment with 1 053 nucleotide positions. Ultrafast bootstrap support values ≥ 50 % are shown above and posterior probabilities ≥ 0.90 from a Bayesian analysis are below branches. <br />Supplementary Fig. S8. EF1α-only maximum likelihood tree. Tree inferred using IQ-TREE v. 2 from a 192 taxon alignment with 879 nucleotide positions. Ultrafast bootstrap support values ≥ 50 % are shown above and posterior probabilities ≥ 0.90 from a Bayesian analysis are below branches. <br />Supplementary Fig. S9. L10-only maximum likelihood tree. Tree inferred using IQ-TREE v. 2 from a 186 taxon alignment with 465 nucleotide positions. Ultrafast bootstrap support values ≥ 50 % are shown above and posterior probabilities ≥ 0.90 from a Bayesian analysis are below branches. <br />Supplementary Fig. S10. HSP90-only maximum likelihood tree. Tree inferred using IQ-TREE v. 2 from a 190 alignment with 933 nucleotide positions. Ultrafast bootstrap support values ≥ 50 % are shown above and posterior probabilities ≥ 0.90 from a Bayesian analysis are below branches.<br /> Supplementary Table S1. Taxa added for Figs. 1, 2.Bourret, Tyler; Abad, Gloria; Burgess, Treena (2023), “Supporting data for taxonomic and phylogenetic revision of the genus Phytophthora based on the types”, Mendeley Data, V1, doi: 10.17632/yg2rmfzstw.1

The global population stru cture of Lacticaseibacillus rhamnosus and its application to an investigation of a rare case of infective endocarditis.

Lacticaseibacillus (formerly Lactobacillus) rhamnosus is widely used in probiotics or food supplements to promote microbiome health and may also be part of the normal microbiota of the human gastrointestinal tract. However, it rarely also causes invasive or severe infections in patients. It has been postulated that these infections may originate from probiotics or from endogenous commensal reservoirs. In this report, we examine the population structure of Lacticaseibacillus rhamnosus and investigate the utility of using bacterial genomics to identify the source of invasive Lacticaseibacillus infections. Core genome phylogenetic analysis was performed on 602 L. rhamnosus genome sequences from the National Center for Biotechnology public database. This information was then used along with newly generated sequences of L. rhamnosus isolates from yogurt to investigate a fatal case of L. rhamnosus endocarditis. Phylogenetic analysis demonstrated substantial genetic overlap of L. rhamnosus isolates cultured from food, probiotics, infected patients, and colonized individuals. This was applied to a patient who had both consumed yogurt and developed L. rhamnosus endocarditis to attempt to identify the source of his infection. The sequence of the isolate from the patient's bloodstream differed at only one nucleotide position from one of the yogurt isolates. Both isolates belonged to a clade, identified here as clade YC, composed of mostly gastrointestinal isolates from healthy individuals, some of which also differed by only a single nucleotide change from the patient's isolate. As illustrated by this case, whole genome sequencing may be insufficient to reliably determine the source of invasive infections caused by L. rhamnosus.

Detection of mtDNA Mutations Using the Amplification Refractory Mutation System-Polymerase Chain Reaction Method in Type II Diabetes Mellitus Patients in Publich Health Center Poasia

Background: Diabetes Mellitus (DM) is a heterogeneous disease caused by hereditary and ecological factors. One form of diabetes mellitus that is related to genetic factors is type 2 DM. type 2 diabetec melitus caused by dysfunction of insulin secretion, due to the presence of inhibition in the production of Adenosine Triphosphate (ATP) necessary in the process of its secretion by cells β glands of the pancreas. The dysfunction is related to the mutation of A to G at the 3243rd nucleotide position of the mitochondrial DNA tRNA gene. The purpose of the study was to find out whether there was a heteroplasmic mtDNA mutation in the genes of respondents to type 2 diabetes mellitus using the ARMS-PCR method. Methods: The type of research used is descriptive research, the sample used in this study is a blood sample in respondents of type 2 diabetes mellitus. The method chosen in this study is the Amplification Refractory Mutation System (ARMS)-PCR Method which is a PCR application that uses a specific primer. Results: According to this study, 20 of the 20 samples evaluated using the ARMS-PCR method were positive for the heteroplasmic A3243G mutation, which is defined by the presence of DNA bands measuring 200 bp on both tubes. These pathogenic mutations are inherited maternally and can cause a variety of disorders. Conclusion: After analyzing all samples, this study has concluded that they all contain hetero plasma mutations. The suggestions provided in this study are expected to be useful for future researchers who employ samples from other public health centers and hospitals.

