Biology subjects

Chatterjee, A.

Publications and source records attributed to Chatterjee, A..

At least 73 records · Page 4Linked to original sources

bioRxiv2023

Precise manipulation of site and stoichiometry of capsid modification enables optimization of functional adeno-associated virus conjugates

The ability to engineer adeno-associated virus (AAV) vectors for targeted infection of specific cell types is critically important to fully harness its potential of human gene therapy. A promising approach to achieve this objective involves chemically attaching retargeting ligands onto the virus capsid. Site-specific incorporation of a bioorthogonal noncanonical amino acid (ncAA) into the AAV capsid proteins provides a particularly attractive strategy to introduce such modifications with exquisite precision. In this study, we show that using ncAA mutagenesis, it is possible to systematically alter the attachment site of a retargeting ligand (cyclic-RGD) on the AAV capsid to create diverse conjugate architectures, and that the site of attachment heavily impacts the retargeting efficiency. We further demonstrate that the performance of these AAV conjugates is highly sensitive to the stoichiometry of capsid labeling (labels per capsid), with an intermediate labeling density ([~]12 per capsid) providing optimal activity. Finally, we developed technology to precisely control the number of attachment sites per AAV capsid, by selectively incorporating a ncAA into the minor capsid proteins with high fidelity and efficiency, such that AAV-conjugates with varying stoichiometry can be synthesized in a homogeneous manner. Together, this platform provides unparalleled control over site and stoichiometry of capsid modification, which will enable the development of next-generation AAV vectors tailored with desirable attributes.

synthetic biology↗

bioRxiv2023

Chimeric identities and reduced stiffness characterise the shoot apex of Arabidopsis stem cell mutants

Stem cell homeostasis in the shoot apical meristem involves a core regulatory feedback loop between the signalling peptide CLAVATA3, produced in stem cells, and the transcription factor WUSCHEL, expressed in the underlying organising centre. clavata mutants display massive meristem overgrowth, which is thought to be caused by stem cell overproliferation, although it is unknown how uncontrolled stem cell divisions lead to this altered morphology. Here we first reveal local buckling defects in mutant meristems, and use analytical models to show how mechanical properties and growth rates may contribute to the phenotype. Indeed, clavata meristems are mechanically more heterogeneous than the wild type, and also display regional growth heterogeneities. Furthermore, stereotypical wild-type meristem organisation is lost in mutants, in which cells simultaneously express distinct fate markers. Finally, cells in mutant meristems are auxin responsive, suggesting that they are functionally different from wild-type stem cells. Thus all benchmarks show that clavata meristem cells are different from wild-type stem cells, suggesting that fasciation is caused by the disruption of a more complex regulatory framework that maintains distinct genetic and functional domains at the shoot apex. Summary statementHeterogeneities in cell mechanics, growth, function and identity contribute to buckling in clavata mutant shoot apices.

plant biology↗

bioRxiv2023

CellSNAP: A fast, accurate algorithm for 3D cell segmentation in quantitative phase imaging

Quantitative phase imaging (QPI) has rapidly emerged as a complementary tool to fluorescence imaging, as it provides an objective measure of cell morphology and dynamics, free of variability due to contrast agents. In particular, three-dimensional (3D) tomographic imaging of live cells has opened up new directions of investigation by providing systematic and correlative analysis of various cellular parameters without limitations of photobleaching and phototoxicity. While current QPI systems allow the rapid acquisition of tomographic images, the pipeline to analyze these raw 3D tomograms is not well-developed. This work focuses on a critical, yet often underappreciated, step of the analysis pipeline, that of 3D cell segmentation from the acquired tomograms. The current method employed for such tasks is the Otsu-based 3D watershed algorithm, which works well for isolated cells; however, it is very challenging to draw boundaries when the cells are clumped. This process is also memory intensive since the processing requires computation on a 3D stack of images. We report the CellSNAP (Cell Segmentation via Novel Algorithm for Phase Imaging) algorithm for the segmentation of QPI images, which outstrips the current gold standard in terms of speed, robustness, and implementation, achieving cell segmentation under 2 seconds per cell on a single-core processor. The implementation of CellSNAP can easily be parallelized on a multi-core system for further speed improvements. For the cases where segmentation is possible with the existing standard method, our algorithm displays an average difference of 5% for dry mass and 8% for volume measurements. We also show that CellSNAP can handle challenging image datasets where cells are clumped and marred by interferogram drifts, which pose major difficulties for all QPI-focused segmentation tools. We envision our work will lead to the broader adoption of QPI imaging for high-throughput analysis, which has, in part, been stymied by a lack of suitable image segmentation tools.

