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bioRxiv2023

Fully automated workflow for integrated sample digestion and Evotip loading enabling high-throughput clinical proteomics

Protein identification and quantification is an important tool for biomarker discovery. With the increased sensitivity and speed of modern mass spectrometers, sample-preparation remains a bottleneck for studying large cohorts. To address this issue, we prepared and evaluated a simple and efficient workflow on the Opentrons OT-2 (OT-2) robot that combines sample digestion, cleanup and Evotip loading in a fully automated manner, allowing the processing of up to 192 samples in 6 hours. Our results demonstrate a highly sensitive workflow yielding both reproducibility and stability even at low sample inputs. The workflow is optimized for minimal sample starting amount to reduce the costs for reagents needed for sample preparation, which is critical when analyzing large biological cohorts. Building on the digesting workflow, we incorporated an automated phosphopeptide enrichment step using magnetic Ti-IMAC beads. This allows for a fully automated proteome and phosphoproteome sample preparation in a single step with high sensitivity. Using the integrated workflow, we evaluated the effects of cancer immune therapy on the plasma proteome in metastatic melanoma patients.

molecular biology↗

bioRxiv2024

Profiling IOP-responsive genes in anterior and posterior ocular tissues in the rat CEI glaucoma model

PurposeThe rat Controlled Elevation of Intraocular pressure (CEI) model allows study of in vivo responses to defined intraocular pressures (IOP). In this study, we use Nanostring technology to investigate in vivo IOP-related gene responses in the trabecular meshwork (TM) and optic nerve head (ONH) simultaneously from the same animals. MethodsMale and female rats (N=35) were subject to CEI for 8-hours at pressures simulating mean, daytime normotensive rat IOP (CEI-20), or 2.5x IOP (CEI-50). Naive animals, receiving no anesthesia or surgical interventions, served as controls. Immediately after CEI, TM and ONH tissues were dissected, RNA isolated, and samples were analyzed with a Nanostring panel containing 770 genes. Post-processing, raw count data were uploaded to Rosalind(R) for differential gene expression analyses. ResultsFor the TM, 45 IOP-related genes were significant in the "CEI-50 vs. CEI-20" and "CEI-50 vs. naive" comparisons, with 15 genes common to both comparisons. Bioinformatics analysis identified Notch and TGF{beta} pathways to be the most up- and down-regulated KEGG pathways, respectively. For ONH, 22 significantly regulated genes were identified in the "CEI-50 vs. naive" comparison. Pathway analysis identified defense response and immune response as two significantly upregulated biological process pathways. ConclusionsThis study demonstrates the ability to assay IOP-responsive genes in both TM and ONH tissues simultaneously. In the TM, downregulation of TGF{beta} pathway genes suggest that TM responses may prevent TGF{beta}-induced extracellular matrix synthesis. For ONH, the initial response to elevated IOP may be protective, with astrocytes playing a key role in these gene responses.

molecular biology↗

bioRxiv2026

A novel long-amplicon rpoB primer pair for high resolution microbiome analysis at the species-level

The 16S rRNA gene is the most widely used genetic marker for microbial community profiling, but its limited sequence divergence often prevents species-level identification. The RNA polymerase {beta}-subunit gene (rpoB) offers higher sequence variability, single-copy occurrence, and stronger phylogenetic consistency, yet its adoption in metataxonomic studies has been constrained by the lack of universal primer sets. Here, we present a novel universal primer pair that amplifies an [~]1,800 bp rpoB region (rpoB_MV) compatible with long-read sequencing platforms. In silico evaluation across 17683 bacterial reference genomes demonstrated high universality, with over 86% of genomes predicted to amplify. Compared with full-length and partial 16S rRNA gene markers, the rpoB_MV amplicon exhibited significantly greater inter-species sequence divergence and improved phylogenetic concordance with core-genome trees. Sequencing of two complementary mock communities confirmed superior species-level identification accuracy, with misclassification rates below 0.01% and no reads assigned to unresolved species clusters. These results establish rpoB_MV as a robust alternative to 16S rRNA gene-based profiling for high-resolution metataxonomic applications. IMPORTANCEMicrobial community studies increasingly require species-level resolution because species within the same genus can differ substantially in pathogenicity, ecological function, and metabolic capacity. Current 16S rRNA gene-based methods frequently fail to distinguish closely related species, collapsing biologically distinct organisms into the same taxonomic assignment and obscuring community differences that matter for clinical diagnostics, food safety, and environmental monitoring. The rpoB_MV primer pair presented here overcomes this limitation by targeting a longer, more variable region of the rpoB gene, enabling accurate species-level identification across diverse bacterial phyla. Combined with advances in long-read sequencing, this approach provides researchers with a practical tool to resolve microbial communities at the species-level.