Quantifying the impact of initial RNA primer length on nucleotide addition by RNA polymerase I

Transient state kinetic studies of eukaryotic DNA-dependent RNA polymerases (Pols) in vitro provide quantitative characterization of enzyme activity at the level of individual nucleotide addition events. Previous work revealed heterogeneity in the rate constants governing nucleotide addition by yeast RNA polymerase I (Pol I) for each position on a template DNA. In contrast, the rate constants that described nucleotide addition by yeast RNA polymerase II (Pol II) were more homogeneous. This observation led to the question, what drives the variability of rate constants governing RNA synthesis by Pol I? Are the kinetics of nucleotide addition dictated by the position of the nascent RNA within the polymerase or by the identity of the next encoded nucleotide? In this study, we examine the impact of nucleotide position (i.e. nascent RNA primer length) on the rate constants governing nine sequential nucleotide addition events catalyzed by Pol I. The results reveal a conserved trend in the observed rate constants at each position for all primer lengths used, and highlight that the 9-nucleotide, or 9-mer, RNA primer provides the fastest observed rate constants. These findings suggest that the observed heterogeneity of rate constants for RNA synthesis by Pol I in vitro is driven primarily by the template sequence.

Mitochondrial DNA 3252A>G mutation presenting as MERRF/MELAS overlapping syndrome: A case report

We report a case of 25 years old male presented with a complex phenotype of myoclonic epilepsy with ragged red fibers (MERRF) and mitochondrial encephalomyopathy, lactic acidosis and stroke- like episodes (MELAS) harbouring m.3252A>G mutation in the mitochondrially encoded tRNA leucine 1 (UUA/G) [MT-TL1] gene encoding the mitochondrial transfer ribonucleic acid (tRNA) for leucine. He presented with frequent myoclonus seizure, stroke-like episodes, elevated blood lactate with muscle biopsy showed numerous ragged-red fibers suggestive of a mitochondrial disorder. Whole mitochondrial genome sequencing revealed no mutations other than the A-to-G transition at nucleotide position 3252. This case report is the first to describe the m.3252A>G mutation in association with the MERRF/MELAS overlap syndrome.

Phylogeographic analysis points toward invasion of the Timanfaya National Park (Lanzarote; Canary Islands) by a translocated native plant (Rumex lunaria)

AbstractRumex lunaria is an endemic shrub of the Canary Islands, which is colonizing the Timanfaya National Park (TNP) in Lanzarote. Whether the arrival of R. lunaria to Lanzarote has been natural or by human intervention is still a matter of debate. To address this question, 100 specimens of R. lunaria were collected from the seven main Canary Islands, and genetic analysis of four chloroplast DNA loci were performed, covering a total length of 4809 nucleotide positions. Multiple alignments revealed 49 nucleotide substitutions, which define 30 different haplotypes. Island-specific haplotypes were found in Tenerife, La Gomera, La Palma and Gran Canaria, with the greatest diversity found in the first island. Interestingly, the unique haplotype detected in El Hierro is shared with almost all plants from Lanzarote (95%), including all individuals sampled in the TNP. The most frequent haplotype present in Gran Canaria was detected in only one sample from Lanzarote (5%). These results were corroborated by a robust phylogenetic analysis, which supports the hypothesis of a common origin of R. lunaria populations from El Hierro and the vast majority of those from Lanzarote. In addition, this study rules out the genetic singularity of the R. lunaria specimens that are colonizing the TNP.

