Homozygous missense variant in POPDC3 causes recessive limb-girdle muscular dystrophy type 26.

BACKGROUND: Limb-girdle muscular dystrophy (LGMD) comprises a heterogeneous group of diseases, affecting different muscles, predominantly skeletal muscles and cardiac muscles of the body. LGMD is classified into two main subtypes A and B, which are further subclassified into eight dominant and thirty recessive subtypes. Three genes, namely POPDC1, POPDC2 and POPDC3, encode popeye domain-containing protein (POPDC), and the variants of POPDC1 and POPDC3 genes have been associated with LGMD. METHODS: In the present study, we performed whole-exome sequencing (WES) analysis on a single-family to investigate the hallmark features of LGMD. The results of WES were further confirmed by Sanger sequencing and 3D protein modeling was also conducted. RESULTS: WES data analysis and Sanger sequencing revealed a homozygous missense variant (c.460A>G; p.Lys154Glu) at a highly conserved amino acid position in the POPDC3. Mutations in the POPDC3 gene have been previously associated with recessive limb-girdle muscular dystrophy type 26. 3D protein modeling further suggested that the identified variant might affect the POPDC3 structure and proper function. CONCLUSIONS: The present study confirms the role of POPDC3 in LGMD, and will facilitate genetic counseling of the family to mitigate the risks of the carrier or affects on future pregnancies.

Targeting Cytotoxin-Associated Antigen A, a Virulent Factor of Helicobacter pylori-Associated Gastric Cancer: Structure-Based In Silico Screening of Natural Compounds.

Gastric cancer is the fifth most frequent cancer and the third major cause of mortality worldwide. Helicobacter pylori, a bacterial infection linked with GC, injects the cytotoxin-associated antigen A (CagA; an oncoprotein) into host cells. When the phosphorylated CagA protein enters the cell, it attaches to other cellular components, interfering with normal cellular signaling pathways. CagA plays an important role in the progression of GC by interacting with phosphatidylserine of the host cell membrane. Therefore, disrupting the CagA-phosphatidylserine connection using small molecules appears to be a promising therapeutic approach. In this report, we screened the natural compounds from ZINC database against the CagA protein using the bioinformatics tools. Hits were initially chosen based on their physicochemical, absorption, distribution, metabolism, excretion, and toxicity (ADMET) characteristics, as well as other drug-like characteristics. To locate safe and effective hits, the PAINS filter, binding affinities estimation, and interaction analysis were used. Three compounds with high binding affinity and specificity for the CagA binding pocket were discovered. The final hits, ZINC153731, ZINC69482055, and ZINC164387, were found to bind strongly with CagA protein, with binding energies of -11.53, -10.67, and -9.21 kcal/mol, respectively, which were higher than that of the control compound (-7.25 kcal/mol). Further, based on binding affinity and interaction pattern, two leads (ZINC153731, ZINC69482055) were chosen for molecular dynamics (MD) simulation analysis. MD results showed that they displayed stability in their vicinity at 100 ns. This study suggested that these compounds could be used as possible inhibitors of CagA protein in the fight against GC. However, additional benchwork tests are required to validate them as CagA protein inhibitors.

Biocomputational Prediction Approach Targeting FimH by Natural SGLT2 Inhibitors: A Possible Way to Overcome the Uropathogenic Effect of SGLT2 Inhibitor Drugs.

The Food and Drug Administration (FDA) approved a new class of anti-diabetic medication (a sodium-glucose co-transporter 2 (SGLT2) inhibitor) in 2013. However, SGLT2 inhibitor drugs are under evaluation due to their associative side effects, such as urinary tract and genital infection, urinary discomfort, diabetic ketosis, and kidney problems. Even clinicians have difficulty in recommending it to diabetic patients due to the increased probability of urinary tract infection. In our study, we selected natural SGLT2 inhibitors, namely acerogenin B, formononetin, (-)-kurarinone, (+)-pteryxin, and quinidine, to explore their potential against an emerging uropathogenic bacterial therapeutic target, i.e., FimH. FimH plays a critical role in the colonization of uropathogenic bacteria on the urinary tract surface. Thus, FimH antagonists show promising effects against uropathogenic bacterial strains via their targeting of FimH's adherence mechanism with less chance of resistance. The molecular docking results showed that, among natural SGLT2 inhibitors, formononetin, (+)-pteryxin, and quinidine have a strong interaction with FimH proteins, with binding energy (∆G) and inhibition constant (ki) values of -5.65 kcal/mol and 71.95 µM, -5.50 kcal/mol and 92.97 µM, and -5.70 kcal/mol and 66.40 µM, respectively. These interactions were better than those of the positive control heptyl α-d-mannopyranoside and far better than those of the SGLT2 inhibitor drug canagliflozin. Furthermore, a 50 ns molecular dynamics simulation was conducted to optimize the interaction, and the resulting complexes were found to be stable. Physicochemical property assessments predicted little toxicity and good drug-likeness properties for these three compounds. Therefore, formononetin, (+)-pteryxin, and quinidine can be proposed as promising SGLT2 inhibitors drugs, with add-on FimH inhibition potential that might reduce the probability of uropathogenic side effects.

