Single-Dose P2 X4R Single-Chain Fragment Variable Antibody Permanently Reverses Chronic Pain in Male Mice

Non-opioid single-chain variable fragment (scFv) small antibodies were generated as pain-reducing block of P2X4R receptor (P2X4R). A panel of scFvs targeting an extracellular peptide sequence of P2X4R was generated followed by cell-free ribosome display for recombinant antibody selection. After three rounds of bio-panning, a panel of recombinant antibodies was isolated and characterized by ELISA, cross-reactivity analysis, and immunoblotting/immunostaining. Generated scFv antibodies feature binding activity similar to monoclonal antibodies but with stronger affinity and increased tissue penetrability due to their ~30% smaller size. Two anti-P2X4R scFv clones (95, 12) with high specificity and affinity binding were selected for in vivo testing in male and female mice with trigeminal nerve chronic neuropathic pain (FRICT-ION model) persisting for several months in untreated BALBc mice. A single dose of P2X4R scFv (4 mg/kg, i.p.) successfully, completely, and permanently reversed chronic neuropathic pain-like measures in male mice only, providing retention of baseline behaviors indefinitely. Untreated mice retained hypersensitivity, and developed anxiety- and depression-like behaviors within 5 weeks. In vitro P2X4R scFv 95 treatment significantly increased the rheobase of larger-diameter (>25 µm) trigeminal ganglia (TG) neurons from FRICT-ION mice compared to controls. The data support use of engineered scFv antibodies as non-opioid biotherapeutic interventions for chronic pain.

Ribosome-Induced Cellular Multipotency, an Emerging Avenue in Cell Fate Reversal

The ribosome, which is present in all three domains of life, plays a well-established, critical role in the translation process by decoding messenger RNA into protein. Ribosomal proteins, in contrast, appear to play non-translational roles in growth, differentiation, and disease. We recently discovered that ribosomes are involved in reverting cellular potency to a multipotent state. Ribosomal incorporation (the uptake of free ribosome by living cells) can direct the fate of both somatic and cancer cells into multipotency, allowing them to switch cell lineage. During this process, both types of cells experienced cell-cycle arrest and cellular stress while remaining multipotent. This review provides a molecular perspective on current insights into ribosome-induced multipotency and sheds light on how a common stress-associated mechanism may be involved. We also discuss the impact of this phenomenon on cancer cell reprogramming and its potential in cancer therapy.

Identification and functional characterization of a fish-specific tlr19 in common carp (Cyprinus carpio L.) that recruits TRIF as an adaptor and induces ifn expression during the immune response

Toll-like receptor 19 (Tlr19) is a fish-specific TLR that plays a critical role in innate immunity. In the present study, we aimed to identify tlr19 from common carp (Cyprinus carpio L.) and explored its expression profile, localization, adaptor, and signaling pathways. A novel tlr19 cDNA sequence (Cctlr19) was identified in common carp. Phylogenetic analysis revealed that CcTlr19 was most closely related to Danio rerio Tlr19. Subcellular localization analysis indicates that CcTlr19 was synthesized in the free ribosome and then transported to early endosomes. Cctlr19 was constitutively expressed in all the examined tissues, with the highest expression in the brain. After poly(I:C) and Aeromonas hydrophila injection, the expression of Cctlr19 was significantly upregulated in immune-related organs. In addition, the expression of Cctlr19 was upregulated in head kidney leukocytes (HKL) upon stimulation with different ligands. Immunofluorescence and luciferase analyses indicate that CcTlr19 recruited TRIF as an adaptor. Furthermore, CcTlr19 can activate the expression of ifn-1 and viperin. Taken together, these findings lay the foundation for future research to investigate the mechanisms underlying fish tlr19.

Pseudouridine-free Ribosome Exhibits Distinct Inter-subunit Movements

Pseudouridine, the most abundant form of RNA modification, is known to play important roles in ribosome function. Mutations in human DKC1, the pseudouridine synthase responsible for catalyzing the ribosome RNA modification, cause translation deficiencies and are associated with a complex cancer predisposition. The structural basis for how pseudouridine impacts ribosome function remains uncharacterized. Here we report electron cryomicroscopy structures of a fully modified and a pseudouridine-free ribosome from Saccharomyces cerevisiae. In the modified ribosome, the rearranged N1 atom of pseudouridine is observed to stabilize key functional motifs by establishing predominately water-mediated close contacts with the phosphate backbone. The pseudouridine-free ribosome, however, is devoid of such interactions and displays conformations reflective of abnormal inter-subunit movements. The erroneous motions of the pseudouridine-free ribosome may explain its observed deficiencies in translation.

