PeerJ

BackgroundIn microbiome research, differential abundance analysis aids in identifying significant differences in microbial taxa across two or more conditions. Statistical approaches used for this purpose include classical tests such as the t-test and Wilcoxon test, as well as methods designed to account for the compositional nature of microbiome data, including ALDEx2, ANCOM-BC2, and metagenomeSeq. In addition, methods originally developed for RNA sequencing data, such as DESeq2 and edgeR, have been frequently applied to microbiome studies. However, the use of these methods has been controversial. One area of concern is whether different modeling frameworks produce accurate p-values when the null hypothesis is true. ResultsWe evaluated eight methods across six publicly available datasets. Four permutation strategies were applied to generate data under the null hypothesis: shuffling sample names, shuffling counts within samples, shuffling counts within taxa, and fully randomizing the counts table. Methods based on the negative binomial distribution (DESeq2 and edgeR) produced p-values that were consistently smaller than expected under the null hypothesis. In contrast, methods that attempt to correct for compositionality (ALDEx2, ANCOM-BC2, and metagenomeSeq) tended to produce larger-than-expected p-values, even when only sample labels were shuffled, a permutation strategy that does not alter compositional structure. These deviations were dependent on dataset characteristics and permutation strategy, suggesting complex interactions between underlying data structure and algorithm performance. Generating data to follow the expected negative binomial distribution did not eliminate the tendency of DESeq2 and edgeR to exaggerate statistical significance. Although similar patterns were observed in RNA sequencing (RNAseq) datasets, the deviations were less pronounced than in microbiome data. In contrast, the classical t-test and Wilcoxon test yielded p-value distributions consistent with theoretical expectations across datasets and permutation strategies. ConclusionsThese results indicate that the performance of several widely used differential abundance methods can be problematic under null conditions and may affect biological interpretation. Our findings emphasize the importance of careful method selection and highlight the robustness of simpler statistical approaches for reliable inference.

The Leray-XT primer pair has been widely used to amplify the mitochondrial cytochrome c oxidase subunit I (COI) gene from animals. In some marine metabarcoding studies, protists have also been amplified and sequenced using these primers. Here, we ask if the Leray-XT COI primer pair is suitable for observing ciliates and radiolarians, which are numerically and ecologically important components of marine protistan communities. We show that while there are sufficient COI reference sequences for ciliates in NCBI for taxonomic assignments, there are currently only two COI reference sequences for radiolarians. Using in-silico analyses, we additionally show that while the reverse primer Leray-XT primer can bind and potentially amplify both ciliates and radiolarians, the forward primer cannot bind to either taxon. These results show that the Leray-XT primer pair is not suitable for observing ciliates and radiolarians, although it may be useful for observing other marine protistan taxa.

Understanding how anthropogenic disturbance and vegetation structure influence bird abundance is important for biodiversity conservation in rapidly changing tropical landscapes. This study evaluated the effects of anthropogenic and vegetation-related variables on the abundance of the Red-cheeked Cordon-bleu (Uraeginthus bengalus) in human settlements and surrounding farmlands in Laminga Village, Jos-East Local Government Area, Plateau State, Nigeria. Bird surveys were conducted using line transects and quadrat-based vegetation assessments during November 2024. Poisson Generalized Linear Models (GLMs) were used to examine the influence of anthropogenic and vegetation predictors on abundance. Among anthropogenic variables, building density significantly reduced abundance ({beta} = -0.141, SE = 0.060, z = -2.333, p = 0.020), whereas human presence ({beta} = -0.073, p = 0.141) and noise level ({beta} = 0.009, p = 0.592) did not significantly influence abundance. Average grass height showed a marginal positive relationship with abundance ({beta} = 2.008, SE = 1.051, z = 1.910, p = 0.056), while hedgerow presence, hedgerow height, grass cover, and bare ground cover were not significant predictors. The vegetation model produced the lowest residual deviance (91.19) and AIC value (297.66), indicating comparatively stronger explanatory performance. The results suggest that structural habitat characteristics and building density may play more important roles in shaping Red-cheeked Cordon-bleu abundance than human activity or noise levels alone. These findings provide insight into species responses to environmental disturbance in human-modified savanna ecosystems.

