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We endeavored to identify objective salivary biomarkers for pain, a subjective sensation with a biological basis, using molecules already described related to pain. The study aimed to analyze inter-individual differences and intersession variability in salivary potential ocular pain biomarkers on healthy subjects, in samples obtained under the influence of controlled environmental conditions. Thirty-four healthy subjects, 20 male, 14 female, median age 35.44 years (range 30-40) were exposed for 30 minutes under standard environmental conditions (T: 22°C, 50% relative humidity) in the Controlled Environmental Research Laboratory (CE-Lab, Vision R&D, Valladolid Spain) in two separate visits (V1, V2) at least 24 hours apart. Saliva was collected after the exposure in each of the visits, and cortisol, α-amylase (sAA), secretory IgA (sIgA), testosterone, and soluble fraction of TNFα receptor II (sTNFαRII) were analyzed by ELISA. Repeatability of inter-subject inter-session measurements was assayed by intraclass correlation coefficient (ICC). There were no significant inter-session differences in testosterone (p = 0.2497), sTNFαRII (p = 0.6451) and sIgA (p = 0.9689) salivary levels. The reproducibility for salivary cortisol, sAA, testosterone, sTNFαRII and sIgA were 0.98 ng/ml, 20.58 U/ml, 21.07 μg/ml, 24.68 pg/ml and 0.19 pg/ml, respectively. Salivary cortisol, sAA, testosterone, sTNFαRII and sIgA yielded the following ICCs: 0.506, 0.569, 0.824, 0.870 and 0.4295, respectively; all these ICCs (except that for cortisol and sIgA) were found to be improved compared to those found previously by our group in a previous study in salivary samples obtained from healthy subjects under non-controlled environmental conditions; Cortisol´s ICC didn´t improve and was in both cases at the limit of acceptability. Environmental factors such as temperature and relative humidity affect the reproducibility of measurement of some salivary molecules which have been proposed as potential pain biomarkers. The exposure of subjects to standard controlled environmental conditions before salivary sample obtention would improve the reproducibility of these molecule measures' as potential biomarkers of chronic ocular pain.

An automated tool for corneal nerve fiber tortuosity quantification from in vivo confocal microscopy (IVCM) is described and evaluated. The method is a multi-stage process based on the splitting of the corneal nerve fibers into individual segments, whose endpoints are an extreme or intersection of white pixels on a binarized image. Individual segment tortuosity is quantified in terms of the arc-chord ratio. Forty-three IVCM images from 43 laser-assisted in situ keratomileusis (LASIK) surgery patients were used for evaluation. Images from symptomatic dry eye disease (DED) post-LASIK patients, with (n=16) and without (n=7) ocular pain, and non-DED post-LASIK controls (n=20) were assessed. The automated tortuosity measure was compared to a manual grading one, obtaining a moderate correlation (Spearman’s rank correlation coefficient = 0.49, p=0.0008). The new tortuosity index was significantly higher in post-LASIK patients with ocular pain than in control patients (p=0.001), while no significant differences were detected with manual measurement (p>0.28). The tortuosity quantification was positively correlated with the ocular surface disease index (OSDI) and a numeric rating scale (NRS) assessing pain (p=0.0012 and p=0.0051, respectively). The results show good performance of the proposed automated methodology for the evaluation of corneal nerve tortuosity.

Caseins are abundant proteins in milk and found in four types (αS1, αS2, β, and κ). There is substantial variation in the allelic and genotypic frequencies of the κ-casein gene in different cattle breeds, although the tropical milking Criollo (TMC) has not yet been investigated. The aim was to determine the allelic and genotypic frequencies in the κ-casein gene for two alleles (A and B) in TMC and further investigate its association to milk production and composition. A total of 180 TMC females were genotyped from blood samples. κ-Casein genotyping was performed using restriction fragment length polymorphism (RFLP) after polymerase chain reaction (PCR)–based amplification of genomic DNA. Allele frequencies were 0.39 for A-allele and 0.61 for B-allele (P < 0.05). Genotype frequencies were 0.09 (AA), 0.60 (AB), and 0.31 (BB) (P < 0.05). The κ-casein genotype in TMC cows did not affect milk yield or composition. In sum, the TMC has high frequencies of the B-allele and AB/BB genotypes, although there are no association of such genotypes and milk traits.

Myostatin is a transforming growth factor-β family member who acts as a negative regulator of skeletal muscle growth. The interference of its biological activity could increase skeletal muscle growth with clinical and animal production applications. A strategy to block the myostatin action is by the induction of an immune response against it. In this work, we evaluated as an immunogen a recombinant myostatin fused to the tetanic toxin T- helper epitopes P2 and P30. Genetic constructs of the chimeric myostatin were cloned in an expression vector and used as a DNA vaccine. Besides, a chimeric genetic construct, P2-miostatin-P30 was expressed in Escherichia coli, obtaining a recombinant chimeric antigen. To find out the functionality of these genetic constructs as a vaccine in inducing muscle growth responses, experimental groups of BALB/c mice were DNA immunized with the myostatin fused to P2, P30 or both. Furthermore, to improve the immune response, a heterologous prime-boost immunization scheme was evaluated where the DNA inoculation was followed by immunization with the recombinant antigen P2-myostatin-P30. The different body segments weight was recorded in control and vaccinated mice groups, finding increased muscle masses in the vaccinated groups. These experiments showed the effectiveness of the P2 and P30 T-helper epitopes in inducing an immune response to the fused myostatin, leading to muscle growth. The heterologous prime-boost immunization protocol is a promising vaccination strategy reducing the time and amount of antigen used to induce a immune response to myostatin.

