Molecular Biology Reports

Aibika (Abelmoschus manihot) is a tropical leafy vegetable with great potential in the prevention of malnutrition. Its high fibre and micronutrient content makes it a valuable complement to staple foods such as rice and starchy crops consumed in South Asia and the Pacific. The present study is the first to report the development of a set of 21 nuclear single sequence repeat (SSR) markers of A. manihot from genomic sequence data obtained using NGS technology. The DNA library was prepared from a pool of four aibika accessions and sequenced using an Illumina MiSeq system. A total of 1,295,217 pair-end reads were generated. Raw data are available in the European Nucleotide Archive (accession number PRJEB88210; https://www.ebi.ac.uk/ena/browser/view/PRJEB88210). The sequences were assembled using the ABySS software. In total 651,320 contigs were generated. Using MISA Perl script and Primer3 software, we identified 8014 SSR motifs, of which 4637 had a suitable primer design. The contigs were blasted with NCBI-BLAST on okra reference genome. After selection based on motif type, repeat length, amplicon size and occurrence blast, we retained 91 candidate SSR loci, which were tested on 23 aibika accessions. Finally, we validated 21 high quality SSR loci by genotyping 45 accessions from three Pacific countries. The number of alleles per locus ranged from 3 to 21 with an average of 7.81 alleles/locus. The 21 selected SSRs were found to be useful in discriminating between accessions and revealing the diversity structure of A. manihot. They will help to optimize genebanks management and breeding programmes, and guide future collection activities.

Acute promyelocytic leukemia is a distinct subtype of acute myeloid leukemia characterized by the t(15;17) translocation, leading to the PML (Promyelocytic leukemia protein)-RARA (Retinoic Acid Receptor Alpha) fusion protein. Although PML-RARA fusion is common, there are 20 more fusion events also reported in APL. All -trans retinoic acid (ATRA) is a standard drug for APL, leading to significant improvement in patient outcomes; nevertheless, a small fraction of patients still experience relapse, and some patients exhibit resistance to the drug. Long non-coding RNAs (LncRNAs) are recognized as promising biomarkers for cancer diagnosis, prognosis, and treatment response. In this study, we used ATRA-Resistant (AP1060) and ATRA -Sensitive (NB4), both treated and untreated cell line transcriptomic data retrieved from the NCBI Gene Expression Omnibus(GEO) database to perform transcriptomic analysis with bioinformatic tools. We utilized the LncRAnalyzer pipeline to predict the lncRNAs, followed by differential expression analysis using DESeq2. Weighted Gene Co-expression Network Analysis (WGCNA) was employed to construct lncRNA co-expression modules associated with ATRA resistance. BEDTools is used to identify cis-acting target genes of lncRNAs.LncRNA -miRNA sponging identified by miRanda algorithm. The identified miRNAs reveal their significant role in APL and other leukemia subtypes. The results of the study show that the identified lncRNAs from the miRNA-LncRNA network are promising biomarkers for ATRA resistance.

Precision-cut lung slices (PCLS) have emerged as a powerful tool for studying the biology of viable human lung tissue. However, the presence of agarose impurities compromises RNA yield and integrity during the extraction process. We tested whether using an alternative Plant kit RNA extraction method to wash agarose impurities or pre-dissolving agarose from PCLS implementing a dissolving buffer for routine RNA isolation in gel-electrophoresis would improve RNA quantity, quality, and integrity. Our results show that RNA quantity and integrity are highly compromised when using a conventional method of RNA extraction. The plant kit and dissolution of agarose increased the RNA quantity to 0.42{+/-}0.11 and 0.65{+/-}0.17 {micro}g/PCLS (measured by the Qubit) and integrity number to 6.60{+/-}0.59 and 9.13{+/-}0.39 (measured by the Bioanalyzer), respectively. The presence of impurities in conventional and Plant kit extractions misled to an overestimation of the RNA quantity and quality using the NanoDrop. The Plant kit and agarose dissolution showed a significant transcript integrity increase in GUSB (p<0.0001) and COL1A1 (p<0.05) expression, validating these methods over conventional extraction. We encourage laboratories applying PCLS experimentation to implement alternative methods to remove agarose impurities during RNA extraction, as well as to rely on sensitive quantitative techniques, such as the Qubit and Bioanalyzer, for RNA quantification and integrity measurements.

