Biology subjects

Kovacevic, A.

Publications and source records attributed to Kovacevic, A..

10 recordsLinked to original sources

bioRxiv2026

XPO1 Inhibition by Selinexor Induces Nuclear p53 and p21 Accumulation, Cell-Cycle Arrest and Apoptosis in Testicular Germ Cell Tumors

Background: Testicular germ cell tumors (TGCTs) are the most common solid malignancies in young men. Despite high cure rates with cisplatin-based chemotherapy, resistance and long-term toxicities remain clinical challenges in TGCTs. Selinexor, an FDA-approved XPO1 inhibitor, has demonstrated anti-tumor activity in other cancers, but its potential in TGCTs remains unknown. Objective: We investigated the antitumor effects of selinexor in TGCT cell lines. Materials and methods: XPO1 RNA expression, protein abundance and localization were evaluated by immunohistochemistry in TGCT tissue microarrays and non-neoplastic testicular tissues, and by meta-analysis of publicly available gene-expression microarray datasets from normal testis, primary TGCT specimens as well as TGCT cell lines. Selinexor effects on cell viability, cell-cycle distribution and apoptosis were evaluated using XTT assay and flow cytometry. p53 and p21 expression and localization were analyzed by Western blotting and Immunofluorescence. Results: XPO1 expression was heterogeneous across normal testis, TGCT tissues and cell lines. Yet, selinexor reduced TGCT cell viability and induced G1 or G2/M cell-cycle arrest, apoptosis, increased total p53 and p21 protein levels, and their nuclear accumulation. Of note, control fibroblast exhibited limited sensitivity to selinexor. Discussion and conclusion: Our findings demonstrate that inhibition of XPO1 with selinexor might be potential therapeutic strategy for TGCTs.

cancer biology↗

bioRxiv2026

From sewage to shoreline: Tracing antibiotic resistance gene trends through tropical island wastewater treatment pathways

Wastewater is a key reservoir and transmission route for antibiotic resistance genes (ARGs), enabling their spread from influent to effluent and into receiving environments. However, how combined selective pressures (antibiotics, biocides, heavy metals, pharmaceuticals) influence resistant bacteria and ARG persistence over space and time remains poorly understood. Likewise, the role of the wastewater microbiome in ARG dynamics is still unclear, as few studies integrate microbiome shifts with chemical and environmental drivers. Here, we investigated how microbiome dynamics, chemical exposures, and environmental conditions shape clinically relevant ARG dynamics from sewage to receiving environments in Guadeloupe, French Caribbean. We analysed data collected from three wastewater continuums, (hospital-based, domestic, touristic) over four campaigns (September 2021-February 2023). We characterised ARG and microbiome composition spatiotemporal patterns and used a mixed-effect model to investigate ARG associations with potential drivers, including exposome factors, microbiome dissimilarity and environmental factors. Several ARGs were negatively associated with microbiome dissimilarity (Bray-Curtis distances) (aac(6)-Ib, aph(3)-III, blaSHV, blaTEM, intI1, qnrS, sul1 and tetM). Negative associations were also observed between upstream-downstream differences in anti-inflammatory drug concentrations and the abundance of aac(6)-Ib, aph(3)-III, blaCTX-M, ermB, intI1, and tetM. In contrast, ARG relative abundance was positively associated with upstream-downstream differences in antibiotic concentrations, suggesting selection along the continuum. These findings indicate that ARG dissemination along wastewater-to-coastal pathways is shaped by opposing processes, with microbiome turnover potentially limiting ARG persistence while chemical gradients promote specific gene enrichment. The outcome is ARG-specific, with implications for antimicrobial resistance risks associated with recreational waters, seafood consumption, and coastal ecosystem interactions.

microbiology↗

bioRxiv2026

Loss of perinuclear theca protein ACTRT2 causes subfertility and acrosome destabilization in mice

