Biology subjects

Chatterjee, A.

Publications and source records attributed to Chatterjee, A..

At least 55 records · Page 3Linked to original sources

bioRxiv2024

Identification and characterization of innate immunity in Actinidia melanandra in response to Pseudomonas syringae pv. actinidiae

Pseudomonas syringae pv. actinidiae biovar 3 (Psa3) has decimated kiwifruit orchards growing susceptible kiwifruit Actinidia chinensis varieties. Effector loss has occurred recently in Psa3 isolates from resistant kiwifruit germplasm, resulting in strains capable of partially overcoming resistance present in kiwiberry vines (A. arguta, A. polygama, and A. melanandra). Diploid male A. melanandra recognises several effectors, sharing recognition of at least one avirulence effector (HopAW1a) with previously studied tetraploid kiwiberry vines. Sequencing and assembly of the A. melanandra genome enabled the characterisation of the transcriptomic response of this non-host to wild-type and genetic mutants of Psa3. A. melanandra appears to mount a classic effector-triggered immunity (ETI) response to wildtype Psa3 V-13, as expected. Surprisingly, the type III secretion (T3S) system-lacking Psa3 V-13 {Delta}hrcC strain did not appear to trigger pattern-triggered immunity (PTI) despite lacking the ability to deliver immunity-suppressing effectors. Contrasting the A. melanandra responses to an effectorless Psa3 V-13 {Delta}33E strain and to Psa3 V-13 {Delta}hrcC suggested that PTI triggered by Psa3 V-13 was based on the recognition of the T3S itself. The characterisation of both ETI and PTI branches of innate immunity responses within A. melanandra further enables breeding for durable resistance in future kiwifruit cultivars.

plant biology↗

bioRxiv2024

Inflammasome-Inhibiting Nanoligomers are Neuroprotective Against Space-Induced Pathology in Healthy and Diseased 3D Human Motor and Pre-Frontal Cortex Brain Organoids

Microgravity and space environment has been linked to deficits in neuromuscular and cognitive capabilities, hypothesized to occur due to accelerated aging and neurodegeneration in space. While the specific mechanisms are still being investigated, spaceflight-associated neuropathology is an important health risk to space astronauts and tourists, and is being actively investigated for the development of appropriate countermeasures. However, such space-induced neuropathology offers an opportunity for accelerated screening of therapeutic targets and lead molecules for treating neurodegenerative diseases. Here we show, a proof-of-concept high-throughput target screening (on Earth), target validation, and mitigation of microgravity-induced neuropathology using our Nanoligomer platform, onboard the 43-day SpaceX CRS-29 mission to the International Space Station (ISS). First, comparing 3D healthy and diseased pre-frontal cortex (PFC, for cognition) and motor neuron (MN, for neuromuscular function) organoids, we assessed space-induced pathology using biomarkers relevant to Alzheimers Disease (AD), Frontotemporal Dementia (FTD), and Amyotrophic Lateral Sclerosis (ALS). Both healthy and diseased PFC and MN organoids showed significantly enhanced neurodegeneration in space, as measured through relevant disease biomarkers, when compared to their respective Earth controls. Second, we tested the top two lead molecules, NI112 which targeted NF-{kappa}B, and NI113 that targeted IL-6. We observed that these Nanoligomers significantly mitigate the AD, FTD, and ALS relevant biomarkers like amyloid beta-42 (A{beta}42), phosphorylated Tau (pTau), Kallikrein (KLK-6), Tar DNA-binding protein 43 (TDP-43), and others. Moreover, the 43-day Nanoligomer treatment of these brain organoids did not appear to cause any observable toxicity or safety issues in the target organoid tissue, suggesting good tolerability for these molecules in the brain at physiologically relevant doses. Together, these results show significant potential for both the development and translation of NI112 and NI113 molecules as potential neuroprotective countermeasures for safer space travel, and demonstrate the usefulness of the space environment for rapid, high-throughput screening of targets and lead molecules for clinical translation. We assert that the use of microgravity in drug development and screening may ultimately benefit millions of patients suffering from debilitating neurodegenerative diseases on Earth.