molecular biology↗

bioRxiv2024

PTTG1-Mediated Pericyte Dysfunction Drives Diabetes-Induced Microvascular Dysfunction

BackgroundPericytes are crucial for the development, stabilization, and functional regulation of microvasculature, especially in the retina. In diabetic retinopathy (DR), early loss of pericytes is a key event that drives microvascular dysfunction. Despite their critical role, the mechanisms underlying the functional heterogeneity of pericytes in DR remain poorly understood, impeding the development of effective therapeutic strategies. MethodsWe employed single-cell RNA sequencing to construct a comprehensive single- cell atlas of non-diabetic and diabetic retinas. Using bioinformatic clustering and subcluster analysis, we identified a specific pericyte subcluster associated with diabetic microvascular complications. Differential gene expression analysis and immunofluorescence validation highlighted PTTG1 as a potential key regulator of pericyte dysfunction. To investigate its functional role, we emplyed CRISPR/Cas9 and adenoviral vectors to modulate PTTG1 expression in vitro and in vivo. Combined transcriptomic and metabolomic approaches were used to explore the mechanistic pathways through which PTTG1 influences pericyte biology and vascular function. ResultsWe identified a novel pericyte subcluster characterized by elevated expression of PTTG1, which was strongly correlated with diabetic microvascular dysfunction. Silencing PTTG1 using CRISPR/Cas9 and siRNA in vitro mitigated pericyte dysfunction under high- glucose conditions. Targeted knockdown of PTTG1 using viral vectors improved retinal vascular integrity and reduced neovascularization in diabetic mice. Transcriptomic and untargeted metabolomic analyses revealed that PTTG1 knockdown reprogrammed pericyte energy metabolism by modulating glycolysis pathway genes, reducing oxidative stress, and restoring pericyte function, ultimately alleviating microvascular dysfunction in DR. ConclusionsPTTG1 plays a critical role in regulating pericyte dysfunction and maintaining vascular homeostasis in diabetic retinopathy. By modulating key metabolic pathways and pericyte phenotypes, PTTG1 represents a promising therapeutic target for treating diabetic microvascular complications. These insights offer a novel molecular framework for developing targeted therapies aimed at restoring retinal vascular health in diabetic patients.

molecular biology↗

bioRxiv2016

PASP - a whole-transcriptome poly(A) tail length determination assay for the Illumina platform

The poly(A) tail, co-transcriptionally added to most eukaryotic RNAs, plays an important role in post-transcriptional regulation through modulating mRNA stability and translational efficiency. The length of the poly(A) tail is dynamic, decreasing or increasing in response to various stimuli through the action of enzymatic complexes, and changes in tail length are exploited in regulatory pathways implicated in various biological processes.\n\nTo date, assessment of poly(A) tail length has mostly relied on protocols targeting only a few transcripts. We present PASP ( poly(A) tail sequencing protocol), a whole-transcriptome approach to measure tail lengths -- including a computational pipeline implementing all necessary analyses. PASP uses direct Illumina sequencing of cDNA fragments obtained through G-tailing of poly(A)-selected mRNA followed by fragmentation and reverse transcription.\n\nAnalysis of reads corresponding to spike-in poly(A) tracts of known length indicated that mean tail lengths can be confidently measured, given sufficient coverage. We further explored the utility of our approach by comparing tail lengths estimated from wild type and {Delta}ccr4-1/pan2 mutant yeasts. The yeast whole-transcriptome tail length distributions showed high consistency between biological replicates, and the expected upward shift in tail lengths in the mutant samples was detected. This suggests that PASP is suitable for the assessment of global polyadenylation status in yeast.\n\nThe correlation of per-transcript mean tail lengths between biological and technical replicates was low (higher between mutant samples). Both, however, reached high values after filtering for transcripts with greater coverage. We also compare our results with those of other methods. We identify a number of improvements that could be used in future PASP experiments and, based on our results, believe that direct sequencing of poly(A) tails can become the method of choice for studying polyadenylation using the Illumina platform

