Genomic DNA Research Articles

Polymeric Vehicles for Nucleic Acid Delivery: Enhancing the Therapeutic Efficacy and Cellular Uptake.

Therapeutic gene delivery may be facilitated by the use of polymeric carriers. When combined with nucleic acids to form nanoparticles or polyplexes, a variety of polymers may shield the cargo from in vivo breakdown and clearance while also making it easier for it to enter intracellular compartments. Polymer synthesis design choices result in a wide variety of compounds and vehicle compositions. Depending on the application, these characteristics may be changed to provide enhanced endosomal escape, longer-lasting distribution, or stronger connection with nucleic acid cargo and cells. Here, we outline current methods for delivering genes in preclinical and clinical settings using polymers. Significant therapeutic outcomes have previously been attained using genetic material- delivering polymer vehicles in both in-vitro and animal models. When combined with nucleic acids to form nanoparticles or polyplexes, a variety of polymers may shield the cargo from in vivo breakdown and clearance while also making it easier for it to enter intracellular compartments. Many innovative diagnoses for nucleic acids have been investigated and put through clinical assessment in the past 20 years. Polymer-based carriers have additional delivery issues due to their changes in method and place of biological action, as well as variances in biophysical characteristics. We cover recent custom polymeric carrier architectures that were tuned for nucleic acid payloads such genomemodifying nucleic acids, siRNA, microRNA, and plasmid DNA. In conclusion, the development of polymeric carriers for gene delivery holds promise for therapeutic applications. Through careful design and optimization, these carriers can overcome various challenges associated with nucleic acid delivery, offering new avenues for treating a wide range of diseases.

A putative new Besnoitia species in the southern black-eared opossum Didelphis aurita

Besnoitia spp. are cyst-forming coccidian parasites with a broad host range, infecting various wild and domestic animal species. Northamerican opossums (Didelphis virginiana) are severely affected by the infection with B. darlingi. This study presents a case of infection with Besnoitia in a road-killed female southern black-eared opossum (Didelphis aurita) in Misiones, Argentina. Many 0.5 to 1 mm cysts were observed in several muscles and visceral organs and were microscopically identified in skeletal muscles, tongue, and heart. Histological analysis disclosed multiple spherical cysts with a myriad of bradyzoites like-cells and a well-defined cyst wall. A small number of degenerate and ruptured cysts, surrounded by mild to moderate inflammation were observed. Genomic DNA from an individual cyst and muscle was extracted and ITS1 marker and 18S rRNA gene fragments from sarcocystid protozoa were successfully amplified by PCR and sequenced. The 18S sequence exhibited 100% identity with sequences of B. darlingi and B. oryctofelisi. Comparison of the complete ITS1 sequence (259 bp) revealed an identity of 99.2% with B. oryctofelisi and 97.7% with B. darlingi. This result together with the phylogeny positioning, suggest that the Besnoitia sp. in the present case differ from B. darlingi, being closely related with B. oryctofelisi.

Identification of Genes Encoded Toxin-Antitoxin System in Mycobacterium Tuberculosis Strains from Clinical Sample.

The toxin-antitoxin system is a genetic element that is highly present in Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis. The toxin-antitoxin system comprises toxin protein and antitoxin protein or non-encoded RNA interacting with each other and inhibiting toxin activity. M. Tuberculosis has more classes of TA loci than non-tubercle bacilli and other microbes, including VapBC, HigBA, MazEF, ParDE, RelBE, MbcTA, PemIK, DarTG, MenTA, one tripartite type II TAC chaperone system, and hypothetical proteins. The study aims to demonstrate the genes encoded toxin-antitoxin system in mycobacterium tuberculosis strains from clinical samples. The pulmonary and extra-pulmonary tuberculosis clinical samples were collected, and smear microscopy (Ziehl-Neelsen staining) was performed for the detection of high bacilli (3+) count, followed by nucleic acid amplification assay. Bacterial culture and growth assay, genomic DNA extraction, and polymerase chain reaction were also carried out. The positive PTB and EPTB samples were determined by 3+ in microscopy smear and the total count of tubercle bacilli determined by NAAT assay was 8.0×1005 in sputum and 1.3×1004 CFU/ml in tissue abscess. Moreover, the genomic DNA was extracted from culture, and the amplification of Rv1044 and Rv1045 genes in 624 and 412 base pairs (between 600-700 and 400-500 in ladder), respectively, in the H37Rv and clinical samples was observed. It has been found that Rv1044 and Rv1045 are hypothetical proteins with 624 and 882 base pairs belonging to the AbiEi/AbiEii family of toxin-antitoxin loci. Moreover, the significant identification of TA-encoded loci genes may allow for the investigation of multidrugresistant and extensively drug-resistant tuberculosis.

