Current Issues in Molecular Biology

Barrett’s esophagus (BE) is an acquired pre-malignant condition that results from chronic gastroesophageal reflux. The malignant transformation occurred in 0.5% of patients/year and was independent of medical and endoscopic conservative treatments. Fatty acid synthase (FAS) is a multifunctional enzyme that catalyzes the synthesis of long-chain fatty acids from acetyl-coenzyme A, malonyl-coenzyme A, a reduced form of nicotinamide adenine dinucleotide phosphate (NADPH), and adenosine triphosphate. Activation of FAS is closely linked to malignant transformation. The aim of the present study was to evaluate the variation of FAS, p53, and Ki67 expressions in two groups of 21 BE patients each, after one year of continuous (group A) or discontinuous (group B) treatment with esomeprazole 40 mg/day in comparison to the initial expression. In both the two groups of BE patients, biopsies were taken from pathologic sites of the mucosa for histological and immuno-histochemical detection of FAS, Ki67, and p53 at entry and after one year of Esomeprazole 40 mg treatment. FAS expression was positive when a strong granular cytoplasmic staining was observed in esophageal cells. Ki67 and p53 were defined as positive when nuclear staining was clearly detected at ×10 magnification. FAS expression was reduced in 43% of patients treated with Esomeprazole continuously in comparison to the 10% of patients treated with Esomeprazole on demand (p = 0.002). Ki67 expression was reduced in 28% of continuously treated patients in comparison to 5% of patients treated on demand (p = 0.001). The p53 expression decreased in 19% of continuously treated patients in comparison to an increase in 2 patients (9%) treated on demand (p = 0.05). Continuously Esomeprazole treatment could help in the diminution of metabolic and proliferative activities in the esophageal columnar epithelium and in part it can help prevent the oxidative damage against cellular DNA, resulting in a diminution in p53 expression. Full article

Herein, we report the major factor for deamination reaction rate acceleration, i.e., hydrophilicity, by using various 5-substituted target cytosines and by carrying out deamination at high temperatures. Through substitution of the groups at the 5′-position of the cytosine, the effect of hydrophilicity was understood. It was then used to compare the various modifications of the photo-cross-linkable moiety as well as the effect of the counter base of the cytosine to edit both DNA and RNA. Furthermore, we were able to achieve cytosine deamination at 37 °C with a half-life in the order of a few hours. Full article

Myocardial infarction (MI) is a common and life-threatening manifestation of ischemic heart diseases (IHD). The most important risk factor for MI is hypertension. Natural products from medicinal plants have gained considerable attention globally due to their preventive and therapeutic effects. Flavonoids have been found to be efficacious in ischemic heart diseases (IHD) by alleviating oxidative stress and beta-1 adrenergic activation, but the mechanistic link is not clear. We hypothesized that antioxidant flavonoid diosmetin is cardioprotective in a rat model of MI induced by beta 1-adrenergic receptor activation. To test this hypothesis, we evaluated the cardioprotective potential of diosmetin on isoproterenol-induced MI in rats by performing lead II electrocardiography (ECG), cardiac biomarkers including troponin I (cTnI) and creatinine phosphokinase (CPK), CK-myocardial band, (CK-MB), lactate dehydrogenase (LDH), alanine aminotransferase (ALT), and aspartate aminotranferase (AST) by using biolyzer 100, as well as histopathological analysis. We found that diosmetin (1 and 3 mg/kg) attenuated isoproterenol-induced elevation in the T-wave and deep Q-wave on the ECG, as well as heart-to-body weight ratio and infarction size. In addition, pretreatment with diosmetin attenuated the isoproterenol-induced increase in serum troponin I. These results demonstrate that flavonoid diosmetin may provide therapeutic benefit in myocardial infarction. Full article