T/A cloning is a popular method for generating recombinant DNA plasmids. This method relies on single A:T nucleotide base pairs between PCR product ends and vector. Theoretically, the directionality of insert ligation with relation to the vector is random. However, we have continuously observed directionality bias using the pGEM-T Vector System for T/A cloning in a Course-based Undergraduate Research Experience (CURE). Cloning of over 400 inserts has shown directional bias higher than 74% (p-value < 0.0005) "sense" to the T7 promoter of the vector. Awareness of biased insertion in our applications reduces time and cost in cloning and downstream analyses.

BackgroundDDX53 (DEAD-box helicase 53, known also as CAGE) is an intronless gene on the X chromosome, which expression shows strong testis specificity. It belongs to the group of cancer-testis (CT) antigens, with most studies to date focusing on its role in cancer, but the precise biological function of DDX53 remains unclear. Previous reports identifying rare DDX53 variants in infertile men provided the rationale for investigating the role of DDX53 in the context of human spermatogenesis. By using the human seminoma cell line (TCam-2) as an in vitro male germline model, we aimed to investigate the function and molecular targets of DDX53. MethodsIn our study, we used transcriptomic and proteomic approaches (RNA sequencing (RNA-seq), enhanced crosslinking and immunoprecipitation (eCLIP), and Co-immunoprecipitation coupled with Mass Spectrometry (Co-IP-MS)) to investigate the role of DDX53 in the context of human spermatogenesis. By using modified TCam-2 cells to express either DDX53-FLAG or GFP-FLAG, we identified regulated genes, RNA targets, and potential protein interactors of DDX53. In addition, we employed Western Blot, RT-qPCR, immunostaining, and confocal microscopy to gain deeper insight into the DDX53 protein. ResultsOur RNA-seq and eCLIP data provide evidence that DDX53 regulates gene expression changes and directly interacts with a broad spectrum of RNA transcripts. Moreover, for the first time, we described RNAs and protein interactors of DDX53 in the context of spermatogenesis. Subcellular localization analysis by confocal microscopy indicated a predominantly cytoplasmic distribution of DDX53, with partial nuclear presence in TCam-2 cells. We also identified DDX53-positive structures that may correspond to germ granule-like assemblies, although their precise nature remains to be determined. Additionally, we confirmed DDX53 presence in human testis using a specific, commercially available anti-DDX53 antibody. ConclusionsThis studys data indicate that DDX53 protein acts as a regulator of RNA metabolism in human cells. Collectively, we show that it participates in transcriptome regulation (including splicing) in male germ cells and exhibits transcriptome-wide RNA interactions, but its wider biological role remains to be clarified.

BackgroundIn acute myeloid leukemia studies, event-free survival (EFS) is defined as time until treatment failure, relapse, or death, whichever occurs first. Since 2020 and 2022, respectively, the US Food and Drug Administration and the European LeukemiaNet recommend analysing treatment failures as day-1 events. This data modification can lead to a potentially large drop in the estimated EFS at day 1. If censoring occurs, the Kaplan-Meier estimator obtained from the recoded data underestimates this drop. Our aim is to obtain an unbiased estimate for EFS as basis for further inference. MethodsWe define "event on day 1" as one event type and " event after day 1" as a competing event in the original data and use the Aalen-Johansen estimator of the cumulative incidence curve to estimate event-specific transition probabilities, which are combined in one EFS estimate. To analyse effects on day 1 treatment failure and other post-day-1 EFS events separately, a formal link to cure models is established by equating treatment failures with the "cured" proportion in cure model terminology. Additionally, a variance estimator, confidence intervals, confidence bands, and simultaneous testing procedures are derived. ResultsOur new estimation method differs from the Kaplan-Meier estimator in settings in which some treatment failures are censored, as in the interim analysis of the AMLSG 09-09 study. If almost no treatment failures are censored, the two estimation methods do not differ. The cure model and simultaneous testing are able to estimate effects on day 1 treatment failure and other post-day-1 EFS events separately and function independently of whether data is modified. ConclusionsThe Kaplan-Meier estimator evaluated on the recoded data underestimates the drop at day 1 if treatment failures are censored. With sufficient follow-up, this bias disappears, and results coincide with our novel approach.