Actin-related protein T2 (ACTRT2) localizes to the perinuclear theca (PT) of male germ cells, yet its functional significance remains unclear. ACTRT2 is evolutionarily conserved and exhibits significant sequence similarity to other testis-specific actin-related proteins, with the highest conservation observed within the canonical actin core domain. We generated Actrt2-deficient mice which displayed male subfertility with pronounced acrosomal malformations originating during the Cap phase of acrosome biogenesis. Actrt2-deficient male mice showed reduced fertilization rate and poor blastocysts quality. Co-immunoprecipitation identified ACTRT2 interactions with PT proteins ACTRT1, ACTRT3, ACTL7A, ACTL9, PFN3, SPEM2 and CCIN while the interaction with CYLC1 was not detected. ACTRT2 overexpression in HEK293T cells altered cell morphology and F-actin distribution. Further, cytoskeletal regulator CFL1 was enriched in testis from Actrt2-deficient mice. We propose that ACTRT2 is a structural component of the PT stabilizing the acroplaxome during spermiogenesis and acrosome biogenesis by modulating actin dynamics. Finally, the high degree of sequence conservation and similarity with ACTRT1 and ACTRT3 together with their similar phenotypes when deleted, indicate that ACTRT2 shares a partial functional redundancy and compensatory capacity with other Arp proteins in testis. Taken together, these findings establish ACTRT2 as a structural regulator of sperm head architecture and male fertility in mice.

molecular biology↗

bioRxiv2026

Male mice heterozygous for Protamine-1 and Protamine-2 are infertile displaying sperm damage and retention of Protamine-2 precursors, transition proteins and histones.

BACKGROUNDDuring spermiogenesis, histones are exchanged by protamines (PRMs) in spermatids, which results in DNA hypercondensation and protection. Rodents and primates express two PRMs (PRM1 and PRM2) in a species-specific ratio. Maintaining this ratio is necessary for functional chromatin reorganization and alteration is associated with sub- or infertility in mice and humans. Prm1 and Prm2 deficient mice are infertile, while Prm1+/- males are subfertile showing a severely altered PRM ratio. Prm2+/- males are fertile and display a protamine ratio comparable to WT. OBJECTIVESHere, we addressed the question whether loss of one allele of Prm1 and one allele of Prm2 affects fertility. MATERIAL AND METHODSDouble heterozygous (dHET) mice lacking one allele of Prm1 and one allele of Prm2 were generated and analyzed RESULTSdHET males were infertile with sperm showing retention of histones and TNPs, high levels of PRM2 precursor and decreased levels of mature PRM2. In mature sperm the PRM ratio and the total PRM content was not altered. However, CMA3 staining revealed incomplete protamination and sperm nuclei appeared more rounded and slightly bigger, suggesting impaired DNA-hypercondensation. In dHET sperm, DNA degradation was apparent, but to a lower level compared to sperm from Prm1 and Prm2 deficient males. Increased 8-OHdG levels suggested oxidative stress in the epididymis of dHET mice. However, a fraction of dHET sperm were capable of fertilization, with embryonic development up to 8-cell stage. DISCUSSION AND CONCLUSIONThese results suggest, that male factor infertility might not be reliably detected by measuring PRM1/PRM2 ratio but rather by determining the level of protamination by e.g. CMA3 analysis and pre-PRM2 retention.

developmental biology↗

bioRxiv2026

Nanoscale Material Size Shapes Distinct Immune Transcriptional States Under Physiological Flow

Nanoplastics interact continuously with circulating immune cells, yet how particle size and exposure complexity shape immune transcriptional organization under physiological flow conditions remains poorly understood. Here, controlled microfluidic exposure was combined with single-cell RNA sequencing to investigate the effects of size-defined polystyrene nanoplastics (PSNPs; 40 nm, 200 nm, and 40 + 200 nm) on primary human peripheral blood mononuclear cells (PBMCs) under dynamic flow conditions. Across immune populations, PSNP exposure induced a conserved ribosome-associated and RNA-regulatory transcriptional program, indicating a shared intracellular adaptive response. Monocytes displayed the strongest transcriptional remodeling, characterized by coordinated modulation of ribosome-associated, metabolic, and inflammatory signaling pathways in a size-dependent manner. Exposure to 40 nm PSNPs negatively enriched (suppressed) mitochondrial metabolic pathways, whereas 200 nm PSNPs enriched inflammatory signaling programs. Combined exposure induced concurrent metabolic and inflammatory pathway engagement without evidence of major immune topology disruption or discrete inflammatory state transitions. In contrast, adaptive immune cells exhibited comparatively modest and lineage-preserving transcriptional modulation. Together, these findings demonstrate that nanoplastic size and exposure complexity shape coordinated immunometabolic adaptation in human immune cells under physiologically relevant flow conditions and establish a framework for studying dynamic material-immune interactions at single-cell resolution.

