Biology subjects

Banerjee, A.

Publications and source records attributed to Banerjee, A..

At least 55 records · Page 3Linked to original sources

bioRxiv2025

FeSBCP Analogue from Cyanobacteria: Insights from in vitro and in silico Studies

The blue light responsive Cryptochrome/Photolyase Family harbours two important photoactivatable proteins - cryptochromes and photolyases. While cryptochromes are essentially photoreceptors with diverse biological activities including circadian rhythm, photolyases repair UV damaged cyclobutane pyrimidine dimers (CPD) or (6-4) pyrimidine-pyrimidone photoadducts. Both cryptochromes and photolyases share a common Photolyase Homology Region (PHR) where they harbour a light harvesting antenna chromophore and redox-active flavin di-nucleotide (FAD). Even though photolyases do not possess an extension in the C-terminal region unlike cryptochromes, a recent discovery of FeS cluster containing bacterial cryptochrome-photolyase proteins (FeSBCPs) present an interesting retreat from the conventional photolyase architecture. They possess an additional modular redox center, [4Fe4S] iron-sulfur cluster connected by conserved cysteine residues. Herein, we characterize a cyanobacterial FeSBCP from Cyanobium sp. using tools from bioinformatics, biochemistry and biophysics. Sequence analysis reveals substitution in the well-conserved aromatic residues, meant for electron transfer. In silico modeling and docking supported by electrophoretic mobility shift assay as well as spectroscopic measurements do suggest efficient binding and repair of CyPhrB to damaged single/double-stranded substrates containing 6-4 photolesion. Considering FeS clusters established roles in DNA binding/repair activities, we speculate the role of FeS clusters in FeSBCPs and existence of FeS-FAD-DNA triangle towards efficient electron transfer.

biochemistry↗

bioRxiv2025

Local homeostasis preserves global neural dynamics compensating for structural loss during human lifespan aging

Aging brain undergoes a structural decline over lifespan accompanied by changes in neurotransmitter levels, leading to altered functional markers. Past studies have reported human resting state brain display a remarkable preservation of coordination among neural assemblies stemming from an underlying neuro-computational principles along aging trajectories, however, the true nature of which remains unknown. Here, we identify the computational mechanisms with which neurotransmitters, such as altered GABA and glutamate concentrations, can preserve functional integration across lifespan aging, despite structural decline. We employ multiscale, biophysically grounded modeling, constrained by the empirically derived anatomical connectome of the human brain, where the neurotransmitter concentrations can be free parameters that are algorithmically adjusted to maintain regional homeostasis and optimal working point. The two estimated neurotransmitters can maintain critical firing rates in the brain region and mimic age-associated functional connectivity patterns, consistent with empirical observations. We identified invariant GABA and reduced glutamate as the principle computational mechanism that can explain the topological variation of functional connectivity along lifespan, validated using graph-theoretic metrics. The results are subsequently replicated on three distinct datasets. Thus, the study offers an operational framework that integrates brain network dynamics at macroscopic and molecular scales, to gain insight into age-associated neural disorders.

neuroscience↗

bioRxiv2025

Small Molecule Activators of Protein Phosphatase 2A Exert Global Stabilising Effects on the Scaffold PR65

Protein phosphatase 2A (PP2A), an important therapeutic target, comprises a scaffold subunit PR65 composed of 15 HEAT (Huntingtin/elongation/A-subunit/TOR1) repeats, a catalytic subunit, and one of many different regulatory subunits that enable binding to specific substrates. Recently, small molecule activators of PP2A (SMAPs) were identified, although their mechanisms of action have not been fully defined. Here we explore the interaction of PR65 with two SMAPs, ATUX-8385 and the non-functional DBK-776, using single-molecule optical tweezers, ensemble methods, and computational analysis. In the absence of SMAP, PR65 shows multiple unfolding and refolding transitions, and the force-extension profiles are very heterogeneous with evidence of misfolding. Similar heterogeneity has been observed for chemical-induced unfolding of tandem-repeat proteins like PR65, a consequence of the internal symmetry of the repeat architecture. In the presence of ATUX-8385, higher unfolding and refolding forces are observed globally, and there is less misfolding, suggesting that ATUX-8385 acts like a pharmacological chaperone. In contrast, DBK-766-binding induces higher unfolding forces for only a few repeats of PR65, suggestive of a more localised effect; moreover, subsequent stretch-relax cycles show that PR65 is irreversibly locked in the unfolded state. Docking and molecular dynamics simulations provide additional insights how SMAP binding modulates PR65 structure and function.

