A Deeper Insight into COL4A3, COL4A4, and COL4A5 Variants and Genotype-Phenotype Correlation of a Turkish Cohort with Alport Syndrome.

Introduction: Alport syndrome (AS) is an inherited, rare, progressive kidney disease that affects the eye and ear physiology. Pathogenic variants of COL4A5 account for 85% of all cases, while COL4A3 and COL4A4 account for the remaining 15%. Methods: Targeted next-generation sequencing of the COL4A3, COL4A4, and COL4A5 genes was performed in 125 Turkish patients with AS. The patients were compared to 45 controls and open-access population data. Results: The incidence of AS variants in patients was found as 21.6%. 27 variants were identified as pathogenic/likely pathogenic, 28 as variant of uncertain significance, and 52 as benign/likely benign. We also found 31 novel variants (14 in COL4A3, 6 in COL4A4, and 11 in COL4A5) of which 27 were classified as pathogenic/likely pathogenic. Pathogenic/likely Pathogenic variants were most commonly found in the COL4A5 gene, consistent with the literature. This study contributed novel variants associated with AS to the literature. Conclusion: Genetic testing is a crucial part for the diagnosis and management of AS. Studies on the genetic etiology of AS are limited for the Turkish population. We believe that this study will contribute to the literature and the clinical decision-making process of patients with AS and emphasize the importance of genetic counseling.

Combination of an Oxindole Derivative with (-)-ß-Elemene Alters Cell Death Pathways in FLT3/ITD+ Acute Myeloid Leukemia Cells.

Acute myeloid leukemia (AML) is one of the cancers that grow most aggressively. The challenges in AML management are huge, despite many treatment options. Mutations in FLT3 tyrosine kinase receptors make the currently available therapies less responsive. Therefore, there is a need to find new lead molecules that can specifically target mutated FLT3 to block growth factor signaling and inhibit AML cell proliferation. Our previous studies on FLT3-mutated AML cells demonstrated that ß-elemene and compound 5a showed strong inhibition of proliferation by blocking the mutated FLT3 receptor and altering the key apoptotic genes responsible for apoptosis. Furthermore, we hypothesized that both ß-elemene and compound 5a could be therapeutically effective. Therefore, combining these drugs against mutated FLT3 cells could be promising. In this context, dose-matrix combination-based cellular inhibition analyses, cell morphology studies and profiling of 43 different apoptotic protein targets via combinatorial treatment were performed. Our studies provide strong evidence for the hypothesis that ß-elemene and compound 5a combination considerably increased the therapeutic potential of both compounds by enhancing the activation of several key targets implicated in AML cell death.

The evidence for repurposing anti-epileptic drugs to target cancer.

Antiepileptic drugs are versatile drugs with the potential to be used in functional drug formulations with drug repurposing approaches. In the present review, we investigated the anticancer properties of antiepileptic drugs and interlinked cancer and epileptic pathways. Our focus was primarily on those drugs that have entered clinical trials with positive results and those that provided good results in preclinical studies. Many contributing factors make cancer therapy fail, like drug resistance, tumor heterogeneity, and cost; exploring all alternatives for efficient treatment is important. It is crucial to find new drug targets to find out new antitumor molecules from the already clinically validated and approved drugs utilizing drug repurposing methods. The advancements in genomics, proteomics, and other computational approaches speed up drug repurposing. This review summarizes the potential of antiepileptic drugs in different cancers and tumor progression in the brain. Valproic acid, oxcarbazepine, lacosamide, lamotrigine, and levetiracetam are the drugs that showed potential beneficial outcomes against different cancers. Antiepileptic drugs might be a good option for adjuvant cancer therapy, but there is a need to investigate further their efficacy in cancer therapy clinical trials.

Vanillin-Based Indolin-2-one Derivative Bearing a Pyridyl Moiety as a Promising Anti-Breast Cancer Agent via Anti-Estrogenic Activity.