Repositioning of aspirin for a more effective breast cancer (BC) treatment requires identification of predictive biomarkers. However, the molecular mechanism underlying the anticancer activity of aspirin remains fully undefined. Cancer cells enhance de novo fatty acid (FA) synthesis and FA oxidation to maintain a malignant phenotype, and the mechanistic target of rapamycin (mTORC1) is required for lipogenesis. We, therefore, aimed to test if the expression of mTORC1 suppressor DNA damage-inducible transcript (DDIT4) affects the activity of main enzymes in FA metabolism after aspirin treatment. MCF-7 and MDA-MB-468 human BC cell lines were transfected with siRNA to downregulate DDIT4. The expression of carnitine palmitoyltransferase 1 A (CPT1A) and serine 79-phosphorylated acetyl-CoA carboxylase 1 (ACC1) were analyzed by Western Blotting. Aspirin enhanced ACC1 phosphorylation by two-fold in MCF-7 cells and had no effect in MDA-MB-468 cells. Aspirin did not change the expression of CPT1A in either cell line. We have recently reported DDIT4 itself to be upregulated by aspirin. DDIT4 knockdown resulted in 1.5-fold decreased ACC1 phosphorylation (dephosphorylation activates the enzyme), 2-fold increased CPT1A expression in MCF-7 cells, and 2.8-fold reduced phosphorylation of ACC1 following aspirin exposure in MDA-MB-468 cells. Thus, DDIT4 downregulation raised the activity of main lipid metabolism enzymes upon aspirin exposure which is an undesired effect as FA synthesis and oxidation are linked to malignant phenotype. This finding may be clinically relevant as DDIT4 expression has been shown to vary in breast tumors. Our findings justify further, more extensive investigation of the role of DDIT4 in aspirin’s effect on fatty acid metabolism in BC cells. Full article

Citrus (Citrus reticulata) is one of the world’s most widely planted and highest-yielding fruit trees. Citrus fruits are rich in a variety of nutrients. The content of citric acid plays a decisive role in the flavor quality of the fruit. There is a high organic acid content in early-maturing and extra-precocious citrus varieties. Reducing the amount of organic acid after fruit ripening is significant to the citrus industry. In this study, we selected a low-acid variety, “DF4”, and a high-acid variety, “WZ”, as research materials. Through WGCNA analysis, two differentially expressed genes, citrate synthase (CS) and ATP citrate-pro-S-lyase (ACL), were screened out, which related to the changing citric acid. The two differentially expressed genes were preliminarily verified by constructing a virus-induced gene-silencing (VIGS) vector. The VIGS results showed that the citric acid content was negatively correlated with CS expression and positively correlated with ACL expression, while CS and ACL oppositely control citric acid and inversely regulate each other. These results provide a theoretical basis for promoting the breeding of early-maturing and low-acid citrus varieties. Full article

Epigenetic studies on the role of DNA-modifying enzymes in HNSCC tumorigenesis have focused on a single enzyme or a group of enzymes. To acquire a more comprehensive insight into the expression profile of methyltransferases and demethylases, in the present study, we examined the mRNA expression of the DNA methyltransferases DNMT1, DNMT3A, and DNMT3B, the DNA demethylases TET1, TET2, TET3, and TDG, and the RNA methyltransferase TRDMT1 by RT-qPCR in paired tumor–normal tissue samples from HNSCC patients. We characterized their expression patterns in relation to regional lymph node metastasis, invasion, HPV16 infection, and CpG73 methylation. Here, we show that tumors with regional lymph node metastases (pN+) exhibited decreased expression of DNMT1, 3A and 3B, and TET1 and 3 compared to non-metastatic tumors (pN0), suggesting that metastasis requires a distinct expression profile of DNA methyltransferases/demethylases in solid tumors. Furthermore, we identified the effect of perivascular invasion and HPV16 on DNMT3B expression in HNSCC. Finally, the expression of TET2 and TDG was inversely correlated with the hypermethylation of CpG73, which has previously been associated with poorer survival in HNSCC. Our study further confirms the importance of DNA methyltransferases and demethylases as potential prognostic biomarkers as well as molecular therapeutic targets for HNSCC. Full article