Studying the genetic diversity of the varicella-zoster virus in selected regions of the Russian Federation using high-throughput sequencing

Introduction. Varicella-zoster virus (VZV), the causative agent of the disease of the same name and herpes zoster, is phylogenetically divided into 8 clades, the distribution of which is characterized by geographic reference to certain regions of the world. For most countries, VZV clades circulating in their territories have been identified, however, such information is almost unavailable for Russia.
 The purpose of the study is to develop an effective method for VZV typing using high-throughput sequencing technologies to identify the prevalence of various VZV clades in Moscow, Moscow Region, and Stavropol Territory.
 Materials and methods. To genotype VZV, it is enough to refer to 7 nucleotide positions. Their unique combinations can be used to assign the virus to one of the clades. Short sections of nucleotide sequences of open reading frames were obtained using a developed set of primers.
 Results. A VZV genotyping technique has been developed and optimized. Using this technique, primary data on the distribution of VZV clades in the studied regions have been obtained. Thus, it has been established that in Moscow and a number of other regions, the 1st, 3rd, and 5th clades of VZV are predominantly distributed.
 Conclusion. The developed technique, including a primer panel and a genotyping algorithm, allows VZV typing in a short time while reducing specimen preparation costs and simultaneously increasing the number of specimens in one sequencing cycle. The results obtained using this assay allow us to assume that in Moscow, Moscow Region, Stavropol Territory, VZV, clades 1, 3, and 5 are the most represented ones. To confirm this hypothesis, it is necessary to include a larger number of clinical specimens in subsequent studies, including from other regions of the country.

Atypical Modes of CTCF Binding Facilitate Tissue-Specific and Neuronal Activity-Dependent Gene Expression States.

Multivalent binding of CTCF to variable DNA sequences is thought to underlie its ability to mediate diverse cellular functions. CTCF typically binds a 20 base-pair consensus DNA sequence, but the full diversity of CTCF binding sites (CBS) within the genome has not been interrogated. We assessed CTCF occupancy in cultured cortical neurons and observed surprisingly that ~ 22% of CBS lack the consensus CTCF motif. We report here that sequence diversity at most of these atypical CBS involves degeneracy at specific nucleotide positions within the consensus CTCF motif, which likely affect the binding of CTCF zinc fingers 6 and 7. This mode of atypical CTCF binding defines most CBS at gene promoters, as well as CBS that are dynamically altered during neural differentiation and following neuronal stimulation, revealing how atypical CTCF binding could influence gene activity. Dynamic CBS are distributed both within and outside loop anchors and TAD boundaries, suggesting both looping-dependent and independent roles for CTCF. Finally, we describe a second mode of atypical CTCF binding to DNA sequences that are completely unrelated to the consensus CTCF motif, which are enriched within the bodies of tissue-specific genes. These tissue-specific atypical CBS are also enriched in H3K27ac, which marks cis-regulatory elements within chromatin, including enhancers. Overall, these results indicate how atypical CBS could dynamically regulate gene activity patterns during differentiation, development, and in response to environmental cues.

Fasciola Infection Unexpectedly Found During Cholecystectomy: Review on How to Avoid Increasing Surgery Interventions in Non-human Endemic Areas

PurposeFascioliasis is caused by Fasciola hepatica of almost worldwide distribution and F. gigantica in wide regions of Asia and Africa. Their adult stage develops in the biliary canals and gallbladder. Infection follows an initial, 3–4 month long invasive, migratory or acute phase, and a several year-long biliary, chronic or obstructive phase.MethodsThe unexpected finding of a fasciolid inside the gallbladder during a cholecystectomy for obstructive lithiasis suspicion in a patient is reported from an area of Iran where human infection had been never reported before and studies on fascioliasis in livestock are absent.ResultsThe fluke obtained was phenotypically classified as F. hepatica by morphometry and genotypically as F. gigantica by mtDNA cox1 fragment sequencing, although with F. hepatica scattered mutations in species-differing nucleotide positions. The clinical, radiological, and biological signs observed at the acute and chronic phases often lead to some misdiagnosis. Serological methods may be useful in cases of negative coprology. Diagnostic techniques with insufficient resolution leading to unnecessary invasive interventions are analyzed. The way to avoid unnecessary surgery is described, including analyses to be made, diagnostic tools to be used, and aspects to be considered.ConclusionReaching a correct diagnosis in the confusing presentations avoids procedure delays and unnecessary surgery. A correct drug treatment may be sufficient. Except in extreme pathological presentations, lesions decrease in number and size and finally disappear or calcify after a successful treatment. Finally, the need to increase awareness of physicians about fascioliasis is highlighted, mainly in non-human endemic areas.