bioinformatics↗

bioRxiv2023

Two successive oligomeric Munc13 assemblies scaffold vesicle docking and SNARE assembly to support neurotransmitter release

The critical presynaptic protein Munc13 serves numerous roles in the process of docking and priming synaptic vesicles. Here we investigate the functional significance of two distinct oligomers of the Munc13 core domain (Munc13C) comprising C1-C2B-MUN-C2C. Oligomer interface point mutations that specifically destabilized either the trimer or lateral hexamer assemblies of Munc13C disrupted vesicle docking, trans-SNARE formation, and Ca2+-triggered vesicle fusion in vitro and impaired neurotransmitter secretion and motor nervous system function in vivo. We suggest that a progression of oligomeric Munc13 complexes couples vesicle docking and assembly of a precise number of SNARE molecules to support rapid and high-fidelity vesicle priming.

neuroscience↗

bioRxiv2023

Pneumatically Controlled Non-Equibiaxial Cell Stretching Device with Live-cell Imaging

ObjectiveAdherent cell behavior is influenced by a complex interplay of factors, including chemical and mechanical signals. In vitro experiments that mimic the mechanical environment experienced by cells in vivo are crucial for understanding cellular behavior and the progression of disease. In this study, we developed and validated a low-cost pneumatically-controlled cell stretcher with independent control of strain in two directions of a membrane, enabling unequal biaxial stretching and realtime microscopy during actuation. MethodsThe stretching was achieved by two independent pneumatic channels controlled by electrical signals. We used finite element simulations to compute the membranes strain field and particle tracking algorithms based on image processing techniques to validate the strain fields and measure the cell orientation and morphology. ResultsThe device can supply uniaxial, equibiaxial, and unequal biaxial stretching up to 15% strain in each direction at a frequency of 1Hz, with a strain measurement error of less than 1%. Through live cell imaging, we determined that distinct stretching patterns elicited differing responses and alterations in cell orientation and morphology, particularly in terms of cell length and area. ConclusionThe device successfully provides a large, uniform, and variable strain field for cell experiments, while also enabling real-time, live cell imaging. SignificanceThis scalable, low-cost platform provides mechanical stimulation to cell cultures by independently controlling strains in two directions. This could contribute to a deeper understanding of cellular response to biorealistic strains and could be useful for future in vitro drug testing platforms.

bioengineering↗

bioRxiv2023

The homeodomain drives favorable DNA binding energetics of prostate cancer target ONECUT2

The ONECUT transcription factors feature a CUT and a homeodomain, evolutionarily conserved elements that bind DNA cooperatively, but the process remains mechanistically enigmatic. Using an integrative DNA binding analysis of ONECUT2, a driver of aggressive prostate cancer, we show that the homeodomain energetically stabilizes the ONECUT2-DNA complex through allosteric modulation of CUT. Further, evolutionarily conserved base-interactions in both the CUT and homeodomain are necessary for the favorable thermodynamics. We have identified a novel arginine pair unique to the ONECUT family homeodomain that can adapt to DNA sequence variations. Base interactions in general, including by this arginine pair, are critical for optimal DNA binding and transcription in a prostate cancer model. These findings provide fundamental insights into DNA binding by CUT-homeodomain proteins with potential therapeutic implications. One-Sentence SummaryBase-specific interactions regulate homeodomain-mediated stabilization of DNA binding by the ONECUT2 transcription factor.