Molecular Biology↗

bioRxiv2020

Circadian clock mechanism driving mammalian photoperiodism

The annual photoperiod cycle provides the critical environmental cue synchronizing rhythms of life in seasonal habitats. In 1936, Bunning proposed a circadian-based coincidence timer for photoperiodic synchronization in plants. Formal studies support the universality of this so-called coincidence timer, but we lack understanding of the mechanisms involved. Here we show in mammals that long photoperiods induce the circadian transcription factor BMAL2, in the pars tuberalis of the pituitary, and triggers summer biology through the eyes absent / thyrotrophin (EYA3 / TSH) pathway. Conversely, long-duration melatonin signals on short photoperiods induce circadian repressors including DEC1, suppressing BMAL2 and the EYA3/TSH pathway, triggering winter biology. These actions are associated with progressive genome-wide changes in chromatin state, elaborating the effect of the circadian coincidence timer. Hence, circadian clock-pituitary epigenetic pathway interactions form the basis of the mammalian coincidence timer mechanism. Our results constitute a blueprint for circadian-based seasonal timekeeping in vertebrates.

molecular biology↗

bioRxiv2022

Strong interactions between highly-dynamic lamina-associated domains and the nuclear envelope stabilize the 3D architecture of Drosophila interphase chromatin

BackgroundInteractions among topologically associating domains (TADs), and between the nuclear envelope (NE) and lamina-associated domains (LADs) are expected to shape various aspects of 3D chromatin structure and dynamics; however, relevant genome-wide experiments that may provide statistically significant conclusions remain difficult. ResultsWe have developed a coarse-grained dynamical model of the Drosophila melanogaster nuclei at TAD resolution that explicitly accounts for four distinct epigenetic classes of TADs and LAD-NE interactions. The model is parameterized to reproduce the experimental Hi-C map of the wild type (WT) nuclei; it describes time evolution of the chromatin over the G1 phase of the interphase. Best agreement with the experiment is achieved when the simulations include an ensemble of nuclei, corresponding to the experimentally observed set of several possible mutual arrangements of chromosomal arms. The model is validated against multiple structural features of chromatin from several different experiments not used in model development, including those that describe changes in chromatin induced by lamin depletion. Predicted positioning of all LADs at the NE is highly dynamic - the same LAD can attach, detach and move far away from the NE multiple times during interphase. The probabilities of LADs to be in contact with the NE vary by an order of magnitude, despite all having the same affinity to the NE in the model. These probabilities are mostly determined by a highly variable local linear density of LADs along the genome which also has a strong effect on the predicted radial positioning of individual TADs. Higher probability of a TAD to be near NE is largely determined by a higher linear density of LADs surrounding this TAD. The distribution of LADs along the chromosome chains plays a notable role in maintaining a non-random average global structure of chromatin. Relatively high affinity of LADs to the NE in the WT nuclei substantially reduces sensitivity of the global radial chromatin distribution to variations in the strength of TAD-TAD interactions compared to the lamin depleted nuclei, where a 0.5 kT increase of cross-type TAD-TAD interactions doubles the chromatin density in the central nucleus region. ConclusionsA dynamical model of the entire fruit fly genome makes multiple genome-wide predictions of biological interest. The distribution of LADs along the chromatin chains affects their probabilities to be in contact with the NE and radial positioning of highly mobile TADs, playing a notable role in creating a non-random average global structure of the chromatin. We conjecture that an important role of attractive LAD-NE interactions is to stabilize global chromatin structure against inevitable cell-to-cell variations in TAD-TAD interactions.