Complete viral protein coding genes NS1 and VP1 sequences and phylogenetic analysis of a novel recombinant Mink Enteritis Virus isolated in China

The primary objective of this study was to detect the presence of MEV in mink samples and investigate the genetic characteristics of the virus. In 2017, a newly identified strain of MEV, named MEV-HLJ, was isolated. The virus’s complete NS1 and VP1 gene sequences were amplified using PCR and subjected to phylogenetic analysis. The results indicated that despite moderate virulence, MEV- HLJ belonged to the attenuated group. Recombination analysis using the GARD programme identified a putative recombination breakpoint at nucleotide position 2,075 between the NS1 and VP1 genes. Separate phylogenetic trees were constructed to confirm the recombinant event. This study provides insights into the genetic diversity and evolution of MEV isolates.

Multiple sequence alignment based on deep reinforcement learning with self-attention and positional encoding.

Multiple sequence alignment (MSA) is one of the hotspots of current research and is commonly used in sequence analysis scenarios. However, there is no lasting solution for MSA because it is a Nondeterministic Polynomially complete problem, and the existing methods still have room to improve the accuracy. We propose Deep reinforcement learning with Positional encoding and self-Attention for MSA, based on deep reinforcement learning, to enhance the accuracy of the alignment Specifically, inspired by the translation technique in natural language processing, we introduce self-attention and positional encoding to improve accuracy and reliability. Firstly, positional encoding encodes the position of the sequence to prevent the loss of nucleotide position information. Secondly, the self-attention model is used to extract the key features of the sequence. Then input the features into a multi-layer perceptron, which can calculate the insertion position of the gap according to the features. In addition, a novel reinforcement learning environment is designed to convert the classic progressive alignment into progressive column alignment, gradually generating each column's sub-alignment. Finally, merge the sub-alignment into the complete alignment. Extensive experiments based on several datasets validate our method's effectiveness for MSA, outperforming some state-of-the-art methods in terms of the Sum-of-pairs and Column scores. The process is implemented in Python and available as open-source software from https://github.com/ZhangLab312/DPAMSA.

Genetic analysis challenges the presence of Ixodes inopinatus in Central Europe: development of a multiplex PCR to distinguish I. inopinatus from I. ricinus

BackgroundIxodes ricinus is an important vector of several pathogens, primarily in Europe. Recently, Ixodes inopinatus was described from Spain, Portugal, and North Africa and then reported from several European countries. In this study, a multiplex polymerase chain reaction (PCR) was developed to distinguish I. ricinus from I. inopinatus and used in the surveillance of I. inopinatus in Algeria (ALG) and three regions in the Czech Republic (CZ).MethodsA multiplex PCR on TROSPA and sequencing of several mitochondrial (16S rDNA, COI) and nuclear markers (TROSPA, ITS2, calreticulin) were used to differentiate these two species and for a subsequent phylogenetic analysis.ResultsSequencing of TROSPA, COI, and ITS2 separated these two species into two subclades, while 16S rDNA and calreticulin could not distinguish I. ricinus from I. inopinatus. Interestingly, 23 nucleotide positions in the TROSPA gene had consistently double peaks in a subset of ticks from CZ. Cloning of these PCR products led to a clear separation of I. ricinus and I. inopinatus indicating hybridization and introgression between these two tick taxa. Based on a multiplex PCR of TROSPA and analysis of sequences of TROSPA, COI, and ITS2, the majority of ticks in CZ were I. ricinus, no I. inopinatus ticks were found, and 10 specimens showed signs of hybridization. In contrast, most ticks in ALG were I. inopinatus, four ticks were I. ricinus, and no signs of hybridization and introgression were detected.ConclusionsWe developed a multiplex PCR method based on the TROSPA gene to differentiate I. ricinus and I. inopinatus. We demonstrate the lack of evidence for the presence of I. inopinatus in Central Europe and propose that previous studies be re-examined. Mitochondrial markers are not suitable for distinguishing I. inopinatus from I. ricinus. Furthermore, our data indicate that I. inopinatus and I. ricinus can hybridize, and the hybrids can survive in Europe.Graphical abstract

