Subtype 1a Research Articles

Impact of Direct-Acting Antiviral Therapy on Liver Fibrosis Regression among People with Chronic HCV Infection: Results from a Real-Life Cohort in Patients Who Achieved Sustained Virological Response.

Background and Objectives: The global prevalence of chronic hepatitis C virus (HCV) infection is 0.8%, affecting around 58 million people worldwide. Treatment with DAAs reduces all-cause HCV mortality by 49-68%. This work aims to determine whether there is liver fibrosis regression (LFR) in patients who achieved Sustained Virological Response (SVR) after treatment with DAAs. Materials and Methods: An analytical, observational, single-center, and cohort study was carried out. The final sample consisted of 248 HCV-infected patients. All started treatment with DAAs between January 2015 and December 2017. Five measurements were performed to determine the fibrotic stage in patients (measured in kilopascals (kPa)) using transient elastography (FibroScan®, Echosens, The Netherlands). Results: Taking the baseline fibrotic stage as a reference, the distribution in subgroups was as follows: 77 F4 patients (31.0%); 55 F3 patients (22.2%); 53 F2 patients (21.4%); and 63 F0/F1 patients (25.4%). There were 40 patients (16.1%) with at least one HCV complication and 13 (5.2%) who developed hepatocellular carcinoma. The overall LFR rate was 77.8% (144 of 185 F2/F3/F4 patients, p = 0.01) at the end of the follow-up period. The highest mean FibroScan® values were observed in patients with: "male gender"; "metabolic syndrome"; "subtype 1a"; "NRP DAA"; "at least one HCV complication"; "death from HCV complications"; and "liver transplantation requirement". Conclusions: Treatment with DAAs achieved high rates of LFR and a decrease in mean FibroScan® values in all subgroups.

Hydrogen sulfide inhibits lipopolysaccharide-based neuroinflammation-induced astrocyte polarization after cerebral ischemia/reperfusion injury

The effect of lipopolysaccharide (LPS)-based neuroinflammation following cerebral ischemia/reperfusion (I/R) on the genotypic transformation of reactive astrocytes and its relationship with endogenous hydrogen sulfide (H2S) were investigated in present study. We found that LPS promoted the cerebral I/R-induced A1 astrocytes proliferation in mouse hippocampal tissues and deteriorated the reduction of hydrogen sulfide (H2S) content in mouse sera, H2S donor NaHS could inhibit A1 astrocytes proliferation. Similarly, knockout of cystathionine γ-lyase (CSE), one of endogenous H2S synthases, likewise up-regulated the cerebral I/R-induced A1 astrocytes proliferation, which could also be blocked by NaHS. Besides, supplement with H2S promoted the A2 astrocytes proliferation in hippocampal tissues of CSE knockout (CSE KO) mice or LPS-treated mice following cerebral I/R. In the oxygen glucose deprivation/reoxygenation (OGD/R) model of astrocytes, H2S also promoted the transformation of astrocytes into A2 subtype. Moreover, we found that H2S could up-regulate the expression of α-subunit of large-conductance Ca2+-activated K+ (BKCa) channels in astrocytes, and the channel opener BMS-191011 likewise promoted the transformation of astrocyte into A2 subtype. In conclusion, H2S inhibits the proliferation of A1 astrocytes induced by LPS-based neuroinflammation following cerebral I/R and promotes the transformation of astrocytes into A2 subtype, which may be related to up-regulation of BKCa channels.

