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There is a dire need to develop any antiviral therapy for the treatment of SARS-CoV-2. Objective: To investigate the potential therapeutic drug agents from Cymbopogon citratus compounds against the main-protease (Mpro) of SARS-CoV-2. Methods: Initial screening was carried out using molecular docking, dynamic simulation followed by ADMET profiling and Lipinski’s physiochemical parameters for prediction of drug likeliness. MOE/PyRx was used for docking before determining the stability of the best complexes through NAMD/VMD softwares. Moreover, SwissADME and admetSAR web-based tools were used for drug likeliness of the best complexes. Results: Out of total 50 compounds, 11 presented the lowest binding energies which includes tannic acid, isoorientin, swertiajaponin, chlorogenic acid, cymbopogonol, warfarin, citral diethyl acetal, citral acetate, luteolin, kaempferol and cianidanol with binding energies of -8.12, -7.38, -7.33, -6.88, -6.48, -6.32, -6.31, -6.18, -6.18, -6.13 and -6.02, respectively. Current studies show isoorientin, chlorogenic acid and tannic acid as the promising drug agents using RMSD, Hbond, heatmap graphs. Conclusion: Further in-vivo experiments are suggested to ascertain the medicinal use of these potential inhibitors against COVID-19.

This study analyzed the effects of the plant extracts (Citrus limon, Solanum lycopersicum, Zingiber officinale, Vitis vinifera and Allium sativum) on the growth of mammalian cells (Vero and MDA-MB-231) and evaluated the most effective plant extract for the expression of specific genes of the JAK/STAT pathway in human breast cancer cells. An antiproliferative bioassay involving neutral red-dye uptake was used to determine the anticancerous potential of plant extracts. In Vero cells, the ginger methanolic extract was least effective; whereas the lemon methanolic extract was more effective with 64 dilutions with IC50 51.42%. In MDA-MB-231 cells, the tomato and ginger methanolic, and grape water extracts were least effective, whereas lemon water extract was most effective with 32 dilutions with IC50 48.67%, by upregulating JAK1, JAK2, TYK2, IRF7 and IRF3 gene expressions of the JAK/STAT pathway. C. limon inhibited the growth of both Vero and MDA-MB 231 cells. It suggested that C. limon has anti-cancer potential by inducing the JAK/STAT pathway.

The advancements in biotechnology over time have led to an increase in the demand of pure, soluble and functionally active proteins. Recombinant protein production has thus been employed to obtain high expression of purified proteins in bulk. E. coli is considered as the most desirable host for recombinant protein production due to its inexpensive and fast cultivation, simple nutritional requirements and known genetics. Despite all these benefits, recombinant protein production often comes with drawbacks, such as, the most common being the formation of inclusion bodies due to improper protein folding. Consequently, this can lead to the loss of the structure-function relationship of a protein. Apart from various strategies, one major strategy to resolve this issue is the use of molecular chaperones that act as folding modulators for proteins. Molecular chaperones assist newly synthesized, aggregated or misfolded proteins to fold into their native conformations. Chaperones have been widely used to improve the expression of various proteins which are otherwise difficult to produce in E. coli. Here, we discuss the structure, function, and role of major E. coli molecular chaperones in recombinant technology such as trigger factor, GroEL, DnaK and ClpB.

Pcal_0768 gene encoding an amylomaltase, a 4-α-glucanatransferase belonging to family 77 of glycosyl hydrolases, from Pyrobaculum calidifontis was cloned and expressed in Escherichia coli. The recombinant protein was produced in E. coli in soluble and active form. However, the expression level was not very high. Analysis of the mRNA of initial seven codons at the 5′-end of the gene revealed the presence of a hair pin like secondary structure. This secondary structure was removed by site directed mutagenesis, without altering the amino acids, which resulted in enhanced expression of the cloned gene. Recombinant Pcal_0768 exhibited optimal amylomaltase activity at 80 °C and pH 6.9. Under these conditions, the specific activity was 690 U/ mg. Recombinant Pcal_0768 was highly thermostable with a half-life of 6 h at 100 °C. It exhibited the highest kcat value among the characterized glucanotransferases. No metal ions were required for activity or stability of the enzyme. Recombinant Pcal_0768 was successfully employed in the synthesis of modified starch for producing thermoreversible gel. To the best of our knowledge, till now this is the most thermostable enzyme among the characterized amylomaltases. High thermostability and starch modification potential make it a novel and distinct amylomaltase.

