Curr. Issues Mol. Biol., Volume 44, Issue 1 (January 2022) – 31 articles

A comprehensive understanding of molecular and biochemical changes during sperm capacitation is critical to the success of assisted reproductive technologies. We reported involvement of the testis-specific isoform of Angiotensin Converting Enzyme (tACE) in bovine sperm capacitation. The objective of this study was to characterize the tACE interactome in fresh and heparin-capacitated bovine sperm through immunoprecipitation coupled with mass spectrometry. These interactions were validated by co-localization of tACE with beta-tubulin as an identified interactome constituent. Although interactions between tACE and several proteins remained unchanged in fresh and capacitated sperm, mitochondrial aldehyde dehydrogenase 2 (ALDH2), inactive serine/threonine protein-kinase 3 (VRK3), tubulin-beta-4B chain (TUBB4B), and tubulin-alpha-8 chain (TUBA8) were recruited during capacitation, with implications for cytoskeletal and membrane reorganization, vesicle-mediated transport, GTP-binding, and redox regulation. A proposed tACE interactional network with identified interactome constituents was generated. Despite tACE function being integral to capacitation, the relevance of interactions with its binding partners during capacitation and subsequent events leading to fertilization remains to be elucidated. Full article

Left ventricular hypertrophy (LVH) is characterized by increased myocardium thickness due to increased oxidative stress and downregulation of cystathione γ lyase (CSE) endothelial nitric oxide synthase (eNOS). Upregulation of CSE by hydrogen sulphide (H₂S) and ENOS by L-arginine can arrest the progression of LVH individually. The present study explored the combined treatment of H₂S and NO in the progression of LVH, and demonstrated that the response is due to H₂S, NO or formation of either new molecule in physiological, pathological, and pharmacological in vivo settings of LVH. Exogenous administration H₂S+NO in LVH significantly reduced (all p < 0.05) systolic blood pressure (SBP) and mean arterial pressure (MAP), LV index, heart index and oxidative stress when compared to the LVH group. There was downregulation of CSE mRNA and eNOS in the heart, and exogenous administration of H₂S+NO groups upregulated eNOS MRNA while CSE MRNA remained downregulated in the hearts of the LVH group. Similar trends were observed with concentrations of H₂S and NO in the plasma and tissue. It can be concluded that combined treatment of LVH with H₂S and NO significantly ameliorate the progression of LVH by attenuating systemic hemodynamic and physical indices, and by decreasing oxidative stress. Molecular expression data in the myocardium of LVH depicts that combined treatment upregulated eNOS/NO while it downregulated CSE/H₂S pathways in in vivo settings, and it is always eNOS/NO pathways which play a major role. Full article

Curcumin (CM), demethoxycurcumin (DMC), and bisdemethoxycurcumin (BDMC) are major curcumin derivatives found in the rhizome of turmeric (Curcuma longa L.), and have yielded impressive properties to halt various diseases. In the present study, we carried out a method validation for curcumin derivatives and analyzed the contents simultaneously using HPLC with UV detection. For validation, HPLC was used to estimate linearity, range, specificity, accuracy, precision, limit of detection (LOD), and limit of quantification (LOQ). Results showed a high linearity of the calibration curve, with a coefficient of correlation (R²) for CM, DMC, and BDMC of 0.9999, 0.9999, and 0.9997, respectively. The LOD values for CM, DMC, and BDMC were 1.16, 1.03, and 2.53 ng/μL and LOQ values were 3.50, 3.11, and 7.67 ng/μL, respectively. Moreover, to evaluate the ability of curcumin derivatives to reduce liver lipogenesis and compare curcumin derivatives’ therapeutic effects, a HepG2 cell model was established to analyze their hepatoprotective properties. Regarding the in vivo study, we investigated the effect of DMC, CM, and BDMC on nonalcoholic fatty liver disease (NAFLD) caused by a methionine choline deficient (MCD)-diet in the C57BL/6J mice model. From the in vitro and in vivo results, curcumin derivatives alleviated MCD-diet-induced lipid accumulation as well as high triglyceride (TG) and total cholesterol (TC) levels, and the protein and gene expression of the transcription factors related to liver adipogenesis were suppressed. Furthermore, in MCD-diet mice, curcumin derivatives suppressed the upregulation of toll-like receptors (TLRs) and the production of pro-inflammatory cytokines. In conclusion, our findings indicated that all of the three curcuminoids exerted a hepatoprotective effect in the HepG2 cell model and the MCD-diet-induced NAFLD model, suggesting a potential for curcuminoids derived from turmeric as novel therapeutic agents for NAFLD. Full article

Schistosomiasis is a chronic parasitic disease caused by trematodes of the genus Schistosoma; it is commonly caused by Schistosoma mansoni, which is transmitted by Bioamphalaria snails. Studies show that more than 200 million people are infected and that more than 90% of them live in Africa. Treatment with praziquantel has the best cost–benefit result on the market. However, hypersensitivity, allergy, and drug resistance are frequently presented after administration. From this perspective, ligand-based and structure-based virtual screening (VS) techniques were combined to select potentially active alkaloids against S. mansoni from an internal dataset (SistematX). A set of molecules with known activity against S. mansoni was selected from the ChEMBL database to create two different models with accuracy greater than 84%, enabling ligand-based VS of the alkaloid bank. Subsequently, structure-based VS was performed through molecular docking using four targets of the parasite. Finally, five consensus hits (i.e., five alkaloids with schistosomicidal potential), were selected. In addition, in silico evaluations of the metabolism, toxicity, and drug-like profile of these five selected alkaloids were carried out. Two of them, namely, 11,12-methylethylenedioxypropoxy and methyl-3-oxo-12-methoxy-n(1)-decarbomethoxy-14,15-didehydrochanofruticosinate, had plausible toxicity, metabolomics, and toxicity profiles. These two alkaloids could serve as starting points for the development of new schistosomicidal compounds based on natural products. Full article

