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Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2026

Impaired proteostasis is an early feature of the diabetic heart in humans and mice

Diabetes and obesity increase cardiac lipid levels leading to cardiomyopathy and heart failure. We hypothesized that intermittent fasting would reduce cardiac lipid levels. Surprisingly, intermittent fasting increased myocardial triglyceride content, but rescued mortality and attenuated cardiomyopathy in mice overexpressing cardiomyocyte acyl-CoA synthetase 1 (MHC-ACSL1). Lipid overload caused cardiomyocyte accumulation of polyubiquitinated protein aggregates containing desmin, a scaffolding intermediate filament protein, which intermittent fasting prevented. Furthermore, intermittent fasting reversed elevated myocardial C16:0 ceramide content, and knockdown of ceramide synthase CerS5 and CerS6 reduced palmitate-induced protein aggregation, highlighting a role for C16:0 ceramides in this pathology. Conversely, impairing aggrephagy with cardiomyocyte-specific p62 ablation induced heart failure in mice fed a high-fat diet, with paradoxically reduced cardiac lipid content. Crucially, non-failing diabetic human hearts also exhibited protein aggregate pathology. Taken together, these results demonstrate that impaired proteostasis characterizes cardiomyopathy from cardiac lipid overload and identify a promising new therapeutic target for this condition.

molecular biology↗

bioRxiv2026

Structures of pUG-fold RNA bound to DNMT1 reveal a mechanism for RNA-mediated epigenetic regulation

Many chromatin-associated proteins have been found to bind RNA as a means of epigenetic regulation. Specifically, DNA methyltransferase 1 (DNMT1), which maintains cytosine methylation at CpG dinucleotides, is inhibited by RNA at transcribed DNA loci in cells. However, the mechanisms by which RNA binds DNMT1 and inhibits its activity remain unknown. Here, we determine a series of cryogenic electron microscopy (cryo-EM) structures of human DNMT1 bound to pUG-fold RNA, a non-canonical G-quadruplex previously observed to inhibit activity, revealing two distinct RNA-binding modes. The pUG-fold RNA binds the surface of DNMT1 in its autoinhibited conformation across a positively charged surface between the methyltransferase domain and the CXXC domain, and it binds directly in the active site of an open DNMT1 conformation. RNA binding is sterically incompatible with substrate DNA engagement in both states. Our 2.5 [A] structure captures the intricate network of hydrogen bonds and electrostatic interactions between amino acids in the methyltransferase domain and the tetrad layers of pUG-fold RNA. Metadynamics molecular dynamics simulations provide an orthogonal view of the conformational landscape of DNMT1, revealing the two distinct RNA-binding modes. Furthermore, our analysis of published DNMT1 RIP-seq and eCLIP-seq data confirms that DNMT1-interacting RNAs in cells exhibit a strong propensity to form non-canonical G-quadruplex RNA structures. Collectively, our study provides the first structural basis for pUG-fold RNA recognition by a protein and illustrates how cryo-EM and AI-based methods for protein and RNA structure prediction synergize to inform the mechanism of RNA-mediated regulation of DNMT1.

biochemistry↗

bioRxiv2026

Background proteome correction promotes confident identification of dynamic protein-protein interactions between different biological contexts

Affinity purification-mass spectrometry (AP-MS) enables the characterization of protein-protein interactions (PPIs), and the ease and sensitivity of such experiments has progressively increased. Beyond steady-state interactions of target proteins, a strong interest has emerged in monitoring how PPIs change upon significant biological perturbations, such as in disease contexts or small molecule modulation of the target protein. These perturbations likely not only induce PPI changes but can also lead to altered expression of proteins not of direct interest. Changes in protein abundance may alter which proteins adsorb to the affinity purification matrix, and due to the sensitivity of modern mass spectrometers, these differential ''background binders'' can masquerade as differential interactors. Contemporary approaches often do not account for differences in the background proteome, potentially inflating the number of false positives and negatives reported. Here, we provide technical considerations for the reliable annotation of dynamic PPIs, using the O-GlcNAc transferase (OGT) as a case study. We describe the installation of affinity epitope tags on endogenous OGT in mouse embryonic stem cells (mESCs), which we then apply for OGT interactor identification via AP-MS. We show that accurate representation of the bead background, which depends on the affinity matrix in use, is critical for elimination of false positive and false negative PPIs. This became even more pertinent as OGT PPI dynamics were measured under OGT catalytic inhibition via OSMI-4, which is known to perturb gene expression. The proteomes of OSMI-4-treated and control-treated mESCs differed, leading to distinct bead backgrounds in which the differential background proteins appeared as interaction gains or losses. These false positives were resolved by incorporating straightforward experimental controls through a practical statistical framework, allowing for a direct and confident comparison between treatment conditions. Incorporating these considerations into workflows investigating PPI dynamics will improve data fidelity and reproducibility.