Background Type 2 diabetes mellitus is a multifactorial disease that escalates the risk of other associated complications such as diabetic neuropathy, retinopathy, and nephropathy. Diabetic nephropathy is a microvascular condition that leads to end-stage renal disease (ESRD). There are several genes involved in disease development and it is a challenging task to investigate all of these. Nonetheless, identifying individual gene roles can assist in evaluating the combinatorial effects with other genes. Angiotensin-1 converting enzyme 2 (ACE2), is the key regulator of blood pressure in the Renin-Angiotensin-Aldosterone System that hydrolyzes angiotensin II (vasoconstrictor) into angiotensin 1–7 (vasodilator). The association of different variants of the ACE2 with the risk of type 2 diabetes mellitus has been determined in various populations with susceptibility to other complications. This study was aimed to investigate the association of Angiotensin-1 converting enzyme 2 polymorphism, G8790A, with the increased risk of type 2 diabetes mellitus (T2DM) development with the complication of diabetic nephropathy (DN) in the Pakistani population. Methods In this case-control study, a total of 100 healthy controls and 100 patients of type 2 diabetes mellitus aged > 40 years, having disease duration ≥ 10 years were compared. The G8790A polymorphism in ACE2 was analyzed by allele-specific polymerase chain reaction (AS-PCR). The urinary albumin excretion (UAE), urinary creatinine, and albumin to creatinine ratios (ACR) were determined to assess renal function status. Pearson bivariate correlation coefficients were calculated to investigate the relationship among all the parameters. Crude and adjusted odds ratios were found to determine any risk association between ACE2 G8790A polymorphisms and disease development. The p-values < 0.05 were considered significant. Results A homogeneity was obtained regarding the distribution of data by sex, BMI, diastolic blood pressure, pulse rate and urinary creatinine levels between case and control groups. The ACR showed a significant correlation with UAE (r = 0.524, p = 0.001), urinary creatinine (r = -0.375, p = 0.001) and random blood sugar levels (r = 0.323, p = 0.005) with the complication of diabetic nephropathy in T2DM patient. Females with the AA genotype had a 10-fold increased risk for the development of type 2 Diabetes (OR = 9.5 [95% CI = 2.00–21.63] p Conclusion The study finding indicated that female AG/AA genotype and male A genotype of G8790A polymorphism in the ACE2 gene were associated with type 2 diabetes mellitus as a genetic risk factor but are not associated with diabetic nephropathy in the Pakistani population.

This study was conducted to determine the heterologous expression of eukaryotic gene in Escherichia coli variable. The expression of Pakistani buffalo (Bubalus bubalis) and cow (Bos taurus) proinsulin genes in BL21 codon plus cells is 30% of total cellular protein. Total RNA was isolated from pancreatic tissues of local (Pakistani) breeds of buffalo (B. bubalis) and cow (B. taurus), converted to cDNA and cloned in a T/A cloning vector. There are five silent mutations: one in B-chain, two each in A-chain and C-peptide encoding regions of local Pakistani buffalo proinsulin DNA sequence as compared to the internationally reported cow proinsulin sequence. The DNA sequence of local (Pakistani) cow proinsulin shows there is one nucleotide encodes C-peptide that has mismatch with the reported cow proinsulin sequence but mismatched nucleotide is same between Pakistani cow and Pakistani buffalo proinsulin sequence. This indicates the genetic similarity of Pakistani cow with Pakistani buffalo. Both genes were expressed in BL21 Codon plus (DE3)-RIL cells with 0.2 mM IPTG at 37°C for 6 to 8 h. Proinsulin was expressed as 30% of total cellular proteins as insoluble inclusion bodies. Key words: Bubalus bubalis, Bos taurus, cDNA, T/A cloning vector, BL21 Codon plus (DE3)-RIL cells.