Zucchini Yellow Mosaic Virus (ZYMV) as a Serious Biotic Stress to Cucurbits: Prevalence, Diversity, and Its Implications for Crop Sustainability.

Zucchini yellow mosaic virus (ZYMV) is a severe threat to cucurbit crops worldwide, including Pakistan. This study was pursued to evaluate the prevalence, geographic distribution, and molecular diversity of ZYMV isolates infecting cucurbits in Pakistan's Pothwar region. Almost all the plant viruses act as a biotic stress on the host plants, which results in a yield loss. These viruses cause losses in single-infection or in mixed-infection cucurbit crops, and we have found a number of mixed-infected samples belonging to the Curubitaceae family. Serological detection of the tested potyviruses in the collected cucurbit samples revealed that ZYMV was the most prevalent virus, with a disease incidence (DI) at 35.2%, followed by Papaya ringspot virus (PRSV) with an incidence of 2.2%, and Watermelon mosaic virus (WMV) having an incidence as little as 0.5% in 2016. In the year 2017, a relatively higher disease incidence of 39.7%, 2.4%, and 0.3% for ZYMV, WMV, and PRSV, respectively, was recorded. ZYMV was the most prevalent virus with the highest incidence in Attock, Rawalpindi, and Islamabad, while PRSV was observed to be the highest in Islamabad and Jhelum. WMV infection was observed only in Rawalpindi and Chakwal. Newly detected Pakistani ZYMV isolates shared 95.8-97.0% nucleotide identities among themselves and 77.1-97.8% with other isolates retrieved from GenBank. Phylogenetic relationships obtained using different ZYMV isolates retrieved from GenBank and validated by in silico restriction analysis revealed that four Pakistani isolates clustered with other ZYMV isolates in group IIb with Chinese, Italian, Polish, and French isolates, while another isolate (MK848239) formed a separate minor clade within IIb. The isolate MK8482490, reported to infect bitter gourd in Pakistan, shared a minor clade with a Chinese isolate (KX884570). Recombination analysis revealed that the recently found ZYMV isolate (MK848239) is most likely a recombinant of Pakistani (MK848237) and Italian (MK956829) isolates, with a recombinant breakpoint between 266 and 814 nucleotide positions. Local isolate comparison and recombination detection may aid in the development of a breeding program that identifies resistant sources against recombinant isolates because the ZYMV is prevalent in a few cucurbit species grown in the surveyed areas and causes heavy losses and economic damage to the agricultural community.

Polymorphism and mutational diversity of virulence (vcgCPI/vcgCPE) and resistance determinants (aac(3)-IIa, (aacC2, strA, Sul 1, and 11) among human pathogenic Vibrio species recovered from surface waters in South-Western districts of Uganda