biochemistry↗

bioRxiv2026

Gaussian accelerated Molecular Dynamics - Thermodynamic Integration (GaMD-TI): Improved alchemical free energy calculations with enhanced sampling

It is valuable to calculate alchemical free energy changes in drug discovery and development. Thermodynamics Integration (TI) has been widely used in computational chemistry for estimating free energy changes with alchemical transformations. However, TI based on usually short Molecular Dynamics (MD) simulations often suffers from insufficient conformational sampling. Here, we have integrated Gaussian accelerated MD and TI (GaMD-TI) to enhance the conformational sampling and improve accuracy of free energy calculations. GaMD-TI has been demonstrated in model systems of alchemical changes in the Valine dipeptide and mutation cycle of the Alanine <-> Valine <-> Isoleucine (AVI) residues. Simulations showed that when GaMD boost potentials followed near-Gaussian distribution, the free energy change could be reweighted accurately through generalized cumulant expansion to the second order. The total free energy change often exhibited faster convergence using Selective GaMD (SGaMD) than using conventional MD (cMD). Accuracy of the free energy estimates from SGaMD-TI simulations was similar to or higher than those from cMD-TI simulations, although the differences were subtle for these small model systems. Meanwhile, dihedral angles in the model systems underwent significantly more frequent conformational transitions in SGaMD than in cMD, indicating improved sampling. Future studies are planned on larger systems with more complicated alchemical changes, such as ligand binding to proteins/nucleic acids and mutations at biomolecular binding interfaces. GaMD-TI should be broadly applicable to alchemical free energy calculations and therapeutic design.

biochemistry↗

bioRxiv2026

Ribosomal proteins are major substrates of starvation-induced endosomal microautophagy in Drosophila.

Maintenance of cellular homeostasis requires tight coordination between protein synthesis and degradation, particularly at old age and under conditions of stress including starvation. Autophagy contributes to sustain this balance by degrading cytoplasmic proteins and organelles. It thus is essential to prevent the accumulation of damaged proteins and organelles and to recycle nutrients. Of the three forms of autophagy, macroautophagy, chaperone mediated autophagy, and (endosomal) microautophagy (e-MI), the latter remains the least well understood. During e-MI, cytosolic substrate proteins are captured into late endosomes via ESCRT-dependent multivesicular body formation and then degraded in late endosomes or lysosomes. e-MI is thought to contribute to protein quality control under basal conditions and under stress. Importantly, very little is known about the endogenous substrates of e-MI in flies and thus about its physiological role. Performing integrative multi-omic analyses in Drosophila larval fat body that has functions similar to mammalian liver and adipose tissue, we identified 153 high-confidence endogenous e-MI substrates with the degradation of ribosomal proteins by e-MI being the most strongly affected functional category. Generally, we found that starvation caused the depletion of proteins involved in translation, aminoacyl-tRNA synthesis, and ribosomal biogenesis, without affecting their level of transcripts. Importantly, we observe a striking specificity between e-MI and macroautophagy, as the two pathways largely target distinct protein sets including different subsets of ribosomal proteins. Our metabolomic analysis further shows that genetic inhibition of e-MI reverses the reduced levels of amino acid caused by starvation. Together, our findings reveal ribosome turnover as a central physiological function of Drosophila e-MI and establish e-MI as a pathway driving metabolic adaptation during starvation.

cell biology↗

bioRxiv2026

Persistent but variable effect of experimental laboratory burns on microbial community resistance, resilience, and function across contrasting boreal forest soils