SARS-CoV-2 spike ectodomain targets α7 nicotinic acetylcholine receptors

Virus entry into animal cells is initiated by attachment to target macromolecules located on host cells. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) trimeric spike glycoprotein targets host angiotensin converting enzyme 2 to gain cellular access. The SARS-CoV-2 glycoprotein contains a neurotoxin-like region that has sequence similarities to the rabies virus and the HIV glycoproteins, as well as to snake neurotoxins, which interact with nicotinic acetylcholine receptor (nAChR) subtypes via this region. Using a peptide of the neurotoxin-like region of SARS-CoV-2 (SARS-CoV-2 glycoprotein peptide [SCoV2P]), we identified that this area moderately inhibits α3β2, α3β4, and α4β2 subtypes, while potentiating and inhibiting α7 nAChRs. These nAChR subtypes are found in target tissues including the nose, lung, central nervous system, and immune cells. Importantly, SCoV2P potentiates and inhibits ACh-induced α7 nAChR responses by an allosteric mechanism, with nicotine enhancing these effects. Live-cell confocal microscopy was used to confirm that SCoV2P interacts with α7 nAChRs in transfected neuronal-like N2a and human embryonic kidney 293cells. The SARS-CoV-2 ectodomain functionally potentiates and inhibits the α7 subtype with nanomolar potency. Our functional findings identify that the α7 nAChR is a target for the SARS-CoV-2 glycoprotein, providing a new aspect to our understanding of SARS-CoV-2 and host cell interactions, in addition to disease pathogenesis.

Abstract 5308: Analysis of extracellular vesicle surface markers as small cell lung cancer biomarkers

Abstract Small Cell Lung Cancer (SCLC) is an aggressive tumor type, usually metastatic at diagnosis, with a median survival of less than a year. SCLC tumors are heterogeneous and are composed of neuroendocrine (A, A2, N) and non-neuroendocrine (Y) subtypes, with the cooperation by heterogeneous cells being key to drive tumor survival. Unfortunately, the current SCLC treatment approach does not take tumor heterogeneity into consideration. Importantly, there are no consensus markers that can identify SCLC subtypes or early tumors. Extracellular vesicles (EVs), small vesicles known to promote cell to cell communication, have been investigated as non-invasive biomarkers for cancer diagnostics, staging, and treatment monitoring. EVs are attractive candidates for biomarker research due to the selective packaging of RNA, DNA, and proteins that are then secreted in the blood. Therefore, our aim was to identify new biomarkers based on EV surface proteins with potential for early detection and tumor characterization. We used differential ultracentrifugation to isolate EVs from four SCLC subtypes A, A2, N, and Y, which were submitted to label-free mass spectrometry (EV-proteomics). We identified a total of 3011 proteins, with the two most distinct subtypes being A2 and N, with 786 differentially expressed EV-proteins. Meanwhile, the 2 most similar subtypes were N and A, with only 75 differentially expressed EV-proteins. Gene set enrichment analysis of EVs derived from the A2 subtype showed an association with pathways in cancer, chemokine signaling, and neuronal systems. In addition, PANTHER analysis identified enrichment in nicotine pharmacodynamic pathways on these EVs, which corroborate the association of A2 cell lines with drug resistance. EVs derived from the N and A subtypes were enriched in spliceosome, RNA processing, and metabolism, while EVs from Y cell lines were enriched in extracellular matrix organization and metabolic pathways, similar to pathways from cell line RNAseq data. 13 proteins were differentially expressed in all group comparisons, including proteins involved in PI3K and nicotinic acetylcholine receptor signaling pathways. Further comparison of EV-proteomics with top genes from CIBERSORT analysis using cell line RNAseq data showed an overlap of 9 surface proteins, with 3 possible SCLC pan markers. In conclusion, EV-proteins isolated from SCLC cell lines presented a unique signature for each subtype, especially EVs derived from A2 subtype, which has a signature associated with drug resistance. We identified surface proteins on SCLC EVs with potential as SCLC pan and tumor characterization, which will be validated on EVs derived from cell lines and patient plasma by Western blotting and single-EV microflow cytometry. Citation Format: Patricia M. Ozawa, Sarah M. Groves, Vito Quaranta, Alissa M. Weaver. Analysis of extracellular vesicle surface markers as small cell lung cancer biomarkers. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5308.

A Systematic Review and Meta-Analysis on Hepatitis E Virus Detection in Farmed Ruminants.