Dynamics of metal/metalloid bioaccumulation and sensitivity in post-larvae shrimp (Macrobrachium rosenbergii) exposed to settleable atmospheric particulate matter from an industrial source

The metallurgy industry is a potent global source of particulate matter (PM) atmospheric emissions. A portion of this PM may settle in aquatic (SePM) carrying metal/metalloid particles and metallic nanoparticles. Surprisingly, this form of contamination has not received due attention from most environmental monitoring agencies. We analyzed the effect of exposure to SePM on shrimp post-larvae, a critical stage for the viability of shrimp populations and for the trophic chain. After acclimation, shrimp were exposed to contaminants using a randomized experimental design—a 4 × 4 factorial arrangement with 2 factors: exposure time (24, 48, 72, and 96 h) and SePM concentration (0.00, 0.01, 0.10, and 1.00 g L−1). The bioaccumulation of metals, contamination rates, mortality, and ROS-related biomarkers (lipid peroxidation – LPO; DNA strand breakage DNA SB and metallothionein content – MTs;) were evaluated. After contamination, the water contained 27 different metals/metalloids. Post-larvae accumulated metals, such as Cd, Pb, Al, As, Se, Sr, Zr, Ba, La, Ce, W, and Hg. However, the rise in SePM did not result in a proportional bioaccumulation rise, indicating that effective biological barriers may work for some metals. Although the different levels of SePM changed mortality dynamics, they resulted in a similar final lethality (60–80 %). SePM caused significant damage to lipids (increased LPO), genetic material (DNA SB), and increased Mts. Such effects may reflect a particularly deleterious ecological problem as it is present at such an early stage of life. These results identified a clear environmental risk since the lower level of exposure used was 102 times lower than that measured in the habitats affected by local industry. Consequently, our results emphasize the need for clear protocols for monitoring the effects of SePM in aquatic environments.

Gonadal development of adultoid reproductive in subterranean termites indicates strong reproductive potential.

The fair allocation of reproductive resources between population growth and outward dispersal plays a crucial role in determining competitive advantage and ecological success of organisms within their environment. Termite colonies demonstrate flexibility in allocating their reproductive resources through wing polymorphism: with alates possessing fully developed wings for dispersal to propagate the colony's genetic material, while adultoids have floppy wings, serving a secondary reproductive to ensure stability and development within the natal colony. However, the extent of reproductive potential exhibited by alates (post-shed-wings called dealate) and adultoids within the colony remains uncertain. In this study, through experimentation, we compared the reproductive capacity of dealate and adultoid. We found that the adultoids can develop to maturity and lay eggs faster after emergence. The reproductive potential including the number of ovariole, the number of oocytes in growth and vitellogenesis stage, and the expression levels of vitellogenin genes were all higher than dealates. More importantly, paired adultoids has a longer oviposition cycle and is able to obtain more eggs and larvae. These results suggest that adultoids possess a greater reproductive potential allowing them to produce a large number of offspring within a short period of time. Our results help to an explanation of the biological significance that the differentiation of adultoids reproductive persist in lower termite colonies.

Date palm (Phoenix dactylifera L.) pollen-based microgel and its promising medical and environmental applications

Pollen, a natural bi-layered porous microcapsule, plays a crucial role in plant reproduction by protecting the plant's genetic material. Due to its unique properties, it has garnered attention for medical and environmental applications. Recent studies have focused on transforming the pollen's rigid structure to soft, gel-like matter, enabling the creation of pollen-based products such as paper and sponge. The main factor in the gelation process involves pectin-to-pectate conversion within the pollen's inner layer. Previously, claimed that this transformation is restricted to eudicot plants, and some others, such as monocot plant pollens do not exhibit this gelation phenomenon. In this study, we successfully produced microgel from date palm (Phoenix dactylifera L.) pollen (DPP), challenging this assertion. A significant portion of these monocot plant pollens exceeds the requirement of date production, presenting an opportunity for large-scale production without environmental concerns. Here, we have prepared pollen-based microgel, paper, and sponge and conducted a comprehensive analysis using various techniques such as optical and fluorescence microscopy, scanning electron microscopy (SEM), Brunauer-Emmett-Teller (BET), elemental CHN analysis, Fourier-transform infrared spectroscopy (FTIR), thermogravimetric differential thermal analysis (TGA/DTA), rheological measurements, mechanical analysis, pH-sensitivity tests, swelling analysis, and oil absorption capacity. By discerning the specific physicochemical properties of these materials, we propose potential medical and environmental applications for further investigation.

