Evolutionary and functional characterization of lagomorph guanylate-binding proteins: a story of gain and loss and shedding light on expression, localization and innate immunity-related functions.

Guanylate binding proteins (GBPs) are an evolutionarily ancient family of proteins that are widely distributed among eukaryotes. They belong to the dynamin superfamily of GTPases, and their expression can be partially induced by interferons (IFNs). GBPs are involved in the cell-autonomous innate immune response against bacterial, parasitic and viral infections. Evolutionary studies have shown that GBPs exhibit a pattern of gene gain and loss events, indicative for the birth-and-death model of evolution. Most species harbor large GBP gene clusters that encode multiple paralogs. Previous functional and in-depth evolutionary studies have mainly focused on murine and human GBPs. Since rabbits are another important model system for studying human diseases, we focus here on lagomorphs to broaden our understanding of the multifunctional GBP protein family by conducting evolutionary analyses and performing a molecular and functional characterization of rabbit GBPs. We observed that lagomorphs lack GBP3, 6 and 7. Furthermore, Leporidae experienced a loss of GBP2, a unique duplication of GBP5 and a massive expansion of GBP4. Gene expression analysis by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and transcriptome data revealed that leporid GBP expression varied across tissues. Overexpressed rabbit GBPs localized either uniformly and/or discretely to the cytoplasm and/or to the nucleus. Oryctolagus cuniculus (oc)GBP5L1 and rarely ocGBP5L2 were an exception, colocalizing with the trans-Golgi network (TGN). In addition, four ocGBPs were IFN-inducible and only ocGBP5L2 inhibited furin activity. In conclusion, from an evolutionary perspective, lagomorph GBPs experienced multiple gain and loss events, and the molecular and functional characteristics of ocGBP suggest a role in innate immunity.

Investigating vertical transmission and maternofoetal outcomes in pregnant women with COVID-19: a case-control study from Pakistan.

Objectives: To estimate the frequency of severe acute respiratory syndrome coronavirus-2 among pregnant women, the impact in terms of obstetrical and clinical outcomes and vertical transmission to the neonates. METHODS: The prospective, case-control study was conducted at Zainab Panjwani Memorial Hospital, Karachi, from March to December 2021, and comprised pregnant women regardless of gestational age who exhibited symptoms or had a suspicion of exposure to any confirmed coronavirus disease-2019 individual. They were screened for severe acute respiratory syndrome coronavirus-2 infection using polymerase chain reaction or serology. Those who tested negative were designated as control group A, while who had a positive serology result along with a negative polymerase chain reaction were taken as recovered case group B1, and those who tested positive for polymerase chain reaction were called the positive case group B2. Groups B1 and B2 were followed up till delivery. The clinical presentation of coronavirus disease-2019 infection in pregnancy and its obstetrical and neonatal outcomes was assessed. Products of conception were tested for the detection of the severe acute respiratory syndrome coronavirus-2 genome. The viral genome from group B2 cases was sequenced to confirm vertical transmission. Data was analysed using GraphPad Prism V8. RESULTS: Of the 139 pregnant women, 74(53.2%) were in group A with mean age 25.87±6.90 years, 49(35.3%) were in group B1 with mean age 25.53±7.02 years, and 16(11.5%) were in group B2 with mean age 27.12±5.03 years. The gestational age at which termination of pregnancy occurred was 38.3±1.26 weeks in group B1 and 38.3±1.85 weeks for group B2. There were 96 neonates across the 3 groups. Of the 11(11.45%) neonates in group B2, 1(9.09%) had postnatal transmission of severe acute respiratory syndrome coronavirus-2 and this mother-neonate case was taken as the Indexed case. The severe acute respiratory syndrome coronavirus-2 genome isolated from the neonate showed similar mutations as the viral strain infecting the mother. Conclusion: The risk of vertical transmission was found to be low. The severe acute respiratory syndrome coronavirus-2 genome was the same for both the mother and the neonate.