biophysics↗

bioRxiv2023

Novel Roles for Diacylglycerol in Synaptic Vesicle Priming and Release Revealed by Complete Reconstitution of Core Protein Machinery

Here we introduce the full functional reconstitution of genetically-validated core protein machinery (SNAREs, Munc13, Munc18, Synaptotagmin, Complexin) for synaptic vesicle priming and release in a geometry that enables detailed characterization of the fate of docked vesicles both before and after release is triggered with Ca2+. Using this novel setup, we discover new roles for diacylglycerol (DAG) in regulating vesicle priming and Ca2+-triggered release involving the SNARE assembly chaperone Munc13. We find that low concentrations of DAG profoundly accelerate the rate of Ca2+-dependent release, and high concentrations reduce clamping and permit extensive spontaneous release. As expected, DAG also increases the number of ready-release vesicles. Dynamic single-molecule imaging of Complexin binding to ready-release vesicles directly establishes that DAG accelerates the rate of SNAREpin assembly mediated by Munc13 and Munc18 chaperones. The selective effects of physiologically validated mutations confirmed that the Munc18-Syntaxin-VAMP2 template complex is a functional intermediate in the production of primed, ready-release vesicles, which requires the coordinated action of Munc13 and Munc18. SIGNIFICANCE STATEMENTMunc13 and Munc18 are SNARE-associated chaperones that act as "priming" factors, facilitating the formation of a pool of docked, release-ready vesicles and regulating Ca2+-evoked neurotransmitter release. Although important insights into Munc18/Munc13 function have been gained, how they assemble and operate together remains enigmatic. To address this, we developed a novel biochemically-defined fusion assay which enabled us to investigate the cooperative action of Munc13 and Munc18 in molecular terms. We find that Munc18 nucleates the SNARE complex, while Munc13 promotes and accelerates the SNARE assembly in a DAG-dependent manner. The concerted action of Munc13 and Munc18 stages the SNARE assembly process to ensure efficient clamping and formation of stably docked vesicles, which can be triggered to fuse rapidly ([~]10 msec) upon Ca2+ influx.

neuroscience↗

bioRxiv2023

Recognition determinants of broad and potent HIV-1 neutralization by an affinity matured antibody from a pediatric elite-neutralizer

The structural and characteristic features of HIV-1 broadly neutralizing antibodies (bnAbs) from chronically infected pediatric donors are currently unknown. Herein, we characterized a heavy chain matured HIV-1 bnAb 44m, identified from a pediatric elite-neutralizer. Interestingly, in comparison to its wild-type AIIMS-P01 bnAb, 44m exhibited moderately higher level of somatic hypermutations of 15.2%. The 44m neutralized 79% of HIV-1 heterologous viruses (n=58) tested, with a geometric mean IC50 titer of 0.36 {micro}g/ml. The cryo-EM structure of 44m Fab in complex with fully-cleaved glycosylated native-like BG505.SOSIP.664.T332N gp140 envelope trimer at 4.4[A] resolution revealed that 44m targets the V3-glycan N332-supersite and GDIR motif to neutralize HIV-1 with improved potency and breadth, plausibly attributed by a matured heavy chain as compared to that of wild-type AIIMS-P01. This study further improves our understanding on pediatric HIV-1 bnAbs and structural basis of broad HIV-1 neutralization by 44m may be useful blueprint for vaccine design in future.

immunology↗

bioRxiv2023

High Throughput Viability Assay for Microbiology

Counting viable cells is a universal practice in microbiology. The colony forming unit (CFU) assay has remained the gold standard to measure viability across disciplines; however, it is time-intensive and resource-consuming. Herein, we describe the Geometric Viability Assay (GVA) that replicates CFU measurements over 6-orders of magnitude while reducing over 10-fold the time and consumables. GVA computes a samples viable cell count based on the distribution of embedded colonies growing inside a pipette tip. GVA is compatible with gram-positive and -negative planktonic bacteria, biofilms, and yeast. Laborious CFU experiments such as checkerboard assays, treatment time-courses, and drug screens against slow-growing cells are simplified by GVA. We therefore screened a drug library against exponential and stationary phase E. coli leading to the discovery of the ROS-mediated, bactericidal mechanism of diphenyliodonium. The ease and low cost of GVA evinces it can accelerate existing viability assays and enable measurements at previously impractical scales.