Plants, as structural elements of habitats, contribute greatly to the maintenance of local biodiversity through their biological interactions. In this study we explore whether their rarity, according to Rabinowitzs (1981) three criteria, is related to the richness and diversity of arthropods and other plants they are associated to, in a gypsum-rich steppe. We first analysed whether the geographic abundance and ecological specialisation of 32 characteristic and dominant plant species are related to the diversity (richness and phylogenetic diversity (MPD)) and degree of local specialisation of arthropods associated with them (1,694 taxa). Then, we focused on a non endemic and non specialized plant in the study area (Krascheninnikovia ceratoides) to explore the effect of population size on two types of interactions: aerial arthropods and plant facilitation. Results indicate that: 1) plant species abundance (geographical range) is not related to the richness or MPD of communities of associated arthropods, 2) plant species ecological specialization (edaphic endemisms or gypsophiles) do not contribute differentially to the maintenance of singular arthropod communities, and 3) the community of aerial arthropods and plants interacting with K. ceratoides in a small population are not necessarily less diverse than those in patches of similar size in a large population. Results also revealed that the two plant species with fewer interactions (one rare, one widespread) do show the highest singularity in their interactions with arthropods. Our study illustrates the important contribution of rare plants to the conservation of local biodiversity.

Dams can significantly alter natural riverine systems, but their impact on movement across rivers for most terrestrial vertebrates is poorly known. The completion of Glen Canyon and Flaming Gorge dams in Arizona and Utah (southwestern United States) profoundly changed the Colorado and Green Rivers and have altered habitat for many species. The common side-blotched lizard (Uta stansburiana) offers an excellent opportunity to examine the effects of riverine impoundments on migration and gene flow in terrestrial biodiversity. To assess these effects, we collected tissue samples from 241 Uta stansburiana above and below Glen Canyon Dam and on both sides of the Colorado river at three separate study areas. We used eight microsatellite loci to estimate genetic exchange in the context of genetic diversity and structure. One study area below Flaming Gorge Dam and above Glen Canyon Dam has annual periods of warmer water temperatures and lower flows that are closer to pre-dam conditions, whereas two study areas below Glen Canyon Dam have cold water temperatures year-round, and less pronounced seasonal low flow episodes. We predicted that warmer water temperatures above Glen Canyon Dam would promote greater genetic exchange among populations than below the dam. However, we found evidence for low levels of genetic exchange between sites both above and below Glen Canyon Dam, and a moderate amount of exchange at a site below this dam where lizards could conceivably move from one side to the other. Our results imply that 1) the changes in water temperature and hydrology in dam-altered rivers are a barrier for this species even when the distance from the dam is great; and 2) genetic exchange may be dependent on river morphology. These results are relevant to other small vertebrates, particularly ectotherms, that occupy habitat proximal to a dammed river and has implications for the conservation management of impounded river systems.

Xenoglossa cressoniana, also known as Tetraloniella cressoniana or Xenoglossodes cressoniana, is a eucerine bee known mainly from the US Great Plains. The species was described from a female collected somewhere in Texas in the early 1900s. Here, we report rediscovery of this species in Texas after over a century with no intervening observations. While surveying north Texas ranches, we collected six specimens, including both males and females, at four sites northwest of the Dallas-Fort Worth Metroplex. Xenoglossa cressonianas range, the Great Plains and parts of the deep South, covers a large proportion of the United States. The southern and northern Great Plains, and deep South, have been historically overlooked by most bee researchers. Our results show the urgent need to increase data from under-sampled regions, even within a heavily sampled country such as the US.

Habitat modification is a major driver of avian population change in tropical savanna ecosystems. This study investigated habitat-related variation in the abundance of the Red-cheeked Cordon-bleu (Uraeginthus bengalus) across human settlements and surrounding farmlands in Laminga Village, Jos-East Local Government Area, Plateau State, Nigeria. Field surveys were conducted over a three-week period in November 2024 using 21 line transects sampled during peak bird activity periods. Bird abundance data were analysed using a Poisson Generalized Linear Model (GLM). Results showed that habitat type significantly influenced abundance, with significantly lower abundance recorded in human settlements compared to farmlands ({beta} = -0.836, SE = 0.192, z = -4.359, p < 0.001). Transect length positively influenced abundance ({beta} = 0.028, SE = 0.008, z = 3.600, p < 0.001). Model performance improved substantially from the null deviance (159.88) to the residual deviance (125.85), with an Akaike Information Criterion (AIC) value of 306.32. The findings suggest that farmlands provide more favourable habitat conditions for the species, likely due to greater vegetation availability and reduced structural disturbance relative to settlement areas. The study highlights the ecological importance of low-intensity agricultural landscapes in supporting avian persistence within human-modified savanna environments.