Heavy metal ATPases (HMAs) are important group of transmembrane proteins involved in homeostasis of metal ions in plant systems. In this study, a comprehensive analysis of genome assembly (VC1973A v7.1) resulted in the identification of nine HMA genes (VrHMA) and their corresponding proteins in Mungbean, an agronomically important legume crop known for its nutritional values. VrHMA proteins were also characterized based on their biomolecular features, conserved domains and motifs arrangement, transmembrane helices, pore-line helices, subcellular location and occurrence of signal peptides. Based on sequence homology, nine VrHMAs were clustered into two major substrate-specific groups: VrHMA1, VrHMA5 and VrHMA7 were categorized under the Zn/Co/Cd/Pb ATPase group, whereas the remaining six VrHMAs belong to the Cu/Ag subgroup. Gene structure analysis and promoter scanning revealed the structural divergence and presence of various stress-responsive cis-acting elements, respectively. The expression analysis of VrHMA genes in root and leaf tissues, in response to heavy metal (Zn, Cd and Cu) stress, indicates their role in the uptake, transport and sequestration of metal ions. Interestingly, VrHMA5 showed incremental upregulation in roots in response to all three heavy metal stresses, whereas its expression was only upregulated in the leaf tissues under Zn stress, which indicates its role in vascular transport in V. radiata. In addition, this study provides valuable insights into the functional roles of VrHMA genes and will lay a foundation for future genetic improvement in mung bean aimed at enhanced heavy metal stress tolerance and micronutrient homeostasis.

Phosphorus Starvation Tolerance 1 in rice (OsPSTOL1, known as Phosphorus uptake 1, Pup1) is a receptor-like cytoplasmic protein kinase that confers tolerance to phosphorus deficiency. The OsPSTOL1 gene possesses a Ser/Thr kinase and shows high amino-acid sequence similarity with the leaf rust receptor-like kinase (OsLrK10). We hypothesize that the putative wheat TaPSTOL1 and TaLrK10 have a common ancestral origin and that putative TaPSTOL1 diverged recently acquiring new structural modifications and biological functions in the process. In this study, we identified all putative TaPSTOL1 homeologs and examine the evolutionary relationship between TaPSTOL1 and TaLrK10 in Triticum species. Our results indicate that the putative TaPSTOL1 diverged recently without possessing the amino-terminal domain, which is a typical characteristic of TaLrK10. We observed numerous conversions tracts between these two genes and the substitution pattern of randomly selected amino acids indicates that dynamic selection pressures acted on both genes. The putative TaPSTOL1 shows high nucleotide diversity compared to TaLrK10 within Triticum species. Further, a multiple-sequence analysis reveals that the third exon of TaLrK10 appears to have been duplicated and diverged as a putative single-exon based TaPSTOL1 in bread wheat. Overall, our comparative analysis indicates that both TaPSTOL1 and TaLrK10 appears to have diverged from a common ancestor, acquiring distinct structural organizations and biological functions.

BackgroundConventional models of human ribosomal DNA (rDNA) array organization have historically depended on transcription-centric boundaries, partitioning the unit into a [~]13 kb rDNA transcription region and a monolithic [~]31 kb intergenic spacer (IGS). While our previous identification of Duplication Segment Units (DSUs) mapped these arrays based on an intuitive analysis of the microsatellite density landscape of the complete reference human genome, our present deep mining of this landscape has revealed a more accurate rDNA Gene Unit Pattern. Methods & ResultsIn this study, we conducted a deep mining analysis of our previously established microsatellite density landscape of the T2T-CHM13 assembly, focusing specifically on nucleolar organizing regions (NORs). We suggest a more accurate rDNA Gene Unit Pattern containing a (CTTT)n microsatellite aggregation ahead of the rDNA gene and a (CT)n microsatellite aggregation behind the gene, rather than a pattern featuring an IGS region inserted between two rDNA genes. ConclusionsA correct rDNA gene pattern of the human genome probably includes a (CTTT)n microsatellite aggregation ahead of the gene and a (CT)n microsatellite aggregation behind it, which possibly constitute cis- and trans-regulating regions; the (CTTT)n and (CT)n microsatellite aggregations may provide two different local stable DNA structures for regulatory protein binding.