Genome characteristics of a MDR Pseudomonas monteilii carrying a novel VIM-type β-lactamase, blaVIM-84

ObjectivesThis study aimed to determine the genetic environment and characterize plasmid carrying a novel VIM-type β-lactamase (VIM-84) in a multidrug-resistant Pseudomonas monteilii isolate obtained from the human gut through whole-genome sequencing. MethodsDNA extraction of P. monteilii L2757hy was performed using the Genomic DNA Isolation Kit (QIAGEN, Hilden, Germany). Whole-genome sequencing was performed by Illumina NovaSeq 6000 and Oxford Nanopore platforms. The transferability of resistance genes was screened single clonal on MHA plates containing rifampicin and meropenem. Verification was performed using MALDI/TOF-MS and PCR with Pseudomonas aeruginosa PAO1Ri as the recipient strain. ResultsL2757hy was identified as P. monteilii through sequencing and ANI analysis. The genome was assigned as ST147 and comprised a 6,130,057 bp chromosome with a GC content of 61.8% and a 49,704 bp plasmid. Several resistance genes, including blaIMP-1, aac(6′)-IIa and tmexCD-toprJ, as well as virulence genes such as iroN, and wzaJ, were identified on the chromosome. A novel VIM-type blaVIM-84 was found on the plasmid, which was previously identified in Pseudomonas aeruginosa. Plasmid harboring blaVIM-84 was untypable, and it could be transferred to P. aeruginosa PAO1Ri and was associated with a class I integron with the genetic environment intI1-blaVIM-84-tniR-tniQ-tniB-tniA, likely derived from Tn402. ConclusionsOur study revealed that the novel blaVIM-84 gene was harbored by P. monteilii rather than P. aeruginosa. We suggested that P. monteilii may serve as a reservoir for resistance genes.

A new isolate of mungbean yellow mosaic India virus in Vigna mungo L. reported from a Dayalbagh field, Agra.

Black gram (Vigna mungo L.) plants showing yellow mosaic symptoms during 2019-2022 crop seasons were collected randomly from a Dayalbagh field, Agra Region of Uttar Pradesh, India. Total genomic DNA was isolated from the infected leaf samples by the Cetyltrimethylammonium bromide (CTAB) method and subjected to PCR. After viral confirmation, the viral genome was amplified by rolling circle amplification following the standard protocol. The DNA A and DNA B subgenomes were cloned individually as a PstI and BamHI fragment in the pUC18 vector. Positive clones were subjected to DNA sequencing. The results revealed that DNA A and DNA B show the closest nucleotide identity with "mungbean yellow mosaic India virus-[Mungbean], DNA-A, the complete sequence" (GeneBank Accession No AF416742.1) with 98.14% identity, and "mungbean yellow mosaic India virus isolate Mu1-Dholi segment DNA-B, the complete sequence" (GeneBank Accession No MW814723.1) with 97.94% identity, respectively. The new isolate of mungbean yellow mosaic India virus (MYMIV) shows sequence similarity with the coat protein gene of various strains of MYMIV. In the new isolate of MYMIV, a point mutation wasobserved at the 2036th nucleotide of DNA B, which disrupts the reading frame to introduce a stop codon and thus leading to a decrease in the size of the movement protein gene. In the present study we are reporting the whole genome sequence of the MYMIV Dayalbagh isolate for the first time.