This study investigated protection against Eimeria tenella following the vaccination of chicks with 5.3 × 106 E. tenella whole-sporozoites emulsified in the nanoparticle adjuvant IMS 1313 N VG Montanide™ (EtSz-IMS1313). One-day-old specific pathogen-free (SPF) chicks were subcutaneously injected in the neck with EtSz-IMS1313 on the 1st and 10th days of age. Acquired immunity was assayed through a challenge with 3 × 104 homologous sporulated oocysts at 21 days of age. The anticoccidial index (ACI) calculated for every group showed the effectiveness of EtSz-IMS1313 as a vaccine with an ACI of 186; the mock-injected control showed an ACI of 18 and the unimmunized, challenged control showed an ACI of −28. In a comparison assay, antibodies from rabbits and SPF birds immunized with EtSz-IMS1313 recognized almost the same polypeptides in the blotting of E. tenella sporozoites and merozoites. However, rabbit antisera showed the clearest recognition pattern. Polypeptides of 120, 105, 94, 70, 38, and 19 kDa from both E. tenella life cycle stages were the most strongly recognized by both animal species. The E. tenella zoite-specific IgG antibodies from the rabbits demonstrated the feasibility for successful B cell antigen identification.

Dry eye disease (DED) is a prevalent condition characterized by ocular surface inflammation and pain. This study evaluated the long-term progression of DED by analyzing clinical and molecular status, considering the impact of chronic ocular pain. Patients with DED were evaluated at two visits (V1 and V2) separated by at least two years. Evaluations included validated symptom questionnaires alongside slit-lamp examination, corneal sensitivity testing, and sub-basal nerve plexus analysis. Basal tear samples were collected for multiplex quantification of 20 cytokines and substance P (SP), and conjunctival cells were obtained to analyze 25 genes and 12 microRNAs (miRNA). Based on the presence or absence of chronic ocular pain, patients were then divided into two groups. Patients improved in DED-related symptoms, with no changes observed in ocular surface signs. Corneal dendritic cell density decreased, along with epidermal growth factor (EGF), fractalkine, and monocyte chemoattractant protein (MCP-1) tear levels, whereas interleukin (IL)-10 and SP tear levels increased. Neurotrophic tyrosine kinase, receptor, type (NTRK)1 gene expression was significantly downregulated, especially in patients without chronic ocular pain. miR-665 expression decreased significantly in DED patients. Monitoring corneal dendritic cells, tear cytokines, and gene/miRNA expression offers promising tools for tracking DED progression. Distinguishing the presence of chronic ocular pain as a separate symptom is crucial to optimizing therapeutic strategies and DED progression.

The avian infectious bronchitis virus (IBV) is a coronavirus that mutates frequently, leading to a contagious and acute disease that results in economic losses to the global poultry industry. Due to its genetic and serological diversity, IBV poses a challenge in preventing and controlling the pathogen. The full-length S1 sequence analysis identifies seven main genotypes (GI–GVII) comprising 35 viral lineages. In addition to the previously described lineage, a new GI lineage (GI-30) and two lineages from novel genotypes (GVIII-1 and GIX-1) have been described in Mexico. To prevent the spread of IBV outbreaks in a specific geographic location and select the suitable vaccine, it is helpful to genetically identify the circulating IBV types. Moreover, sequencing genomes can provide essential insights into virus evolution and significantly enhance our understanding of IBV variability. However, only genomes of previously described lineages (GI-1, GI-9, GI-13, and GI-17) have been reported for Mexican strains. Here, we sequenced new genomes from Mexican lineages, including the indigenous GI-30, GVIII-1, and GIX-1 lineages. Comparative genomics reveals that Mexico has relatively homogenous lineages (i.e., GI-13), some with greater variability (i.e., GI-1 and GI-9), and others extremely divergent (GI-30, GVIII-1, and GIX-1). The circulating lineages and intra-lineage variability support the unique diversity and dynamic of Mexican IBV.

