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Potato tubers are reproductive and storage organs, enabling their survival. Unraveling the molecular mechanisms that regulate tuberization is crucial for understanding how potatorespond to environmental stress situations and for potato breeding. Previously, we did a transcriptomic analysis of potato microtuberization without light. This showed that important cellular processes like ribosomal proteins, cell cycle, carbon metabolism, oxidative stress, fatty acids, and phytosterols (PS) biosynthesis were closely connected in a protein-protein interaction (PPI) network. Research on PS function during potato tuberization has been scarce. PS plays a critical role in regulating membrane permeability and fluidity, and they are biosynthetic precursors of brassinosteroids (BRs) in plants, which are critical in regulating gene expression, cell division, differentiation, and reproductive biology. Within a PPI network, we found a module of 15 genes involved in the PS biosynthetic process. Darkness, as expected, activated the mevalonate (MVA) pathway. There was a tight interaction between three coding gene products for HMGR3, MVD2, and FPS1, and the gene products that synthetize PS, including CAS1, SMO1, BETAHSD, CPI1, CYP51, FACKEL, HYDRA1, SMT2, SMO2, STE1, and SSR1. Quantitative real-time polymerase chain reaction (qRT-PCR) confirmed the expression analysis of ten specific genes involved in the biosynthesis of PS. This manuscript discusses the potential role of genes involved in PS biosynthesis during microtuber development.

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Viral infection disrupts the normal regulation of the host gene's expression. In order to normalise the expression of dysregulated host genes upon virus infection, analysis of stable reference housekeeping genes using quantitative real-time-PCR (qRT-PCR) is necessary. In the present study, healthy and African swine fever virus (ASFV) infected porcine tissues were assessed for the expression stability of five widely used housekeeping genes (HPRT1, B2M, 18 S rRNA, PGK1 and H3F3A) as reference genes using standard algorithm. Total RNA from each tissue sample (lymph node, spleen, kidney, heart and liver) from healthy and ASFV-infected pigs was extracted and subsequently cDNA was synthesized, and subjected to qRT-PCR. Stability analysis of reference genes expression was performed using the Comparative delta CT, geNorm, BestKeeper and NormFinder algorithm available at RefFinder for the different groups. Direct Cycle threshold (CT) values of samples were used as an input for the web-based tool RefFinder. HPRT1 in spleen, 18 S rRNA in liver and kidney and H3F3A in heart and lymph nodes were found to be stable in the individual healthy tissue group (group A). The majority of the ASFV-infected organs (liver, kidney, heart, lymph node) exhibited H3F3A as stable reference gene with the exception of the ASFV-infected spleen, where HPRT1 was found to be the stable gene (group B). HPRT1 was found to be stable in all combinations of all CT values of both healthy and ASFV-infected porcine tissues (group C). Of five different reference genes investigated for their stability in qPCR analysis, the present study revealed that the 18 S rRNA, H3F3A and HPRT1 genes were optimal reference genes in healthy and ASFV-infected different porcine tissue samples. The study revealed the stable reference genes found in healthy as well as ASF-infected pigs and these reference genes identified through this study will form the baseline data which will be very useful in future investigations on gene expression in ASFV-infected pigs.

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BACKGROUND: Phospholipase A2 (PLA2) is a large family of enzymes involved in the inflammatory process that catalyzes the hydrolysis of membrane phospholipids, leading to the production of free fatty acids and lysophospholipids, starting the arachidonic acid cascade. Their expression has been related to the behavior of several cancers. Our objective is to search for PLA2 expression in prostate cancer (PCa) tissue that correlates with prognosis and survival. METHODS: Using qRT-PCR, we analyzed the expression levels of PLA2G1B, PLA2G2A, PLA2G2D, PLA2G4A, PLA2G4B, PLA2G4C, PLA2G4D, PLA2G4E, PLA2G4F, PLA2G6, PLA2G7, PLA2G16, PNPLA1, and PNPLA2 in PCa tissue from 108 patients submitted to radical prostatectomy, followed by a mean time of 163 months. RESULTS: All PLA2 was overexpressed in PCa compared to normal tissue. Interestingly, higher expression of some PLA2 was related to favorable prognostic factors: lower levels of PSA (PLA2G2A, PLA2G4D), lower rates of lymph node metastasis (PLA2G16 and PLA2G1B), and organ-confined disease (PLA2G4A). Most importantly, PLAG4B was independently related to longer disease-free survival. CONCLUSION: This is the first study exploring comprehensively the expression levels of PLA2 in PCa, showing that the higher expression of some PLA2 should be used as biomarkers of good prognosis and longer disease-free survival.

