Biology subjects

Srivastava, R.

Publications and source records attributed to Srivastava, R..

At least 19 recordsLinked to original sources

bioRxiv2026

Engineering Endogenous T Cell Receptors to Recognize Cancer Neoantigens Using a Hybrid Physics-AI Approach

T cell receptors (TCRs) are critical for immune surveillance and successful adaptive immune response against foreign antigens. TCRs drive this key arm of the immune system through recognition of peptide epitopes presented on MHC complexes. However, they are limited due to their stochastic nature and generation via genetic recombination. In silico design of functional TCRs that target defined peptide epitopes would be of considerable utility but has up until now been unsuccessful. Here, we develop an artificial intelligence (AI)-powered approach using a hybrid physics-based simulation and generative AI that successfully engineers TCRs against defined epitopes presented by MHC-I. We use this approach to design TCRs against two cancer antigens, a HERC1 neoantigen and an immunogenic neoepitope in mutant EGFR. We engineer multiple TCRs against the HERC1 neoantigen which activate T cells in response to exposure to peptide-MHC I and kill cancer cells more effectively than a patient-derived TCR. In addition, we used generative AI to design functional TCRs that target the EGFR T790M neoantigen, engineering greater specificity against the mutant sequence. We present an AI-based approach to TCR design with broad utility for efforts to engineer TCRs and for the development of new cell therapies. One sentence summaryArtificial intelligence-based approach enables the directed engineering of functional TCRs with enhanced features that target cancer neoantigens.

immunology↗

bioRxiv2025

Redefined Strategies to generate Conditional miR-141/200c miRNA cluster Knockout mice to eliminate confounding neo cassettes

MicroRNAs (miRNAs) of the miR-200 family specifically miR-141 and miR-200c regulate neurogenesis, differentiation, and epithelial-mesenchymal transitions in development. Dysregulation of these miRNAs is associated with several diseases including cancer and stroke. The Mirc13tm1Mtm/Mmjax mouse line, which targets the miR-141/200c cluster, was originally generated and described by Park et al. 2012 as a knockout-first, reporter-tagged insertion with conditional potential (conditional-ready) mouse line. Harnessing its full potential requires a two-step breeding process: breeding with FLP mice to excise the lacZ/neo cassette, then breeding with Cre to delete the floxed miRNA cluster (Park, et al. 2012). However, many studies either bypassed removal of the lacZ/Neo cassettes and treated the mouse line as Mirc13 knockouts or bred directly with Cre mouse lines, which could lead to unpredictable recombination and genotypes. Here we show that retention of the lacZ/Neo cassette is associated with reduced expression of the neighboring genes Ptpn6, Phb2 and Atn1 in the olfactory bulb, and that these genes are expressed normally once the cassette is excised. We therefore recommend a validated two-step FLPo-then-Cre breeding plan for this line, together with case-by-case allele validation for other knockout-first, reporter-tagged mouse lines.

neuroscience↗

bioRxiv2025

Attenuated CaV1.2-BK channel protein interaction in bipolar disorder

Bipolar disorder (BP) is a severe mental illness featured with potential cycling of depressive and manic episodes. The recurrent cycling is known to be associated with poor prognosis. Thus, it is important to decipher the pathophysiological mechanism that may possibly underlie the cycling. In our study, by using BP patient-derived neurons, we discovered the delay of time-to-peak in depolarization-associated intracellular calcium dynamics. This cellular deficit is selectively associated with the number of manic episodes in BP patients, suggesting that this deficit may participate in the mechanism of clinical cycling. We also observed an attenuated protein interaction between the calcium and voltage-activated potassium (BK) channel and the CaV1.2 L-type VGCC in BP patients. Pharmacological and molecular interventions that target CaV1.2-BK protein interaction, directly to BK channel for the amelioration of this attenuated protein interaction rescues the disease-associated intracellular calcium deficit. To test the involvement of BK channel in cycling-associated behaviors, we generated a novel animal model.