BackgroundAnthracyclines are central to childhood cancer therapy but predispose patients to cardiotoxicity leading to long-term cardiovascular risk. Endothelial injury and impaired repair contribute to this, yet pediatric data remain limited. ObjectiveTo longitudinally assess endothelial injury and repair in childhood cancer patients treated with anthracyclines by quantifying circulating endothelial cells (CECs) and endothelial progenitor cells (EPCs). MethodsIn a single-centre retrospective cohort, children (<18 years) diagnosed with leukemia (n=35) or lymphoma (n=13) were studied at four timepoints: pre-treatment ("Pre"), [~]1-month- ("End"), 3 months- (3M), and 1 year- (1Y) post-treatment. Peripheral blood mononuclear cells were analyzed by flow cytometry to quantify CECs and EPCs, and EPC fate was assessed by p16 (senescence) and Annexin V (apoptosis). Cardiac injury biomarkers and left ventricular function were assessed at each timepoint. ResultsLongitudinal trends of CEC and EPC counts were similar between leukemia and lymphoma participants. CECs were highest at pre-treatment and declined significantly thereafter, though they remained marginally elevated during remission compared with healthy controls, indicating that endothelial damage had largely subsided following treatment. EPCs were also highest at pre-treatment and decreased to levels below healthy controls during remission, suggesting impaired baseline endothelial maintenance and repair. Furthermore, EPCs were predominantly senescent up to 1-year post-treatment. ConclusionsEndothelial injury resolves by treatment completion, but repair remains impaired during remission with EPC pools dominated by senescent cells. This suggests defective endothelial regeneration, rather than persistent injury, drives long-term cardiovascular complications and underscores the need to restore EPC viability and function in childhood cancer survivors.

ObjectiveHyperhomocysteinemia (HHcy) affects approximately 75% of the population in China, and there is currently controversy regarding whether HHcy increases the risk of hemorrhagic stroke. This study aims to investigate the effects of high homocysteine (Hcy) levels on cerebral hemorrhage in hypertensive mice by administering homocysteine to them. MethodsMale C57BL/6 mice at 8 months of age were used in the experiment. The study was divided into two groups: the Hcy + AngII + L - NAME group and the AngII + L - NAME group. Magnetic resonance imaging (MRI) was performed when the mice exhibited signs of cerebral hemorrhage.After the hemorrhage, anesthesia was induced to euthanize the animals, and then the brain tissue was fixed. The total rearing period was 18 weeks. The relationship between homocysteine and stroke was described by plotting survival curves. The location and quantity of cerebral hemorrhage were determined through histopathological staining. ResultsThe serum Hcy concentration of mice fed with Hcy for 6 weeks increased to 23.07 mol/L, and the blood pressure ranged from 170 to 180 mmHg. The number of deaths due to cerebral hemorrhage was 10 in both the AngII + L - NAME + Hcy group and the AngII + L - NAME group. The p - value of the survival curves between the two groups was 0.162, indicating no statistically significant difference. ConclusionThe results demonstrated that elevated homocysteine levels did not influence the incidence of intracerebral hemorrhage in hypertensive mice. Hyperhomocysteinemia does not increase the risk of intracerebral hemorrhage in hypertensive mice