Swine are widely recognized as the main reservoir of zoonotic HEV; however, a growing body of data on the HEV prevalence in farmed ruminants of different species also points to a potential route for HEV transmission through ruminants and ruminant products and by-products. Definite information on the zoonotic potential of ruminants is still absent or unclear, compelling the necessity for increasing knowledge on this. The aim of the current study was to analyze the state-of-the-art in this research topic and provide a summary of HEV detection and characterization in farmed ruminants. A total of 1567 papers were retrieved from four search databases that resulted in 35 eligible papers after application of exclusion/inclusion criteria. Studies on HEV in farmed ruminants were mainly based on the detection of HEV RNA and were reported in Africa (n = 1), America (n = 3), Asia (n = 18) and Europe (n = 13), and focused on a variety of ruminants species, namely cow, goat, sheep, deer, buffalo and yak. The overall pooled prevalence of HEV was 0.02% (0.01-0.03, 95% CI). The subgroup pooled prevalence of HEV RNA was 0.01% (0.00-0.02, 95% CI) in cow milk, stool, serum, liver, intestinal, bile, blood, spleen and rectal swab samples; 0.09% (0.02-0.18, 95% CI) in goat serum, bile, stool, milk, liver, rectal swab and blood samples; 0.01% (0.00-0.04, 95% CI) in sheep stool, serum, milk, blood and liver samples. Most of the HEV genotypes found in farmed ruminants belonged to the zoonotic HEV-3 (subtypes 3a, 3c) and HEV-4 (subtype 4d, 4h), with Rocahepevirus also found. The wide HEV circulation observed in different farmed ruminants raises concerns for the possibility of HEV transmission through products from infected ruminants and alerts for the potential zoonotic route for HEV in ruminant products, such as meat and dairy products. Also, contact exposure to infected farmed animals could be a risk factor. Further research should be conducted in order to understand the circulation of HEV in these animals and its zoonotic potential, as there is currently a lack of data on this topic.

Genetic diversity of the human immunodeficiency virus of type 1 in Gabonese transfusional settings

BackgroundThe high endemicity of transfusion-transmissible infections (TTIs) in sub-Saharan Africa is a real public health problem. To reduce the risk of HIV transmission through blood donation, the NBTC of Gabon has launched in recent years a reorganization of its blood transfusion system. This study aims to characterize the molecular strains of HIV-1 circulating in donors and to estimate the risk of viral transmission.Materials and methodsA cross-sectional study was carried out during the period from August 2020 to August 2021 among 381 donors who had agreed to donate blood at the National Blood Transfusion Center (NBTC). Viral load was determined by Abbott Real-Time (Abbott m2000®, Abbott) and sequencing by the Sanger method (ABI 3500 Hitachi®). The phylogenetic tree was constructed by MEGA X software. Data were checked, entered, and analyzed using SPSS version 21.0 software, with p ≤ 0.05 considered statistically significant.ResultsA total of 381 donors were enrolled in the study. Among the 359 seronegative donors, five (5) seronegative donors were detected positive for HIV-1 using Real-Time PCR. The residual risk was 648 per 1,000,000 donations. The prevalence of residual infection was 1.4% [0,01; 0,03]. Sixteen (16) samples were sequenced. The strains obtained were CRF02_AG (50%), subtype A1 (18.8%), subtype G (12.5%), CRF45_cpx (12.5%) and subtype F2 (6.2%). Six sequences clustered with A1, G, CRF02_AG, and CRF45_cpx subtypes.ConclusionThe residual risk of HIV-1 transmission by blood transfusion remains a concern in the Gabonese transfusional settings. A policy based on improving the current screening strategy would involve the implementation of the nucleic acid test (NAT) in order to optimize the safety of the donation by detecting the HIV-1 subtypes in circulation in the donors.

Discovery of novel virulence mechanisms in Clostridium botulinum type A3 using genome-wide analysis

