Abstract: A cell-based screening system for viral protease inhibitors was developed using firefly luciferase fragment complementation and validated on the SARS-CoV-2 3CLpro model. The optimal luciferase variant incorporating the VLQSGF proteolytic site (Luc III) retained 88% of its native activity. A critical requirement for system performance was the use of an extended nsp4–nsp6 fragment of the viral polyprotein rather than the mature protease, underscoring the importance of the native context for 3CLpro activity. The bicistronic construct pCAG-Luc-III-IRES-nsp4-6 enables coordinated expression of the reporter and protease, thereby increasing assay reproducibility. IC50 values obtained in this system for nirmatrelvir and GC376 correlated with live-virus assay data but differed significantly from those of a cell-free FRET assay, reflecting the impact of cellular barriers. This approach combines simplicity, a standard substrate, and high reproducibility, making it promising for high-throughput screening in basic laboratory settings and adaptable to other viral proteases.

Abstract: Percutaneous cable infection of left ventricular assist device (LVAD) patients is a significant source of morbidity, often caused by biofilm producing or multidrug resistant bacteria. We hypothesized that bacteriophage viruses can be identified from biological samples of patients with active driveline infection. Six patients with local percutaneous lead infections were enrolled. Microbiological samples were collected from the infected wound and other skin regions. The isolated viral strains and phages from wastewater samples were then tested against the pathogen bacterial cultures in vitro. Biofilm disruption assay and genetic analysis of the strains were also performed. Bacteriophages with lytic activity could be identified from samples of two patients. One patient contained four strains showing strong efficacy against his own Staphylococcus epidermidis. Furthermore, this bacterium was susceptible to phages identified from another patient and strains from wastewater samples. Genomic analysis suggested lysogenic lifestyle of the phages. However, none of them have shown any microbiological signs of lysogeny. In conclusion, we have been able to prove in vitro lytic activity of bacteriophages originating from the same LVAD patient. We also found effective phages in biological samples of other patients and wastewater samples, suggesting that patients implanted in the same center may share bacteriophage flora.

Abstract: Vascular endothelial cells (ECs) coordinate with osteogenic processes to establish the specialized vasculature of bone tissue, where endothelial cells and bone cells interact, and bone cells regulate EC proliferation and differentiation. However, it remains unclear how ECs and bone cells are coordinated during early bone formation and whether these interactions differ between endochondral ossification (e.g., femur) and intramembranous ossification (e.g., skull). To address this question, we analyzed endothelial and osteogenic marker expression in the femur and skull between postnatal days 3 and 39. We identified distinct expression patterns of endothelial markers (Endomucin, VE-cadherin and CD31) and osteogenic markers (Osterix, Cbfa1 and BGLP) during osteogenesis in these tissues. In the femurs, endothelial marker expression alternated with the expression of osteogenic markers, suggesting potential reciprocal regulation. In contrast, in the skull, endothelial and osteogenic markers exhibited similar temporal expression patterns without alternation. We also analyzed the expression of VEGF and its receptor FLK1. In the femur, VEGF expression paralleled osteogenic marker expression, whereas in the skull VEGF expression differed from both osteogenic and endothelial marker patterns. Together, these results demonstrate that the coordination of endothelial and osteogenic marker expression, as well as VEGF signaling, differs between endochondral and intramembranous ossification, suggesting distinct modes of interaction between endothelial and bone cells during the formation of long and flat bones.