Many years and billions spent for research did not yet produce an effective answer to prostate cancer (PCa). Not only each human, but even each cancer nodule in the same tumor, has unique transcriptome topology. The differences go beyond the expression level to the expression control and networking of individual genes. The unrepeatable heterogeneous transcriptomic organization among men makes the quest for universal biomarkers and “fit-for-all” treatments unrealistic. We present a bioinformatics procedure to identify each patient’s unique triplet of PCa Gene Master Regulators (GMRs) and predict consequences of their experimental manipulation. The procedure is based on the Genomic Fabric Paradigm (GFP), which characterizes each individual gene by the independent expression level, expression variability and expression coordination with each other gene. GFP can identify the GMRs whose controlled alteration would selectively kill the cancer cells with little consequence on the normal tissue. The method was applied to microarray data on surgically removed prostates from two men with metastatic PCas (each with three distinct cancer nodules), and DU145 and LNCaP PCa cell lines. The applications verified that each PCa case is unique and predicted the consequences of the GMRs’ manipulation. The predictions are theoretical and need further experimental validation. Full article

CD147 (basigin, BSG) is a membrane-bound glycoprotein involved in energy metabolism that plays a role in cancer cell survival. Its soluble form is a promising marker of some diseases, but it is otherwise poorly studied. CD147 is overexpressed in multiple myeloma (MM) and is known to affect MM progression, while its genetic variants are associated with MM survival. In the present study, we aimed to assess serum soluble CD147 (sCD147) expression as a potential marker in MM. We found that sCD147 level was higher in MM patients compared to healthy individuals. It was also higher in patients with more advanced disease (ISS III) compared to both patients with less advanced MM and healthy individuals, while its level was observed to drop after positive response to treatment. Patients with high sCD147 were characterized by worse progression-free survival. sCD147 level did not directly correlate with bone marrow CD147 mRNA expression. In conclusion, this study suggests that serum sCD147 may be a prognostic marker in MM. Full article

Schizophrenia is a serious and chronic mental illness, the symptoms of which usually appear for the first time in late adolescence or early adulthood. To date, much research has been conducted on the etiology of schizophrenia; however, it is still not fully understood. Oxytocin and vasopressin as neuromodulators that regulate social and emotional behavior are promising candidates for determining the vulnerability to schizophrenia. The aim of this study was to evaluate the expression of OXT, OXTR, AVP, and AVPR1a genes at the mRNA and protein levels in patients with schizophrenia. Due to the neurodegenerative nature of schizophrenia, the study group was divided into two subgroups, namely, G1 with a diagnosis that was made between 10 and 15 years after the onset of the illness, and G2 with a diagnosis made up to two years after the onset of the illness. Moreover, the relationship between the examined genes and the severity of schizophrenia symptoms, assessed using PANSS (Positive and Negative Syndrome Scale) and CDSS scales (Clinical Depression Scale for Schizophrenia) was evaluated. The analysis of the expression of the studied genes at the mRNA and protein levels showed statistically significant differences in the expression of all the investigated genes. OXT and AVPR1a gene expression at both the mRNA and protein levels were significantly lower in the schizophrenia group, and OXTR and AVP gene expression at both the mRNA and protein levels was higher in the schizophrenia subjects than in the controls. Furthermore, a significant correlation of OXT gene expression at the mRNA and protein levels with the severity of depressive symptoms in schizophrenia as assessed by CDSS was found. Full article

The aim of this study is to investigate the circulating variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from Athens and from rural areas in Greece during July and August 2021. We also present a rapid review of literature regarding significant SARS-CoV-2 mutations and their impact on public health. A total of 2500 nasopharyngeal swab specimens were collected from suspected COVID-19 cases (definition by WHO 2021b). Viral nucleic acid extraction was implemented using an automatic extractor and the RNA recovered underwent qRT-PCR in order to characterize the specimens as positive or negative for SARS-CoV-2. The positive specimens were then used to identify specific Spike gene mutations and characterize the emerging SARS-CoV-2 variants. For this step, various kits were utilized. From the 2500 clinical specimens, 220 were tested positive for SARS-CoV-2 indicating a prevalence of 8.8% among suspected cases. The RT-PCR Ct (Cycle threshold) Value ranged from 19 to 25 which corresponds to medium to high copy numbers of the virus in the positive samples. From the 220 positive specimens 148 (67.3%) were from Athens and 72 (32.7%) from Greek rural areas. As far as the Spike mutations investigated: N501Y appeared in all the samples, D614G mutation appeared in 212 (96.4%) samples with a prevalence of 87.2% in Athens and 98.6% in the countryside, E484K had a prevalence of 10.8% and 12.5% in Athens and the rural areas, respectively. K417N was found in 18 (12.2%) samples from Athens and four (5.6%) from the countryside, P681H was present in 51 (34.5%) Athenian specimens and 14 (19.4%) specimens from rural areas, HV69-70 was carried in 32.4% and 19.4% of the samples from Athens and the countryside, respectively. P681R had a prevalence of 87.2% in Athens and 98.6% in rural areas, and none of the specimens carried the L452R mutation. 62 (28.2%) samples carried the N501Y, P681H, D614G and HV69-70 mutations simultaneously and the corresponding variant was characterized as the Alpha (UK) variant (B 1.1.7). Only six (2.7%) samples from the center of Athens had the N501Y, E484K, K417N and D614G mutations simultaneously and the virus responsible was characterized as the Beta (South African) variant (B 1.351). Our study explored the SARS-CoV-2 variants using RT-PCR in a representative cohort of samples collected from Greece in July and August 2021. The prevalent mutations identified were N501Y (100%), D614G (96.4%), P681R (90.1%) and the variants identified were the Delta (90.1%), Alpha (28.2%) and Beta (2.7%). Full article

