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Chakraborty, D.

Publications and source records attributed to Chakraborty, D..

At least 19 recordsLinked to original sources

Rotavirus NSP1 activates human NLRP1 inflammasome

While inflammasomes detect and defend against pathogens, the precise mechanisms by which enteric viruses trigger these complexes, and the specific viral components involved remain incompletely understood. In this study, we demonstrate that rotavirus infection robustly induced human NLRP1 inflammasome activation via p38 mitogen-activated protein kinase (MAPK)-dependent and -independent pathways. Utilizing cell lines reconstituted with NLRP1 signaling and reverse genetics-rescued recombinant rotaviruses, we identified non-structural protein 1 (NSP1), a well-characterized viral antagonist of host interferon signaling, as an activator of the NLRP1 inflammasome in the absence of double-stranded RNA. Domain mapping analysis revealed that the C-terminus of NSP1 but not the C-terminal IRF3-binding site was essential for triggering inflammasome activation. Mechanistically, we show that NSP1 induced the proteasomal degradation of NLRP1 to elicit its activation, a process dependent on host neddylation pathways and the Cullin-3/Elongin B/C ubiquitin ligase complex. Together, these findings suggest that RV engages parallel NLRP1 surveillance pathways, including a p38-dependent response to infection, and a novel NSP1-dependent pathway involving the ubiquitin-proteosome system, where a viral strategy aimed at host protein degradation inadvertently trips the human NLRP1 inflammasome wire.

microbiology↗

Mutation in Ralstonia pseudosolanacearum F1C1 cytochrome p450 homologue impairs virulence through root but not through shoot of tomato plants

Ralstonia pseudosolanacearum causes a lethal bacterial wilt disease in many plants. The pathogen enters through roots and systemically colonizes the xylem tissue, resulting in wilting and eventual death of the host. Using transposon mutagenesis followed by screening in tomato seedlings, we identified a mutant exhibiting virulence deficiency by root inoculation, but not by leaf inoculation. The mutation was recreated in the wild-type genome via natural transformation to prove the linkage of virulence deficiency with the transposon insertion. Whole genome sequencing revealed that the transposon insertion was in a cyp450 homologue, encoding cytochrome P450 monooxygenase. Two independent targeted mutations were generated in the cyp450 gene, and both the mutants recapitulated the root-specific virulence deficiency. The mutants rarely cause disease in mature plants following soil drenching but remained fully virulent upon petiole pricking inoculation. Complementation with the full length cyp450 gene amplified from the wild-type strain F1C1 restored virulence in the mutant. Compared to the wild-type F1C1, the cyp450 mutants exhibited greater sensitivity to tomato root extracts. HR-LCMS of tomato root extracts revealed the occurrence of several antimicrobial compounds. Cytochrome P450 enzymes are known to mediate the degradation or biosynthesis of diverse metabolites in bacteria. Considering the virulence difference due to the mode of inoculation, our findings indicate that cyp450 contributes to tissue-specific adaptation of the pathogen during infection has opened an avenue for integrating tissue metabolomics with bacterial wilt susceptibility research.

pathology↗

SOP-Multi-π: A Two-Site Coarse-Grained Model with Rescaled Aromatic Interactions to Bridge Single-Chain Ensembles and Condensate Phase Behavior of IDPs and Multidomain Proteins

