Gene Fragments Research Articles

Restoration of the spike architectonics in ancient barley excavated at the twelfth-century settlement of Usvyaty

Background. The data are presented on the architectonics of ancient barley spikes from the 12th century, excavated in 2019 at Usvyaty Settlement. Modern molecular genetics approaches were used to study domestication genes (Btr1, Btr2, and Vrs) in ancient and contemporary barleys (germplasm accessions preserved at VIR).Materials and methods. The carbonized kernels found by archaeologists during the excavations at Usvyaty were analyzed. Primers for domestication genes were designed, and PCR was performed on contemporary and ancient barley grains. Ancient kernels were studied in accordance with the rules established for organizing a paleogenetics laboratory, which excluded any contamination with contemporary DNA. Fragments of domestication genes from contemporary and ancient barley grain samples underwent Sanger sequencing. Results. Ancient DNA was isolated and enriched. The analysis of domestication gene sequences made it possible to reconstruct the ancient barley spike’s features. Conclusion. The ancient cereal crop architectonics was restored to ascertain a brittle two-row spike of ancient barley

Genetic Identification of Putative Hybrids Between Grey Wolf and Golden Jackal

We describe the results of genetic analysis of 11 phenotypically deviant individuals of grey wolf (Canis lupus Linnaeus, 1758) sensu lato collected in Voronezh State Nature Biosphere Reserve (Chernozem zone of European Russia) and Dagestan (Northern Caucasus, Russia) putatively identified morphologically as hybrids between grey wolf and golden jackal (Canis aureus Linnaeus, 1758). By means of maternally inherited mtDNA (sequences of cytochrome b gene fragment) and paternal lineage markers ZfY no F1 wolf-jackal hybrids were identified. As well, possibility of classification of the studied individuals to next generation hybrids from crosses between different wolf-jackal F1s. However, attribution of these animals to complex hybrids such as various backcrosses cannot be rejected. From the results of analysis by a set of autosomal microsatellite loci we putatively diagnosed a single F2 hybrid. As well, we obtained data that can be considered as traces of hybridization between wolf and jackal in southern regions of European Russa, albeit direct indications of introgression between these species not found. At the same time, the results of both genetic and craniological studies could be interpreted as indication to hybridization between wolves and domestic dogs on the same territories.

Molecular Detection of Anaplasma marginale in Capybara (Hydrochoerus hydrochaeris) from Corrientes, Argentina.

Monitoring wildlife health is essential for understanding global disease patterns, particularly as vector-borne infections extend the geographic ranges and thereby hosts due to environmental shifts. Anaplasma marginale, primarily impacting cattle, has economic implications and has been found in diverse hosts, yet its presence in capybaras (Hydrochoerus hydrochaeris), influential in tick-borne pathogen spread, lacks comprehensive understanding. From 2015 to 2022, 14 capybaras were surveyed across two different areas of northeastern Argentina. In 1 of 14 (7%) capybaras, the presence of A. marginale was confirmed through the amplification of specific genes, msp5 and msp1β. In addition, A. marginale DNA was detected in the capybara's blood sample through quantitative PCR, with a cycle threshold value of 30.81 (800 copies per reaction). Amplification of a fragment of the msp1α gene revealed PCR products of three different sizes, suggesting the presence of at least three coinfecting A. marginale variants in the capybara host. This study suggests that capybaras are wild hosts for A. marginale in the Ibera Wetlands in Argentina, potentially influencing the infection dynamics of both domestic and wild species. This finding highlights the necessity for thorough studies on the role of capybaras in disease dynamics, crucial for understanding wildlife health and the spread of disease.

Evidence of the involvement of Xylella fastidiosa in the occurrence of walnut leaf scorch in Xinjiang, China.

