Adolescent ethanol exposure promotes mechanical allodynia and alters dopamine transmission in the nucleus accumbens shell.

ABSTRACT: Excessive alcohol consumption in adolescence can disrupt neural development and may augment pain perception. Recent studies have shown that the nucleus accumbens (NAc) shell is involved in mediating pain sensitivity after peripheral inflammation in rodent models of chronic pain and alcohol use disorder. Interestingly, there have been very few studies examining the impact of chronic ethanol exposure during adolescence on pain sensitivity in adulthood. Therefore, in this project, we investigated the impact of adolescent chronic intermittent ethanol (aCIE) exposure on mechanical allodynia. Furthermore, given the involvement of the NAc shell in pain processing and chronic ethanol-mediated changes, we measured changes in accumbal dopamine kinetics during protracted withdrawal. We found that both male and female aCIE rats show mechanical allodynia during withdrawal. Furthermore, male and female aCIE rats show greater evoked tonic dopamine release, maximal rate of dopamine reuptake, and dopamine affinity to the dopamine transporter in the NAc shell compared with controls. With phasic stimulation, aCIE rats also showed greater dopamine release compared with AIR-exposed rats. Inhibition of dopamine transmission targeted in the NAc shell reversed the aCIE-associated facilitation of mechanical allodynia in both sexes. These data suggest that aCIE exposure exacerbates pain sensitivity during withdrawal in an accumbal dopamine-dependent manner.

The smallest functional antibody fragment: Ultralong CDR H3 antibody knob regions potently neutralize SARS-CoV-2.

Cows produce antibodies with a disulfide-bonded antigen-binding domain embedded within ultralong heavy chain third complementarity determining regions. This "knob" domain is analogous to natural cysteine-rich peptides such as knottins in that it is small and stable but can accommodate diverse loops and disulfide bonding patterns. We immunized cattle with SARS-CoV-2 spike and found ultralong CDR H3 antibodies that could neutralize several viral variants at picomolar IC50 potencies in vitro and could protect from disease in vivo. The independent CDR H3 peptide knobs were expressed and maintained the properties of the parent antibodies. The knob interaction with SARS-CoV-2 spike was revealed by electron microscopy, X-ray crystallography, NMR spectroscopy, and mass spectrometry and established ultralong CDR H3-derived knobs as the smallest known recombinant independent antigen-binding fragment. Unlike other vertebrate antibody fragments, these knobs are not reliant on the immunoglobulin domain and have potential as a new class of therapeutics.

A critical review of effects on ethanol intake of the dynorphin/kappa opioid receptor system in the extended amygdala: From inhibition to stimulation.

The dynorphin (DYN)/kappa opioid receptor (KOR) system has increasingly been investigated as a possible pharmacotherapeutic target for alcohol use disorder, but findings on the direction of its effects have been mixed. Activation of KORs by DYN has been shown to elicit dysphoric effects, and the DYN/KOR system has canonically been considered particularly important in driving alcohol intake through negative reinforcement in dependent states. However, this review also highlights its activity in opposing the positive reinforcement that drives alcohol intake at earlier stages. Both DYN and KORs are concentrated in the extended amygdala, a set of interconnected regions that includes the bed nucleus of the stria terminalis, central nucleus of the amygdala, and nucleus accumbens shell. This review focuses on the role of the DYN/KOR system in the extended amygdala in ethanol use. It begins by examining the effects of ethanol on the expression of DYN/KOR in the extended amygdala, expression of DYN/KOR in alcohol-preferring and alcohol-avoiding animals, and the effects of knocking out DYN/KOR genes on ethanol intake. Then, it examines the effects on ethanol use in both dependent and nondependent states from systemic pharmacological manipulations of DYN/KOR and from specific manipulation of this system in regions of the extended amygdala. We propose that greater expression and binding of DYN/KOR, by reducing the positive reinforcement that drives early stages of intake, initially acts to prevent the escalation of ethanol drinking. However, prolonged, binge-like, or intermittent ethanol intake enhances levels of DYN/KOR in the extended amygdala such that the system ultimately facilitates the negative reinforcement that drives later stages of ethanol drinking. This review highlights the potential of the DYN/KOR system as a target that can affect different outcomes across different stages of ethanol drinking and the development of alcohol use disorder.

