Sequencing Of PCR Products Research Articles

Ichthyobodo necator infection as a first record in cultured Dicentrarchus labrax: morphomolecular characterization and infestation patterns in correlation to water quality parameters

Abstract Ichthyobodo necator is a damaging and economically important protozoan parasite; however, its clinical patterns and incidence, correlated to environmental factors, are still poorly understood. This study provides the first report of Ichthyobodo necator infestation among Dicentrarchus labrax. We recorded significant mortalities in the cultured D. labrax of average size (5.92 g weight, and 11.55 cm length) in El-Max Station, National Institute of Oceanography and Fisheries, NIOF, Egypt, during the period extending from early summer to early fall (June to October) 2023. Behavioral and gross lesions of the infested fish were recorded; besides, dead and moribund fish were collected. Wet smears of the skin and gill of the affected fish were examined by compound light microscopy. Moreover, water samples from the affected ponds were collected and examined for their physicochemical parameters. The bodonid biflagellated Ichthyobodo necator (Henneguy) C. Pinto, 1928 was isolated from the skin and gills and identified based on specific morphological characteristics. DNA sequencing of PCR products and phylogenetic analysis confirmed identification, with clustering of the current isolates with I. necator isolates from different fish species. The multivariate analysis revealed a positive correlation between the prevalence and infestation intensity of Ichthyobodo, increased temperature levels, decreased dissolved oxygen DO, and also correlated with higher ammonia, and pH values. Additionally, the elevated concentrations of iron, calcium, magnesium, zinc, and copper in pond water were positively correlated with infestation and mortalities. These water parameter levels promote respiratory distress, accelerated prevalence, and infestation intensities of I. necator, particularly during the infestation peak in August and September. This study bridges the knowledge gap regarding I. necator parasite infestations and their correlation with water quality parameters.

RNA-First Approach Identifies Deep Intronic PHEX Variants in X-Linked Hypophosphatemic Rickets.

Up to 20% of patients with X-linked hypophosphatemic rickets (XLH) have no causative variant identified on routine molecular diagnostic testing. To identify intronic variants causing PHEX mis-splicing in patients with XLH. The metabolic bone clinic of a paediatric orthopedic hospital. Four patients (age 6 to 12 years; 3 girls) with clinically diagnosed XLH and no PHEX variant on routine testing. RNA and DNA sequence analysis of PHEX. Urine-derived cells were cultured, and mRNA was extracted and transcribed to cDNA. PHEX cDNA was amplified by PCR, followed by sequencing of PCR products. Sequencing of PHEX intronic DNA regions was performed to identify variants causing mis-splicing observed on RNA analysis. PHEX mis-splicing was identified in 3 of the 4 participants, and an intronic variant was identified in all 3 cases. In a 12-year-old boy, transcript analysis showed skipping of PHEX exon 13, while sequencing of PHEX intronic regions revealed a de novo 18 bp deletion in intron 13. In a 7-year-old girl, a pseudoexon in PHEX intron 17 was found, associated with a de novo deep intronic variant (c.1768+173A>G) that activated a cryptic splice donor site. Finally, an 84 bp pseudoexon in PHEX intron 21 caused by a recurrent de novo deep intronic variant (c.2147+1197A>G) was identified in an 11-year-old girl. Analysis of RNA from urine-derived cells combined with sequencing of PHEX introns can identify deep intronic variants in individuals with XLH without a detectable PHEX variant in routine exon-centric molecular diagnosis.

Knockout of Caspase-1 Disrupts Pyroptosis and Protects Photoreceptor Cells from Photochemical Damage

Retinal photochemical damage (RPD) plays a significant role in the development of various ocular diseases, with Caspase-1 being a key contributor. This study investigates the protective effects of Caspase-1 gene-mediated pyroptosis against RPD. Differentially expressed genes (DEGs) associated with RPD were identified through the analysis of two expression profiles from the GEO database. Correlation analysis was used to pinpoint pyroptosis-related genes (PRGs) linked to RPD. A Caspase-1 knockout 661W cell line was generated via CRISPR-Cas9 gene editing, and single-cell colonies were screened and purified. Validation of knockout cells was performed through RT-qPCR, gene sequencing, and Western blot analysis. Comparative assays on cell proliferation, intracellular reactive oxygen species (ROS), and cytotoxicity were conducted between wild-type and Caspase-1 knockout cells under light exposure. Further RT-qPCR and Western blot experiments examined changes in the mRNA and protein levels of key pyroptosis pathway components. Significant alterations in Caspase-1 expression were observed among PRGs. Homozygous Caspase-1 knockout cell lines were confirmed through RT-qPCR, genomic PCR product sequencing, and Western blot analysis. Compared to wild-type 661W cells, Caspase-1 knockout cells exhibited higher viability and proliferation rates after 24 hours of light exposure, alongside reduced LDH release. The expression of downstream pyroptosis factors at both the mRNA and protein levels was markedly decreased in the knockout group. CRISPR/Cas9-mediated Caspase-1 knockout enhanced the resistance of 661W cells to photochemical damage, suggesting that Caspase-1 may serve as a potential therapeutic target for RPD-related diseases.