Nodule number regulation in legumes is controlled by a feedback loop that integrates nutrient and rhizobia symbiont status signals to regulate nodule development. Signals from the roots are perceived by shoot receptors, including a CLV1-like receptor-like kinase known as SUNN in Medicago truncatula. In the absence of functional SUNN, the autoregulation feedback loop is disrupted, resulting in hypernodulation. To elucidate early autoregulation mechanisms disrupted in SUNN mutants, we searched for genes with altered expression in the loss-of-function sunn-4 mutant and included the rdn1-2 autoregulation mutant for comparison. We identified constitutively altered expression of small groups of genes in sunn-4 roots and in sunn-4 shoots. All genes with verified roles in nodulation that were induced in wild-type roots during the establishment of nodules were also induced in sunn-4, including autoregulation genes TML2 and TML1. Only an isoflavone-7-O-methyltransferase gene was induced in response to rhizobia in wild-type roots but not induced in sunn-4. In shoot tissues of wild-type, eight rhizobia-responsive genes were identified, including a MYB family transcription factor gene that remained at a baseline level in sunn-4; three genes were induced by rhizobia in shoots of sunn-4 but not wild-type. We cataloged the temporal induction profiles of many small secreted peptide (MtSSP) genes in nodulating root tissues, encompassing members of twenty-four peptide families, including the CLE and IRON MAN families. The discovery that expression of TML2 in roots, a key factor in inhibiting nodulation in response to autoregulation signals, is also triggered in sunn-4 in the section of roots analyzed, suggests that the mechanism of TML regulation of nodulation in M. truncatula may be more complex than published models. Full article

Bacillus subtilis S-16 isolated from sunflower-rhizosphere soil is an effective biocontrol agent for preventing soilborne diseases in plants. Previous research revealed that the volatile organic compounds (VOCs) produced by the S-16 strain have strong inhibitory effects on Sclerotinia sclerotiorum. The identification of the VOCs of S-16 using gas chromatography-tandem mass spectrometry (GC-MS/MS) revealed 35 compounds. Technical-grade formulations of four of these compounds were chosen for further study: 2-pentadecanone, 6,10,14-trimethyl-2-octanone, 2-methyl benzothiazole (2-MBTH), and heptadecane. The major constituent, 2-MBTH, plays an important role in the antifungal activity of the VOCs of S-16 against the growth of Sclerotinia sclerotiorum. The purpose of this study was to determine the impact of the thiS gene’s deletion on the 2-MBTH production and to conduct an antimicrobial activity analysis of the Bacillus subtilis S-16. The thiazole-biosynthesis gene was deleted via homologous recombination, after which the contents of 2-MBTH in the wild-type and mutant S-16 strains were analyzed using GC-MS. The antifungal effects of the VOCs were determined using a dual-culture technique. The morphological characteristics of the Sclerotinia sclerotiorum mycelia were examined via scanning-electron microscopy (SEM). Additionally, the lesion areas on the sunflower leaves with and without treatment with the VOCs from the wild-type and mutant strains were measured to explore the effects of the VOCs on the virulence of the Sclerotinia sclerotiorum. Moreover, the effects of the VOCs on the sclerotial production were assessed. We showed that the mutant strain produced less 2-MBTH. The ability of the VOCs produced by the mutant strain to inhibit the growth of the mycelia was also reduced. The SEM observation showed that the VOCs released by the mutant strain also caused more flaccid and gapped hyphae in the Sclerotinia sclerotiorum. The Sclerotinia sclerotiorum treated by the VOCs produced by the mutant strains caused more damage to the leaves than that treated by the VOCs produced by the wild type and the mutant-strain-produced VOCs inhibited sclerotia formation less. The production of 2-MBTH and its antimicrobial activities were adversely affected to varying degrees by the deletion of thiS. Full article