Mastitis, inflammation of the mammary gland, is the most prevalent disease in dairy cattle that has a potential impact on profitability and animal welfare. Specifically designed multi-omics studies can be used to prioritize candidate genes and identify biomarkers and the molecular mechanisms underlying mastitis in dairy cattle. Hence, the present study aimed to explore the genetic basis of bovine mastitis by integrating microarray and RNA-Seq data containing healthy and mastitic samples in comparative transcriptome analysis with the results of published genome-wide association studies (GWAS) using a literature mining approach. The integration of different information sources resulted in the identification of 33 common and relevant genes associated with bovine mastitis. Among these, seven genes—CXCR1, HCK, IL1RN, MMP9, S100A9, GRO1, and SOCS3—were identified as the hub genes (highly connected genes) for mastitis susceptibility and resistance, and were subjected to protein-protein interaction (PPI) network and gene regulatory network construction. Gene ontology annotation and enrichment analysis revealed 23, 7, and 4 GO terms related to mastitis in the biological process, molecular function, and cellular component categories, respectively. Moreover, the main metabolic-signalling pathways responsible for the regulation of immune or inflammatory responses were significantly enriched in cytokine–cytokine-receptor interaction, the IL-17 signaling pathway, viral protein interaction with cytokines and cytokine receptors, and the chemokine signaling pathway. Consequently, the identification of these genes, pathways, and their respective functions could contribute to a better understanding of the genetics and mechanisms regulating mastitis and can be considered a starting point for future studies on bovine mastitis. Full article

Immunocytokines, antibody-cytokine fusion proteins, have the potential to improve the therapeutic index of cytokines by delivering the cytokine to the site of localized tumor cells using antibodies. In this study, we produced a recombinant anti-programmed death-ligand 1 (PD-L1) scFv, an antibody fragment against PD-L1 combined with a Neo2/15, which is an engineered interleukin with superior function using an E. coli expression system. We expressed the fusion protein in a soluble form and purified it, resulting in high yield and purity. The high PD-L1-binding efficiency of the fusion protein was confirmed via enzyme-linked immunosorbent assay, suggesting the application of this immunocytokine as a cancer-related therapeutic agent. Full article

The aim of the study was to develop a new diagnostic biomarker for identifying serum exosomal miRNAs specific to epithelial ovarian cancer (EOC) and to find out target gene of the miRNA for exploring the molecular mechanisms in EOC. A total of 84 cases of ovarian masses and sera were enrolled, comprising EOC (n = 71), benign ovarian neoplasms (n = 13). We detected expression of candidate miRNAs in the serum and tissue of both benign ovarian neoplasm group and EOC group using real-time polymerase chain reaction. Immunohistochemistry were constructed using formalin fixed paraffin embedded (FFPE) tissue to detect expression level of suppressor of cytokine signaling 4 (SOCS4). In the EOC group, miRNA-1290 was significantly overexpressed in serum exosomes and tissues as compared to benign ovarian neoplasm group (fold change ≥ 2, p < 0.05). We observed area under the receiver operating characteristic curve (AUC) for miR-1290, using a cut-off of 0.73, the exosomal miR-1290 from serum had AUC, sensitivity, and specificity values of 0.794, 69.2 and 87.3, respectively. In immunohistochemical study, expression of SOCS4 in EOC was lower than that in benign ovarian neoplasm. Serum exosomal miR-1290 could be considered as a biomarker for differential diagnosis of EOC from benign ovarian neoplasm and SOCS4 might be potential target gene of miR-1290 in EOC. Full article

A retrospective case control study was undertaken at the molecular biology department of a private center for reproductive medicine in order to determine whether any correlation exists between mitochondrial DNA (mtDNA) content of cleavage-stage preimplantation embryos and their developmental potential. A total of 69 couples underwent IVF treatment (averaged women age: 36.5, SD 4.9) and produced a total of 314 embryos. A single blastomere was biopsied from each embryo at the cleavage stage (day-3 post-fertilization) subjected to low-pass next generation sequencing (NGS), for the purpose of detecting aneuploidy. For each sample, the number of mtDNA reads obtained after analysis using NGS was divided by the number of reads attributable to the nuclear genome. The mtDNA copy number amount was found to be higher in aneuploid embryos than in those that were euploid (mean mtDNA ratio ± SD: 6.3 ± 7.5 versus 7.1 ± 5.8, p < 0.004; U Mann–Whitney test), whereas no statistically significant differences in mtDNA content were seen in relation to embryo morphology (6.6 ± 4.8 vs. 8.5 ± 13.6, p 0.09), sex (6.6 ± 4.1 vs. 6.2 ± 6.8, p 0.16), maternal age (6.9 ± 7.8 vs. 6.7 ± 4.5, p 0.14) or its ability to implant (7.4 ± 6.6 vs. 5.1 ± 4.6, p 0.18). The mtDNA content cannot serve as a useful biomarker at this point in development. However, further studies investigating both quantitative and qualitative aspects of mtDNA are still required to fully evaluate the relationship between mitochondrial DNA and human reproduction. Full article