Justification: Accidental lab-acquired infections (LAIs) with potential pandemic pathogens (PPPs) in high-biosafety research facilities risk causing a pandemic. Routine testing of lab workers for LAIs coupled with isolation of infected workers could reduce the risk, but the impact of such an intervention may depend on pathogens' epidemiological characteristics. Objective: This study aims to understand how the epidemiological characteristics of PPPs moderate the efficacy of a routine testing and isolation intervention in preventing larger outbreaks after an LAI. Methods: We employed a discrete-time stochastic network infectious disease model to run 625,000 epidemic simulations encompassing 625 unique combinations of five parameters of interest: test frequency, pathogen transmissibility, the self-isolation rate for symptomatic cases, the percentage of cases that are asymptomatic, and the percentage of infectious time that is spent in the pre-symptomatic state among those who show symptoms. To summarize the Monte Carlo simulations, we paired visual analysis with logistic regression for formal hypothesis testing, with an emphasis on the interaction terms that capture the moderating effect of epidemiological parameters on the impact of test frequency. Main Results: There were four main findings. First, the relative reductions in risk of outbreak that were caused by increased test frequency were inversely correlated with pathogen transmissibility. Second, the effect of test frequency was magnified at higher asymptomatic shares when the symptomatic self-isolation rate was high, but minimally when the self-isolation rate is low. Third, the direction of how the symptomatic self-isolation rate moderated the effect of increased test frequency depended on the asymptomatic share. Fourth, as the pre-symptomatic share of infectious time increased, the effect of test frequency on the probability of an outbreak was strongly magnified largely independent of symptomatic self-isolation rates. Conclusions: Routine testing and isolation could significantly mitigate the risk of catastrophic PPP escapes, with the intervention's success varying based on pathogen characteristics. High shares of asymptomatic and pre-symptomatic transmission notably increased the relative risk reductions achieved by the intervention. These findings suggest prioritizing testing interventions for pathogens with high asymptomatic and pre-symptomatic transmission and highlight the symptomatic self-isolation rate as a policy intervention target.

Ethyl-iophenoxic acid (Et-IPA) is widely recognized as a useful biomarker to confirm oral bait consumption in eutherian species. In historical studies on marsupials, Et-IPA was rapidly eliminated from brushtail possums (Trichosurus vulpecula) and swamp wallabies (Wallabia bicolor) suggesting limited use for marsupial species. However, a 1 mg oral dose of Et-IPA was detectable in the marsupial Tasmanian devils (Sarcophilus harrisii) for [&ge;] 56 days suggesting the biomarker can be used in a devil bait vaccine program. To assess Et-IPA marking in off-target marsupials that may consume baits, we administered 1 mg oral doses of Et-IPA to brushtail possums, forester kangaroos (Macropus giganteus tasmaniensis), spotted-tailed quolls (Dasyurus maculatus) and eastern quolls (Dasyurus viverrinus). Liquid chromatography with tandem mass spectrometry was used to detect and quantify serum Et-IPA. Et-IPA was detected in the serum on day 2 but was not detected by day 14 in any of the species tested, including the two quoll species which are in the same carnivorous Dasyuridae family as the devils. The rapid elimination of Et-IPA in the marsupials included in this study suggests it is not useful as a biomarker for these species. Furthermore, rapid elimination in the kangaroos and possums suggests that Et-IPA is unlikely to accumulate in the food chain following distribution of Et-IPA-marked oral bait vaccines for Tasmanian devils. Short summary for non-expertsA recent study in Tasmanian devils (Sarcophilus harrisii) challenged the concept that ethyl iophenoxic acid (Et-IPA) is not a useful serum biomarker for marsupials. Using the same sensitive liquid chromatography-tandem mass spectrometry method we detected serum Et-IPA in four marsupial species on day two post-ingestion but by day 14, serum Et-IPA was undetectable. These findings indicate that Et-IPA is an unsuitable biomarker for these species and suggest that Et-IPA from devil bait vaccines is unlikely to bioaccumulate in the Tasmanian environment.