ObjectiveClostridium botulinum type A is a neurotoxin-producing, spore-forming anaerobic bacterium that causes botulism in humans. The evolutionary genomic context of this organism is not yet known to understand its molecular virulence mechanisms in the human intestinal tract. Hence, this study aimed to investigate the mechanisms underlying virulence and pathogenesis by comparing the genomic contexts across species, serotypes, and subtypes. MethodsA comparative genomic approach was used to analyze evolutionary genomic relationships, intergenomic distances, syntenic blocks, replication origins, and gene abundance with phylogenomic neighbors. ResultsType A strains have shown genomic proximity to group I strains with distinct accessory genes and vary even within subtypes. Phylogenomic data showed that type C and D strains were distantly related to a group I and group II strains. Synthetic plots indicated that orthologous genes might have evolved from Clostridial ancestry to subtype A3 strains, whereas syntonic out-paralogs might have emerged between subtypes A3 and A1 through α-events. Gene abundance analysis revealed the key roles of genes involved in biofilm formation, cell–cell communication, human diseases, and drug resistance compared to the pathogenic Clostridia. Moreover, we identified 43 unique genes in the type A3 genome, of which 29 were involved in the pathophysiological processes and other genes contributed to amino acid metabolism. The C. botulinum type A3 genome contains 14 new virulence proteins that can provide the ability to confer antibiotic resistance, virulence exertion and adherence to host cells, the host immune system, and mobility of extrachromosomal genetic elements. ConclusionThe results of our study provide insight into the understanding of new virulence mechanisms to discover new therapeutics for the treatment of human diseases caused by type A3 strains.

α7 Nicotinic acetylcholine receptors regulate translocation of HIF-1α to the cell nucleus and mitochondria upon hypoxia

Nicotinic acetylcholine receptors (nAChRs), initially characterized as ligand-gated ion channels mediating fast synaptic transmission, are now found in many non-excitable cells and mitochondria where they function in ion-independent manner and regulate vital cellular processes like apoptosis, proliferation, cytokine secretion. Here we show that the nAChRs of α7 subtype are present in the nuclei of liver cells and astrocytoma U373 cell line. As shown by lectin ELISA, the nuclear α7 nAChRs are mature glycoproteins that follow the standard rout of post-translational modifications in Golgi; however, their glycosylation profile is non-identical to that of mitochondrial nAChRs. They are exposed on the outer nuclear membrane and are found in combination with lamin B1. The nuclear α7 nAChRs are up-regulated in liver within 1 h after partial hepatectomy and in H2O2-treated U373 cells. As shown both in silico and experimentally, the α7 nAChR interacts with hypoxia-inducible factor HIF-1α and this interaction is impaired by α7-selective agonists PNU282987 and choline or type 2 positive allosteric modulator PNU120596, which prevent HIF-1α accumulation in the nuclei. Similarly, HIF-1α interacts with mitochondrial α7 nAChRs in U373 cells treated with dimethyloxalylglycine. It is concluded that functional α7 nAChRs influence HIF-1α translocation into the nucleus and mitochondria upon hypoxia.

Botulinum Neurotoxin A4 Has a 1000-Fold Reduced Potency Due to Three Single Amino Acid Alterations in the Protein Receptor Binding Domain.

Botulinum neurotoxin subtype A4 (BoNT/A4) is ~1000-fold less potent than BoNT/A1. This study addresses the basis for low BoNT/A4 potency. Utilizing BoNT/A1-A4 and BoNT/A4-A1 Light Chain-Heavy Chain (LC-HC) chimeras, HC-A4 was responsible for low BoNT/A4 potency. Earlier studies showed BoNT/A1-receptor binding domain (Hcc) bound a β-strand peptide (556-564) and glycan-N559 within Luminal Domain 4 (LD4) of SV2C, the BoNT/A protein receptor. Relative to BoNT/A1, the Hcc of BoNT/A4 possesses two amino acid variants (D1141 and N1142) within the β-peptide binding interface and one amino acid variant (R1292) located near the SV2C glycan-N559. Introduction of BoNT/A4 β-strand peptide variant (D1141 and N1142) into BoNT/A1 reduced toxin potency 30-fold, and additional introduction of the BoNT/A4 glycan-N559 variant (D1141, N1142, and R1292) further reduced toxin potency to approach BoNT/A4. While introduction of BoNT/A1 glycan-N559 variant (G1292) into BoNT/A4 did not alter toxin potency, additional introduction of BoNT/A1 β-strand peptide variants (G1141, S1142, and G1292) resulted in potency approaching BoNT/A1 potency. Thus, outcomes from these functional and modeling studies indicate that in rodent models, disruption of Hcc -SV2C β-peptide and -glycan-N559 interactions mediate low BoNT/A4 potency, while in human motor neurons, disruption of Hcc-SV2C β-peptide alone mediates low BoNT/A4 potency, which link to a species-specific variation at SV2C563.