Abstract: Alzheimer’s disease (AD) disproportionately affects women, with risk increasing sharply during and after the menopausal transition. While declines in estrogen have traditionally been emphasized, emerging evidence suggests that elevations in follicle-stimulating hormone (FSH) may represent a critical and underappreciated driver of neurodegenerative vulnerability. This review synthesizes current evidence linking reproductive aging to AD pathobiology, with a focus on endocrine, metabolic, and inflammatory mechanisms. We examine how sustained FSH elevation interacts with key molecular pathways implicated in AD, including C/EBPβ–δ-secretase signaling, mitochondrial dysfunction, impaired glucose metabolism, and disruptions in autophagic and lysosomal clearance. These processes converge to promote amyloid-β accumulation, tau pathology, and chronic neuroinflammation. In parallel, FSH influences apolipoprotein biology - particularly ApoE - through effects on lipid metabolism, protein lipidation, and clearance dynamics, thereby modulating both amyloid kinetics and inflammatory responses in an isoform-dependent manner. Reproductive aging is further characterized by systemic shifts in vascular integrity, blood–brain barrier function, and immunometabolic regulation, all of which may amplify susceptibility to neurodegenerative processes. Importantly, these upstream disturbances precede classical pathological hallmarks, reframing amyloid and tau accumulation as downstream manifestations of broader regulatory failure. Collectively, this work positions FSH not merely as a biomarker of ovarian decline, but as an active endocrine mediator of neurodegeneration. Targeting FSH signaling and its downstream pathways may therefore represent a promising and mechanistically grounded approach for mitigating AD risk, particularly in women.

Abstract: Background: The growing demand for versatile laboratory automation is exemplified in the context of liquid biopsy, where multi-analyte approaches are increasingly recognised for their potential to enhance diagnostic sensitivity in oncology. However, current practice often necessitates the use of dedicated instruments and workflows for the extraction of each analyte, posing financial and logistical barriers for automated multi-analyte liquid biopsy. Methods: Here, we present Robotic Centrifugal Microfluidics (RoCM), an all-in-one platform that combines the versatility of centrifugal microfluidics and operational flexibility of robotic liquid handling. This combination enables the automation of complex micro- and macrofluidic protocols, realised through the use of (1.) exchangeable microfluidic cartridges and (2.) programmable robotic operations such as in-rotation liquid supply, magnetic bead manipulation, or microfluidic valving. In-rotation robotic liquid manipulation maintains fluid control under centrifugal forces and reduces the cartridge footprint associated with pre-loaded liquid reservoirs. Platform applicability was demonstrated using two exemplary liquid biopsy workflows: the extraction of cell-free DNA (cfDNA) from blood plasma using RoCM-cfDNA slices and the extraction of extracellular vesicles (EVs) from blood plasma using RoCM-EV slices. Results: In a pilot study with patient samples from different cancer entities, the RoCM-cfDNA slices yielded comparable variant allele frequencies to a commercial bead-based instrument, while the RoCM-EV slices achieved a recovery of a greater diversity of EV subpopulations and up to one magnitude higher recovery than semi-automated size exclusion chromatography. Conclusion: By simply exchanging cartridges, RoCM enables the extraction of diverse analytes within a single automated system. Its application can be extended to further analytes, such as circulating tumor cells (CTCs), or to applications beyond liquid biopsies, where versatile micro- and macrofluidic protocols benefit from implementation in a single automation instrument.

Abstract:

The alkaline pectate lyase A from Paenibacillus barcinonensis, encoded by pelA (GenBank accession no. CAB40884), is an enzyme with high activity on pectin and potential application in sustainable industrial biotechnology. In this study, pelA was expressed in Saccharomyces cerevisiae by using different domains of the cell wall protein Pir4 as translational fusion partners. Given the presence of five potential N-glycosylation sites in the amino acid sequence coded by pelA, two of them in conserved regions of class III pectate lyases, the effect of glycosylation on the enzymatic activity of the recombinant enzyme was investigated by expressing the recombinant fusion proteins in both, standard and glycosylation deficient strains of S. cerevisiae. Correct targeting of the recombinant fusion proteins was confirmed by Western blot analysis using Pir-specific antibodies, whilst enzymatic activity on polygalacturonic acid was demonstrated on both plate assays and colorimetric assays. Hyper- glycosylation of the enzyme when expressed in the standard strain of S. cerevisiae did not occur, however maximum activities were over two and a half times higher when the enzyme was expressed in the glycosylation deficient strain, suggesting a better adaptation of this strain to the secretion of the functional enzyme. Notably, pectate lyase activity was approximately fourfold higher when the pelA gene was expressed in this yeast strain compared to its expression in a prokaryotic host such as Bacillus subtilis or Escherichia coli.