neuroscience↗

bioRxiv2025

DIA-PASEF Proteomic Profiling Reveals MpkA-Dependent Iron Stress Responses and Siderophore Biosynthesis in Aspergillus nidulans

Filamentous faungi play essential roles in biotechnology as producers of valuable bioproducts and, conversely, as opportunistic pathogens. Aspergillus nidulans is a widely used model organism for fungal genetics and cell biology; however, comprehensive proteomic references for this species remain limited. In this study, we applied a data-independent acquisition-parallel accumulation serial fragmentation (DIA-PASEF) approach to enable efficient and in-depth proteome profiling of A. nidulans. Leveraging ion mobility-based ion cloud information from DDA-PASEF experiments, we developed DIA-PASEF methods that identified 3,904 proteins across biological triplicates grown in rich medium. Compared to prior studies, this represents an increase in protein identifications of more than 140% and was achieved with more than five-fold reduction in analysis time. We employed this newly developed DIA-PASEF methodology to conduct both proteomic and phosphoproteomic analyses under iron-depleted conditions in an MpkA protein-kinase deficient mutant ({Delta}mpkA). The {Delta}mpkA strain exhibited expression of approximately 500 additional proteins and occupancy of over 1,800 additional phosphosites relative to a control. Differentially expressed and phosphorylated proteins increased by more than an order of magnitude in the {Delta}mpkA mutant across both iron-replete and iron-deplete conditions. Gene Ontology (GO) enrichment analysis revealed broader and distinct biological processes under iron-depleted conditions, highlighting adaptive responses specific to iron limitation and MAPK pathway disruption. This work establishes a high-coverage proteomic resource for A. nidulans and provides novel insights into fungal stress responses and signaling network perturbation. Importantly, high-throughput proteomic profiling reveals that limited iron availability and MAPK pathway disruption increases siderophore biosynthesis.

systems biology↗

bioRxiv2025

PLGA Soya Lecithin Based Hybrid Nanocomposite for Targeted Topical Delivery of Resveratrol in Psoriasis Management

Psoriasis is a recurring autoimmune-driven inflammatory skin disorder characterised by red, scaly, dry skin patches, keratinocyte hyperproliferation and infiltration of inflammatory cells, impacting millions globally. Patients with psoriasis often require repeated treatments with corticosteroids, which are often associated with significant side effects and comorbidities. Therefore, there is a growing need for advanced nanomedicine-based therapeutic approaches. Resveratrol (RSV), a plant-derived polyphenol, has shown promising anti-inflammatory and antioxidant properties for the treatment and management of psoriasis. However, its therapeutic potential is limited due to its poor solubility, low stability, and reduced skin penetration. To overcome these challenges, we have developed dual-carrier conjugated nanoparticles (RSVNPL) composed of soya lecithin and poly(lactic-co-glycolic acid) (PLGA). To further enhance its ease of application and prolong skin residence time, RSVNPL was incorporated into a carbomer 974P-based hydrogel system, forming RSVNPGel. Our formulation demonstrated improved physicochemical properties, including enhanced spreadability, superior skin adhesiveness and stability. Ex vivo skin permeation studies confirmed a significantly higher accumulation of RSV in the epidermis and dermis compared to free drugs. In vivo, efficacy was investigated using imiquimod (IMQ)-induced psoriatic mouse model, where RSVNPGel treatment led to a notable reduction in epidermal thickness, inflammatory cell infiltration, and key histopathological features associated with psoriasis. Additionally, serum biochemical analysis and organ histology established the biocompatibility and safety of the formulation. Overall, RSVNPGel presents a promising nanocarrier-based strategy for the effective topical management of psoriasis, enhancing RSV delivery and therapeutic efficacy while minimizing systemic exposure. Research HighlightsO_LIHybrid nanoparticles composite of soya lecithin and PLGA were developed for RSV delivery. C_LIO_LIThe formulation enhanced skin penetration, retention and created a better skin adhesive profile in psoriatic lesions. C_LIO_LISignificant anti-psoriatic effects were achieved in a preclinical model. C_LIO_LIThe system showed excellent safety and biocompatibility. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=101 SRC="FIGDIR/small/648585v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@e28c4corg.highwire.dtl.DTLVardef@1690c80org.highwire.dtl.DTLVardef@1b3e4bforg.highwire.dtl.DTLVardef@16ec0ad_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

The impact of fungal developmental structures on mechanical properties of mycelial materials.