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Alzheimer's Disease (AD) is an age-related neurodegenerative disorder characterized by progressive memory loss and cognitive impairment, affecting 35 million individuals worldwide. Intracerebroventricular (ICV) injection of low to moderate doses of streptozotocin (STZ) in adult male Wistar rats can reproduce classical physiopathological hallmarks of AD. This biological model is known as ICV-STZ. Most studies are focused on the description of behavioral and morphological aspects of the ICV-STZ model. However, knowledge regarding the molecular aspects of the ICV-STZ model is still incipient. Therefore, this work is a first attempt to provide a wide proteome description of the ICV-STZ model based on mass spectrometry (MS). To achieve that, samples from the pre-frontal cortex (PFC) and hippocampus (HPC) of the ICV-STZ model and control (wild-type) were used. Differential protein abundance, pathway, and network analysis were performed based on the protein identification and quantification of the samples. Our analysis revealed dysregulated biological pathways implicated in the early stages of late-onset Alzheimer's disease (LOAD), based on differentially abundant proteins (DAPs). Some of these DAPs had their mRNA expression further investigated through qRT-PCR. Our results shed light on the AD onset and demonstrate the ICV-STZ as a valid model for LOAD proteome description.

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The genus Paracoccidioides includes Paracoccidioides lutzii and the Paracoccidioides brasiliensis complex, which comprises four phylogenetic species. A key feature distinguishing planktonic growth from biofilm is the presence of a 3D extracellular matrix (ECM). Therefore, in this study, we analyzed biofilm formation in different species of Paracoccidioides yeast phase, characterized the structural elements of the matrix of P. brasiliensis (Pb18), P. lutzii (Pl01 and 8334) and P. restrepiensis (339 and 192) and evaluated the expression of glucan genes, according to the stage of biofilm evolution for P. brasiliensis. The strains were cultivated in planktonic and biofilm form for 24-144 h. The fungi biomass and metabolic activity were determined by crystal violet and tetrazolium salt reduction (XTT) tests and colony-forming unit (CFU) by plating. The biofilm structure was designed using scanning electron microscopy and confocal laser scanning microscopy techniques. The extracellular matrix of P. brasiliensis and P. lutzii biofilms was extracted by sonication, and polysaccharides, proteins, and extracellular DNA (eDNA) were quantified. The RNA was extracted with the Trizol® reagent and quantified; then, the cDNA was synthesized to analyze the enolase expression, 14-3-3, FKS1, AGS1, GEL3, and KRE6 genes by real-time PCR. All strains of Paracoccidioides studied form a biofilm with more significant metabolic activity and biomass values in 144 h. The extracellular matrix of P. brasiliensis and P. lutzii had a higher content of polysaccharides in their composition, followed by proteins and eDNA in smaller quantities. The P. brasiliensis biofilm kinetics of formation showed greater expression of genes related to glucan's synthesis and its delivery to the external environment in addition adhesins during the biofilm's adhesion, initiation, and maturation. The GEL3 and enolase genes increased in expression within 24 h and during the biofilm maturation period, there was an increase in 14-3-3, AGS1, and FKS1. Furthermore, at 144 h, there was a decrease in KRE6 expression and an increase in GEL3. This study highlights the potential for biofilm formation for three species of Paracoccidioides and the main components of the extracellular matrix that can contribute to a better understanding of biofilm organization.