Two Sporadic Cases of Legionellosis Associated with the Use of Domestic Ultrasonic Humidifiers

Two sporadic cases of legionellosis occurring in consecutive years were confirmed by positive antigenuria to Legionella pneumophila serogroup 1 in individuals with limited mobility who were confined to their homes. Both cases had a history of using ultrasonic humidifiers and of low exposure to other possible sources of infection. This study was conducted through an expanded epidemiological survey and home inspection. Samples were collected from domestic hot water and humidifiers. Environmental isolates were characterised by immunoagglutination and immunofluorescence. The Dresden panel is employed for the determination of groups and subgroups of serogroup 1. The amoebae were isolated by water filtration and subsequent cultivation of the filters. Identification of the isolates was conducted through the sequencing of PCR products. In both cases, epidemiological studies identified the ultrasonic humidifiers as the probable source of infection. The presence of Legionella pneumophila was confirmed in the sanitary water installation and in the humidifiers, where high values of Legionella pneumophila serogroup 1 counts were also obtained. The isolates were characterised as Olda Oxford and Olda OLDA. Furthermore, in addition to the Legionella isolates, the presence of Vermamoeba vermiformis was also identified in samples obtained from domestic hot water and humidifiers in one of the residential addresses under investigation. It has been demonstrated that humidifiers can act as an amplification mechanism for pathogenic microorganisms when they are not cleaned and maintained correctly. This can pose a health danger, especially to people with previous respiratory pathologies, the immunosuppressed, and the elderly. Therefore, it is of the utmost importance that professionals who recommend the therapeutic use of this equipment should issue warnings regarding the importance of its correct use, cleaning, and disinfection. Finally, humidifiers should be considered in epidemiological surveys and in the study of sporadic cases.

Identification of two effective sex-specific DNA markers in silver arowana (Osteoglossum bicirrhosum)

The silver arowana (Osteoglossum bicirrhosum), often referred to as a living fossil, presents significant challenges in sex identification based on morphological characteristics, with its sex determination mechanism remaining poorly understood. This issue poses considerable obstacles to its captive breeding efforts. Given the species' substantial value for both consumption and ornamental purposes, it is a critical candidate for aquaculture. Through genome sequencing, read mapping, and alignment analysis of five male and five female silver arowanas, along with one mixed pool of males, we successfully identified two sex-specific SNPs and confirmed a ZW model of sex determination in this ancient species. These two SNPs demonstrated remarkable accuracy, achieving 100 % success in genetic sex identification, as validated by Sanger sequencing of PCR products from 135 individuals. Both sex-specific SNPs are located approximately 26 kb upstream of the foxl2 gene, indicating a potentially pivotal role for the foxl2 gene in the sex-determination process of the silver arowana. Consequently, we have developed precise molecular markers for sex identification in the silver arowana, significantly enhancing reproductive efficiency and promoting further industrial development. This achievement lays a foundational basis for investigating the sex-determination mechanism of the silver arowana and other osteoglossid fish, thereby advancing aquaculture practices within this species.

Cryptosporidium species and subtypes in Norway: Predominance of C. parvum and emergence of C. mortiferum

PCR-based diagnostics has revealed the previously largely unknown Cryptosporidium transmission and infections in high-income countries. This study aimed to determine domestic and imported subtypes of Cryptosporidium species in Norway, evaluate their demographic distribution, and identify potential small outbreaks. Cryptosporidium-positive human faecal samples were obtained from six medical microbiology laboratories between February 2022 and January 2024, together with 22 Cryptosporidium-positive animal samples. Species and subtypes were identified by sequencing PCR products from gp60 and SSU rRNA genes. Most cryptosporidiosis cases occurred during late summer/early autumn, primarily in children and young adults. Of 550 human samples, 359 were successfully characterized molecularly (65%), revealing infection with 10 different Cryptosporidium species. C. parvum occurred in 245 (68%) human isolates with IIa and IId being major allele families, with distinct regional distribution patterns of common subtypes. A kindergarten outbreak with 5 cases was due to C. parvum IIaA14G1R1. C. mortiferum was identified in 33 (9.2%) human cases of which 24 were known to be of domestic origin, making it the second most common species in human autochthonous cases in Norway. All C. mortiferum isolates were of the same genotype; XIVaA20G2T1, including 13 cases from a suspected small outbreak in Trøndelag. C. hominis occurred in 68 typed cases (19%), but mostly in infections acquired abroad, with allele families Ib and If occurring most often. In conclusion, this study of recent Cryptosporidium spp. and subtypes in Norway, highlights the predominance of C. parvum and the emergence of C. mortiferum among autochthonous cases.

First Report of Cacao Mild Mosaic Virus Associated with Cacao (Theobroma cacao) in Hawai'i, USA.