This study explores how suppressing asexual development in Aspergillus nidulans enhances the mechanical properties of mycelial materials. Using four aconidial mutants ({Delta}brlA, {Delta}flbA, {Delta}fluG, and fadAG42R) that lack asexual development and a control strain (A28) that undergoes typical asexual development, we found that the absence of asexual development significantly improves mechanical strength. All mutants exhibited higher ultimate tensile strength (UTS) than the control, with {Delta}fluG and {Delta}brlA (fluffy nonsporulating, FNS phenotype) showing the highest UTS. Additionally, fadAG42R and {Delta}flbA (fluffy autolytic dominant, FAD phenotype) demonstrated significantly higher strain at failure (SF), linked to increased autolysis and lower dry cell mass compared to the control and FNS mutants. Solid-state NMR analysis revealed that autolysis in FAD mutants disrupts galactofuranose-related metabolic processes, altering cell wall composition and contributing to higher elasticity. These findings suggest that suppressing asexual development enhances mycelial material strength, while autolysis mechanisms influence elasticity. This research highlights the potential for genetic manipulation in fungi to engineer advanced mycelial-based materials with tailored mechanical properties.

bioengineering↗

bioRxiv2025

Synthetic Generation of Dynamic Omics Data Demonstrates Aspergillus nidulans BrlA Paradoxical Wall Stress Response

We propose a method to generate additional dynamic omics trajectories which could support pathway analysis methods such as enrichment analysis, genetic programming, and machine learning. Using long short-term memory neural networks, we can effectively predict an organisms dynamic response to a stimulus based on an initial dataset with relatively few samples. We present both an in silico proof of principle, based on a model that simulates viral propagation, and an in vitro case study, tracking the dynamics of Aspergillus nidulans BrlA transcript in response to antifungal agent micafungin. Our silico experiment was conducted using a highly noisy dataset with only 25 replicates. This proof of principle shows that this method can operate on biological datasets, which often have high variance and few replicates. Our in silico validation achieved a maximum R2 value of approximately 0.95 on our highly noisy, stochastically simulated data. Our in vitro validation achieves an R2 of 0.71. As with any machine learning application, this method will work better with more data; however, both of our applications attain acceptable validation metrics with very few biological replicates. The in vitro experiments also revealed a novel paradoxical dose-response effect: transcriptional upregulation by Aspergillus nidulans BrlA is highest at an intermediate dose of 10 ng/mL and is reduced at both higher and lower concentrations of micafungin.

genomics↗

bioRxiv2024

Aspergillus nidulans Transcription Factor BrlA is Utilized in a Conidiation-Independent Response to Cell-Wall Stress

Under synchronized conidiation, over 2500 gene products show differential expression, including transcripts for both brlA and abaA, which increase steadily over time. In contrast, during wall-stress induced by the echinocandin micafungin, the brlA transcript is upregulated while the abaA transcript is not. In addition, when mpkA (last protein kinase in the cell wall integrity signaling pathway) is deleted, brlA expression is not upregulated in response to wall stress. Together, these data imply BrlA may play a role in a cellular stress-response which is independent of the canonical BrlA-mediated conidiation pathway. To test this hypothesis, we performed a genome-wide search and found 332 genes with a putative BrlA response element (BRE) in their promoter region. From this set, we identified 28 genes which were differentially expressed in response to wall-stress, but not during synchronized conidiation. This set included seven gene products whose homologues are involved in transmembrane transport and 14 likely to be involved in secondary metabolite biosynthesis. We selected six of these genes for further examination and find that they all show altered expression behavior in the brlA deletion strain. Together, these data support the idea that BrlA plays a role in various biological processes outside asexual development. ImportanceThe Aspergillus nidulans transcription factor BrlA is widely accepted as a master regulator of conidiation. Here, we show that in addition to this function BrlA appears to play a role in responding to cell-wall stress. We note that this has not been observed outside A. nidulans. Further, BrlA-mediated conidiation is highly conserved across Aspergillus species, so this new functionality is likely relevant in other Aspergilli. We identified several transmembrane transporters that have altered transcriptional responses to cell-wall stress in a brlA deletion mutant. Based on our observation, together with what is known about the brlA gene locus regulation, we identify brlA{beta} as the likely intermediary in function of brlA in the response to cell-wall stress.