Evolution of immunogenetic components encoding ultralong CDR H3.

The genomes of most vertebrates contain many V, D, and J gene segments within their Ig loci to construct highly variable CDR3 sequences through combinatorial diversity. This nucleotide variability translates into an antibody population containing extensive paratope diversity. Cattle have relatively few functional VDJ gene segments, requiring innovative approaches for generating diversity like the use of ultralong-encoding IGHV and IGHD gene segments that yield dramatically elongated CDR H3. Unique knob and stalk microdomains create protracted paratopes, where the antigen-binding knob sits atop a long stalk, allowing the antibody to bind both surface and recessed antigen epitopes. We examined genomes of twelve species of Bovidae to determine when ultralong-encoding IGHV and IGHD gene segments evolved. We located the 8-bp duplication encoding the unique TTVHQ motif in ultralong IGHV segments in six Bovid species (cattle, zebu, wild yak, domestic yak, American bison, and domestic gayal), but we did not find evidence of the duplication in species beyond the Bos and Bison genera. Additionally, we analyzed mRNA from bison spleen and identified a rich repertoire of expressed ultralong CDR H3 antibody mRNA, suggesting that bison use ultralong IGHV transcripts in their host defense. We found ultralong-encoding IGHD gene segments in all the same species except domestic yak, but again not beyond the Bos and Bison clade. Thus, the duplication event leading to this ultralong-encoding IGHV gene segment and the emergence of the ultralong-encoding IGHD gene segment appears to have evolved in a common ancestor of the Bos and Bison genera 5-10 million years ago.

Diversity of ACE2 and its interaction with SARS-CoV-2 receptor binding domain.

COVID-19, the clinical syndrome caused by the SARS-CoV-2 virus, has rapidly spread globally causing hundreds of millions of infections and over two million deaths. The potential animal reservoirs for SARS-CoV-2 are currently unknown, however sequence analysis has provided plausible potential candidate species. SARS-CoV-2 binds to the angiotensin I converting enzyme 2 (ACE2) to enable its entry into host cells and establish infection. We analyzed the binding surface of ACE2 from several important animal species to begin to understand the parameters for the ACE2 recognition by the SARS-CoV-2 spike protein receptor binding domain (RBD). We employed Shannon entropy analysis to determine the variability of ACE2 across its sequence and particularly in its RBD interacting region, and assessed differences between various species' ACE2 and human ACE2. Recombinant ACE2 from human, hamster, horseshoe bat, cat, ferret, and cow were evaluated for RBD binding. A gradient of binding affinities were seen where human and hamster ACE2 were similarly in the low nanomolar range, followed by cat and cow. Surprisingly, horseshoe bat (Rhinolophus sinicus) and ferret (Mustela putorius) ACE2s had poor binding activity compared with the other species' ACE2. The residue differences and binding properties between the species' variants provide a framework for understanding ACE2-RBD binding and virus tropism.

Diversity of ACE2 and its interaction with SARS-CoV-2 receptor binding domain.

COVID-19, the clinical syndrome caused by the SARS-CoV-2 virus, has rapidly spread globally causing tens of millions of infections and over a million deaths. The potential animal reservoirs for SARS-CoV-2 are currently unknown, however sequence analysis has provided plausible potential candidate species. SARS-CoV-2 binds to the angiotensin I converting enzyme 2 (ACE2) to enable its entry into host cells and establish infection. We analyzed the binding surface of ACE2 from several important animal species to begin to understand the parameters for the ACE2 recognition by the SARS-CoV-2 spike protein receptor binding domain (RBD). We employed Shannon entropy analysis to determine the variability of ACE2 across its sequence and particularly in its RBD interacting region, and assessed differences between various species' ACE2 and human ACE2. As cattle are a known reservoir for coronaviruses with previous human zoonotic transfer, and has a relatively divergent ACE2 sequence, we compared the binding kinetics of bovine and human ACE2 to SARS-CoV-2 RBD. This revealed a nanomolar binding affinity for bovine ACE2 but an approximate ten-fold reduction of binding compared to human ACE2. Since cows have been experimentally infected by SARS-CoV-2, this lower affinity sets a threshold for sequences with lower homology to human ACE2 to be able to serve as a productive viral receptor for SARS-CoV-2.