Cacao, Theobroma cacao, is an important tropical agricultural crop and the key ingredient of chocolate, which has an international trade value of $103 billion/year (Marelli et al. 2019). Cacao mild mosaic virus (CaMMV) is a badnavirus that causes mild symptoms compared with some of the closely related species of Cacao swollen shoot virus (CSSV), the latter of which are currently only found in West Africa (Marelli et al. 2019; Ullah et al. 2021). CaMMV was recently found in symptomatic commercial cacao trees in Mayagüez, Puerto Rico (Puig et al. 2020), and subsequently detected in a USDA ARS (Agricultural Research Service) quarantine greenhouse in Miami, FL (Puig 2021). The USDA ARS germplasm repository in Hilo, Hawai'i, USA serves as a backup collection for Puerto Rico's cacao germplasm, and field trials have been established from cacao germplasm from Miami to evaluate select varieties. To determine if CaMMV is present in the collection in Hilo, greenhouse and field accessions were tested. Using an optimized sampling and PCR protocol established by Puig (2021), three young cacao petioles per tree or seedling were collected and pooled, and DNA was extracted using the NucleoSpin Plant II commercial kit (Macherey-Nagel, Düren, Germany) following the manufacturer's instructions. Samples were molecularly identified via end point PCR, gel electrophoresis, and Sanger sequencing. PCR amplification of CaMMV using the virus-specific primer set Mia-1396F (5'-ACCGTGTCTAYCAGCACTGGA-3') and Mia-1667R (5'-GACCACCGTCAGCCAGAC-3') produced 289 bp amplicons. Of 230 plants sampled, 26 CaMMV positive detections were discovered in greenhouse and field plantings in Hilo. Most CaMMV+ plants contained some form of leaf chlorosis (96.2%). The sequenced PCR products from Hawai'i were deposited in GenBank (accession nos. OQ692890-OQ692891) and showed 99.2% nucleotide identity to CaMMV accessions from Puerto Rico (MW052520; n = 23) and 98.1% nucleotide identity to CaMMV accessions from Florida, USA (MZ409692; n = 3) in BLASTn analysis. For species-level confirmation, the RT-RNase H domain was amplified from 9 isolates using CaMMV-specific primers (Mia5385F, 5'-AGGACAACGGCTTTCTTGGT-3'/Mia6616R, 5'-GAGACTAACTTGGTTAGGGCT-3'), sequenced, and deposited in GenBank (accession nos. PP997461-PP997462). Sequences matched most closely to GenBank isolates from Puerto Rico (MT253659; 98.0%; n = 7) and Trinidad and Tobago (NC_033738; 97.1%; n = 2). CaMMV, previously known as cacao Trinidad virus A, was first reported in 1943 and was associated with 7 to 33% yield reduction, loss of vigor, and tree decline (Posnette 1944; Swarbrick 1961). CaMMV is known to exist in the Americas and was most recently detected in Brazil and Indonesia (Kandito et al. 2022; Ramos‑Sobrinho et al. 2021). To the best of our knowledge, this is the first report of CaMMV infecting cacao in Hawai'i. The cacao industry continues to expand in Hawai'i, and cultivation occurs on at least four of the main islands including Hawai'i Island, O'ahu, Maui, and Kaua'i. To develop disease management strategies, further investigation is needed to define CaMMV symptomology, and determine the distribution and effect this virus has on production in Hawai'i. In the meantime, cacao will be screened regardless of visible symptomology to maintain pathogen-free accessions and avoid the transfer of virus-containing germplasm.

First report of leaf spot caused by Paramyrothecium roridum on Coffea arabica in Hawai'i, USA.

During the 2022-2023 season, the harvested coffee crop in Hawai'i (Coffea arabica) was valued at $57.1 million (USDA NASS 2023). In September 2022, coffee leaf samples with foliar leaf spots affecting the Kona Typica variety were collected from Hōnaunau, Hawai'i, incidence <10%. The symptoms were circular, necrotic leaf spots with yellow margins, which merged, resulting in complete leaf blade coverage and subsequent leaf drop. Sporodochia were present on the abaxial leaf surface. Symptomatic leaf tissue was disinfected in 10% bleach solution for 60 seconds and chlorotic leaf tissue from the spot margins were excised and placed onto water agar and potato dextrose agar (PDA; Difco, USA). After a 7-day incubation period, pure cultures with white aerial mycelium having sporodochia arranged in concentric rings with olivaceous to black conidial masses were isolated. The conidia were aseptate, hyaline, smooth, cylindrical with rounded ends, measuring 5.1 to 6.8 μm long and 1.7 to 2.3 μm wide (n=50). Based on symptomology and cultural/morphological characteristics (Huaman-Pilco et al. 2023; Lombard et al. 2016; Pelayo-Sanchez et al. 2017), the isolates were initially identified as Paramyrothecium roridum (Tode) L. Lombard & Crous, comb. nov. (syn. Myrothecium roridum Tode). Fungal identification of isolate P22-81-2 was further confirmed using BLAST analysis of bulk sequenced PCR products of the ribosomal DNA internal transcribed spacer (ITS) region (White et al. 1990), β-tubulin (βtub), RNA polymerase II (RPB2), and calmodulin genes (Lombard et al., 2016; Huaman-Pilco et al., 2023). The gene sequences (GenBank accession nos. PP211198, PQ192517-19) were >98.4% identical to the P. roridum type specimen (CBS 357.89). A multilocus maximum likelihood phylogenetic analysis incorporating sequence data from previous relevant studies (Lombard et al., 2016; Pinruan et al. 2022) confirmed species identification. To prove pathogenicity, four, 26-month-old Kona Typica variety seedlings were foliar inoculated with a 1 X 106 conidia/ml suspension using a perfume atomizer. An additional four plants were inoculated in a similar manner with sterile water which served as controls. All plants were sprayed to drip on both the upper and lower leaf surfaces and incubated in a clear plastic bag to keep the humidity levels between 90 to 100% for 48 hours at 24°C. After 48 hours, the plants were removed from the bags, placed on a greenhouse bench, and observed weekly for symptom development. Within seven days light brown sunken spots had developed on all inoculated plants. The spots continued to enlarge having a dark distinct margin, light tan center, chlorotic halo, and formed concentric rings, which were identical to the original diseased samples. Leaf spots were not present on any of the control plants. The test was conducted twice. A fungus was consistently reisolated from the leaf spot margins of inoculated plants and morphologically (PDA) and molecularly (ITS, βtub, RPB2, calmodulin) identified as P. roridum, thus fulfilling Koch's Postulates. To the best of our knowledge, this is the first report of P. roridum causing leafspots on C. arabica plants in Hawai'i. This pathogen has been reported on coffee in other parts of the world including Colombia, Costa Rica, Guatemala, Puerto Rico, and Mexico (USDA Fungus-Host Database). Under the right conditions, P. roridum has the potential to cause leafspots and defoliation resulting in economic losses for coffee growers in Hawai'i.

