Amplification Of Region Research Articles

Fungi Associated with Olive Tree (cv. ‘Nocellara del Belice’) Decay in Trapani Province (Sicily, Italy)

Recently, in several locations in the province of Trapani (Sicily, Italy), olive growers have reported cases of decaying olive trees of cv. ‘Nocellara del Belice’, showing symptoms of defoliation, branch drying, xylem browning, and reduced production. Internal symptoms include white and brown wood rot, starting from the base of the trunk. These alterations have been observed in trees irrigated using a pipe system at the trunk with spray sprinklers. To identify the causal agents of decay, some trees were eradicated and dissected, and woody samples were processed to isolate and identify the associated fungal micro-organisms. The most common colonies were identified using morphological (macro- and microscopical observation) and molecular (PCR amplification of the rDNA-ITS region) analyses. Nine fungal taxa were identified, of which four were associated with this decay syndrome (Coriolopsis gallica, Fomitiporia mediterranea, Kirschsteiniothelia sp., and Pleurostoma richardsiae), three were considered ubiquitous and opportunistic fungi (Alternaria spp., Aspergillus amstelodami, and Trichoderma sp.), and the other two were mycelia sterilia. Artificial inoculation satisfied Koch’s postulates, confirming the pathogenicity of the aforementioned fungi, even though the infections in the fields seem to be related to the irrigation system. This hypothesis would seem to be confirmed by the progression of decay over time in the trees subjected to the irrigation system described but not reported in olive groves differently managed. It is therefore considered appropriate to conduct further and more in-depth investigations aimed at studying the correlation between the irrigation system, presence of fungal agents, and manifestation of the syndrome. A further ongoing investigation is aimed at the use of biostimulants (Agrusaver, Savory Sun, VA LLC) on symptomatic trees, with the aim of both improving the vegetative performance of the host and limiting the symptoms detected in the field.

Design and shock isolation performances of two-layered ring-spring meta-isolation systems

Two-layered ring-spring-resonator (RSR) based meta-isolation system (RSR-M) has been developed to improve shock isolation effect. In the numerical calculations, shock acceleration ratio (SAR), shock displacement ratio (SDR) and relative displacement ratio (RDR) were introduced to evaluate the shock isolation performances of the conventional isolation system (CON) and the RSR-M, considering the effects of the excitation amplitude, the weight of the middle mass block and the stiffness of the lower isolators. The results demonstrate that the SAR of the RSR-M is always smaller than that of the CON, and the SDR of the RSR-M is first larger than that of CON in the shock amplification region and then smaller in the isolation region. The weight of middle mass blocks has little influence on the isolation performance of the RSR-M because of the softening behavior of the isolators. Finally, shock isolation experiments were also carried out to measure the damping ratios of the selected wire rope isolators and further analyze the isolation performance of the RSR-Ms. In general, meta-isolation system provides a new reference and idea for the design of the isolation system of engineering structures in some sense.

Optimal 16S rRNA gene amplicon sequencing analysis for oral microbiota to avoid the potential bias introduced by trimming length, primer, and database.

16S rRNA gene amplicon sequencing analysis is used to investigate bacterial communities; however, the estimated bacterial composition can differ from the original due to experimental and analytical biases. Therefore, this study determines the optimal conditions to minimize the potential biases in 16S rRNA gene amplicon sequencing analysis using different DNA samples, trimming lengths, primers, and databases. The results of the mock1 community with 250 and 300 bp paired-end (PE), comprising 15 bacteria from various environments, showed the highest similarity between the theoretical value and the data using the Greengenes2 and the V3 region at the genus level and V5-V6 at the species level. In the 300 bp PE sequencing analysis of the mock2 community, comprising six major oral bacteria, the data using the V3-V4, V4, and V5-V6 regions with the SILVA, Greengenes2, and the Human Oral Microbiome Database (HOMD) showed the highest similarity to the theoretical values at the genus level. At the species level, the data using the V3-V4 and V4 regions with Greengenes2 and the data using the V1-V2 with HOMD exhibited the highest alignment with the theoretical values. In the species analysis of the dental calculus samples with 300 bp PE, the Shannon index value was higher with the V1-V2 region and HOMD than with others. Our results suggest that the optimal conditions for oral microbiome analysis are the combinations of 300 bp PE and the V3-V4 or V4 region with the Greengenes2 and the V1-V2 with HOMD.IMPORTANCEThe 16S rRNA gene amplicon sequencing analysis is frequently used in oral microbiome research. However, this method can have biases that distort the experimental data, which depend on the methodological steps, including sequencing length, trimming length, selected amplification regions, and referenced databases. In this study, a combination of 300 bp PE and the primer targeting the V3-V4 or V4 regions with the Greengenes2 and the V1-V2 region with the HOMD was the most bias-minimizing condition for oral microbiota analysis. In addition, this is the first report of such analyses in modern Japanese dental calculus. The methods used in this study will aid in setting appropriate conditions for sequence analysis of microbiota obtained not only from the oral cavity but also from any environment.

