Biology subjects

Hirsch, A.

Publications and source records attributed to Hirsch, A..

9 recordsLinked to original sources

bioRxiv2026

Evidence for a rod-to-cone lactate shuttle in the mammalian retina

In his seminal 1920s studies, Otto Warburg found the retina to generate large amounts of lactate. However, it was unclear what retinal cells produced lactate and whether it was a metabolic waste product or used further. Here, we show that lactate produced by rod photoreceptors fuels the energy-intensive function and viability of cone photoreceptors. In an initial expression analysis, we found monocarboxylate transporter-1 (MCT1), lactate-producing lactate-dehydrogenase-A (LDHA), and pyruvate carboxykinase-1 (PCK1) localized to rod photoreceptors, while high-affinity MCT2, pyruvate-producing LDHB, and PCK2 were expressed in cones. We then exposed retina to defined media containing either glucose or lactate as caloric component, and applied specific MCT inhibitors. In glucose-containing medium, 1H-NMR metabolomics showed rod MCT1 inhibition to increase retinal lactate, suggesting rods as a major source of lactate. In lactate-only medium, functional recordings using micro-electroretinography showed decreased rod function, while cone function was maintained. In glucose-containing medium, blocking rod MCT1 abolished cone function. Long-term treatment with MCT inhibitors selectively decreased photoreceptor viability. Conversely, supplementing the defined medium with lactate preserved cone viability in the rd1 mouse model for Retinitis Pigmentosa. Together, our data suggest that lactate shuttling from rods is crucial for cone function and viability. This may explain cone degeneration seen in various retinal diseases and provides an entirely new avenue for metabolism-based treatment development. The discovery of a lactate-shuttle between two functionally similar, yet distinct types of neurons may have far-reaching implications for our understanding of the central nervous system in general.

neuroscience↗

bioRxiv2026

Generative AI designs functional thiolation domains for reprogramming non-ribosomal peptide synthetases

Large language models and generative protein design promise to accelerate biotechnology, but it remains unclear whether they can engineer dynamic megasynth(et)ases whose activity depends on transient, context-specific domain interfaces. Non-ribosomal peptide synthetases (NRPSs) are an especially demanding target, yet a high-value one because they produce many clinically important natural products and offer a route to analogs that are often difficult or impractical to access by chemical synthesis. Here we integrate pretrained generative models (ESM3, ProteinMPNN and EvoDiff) with design-build-test-learn cycles and data-guided prioritization to generate 76 de novo thiolation (T) domains. We built and tested 578 recombinant NRPS variants in vivo spanning minimal, full-length and hybrid assembly lines. AI-designed T-domains supported product formation across architectures, enabled catalytically active hybrids at recombined junctions and increased yields by up to [~]3-fold relative to NRPSs carrying the native T-domain. A representative design showed improved soluble expression, refolding, and a 12 {degrees}C higher melting temperature, while molecular dynamics simulations indicated preserved global stability but reshaped, state-dependent interdomain contact networks. Together, these results establish generative design as an effective route to context-conditioned optimization and reprogramming of biosynthetic assembly lines.

synthetic biology↗

bioRxiv2025

Tracheal tuft cell-released leukotrienes promote antibacterial immune responses

Tuft cells act as crucial sentinels in the airways that detect bacterial metabolites. In response, tuft cells release signaling molecules that trigger immune responses essential for clearing the infection. The molecular mechanisms driving immune cell activation following tuft cell stimulation in pneumonia are still not fully understood. Here, we identify tuft cells as the primary source of proinflammatory leukotrienes (LTs), which are released in the presence of pathogenic bacteria in the airways. We show that tracheal tuft cells discriminate pathogenic from non-pathogenic bacteria by sensing adenosine triphosphate (ATP) released from pathogens such as Pseudomonas aeruginosa and Rodentibacter pneumotropicus within the first 4 h of invasion, and recruit neutrophils and macrophages to the trachea and alveolar spaces. Taste signaling through the chemosensory transient receptor potential cation channel subfamily M member 5 (Trpm5) channel was essential for tuft cell activation and LT release. Mice lacking Trpm5 were not capable of detecting bacteria-released ATP and became colonized upon R. pneumotropicus infection. In contrast, Trpm5+/+ mice cleared the pathogen. We uncover a critical tuft cell-dependent sensing mechanism in pneumonia and establish tracheal tuft cells as both detectors of bacterial extracellular ATP and triggers of acute innate immune responses.