While induced pluripotent stem cells (iPSCs) have gained popularity in studying neurodegenerative diseases, the heterogeneity of stem cells used across studies impacts cross-study comparison. The iPSC Neurodegenerative Disease Initiative (iNDI) selected the KOLF2.1J cell line and prioritized its use as a reference standard for studying the effects of pathogenic variants on cell biology due to its stability and neutral neurodegenerative disease genetic risk. This cell line, and its derivatives expressing over 100 variants related to Alzheimers disease, Parkinsons disease, and other neurological diseases, are available for academic and industry access. Current genomic data analyses are limited by the use of a human reference genome that does not capture the complete genetic background of a given iPSC line. While in the future this issue may be partially mitigated by the creation of a comprehensive human pangenome, previous work has shown that generating custom genomes is of value both to characterize the variation present and to serve as a more appropriate genomic reference. Here, we generated and characterized a custom complete genome assembly from KOLF2.1J. Mapping of sequencing reads to a personalized diploid assembly results in more comprehensive mapping compared to traditional linear references (i.e GRCh38). In addition, we provide a comprehensive custom gene annotation along with isoform expression and differential methylation analyses across multiple cell types. The assembly and all additional data is browsable and publicly available. This resource will enable more accurate investigation of the KOLF2.1J cell line and any genomics data generated compared to using traditional generalized references, while also serving as a foundational approach for establishing custom reference assemblies for other high-value iPSC lines.

The paper presents the results of a comparative analysis of gene networks activated by water stress and low temperatures in extensive (Saratovskaya 29, S29) and intensive (Yanetskis Probat, YP) wheat varieties during the seedling development stage. It is concluded that the creation of the S29 variety, which occurred through complex stepwise hybridization and selection for morphological traits, productivity, and grain quality traits, resulted in the emergence and inheritance of regulatory gene networks involving proteins with the CC domain, as well as the BTB/POZ and TAZ domains, which have an increased affinity for transcription factors involved in the transcriptomic response to changing external conditions. It was established that, at the transcriptomic level, the S29 variety is characterized by a transition to an energy saving mode to maintain the activity of the Calvin-Benson cycle under the water deficit conditions and the inhibition of proteolytic processes at low temperatures. The transcriptional response of the high-yielding YP variety to 24-hour low-temperature treatment was more active and involved a larger number of genes compared to the S29 variety. Identifying varietal variability in molecular genetic mechanisms of resistance to abiotic stressors facilitates the development of marker-assisted and genomic selection technologies for common wheat. Key messageThe extensive S29 variety was characterized by its transition to energy-saving mode to maintain the Calvin-Benson cycle under water deficit and a reduction in proteolytic processes under low temperature.

Garcinia binucao (Blanco) Choisy is an indigenous species endemic to the Philippines. Its fruit is traditionally used as a souring agent in local cuisine and has been reported to possess nutritional and medicinal properties. Despite its ethnobotanical significance and promising bioactive properties, the species remains underutilized. To date, no genomic resources have been published for G. binucao, limiting its application in food systems, genetic studies, and conservation programs. This study reports the first complete chloroplast genome of G. binucao from an accession conserved at the Institute of Plant Breeding, University of the Philippines Los Banos. The assembled plastome is circular with a length of 156,570 base pairs (bp). It displays the typical quadripartite structure of most angiosperms, consisting of a large single-copy (LSC) region (85,357 bp), a small single-copy (SSC) region (17,129 bp), and a pair of inverted repeats (IR), each 27,042 bp in size. A total of 128 genes were annotated, including 83 protein-coding genes, 37 transfer RNAs (tRNAs), and eight ribosomal RNAs (rRNAs), consistent with the majority of Garcinia species. Of the protein-coding genes, 45 are involved in photosynthesis, 28 genes for self-replication, five genes with conserved open reading frames, and five genes are associated with other functions. The GC content was 36.2%. Leucine (10.6%) was the most abundant amino acid, with a codon usage bias toward UUA. Additionally, 98 simple sequence repeats (SSRs) were detected, 88.78% consisting of A/T motifs. Phylogenomic analysis based on assembled plastome and publicly available cpDNA sequences of 17 other species in the order Malpighiales revealed that G. indica is the closest relative of G. binucao. These findings provide a framework for future research on the species, including its conservation and potential use as a genetic resource.