The structure-based design introduced indoles as an essential motif in designing new selective estrogen receptor modulators employed for treating breast cancer. Therefore, here, a series of synthesized vanillin-substituted indolin-2-ones were screened against the NCI-60 cancer cell panel followed by in vivo, in vitro, and in silico studies. Physicochemical parameters were evaluated with HPLC and SwissADME tools. The compounds demonstrated promising anti-cancer activity for the MCF-7 breast cancer cell line (GI = 6-63%). The compound with the highest activity (6j) was selective for the MCF-7 breast cancer cell line (IC50 = 17.01 µM) with no effect on the MCF-12A normal breast cell line supported by real-time cell analysis. A morphological examination of the used cell lines confirmed a cytostatic effect of compound 6j. It inhibited both in vivo and in vitro estrogenic activity, triggering a 38% reduction in uterine weight induced by estrogen in an immature rat model and hindering 62% of ER-α receptors in in vitro settings. In silico molecular docking and molecular dynamics simulation studies supported the stability of the ER-α and compound 6j protein-ligand complex. Herein, we report that indolin-2-one derivative 6j is a promising lead compound for further pharmaceutical formulations as a potential anti-breast cancer drug.

Discovery of oxindole-based FLT3 inhibitors as a promising therapeutic lead for acute myeloid leukemia carrying the oncogenic ITD mutation.

FMS-like tyrosine kinase 3 (FLT3) mutations occur in approximately 30% of acute myeloid leukemia (AML) patients. In the current study, the oxindole chemotype is employed as a structural motif for the design of new FLT3 inhibitors as potential hits for AML irradiation. Cell-based screening was performed with 18 oxindole derivatives and 5a-c inhibited 68%-73% and 83%-91% of internal tandem duplication (ITD)-mutated MV4-11 cell growth for 48- and 72-h treatments while only 0%-2% and 27%-39% in wild-type THP-1 cells. The most potent compound 5a inhibited MV4-11 cells with IC50 of 4.3 µM at 72 h while it was 8.7 µM in THP-1 cells, thus showing two-fold selective inhibition against the oncogenic ITD mutation. The ability of 5a to modulate cell death was examined. High-throughput protein profiling revealed low levels of the growth factors IGFBP-2 and -4 with the blockage of various apoptotic inhibitors such as Survivin. p21 with cellular stress mechanisms was characterized by increased expression of HSP proteins along with TNF-ß. Mechanistically, compounds 5a and 5b inhibited FLT3 kinase with IC50 values of 2.49 and 1.45 µM, respectively. Theoretical docking studies supported the compounds' ability to bind to the FLT3 ATP binding site with the formation of highly stable complexes as evidenced by molecular dynamics simulations. The designed compounds also provide suitable drug candidates with no violation of drug likeability rules.

The SARS-CoV-2 mutations versus vaccine effectiveness: New opportunities to new challenges.

BACKGROUND: The SARS-CoV-2 coronavirus epidemic is hastening the discovery of the most efficient vaccines. The development of cost-effective vaccines seems to be the only solution to terminate this pandemic. However, the vaccines' effectiveness has been questioned due to recurrent mutations in the SARS-CoV-2 genome. Most of the mutations are associated with the spike protein, a vital target for several marketed vaccines. Many countries were highly affected by the 2nd wave of the SARS-CoV-2, like the UK, India, Brazil and France. Experts are also alarming the further COVID-19 wave with the emergence of Omicron, which is highly affecting the South African populations. This review encompasses the detailed description of all vaccine candidates and COVID-19 mutants that will add value to design further studies to combat the COVID-19 pandemic. METHODS: The information was generated using various search engines like google scholar, PubMed, clinicaltrial.gov.in, WHO database, ScienceDirect, and news portals by using keywords SARS-CoV-2 mutants, COVID-19 vaccines, efficacy of SARS-CoV-2 vaccines, COVID-19 waves. RESULTS: This review has highlighted the evolution of SARS-CoV-2 variants and the vaccine efficacy. Currently, various vaccine candidates are undergoing several phases of development. Their efficacy still needs to check for newly emerged variants. We have focused on the evolution, multiple mutants, waves of the SARS-CoV-2, and different marketed vaccines undergoing various clinical trials and the design of the trials to determine vaccine efficacy. CONCLUSION: Various mutants of SARS-CoV-2 arrived, mainly concerned with the spike protein, a key component to design the vaccine candidates. Various vaccines are undergoing clinical trial and show impressive results, but their efficacy still needs to be checked in different SARS-CoV-2 mutants. We discussed all mutants of SARS-CoV-2 and the vaccine's efficacy against them. The safety concern of these vaccines is also discussed. It is important to understand how coronavirus gets mutated to design better new vaccines, providing long-term protection and neutralizing broad mutant variants. A proper study approach also needs to be considered while designing the vaccine efficacy trials, which further improved the study outcomes. Taking preventive measures to protect from the virus is also equally important, like vaccine development.