microbiology↗

bioRxiv2022

High resolution cryo-EM structures of two potently SARS-CoV-2 neutralizing monoclonal antibodies of same donor origin that vary in neutralizing Omicron variants

While vaccines have by large been found to effective against the evolving SARS-CoV-2 variants, the profound and rapid effectivity of monoclonal antibodies (mAbs) in significantly reducing hospitalization to severe disease outcomes have also been demonstrated. In the present study, by high resolution cryo-electron microscopy (cryo-EM), we examined the structural insights of two trimeric spike (S) protein bound mAbs isolated from an Indian convalescent individual infected with ancestral SARS-CoV-2 which we recently reported to potently neutralize SARS-CoV-2 from its ancestral form through highly virulent Delta form however different in their ability to neutralize Omicron variants. Our findings showed binding and conformational heterogeneities of both the mAbs (THSC20.HVTR04 and THSC20.HVTR26) bound to S trimer in its apo and hACE-2 bound forms. Additionally, cryo-EM resolved structure assisted modeling highlighted key residues associated with the ability of these two mAbs to neutralize Omicron variants. Our findings highlighted key interacting features modulating antigen-antibody interacting that can further aid in structure guided antibody engineering to enhance their breadth and potency. HighlightsO_LITwo potent human mAbs obtained from a single donor differ binding to Omicron spikes C_LIO_LIPattern of binding and conformation of these mAbs bound to full length spike differs C_LIO_LIAntibody binding alters the conformational states of S trimer in its apo and hACE-2 bound forms. C_LIO_LICryo-EM structure guided modeling highlighted correlates of interacting residues associated with resistance and sensitivity of BA.1, BA.2, BA.4/BA.5 resistance and sensitivity against these mAbs. C_LI

biophysics↗

bioRxiv2022

A Synthetic ERRα Agonist Induces an Acute AerobicExercise Response and Enhances Exercise Capacity

Repetitive physical exercise induces physiological adaptations in skeletal muscle that improves exercise performance and is effective for the prevention and treatment of several diseases. Here we report the identification of a synthetic agonist for the orphan nuclear receptor ERR (estrogen receptor-related receptor ), SLU-PP-332, that activates an acute aerobic exercise genetic program in skeletal muscle in an ERR-dependent manner. SLU-PP-332 increases mitochondrial function and cellular respiration consistent with induction of this genetic program. When administered to mice, SLU-PP-332 increased the type IIa oxidative skeletal muscle fibers and enhanced exercise endurance. These data indicate the feasibility of targeting ERR for development of compounds that act as exercise mimetics that may be effective in treatment of numerous metabolic disorders and to improve muscle function in the aging.

pharmacology and toxicology↗

bioRxiv2022

Atypical peripheral actin band formation via overactivation of RhoA and Non-muscle myosin II in Mitofusin 2 deficient cells

Cell spreading and migration play central roles in many physiological and pathophysiological processes. We have previously shown that MFN2 regulates the migration of human neutrophillike cells via suppressing Rac activation. Here, we show that in mouse embryonic fibroblasts, MFN2 suppresses RhoA activation and supports cell polarization. After the initial spreading period, the wild-type cells polarize and migrate, whereas the Mfn2-/- cells maintain a circular shape. Increased cytosolic Ca2+ resulting from the loss of Mfn2 is directly responsible for this phenotype, which can be rescued by expressing an artificial tether to bring mitochondria and ER to close vicinity. Elevated cytosolic Ca2+ activates Ca2+/calmodulin-dependent protein kinase II, RhoA, and Myosin light-chain kinase, causing an over-activation of non-muscle myosin II and a formation of a prominent F-actin ring at the cell periphery and increased cell contractility. The formation of the peripheral actin band alters cell physics and is dependent on substrate rigidity. Our results provide a novel molecular basis to understand how MFN2 regulates distinct signaling pathways in different cells and tissue environments, which is instrumental in understanding and treating MFN2-related diseases.