Impact of prescribed fire on fungal communities in Scots pine (Pinus sylvestris) forests in Mediterranean transitional zones

The Mediterranean region is renowned for its natural susceptibility to wildfires. In recent years, this risk has intensified due to various factors, including climate change and rural abandonment. Castilla y Leon stands out as one of the most severely impacted areas grappling with rural exodus. This evolving scenario accentuates the urgency of implementing forest management strategies to mitigate the escalating threat of wildfires, with a primary focus on fuel reduction. Although prescribed fires represent an efficient and cost-effective tool for wildfire prevention, they remain a contentious subject in Europe. Fungi not only contribute significantly to rural economies but also play a pivotal role in maintaining the equilibrium of forest ecosystems. The main objective of this study was to examine the effects of prescribed burning on Pinus sylvestris soils, with a specific focus on the recovery of fungal populations after such fires. To assess the short-term effects of prescribed fires on soil fungal communities, we collected soil samples from both burned and unburned plots 12 months post-burning to perform genomic DNA analyses. Our findings indicate that prescribed burning does not significantly alter fungal diversity or composition, with only litter saprotrophs showing significantly higher levels of abundance in burned areas than in unburned areas. Valuable edible fungi persisted post-burning, suggesting that prescribed burning could be used to reduce wildfire fuel loads while preserving fungal biodiversity and valuable edible fungi. These results advocate for the use of prescribed burning as a viable, myco-friendly forest management practice, offering a balance between fire prevention and ecological conservation.

Dynamic change of gut microbiota in head and neck concurrent chemoradiotherapy patients and its potential value in the prediction of acute oral mucositis grade as well as quality of life.

Radiotherapy is the major therapy for head and neck squamous cell carcinoma (HNSCC). However, whether gut microbiota changes in HNSCC patients who received concurrent chemoradiotherapy remains unclear. This study aimed to investigate the dynamic change of gut microbiota composition, construct the first radiotherapy-related gut microbiota database in these patients and identify the potential value of the gut microbiota changing in the prediction of acute oral mucositis grade as well as patients' life quality. We enrolled 47 HNSCC patients who scheduled with concurrent chemoradiotherapy. The field was irradiated with a total dose of 66-70Gy in 33-35 fractions. All the patients received 2-3 cycles of platinum-based chemotherapy. After feces specimens collected, bacterial genomic DNA was isolated using magnetic beads and then analyzed by the Illumina MiSeq Sequencing System based on the V3-V4 hypervariable regions of the 16S rRNA gene. 194 genera which belonged to 27 phyla were found in 141 samples. Increased abundance of microbiota in diversity and richness was observed in mid-radiotherapy group. Bacteroides, Blautia, Phascolarctobacterium were three main genera in all three groups and the mid-radiotherapy group had the highest relative abundance of Phascolarctobacterium. What is more, most significantly altered bacteria shared the same variation pattern which was increased in mid-radiotherapy while decreased to the almost same level of as pre-radiotherapy in post-radiotherapy group. Further analysis indicated that Bacteroidetes showing an upward trend while Proteobacteria declining in higher grade of acute mucositis. Moreover, relatively low abundant Proteobacteria was significantly correlated with high-grade acute oral mucositis. As for the quality of life, Lactobacillales and Actinomycetales were specifically found in better life quality group. However, Clostridia_UCG_014, Eubacteriaceae, UCG_010 and Moraxellaceae were unique abundantly present in worse life quality group. Chemoradiotherapy can affect the composition of the gut microbiota in HNSCC patients during the mid-term of treatment. Yet self-stabilized ability maintained the gut microbiota homeostasis. Dynamic change of specific species could help predict acute oral mucositis grade and characterize different quality of life group in these patients.

In silico screening and identifying phytoconstituents of Withania somnifera as potent inhibitors of BRCA1 mutants: A therapeutic against breast cancer

Breast CAncer gene 1 (BRCA1) is an anti-oncogene that helps the cell repair damaged DNA and preserve genetic material. BRCA1 also acts as a cell growth suppressor and produces tumor suppressor gene (TSG) proteins, i.e., BRCA1 protein. Remarkably, BRCA1 mutations account for 90 % of hereditary breast cancer and a majority of hereditary ovarian cancer. Hence, we have considered three mutants of BRCA1 (R1699W, R1699Q, T1700A) in this study and adopted an in-silico approach to find the best possible phytochemical to inhibit these mutated proteins, enabling early breast cancer diagnosis. Perceiving the importance, many natural molecules from ancient medicinal plants are considered for molecular docking. Our findings suggest that though many molecules bind actively with the receptor's active site, the top three phytoconstituents (27-Deoxy-14-hydroxywithaferin A, Withacoagulin, Somniferanolide) of Withania somnifera, commonly known as Ashwagandha, have high binding affinities and suitable pharmacokinetic properties, making these natural compounds potential drug candidates. Further, molecular dynamics (MD) simulation and the binding free energy calculation show stability and thermodynamically favourable. We can, therefore, draw the conclusion that these lead compounds act as potential inhibitors against BRCA1. However, wet lab experiments and clinical trials are recommended to ascertain its efficacy, hence the development of novel BRCA1 inhibitors.