Walnut (Juglans regia Linn.) leaf scorch (WLS) was first reported in 2012 in Hotan, Xinjiang, China (Zhang et al., 2012). Initially, brown spots appear at the apex of the leaflets with slight shrinking. These spots spread inward along the leaf margins in a flame-like pattern. The scorched areas curl inward with a yellow halo. No powdery or bacterial signs were observed on the leaf surface. In severe cases, the leaves dried up and shrank, affecting the entire tree. However, new leaves did not show any signs of scorching. We collected 300 symptomatic leaf samples from 10-12 year-old trees of the susceptible WLS species Wen185, located in Daryaboyi (40°72'N, 80°49'E), Xakal (40°69'N, 80°52'E), and Karatal (40°73'N, 80°39'E) for X. fastidiosa PCR detection analysis. X. fastidiosa was detected in asymptomatic leaves of trees with severe WLS, as well as in asymptomatic leaves of trees exhibiting mild WLS symptoms, and it was even found in asymptomatic leaves of trees without any WLS symptoms.To isolate X. fastidiosa, walnut leaves with petioles were disinfected with 3% bleach for 10 minutes, followed by four washes in autoclaved deionized water. The midrib and petiole of the leaf were cut off with a sterile blade, and a 2 mm to 3 mm section was excised. Then, the cut ends were squeezed with a pair of pliers, and the sap was blotted onto Periwinkle Wilt (PW) plates (Davis et al. 1983). The plates were sealed with parafilm and incubated at 28°C in the dark, with daily observations for the development of individual colonies. The sap collected from 5 out of 20 leaf samples with scorch symptoms and from 20 leaves without symptoms never showed bacterial growth in PW.. Six colonies were tested and confirmed positive for X. fastidiosa using the RST31 and RST33 primer pair (Minsavage et al., 1994). The uploaded sequence accession numbers are PP871340-PP871342. Blast analysis of Multilocus Sequence Typing (MLST) sequences from the isolated strain using the X. fastidiosa MLST Database (http://pubmlst.org/xfastidiosa) revealed a perfect match with sequences of alleles leuA_3, petC_3, malF_, cysG_3, holC_4, nuoL_3, and gltT_3 (PP871343-PP871349). Using MEGA software to concatenate the MLST single gene fragments and construct a ML multi-gene fragment phylogenetic tree through Maximum Likelihood (ML) analysis, the bacterium was identified as X. fastidiosa subsp. multiplex To fulfill Koch's postulates, a purified colony from the PW plate was suspended in 500 μL of deionized water at 1×10^8 CFU·mL-1. A sterilized 5 ml medical needle was used to prick a small hole on the main branch, 5 cm from the base, of three-year-old walnut seedlings, and the bacterial solution was injected into the small hole. Three biological replicates were performed for each treatment (5 μL and 10 μL of bacterial solution, deionized water), and the experiment was repeated three times. Scorching symptoms were observed three months after inoculation in 11 out of the 15 seedlings inoculated with a 10 μL bacterial solution, and isolation of X. fastidiosa occurred in leaf samples from 5 of these seedlings, completing Koch's postulates. Walnut seedlings with water stayed asymptomatic, PCR negative. To our knowledge, X. fastidiosa has been found infecting grapes (Chu, 2001) in Shaanxi, China and pears (Su et al., 2016) in Taiwan, China. This is the first report of X. fastidiosa involvement in WLS in China. Further research on the occurrence of the disease will help prevent the spread of the disease.

ONT sequencing identifies a high prevalence of crt sensitive, triple mutant dhfr and single mutant dhps parasites within an ANC population in Nigeria.

Malaria in pregnancy is a major public health issue, particularly among vulnerable populations in malaria-endemic sub-Saharan African countries. To mitigate its risks, WHO recommends sulphadoxine-pyrimethamine (SP) for chemoprevention and artemisinin-based combination therapy (ACT) to treat uncomplicated Plasmodium falciparum malaria. These interventions have helped to alleviate the risk associated with malaria in pregnancy; however, in the context of the emergence of SP- and ACT-resistant P. falciparum, maintained efficacy is under threat. Molecular surveillance is a reliable tool to monitor the emergence of resistance where molecular markers are known. Thus, the objective of the study was to use a multiplexed amplicon Oxford Nanopore sequencing approach to assess the molecular markers for antimalarial resistance among pregnant women in Nigeria. Dried blood spots (DBS) were collected from pregnant women who received IPTp-SP at the enrollment and follow-up visits. P. falciparum genomic DNA was extracted by the Chelex® method and Pf18S qPCR was used to detect parasite DNA in each sample. With nested PCR assays, fragments of Pfdhps, Pfdhfr, Pfmdr1, Pfcrt, Pfk13 and Pfama1 genes were amplified and multiplexed amplicon-based sequencing was conducted on the minION Oxford Nanopore Technology. In total, 251 pregnant women were enrolled in the study and 457 DBS samples were collected. P. falciparum genomic DNA was detected in 12% (56/457) of the samples, 31 at baseline and the remaining during the follow-up visits. Pfama1, pfk13, Pfdhps, Pfdhfr, Pfmdr1 and Pfcrt were successfully sequenced in a single run. Notably, k13 artemisinin resistance mutations were absent, the frequencies of Pfdhfr and Pfdhps SP resistance haplotypes, IRN for pyrimethamine resistance and ISGKA/IAGKA associated with sulphadoxine resistance were 82% (36/44) and 64% (27/42), respectively, and the Pfcrt CVIET resistant haplotype was at approximately 22% (7/32). Here a multiplexed amplicon-based ONT assay established that triple mutant Pfdfhr-IRN, double mutant Pfdhps-SG haplotypes and the chloroquine sensitive strain were prevalent among pregnant women in Nigeria.