Covalently Binding Adenosine A3 Receptor Agonist ICBM Irreversibly Reduces Voltage-Gated Ca2+ Currents in Dorsal Root Ganglion Neurons

Interest has been focused in recent years on the analgesic effects exerted by adenosine and its receptors, A1, A2A, A2B, and A3 adenosine receptor (AR) subtypes, in different in vivo models of chronic pain. In particular, it was demonstrated that selective A3AR agonists reduced pro-nociceptive N-type Ca2+ channels in dorsal root ganglion (DRG) neurons isolated from rats and, by this mechanism, inhibit post inflammatory visceral hypersensitivity. In the present study, we investigate the effect of a previously reported irreversibly binding A3AR agonist, ICBM, on Ca2+ currents (ICa) in rat DRG neurons. Present data demonstrate that ICBM, an isothiocyanate derivative designed for covalent binding to the receptor, concentration-dependently inhibits ICa. This effect is irreversible, since it persists after drug removal, differently from the prototypical A3AR agonist, Cl-IB-MECA. ICBM pre-exposure inhibits the effect of a subsequent Cl-IB-MECA application. Thus, covalent A3AR agonists such as ICBM may represent an innovative, beneficial, and longer-lasting strategy to achieve efficacious chronic pain control versus commonly used, reversible, A3AR agonists. However, the possible limitations of this drug and other covalent drugs may be, for example, a characteristic adverse effect profile, suggesting that more pre-clinical studies are needed.

Anatomical variations of left hepatic vein and outflow reconstruction techniques in pediatric living donor liver transplantation

Anatomical variations of left hepatic vein are observed in nearly a third of left lateral segment (LLS) donors in living donor liver transplantation. However, there is a paucity of studies and no structured algorithm for customized outflow reconstruction in LLS grafts with variant anatomy. Analysis of a prospectively collected database of 296 LLS pediatric living donor liver transplantation was done to identify different venous drainage patterns of segments 2 (V2) and 3 (V3). Left hepatic vein anatomy was classified into 3 types: type 1 (n = 270, 91.2%): V2 and V3 joined to form a common trunk which drains into the middle hepatic vein/inferior vena cava (IVC), subtype 1a length of trunk ≥9 mm and subtype 1b length of trunk <9 mm; type 2(n = 6, 2%): V2 and V3 drain independently into IVC; type 3 (n = 20, 6.8%): V2 and V3 drain into IVC and middle hepatic vein respectively. Analysis of postoperative outcomes between LLS grafts with single and reconstructed multiple outflows showed no difference in the occurrence of hepatic vein thrombosis/stenosis, major morbidity (P = .91), and 5-year survival (log-rank P = .562). This classification is a simple yet effective tool for preoperative donor assessment, and we propose a schema for the customized reconstruction of LLS grafts with excellent and consistently reproducible outcomes.

Suppression of the vitamin D metabolizing enzyme CYP24A1 provides increased sensitivity to chemotherapeutic drugs in breast cancer.

Vitamin D is an essential nutrient for the human body not only for the metabolism of calcium but also for homeostasis. Vitamin D contributes to cell fate decisions, including cell proliferation, differentiation and viability. Accumulated epidemiological data suggest a relationship between vitamin D deficiency and carcinogenesis in numerous organs. Furthermore, it is known that the expression of the vitamin D metabolizing enzyme, cytochrome P450 family 24 subtype A1 (CYP24A1), is increased in different types of human malignancy including breast carcinoma. However, the pathological relevance of elevated CYP24A1 expression level requires further clarification. In the present study, it was demonstrated that CYP24A1 promoted the oncogenic property of breast carcinoma cells. Consistent with previous reports, it was demonstrated that the expression of CYP24A1 was elevated in invasive breast carcinoma and significantly decreased the overall survival of patients with invasive breast carcinoma. Importantly, suppression of CYP24A1 expression significantly enhanced cell death sensitivity to two anticancer drugs with pharmacologically different modes of action, cisplatin and gefitinib. The results of the present study suggest the possibility of CYP24A1‑inhibiting therapy as a novel therapy in breast cancer with overexpression of CYP24A1.