Background/Objectives: The purpose of this study was to clinically characterize the lacrimal functional unit (LFU) of patients with chronic ocular pain associated with dry eye disease (DED). Methods: Ninety-three participants were included in this cross-sectional study: 28 patients with chronic ocular pain associated with DED (pain-DED), 35 patients with DED but no pain (no pain-DED), and 30 subjects without DED or ocular pain (controls). The following examinations were performed: symptom questionnaires, visual function assessment, tear meniscus, ocular surface evaluation, meibography, corneal sensitivity, Schirmer test, and in vivo corneal confocal microscopy. Results: Both DED groups presented increased DED-related symptoms (p < 0.001), corneal staining (p < 0.001), Meibomian gland loss (p < 0.010), and dendritic cell density (p < 0.001) compared with controls. Comparing both DED groups, the pain-DED group showed higher DED-related symptoms (p < 0.002) and increased microneuroma density (p < 0.001). Additionally, significant positive correlations were observed between symptom questionnaires and corneal staining (vs. OSDI: r = 0.514, p < 0.001; vs. m-SIDEQ: r = 0.504, p < 0.001; vs. NRS: r = 0.361, p < 0.001; vs. WBFPRS: r = 0.317, p = 0.002), dendritic cell density (vs. OSDI: r = 0.429, p < 0.001; vs. m-SIDEQ: r = 0.440, p < 0.001), and microneuroma density (vs. NRS: r = 0.405, p < 0.001; vs. WBFPRS: r = 0.416, p < 0.001). Conclusions: Differences in the LFU, especially in the morphology of sub-basal corneal nerves, are related to the presence of DED and chronic ocular pain and, along with ocular clinical questionnaires, can help phenotype these patients.

Coccidiosis, caused by parasites of numerous species, has long been recognized as an economically significant disease in the chicken industry worldwide. The rise of anti-coccidian resistance has driven a search for other parasite management techniques. Recombinant antigen vaccination presents a highly feasible alternative. Properly identifying antigens that might trigger a potent immune response is one of the major obstacles to creating a viable genetically modified vaccine. This study evaluated a reverse immunology approach for the identification of B-cell epitopes. Antisera from rabbits and hens inoculated with whole-sporozoites of were used to identify Western blot antigens. The rabbit IgG fraction from the anti-sporozoite serum exhibited the highest reactogenicity; consequently, it was purified and utilized to screen two random Phage-display peptide libraries (12 mer and c7c mer). After three panning rounds, 20 clones from each library were randomly selected, their nucleotide sequences acquired, and their reactivity to anti-sporozoite serum assessed. The selected peptide clones inferred amino acid sequences matched numerous proteins. The extracellular domain of the epidermal growth factor-like (EGF-like) repeats, and the thrombospondin type-I (TSP-1) repeats of micronemal protein 4 (EtMIC4) matched with the c7c mer selected clones CNTGSPYEC (2/20) and CMSTGLSSC (1/20) respectively. The clone CSISSLTHC that matched with a conserved hypothetical protein of was widely selected (3/20). Selected clones from the 12-mer phage display library AGHTTQFNSKTT (7/20), GPNSAFWAGSER (2/20) and HFAYWWNGVRGP (8/20) showed similarities with a cullin homolog, elongation factor-2 and beta-dynein chain a putative protein, respectively. Four immunodominant clones were previously selected and used to immunize rabbits. By ELISA and Western blot, all rabbit anti-clone serums detected native antigens. Thus, selected phagotopes contained recombinant antigen peptides. Using antibodies against sporozoites, this study demonstrated the feasibility of screening Phage-display random peptide libraries for true immunotopes. In addition, this study looked at an approach for finding novel candidates that could be used as an recombinant epitope-based vaccine.

New vaccine design approaches, platforms, and immunization strategies might foster antiviral mucosal effector and memory responses to reduce asymptomatic infection and transmission in vaccinated individuals. Here, we investigated a combined parenteral and mucosal immunization scheme to induce local and serum antibody responses, employing the epitope-based antigens 3BT and NG19m. These antigens target the important emerging and re-emerging viruses PRRSV-2 and SARS-CoV-2, respectively. We assessed two versions of the 3BT protein, which contains conserved epitopes from the GP5 envelope protein of PRRSV-2: soluble and expressed by the recombinant baculovirus BacDual-3BT. On the other hand, NG19m, comprising the receptor-binding motif of the S protein of SARS-CoV-2, was evaluated as a soluble recombinant protein only. Vietnamese mini-pigs were immunized employing different inoculation routes: subcutaneous, intranasal, or a combination of both (s.c.-i.n.). Animals produced antigen-binding and neut1ralizing antibodies in serum and mucosal fluids, with varying patterns of concentration and activity, depending on the antigen and the immunization schedule. Soluble 3BT was a potent immunogen to elicit binding and neutralizing antibodies in serum, nasal mucus, and vaginal swabs. The vectored immunogen BacDual-3BT induced binding antibodies in serum and mucosae, but PRRSV-2 neutralizing activity was found in nasal mucus exclusively when administered intranasally. NG19m promoted serum and mucosal binding antibodies, which showed differing neutralizing activity. Only serum samples from subcutaneously immunized animals inhibited RBD-ACE2 interaction, while mini-pigs inoculated intranasally or via the combined s.c.-i.n. scheme produced subtle neutralizing humoral responses in the upper and lower respiratory mucosae. Our results show that intranasal immunization, alone or combined with subcutaneous delivery of epitope-based antigens, generates local and systemic binding and neutralizing antibodies. Further investigation is needed to evaluate the capability of the induced responses to prevent infection and reduce transmission.