immunology↗

bioRxiv2025

An Enantioselective Chemical Probe for Chikungunya nsP2 Helicase with Antialphaviral Activity

Chikungunya virus (CHIKV) replication relies on the multifunctional nsP2 protein, making it an attractive target for antiviral drug discovery. Here, we report the resolution of oxaspiropiperidine 1, a first-in-class inhibitor of the CHIKV nsP2 RNA helicase (nsP2hel), into its constitutive enantiomers and characterization of their antiviral activity. The enantiomer (R)-1 exhibited potent inhibition of viral replication, nsP2hel ATPase activity, and dsRNA unwinding, while the (S)-1 enantiomer was >100-fold less active. The (R)-1 enantiomer also demonstrated high selectivity for CHIKV over other RNA viruses and for nsP2hel over other RNA helicases. Direct binding of (R)-1 to nsP2hel protein was confirmed by 19F NMR. Biophysical and structural studies revealed conformational polymorphism in the spirocyclic scaffold of (R)-1, suggesting a potential role of thermal mobility of the ligand in allosteric inhibition of nsP2hel. Collectively, these findings designate (R)-1 (RA-NSP2-1) as a high-quality chemical probe and (S)-1 (RA-NSP2-1N) as a negative control for probing the biology of alphavirus RNA helicases.

pharmacology and toxicology↗

bioRxiv2023

NRF2 activators inhibit influenza A virus replication by interfering with nucleo-cytoplasmic export of viral RNPs in an NRF2-independent manner

In addition to antioxidative and anti-inflammatory properties, activators of the cytoprotective nuclear factor erythroid-2-like-2 (NRF2) signaling pathway have antiviral effects, but the underlying antiviral mechanisms are incompletely understood. We evaluated the ability of the NRF2 activators 4-octyl itaconate (4OI), bardoxolone methyl (BARD), sulforaphane (SFN), and the inhibitor of exportin-1 (XPO1)-mediated nuclear export selinexor (SEL) to interfere with influenza virus A/Puerto Rico/8/1934 (H1N1) infection of human cells. All compounds reduced viral titers in supernatants from A549 cells and vascular endothelial cells in the order of efficacy SEL>4OI>BARD=SFN, which correlated with their ability to prevent nucleo-cytoplasmic export of viral nucleoprotein and the host cell protein p53. In contrast, intracellular levels of viral HA mRNA and nucleocapsid protein (NP) were unaffected. Knocking down mRNA encoding KEAP1 (the main inhibitor of NRF2) or inactivating the NFE2L2 gene (which encodes NRF2) revealed that physiologic NRF2 signaling restricts IAV replication. However, the antiviral effect of all compounds was NRF2-independent. Instead, XPO1 knock-down greatly reduced viral titers, and incubation of Calu3 cells with an alkynated 4OI probe demonstrated formation of a covalent complex with XPO1. Ligand-target modelling predicted covalent binding of all three NRF2 activators and SEL to the active site of XPO1 involving the critical Cys528. SEL and 4OI manifested the highest binding energies, whereby the 4-octyl tail of 4OI interacted extensively with the hydrophobic groove of XPO1, which binds nuclear export sequences on cargo proteins. Conversely, SEL as well as the three NRF2 activators were predicted to covalently bind the functionally critical Cys151 in KEAP1. Blocking XPO1-mediated nuclear export may, thus, constitute a "noncanonical" mechanism of anti-influenza activity of electrophilic NRF2 activators that can interact with similar cysteine environments at the active sites of XPO1 and KEAP1. Considering the importance of XPO1 function to a variety of pathogenic viruses, compounds that are optimized to inhibit both targets may constitute an important class of broadly active host-directed treatments that embody anti-inflammatory, cytoprotective, and antiviral properties. Author summaryVirus infections often cause organ damage via excessive inflammation and oxidative stress. The identification of host-directed treatments that reduce inflammation, accumulation of reactive oxygen species, and viral infectivity is an important goal of antiviral drug development. One advantage of host-directed antivirals is that their targets are encoded by the stable host genome, making emergence of viral resistance less likely. The KEAP1/NRF2 signaling pathway is the most important pathway in humans that protects cells from oxidative stress, and it also induces antiviral and anti-inflammatory responses. NRF2 activators, therefore, are promising candidates for development of host-directed antivirals. We evaluated three NRF2-activating compounds as host-directed treatments for influenza A virus (IAV) infection. All three compounds reduced viral replication, cellular inflammation, and reactive oxygen species. Surprisingly, these effects were completely independent of NRF2 signaling. Instead, we found that these compounds (particularly 4-octyl itaconate) interfere with export of viral RNA/protein complexes from the nucleus, thereby reducing release of viral particles. The most plausible explanation is that the "natural" target of these compounds, KEAP1 (which limits NRF2 signaling), and the nuclear export factor XPO1 (which is required for egress of IAV from the nucleus) contain similar binding sites, thus allowing "NRF2 activators" to also inhibit XPO1.