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OBJECTIVE: Sperm Associated Antigen 11A (SPAG11A) protein is a family of the epididymis-specific secretory proteins implicated in sperm maturation and function. Varicocele might cause pathophysiological difficulties in the testis and epididymis, with a harmful effect on the environment for spermatogenesis and sperm maturation. The aim of this study was to evaluate the expression level of the SPAG11A gene and sperm parameters in infertile men with grade 1 and 2 varicocele before and after treatment. METHODS: Semen specimens were collected from 20 infertile men with varicocele pre-and post-treatment and 10 healthy volunteers. Semen analysis was conducted according to world health organization guidelines. Real time PCR (qRT-PCR) reaction was applied for determination of SPAG11A mRNA expression. RESULTS: The results showed that there was a significant difference between the concentration and normal morphology between pre- and post-treatment groups and the controls. There were significant differences between pre-treatment and control groups in terms of progressive and non-progressive mobility. SPAG11A mRNA levels were significantly lower in the pre-treatment group than in healthy control subjects (p=0.007). There was no statistically significant difference in the expression of SPAG11A as well as semen parameters in the post-treatment group compared to the pre-treatment group. CONCLUSIONS: SPAG11A gene expression and semen parameters may be affected by varicocele. Whether varicocele treatment is an effective approach to reduce the adverse effect of this disease on SPAG11A expression and semen parameters needs further investigation.

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Oxidative stress has been associated with different diseases, and different medicinal plants have been used to treat or prevent this condition. The leaf ethanolic extract (EE) and aqueous extract (AE) from Coccoloba alnifolia have previously been characterized to have antioxidant potential in vitro and in vivo. In this study, we worked with EE and AE and two partition phases, AF (ethyl acetate) and BF (butanol), from AE extract. These extracts and partition phases did not display cytotoxicity. The EE and AE reduced NO production and ROS in all three concentrations tested. Furthermore, it was observed that EE and AE at 500 µg/mL concentration were able to reduce phagocytic activity by 30 and 50%, respectively. A scratch assay using a fibroblast cell line (NHI/3T3) showed that extracts and fractions induced cell migration with 60% wound recovery within 24 h, especially for BF. It was also observed that AF and BF had antioxidant potential in all the assays evaluated. In addition, copper chelation was observed. This activity was previously not detected in AE. The HPLC-DAD analysis showed the presence of phenolic compounds such as p-cumaric acid and vitexin for extracts, while the GNPS annotated the presence of isoorientin, vitexin, kanakugiol, and tryptamine in the BF partition phase. The data presented here demonstrated that the EE, AE, AF, and BF of C. alnifolia have potential immunomodulatory effects, antioxidant effects, as well as in vitro wound healing characteristics, which are important for dynamic inflammation process control.

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The microRNA (miRNA) expression profile by qRT-PCR depends directly on the most appropriate normalization strategy adopted; however, currently there is no universally adequate reference gene. Therefore, this study aimed to determine, considering RNA-Seq results, the most adequate endogenous normalizer for use in the relative quantification of urine miRNAs from head and neck cancer patients, treated with cisplatin chemoradiotherapy. The massive sequencing was performed to identify the miRNAs differentially expressed between the group with cisplatin nephrotoxicity (n = 6) and the one without (n = 6). The candidate endogen normalizer was chosen according to four criteria: (1) the miRNA must be expressed in most samples; (2) the miRNA must have a fold change value between 0.99 and 1.01; (3) the miRNA must have a p-value ≥ 0.98; and (4) the miRNA must not be commented on by the final GeneGlobe (Qiagen, Hilden, Germany) analysis. Four miRNAs met all the criteria (hsa-miR-363-5p, hsa-miR-875-5p, hsa-miR-4302, and hsa-miR-6749-5p) and were selected for validation by qRT-PCR in a cohort of 49 patients (including the 12 sequencing participants). Only hsa-miR-875-5p was shown to be an adequate normalizer for the experimental condition under investigation, as it exhibited invariant expression between the two groups.