Reverse Transcription-polymerase Chain Reaction (RT-PCR) Based Detection and Identification of Monopartite Bean Common Mosaic Virus in Mungbean

Background: Bean common mosaic virus (BCMV) which belongs to the genus Potyvirus and the family Potyviridae, causes significant yield losses in legumes across the globe. Traditional methods for BCMV detection and diagnosis, such as visual inspection and serological assays, are often time-consuming, labour-intensive and may lack the sensitivity required for early detection. Advances in molecular techniques have made RT-PCR a powerful tool for the accurate detection of BCMV-infected plants. Methods: Three pairs of specific primers that flank the BCMV coat protein and polyprotein gene were designed using the Primer 3web tool to amplify fragments approximately ranging from 191 to 205 bp. The RT-PCR protocol was then standardized, including the optimization of annealing temperatures for all three primers, followed by the sequencing of the amplified PCR products. The, specificity and sensitivity of primer pair BCMV2 were then established. Result: Three pairs of specific primers were designed and RT-PCR protocol was standardized. The sequenced PCR products showed 98.03% to 99.50 % nucleotide similarity with the corresponding sequences of BCMV isolates used in primer designing. Further, the BCMV2 primer pair specifically amplified the target amplicon (200 bp) from the suspected leaf sample and no amplicon was observed when tested against other pathotypes of potyvirus such as BCMNV, BYMV, CABMV, SMV and PeMoV. The designed primer was sensitive enough to detect 0.05% of target cDNA. Additionally, the RT-PCR protocol was validated using field samples collected from Hyderabad, India. ELISA in combination with RT-PCR using the developed specific BCMV primer pair will ensure a foolproof, sensitive and rapid, procedure for diagnosis of BCMV in the quarantine processing of imported germplasm.

Approach for Phased Sequence-Based Genotyping of the Critical Pharmacogene Dihydropyrimidine Dehydrogenase (DPYD).

Pre-treatment genotyping of four well-characterized toxicity risk-variants in the dihydropyrimidine dehydrogenase gene (DPYD) has been widely implemented in Europe to prevent serious adverse effects in cancer patients treated with fluoropyrimidines. Current genotyping practices are largely limited to selected commonly studied variants and are unable to determine phasing when more than one variant allele is detected. Recent evidence indicates that common DPYD variants modulate the functional impact of deleterious variants in a phase-dependent manner, where a cis- or a trans-configuration translates into different toxicity risks and dosing recommendations. DPYD is a large gene with 23 exons spanning nearly a mega-base of DNA, making it a challenging candidate for full-gene sequencing in the diagnostic setting. Herein, we present a time- and cost-efficient long-read sequencing approach for capturing the complete coding region of DPYD. We demonstrate that this method can reliably produce phased genotypes, overcoming a major limitation with current methods. This method was validated using 21 subjects, including two cancer patients, each of whom carried multiple DPYD variants. Genotype assignments showed complete concordance with conventional approaches. Furthermore, we demonstrate that the method is robust to technical challenges inherent in long-range sequencing of PCR products, including reference alignment bias and PCR chimerism.

Large-Scale Molecular Epidemiological Survey of Blastocystis sp. among Herbivores in Egypt and Assessment of Potential Zoonotic Risk.