The World Health Organization has estimated the annual occurrence of approximately 392 million dengue virus (DENV) infections in more than 100 countries where the virus is endemic, which represents a serious threat to humanity. DENV is a serologic group with four distinct serotypes (DENV-1, DENV-2, DENV-3, and DENV-4) belonging to the genus Flavivirus, in the family Flaviviridae. Dengue is the most widespread mosquito-borne disease in the world. The ~10.7 kb DENV genome encodes three structural proteins (capsid (C), pre-membrane (prM), and envelope (E)) and seven non-structural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The NS1 protein is a membrane-associated dimer and a secreted, lipid-associated hexamer. Dimeric NS1 is found on membranes both in cellular compartments and cell surfaces. Secreted NS1 (sNS1) is often present in patient serum at very high levels, which correlates with severe dengue symptoms. This study was conducted to discover how the NS1 protein, microRNAs-15/16 (miRNAs-15/16), and apoptosis are related during DENV-4 infection in human liver cell lines. Huh 7.5 and HepG2 cells were infected with DENV-4, and miRNAs-15/16, viral load, NS1 protein, and caspases-3/7 were quantified after different durations of infection. This study demonstrated that miRNAs-15/16 were overexpressed during the infection of HepG2 and Huh 7.5 cells with DENV-4 and had a relationship with NS1 protein expression, viral load, and the activity of caspases-3/7, thus making these miRNAs potential injury markers during DENV infection in human hepatocytes. Full article

Alzheimer’s Disease (AD) is characterized by synapse and neuronal loss and the accumulation of neurofibrillary tangles and Amyloid β plaques. Despite significant research efforts to understand the late stages of the disease, its etiology remains largely unknown. This is in part because of the imprecise AD models in current use. In addition, little attention has been paid to neural stem cells (NSC), which are the cells responsible for the development and maintenance of brain tissue during an individual’s lifespan. Thus, an in vitro 3D human brain tissue model using induced pluripotent stem (iPS) cell-derived neural cells in human physiological conditions may be an excellent alternative to standard models to investigate AD pathology. Following the differentiation process mimicking development, iPS cells can be turned into NSCs and, ultimately, neural cells. During differentiation, the traditionally used xenogeneic products may alter the cells’ physiology and prevent accurate disease pathology modeling. Hence, establishing a xenogeneic material-free cell culture and differentiation protocol is essential. This study investigated the differentiation of iPS cells to neural cells using a novel extracellular matrix derived from human platelet lysates (PL Matrix). We compared the stemness properties and differentiation efficacies of iPS cells in a PL matrix against those in a conventional 3D scaffold made of an oncogenic murine-matrix. Using well-defined conditions without xenogeneic material, we successfully expanded and differentiated iPS cells into NSCs via dual-SMAD inhibition, which regulates the BMP and TGF signaling cascades in a manner closer to human conditions. This in vitro, 3D, xenogeneic-free scaffold will enhance the quality of disease modeling for neurodegenerative disease research, and the knowledge produced could be used in developing more effective translational medicine. Full article

In recent years, various forms of caloric restriction (CR) and amino acid or protein restriction (AAR or PR) have shown not only success in preventing age-associated diseases, such as type II diabetes and cardiovascular diseases, but also potential for cancer therapy. These strategies not only reprogram metabolism to low-energy metabolism (LEM), which is disadvantageous for neoplastic cells, but also significantly inhibit proliferation. Head and neck squamous cell carcinoma (HNSCC) is one of the most common tumour types, with over 600,000 new cases diagnosed annually worldwide. With a 5-year survival rate of approximately 55%, the poor prognosis has not improved despite extensive research and new adjuvant therapies. Therefore, for the first time, we analysed the potential of methionine restriction (MetR) in selected HNSCC cell lines. We investigated the influence of MetR on cell proliferation and vitality, the compensation for MetR by homocysteine, the gene regulation of different amino acid transporters, and the influence of cisplatin on cell proliferation in different HNSCC cell lines. Full article

Glucagon-like peptide 1 receptor agonists (GLP-1RAs) have been shown to improve glucose and lipid homeostasis, promote weight loss, and reduce cardiovascular risk factors. They are a promising therapeutic option for non-alcoholic fatty liver disease (NAFLD), the most common liver disease, associated with T2DM, obesity, and metabolic syndrome. GLP-1RAs have been approved for the treatment of T2DM and obesity, but not for NAFLD. Most recent clinical trials have suggested the importance of early pharmacologic intervention with GLP-1RAs in alleviating and limiting NAFLD, as well as highlighting the relative scarcity of in vitro studies on semaglutide, indicating the need for further research. However, extra-hepatic factors contribute to the GLP-1RA results of in vivo studies. Cell culture models of NAFLD can be helpful in eliminating extrahepatic effects on the alleviation of hepatic steatosis, modulation of lipid metabolism pathways, reduction of inflammation, and prevention of the progression of NAFLD to severe hepatic conditions. In this review article, we discuss the role of GLP-1 and GLP-1RA in the treatment of NAFLD using human hepatocyte models. Full article