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The COVID-19 pandemic has caused an unprecedented health and economic crisis, highlighting the importance of developing new molecular tools to monitor and detect SARS-CoV-2. Hence, this study proposed to employ the carrageenan extracted from Gigartina skottsbergii algae as a probe for SARS-CoV-2 virus binding capacity and potential use in molecular methods. G. skottsbergii specimens were collected in the Chilean subantarctic ecoregion, and the carrageenan was extracted -using a modified version of Webber's method-, characterized, and quantified. After 24 h of incubation with an inactivated viral suspension, the carrageenan's capacity to bind SARS-CoV-2 was tested. The probe-bound viral RNA was quantified using the reverse transcription and reverse transcription loop-mediated isothermal amplification (RT-LAMP) methods. Our findings showed that carrageenan extraction from seaweed has a similar spectrum to commercial carrageenan, achieving an excellent proportion of binding to SARS-CoV-2, with a yield of 8.3%. Viral RNA was also detected in the RT-LAMP assay. This study shows, for the first time, the binding capacity of carrageenan extracted from G. skottsbergii, which proved to be a low-cost and highly efficient method of binding to SARS-CoV-2 viral particles.

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FOXO3a dysregulation is frequently implicated in tumorigenesis, and its inhibition can occur by several molecular mechanisms. Among these, post-transcriptional suppression by miRNAs has been associated with various cancers initiation. Here, we assessed the expression profiles of the most relevant miRNAs for breast tumorigenesis, using Luminal A (LA) and Triple-Negative (TN) breast cancer from Brazilian patients, by the quantitative real time-PCR method. Their potential prognostic role for the patients was also evaluated. We identified the miRNAs miR-96-5p and miR-182-5p, de-scribed as negative regulators of FOXO3A, with differential expression both in LA and TN tumors when compared to normal tissue. The miR-96-5p and miR-182-5p miRNAs were upregulated in LA (7.82 times, p < 0.005; 6.12 times, p < 0.005, respectively) and TN breast cancer samples (9.42 times, p < 0.0001; 8.51 times, p < 0.0001) compared to normal tissues. The samples with higher miR-96-5p and miR-182-5p expression (FR ≥ 4) were submitted for FOXO3a immunostaining. Reduced protein detection was observed in all of the tumors compared to normal tissues. The most prominent miRNA expression and FOXO3a protein suppression were observed in TN samples (p < 0.001), indicating the relevant role of these molecules in this tumor biology and clinical behavior. Our results corroborate the literature regarding to the relevance of FOXO3a in the breast cancer, and they open new perspectives for alternative target therapy options for Brazilian patients expressing both FOXO3a and its regulatory miRNAs.

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Abstract FOXO3a dysregulation is frequently implicated in tumorigenesis, and its inhibition can occur by several molecular mechanisms. Among these, post-transcriptional suppression by miRNAs has been associated with various cancers initiation. Here, we assessed the expression profiles of the most relevant miRNAs for breast tumorigenesis, using Luminal A (LA) and Triple-Negative (TN) breast cancer from Brazilian patients, by the quantitative real time-PCR method. Their potential prognostic role for the patients was also evaluated. We identified the miRNAs miR-96-5p and miR-182-5p, de-scribed as negative regulators of FOXO3A, with differential expression both in LA and TN tumors when compared to normal tissue. The miR-96-5p and miR-182-5p miRNAs were upregulated in LA (7.82 times, p < 0.005; 6.12 times, p < 0.005, respectively) and TN breast cancer samples (9.42 times, p < 0.0001; 8.51 times, p < 0.0001) compared to normal tissues. The samples with higher miR-96-5p and miR-182-5p expression (FR ≥ 4) were submitted for FOXO3a immunostaining. Reduced protein detection was observed in all of the tumors compared to normal tissues. The most prominent miRNA expression and FOXO3a protein suppression were observed in TN samples (p < 0.001), indicating the relevant role of these molecules in this tumor biology and clinical behavior. Our results corroborate the literature regarding to the relevance of FOXO3a in the breast cancer, and they open new perspectives for alternative target therapy options for Brazilian patients expressing both FOXO3a and its regulatory miRNAs.

