Biology subjects

Jackson, M.

Publications and source records attributed to Jackson, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Heatwaves and turbidity influxes have interacting and lasting effects on the foraging behaviour of three-spined stickleback Gasterosteus aculeatus

Freshwater ecosystems support a disproportionately high share of fish species and biodiversity yet are among the most threatened ecosystems globally and are increasingly being exposed to multiple, co-occurring environmental stressors. In addition, predicting the combined impacts of environmental stressors remains a major challenge, particularly when stressors differ in how quickly they arise and how long their effects persist. Understanding how these stressors interact across time is critical for predicting their ecological consequences. Here, we examine how warming and turbidity, two widespread and intensifying stressors, interact to influence predator-prey dynamics in the three-spined stickleback (\textit{Gasterosteus aculeatus}). Using a fully factorial experiment, fish were exposed to ambient, warmed, turbid, or combined conditions for short (one week) or prolonged (six weeks) periods, before feeding rates were quantified across prey densities using a novel stochastic Bayesian model. We found that foraging behaviour was shaped primarily by prior exposure rather than the immediate experimental conditions. Short-term exposure to both stressors increased feeding rates, whereas prolonged warming reduced them. In contrast, turbidity continued to increase feeding rates over both time periods and, when combined with warming, offset the negative effects of extended heat exposure. Changes in feeding performance at the individual level may propagate through food webs, potentially triggering trophic cascades that reduce biodiversity. Given the exceptional biodiversity of freshwater systems, such dynamics are of particular concern. We also found that behavioural changes persisted even after stressors were removed, indicating that exposure history can have lasting effects. These findings suggest that short-term experiments, such as the functional response, may actually be measuring subject experiential history rather than responses to specific experimental conditions.

animal behavior and cognition↗

bioRxiv2026

Post-transcriptional dysregulation precedes morphological transformation in chemically induced liver cancer

DNA lesions impede RNA polymerase II, yet the transcriptional consequences of that stress are less understood than the lesions themselves. We reanalysed total RNA and matched whole genomes from 438 liver lesions across four divergent mouse backgrounds, one of them the separate species M. caroli, 428 of them induced by a single dose of diethylnitrosamine (DEN) and 10 arising spontaneously, together with untreated liver and, critically, histologically normal liver from treated, tumour-bearing animals (paired normal liver). Three post-transcriptional changes accompany transformation in every background. Canonical splicing efficiency falls by 3.6 to 4.8 percentage points, and in genes that acquire them, cancer-specific junctions account for a median of 20-25% of junctions and arise at weaker splice signals. Output shifts toward circular RNA: a five-method consensus pipeline identifies 56,418 recurrent backsplice junctions, 9,718 of them detected in all four backgrounds, with ~2-fold enrichment in circRNA abundance and an expanded repertoire of distal configurations. Adenosine-to-inosine editing sites fall 2.2- to 3.8-fold, most steeply at intronic sites. Exposure changes not only these outputs but their coordination. In untreated C57BL/6J liver, splicing efficiency, mean relative backsplice span and editing co-vary positively (pairwise {rho} +0.78 to +0.87). That coordination is lost in exposed tissue, and paired normal liver shows the same loss as tumour. Pooling conditions obscures the effect because opposing estimates cancel. In C57BL/6J, paired normal liver already shows tumour-like splicing, circRNA abundance and editing roughly eight months after one exposure, while backsplice architecture has begun to reorganise. These samples carry a median of 337 callable somatic single-nucleotide variants against 78,843 in tumours and show no elevation of Mki67 or cell-cycle programmes. The post-transcriptional phenotype is therefore a durable property of damaged tissue established before morphological transformation.

cancer biology↗

bioRxiv2026

NAMPT activation uncovers a senescence-specific vulnerability and promotes healthy aging in combination with NAM