Anatomy of the Liver of Mizoram Local Pig (Zovawk)

Background: The present study was aimed for the promotion and advancement of the anatomical knowledge at the gross, light microscopic and electron microscopic level in Zovawk (mizo local pig). Methods: The current investigation was done at the Instructional livestock farm, Department of Veterinary Anatomy and Histology, College of Veterinary Sciences and Animal Husbandry, Central Agricultural University, Selesih, Aizawl, Mizoram and Sophisticated Analytical Instrument Facility (SAIF), North Eastern Hill University (NEHU), Shillong, Meghalaya. Six liver samples were collected from six apparently healthy Zovawk animal of either sex and gross morphological observations were done directly after collection. Thereafter tissue samples were collected as such and were preserved in neutral buffer formalin (NBF) and in Karnovsky’s fixative for routine histology and transmission electron microscopic examination, respectively.Result: Zovawk liver shows four distinct borders, i.e. medial, lateral, dorsal and ventral border, two surfaces, i.e. parietal surface or diaphragmatic surface and caudal or visceral surface and six distinct lobes. The average weight of Zovawk liver was 1.402 kg. Weight of the liver was highly correlated with body weight of animal. Histologically, Zovawk liver was characterized with thick Glisson’s capsule and thick connective tissue septa emerging from it, which gives the hepatic lobules its hexagonal shape. Sinusoids of adjacent hepatocytes were lined by stellate shaped Kupffer cells. The ultra-structural examination of liver shows that, the hepatocytes were rich in mitochondria, endoplasmic reticulum and glycogen granules. Free ribosome and well developed rER and dense lysosomal granules were common in those hepatocyte.

A two-state ribosome and protein model can robustly capture the chemical reaction dynamics of gene expression

We derive phenomenological models of gene expression from a mechanistic description of chemical reactions using an automated model reduction method. Using this method, we get analytical descriptions and computational performance guarantees to compare the reduced dynamics with the full models. We develop a new two-state model with the dynamics of the available free ribosomes in the system and the protein concentration. We show that this new two-state model captures the detailed mass-action kinetics of the chemical reaction network under various biologically plausible conditions on model parameters. On comparing the performance of this model with the commonly used mRNA transcript-protein dynamical model for gene expression, we analytically show that the free ribosome and protein model has superior error and robustness performance.

The protein translation machinery is expressed for maximal efficiency in Escherichia coli

Protein synthesis is the most expensive process in fast-growing bacteria1,2. The economic aspects of protein synthesis at the cellular level have been investigated by estimating ribosome activity3–5 and the expression of ribosomes3,6, tRNA7–9, mRNA2, and elongation factors10,11. The observed growth-rate dependencies form the basis of powerful phenomenological bacterial growth laws5,12–16; however, a quantitative theory allowing us to understand these phenomena on the basis of fundamental biophysical and biochemical principles is currently lacking. Here, we show that the observed growth-rate dependence of the concentrations of ribosomes, tRNAs, mRNA, and elongation factors in Escherichia coli can be predicted accurately by minimizing cellular costs in a detailed mathematical model of protein translation; the mechanistic model is only constrained by the physicochemical properties of the molecules and requires no parameter fitting. We approximate the costs of molecule species through their masses, justified by the observation that cellular dry mass per volume is roughly constant across growth rates17 and hence represents a limited resource. Our results also account quantitatively for observed RNA/protein ratios and ribosome activities in E. coli across diverse growth conditions, including antibiotic stresses. Our prediction of active and free ribosome abundance facilitates an estimate of the deactivated ribosome reserve14,18,19, which reaches almost 50% at the lowest growth rates. We conclude that the growth rate dependent composition of E coli’s protein synthesis machinery is a consequence of natural selection for minimal total cost under physicochemical constraints, a paradigm that might generally be applied to the analysis of resource allocation in complex biological systems.