Clinical Manifestations and Molecular Identification of Giardia duodenalis in Pediatric and Adolescent Cancer Patients in Southwestern Iran.

This study aimed to investigate the clinical and molecular characteristics of Giardia duodenalis (G. duodenalis) infection and identify potential risk factors in children and teenagers with malignancies in Shiraz, southwestern Iran. A total of 200 fresh fecal samples were collected from children and adolescents suffering from 32 different cancer types at Amir, Nemazee, and Saadi hospitals affiliated with Shiraz University of Medical Sciences between October 2021 and May 2022. Direct microscopy using saline and iodine wet mount was conducted, and all fecal samples were rechecked by SSU-PCR. Subsequently, a specific fragment of the tpi gene was amplified on all samples for prevalence, sequencing, and assemblage identification. Our study found a 4% (8/200) prevalence of G. duodenalis using microscopy and PCR. The molecular findings were consistent with the microscopic results. All eight positive samples with SSU-rRNA gene were also detected as positive with tpi gene and were correctly sequenced. Among the examined cancer patients, two assemblages were identified: A [sub-assemblage AI (2/8, 25%) and sub-assemblage AII (3/8, 37.5%)] and B [sub-assemblage BIV (3/8, 37.5%)]. Notably, patients were more vulnerable to G. duodenalis infection after receiving at least 8 treatment episodes (p < 0.05) and displaying gastrointestinal symptoms (p > 0.05). The demographic characteristics of cancer patients with G. duodenalis infection and the statistical conclusions were separately detailed. The small sample size and low prevalence rate in this study hindered precise epidemiological conclusions. Nonetheless, the results suggest that G. duodenalis infection among cancer patients in Shiraz city originates from humans, without any specific animal groups (C-H) involved.

Functional characterization of chalcone reductase gene CHR3 through RNAi

In this experiment, the soybean genome "Jilin 30" was used as a template to clone the target gene CHR3 using specific primers. The sense fragment, antisense fragment, and intron of the target gene were ligated into pCAMBIA3300 through double enzyme digestion to form a stem loop structure. We then transferred the constructed RNAi vector pCAMBIA3301-CHR3 RNAi into the recipient soybean genome via Agrobacterium mediated method, specifically reducing the expression of the target gene CHR3. By genetic transformation, positive plants were obtained, and molecular biology methods such as Southern blotting, real-time quantitative PCR, isoliquiritigenin analysis, and identification of resistance to Phytophthora root rot were used to detect the expression level of the CHR3 gene in the positive plants and study its function.The RNAi expression vector pCAMBIA3300-CHR3-RNAi was successfully constructed using the sense and antisense fragments of 428 bp CHR3 gene and an intron of 110 bp was inserted to form the stem-loop structure of the RNAi vector. PCR was used to detect 6 lines of the T1 generation and 15 lines of the T2 generation. The results of Southern analysis confirmed the integration of CHR3 and marker genes into the soybean genome. The expression CHR3 gene in the T1 genotypes decreased by 13.1 to 58.3%; the expression in the root decreased by 0.9 to 47.4%; the expression in the leaf decreased by 20 to 44.2%; and the expression in the stem decreased by 7.6 to 23%. The content of isoliquiritigenin decreased by 26.7% as transgenic shoots had a lower content of isoliquiritigenin and reduced resistance to Phytophthora sojae. Bangladesh J. Bot. 53(3): 627-633, 2024 (September) Special

Genetic characterization of Neisseria meningitidis isolates recovered from patients with invasive meningococcal disease in Lithuania.