Metabolic profile changes of kidney aging and protective effects of Polygonatum sibiricum polysaccharides on D-galactose-induced aging mice / 数字中医药（英文）

Objective@#To investigate the metabolic trajectory of kidney aging and the effects of Polygonatum sibiricum polysaccharides (PSP) against kidney aging in D-galactose (D-gal)-induced aging mice, based on ultra-performance liquid chromatography/Q-Exactive Orbitrap mass spectrometry (UPLC-Q-Exactive MS/MS). @*Methods@#A total of 36 C57 BL/6J mice were randomly allocated to six groups: control (CON), model (MOD), PSP low-dose (PSP-L), PSP medium-dose (PSP-M), PSP high-dose (PSP-H), and positive drug ascorbic acid (VC) groups. To create models of aging mice, D-gal was intraperitoneally administered to all other groups of mice except the CON group. After modeling, the appropriate Chinese medicine [PSP-L: 150 mg/(kg·d), PSP-M: 300 mg/(kg·d), PSP-H: 600 mg/(kg·d)] or positive drug [ascorbic acid, 300 mg/(kg·d)] was administered for intervention. Key markers of renal function in urine and serum of mice in each group, such as creatinine (Crea), urea nitrogen (BUN), and uric acid (UA) levels, as well as key indicators of oxidative stress in serum and kidney, including superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), and glutathione peroxidase (GSH-Px) were determined to validate the successful establishment of kidney aging models and to estimate the effects of PSP. Hematoxylin and eosin (HE), periodic acid Schiff (PAS), and β-galactosidase staining were used to assess the renal pathological changes. The metabolic profiles of serum, kidney, and urine samples from CON, MOD, and PSP-H groups were analyzed by UPLC-Q-Exactive MS/MS, and pattern recognition methods were used to outline the metabolic trajectory of kidney aging and to identify the characteristic metabolites. @*Results@#Age-related alterations in renal histopathology and impaired renal function in mice were also associated with oxidative stress indicators. Following the injection of PSP [PSP-H: 600 mg/(kg·d)], the pathological indices associated with aging were adjusted to normal levels, renal function and oxidative stress were improved in aging mice, and renal pathological damage was markedly improved. Meanwhile, the potential biomarkers were identified by UPLC-Q-Exactive MS/MS analysis and were further analyzed to form related metabolic pathways, with P < 0.05 as a threshold. The results showed that purine, sphingolipid, glycerophospholipid, tryptophan, and riboflavin metabolisms were the main metabolic pathways associated with aging. After administration of PSP, these pathological indices returned to normal levels, and biomarkers related to the aging process, such as adenosine monophosphate (AMP), tryptophan, and 5-hydroxytryptophan, also demonstrated, to some degree, reverse regulation (promoting synthesis). @*Conclusion@#Metabolomics methods based on UPLC-Q-Exactive MS/MS and multivariate statistical analysis can be adopted to establish metabolic profiles in aging mice. PSP has been shown to protect against kidney aging by interfering with the purine, sphingolipid, glycerophospholipid, tryptophan, and riboflavin metabolisms in the kidney.

Anti-glycemic potential of benzophenone thio/semicarbazone derivatives: synthesis, enzyme inhibition and ligand docking studies.