molecular biology↗

bioRxiv2023

SAP domain facilitates efficient loading of Ku onto DNA ends

Recognition and processing of DNA ends play a central role in maintaining genome integrity. The evolutionarily conserved DNA repair complex Ku serves as the primary sensor of free DNA ends in eukaryotic cells. Its rapid association with DNA ends is crucial for several cellular processes, including non-homologous end joining (NHEJ) DNA repair and telomere protection. In this study, we conducted a transient kinetic analysis to investigate the impact of the SAP domain on individual phases of the Ku-DNA interaction. Specifically, we examined the initial binding, the subsequent docking of Ku onto DNA, and the sliding of Ku along DNA. Our findings revealed that the C-terminal domain of Ku70, known as SAP ((SAF-A/B, Acinus and PIAS), facilitates the initial phases of Ku-DNA interaction, but does not affect the sliding process. This suggests that SAP may either establish the first interactions with DNA, or stabilize these initial interactions during loading. To assess the biological role of SAP, we generated Arabidopsis plants expressing Ku lacking the SAP domain ({Delta}SAP). Intriguingly, despite the decreased efficiency of the {Delta}SAP Ku complex in loading onto DNA, the mutant plants exhibited full proficiency in classical NHEJ and telomere maintenance. This indicates that the speed of Ku loading onto telomeres or DNA double-strand breaks (DSBs) is not the decisive factor in stabilizing these DNA structures.

molecular biology↗

bioRxiv2024

An intra-family conserved high-order RNA structure within the M ORF is important for arterivirus subgenomic RNA accumulation and infectious virus production

Synthesis of subgenomic RNAs is a strategy commonly used by polycistronic positive sense single-stranded RNA viruses to express 3'-proximal genes. Members of the order of Nidovirales, including coronaviruses and arteriviruses, use a unique discontinuous transcription strategy to synthesize subgenomic RNAs. In this study, in silico synonymous site conservation analysis and RNA structure folding predicted the existence of intra-family conserved high-order RNA structure within the M ORF of arteriviral genomes, which was further determined to be important for the transcription/accumulation of subgenomic RNAs and production of infectious viral particles. Mutations disrupting the stability of the RNA structures significantly decreased the accumulation of multiple subgenomic RNAs. In contrast, the impact of mutagenesis on full-length genomic RNA accumulation was limited. The degree to which wild-type levels of subgenomic RNA accumulation were maintained was found to correlate with the efficiency of infectious virus production. Moreover, the thermo-stability of stems within the high-order RNA structure is also well correlated with viral replication capacity and the maintenance of subgenomic RNA accumulation. This study is the first to report an intra-Arteriviridae conserved high-order RNA structure that is located in a protein-coding region and functions as an important cis-acting element to control the accumulation/transcription of arteriviral subgenomic RNAs. This work suggests a complex regulation mechanism between genome replication and discontinuous transcription in nidoviruses. IMPORTANCEArteriviruses are a group of RNA viruses that infect different animal species. They can cause diseases associated with respiratory/reproductive syndromes, abortion, or haemorrhagic fever. Among arteriviruses, porcine reproductive and respiratory syndrome virus (PRRSV) and equine arteritis virus (EAV) are economically important veterinary pathogens. The challenge in control of arterivirus infection reflects our limited knowledge of viral biology. In this study, we conducted a comprehensive bioinformatical analysis of arteriviral genomes and discovered intra-family conserved regions in the M ORF with a high-order RNA structure. The thermo-stability of the RNA structure influences sgRNA transcription/accumulation and correlates with the level of infectious virus production. Our studies provide a new insight on arterivirus replication mechanism, which may have implications in developing disease control and prevention strategies.

molecular biology↗

bioRxiv2019

Transcriptome Landscape Reveals Underlying Mechanisms of Ovarian Cell Fate Differentiation and Primordial Follicle Assembly