We present SOP-Multi-{pi}, an enhanced version of the two-bead-per-amino-acid resolution Self-Organized Polymer (SOP) model designed to investigate the conformational dynamics of Intrinsically Disordered Proteins (IDPs) and their phase behavior in biomolecular condensates. The original SOP-IDP and SOP-Multi models work well for chains in good solvents that predominantly adopt expanded conformations. However, they fail to reproduce the single-chain properties and phase-separation behavior of IDPs such as hnRNPA1 and its variants, which have a distinct pattern of aromatic residues. We therefore reparameterized the SOP-IDP force field to explicitly capture {pi}-{pi} and cation-{pi} interactions at the coarse-grained (CG) resolution. These interactions are weak but important for biomolecular recognition and for driving and stabilizing biomolecular condensates. Dimensionless rescaling prefactors ({lambda}) were applied exclusively to the aromatic {pi}-{pi} (Phe, Tyr, Trp) and cation-{pi} (Arg/Lys with Phe, Tyr, Trp) pairs. They were optimized iteratively against experimental radii of gyration of the hnRNPA1 low-complexity domain (A1-LCD) and its 16 aromatic and charge mutants. This restored a physically meaningful Tyr > Phe interaction hierarchy, and the Pearson correlation for Rg improved from r = 0.07 (SOP-IDP) to r = 0.83 (RMSD = 0.08 nm). The same parameters, without further tuning, gave r = 0.93 for 34 additional IDPs (24-198 residues) and reproduced Flory scaling ( {nu} ~ 0.588). We back-mapped microsecond-scale CG ensembles to all-atom representations and computed SAXS and Kratky profiles for direct comparison with experiments on IDPs and on the multidomain proteins hnRNPA1, TDP-43, G3BP1, and FUS. In direct-coexistence slab simulations, the model captures the phase behavior of A1-LCD condensates and their variants, with critical temperatures that correlate strongly with experiment (r = 0.88) but are somewhat underestimated in absolute terms. The model is implemented in LAMMPS, and a detailed user manual is provided for setting up single-chain and multi-chain condensate simulations of full-length multidomain proteins.

biophysics↗

Effect of Glycosylation on the Free Energy Landscape of the Catalytic Domain of Human Carbonic Anhydrase IX

N-linked glycosylation is known to modulate the catalytic function of human carbonic anhydrase (HCA) IX, yet its influence on the underlying free-energy landscape remains largely unexplored. In the present work, we combine extensive all-atom molecular dynamics simulations with kinetic transition network analysis to investigate the effect of glycosylation on the conformational organization of the catalytic domain of HCA IX in both monomeric and dimeric forms. The multidimensional conformational space is discretized into distinct free energy minima using the distribution of reciprocal interatomic distances (DRID), and the effective barriers separating them are estimated using the max flow-min cut formalism. The corresponding free energy landscapes are visualized in terms of disconnectivity graphs, which provide a faithful representation of underlying kinetics. Minimum free energy paths, mean first passage times, as well as frustration metrics are computed to further quantify the effect of glycosylation on landscape topography. Unglycosylated systems are found to exhibit predominantly funnel-like landscapes, with a limited number of metastable states in the vicinity of the native protein fold. In contrast, glycosylation enhances landscape complexity, resulting in a wide array of relaxation timescales. Strikingly, the two glycan chains affect the landscape topography in distinct ways, despite having closely matching sequences. Dimerization couples the glycan chain dynamics, with transitions between key metastable states involving coordinated motions of both the chains. Our work illustrates that interpretation in terms of disconnectivity graphs and transition networks could reveal important insights into the organization of glycoprotein energy landscapes.

biophysics↗

A Robust and Scalable Workflow for the Production of Circular Single-Stranded DNA for Genome Engineering Applications

Circular single-stranded DNA (cssDNA) is a versatile biomolecule with applications spanning genome editing, DNA nanotechnology, synthetic biology, molecular diagnostics, and aptamer development. Compared with linear single-stranded DNA, cssDNA offers enhanced structural stability, resistance to in-cellulo degradation by exonucleases and enables the generation of long, sequence-defined DNA molecules that are difficult to obtain through conventional chemical synthesis methods. Despite its growing utility, widespread adoption of cssDNA has been limited by the lack of accessible, scalable, and cost-effective production methods, with many existing workflows relying on specialised reagents, extensive optimisation, or commercially synthesised DNA. Here, we present a streamlined, end-to-end protocol for the laboratory-scale production of high-purity cssDNA using an M13 phagemid-based system and standard molecular biology laboratory infrastructure. The workflow encompasses bacterial culture, phage amplification, nuclease treatment, phage precipitation, anion-exchange purification, and quality control, with practical optimisations to improve yield, reproducibility, and scalability. Using this approach, yields range from 120-195 {micro}g of purified cssDNA from 300 mL of culture supernatant. The protocol provides detailed guidance on critical process parameters, troubleshooting, and quality assessment, enabling reliable production of cssDNA suitable for a wide range of downstream molecular biology and genome engineering applications.