Multitargeted inhibitory effect of Mitoxantrone 2HCl on cervical cancer cell cycle regulatory proteins: a multitargeted docking-based MM\GBSA and MD simulation study.

Cervical cancer remains a significant global health concern that starts in the cervix, the lower part of the uterus that connects to the vagina and is caused by the human papillomavirus (HPV), necessitating the development of effective multitargeted effective and resistance-proof therapies. In early-stage cervical cancer may not show any symptoms, however, as the cancer progresses, some people may experience- abnormal vaginal bleeding, watery or bloody vaginal discharge, pain in the pelvis or lower back, pain during sex, and frequent and painful urination. In this study, we screened the complete FDA-approved drug library using a multitargeted inhibitory approach against four cervical cancer proteins, namely mitotic arrest deficient -2, DNA polymerase epsilon B-subunit, benzimidazole-related -1, and threonine-protein kinase-1 which crucially plays its role for the in its development process. We employed the HTVS, SP and XP algorithms for efficient filtering and screening that helped to identify Mitoxantrone 2HCl against all of them with docking and MM\GBSA scores ranging from - 11.63 to - 7.802 kcal/mol and - 74.38 to - 47.73 kcal/mol, respectively. We also evaluated the interaction patterns of each complex and the pharmacokinetics properties that helped gain insight into interactions. Subsequently, we performed multiscale MD simulations for 100 ns to understand the dynamic behaviour and stability of the Mitoxantrone 2HCl -protein complexes that revealed the formation of stable drug-protein complexes and provided insights into the molecular interactions that contribute to Mitoxantrone's inhibitory effects on these proteins and can be a better drug for cervical cancer. However, experimental studies of these findings could pave the way for therapies to combat cervical cancer effectively.

Nanocarriers for anticancer drugs: Challenges and perspectives.

Cancer is the second most common cause of death globally, surpassed only by cardiovascular disease. One of the hallmarks of cancer is uncontrolled cell division and resistance to cell death. Multiple approaches have been developed to tackle this disease, including surgery, radiotherapy and chemotherapy. Although chemotherapy is used primarily to control cell division and induce cell death, some cancer cells are able to resist apoptosis and develop tolerance to these drugs. The side effects of chemotherapy are often overwhelming, and patients can experience more adverse effects than benefits. Furthermore, the bioavailability and stability of drugs used for chemotherapy are crucial issues that must be addressed, and there is therefore a high demand for a reliable delivery system that ensures fast and accurate targeting of treatment. In this review, we discuss the different types of nanocarriers, their properties and recent advances in formulations, with respect to relevant advantages and disadvantages of each.

Vorinostat enhances the therapeutic potential of Erlotinib via MAPK in lung cancer cells.

BACKGROUND: Lung cancer is the second most common cancer in both men and women, with an estimated 235,760 new cases and 131,880 deaths in 2021 in the US. Despite the modern therapies being available such as radiotherapy and chemotherapy, death rates are still increasing. Erlotinib (ERL) is one of the treatment options for lung cancer, although the probability for the patients to develop resistance to ERL constrains its reliability. The aim of the present study is to assess the synergetic effect of combining ERL with vorinostat (SAHA) on the progression of lung cancer cells. RESULTS: Adenocarcinoma alveolar basal epithelial cells (A549) were treated with either ERL, SAHA as mono drugs or with the combination of them for 24 h. Cytotoxicity assay and cell cycle analysis along with apoptosis detection were investigated. The expression profile of CDH1, TGF1, and MAPK was also assessed. Results showed an elevation in the apoptosis level in all treatments compared to WISH; the normal human amnion-derived cells. Furthermore, the treatments caused the cell cycle to arrest at G2/M, indicating its cytotoxic activity. CONCLUSION: The combination of SAHA and ERL significantly increased the level of apoptosis in lung cancer cells. Meanwhile, this combination treatment downregulated MAPK compared to the mono drugs and the control cells, suggesting the potential role of MAPK in regulating apoptosis and cell cycle machinery in lung cancer.