Neisseria meningitidis is a gram-negative bacterium responsible for life-threatening invasive infections known as invasive meningococcal disease and is associated with high fatality rates and serious lifelong disabilities among survivors. This study aimed to characterize N. meningitidis isolates cultured from blood and cerebrospinal fluid collected between 2009 and 2021 in Lithuania, assess their genomic relationships with European strains, and evaluate the possibility of using a cost-effective method for strain characterization, thus improving the national molecular surveillance of invasive meningococcal disease. In total, 321 N. meningitidis isolates were collected and analyzed using multilocus restriction typing (MLRT). Amplification of the penA gene and restriction fragment length polymorphism analysis were performed to identify the modified penA genes. Based on the MLRT genotyping results, we selected 10 strains for additional analysis using whole-genome sequencing. The sequenced genomes were incorporated into a dataset of publicly available N. meningitidis genomes to evaluate genomic diversity and establish phylogenetic relationships within the Lithuanian and European circulating strains. We identified 83 different strains using MLRT genotyping. Genomic diversity of N. meningitidis genomes analysed revealed 21 different sequence types (STs) circulating in Lithuania. Among these, ST34 was the most prevalent. Notably, three isolates displayed unique combinations of seven housekeeping genes and were identified as novel STs: ST16969, ST16901, and ST16959. The analyzed strains were found to possess virulence factors not commonly found in N. meningitidis. Six distinct penA profiles were identified, each with different frequencies. In the present study, we also identified N. meningitidis strains with new penA, NEIS0123, NEIS1320, NEIS1525, NEIS1600, and NEIS1753 loci variants. In our study, using the cgMLST scheme, Minimum Spanning Tree (MST) analysis did not identify significant geographic relationships between Lithuanian N.meningitidis isolates and strains from Europe. Discussion: To our knowledge, this is the first study to employ whole genome sequencing (WGS) method for a comprehensive genetic characterization of invasive N. meningitidis isolates from Lithuania. This approach provides a more detailed and precise analysis of genomic relationships and diversity compared to prior studies relying on traditional molecular typing methods and antigen analysis.

First report of lily mottle virus naturally infecting lily (Lilium asiatica) in Texas, USA.

Lily (Lilium asiatica) is an important plant grown for its range of flower colors and heavy scent. In March 2024, potyvirus-like symptoms consisting of light-yellow mottling and mosaic were noticed on 12/20 lily plants in a private property in Weslaco, Hidalgo County, Texas. Two symptomatic plants (WTX1 and WTX2) were sampled randomly for virus diagnosis. The leaf extracts of both samples were negative for the potyvirus group using Agdia's Poty ImmunoStrip® (Agdia, Inc., Elkhart, IN, USA). However, rub-inoculation of the extracts onto healthy Nicotiana benthamiana and Vigna unguiculata plants (n=4, each) induced mild mottle symptoms on the systemic leaves of both herbaceous test plants 20 to 28 days post inoculation, indicating the presence of a mechanically transmissible agent in the samples. No virus-like symptoms were observed on the mock-inoculated plants (n=1, each) of both species. To test for suspected potyvirus infection, 2-µg of total nucleic acid extracts (Dellaporta et al. 1983) from WTX1 and WTX2 were used for complimentary DNA (cDNA) synthesis with Oligo(dT) primer and the PrimeScript 1st strand cDNA synthesis kit (Takara Bio, USA). One microliter aliquot of each cDNA served as template in 12.5-µl conventional PCR reaction volumes with 5 U Taq polymerase (Roche Diagnostics, Indianapolis, IN), and two pairs of degenerate primers targeting the partial cylindrical inclusion (CI) gene and the helper component protease (HC-Pro) of potyviruses (Ha et al. 2008). The expected ~700-bp DNA product of each gene target was amplified from both samples. The amplicons were excised, gel eluted (Zymoclean™ Gel DNA Recovery kit) and cloned individually into the pJET1.2/Blunt vector (Life Technologies). Recombinant plasmids from two transformed Escherichia coli cells per cloned DNA insert were Sanger sequenced. BLASTn analysis of each gene-specific nucleotide (nt) sequence fragment revealed their identities (83 to 92 % nt) as lily mottle virus (LMoV; genus Potyvirus) at 93 to 98% query coverage. In pairwise comparison, the obtained sequences of LMoV isolates from TX specific to the CI (GenBank accession nos. PQ037260-61) and the HC-Pro (PQ037262-63) shared ~99% nt and 100% amino acid (aa) identities with each other and ~91-92% nt and 98-100% aa identities with the closest isolate, Bate5 (JN127341) from Australia. Based on these results, a pair of LMoV-specific primers (LM_v109-F: 5'-GGCCAGTAATGTGCACAAGC-3' and LM_c527-R: 5'-TCGCTGTAGCTAGCGACGTAC-3') was newly designed to target the partial CI gene, internal to the 700-bp fragment obtained above with the generic primers. The primer pair was used to screen each of the mechanically inoculated test plants by RT-PCR as previously described above. The expected 438-bp fragment of the LMoV CI gene was amplified from all the inoculated plants of both N. benthamiana and V. unguiculata; the results were confirmed by Sanger sequencing as described above. The mock-inoculated plant of each experimental host plant tested negative for LMoV. LMoV is a quarantine pest and was previously reported in the U.S. (Brierley and Smith, 1944) and other lily-producing parts of the world, such as the Netherlands, China, Korea, and Brazil. To our knowledge, this is the first confirmed report of the virus in Texas, thus expanding its geographical range. Testing of lily foundation stocks before propagation is essential to prevent further spread of LMoV via planting material.