Aging is driven by multiple interacting processes, suggesting that effective strategies to promote healthy aging may require simultaneous targeting of more than one underlying mechanism. Here we identify a strategy that couples restoration of nicotinamide adenine dinucleotide (NAD+) homeostasis with selective targeting of senescent cells, two mechanistically linked features of aging. Senescent cells express elevated intracellular levels of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the nicotinamide (NAM) salvage pathway for NAD+ biosynthesis. Despite increased NAMPT abundance, isotope-tracing studies revealed decreased NAD+ biosynthesis and consumption, indicating that elevated NAMPT abundance was not accompanied by a corresponding increase in NAD+ metabolic flux. Treatment with the NAMPT activator SBI-0802162 engaged the spare enzymatic capacity of NAMPT in senescent cells and produced a marked rise in intracellular NAD+ that, when sustained, disrupted their transcriptional program and selectively reduced the viability of senescent cells but not proliferating cells. In mice, SBI-0802162 reduced circulating NAM levels, suggesting that sustained NAMPT activation may be limited by substrate availability. This observation prompted the development of a combination approach using SBI-0802162 together with dietary NAM supplementation. Co-administration of SBI-0802162 and NAM robustly increased tissue NAD+, suppressed select age-associated inflammatory signatures and markers of cellular senescence in a tissue-specific manner. These molecular effects occurred alongside preserved physical performance in aged mice and reductions in food intake and body weight, which were observed whether SBI-0802162 was present in the chow or administered by oral gavage. Together, these findings establish a mechanistically integrated approach to target two convergent features of aging, NAD+ dysregulation and senescent cell accumulation, and support combined NAMPT activation and NAM supplementation as a strategy to promote healthy aging.

molecular biology↗

bioRxiv2026

Predicting the protein interaction landscape of a mycobacterial pathogen

High-dimensional phenotypic screens of bacterial loss-of-function mutant libraries have determined gene-gene connections and specific phenotypes for thousands of bacterial genes in many species, but deciphering the underlying mechanisms remains decidedly low-throughput. Here, we demonstrate the utility of proteome-wide AI-based protein-protein interaction (PPI) predictions for overcoming this gap by using pooled-AlphaFold3 to assess all [~]1.3 million possible pairwise interactions in the proteome of Mycobacterium leprae. We identify [~]2,000 strong and intermediate PPIs that underlie a significant fraction of phenotypes and gene-gene connections observed in large-scale chemical genomics screens from Mycobacterium tuberculosis, Mycobacterium smegmatis, and Corynebacterium glutamicum. This combined approach predicts specific functions for dozens of previously uncharacterized core, conserved, and essential mycobacterial proteins. We highlight new information derived from the study, including insights into mycobacterial envelope assembly, peptidoglycan remodeling, and new modulators of the central dogma enzymes RNA polymerase and DNA gyrase. These data establish combined pooled-AlphaFold3 PPI prediction and high-throughput genomics approach as the gold standard for large-scale characterization of protein function.

systems biology↗

bioRxiv2026

Distinct senescent β-cell senotypes differentially drive islet aging and dysfunction

Biological aging greatly impacts the bodys ability to handle glucose, and represents a major risk factor the development and progression of type 2 diabetes (T2D). Nonetheless, despite advances in cellular senescence research and the development of new senolytic therapies, the heterogeneity of cellular senescence in the human endocrine pancreas, as well as its roles in normal aging, remains to be elucidated at the single-cell level. Here, we performed single-cell-resolved spatial proteomics and transcriptomics on intact pancreas from 26 donors (ages 20-80) and multiplexed single-cell RNA sequencing and functional assays on dispersed islets from 14 donors (ages 34-69). We identify two discrete SnC subpopulations distinguished by relative expression of CDKN1A and CDKN2A. CDKN1A senescent cells (SnCs) exhibit loss of {beta}-cell identity, impaired insulin secretion, and a proinflammatory SASP associated with increased islet immune infiltration. In contrast, CDKN2A SnCs retain transcriptional identity and functional competence, with lower inflammatory signaling. Together, these findings identify heterogeneous and functionally divergent senotypes in the human pancreas, distinguishing an adaptive (CDKN2A) from a maladaptive (CDKN1A) senescence program, thus providing a mechanism-guided framework for senescence-targeted therapies in T2D.

cell biology↗

bioRxiv2026

MeCP2 is Necessary in Cerebellar Purkinje Cells for Precise Network Dynamics During Associative Motor Learning