Vascular dementia (VaD) is characterized by a time-dependent memory deficit and essentially combined with evidence of neuroinflammation. Thus, polyphenol-rich natural plants, which possess anti-inflammatory properties, have received much scientific attention. This study investigated whether Perilla frutescens leaf extract (PFL) exerts therapeutic efficacy against VaD. Sprague Dawley rats were divided into five groups: SO, sham-operated and vehicle treatment; OP, operated and vehicle treatment; PFL-L, operated and low-dose (30 mg/kg) PFL treatment; PFL-M, operated and medium-dose (60 mg/kg) PFL treatment; and PFL-H, operated and high-dose (90 mg/kg) PFL treatment. Two-vessel occlusion and hypovolemia (2VO/H) were employed as a surgical model of VaD, and PFL was given orally perioperatively for 23 days. The rats underwent the Y-maze, Barnes maze, and passive avoidance tests and their brains were subjected to histologic studies. The OP group showed VaD-associated memory deficits, hippocampal neuronal death, and microglial activation; however, the PFL-treated groups showed significant attenuations in all of the above parameters. Using lipopolysaccharide (LPS)-stimulated BV-2 cells, a murine microglial cell line, we measured PFL-mediated changes on the production of nitric oxide (NO), TNF-α, and IL-6, and the activities of their upstream MAP kinases (MAPKs)/NFκB/inducible NO synthase (iNOS). The LPS-induced upregulations of NO, TNF-α, and IL-6 production and MAPKs/NFκB/iNOS activities were globally and significantly reversed by 12-h pretreatment of PFL. This suggests that PFL can counteract VaD-associated structural and functional deterioration through the attenuation of neuroinflammation. Full article

Liquidambar formosana is important for its ornamental value in China; it is increasingly used for landscaping and gardening trees due to its diverse leaf colors and seasonal changes. Varieties including either a fixed leaf color, the purplish ‘Fuluzifeng’ (ZF), or seasonal changes in leaf color, the reddish ‘Nanlinhong’ (NLH) have been bred and registered as new plant varieties under the International Union for the Protection of New Plant Varieties (UPOV) system. To gain practical insights into the anthocyanin biosynthetic process, transcriptome sequencing (Illumina) was performed to clarify the metabolic pathways present in the three seasonal changes in leaf colors in NLH and in the springtime purple-red color of ZF. qRT-PCR was used to verify the speculation. Based on the differentially expressed genes and flavonoids analyses, the spring, summer, and autumn leaves of NLH were compared to study the seasonal differences. NLH and ZF were compared to study the formation mechanism of the two leaf colors in spring. Transcriptome sequencing produced a total of 121,216 unigenes from all samples, where 48 unigenes were differentially expressed and associated with the anthocyanidin pathway. The expression levels of LfDFR and LfANS genes corresponded to the accumulation of concentrations of cyanidins in spring (NLHC) and autumn leaves (NLHQ), respectively, with different shades of red. Moreover, the LfF3′5′H gene corresponded to the accumulation of flavonols and delphinidins in purple-red leaves (ZFC). Cyanidin and peonidin were the key pigments in red and dark-red leaves, and purple-red leaves were co-pigmented by cyanidins, pelargonidins, and delphinidins. Full article

According to a report from the World Health Organization (WHO), the mortality and disease severity induced by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are significantly higher in cancer patients than those of individuals with no known condition. Common and cancer-specific risk factors might be involved in the mortality and severity rates observed in the coronavirus disease 2019 (COVID-19). Similarly, various factors might contribute to the aggravation of COVID-19 in patients with cancer. However, the factors involved in the aggravation of COVID-19 in cancer patients have not been fully investigated so far. The formation of metastases in other organs is common in cancer patients. Therefore, the present study investigated the association between lung metastatic lesion formation and SARS-CoV-2 infectivity. In the pulmonary micrometastatic niche of patients with ovarian cancer, alveolar epithelial stem-like cells were found adjacent to ovarian cancer. Moreover, angiotensin-converting enzyme 2, a host-side receptor for SARS-CoV-2, was expressed in these alveolar epithelial stem-like cells. Furthermore, the spike glycoprotein receptor-binding domain (RBD) of SARS-CoV-2 was bound to alveolar epithelial stem-like cells. Altogether, these data suggested that patients with cancer and pulmonary micrometastases are more susceptible to SARS-CoV-2. The prevention of de novo niche formation in metastatic diseases might constitute a new strategy for the clinical treatment of COVID-19 for patients with cancer. Full article