neuroscience↗

bioRxiv2024

Multiscale Correlations between Joint and Tissue-Specific Biomechanics and Anatomy in Postmortem Ovine Stifles

The stability of the knee joint is an important indicator of its overall health and function. Joint stability depends on multiple structural and functional properties that include the anatomy of the underlying bones, the geometry and stiffness of the joint capsule and the soft tissues within like tendon, ligaments, cartilage and meniscus. The multiscale biomechanical relationships between the form and function of the whole joint and individual tissues can provide useful insights on the physiological condition of the knee and require further exploration. To better understand these relationships, in this study we compare multiple structural and mechanical parameters in healthy ovine stifles (n = 6). Specifically, we have evaluated joint laxity, joint morphology, individual tissue T2* relaxation and mechanical properties of the anterior and posterior cruciate ligaments (ACL, PCL), medial and lateral collateral ligaments (MCL, LCL), the patellar tendon, menisci, and cartilage from the femoral condyles. Using mechanical testing at two length scales along with anatomical and quantitative magnetic resonance imaging (MRI) scans, we investigate the correlation between individual tissue and whole joint mechanical properties. We also performed statistical tests to measure the strength of correlation coefficients between the measured metrics at multiple levels among whole joint mechanics, joint size, and individual tissue properties. We observe positive correlations between the joint laxity forces and the epicondyle-to-epicondyle distance measured as an anatomical marker. We also find that the viscoelastic properties of the tendons and ligaments correlate positively with joint laxity forces. No such correlations were observed between the cartilage and meniscus properties and the joint laxity forces. Further, we found a weak inverse correlation between the tissue viscoelastic properties and T2* for MCL; strong and moderately positive correlations for cartilage samples from both femoral condyles and the menisci, LCL and PCL respectively. These results provide useful insights into the differential role of individual tissue properties that can be used to predict the whole joint responses that are key indicators of knee health and performance.

bioengineering↗

bioRxiv2024

Brain-Penetrant NF-κB and NLRP3 Targeting Nanoligomers are Therapeutic in Amyotrophic Lateral Sclerosis (ALS) and Alzheimers Disease (AD) Human Organoid and Mouse Models

Millions of people suffer worldwide from neurodegenerative diseases ranging from rapidly progressing and fatal motor neuron diseases like Amyotrophic Lateral Sclerosis (ALS) to more chronic illnesses such as frontotemporal dementia (FTD) and Alzheimers disease (AD). A growing number of studies have implicated neuroinflammation as a key and causative phenomenon and an important target for novel therapeutics for these diseases. Neuroinflammation is characterized by reactive glial cells that produce pro-inflammatory neurotoxic cytokines. Our previous studies have shown a brain-penetrant Nanoligomer cocktail (NI112) inhibiting the neuroinflammation mediators nuclear factor kappa-light-chain-enhancer of activated B cells (NF-{kappa}B) and NOD-like receptor family, pyrin domain containing 3 (NLRP3) is a safe, targeted, and effective neurotherapeutic drug. Here, we show that a four-week NI112 treatment is therapeutic using: 1) an ALS-FTD 3D human motor neuron organoid model of tar DNA binding protein 43 (TDP-43, a key contributor to ALS pathology) overexpression (knock-in); 2) an AD model of APOE4/APOE4 (AD risk allele) double mutation in human neurons comprising a 3D human prefrontal cortex (PFC) organoid; and 3) multiple in vivo (mouse models) of the same/related conditions. In 3D organoids made from healthy motor neurons (HMN negative control) and TDP-43 overexpressing (or ALS organoids), we monitored the mean firing rate using calcium signaling as a functional output, while measuring TDP-43 and other key neurodegeneration biomarkers. After 4 weeks, we observed a massive improvement in the mean firing rate of NI112-treated ALS organoids compared to untreated ALS organoids, which was more comparable to healthy HMN organoids. Similarly, we found a significant decrease in neurodegeneration markers like amyloid beta 42 (A{beta}42) in NI112-treated AD organoids compared to untreated AD organoids (A{beta}42 comparable to healthy PFC organoids). In the mouse ALS (SOD1-G93A) model, we observed behavioral improvements and restoration of motor function (e.g., grip strength) in NI112-treated mice, and in mouse AD model mice (radiation-induced accelerated neuropathology in APP/PS1, and rTg4510 phospho-tau), we observed improved cognition. In both models, we also found an accompanying reduction in neuroinflammation and reduced neuropathology. These results show the promise for further testing and development of neuroinflammation-targeting Nanoligomers to benefit patients suffering from debilitating neurodegenerative diseases like ALS, FTD, and AD.