Loss-of-function variants of MECP2 cause Rett syndrome; however, their impact on cerebellar computations for learning remains poorly understood. Here, we show that Mecp2 deletion specifically in Purkinje cells does not broadly disrupt cerebellar-dependent behaviors but selectively compromises those that require precise timing, coordination, and associative updating in the cerebellar cortex. Mecp2 loss altered the emergence of learning-related Purkinje cell activity in vivo and disrupted their intrinsic and synaptic properties that support adaptive cerebellar output. These findings identify MeCP2 as a critical player in Purkinje cell function to regulate cerebellar learning signals and suggest that Rett syndrome-related motor dysfunction reflects impaired adaptive computation rather than a generalized loss of motor capacity.

neuroscience↗

bioRxiv2026

Suidae iPSC-derived macrophages as models for investigating susceptibility and resilience to African swine fever virus

African swine fever virus (ASFV) causes a lethal haemorrhagic fever in pigs and spread of this disease threatens many pig species (Suidae) globally. By contrast, ASFV infections in the natural evolved hosts, the warthog and bushpig, are subclinical. The macrophage (M{varphi}) is the primary target of ASFV and species-dependent responses in M{varphi}s are presumed to influence disease susceptibility. In an attempt to model these differences in vitro, we generated transgene-regulated induced pluripotent stem cells (iPSCs) from domestic pig, wild boar, red river hog and warthog, and confirmed that their corresponding iPSC-derived M{varphi}s (iPSCdMs) supported infection and replication of ASFV. In contrast to the other species, however, warthog iPSCdMs did not induce interferon upon infection by either virulent or attenuated ASFV. iPSCdMs may therefore represent an experimental system to understand how ASFV infection of M{varphi}s contributes to disease and aid development of strategies to combat this economically and environmentally devastating pathogen.

developmental biology↗

bioRxiv2025

Automation workflow for high-throughput arrayed plasmid DNA preparation and quantification.

High-throughput generation of arrayed plasmid DNA library using commercially available miniprep kits remains labor-intensive and costly. The yield and quality of plasmid preparations directly affect downstream applications, including arrayed viral library production and CRISPR library screening. Insufficient plasmid yield or DNA concentration often requires repeated preparations or additional DNA concentration steps to obtain adequate quantities. Similarly, higher variations in yield or quality across wells or plates can render an entire library unsuitable for subsequent experiments. To increase productivity and mitigate human intervention and errors, the present study established an automated workflow for high-throughput plasmid DNA preparation and quantification. The workflow was carried out by the Biomek i7 Hybrid automated workstation, synergizing a robotic liquid handler and multiple peripheral instruments to produce and measure plasmid DNA in a 96-well plate format. Bacterial competent cells were alkaline lysed and plasmid DNA was purified using magnetic beads, followed by quantification with the PicoGreen assay. The PicoGreen assay reported median and average yields of approximately 9.5 and 10 {micro}g per sample, respectively, which are equivalent to 7.6 and 8 {micro}g/mL of bacterial culture. Plasmid DNA concentrations measured by the PicoFluor fluorometer were consistently lower than those obtained using the NanoDrop UV spectrophotometer. The comparison demonstrated robust positive correlation between PicoGreen assay and NanoDrop measurements (R2 > 0.8). Among 480 plasmid DNA samples, average and median yields measured by the NanoDrop reached approximately 24 and 25 {micro}g per sample per well, corresponding to 19 and 20 {micro}g/mL of bacterial culture. Over 98% of samples exceeded the high-yield threshold of 10 {micro}g/mL of culture, with high plasmid quality validated through DNA gel electrophoresis. Collectively, this study demonstrated a robust, scalable, and cost-effective automation platform for high throughput arrayed plasmid library generation and quantification.

molecular biology↗

bioRxiv2025

Automation of high-throughput workflow for arrayed CRISPR activation library screening