Colon cancer is the third most important cancer type, leading to a remarkable number of deaths, indicating the necessity of new biomarkers and therapeutic targets for colon cancer patients. Several transmembrane proteins (TMEMs) are associated with tumor progression and cancer malignancy. However, the clinical significance and biological roles of TMEM211 in cancer, especially in colon cancer, are still unknown. In this study, we found that TMEM211 was highly expressed in tumor tissues and the increased TMEM211 was associated with poor prognosis in colon cancer patients from The Cancer Genome Atlas (TCGA) database. We also showed that abilities regarding migration and invasion were reduced in TMEM211-silenced colon cancer cells (HCT116 and DLD-1). Moreover, TMEM211-silenced colon cancer cells showed decreased levels of Twist1, N-cadherin, Snail and Slug but increased levels of E-cadherin. Levels of phosphorylated ERK, AKT and RelA (NF-κB p65) were also decreased in TMEM211-silenced colon cancer cells. Our findings indicate that TMEM211 regulates epithelial–mesenchymal transition for metastasis through coactivating the ERK, AKT and NF-κB signaling pathways, which might provide a potential prognostic biomarker or therapeutic target for colon cancer patients in the future. Full article

Among genetically engineered mouse models of breast cancer, MMTV-PyVT is a mouse strain in which the oncogenic polyoma virus middle T antigen is driven by the mouse mammary tumor virus promoter. The aim of the present study was to perform morphologic and genetic analyses of mammary tumors arising from MMTV-PyVT mice. To this end, mammary tumors were obtained at 6, 9, 12, and 16 weeks of age for histology and whole-mount analyses. We conducted whole-exome sequencing to identify constitutional and tumor-specific mutations, and genetic variants were identified using the GRCm38/mm10 mouse reference genome. Using hematoxylin and eosin analysis and whole-mount carmine alum staining, we demonstrated the progressive proliferation and invasion of mammary tumors. Frameshift insertions/deletions (indels) were noted in the Muc4. Mammary tumors showed small indels and nonsynonymous single-nucleotide variants but no somatic structural alterations or copy number variations. In summary, we validated MMTV-PyVT transgenic mice as a multistage model for mammary carcinoma development and progression. Our characterization may be used as a reference for guidance in future research. Full article

It is well-established that tumor antigens and molecules expressed and secreted by cancer cells trigger innate and adaptive immune responses. These two types of anti-tumor immunity lead to the infiltration of the tumor’s microenvironment by immune cells with either regulatory or cytotoxic properties. Whether this response is associated with tumor eradication after radiotherapy and chemotherapy or regrowth has been a matter of extensive research through the years, mainly focusing on tumor-infiltrating lymphocytes and monocytes and their subtypes, and the expression of immune checkpoint and other immune-related molecules by both immune and cancer cells in the tumor microenvironment. A literature search has been conducted on studies dealing with the immune response in patients with rectal cancer treated with neoadjuvant radiotherapy or chemoradiotherapy, assessing its impact on locoregional control and survival and underlying the potential role of immunotherapy in the treatment of this cancer subtype. Here, we provide an overview of the interactions between local/systemic anti-tumor immunity, cancer-related immune checkpoint, and other immunological pathways and radiotherapy, and how these affect the prognosis of rectal cancer patients. Chemoradiotherapy induces critical immunological changes in the tumor microenvironment and cancer cells that can be exploited for therapeutic interventions in rectal cancer. Full article