Wildlife Infection of Peste des Petits Ruminants Detected in China, 2024.

In 2013, the second outbreak of peste des petits ruminants occurred in China, leading to a spillover in more than 20 provinces and municipalities over the next few months. Thereafter, the epidemic situation was stable owing to strict prevention and control measures. In February 2024, several bharals and argali with suspected symptoms of PPR were discovered in Rutog country, Tibet Autonomous Region. Samples collected from these animals were delivered to our laboratory for diagnosis; the results of fluorescence quantitative reverse-transcription (RT) PCR indicated that all samples were positive for PPR viral RNA. The N and F gene fragments were amplified successfully via RT-PCR, and these results confirmed that these animals were infected with PPRV. A PPRV strain (subsequently named ChinaTibet2024) was sequenced, and its genome length was 15,954 nucleotides. A phylogenetic tree analysis using N and F genes and viral genomes showed that the ChinaTibet2024 genome was classified into lineage IV of the PRRV genotypes. The genome of the ChinaTibet2024 strain was found to be closely related to PPRVs isolated in China between 2013 and 2014. A base insertion and a base deletion were detected in the M gene 5' untranslated region. Results indicated that the prevalent PPRV strains in China did not show significant changes and that special attention should be paid to the surveillance of wild animals as an important part of PPR prevention and control.

Prevalence and genotyping of Toxoplasma gondii in questing Ixodes ricinus ticks from forest areas of Northern Poland.

Toxoplasma gondii occurs in a wide range of intermediate hosts, whose blood may be a meal for different tick species. A few studies have examined the role of ticks in the life cycle of T. gondii. This one includes the largest number and all stages of Ixodes ricinus collected from the widest area, covering seven recreational localities within a forest biotope in Northern Poland. This study aimed to determine the prevalence of T. gondii DNA in 2144 collected questing ticks to establish whether they may be involved in T. gondii life cycle. The additional goal was to genotype the detected T. gondii, as knowledge about its genotypes occurring in European ticks is insufficient. A further purpose was to detect coinfection with T. gondii and Borreliaceae in the collected ticks, as all of them have previously been tested for the presence of bacteria DNA. Nested PCR and sequencing of the obtained B1 gene fragment were conducted. T. gondii DNA was detected in 0.9% of all ticks (1.1% of nymphs and 0.7% of larvae). The presence of T. gondii in unfed larvae and nymphs may indicate the possibility of its vertical transmission. The prevalence of T. gondii DNA in ticks collected from individual sites was focal (0-4.3%) and seems to depend on local climatic conditions. Among all examined ticks, 0.3% were coinfected with T. gondii and Borreliella spp., vs. 0.6% of specimens with a single T. gondii infection. The obtained B1 sequences showed the greatest similarity (99.71-100%) to the sequence representing type III.

Blastocystis occurrence and subtype diversity in European wild boar (Sus scrofa) from the Iberian Peninsula.

The ongoing increase in wild boar populations across Europe has fostered human-wildlife conflicts, including the transmission of emerging pathogens with zoonotic importance. Blastocystis is a ubiquitous, faecal-oral transmitted protist that can cause gastrointestinal illnesses and is observed in humans and animals worldwide. The role of wildlife in the epidemiology of Blastocystis is insufficiently understood. Thus, we investigated the occurrence and subtype diversity of Blastocystis in free-ranging wild boars from the Iberian Peninsula using conventional PCR and next-generation amplicon sequencing of a fragment of the ssu RNA gene. A total of 459 wild boar faecal samples were collected across Spain (n = 360) and Portugal (n = 99) between 2014 and 2021. Blastocystis was present in 15.3% (70/459; 95% CI 12.1-18.9) of the wild boars analysed, and its occurrence was significantly higher in Portugal (34.3%, 34/99; 95% CI 25.1-44.6) than in Spain (10.0%, 36/360; 95% CI 7.1-13.6). Seven Blastocystis subtypes (ST5, ST10b, ST13-ST15, ST24b, and ST43) were detected among the surveyed wild boar populations, with greater variability detected in Portuguese samples. ST5 was identified in all the Blastocystis-positive animals, whereas 14.3% of them harboured ST mixed colonisations. Our results demonstrate that Blastocystis ST5 is particularly adapted to infect wild boars. The additional identification of zoonotic STs reinforces the role of wild boars as spreaders of zoonotic infections with public health significance.