Signaling Molecules in Pseudomonas aeruginosa Response to Antibiotics at Sub-Inhibitory Concentrations

Signaling Molecules are N-acylhomoserine lactones (AHLs) that form the Pseudomonas aeruginosa cell information system which determines gene expressions in a population dependent manner called quorum sensing (QS). Signal molecules, which are chemically varied, control genes expressions to antibiotics resistance, pathogenicity, motility, biofilm development, bioluminescence, secondary metabolite production, and plasmid transfer. This research was aimed to identify the signaling molecules in Pseudomonas aeruginosa response to antibiotics at sub-inhibitory concentrations. One hundred and fifty (150) clinical swab specimens were collected from urinary catheter wound and ear infection of patients and the swabs inoculated using standard microbiology method. The isolates were characterized based on the bacteriological methods such as morphology and biochemical tests. The isolates were further confirmed by species specific PCR by amplification of 16S rRNA and the amplicons were analyzed by gel electrophoresis; and further genomic sequencing was done and blast with NCBI database mining. Disc diffusion method was applied for the antibiotics susceptibility pattern. The isolates cell suspensions were analyzed by Gas Chromatography-Mass Spectrometry (GC-MS). The result of the isolation showed 22(59.46%) from wound infections, 12(32.43%) from ear infections and 3(8.11%) from urinary catheter. The isolates were Gram negative, produced β-hemolysis on blood agar and the morphology is small pigmented circular in form. The isolates showed positive result to catalase, oxidase, citrate, nitrate and indole tests. The amplification of 16S rRNA gene region resulted in the band size of 1500bp PCR product and the BLAST analysis gave 99% similarity. The resistant antibiotics susceptibility showed that Pseudomonas aeruginosa is multidrug resistant bacteria. The GC-MS results obtained from urinary catheter isolates showed four (4) signaling molecules such as N-Octanoyl-L-homoserine Lactone, N-Decanoyl-DL-Homoserine Lactone, N-Dodecanoyl-DL-Homoserine Lactone and N-Tetradecanoyl-L-Homoserine Lactone. Three (3) signaling molecules such as N-Octanoyl-L-homoserine Lactone, N-Decanoyl-DL-Homoserine Lactone and N-Tetradecanoyl-L-Homoserine Lactone were obtained from wound isolates. N-Tetradecanoyl-L-Homoserine Lactone was the only signaling molecule obtained from ear isolates. Further research should be done to develop rapid tests that could be possible to detect acyl homoserine specific to Pseudomonsa aeruginosa present to human samples and deliver results in real time. Signaling molecules inhibitors (SMI) are possible potential therapeutics that could produce the subsequent class of antibacterial drugs. The fundamental role of each signal molecule in the production of biofilms and virulent factors should also be investigated, as well as whether Pseudomonas aeruginosa needs one or more signaling molecules to form quorum sensing. Identification of the signals molecules (AHLs) of P. aeruginosa and developing signaling molecules inhibitors (SMI) can have an important prognostic, diagnostic and therapeutic value in human medicine for the treatment of P. aeruginosa infections.

Exploring the antifungal activity of clove oil (Syzygium aromaticum) against wilt disease caused by Fusarium equiseti in bitter gourd (Momordica charantia L.)