Testicular cancer (TC) is a rare malignancy worldwide and is the most common malignancy in males aged 15–44 years. The Wnt/β-catenin signaling pathway mediates numerous essential cellular functions and has potentially important effects on tumorigenesis and cancer progression. The search for drugs to inhibit this pathway has identified a small molecule, PNU-74654, as an inhibitor of the β-catenin/TCF4 interaction. We evaluated the therapeutic role of PNU-74654 in two TC cell lines, NCCIT and NTERA2, by measuring cell viability, cell cycle transition and cell death. Potential pathways were evaluated by protein arrays and Western blots. PNU-74654 decreased cell viability and induced apoptosis of TC cells, with significant increases in the sub G1, Hoechst-stained, Annexin V-PI-positive rates. PNU-74654 treatment of both TC cell lines inhibited the TNFR1/IKB alpha/p65 pathway and the execution phase of apoptosis. Our findings demonstrate that PNU-74654 can induce apoptosis in TC cells through mechanisms involving the execution phase of apoptosis and inhibition of TNFR1/IKB alpha/p65 signaling. Therefore, small molecules such as PNU-74654 may identify potential new treatment strategies for TC. Full article

With the wide application of microwave technology, concerns about its health impact have arisen. The signal transmission mode of the central nervous system and neurons make it particularly sensitive to electromagnetic exposure. It has been reported that abnormal release of amino acid neurotransmitters is mediated by alteration of p-SYN1 after microwave exposure, which results in cognitive dysfunction. As the phosphorylation of SYN1 is regulated by different kinases, in this study we explored the regulatory mechanisms of SYN1 fluctuations following microwave exposure and its subsequent effect on GABA release, aiming to provide clues on the mechanism of cognitive impairment caused by microwave exposure. In vivo studies with Timm and H&E staining were adopted and the results showed abnormality in synapse formation and neuronal structure, explaining the previously-described deficiency in cognitive ability caused by microwave exposure. The observed alterations in SYN1 level, combined with the results of earlier studies, indicate that SYN1 and its phosphorylation status (ser-553 and ser62/67) may play a role in the abnormal release of neurotransmitters. Thus, the role of Cdk5, the upstream kinase regulating the formation of p-SYN1 (ser-553), as well as that of MEK, the regulator of p-SYN1 (ser-62/67), were investigated both in vivo and in vitro. The results showed that Cdk5 was a negative regulator of p-SYN1 (ser-553) and that its up-regulation caused a decrease in GABA release by reducing p-SYN1 (ser-553). While further exploration still needed to elaborate the role of p-SYN1 (ser-62/67) for neurotransmitter release, MEK inhibition had was no impact on p-Erk or p-SYN1 (ser-62/67) after microwave exposure. In conclusion, the decrease of p-SYN1 (ser-553) may result in abnormalities in vesicular anchoring and GABA release, which is caused by increased Cdk5 regulated through Calpain-p25 pathway after 30 mW/cm² microwave exposure. This study provided a potential new strategy for the prevention and treatment of microwave-induced cognitive dysfunction. Full article

Background. Humoral immunity requires interaction between B cell and T follicular helper cells (Tfh) to produce effective immune response, but the data regarding a role of B cells and Tfh in SARS-CoV-2 defense are still sparse. Methods. Blood samples from patients with acute COVID-19 (n = 64), convalescents patients who had specific IgG to SARS-CoV-2 N-protein (n = 55), and healthy donors with no detectable antibodies to any SARS-CoV-2 proteins (HC, n = 44) were analyses by multicolor flow cytometry. Results. Patients with acute COVID-19 showed decreased levels of memory B cells subsets and increased proportion plasma cell precursors compared to HC and COVID-19 convalescent patients, whereas for the latter the elevated numbers of virgin naïve, Bm2′ and “Bm3+Bm4” was found if compared with HC. During acute COVID-19 CXCR3+CCR6− Tfh1-like cells were decreased and the levels of CXCR3−CCR6+ Tfh17-like were increased then in HC and convalescent patients. Finally, COVID-19 convalescent patients had increased levels of Tfh2-, Tfh17- and DP Tfh-like cells while comparing their amount with HC. Conclusions. Our data indicate that COVID-19 can impact the humoral immunity in the long-term. Full article

Pecan is one of the most popular nut species in the world. The fruit drop rate of the pecan ‘Pawnee’ is more than 57%, with four fruit drop stages, which is very serious. In this study, we conducted transcriptomic profiling of persisting and abscised fruitlets in early fruit development by RNA-seq. A total of 11,976 differentially expressed genes (DEGs) were identified, 3012 upregulated and 8964 downregulated, in a comparison of abscised vs. persisting fruitlets at 35 days after anthesis (DAA). Our transcriptomic data suggest that gene subsets encoding elements involving the biosynthesis, metabolism, perception, signal transduction, and crosstalk of the plant hormones abscisic acid (ABA), auxin, cytokinin, ethylene, and gibberellin (GA) and plant growth regulators jasmonates, salicylic acid, and brassinosteroids were differentially expressed. In addition, the majority of transcriptionally activated genes involved in hormone signaling (except for ethylene and salicylic acid signaling) were downregulated in abscised fruitlets. The differential expression of transcripts coding for enzymes involved in sucrose, glucose, trehalose, starch, galactose, and galactinol metabolism shows that sucrose, galactinol, and glucose synthesis and starch content were reduced as starch biosynthesis was blocked, and retrogradation and degradation intensified. These results suggest that the abscised pecan fruitlets stopped growing and developing for some time before dropping, further indicating that their sugar supply was reduced or stopped. The transcriptome characterization described in this paper contributes to unravelling the molecular mechanisms and pathways involved in the physiological abscission of pecan fruits. Full article