Molecular genetic methods for quality control of inactivated vaccines using a Chikungunya virus model: vaccine strain identification and completeness of virus inactivation

INTRODUCTION. The completeness of virus inactivation and the identity of the vaccine strain are essential parameters for the safety and quality of inactivated virus vaccines, which should be controlled during vaccine development and production. Currently, the most promising quality control methods for inactivated virus vaccines are molecular genetic methods, which provide rapid results with high sensitivity and specificity.AIM. The aim of this study was the development of a real-time quantitative polymerase chain reaction (qPCR) method and an integrated cell culture real-time quantitative polymerase chain reaction (ICC-qPCR) method to assess the completeness of virus inactivation, as well as a reverse-transcription polymerase chain reaction assay coupled with restriction fragment length polymorphism analysis (RT-PCR-RFLP) to confirm the identity of the vaccine virus strain.MATERIALS AND METHODS. This study used RNA of CHIKV genotypes (three strains of each of the four CHIKV genotypes, including Asian, West African (WAf), and East/Central/South African (ECSA) genotypes, and the Indian Ocean Lineage of the ECSA genotype (ECSA-IOL), which were identified by sequencing prior to analysis). Additionally, the study used the Nika21 CHIKV strain (ECSA genotype), the Nika21 CHIKV strain inactivated with β-propiolactone, and the Nika21 CHIKV strain antigen adsorbed on aluminium hydroxide. The methods used included real-time qPCR, RT-PCR-RFLP, and virus neutralisation.RESULTS. The study identified a 218 bp fragment of the nsP1 gene (positions 789 to 1006) with restriction endonuclease recognition sites. These sites were present or absent in combinations specific to each of the four CHIKV genotypes. The authors selected primers for amplification of the specified gene region and tested the conditions for real-time qPCR and RT-PCR-RFLP. The study demonstrated the possibility of using the ICC-qPCR method to confirm the completeness of virus inactivation and the RT-PCR-RFLP method to identify the vaccine strain.CONCLUSIONS. The study showed the advantages of using the ICC-qPCR method to confirm the completeness of antigen inactivation and the RT-PCR-RFLP method to identify the vaccine strain. These methods are more sensitive and faster than traditional culture methods. ICC-qPCR and RT-PCR-RFLP can be used at any stage of the production process for inactivated vaccines.

Identification and genotyping of Chikungunya virus using reverse transcription polymerase chain reaction and restriction fragment length polymorphism methods

INTRODUCTION. Chikungunya virus (CHIKV) genotyping involves sequencing fractions of genes encoding E1, E2, and nsP1 proteins or the entire genome of the virus. Available reagent kits or polymerase chain reaction protocols cannot be used for CHIKV genotyping, and nucleic acid sequencing requires expensive equipment and materials, which are not always available. Therefore, it seems promising to use a simpler and more cost-effective restriction fragment length polymorphism (RFLP) method, which has not previously been used for CHIKV genotyping.AIM. This study aimed to investigate the possibility of using reverse transcription polymerase chain reaction (RT-PCR) and RFLP for CHIKV identification and genotyping.MATERIALS AND METHODS. The experimental study used RNA from CHIKV strains of four genotypes, including the Asian, West African (WAf), and East/Central/South African (ECSA) genotypes, and the Indian Ocean Lineage of the ECSA genotype (ECSA-IOL). The study used RT-PCR followed by DNA restriction and restriction fragment length analysis.RESULTS. The nsP2 gene fragment of 648 bp in length (positions 3806 to 4453) contains recognition sites for the restriction endonucleases PspEI, PvuII, and DraI. The presence or absence of these sites generates a different combination specific to each of the four CHIKV genotypes. The authors designed primers for amplification of the selected gene region and performed RTPCR and RFLP.CONCLUSIONS. The RFLP method can be used for rapid CHIKV identification and genotyping. The method provides results within a few hours and does not require high-tech equipment.