Crouching bacteria, hidden tetA genes in natural waters: intracellular damage via double persulfate activation (UVA/Fe2+/PDS) effectively alleviates the spread of antibiotic resistance

In this study, we elucidated the chemical and biological inactivation mechanisms of peroxydisulfate (PDS) activated by UVA and Fe2+ (UVA/Fe2+/PDS) in wild-type antibiotic-resistant bacteria (ARB) isolated from a river in Inner Mongolia. Among the screened wild-type ARB, the relative abundance of unidentified Enterobacteriaceae, Stenotrophomonas, and Ralstonia was high. A ratio of 1:1 for Fe2+ and PDS under 18W·m-2 UVA radiation (sunny days) completely inactivated the environmental ARB isolates. In the macro view of the inactivation process, Fe2+ first activates PDS rapidly, and later the UVA energy accumulated starts to activate PDS; HO• then becomes the main active species at a rate-limiting step. From a micro perspective, damage to the cell wall, intracellular proteins, inactivation of antioxidant enzymes, and genetic material degradation are the inactivation series of events by UVA/Fe2+/PDS, contributing to the 97.8% inactivation of ARB at the initial stage. No regrowth of sublethal ARBs was observed. The transfer of tetracycline resistance genes from ARB to lab E. coli was evaluated by horizontal gene transfer (HGT), in which no HGT occurred when ARB was eliminated by UVA/Fe2+/PDS. Moreover, the sulfate and iron residuals in the effluents of treated water were lower than the drinking water standards. In summary, PDS, UVA, and Fe2+ activation effectively inactivated wild ARB with a low concentration of reagents, while inhibiting their regrowth and spread of resistance due to the contribution of intracellular inactivation pathways. Environmental ImplicationAntibiotic resistance bacteria (ARB) were isolated from the feces and river of Inner Mongolian Plateau, which indicated that the grassland system and surface water had been polluted by the antibiotic resistance. In our previous survey, most people living in remote pasture lands, are using surface water for drinking.Hence, we applied UVA/Fe2+/PDS to control the antibiotic resistance, which has great oxidative ability and lower input requires. ARB and antibiotic resistance genes (ARGs) were well eliminated, and the chemicals of effluent were under the drinking water requires. UVA/Fe2+/PDS is an environmental-friendly disinfection method, which effectively protect the ecological and human health.

Embryologie und Wirbelsäule – Teil 1: Entstehung und Entwicklung der Somiten als Anlage der Wirbelsäule

The early embryonic segmentation and vertebral column arise from the genetic material of the somites and, as in every other organ in the human body, they are part of, and inseparable from, the overall development of the embryo. The first somites and their subcomponents arise from the end of the 3rd week of development in a constructively logical succession as a gradual differentiation of the unsegmented mesenchyme. Following the development of the somites, a closed series of formative processes then leads to the development of the vertebral column. Resegmentation of somites does not occur in the formation of the vertebral column. This is the first time that we have described the basic embryological principles according to Blechschmidt in this way.

Enhanced ePAD-DNA biosensor incorporating ZnO-NRs: Rapid and reliable electrochemical detection of field-isolated Pasteurella multocida