This study investigates the isolation and characterization of Fusarium equiseti, a fungal pathogen causing wilt in bitter gourd. It explores the antifungal activity of essential oils, particularly clove oil, as a natural management approach. The pathogen was isolated from wilt-infected bitter gourd stems and it was confirmed as F. equiseti morphologically by the presence of three-septate falcate macroconidia and globose microconidia and also confirmed molecularly by PCR amplification of the ITS region (PP501044.1). Among the tested essential oils, clove oil demonstrated the highest mycelial growth inhibition (100%) at a concentration of 0.5%, followed by peppermint, wintergreen and tea tree oils. In comparison, neem oil exhibited the least inhibition (15.54%). GC-MS analysis of clove oil revealed 38 compounds, where eugenol is the predominant compound that played a crucial role in antifungal activity. Additionally, metabolic analysis revealed several enriched pathways in F. equiseti during its interaction with clove oil, particularly those involved in lipid metabolism and energy production. Pot culture experiments confirmed the efficacy of clove oil in reducing disease severity and incidence in bitter gourd, achieving a 66.99 % reduction when combined with soil application.Furthermore, clove oil-treated plants showed increased activity of defense-related enzymes, including peroxidase (PO), polyphenol oxidase (PPO) and phenylalanine ammonia-lyase (PAL), suggesting the induction of systemic resistance. This study highlights the potential of clove oil as an eco-friendly alternative to synthetic fungicides for managing F. exquisite-induced wilt in bitter gourd. Future research should focus on optimizing clove oil application methods and understanding its interactions with fungal pathogens at the molecular level to enhance its efficacy in sustainable disease management.

Structural alterations of the EGFR gene in glioblastoma samples as a prognostic factor and molecular target for therapy

Introduction. Epidermal growth factor receptor (EGFR) is a transmembrane protein of the receptor tyrosine kinase family that is activated in various cancers (non-small cell lung cancer, colorectal cancer, head and neck tumors). In glial brain tumors, increased EGFR expression levels are characteristic of the most aggressive subtype, glioblastoma. Frequent structural changes of EGFR in glioblastoma are amplification of the chromosome region where the EGFR gene is located, point mutations, as well as deletion of exons 2–7 of the EGFR gene leading to the formation of EGFRvIII transcript.Aim. To determine structural changes of the EGFR gene (point mutations and amplification of the EGFR gene, EGFRvIII transcript) in tumor samples using different methods and to evaluate their potential clinical significance.Materials and methods. The study included 75 patients with brain gliomas (70 of them glioblastoma) aged 34 to 78 years (mean age 56 years). DNA and RNA isolation was performed from fresh frozen tumor tissue, as well as from peripheral blood leukocytes. EGFR gene mutations were determined by next-generation sequencing (NGS), and β allele frequency (BAF) comparative analysis (normal-tumor) was performed to determine the copy number of chromosome 7 regions. Quantitative polymerase chain reaction was used to confirm the EGFR gene amplification in tumor samples, and reverse transcription-PCR was used to detect EGFRvIII variant.Results. The NGS method revealed 11/70 (16 %) mutations in coding regions of EGFR gene in glioblastoma samples, the EGFR gene amplification was detected in 26/70 (37 %) cases; no structural changes of the EGFR gene were detected in 5 glioma samples (astrocytoma, oligodendroglioma). All cases of EGFR gene amplification detected by NGS were confirmed by quantitative polymerase chain reaction. To search for EGFRvIII transcript, 31 tumor RNA samples were examined, of which EGFR amplification was present in 12 samples. EGFRvIII transcript was detected only in samples with EGFR gene amplification – 4/12 (33 %). To assess the clinical significance of structural gene alterations, the frequency of occurrence in primary and recurrent glioblastoma samples was compared.Conclusion. The NGS method allows to detect both point mutations and amplification of the EGFR gene. The EGFR gene amplification was associated with EGFRvIII mutation in 33 % of cases. No statistically significant differences in the frequency of structural changes in the EGFR gene between primary and relapsed glioblastomas were found.

Sequentially Activated-Dumbbell DNA Nanodevices for Accurate Detection of Uracil-DNA Glycosylase via PER-Based Orthogonal Signal Outputs.

Accurate and reliable detection of uracil-DNA glycosylase (UDG) activity is crucial for clinical diagnosis and prognosis assessment. However, current techniques for accurately monitoring UDG activity still face significant challenges due to the single input or output signal modes. Here, we develop a sequentially activated-dumbbell DNA nanodevice (SEAD) that enables precise and reliable evaluation of UDG activity through primer exchange reactions (PER)-based orthogonal signal output. The SEAD incorporates a double-hairpin structure with a stem containing two deoxyuridine (dU) sites for target recognition and two preblocked primer binding regions for target amplification and signal output. Upon UDG recognition of dU, the SEAD can be cleaved by apurinic/apyrimidinic endonuclease 1 (APE1), generating two different hairpins with exposed primer binding regions. These hairpins serve as templates to initiate the parallel PER, enabling the extending of two different amplification products: a long single-stranded DNA (ssDNA) with repetitive sequences and a short ferrocene-labeled ssDNA with complementary sequences. These products further self-assemble into DNA nano-strings in an orthogonal manner that act as an electrochemiluminescence signal switch, enabling precise detection of low-abundance UDG. This work develops a sequential input and orthogonal output strategy for accurately monitoring UDG activity, highlighting the significant potential in cancer diagnosis and treatment.