Given the proven zoonotic potential of the intestinal protozoan Blastocystis sp., a fast-growing number of surveys are being conducted to identify potential animal reservoirs for transmission of the parasite. Nevertheless, few epidemiological studies have been conducted on farmed animals in Egypt. Therefore, a total of 1089 fecal samples were collected from herbivores (sheep, goats, camels, horses, and rabbits) in six Egyptian governorates (Dakahlia, Gharbia, Kafr El Sheikh, Giza, Aswan, and Sharqia). Samples were screened for the presence of Blastocystis sp. by real-time PCR followed by sequencing of positive PCR products and phylogenetic analysis for subtyping of the isolates. Overall, Blastocystis sp. was identified in 37.6% of the samples, with significant differences in frequency between animal groups (sheep, 65.5%; camels, 62.2%; goats, 36.0%; rabbits, 10.1%; horses, 3.3%). Mixed infections were reported in 35.7% of the Blastocystis sp.-positive samples. A wide range of subtypes (STs) with varying frequency were identified from single infections in ruminants including sheep (ST1-ST3, ST5, ST10, ST14, ST21, ST24, ST26, and ST40), goats (ST1, ST3, ST5, ST10, ST26, ST40, ST43, and ST44), and camels (ST3, ST10, ST21, ST24-ST26, ST30, and ST44). Most of them overlapped across these animal groups, highlighting their adaptation to ruminant hosts. In other herbivores, only three and two STs were evidenced in rabbits (ST1-ST3) and horses (ST3 and ST44), respectively. The greater occurrence and wider genetic diversity of parasite isolates among ruminants, in contrast to other herbivores, strongly suggested that dietary habits likely played a significant role in influencing both the colonization rates of Blastocystis sp. and ST preference. Of all the isolates subtyped herein, 66.3% were reported as potentially zoonotic, emphasizing the significant role these animal groups may play in transmitting the parasite to humans. These findings also expand our knowledge on the prevalence, genetic diversity, host specificity, and zoonotic potential of Blastocystis sp. in herbivores.

Limited potexvirus diversity in eastern Gulf of Mexico seagrass meadows.

Turtlegrass virus X, which infects the seagrass Thalassia testudinum, is the only potexvirus known to infect marine flowering plants. We investigated potexvirus distribution in seagrasses using a degenerate reverse transcription polymerase chain reaction (RT-PCR) assay originally designed to capture potexvirus diversity in terrestrial plants. The assay, which implements Potex-5 and Potex-2RC primers, successfully amplified a 584 nt RNA-dependent RNA polymerase (RdRp) fragment from TVX-infected seagrasses. Following validation, we screened 74 opportunistically collected, apparently healthy seagrass samples for potexviruses using this RT-PCR assay. The survey examined the host species T. testudinum, Halodule wrightii, Halophila stipulacea, Syringodium filiforme, Ruppia maritima, and Zostera marina. Potexvirus PCR products were successfully generated only from T. testudinum samples and phylogenetic analysis of sequenced PCR products revealed five distinct TVX sequence variants. Although the RT-PCR assay revealed limited potexvirus diversity in seagrasses, the expanded geographic distribution of TVX shown here emphasizes the importance of future studies to investigate T. testudinum populations across its native range and understand how the observed fine-scale genetic diversity affects host-virus interactions.

The impact of SLCO1B1 single nucleotide polymorphism and non-genetic factors on statin-induced myopathy

Abstract Funding Acknowledgements None. Background Statins, also known as HMG CoA Reductase Inhibitors, are a class of drugs considered the drug of choice for treating hypercholesterolemia. Statins are well tolerated; however, they may cause severe myopathy in 20% to 30% of patients, due to the individual variation regarding its efficacy and related adverse effects. Of these, comes the Single Nucleotide Polymorphism (SNP) T521C of the SLCO1B1 gene that encodes the protein involved in the trafficking of statins from the blood to hepatocytes which is called Organic Anion Transport Protein 1B1 (OATP1B1). The T521C single nucleotide polymorphism (SNP) results in a substitute of valine with alanine resulting in a decreased or lack of functional protein. Purpose The aim is to determine the frequency of the high-risk C allele and to define its impact on statin-induced myopathy in Syrian patients on statins. In addition to the non-genetic factors that may affect the ability to develop statin-induced myopathy. Methods This was an observational case-control study that had ethical approval from Damascus University (No.433/ 2020). The case group included 44 patients on statins who experienced myopathy, 18 patients who had increased creatinine kinase levels (CK), and 26 patients with normal CK levels, while the control group encompassed 56 patients who were taking statins and had no muscular adverse events. The DNA was extracted from each sample and then genotyped using standard sequencing of specific PCR products using a specific pair of primers. Results The age of patients in the case arm ranged between 27 and 84 years with an average ± SD of (58.2 ± 12.7 years), 70% of them were males. In comparison with (53.34 ± 12.4 years) in the control arm, 66% of them were males without a statistically significant differences (p=0.672, 0.057) respectively. The statin type and dose have a statistically non-significant effect, the average statin dose in the case arm and the control arm was 26.02 mg and 31.27 mg respectively (p=0.072). While the statin type treatment varied as 59% used atorvastatin and 39% used rosuvastatin in the case group compared with 71% used atorvastatin and 27% used rosuvastatin in the control group, without a statistically significant difference (p=0.252). The frequency of the C allele reaches 19.3% and is distributed in three genotypes: wild-type TT (68%), heterozygote TC (25%), and homozygote CC (7%) in the case arm, while it does not exceed 8% in the control arm with the absence of the CC genotype. Only a statistically significant difference was found in the frequency of the CC genotype between these two groups (p=0.047). Conclusion Our study is the first to identify the frequency of the 521T&amp;gt;C SNP of the SLCO1B1 gene in Syrian patients taking statins. Our data reveals a high prevalence of risk allele C in the patients who suffer from statin-induced myopathy with a statically significant difference from the control arm (p=0.026).