Parkinson’s disease is a severe neurodegenerative disorder. Currently, deep brain electrical stimulation (DBS) is the first line of surgical treatment. However, serious neurological impairments such as speech disorders, disturbances of consciousness, and depression after surgery limit the efficacy of treatment. In this review, we summarize the recent experimental and clinical studies that have explored the possible causes of neurological deficits after DBS. Furthermore, we tried to identify clues from oxidative stress and pathological changes in patients that could lead to the activation of microglia and astrocytes in DBS surgical injury. Notably, reliable evidence supports the idea that neuroinflammation is caused by microglia and astrocytes, which may contribute to caspase-1 pathway-mediated neuronal pyroptosis. Finally, existing drugs and treatments may partially ameliorate the loss of neurological function in patients following DBS surgery by exerting neuroprotective effects. Full article

Mitochondria have made a long evolutionary path from ancient bacteria immigrants within the eukaryotic cell to become key players for the cell, assuming crucial multitasking skills critical for human health and disease. Traditionally identified as the powerhouses of eukaryotic cells due to their central role in energy metabolism, these chemiosmotic machines that synthesize ATP are known as the only maternally inherited organelles with their own genome, where mutations can cause diseases, opening up the field of mitochondrial medicine. More recently, the omics era has highlighted mitochondria as biosynthetic and signaling organelles influencing the behaviors of cells and organisms, making mitochondria the most studied organelles in the biomedical sciences. In this review, we will especially focus on certain ‘novelties’ in mitochondrial biology “left in the shadows” because, although they have been discovered for some time, they are still not taken with due consideration. We will focus on certain particularities of these organelles, for example, those relating to their metabolism and energy efficiency. In particular, some of their functions that reflect the type of cell in which they reside will be critically discussed, for example, the role of some carriers that are strictly functional to the typical metabolism of the cell or to the tissue specialization. Furthermore, some diseases in whose pathogenesis, surprisingly, mitochondria are involved will be mentioned. Full article

Rapeseed is one of the most important oil crops in the world. Increasing demand for oil and limited agronomic capabilities of present-day rapeseed result in the need for rapid development of new, superior cultivars. Double haploid (DH) technology is a fast and convenient approach in plant breeding as well as genetic research. Brassica napus is considered a model species for DH production based on microspore embryogenesis; however, the molecular mechanisms underlying microspore reprogramming are still vague. It is known that morphological changes are accompanied by gene and protein expression patterns, alongside carbohydrate and lipid metabolism. Novel, more efficient methods for DH rapeseed production have been reported. This review covers new findings and advances in Brassica napus DH production as well as the latest reports related to agronomically important traits in molecular studies employing the double haploid rapeseed lines. Full article

Kernel number per row (KNR) is an essential component of maize (Zea mays L.) grain yield (GY), and understanding its genetic mechanism is crucial to improve GY. In this study, two F₇ recombinant inbred line (RIL) populations were created using a temperate–tropical introgression line TML418 and a tropical inbred line CML312 as female parents and a backbone maize inbred line Ye107 as the common male parent. Bi-parental quantitative trait locus (QTL) mapping and genome-wide association analysis (GWAS) were then performed on 399 lines of the two maize RIL populations for KNR in two different environments using 4118 validated single nucleotide polymorphism (SNP) markers. This study aimed to: (1) detect molecular markers and/or the genomic regions associated with KNR; (2) identify the candidate genes controlling KNR; and (3) analyze whether the candidate genes are useful in improving GY. The authors reported a total of 7 QTLs tightly linked to KNR through bi-parental QTL mapping and identified 21 SNPs significantly associated with KNR through GWAS. Among these, a highly confident locus qKNR7-1 was detected at two locations, Dehong and Baoshan, with both mapping approaches. At this locus, three novel candidate genes (Zm00001d022202, Zm00001d022168, Zm00001d022169) were identified to be associated with KNR. These candidate genes were primarily involved in the processes related to compound metabolism, biosynthesis, protein modification, degradation, and denaturation, all of which were related to the inflorescence development affecting KNR. These three candidate genes were not reported previously and are considered new candidate genes for KNR. The progeny of the hybrid Ye107 × TML418 exhibited strong heterosis for KNR, which the authors believe might be related to qKNR7-1. This study provides a theoretical foundation for future research on the genetic mechanism underlying KNR in maize and the use of heterotic patterns to develop high-yielding hybrids. Full article