Oral administration of a recombinant modified RBD antigen of SARS-CoV-2 as a possible immunostimulant for the care of COVID-19.

BACKGROUND: Developing effective vaccines against SARS-CoV-2 that consider manufacturing limitations, equitable access, and acceptance is necessary for developing platforms to produce antigens that can be efficiently presented for generating neutralizing antibodies and as a model for new vaccines. RESULTS: This work presents the development of an applicable technology through the oral administration of the SARS-CoV-2 RBD antigen fused with a peptide to improve its antigenic presentation. We focused on the development and production of the recombinant receptor binding domain (RBD) produced in E. coli modified with the addition of amino acids extension designed to improve antigen presentation. The production was carried out in shake flask and bioreactor cultures, obtaining around 200 mg/L of the antigen. The peptide-fused RBD and peptide-free RBD proteins were characterized and compared using SDS-PAGE gel, high-performance chromatography, and circular dichroism. The peptide-fused RBD was formulated in an oil-in-water emulsion for oral mice immunization. The peptide-fused RBD, compared to RBD, induced robust IgG production in mice, capable of recognizing the recombinant RBD in Enzyme-linked immunosorbent assays. In addition, the peptide-fused RBD generated neutralizing antibodies in the sera of the dosed mice. The formulation showed no reactive episodes and no changes in temperature or vomiting. CONCLUSIONS: Our study demonstrated the effectiveness of the designed peptide added to the RBD to improve antigen immunostimulation by oral administration.

Environmental enrichment attenuates conditioned taste aversion through the restoration of BDNF levels in the insular cortex.

It has been shown that exposure to an enriched environment (EE) can modulate the physiological impact of aversive stimuli in animals, promoting adaptive attitudes, as well as the development of resilience to stressful situations. These changes are known to be related to increased levels of some trophic factors, such as brain-derived neurotrophic factor (BDNF), which has been considered a regulatory protein for synaptic plasticity in the adult brain. Our previous studies have demonstrated that in the insular cortex (IC), a brain region of the temporal lobe implicated in the acquisition, consolidation, and retention of conditioned taste aversion (CTA) task, BDNF can reverse the CTA memory deficit caused by a protein synthesis inhibitor. Likewise, our research group have also shown that BDNF is required for the maintenance of CTA long-term memory. Here we evaluate the effects of the exposure to an enriched environment on the CTA memory strength, using a weak and strong version of this paradigm. The exposure to an EE for 21 days was able to attenuate the strong-CTA response through the restoration of BDNF levels in the IC of adult rats. These results provide evidence that environmental enrichment is capable of reducing the strength of an aversive memory trace, restoring the BDNF levels in a neocortical region of the adult brain.

Neotropical Rattlesnake (Crotalus simus) Venom Pharmacokinetics in Lymph and Blood Using an Ovine Model.

The most abundant protein families in viper venoms are Snake Venom Metalloproteases (SVMPs), Snake Venom Serine Proteases (SVSPs) and Phospholipases (PLA2s). These are primarily responsible for the pathophysiology caused by the bite of pit-vipers; however, there are few studies that analyze the pharmacokinetics (PK) of whole venom (WV) and its protein families. We studied the pathophysiology, PK profile and differential absorption of representative toxins from venom of Neotropical Rattlesnake (Crotalus simus) in a large animal model (ovine). Toxins studied included crotoxin (the main lethal component), which causes moderate to severe neurotoxicity; SVSPs, which deplete fibrinogen; and SVMPs, which cause local tissue damage and local and systemic hemorrhage. We found that Whole Venom (WV) was highly bioavailable (86%) 60 h following intramuscular (IM) injection, and extrapolation suggests that bioavailability may be as high as 92%. PK profiles of individual toxins were consistent with their physicochemical properties and expected clinical effects. Lymph cannulated animals absorbed 1.9% of WV through lymph during the first 12 h. Crotoxin was minimally detectable in serum after intravenous (IV) injection; however, following IM injection it was detected in lymph but not in blood. This suggests that crotoxin is quickly released from the blood toward its tissue targets.

Prognostic Value of the Pretreatment Neutrophil-to-Lymphocyte Ratio in Different Phenotypes of Locally Advanced Breast Cancer During Neoadjuvant Systemic Treatment.