CRISPR-mediated gene activation (CRISPRa) is among the most efficient and reliable strategies for mimicking sustained activation of endogenous promoters and their corresponding genes at physiological levels. By leveraging guide-RNA (gRNA) library design, CRISPRa screens can be applied on a whole-genome scale and are compatible with both arrayed and pooled formats, depending on assay requirements. Compared with conventional arrayed CRISPRa libraries that use single or dual gRNAs and often require multiple gRNA candidates per target, a recently developed CRISPRa library (termed T. gonfio) incorporates four tandem gRNAs per lentivector per target, thereby reducing library complexity and representing the smallest arrayed genome-wide CRISPRa library. To streamline genome-wide arrayed CRISPRa screening, this study developed a high-throughput automated workflow using the Biomek i7 Hybrid liquid-handling platform, integrated with multiple peripheral instruments. The workflow comprises three pipelines: lentiviral library transduction, cell library passaging, and assay processing. These pipelines together establish and maintain the transduced cell library for extended screening times. This enables assay processing at desired extended time points and improves the likelihood of identifying phenotypes that require longer time to develop, making the workflow suitable even for rapidly proliferating cell models. In a pilot arrayed screen using a T. gonfio mini-library targeting kinases and phosphatases, activation of the EPHA2 receptor promoter induced a growth reduction phenotype in the HEK293 cell model. This phenotype was recapitulated in a parallel pooled CRISPRa screen using the same mini-library and further validated in a co-culture assay.

cancer biology↗

bioRxiv2025

Dysregulation of U12-Type Splicing in Lupus Neutrophils

ObjectiveNeutrophil dysfunction is a hallmark of systemic lupus erythematosus (SLE), but its molecular basis remains unclear. This study explores transcriptional and post-transcriptional changes in low-density granulocytes (LDGs), a proinflammatory neutrophil subset expanded in SLE, focusing on NADPH oxidase (Nox) function and minor intron splicing. MethodsLDGs and normal-density neutrophils (NDGs) were isolated from SLE patients and healthy controls (HCs). CYBA (P22phox) expression was evaluated at transcript and protein levels. Nox activity was measured using luminol assays. Bulk RNA sequencing and rMATS software were used to assess alternative splicing, particularly of U12-type intron-containing genes. ResultsCYBA expression was reduced in SLE LDGs (n=11) compared to SLE NDGs and HCs (n=6), with levels resembling those in chronic granulomatous disease neutrophils. SLE LDGs exhibited impaired Nox activity (n=7 SLE, n=12 HC). CYBA is a U12 intron-containing gene, and transcriptomic analysis revealed broad downregulation of this gene class in SLE LDGs, suggesting minor spliceosome dysfunction. rMATS analysis showed increased U12-type intron retention and widespread splicing defects-- including exon skipping and mutually exclusive exon use--in genes such as GBP5, MAEA and STX10. These abnormalities were validated in an independent long-read RNA-seq dataset from SLE PBMCs. Importantly, splicing disruptions correlated with disease activity and autoantibody profiles. ConclusionImpaired U12-dependent splicing may contribute to neutrophil dysfunction in SLE, potentially via defective oxidative burst and altered immune regulation. These findings highlight the minor spliceosome as a novel player in lupus pathogenesis.

immunology↗

bioRxiv2025

Gram-positive bacterial cell wall components inhibit herpes simplex virus infection.

The role of the mucosal microbiome in viral infections remains unclear. Genital herpes, caused by herpes simplex virus 1 and 2 (HSV-1 and HSV-2), is among the most prevalent sexually transmitted infections worldwide. Despite evidence linking vaginal Lactobacillus to protection against sexually transmitted viruses, the specific microbial components and mechanisms that mediate this defense are not well understood. Here, we show that multiple cell wall components from diverse gram-positive bacteria, including lactobacilli, inhibit HSV-1 and HSV-2 infection in cells and in a mouse model of genital herpes infection. Peptidoglycan (PG) and lipoteichoic acid (LTA), both major components of the gram-positive bacterial cell wall, significantly reduced HSV infectivity in vitro and improved survival and disease outcomes in mice. We further showed that Lactobacillus crispatus surface layer proteins SlpA and SlpB bind HSV-1 and inhibit infection. Antiviral effects of cell wall components were dose-dependent, relied on intact PG structure, and, in the case of PG and LTA, were independent of TLR2-mediated host signaling. Collectively, our findings identify a species-independent antiviral function for gram-positive bacterial cell wall components against HSV and suggest that the composition of the mucosal microbiome may play an underappreciated role in suppressing mucosal herpes infection in humans.