Boreal forests stretch across vast swaths of the northern hemisphere, are shaped by wildfire, and play an important role in the global carbon cycle. Microorganisms play a critical role in soil nutrient cycling in these ecosystems, yet there are many open questions about the impacts of wildfire on microbially mediated soil biogeochemical cycles. In this study, we used laboratory burns and soil incubations of intact soil cores collected from two distinct soil types -- Histosols and Gleysols -- from boreal forest within Wood Buffalo National Park, Alberta, Canada, to assess burn effects on soil bacterial and fungal community composition and function. We compared resistance and resilience to burning for microbial communities vs. resistance and resilience to burning for soil pH and soil respiration to assess the relationships between burn-induced shifts in microbial community composition, the soil environment, and microbial activity. To link shifts in microbial community composition to potential community function, we measured glucose-specific carbon use efficiency (CUE) and assessed its relationship with weighted mean predicted 16S rRNA gene copy numbers for bacterial communities and FUNGuild-estimated relative abundance of putative symbiotrophic and saprotrophic fungi in burned and unburned soils. Microbial community resistance and resilience to burning varied across soil type with higher resistance of both bacterial and fungal communities from Histosols compared to the O horizons of Gleysols. This may be explained by a larger impact of burning on microbes in the thinner Gleysol O horizons. The relatively low resilience of bacterial and fungal communities to burning as well as the failure of resilience to increase with time since burning supports previous reports of post-burn microbial community recovery occurring over years rather than months. Burning caused a decrease in CUE with larger decreases following longer, hotter burns, which correlated with an increase in weighted mean predicted 16S rRNA gene copy number, raising the possibility that copy number could serve as a proxy for post-fire CUE in boreal forest soils, though more research is needed to constrain the effects of environmental conditions, substrates, and time since fire on this relationship. These findings suggest several ways in which burn-induced shifts in microbial community composition reflect altered microbial community function in meaningful ways for soil carbon cycling.

ecology↗

bioRxiv2026

Proteomic analysis of Stony Coral Tissue Loss Disease demonstrates coral-algal dysbiosis during disease progression

Stony Coral Tissue Loss Disease (SCTLD) has devastated Caribbean reefs, yet the host molecular response to infection is poorly understood. Previous gene expression studies of diseased corals identified shifts in the immune response, apoptosis, and coral-algal dysbiosis. Here, we characterized the proteomic response of Diploria labyrinthiformis to SCTLD by comparing protein abundance in healthy tissue from uninfected colonies and apparently healthy and neighboring diseased tissue from infected colonies. These results were compared with existing metagenomic data from the same samples that previously demonstrated significant shifts in the coral microbiome due to SCTLD. We identified 480 differentially abundant proteins when comparing diseased lesion and healthy tissues, but only 12 between apparently healthy and healthy tissues. Pathway-level analysis provides evidence of immune suppression in apparently healthy tissue, suggesting that host molecular responses precede visible disease progression. Diseased lesion tissues showed wound-healing responses combined with a decreased abundance of proteins involved in symbiosome maintenance and increased oxidative stress responses, consistent with host-algal dysbiosis. This result correlates with the previous metagenomic analysis of these samples which found that infected colonies exhibit distinct algal symbiont communities dominated by Symbiodinium necroappetens, whereas healthy colonies are dominated by Durusdinium trenchii and Breviolum spp. Comparison with existing transcriptomic studies revealed both shared and distinct molecular responses, underscoring the importance of integrating multi-omics approaches to understand coral diseases. Our results suggest that SCTLD in D. labyrinthiformis is associated with early immune suppression, coral-algal dysbiosis, oxidative stress, and subsequent wound-healing responses.

ecology↗

bioRxiv2026

Effects of spectral light quality on growth, photosynthetic pigments and bioactive compounds in Brassicaceae microgreens