Drugs repurposed: An advanced step towards the treatment of breast cancer and associated challenges.

Breast cancer (BC) is mostly observed in women and is responsible for huge mortality in women subjects globally. Due to the continued development of drug resistance and other contributing factors, the scientific community needs to look for new alternatives, and drug repurposing is one of the best opportunities. Here we light upon the drug repurposing with a major focus on breast cancer. BC is a division of cancer known as the leading cause of death of 2.3 million women globally, with 685,000 fatalities. This number is steadily rising, necessitating the development of a treatment that can extend survival time. All available treatments for BC are very costly as well as show side effects. This unfulfilled requirement of the anti-cancer drugs ignited an enthusiasm for drug repositioning, which means finding out the anti-cancer use of already marketed drugs for other complications. With the advancement in proteomics, genomics, and computational approaches, the drug repurposing process hastens. So many drugs are repurposed for the BC, including alkylating agents, antimetabolite, anthracyclines, an aromatase inhibitor, mTOR, and many more. The drug resistance in breast cancer is rising, so reviewing how the challenges in breast cancer can be combated with drug repurposing. This paper provides the updated information on all the repurposed drugs candidates for breast cancer with the molecular mechanism responsible for their anti-tumor activity. Additionally, all the challenges that occur during the repurposing of the drugs are discussed.

Role of autophagy and evaluation the effects of microRNAs 214, 132, 34c and prorenin receptor in a rat model of focal segmental glomerulosclerosis.

AIMS: Focal segmental glomerulosclerosis (FSGS) is the common cause of chronic renal disease worldwide. Although there are many etiologic factors which have common theme of podocyte injury conclusive etiology is not clearly understood. In this study, we aimed to explore the role of autophagy in the pathogenesis of podocyte injury, which is the key point in disease progression, and the roles of intrarenal microRNAs and the prorenin receptor (PRR) in the 5/6 nephrectomy and adriamycin nephropathy models of FSGS. MAIN METHODS: For experimental FSGS model, 5/6 nephrectomy and adriamycin nephropathy models were created and characterized in adult Sprague Dawley rats. Microarray analysis was performed on FSGS and control groups that was confirmed by q-RT-PCR. Beclin1, LC3B, PRR, ATG7 and ATG5 expression were evaluated by western blotting and immunohistochemistry. Also, Beclin1 and PRR expression were measured by ELISA. Glomerular podocyte isolation was performed and autophagic activity was evaluated in podocytes before and after transfection with miRNA mimic and antagonists. KEY FINDINGS: Glomerular expression of Beclin1, LC3B, PRR, ATG7 and ATG5 were significantly lower in the 5/6 nephrectomy than adriamycin nephropathy group and in both groups lower when compared to control groups. Western blot results were consistent with immunohistochemical data. Electron microscopy revealed signs of impaired autophagy in FSGS. Autophagic activity decreased significantly after miR-214, miR-132 and miR-34c mimics and increased after transfection with antagonists. SIGNIFICANCE: These results showed that the role of autophagic activity and decreased expression of PRR in FSGS pathogenesis and miR-34c, miR-132 and miR-214 could be a potential treatment strategy by regulating autophagy.

Chemical, biological and molecular modelling analyses to probe into the pharmacological potential of Antidesma madagascariense Lam.: A multifunctional agent for developing novel therapeutic formulations.