Conserved allomorphs of MR1 drive the specificity of MR1-restricted TCRs.

Major histocompatibility complex class-1-related protein (MR1), unlike human leukocyte antigen (HLA) class-1, was until recently considered to be monomorphic. MR1 presents metabolites in the context of host responses to bacterial infection. MR1-restricted TCRs specific to tumor cells have been described, raising interest in their potential therapeutic application for cancer treatment. The diversity of MR1-ligand biology has broadened with the observation that single nucleotide variants (SNVs) exist within MR1 and that allelic variants can impact host immunity. The TCR from a MR1-restricted T-cell clone, MC.7.G5, with reported cancer specificity and pan-cancer activity, was cloned and expressed in Jurkat E6.1 TCRαβ- β2M- CD8+ NF-κB:CFP NFAT:eGFP AP-1:mCherry cells or in human donor T cells. Functional activity of 7G5.TCR-T was demonstrated using cytotoxicity assays and by measuring cytokine release after co-culture with cancer cell lines with or without loading of previously described MR1 ligands. MR1 allele sequencing was undertaken after the amplification of the MR1 gene region by PCR. In vivo studies were undertaken at Labcorp Drug Development (Ann Arbor, MI, USA) or Epistem Ltd (Manchester, UK). The TCR cloned from MC.7.G5 retained MR1-restricted functional cytotoxicity as 7G5.TCR-T. However, activity was not pan-cancer, as initially reported with the clone MC.7.G5. Recognition was restricted to cells expressing a SNV of MR1 (MR1*04) and was not cancer-specific. 7G5.TCR-T and 7G5-like TCR-T cells reacted to both cancer and healthy cells endogenously expressing MR1*04 SNVs, which encode R9H and H17R substitutions. This allelic specificity could be overcome by expressing supraphysiological levels of the wild-type MR1 (MR1*01) in cell lines. Healthy individuals harbor T cells reactive to MR1 variants displaying self-ligands expressed in cancer and benign tissues. Described "cancer-specific" MR1-restricted TCRs need further validation, covering conserved allomorphs of MR1. Ligands require identification to ensure targeting MR1 is restricted to those specific to cancer and not normal tissues. For the wider field of immunology and transplant biology, the observation that MR1*04 may behave as an alloantigen warrants further study. .

PoxtA amplification and mutations in 23S rRNA confer enhanced linezolid resistance in Enterococcus faecalis.

This study aimed to explore the evolutionary patterns and resistance mechanisms of an Enterococcus faecalis strain harbouring poxtA under linezolid exposure. A poxtA-carrying E. faecalis electrotransformant DJH702 with a linezolid minimum inhibitory concentration of 4 mg/L was exposed to increasing concentrations of linezolid (8-64 mg/L). The derived strains growing at 8, 16, 32 and 64 mg/L, designed DJH702_8, DJH702_16, DJH702_32 and DJH702_64, were obtained. The amplification and overexpression of poxtA were measured using sequencing and RT-PCR, the fitness cost by competition assays and the stability of the repeat units by serial passage. In all derived strains, high-level linezolid resistance develops through poxtA amplification. The relative copy numbers and transcription levels of poxtA were significantly increased. However, in the presence of higher linezolid concentrations, DJH702_32 and DJH702_64 showed reduced poxtA copy numbers and transcription levels compared with DJH702_8 and DJH702_16, but additional mutations in the 23S rRNA (G2505A). IS1216E-mediated formation of translocatable units with subsequent tandem amplification of these translocatable units supported the gain of poxtA segments. However, these amplicons were not stable and were lost frequently in the absence of a linezolid selection pressure. The amplification of the poxtA region did not result in a fitness cost, but mutations in 23S rRNA did. poxtA-carrying E. faecalis electrotransformants used two distinct mechanisms to resist linezolid selection pressure: at lower concentrations, strains prioritized increasing poxtA expression levels, while at higher concentrations, a combination of increased poxtA expression and mutations in 23S rRNA was observed.