Template-free preparation of iron oxide loaded hollow silica spheres and their anticancer proliferation capabilities.

Hollow silica spheres (HSS) exhibited high-specific surface area, low toxicity, low density, and good biocompatibility. The effectivity of HSS material can be improved further by loading nanoparticles for smart biological applications. In this work, magnetic nanoparticle (iron oxide; Fe3O4)-loaded pure HSS (c-HSS-Fe) were synthesized successfully using a template-free chemical route and investigated for their anticancer cell proliferation capabilities against cancerous cell lines: human colorectal carcinoma cells (HCT-116). The structure, morphology, chemical bonding, and thermal stability of the prepared HSS derivatives were studied using spectroscopic and microscopic techniques. Our analyses confirmed the successful preparation of Fe3O4 loaded HSS material (sphere diameter ∼515 nm). The elemental analysis revealed the existence of Fe along with Si and O in the Fe3O4 loaded HSS material, thus reaffirming the production of the c-HSS-Fe product. The effects of silica spheres on HCT-116 cells were examined microscopically and by MTT assays. It was observed that the c-HSS-Fe demonstrated dose-dependent behavior and significantly reduced the cancer cell proliferation at higher doses. Our results showed that c-HSS-Fe was more effective and profound in reducing the cancer cells' activities as compared to unloaded HSS material where the cancer cells have undergone nuclear disintegration and fragmentation. It is concluded that c-HSS-Fe is a powerful bio-active material against cancerous cells.

Novel Feather Degrading Keratinases from Bacillus cereus Group: Biochemical, Genetic and Bioinformatics Analysis.

In this study, five keratinolytic bacteria were isolated from poultry farm waste of Eastern Province, Saudi Arabia. The highest keratinase activity was obtained at 40-45 °C, pH 8-9, feather concentration 0.5-1%, and using white chicken feather as keratin substrate for 72 h. Enhancement of keratinase activity through physical mutagen UV radiation and/or chemical mutagen ethyl methanesulfonate (EMS) resulted in five mutants with 1.51-3.73-fold increased activity over the wild type. When compared with the wild type, scanning electron microscopy validated the mutants' effectiveness in feather degradation. Bacterial isolates are classified as members of the S8 family peptidase Bacillus cereus group based on sequence analysis of the 16S rRNA and keratinase genes. Interestingly, keratinase KerS gene shared 95.5-100% identity to keratinase, thermitase alkaline serine protease, and thermophilic serine protease of the B. cereus group. D137N substitution was observed in the keratinase KerS gene of the mutant strain S13 (KerS13uv+ems), and also seven substitution variations in KerS26 and KerS26uv of strain S26 and its mutant S26uv. Functional analysis revealed that the subtilisin-like serine protease domain containing the Asp/His/Ser catalytic triad of KerS gene was not affected by the predicted substitutions. Prediction of physicochemical properties of KerS gene showed instability index between 17.5-19.3 and aliphatic index between 74.7-75.7, which imply keratinase stability and significant thermostability. The docking studies revealed the impact of substitutions on the superimposed structure and an increase in binding of mutant D137N of KerS13uv+ems (affinity: -7.17; S score: -6.54 kcal/mol) and seven mutants of KerS26uv (affinity: -7.43; S score: -7.17 kcal/mol) compared to the wild predicted structure (affinity: -6.57; S score: -6.68 kcal/mol). Together, the keratinolytic activity, similarity to thermostable keratinases, and binding affinity suggest that keratinases KerS13uv+ems and KerS26uv could be used for feather processing in the industry.

Case Report: Biallelic Variant in the tRNA Methyltransferase Domain of the AlkB Homolog 8 Causes Syndromic Intellectual Disability.

Intellectual disability (ID) has become very common and is an extremely heterogeneous disorder, where the patients face many challenges with deficits in intellectual functioning and adaptive behaviors. A single affected family revealed severe disease phenotypes such as ID, developmental delay, dysmorphic facial features, postaxial polydactyly type B, and speech impairment. DNA of a single affected individual was directly subjected to whole exome sequencing (WES), followed by Sanger sequencing. Data analysis revealed a novel biallelic missense variant (c.1511G>C; p.(Trp504Ser)) in the ALKBH8 gene, which plays a significant role in tRNA modifications. Our finding adds another variant to the growing list of ALKBH8-associated tRNA modifications causing ID and additional phenotypic manifestations. The present study depicts the key role of the genes associated with tRNA modifications, such as ALKBH8, in the development and pathophysiology of the human brain.