microbiology↗

bioRxiv2025

Dynamic proteome profiling uncovers age-related impairments in proteostasis and the protective effects of resistance exercise in human skeletal muscle

A loss of proteostasis is a primary hallmark of ageing that has emerged from mechanistic studies in model organisms, but little is currently known about changes to proteostasis in the muscle of older humans. We used stable isotope labelling (deuterium oxide; D2O) in vivo, and peptide mass spectrometry of muscle samples to investigate differences in proteome dynamics between the muscle of younger (28 {+/-} 5 y; n=4) and older (69 {+/-} 3 y; n=4) men during either habitual activity or resistance exercise training. We quantified the abundance of 1787 proteins and the turnover rate of 1046 proteins in bi-lateral samples of vastus lateralis (n=32 samples total) taken before and after a 15-day program including 5 sessions of unilateral leg-press exercise (3 sets of 10 repetitions at 90% of 10 RM). Our protein abundance profiling revealed a stoichiometric imbalance within the proteostasis network in aged skeletal muscle, including subunits of eIF3, subunits of 40S and 60S ribosomal proteins. The rate of bulk, mixed-protein synthesis was not different between younger and older men, but most ribosomal proteins were less abundant in the muscle of older participants, suggesting ribosomes in older muscle may exhibit increased translational efficiency to maintain similar levels of protein turnover compared to ribosomes in younger muscle. Resistance exercise partially restored age-related disruptions to the proteostasis network. In older skeletal muscle, resistance exercise specifically increased the absolute turnover rate (ATR) of mixed mitochondrial proteins, with increased fractional turnover rate (FTR) of prohibitin 1 (PHB1) and profilin-1 (PROF1), and increased abundance of prohibitin 2 (PHB2). These adaptations may suggest resistance exercise promotes mitochondrial proteostasis by facilitating the synthesis and maintenance of key mitochondrial proteins. Thus, our Dynamic Proteome Profiling data provide an impetus for further exploration of the role of proteostasis in maintaining skeletal muscle quality and supports resistance exercise as a potential therapeutic strategy to promote healthy skeletal muscle ageing in humans. In BriefNishimura et al. used Dynamic Proteome Profiling to uncover whether the distorted proteomic landscape of ageing skeletal muscle is associated with altered turnover of specific proteins. Basal muscle from older men exhibits a divergence in protein abundance between subunits of eIF3 and subunits of 40S and 60S ribosomal proteins, whereas resistance exercise partially restored age-related disruptions in the muscle proteome. In older muscle, protein-specific turnover generally increases after resistance exercise, independent of changes in protein abundance, suggesting improved protein quality and renewal. Created in BioRender. Nishimura, Y. (2025) https://BioRender.com/p2a1aio HighlightsO_LIDynamic Proteome Profiling in human skeletal muscle ageing C_LIO_LIAgeing alters muscle proteostasis C_LIO_LIMixed-muscle protein synthesis does not differ between younger and older men C_LIO_LIResistance exercise increased mitochondrial protein turnover specifically in older muscle C_LIO_LIProtein-specific responses to resistance exercise differed between age groups C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/684531v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@11d0c8corg.highwire.dtl.DTLVardef@22f451org.highwire.dtl.DTLVardef@d62118org.highwire.dtl.DTLVardef@16eda9_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2025

Intercellular signaling drives robust cell fate and patterning in multicellularsystems