cell biology↗

bioRxiv2024

Using flux theory in dynamic omics data sets to identify differentially changing signals using DPoP

Derivative profiling (DP) is a novel approach to identify differential signals from dynamic omics data sets. This approach applies variable step-size differentiation to time dynamic omics data. This work assumes that there is a general omics derivative that is a useful and descriptive feature of dynamic omics experiments. We assert that this omics derivative, or omics flux, is a valuable descriptor that can be used instead of, or with, fold change calculations. The results of derivative profiling are compared to established methods such as Multivariate Adaptive Regression Splines (MARS), significance versus fold change analysis (Volcano), and an adjusted ratio over intensity (M/A) analysis to find that there is a statistically significant similarity between the results. This comparison is repeated for transcriptomic and phosphoproteomic expression profiles previously characterized in Aspergillus nidulans. This method has been packaged in an open-source, GUI-based MATLAB app, the Derivative Profiling omics Package (DPoP). Gene Ontology (GO) term enrichment has been included in the app so that a user can automatically/programmatically describe the over/under-represented GO terms in the derivative profiling results using domain specific knowledge found in their organisms specific GO database file. The advantage of the DPoP analysis is that it is computationally inexpensive, it does not require fold change calculations, it describes both instantaneous as well as overall behavior, and it achieves statistical confidence with signal trajectories of a single bio-replicate over four or more points. While we apply this method to time dynamic transcriptomic and phosphoproteomic datasets, it is a numerically generalizable technique that can be applied to any organism and any field interested in time series data analysis. The app described in this work enables omics researchers with no computer science background to apply derivative profiling to their data sets, while also allowing multidisciplined users to build on the nascent idea of profiling derivatives in omics.

bioinformatics↗

bioRxiv2024

In situ deposition of nanobodies by an engineered commensal microbe promotes survival in a mouse model of enterohemorrhagic E. coli

Engineered smart microbes that deliver therapeutic payloads are emerging as treatment modalities, particularly for diseases with links to the gastrointestinal tract. Enterohemorrhagic E coli (EHEC) is a causative agent of potentially lethal hemolytic uremic syndrome. Given concerns that antibiotic treatment increases EHEC production of Shiga toxin (Stx), which is responsible for systemic disease, novel remedies are needed. EHEC encodes a type III secretion system (T3SS) that injects Tir into enterocytes. Tir inserts into the host cell membrane, exposing an extracellular domain that subsequently binds intimin, one of its outer membrane proteins, triggering the formation of attaching and effacing (A/E) lesions that promote EHEC mucosal colonization. Citrobacter rodentium (Cr), a natural A/E mouse pathogen, similarly requires Tir and intimin for its pathogenesis. Mice infected with Cr({Phi}Stx2dact), a variant lysogenized with an EHEC-derived phage that produces Stx2dact, develop intestinal A/E lesions and toxin-dependent disease. Stx2a is more closely associated with human disease. By developing an efficient approach to seamlessly modify the C. rodentium genome, we generated Cr_Tir-MEHEC({Phi}Stx2a), a variant that expresses Stx2a and the EHEC extracellular Tir domain. We found that mouse pre-colonization with HS-PROT3EcT-TD4, a human commensal E. coli strain (E. coli HS) engineered to efficiently secrete- an anti-EHEC Tir nanobody, delayed bacterial colonization and improved survival after challenge with Cr_Tir-MEHEC({Phi}Stx2a). This study provides the first evidence to support the efficacy of engineered commensal E. coli to intestinally deliver therapeutic payloads that block essential enteric pathogen virulence determinants, a strategy that may serve as an antibiotic-independent antibacterial therapeutic modality. Significance StatementEngineered smart microbes that secrete therapeutics are emerging as treatment modalities, particularly for gut-based diseases. With the growing threat of multidrug-resistant infection, non-antibiotic treatments are urgently needed. The gastrointestinal pathogen enterohemorrhagic E coli (EHEC) can cause the potentially lethal hemolytic uremic syndrome, a toxin-driven disease. Given concerns that antibiotics increase toxin release, treatment is largely limited to supportive care. Here, we show that pre-treatment with a commensal E. coli (HS-PROT3EcT) engineered to secrete an antibody that blocks an essential EHEC virulence factor delays the establishment of an EHEC-like infection in mice. This study strongly suggests that smart microbes that deliver payloads that block colonization factors of gut pathogens can be developed as critically needed alternatives to antibiotics for fighting bacterial infections.