Abstract:

Polyols are widely used as non-cariogenic sweeteners in foods and oral care products, yet their comparative activity against diverse oral microbes and their potential relevance to the oral–systemic axis remain incompletely defined. Here, we performed an in vitro, optical-density (OD)-based screening of four polyols—allulose, D-mannose, erythritol, and xylitol—against Streptococcus mutans, Streptococcus anginosus, Candida albicans, and Fusobacterium nucleatum. Cultures were grown with polyols at 1–20% (w/v), and OD600 was recorded at organism-specific endpoints (~24 h). Allulose, erythritol, and xylitol produced strong, concentration-dependent suppression of streptococcal growth at ≥5–10%, whereas C. albicans showed minimal changes across the tested range. F. nucleatum was highly sensitive to allulose, D-mannose, and xylitol at ≥5% (reducing OD to ≤13% of untreated control), while low concentrations of D-mannose and erythritol increased OD above control, suggesting species-specific utilization or stress responses. One-way ANOVA with Tukey’s HSD post hoc testing supported significant between-polyol differences for most concentrations in Streptococcus spp. and F. nucleatum. Collectively, these results identify polyol- and taxon-specific growth phenotypes that can inform the formulation of swallow-safe oral hygiene products and motivate follow-up work in polymicrobial biofilm models and clinical studies targeting oral inflammation and downstream systemic risk.

Abstract: Background: Deep learning has become an important tool for predicting mutation-induced changes in binding free energy (ΔΔG). However, most current state-of-the-art methods rely heavily on paired wild-type (WT) and mutant (MT) complex structures during both training and inference. This dependence on post-mutation structural information substantially limits their practical utility in real-world scenarios, such as clinical diagnosis and early-stage drug screening, where mutant structures are difficult to obtain experimentally in a timely manner. Methods: To evaluate model performance in more realistic and challenging translational settings, we conducted a systematic benchmark of graph-based deep learning models under a WT-only inductive setting. We constructed a full-protein heterogeneous graph framework that incorporates long-range spatial constraints to implicitly infer mutational effects from static wild-type structures. We compared it against a sequence-based vector baseline model. Results: Through a systematic evaluation on the MdrDB dataset, we revealed a critical generalization gap. Although random splitting yielded relatively high predictive correlation due to homologous data leakage (Pearson R ≈ 0.55), model performance dropped sharply under a strict UniProt-based cross-protein split designed to simulate prediction on truly unseen targets (Pearson R ≈ 0.15). Although the absolute performance remained limited, the graph-based model showed a weak but consistent improvement over the sequence baseline, which was close to random guessing (Pearson R ≈ 0.04). Conclusions: Further analyses suggest that the performance bottleneck may partly arise from intrinsic experimental noise in the dataset (i.e., label inconsistency) and from the absence of conformational entropy (dynamic) information in static WT structures. This study indicates that random splitting can lead to substantial overestimation of model generalizability. It highlights the need to integrate physical priors and dynamic features to overcome the current limitations of drug resistance prediction when explicit mutant structures are unavailable.

Abstract: Background: Currently marketed hepatitis B vaccines are primarily recombinant protein vaccines. However, their antigen immunogenicity is relatively weak, requiring combination with effective adjuvants to enhance the immune response. The development of novel, highly effective adjuvants is a key strategy for optimizing vaccine performance. Polyinosinic-polycytidylic acid (PolyI:C), a synthetic double-stranded RNA analog, activates TLR3/RLR pathways to enhance T-cell priming and cellular immunity. However, its utility as a sole adjuvant is limited by rapid nuclease degradation and poor cytosolic delivery. Lipid nanoparticles (LNPs), a mature delivery platform, enable high encapsulation efficiency, efficient cellular uptake, and endosomal escape. Objectives: This study aimed to evaluate the adjuvant effect of LNP-encapsulated PolyI:C (LNP-PolyI:C) on the immunogenicity of hepatitis B surface antigen (HBsAg) in vivo. Methods: The colloidal stability of LNP-PolyI:C stored at 2–8°C for 9 months was monitored using dynamic light scattering (DLS) on a Zetasizer Lab instrument. Serum levels of HBsAg-specific IgG, IgG1, and IgG2a antibodies in immunized Kunming mice were measured by enzyme-linked immunosorbent assay (ELISA). The secretion of HBsAg-specific cytokines by splenocytes was analyzed using flow cytometry and enzyme-linked immunospot (ELISpot) assay. Results: The results demonstrated that the LNP-encapsulated PolyI:C adjuvant significantly increased the secretion of HBsAg-specific IFN-γ, IL-2, and TNF-α by murine splenocytes, indicating a Th1-biased and cytotoxic T lymphocyte (CTL)-mediated cellular immune response. In addition, this formulation markedly elevated serum titers of HBsAg-specific IgG, IgG1, and IgG2a. Notably, the increased IgG2a/IgG1 ratio highlights a robust enhancement of the humoral immune response. Conclusions: These findings underscore the advantages of the LNP-PolyI:C adjuvant in enhancing both humoral and cellular immunity, demonstrating its considerable potential as a novel adjuvant.