Cells do not act in isolation; they communicate with each other through a complex network of signals. In multicellular organisms, cell signaling buffers against fluctuations in gene expression. Its effect, however, on multistability--the ability of genetically identical cells to take on and maintain diverse stable states or phenotypes--is unclear. We develop spatially explicit stochastic models that couple fine-grained gene regulatory dynamics with intercellular signaling to study cell fate control in multicellular tissues. We show that intercellular signaling acts as a switch-like controller of cell fate, driving transitions from transient to stable states across tissues. Even weak signaling stabilizes short-lived expression states, and we derive mathematical expressions for the threshold signaling strength required to trigger tissue-wide stabilization. We also derive a universal spatial limit: stable region size grows only sublinearly with signaling strength, implying that large stable regions are prohibitively costly to maintain. The resulting trade-off between robustness and signaling in cell fate control highlights the delicate balance required during developmental processes to maintain spatial precision and provides insight into how organisms regulate tissue structure. OverviewCell fate decisions in developing tissues are governed by gene regulatory networks, but how communication between neighboring cells influences these decisions is poorly understood. We develop a mathematical framework showing that intercellular signaling acts as a controller of cell fate, suppressing spontaneous switching, and inducing stable, coordinated behaviors across tissues. Even weak signaling induces a sharp transition from unstable to stable population-wide dynamics. We also derive fundamental scaling laws showing that the size of stable regions increases sublinearly with signaling strength, revealing a trade-off between developmental robustness and resource cost. These results offer insight into how organisms achieve reliable spatial organization during development, despite molecular noise, and provide design principles for engineering synthetic tissues that balance stability, scalability, and resource efficiency.

systems biology↗

bioRxiv2025

Urbanization drives genetic erosion and population structure in a historically connected carnivore population

Urbanization is a dominant driver of habitat fragmentation globally, creating small, isolated wildlife populations vulnerable to accelerated genetic drift, reduced genetic diversity, and increased population differentiation. We investigated how urban development affects the genetic composition and structure of caracals (Caracal caracal) in Cape Town, South Africa using microsatellites and mitochondrial DNA sequence data. Sampling across four geographically disparate urban and rural populations revealed contrasting temporal patterns: mitochondrial markers indicated historical genetic connectivity among populations, while microsatellite data demonstrated recent genetic structuring driven primarily by urbanization. An extensively isolated urban population showed reduced allelic richness and pronounced genetic differentiation, reflecting urbanization as a strong barrier to gene flow. Within the isolated urban population, GPS-collared caracals demonstrated a degree of spatial genetic organization, with related individuals maintaining significantly higher home range overlap despite inhabiting a severely fragmented urban landscape. This kin-structured space use occurred despite caracals in the system having large home ranges compressed within a relatively small, isolated environment. Our findings reveal that urbanization has rapidly disrupted gene flow in this otherwise geographically widespread and adaptable carnivore, imposing a sufficient barrier to generate detectable genetic consequences within contemporary timeframes. The contrasting signals from historical versus contemporary molecular markers highlight urbanizations role in fragmenting previously connected populations and demonstrates the value of multi-marker approaches for detecting anthropogenic impacts on wildlife populations. These results underscore urbanizations capacity to rapidly alter population genetic dynamics, even in a highly mobile and adaptable carnivore.

genetics↗

bioRxiv2025

Enhanced splicing modulation by NMA-modified antisense oligonucleotides

Aberrant RNA splicing contributes to many human diseases, and splice-switching antisense oligonucleotides (SSOs) are ideally suited as a therapeutic strategy to modulate splicing and restore normal gene expression. Nusinersen (Spinraza) has revolutionized the treatment of spinal muscular atrophy. It is a splice-switching oligonucleotide (SSO) that is modified with 2-O-methoxyethyl (MOE) modifications. Here, we introduce a next-generation ribose modification, 2'-O-[2-(methyl-amino)-2-oxoethyl] (NMA), which enhances the pharmacological properties of SSOs. We identified a long-lasting NMA-modified human candidate SSO, salanersen, that is 3-4-fold more potent than nusinersen in human SMN2 transgenic mice. To evaluate the generality of the NMA chemistry, we applied it to modulation of SCN1A exon 20N splicing, a therapeutic strategy for Dravet syndrome. An NMA-modified SSO is 3.5-fold more potent than STK-001, a MOE-modified SSO currently in clinical trials. Our data establish the NMA chemistry as a broadly applicable ribose modification that markedly improves the pharmacological profile of SSOs, supporting its development as a next-generation platform for splicing modulation therapies. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=69 SRC="FIGDIR/small/680653v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@122e426org.highwire.dtl.DTLVardef@1b0a56eorg.highwire.dtl.DTLVardef@3ce4e8org.highwire.dtl.DTLVardef@1d86378_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2025