Genomic Association and Prediction Study for Yield Traits in a Sugarcane (Saccharum spp. Hybrids) Mapping Population ‘LCP 85‐384’

ABSTRACTSugarcane (Saccharum spp. hybrids) are complex polyploid and aneuploid interspecific hybrids with 110–130 chromosomes. A traditional sugarcane breeding cycle takes 13–15 years and involves multiple years and locations testing of yield. To identify molecular markers associated with yield‐related traits, the LCP 85‐384 cultivar and its mapping population of 263 self‐progenies were planted in two randomly replicated field plots. The mapping population was genotyped with amplified fragment length polymorphism (AFLP), simple sequence repeats (SSR) and target region amplification polymorphism (TRAP) markers. Data on plant height, stalk number, stalk diameter and stalk weight were collected. A large variation was observed for each trait. A genome‐wide association study (GWAS) was conducted using mixed linear model (MLM), generalized linear model (GLM) and single marker regression (SMR) programmes of TASSEL 5 and FarmCPU of GAPIT 3. A total of 64 yield trait‐associated alleles were identified, including 11 for stalk number, 36 for stalk weight, 21 for stalk diameter and 5 for plant height. Of the 64 alleles, seven were linked to two traits and one to three traits. Genomic prediction (GP) was also performed by cross‐prediction with five models, namely, ridge regression best linear unbiased prediction (rrBLUP), Bayesian ridge regression (BRR), Bayesian A (BA), Bayesian B (BB), and Bayesian least absolute shrinkage and selection operator (BL). Prediction accuracy (r value) reached 0.40 for plant height, 0.36 for stalk number, 0.44 for stalk diameter and 0.54 for stalk weight with the standard errors from 0.009 to 0.012. Once verified, these markers will be a valuable tool to aid in the selection of yield‐related traits in sugarcane improvement programmes.

Comparative analysis of size-fractional eukaryotic microbes in subtropical riverine systems inferred from 18S rRNA gene V4 and V9 regions

Eukaryotic microbes play key ecological roles in riverine ecosystems. Amplicon sequencing has greatly facilitated the identification and characterization of eukaryotic microbial communities. Currently, 18S rRNA gene V4 and V9 hypervariable regions are widely used for sequencing eukaryotic microbes. Identifying optimal regions for the profiling of size-fractional eukaryotic microbial communities is critical for microbial ecological studies. In this study, we spanned three rivers with typical natural-human influenced transition gradients to evaluate the performance of the 18S rRNA gene V4 and V9 hypervariable regions for sequencing size-fractional eukaryotic microbes (>180 μm, 20-180 μm, 5-20 μm, 3-5 μm, 0.8-3 μm). Our comparative analysis revealed that amplicon results depend on the specific species and microbial size. The V9 region was most effective for detecting a broad taxonomic range of species. The V4 region was superior to the V9 region for the identification of microbes in the minor 3 μm and at the family and genus levels, especially for specific microbial groups, such as Labyrinthulomycetes. However, the V9 region was more effective for studies of diverse eukaryotic groups, including Archamoebae, Heterolobosea, and Microsporidia, and various algae, such as Haptophyta, Florideophycidae, and Bangiales. Our results highlight the importance of accounting for potential misclassifications when employing both V4 and V9 regions for the identification of microbial sequences. The use of optimal regions for amplification could enhance the utility of amplicon sequencing in environmental studies. The insights gained from this work will aid future studies that employ amplicon-based identification approaches for the characterization of eukaryotic microbial communities and contribute to our understanding of microbial ecology within aquatic systems.