The number of patients with neurodegenerative diseases, particularly Alzheimer’s disease (AD), continues to grow yearly. Cholinesterase inhibitors (ChEIs) represent the first-line symptomatic drug treatment for mild-to-moderate AD; however, there is an unmet need to produce ChEIs with improved efficacy and reduced side effects. Herein, phytochemicals with reported anti-acetylcholinesterase (AChE) activity were ranked in silico for their anti-AChE potential. Ligands with a similar or higher binding affinity to AChE than galantamine were then selected for the design of novel dual-binding site heterodimeric drugs. In silico molecular docking of heterodimers with the target enzymes, AChE and butyrylcholinesterase (BuChE), were performed, and anti-cholinesterase binding affinities were compared with donepezil. Drug-likeliness properties and toxicity of the heterodimers were assessed using the SwissADME and ProTox-II webservers. Nine phytochemicals displayed similar or higher binding affinities to AChE than galantamine: sanguinarine > huperzine A > chelerythrine > yohimbine > berberine > berberastine > naringenin > akuammicine > carvone. Eleven heterodimeric ligands were designed with phytochemicals separated by four- or five-carbon alkyl-linkers. All heterodimers were theoretically potent AChE and BuChE dual-binding site inhibitors, with the highest affinity achieved with huperzine-4C-naringenin, which displayed 34% and 26% improved affinity to AChE and BuChE, respectively, then the potent ChEI drug, donepezil. Computational pharmacokinetic and pharmacodynamic screening suggested that phytochemical heterodimers would display useful gastrointestinal absorption and with relatively low predicted toxicity. Collectively, the present study suggests that phytochemicals could be garnered for the provision of novel ChEIs with enhanced drug efficacy and low toxicity. Full article

The conventional approach in bioactive peptides discovery, which includes extensive bioassay-guided fractionation and purification processes, is tedious, time-consuming and not always successful. The recently developed bioinformatics-driven in silico approach is rapid and cost-effective; however, it lacks an actual physiological significance. In this study a new integrated peptidomics and in silico method, which combines the advantages of the conventional and in silico approaches by using the pool of peptides identified in a food hydrolysate as the starting point for subsequent application of selected bioinformatics tools, has been developed. Pinto bean protein extract was in vitro digested and peptides were identified by peptidomics. The pool of obtained peptides was screened by in silico analysis and structure–activity relationship modelling. Three peptides (SIPR, SAPI and FVPH) were selected as potential inhibitors of the dipeptidyl-peptidase-IV (DPP-IV) enzyme by this integrated approach. In vitro bioactivity assay showed that all three peptides were able to inhibit DPP-IV with the tetra-peptide SAPI showing the highest activity (IC50 = 57.7 μmol/L). Indeed, a new possible characteristic of peptides (i.e., the presence of an S residue at the N-terminus) able to inhibit DPP-IV was proposed. Full article

Primary aldosteronism is most often caused by aldosterone-producing adenoma (APA) and bi-lateral adrenal hyperplasia. Most APAs are caused by somatic mutations of various ion channels and pumps, the most common being the inward-rectifying potassium channel KCNJ5. Germ line mutations of KCNJ5 cause familial hyperaldosteronism type 3 (FH3), which is associated with severe hyperaldosteronism and hypertension. We present an unusual case of FH3 in a young woman, first diagnosed with primary aldosteronism at the age of 6 years, with bilateral adrenal hyperplasia, who underwent unilateral adrenalectomy (left adrenal) to alleviate hyperaldosteronism. However, her hyperaldosteronism persisted. At the age of 26 years, tomography of the remaining adrenal revealed two different adrenal tumors, one of which grew substantially in 4 months; therefore, the adrenal gland was removed. A comprehensive histological, immunohistochemical, and molecular evaluation of various sections of the adrenal gland and in situ visualization of aldosterone, using matrix-assisted laser desorption/ionization imaging mass spectrometry, was performed. Aldosterone synthase (CYP11B2) immunoreactivity was observed in the tumors and adrenal gland. The larger tumor also harbored a somatic β-catenin activating mutation. Aldosterone visualized in situ was only found in the subcapsular regions of the adrenal and not in the tumors. Collectively, this case of FH3 presented unusual tumor development and histological/molecular findings. Full article

Hepcidin and ferritin are key proteins of iron homeostasis in mammals. In this study, we characterize a chimera by fusing camel hepcidin to a human ferritin H-chain to verify if it retained the properties of the two proteins. The construct (HepcH) is expressed in E. coli in an insoluble and iron-containing form. To characterize it, the product was incubated with ascorbic acid and TCEP to reduce and solubilize the iron, which was quantified with ferrozine. HepcH bound approximately five times more iron than the wild type human ferritin, due to the presence of the hepcidin moiety. To obtain a soluble and stable product, the chimera was denatured and renatured together with different amounts of L-ferritin of the H-chain in order to produce 24-shell heteropolymers with different subunit proportions. They were analyzed by denaturing and non-denaturing PAGE and by mass spectroscopy. At the 1:5 ratio of HepcH to H- or L-ferritin, a stable and soluble molecule was obtained. Its biological activity was verified by its ability to both bind specifically cell lines that express ferroportin and to promote ferroportin degradation. This chimeric molecule showed the ability to bind both mouse J774 macrophage cells, as well as human HepG2 cells, via the hepcidin–ferroportin axis. We conclude that the chimera retains the properties of both hepcidin and ferritin and might be exploited for drug delivery. Full article