neuroscience↗

bioRxiv2024

Characterization of Composite Agarose-Collagen Hydrogels for Chondrocyte Culture

To elucidate the mechanisms of cellular mechanotransduction, it is necessary to employ biomaterials that effectively merge biofunctionality with appropriate mechanical characteristics. Agarose and collagen separately are common biopolymers used in cartilage mechanobiology and mechanotransduction studies but lack features that make them ideal for functional engineered cartilage. In this study, agarose (8% w/v and 4% w/v) is blended with collagen type I (4mg/mL) to create composites. We hypothesized that a higher stiffness, composite hydrogel would promote native cartilage-like conditions. To address these questions, acellular and cell-laden studies were completed to assess rheologic and compressive properties, contraction, and structural homogeneity in addition to matrix mechanics, cell proliferation, and glycosaminoglycan production. Over 21 days in culture, cellular 4% agarose - 2mg/mL collagen I hydrogels displayed good structural and bulk mechanical properties, cell proliferation, and continual glycosaminoglycan production, indicating promise towards the development of an effective hydrogel for chondrocyte mechanotransduction and mechanobiology studies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/583023v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@133f6aborg.highwire.dtl.DTLVardef@876594org.highwire.dtl.DTLVardef@11e05faorg.highwire.dtl.DTLVardef@1a9914_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

NF-kB/NLRP3 Translational Inhibition by Nanoligomer Therapy Mitigates Ethanol and Advanced Age-Related Neuroinflammation

Binge alcohol use is increasing among aged adults (>65 years). Alcohol-related toxicity in aged adults is associated with neurodegeneration, yet the molecular underpinnings of age-related sensitivity to alcohol are not well described. Studies utilizing rodent models of neurodegenerative disease reveal heightened activation of Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-{kappa}B) and Nod like receptor 3 (NLRP3) mediate microglia activation and associated neuronal injury. Our group, and others, have implicated hippocampal-resident microglia as key producers of inflammatory mediators, yet the link between inflammation and neurodegeneration has not been established in models of binge ethanol exposure and advanced age. Here, we report binge ethanol increased the proportion of NLRP3+ microglia in the hippocampus of aged (18-20 months) female C57BL/6N mice compared to young (3-4 months). In primary microglia, ethanol-induced expression of reactivity markers and NLRP3 inflammasome activation were more pronounced in microglia from aged mice compared to young. Making use of an NLRP3-specific inhibitor (OLT1177) and a novel brain- penetrant Nanoligomer that inhibits NF-{kappa}B and NLRP3 translation (SB_NI_112), we find ethanol- induced microglial reactivity can be attenuated by OLT1177 and SB_NI_112 in microglia from aged mice. In a model of intermittent binge ethanol exposure, SB_NI_112 prevented ethanol-mediated microglia reactivity, IL-1{beta} production, and tau hyperphosphorylation in the hippocampus of aged mice. These data suggest early indicators of neurodegeneration occurring with advanced age and binge ethanol exposure are NF-{kappa}B- and NLRP3-dependent. Further investigation is warranted to explore the use of targeted immunosuppression via Nanoligomers to attenuate neuroinflammation after alcohol consumption in the aged.

neuroscience↗

bioRxiv2024

Directed evolution of a bacterial leucyl tRNA in mammalian cells for enhanced noncanonical amino acid mutagenesis