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BACKGROUND: Soil salinity is a problem in more than 100 countries across all continents. It is one of the abiotic stress that threatens agriculture the most, negatively affecting crops and reducing productivity. Transcriptomics is a technology applied to characterize the transcriptome in a cell, tissue, or organism at a given time via RNA-Seq, also known as full-transcriptome shotgun sequencing. This technology allows the identification of most genes expressed at a particular stage, and different isoforms are separated and transcript expression levels measured. Once determined by this technology, the expression profile of a gene must undergo validation by another, such as quantitative real-time PCR (qRT-PCR). This study aimed to select, annotate, and validate stress-inducible genes-and their promoters-differentially expressed in the leaves of oil palm (Elaeis guineensis) plants under saline stress. RESULTS: The transcriptome analysis led to the selection of 14 genes that underwent structural and functional annotation, besides having their expression validated using the qRT-PCR technique. When compared, the RNA-Seq and qRT-PCR profiles of those genes resulted in some inconsistencies. The structural and functional annotation analysis of proteins coded by the selected genes showed that some of them are orthologs of genes reported as conferring resistance to salinity in other species. There were those coding for proteins related to the transport of salt into and out of cells, transcriptional regulatory activity, and opening and closing of stomata. The annotation analysis performed on the promoter sequence revealed 22 distinct types of cis-acting elements, and 14 of them are known to be involved in abiotic stress. CONCLUSION: This study has helped validate the process of an accurate selection of genes responsive to salt stress with a specific and predefined expression profile and their promoter sequence. Its results also can be used in molecular-genetics-assisted breeding programs. In addition, using the identified genes is a window of opportunity for strategies trying to relieve the damages arising from the salt stress in many glycophyte crops with economic importance.

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Wastewater-based epidemiology (WBE) is a tool involving the analysis of wastewater for chemicals and pathogens at the community level. WBE has been shown to be an effective surveillance system for SARS-CoV-2, providing an early-warning-detection system for disease prevalence in the community via the detection of genetic materials in the wastewater. In numerous nation-states, studies have indicated the presence of SARS-CoV-2 in wastewater. Herein, we report the primary time-course monitoring of SARS-CoV-2 RNA in wastewater samples in São José do Rio Preto-SP/Brazil in order to explain the dynamics of the presence of SARS-CoV-2 RNA during one year of the SARS-CoV-2 pandemic and analyze possible relationships with other environmental parameters. We performed RNA quantification of SARS-CoV-2 by RT-qPCR using N1 and N2 targets. The proportion of positive samples for every target resulted in 100% and 96.6% for N1 and N2, respectively. A mean lag of -5 days is observed between the wastewater signal and the new SARS-CoV-2-positive cases reported. A correlation was found between the air and wastewater temperatures and therefore between the SARS-CoV-2 viral titers for N1 and N2 targets. We also observed a correlation between SARS-CoV-2 viral titers and media wastewater flow for the N1 target. In addition, we observed higher viral genome copies within the wastewater samples collected on non-rainy days for the N1 target. Thus, we propose that, based on our results, monitoring raw wastewater may be a broadly applicable strategy that might contribute to resolving the pressing problem of insufficient diagnostic testing; it may represent an inexpensive and early-warning method for future COVID-19 outbreaks, mainly in lower- and middle-income countries.

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Severe acute respiratory syndrome-related coronavirus (SARS-CoV-2) transmission occurs even among fully vaccinated individuals; thus, prompt identification of infected patients is central to control viral circulation. Antigen rapid diagnostic tests (Ag-RDTs) are highly specific, but sensitivity is variable. Discordant RT-qPCR vs. Ag-RDT results are reported, raising the question of whether negative Ag-RDT in positive RT-qPCR samples could imply the absence of infectious viruses. To study the relationship between negative Ag-RDT results with virological, molecular, and serological parameters, we selected a cross-sectional and a follow-up dataset and analyzed virus culture, subgenomic RNA quantification, and sequencing to determine infectious viruses and mutations. We demonstrated that RT-qPCR positive while SARS-CoV-2 Ag-RDT negative discordant results correlate with the absence of infectious virus in nasopharyngeal samples. A decrease in sgRNA detection together with an expected increase in detectable anti-S and anti-N IgGs was also verified in these samples. The data clearly demonstrate that a negative Ag-RDT sample is less likely to harbor infectious SARS-CoV-2 and, consequently, has a lower transmissible potential.