LED spectral composition is an important tool for improving the growth and nutritional quality of microgreens cultivated in controlled environments. This study evaluated the effects of three LED light treatments on growth, morphology, pigments, primary metabolites, phenolic composition, and antioxidant capacity in arugula (Eruca sativa), mustard (Brassica juncea), and radish (Raphanus sativus) microgreens. Microgreens were cultivated under controlled environmental conditions and exposed to broad-spectrum white (W), blue-enriched white (WB), and red-enriched white (R) light at a photosynthetic photon flux density of 200 micromol/m2/s. Light quality did not affect yield in any species. However, R increased cotyledon area in arugula by 50 to 60% and promoted hypocotyl elongation in both arugula and radish, whereas W resulted in the longest hypocotyls in mustard. Photosynthetic pigment composition responded differently among species. In mustard, WB increased the chlorophyll a/b ratio (1.12 to 1.18), whereas lutein concentration decreased from 7.06 to 4.20 mg 100 g/FW. Primary metabolism also responded to light treatments in a species-dependent manner. In mustard, W increased glucose (0.43 vs. 0.26 and 0.29 g 100 g/ FW) and fructose (0.33 vs. 0.20 and 0.22 g 100 g/ FW) concentrations compared with WB and R. Organic acid composition was more responsive to light treatments in radish, with higher concentrations under R. Phenolic metabolism also responded in a species-dependent manner. In mustard, W increased total phenolic content to 0.25 mg GAE g/FW compared with 0.15 mg GAE g/FW under WB and R, and ABTS antioxidant capacity to 1.17 mg TE g/FW compared with 0.74 and 0.75 mg TE g/FW under WB and R, respectively. Individual phenolic compounds were also affected by light treatments, particularly in arugula and mustard. These findings demonstrate that the effects of LED spectral composition on microgreen quality are highly species-dependent. Therefore, LED light spectra should be optimized according to the target species and the desired quality attributes rather than applying a single lighting strategy to all Brassicaceae microgreens.

plant biology↗

bioRxiv2026

HRV-GUI: A MATLAB Graphical User Interface for Heart Rate Variability Analysis and Validation Using Human, Rodent, and Clinical Diabetic Gastroparesis Data

Background and Objective: Heart rate variability (HRV) analysis provides a non-invasive method for quantifying autonomic modulation from electrocardiographic recordings. However, practical HRV analysis often depends on fragmented workflows, limited signal-quality review, and software tools optimized for either human or preclinical recordings, but not both. This study developed and evaluated HRV-GUI, a MATLAB-based graphical interface for electrocardiogram (ECG)-derived HRV analysis in translational biomedical research. Methods: The HRV-GUI integrates electrocardiographic and RR interval loading, human and rat analysis modes, preprocessing, segment selection, automated R-peak detection, manual peak correction, RR interval generation, multi-domain HRV computation, diagnostic visualization, result export, and session saving/loading. The software was evaluated using deterministic synthetic RR interval datasets, baseline recordings from healthy human controls and healthy rats, and a clinical use-case comparison between healthy controls and patients with diabetic gastroparesis. Results: The HRV-GUI produced expected outputs in synthetic RR validation tests, including constant RR sequences, alternating RR sequences, outlier-containing RR sequences, and low-frequency- or high-frequency-dominant sinusoidal RR modulation. The software generated physiologically plausible HRV profiles in both human and rat recordings. In the clinical use-case analysis, patients with diabetic gastroparesis showed higher heart rate and sympathetic index, together with lower respiratory sinus arrhythmia, absolute low- and high-frequency spectral power, standard deviation of normal-to-normal intervals (SDNN), root mean square of successive differences (RMSSD), percentage of successive RR intervals differing by more than 50 ms (pNN50), Poincare short-term variability (SD1), and Poincare long-term variability (SD2) compared with healthy controls. Conclusions: HRV-GUI provides an integrated biomedical software workflow for ECG-derived HRV analysis. The validation results support its use for controlled RR testing, human and rodent ECG recordings, and clinical autonomic assessment in diabetic gastroparesis.

bioengineering↗

bioRxiv2026

Structural mechanism governing radiationless energy transfer in Renilla bioluminescence