The E. coli leucyl-tRNA synthetase (EcLeuRS)/tRNAEcLeu pair has been engineered to genetically encode a structurally diverse group of enabling noncanonical amino acids (ncAAs) in eukaryotes, including those with bioconjugation handles, environment-sensitive fluorophores, photocaged amino acids, and native post-translational modifications. However, the scope of this toolbox in mammalian cells is limited by the poor activity of tRNAEcLeu. Here, we overcome this limitation by evolving tRNAEcLeu directly in mammalian cells using a virus-assisted selection scheme. This directed evolution platform was optimized for higher throughput such that the entire acceptor stem of tRNAEcLeu could be simultaneously engineered, which resulted in the identification of several variants with remarkably improved efficiency for incorporating a wide range of ncAAs. The advantage of the evolved leucyl tRNAs was demonstrated by expressing ncAA mutants in mammalian cells that were challenging to express before using the wild-type tRNAEcLeu, by creating viral vectors that facilitated ncAA mutagenesis at a significantly lower dose, and by creating more efficient mammalian cell lines stably expressing the ncAA-incorporation machinery.

synthetic biology↗

bioRxiv2024

Microbiome- and Host Inflammasome-Targeting Inhibitor Nanoliogmers are Therapeutic in Murine Colitis Model

Autoimmune and autoinflammatory diseases account for more than 80 chronic conditions affecting more than 24 million people in the US. Amongst these autoinflammatory diseases, non-infectious chronic inflammation of the gastrointestinal (GI) tract causes inflammatory bowel diseases (IBD), primarily Crohns and Ulcerative Colitis (UC). IBD is a complex disease, and one hypothesis is that these are either caused or worsened by compounds produced by bacteria in the gut. While traditional approaches have focused on pan immunosuppressive techniques (e.g., steroids), low remission rates, prolonged illnesses, and increased frequency of surgical procedures have prompted the search for more targeted and precision therapeutic approaches. IBD is a complex disease resulting from both genetic and environmental factors, but several recent studies have highlighted the potential pivotal contribution of gut microbiota dysbiosis. Gut microbiota are known to modulate the immune status of the gut by producing metabolites that are encoded in biosynthetic gene clusters (BGCs) of the bacterial genome. Here we show, a targeted and high-throughput screening of more than 90 biosynthetic genes in 41 gut anaerobes, through down selection using available bioinformatics tools, targeted gene manipulation in these genetically intractable organisms using Nanoligomer platform, and identification and synthesis of top microbiome-targets as a Nanoligomer BGC cocktail (SB_BGC_CK1, abbreviated as CK1) as a feasible precision therapeutics approach. Further, we used a host-directed immune-target screening to identify NF-{kappa}B and NLRP3 cocktail SB_NI_112 (or NI112 for short) as a targeted inflammasome inhibitor. We used these top two microbe- and host-targeted Nanoligomer cocktails in acute and chronic dextran sulfate sodium (DSS) mouse colitis and in TNF{Delta}ARE/+ transgenic mice that develop spontaneous Crohns like ilitis. The mouse microbiome was humanized to replicate that in human IBD through antibiotic treatment followed by mixed fecal gavage from 10 human donors and spiked with IBD-inducing microbial species. Following colonization, colitis was induced in mice using one week of 3% DSS (acute), or six weeks of 3 rounds of 2.5% DSS induction for a week followed by one week of no DSS (chronic colitis model). Both Nanoligomer cocktails (CK1 and NI112) showed a strong reduction in disease severity, significant improvement in disease histopathology, and profound downregulation of disease biomarkers in colon tissue as assessed by multiplexed ELISA. Further, we used two different formulations of intraperitoneal injections (IP) and Nanoligomer pills in the chronic DSS colitis model. Although both formulations were highly effective, the oral pill formulation demonstrated a greater reduction in biochemical markers compared to IP. A similar therapeutic effect was observed in the TNF{Delta}ARE/+ model. Overall, these results point to the potential for further development and testing of these inflammasome-targeting host-directed therapy (NI112), and more personalized microbiome-cocktails (CK1) for patients with recalcitrant IBD. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/581256v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@834b11org.highwire.dtl.DTLVardef@1b5539corg.highwire.dtl.DTLVardef@1844356org.highwire.dtl.DTLVardef@60f51e_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOTOC GRAPHICC_FLOATNO C_FIG

molecular biology↗

bioRxiv2024

Large- and Small-Animal Studies of Safety, Pharmacokinetics (PK), and Biodistribution of Inflammasome-Targeting Nanoligomer in the Brain and Other Target Organs