Distribution of toxigenic cyanobacteria in Alpine lakes and rivers as revealed by molecular screening

The increasing frequency of cyanobacteria blooms in waterbodies caused by ecosystem eutrophication could endanger human health. This risk can be mitigated by effective monitoring incorporating molecular methods. To date, most molecular studies on toxigenic cyanobacteria have been limited to microcystins (MCs), disregarding other cyanotoxins, to freshwater planktic habitats while ignoring benthic habitats, and to limited geographic areas (usually one or a few specific waterbodies). In this study, we used PCR-based methods including PCR product sequencing and chemical-analytical methods (LC-MS/MS) to screen many plankton (n = 123) and biofilm samples (n = 113) originating from 29 Alpine lakes and 18 rivers for their cyanotoxin production potential. Both mcyE (indicating MC synthesis) and anaC (indicating anatoxin (ATX) synthesis) gene fragments were able to qualitatively predict MC or ATX occurrence. The abundance of mcyE gene fragments was significantly related to MC concentrations in plankton samples (R2 = 0.61). mcyE gene fragments indicative of MC synthesis were most abundant in planktic samples (65 %) and were assigned to the genera Planktothrix and Microcystis. However, mcyE rarely occurred in biofilms of lakes and rivers, i.e., 4 % and 5 %, respectively, and were assigned to Microcystis, Planktothrix, and Nostoc. In contrast, anaC gene fragments occurred frequently in planktic samples (14 % assigned to Tychonema, Phormidium (Microcoleus), and Oscillatoria), but also in biofilms of lakes (49 %) and rivers (18 %) and were assigned to the genera Phormidium, Oscillatoria, and Nostocales. The cyrJ gene fragment indicating cylindrospermopsin synthesis occurred only once in plankton (assigned to Dolichospermum), while saxitoxin synthesis potential was not detected. For plankton samples, monomictic and less eutrophic conditions were positively related to mcyE/MC occurrence frequency, while oligomictic conditions were related to anaC/ATX frequency. The anaC/ATX frequency in biofilm was related to the lake habitats generally showing higher biodiversity as revealed from metabarcoding in a parallel study.

Molecular Detection of Anaplasma phagocytophilum in Small Mammals and Infesting Ticks in Laikipia County, Kenya.

Anaplasmosis is a set of disease conditions of various mammals caused by bacteria species of the genus Anaplasma. These are sub-microscopic, Gram-negative, obligate intracellular pathogens that infect both vertebrate and invertebrate hosts. Significant species that infect domestic and wildlife animals include Anaplasma marginale, Anaplasma ovis, Anaplasma mesaeterum, Anaplasma platys, and Anaplasma phagocytophilum. Although A. phagocytophilum has a widespread distribution, there are only a few epidemiological reports from sub-Saharan Africa. This study focused on molecular detection and characterization of A. phagocytophilum in small mammals and their infesting ticks in Laikipia County, Kenya. A total of 385 blood and 84 tick archival samples from small mammals (155 females and 230 males) were analyzed. The blood samples were subjected to a nested PCR-HRM melt analysis using species-specific primers to amplify the 16S ribosomal RNA genes. The ticks were also subjected to nested PCR-HRM involving 16S rRNA gene primers. Anaplasma phagocytophilum DNA was detected in 19 out of 385 samples using species-specific 16S rRNA gene primers giving a prevalence of 4.9% for A. phagocytophilum. Analysis of the tick's samples using 16S rRNA gene species-specific primers also detected A. phagocytophilum in 3 samples from Haemaphysalis leachi ticks (3/84) equivalent to prevalence of 3.6%. Sequencing of 16S rRNA PCR products confirmed A. phagocytophilum in small mammals and ticks' samples. Phylogenetic analysis of the haplotype from this study demonstrated a close ancestral link with strains from Canis lupus familiaris, Alces alces, Apodemus agrarius, and ticks (Haemaphysalis longicornis) reported in Europe, China, and Africa. Comparison was also made with a known pathogenic A. phagocytophilum variant HA and a nonpathogenic variant 1 that were clustered into a distinctive clade different form haplotypes detected in this study. All the haplotype sequences for A. phagocytophilum from this study were submitted and registered in GenBank under the accession numbers OQ308965-OQ308976. Our study shows that small mammals and their associated ticks harbor A. phagocytophilum. The vector competence for H. leachi in A. phagocytophilum transmission should further be investigated.

Exome Sequencing for the Diagnostics of Osteogenesis Imperfecta in Six Russian Patients.

Osteogenesis imperfecta (OI) is a group of inherited disorders of connective tissue that cause significant deformities and fragility in bones. Most cases of OI are associated with pathogenic variants in collagen type I genes and are characterized by pronounced polymorphisms in clinical manifestations and the absence of clear phenotype-genotype correlation. The objective of this study was to conduct a comprehensive molecular-genetic and clinical analysis to verify the diagnosis of OI in six Russian patients with genetic variants in the COL1A1 and COL1A2 genes. Clinical and laboratory data were obtained from six OI patients who were observed at the Medical Genetics Center in Saint Petersburg from 2016 to 2023. Next-generation sequencing on MGISEQ G400 (MGI, China) was used for DNA analysis. The GATK bioinformatic software (version 4.5.0.0) was used for variant calling and hard filtering. Genetic variants were verified by the direct automatic sequencing of PCR products using the ABI 3500X sequencer. We identified six genetic variants, as follows pathogenic c.3505G>A (p. Gly1169Ser), c.769G>A (p.Gly257Arg), VUS c.4123G>A (p.Ala1375Thr), and c.4114A>T (p.Asn1372Tyr) in COL1A1; and likely pathogenic c.2035G>A (p.Gly679Ser) and c.739-2A>T in COL1A2. In addition, clinical cases are presented due to the presence of the c.4114A>T variant in the COL1A2 gene. Molecular genetics is essential for determining different OI types due to the high similarity across various types of the disease and the failure of unambiguous diagnosis based on clinical manifestations alone. Considering the variable approaches to OI classification, an integrated strategy is required for optimal patient management.