Abstract: Caffeine is a widely consumed psychostimulant known to affect memory, yet its dual role in impairing long-term potentiation (LTP) while enhancing cognitive performance remains unresolved. This study aimed to clarify this paradox by investigating the differential effects of caffeine on distinct forms of synaptic plasticity in the hippocampus. Using extracellular recordings in mouse hippocampal slices, we assessed long-term (LTP and E-S potentiation), short-term plasticity, and neuronal excitability under 30 μM caffeine exposure – a physiologically relevant concentration. Our findings confirm that caffeine suppresses LTP but does not inhibit E-S potentiation; instead, it enhances it. Furthermore, caffeine alters excitability in a form-dependent manner, reducing it following LTP and increasing it following E-S potentiation. We also show that caffeine blocks short-term synaptic plasticity regardless of prior LTP induction. These results suggest that E-S potentiation may serve as a caffeine-resistant mechanism for memory formation, potentially mediated by selective modulation of adenosine receptors. This study provides new insight into how caffeine influences synaptic processes underlying learning and memory.

Abstract: Background/Objectives: Among the most common hereditary neurodegenerative disorders in people are the neuronal ceroid lipofuscinoses (NCLs), a subgroup of lysosomal storage disorders. For most cases of NCL, the genes containing the causative variants have been identified. NCLs also occur in dogs, and in most instances variants responsible for the canine NCLs occur in genes orthologous to those associated with the human disorders. An adult Miniature Dachshund presented with clinical signs consistent with NCL. Studies were undertaken to determine whether the disease phenotype supported classification of the disease as an NCL and to identify potential causal DNA sequence variants. Methods: The proband underwent complete neurologic and ophthalmological examinations followed by euthanasia. Tissues were examined for NCL-like pathology. Whole genome sequence analysis (WGS) was performed. Results: The clinical signs and tissue pathology were consistent with those of NCL disease, although with some features distinct from previously described forms of canine NCL. The proband was uniquely homozygous for variants in 5 genes associated with lysosomal function, 4 of which have not previously been associated with the NCLs. Conclusion: The proband suffered from a novel NCL-like disorder. Determining whether one or a combination of more than one of the 5 potentially causal DNA sequence variants was responsible for the disease will require evaluation of additional cases.

Abstract: Microbial-based products are essential for sustainable agriculture, yet inconsistent performance and limited mechanistic understanding constrain their adoption. While terminology varies globally—from "bioinputs" to "microbial products"—this linguistic diversity reflects a deeper conceptual gap. Historically, the sector has relied on a successful but empirical Bioinputs 1.0 paradigm, based on phenotypic screening and a "black box" approach to efficacy. We propose Bioinputs 2.0 as an evolutionary framework grounded in genomics, functional biology, and advanced formulation. This paradigm integrates microbial ecology, metabolite-driven bioactivity, and systems-level interactions, positioning formulation as an integral design component rather than a secondary step. Transitioning from empirical discovery to knowledge-driven design is necessary to ensure reliable, scalable applications. While particularly evident in biocontrol, this shift provides a stronger basis for interpreting field responses in plant growth-promoting microorganisms. Overall, Bioinputs 2.0 emphasizes integrated, context-dependent biological systems to bridge the gap between laboratory insights and consistent field performance.