cell biology↗

bioRxiv2022

Targeting neuroinflammatory pathways using Nanoligomer is neuroprotective in prion disease

Neuroinflammation plays a crucial role in the development of neurodegenerative protein misfolding disorders. This category of progressive diseases includes, but is not limited to, Alzheimers disease, Parkinsons disease, and prion diseases. Shared pathogenesis involves the accumulation of misfolded proteins, chronic neuroinflammation, and synaptic dysfunction, ultimately leading to irreversible neuronal loss, measurable cognitive deficits, and death. Presently, there are little to no effective treatments to halt the advancement of neurodegenerative diseases. We hypothesized directly targeting neuroinflammation by downregulating the transcription factor, NF-{kappa}B and the inflammasome protein, NLRP3, with the brain-penetrant, non-toxic, SB_NI_112, would be neuroprotective. To achieve this, we used a cocktail of RNA targeting therapeutics (SB_NI_112) shown to be brain-penetrant, non-toxic, and targeting both NF-|B and NLRP3. We utilized a mouse-adapted prion strain as a model for neurodegenerative diseases to assess aggregation of misfolded proteins, glial inflammation, neuronal loss, cognitive deficits, and lifespan. Prion-diseased mice were treated either intraperitoneally or intranasally with SB_NI_112. Behavioral and cognitive deficits were significantly protected by this combination of NF-{kappa}B and NLRP3 down-regulators. Treatment reduced glial inflammation, protected against neuronal loss, prevented spongiotic change, rescued cognitive deficits, and significantly lengthened lifespan of prion-diseased mice. We have identified a non-toxic, systemic pharmacologic that down-regulates NF-|B and NLRP3, prevents neuronal death and slows the progression of neurodegenerative disease. Though mouse models do not always predict human patient success, and the study was limited due to sample size and number of dosing methods utilized, these findings serve as a proof of principle for continued translation of the therapeutic SB_NI_112 for prion disease and other neurodegenerative diseases. Based on success in a murine prion model, we will be continual testing SB_NI_112 in a variety of neurodegenerative disease models, including Alzheimers Disease and Parkinsons Disease.

neuroscience↗

bioRxiv2022

Genetically encoded non-canonical amino acids reveal asynchronous dark reversion of chromophore, backbone and side-chains in EL222

Photoreceptors containing the light-oxygen-voltage (LOV) domain elicit biological responses upon excitation of their flavin mononucleotide (FMN) chromophore by blue light. The mechanism and kinetics of dark-state recovery are not well understood. Here we incorporated the non-canonical amino acid p-cyanophenylalanine (CNF) by genetic code expansion technology at forty-five positions of the bacterial transcription factor EL222. Screening of light-induced changes in infrared (IR) absorption frequency, electric field and hydration of the nitrile groups identified residues CNF31 and CNF35 as reporters of monomer/oligomer and caged/decaged equilibria, respectively. Time-resolved multi-probe UV/Visible and IR spectroscopy experiments of the lit-to-dark transition revealed four dynamical events. Predominantly, rearrangements around the A helix interface (CNF31 and CNF35) precede FMN-cysteinyl adduct scission, folding of -helices (amide bands), and relaxation of residue CNF151. This study illustrates the importance of characterizing all parts of a protein and suggests a key role for the N-terminal A extension of the LOV domain in controlling EL222 photocycle length. SignificanceThe kinetics of fold switching between non-illuminated and blue-light-irradiated states in the transcription factor EL222 is important for understanding the signal transduction mechanism of LOV photoreceptors. Here we combine two native probes, the FMN chromophore (absorption bands in the UV/Visible region) and the protein backbone (amide bands in the infrared region), with genetically encoded cyano (C{equiv}N)-containing phenylalanine residues as infrared reporters of protein microenvironments. EL222 structural dynamics is more complex than expected if using a single type of probe. Local changes around residues 31 and 35 precede FMN-protein adduct rupture, which in turn precedes the global protein conformational relaxation. Our findings point the way forward to obtaining comprehensive descriptions of kinetic transitions in LOV and other photosensors.