Primordial follicle assembly in mammals occurs at perinatal ages and largely determines the ovarian reserve available to support the reproductive lifespan. The primordial follicle structure is generated by a complex network of interactions between oocytes and ovarian somatic cells that remain poorly understood. In the present research, using single-cell RNA sequencing performed over a time-series on mouse ovaries coupled with several bioinformatics analyses, the complete dynamic genetic programs of germ and granulosa cells from E16.5 to PD3 are reported for the first time. The time frame of analysis comprises the breakdown of germ cell cysts and the assembly of primordial follicles. Confirming the previously reported expression of genes by germ cells and granulosa cells, our analyses identified ten distinct gene clusters associated to germ cells and eight to granulosa cells. Consequently, several new genes expressed at significant levels at each investigated stage were assigned. Building single-cell pseudo temporal trajectories five states and two branch points of fate transition for the germ cells, and three states and one branch point for the granulosa cells were revealed. Moreover, GO and ClueGO term enrichment enabled identifying biological processes, molecular functions and cellular components more represented in germ cells and granulosa cells or common to both cell types at each specific stage. Finally, by SCENIC algorithm, we were able to establish a network of regulons that can be postulated as likely candidates for sustaining germ cell specific transcription programs throughout the investigated period.

developmental biology↗

bioRxiv2026

Male Age and Sexual Maturity: Lipopolysaccharide-induced tumor necrosis factor influences sperm quality and reproduction in Anopheles culicifacies

Elucidating the biological and molecular mechanisms that govern male fertility and mating behavior in mosquitoes is critical for optimizing genetic and sterile insect technique-based vector control strategies. Here, we examined age-related changes in male reproductive capacity in Anopheles culicifacies, using female egg output as an indirect indicator of male fertility. Our results demonstrated that male reproductive age follows a non-linear pattern of fertility. Morphometric analysis from emergence to day 13 post-eclosion revealed a strong correlation between seminal vesicle capacity and female fecundity, suggesting that age-dependent gonadal development directly influences reproductive potential. At the molecular level, we identified AcLITAF6 as a key regulator of male reproductive homeostasis. RNAi-mediated knockdown of AcLITAF6 impaired apoptosis-associated and phagocytic clearance, reduced sperm viability, and decreased female productive outcomes. Conclusively, we reveal a previously unrecognized role of LITAF in sperm quality control and male reproductive fitness, highlighting AcLITAF6 as a potential target for mosquito population suppression strategies.

developmental biology↗

bioRxiv2023

Whole-genome Omics delineates the function of CCM1 within the CmPn networks

IntroductionCerebral cavernous malformations (CCMs) are abnormal dilations of brain capillaries that increase the risk of hemorrhagic strokes. Mutations in the KRIT1, MGC4607, and PDCD10 genes cause CCMs, with mutations in CCM1 accounting for about 50% of familial cases. The disorder exhibits incomplete penetrance, meaning that individuals with CCM may appear normal initially, but once symptoms manifest, their brains have already suffered irreversible damage. Compromised blood-brain barrier (BBB) is crucial in regulating the flow of substances between the blood and the central nervous system, which can result in hemorrhagic CCMs. Progesterone and its derivatives have been studied for their impact on maintaining BBB integrity. CCM2 interacts with CCM1 and CCM3, forming the CCM signaling complex (CSC), which connects classic and non-classic progesterone signaling to establish the CmPn signaling network, vital in preserving BBB integrity. MethodsThe study aimed to explore the relationship between CCM1 and key pathways of the CmPn signaling network, utilizing a toolset comprising three mouse embryonic fibroblast lines (MEFs) with distinct CCM1 expression levels. Omics and systems biology analysis were performed to investigate Ccm1-mediated signaling within the CmPn signaling network. ResultsThe findings suggest that CCM1 plays a critical role in controlling cellular processes in response to different progesterone-mediated actions within CmPn/CmP signaling networks, partly by regulating gene transcription. This function is crucial for preserving the integrity of microvessels, indicating that targeting CCM1 could hold promise as a therapeutic approach for this condition.