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BACKGROUND: SARS-CoV-2 emerged in 2019 and resulted in a pandemic causing millions of infections worldwide. Gold-standard for SARS-CoV-2 detection uses quantitative RT-qPCR on respiratory secretions to detect viral RNA (vRNA). Acquiring these samples is invasive, can be painful for those with xerostomia and other health conditions, and sample quality can vary greatly. Frequently only symptomatic individuals are tested even though asymptomatic individuals can have comparable viral loads and efficiently transmit virus. METHODS: We utilized a non-invasive approach to detect SARS-CoV-2 in individuals, using polyvinyl alcohol (PVA) strips embedded in KN95 masks. PVA strips were tested for SARS-CoV-2 vRNA via qRT-PCR and infectious virus. RESULTS: We show efficient recovery of vRNA and infectious virus from virus-spiked PVA with detection limits comparable to nasal swab samples. In infected individuals, we detect both human and SARS-CoV-2 RNA on PVA strips, however, these levels are not correlated with length of time mask was worn, number of times coughed or sneezed, or level of virus in nasal swab samples. We successfully cultured and deep-sequenced PVA-associated virus. CONCLUSIONS: These results demonstrate the feasibility of using PVA-embedded masks as a non-invasive platform for detecting SARS-CoV-2 in exhaled air in COVID-positive individuals regardless of symptom status.

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BACKGROUND: Mesial temporal lobe epilepsy (MTLE) is a symptomatic epilepsy syndrome clinically characterized by high prevalence, pharmacoresistance, good surgical prognosis and hippocampal sclerosis (HS); however, no singular criteria can be considered sufficient for the MTLE-HS diagnosis. MicroRNAs (miRNAs) are small non-coding molecules that act as important gene-expression regulators at post-transcriptional level. Evidences on the involvement of miRNAs in epilepsy pathogenesis as well as their potential to be employed as biomarkers claim for investigations on miRNAs' applicability as epilepsy diagnosis and prognosis biomarkers. Consequently, the present study aimed to evaluate the applicability of three specific miRNAs as biomarkers of diagnosis and surgical outcomes in adult patients with MTLE-HS. METHOD: Hippocampus, amygdala and blood samples from 20 patients with MTLE-HS were analyzed, 10 with favorable surgical prognosis (Engel I) and 10 with unfavorable surgical prognosis (Engel III-IV). For the control groups, hippocampus and amygdala from necropsy and blood samples from healthy individuals were adopted. The miRNAs expression analysis was performed using Real-Time Quantitative Polymerase Chain Reaction for miRNAs highlighted from microarray as being involved in GABAergic neurotransmission. RESULTS: The miRNAs miR-629-3p, miR-1202 and miR-1225-5p were found to be hyper-expressed in MTLE-HS patients' blood. CONCLUSIONS: Our data suggest the existence of three circulating miRNAs (miR-629-3p, miR-1202 and miR-1225-5p) that could possibly act as additional tools in the set of factors that contribute to MTLE-HS diagnose.

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Governments have implemented measures to minimize SARS-CoV-2 spread. However, these measures were relaxed, and the appearance of new variants has prompted periods of high contagion known as waves. In Mexico, four waves distributed between July and August 2020, January and February 2021, August and September 2021, and January and February 2022 have appeared. Current health policies discourage mass sampling, preferring to focus on the corrective treatment of severe cases. Outpatients are only advised to undergo brief voluntary confinement and symptomatic treatment, with no follow-up. Therefore, the present study aimed to analyze sex, age, and viral load in outpatients during the four waves in a medium-sized city in Mexico. For each wave, the date of peak contagion was identified, and data were collected within ±15 days. In this regard, data from 916 patients (434 men and 482 women) were analyzed. The age range of positive patients (37-45 years) presented a higher frequency during the first and third waves, while 28-36 years was the most frequent age range during the second and fourth waves, while the viral load values were significantly higher, for both sexes, during the fourth wave. Obtained data of COVID-19 prevalence in population segments can be used for decision-making in the design of effective public health policies.