PURPOSE: Neutrophils are among the key cellular players in the inflammatory milieu produced in patients with breast cancer (BC), and strong evidence exists in terms of the prognostic value of assessing the neutrophil-to-lymphocyte ratio (NLR) in patients with BC. In this study we sought to determine whether the baseline NLR correlates with pathological complete response (pCR), disease-free survival (DFS), and overall survival (OS) in patients with locally advanced BC in the neoadjuvant chemotherapy (NAC) setting. METHODS: We analyzed the pretreatment NLR from the first blood count of patients treated from 2007 to 2015 in terms of pCR, DFS, and OS in patients with locally advanced BC. Patients received standard medical care based on national guidelines. RESULTS: A total of 1519 patients were included in the study. Median age was 49 years (22-88). The cutoff point for NLR was 2.0. NLR was not associated with pCR or DFS. However, patients with high NLR had worse OS in the presence of triple-negative BC (105.9 months; 95% confidence interval [CI], 100.2-111.5] vs. 98.7 months; 95% CI, 91.1-106.3; P = .029), Her2 overexpression (114.0 months; 95% CI, 110.5-118.0 vs. 100.8 months; 95% CI 95.7-105.9; P = .019), and residual disease after NAC for both phenotypes. Multivariate analysis showed that NLR was independently associated with OS (hazard ratio, 1.4; 95% CI, 1.02-1.95; P = .037). CONCLUSIONS: Pretreatment NLR in patients with locally advanced BC correlates with OS as an independent prognostic factor. This influence depends on phenotype and residual disease. Routine assessment of this parameter could be an easy and affordable tool for defining prognosis.

BDNF induces in vivo long-lasting enhancement of synaptic transmission and structural reorganization at the hippocampal mossy fibers in a transcription and translation-independent manner.

Brain-derived neurotrophic factor (BDNF) is an essential product of protein synthesis with a prominent impact on brain signaling and synaptic plasticity. Exogenous application of this neurotrophin is able to induce long-term potentiation (LTP) in several brain structures such as the hippocampus along with increases in gene transcription and translation of proteins involved in functional and structural plasticity. In this regard, our previous studies have demonstrated that acute intrahippocampal administration of BDNF induces long-lasting enhancement of synaptic transmission at the mossy fibers projection (MF) accompanied by a structural reorganization at the CA3 hippocampus area. Thus, considering the non-canonical molecular mechanisms underlying MF-CA3-LTP and the high expression of this neurotrophin in the CA3 area, we wonder whether transcriptional and translational inhibition interferes with the persistence of the MF functional and structural synaptic plasticity elicited by BDNF in adult rats in vivo. Our results show that BDNF is able to induce a lasting potentiation of synaptic efficacy at the MF projection accompanied by a structural reorganization at the CA3 area in an mRNA synthesis and protein translation-independent manner. The present findings support the idea that BDNF is an essential plasticity related product, which is necessary and sufficient to induce and maintain functional and structural synaptic plasticity at the MF-CA3 pathway.

Consolidation of an aversive taste memory requires two rounds of transcriptional and epigenetic regulation in the insular cortex.

The current view of the neurobiology of learning and memory suggests that long-term memory (LTM) depends not only on the de novo protein synthesis but also on the synthesis of mRNA even hours after the acquisition of memory, as well as that the regulation of transcription through the histone acetylation is essential for the memory establishment. Our previous studies showed that protein synthesis inhibition around the time of training and 5-7 hours after acquisition in the insular cortex (IC) prevents the consolidation of conditioned taste aversion (CTA), a well-established learning and memory paradigm in which an animal learns to associate a novel taste with nausea. However, the participation of mRNA synthesis and the epigenetic regulation through histone acetylation in this process remains unexplored. In the present study we evaluated the effect of the inhibition of transcription as well as deacetylation of histones at two temporal windows on the consolidation of CTA. Thus, immediately or seven hours after CTA acquisition animals received a microinfusion of 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) or MS-275 in the IC, respectively. The present results show that transcription inhibition immediately and 7 h after acquisition impairs the CTA memory consolidation, whereas the inhibition of histone deacetylation strengths this memory at those temporal windows. These findings reveal that CTA memory requires recurrent rounds of transcriptional modulation events in the IC in order to consolidate this memory trace, demonstrating that transcriptional and epigenetic modulation substantially contribute to memory-consolidation-related functions performed by a neocortical area even several hours after memory acquisition.

Extinction of aversive taste memory homeostatically prevents the maintenance of in vivo insular cortex LTP: Calcineurin participation.