bioinformatics↗

bioRxiv2026

Tipping the balance: Synthesis and evaluation of centrinone-based degraders of polo-like kinase 4

Polo-like kinase 4 (PLK4) is a serine/threonine-protein kinase that plays a pivotal role in centriole biogenesis and, as such, represents a master regulator of centriole duplication. Due to its importance in cancer development and progression, PLK4 represents an attractive target for the development of novel therapeutics. Herein, we present a series of molecular degraders of PLK4, based on the highly selective PLK4 inhibitor centrinone, with the aim of targeting PLK4 for degradation via the ubiquitin-proteasome system. While all synthesized degraders retained low nanomolar binding affinities to the kinase domain of PLK4, large differences were found with respect to their ability to change cellular PLK4 levels. We uncover a complex pharmacological profile of the most potent degraders, D6 and D10, consisting of concomitant lowering of PLK4 levels through degradation, and enhancing PLK4 levels through inhibition of its autoregulation - dependent on its localization at the centrioles.

cell biology↗

bioRxiv2025

Impacts of wastewater treatment and the exposome on antibiotic resistance gene dynamics: Insights from Guadeloupe, a French Caribbean Island

Wastewater treatment plants (WWTPs) are global hotspots for the dissemination of antibiotic resistance genes (ARGs), primarily driven by different anthropogenic activities. Chemical compounds commonly found in wastewater, including antibiotics, biocides, non-steroidal anti-inflammatory drugs (NSAIDs), and heavy metals, along with environmental factors such as temperature and precipitation, might enhance the selection of antibiotic-resistant bacteria (ARB) even at low concentrations, inadvertently promoting the proliferation of ARGs. Since WWTPs can vary in their removal efficiency and influent compositions, it is important to assess their impact in influencing the relative abundance of ARGs and pollutant concentrations in discharged effluent. We analyzed data collected in Guadeloupe, French Caribbean, using random forest and lasso to investigate changes in the resistome and exposome within untreated wastewater influents and treated effluents from three WWTPs. These WWTPs represented three distinct wastewater continuums--hospital-based, urban non-touristic, and urban touristic--each with unique influent compositions. The study, which spanned four sampling campaigns from September 2021 to January 2023, revealed that the reduction in ARG abundance by the WWTPs was lower than expected. Of the 16 clinically relevant genes and mobile genetic elements examined, the relative abundance of aph(3)-III, blaOXA, blaSHV, blaTEM, ermB, intI1, qnrS, and tetM was reduced by 59.8% to 89.9% across the three WWTPs. An increasing trend in the relative abundance of the mcr-1 gene was observed, showing a 9.52-fold increase in the treated effluent in the hospital continuum. Of the nine antibiotics tested, trimethoprim levels dropped by 70.8% in the touristic continuum and 78.8% in the non-touristic, while ciprofloxacin saw a significant 64.4% reduction only in the touristic continuum, while no other antibiotics or biocides among the eight tested were effectively removed. Regarding heavy metals, significant reductions were observed only in the non-touristic continuum, with cadmium (Cd), copper (Cu), and mercury (Hg) concentrations reduced by 32.5%, 21.1%, and 36.2%, respectively. Our findings provide insights into the associations between environmental factors and the relative abundance of ARGs in Guadeloupe. Generally, depending on the continuum, biocides, the antibiotic erythromycin, certain heavy metals (As, Cd, Cu, Cr, and Hg) and water temperature were associated with the relative abundance of clinically relevant ARGs. This study was not able to assess the impact of climate. Future research should include longitudinal studies with more frequent sampling to better investigate the evolution of antibiotic resistance in bacterial species and its associated drivers, including pollutants and climate-related factors.

microbiology↗

bioRxiv2025

Actin-related protein M1 (ARPM1) required for acrosome biogenesis and sperm function in mice