The genus Trichiurus is the most economically valuable fish in the family Trichiuridae, currently recognized to include 10 valid species. However, historically numerous morphologically similar congeners have been erroneously assigned as synonyms or subspecies of T. lepturus. In this study, we examined 16 hairtail specimens collected from the southern waters of Java Island, Indonesia. Integrated morphological and mitochondrial phylogenetic analyses (COX1 and 16S rRNA), compared against global Trichiurus sequences, revealed that these specimens form an independent lineage that diverged early from other congeners. Consequently, we describe this lineage as a previously undescribed cryptic species. Diagnostic characters include: first anal-fin spine below 36th-37th dorsal-fin rays; anus below 35th-36th dorsal-fin rays; anteriormost tip of supraoccipital well posterior to posterior distal margin of eye; anterior margin of the pectoral-fin spine non-serrated; fangs on both jaws with barb-like processes; upper jaw long, mean 16.6% (15.5-17.6%) of preanal length; snout short, 12.0% (10.9-13.1%) of preanal length; eye small, diameter 5.3% (4.3-5.7%) of preanal length; and absence of hyperostosis on dorsal cranium. We herein propose the name Trichiurus javaensis sp. nov., and provide a formal morphological description and diagnostic characterization of this species.

The role of passive muscle mechanical properties in explosive performance is important to understand to maximize training and performance in strength sports. The objective of this study was to create and test a Structural Equation Model (SEM) to analyze the interrelations between muscle mechanical properties and neuromuscular performance in competitive weightlifters. The MyotonPRO was used to measure muscle stiffness, muscle tone, muscle elasticity, relaxation time and creep of four major muscles: Quadriceps Femoris, Hamstrings, Trapezius and Biceps Brachii, on thirty elite male weightlifters. This involved performance metrics of the rate of force development (RFD), countermovement jump (CMJ) and time to contraction threshold (TCT). AMOS was used to analyze the direct and indirect relationships between variables through SEM analysis. These findings indicated that muscle stiffness and relaxation time were significant predictors of explosive performance measures (p < 0.05) but there were weak or no relationships between tone, elasticity and creep. The model proposed had good fit indices (CFI = 0.97, RMSEA = 0.049) indicating its structural soundness. These results present the significance of muscle stiffness and relaxation time as important predictors of neuromuscular performance. The proposed model suggests an effective structure of monitoring athletes, their performance optimization, and individual training design in strength-based sports.

Gastropods are a highly diverse and often overlooked taxonomic group of significant ecological and economic importance. Some terrestrial gastropods are critical pests of commercial agriculture and home gardens worldwide. Malacopathogenic nematodes offer an effective biological control method of managing pest slugs and snails as a natural enemy. Pellioditis (syn. Phasmarhabditis) hermaphrodita and Pellioditis (syn. Phasmarhabditis) californica are two species of biocontrol nematodes that have been commercialized, sold as Nemaslug(R) and Nemaslug(R) 2.0 respectively on three continents. Although there is interest in bringing Nemaslug(R) products to the US, they are currently not permitted due to limited knowledge on their North American distribution and effects on non-target and native species. In this study, we investigated the impact of P. hermaphrodita and P. californica on Ariolimax columbianus across two slug-host life stages, in laboratory infectivity assays. The objectives were to 1. determine whether P. hermaphrodita and P. californica nematodes impact survival of A. columbianus, and 2. evaluate whether there are differential effects on survival in juvenile and adult life stages of A. columbianus, in laboratory infectivity trials. We found that P. hermaphrodita caused significant mortality in A. columbianus with 100% mortality observed in both juvenile and adult slug hosts. The P. californica treatment had significant effects on the juvenile A. columbianus group only, with 80% mortality. By contrast, only 16% of unexposed control juveniles and 4% of control adult slugs died during the experiment. These results indicate that P. hermaphrodita and P. californica are lethal to the native, non-target Pacific banana slug (A. columbianus) under laboratory conditions, with mortality differing between juvenile and adult host life stages. Given the ecological importance of A. columbianus, these findings raise concerns for potential non-target effects of P. californica and P. hermaphrodita on terrestrial gastropod communities and emphasize the need for testing biocontrol agents against multiple life stages.