biophysics↗

bioRxiv2025

Coronavirus protein interaction mapping in bat and human cells identifies molecular and genetic switches for immune evasion and replication

Coronaviruses, including SARS-CoV-2, can cause severe disease in humans, whereas reservoir hosts like Rhinolophus bats remain asymptomatic. To investigate how host-specific protein-protein interactions (PPIs) influence infection, we generated comparative PPI maps for SARS-CoV-2 and its bat-origin relative RaTG13 using affinity purification-mass spectrometry (AP-MS) in human and Rhinolophus ferrumequinum (RFe) bat cells. This approach identified both conserved and virus- and host-specific interactions that regulate infection dynamics. Notably, SARS-CoV-2 required a non-synonymous mutation in nucleocapsid to replicate in bat cells expressing human ACE2 and TMPRSS2. Analysis of the viral protein Orf9b revealed differential interactions with mitochondrial proteins Tom70 and MTARC2. A single residue difference in Orf9b between SARS-CoV-2 and RaTG13 functions as a molecular switch, weakening Tom70 binding and immune evasion in human cells while enhancing interaction with the bat-specific restriction factor MTARC2. These findings demonstrate how a single-residue substitution can reshape virus-host interactions and contribute to immune evasion and host adaptation.

systems biology↗

bioRxiv2025

Beta Desynchronization Mediates The Impact Of Internally Directed Attention In A Dual-Task Working Memory Paradigm

Internally directed attention (IDA), whether spontaneous or intentional, has been associated with impaired performance on externally directed cognitive tasks. Yet, the neurophysiological mechanisms underpinning this disruption remain poorly understood. In the present study, we characterized the neural correlates of IDA and identified how they impact on performance in a color-recall working memory task using electroencephalography (EEG). Participants performed a novel dual-task paradigm involving either self-referential (IDA) or perceptual processing of adjectives, involving externally directed attention (EDA) followed by a color-recall task. IDA enhanced late positive potentials (LPP) in EEG over medial-frontal electrodes as a marker of sustained affective and self-referential engagement. Time-frequency analyses further revealed increased event-related desynchronization in alpha and beta bands during stimulus encoding in the IDA condition, as well as increased alpha synchronization during the delay period, consistent with internal attention maintenance. To capture trial-level variability in task performance, we applied conditional quantile regression on the single trial data. Results showed that beta desynchronization in interaction with condition type during encoding influenced performance significantly in trials with low errors, whereas trials with high error in color recall were better explained by increased reaction times. These findings provide converging electrophysiological evidence for distinct neural signatures of internally directed attention and highlight their behavioral consequences in working memory performance.

neuroscience↗

bioRxiv2025

A deep learning framework for understanding cochlear implants

Sensory prostheses replace dysfunctional sensory organs with electrical stimulation but currently fail to restore normal perception. Outcomes may be limited by stimulation strategies, neural degeneration, or suboptimal decoding by the brain. We propose a deep learning framework to evaluate these issues by estimating best-case outcomes with task-optimized decoders operating on simulated prosthetic input. We applied the framework to cochlear implants - the standard treatment for deafness - by training artificial neural networks to recognize and localize sounds using simulated auditory nerve input. The resulting models exhibited speech recognition and sound localization that was worse than that of normal hearing listeners, and on par with the best human cochlear implant users, with similar results across the three main stimulation strategies in current use. Speech recognition depended heavily on the extent of decoder optimization for implant input, with lesser influence from other factors. The results identify performance limits of current devices and demonstrate a model-guided approach for understanding the limitations and potential of sensory prostheses.