Abstract: The problem of aligning humans and artificial intelligences can be understood in terms of minimizing externalities between them. However, economics cannot define externality because it contradicts the rationality assumption. This paper applies the homeostatic principles, from anatomical homeostasis to its disorder – cancer, to define externality. Drawing upon the perspective of cancer as a problem of scaling cellular collectives, this paper shows how to redefine both externality and rationality in terms of cognitive light cones (which demarcate the scale of goals any agent can pursue). We propose that cognitive light cones are constructed out of interoceptive signals for the purpose of anatomical homeostasis. We show that externalities can be understood in terms of anatomical homeostasis and derive some important implications for AI alignment, including the possibility of using market mechanisms enable the mutual co-construction of alignment between artificial intelligences and humans.

Abstract: Background/Objectives: The aim of the paper is to assess the impact of HPV DNA on the survival and cure rates of patients with newly diagnosed head and neck cancers who underwent surgery and subsequent adjuvant radiotherapy. Methods: The study group consisted of 28 patients (55%) with squamous cell carcinoma of the oral cavity and 23 patients (45%) with squamous cell carcinoma of the oropharynx. All 51 patients underwent primary surgical treatment, extended by adjuvant radiotherapy in 33 patients (65%). Cases with cT2 (n=23; 45%) and cN0 (n=25; 49%) were the most common. Twenty-seven patients (53%) were HPV-positive, including 14 (27%) patients with squamous cell carcinoma of the oral cavity and 13 (25%) with squamous cell carcinoma of the oropharynx. Twenty-one patients (41%) were HPV positive p 16 (+). The Kaplan-Meier method was used to assess 3-year overall survival (OS) and 3-year locoregional control (LRC) in the study group, depending on tumor location and HPV status. Results: The 3-year OS rates in the group of HPV-positive and HPV-negative patients with oral cavity tumors were 80% and 82%, respectively; p=0.965, while in the group of HPV-positive and HPV-negative patients with oropharyngeal cancer, the 3-year OS rates were 100% and 67.5%, respectively; p=0.045. In patients with oropharyngeal cancer, the 3-year LRC rates for HPV-positive and HPV-negative patients were 100% and 59%, respectively; p=0.059. In the group of patients with oral cavity cancer, 3-year LRC rates for HPV-positive and HPV-negative subjects were 55% and 52%, respectively; p=0.771. 3-year OS for HPV-positive p16 (+) and HPV-positive p16 (-) was 88% and 78%, respectively; (p=0.965). In the whole study group, HPV-negative patients were more often offered adjuvant treatment compared to HPV-positive patients. Conclusions: The 3-year OS rates of patients with cancers of the oral cavity and the oropharynx treated with combined therapy in the study group did not differ statistically significantly depending on HPV status. HPV-positive patients with oropharyngeal cancer had better 3-year OS and LRC rates compared to HPV-negative patients. Patients with HPV-dependent oropharyngeal cancer, no statistically significant improvement in 3-year OS was observed compared to patients with HPV p16 (+) vs HPV p 16 (-). However, in the group of patients with oral cavity cancer, HPV status did not show prognostic significance.

Abstract: To understand tropical avian ectoparasitism, we analyzed bird–tick data and remote-sensing habitat metrics across the Colombian Andes (2008-2019), sampling 1,164 birds (204 species) across 15 localities (538-3121 m a.s.l.). Macrohabitat variables, land use/land cover (LULC), forest structure, and bioclimatic data were derived from Sentinel-2/CIAT raster products (5 km buffers); microhabitat variables comprised avian morphological and behavioral traits. Random Forest Models (RFMs) linked host and habitat factors to tick presence. Tick prevalence and host assemblages varied significantly by elevation and Andean region. Eastern Andean sites had the highest bird richness and tick infestation rates, whereas middle elevations had the lowest. Remote sensing revealed major LULC shifts over the study period. Forest decline and increased cropland/grassland, especially in the Western Andes, while Eastern sites showed a mixture of habitat loss and recovery. Macrohabitat models identified elevation and vegetation structure (Barren/Sparse vegetation, Closed shrublands) as strong predictors of tick presence (AUC = 95; R² = 62.3%). Microhabitat models highlighted host behavior and morphology (mixed-flock participation, bill shape, foraging strata) (AUC = 82.5%). The integrated model performed best (AUC = 98; R² = 76.4%), confirming that tick–bird dynamics are driven by combined environmental gradients and host traits. Our findings emphasize the value of multi-scale, remote-sensing-informed frameworks for predicting ectoparasite risk in complex tropical systems.