Actin-related proteins (Arps) are a superfamily of proteins which share sequence similarities with conventional actin and are involved in different cellular processes. Actin-related protein M1 (ARPM1) also known as actin-related protein T3 (ACTRT3) is a testis-enriched Arp which can be found in the perinuclear theca (PT) of murine round and elongating spermatids. ARPM1 forming a complex with Profilin 3 (PFN3) is lost in Pfn3-deficient sperm. We generated a mouse model deficient for Arpm1 and demonstrate that Arpm1-/- male mice are subfertile, with morphological aberrations of the acrosome. During spermiogenesis, defects become apparent in Cap phase of acrosome biogenesis when abnormal acrosomal granules are observed. Arpm1-deficiency causes deregulation of GM130 and TGN46 suggestive of defects in cis- and trans-Golgi trafficking required for acrosome development. Co-immunoprecipitation revealed that ARPM1 interacts with the PT-specific proteins ACTRT1, ACTRT2, ACTL7A and the sperm surface protein ZPBP, additionally to its already shown interaction with PFN3. We propose that ARPM1 acts as a structural component of the PT contributing to the cytoskeletal network connecting acrosome and nucleus. In addition, ARPM1 mediates the localization of ZPBP to enable fertilization and it tethers PFN3 to properly regulate Golgi-related acrosome development.

developmental biology↗

bioRxiv2022

Cylicins are a structural component of the sperm calyx being indispensable for male fertility in mice and human

Cylicins are testis-specific proteins, which are exclusively expressed during spermiogenesis. In mice and humans, two Cylicins, the gonosomal X-linked Cylicin 1 (Cylc1/CYLC1) and the autosomal Cylicin 2 (Cylc2/CYLC2) genes have been identified. Cylicins are cytoskeletal proteins with an overall positive charge due to lysine-rich repeats. While Cylicins have been localized in the acrosomal region of round spermatids, they resemble a major component of the calyx within the perinuclear theca at the posterior part of mature sperm nuclei. However, the role of Cylicins during spermiogenesis has not yet been investigated. Here, we applied CRISPR/Cas9-mediated gene-editing in zygotes to establish Cylc1- and Cylc2-deficient mouse lines as a model to study the function of these proteins. Cylc1 deficiency resulted in male subfertility, whereas Cylc2-/-, Cylc1-/y Cylc2+/-, and Cylc1-/y Cylc2-/- males were infertile. Phenotypical characterization revealed that loss of Cylicins prevents proper calyx assembly during spermiogenesis. This results in decreased epididymal sperm counts, impaired shedding of excess cytoplasm, and severe structural malformations, ultimately resulting in impaired sperm motility. Furthermore, exome sequencing identified an infertile man with a hemizygous variant in CYLC1 and a heterozygous variant in CYLC2, displaying morphological abnormalities of the sperm including the absence of the acrosome. Thus, our study highlights the relevance and importance of Cylicins for spermiogenic remodeling and male fertility in human and mouse, and provides the basis for further studies on unraveling the complex molecular interactions between perinuclear theca proteins required during spermiogenesis.

developmental biology↗

bioRxiv2022

COVID-19 pandemic responses may impact the spread of antibiotic-resistant bacteria: a modelling study

Non-pharmaceutical interventions implemented to block SARS-CoV-2 transmission in early 2020 led to global reductions in the incidence of invasive pneumococcal disease (IPD). By contrast, most European countries reported an increase in antibiotic resistance among invasive Streptococcus pneumoniae isolates from 2019 to 2020, while an increasing number of studies reported stable pneumococcal carriage prevalence over the same period. To disentangle the impacts of the COVID-19 pandemic on pneumococcal epidemiology in the community setting, we propose a mathematical model formalizing simultaneous transmission of SARS-CoV-2 and antibiotic-sensitive and -resistant strains of S. pneumoniae. To test hypotheses underlying these trends five mechanisms were built in into the model and examined: (1) a population-wide reduction of antibiotic prescriptions in the community, (2) lockdown effect on pneumococcal transmission, (3) a reduced risk of developing an IPD due to the absence of common respiratory viruses, (4) community azithromycin use in COVID-19 infected individuals, (5) and a longer carriage duration of antibiotic-resistant pneumococcal strains. Among 31 possible pandemic scenarios involving mechanisms individually or in combination, model simulations surprisingly identified only two scenarios that reproduced the reported trends in the general population. They included factors (1), (3), and (4). These scenarios replicated a nearly 50% reduction in annual IPD, and an increase in antibiotic resistance from 20% to 22%, all while maintaining a relatively stable pneumococcal carriage. Exploring further, higher SARS-CoV-2 R0 values and synergistic within-host virus- bacteria interaction mechanisms could have additionally contributed to the observed antibiotic resistance increase. Our work demonstrates the utility of the mathematical modeling approach in unraveling the complex effects of the COVID-19 pandemic responses on AMR dynamics.

evolutionary biology↗