biophysics↗

bioRxiv2022

Screening Immunotherapy Targets to Counter Radiation-Induced Neuroinflammation

Galactic cosmic rays (GCR) in space induce increase in cerebral amyloid-{beta} levels and elevated levels of microgliosis and astrocytosis, causing accelerated neurodegeneration from this increased neuroinflammation. Even exposure to low-levels of high-Z high-energy (HZE) radiation (50 cGy) has been shown to induce biochemical and immunohistochemical changes in short-term leading to degradation in cognition, motor skills, and development of space-induced neuropathy. There is lack of effective neuroinflammation countermeasures, and current experimental therapies require invasive intracerebral and intrathecal delivery due to difficulty associated with therapeutic crossover between blood-brain barrier. Here, we present a new countermeasure development approach for neurotherapeutics using high-throughput drug-discovery, target validation, and lead molecule identification with nucleic acid-based molecules. These Nanoligomer molecules are rationally designed using a bioinformatics and AI-based ranking method and synthesized as a single-modality combining 6-different design elements to up- or down-regulate gene expression of target gene at will, resulting in elevated or diminished protein expression of intended target. This platform approach was used to perturb and identify most effective upstream regulators and canonical pathways for therapeutic intervention to reverse radiation-induced neuroinflammation. The lead Nanoligomer and corresponding target granulocyte-macrophage colony-stimulating factor (GM-CSF) were identified using in vitro cell-based screening in human astrocytes and donor derived peripheral blood mononuclear cells (PBMCs) and further validated in vivo using a mouse model of radiation-induced neuroinflammation. GM-CSF transcriptional downregulator Nanoligomer 30D.443_CSF2 downregulated proinflammatory cytokine GM-CSF (or CSF2) using simple intraperitoneal injection of low-dose (3mg/kg) and completely reversed expression of CSF2 in cortex tissue, as well as other neuroinflammation markers. These results point to the broader applicability of this approach towards space countermeasure development, and potential for further investigation of lead neurotherapeutic molecule as a reversible gene therapy.

neuroscience↗

bioRxiv2022

Identifying Optimal Neuroinflammation Treatment Using Nanoligomer Discovery Engine

Acute activation of innate immune response in the brain, or neuroinflammation, protects this vital organ from a range of external pathogens and promotes healing after traumatic brain injury. However, chronic neuroinflammation leads to the activation of immune cells like microglia and astrocytes causes damage to the nervous tissue, and is causally linked to a range of neurodegenerative diseases such as Alzheimers diseases (AD), Multiple Sclerosis (MS), Parkinsons diseases (PD), and many others. While neuroinflammation is a key target for a range of neuropathological diseases, there is a lack of effective countermeasures to tackle it, and existing experimental therapies require fairly invasive intracerebral and intrathecal delivery due to difficulty associated with the therapeutic crossover between the blood-brain barrier (BBB), making such treatments impractical to treat neuroinflammation long-term. Here, we present the development of an optimal neurotherapeutic using our Nanoligomer discovery engine, by screening downregulation of several proinflammatory cytokines (e.g., Interleukin-1{beta} or IL-1{beta}, tumor necrosis factor-alpha or TNF-, TNF receptor 1 or TNFR1, Interleukin 6 or IL-6), inflammasomes (e.g., NLRP1), key transcription factors (e.g., nuclear factor kappa-B or NF-{kappa}{beta}) and their combinations, as upstream regulators and canonical pathway targets, to identify and validate the best-in-class treatment. Using our high-throughput drug discovery, target validation, and lead molecule identification via a bioinformatics and AI-based ranking method to design sequence-specific peptide molecules to up-or down-regulate gene expression of the targeted gene at will, we used our discovery engine to perturb and identify most effective upstream regulators and canonical pathways for therapeutic intervention to reverse neuroinflammation. The lead neurotherapeutic was a combination of Nanoligomers targeted to NF-{kappa}{beta} (SB.201.17D.8_ NF-{kappa}{beta}1) and TNFR1 (SB.201.18D.6_TNFR1), which were identified using in vitro cell-based screening in donor-derived human astrocytes, and further validated in vivo using a mouse model of lipopolysaccharide (LPS)-induced neuroinflammation. The combination treatment SB_NI_111 was delivered without any special formulation using a simple intraperitoneal injection of low-dose (5mg/kg) and was found to significantly suppress the expression of LPS-induced neuroinflammation in mouse hippocampus. These results point to the broader applicability of this approach towards the development of therapies for chronic neuroinflammation-linked neurodegenerative diseases, sleep countermeasures, and others, and the potential for further investigation of the lead neurotherapeutic molecule as reversible gene therapy.