An electrochemical sensor has received much attention due to its importance for early infection identification, hinting at its critical relevance in diagnostic applications. For the detection of field-isolated strains of Pasteurella multocida, this paper reports the development and fabrication of a DNA-based electrochemical biosensor by integrating zinc oxide (ZnO) nanorods (NRs) into an electrochemical paper-based analytical device (ePAD). One significant improvement over the state-of-the-art features of the sensor is the using paper, an economically viable substrate that can be manufactured in large numbers. These sensors, being categorized under precision instruments with an ecologically friendly design, are ideal for mass production of eco-sensitive substrates. The biosensor is more sensitive and specific as compared to the conventional PCR-based sensing techniques. Moreover, the biosensor exploits the inherent properties of ZnO-NRs to amplify the signal output, whereas ePAD limits detection cost. In addition, a probe targeting the KMT gene of P. multocida was first characterized using conventional PCR and then immobilized onto the ZnO nanorods-modified ePAD surface, which improved the biosensor's ability for the rapid and selective genetic material of the pathogen detection. In addition, the sensor was validated using the methods of Cyclic Voltammetry (CV) and Linear Sweep Voltammetry (LSV). The reported biosensor it provides Linear Response of 0.001-10 μg/ml with LOD 0.001 μg/ml within a response time of 30 s. Subsequently, the biosensor reveals fast kinetics of the reaction as a powerful tool for timely and accurate diagnostics, pointing to its contribution to further development in biosensing technologies in the diagnostic area of infectious diseases at hospitals and clinics.

Exome sequencing in Nigerian children with early-onset epilepsy syndromes.

Nigeria, along with other Sub-Saharan African countries, bears the highest burden of epilepsy worldwide. This high prevalence is attributed to a combination of factors, including a significant incidence of infectious diseases, perinatal complications, and genetic etiologies. Genetic testing is rarely available and is not typically included in the routine diagnostic work-up for individuals with infantile and childhood epilepsy syndromes in these regions. Exome sequencing (ES) offers a diagnostic yield of 24%-62%, but these figures primarily reflect data from high-income countries (HICs) and may not be applicable to low- and middle-income countries (LMICs). In this study, we employed ES to investigate the genetic basis of early-onset epilepsy in 22 affected children from Nigeria. The study involved sampling of patients diagnosed with early-onset epilepsy syndromes at the Lagos State University Teaching Hospital (LASUTH) Neurology clinic. Venous blood samples were collected, and genomic DNA was isolated and purified. Molecular analysis included DNA fragmentation, ligation, target enrichment, library preparation, and whole-exome sequencing. Computational analysis involved variant calling, curation, and classification using specialized tools and databases. Pathogenic variants were identified in 6 out of 22 individuals, equaling a diagnostic yield of 27.3% and comprising variants in BPTF, NAA15, SCN1A, TUBA1A and twice in CACNA1A. In this study, we present the first exome study on early-onset epilepsy syndromes from West Africa, facilitated by a Nigerian-German research collaboration. Our findings reveal a genetic diagnostic yield comparable to that of HICs. The integration of genomic medicine into epilepsy management in Nigeria holds promising prospects for improving patient care and reducing mortality rates. This study represents the first published exome findings in Nigerian children with early-onset epilepsy, revealing a genetic diagnosis in 27% of cases. Pathogenic variants were identified in five genes amongst 6 of 22 patients, underscoring the potential of genetic testing to enhance epilepsy management in developing nations like Nigeria.

A Latent Cardiomyocyte Regeneration Potential in Human Heart Disease.

Cardiomyocytes in the adult human heart show a regenerative capacity, with an annual renewal rate of ≈0.5%. Whether this regenerative capacity of human cardiomyocytes is employed in heart failure has been controversial. We determined cardiomyocyte renewal in 52 patients with advanced heart failure, 28 of whom received left ventricular assist device support. We measured the concentration of nuclear bomb test-derived 14C in cardiomyocyte genomic DNA and performed mathematical modeling to establish cardiomyocyte renewal in heart failure with and without LVAD unloading. We show that cardiomyocyte generation is minimal in end-stage heart failure patients at rates 18 to 50× lower compared with the healthy heart. However, patients receiving left ventricle support device therapy, who showed significant functional and structural cardiac improvement, had a >6-fold increase in cardiomyocyte renewal relative to the healthy heart. Our findings reveal a substantial cardiomyocyte regeneration potential in human heart disease, which could be exploited therapeutically.

First Report of Neocosmospora sp., Causing Stem Rot on Agave potatorum Zucc., in Mexico.