microbiology↗

bioRxiv2024

Eugenol loaded lipid nanoparticles derived hydrogels ameliorate psoriasis-like skin lesions by lowering oxidative stress and modulating inflammation

Psoriasis is a chronic T-cell-mediated autoimmune skin disorder characterized by excessive epidermal thickening, keratinocyte over-proliferation, disruption of epidermal cell differentiation, and increased blood vessel growth in the dermal layer. Despite the common use of corticosteroids in psoriasis treatment, their limited efficacy and numerous side effects pose significant challenges. This research introduces a promising alternative approach by presenting hydrogels loaded with Eugenol (EU) in combination with Carbopol 974P (EUNPGel) for potential psoriasis management. EUN-loaded lipid nanoparticles (EUNPs) exhibit superior drug loading, enhanced release kinetics, long-term stability, and the ability to scavenge reactive oxygen species (ROS). Furthermore, EUNPs have been shown to inhibit keratinocyte proliferation, induce apoptosis, and augment the uptake of IL-6-mediated inflammation in human keratinocyte cells. Application of EUNPs-loaded gels (EUNPGel) to imiquimod-induced psoriatic lesions has demonstrated effective dermal penetration, suppressing keratinocyte hyperplasia and restoring epidermal growth. This led to a remarkable reduction in the Psoriasis Area and Severity Index (PASI) score from 3. 75 to 0. 5 within five days. These findings highlight the potential of EUNPGel as an innovative nanomedicine for treating inflammation. This novel approach enhances ROS scavenging capacity, improves cellular uptake, facilitates skin penetration and retention, reduces the activity of hyperactive immune cells, and suggests potential applications for treating other immune-related disorders such as acne and atopic dermatitis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/599274v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@1f12fe6org.highwire.dtl.DTLVardef@17fa70forg.highwire.dtl.DTLVardef@1b0f28forg.highwire.dtl.DTLVardef@4ae07_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIHydrogel loaded with eugenol is an innovative alternative for psoriasis management. C_LIO_LISuperior drug loading, release kinetics, stability, and ROS scavenging capacity. C_LIO_LICurb (human keratinocyte) HaCaT cells proliferation, induce apoptosis, lower IL-6 mediated inflammation. C_LIO_LIEffective dermal penetration and retention both in vivo and ex vivo. C_LI

immunology↗

bioRxiv2024

Transcutaneous delivery of disease specific PI3K/Akt/mTOR inhibitor based hybrid nanoparticles in hydrogel system for the management of psoriasis: Insights from in vivo studies