At the Wisconsin National Primate Research Center, we have identified a family of rhesus carrying the microtubule-associated protein tau (MAPT) R406W mutation linked to frontotemporal dementia (FTD). Rhesus induced pluripotent stem cells (RhiPSCs) derived from these monkeys present a unique opportunity for in vitro modeling and comparison with cells derived from MAPT R406W human carriers. Here, we report the development of a reproducible method to generate RhiPSCs compliant with the standards of the International Society for Stem Cell Research (ISSCR) to support in vitro modeling of FTD-MAPT R406W. Our stepwise approach identified efficient methods for fibroblast derivation, fibroblast reprogramming to RhiPSC, and RhiPSC maintenance over continued culture. To derive fibroblasts from MAPT wild type (WT) and R406W monkeys, a combination of manual processing and overnight enzymatic digestion was required to maximize the number of low passage fibroblasts available for reprogramming. Fibroblast reprogramming to RhiPSC using Sendai viral vectors versus oriP/EBNA1 episomal plasmids revealed the latter as most efficient. Electroporation conditions for oriP/EBNA1 reprogramming were optimized to maximize plasmid uptake and cell survival. Ultimately, eight RhiPSC lines were derived from 4 donor rhesus monkeys (n=2 WT, n=2 R406W; two clonal lines per donor) and fully characterized according to ISSCR standards. RhiPSC stemness and genetic stability was best maintained on mouse embryonic fibroblast feeders in Universal Primate Pluripotency Stem Cell medium, as opposed to Essential 12 medium supplemented with IWR1, which produced cytogenetic abnormalities. Rhesus neural progenitor cells were generated using a monolayer protocol and expressed PAX6 and NESTIN after 21 days of differentiation. Our reliable method will be useful to labs seeking to derive RhiPSCs for preclinical studies. Overall, the RhiPSCs generated from MAPT R406W carriers will be a critical resource for evaluating the molecular underpinnings of tau-related neurodegeneration across primate species.

The Receptor for Advanced Glycation End-products (RAGE) has been implicated in driving cancer growth, aggression, and metastasis through the fueling of chronic inflammation in the tumor microenvironment. This systematic review and meta-analysis summarize and analyze current clinical and preclinical data to provide insight into the relationship between RAGE and cancer, cancer grade, metastasis, patient survival, and cellular processes. A multi-database search was performed to identify original clinical and preclinical research studies examining RAGE expression in cancer. After screening and review, 53 clinical and 233 preclinical studies were included. Associations of RAGE with clinical cancer outcomes were estimated using odds ratio (OR) and associated 95% confidence intervals (CI). The meta-analysis found that RAGE expression was highly correlated with cancerous tissue when compared to controls; high-grade tumors; regional lymph node invasion; and was somewhat negatively associated with patient survival. In addition, meta-analysis estimates of preclinical studies found positive associations between RAGE expression/activation and cancer growth, metastatic potential, evasion of apoptosis, and activated NF-{kappa}B expression. This systematic review and meta-analysis is the first comprehensive study through which both preclinical and clinical research in all available cancer types are assessed for correlations with RAGE expression and activation, demonstrating that RAGE does indeed play a significant role in cancer progression and that further research is warranted.

BackgroundWnt/{beta}-catenin signalling is dysregulated in acute myeloid leukaemia (AML), where it lacks effective targeting strategies. Previously, we discovered that {beta}-catenin interacts with several RNA-binding proteins (RBP), indicating post-transcriptional influence which is yet to be therapeutically interrogated in AML. MethodsCo-immunoprecipitation confirmed protein interactions, and TCF/LEF reporters were used to assess Wnt signalling output in leukaemia cells. Regulatory crosstalk was assessed using immunoblotting and RT-qPCR approaches following lentiviral transduction of myeloid cell lines. Targeting of {beta}-catenin and LIN28B was tested through combinations of genetic and pharmacological inhibition in AML cells. ResultsThe most frequent RBP-binding motif amongst {beta}-catenin-bound mRNAs was the GGAG motif targeted by oncofetal miRNA-regulating RBP; LIN28B. {beta}-Catenin:LIN28B interactions were detected in lymphoid and myeloid cell lines, plus primary human CD34 fetal-liver HSCs. LIN28B positively regulated Wnt signalling output through LEF1 regulation involving a post-transcriptional let7 miRNA mechanism. Further miRNA sequencing of {beta}-catenin- and LIN28B-depleted myeloid cells revealed potential cooperative and antagonistic function in miRNA regulation. Finally, dual-targeting both {beta}-catenin and LIN28B through either genetic and/or pharmacological means preferentially reduced AML cell viability. ConclusionThe {beta}-catenin:LIN28B axis could represent a novel synthetically lethal relationship in AML which could be exploited in rare subtypes where LIN28B expression becomes reactivated.