Abstract: Cockroaches display a double symbiosis: an obligate intracellular one with Blattabacterium cuenoti, and a complex extracellular intestinal non-vertically transmitted microbiota, that may be affected by horizontally transmitted factors. Four experiments using 16S rRNA gene amplicon sequencing analyzed the microbiota of the hindgut and feces of adult cockroaches to understand the influence of the environment, feces, and host genetic background on hindgut microbiota acquisition and development. We observed that sample type, rearing conditions, and host influenced microbiota composition. Furthermore, the induction of germ-free cockroaches placed in non-sterile conditions had a greater impact on microbiota than rearing conditions, also showing that in absence of fecal inputs the cockroach gut microbiota is strongly diminished. Moreover, when exploring fecal microbiota differences between three cockroach species, the greatest divergence was found between Periplaneta americana and Blattella germanica, with Blatta orientalis being placed in an intermediate position. Therefore, P. americana was selected for fecal transplantation on B. germanica. This transplantation experiment indicates that host species clearly influence intestinal bacterial selection, limiting full integration of donor-derived communities. Overall, these results suggest that beyond other factors the host species had the strongest influence on shaping the cockroach gut microbiota.

Abstract: Reaction time (RT) is widely used as a fundamental indicator of central nervous system processing speed. Numerous studies have shown that RT increases with age, generally in-terpreted as a decline in information processing efficiency. However, most previous studies have focused on absolute RT values, and it remains unclear whether aging also alters the relative relationships between responses under different task conditions. The present study investigated whether aging affects the relative difference between inside and outside pedal reaction times in a Foot Psychomotor Vigilance Test (Foot PVT). A total of 44 participants were analyzed, including 20 younger adults (24 ± 3 years) and 24 older adults (73 ± 5 years). Participants responded to visual stimuli by pressing either the left or right pedal with the right foot. The difference between inside and outside RT (dRT) was calculated for each participant as an index of relative response structure. Group compari-sons and correlation analyses were conducted to examine associations with age, height, physical activity level (PAL), and sleep-related factors. As expected, RTs were consistently longer in older adults across conditions. In contrast, dRT did not differ significantly be-tween younger and older groups, with negligible effect sizes(|d|< 0.1). Furthermore, dRT showed no significant correlations with height, PAL, or sleep-related indices. These find-ings indicate that while aging affects the overall speed of motor responses, the relative temporal structure between response conditions is preserved. This dissociation between global slowing and stable response structure may represent a fundamental characteristic of neuromotor aging.

Abstract: Periodontal disease (PD) affects a large proportion of adults and is increasingly associated with systemic inflammation and neurodegenerative risk. However, current therapies have limited efficacy in disrupting biofilms and modulating systemic responses. In this pilot study, we evaluated epigallocatechin-3-gallate-palmitate (EGCG-palmitate or EC16) nanoparticles (NPs), a lipid-soluble derivative of epigallocatechin-3-gallate (EGCG), generated using Facilitated Self-Assembling Technology (FAST), a green nanotechnology that enables spontaneous formation of stable nanoparticles without surfactants or carrier materials. We hypothesized that EC16 NPs could inhibit periodontal pathogens and modulate neuroinflammatory responses. Antimicrobial activity was assessed in vitro, and potential therapeutic effects were evaluated in a ligature + pathogen-induced mouse model of periodontitis. EC16 NPs inhibited the growth of Porphyromonas gingivalis. Oral administration of EC16 NPs (0.02% w/v equivalent to 16-20 mg/kg) significantly reduced bacterial load and decreased alveolar bone loss by approximately 50% compared with controls. Importantly, biodistribution analysis using Cy5-labeled EC16 NPs demonstrated detectable signals in mouse brain tissue following oral gavage, indicating EC16 NPs can cross the blood–brain barrier. This represents, to our knowledge, the first evidence that an orally administered EGCG derivative in nanoparticle form reaches the central nervous system and induces biological responses. In addition, EC16 NP treatment was associated with increased regulatory T cell (Treg) populations in cervical lymph nodes and reduced expression of inflammatory (IL-1β) and senescence-related markers (p16, p53) in brain tissue. These findings demonstrate that EC16 nanoparticles possess dual local and systemic activity and support further investigation of FAST-enabled nanoformulations as a novel therapeutic strategy for periodontal disease and inflammation-related brain conditions.