molecular biology↗

Edge-First Ground Reaction Force Estimation with Consumer Smartwatches

Ground reaction force (GRF) measurement remains largely confined to instrumented laboratories, limiting longitudinal monitoring in daily life. This article presents an edge-first wearable system for estimating vertical GRF from consumer smartwatches. Two Apple Watch Series 6 devices worn at the wrist and waist stream 12-channel inertial data at 100 Hz to an iPhone, where preprocessing, storage, and inference occur locally without cloud dependence. The proposed GRFNet-MultiScale model is a compact temporal convolutional network with four dilated residual blocks and a global context branch. Under leave-one-subject-out evaluation on 539 stance windows from 10 healthy participants, the dual-sensor system achieved a mean Pearson correlation of 0.798 with an RMSE of 257 N, while a wrist-only configuration retained 82.5% of dual-sensor correlation. Temporal attribution remained stable across validation folds and identified early-stance wrist acceleration as the dominant reproducible signal. The system is strongest for cyclic locomotion.

bioengineering↗

Dose-Dependent Softening of Bacterial Model Membranes by Structurally Distinct Antimicrobial Peptides: A Coarse-Grained Molecular Dynamics Study

Antimicrobial peptides (AMPs) act at the membrane interface, where they remodel lipid packing defects and redistribute lateral stresses, yet a quantitative, dose-dependent understanding of how they alter membrane mechanical properties remains incomplete. We use coarse-grained MARTINI 3 molecular dynamics simulations to systematically characterize the mechanical and microstructural response of a 70:30 POPE:POPG bilayer to three AMPs spanning distinct structural classes: aedesin (alpha-helical, 2MMM), arenicin-1 (beta-hairpin, 2JSB), and indolicidin (disordered, 1G89). For each peptide we vary the surface loading from one to four peptides per leaflet and extract the bending modulus Kc, the area compressibility modulus KA, peptide localization depth, bilayer thickness, peptide-lipid and peptide-peptide spatial organization, and leaflet-resolved lipid packing defect distributions. All three peptides soften Kc monotonically with loading, but at per-peptide rates that span a threefold range and order systematically by structural class: - 1.39 {+/-} 0.09, - 0.66 {+/-} 0.04, and - 0.44 {+/-} 0.01 kBT per peptide for aedesin, arenicin-1, and indolicidin, respectively. The tilt and twist moduli remain invariant across all conditions, indicating that the perturbation operates selectively on long-wavelength collective deformation modes. KA softens for the two structured peptides but is statistically indistinguishable from the control for indolicidin, a dissociation we trace to a supraphosphate adsorption versus interfacial insertion dichotomy: structured peptides sit above the phosphate plane and act as supraphosphate wedges, while the disordered peptide threads into the interface without coherently displacing lipids. Independent geometric, spatial-organization, and microstructural observables corroborate this framework, with the deep versus shallow defect remodeling asymmetry providing a clean microstructural counterpart to the Kc-KA dichotomy. Acyl chain order parameters resolve the per-lipid splay from the bilayer-averaged response and show that the per-lipid perturbation tracks conformational state rather than peptide length: the two structured peptides impose comparable per-lipid chain disordering despite differing in length, while the disordered peptide imposes far less. These findings establish a quantitative connection between peptide-induced defect remodeling and the elastic response of the bilayer, and suggest a design principle in which conformational restriction maximizes the per-peptide membrane perturbation, motivating experimental tests on stapled-peptide AMP analogs.

biophysics↗

StickForStats: automated statistical assumption validation for reproducible computational biology