Agave potatorum Zucc., also known as "agave tobalá", is an endemic species in the Mexican states of Oaxaca and Puebla and is one of the most important wild species for producing the Mexican alcoholic distillate called mezcal. In September 2021, symptoms of dry stem rot and subsequent death of plants were observed, in a commercial field of 10 acres located in the municipality of Villa Sola de Vega, Oaxaca, Mexico. The disease incidence was 45%. Twenty symptomatic agave stem plants were sampled to isolate the pathogen. Pieces (5 × 5 mm) of the symptomatic tissues from diseased plants were surface disinfested with 1% NaOCl (60 s) and 70% ethanol (1 min), rinsed with sterile water (2 × 60 s), placed on potato dextrose agar (PDA Bioxon®, Mexico), and incubated at 25°C for 6 days with natural light. Next, we prepared single-spore derived cultures on PDA and carnation leaf-piece agar (CLA). Fusarium-like colonies were consistently isolated on potato dextrose agar (PDA) and 14 monoconidial isolates were obtained. On PDA, aerial mycelium was white to beige with diffuse pink pigment in the center of the culture and colonies were appressed with a regular shape after 7 days of incubation at 25℃. Macroconidia (n=100) were falcate, almost straight, 1 to 4-septate, 18.5 to 47.9 × 3.9-5.7 μm. Microconidia (n=100) were ellipsoidal to subcylindrical, slightly curved, hyaline, 8 to 16 × 3.5 to 4.5 μm, 0 to 1-septate, and grouped in false heads. Chlamydospores were globose to subglobose, terminal or intercalary in hyphae, and were solitary or in chains. Morphological features were consistent with the description of Neocosmospora spp. (Crous et al. 2021; Sandoval-Denis et al. 2019). Three isolates were selected for multilocus phylogenetic analysis and pathogenicity tests. Isolates were deposited as IPN-AP1, IPN-AP2, and IPN-AP3 in the Culture Collection of Phytopathogenic Fungi of Plant Pathology Laboratory of the CIIDIR-Oaxaca of the Instituto Politécnico Nacional. For molecular identification, genomic DNA was extracted, and partial sequences of translation elongation factor 1-alpha (EF1-α) and the second largest subunit of RNA polymerase II (rpb2) genes were amplified and sequenced with the primer sets EF1-728F/EF1-986R (Carbone and Kohn 1999) and RBP2-5F/RPB2-7R (Liu et al. 1999), respectively. A phylogenetic tree using concatenated EF1-α and rpb2 sequences was constructed for Neocosmospora spp. based on Maximum Likelihood. The phylogenetic analysis suggested that the three isolates belong to a new species, closely related to N. noneumartii, as they formed a sister clade with this species. The sequences were deposited in GenBank with accession nos. OP902594 to OP902596 for EF1-α, and OP902591 to OP902593 for rpb2. A pathogenicity test for each isolate was performed by inoculating five healthy A. potatorum plants (1-year-old) grown in pots. Conidia were cultured on PDA for 7 days and the conidial solution was prepared with sterile water and adjusted to 1×105 spores/ml. A total of 1 ml of the conidial suspension was placed on the basal stem of each plant. Five plants mock-inoculated with sterilize water served as the controls. All plants were kept in a greenhouse for 45 days at temperatures ranging from 20 to 30°C. At 35 days after inoculation, dry stem rot was visible on the inoculated plants, whereas the control plants remained asymptomatic. The pathogenicity test was repeated twice with similar results. The fungi were reisolated from the infected plants and found to be morphologically identical to the isolates used for inoculation, fulfilling Koch's postulates. Neocosmospora spp., has been previously associated with dry root rot in Citrus sinensis in South Africa (Guarnaccia et al. 2021) as well as rot of Cactaceae and other succulent plants in Iran (Kamali-Sarvestani et al. 2022). To our knowledge, this is the first report of Neocosmospora sp. causing stem rot of A. potatorum in Mexico and worldwide. Further studies should focus on determining the distribution, impact, and management of this disease in the production areas of agave tobalá in Mexico.

Epidemiology and genetic diversity of Echinococcus granulosus sensu stricto in the East Tianshan Mountains, Xinjiang, China.