Immune malfunction or misrecognition of healthy cells and tissue, termed autoimmune disease, is implicated in more than 80 disease conditions and multiple other secondary pathologies. While pan-immunosuppressive therapies like steroids offer some relief for systemic inflammation for some organs, many patients never achieve remission and such drugs do not cross the blood-brain barrier making them ineffective for tackling neuroinflammation. Especially in the brain, unintended activation of microglia and astrocytes is hypothesized to be directly or indirectly responsible for Multiple Sclerosis (MS), Amyotrophic Lateral Sclerosis (ALS), Parkinsons Disease (PD), and Alzheimers Disease (AD). Recent studies have also shown that targeting inflammasome and specific immune targets can be beneficial for these diseases. Further, our previous studies have shown targeting NF-{kappa}B and NLRP3 through brain penetrant Nanoligomer cocktail SB_NI_112 (abbreviated to NI112) can be therapeutic for several neurodegenerative diseases. Here we show safety-toxicity studies, followed by pharmacokinetics (PK) and biodistribution in small- (mice) and large-animal (dog) studies of this inflammasome-targeting Nanoligomer cocktail NI 112. We conducted studies using four different routes of administration: intravenous (IV), subcutaneous (SQ), intraperitoneal (IP), and intranasal (IN), and identified the drug concentration over time using inductively coupled plasma mass spectrometry (ICP-MS) in the blood serum, the brain (including different brain regions), and other target organs like liver, kidney, and colon. Our results indicate the Nanoligomer cocktail has a strong safety profile, and shows high biodistribution (F [~]0.98) and delivery across multiple routes of administration. Further analysis showed high brain bioavailability with a ratio of NI112 in brain tissue to blood serum [~]30%. Our model accurately shows dose scaling, translation between different routes of administration, and interspecies scaling. These results provide an excellent platform for human clinical translation and predicting therapeutic dosage between different routes of administration.

pharmacology and toxicology↗

bioRxiv2024

Nanoligomers targeting NF-κB and NLRP3 reduce neuroinflammation and improve cognitive function with aging and tauopathy

Neuroinflammation contributes to impaired cognitive function in brain aging and neurodegenerative disorders like Alzheimers disease, which is characterized by the aggregation of pathological tau. One major driver of both age- and tau-associated neuroinflammation is the NF-{kappa}B and NLRP3 signaling axis. However, current treatments targeting NF-{kappa}B or NLRP3 may have adverse/systemic effects, and most have not been clinically translatable. In this study, we tested the efficacy of a novel, nucleic acid therapeutic (Nanoligomer) cocktail specifically targeting both NF-{kappa}B and NLRP3 in the brain for reducing neuroinflammation and improving cognitive function in old (aged 19 months) wildtype mice, and in rTg4510 tau pathology mice (aged 2 months). We found that 4 weeks of NF-{kappa}B/NLRP3-targeting Nanoligomer treatment strongly reduced neuro-inflammatory cytokine profiles in the brain and improved cognitive-behavioral function in both old and rTg4510 mice. These effects of NF-{kappa}B/NLRP3-targeting Nanoligomers were also associated with reduced glial cell activation and pathology, favorable changes in transcriptome signatures of glia-associated inflammation (reduced) and neuronal health (increased), and positive systemic effects. Collectively, our results provide a basis for future translational studies targeting both NF-{kappa}B and NLRP3 in the brain, perhaps using Nanoligomers, to inhibit neuroinflammation and improve cognitive function with aging and neurodegeneration.

physiology↗

bioRxiv2024

Curtailed CaSR expression in peripheral blood mononuclear cells is associated with hypertension