Nisin: Prerequisites and Prospects for Application in the Medical Field

Nisin is a typical representative of class I bacteriocins belonging to subtype A1 lantibiotics. This lantibiotic is produced by gram-positive bacteria of the genera Lactococcus, Streptococcus, Staphylococcus, and Blautia. Currently, at least twelve natural variants of nisin are known. They differ in some physicochemical properties and biological activity. Nisin is generally recognized as safe (GRAS) and has been used as a food additive for seven decades. The current review presents the data from scientific reports over the past decade supporting the value of this lantibiotic as a therapeutic agent. Most research works demonstrate a high inhibitory potential of nisin against antibiotic-resistant bacteria when using alone and in combination with antibiotics. A number of studies prove the ability of nisin to modulate the composition of oral and intestinal microbiomes, to inhibit the growth of both bacteria causing infectious processes and bacterial pathogens associated with cancer. Nisin has been shown to have its own antitumor potential and to be able to enhance the therapeutic effect of chemotherapeutic drugs. There is convincing evidence of its immunomodulatory and anti-inflammatory activity. The reasons limiting its widespread application in clinical practice can be successfully eliminated via modern bioengineering technologies that improve the functional characteristics and stability of nisin in various physiological conditions.

Atretic cephalocele and encephalocele: A single-institution clinicopathological study.

Encephaloceles are neural tube defects characterized by herniation of meninges, neural tissue and cerebrospinal fluid, while atretic cephaloceles denote a rudimentary connection to the intracranial space with absence of herniated neural tissue and represent an infrequent dermatopathologic diagnosis. Limited reports of these entities confound the challenge in their histopathologic distinction. Accurate classification is important given associated anomalies and neurologic manifestations that impact prognosis. We describe the clinicopathological and immunohistochemical [glial fibrillary acidic protein (GFAP), S100, epithelial membrane antigen (EMA), and somatostatin receptor subtype 2A (SSTR2A)] features in a retrospective series encountered at a single institution between 1994 and 2020. We identified 13 cases classified as atretic cephalocele (n=11) and encephalocele (n=2). Hamartomatous changes and multinucleated cells were unique to atretic cephaloceles while myxoid areas were unique to encephaloceles. At least focal staining for SSTRA was seen in all atretic cephaloceles with the majority (87.5%) staining for EMA; negative staining for GFAP and S100 confirmed absence of neural tissue. Encephaloceles were GFAP and S100 positive, and negative for SSTR2 and EMA. Atretic cephaloceles had a favorable prognosis compared to encephaloceles, with severe morbidity present in both encephalocele cases. Our study raises awareness of atretic cephalocele and encephalocele among dermatopathologists and reveals a mutually exclusive immunophenotype that facilitates their distinction for prognostication and management.

Adenosine Receptors Expression in Human Retina and Choroid with Age-related Macular Degeneration.

Adenosine signaling modulates ocular inflammatory processes, and its antagonism mitigates neovascularization in both newborns and preclinical models of ocular neovascularization including age-related macular degeneration (AMD). The adenosine receptor expression patterns have not been well characterized in the human retina and choroid. Here we examined the expression of adenosine receptor subtypes within the retina and choroid of human donor eyes with and without AMD. Antibodies specifically targeting adenosine receptor subtypes A1, A2A, A2B, and A3 were used to assess their expression patterns. Quantitative real-time PCR analysis was used to confirm gene expression of these receptors within the normal human retina and choroid. We found that all four receptor subtypes were expressed in several layers of the retina, and within the retinal pigment epithelium and choroid. The expression of A1 receptors was more prominent in the inner and outer plexiform layers, where microglia normally reside, and supported by RNA expression in the retina. A2A and A2B showed similar expression patterns with prominent expression in the vasculature and retinal pigment epithelium. No dramatic differences in expression of these receptors were observed in eyes from patients with dry or wet AMD compared to control, with the exception A3 receptors. Eyes with dry AMD lost expression of A3 in the photoreceptor outer segments compared with eyes from control or wet AMD. The ocular presence of adenosine receptors is consistent with their proposed role in modulation of inflammation in both the retina and choroid, and their potential targeting for AMD treatment.