neuroscience↗

bioRxiv2025

Comparative evaluation on the fermentative potential of single and assorted fruit and vegetable waste for production of bioethanol

Fruit and vegetable waste (FVW) constitute a large part of municipal solid waste (MSW) and create significant environmental challenges by emitting the greenhouse gas methane. In contrast, these are a big source of fermentable sugar to produce bioenergy. Therefore, in this study, the Scanning Electron Microscopy (SEM), Attenuated total reflectance-fourier-transform infrared spectroscopy (ATR-FTIR), thermogravimetric analysis (TGA), X-ray diffraction (XRD), and biochemical quantification of -cellulose and acid-insoluble lignin contents were carried out to compare fermentative potential between single (potato peel vs. orange peel) and assorted vegetable and fruit wastes. Collectively, the study indicates that assorted vegetable waste holds enormous biofuel potential by having low moisture content (6.78 %), moderately low decomposition temperature (Tmax: 299.36 {degrees}C), low crystallinity index (CrI: 19.12%), and high -cellulose contents (32.35 - 47.73 gm%) yielding high sugar (325.65 mg/g). The study also optimized several key fermentation parameters such as pH, sugar concentration and the time to produce bioethanol. It was observed that maximum production of ethanol (2.14%) was achieved at 28{degrees}C, at pH 6.3 with a higher initial sugar concentration of 200mg/g by using Saccharomyces cerevisiae strain NCIM 3594. This study supports the likely utility of unsorted vegetable waste to obtain bioethanol at a mass scale.

plant biology↗

bioRxiv2025

Proximity-based super-resolution imaging enabled by DNA base-stacking interactions

Super-resolution imaging has gained significant traction in recent years due to its unprecedented ability to visualize target biomolecules at nanometer resolution. Here, we demonstrate the capability to detect target pairs that are in close proximity by exploiting base-stacking interactions between two DNA strands, each labelling one target. Our DNA probes hybridize transiently with the each other only when both target molecules are proximal, thus creating a hybridization site for fluorophore-conjugated DNA strand, called imager. In this design, hybridization and co-axial base-stacking act synergistically to enable imager binding, with stacking interactions providing essential stabilization that allows for transient hybridization. This synergy generates the stochastic binding events required for DNA-PAINT imaging, which we call Stack-Proximity-PAINT (Stack-pPAINT). To gain mechanistic insights into hybridization of our probe, we performed atomistic equilibrium and steered molecular dynamics (MD) simulations. The simulations reveal that DNA base-stacking and fluorophore stacking together stabilize the imager. We utilized programmable DNA nanostructures to benchmark the applicability of Stack-pPAINT. As a cellular proof of concept, we visualized microtubular structures using Stack-pPAINT with antibodies targeting both alpha- and beta-tubulin molecules. This probe technology offers promising applications in cell biology research aimed at elucidating spatial interactomes at high resolution within cells.

biophysics↗

bioRxiv2025

Commensal to pathogen switch in Streptococcus pneumoniae is governed by a thermosensing master regulator