Accumulating evidence indicates that homeostatic plasticity mechanisms dynamically adjust synaptic strength to promote stability that is crucial for memory storage. Our previous studies have shown that prior training in conditioned taste aversion (CTA) prevents the subsequent induction of long-term potentiation (LTP) in the projection from the basolateral nucleus of the amygdala (Bla) to the insular cortex (IC) in vivo. We have also reported that induction of LTP in the Bla-IC pathway modifies the CTA extinction. Memoryextinction involves the formation of a new associativememorythat inhibits a previously conditioned association. The aim of the present study was to analyze the effect of CTA extinction on the ability to induce subsequent LTP in the Bla-IC projection in vivo. Thus, 48 h after CTA extinction animals received high frequency stimulation in order to induce IC-LTP. Our results show that extinction training allows the induction but not the maintenance of IC-LTP. In addition, with the purpose of exploring part of the mechanisms involved in this process and since a body of evidence suggests that protein phosphatase calcineurin (CaN) is involved in the extinction of some behavioral tasks, we analyzed the participation of this phosphatase. The present results show that extinction training increases the CaN expression in the IC, as well as that the inhibition of this phosphatase reverts the effects of the CTA-extinction on the IC-LTP. These findings reveal that CTA extinction promotes a homeostatic regulation of subsequent IC synaptic plasticity maintenance through increases in CaN levels.

Production of a recombinant phospholipase A2 in Escherichia coli using resonant acoustic mixing that improves oxygen transfer in shake flasks.

BACKGROUND: Shake flasks are widely used during the development of bioprocesses for recombinant proteins. Cultures of recombinant Escherichia coli with orbital mixing (OM) have an oxygen limitation negatively affecting biomass growth and recombinant-protein production. With the aim to improve mixing and aeration in shake flask cultures, we analyzed cultures subjected to OM and the novel resonant acoustic mixing (RAM) by applying acoustic energy to E. coli BL21-Gold (DE3): a producer of recombinant phospholipase A2 (rPLA2) from Micrurus laticollaris snake venom. RESULTS: Comparing OM with RAM (200 rpm vs. 7.5g) at the same initial volumetric oxygen transfer coefficient (kLa ≈ 80 h-1) ~69% less biomass was obtained with OM compared with RAM. We analyzed two more conditions increasing agitation until maximal speed (12.5 and 20g), and ~1.6- and ~1.4-fold greater biomass was obtained as compared with cultures at 7.5g. Moreover, the specific growth rate was statistically similar in all cultures carried out in RAM, but ~1.5-fold higher than that in cultures carried out under OM. Almost half of the glucose was consumed in OM, whereas between 80 and 100% of the glucose was consumed in RAM cultures, doubling biomass per glucose yields. Differential organic acid production was observed, but acetate production was prevented at the maximal RAM (20g). The amount of rPLA2 in both, OM and RAM cultures, represented 38 ± 5% of the insoluble protein. A smaller proportion of α-helices and ß-sheet of purified inclusion bodies (IBs) were appreciated by ATR-FTIR from cultures carried out under OM, than those from RAM. At maximal agitation by RAM, internal E. coli localization patterns of protein aggregation changed, as well as, IBs proteolytic degradation, in conjunction with the formation of small external vesicles, although these changes did not significantly affect the cell survival response. CONCLUSIONS: In moderate-cell-density recombinant E. coli BL21-Gold (DE3) cultures, the agitation increases in RAM (up to the maximum) was not enough to avoid the classical oxygen limitation that happens in OM shake flasks. However, RAM presents a decrease of oxygen limitation, resulting in a favorable effect on biomass growth and volumetric rPLA2 production. While under OM a higher recombinant protein yield was obtained.

CaMKII requirement for the persistence of in vivo hippocampal mossy fiber synaptic plasticity and structural reorganization.

CaMKII has been proposed as a molecular substrate for long-term memory storage due to its capacity to maintain an active autophosporylated state even after the decay of the external stimuli. The hippocampal mossy fiber-CA3 pathway (MF-CA3) is considered as a relevant area for acquisition and storage of different learning tasks. MF-CA3 pathway exhibits a form of LTP characterized by a slow initial increase in the EPSP slope that is independent of NMDA receptors activation. Our previous studies show that application of high frequency stimulation sufficient to elicit MF-CA3 LTP produces structural reorganization, in a manner independent of LTP induction, at the stratum oriens of hippocampal CA3 area 7days after stimulation. However, the molecular mechanisms that underlie the maintenance of MF-CA3 LTP as well as the concomitant structural reorganization in this area remain to be elucidated. Here we show that acute microinfusion of myr-CaMKIINtide, a noncompetitive inhibitor of CaMKII, in the hippocampal CA3 area of adult rats during the late-phase of in vivo MF-CA3 LTP blocked its maintenance and prevented the accompanying morphological reorganization in CA3 area. These findings support the idea that CaMKII is a key molecular substrate for the long-term hippocampal synaptic plasticity maintenance.