The role of human papillomavirus in laryngeal carcinoma using PCR

<b>Introduction:</b> The etiology of laryngeal carcinoma is multifactorial. Smoking and alcoholism are well-known risk factors; however, recently oncogenic human papillomavirus has been suggested to promote carcinogenesis. PCR has had the most dramatic impact on molecular biology, enabling the amplification of specific regions of interest and detection of gene sequences.<b>Aim:</b> The aims are to evaluate the possible relationship between laryngeal carcinoma and human papillomavirus and its impact on the socio-demographic findings and the clinicopathological presentation.<b>Material and method:</b> Twenty-five patients with laryngeal carcinoma were included. Two biopsies were taken from every patient, the first from the main bulk of the tumor, the second from the normal surrounding mucosal tissues and considered as a control group. Virus detection was done by PCR test. The HPV screen test is an <i>in vitro</i> nucleic acid amplification test for qualitative detection of HPV type (16, 18, 31, 33, 35, 39, 45, 52, 53, 56, 58, 59, 66, and 70) and β-globin gene used as internal control. Samples were transferred into the thermal cycler, in which denaturation, annealing, and chain elongation occurred. Specific bands of HPV were detected using a high-resolution Cannon camera.<b>Results:</b> The average age of the patients was 60.8 years. Twenty-three patients (92%) were males. Viral DNA was detected in 8% of patients, representing genotypes (16, 18, 31, 33, 35, 39, 45, 52, 53, 56, 58, 59, 66, and 70). There is no statistically significant relationship between the presence of the virus and epidemiological and clinic-pathological features except for female gender distribution (P-value = 0.022).<b>Conclusions:</b> There is no significant relationship between laryngeal carcinoma and human papillomavirus, nor a significant impact on socio-demographic findings and clinicopathological presentation.

Identification of HPV16 Lineages in South African and Mozambican Women with Normal and Abnormal Cervical Cytology.

Human papillomavirus 16 (HPV16) is an oncogenic virus responsible for the majority of invasive cervical cancer cases worldwide. Due to genetic modifications, some variants are more oncogenic than others. We analysed the HPV16 phylogeny in HPV16-positive cervical Desoxyribonucleic Acid (DNA) samples collected from South African and Mozambican women to detect the circulating lineages. Polymerase chain reaction (PCR) amplification of the long control region (LCR) and 300 nucleotides of the E6 region was performed using HPV16-specific primers on HPV16-positive cervical samples collected in women from South Africa and Mozambique. HPV16 sequences were obtained through Next Generation Sequencing (NGS) methods. Geneious prime and MEGA 11 software were used to align the sequences to 16 HPV16 reference sequences, gathering the A, B, C, and D lineages and generating the phylogenetic tree. Single nucleotide polymorphisms (SNPs) in the LCR and E6 regions were analysed and the phylogenetic tree was generated using Geneious Prime software. Fifty-eight sequences were analysed. Of these sequences, 79% (46/58) were from women who had abnormal cervical cytology. Fifteen SNPs in the LCR and eight in the E6 region were found to be the most common in all sequences. The phylogenetic analysis determined that 45% of the isolates belonged to the A1 sublineage (European variant), 34% belonged to the C1 sublineage (African 1 variant), 16% belonged to the B1 and B2 sublineage (African 2 variant), two isolates belonged to the D1-3 sublineages (Asian-American variant), and one to the North American variant. The African and European HPV16 variants were the most common circulating lineages in South African and Mozambican women. A high-grade squamous intraepithelial lesion (HSIL) was the most common cervical abnormality observed and linked to European and African lineages. These findings may contribute to understanding molecular HPV16 epidemiology in South Africa and Mozambique.

DNA Sequencing Accurately Diagnosed 146 Cases of Superficial Mycosis

Objective: Traditional fungal detection methods, such as fungal microscopy and cultivation, often have drawbacks such as high false negative rates and time-consuming cultivation. Using molecular biology methods for diagnosis can not only be used for identifying fungal strains in cultured colonies, but also for diagnosing diseased tissues, which can shorten the diagnosis time. There is a lack of systematic research on the clinical characteristics, susceptibility factors, and the composition and distribution of pathogenic fungi of superficial mycosis in Shiyan area. In order to understand the relevant situation of superficial mycosis and pathogenic fungi in this area, this study conducted a molecular epidemiological investigation on 146 patients with superficial mycosis who visited our outpatient department. Methods: From January 2022 to December 2022, the typical clinical manifestations of outpatient visits in our department were collected. 146 cases of superficial fungal patients with positive fungal microscopy were cultured and DNA was extracted. PCR technology was applied to compare the products in GeneBank after the amplification of ITS region. Results: A total of 23 pathogenic strains were obtained, including 112 strains of <i>Trichophyton rubrum</i> (76.71%), 5 strains of other dermatophytes (3.42%), 6 strains of <i>Candida</i> species (4.11%), 4 strains of <i>Aspergillus</i> species (2.74%), 8 strains of <i>Cladosporium</i> species (5.48%), and 11 strains of other fungi (7.53%). Conclusions: DNA sequencing combined with traditional fungal microscopic culture is helpful for more accurate diagnosis of superficial mycosis.