Cancer metastasis is a stage of the disease where therapy is mostly ineffective; hence, the need to find reliable markers of its onset. The metalloproteinase-9 (MMP-9, gelatinase B) in its 82 kDa active form, is a good candidate, but here we show that the correspondent little known 65 kDa active MMP-9 isoform, often misrepresented with the other gelatinase MMP-2, is a more suitable marker. Sera from patients with lung and breast cancer were analyzed by bidimensional zymography to detect the activity of MMP-9 and MMP-2. Enzyme identity was confirmed by comparison with MMP-9 standards and by western blotting. The 65 kDa isoform of MMP-9 is a suitable biomarker to monitor tumor progression from tissue neoplasms to metastatic stage, as its activity begins to appear when disease severity increases and becomes very high in metastasis. Moreover, the 65 kDa MMP-9, which derives from the 82 kDa MMP-9, no longer responds to natural MMP-9 inhibitors. As its activity cannot be controlled, its appearance may warn that the pathological process is becoming irreversible. Identification and inhibition of the enzymes converting the inhibitor-sensitive 82 kDa MMP-9 into the corresponding “wild” 65 kDa MMP-9 may allow to develop therapies capable of blocking metastases. Full article

The use of metals in medicine has grown in popularity in clinical and commercial settings. In this study, the immune-protecting effects and the hypoglycemic and antioxidant activity of vanadyl sulfate (VOSO₄) and/or selenium tetrachloride (Se) on oxidative injury, DNA damage, insulin resistance, and hyperglycemia were assessed. Fifty male albino rats were divided into five groups, and all treatments were administrated at 9:00 a.m. daily for 60 successive days: control, STZ (Streptozotocin; 50 mg/kg of STZ was given to 6 h fasted animals in a single dose, followed by confirmation of diabetic state occurrence after 72 h by blood glucose estimation at >280 mg/dl), STZ (Diabetic) plus administration of VOSO₄ (15 mg/kg) for 60 days, STZ (Diabetic) plus administration of selenium tetrachloride (0.87 mg/Kg), and STZ plus VOSO₄ and, after 1/2 h, administration of selenium tetrachloride at the above doses. The test subjects’ blood glucose, insulin hormone, HbA1C, C-peptide, antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase, myeloperoxidase, and xanthine oxidase), markers of lipid peroxidation (MDA), and histological sections of pancreatic tissues were evaluated, and a comet assay was performed. Histological sections in pancreas tissues were treated as indicators of both VOSO₄ and selenium tetrachloride efficacy, either alone or combined, for the alleviation of STZ toxicity. The genotoxicity of diabetes mellitus was assessed, and the possible therapeutic roles of VOSO₄ or selenium tetrachloride, or both, on antioxidant enzymes were studied. The findings show that the administration of VOSO₄ with selenium tetrachloride reduced oxidative stress to normal levels, lowered blood glucose levels, and elevated insulin hormone. Additionally, VOSO₄ with selenium tetrachloride had a synergistic effect and significantly decreased pancreatic genotoxicity. The data clearly show that both VOSO₄ and selenium tetrachloride inhibit pancreatic and DNA injury and improve the oxidative state in male rats, suggesting that the use of VOSO₄ with selenium tetrachloride is a promising synergistic potential ameliorative agent in the diabetic animal model. Full article

In aging males, androgen production by testicular Leydig cells decreases at a rate of approximately 1% per year. Phenolic compounds may enhance testosterone biosynthesis and delay the onset of male hypogonadism. Gigantol is a bibenzyl compound isolated from several types of orchids of the genus Dendrobium. This compound has various biological activities, including antioxidant activity. However, its capacity to regulate gene expression and steroid production in testicular Leydig cells has never been evaluated. We investigated the effect of gigantol on MA-10 Leydig cells’ gene expression using an RNA-Seq approach. To further investigate the structure-function relationship of the hydroxy-methoxyphenyl moiety of gigantol, experiments were also performed with ferulic acid and isoferulic acid. According to transcriptomic analysis, all genes coding for cholesterol biosynthesis-related enzymes are increased in response to gigantol treatment, resulting in increased lipid droplets accumulation. Moreover, treatments with 10 μM gigantol increased StAR protein levels and progesterone production from MA-10 Leydig cells. However, neither ferulic acid nor isoferulic acid influenced StAR protein synthesis and progesterone production in MA-10 Leydig cells. Thus, our findings indicate that gigantol improves cholesterol and steroid biosynthesis within testicular Leydig cells. Full article

Chemotherapy is the treatment of choice for gastric cancer, but the currently available therapeutic drugs have limited efficacy. Studies have suggested that gastric cancer stem cells may play a key role in drug resistance in chemotherapy. Therefore, new agents that selectively target gastric cancer stem cells in gastric tumors are urgently required. Sirtuin-3 (SIRT3) is a deacetylase that regulates mitochondrial metabolic homeostasis to maintain stemness in glioma stem cells. Targeting the mitochondrial protein SIRT3 may provide a novel therapeutic option for gastric cancer treatment. However, the mechanism by which stemness is regulated through SIRT3 inhibition in gastric cancer remains unknown. We evaluated the stemness inhibition ability of the SIRT3 inhibitor 4′-bromo-resveratrol (4-BR), an analog of resveratrol in human gastric cancer cells. Our results suggested that 4-BR inhibited gastric cancer cell stemness through the SIRT3-c-Jun N-terminal kinase pathway and may aid in gastric cancer stem-cell–targeted therapy. Full article