neuroscience↗

bioRxiv2022

An efficient opal-suppressor tryptophanyl pair creates new routes for simultaneously incorporating up to three distinct noncanonical amino acids into proteins in mammalian cells

The site-specific incorporation of multiple distinct noncanonical amino acids (ncAAs) into proteins in mammalian cells is an emergent technology with much potential. For each different ncAA to be incorporated, this technology requires a distinct orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pair that recognizes a distinct nonsense codon. The aaRS/tRNA pairs currently available for ncAA mutagenesis in eukaryotes are all traditionally used to decode the TAG nonsense codon. Unfortunately, these pairs suppress the other two nonsense codons, TGA or TAA, at a significantly lower level, compromising the scope of multi-ncAA mutagenesis. Here we report that the bacteria-derived tryptophanyl (EcTrp) pair is an excellent TGA-suppressor in mammalian cells. Additionally, we show that this pair does not cross-react with any of the three previously established aaRS/tRNA pairs. Consequently, the TGA-suppressing EcTrp pair can be combined with TAG-suppressing pyrrolysyl (archaeal), tyrosyl (bacterial), or leucyl (bacterial) pairs to develop three new routes for dual-ncAA incorporation in mammalian cells. We show that all three platforms enable site-specific incorporation of two distinct ncAAs into proteins - including a full-length humanized antibody - with excellent fidelity and good efficiency. Finally, we combined the EcTrp pair with the bacterial Tyr pair and the archaeal pyrrolysyl pair to site-specifically incorporate different combinations of three distinct ncAAs into a reporter protein in mammalian cells.

synthetic biology↗

bioRxiv2022

Stimulus duration encoding occurs early in the moth olfactory pathway

Pheromones convey rich ethological information and guide insects search behavior. Insects navigating in turbulent environments are tasked with the challenge of coding the temporal structure of an odor plume, obliging recognition of the onset and offset of whiffs of odor. The coding mechanisms that shape odor offset recognition remain elusive. We designed a device to deliver sharp pheromone pulses and simultaneously measured the response dynamics from pheromone-tuned olfactory receptor neurons (ORNs) in male moths and Drosophila. We show that concentration-invariant stimulus duration encoding is implemented in moth ORNs by spike frequency adaptation at two time scales. A linear-nonlinear model fully captures the underlying neural computations and offers an insight into their biophysical mechanisms. Drosophila use pheromone cis-vaccenyl acetate (cVA) only for very short distance communication and are not faced with the need to encode the statistics of the cVA plume. Their cVA-sensitive ORNs are indeed unable to encode odor-off events. Expression of moth pheromone receptors in Drosophila cVA-sensitive ORNs indicates that stimulus-offset coding is receptor independent. In moth ORNs, stimulus-offset coding breaks down for short (<200 ms) whiffs. This physiological constraint matches the behavioral latency of switching from the upwind surge to crosswind cast flight upon losing contact with the pheromone.

neuroscience↗