Antidesma madagascariense Lam. (AM), an indigenous medicinal plant to the Mascarene Islands, is used for the treatment of several diseases. We endeavoured to validate its use via evaluating the kinetics of inhibition of crude aqueous extract (CAE) and crude methanol extract (CME) of AM against key metabolic enzymes (pancreatic lipase, cholesterol esterase [CEase], acetylcholinesterase [AChE], and urease). In vitro antiglycation, antioxidant, cytotoxicity using iCELLigence real time cell analysis system and WST-1 methods, were used. LC-ESI-MS/MS was employed to determine the phenolic composition of the extracts and interaction of selected compounds to the studied enzymes was determined using in silico docking. AChE was inhibited by the CME of AM and CEase by the CAE. Both extracts were active inhibitors of urease and pancreatic lipase. Hyperoside (271.97 µg/g extract), present in large amount in the CME, docked to the enzymatic pocket of urease and CEase. The extracts showed competitive and mixed inhibition of urease and pancreatic lipase, respectively. The antioxidant capacity of the CME (6.61 µg GAE/mg crude extract) was higher compared to CAE (2.20 µg GAE/mg crude extract). AM extracts were significantly (p < 0.05) less potent than aminoguanidine in preventing advanced glycation end products formation. Toxicological screening revealed that both extracts were non-toxic on HEK-293 cells. AM crude extracts at concentrations ranging from 78 to 312 µg/ml did not cause a visible change in cell morphology compared to control. This study supports the safe use of AM as a biomedicine for the management and/or treatment of common non-communicable diseases.

Integration of in vitro and in silico perspectives to explain chemical characterization, biological potential and anticancer effects of Hypericum salsugineum: A pharmacologically active source for functional drug formulations.

The genus Hypericum is one of the most popular genera in both traditional medicine and scientific platform. This study is designed to provide conceptual insights on the biological potential and chemical characterization of H. salsugineum, which is endemic to Turkey. The qualitative and quantitative phenolic content of the extracts was characterized by HPLC-ESI-MSn. Biological efficiency was investigated by enzyme inhibitory assays (cholinesterases, tyrosinase, amylase, and glucosidase) and anti-cancer efficacy tests (anti-proliferative activities with the iCELLigence technology, colony formation and wound healing scratch assays). Phenolic acids (3-O-caffeoylquinic, 5-O-caffeoylquinic, and 4-O-caffeoylquinic acids) were the predominant group in the studied extracts, although several flavonoids were also detected and quantified. The extracts exhibited good inhibitory effects on tyrosinase and glucosidase, while they had weak ability against cholinesterases and amylase. Computational studies were also performed to explain the interactions between the major phenolics and these enzymes. The extracts displayed significant anti-cancer effects on breast carcinoma cell lines. Our findings suggest that Hypericum salsugineum could be valued as a potential source of biologically-active compounds for designing novel products.

UHPLC-QTOF-MS analysis of bioactive constituents from two Romanian Goji (Lycium barbarum L.) berries cultivars and their antioxidant, enzyme inhibitory, and real-time cytotoxicological evaluation.

Goji (Lycium barbarum L.) berries are emphasized as healthy food or are used widely as dietary supplements. In the present study, the antioxidant and enzyme inhibitory activities of berries extracts from two selected Romanian cultivars (cv. Erma and cv. Biglifeberry) have been evaluated. UHPLC-QTOF-MS analysis results revealed the presence of organic acids, phenolic acids, flavonoids, fatty acids (oxylipins), and spermidine derivatives. In particular, cv. Erma showed an important tyrosinase-inhibitory effect, whereas, cv. Biglifeberry had a superior antioxidant capacity. Particularly, results provided by the CUPRAC assay showed the highest antioxidant capacity values (26.91 mg TE/g and 35.41 mg TE/g, for 'Erma' and 'Biglifeberry', respectively). Toxicological properties of the extracts were evaluated with human embryonic kidney cells (HEK-293) using the cytotoxicity analysis platform iCELLigence, real-time and label-free impedance technology. No cytotoxic effects were observed in a time- and dose-dependent manner. Overall, Goji berries are a rich source of bioactive compounds with functional properties that need further risk/benefit evaluation when used in foods or health-promoting formulations.

Functional analysis of ESM1 by siRNA knockdown in primary and metastatic head and neck cancer cells.