Background MicroRNAs (miRNAs) are small RNA molecules that modulate post-transcriptional gene regulation. They are often used as promising non-invasive biomarkers for the early diagnosis of cancer. However, their roles in assisted reproduction are still unknown. Methods This prospective study was designed to evaluate the expression profiles of seven extracellular miRNAs (miR-7-5p, miR-202-5p, miR-378-3p, miR-224, miR-320a, miR-212-3p, and miR-21-5p) in human follicular fluid (FF) to explore the outcomes of in vitro fertilization (IVF). Of 255 women, 145 were without polycystic ovary syndrome (PCOS), and their ovarian assets were normal (NOR), while 110 were with normo-androgenic PCOS. Results The combination of six FF miRNAs expression profile discriminated between PCOS and NOR women with a sensitivity of 79.2% and a specificity of 87.32% (AUC = 0.881 [0.61; 0.92], p = 0.001). MiR-202-5p significantly had a lower abundance level, and miR-378-3p had a high abundance level in pooled FF samples from patients treated with human menopausal gonadotropin (hMG) than those treated with recombinant follicle-stimulating hormone (rFSH) (p < 0.001). Our results showed that miRNA-320a was significantly different in top-quality embryos versus non-top-quality embryos on day 3 in NOR patients with a sensitivity of 80% and specificity of 71%, (AUC = [0.753 (0.651; 0.855)], p = 0.001). For clinical pregnancy outcome prediction, FF miRNA-21 exhibited high sensitivity (74.8%) and specificity (83.7%) with the AUC value of 0.774 (0.682; 0.865). Conclusion Conclusively, our results provide evidence that miR-7-5p, miR-378-3p, miR-224, miR-212-3p were a differentially high expression in normo-androgenic PCOS patients than NOR patients. While miRNA-320a was significantly different in top-quality embryos versus non-top-quality embryos on day 3 (p = 0.001). The expression level of FF miR-212-3p was significantly related to the probability of embryos to develop into a high-quality blastocyst in patients with normal ovarian reserve., Lahore institute of Fertility and Endocrinology, Hameed Latif Hospital, Lahore, Pakistan, Lahore institute of Fertility and Endocrinology, Hameed Latif Hospital, Lahore, Pakistan.

Additional file 1 : Table S1. Fold change in the FF miRNAs at oocyte retravel day between women with normo-androgenic PCOS (n = 110) versus NOR women (n = 145). Table S2. Univariate analysis exhibiting association of specific FF miRNAs with blastocyst formation and pregnancy outcome.

Background Non-Small Cell Lung Carcinoma is one of the major cause of morbidity and mortality worldwide with an incidence rate of 1.3 million cases per year. Heat shock protein 90 (HSP90) is a promising drug target in cancer treatment. HSP90 is required to activate numerous eukaryotic proto-oncogenic protein kinases, hence play a prominent role in cancer. Method We reviewed fifty-five articles to highlight the importance of HSP90 in NSCLC and the recent developments of its inhibitors. Results This review showed that HSP90 inhibitors i.e. Ganestespib have shown great potential in the treatment of non-small cell lung carcinoma (NSCLC). Different HSP90 inhibitors has been designed till date that acts as effective drugs in tumour suppression. However, the utility of these drugs has been limited due to several drawbacks including hepato-toxicity, poor solubility, and poorly tolerated formulations. Conclusion The goal of this review is to present the data in support of use of HSP90 inhibitors in NSCLC and to provide an overview of the on-going clinical trials involving new-generation HSP90 inhibitors.