We have reported robust and scalable lipid polymeric conjugated hybrid nanoparticles comprised of phospholipid shell and polymeric core that combine the benefit of both polymeric and lipid nanoparticles. Rapamycin, a PI3K/Akt/mTORC1 inhibitor, was encapsulated inside the lipid-polymeric conjugated spherical-shaped hybrid nanoparticles (RPMN), having an encapsulation efficiency of {approx} 83% and particle size {approx} 277.6 nm. Further, RPMN was converted into the carpool-based hydrogel system (RPMNGel), which enhanced release kinetics, long-term stability and skin residence time. Specifically, in an in-vivo imiquimod-induced psoriatic model, RPMNGel showed high accumulation and deeper penetration inside the epidermis, slowly diffusing away inside the psoriatic skin without causing any side effect to normal skin. This leads to longer and sustained retention over more than three days without being affected by sweat, humidity or wiping due to adherence between the stratum corneum and epidermis. Similarly, the cumulative PASI score was also reduced from 10.25 to 1.75 on day 7 in the group treated with RPMNGel. Overall, RPMNGel has a potential role in treating and managing psoriasis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/599287v2_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1366d88org.highwire.dtl.DTLVardef@51255forg.highwire.dtl.DTLVardef@16ca71dorg.highwire.dtl.DTLVardef@4f93e9_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2024

Transpiration efficiency variations in the pearl millet reference collection PMiGAP

Transpiration efficiency (TE), the biomass produced per unit of water transpired, is a key trait for crop performance under limited water. As water becomes scarce, increasing TE would contribute to increase crop drought tolerance. This study is a first step to explore pearl millet genotypic variability for TE on a large and representative diversity panel. We analyzed TE on 538 pearl millet genotypes, including inbred lines, test-cross hybrids, and hybrids bred for different agroecological zones. Three lysimeter trials were conducted in 2012, 2013 and 2015, to assess TE both under well-watered and terminal-water stress conditions. We recorded grain yield to assess its relationship with TE. Up to two-fold variation for TE was observed over the accessions used. Mean TE varied between inbred and testcross hybrids, across years and was slightly higher under water stress. TE also differed among hybrids developed for three agroecological zones, being higher in hybrids bred for the wetter zone, underlining the importance of selecting germplasm according to the target area. Environmental conditions triggered large genotype-by-environment interactions, although TE showed some moderately high heritability. Transpiration efficiency was the second contributor to grain yield after harvest index, hence its relevance in pearl millet breeding programs. Future research on TE in pearl millet should focus (i) on investigating the causes of its plasticity i.e. the GxE interaction (ii) on studying its genetic basis and its association with other important physiological traits.

plant biology↗

bioRxiv2023

Lack of Purple Acid Phosphatase SlPAP26b compromises the phosphorous starvation response in tomato independent of SlPHR1 and SlPHL1

The scarcity of soil phosphorus (P), an essential macronutrient, often limits plant growth and development. Enhanced secretion of intracellular and secretory acid phosphatases is essential to maintain cellular inorganic P (Pi) homeostasis in plants. Herein, using transcriptomics and proteomics approach, we observed upregulation of several purple acid phosphatases (PAPs), including SlPAP1, SlPAP10b, SlPAP12, SlPAP15, SlPAP17b, SlPAP26a, and SlPAP26b in Pi-deficient tomato seedlings. Higher transcript levels of SlPAP17b and SlPAP26b in the older senescing leaves than the younger leaves indicated active involvement of these PAPs in Pi remobilization. Subsequent detailed characterization of SlPAP17b, SlPAP26a, and SlPAP26b revealed a prominent role of SlPAP26b in Pi homeostasis. Silencing of SlPAP26b led to an exacerbated P starvation response as these plants exhibited smaller shoots, lower soluble Pi, total P levels, and higher sucrose than their EV controls under Pi deprivation. SlPAP26b-silenced plants also showed misregulation of P starvation inducible genes such as phosphate transporters and glycerolipid remodellers, even under Pi-sufficient conditions. Whereas SlPAP26b levels were induced by external sucrose, its expression was found to be independent of the Myb class master regulators of P starvation response, SlPHR1 and SlPHL1. Altogether, this study identifies a prominent role of SlPAP26b in the Pi compensation network in tomato seedlings.