An ELISA to Detect Antibodies to Bovine Alphaherpesviruses 1 and 5 and Bubaline Alphaherpesvirus 1 in Cattle Sera

Bovine alphaherpesvirus 1 (subtypes 1.1, 1.2a, and 1.2b), type 5 (subtypes 5a, 5b, and 5c), and bubaline herpesvirus 1 (BuHV-1) induce highly, though not fully cross-reactive serological responses. Most types and subtypes of these viruses circulate particularly in countries of the southern hemisphere, notably Brazil and Argentina. Therefore, the detection of infected animals is important in defining prevention and control strategies, particularly when flocks are destined for international trade. Identification of infected herds is most often achieved by assays that detect antibodies, such as enzyme immunoassays (ELISAs). However, to date, no ELISA has been evaluated in its capacity to detect antibodies to these alphaherpesviruses. Here, an ELISA was developed to detect antibodies to all currently recognized BoAHV-1, BoAHV-5, and BuAHV-1 types/subtypes, and its sensitivity and specificity were determined. Six hundred bovine sera were screened in serum neutralization tests (SN) against the seven viruses. ELISAs prepared with each of the viruses were compared to SN. Subsequently, a combined assay with multiple antigens LISA was prepared by mixing five viral antigens, chosen for their highest sensitivity in the preparative assays. In comparison to SN, the mAgELISA sensitivity was 96.5% with 96.1% specificity (κ = 0.93; PPV = 95.0%; NPV = 97.3%). The findings reveal that the mAgELISA developed here is highly suitable for the detection of antibodies, comparable in sensitivity and specificity to that of SN when performed with all known types and subtypes of bovine and bubaline alphaherpesviruses.

Design and optimization of nicotinic acetylcholine receptor subtype selective peptides.

Nicotinic acetylcholine receptors (nAChRs) are widespread throughout the brain, express as distinct subtypes, and play pivotal roles in neurologic processes. Chronic neurological pain and depression are in part mediated by distinct nAChRs, including the α9α10 and α7 subtypes, which are in the central and peripheral nervous systems. Cell penetrating peptides (CPPs) derived from the rabies virus glycoprotein (RVG) interact non-selectively with nAChRs and can deliver cargo past the murine blood brain barrier (BBB). We hypothesize subtype selective chimeric CPPs can be generated from regions of the RVG and proteins with endogenous nAChR subtype selectivity that can cross the human BBB. We expressed distinct human nAChR subtypes in Xenopus laevis oocytes by injecting biased ratios of subunit cRNA. Two-electrode voltage clamp (TEVC) electrophysiology was used to determine functional changes in the acetylcholine-induced response when oocytes were pre-exposed to increasing concentration of each tested peptide. To assess selectivity, a high inhibiting peptide concentration (100 μM) was applied. For peptides that displayed enhanced selectivity, the potency was determined from concentration-response profiles. We initially tested nine chimeric peptides generated from three unique proteins with different nAChR subtype targets. One chimeric peptide revealed high selectivity for α7-, as well as α9α10-nAChRs. An alanine mutation screening protocol is being utilized to characterize residues integral to potency and selectivity of this lead peptide. Of the eight tested residues, one residue caused a two-fold increase in potency. This mutation will be incorporated during refinement of our lead peptide. Four additional residues reduced potency by 3 to 8-fold. These hydrophobic or electrostatic residues will be further mutated with the goal of enhancing potency and selectivity. These peptides have the potential to be beneficial in the treatment of the co-morbid conditions chronic pain and depression.

Dual-Toxin ("Bivalent") Infant Botulism in California, 1976-2020: Epidemiologic, Clinical, and Laboratory Aspects.