Abstract:

The black soldier fly (BSF; Hermetia illucens) is rapidly emerging as a model for evolutionary biology and insect biotechnology. Although larval biology has been extensively characterised, the reproductive biology of adults remains comparatively understudied. In this review, we synthesise the most recent empirical work on physiology, behavioural and chemical ecology to open the “black box” of BSF reproduction, focusing on processes that span eclosion to senescence. We highlight pre- and post-mating mechanisms that determine overall reproductive fitness: from mating latency, lekking dynamics, courtship and copulation, to sperm transfer, storage and oviposition. We discuss these processes within the framework of sexual selection theory. Several notable characteristics of BSF reproduction differ from traditional insect models. These include a hybrid capital-income breeding strategy (adults do not need to feed but can benefit from supplemental nutrition), protandry (early male emergence), sex-specific longevity that varies with mating status and a lek-like mating system. In addition, females possess morphologically complex sperm‑storage organs, providing ample opportunity for intense post‑copulatory sexual selection. Recent work shows that environmental factors such as light, humidity, temperature, substrate volatiles and rearing design strongly influence reproductive output in industrial settings, highlighting the potential for BSF to bridge fundamental and applied research. We propose a novel conceptual framework that integrates these elements and outline key unresolved questions (e.g., mechanisms of sperm precedence, female control of fertilization, reproductive barriers, drivers of speciation etc.). This interdisciplinary model supports both fundamental insights into the evolution of reproductive traits and provides practical improvements for optimizing industrial mass-rearing.

Abstract: Oncomodulin (OCM) is the most abundant Ca2+ buffering protein found in mature outer hair cells (OHCs). Cadherin 23 (CDH23) is a crucial component of the tip-links in hair cell stereocilia. The absence or dysfunction of these two proteins contributes to the early onset of age-related hearing loss (AHL). In this study, we investigated the effects of the Cdh23^753G→A mutation on OHC function using new Ocm-knockout (KO) mouse models (Ocm^tm1a/tm1a) with or without the Cdh23^753G→A mutation. Despite having the same genetic background, Ocm-KO mice carrying the Cdh23^753G→A mutation displayed significant hearing loss and a notable decline in OHC function across all measured frequencies as early as three months of age. In contrast, Ocm-KO mice without the Cdh23^753G→A mutation did not exhibit comparable hearing loss until they reached twelve months of age. Additionally, we examined the role of OCM in preserving OHC function under ototoxic stress induced by HPβCD (2-hydroxypropyl-β-cyclodextrin). The administration of HPβCD resulted in a more pronounced decline in OHC function in Ocm-KO mice compared to wild-type (WT) mice, as indicated by distortion product otoacoustic emissions (DPOAE) measurements. Furthermore, HPβCD treatment led to greater structural deterioration and more rapid rupture events in OHCs from Ocm-KO mice than in those from WT mice, as demonstrated by in vitro live imaging. These findings suggest that the Cdh23^753G→A mutation, rather than other potential strain-specific genetic factors associated with AHL, solely exacerbates the early onset of AHL phenotypes in Ocm-KO mice. Furthermore, our data indicates that OCM proteins in OHCs enhance their ability to withstand ototoxic stimuli.