Opportunistic pathogens switch from a commensal to pathogenic state by sensing and responding to a variety of environmental cues, including temperature fluctuations. Minor temperature oscillations can alert the pathogen to a changing niche ecosystem, necessitating efficient sensing and rapid integration to trigger behavioral change. This is typically achieved through master regulators, which act as umbrella systems dictating pleiotropic phenotypes. Here, we uncover a pivotal role of minor temperature shifts in transition of Streptococcus pneumoniae (SPN) from commensal to virulent lifestyles, mediated via an RNA thermosensing (RNAT) element within the untranslated region of the global regulator CiaR. By positively regulating the expression of the surface adhesin, Phosphorylcholine (PCho), in response to elevated temperature, CiaR potentiates pneumococcal infection. Engineering the RNAT structure to create translation restrictive or permissive versions allowed us to demonstrate how modulation in expression of CiaR could alter pneumococcal invasion capability, influencing infection outcomes. Moreover, intranasal administration of PCho mitigated SPN-induced bacteraemic pneumonia. Since a majority of opportunistic respiratory bacterial pathogens decorate their surface with PCho, this signaling arm could be exploited for anti-infective interventions. Significance statementOur nasopharyngeal area is dominated by opportunistic pathogens, such as Streptococcus pneumoniae (SPN), which typically reside as harmless bystanders. However, in individuals with heightened nasopharyngeal inflammation, resulting from allergy, viral infection or an immature or senescent immune system, these seemingly innocuous microbes, including SPN, switch to virulent lifestyles. We reveal an elegant mechanism for rewiring of SPN virulence genes upon sensing temperature oscillations in the nasopharynx by the master regulator CiaR. Elevated nasopharyngeal temperatures due to pathologic conditions is sensed by thermosensing ciaR mRNA. This primarily promotes SPN surface decoration by PCho which facilitates improved invasion, triggering virulence phenotypes. Our findings point towards adoption of a common mechanism for switch to virulent lifestyles by variety of microbes sharing the specific respiratory niche.

microbiology↗

bioRxiv2025

Identification and In Silico Characterization of Novel lncRNAs Associated with Dengue Infection

BackgroundLong noncoding RNAs (lncRNAs) are essential regulators of various biological processes in viral infections. In our previous study, we described the association of known lncRNAs with dengue disease progression. However, little is known about novel lncRNAs in the context of dengue infection. In this study, we aimed to identify novel lncRNAs and characterize their interaction with mRNAs during different stages of dengue infection using RNA sequencing (RNA-seq) technology to better understand their role in dengue pathogenesis. MethodsWe analyzed RNA-seq data (GSE94892) obtained from peripheral blood mononuclear cells (PBMCs) of 39 dengue-infected patients. RNA-seq data analysis was performed using the Tuxedo protocol, and the coding potential of novel transcripts was examined using CPC and CPAT tools. LncRNAs and cis-regulated mRNAs were identified through location-expression analysis within a 20 kbp genomic region surrounding the identified lncRNAs. ResultsWe identified and characterized a total of 65 novel lncRNAs differentially expressed during dengue disease progression. These included 34 long intergenic lncRNAs, 2 antisense lncRNAs, and 25 alternatively spliced lncRNAs classified based on their localization. Bioinformatics analysis predicted potential cis-regulated target genes. Co-expression analysis revealed a strong correlation between lncRNAs and genes enriched in pathways related to megakaryocyte development and platelet production, suggesting a potential role in dengue pathogenesis. Furthermore, comparison with miRBase-21 indicated that some of the identified lncRNAs could act as miRNA precursors, highlighting a novel function of lncRNAs in gene regulation. ConclusionWe developed an analytical pipeline to identify novel lncRNAs from RNA-seq data. Our analyses led to the identification of novel lncRNAs and their associated genes involved in dengue disease progression. These findings provide insights into the functional role of lncRNAs in dengue pathogenesis.

bioinformatics↗

bioRxiv2025

Green Synthesis of a Novel Gelatin Crosslinked Oxidized Pullulan-Lignin biocomposite film for active food packaging

Biopolymer-based plastics, made from plants and microbes, are gaining popularity in food packaging due to their eco-friendliness, but degrade upon contact with water, thus requiring the addition of water-resistant materials. Lignin was extracted from cotton stalk using the acid-alkali method and was characterized through FTIR and 2D HSQC NMR to evaluate its structure and purity. Pullulan dialdehyde, gelatin, and lignin were blended using a green process to make water-stable PGL hydrogel film. Incorporation of Lignin into PDA/Gelatin blend, having imine bonds, increased network strength and solvent-resistance by extensive physical interactions like H-bonding and electrostatic interactions, which were confirmed through FTIR and XPS analysis. These films were cured at 60{degrees}C, making the process energy efficient. PGL film exhibited water stability, high mechanical strength, moisture barrier, and bioactive properties ideal for fruit preservation. Lignin improved hydrophobicity, hydrogel strength, biodegradability, and non-flammability relative to the control. The films effectiveness for food packaging was assessed by analyzing the impact on the preservation and shelf life of kinnow oranges. Physical properties, such as weight loss, firmness, and chemical properties, including pH, titratable acidity, lipid oxidation, sucrose content, antioxidant activity, and ethylene concentration of oranges before and after packaging, were monitored. PGL film showed high stability in hot and ambient water, with a water swelling index of 303%, impressive tensile strength of 8.5 MPa, and elongation at break exceeding 72%. PGL-packed oranges produced 0.034{+/-}0.0032 ppm/g of ethylene after seven days, which is 4.2-fold lower than unpacked oranges, signifying the fruit preservation ability of PGL films.