Reproducible computational biology depends on statistical decisions that routine workflows often skip: verifying that a differential-expression tests assumptions hold across all genes, that a strategy-comparison ANOVA is robust to non-normality, or that a meta-analysis is not distorted by publication bias. Surveys consistently find that fewer than 20% of published biomedical studies report checking these assumptions, and existing statistical software leaves validation to the analyst as an optional step. We present StickForStats, an open-source web platform that reframes assumption validation as a default precondition for every analysis. Its Guardian system--a middleware pipeline of eight validators (normality, variance homogeneity, independence, outliers, sample size, modality, linearity, homoscedasticity)--checks assumptions before execution and, on critical violations, reroutes to an appropriate nonparametric alternative with a documented decision trail. At genome scale, applying Guardian to a 91-sample synovial-sarcoma RNA-seq study (GSE271517) cascaded 90.6% of 27,221 genes to a rank-based test and flipped the differential-expression verdict for 553 genes--479 rescued from an under-powered t-test and 74 outlier-driven false positives rejected--materially changing the gene list a biologist would act on. The same automatic validation generalizes across domains: a CRISPR editing-strategy comparison (ANOVA F = 1122, with Guardian recommending Kruskal-Wallis H = 36.6), an ordinal correlation (Pearson r = 0.476 corrected to Spearman {rho} = 0.479), and a sixteen-trial clinical meta-analysis revealing severe publication bias (Eggers t = -5.78, p < 0.001); a complementary module extends the same validators to published manuscripts, checking claims against CONSORT, STROBE, ICH-E9, and JARS-Quant reporting standards. By making assumption validation automatic and transparent, StickForStats targets a tractable, under-served contributor to irreproducibility. The platform is MIT-licensed, validated against SciPy and R, and freely available at https://github.com/visvikbharti/stickforstats_new. Author summaryMost scientific conclusions rest on statistical tests, and every test comes with fine print: assumptions about the data that must hold for the result to be trustworthy. In practice, this fine print is often left unchecked. Surveys find that fewer than one in five published studies reports verifying these assumptions, partly because popular software treats the check as an optional extra that busy researchers easily skip. When the assumptions are ignored, a study can report a difference that is not really there. We built StickForStats to make this checking automatic. Before it runs any statistical test, our platform inspects the data, reports whether each assumption is met, and--if a serious problem is found--switches to a more appropriate method and records why. On four real biomedical datasets, including a gene-editing comparison and a large gene-expression study, we show that this safety net changes which findings are flagged as reliable. A companion tool applies the same checks to finished manuscripts, helping catch reporting problems before publication. By turning assumption checking from something you must remember into something that happens by default, we aim to make everyday analyses more reproducible.

bioinformatics↗

Exploring the Energy Landscape of Hairpin Folding using the TIS-DNA model

Coarse-grained (CG) modeling of DNA has been gathering steam in recent years due to the limitations imposed by current computer hardware, and deficiencies of atomistic force-fields. Specifically, CG simulations have emerged as a potent tool for exploring complex energy landscapes underlying biochemical processes, such as DNA transcription, as well as material design based on programmable self-assembly. In this chapter, we illustrate how the Three Interaction Site (TIS) model for DNA, a robust coarse-graining framework, can be used to study the folding landscape of DNA hairpins. We show that despite its simplicity, the TIS-DNA model quantitatively describes the hairpin folding thermodynamics and recapitulates many features of the kinetics, including the multiplicity of pathways. The free energy landscape exhibits single-funnel character with a distinct bias towards the folded state. It is likely that folding initiates through non-specific collapse of the DNA chain, involving multiple excursions on the energy landscape, until the opposing strands are approximately aligned. Subsequently, the loop region becomes more ordered, and after the first native-contact nucleates, the rest of the process becomes essentially downhill.

biophysics↗

Sphingomyelin-Cholesterol Synergy as a Lipid Chaperone Driving Cytolysin A Pore Formation

Bacterial pathogens rely on pore-forming toxins (PFTs) to breach host barriers, a feat that requires water-soluble monomers to spontaneously metamorphose into membrane-inserted pores. In the extracellular milieu, these toxins must navigate complex folding landscapes without ATP-dependent chaperones. Here, we show that the synergistic interplay of host sphingomyelin (SM) and cholesterol (CHOL) acts as an intrinsic "lipid chaperone" that drives the conformational maturation of the bacterial toxin Cytolysin A (ClyA). Using leakage assays on vesicles and suspended lipid bilayer (SuLBs) arrays, atomistic molecular dynamics (MD) simulations, cell membrane permeabilization assays and biophysical measurements, we demonstrate that SM-CHOL synergy accelerates the rate-limiting unfolding of the toxins membrane bound {beta}-tongue motif into a reactive "molten globule" intermediate and guides its subsequent refolding into functional -helical conformation crucial for pore formation. We identify conserved lysine residues (K175 and K206) as critical molecular sensors that detect this specific lipid signature to drive productive transformation. This assembly process reciprocally remodels the host membrane and dismantling liquid-ordered domains, suggesting a mechanical coupling between toxin folding and the disruption of host lipid homeostasis. Our findings establish a paradigm of lipid-mediated chaperoning, revealing how pathogens co-evolve to exploit host lipid complexity to overcome the energetic barriers of membrane insertion.

biophysics↗

Dengue virus suppression by 25-hydroxycholesterol is mediated by a combination of membrane fusion inhibition, innate immune activation and cholesterol biosynthesis dysregulation