Recent Developments in Using Microneedle Patch Technology as a More Efficient Drug Delivery System for Treating Skin Photoaging.

Skin photoaging, resulting from prolonged exposure to ultraviolet (UV) radiation, is characterized by intricate biological changes involving oxidative damage and structural alterations. Despite an increasing demand for effective interventions, the current therapeutic options for treating skin photoaging are limited. We discovered through literature data search on PubMed that recent research has shifted its focus to the application of microneedle patches as an innovative approach to address this concern. Microneedle patches, serving as a novel transdermal delivery system, exhibit the potential to deliver bioactive substances such as cytokines, cellular vesicles, gene fragments and even alive algae to mitigate the effects of skin photoaging. This review aims to provide a comprehensive overview of recent advancements in research about utilizing microneedle patches for the treatment of skin photoaging and potential future directions in leveraging microneedle patches as clinical therapeutic agents for skin rejuvenation. Ultimately, we believe that microneedle patches have a broader application prospect in the fields of medical cosmetology and anti-photoaging.

Constructing phylogenetic trees for microbiome data analysis: A mini-review

As next-generation sequencing technologies advance rapidly and the cost of metagenomic sequencing continues to decrease, researchers now face an unprecedented volume of microbiome data. This surge has stimulated the development of scalable microbiome data analysis methods and necessitated the incorporation of phylogenetic information into microbiome analysis for improved accuracy. Tools for constructing phylogenetic trees from 16S rRNA sequencing data are well-established, as the highly conserved regions of the 16S gene are limited, simplifying the identification of marker genes. In contrast, metagenomic and whole genome shotgun (WGS) sequencing involve sequencing from random fragments of the entire gene, making identification of consistent marker genes challenging owing to the vast diversity of genomic regions, resulting in a scarcity of robust tools for constructing phylogenetic trees. Although bacterial sequence tree construction tools exist for upstream bioinformatics, many downstream researchers—those integrating these trees into statistical models or machine learning—are either unaware of these tools or find them difficult to use due to the steep learning curve of processing raw sequences. This is compounded by the fact that public datasets often lack phylogenetic trees, providing only abundance tables and taxonomic classifications. To address this, we present a comprehensive review of phylogenetic tree construction techniques for microbiome data (16S rRNA or whole-genome shotgun sequencing). We outline the strengths and limitations of current methods, offering expert insights and step-by-step guidance to make these tools more accessible and widely applicable in quantitative microbiome data analysis.

Frequency of chloroquine-resistant haplotype of Plasmodium falciparum (CVIET) in Ibadan, Southwest Nigeria 17 years post-chloroquine withdrawal

The replacement of chloroquine with artemisinin-based combination therapies (ACTs) for over a decade has had varying impacts on the ability of the malaria parasite to sustain its chloroquine resistance prowess in different malaria-endemic regions. We evaluated the frequency of Plasmodium falciparum chloroquine resistance transporter (PfCRT) mutations in Ibadan, Nigeria 17 years after the replacement of chloroquine with ACTs for malaria treatment. Fragments of PfCRT gene from genomic DNA of microscopically confirmed P. falciparum-infected patients were amplified and sequenced. There were 19% CVIET mutant and 81% CVMNK wild-type haplotypes on residues 72–76. A220S change were found in 16.7% of samples occurring concurrently with the CVIET haplotype, while a Q271E mutation occurred in a PfCRT wild-type isolate. The reduced prevalence of the PfCRT mutant alleles in this study compared to previous reports suggests a gradual disappearance of chloroquine-resistant malaria parasites following reduced drug pressure. It may also be a result of fitness demand on the parasites in attempts to evolve resistance against the current first-line regimen. However, evaluating the prevalence of other chloroquine resistance markers such as Plasmodium falciparum multidrug resistance 1 gene mutations in this population, and a more robust sample size will help to consolidate these findings.

Antimicrobial resistance profiles of and associated risk factors for Pseudomonas aeruginosa nosocomial infection among patients at two tertiary healthcare facilities in Lusaka and Copperbelt Provinces, Zambia.