The calcium-sensing receptor (CaSR) is a GPCR family member. CaSR is abundantly expressed in the parathyroid gland and controls calcium homeostasis. There are no particular discoveries involving CaSR expression in hypertensive peripheral blood mononuclear cells. We investigated lower CaSR expression in hypertensive human and animal models. Nitric oxide (NO) has an important role in vascular function. Impaired NO production disrupts CaSR-mediated fine adjustment of the intra- and extracellular [Ca2+] gradients. According to the law Ca2+ transportation through Ca channels maintains its connection from the circulatory micro-vascular bed to interstitial space, influenced by capillary and interstitial fluid hydrostatic pressure, as well as oncotic pressure. In that situation, the alterations in Ca2+ microenvironment and dynamics remain unknown. CaSR on circulatory mononuclear cells may detect changes in the Ca2+ microenvironment in the ISF of endothelial cells-VSMC and endothelium lining in hypertension. In this experiment, the decreased expression of CaSR revealed a molecular sensor in the presence of elevated blood pressure.

biochemistry↗

bioRxiv2024

Targeted-Neuroinflammation Mitigation Using Inflammasome-Inhibiting Nanoligomers is Therapeutic in Experimental Autoimmune Encephalomyelitis (EAE) Mouse Model

Multiple Sclerosis (MS) is a debilitating autoimmune disease that impacts millions of patients worldwide, disproportionately impacting women (4:1), and often presenting at highly productive stages of life. This disease affects the spinal cord and brain, and is characterized by severe neuroinflammation, demyelination, and subsequent neuronal damage, resulting in symptoms like loss of mobility. While untargeted and pan-immunosuppressive therapies have proven to be disease-modifying and manage (or prolong the time between) symptoms in many patients, a significant fraction are unable to achieve remission. Recent work has suggested more targeted neuroinflammation mitigation through selective inflammasome-inhibition can offer relief to patients, while preserving key components of immune function. Here we show a screening of potential therapeutic targets using inflammasome-inhibiting Nanoligomers (NF-{kappa}B1, TNFR1, TNF-, IL-6), that meet or far-exceed commercially available small-molecule counterparts like Ruxolitinib, MCC950, and Deucravacitinib. Using the human brain organoid model, top Nanoligomer combinations (NF-{kappa}B1+TNFR1: NI111, and NF-{kappa}B1+NLRP3: NI112) were shown to significantly reduce neuroinflammation, without any observable negative impact on organoid function. Further testing of these top Nanoligomer combinations in an aggressive Experimental Autoimmune Encephalomyelitis (EAE) mouse model for MS using intraperitoneal (IP) injections showed that NF-{kappa}B1and NLRP3 targeting Nanoligomer combination NI112 rescues mice without observable loss of mobility or disability, minimal inflammation in brain and spinal cord histology, and minimal to no immune cell infiltration of the spinal cord and no demyelination, similar to or at par with mice that received no EAE injections (negative control). Mice receiving NI111 (NF-{kappa}B1+TNFR1) also showed reduced neuroinflammation compared to saline (sham) treated EAE mice and at par/similar to other inflammasome-inhibiting small molecule treatments, although it was significantly higher than NI112 leading to subsequent worsening clinical outcomes. Furthermore, treatment with an oral formulation of NI112 at lower doses showed a significant reduction in EAE severity, albeit with higher variance owing to administration and formulation/fill-and-finish variability. Overall, these results point to the potential of further development and testing these inflammasome targeting Nanoliogmers as an effective neuroinflammation treatment for multiple neurodegenerative diseases, and potentially benefit several patients suffering from such debilitating autoimmune diseases like MS.

neuroscience↗

bioRxiv2024

Northeast India: Genetic Inconsistency across Ethnicity and Geography

The genetic landscape of South Asia is intriguing. In a vast geographical region like India, mostly genes follow geography, whereas in a smaller geographical region e.g., Nepal, the genetic landscape is more akin to languages. Moreover, in neighboring regions like East and Southeast Asia, language is the major denominator of the genes. However, these are not just the two alternatives, in many geographical pockets, ethnicity plays a stronger role than either of these. Being a crossroads of South and Southeast Asia, the Himalayan region is mostly populated by people speaking the Trans-Himalayan language. Archaeological and anthropological studies suggest migration and cultural diffusion in this region with the Tibetan plateau in the North. To understand this complex pattern, we have performed a fine-grained genetic analysis of the major Mizo population and its various clans living in the Himalayan geography. We have investigated 110 individuals belonging to seven different clans of Mizo people for a hundred thousand autosomal markers. We have used various statistical methods and tested the role of ethnicity and geography in shaping the genetic landscape of the Himalayan region. In contrast to the East and Southeast Asian genetic landscape, our results suggested that the fine-scaled genetic structure of Northeast India is much more complex and a mixture of interplay between languages, ethnicity, and geography. Allelic frequency-based analyses indicated that a novel Trans-Himalayan ancestry unite all the populations of this region.