Understanding behavioral differences between non-native and closely related endangered species could be important to aid conservation management. In volume 169 of Zoology, Bels et al. (2025) reported on their comparison of display-action-patterns (DAP) between native Iguana delicatissima and non-native iguanas present on islands of the Guadeloupe Archipelago in the Caribbean Lesser Antilles. Here, we address conceptual and methodological concerns about their work and reanalyze their data given our proposed corrections, primarily a literature-informed adjustment of their "species" category. We additionally utilize online videos from South American mainland I. iguana populations, from where the non-native iguanas in the Guadeloupe Archipelago originate, to better understand the different DAPs between native and non-native iguanas in the Guadeloupe Archipelago. Significant differences in DAP characteristics among "species" categories (native I. delicatissima, non-native iguanas, and hybrids) show that Bels et al. (2025) oversimplified their data analyses by merging all non-native populations into one group. This result indicates the presence of behavioral variation among subpopulations within widely hybridizing iguanid populations, which has been poorly studied. Additionally, videos from mainland populations across two major mitochondrial clades of Iguana iguana show that non-native iguanas on Guadeloupe retained DAP characteristics of those populations from which they originate. We discuss these findings in light of the proposed hypotheses put forward by Bels et al. (2025), of which two can be excluded. Overall, our reanalysis shows that studies focusing on characteristics within settings of complex hybridization in diverse species should acknowledge this complexity.

Background & AimsColorectal cancer liver metastasis is the leading cause of mortality in affected patients, with liver sinusoidal endothelial cells playing a pivotal role in metastatic niche formation. Proprotein convertase subtilisin/kexin type 9 has emerged as a regulator of tumor biology, but its function in the hepatic microenvironment remains poorly defined. This study aimed to characterize the role and subcellular localization of PCSK9 in liver sinusoidal endothelial cells and to evaluate the therapeutic potential of its endothelial-specific inhibition in colorectal liver metastasis. MethodsIn vitro studies were performed using human and murine liver sinusoidal endothelial cells stimulated with conditioned media from metastatic colorectal cancer cells and cancer stem cells. Subcellular localization was assessed by immunofluorescence, immunogold electron microscopy, and biochemical fractionation. Protein interactions were investigated using co-immunoprecipitation and proteomic analyses. For in vivo validation, a murine model of colorectal liver metastasis was generated by intrasplenic injection of tumor cells, followed by systemic administration of chondroitin sulfate-targeted nanoparticles delivering PCSK9 siRNA every 5 days for 18 days. ResultsPCSK9 was consistently expressed in liver sinusoidal endothelial cells and displayed a predominant nuclear localization, which increased upon tumor-induced activation. Proteomic integration identified multiple candidate interacting proteins involved in metabolic and tumor-related pathways. Targeted nanoparticle-mediated delivery achieved efficient PCSK9 silencing in vitro. In vivo, endothelial-specific PCSK9 inhibition significantly reduced liver metastatic tumor burden compared with control groups, whereas free siRNA showed no significant effect. ConclusionsPCSK9 exhibits a novel nuclear localization in liver sinusoidal endothelial cells and potentially interacts with proteins implicated in tumor mediated pathways. Selective inhibition of endothelial PCSK9 using targeted nanoparticles significantly reduces colorectal liver metastasis, highlighting a novel therapeutic strategy focused on the hepatic microenvironment. Impact and ImplicationsThis study provides mechanistic insight into how PCSK9 contributes to colorectal liver metastasis by identifying its novel nuclear localization and potential function in liver sinusoidal endothelial cells. These findings are important for researchers and clinicians seeking to understand microenvironment-driven metastasis and resistance to current therapies. The demonstration that endothelial-specific targeting of PCSK9 reduces metastatic burden suggests a new avenue for therapeutic development beyond systemic inhibition. Such strategies could be translated into precision nanomedicine approaches to improve outcomes in patients with metastatic colorectal cancer while minimizing off-target effects.