bioengineering↗

bioRxiv2025

Frontal Vulnerability vs. Temporal Resilience: Neuro-compensatory mechanisms underline differential language aging trajectories

Age-related decline underlies cognitive functions such as sensorimotor control, executive functioning, memory, and language production (LP), whereas language comprehension (LC) tends to remain intact or improve across healthy adult lifespan. This preservation likely stems from structural and functional integrity within core language network (cLAN) regions. To investigate this hypothesis, we analyzed the relationships among brains resting-state functional connectivity (FC), structural connectivity (SC), and language behaviour (LC and LP) using a cross-sectional cohort of healthy adults (N = 652; M/F = 322/330; aged 18-88) from the Cambridge Centre for Ageing and Neuroscience (Cam-CAN) dataset. Six cognitive tasks assessing LC and LP were employed, with neuroimaging measures focused on region-specific connections within the cLAN. Using generalized additive mixed models (GAMMs), complex brain-behaviour interactions were identified. Behavioral analyses revealed established age-related dichotomy, LC abilities in vocabulary and proverb comprehension improved and in syntactic and semantic comprehension remained stable, whereas LP tasks, e.g., verbal fluency, picture priming, and tip of tongue exhibited significant decline across the lifespan. SC exhibited decline in both intra-and inter-hemispheric fronto-temporal and frontal lobe connections, contrasted by preserved or enhanced temporal lobe connectivity, supporting a pattern of frontal vulnerability concomitant with temporal resilience. Age-related FC patterns demonstrated overall preservation, reflecting compensatory mechanisms to sustain functional integrity despite structural degradation. GAMM analyses revealed complex relationships between brain connectivity and language performance across age. Thus, integrating knowledge of brain structure, function, and language abilities, we identified the brain network mechanisms associated with dichotomous language behavior along lifespan. SignificanceUnderstanding age-related changes in language abilities is vital for early detection of cognitive decline and intervention development. This study reveals heterogeneous language aging trajectories: language production often declines, while comprehension remains stable or improves. Notably, comprehension tasks with high executive and working memory demands also show resilience, suggesting compensatory mechanisms beyond crystallized knowledge. Using multimodal neuroimaging in a large adult lifespan cohort, we link behavioral patterns to distinct network dynamics within the core language system. We uncover relative vulnerability in frontal brain regions, with pronounced structural decline in fronto-temporal pathways, contrasted by resilience in temporal connectivity, which is preserved or strengthened. These findings highlight regionally specific vulnerabilities and compensatory functional adaptations, refining current models and providing a framework to guide research promoting healthy cognitive aging.

neuroscience↗

bioRxiv2025

Parametric Modulation of a Shared Midbrain Circuit Drives Distinct Vocal Modes in a Singing Mouse

The ability of neural circuits to generate multiple outputs is critical for behavioral flexibility. Here, we leverage the rich vocal behavior of the singing mouse (Scotinomys teguina) to investigate the organizational logic of multifunctional motor circuits. We show that two distinct vocal modes--soft, unstructured ultrasonic vocalizations (USVs) for short-range and loud, rhythmic songs for long-range communication--arise not via parallel pathways but through shared brainstem phonatory circuitry involving the caudolateral periaqueductual gray (clPAG). Using a three-parameter linear model of song rhythm, we demonstrate that synaptic silencing of clPAG progressively alters song duration through a single parameter controlling its termination. This parameter also explains sexual dimorphism in songs, identifying clPAG as a key locus for driving natural behavioral variability. Our findings reveal how parametric modulation of a central circuit node can produce distinct behavioral modes, providing a mechanistic basis for rapid behavioral evolution in mammals.