evolutionary biology↗

bioRxiv2023

Role of Ethanolamine Utilization and Bacterial Microcompartment Formation in Listeria monocytogenes Intracellular Infection

Ethanolamine (EA) affects the colonization and pathogenicity of certain human bacterial pathogens in the gastrointestinal tract. However, EA can also affect the intracellular survival and replication of host-cell invasive bacteria such as Listeria monocytogenes (LMO) and Salmonella enterica serovar Typhimurium (S. Typhimurium). The EA utilization (eut) genes can be categorized as regulatory, enzymatic, or structural, and previous work in LMO showed that loss of genes encoding functions for the enzymatic breakdown of EA inhibited LMO intracellular replication. In this work, we sought to further characterize the role of EA utilization during LMO infection of host cells. Unlike what was previously observed for S. Typhimurium, in LMO, an EA regulator mutant ({Delta}eutV) was equally deficient in intracellular replication compared to an EA metabolism mutant ({Delta}eutB), and this was consistent across Caco-2, RAW 264.7 and THP-1 cell lines. The structural genes encode proteins that self-assemble into bacterial microcompartments (BMCs) that encase the enzymes necessary for EA metabolism. For the first time, native EUT BMCs were fluorescently tagged, and EUT BMC formation was observed in vitro, and in vivo. Interestingly, BMC formation was observed in bacteria infecting Caco-2 cells, but not the macrophage cell lines. Finally, the cellular immune response of Caco-2 cells to infection with eut mutants was examined, and it was discovered that{Delta} eutB and{Delta} eutV mutants similarly elevated the expression of inflammatory cytokines. In conclusion, EA sensing and utilization during LMO intracellular infection are important for optimal LMO replication and immune evasion but are not always concomitant with BMC formation.

microbiology↗

bioRxiv2023

A translation-independent directed evolution strategy to engineer aminoacyl-tRNA synthetases

Using directed evolution, engineered aminoacyl-tRNA synthetases (aaRS) have been developed that enable co-translational incorporation of numerous noncanonical amino acids (ncAAs) into proteins in living cells. Until now, the selection of such novel aaRS mutants has relied on coupling their activity to the expression of a reporter protein with a selectable phenotype. However, such translation-dependent selection schemes are incompatible with exotic monomers that diverge structurally from canonical -amino acids and are suboptimal substrates for the ribosome. To enable the ribosomal incorporation of such exotic monomers, a two-step solution is needed: A) Engineering an aaRS to acylate its cognate tRNA with the exotic monomer, without relying on ribosomal translation as a readout, and B) Subsequent engineering of the ribosome to accept the resulting acylated tRNA for translation. Here, we report a platform for aaRS engineering that directly selects for tRNA-acylation without ribosomal translation (START). In START, each distinct aaRS mutant is correlated to a cognate tRNA containing a unique sequence barcode. Acylation by an active aaRS mutant protects the associated barcode-containing tRNAs from an oxidative treatment designed to damage the 3'-terminus of the uncharged tRNAs. Sequencing of these surviving barcode-containing tRNAs is then used to reveal the identity of aaRS mutants that acylated the correlated tRNA sequences. The efficacy of START was demonstrated by identifying novel mutants of the M. alvus pyrrolysyl-tRNA synthetase from a naive library that charge noncanonical amino acids.

synthetic biology↗

bioRxiv2023

Cytosolic RNA binding of the mitochondrial TCA cycle enzyme malate dehydrogenase (MDH2)