plant biology↗

bioRxiv2023

Rewiring native post-transcriptional carbon regulators to build multi-layered genetic circuits and optimize engineered microbes for bioproduction

As the bioprocessing and synthetic biology spaces rapidly expand, post-transcriptional regulation is emerging as driver for maximizing signal response rate and for minimizing cost per protein within cells. Designing robust post-transcriptional control systems that have precise tunability and can achieve diverse regulatory outcomes are paramount to advance the field. Herein, we develop a new approach for engineered post-transcriptional control in bacteria by rewiring a native regulatory system in Escherichia coli, the Carbon Storage Regulatory (Csr) Network, to create tunable, complex genetic circuits. First, by co-opting native components of the Csr Network to regulate translation of a target mRNA transcript, we establish a Csr-regulated Buffer Gate. Next, by rationally engineering the interactions between our synthetic construct and the native components of the Csr Network, we expand our original design into a genetic toolbox of 12 Buffer Gates that achieve precise tunability across a 10-fold range of target gene expression. Subsequently, to further regulatory capabilities using this approach, we develop a Csr-regulated NOT Gate through engineering a Csr-activated sequence into our synthetic constructs. We then build upon the Csr Buffer and Not Gates to create post-transcriptional dual input Boolean OR, NOR, AND and NAND Logic Gates, as well as a genetic pulse circuit. As a third step, we demonstrate portability of our Csr-regulated Buffer Gates into three industrially relevant bacteria by recapitulating Buffer Gate activity simply by leveraging the conserved homologous Csr Network in each species. Lastly, as a demonstration of downstream application, we apply our system to a proof-of-concept synthetic mevalonate pathway. Using our engineered constructs, we optimize mevalonate production in E. coli resulting in a three-fold increase in production relative to a transcriptionally controlled mevalonate pathway. As a whole, we establish a novel approach to rewire post-transcriptional regulatory networks for complex bacterial computation that can be utilized for efficient bioproduction in engineered microbes.

bioengineering↗

bioRxiv2023

Anti-Viral and Anti-Inflammatory Therapeutic Effect of RAGE-Ig Protein Against Multiple SARS-CoV-2 Variants of Concern Demonstrated in K18-hACE2 Mouse and Syrian Golden Hamster Models

SignificanceSARS-CoV-2 Variants of Concern (VOCs) continue to evolve and re-emerge with chronic inflammatory long-COVID sequelae necessitating the development of anti-inflammatory therapeutic molecules. Therapeutic effects of the Receptor for Advanced Glycation End products (RAGE) were reported in many inflammatory diseases. However, a therapeutic effect of the RAGE in COVID-19 has not been reported. In the present study, we investigated whether and how the RAGE-Ig fusion protein would have an anti-viral and anti-inflammatory therapeutic effect in the COVID-19 system. MethodsThe protective therapeutic effect of RAGE-Ig was determined in vitro in K18-hACE2 transgenic mice and Syrian golden hamsters infected with six various VOCs of SARS-CoV-2. The underlying anti-viral mechanism of RAGE-Ig was determined in vitro in SARS-CoV-2-infected human lung epithelial cells (BEAS-2B). ResultsFollowing treatment of K18-hACE2 mice and hamsters infected with various SARS-CoV-2 VOCs with RAGE-Ig, we demonstrated: (i) significant dose-dependent protection (i.e. greater survival, less weight loss, lower virus replication in the lungs); (ii) a reduction of inflammatory macrophages (F4/80+/Ly6C+) and neutrophils (CD11b+/Ly6G+) infiltrating the infected lungs; (iii) a RAGE-Ig dose-dependent increase in the expression of type I interferons (IFN-, and IFN-{beta}) and type III interferon (IFN{lambda}2) and a decrease in the inflammatory cytokines (IL-6 and IL-8) in SARS-CoV-2-infected human lung epithelial cells; and (iv) a dose-dependent decrease in the expression of CD64 (FcgR1) on monocytes and lung epithelial cells from symptomatic COVID-19 patients. ConclusionOur pre-clinical findings revealed type I and III interferons-mediated anti-viral and anti-inflammatory therapeutic effects of RAGE-Ig protein against COVID-19 caused by multiple SARS-CoV-2 VOCs.