Automation of high-throughput arrayed lentivirus production and titration

Generation of arrayed genome-wide CRISPR libraries in a ready-to-transduce lentiviral format remains laborious, time-consuming, and costly. To address these limitations, the present study developed a fully automated lentivirus production and titration workflow using a Biomek i7 Hybrid automated workstation, integrated with multiple instruments and managed by SAMI EX software. The workflow produced and titrated viruses in 96 and 384-well plate formats, respectively. It employed reverse transfection and triplicate wells per lentivector to reduce variability and yielded an average of three viral particles in transduction unit (TU) per producing HEK293T cell. Titration was performed using U937-mCherry suspension cells, with the percentage of transduced cells converted from U937 (X%) to HEK293T (Y%) values via a linear regression equation (Y% = 4.3X% + 9.3%). The titer calculation was based on the initial seeding cell number, the converted percentage of HEK293T transduced cells, and virus input volume. The titration demonstrated strong reproducibility across LSRFortessa (BD) and Aurora (Cytek) flow cytometers (R2 = 0.9). Among 1,760 unconcentrated virus preparations, median and mean titers reached approximately 1.2 x 106 TU/mL, with over 97% of samples exceeding the high-titer threshold of 2x105 TU/mL, thus demonstrating a robust, scalable, and cost-effective automation platform for high throughput arrayed lentiviral library production and titration.

microbiology↗

bioRxiv2025

Automation of high-throughput arrayed mammalian cell line cultivation

Cell culture automation has traditionally been limited to basic tasks at low throughput, which are insufficient for passaging rapidly proliferating cell lines or for generating stable clonal lines. To address unmet needs, this study implemented a Biomek i7 Hybrid automated workstation, integrated with peripheral instruments and coordinated by SAMI EX software, to enable automated, high throughput mammalian cell culture workflows. The workflows support cell density monitoring, arrayed passaging, sample cherry-picking, plate reformatting, cell density normalization, and cryopreservation in 96-well plates. Integration with the CloneSelect imager allows rapid confluency monitoring and monoclonality assessment (<100 sec per plate). Cell passaging and density normalization require 32 minutes for one plate and 61 minutes for two plates. Workflow consistency was demonstrated across multiple cell lines and biological replicates, with wells showing comparable confluency within three standard deviations, lower coefficient of variation, and substantially narrower interquartile ranges after a single cell passage and density normalization. Four automation pipelines, including monoclonality screening, cell passaging and cherry-picking, density normalization, and cryopreservation, collectively enable clonal line establishment. Depending on scale, one to eight 384-well plates were processed in 69 to 355 minutes, yielding an average of 35 clonal lines per plate suitable for downstream genomic DNA sequence confirmation.

cell biology↗

bioRxiv2025

Context-dependent presynaptic inhibition of somatostatin interneuron inputs to Layer 1 of the visual cortex

Layer 1 of the cortex is a critical site for integrating top-down inputs onto the distal dendrites of pyramidal neurons, where inhibitory neurons modulate these inputs to enable context-dependent sensory processing. Yet, it remains unclear how behavioral context dynamically regulates inhibition in Layer 1. We discover a circuit motif in which NDNF cortical interneurons (cINs) presynaptically inhibit the axonal outputs of somatostatin (SST) cINs in Layer 1 of the visual cortex. Using combinatorial genetics, monosynaptic retrograde tracing, super-resolution imaging, optogenetics, slice electrophysiology, and in vivo calcium imaging, we show that NDNF cINs form direct contacts onto SST axons and suppress their output, thereby modulating inhibitory responses in L2/3 pyramidal neurons. This presynaptic inhibitory circuit is preferentially engaged during locomotion and low-contrast visual conditions. By dynamically modulating Layer 1-mediated inhibitory output onto pyramidal neurons, this circuit motif enables context-dependent modulation of visual processing.

neuroscience↗