microbiology↗

bioRxiv2022

Structural analysis of 1-deoxy-D-xylulose 5-phosphate synthase from Pseudomonas aeruginosa and Klebsiella pneumoniae reveals conformational changes upon cofactor binding

Isoprenoid precursor biosynthesis is an essential part of primary metabolism in all living organisms. While eukaryotes utilize the mevalonate (MEV) pathway for isoprenoid-precursor biosynthesis, the most important bacterial pathogens rely on the methylerythritol-phosphate (MEP) pathway. Therefore, enzymes involved in the MEP pathway are potentially valuable targets for the development of novel antibacterials to tackle the current antimicrobial resistance crisis. Within the MEP pathway, the enzyme 1-deoxy-D-xylulose-5-phosphate synthase (DXPS) represents a crucial, rate-limiting first step and a branch point in the biosynthesis of the vitamins B1 and B6. Herein, we present two novel, high-resolution DXPS crystal structures of the important ESKAPE pathogens Pseudomonas aeruginosa and Klebsiella pneumoniae in both the co-factor-bound and apo forms. We demonstrate that the absence of the cofactor ThDP leads to a disordered loop close to the active site and may be important for the design of potent DXPS inhibitors, albeit being different in both structures. In addition, we report the complex structure of paDXPS with a fluoropyruvate adduct, shedding more light on the structural basis of DXPS catalysis. Lastly, we have determined a complex structure of paDXPS with a thiamine analogue, opening up a route for structure-based drug design of this essential enzyme of the MEP pathway.

molecular biology↗

bioRxiv2019

Impact of soil salinity on the cowpea nodule-microbiome and the isolation of halotolerant PGPR strains to promote plant growth under salinity stress

Four soil samples (SS-1--SS-4) isolated from semi-arid soils in Punjab, Pakistan were used as inocula for cowpea (Vigna unguiculata L.) grown under salinity stress to analyze the composition of bacteria in the rhizosphere and within nodules through cultivation-dependent and cultivation-independent methods. Two cowpea varieties, 603 and the salt-tolerant CB 46, were each inoculated with four different native soil samples, and data showed that plants inoculated with soil samples SS-2 and SS-4 grew better than plants inoculated with soil samples SS-1 and SS-3. Bacteria were isolated from both soils and nodules, and 34 of the 51 original isolates tested positive for PGPR traits in plate assays with many exhibiting multiple plant growth-promoting properties. A number of isolates were positive for all PGPR traits tested. For the microbiome studies, environmental DNA (eDNA) was isolated from SS-1 and SS-4, which represented the extremes of the Pakistan soils to which the plants responded, and by 16S rRNA gene sequencing analysis were found to consist mainly of Actinobacteria, Firmicutes, and Proteobacteria. However, sequencing analysis of eDNA isolated from cowpea nodules established by the trap plants grown in the four Pakistan soils indicated that the nodule microbiome consisted almost exclusively of Proteobacterial sequences, particularly Bradyrhizobium. Yet, many other bacteria including Rhizobium, Mesorhizobium, Pseudomonas, as well as Paenibacillus, Bacillus as well as non-proteobacterial genera were isolated from the nodules of soil-inoculated cowpea plants. This discrepancy between the bacteria isolated from cowpea nodules (Proteobacteria and non-Proteobacteria) versus those detected in the nodule microbiome (Proteobacteria) needs further study.