molecular biology↗

bioRxiv2019

Transcriptional changes are regulated by metabolic pathway dynamics but decoupled from protein levels

Transcription is necessary for the synthesis of new proteins, often leading to the assumption that changes in transcript levels lead to changes in protein levels which directly impact a cells phenotype. Using a synchronized biological rhythm, we show that despite genome-wide partitioning of transcription, transcripts and translation levels into two phase-shifted expression clusters related to metabolism, detectable protein levels remain constant over time. This disconnect between cycling translation and constant protein levels can be explained by slow protein turnover rates, with overall protein levels maintained by low level pulses of new protein synthesis. Instead, rhythmic post-translational regulation of the activities of different proteins, influenced by the metabolic state of the cells, appears to be key to coordinating the physiology of the biological rhythm with cycling transcription. Thus, transcriptional and translational cycling reflects, rather than drives, metabolic and biosynthetic changes during biological rhythms. We propose that transcriptional changes are often the consequence, rather than the cause, of changes in cellular physiology and that caution is needed when inferring the activity of biological processes from transcript data. O_LIChanges in protein levels do not explain the changing states of a biological rhythm C_LIO_LISlow protein turnover rates decouple proteins levels from a rhythmic transcriptome C_LIO_LIMetabolites determine protein activity via rhythmic post-translational modifications C_LIO_LICycling protein activity explains rhythmic transcription and ribosome biogenesis C_LIO_LIA cycling transcriptome is a consequence, not a cause, of physiological changes C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/833921v2_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@25cad0org.highwire.dtl.DTLVardef@1265735org.highwire.dtl.DTLVardef@2779fdorg.highwire.dtl.DTLVardef@1b9efe5_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

Dynamic Ribosomal RNA Methylation Regulates Translation in the Hematopoietic System and is Essential for Stem Cell Fitness

Self-renewal and differentiation are at the basis of hematopoiesis. While it is known that tight regulation of translation is vital for hematopoietic stem cells (HSCs) biology, the mechanisms underlying translation regulation across the hematopoietic system remain obscure. Here we reveal a novel mechanism of translation regulation in the hematopoietic hierarchy, which is mediated by ribosomal RNA (rRNA) methylation dynamics. Using ultra-low input ribosome-profiling, we characterized cell-type-specific translation capacity during erythroid differentiation. We found that translation efficiency changes progressively with differentiation and can distinguish between discrete cell populations as well as to define differentiation trajectories. To reveal the underlying mechanism, we performed comprehensive mapping of the most abundant rRNA modification - 2-O-methyl (2OMe). We found that, like translation efficiency, 2OMe dynamics followed a distinct trajectory during erythroid differentiation. Genetic perturbation of individual 2OMe sites demonstrated their distinct roles in modulating proliferation and differentiation. By combining CRISPR screening, molecular and functional analyses, we identified a specific methylation site, 28S-Gm4588, which is progressively lost during differentiation, as a key regulator of HSC self-renewal. We showed that low methylation at this site led to translational skewing, mediated mainly by codon frequency, which promoted differentiation. Functionally, HSCs with diminished 28S-Gm4588 methylation exhibited impaired self-renewal capacity ex-vivo, and loss of fitness in-vivo in bone marrow transplantations. Extending our findings beyond the hematopoietic system, we also found distinct dynamics of 2OMe profiles during differentiation of non-hematopoietic stem cells. Our findings reveal rRNA methylation dynamics as a general mechanism for cell-type-specific translation, required for cell function and differentiation. KEY POINTSO_LIHematopoietic differentiation is associated with rRNA methylation dynamics to control cell-type-specific translation. C_LIO_LITranslation efficiency can distinguish discrete cell types and define differentiation trajectories. C_LIO_LIHSC fitness is regulated by a single rRNA methylation. C_LI