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The COVID-19 pandemic highlighted health systems vulnerabilities, as well as thoughtlessness by governments and society. Due to the nature of this contingency, the use of geographic information systems (GIS) is essential to understand the SARS-CoV-2 distribution dynamics within a defined geographic area. This work was performed in Tepic, a medium-sized city in Mexico. The residence of 834 COVID-19 infected individuals was georeferenced and categorized by viral load (Ct). The analysis took place during the maximum contagion of the first four waves of COVID-19 in Mexico, analyzing 158, 254, 143, and 279 cases in each wave respectively. Then heatmaps were built and categorized into five areas ranging from very low to very high risk of contagion, finding that the second wave exhibited a greater number of cases with a high viral load. Additionally, a spatial analysis was performed to measure urban areas with a higher risk of contagion, during this wave this area had 19,203.08 km2 (36.11% of the city). Therefore, a kernel density spatial model integrated by meaningful variables such as the number of infected subjects, viral load, and place of residence in cities, to establish geographic zones with different degrees of infection risk, could be useful for decision-making in future epidemic events.

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We investigated whether the recombinant leptin (1, 10, 100 ng/mL) influences the meiotic maturation of goat oocytes, whether the MAPK and JAK2/STAT3 pathways mediate the effects of leptin during in-vitro maturation, and whether leptin differently affects the abundance of mRNAs relevant to leptin signal transduction and apoptosis in oocytes and cumulus cells. The addition of leptin to the maturation medium positively affected the number of oocytes that completed nuclear maturation. Nuclear oocyte maturation stimulated by leptin was significantly impaired when we added the specific inhibitors of MAPK (U0126) and JAK2/STAT3 (AG490) to the maturation medium. The addition of leptin (10 ng/mL) during maturation did not affect the expression of AMPKα1, PPARα, Caspase 3, and BCL2 genes in oocytes or cumulus cells. The PPARγ and BAX mRNA abundances were significantly reduced in cumulus cells in the leptin group compared to the control group. Our results demonstrate that supplementation of the in-vitro maturation medium with leptin significantly improves nuclear maturation and reveal the important role of the MAPK and JAK2/STAT3 signaling pathways in establishing the leptin-mediated nuclear maturation of goat oocytes. Moreover, leptin treatment affects PPARγ and BAX gene expression in cumulus cells.

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Until mass vaccination befalls, control of the new betacoronavirus-associated severe acute respiratory syndrome pandemic (SARS-CoV-2) is based on decreasing virus circulation by social distancing and blocking transmission foci after diagnosis. Globally adopted SARS-CoV-2 diagnostic criteria embrace viral RNA detection by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) on nasopharynx secretions, which requires healthcare facilities and specialized personnel for sample collection. To develop an alternative protocol, hydrophilic cotton as the material and saliva as the source for biological sample collection in qRT-PCR/RT-endpoint-PCR SARS-CoV-2 diagnostic methods prepared with local consumables were evaluated using 99 archived nasopharynx samples previously diagnosed as positive for SARS-CoV-2 and 111 prospective saliva samples pared with nasopharynx samples from patients attending the local reference ABC Medical School diagnostic laboratory. The kappa agreement coefficient between the SARS-CoV-2 qRT-PCR and RT-endpoint-PCR was k = 0.97 (95 % CI 0.92-1.00) and k = 0.90 (95 % CI 0.81-0.99), respectively, on SARS-CoV-2-positive archived samples, with the initial qRT-PCR CT under 25. The agreement coefficient of the SARS-CoV-2 alternative saliva diagnostic protocol, when used to test the paired nasopharynx samples, was k = 0.79 (95 % CI 0.56-1,00). These data support that the SARS-CoV-2 diagnostic assay based on self-collected saliva on cotton represents an alternative protocol for mass diagnosis and epidemiological studies in low-income regions.

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