Conditioned taste aversion prevents the long-lasting BDNF-induced enhancement of synaptic transmission in the insular cortex: A metaplastic effect.

Homeostatic plasticity mechanisms dynamically adjust synaptic strengths to promote stability that is crucial for memory storage. Metaplasticity is an example of these forms of plasticity that modify the capacity of synapses to experience subsequent Hebbian modifications. In particular, training in several behavioral tasks modifies the ability to induce long-term potentiation (LTP). Recently, we have reported that prior training in conditioned taste aversion (CTA) prevents the subsequent induction of LTP generated by high frequency stimulation in the projection from the basolateral nucleus of the amygdala (Bla) to the insular cortex (IC). One of the key molecular players that underlie long-term synaptic plasticity is brain-derived neurotrophic factor (BDNF). Previous studies from our group reported that acute microinfusion of BDNF in the IC induces a lasting potentiation of synaptic efficacy at the Bla-IC projection. Thus, the aim of the present study was to analyze whether CTA training modifies the ability to induce subsequent BDNF-induced potentiation of synaptic transmission in the Bla-IC projection in vivo. Accordingly, CTA trained rats received intracortical microinfusion of BDNF in order to induce lasting potentiation 48h after the aversion test. Our results show that CTA training prevents the induction of in vivo BDNF-LTP in the Bla-IC projection. The present results provide evidence that CTA modulates BDNF-dependent changes in IC synaptic strength.

Intraspecific differences in the immunochemical reactivity and neutralization of venom from Argentinean Bothrops (Rhinocerophis) alternatus by specific experimental antivenoms.

The venoms of Bothrops (Rhinocerophis) alternatus (B.a.) from different regions of Argentina have shown biochemical, toxicological and immunological variations. Considering these variations, we produced nine experimental antisera (rabbit, IgG) against venoms from snakes of nine different regions and a pool of venom, comprised of equal amounts of venoms from each region. The immunologic studies (ELISA, Westernblot) showed significant cross reactivity among all regional antivenoms with all regional venoms, with no significant differences regarding the specificity of the immunogens used for the production of antivenom. Neutralization of hemorrhage was variable (although all the antivenoms neutralized this activity in all venoms) and the neutralization of coagulant and phospholipase activities were evident in all cases. Some antivenoms neutralized toxic activities that were absent or very low in the venoms used as immunogen, on other non-homologous venoms (e.g. thrombin like activity). Despite the different toxic potencies of regional venoms, antivenoms developed using venoms of snakes from a particular region showed high immunochemical reactivity and cross-neutralizing capacity on snake venoms from different and distant regions, in occasions over those of the homologous antivenoms. These findings could be used to improve the generation of pools of venoms for the production of antivenoms.

Identification, cDNA cloning and heterologous expression of a hyaluronidase from the tarantula Brachypelma vagans venom.

Hyaluronidases (Hyal) present in the venom of poisonous animals have been considered as "spreading factors" that facilitate a fast penetration of the venom in the prey. We have found that hyaluronidase from the tarantula Brachypelma vagans venom (BvHyal) displays a substrate-specific Hyal activity against hyaluronan. By using a combined strategy based on peptide sequencing and RT-PCR, we have cloned a BvHyal cDNA. Active recombinant BvHyal was efficiently expressed in a baculovirus system in insect cell.

[Poland's syndrome. Case report]. / Síndrome de Poland. Informe de un caso.

OBJECTIVE: To report a case of Poland syndrome in a 67-year-old woman. CLINICAL CASE: The patient was questioned and examined for a history of malformations characteristic of this syndrome on a routine medical visit. Thorax, bone thorax, and comparative hand X-ray were taken. Mammary ultrasound, mastography, and computed axial tomography were taken also. The right hemithorax showed a thin subcutaneous cell tissue. Axillary alopecia, mammary hypoplasia and areola-nipple complex retraction, hypoplasia of major pectoral muscles was observed. In the right upper limb, there was forearm shortening, hypoplasia of the second phalanx in all five fingers, and absence of distal phalanx in the index finger. CONCLUSIONS: The patient showed 4 out of the 5 characteristics considered as typical by Hester and Bostwick. In terms of hand anomalies, she was classified as type 1 according to Gausewitz and according with Al-Qaifan, the patient belongs to type 2.