The membrane-active nature of phospholipase A₂-derived peptides makes them potential candidates for antineoplastic and antibacterial therapies. Two short 13-mer C-terminal fragments taken from snake venom Lys49-PLA₂ toxins (p-AppK and p-Acl), differing by a leucine/phenylalanine substitution, were synthesized and their bioactivity was evaluated. Their capacity to interfere with the survival of Gram-positive and Gram-negative bacteria as well as with solid and liquid tumors was assessed in vitro. Toxicity to red blood cells was investigated via in silico and in vitro techniques. The mode of action was mainly studied by molecular dynamics simulations and membrane permeabilization assays. Briefly, both peptides have dual activity, i.e., they act against both bacteria, including multidrug-resistant strains and tumor cells. All tested bacteria were susceptible to both peptides, Pseudomonas aeruginosa being the most affected. RAMOS, K562, NB4, and CEM cells were the main leukemic targets of the peptides. In general, p-Acl showed more significant activity, suggesting that phenylalanine confers advantages to the antibacterial and antitumor mechanism, particularly for osteosarcoma lines (HOS and MG63). Peptide-based treatment increased the uptake of a DNA-intercalating dye by bacteria, suggesting membrane damage. Indeed, p-AppK and p-Acl did not disrupt erythrocyte membranes, in agreement with in silico predictions. The latter revealed that the peptides deform the membrane and increase its permeability by facilitating solvent penetration. This phenomenon is expected to catalyze the permeation of solutes that otherwise could not cross the hydrophobic membrane core. In conclusion, the present study highlights the role of a single amino acid substitution present in natural sequences towards the development of dual-action agents. In other words, dissecting and fine-tuning biomembrane remodeling proteins, such as snake venom phospholipase A₂ isoforms, is again demonstrated as a valuable source of therapeutic peptides. Full article

Introduction: according to the World Health Organization (WHO), COVID-19 is an infectious disease caused by the SARS-CoV-2 virus, responsible for an increasing number of cases and deaths. From a preventive and therapeutic point of view, there are two concerns that affect institutions and healthcare professionals: global immunization (which is still far from being achieved) and the availability of drugs capable of preventing its consequences in the infected patient. In this sense, the role that melatonin can play is has been assessed in the recent literature. Justification and Objectives: the serious health, social and economic consequences of COVID-19 have forced an urgent search for preventive methods, such as vaccines, among others, and therapeutic methods that could be alternatives to the drugs currently used. In this sense, it must be accepted that one of the most recommended has been the administration of melatonin. The present study proposes to carry out a systematic review of its possible role in the treatment and/or prevention of COVID-19. Material and methods: a systematic review of the literature related to the prevention of COVID-19 through the administration of melatonin was carried out, following the sequence proposed by the Prisma Declaration regarding the identification and selection of documents, using the specialized health databases Trip Medical Database, Cochrane Library, PubMed, Medline Plus, BVS, Cuiden and generic databases such as Dialnet, Web of Science and Google Scholar for their retrieval. Appropriate inclusion and exclusion criteria are described for the articles assessed. The main limitation of the study has been the scarcity of works and the lack of defining a specific protocol in terms of dosage and administration schedule. Results: once the selection process was completed, and after an in-depth critical analysis, 197 papers were selected, and 40 of them were finally used. The most relevant results were: (1) melatonin prevents SARS-CoV-2 infection, (2) although much remains to be clarified, at high doses, it seems to have a coadjuvant therapeutic effect in the treatment of SARS-CoV-2 infection and (3) melatonin is effective against SARS-CoV-2 infection. Discussion: until group immunization is achieved in the population, it seems clear that we must continue to treat patients with SARS-CoV-2 infection, and, in the absence of a specific and effective antiviral therapy, it is advisable to continue researching and providing drugs that demonstrate validity based on the scientific evidence. In this regard, we believe that the available studies recommend the administration of melatonin for its anti-inflammatory, antioxidant, immunomodulatory, sleep-inducing, CD147, Mpro, p65 and MMP9 protein suppressing, nephrotoxicity-reducing and highly effective and safe effects. Conclusions: (1) melatonin has anti-inflammatory, antioxidant, immunomodulatory, and Mpro and MMP9 protein-inhibitory activity. (2) It has been shown to have a wide margin of safety. (3) The contributions reviewed make it an effective therapeutic alternative in the treatment of SARS-CoV-2 infection. (4) Further clinical trials are recommended to clearly define the administration protocol. Full article

Cells have developed a highly integrated system responsible for proteome stability, namely the proteostasis network (PN). As loss of proteostasis is a hallmark of aging and age-related diseases, the activation of PN modules can likely extend healthspan. Here, we present data on the bioactivity of an extract (SA223-S2BM) purified from the strain Salinispora arenicola TM223-S2 that was isolated from the soft coral Scleronephthya lewinsohni; this coral was collected at a depth of 65 m from the mesophotic Red Sea ecosystem EAPC (south Eilat, Israel). Treatment of human cells with SA223-S2BM activated proteostatic modules, decreased oxidative load, and conferred protection against oxidative and genotoxic stress. Furthermore, SA223-S2BM enhanced proteasome and lysosomal-cathepsins activities in Drosophila flies and exhibited skin protective effects as evidenced by effective inhibition of the skin aging-related enzymes, elastase and tyrosinase. We suggest that the SA223-S2BM extract constitutes a likely promising source for prioritizing molecules with anti-aging properties. Full article