Physicochemical properties and composition of cellular membranes are crucial for regulating broad cellular responses including signaling and defense against pathogens. Dengue virus (DENV) exploits cholesterol-rich membranes and host lipid pathways, such as cholesterol biosynthesis, lipid raft organization, and lipid droplet formation, for entry, replication, and assembly. Additionally, lipid-based plasma membrane signaling can trigger innate immune responses that attenuate viral growth, underscoring the dual role of lipids in facilitating and restricting DENV infection. Here, we demonstrate that 25-hydroxycholesterol (25-HC), an oxidized cholesterol metabolite, inhibits DENV infection through a multifaceted mechanism. 25-HC disrupts viral membrane fusion by altering cholesterol distribution and lipid raft organization, impairing the binding and fusion of the DENV envelope (E) protein with host membranes. Additionally, 25-HC modulates host cholesterol metabolism by suppressing biosynthesis pathways essential for viral replication while enhancing lipid droplet formation and stress-response pathways. Transcriptomic analyses reveal that 25-HC primes innate immune responses, activating proinflammatory pathways such as the NLRP3 inflammasome and MAPK signaling, while selectively modulating interferon-stimulated gene expression. Notably, 25-HC exhibits synergistic antiviral effects when combined with direct-acting antivirals like Remdesivir, underscoring its potential in combination therapies. These findings establish 25-HC as a promising candidate for host-directed antiviral strategies against DENV and other enveloped viruses.

cell biology↗

Highly pathogenic avian influenza management in high-density poultry farming areas

The continuous spread of highly pathogenic avian influenza H5 viruses poses significant challenges, particularly in regions with high poultry farm densities where conventional control measures are less effective. Using phylogeographic and phylodynamic tools, we analysed virus spread in Southwestern France in 2020-21, a region with recurrent outbreaks. Following a single introduction, the virus spread regionally, mostly affecting duck farms, peaking in mid-December with a velocity of 27.8 km/week and an effective reproduction number between farms (Re) of 3.8, suggesting the virus can spread beyond current control radii. Transmission declined after late December following preventive culling. Farm infectiousness was estimated around 9 days. Duck farm density was the main driver of virus spread and we identified farm density and proximity thresholds required to maintain effective control (Re < 1). These findings offer actionable guidance to support regional biosecurity and to improve the robustness of the poultry sector to mitigate future outbreaks.

evolutionary biology↗

A Unified Framework to Dissect Robustness, Plasticity, Evolvability and Canalisation of Biological Function

A unique balance of seemingly contradictory properties like robustness and plasticity, or evolvability and functional canalisation, characterises biological systems. To understand the basis of these properties, we investigate gene regulation, which is at the core of biological function. We simulate dynamical models of over 190 million genetic circuits covering all possible three-gene circuit structures. Our computational pipeline classifies these circuits into functional clusters by matching their temporal response shapes. Thus, we generate a dataset linking circuit structure, parameters and a corresponding functional label. Our key finding is a finite list of 20 functions that three-node genetic circuits can perform. Moreover, the structure-parameter space for these circuits tends to be primed for responses that stabilise over time following a perturbation. Every structure exhibits potential for multifunctionality with a range of 2-17 functions contingent upon parameters. We quantify network degeneracy, showing that many structural changes can be made to circuits without altering function. We define three quantities: structural, parametric, and functional diversities. Using these diversities, we construct a UNified FramewOrk for reguLatory Dynamics (UNFOLD) to analyse four key biological properties--robustness, plasticity, evolvability, and functional canalisation. Using UNFOLD, we identify that only 6.5% of network structures are non-plastic, while parameter sets enabling parametric robustness exist for every three-node network. We identify functionally canalised circuits from structure pairs that can be interchanged for a large number of parameter sets without a change in function. Overall, our framework offers insights into the fundamental organisation of biological networks by thorough analysis of three-node networks. Significance StatementBiological systems exhibit remarkable properties like robustness, plasticity, evolvability, and canalisation. This study presents a unified computational framework to understand these properties by exhaustively exploring the design space of three-node genetic circuits, identifying that only 20 functions are achievable, and revealing a natural bias toward stability. We uncover key principles of network degeneracy and multifunctionality, highlighting the versatility of genetic circuits. By analysing structural, parametric, and functional diversities, we identify structural and parametric changes that can transition a genetic circuit from robust behaviour to plasticity or from being canalised to becoming evolvable. Our work advances theoretical insights into biological function. It provides a method to identify alternate designs and parametric conditions for genetic circuits, paving the way for the design of reliable synthetic genetic circuits.