neuroscience↗

bioRxiv2025

The experimentally evolved fluconazole-resistant Clade II isolates of Candidozyma auris exhibit a distinct lipid compositional landscape, highlighting intra-clade sphingolipid heterogeneity

The intrinsic resistance of Candidozyma auris to antifungal drugs poses a major therapeutic challenge, with conventional resistance mechanisms providing only partial explanations. Sphingolipids (SLs), known for their interclade heterogeneity, play a crucial role in antifungal resistance. This study examined the SL landscape in two drug-susceptible clade II isolates, C-line and P-line, from distinct geographical origins, which were experimentally evolved to develop stable fluconazole (FLC) resistance. The progenitors displayed distinct SL profiles, P1 had higher PhytoCer and OHPhytoCer, indicating a more active acidic SL biosynthesis branch, whereas C1 exhibited elevated OHGlcCer, OHCer, and LCBs, reflecting a greater role of the neutral biosynthesis branch. The principal component analysis (PCA) also confirmed distinct segregation of the two progenitors. Upon evolution, P1.1 and C1.1 adaptors showed significant SL alterations. P1.1 exhibited PhytoCer enrichment, while C1.1 showed reduced OHGlcCer alongside increased PhytoCer, dhCer and OHPhytoCer levels. Notably, OHGlcCer remained unchanged in P1.1, whereas LCBs and OHPhytoCer decreased compared to P1. Despite these lineage-specific differences between the progenitors, both evolved replicates exhibited increased PhytoCer as a common denominator like what is also observed in clinical FLC-resistant isolates. These findings highlight intra-clade SL variability and suggest that specific SLs contribute to FLC resistance in C. auris.

microbiology↗

bioRxiv2025

Biological subtyping of autism via cross-species fMRI

It is frequently assumed that the phenotypic heterogeneity in autism spectrum disorder reflects underlying pathobiological variation. However, direct evidence in support of this hypothesis is lacking. Here, we leverage cross-species functional neuroimaging to examine whether variability in brain functional connectivity reflects distinct biological mechanisms. We find that fMRI connectivity alterations in 20 distinct mouse models of autism (n=549 individual mice) can be clustered into two prominent hypo- and hyperconnectivity subtypes. We show that these connectivity profiles are linked to distinct signaling pathways, with hypoconnectivity being associated with synaptic dysfunction, and hyperconnectivity reflecting transcriptional and immune-related alterations. Extending these findings to humans, we identify analogous hypo- and hyperconnectivity subtypes in a large, multicenter resting state fMRI dataset of n=940 autistic and n=1036 neurotypical individuals. Remarkably, hypo- and hyperconnectivity autism subtypes are replicable across independent cohorts (accounting for 25.1% of all autism data), exhibit distinct functional network architecture, are behaviorally dissociable, and recapitulate synaptic and immune mechanisms identified in corresponding mouse subtypes. Our cross-species investigation, thus, decodes the heterogeneity of fMRI connectivity in autism into distinct pathway-specific etiologies, offering a new empirical framework for targeted subtyping of autism.

neuroscience↗

bioRxiv2025

Fluoride triggers lysis in Streptococcus mutans by inhibition of Clp protease complex leading to an unabated competence cascade