Increasing human activities along the US west coast are of concern for populations of cetaceans and particularly for a number of large whale species that are recovering from overexploitation during the era of commercial whaling. New rapid monitoring tools, such as satellite imagery analysis powered by recent advances in artificial intelligence, have potential to provide additional broad-scale and near real-time capacities for survey and monitoring. This paper investigates and demonstrates the feasibility of automatic detection of gray whales in sub-meter satellite imagery off the coast of California, USA. Observations and statistical analysis of regional imagery allowed not only an assessment of their detectability but also the development of robust signal processing and machine learning-based solutions for automated detection. To that end, a regional dataset of 221 gray whales was created using signal processing to inform a deep-learning-based detection framework, and 20 different large neural network architectures for feature extraction followed by a support vector machine algorithm for classification were evaluated for their detection performance. Neural network backbones included 19 convolutional neural networks and 1 transformer network. The best architecture generally achieved satisfying performance with an average balanced accuracy reaching up to 99.90%. It is also demonstrated that panchromatic imagery, in spite of the lesser amount of information provided, can be used to perform detection with a relatively high accuracy of 87.05%, allowing wider spatial and temporal coverage. Large-scale deployment of the best performing models over a broad range of regional satellite imagery resulted in the detection of 3353 gray whales, as well as opportunistic detections of humpback, blue and fin whales, in and going from December 28th 2009 to March 26th 2023. It also provided meaningful data points concerning the migration routes of gray whales within the Channel Islands and Southern California Bight. The large number of high-confidence detections indicates the capacity for a large-scale monitoring approach to support state and federal conservation policies such as gear mitigation, vessel speed reduction programs, or shipping lane redefinition that could also be expanded to other areas and for other species.

Purpose This study aimed to evaluate the efficacy and safety of neoadjuvant chemotherapy (NAC) combined with the programmed death protein 1 (PD-1) inhibitor sintilimab versus NAC alone in patients with triple-negative breast cancer (TNBC). Materials and Methods In this retrospective cohort study, we collected clinical data from 61 patients with triple-negative breast cancer (TNBC) who received neoadjuvant therapy at The First Hospital of Lanzhou University between July 2024 and July 2025. These patients were divided into two groups: the neoadjuvant chemotherapy (NAC) plus sintilimab group (n=27) and the NAC-alone group (n=34). The primary endpoint was the pathological complete response (pCR) rate. Secondary endpoints included objective response rate (ORR), safety, and changes in tumor markers. Results The combination therapy group showed significantly higher ORR (85.2% vs. 58.8%) and pCR rates (59.3% vs. 32.4%) compared to the NAC alone group (both P<0.05). Post-treatment Ki-67 levels were also significantly lower in the combination group (P<0.05). The overall incidence of adverse events was comparable between groups (P>0.05), although leukopenia was more frequent with sintilimab (P<0.05). Conclusion In the neoadjuvant setting for TNBC, the addition of sintilimab to NAC significantly improves ORR and pCR rates, effectively reduces the tumor proliferation index Ki-67, and does not significantly increase the overall burden of adverse events. The combination regimen shows a manageable safety profile and demonstrates positive clinical value. Keywords Triple Negative Breast Cancer, Immunotherapy, Sintilimab, Combination neoadjuvant chemotherapy, Efficacy, Real-World data.

Estimating taxonomic profiles is a central task in microbiome research. Several bioinformatic tools have been developed for this purpose, differing in algorithmic strategy, reference database flexibility, sensitivity parameters, and the type of abundance they estimate. As a result, taxonomic profiles carry an unwanted methodological signal whose driving characteristics remains understudied. While benchmarks have evaluated the performance of some of these tools, they rely on simulated data; little work has been done to compare them using real metagenomes in the presence of noise and uncharacterised diversity. Overall, the impact of taxonomic profiler choice and parameterisation on scientific conclusions remains poorly understood. Here, we provide a much-needed characterisation of four taxonomic profilers to help researchers better understand the available bioinformatic tools and inform their methodological choices. Then, we leverage 1,211 shotgun metagenomes from eight datasets to compare these taxonomic profilers across 13 methodological designs. Based on diversity indices, we found substantial variability in estimated taxonomic composition depending on methodological features such as reference database and algorithmic strategy. Alpha diversity and its analysis varied substantially with tool choice (particularly among k-mer-based tools) and with reference database. Beta diversity showed sensitivity to both database and parameter choices, yet this variability barely affected statistical inference. This work raises awareness about the causes of variability in metagenome analysis attributable to choices in taxonomic profiling methodology. Our findings highlight the sensitivity of taxonomic diversity analyses to these choices and the importance for researchers to consider assessing the robustness of their results to choice of tool, parameter, and reference database. Crucially, differences in sample diversity across methodologies are symptomatic of differences in estimated taxonomic composition, which can affect any analysis based on taxonomic abundances. Overall, this study underscores the importance of tool selection and parametrisation, and of conducting sensitivity analyses to support robust and reliable scientific conclusions.