systems biology↗

Development of Severity Index for Oral Epithelial Dysplasia Using Fuzzy Group Decision Making Algorithm

BackgroundOral epithelial dysplasia (OED) grading suffers from several levels of uncertainties and imprecision. An index is preferred in this context to improve reliability and reduce subjectivity in diagnostic decision making. In this study, a fuzzy logic-based disease severity index (SI) is formulated considering standard histopathological features used in OED grading. MethodsOral onco-pathologists were asked to independently provide weights of different features, according to their clinical significance in the context of dysplasia. Aggregated weight of each feature was calculated from individual assessment of different onco-pathologists by a fuzzy logic-based group decision making algorithm. Confidence levels of experts were also included to improve robustness of the method. SI was generated by integrating abnormality score of each feature with its weight. Abnormality degree of each feature was expressed in linguistic terms by onco-pathologists which were subsequently represented by a triangular fuzzy number. Fuzzy membership function was used as it can capture the ambiguity of experts opinion regarding abnormality of individual feature. Finally, defuzzification was used to get a crisp index from weighted sum of all features. ResultSI was found to be statistically different (p<0.01) for different grades of dysplasia i.e mild, moderate and severe with added advantage of stratifying each grade in low and high subcategory. ConclusionKey contribution of our work is that we have developed a fuzzy logic-based group consensus process regarding weights of histopathological features for OED grading. Present methodology of developing SI can be applied to other medical decision-making problems as well. HighlightsO_LIA severity index is proposed to improve reproducibility of oral epithelial dysplasia grading. C_LIO_LIClinical significance of each histopathological and cytopathological feature in the context of dysplasia is reflected by its weight. C_LIO_LIA fuzzy logic-based group decision making algorithm is used to reduce subjectivity of features weights. C_LIO_LIConfidence level of oral onco-pathologists are given due importance in derivation of aggregated weight of each feature. C_LIO_LIAbnormality score of each feature is evaluated in fuzzy scale to capture clinicians ambiguity. C_LIO_LIPresent methodology may be useful for developing indices of other diseases. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/633806v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@5a5e28org.highwire.dtl.DTLVardef@dba3a9org.highwire.dtl.DTLVardef@1d5cc7corg.highwire.dtl.DTLVardef@16404b5_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG

cancer biology↗

Modeling Retinitis Pigmentosa 59: Dhdds T206A and Dhdds K42E knock-in mutant mice are phenotypically similar.

Dehydrodolichyl diphosphate synthase (DHDDS) is an essential enzyme required for several forms of protein glycosylation in all eukaryotic cells. Surprisingly, three mutant alleles, (DhddsK42E/K42E (K42E/K42E), DhddsT206A/K42E (T206A/K42E), and found in only one patient, DhddsR98W/K42E (R98W/K42E) have been reported that cause non-syndromic retinitis pigmentosa (RP59), an inherited retinal degeneration (IRD). Because T206A was only observed heterozygously with the K42E allele in RP59 patients, we used CRISPR/CAS9 technology to generate T206A/T206A, and subsequently T206A/K42E alleles in mice to assess the contribution of the T206A allele to the disease phenotype, to model the human disease, and to compare resulting phenotypes to our homozygous K42E mouse model. By postnatal (PN) 12-mo, T206A/K42E mice exhibit significant reduction of inner nuclear layer thickness as was observed in K42E/K42E mice. No change in outer nuclear layer thickness is observed in all mutant phenotypes up to PN 12 mo. Electroretinography (ERG) showed a significantly reduced b-wave without a-wave decrement and by PN 3-mo, ERG c- and d-wave responses were significantly attenuated in all phenotypes. Consistent with a reduction in inner nuclear layer thickness seen by OCT and cell loss observed by histology, bipolar and amacrine cell densities were reduced in all Dhdds mutant phenotypes compared to PN 8-12 mo age-matched controls. These results indicate that the DHDDS T206A allele causes retinal disease independent of the K42E allele, and that there likely is a common disease mechanism involving RP59-associated DHDDS mutations. We conclude that the physiological basis of retinal dysfunction in RP59 involves defective signaling in the inner retina resulting in bipolar/amacrine cell degeneration.