BACKGROUND: Genetic factors play a large role in cancer, and thus, there is a great desire to understand the effects of different genes in cancer and to also develop gene therapy for better treatments. Therefore, the development of alternative diagnosis and therapy modalities is of utmost importance. The aim of our study was to illuminate the role of ESM1 (endothelial cell-specific molecule-1, also known as Endocan) in proliferation and migration of head and neck cancer, thus helping to pave the way for new treatment modalities and predictive biomarkers. METHODS: ESM1 expression was shown with immunofluorescence assay using confocal laser scanning microscope in primary and metastatic head and neck cancer cells. ESM1 expression was knocked down by RNA interference in head and neck cancer cells. Knockdown efficiency was evaluated by quantitative real-time RT-PCR and Western blot. Cell proliferation and migration assays were performed by xCELLigence real-time cell analysis system. RESULTS: Immunofluorescence assay showed nuclear localization and high expression of ESM1 in primary and metastatic head and neck cancer cells. ESM1 mRNA and protein levels were significantly decreased in ESM1-knockdown cells compared to control. ESM1-knockdown cells showed reduced proliferation and migration activity when compared to control cells. CONCLUSION: These findings suggest that ESM1 has roles on proliferation and migration of head and neck cancer cells.

Traditionally Used Lathyrus Species: Phytochemical Composition, Antioxidant Activity, Enzyme Inhibitory Properties, Cytotoxic Effects, and in silico Studies of L. czeczottianus and L. nissolia.

Members of the genus Lathyrus are used as food and as traditional medicines. In order to find new sources of biologically-active compounds, chemical and biological profiles of two Lathyrus species (L. czeczottianus and L. nissolia) were investigated. Chemical profiles were evaluated by HPLC-ESI-MSn, as well as by their total phenolic and flavonoid contents. In addition, antioxidant, enzyme inhibitory, and cytotoxic effects were also investigated. Antioxidant properties were tested by using different assays (DPPH, ABTS, CUPRAC, FRAP, phosphomolybdenum, and metal chelation). Cholinesterases (AChE and BChE), tyrosinase, α-amylase, and α-glucosidase were used to evaluate enzyme inhibitory effects. Moreover, vitexin (apigenin-8-C-glucoside) and 5-O-caffeoylquinic acid were further subjected to molecular docking experiments to provide insights about their interactions at molecular level with the tested enzymes. In vitro cytotoxic effects were examined against human embryonic kidney cells (HEK293) by using iCELLigence real time cell analysis system. Generally, L. czeczottianus exhibited stronger antioxidant properties than L. nissolia. However, L. nissolia had remarkable enzyme inhibitory effects against cholinesterase, amylase and glucosidase. HPLC-ESI-MSn analysis revealed that flavonoids were major components in these extracts. On the basis of these results, Lathyrus extracts were rich in biologically active components; thus, these species could be utilized to design new phytopharmaceutical and nutraceutical formulations.

Multiple pharmacological targets, cytotoxicity, and phytochemical profile of Aphloia theiformis (Vahl.) Benn.

Aphloia theiformis (Vahl.) Benn. (AT) is traditionally used in Sub-Saharan African countries including Mauritius as a biomedicine for the management of several diseases. However, there is a dearth of experimental studies to validate these claims. We endeavoured to evaluate the inhibitory effects of crude aqueous extract as traditionally used together with the crude methanol extracts of AT leaves on urease, angiotensin (I) converting enzyme (ACE), acetylcholinesterase (AChE), cholesterol esterase (CEase), glycogen phosphorylase a (GPa), and glycation in vitro. The crude extract showing potent activity against the studied enzymes was further partitioned using different solvents of increasing polarity. The enzyme inhibitory and antiglycation activities of each fraction was assessed. Kinetic of inhibition of the active crude extract/fractions on the aforementioned enzymes was consequently determined using Lineweaver-Burk plots. An ultra-high performance liquid chromatography (UHPLC-UV/MS) system was used to establish the phytochemical profile of AT. The real time cell analysis system (iCELLigence™) was used to monitor any cellular cytotoxicity of AT. Crude methanolextract (CME) was a potent inhibitor of the studied enzymes, with IC50 ranging from 696.22 to 19.73µg/mL. CME (82.5%) significantly (p<0.05) inhibited glycation and was comparable to aminoguanidine (81.5%). Ethyl acetate and n-butanol fractions of CME showed non-competitive, competitive, and uncompetitive mode of inhibition against ACE, CEase, and AChE respectively. Mangiferin, a xanthone glucoside was present in CME, ethyl acetate, and n-butanol fractions. Active extract/fractions were found to be non-cytotoxic (IC50>20µg/mL) according to the U.S National Cancer Institute plant screening program. This study has established baseline data that tend to justify the traditional use of AT and open new avenues for future biomedicine development.