In ovo Inoculation of Bacillus subtilis and Raffinose Affects Growth Performance, Cecal Microbiota, Volatile Fatty Acid, Ileal Morphology and Gene Expression, and Sustainability of Broiler Chickens (Gallus gallus).

Banning antibiotic growth promoters has negatively impacted poultry production and sustainability, which led to exploring efficient alternatives such as probiotics, probiotics, and synbiotics. Effect of in ovo injection of Bacillus subtilis, raffinose, and their synbiotics on growth performance, cecal microbial population and volatile fatty acid concentration, ileal histomorphology, and ileal gene expression was investigated in broilers (Gallus gallus) raised for 21 days. On 300 h of incubation, a total of 1,500 embryonated eggs were equally allotted into 10 groups. The first was non-injected (NC) and the remaining in ovo injected with sterile distilled water (PC), B. subtilis 4 × 105 and 4 × 106 CFU (BS1 and BS2), Raffinose 2 and 3 mg (R1 and R2), B. subtilis 4 × 105 CFU + raffinose 2 mg (BS1R1), B. subtilis 4 × 105 CFU + raffinose 3 mg (BS1R2), B. subtilis 4 × 106 CFU + raffinose 2 mg (BS2R1), and B. subtilis 4 × 106 CFU + raffinose 3 mg (BS2R2). At hatch, 60 chicks from each group were randomly chosen, divided into groups of 6 replicates (10 birds/replicate), and fed with a corn-soybean-based diet. In ovo inoculation of B. subtilis and raffinose alone or combinations significantly improved body weight, feed intake, and feed conversion ratio of 21-day-old broilers compared to NC. Cecal concentrations of butyric, pentanoic, propionic, and isobutyric acids were significantly elevated in R1, R2, BS2R1, and BS2R2, whereas isovaleric and acetic acids were significantly increased in R1 and BS2R1 compared to NC. Cecal microbial population was significantly altered in treated groups. Ileal villus height was increased (p < 0.001) in BS1, R2, and BS2R2 compared to NC. The mRNA expression of mucin-2 was upregulated (p < 0.05) in synbiotic groups except for BS1R1. Vascular endothelial growth factor (VEGF) expression was increased (p < 0.05) in BS2, R1, BS1R1, and BS1R2 compared to NC. SGLT-1 expression was upregulated (p < 0.05) in all treated birds except those of R1 group compared to NC. The mRNA expressions of interleukin (IL)-2 and toll-like receptor (TLR)-4 were downregulated (p < 0.05) in BS2 and R1 for IL-2 and BS1R1 and BS2R2 for TLR-4. It was concluded that in ovo B. subtilis, raffinose, and synbiotics positively affected growth performance, cecal microbiota, gut health, immune responses, and thus the sustainability of production in 21-day-old broilers.

Temozolomide modulates the expression of miRNAs in colorectal cancer.

Cancer is the second leading cause of death globally, where nearly 1 in 6 deaths is due to cancer, with 70% of all deaths from cancer occur in low- and middle-income countries. The overall lifetime risk of developing colorectal cancer is 1 in 22 in men and 1 in 24 in women. In this work, we aimed to evaluate the role of temozolomide (TMZ) in controlling colon cancer cells (CRC) via regulating the miRnome. For this purpose, CRC cells (CaCo-2) were treated with 50 µM of TMZ for 48 h. Cell count using trypan test and cytotoxicity using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were carried out, and the obtained results indicated a significant decrease in cell count (p = 0.029), and in the cell viability (p = 0.0019). Cell cycle analysis was performed using flow cytometer, and results showed that TMZ arrested CRC cells at G2/M phase. A total of 84 miRNAs were profiled using real time PCR, and the results indicated that TMZ treatment upregulated 15 of 84 miRNAs panel profiled and downregulated the rest. The TMZ-upregulated/downregulated miRNAs were predicted to interact with many epigenetic-related proteins i.e., DNMTs, EZH2, and SUV31H1. This study shed some light on the role of TMZ in regulating the miRnome of CRC and hence in different types of cancers.

In vitro and in silico characterization of alkaline serine protease from Bacillus subtilis D9 recovered from Saudi Arabia.