cell biology↗

Endogenous gene tagging with FnCas9 to track and sort neural lineages from 3D cortical organoids

Neural lineage tracing, or molecular dissection of lineage-specific brain cell types, is used in many labs to learn how neurons grow and mature. However, these studies depend on the growth and characterization of pure cultures, which takes a long time because of biochemical or fluorescence-based isolation through cell surface markers that overlap. These lineage-specific cells, however, can be efficiently sorted using endogenously expressed, fluorescently labeled marker genes. The labeled cell lines can be used not only to differentiate and purify different types of neurons but also to study the long-term development of neural lineages in two- and three-dimensional development models. In this study, we used an orthogonal Cas protein to generate human embryonic stem cell (hESC) lines with genetically labeled fluorescent barcodes for discrete neural lineages. We use these lines to successfully demonstrate spatial and temporal tracing of DCX-positive neuroblasts and immature neuronal cells within 2D neural cultures and 3D cortical organoids derived from human embryonic stem cells. This allowed the purification of endogenously tagged live neural cells from heterogeneous cortical organoids across multiple stages of development.

neuroscience↗

MLC1 alteration in iPSCs give rise to disease-like cellular vacuolation phenotype in the astrocyte lineage

BackgroundMegalencephalic leukoencephalopathy with subcortical cysts (MLC), a rare and progressive neurodegenerative disorder involving the white matter, is not adequately recapitulated by current disease models. Somatic cell reprogramming, along with advancements in genome engineering, may allow the establishment of in-vitro human models of MLC for disease modeling and drug screening. In this study, we utilized cellular reprogramming and gene-editing techniques to develop induced pluripotent stem cell (iPSC) models of MLC to recapitulate the cellular context of the classical MLC-impacted nervous system. MethodsSomatic cell reprogramming of peripheral patient-derived blood mononuclear cells (PBMCs) was used to develop iPSC models of MLC. CRISPR-Cas9 system-based genome engineering was also utilized to create the MLC1 knockout model of the disease. Directed differentiation of iPSCs to neural stem cells (NSCs) and astrocytes was performed in a 2D cell culture format, followed by various cellular and molecular biology approaches, to characterize the disease model. ResultsMLC iPSCs established by somatic cell reprogramming and genome engineering were well characterized for pluripotency. iPSCs were subsequently differentiated to disease-relevant cell types: neural stem cells (NSCs) and astrocytes. RNA sequencing profiling of MLC NSCs revealed a set of differentially expressed genes related to neurological disorders and epilepsy, a common clinical finding within MLC disease. This gene set can serve as a target for drug screening for the development of a potential therapeutic for this disease. Upon differentiation to the more disease relevant cell type-astrocytes, MLC-characteristic vacuoles were clearly observed, which were distinctly absent from controls. This emergence recapitulated a distinguishing phenotypic marker of the disease. ConclusionThrough the creation and analyses of iPSC models of MLC, our work addresses a critical need for relevant cellular models of MLC for use in both disease modeling and drug screening assays. Further investigation can utilize MLC iPSC models, as well as generated transcriptomic data sets and analyses, to identify potential therapeutic interventions for this debilitating disease.

cell biology↗

Ex Vivo Delivery of mRNA to Immune Cells Via a Non-Endosomal Route Obviates the Need for Nucleoside Modification

Base modification and the use of lipid nanoparticles (LNPs) are thought to be essential for efficient in vivo delivery and expression of mRNA. However, for ex vivo immune cell engineering, the need for either of the two is unclear. Previous reports have suggested that nucleic acids may be efficiently delivered to immune cells ex vivo, through a non-endosomal delivery route, but the need for base modification has not been determined. Herein, we demonstrate that when a non-endosomal delivery method is used, unmodified mRNA performs equally well to the commonly used base-modified mRNA, including the N1 methyl pseudo-uridine modification, in terms of protein expression and inflammatory response in cells. However, if an endosomal delivery route is used, then N1 methyl pseudo-uridine modification is necessary for high expression and low inflammatory response, as demonstrated by others as well. Overall, we show that non-endosomal mRNA delivery renders nucleoside modifications non-essential, and that unmodified mRNA combined with non-endosomal delivery route may be used for efficient ex vivo mRNA-based engineering of immune cells.

bioengineering↗