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.

BackgroundCodon usage bias, the non-random usage of synonymous codons in coding sequences, represents a fundamental feature of genomic organization that has been largely understudied in cancer biology. Pancreatic ductal adenocarcinoma (PDAC), the predominant subtype of pancreatic cancer, is characterized by aggressive disease progression and limited therapeutic options, necessitating novel approaches to understand its molecular pathogenesis. Leveraging publicly available single-cell RNA sequencing data, we performed comprehensive codon usage analyses across different cellular populations in PDAC. ResultsEmploying a variety of computational codon usage indices uncovered the connections between cancer-specific cellular state features and codon usage signatures. Our findings reveal that malignant pancreatic cells express genes with significantly higher GC content, demonstrate preferential usage of optimal codons through increased frequency of preferred synonymous codons, and exhibit a marked preference for more cost-effective amino acids. Analysis of transcript-level bulk RNA-seq data from PDAC tumors revealed that these codon optimization patterns extend to alternative isoform usage, with highly expressed isoforms displaying increased codon optimality and enhanced mRNA stability. ConclusionThese codon usage-dependent adaptations operating at both gene expression and transcript isoform levels may enable malignant cells to enhance gene expression rates, potentially leading to increased translational efficiency and protein production. These insights into the codon usage landscape of PDAC may provide potential biomarkers for disease monitoring and treatment response prediction.

Background and aimTrauma healing with low-intensity ultrasound is effective for different types of injuries affecting both soft tissues and bones. The work aimed to disclose the healing potential of a new type of ultrasound, ultra-wideband low-intensity mechanical waves (UMUS), and to compare its effects with those of low-intensity pulsed ultrasound (LIPUS) in a model of trauma. Material and methodsThe work was performed on 2-to 3-month-old male Wistar rats. The model of tail amputation was created, and a transducer emitting UMUS (1-7 MHz, 0.22 mW/cm2) was applied daily for 10 days to the surface of the trauma site in animals that were timely immobilized. LIPUS (1.5 mHz, 30.0 mW/cm2) was used in a separate group of animals. Sham-stimulated rats were used as a control. The intensity of collagen expression in the subdermal tissue was assessed in van Gieson-stained sections, whereas in the UMUS group, expression of CD31, CD34, VEGF, and Ki67 was analyzed. ResultsStarting on the 20th day after trauma, UMUS-treated animals demonstrated a statistically significant decrease in the surface area of the traumatic zone compared to the control, whereas LIPUS-treated rats showed this difference on the 30th day of observation. Starting from the 30th day, a significantly greater reduction in the surface of trauma was observed in UMUS, with complete closure achieved in 6 out of 9 rats (P=0.019 vs control), whereas in LIPUS-treated animals, a similar result was observed in 2 out of 8 rats (P>0.05). In UMUS-treated rats, heightened expression of collagen in animals with LIPUS exceeded control data by 7.84% (P=0.034), while the expression in rats with UMUS exceeded data in LIPUS-treated rats by 14.71% (P=0.013). Increased expression of CD31, CD34, VEGF, and Ki67 was observed in UMUS-treated rats. ConclusionsUMUS treatment accelerated healing and reduced wound size, and increased the expression of collagen, CD31, VEGF, CD34, and Ki67, supporting angiogenesis and collagen formation. Effects are more pronounced compared to LIPUS treatment. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=176 HEIGHT=200 SRC="FIGDIR/small/717366v1_ufig1.gif" ALT="Figure 1"> View larger version (75K): org.highwire.dtl.DTLVardef@936775org.highwire.dtl.DTLVardef@16d505eorg.highwire.dtl.DTLVardef@1b75d7dorg.highwire.dtl.DTLVardef@15ae99c_HPS_FORMAT_FIGEXP M_FIG C_FIG