In this study, we have isolated and characterized proteolytic soil bacteria and their alkaline protease. Based on 16S rRNA sequence analysis, 12 isolates with the highest protease activity were classified as B. subtilis and B. cereus groups. B. subtilis D9 isolate showing the highest protease activity was selected for in vitro and in silico analysis for its ِِAKD9 protease. The enzyme has a molecular mass of 48 kDa, exhibiting optimal activity at 50 °C pH 9.5, and showed high stability till 65 °C and pH 8-11 for 1 h. Fe3+ stimulated, but Zn2+ and Hg2+ strongly inhibited the protease activity. Also, the maximum inhibition with PMSF indicated serine protease-type of AKD9 protease. AkD9 alkaline serine protease gene showed high sequence similarity and close phylogenetic relationship with AprX serine protease of B. subtilis isolates. Functional prediction of AKD9 resulted in the detection of subtilase domain, peptidase_S8 family, and subtilase active sites. Moreover, prediction of physicochemical properties indicated that AKD9 serine protease is hydrophilic, thermostable, and alkali-halo stable. Secondary structure prediction revealed the dominance of the coils enhances AKD9 activity and stability under saline and alkaline conditions. Based on molecular docking, AKD9 showed very promising binding affinities towards casein substrate with expected intrinsic proteolytic activities matching our obtained in vitro results. In conclusion, AKD9 alkaline serine protease seems to be a significant candidate for industrial applications because of its stability, hydrophilicity, enhanced thermostability, and alkali-halo stability.

A Novel Homozygous Missense Mutation in the Zinc Finger DNA Binding Domain of GLI1 Causes Recessive Post-Axial Polydactyly.

Background: Polydactyly is a prevalent digit abnormality characterized by having extra digits/toes. Mutations in eleven known genes have been associated to cause nonsyndromic polydactyly: GLI3, GLI1, ZRS regulating LMBR1, IQCE, ZNF141, PITX1, MIPOL1, FAM92A, STKLD1, KIAA0825, and DACH1. Method: A single affected family member (IV-4) was subjected to whole-exome sequencing (WES) to identify the causal gene. Bi-directional Sanger sequencing was performed to segregate the identified variant within the family. In silico analysis was performed to investigate the effect of the variant on DNA binding properties. Results: whole-exome sequencing identified a bi-allelic missense variant (c.1010C > T; p. Ser337Leu) in exon nine of GLI1 gene located on chromosome 12q13.3. With the use of Sanger sequencing, the identified variant segregated perfectly with the disease phenotype. Furthermore, in silico analysis of this DNA binding protein revealed that the variant weakened the DNA binding interaction, resulting in indecorous GLI1 function. Conclusion: Herein, we report a novel variant in GLI1 gene, causing autosomal recessive post-axial polydactyly type A (PAPA) type 8. This confirms the critical role of GLI1 in digit development and might help in genotype-phenotype correlation in the future.

CRISPR/Cas9-mediated activation of CDH1 suppresses metastasis of breast cancer in rats

BACKGROUND Cancer is a life-threatening disease that affects approximately 18 million individuals worldwide. Breast cancer is the most common female neoplasm globally with more than 276,480 new cases of invasive breast cancer expected to be diagnosed in women in the U.S. alone in 2020. Genetic and epigenetic factors play role in the carcinogenesis and progression of this disease. In this study, MCF-7 adenocarcinoma cells were transfected with CRISPR/Cas9 plasmid to either knock out CDK11 or to activate CDH1. Treated cells were allografted into the mammary glands of female rats (150­190 g, 6­8 weeks) to evaluate the capability of these cells to control cancer progression and metastasis. RESULTS qPCR data revealed a significant downregulation of CDK11 and upregulation of CDH1. Cell cycle analysis and apoptosis assays indicated the knockout of CDK11 and simultaneous activation of CDH1 resulted in cell cycle arrest at G2/M phase and accumulation of cells at G2. Meanwhile, the percentage of cells that underwent late apoptosis increased in both genome editing hits. Histopathological sectioning data indicated that untransfected MCF-7 cells were capable of developing tumors in the mammary gland and initiation g angiogenesis. Transfected cells significantly restricted cancer cell infiltration/invasion by minimally localizing tumors and inhibiting angiogenesis. CONCLUSIONS Although further investigation is needed, the present data indicate the potentiality of using CRISPR/Cas9-based therapy as a promising approach to treat breast cancer. Impact: these data indicate targeting cancer-related genes via any genome editing tool might represent a novel approach to combat cancer.