Several enzymes of intermediary metabolism have been identified to bind RNA in cells, with potential consequences for the bound RNAs and/or the enzyme. In this study, we investigate the RNA-binding activity of the mitochondrial enzyme malate dehydrogenase 2 (MDH2), which functions in the tricarboxylic acid (TCA) cycle and the malate-aspartate shuttle. We confirmed in cellulo RNA-binding of MDH2 using orthogonal biochemical assays and performed enhanced crosslinking and immunoprecipitation (eCLIP) to identify the cellular RNAs associated with endogenous MDH2. Surprisingly, MDH2 preferentially binds cytosolic over mitochondrial RNAs, although the latter are abundant in the milieu of the mature protein. Subcellular fractionation followed by RNA-binding assays revealed that MDH2-RNA interactions occur predominantly outside of mitochondria. We also found that a cytosolically-retained N-terminal deletion mutant of MDH2 is competent to bind RNA, indicating that mitochondrial targeting is dispensable for MDH2-RNA interactions. MDH2 RNA binding increased when cellular NAD+ levels (MDH2s co-factor) was pharmacologically diminished, suggesting that the metabolic state of cells affects RNA binding. Taken together, our data implicate an as yet unidentified function of MDH2 binding RNA in the cytosol.

cell biology↗

bioRxiv2023

Backbone extension acyl rearrangements enable cellular synthesis of proteins with internal β2-peptide linkages

Proteins and polypeptides containing extended backbone monomers embody highly desirable structures and functions, but they cannot yet be biosynthesized in cells. There are two challenges at work. First is the ribosome, whose ability to promote rapid bond-forming reactions to and from anything other than an -amino acid or -hydroxy acid is unknown. The second challenge is the absence of orthogonal enzymes that acylate tRNA with extended backbone monomers. Here we describe a general approach to the programmed cellular synthesis of proteins containing extended backbone monomers that circumvents both of these challenges. Rather than relying on direct and uncharacterized reactions of non--amino acid monomers within the ribosomal PTC, we develop a proximity-guided intramolecular rearrangement that effectively edits the protein backbone post-translationally. The method relies on the ability of PylRS-like aminoacyl-tRNA synthetase enzymes to accept diverse -hydroxy acid monomers, including those whose side chains contain masked nucleophiles. Introduction of such an -hydroxy acid monomer into a protein translated in vivo, followed by nucleophile unmasking, sets up a thermodynamically favored and quantitative intramolecular Backbone Extension Acyl Rearrangement (BEAR) reaction that edits the protein backbone to install an extended backbone monomer. In the examples described here, the intramolecular rearrangement converts an -peptide backbone directly into a {beta}-backbone. As far as we know, this report represents the first example in which a much-desired expanded backbone {beta}-amino acid linkage has been introduced site-selectively into a protein in a cell.

synthetic biology↗

bioRxiv2023

NbPTR1 confers resistance against Pseudomonas syringae pv. actinidiae in kiwifruit

Pseudomonas syringae pv. actinidiae biovar 3 (Psa3) causes a devastating canker disease in yellow-fleshed kiwifruit (Actinidia chinensis). The effector HopZ5, which is present in all isolates of Psa3 causing global outbreaks of pandemic kiwifruit canker disease, triggers immunity in Nicotiana benthamiana and is not recognised in susceptible A. chinensis cultivars. In a search for N. benthamiana non-host resistance genes against HopZ5, we found that the nucleotide-binding leucine-rich repeat receptor NbPTR1 recognised HopZ5. RPM1-interacting protein 4 (RIN4) orthologues from multiple plants, including kiwifruit, were associated with NbPTR1-mediated autoimmunity suppression and recognition of HopZ5. No functional orthologues of NbPTR1 were found in A. chinensis. NbPTR1 transformed into Psa3-susceptible A. chinensis var. chinensis Hort16A plants introduced HopZ5-specific resistance against Psa3. Altogether, this study suggested that expressing NbPTR1 in Psa3-susceptible kiwifruit is a viable approach to acquiring resistance to Psa3 and it provides valuable information for engineering resistance in otherwise susceptible kiwifruit genotypes.

plant biology↗