Genomic profiles and clinical presentation of chordoma

Chordoma is a rare bone cancer with variable clinical outcomes. Here, we recruited 184 sporadic chordoma patients from the US and Canada and collected their clinical and treatment data. The average age at diagnosis was 45.5 years (Range 5–78) and the chordoma site distribution was 49.2% clivus, 26.2% spinal, and 24.0% sacral. Most patients (97.5%) received surgery as the primary treatment, among whom 85.3% also received additional treatment. Except for the most prevalent cancers like prostate, lung, breast, and skin cancer, there was no discernible enrichment for any specific cancer type among patients or their family members. Among a subset of patients (N = 70) with tumor materials, we conducted omics analyses and obtained targeted panel sequencing and SNP array genotyping data for 51 and 49 patients, respectively. The most recurrent somatic driver mutations included PIK3CA (12%), followed by chromatin remodeling genes PBRM1 and SETD2. Amplification of the 6q27 region, containing the chordoma susceptibility gene TBXT, was detected in eight patients (16.3%). Clival patients appeared to be less likely to carry driver gene mutations, chromosome arm level deletion events (e.g., 5p, 5p, and 9p), or 6q27 amplification compared to sacral patients. After adjusting for age, sex, tumor site, and additional treatment, patients with somatic deletions of 14q (OR = 13.73, 95% CI 1.96–96.02, P = 0.008) and 18p (OR = 13.68, 95% CI 1.77–105.89, P = 0.012) were more likely to have persistent chordoma. The study highlights genomic heterogeneity in chordoma, potentially linked to location and clinical progression.

Nonlinear dynamics of inverse tapered granular chains for mechanical amplification in voice frequency range.

Here, the dynamics of inverse tapered granular chains, which are alignments of beads with progressively increasing radii, are investigated. These chains are subjected to harmonic excitation by a moving wall and probed at the tapered end. Intermediate taperings using Ni-Ti (metal) and Teflon (plastic) granules are considered. The proposed chain is based on the frequency responses. The incident signal is set between 1 Hz and 3 kHz. The system transmits or filters incident signals at low or high driving frequencies, respectively. For intermediate frequencies, similar to the human voice and most musical instruments, the driving frequency is amplified, presenting a median gain of 24 dB (an increase in energy by a factor of 250) over long durations. A transition map, showing regions of transmission, amplification, and filtration as a function of driving amplitude and frequency, is provided. This system could serve as a starting point for studies on natural voice amplifiers, which are important for addressing hearing loss and vocal disorders, as well as other applications requiring mechanical signal amplification. The findings suggest that a proper granular arrangement could enable the amplification of low or extremely low driving frequencies.

Genotype classification and pathogenicity of infectious bursal disease virus circulating in vaccinated broiler chicken farms

This study investigated the genotype classification and pathogenicity of infectious bursal disease virus (IBDV) circulating in vaccinated broiler chicken farms in Egypt. A total of 150 samples were collected from 30 vaccinated commercial broiler chicken farms and pooled into 30 working samples. IBDV was tested using reverse transcriptase polymerase chain reaction (RT-PCR) amplification of the hypervariable region of the viral protein 2 (hvVP2) and the VP1 gene 5’ extremity. Both RT-PCR fragments were sequenced from six samples, and then the obtained nucleotide sequences were analyzed. The IBDV genotypes were identified using nucleotide sequences. Five sequences of the six strains examined were classified as genotype A3B2 for the highly virulent segments A and B (vv-A/vv-B IBDV). Interestingly, this study identified and classified a novel segment-reassortant strain as the A1B2 genotype. Specifically, it involved the segment reassortment of classical virulent segment A (cv-A) with vv-B producing cv-A/vv-B reassortant IBDV. Subsequently, we compared the pathogenicity of reassortant (cv-A/vv-B) IBDV and vvIBDV strains identified in this study. Both strains developed typical IBD clinical signs, postmortem lesions, histopathology, immunohistochemistry, and lesion scores, which were more severe in vvIBDV than reassortant IBDV. In conclusion, this is the first report of the genotype classification based on both genome segments (hvVP2 and VP1) with pathogenicity of IBDV circulating in vaccinated broiler chicken farms and this pathogenicity is more severe in vvIBDV strain than a novel reassortant IBDV strain.