plant biology↗

bioRxiv2019

Inoculation with a microbe isolated from the Negev Desert enhances corn growth

Corn (Zea mays L.) is not only an important food source, but also has numerous uses, including for biofuels, fillers for cosmetics, glues, and so on. The amount of corn grown in the U.S. has significantly increased since the 1960s and with it, the demand for synthetic fertilizers and pesticides/fungicides to enhance its production. However, the downside of the continuous use of these products, especially N and P fertilizers, has been an increase in N2O emissions and other greenhouse gases into the atmosphere as well as run-off into waterways that fuel pollution and algal blooms. These approaches to agriculture, especially if exacerbated by climate change, will result in decreased soil health as well as human health. We searched for microbes from arid, native environments that are not being used for agriculture because we reasoned that indigenous microbes from such soils could promote plant growth and help restore degraded soils. Employing cultivation-dependent methods to isolate bacteria from the Negev Desert in Israel, we tested the effects of several microbial isolates on corn in both greenhouse and small field studies. One strain, Dietzia cinnamea 55, originally identified as Planomicrobium chinensis, significantly enhanced corn growth over the uninoculated control in both greenhouse and outside garden experiments. We sequenced and analyzed the genome of this bacterial species to elucidate some of the mechanisms whereby D. cinnamea 55 promoted plant growth. In addition, to ensure the biosafety of this previously unknown plant growth promoting bacterial (PGPB) strain as a potential bioinoculant, we tested the survival and growth of Caenorhabditis elegans (a test for virulence) in response to D. cinnamea 55. We also looked for genes for potential virulence determinants as well as for growth promotion.

plant biology↗

bioRxiv2017

Isoxanthopterin: An Optically Functional Biogenic Crystal in the Eyes of Decapod Crustaceans

The eyes of some aquatic animals form images through reflective optics. Shrimp, lobsters, crayfish and prawns possess reflecting superposition compound eyes, composed of thousands of square-faceted eye-units (ommatidia). Mirrors in the upper part of the eye (the distal mirror) reflect light collected from many ommatidia onto the underlying photosensitive elements of the retina, the rhabdoms. A second reflector, the tapetum, underlying the retina, back-scatters dispersed light onto the rhabdoms. Using microCT and cryo-SEM imaging accompanied by in situ micro-X-ray diffraction and micro-Raman spectroscopy, we investigated the hierarchical organization and materials properties of the reflective systems at high resolution and under close to physiological conditions. We show that the distal mirror consists of three or four layers of sparse plate-like nano-crystals. The tapetum is a diffuse reflector composed of hollow nanoparticles constructed from concentric lamellae of crystals. Isoxanthopterin, a pteridine analog of guanine, forms both the reflectors in the distal mirror and in the tapetum. The crystal structure of isoxanthopterin was determined from crystal structure prediction calculations and verified by comparison with experimental X-ray diffraction. The extended hydrogen bonded layers of the molecules results in an extremely high calculated refractive index in the H-bonded plane, n = 1.96, which makes isoxanthopterin crystals an ideal reflecting material. The crystal structure of isoxanthopterin, together with a detailed knowledge of the reflector superstructures, provide a rationalization of the reflective optics of the crustacean eye.\n\nSignificanceAquatic animals use reflectors in their eyes either to form images or to increase photon capture. Guanine is the most widespread molecular component of these reflectors. Here we show that crystals of isoxanthopterin, a pteridine analogue of guanine, form both the image-forming distal mirror and the intensity-enhancing tapetum reflector in the compound eyes of some decapod crustaceans. The crystal structure of isoxanthopterin was determined, providing an explanation for why these crystals are so well suited for efficient reflection. Pteridines were previously known only as pigments and our discovery raises the question of which other organic molecules may be used to form crystals with superior reflective properties either in organisms or in artificial optical devices.

bioengineering↗