Optimized protoplast isolation and transfection with a breakpoint: accelerating Cas9/sgRNA cleavage efficiency validation in monocot and dicot

The CRISPR-Cas genome editing tools are revolutionizing agriculture and basic biology with their simplicity and precision ability to modify target genomic loci. Software-predicted guide RNAs (gRNAs) often fail to induce efficient cleavage at target loci. Many target loci are inaccessible due to complex chromatin structure. Currently, there is no suitable tool available to predict the architecture of genomic target sites and their accessibility. Hence, significant time and resources are spent on performing editing experiments with inefficient guides. Although in vitro-cleavage assay could provide a rough assessment of gRNA efficiency, it largely excludes the interference of native genomic context. Transient in-vivo testing gives a proper assessment of the cleavage ability of editing reagents in a native genomic context. Here, we developed a modified protocol that offers highly efficient protoplast isolation from rice, Arabidopsis, and chickpea, using a sucrose gradient, transfection using PEG (polyethylene glycol), and validation of single guide RNAs (sgRNAs) cleavage efficiency of CRISPR-Cas9. We have optimized various parameters for PEG-mediated protoplast transfection and achieved high transfection efficiency using our protocol in both monocots and dicots. We introduced plasmid vectors containing Cas9 and sgRNAs targeting genes in rice, Arabidopsis, and chickpea protoplasts. Using dual sgRNAs, our CRISPR-deletion strategy offers straightforward detection of genome editing success by simple agarose gel electrophoresis. Sanger sequencing of PCR products confirmed the editing efficiency of specific sgRNAs. Notably, we demonstrated that isolated protoplasts can be stored for up to 24/48 h with little loss of viability, allowing a pause between isolation and transfection. This high-efficiency protocol for protoplast isolation and transfection enables rapid (less than 7 days) validation of sgRNA cleavage efficiency before proceeding with stable transformation. The isolation and transfection method can also be utilized for rapid validation of editing strategies, evaluating diverse editing reagents, regenerating plants from transfected protoplasts, gene expression studies, protein localization and functional analysis, and other applications.

First report of Neocosmospora pisi causing blight on Ammopiptanthus mongolicus in China.

Ammopiptanthus mongolicus is the only evergreen broad-leaved shrub in the desert region of Northwest China, which is one of the dominant species in the desert vegetation of the region, playing an important role in maintaining the stability of the local desert ecosystem. A. mongolicus is also very hardy and drought resistant and can survive extreme temperatures (Liu et al. 2013; Yang et al. 2022). The large-scale death of A. mongolicus could cause desertification in the region. Two months after the discovery of Fusarium verticillioides causing blight on A. mongolicus in Etuoke county, Inner Mongolia Autonomous Region in September 2023 (Yang et al. 2024), a large number of A. mongolicus plants with symptoms of blights were found in Lingwu city, Ningxia Hui Autonomous Region, China (106.442368°E, 37.734026°N) in November 2023. The incidence of diseased plants in this field was about 30%. The field symptoms in Lingwu city were similar to those observed in Etuoke county. The diseased leaves initially turned yellow, then wilted and dehisced, eventually resulting in plant death (Figure 1). The roots of the diseased plants were cut diagonally and the central cylinder showed a brown color (Figure 2). In order to investigate whether the death of A. mongolicus was caused by the same pathogen as those identified previously, 30 roots were collected from 10 diseased plants. After rinsing and surface sterilization (70% ethanol for 3 min and 2.5% NaClO for 5 min, rinsed 3 times with sterile distilled water), diseased tissues (10×10 mm) were placed on potato dextrose agar (PDA) (3 pieces per plate) and incubated from 3 to 5 days at 25°C. The strain AmP5 was isolated and used for further study. After 3 days on PDA medium, fungal colonies were white to milky, the undersides of the cultures were yellowish to orange-brown (Figure 3). After 7 days on synthetic nutrient-poor agar (SNA), microconidia were ovoidal or with a rounded apex and truncate base, 10.5 ± 1.5 μm × 1.6 ± 0.2 μm (×400). The macroconidia were slightly curved or arcuate, 40.5 ± 3.5 μm × 5 ± 0.5 μm (×400) (Figure 4) (Šišić et al. 2018). The pathogen was confirmed to be Neocosmospora pisi by multigene phylogenetic analysis of TEF, RPB1 and RPB2 genes using primers EF1/EF2, F5/G2R and 5F2/11AR, respectively (O'Donnell et al. 2022). The sequences of PCR products were deposited in GenBank with accession numbers OR944631 (RPB1), OR988086 (TEF) and OR988087 (RPB2), respectively. The results of pairwise alignment in Fusarioid-ID database (Crous et al. 2021) showed 99.84% similarity and 83.96% overlap of the EF1-α sequence to the corresponding sequence LR583636 of ex-epitype CBS 123669 of Neocosmospora pisi (syn. Fusarium solani f. sp. pisi), 99.72% similarity and 85.66% overlap of the RPB1 sequence to the corresponding sequence MW834242 of ex-epitype CBS 123669 of N. pisi, and 99.47% similarity and 78.26% overlap of RPB2 sequence to the corresponding sequence LR583862 of ex-epitype CBS 123669 of N. pisi. Moreover, the result of polyphasic identification in the Fusarioid-ID database also showed EF1-a, RPB1, and RPB2 sequences had 99.15% similarity to the corresponding sequences of CBS 1233669. The pathogenicity of AmP5 was tested on potted 64 days old seedlings A. mongolicus plants. The roots of 3 seedlings were inoculated with conidial suspension (1×106 /ml), and another 3 used as controls were inoculated with sterile water, by gently peeling off the soil around the roots during inoculation, and pouring the conidial suspension around the roots (10 ml/seedling). All plants were placed in a growth chamber at 18-25℃ (10 h light; 14 h dark). After incubation for 3-5 days, the symptoms similar to those observed in the field (Figure 5), including brown rot of steles (Figure 6), developed on plants inoculated with conidial suspension, whereas no symptoms were observed on the control plants. The same pathogen was reisolated from inoculated roots and confirmed as N. pisi based on morphological and molecular analyses (TEF, RPB1 and RPB2). To our knowledge, this is the first report of blight on A. mongolicus caused by N. pisi in China. This study also indicates that blight on A. mongolicus can be caused by different fungal pathogens. Blight caused by different pathogens may have different in terms of control measures and pathogenic mechanisms, so the study of blight caused by different pathogens is of profound value.