Banana leaf diseases are a significant threat to Cavendish banana production. In the Philippines, the main disease has been diagnosed as Black sigatoka disease caused by Pseudocercospora fijiensis based on symptoms. However, our study showed that the main pathogen in Mindanao island, the largest banana-producing region in the Philippines, belongs to the genus Nigrospora, contradicting previous assumptions. We clarified the phylogenetic positions of 160 Nigrospora isolates based on molecular phylogenetic analyses using ITS, {beta}-tubulin, and tef1 sequences, and compared their morphology with known species. Molecular phylogenetic and morphological analysis revealed that Nigrospora isolates comprised N. chinensis, N. lacticolonia, N. cf. singularis, N. sphaerica, N. vesicularifera, and a novel species, N. nigrocolonia. Pathogenicity tests on banana leaves confirmed that these species are pathogenic. Species other than N. sphaerica were for the first time reported as pathogens of banana leaf. The results of the fungicide sensitivity test using 14 fungicides, including pyrimethanil, spiroxamine, and tebuconazole, for the Sigatoka disease showed 100% inhibition of all isolates at 100 ppm of active ingredients. However, low-sensitivity isolates were observed for the remaining 11 fungicides. Our findings indicated the need for a comprehensive review of banana leaf disease prevention strategies.

MicroRNAs (miRNAs) play essential roles in the posttranscriptional regulation of gene expression in organisms. In the process of synthesizing mature miRNAs from miRNA precursors, the miRNA precursors are cleaved via Dicer at their loop structure, after which the miRNA precursors become mature and regulate transcription. However, the consequences of altering the loop sequence are not fully understood. The silkworm Bombyx mori is a lepidopteran insect with many genetic strains. We identified a mutant of the miRNA miR-3260 whose the part of the loop structure was lacking in a silkworm strain with translucent larval skin. Here, we aimed to analyze the role of wild-type miR-3260 and the influence of the mutation of the loop structure in B. mori. First, we identified the genomic region responsible for the translucent larval skin phenotype and determined that the mutated miR-3260 nucleotide sequences. Then, we predicted the binding partners of wild-type miR-3260 using the RNA hybrid tool and found two juvenile hormone (JH)-related genes as targets of wild-type miR-3260. Next, we assessed the relationships between miR-3260 and JH and found that miR-3260 was highly expressed in the Corpora allata and its expression responded to JH treatment. Meanwhile, miR-3260 mimic and inhibitor did not induce the typical phenotypes associated with JH in B. mori. Then, we compared the dicing products from wild-type and mutant miR-3260 precursors and observed that neither form underwent Dicer-mediated cleavage when the loop structure was altered. These results suggest that loop mutations in the miR-3260 precursor may not influence dicing activity, consistent with the lack of observable phenotypic effects.

Natural extracts could improve sperm storage and artificial insemination (AI). This study, for the first time, evaluates the suitability of a blueberry extract (Vaccinium corymbosum) obtained from pomace using a sustainable methodology as a supplement for bull semen extenders. Cryopreserved semen doses from eight bulls were combined in 9 pools (3 bulls/pool), supplemented with 0%, 1%, 5%, or 10% extract, and incubated up to 5 h at 38 {degrees}C. Motility was assessed hourly using OpenCASA, and the effects of treatment and time were evaluated using linear mixed-effects models. Motility was significantly better preserved with 1% extract (total and progressive motility, improved linear velocity and linearities, and decreased BCF and fractal dimension, related to hyperactivation). The effect of 5% was overall positive, but it was below 1%, whereas 10% mostly showed a negative effect. These results show that this natural extract could safely supplement bull semen extenders at least between 1% to 5%, and even help improve sperm motility. Therefore, this extract offers an opportunity to enhance cattle semen extenders using a sustainable approach, potentially improving reproductive outcomes.