Heterologous expressed toxic and non-toxic peptide variants of toxin CssII are capable to produce neutralizing antibodies against the venom of the scorpion Centruroides suffusus suffusus.

Two toxic and one non-toxic recombinant peptide variants of the mammalian neurotoxin CssII was cloned into the expression vector pQE30 containing a 6His-tag and a Factor Xa proteolytic cleavage site. The toxic recombinant peptides rCssII, HisrCssII and the non-toxic rCssIIE15R were expressed under induction with isopropyl thiogalactoside (IPTG), isolated using chromatographic techniques and folded correctly in vitro. The three recombinant variants showed similar secondary structures as the native CssII, but only the rCssIIE15R was not toxic to mice at concentrations up to 30microg/20g mouse body weight when injected intraperitoneally. All three recombinant peptides were capable of displacing the native CssII from their receptor sites in rat brain synaptosomes, suggesting that they had similar structural and functional characteristics of the native peptides. The three recombinant variants of CssII and the native one were used as antigens for immunization of New Zealand rabbits. The antibodies present in the rabbit antisera were able to recognize the native CssII. Additionally and more importantly, the sera of the immunized rabbits were able to neutralize both the native toxin CssII and the whole soluble venom of the scorpion Centruroides suffusus suffusus. These results indicate that the recombinant peptides can be used to produce antidotes against the venom of this species of scorpion.

Toxicity of two North American Loxosceles (brown recluse spiders) venoms and their neutralization by antivenoms.

The toxic, biochemical, and immunological characteristics of L. boneti and L. reclusa venoms and its neutralization by anti-L. boneti and anti-L. reclusa antivenoms were studied. The electrophoretic profile showed very similar patterns and the toxic activities were very close. Immunological studies showed cross-reactivity among L. boneti and L. reclusa venoms, with L. boneti and L. reclusa experimental antivenoms, and anti-L. gaucho and anti-L. laeta antivenoms. The venom of L. laeta showed low immunological reactivity with the North American Loxosceles antivenoms. Experimental anti-North American Loxosceles antivenoms protected mice of the systemic toxicity and were able to prevent necrosis in rabbit skin after the injection of the venom. Both antivenoms displayed cross neutralization. The results showed that both Loxosceles venoms have very close toxic, biochemical, and immunological characteristics, and that either monospecific antivenoms or an antivenom raised with L. boneti and L. reclusa venoms as immunogens could be useful for treating bites by North American Loxosceles spiders.

North and South American Loxosceles spiders: development of a polyvalent antivenom with recombinant sphingomyelinases D as antigens.

We report the cloning of sphingomyelinase D (SMD) cDNA from Loxosceles reclusa, Loxosceles boneti and Loxosceles laeta into bacterial expression systems, as well as optimization of expression conditions so as to obtain soluble and active recombinant enzymes. The recombinant mature SMDs, tagged with a histidine tail at the N- or C-termini, were compared in terms of toxicity and enzymatic activity, and were used as immunogens for the production of monovalent antisera in rabbits and F(ab')(2) preparations in animals used for commercial antivenom production (horses). We performed studies on in vitro inhibition of enzymatic activity of natural venom preparations by antibodies generated against the tagged proteins. We also present and discuss the results of studies on the specific and para-specific in vivo protective potential of the rabbit and equine antibody preparations against the recombinant proteins themselves and natural venom preparations. Our conclusions support the feasibility of using recombinant SMDs for production and evaluation of polyvalent anti-Loxosceles antivenoms, and we offer data on the potential of paraspecific neutralization in the context of the antigenic groupings and the molecular phylogeny of those active SMDs for which amino acid sequence information is available.

Genetic and enzymatic characterization of sphingomyelinase D isoforms from the North American fiddleback spiders Loxosceles boneti and Loxosceles reclusa.

In this study we report the isolation and characterization of several sphingomyelinase D isoforms from the venoms of the North American spiders Loxosceles boneti and Loxosceles reclusa, from Mexico and the United States, respectively. We have measured their enzymatic activity, their capacity to induce necrotic lesions in rabbits, cloned the cDNAs coding for the mature forms of two of the isoforms from L. boneti and two of L. reclusa based on N-terminal sequence information of the purified proteins, and performed a comprehensive comparison of the sequence data generated by us with that reported for other sphingomyelinase genes to date.