Fluoride has long been known to possess antimicrobial properties, triggering extensive lysis in diverse bacterial species. Surprisingly, given the ubiquity of fluoride in oral healthcare, the underlying mechanisms by which fluoride kills bacteria remain unknown. Using dental pathogen Streptococcus mutans as a model, we show that fluoride elicits an uncontrolled stress response characterized by upregulation of competence pathways and extensive cell wall degradation. While limited autolysis is adaptive, we show that fluoride subverts the typical tight temporal control of the competence-associated alternative sigma factor ComX by specifically inhibiting assembly and activity of Clp ATPases, thus disrupting proteolytic degradation of ComX. Phenylalanine partially restores Clp protease activity, explaining the frequent presence of a gene encoding chorismate mutase in fluoride-responsive operons. Together, these findings reveal the molecular mechanism that underlie fluoride-dependent lysis in bacteria, and identify new pathways that could be exploited to potentiate the antimicrobial effects of oral fluoride.

microbiology↗

bioRxiv2025

Impact of cell culture model systems on SARS-CoV-2 and MERS-CoV infection dynamics and antiviral responses

Cell cultures are widely used to study infectious respiratory diseases and to test therapeutics; however, they do not faithfully recapitulate the architecture and complexity of the human respiratory tract. Lung organoids have emerged as an alternative model that partially overcomes this key disadvantage. Lung organoids can be cultured in various formats that offer potential for studying highly pathogenic viruses. However, the effects of these different formats on virus infection remain unexplored, leaving their relative value unclear. In this study, we generated primary lung organoids from human donor cells and used them to derive monolayers and air-liquid interface (ALI) cultures with the goal of comparing the replication kinetics of two circulating highly pathogenic coronaviruses, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Middle East respiratory syndrome coronavirus (MERS-CoV). Infection studies revealed that organoid-derived monolayers displayed limited infection and the innate immune response was impaired against bacterial lipopolysaccharide (LPS) but not against virus-like double-stranded dsRNA or poly(I:C). Meanwhile, organoids and organoid-derived ALI cultures retained viral permissivity, with ALI cultures displaying diverse antiviral immune responses against both coronaviruses. SARS-CoV-2 and MERS-CoV demonstrated differential replication kinetics in organoid and organoid-derived ALI cultures. Therefore, primary organoid-derived cells in two-dimensional monolayer or three-dimensional ALI formats influence virus infection and host antiviral responses. Our study informs the selection of culture conditions for organoid-based respiratory disease research and therapeutic testing. ImportanceThe COVID-19 pandemic heralded the upsurge in human-derived lung organoid based studies due to their cellular heterogeneity that emulates the cellular complexity of the respiratory tract. A major disadvantage of organoid models resides in their apical-in conformation that "hides" cells and proteins that are typically exposed to the air-liquid interface (ALI) in the airways and are targets of viruses. Here, we generated monolayers and ALI cultures to facilitate cell exposure to highly relevant pathogens and compared them to parental organoids. Organoids at the ALI captured infection and immune responses better than organoids and organoid-derived monolayer cultures. Organoids at the ALI are a viable approach to improve identification and characterization of virus infection, host responses, and therapeutic testing.

microbiology↗

bioRxiv2025

Axially decoupled photo-stimulation and two photon readout (ADePT) for mapping functional connectivity of neural circuits

All-optical in vivo physiology enables three-dimensional interrogation of neural circuits function. Here, we introduce ADePT (Axially-Decoupled Photo-stimulation and Two-photon readout), a strategy that combines flexible optogenetic stimulation with neural activity imaging. ADePT achieves axially contained widefield patterned stimulation by coupling a digital micro-mirror device illuminated by a solid-state laser to a motorized holographic diffuser, while in parallel performing multiphoton imaging across different z-planes. As a proof of principle, we apply ADePT to map excitatory and inhibitory functional connectivity in the mouse early olfactory system. By controlling activity in individual glomeruli on the olfactory bulb surface and recording responses from output mitral and tufted cells in deeper layers, we identify cohorts of sister cells driven by the same parent glomerulus and analyze their distinct connectivity patterns. We further report specificity in the inhibitory circuitry, as we find that GABAergic/dopaminergic (DAT+) interneurons associated with different glomeruli selectively suppress baseline and odor-evoked activity in distinct mitral and tufted cell populations. Together, these results establish ADePT as a platform for high-throughput functional connectivity mapping in optically accessible brain regions.

neuroscience↗