molecular biology↗

bioRxiv2021

Histone deacetylase 8 interacts with the GTPase SmRho1 in Schistosoma mansoni

BACKGROUNDSchistosoma mansoni histone deacetylase 8 (SmHDAC8) is a privileged target for drug discovery. Invalidation of its transcription by RNAi leads to impaired survival of the worms in infected mice and its inhibition causes cell apoptosis and death. To determine why it is a promising therapeutic target the study of the currently unknown cellular signaling pathways involving this enzyme is essential. Protein partners of SmHDAC8 have been identified by yeast two-hybrid (Y2H) cDNA library screening and by mass spectrometry (MS) analysis. Among these partners we characterized SmRho1, the schistosome orthologue of human RhoA GTPase, which is involved in the regulation of the cytoskeleton. In this work, we validated the interaction between SmHDAC8 and SmRho1 and explored the role of the lysine deacetylase in cytoskeletal regulation. METHODOLOGY/PRINCIPAL FINDINGSWe characterized two isoforms of SmRho1, SmRho1.1 and SmRho1.2. Co-IP/Mass Spectrometry analysis identified SmRho1 partner proteins and we used two heterologous expression systems (Y2H assay and Xenopus laevis oocytes) to study interactions between SmHDAC8 and SmRho1 isoforms. To confirm SmHDAC8 and SmRho interaction in adult worms and schistosomula, we performed co-immunoprecipitation (Co-IP) experiments and additionally demonstrated SmRho1 acetylation using a Nano LC-MS/MS approach. A major impact of SmHDAC8 in cytoskeleton organization was documented by treating adult worms and schistosomula with a selective SmHDAC8 inhibitor or using RNAi followed by confocal microscopy. CONCLUSIONS/SIGNIFICANCEOur results suggest that SmHDAC8 is involved in cytoskeleton organization via its interaction with the SmRho1.1 isoform. A specific interaction between SmHDAC8 and the C-terminal moiety of this isoform was demonstrated, and we showed that SmRho1 is acetylated on lysine K136. SmHDAC8 inhibition or knockdown using RNAi caused massive disruption of schistosomula actin cytoskeleton. A specific interaction between SmRho1.2 and SmDia suggested the existence of two signaling pathways that could regulate cytoskeleton organization via the two SmRho1 isoforms. Author summarySchistosoma mansoni is the major parasitic platyhelminth species causing intestinal schistosomiasis, for which around 200 million people are in need of treatment. Currently one drug, praziquantel, is the treatment of choice and its use in mass treatment programs, rendered imperative the development of new therapeutic agents. As new potential targets, we have focused on lysine deacetylases, and in particular Schistosoma mansoni histone deacetylase 8 (SmHDAC8). Previous studies showed that invalidation of the transcription of SmHDAC8 by RNAi led to the impaired survival of the worms after the infection of mice. The analysis of the 3D structure of SmHDAC8 by X-ray crystallography showed that the catalytic domain structure diverges significantly from that of human HDAC8 and this was exploited to develop novel anti-schistosomal drugs. Biological roles of SmHDAC8 are unknown. For this reason, we previously characterized its protein partners and identified the schistosome orthologue of the human RhoA GTPase, suggesting the involvement of SmHDAC8 in the modulation of cytoskeleton organization. Here, we investigated the interaction between SmHDAC8 and SmRho1 and identified two SmRho1 isoforms (SmRho1.1 and SmRho1.2). Our study showed that SmHDAC8 is indeed involved in schistosome cytoskeleton organization.

molecular biology↗

bioRxiv2023

A Key Piece of the Puzzle: The central tetramer of the Saccharomyces cerevisiae septin protofilament and Its Implications for Self-Assembly