The nonradiative transport of electronic excitation from one chromophore to another, known as resonance energy transfer, lies at the root of photochemical processes in biology. Unlike photosynthesis, bioluminescence converts chemical energy into light through an enzymatic oxygenation of an energy-rich luciferin. In glowing cnidarians, the energy is relocated from an excited oxyluciferin to a fluorescent protein, shifting the colour and enhancing the quantum yield of a photogenic reaction. How protein-chromophore complexes assemble during this interplay in real space, and what this association entails for function, are unknown. Here, we report co-crystal structures of a 120-kilodalton energy-transfer complex from the luminescent soft coral Renilla reniformis. We find a heterotetrameric 2:2 assembly composed of two coelenteramide-loaded luciferases (RrLuc) docked at opposite sides of a head-to-tail dimer of green fluorescent protein (RrGFP). The edge-to-edge distance between donor and acceptor chromophores is below 3 nm, favouring the Forster-type radiationless energy transfer. Furthermore, RrGFP serves not only as a colour-switchable antenna and luminescence amplifier but also tunes the efficiency of luciferase catalysis by controlling its inherent dynamics. Our results provide detailed spatial information about intermolecular dipole-dipole coupling in Renilla bioluminescence, including the arrangement of donor-acceptor pairs that secure excited-state energy transfer with exquisite precision.

biochemistry↗

bioRxiv2026

Spatial profiling and neurovascular communication in the developing and adolescent cortex following prenatal alcohol exposure

Fetal alcohol spectrum disorders (FASD) constitute a wide range of developmental, cognitive, and behavioral impairments caused by prenatal alcohol exposure (PAE). Although neuronal and vascular consequences of PAE have been studied, how alcohol affects the cerebrovasculature within the framework of the neurovascular unit (NVU) across development remains poorly understood. At minimum, the NVU comprises neurons, astrocyte endfeet, and endothelial cells (ECs), which coordinate to maintain brain homeostasis. Here, we used the NanoString Digital Spatial Profiling platform to characterize spatial transcriptomic data from neurons, astrocytes, and ECs from PAE and saccharin (SAC) control cortices at embryonic day 18 (E18) and postnatal day 28 (P28). Differentially expressed genes were then used for Ingenuity Pathway Analysis (IPA) to identify altered biological pathways and perform comparison analyses across developmental time points, while CellChat was used to infer cell cell communication networks. We uncovered thousands of differentially expressed genes and numerous altered pathways and biological processes in PAE cortices across development. Both IPA and CellChat analyses implicated dysregulation of vascular and extracellular matrix (ECM) remodeling, cell adhesion, and neuroinflammatory signaling. CellChat further predicted the loss of several key bidirectional relationships and altered ligand-receptor interactions among neurovascular cell types at E18 and P28. Overall, these findings identify PAE associated alterations in neurovascular gene expression and intercellular signaling across development, providing potential mechanisms by which PAE may disrupt neurodevelopment.

molecular biology↗

bioRxiv2026

Rewiring of Integrin Signaling and Cell-cycle Deregulation Drive SMARCB1-Deficient Epithelioid Sarcoma

Epithelioid sarcoma (EPS) is an aggressive soft-tissue sarcoma characterized by loss of the chromatin-remodeling subunit SMARCB1. The oncogenic programs driving EPS remain poorly understood. Through CRISPR loss-of-function screens, we identified conserved dependencies on integrin signaling components and cyclin-dependent kinases (CDKs). Genetic disruption of integrin subunit alpha V (ITGAV)-centered signaling impaired epithelioid cluster formation and reduced MYC expression. SMARCB1 re-expression phenocopied these effects and revealed that SMARCB1 loss selectively represses context-dependent integrin subunits while preserving an ITGAV-centered pro-survival axis, associated with altered BAF complex occupancy. Analysis of EPS cell lines and primary tumors revealed frequent genetic or epigenetic inactivation of CDKN2A/p16, indicating that loss of cell-cycle control is a key cooperating event in EPS development and providing a mechanistic rationale for targeting CDK4/6. Together, these findings establish integrin-driven oncogenic signaling coupled with disruption of cell-cycle control as a central oncogenic program in EPS and identify actionable therapeutic vulnerabilities.

cancer biology↗

bioRxiv2026

Why are fishers retaining manta and devil ray bycatch?