immunology↗

bioRxiv2023

The Prolonged Terminal Phase of Human Life Induces Survival Response in the Skin Transcriptome

Human death marks the end of organismal life under conditions such that the components of the human body continue to be alive. Such postmortem cellular survival depends on the nature (Hardy scale of slow-fast death) of human death. Slow and expected death typically results from terminal illnesses and includes a prolonged terminal phase of life. As such organismal death process unfolds, do cells of the human body adapt for postmortem cellular survival? Organs with low energy cost-of-living, such as the skin, are better suited for postmortem cellular survival. In this work, the effect of different durations of terminal phase of human life on postmortem changes in cellular gene expression was investigated using RNA sequencing data of 701 human skin samples from the Genotype-Tissue Expression (GTEx) database. Longer terminal phase (slow-death) was associated with a more robust induction of survival pathways (PI3K-Akt signaling) in postmortem skin. Such cellular survival response was associated with the upregulation of embryonic developmental transcription factors such as FOXO1, FOXO3, ATF4 and CEBPD. Upregulation of PI3K-Akt signaling was independent of sex or duration of death-related tissue ischemia. Analysis of single nucleus RNA-seq of post-mortem skin tissue specifically identified the dermal fibroblast compartment to be most resilient as marked by adaptive induction of PI3K-Akt signaling. In addition, slow death also induced angiogenic pathways in the dermal endothelial cell compartment of postmortem human skin. In contrast, specific pathways supporting functional properties of the skin as an organ were downregulated following slow death. Such pathways included melanogenesis and those representing the skin extracellular matrix (collagen expression and metabolism). Efforts to understand the significance of death as a biological variable (DABV) in influencing the transcriptomic composition of surviving component tissues has far-reaching implications including rigorous interpretation of experimental data collected from the dead and mechanisms involved in transplant-tissue obtained from dead donors.

genomics↗

bioRxiv2023

Development of a human glioblastoma model using humanized DRAG mice for immunotherapy

Glioblastoma (GBM) is the most common and lethal primary brain tumor with high mortality rates and a short median survival rate of about 15 months despite intensive multimodal treatment of maximal surgical resection, radiotherapy, and chemotherapy. Although immunotherapies have been successful in the treatment of various cancers, disappointing results from clinical trials for GBM immunotherapy represent our incomplete understanding. The development of alternative humanized mouse models with fully functional human immune cells will potentially accelerate the progress of GBM immunotherapy. In this study, we developed a humanized DRAG (NOD.Rag1KO.IL2R{gamma}cKO) mouse model, in which the human hematopoietic stem cells (HSCs) were well-engrafted and subsequently differentiated into a full lineage of immune cells. Using this humanized DRAG mouse model, GBM patient-derived tumorsphere lines were successfully engrafted to form xenografted tumors, which can recapitulate the pathological features and the immune cell composition of human GBM. Importantly, the administration of anti-human PD-1 antibodies in these DRAG mice bearing a GBM patient-derived tumorsphere line resulted in decreasing the major tumor-infiltrating immunosuppressive cell populations, including CD4+PD-1+ and CD8+PD-1+ T cells, CD11b+CD14+HLA-DR+ macrophages, CD11b+CD14+HLA-DR-CD15- and CD11b+CD14- CD15+ myeloid-derived suppressor cells, indicating the humanized DRAG mouse model as a useful model to test the efficacy of immune checkpoint inhibitors in GBM immunotherapy. Together, these results suggest that humanized DRAG mouse models are a reliable preclinical platform for brain cancer immunotherapy and beyond.

cancer biology↗