BackgroundVibrio species are among the autochthonous bacterial populations found in surface waters and associated with various life-threatening extraintestinal diseases, especially in human populations with underlying illnesses and wound infections. Presently, very diminutive information exists regarding these species’ mutational diversity of virulence and resistance genes. This study evaluated variations in endonucleases and mutational diversity of the virulence and resistance genes of Vibrio isolates, harboring virulence-correlated gene (vcgCPI), dihydropteroate synthase type 1 and type II genes (Sul 1 and 11), (aadA) aminoglycoside (3′′) (9) adenylyltransferase gene, (aac(3)-IIa, (aacC2)a, aminoglycoside N(3)-acetyltransferase III, and (strA) aminoglycoside 3′-phosphotransferase resistance genes. MethodsUsing combinations of molecular biology techniques, bioinformatics tools, and sequence analysis. ResultsOur result revealed various nucleotide variations in virulence determinants of V. vulnificus (vcgCPI) at nucleotide positions (codon) 73–75 (A → G) and 300–302 (N → S). The aminoglycosides resistance gene (aadA) of Vibrio species depicts a nucleotide difference at position 482 (A → G), while the aminoglycosides resistance gene (sul 1 and 11) showed two variable regions of nucleotide polymorphism (102 and 140). The amino acid differences exist with the nucleotide polymorphism at position 140 (A → E). The banding patterns produced by the restriction enzymes HinP1I, MwoI, and StyD4I showed significant variations. Also, the restriction enzyme digestion of protein dihydropteroate synthase type 1 and type II genes (Sul 1 and 11) differed significantly, while enzymes DpnI and Hinf1 indicate no significant differences. The restriction enzyme NlaIV showed no band compared to reference isolates from the GenBank. However, the resistant determinants show significant point nucleotide mutation, which does not produce any amino acid change with diverse polymorphic regions, as revealed in the restriction digest profile. ConclusionThe described virulence and resistance determinants possess specific polymorphic locus relevant to pathogenomics studies, pharmacogenomic, and control of such water-associated strains.

SAT217 Chronic Asymptomatic Hypoparathyroidism Due To Two Separate Single Nucleotide Heterozygous Mutations In The CaSR Gene

Abstract Disclosure: M. Antony: None. S. Gundlapally: None. S. Storm: None. P. Russell: None. V. Verma: None. R. Kant: None. Introduction: The most common cause for acquired hypoparathyroidism is either post-surgical or autoimmune destruction of the parathyroid glands. The clinical presentation can range from an asymptomatic state to severe life-threatening symptoms that require intensive therapeutic management with intravenous and/or oral calcium, calcitriol and in some cases, recombinant parathyroid hormone therapy. Hypoparathyroidism secondary to CASR (calcium sensing gene receptor) gene mutation is an uncommon etiology that can also have a varied clinical presentation as above. Clinical Case: A 60-year-old Caucasian male with no significant past medical history was found to have chronic hypocalcemia. He denied experiencing symptoms such as numbness, tingling or cramping sensation around face, lips, or upper extremities. He denied a history of movement disorder or dental problems. He denied a history of neck surgeries or neck radiation exposure. He denied a family history of calcium disorders. He was on calcium carbonate/Vitamin D3 600 mg/400 I.U once daily. The first documented low corrected serum calcium was 6.9 mg/dl (8.6-10.0 mg/dl) in November of 2017. Since then, there have been multiple low corrected serum calcium levels ranging between 7 to 7.6 mg/dl. Patient underwent endocrinology work-up after withholding the above calcium/Vitamin D3 supplementation and it revealed a low corrected serum calcium of 7.6 mg/dl with an inappropriately normal serum PTH level of 37 pg/ml (15-65 pg/ml), normal vitamin D level of 45.1 ng/ml (> 30.0 ng/ml), normal serum phosphorus 3.8 mg/dl (2.5-4.5 mg/dl), and normal serum magnesium 1.8 mg/dl (1.6-2.6 mg/dl). The 24-hour urine calcium was 93 mg/24 hour with a corresponding 24-hour urine creatinine of 1836 mg (1040-2350 mg/24hour) and urine volume of 1625 ml. CASR gene analysis revealed two separate single nucleotide heterozygous mutations. The first mutation was at Exon 4/7, nucleotide position was c.614G>A and Amino Acid position was p.Arg205His. The second mutation was at Exon 7/7, nucleotide position was c.2546T>C and Amino Acid Position is p.lle849Thr. Due to lack of specific symptoms, patient was not initiated on specific therapies such as calcitriol or Natpara (recombinant PTH) and was advised to continue taking the daily calcium/vitamin D3 supplementation. He is being currently monitored every 4 to 6 months to assess for any clinical symptoms along with pertinent laboratory work-up. Conclusion: Hypoparathyroidism due to CASR gene mutation has a varied clinical presentation. One-third of cases do not develop any clinical symptoms, such as our patient and hence do not warrant any specific treatment other than periodic clinical and biochemical assessment. Presentation: Saturday, June 17, 2023