Increasing fishing pressure, including from small-scale fisheries, has caused declines in more than one-third of all elasmobranch species. Tackling conservation issues in small-scale fisheries requires interdisciplinary approaches due to the coastal community's interdependence on marine resources. To support inclusive policy change and fisher engagement, an understanding of the motivations driving fishers' operational choices (i.e., the retention of elasmobranch bycatch) is needed. We assess the motivational drivers behind bycatch retention of one of the slowest-growing and most vulnerable elasmobranch groups, manta and devil rays (collectively, mobulids), through a case study in India, their largest fishery in the world. We conducted a best-worst scaling survey in the fishery-intensive states of Tamil Nadu and Andhra Pradesh, which make significant contributions to mobulid landings on India's east coast. Our results suggest that fishers exhibit varied motivations for retaining mobulid bycatch across states. Financial motivation to sell mobulids for additional revenue was the most important motivator for bycatch retention in both states. In Tamil Nadu, the top three motivators were all financially driven, whereas in Andhra Pradesh, the top three motivators included both financial and non-financial attributes, such as nutritional importance and storage optimisation. As the first socio-economic study of mobulid fisheries in India, we show that motivations underlying bycatch retention decisions vary geographically and may be influenced by cultural differences between states and the socio-economic characteristics of decision makers. Based on identified fisher motivations, we provide context-specific recommendations to align conservation strategies with the values fishers derive from the mobulid fishery and encourage participation in conservation. These include subsidies for net repair to encourage mobulid release; promotion of a minimum price measure for sustainably sourced alternative species; quality improvement of target species; and increased awareness of national and international regulatory obligations (e.g., CITES, CMS, IOTC).

ecology↗

bioRxiv2026

Adeno-Associated Virus Mediated Expression of Bcl-xL Attenuates Apoptosis in Fuchs Endothelial Corneal Dystrophy

Fuchs Endothelial Corneal Dystrophy (FECD) is characterized by progressive corneal endothelial cell loss and the formation of corneal guttae. Currently, there is a global shortage of donor corneas and new strategies are needed to reduce the need for corneal transplantation. While adeno-associated viruses (AAVs) have the capacity to deliver anti-apoptotic genes to human corneal endothelial cells (CECs), this has not been fully explored as a therapeutic strategy for FECD. In this study, we evaluated the transduction efficiency of self-complementary (sc-) and single-stranded (ss-) AAV2 serotypes in human CECs and ex vivo tissues and assessed whether AAV-mediated expression of Bcl-xL could attenuate apoptosis in FECD. Seventeen scAAV2 serotypes were screened for transduction efficiency in normal human CECs via green fluorescent protein (GFP) expression. The top 4 AAV2 serotypes were further evaluated in FECD cell lines, healthy cadaveric donor specimens, and FECD patient specimens. FECD cell lines were transduced with anti-apoptotic ssAAV2/5-Bcl-xL (AAV2/5-CAG-eGFP-P2A-BCLXL) and treated with etoposide to induce apoptosis. We found that scAAV2/5 demonstrated high transduction efficiency across all normal and FECD cell lines and tissues. We observed that ssAAV2/5-mediated expression of Bcl-xL provided significant protection against etoposide-induced apoptosis in FECD CECs (71.68%{+/-}0.69 vs 23.96%{+/-}8.88%, p=0.018). Our findings show that AAVs have the potential for therapeutic gene delivery to the human corneal endothelium, and that targeting the Bcl-xL mediated apoptotic pathway can be further explored as a therapeutic for FECD.

cell biology↗

bioRxiv2026

Structural basis for tetraspanin-dependent surface export and adhesive function of integrin α3β1

Integrin 3{beta}1 (ITG3{beta}1) is a member of an integrin subfamily that binds to laminin proteins and promotes attachment of epithelial cells to the basement membrane. ITG3{beta}1 forms a complex with the tetraspanin CD151, and loss-of-function mutations in both ITG3 and CD151 cause epidermolysis bullosa, a severe skin blistering disease resulting from a defect in basement membrane attachment. Here, we report the cryoEM structure of an ITG3{beta}1 complex with CD151 and show that mutation of CD151 at the binding interface disrupts complex formation in cells. Strikingly, CRISPR-mediated knockout of CD151 leads to a variably penetrant ITG3{beta}1 surface export defect that is restored by re-expression of wild-type but not interface-mutated CD151. Together, these studies define the molecular basis for binding of CD151 to ITG3{beta}1, and show that CD151 promotes ITG3{beta}1 surface export, providing a biochemical explanation for the CD151 loss-of-function phenotype in epidermolysis bullosa.