Inhibition of dipeptidyl peptidase-IV (DPP-IV) has been identified as a promising approach for the treatment of type 2 diabetes mellitus (T2DM). Therefore, development of DPP-IV inhibitors with new chemical scaffold is of utmost importance to medicinal chemistry. In the present study, we identified benzophenone thio- and semicarbazone scaffolds as novel DPP-IV inhibitors. For that purpose, benzophenone thio- and semicarbazone were synthesized through a 2-step reaction. These newly synthetic derivatives were characterized by different spectroscopic techniques, including HREI-MS and NMR. whereas stereochemistry of the iminic bond was predicted by NOESY experiments. Thio- and semicarbazones derivatives were evaluated for their DPP-IV inhibitory potential and found to exhibit a good to moderate enzyme inhibitory activity. Most active and non-cytotoxic derivatives were further evaluated for their DPP-IV inhibitory potential in in cellulo model. The binding sites as well as affinity of active compounds for DPP- IV enzyme were predicted by in silico studies, and compared to a standard drug, sitagliptin. Pharmacophore studies of thio- and semicarbazones derivatives 1-29 suggest that substitution of aryl group, particularly a lipophilic substituents at C-4″ of benzene ring, and a hydroxyl at C-4' strongly influenced the DPP-IV inhibitory activity. Compound 9 showed the highest inhibitory activity (IC50 = 15.0 ± 0.6 µM), whereas compounds 10, 17, 12, 14 and 23 showed a moderate activity with IC50 values in the range of 28.9-39.2 µM. This study identifies thio- and semicarbazones as new classes of DPP-IV inhibitors which may translate into safe and effective therapeutics for a better management of type 2 diabetes.Communicated by Ramaswamy H. Sarma.

Megestrol acetate induced proliferation and differentiation of osteoblastic MC3T3-E1 cells: A drug repurposing approach.

AIMS: Drug repurposing or repositioning i.e.; identifying new indications for existing drugs have recently accelerated the process of drug discovery and development. Megestrol acetate (1) is a well-known progestin. It is commonly used as an appetite stimulant, and also in the treatment of breast, and endometrial cancers. The aim of this study is to investigate the effect of megestrol acetate (1) in osteoblast differentiation, and to determine the possible mechanism involved in megestrol acetate (1) induced osteoblast differentiation. MAIN METHODS: Cytotoxicity of different steroidal drugs was evaluated using MTT assay. Alkaline phosphatase (ALP) activity was also determined, and alizarin red S (ARS) staining was performed to measure extracellular mineralization. Osteogenic protein levels were determined using Western blot analysis. KEY FINDINGS: Results of the current study indicated that the megestrol acetate (1) enhanced the proliferation and differentiation of osteoblast cells at 1, 0.2, and 0.04 µM. This stimulatory effect of the megestrol acetate (1) was more prominent at 0.2 µM for cell proliferation, while the maximum cell differentiation (ALPase activity, and calcification) was observed at 0.04 µM. Western blot analysis also showed that megestrol acetate (1) altered the expression of bone morphogenic protein-2 (BMP2), p38, and pJNK proteins. Hence, only moderate doses of MGA (1) can enhance osteoblast proliferation and differentiation. SIGNIFICANCE: Our results identified that megestrol acetate (1) could be a potential lead for further research towards bone fragility related disorders.

Integrated 16S rRNA Sequencing, Metagenomics, and Metabolomics to Characterize Gut Microbial Composition, Function, and Fecal Metabolic Phenotype in Non-obese Type 2 Diabetic Goto-Kakizaki Rats.

Type 2 diabetes mellitus (T2DM) is one of the most prevalent endocrine diseases in the world. Recent studies have shown that dysbiosis of the gut microbiota may be an important contributor to T2DM pathogenesis. However, the mechanisms underlying the roles of the gut microbiome and fecal metabolome in T2DM have not been characterized. Recently, the Goto-Kakizaki (GK) rat model of T2DM was developed to study the clinical symptoms and characteristics of human T2DM. To further characterize T2DM pathogenesis, we combined multi-omics techniques, including 16S rRNA gene sequencing, metagenomic sequencing, and metabolomics, to analyze gut microbial compositions and functions, and further characterize fecal metabolomic profiles in GK rats. Our results showed that gut microbial compositions were significantly altered in GK rats, as evidenced by reduced microbial diversity, altered microbial taxa distribution, and alterations in the interaction network of the gut microbiome. Functional analysis based on the cluster of orthologous groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations suggested that 5 functional COG categories belonged to the metabolism cluster and 33 KEGG pathways related to metabolic pathways were significantly enriched in GK rats. Metabolomics profiling identified 53 significantly differentially abundant metabolites in GK rats, including lipids and lipid-like molecules. These lipids were enriched in the glycerophospholipid metabolic pathway. Moreover, functional correlation analysis showed that some altered gut microbiota families, such as Verrucomicrobiaceae and Bacteroidaceae, significantly correlated with alterations in fecal metabolites. Collectively, the results suggested that an altered gut microbiota is associated with T2DM pathogenesis.