To assess the consequences of infant botulismthat result from Clostridium botulinum strains that produce 2 botulinum toxin serotypes, termed "bivalent." Epidemiologic investigations used a standard questionnaire. Clostridium botulinum strains were isolated by standard methods. Botulinum neurotoxin (BoNT) serotypes and the relative amounts of toxins produced were identified using the standard mouse bioassay. BoNT subtypes and genomic locations were identified by DNA nucleotide sequencing. Thirty bivalent cases of infant botulism occurred in the 45years (1976-2020), representing 2.0% of all California infant botulism cases, in the 3 geographic regions of southern California, the southern Central Valley, and mid-northern California. Toxin serotype combinations were Ba (n=22), Bf (n=7), and Ab (n=1). More patients with illness caused by bivalent C botulinum Ba and Bf strains needed endotracheal intubation at hospital admission, 60.0% (18/30), than did patients with illness caused by monovalent BoNT/B strains, 34.3% (152/443). The Cbotulinum Ba and Bf strains produced BoNT/B5 and either BoNT/A4 or /F2. The Ab strain produced BoNT/A2 and /B1. All toxin gene clusters were on plasmids. Infant botulism caused by bivalent Cbotulinum strains occurs sporadically and in diverse locations in California. Affected patients with bivalent Ba and Bf strains lacked distinguishing epidemiological features but appeared to be more severely paralyzed at hospital presentation than patients with illness caused by only BoNT/B. These bivalent strains produced BoNT subtypes A2, A4, B1, B5, and F2, and all toxin gene clusters were on plasmids.

Genetic Diversity of the LTR Region of Polish SRLVs and Its Impact on the Transcriptional Activity of Viral Promoters.

A long terminal repeat (LTR) plays an indispensable role in small ruminant lentivirus (SRLV) gene expression. In this study, we present the LTR sequence of Polish SRLVs representing different subtypes, and analyzed their impact on SRLV promoter activity, as measured in transient transfection assays. Although certain nucleotide motifs (AML(vis), TATA box and the polyadenylation site (AATAAA)) were conserved across sequences, numerous mutations within the LTR sequences have been identified. Single nucleotide polymorphisms (SNPs) were detected in both regulatory (AP-1, AP-4, Stat and Gas) and non-regulatory sequences, and subtype-specific genetic diversity in the LTR region of Polish SRLVs was observed. In vitro assays demonstrated subtype-specific functional differences between the LTR regions of distinct SRLV subtypes. Our results revealed that the promoter activity of Polish strains was lower (1.64-10.8-fold) than that noted for the K1514 reference strain; however, the differences in most cases were not statistically significant. The lowest promoter activity was observed for strains representing subtype A5 (mean 69.067) while the highest promoter activity was observed for strain K1514 representing subtype A1 (mean 373.48). The mean LTR activities of strains representing subtypes A12, A17, A23, A18 and A24 were 91.22, 137.21, 178.41, 187.05 and 236.836, respectively. The results of the inter-subtype difference analysis showed that the promoter activity of strains belonging to subtype A5 was significantly lower than that for subtype A12 strains (1.32-fold; p < 0.00). The promoter activities of the A5 strain were 1.98-fold and 2.58-fold less active than that of the A17 and A23 strains, and the promoter activities of A12 strains were 1.955 and 1.5 times lower than the promoter activity of A23 and A17 strains, respectively. Furthermore, the promoter activity of A17 strains was 1.3 lower than the promoter activity of A23 strains. Our findings suggest that subtype-specific genetic diversity, mainly in the transcription factor's binding sites, has an impact on their transcriptional activity, producing a distinct activity pattern for the subtypes. This study provides new information that is important for better understanding the function of the SRLV LTR. However, further research including more strains and subtypes as well as other cell lines is needed to confirm these findings.

α7 nicotinic acetylcholine receptor in memory processing.

Information storage in the brain involves different memory types and stages that are processed by several brain regions. Cholinergic pathways through acetylcholine receptors actively participate on memory modulation, and their disfunction is associated with cognitive decline in several neurological disorders. During the last decade, the role of α7 subtype of nicotinic acetylcholine receptors in different memory stages has been studied. However, the information about their role in memory processing is still scarce. In this review, we attempt to identify brain areas where α7 nicotinic receptors have an essential role in different memory types and stages. In addition, we discuss recent work implicating-or not-α7 nicotinic receptors as promising pharmacological targets for memory impairment associated with neurological disorders.