biochemistry↗

bioRxiv2026

Deep sequencing artificially inflates estimates of microbial diversity

Sequencing artifacts challenge accuracy and reproducibility when quantifying microbial diversity. To track error propagation in microbiome analyses, we analyze no-diversity amplicons, which are amplified from host genes with limited genetic diversity or from synthetic spike-ins. We find that sequencing at greater than 104 reads exponentially increased no-diversity amplicon sequence variant (ASV) richness, with hundreds of ASVs observed per sample. This striking pattern was shared with microbial amplicons (16S rRNA, ITS, gyrB, rpoB), which revealed inflated Shannon diversity with an increase in read counts for both the community and within taxa. Comparing sequencing error profiles between no-diversity and microbial amplicons showed that truncating reads to shorter lengths and use of the AVITI Element platform can mitigate, but not abolish, the impacts of artificial inflation; we recommend caution when read depths vary orders of magnitude between samples. Overall, utilizing no-diversity amplicons can help optimize parameters to improve estimates of microbial diversity.

ecology↗

bioRxiv2026

Cryo-EM structures reveal the mechanism of phosphatidylserine remodeling by membrane-bound glycerophospholipid O-acyltransferase 1

Lands cycle remodeling of glycerophospholipid acyl chains is crucial for cells to maintain appropriate membrane composition. Glycerophospholipids are cleaved at the glycerol sn2-position by phospholipase A. The lysophospholipids are reacylated by enzymes of the membrane-bound O-acyltransferase (MBOAT) family to incorporate specific fatty-acyl chains to adjust membrane properties. How MBOAT enzymes recognize specific acyl-CoA donors, select lysophospholipid acceptors, and release products is unclear. Phosphatidylserine (PS), a critical anionic phospholipid, controls membrane surface charge, signaling-protein recruitment, and cell-death-associated membrane recognition, and PS acyl-chain remodeling is linked to ferroptosis resistance. Here, we showed that MBOAT1 preferentially generates monounsaturated fatty acid-containing PS from lyso-PS. High-resolution cryo-electron microscopy structures of human MBOAT1 captured distinct binding poses of the fatty acyl donor, lyso-PS acceptor, and PS product. With lipidomics, enzymology and molecular dynamics simulations, these structures reveal the mechanism and pathway of MBOAT1-dependent PS remodeling.

biochemistry↗

bioRxiv2026

Combined Image-Based Profiling and Biochemical Analysis of GCaMP Overexpression Effects on Mammalian Cells

Protein-based fluorescent sensors are a powerful addition to the biology toolbox for their ability to be stably expressed within living organisms, tissues, cells, and subcellular compartments, with the capacity to report on the presence of specific target molecules or other analytes. At the same time, sensor components will unavoidably present opportunities for unintended interaction with endogenous cellular machinery, potentially confounding both sensor function and cell health. Interactions with host components may not be readily predictable during the sensor design process, especially when simultaneously optimizing many other sensor parameters such as fluorescence response, dynamic range, and kinetics. Characterizing effects of sensor expression on cells is currently a laborious ad hoc process; new methods to characterize the cell expression effects of sensors and their variants could dramatically improve sensor design pipelines, laying the groundwork to recognize potentially problematic expression side effects earlier in the iterative design and testing workflow. Here, we take a dual high-content imaging-based and biochemical approach to examine sensor interactions with native cell biology, focusing on the widely used GCaMP calcium sensor. We identify a morphology-based signature of the cellular effects of high sensor expression in a neuroblastoma cell line. Subsequently, we identify biochemical interactions between GCaMP and a component of the mammalian cytoskeleton and track morphological features in sensor-expressing cells that lack these structural components. Our findings present an entry point for engineering new minimally cross-reactive sensor versions given a contextual biological understanding of sensor overexpression. We anticipate that as this and related workflows are incorporated into sensor engineering pipelines, bioorthogonality can be more systematically assessed and prioritized in diverse sensor scaffolds.

cell biology↗