In Xinjiang, previous reports of cystic echinococcosis (CE) in livestock have focused on West and Middle Tianshan Mountains. In contrast, there is an absence of research on CE in the East Tianshan Mountains. To determine the epidemiology and genetic diversity of Echinococcus granulosus sensu stricto (s.s.) in domestic animals in the East Tianshan Mountains, Xinjiang, China, the livers and lungs of 1773 domestic animals were examined, between January 2023 and March 2023, and 40 cysts were collected. Polymerase chain reaction was used for the molecular diagnosis of the cysts. Statistical results showed that the overall prevalence of echinococcosis in sheep was 6.92% (114/1646), which was significantly higher than that in cattle (1.57%, 2/127). A total of 40 cyst isolates were obtained, including 38 from sheep and 2 from cattle. Genomic DNA was extracted, and the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene was amplified by PCR to obtain the target 850bp fragment. The results revealed that all the isolates had the G1 genotype, with similarities ranging from 98.88-100%. Haplotype network analysis revealed 32 haplotypes of the cox1 gene, among which Hap_7 was the main haplotype. Furthermore, haplotype diversity (Hd = 0.313 ± 0.093) and nucleotide diversity (π = 0.00173 ± 0.00079) were lower in the East Tianshan Mountains than in other regions, indicating that the populations are genetically less differentiated. Tajima's D and Fu's Fs tests were negative (p ≤ 0.01), indicating an expansion of the population in the East Tianshan Mountains of Xinjiang, similar to the results previously reported for Xinjiang. The low fixation index (Fst) ranged from negative values (Gansu Province) to 0.30346 (Mongolia), indicating that the genetic differentiation between the East Tianshan Mountains and Gansu, and Tibet, Xinjiang was relatively low, with frequent gene flow. In this survey, two 'new' haplotypes were identified in the East Tianshan livestock. In addition, two different haplotypes of liver and lung infections were found in one cattle. This survey provides information on the epidemiology and genetic diversity of E. granulosus s.s. in the East Tianshan Mountains of Xinjiang, China.

L1-ORF1p nucleoprotein can rapidly assume distinct conformations and simultaneously bind more than one nucleic acid.

LINE-1 (L1) is a parasitic retrotransposable DNA element, active in primates for the last 80-120 Myr. L1 has generated nearly one-third of the human genome by copying its transcripts, and those of other genetic elements (e.g. Alu and SVA), into genomic DNA by target site-primed reverse transcription (TPRT) and remains active in modern humans. L1 encodes two proteins that bind their encoding transcript (cis preference) to form an L1 ribonucleoprotein (RNP) that mediates retrotransposition. ORF2p provides reverse transcriptase and endonuclease activity. ORF1p, its major component, is a homo-trimeric phospho-protein that binds single-stranded nucleic acid (ssNA) with high affinity and exhibits nucleic acid (NA) chaperone activity. We used optical tweezers to examine ORF1p binding to individual single-stranded DNA (ssDNA) molecules and found that the arrangement of ORF1p on the ssDNA depends on their molar ratio. When the concentration of ORF1p is just sufficient to saturate the entire NA molecule, the nucleoprotein (NP) is compact and stable. However, additional ORF1p binds and destabilizes the compacted NP, allowing it to engage a second ssDNA. Our results suggest that ORF1p displaced from its RNA template during TPRT could bind and destabilize remaining downstream L1 RNP, making them susceptible to hijacking by non-L1 templates, and thereby enable retrotransposition of non-L1 transcripts.

Rapid and visual detection of Pyricularia oryzae using coupled recombinase polymerase amplification-lateral flow dipstick assay.

Rice blast, caused by Pyricularia oryzae, is one of the most destructive fungal diseases in rice, severely impacting rice production worldwide every year. Rapid, accurate and visual detection of P. oryzae is essential for more effective prevention and control. In this study, we developed a recombinase polymerase amplification-lateral flow dipstick (RPA-LFD) assay to detect P. oryzae. Species-specific RPA primer pairs and probe were designed based on target gene MGG_15975. The optimized reaction temperature and time were set at 37 °C and 25 min, respectively. Specificity analysis showed that the assay could specifically detect P. oryzae isolates from rice, whereas other fungal species or Pyricularia species from grasses were not detected. Additionally, this assay demonstrated highly sensitivity, capable of detecting as low as 10-2 ng/µL of P. oryzae genomic DNA, which was found to be 100 times more sensitive than conventional PCR. Furthermore, using this assay, P. oryzae was effectively detected in diseased leaves in rice fields, and could also be identified at an early stage of infection before obvious lesions appeared in artificially inoculated rice seedlings. Therefore, the RPA-LFD assay developed in our study for the detection of P. oryzae is rapid, highly sensitive and efficient, which has the potential application for early diagnosis of P. oryzae infection in rice fields.