Tilletia laevis caused common bunt disease of wheat in district Swat, Khyber Pakhtunkhwa-Pakistan

Wheat common bunt caused by Tilletia laevis (Kühn) and T. caries (Bjerk.) Wint., is one of the world's most destructive diseases (He et al., 2022). In June 2022, we observed wheat common bunt symptoms in ten fields of city Mingora in Swat District, Khyber Pakhtunkhwa, Pakistan (35.2227°N, 72.4258°E) with spring cultivar Galaxy 2013. The corresponding disease heads were collected to isolate and characterized the causal agent of wheat common bunt. Teliospores morphology, germination, and disease symptoms revealed that fungal isolates belong to Tilletia laevis, which cause common bunt disease of wheat. After Koch's postulates, the artificially inoculated plants showed similar symptoms to those observed in the fields with same teliospores morphology and germination. Tilletia laevis was also confirmed through molecular characterization using sequence characterized amplification region (SCAR) markers specific for T. laevis and obtained 660 bp bands from leaves, stem and heads from artificially inoculated plants. Therefore, to the best of our knowledge, this is the first report of T. laevis causing common bunt in Mingora, Swat, Khyber Pakhtunkhwa, Pakistan.

Timber DNA release using focused ultrasound extraction (FUSE) for genetic species identification

The use of genetic data for timber species and population assignment is a powerful tool for combating the illegal timber trade, but the challenges of extracting DNA from timber have prevented the routine use of genetics as a supply chain management tool. To overcome these challenges, we explored the feasibility of focused ultrasound extraction (FUSE) for rapid DNA release from timber. Using high-pressure ultrasound pulses, FUSE generates a cavitation bubble cloud that disintegrates samples into acellular debris, resulting in the mechanical release of DNA. In this work, FUSE was applied to white oak (Quercus alba) timber shavings to test the feasibility of using FUSE for timber DNA extraction for the first time. Results showed that FUSE processing disintegrated the tissue samples and released significant quantities of DNA. After five minutes of tissue processing DNA quantities of 0.21 ± 0.02 ng/mg, 0.99 ± 0.32 ng/mg, and 0.14 ± 0.01 ng/mg, were released from medium, coarse, and combination shaving groups, respectively. Amplification and sequencing of regions within the matK and rbcL chloroplast genes confirmed that the quality of DNA prepared with FUSE was suitable for PCR and short-read sequencing applications. Overall, these results show that FUSE can serve as a DNA sample preparation method capable of releasing high-quality DNA from timber in a fraction of the time required by conventional extraction methods. Based on the improved efficiency of DNA release with FUSE, ongoing work aims to develop this technology into portable systems that can be used to rapidly prepare timber samples for genetic species identification.

Phylogenetic and Evolutionary Analysis of Porcine Epidemic Diarrhea Virus in Guangxi Province, China, during 2020 and 2024.

The variant porcine epidemic diarrhea virus (PEDV) has caused considerable economic losses to the global pig industry since 2010. In this study, a total of 5859 diarrhea samples were collected from different pig farms in China's Guangxi province during January 2020 and March 2024 and tested for PEDV using RT-qPCR. The positivity rate of PEDV was 11.90% (697/5859). Ninety-two PEDV-positive samples were selected based on sampling time, and the sampling region for amplification, sequencing, and analysis of the S1, M, and N genes. Phylogenetic analysis of the S1 gene revealed that all strains from Guangxi province were distributed in three subgroups, i.e., 81.5% (75/92) in the G2a subgroup, 4.3% (4/92) in the G2b subgroup, and 14.1% (13/92) in the G2c subgroup. The sequence analysis revealed that the S1 gene sequences from Guangxi province had higher homology with the variant strains than with the classical strains, showing as high as 99.2% with the variant strain AJ1102 and only 94.3% with the classical strain CV777. Recombination analysis revealed that the GX-BS08-2023 strain (G2c) from Guangxi province originated from inter-lineage recombination between the GX-BS09-2023 (G2a) and CH-JN547228-2011 (G1a) strains. In addition, the S1 gene of the G2a and G2b subgroup strains shared many mutations and insertions. There were common mutations of N143D and P235L in the G2a subgroup. Evolutionary analysis revealed that all Guangxi strains belonged to the G2 genotype. These strains have spread rapidly since the PEDV variant strains that emerged in 2010, weakened until 2021, and then remained stable. In conclusion, the results revealed the latest genetic evolution of circulating PEDV strains in Guangxi province in recent years, providing important information for preventing and controlling PEDV infection. Currently, the G2a subgroup strains are the predominant strains circulating in pig herds in Guangxi province, southern China.