Molecular identification of Fusarium oxysporum f. sp. cubense (Foc) causing Fusarium wilt disease of Tieu hong banana cultivar in the Red River Delta of Vietnam

The Red River Delta is one of primary banana-producing regions in Vietnam, and it has been afflicted by Fusarium wilt disease, caused by Fusarium oxysporum f. sp. cubense (Foc). This study aimed to identify Foc strains from banana-diseased samples collected from six different provinces in this region. Foc’s identity was confirmed based on morphological characteristics, artificial inoculation on the Tieu hong banana cultivar, and through DNA sequencing and phylogenetic analysis of PCR products amplified by race-specific primer pairs (Foc-1/Foc-2, Foc TR4-F/FocTR4-R). Ten Foc TR4 strains were identified, including VP1(2), VP1(5), and VP2(6) strains were found in Vinh Phuc Province, ML02.2 strain in Ha Noi City, MS1T2 strain in Hung Yen province, BV03 and BV06 strains in Hai Phong City, and QG1(R), QG4(T), QH2(R) strains in Thai Binh province. This discovery underscores the importance of appropriately identifying causal pathogens for integrated pest management of Fusarium wilt disease in the Red River Delta.

Molecular Identification and Subtype Analysis of Blastocystis sp. Isolates from Wild Mussels (Mytilus edulis) in Northern France.

Blastocystis sp. is the most common single-celled eukaryote colonizing the human gastrointestinal tract worldwide. Because of the proven zoonotic potential of this protozoan, sustained research is therefore focused on identifying various reservoirs of transmission to humans, and in particular animal sources. Numerous groups of animals are considered to be such reservoirs due to their handling or consumption. However, some of them, including mollusks, remain underexplored. Therefore, a molecular epidemiological survey conducted in wild mussels was carried out in Northern France (Hauts-de-France region) to evaluate the frequency and subtypes (STs) distribution of Blastocystis sp. in these bivalve mollusks. For this purpose, 100 mussels (Mytilus edulis) were randomly collected in two sampling sites (Wimereux and Dannes) located in the vicinity of Boulogne-sur-Mer. The gills and gastrointestinal tract of each mussel were screened for the presence of Blastocystis sp. by real-time polymerase chain reaction (qPCR) assay followed by direct sequencing of positive PCR products and subtyping through phylogenetic analysis. In parallel, sequences of potential representative Blastocystis sp. isolates that were previously obtained from temporal surveys of seawater samples at marine stations offshore of Wimereux were integrated in the present analysis. By taking into account the qPCR results from all mussels, the overall prevalence of the parasite was shown to reach 62.0%. In total, more than 55% of the positive samples presented mixed infections. In the remaining mussel samples with a single sequence, various STs including ST3, ST7, ST14, ST23, ST26 and ST44 were reported with varying frequencies. Such distribution of STs coupled with the absence of a predominant ST specific to these bivalves strongly suggested that mussels might not be natural hosts of Blastocystis sp. and might rather be carriers of parasite isolates from both human and animal (bovid and birds) waste. These data from mussels together with the molecular identification of isolates from marine stations were subsequently discussed along with the local geographical context in order to clarify the circulation of this protozoan in this area. The identification of human and animal STs of Blastocystis sp. in mussels emphasized the active circulation of this protozoan in mollusks and suggested a significant environmental contamination of fecal origin. This study has provided new insights into the host/carrier range and transmission of Blastocystis sp. and emphasized its potential as an effective sentinel species for water quality and environmental contamination.