Antimicrobial resistance (AMR) of pathogens such as Pseudomonas aeruginosa is among the top 10 threats to global health. However, clinical and molecular data are scarce in Zambia. We, therefore, evaluated the AMR profiles of P. aeruginosa nosocomial infections (NIs). A year-long hospital-based cross-sectional study was conducted at two large tertiary-level hospitals in Zambia. Patients with current or previous hospital contact were screened for NIs. The current study focused on patients diagnosed with P. aeruginosa NIs. Clinical specimens were collected for bacteriological culture, and PCR amplification of 16S rRNA gene fragments was performed on pure isolates. Hospital or NIs were defined as infections that arise during hospitalization, occurring at least 48 h after admission. The Kirby-Bauer's disk diffusion method was used to evaluate antibiotic resistance patterns. The association between AMR and risk factors was analysed using the χ2 test. Eight hundred and forty-one patients were screened, and clinical specimens were collected and analysed. Of them, 116 (13.7%) were diagnosed with P. aeruginosa NIs. The participants' ages ranged from 15 to 98 years, with a mean of 51 (SD ± 18). Catheter-associated urinary tract infections (57%) were the most common, followed by pressure sores (38.7%). P. aeruginosa isolates were primarily susceptible to amikacin, which had the highest resistance to FEP. We observed a high prevalence of multidrug resistance (73.6%). The AMR was associated with carbapenem-hydrolysing β-lactamase gene blaOXA-51 and surgical care. This study has demonstrated that multidrug-resistant P. aeruginosa is prevalent in hospitals in Zambia's Lusaka and Ndola districts and possibly countrywide.

Design of a portable bioassay system based on highly efficent nanozyme and Au-DNA nanocluster: Toward current/colorimetric dual-signal detection of sugarcane smut

Sugarcane smut is a major agricultural pest affecting sugarcane yield and quality, posing a significant threat to the sustainable and healthy development of the global sugar industry. The best strategy for controlling Sugarcane smut is early and precise diagnosis. An intelligent mobile bio-platform is constructed based on Au-Co/Zn ZIF nanozymes and Au-DNA nanoclusters for current/colorimetric dual signal detection of the sugarcane smut pathogen. Efficient and specific cycling of the specific gene fragment (bE4′) for Sugarcane smut is achieved by strand displacement reaction. The output strand then forms Au-DNA nanoclusters with AuNPs-DNA, and the bioconjugate is captured by H2 probe, which results in the formation of more double-stranded structures, causing methylene blue (MB) to adsorb on electrode. The anodic nanozyme efficiently catalyzes the oxidation of glucose, generating a large number of electrons, and MB in the system is therefore reduced to MBH, causing a color change. This results in distinct current/colorimetric dual output signals, significantly enhancing the accuracy. A capacitor is further coupled with the system to improve the sensitivity. The detection signals are read in real-time by a smartphone. The developed method for sugarcane smut genes shows good linearity within a concentration range of 0.0001–10000 pM, with a detection limit as low as 23.59 aM (S/N = 3). This work integrates advanced technologies from various fields to create a portable sensing device with extremely high sensitivity and precision for detecting sugarcane smut. It provides a reliable new option for early and precise diagnosis of the disease and offers valuable theoretical and technical insights for early prevention, intelligent detection, and real-time monitoring of other plant diseases.

Study on the role of RPL23 gene in active immunity of termite Reticulitermes chinensis against Metarhizium anisopliae

Ribosomal proteins are considered to be involved in the immunity of different animals against pathogens. The protein level of RPL23 increased after fungal infection in termites, but how it influence active immunity in termites is unknown. The role of RPL23 gene was studied to evaluate its impact on active immunity of termite Reticulitermes chinensis against entomopathogenic fungus (EPF) Metarhizium anisopliae. The RPL23 gene fragment (414 bp) was cloned and phylogenetic analysis revealed that it’s very close to termite Coptotermes formosanus. Expression of RPL23 gene was significantly higher in abdomen as compared to thorax and head. Silencing RPL23 gene had no significant impact on the frequency and time of allogrooming towards fungus exposed termites from nestmates, which showed that nestmates acquired spores from infected termites through allogrooming. Expression of immune genes (GNBP1, GNBP2 and phenoloxidase) and apoptosis related genes (TNF-α, caspase 1, caspase 3 and caspase 8) decreased significantly in nestmates of fungus-treated termites after silencing of RPL23 gene as compared to control. Antifungal activity and survival of RPL23 silenced nestmates of fungus-treated termites also decreased. To sum up, this study found that silencing of RPL23 gene broke the active immunity against M. anisopliae infection, reduced the antifungal activity of termites, weakened cell apoptosis, and led to increased mortality of termites, which may help to find a potential alternative for chemical insecticides to control termites.