Septins, often described as the fourth component of the cytoskeleton, are structural proteins found in a vast variety of living beings. They are related to small GTPases and thus, generally, present GTPase activity which may play an important (although incompletely understood) role in their organization and function. Septins polymerase into long non-polar filaments, in which each subunit interacts with two others by alternating interfaces, NC and G. In Saccharomyces cerevisiae four septins are organized in the following manner, [Cdc11-Cdc12-Cdc3-Cdc10- Cdc10-Cdc3-Cdc12-Cdc11]n in order to form filaments. Although septins were originally discovered in yeast and much is known regarding their biochemistry and function, only limited structural information about them is currently available. Here we present crystal structures of Cdc3/Cdc10 which provide the first view of the physiological interfaces formed by yeast septins. The G-interface has properties which place it in between that formed by SEPT2/SEPT6 and SEPT7/SEPT3 in human filaments. Switch I from Cdc10 contributes significantly to the interface, whereas in Cdc3 it is largely disorded. However, the significant negative charge density of the latter suggests it may have a unique role. At the NC-interface, we describe an elegant means by which the sidechain of a glutamine from helix 0 imitates a peptide group in order to retain hydrogen-bond continuity at the kink between helices 5 and 6 in the neighbouring subunit, thereby justifying the conservation of the helical distortion. Its absence from Cdc11, along with this structures other unusual features are critically discussed by comparison with Cdc3 and Cdc10. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=61 SRC="FIGDIR/small/537027v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@10c1eccorg.highwire.dtl.DTLVardef@ba0dacorg.highwire.dtl.DTLVardef@d7b866org.highwire.dtl.DTLVardef@8b6d7e_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LIThe first crystal structure of a yeast septin heterodimer (Cdc3-Cdc10) provides important insights into their structural biology. C_LIO_LIIdentification of common features and differences between yeast and human septins, sheds light on the unique characteristics of yeast septin filaments. C_LIO_LIThe Cdc3G-Cdc10{Delta}1-10 crystal structure could be a crucial piece of the puzzle towards obtaining a high-resolution cryo-EM structure of the yeast septin octamer. C_LI

molecular biology↗

bioRxiv2026

BioReason-Pro: Advancing Protein Function Prediction with Multimodal Biological Reasoning

Protein function annotation is fundamental to understanding biological mechanisms, designing therapeutics, and advancing biomedical research. Current computational methods either rely on shallow sequence similarity or treat function prediction as isolated classification tasks, failing to capture the integrative reasoning across sequence, structure, domains, and interactions that expert biologists perform to infer function. We introduce BioReason-Pro, the first multimodal reasoning large language model (LLM) for protein function prediction that integrates protein embeddings with biological context to generate structured reasoning traces. A key input into BioReason-Pro is the set of GO term predictions made by GO-GPT, our autoregressive transformer that captures hierarchical and cross-aspect dependencies of GO terms. BioReason-Pro is trained via supervised fine-tuning on synthetic reasoning traces generated by GPT-5 for over 130K proteins and further optimized through reinforcement learning. It achieves 73.6% Fmax on GO term prediction and an LLM judge score of 8/10 on functional summaries, substantially outperforming previous methods. Evaluations with human protein experts show that BioReason-Pro annotations are preferred over ground truth UniProt annotations in 79% of cases. Remarkably, BioReason-Pro predicted a novel interaction partner for the renal cancer biomarker RCDG1, which we confirmed in the lab by co-immunoprecipitation. In other binding-partner predictions, its per-residue attention localized to the exact contact residues resolved in cryo-EM structures. Together, GO-GPT and BioReason-Pro establish a framework for protein function prediction that combines precise ontology modeling with interpretable biological reasoning.

molecular biology↗

bioRxiv2025

Protein Hunter: exploiting structure hallucination within diffusion for protein design

1Interactions between proteins and other biomolecules underlie nearly all biological processes, yet designing such interactions de novo remains challenging. Capturing their specific interactions and co-optimizing sequence and structure are difficult and often require extensive computation. We present Protein Hunter, a fast, fine-tuning-free framework for de novo protein design. Starting from an all-X sequence, we find diffusion-based structure prediction models hallucinate reasonable looking structures that can be further improved through iterative sequence re-design and structure re-prediction. This lightweight strategy achieves high AlphaFold3 in silico success rates across both unconditional and conditional generation tasks, including binders to proteins, cyclic peptides, small molecules, DNA, and RNA. Protein Hunter also supports multi-motif scaffolding and partial redesign, providing a general and efficient platform for de novo protein design across diverse molecular targets.

molecular biology↗