Small Molecular Leads Differentially Active Against HER2 Positive and Triple Negative Breast Cancer Cell Lines.

BACKGROUND: Breast cancer is the most prevalent cancer type in women globally. It is characterized by distinct subtypes depending on different gene expression patterns. Oncogene HER2 is expressed on the surface of cell and is responsible for cell growth regulation. Increase in HER2 receptor protein due to gene amplification, results in aggressive growth, and high metastasis in cancer cells. METHODS: The current study evaluates and compares the anti-breast cancer effect of commercially available compounds against HER2 overexpressing BT-474, and triple negative MDA-MB-231 breast cancer cell lines. RESULTS: Preliminary in vitro cell viability assays on these cell lines identified 6 lead molecules active against breast cancer. Convallatoxin (4), a steroidal lactone glycoside, showed the most potent activity with IC50 values of 0.63 ± 0.56, and 0.69 ± 0.59 µM against BT-474 and MDA-MB-231, respectively, whereas 4-[4-(Trifluoromethyl)-phenoxy] phenol (3) a phenol derivative, and Reserpine (5) an indole alkaloid selectively inhibited the growth of BT-474, and MDA-MB-231 breast cancer cells, respectively. CONCLUSION: These results exhibited the potential of small molecules in the treatment of HER2 amplified and triple negative breast cancers in vitro.

Synthesis of novel bisindolylmethanes: New carbonic anhydrase II inhibitors, docking, and 3D pharmacophore studies.

In this study, 45 bisindolylmethanes having sulfonamide moiety had been synthesized through 3 steps. In vitro assay for inhibition of carbonic anhydrase showed that some of the compounds having sulfonamide moiety are capable of inhibiting carbonic anhydrase II. Bisindoles having halogens at fifth position showed better inhibitory activity as compared to unsubstituted bisindoles. The results obtained from in vitro inhibitory activity were subjected through 3D QSAR and docking studies to identify important features contributing to the activity and further improve the structure. Pharmacophore studies suggest that bisindolylmethane moiety is contributing significantly towards the inhibition activity. Docking studies showed that compounds having nitro substituent (5g and 5i) were found to be able interact with Zn(2+) ion, Thr199, His94, His96, and His119, which interferes with the ZnOHThr199Glu106 hydrogen bond network. Bulky nitro substituent at ortho position for compound 5g prevents the compound from interacting with other residues like Thr199 and Thr200. Methyl substituent at ortho position for Compound 5i induces less steric hindrance effect, thus allowing second oxygen atom of sulfonamide to interact with Thr199 (2.51Å). Hydrogen bonding between NH on indole ring with Glu69 might have increased stability of ligand-receptor complex.

Synthesis, biological evaluation, and docking studies of novel thiourea derivatives of bisindolylmethane as carbonic anhydrase II inhibitor.

This article describes discovery of 29 novel bisindolylmethanes consisting of thiourea moiety, which had been synthesized through three steps. These novel bisindolylmethane derivatives evaluated for their potential inhibitory activity against carbonic anhydrase (CA) II. The results for in vitro assay of carbonic anhydrase II inhibition activity showed that some of the compounds are capable of suppressing the activity of carbonic anhydrase II. Bisindoles having halogen at fifth position showed better inhibitory activity as compared to unsubstituted bisindoles. Derivatives showing inhibition activity docked to further, understand the binding behavior of these compounds with carbonic anhydrase II. Docking studies for the active compound 3j showed that nitro substituent at para position fits into the core of the active site. The nitro substituent of compound 3j is capable of interacting with Zn ion. This interaction believed to be the main factor causing inhibition activity to take place.