Human M-proinsulin was cleaved by trypsin at the R(31)R(32)-E(33) and K(64)R(65)-G(66) bonds (B/C and C/A junctions), showing the same cleavage specificity as exhibited by prohormone convertases 1 and 2 respectively. Buffalo/bovine M-proinsulin was also cleaved by trypsin at the K(59)R(60)-G(61) bond but at the B/C junction cleavage occurred at the R(31)R(32)-E(33) as well as the R(31)-R(32)E(33) bond. Thus, the human isoform in the native state, with a 31 residue connecting C-peptide, seems to have a unique structure around the B/C and C/A junctions and cleavage at these sites is predominantly governed by the structure of the proinsulin itself. In the case of both the proinsulin species the cleavage at the B/C junction was preferred (65%) over that at the C/A junction (35%) supporting the earlier suggestion of the presence of some form of secondary structure at the C/A junction. Proinsulin and its derivatives, as natural substrates for trypsin, were used and mass spectrometric analysis showed that the k(cat.)/K(m) values for the cleavage were most favourable for the scission of the bonds at the two junctions (1.02±0.08×10(5)s(-1)M(-1)) and the cleavage of the K(29)-T(30) bond of M-insulin-RR (1.3±0.07×10(5)s(-1)M(-1)). However, the K(29)-T(30) bond in M-insulin, insulin as well as M-proinsulin was shielded from attack by trypsin (k(cat.)/K(m) values around 1000s(-1)M(-1)). Hence, as the biosynthetic path follows the sequence; proinsulin→insulin-RR→insulin, the K(29)-T(30) bond becomes shielded, exposed then shielded again respectively.

Insulin plays an important role in maintaining the blood glucose level of the body. The β-cells of pancreas produce insulin in the form of precursor that is preproinsulin. The gene of preproinsulin provides an interesting system for addressing question related to molecular evolution. Recombinant DNA technology has made it possible to isolate and sequence the chromosomal genes coding for unique protein products. Although preproinsulin of various organism has been isolated and cloned, but there is no report from buffalo (Bubalus bubalis) that is our major livestock. The genomic DNA of buffalo was isolated using Laura-Lee-Boodram method. The part of preproinsulin gene (596bp and 520bp) using BPPI-UPS and bpiful_F as forward and BC1-C as reverse primer was amplified. Cloning of amplified fragments of gene were performed in pCR 2.1 vector. Positive clones were screened on the basis of blue white selection. The band obtained on 596bp and 520bp after colony PCR confirmed the successful cloning of preproinsulin gene in pCR 2.1 vector.

Buffalo proinsulin cDNA was isolated, sequenced and shown to differ from that of its bovine counterpart in six nucleotides. However, at the protein level the predicted sequences of the two species are identical. Buffalo M-proinsulin, containing the initiation methionine, was produced in Escherichia coli and purified to give Mr of 8812. Following the replacement of 99% of the exchangeable hydrogen atoms with deuterons a preparation containing 131 D atoms was obtained. Buffer exchange of the latter into a protio medium led to, the immediate release of 109 (±1) D atoms into the medium and the retention of 22 (±1) D atoms in the protein. The slow exchange of these D atoms was studied at 0 °C/pH 2.8. Insulin derived from buffalo proinsulin as well as bovine when deuteriated and buffer exchanged, similarly, gave the retention of 25 (±1) D atoms. The data show that the secondary structure of the insulin core present within buffalo/bovine proinsulin contains 5 (±1) fewer slow exchanging hydrogen atoms than are present in the final hormone. This effect is attributed, predominantly, to the long range influence of the C-peptide, composed of 26 residues, on the insulin core of buffalo proinsulin. In contrast, in the case of human proinsulin, comprising 31 amino acids in the C-peptide, the secondary structure of the insulin core within human proinsulin is closer to that of insulin itself.

Secondary structure elements of human proinsulin and of its tryptic products were compared by H/D exchange, in a single-pot, using mass spectrometry. Human proinsulin containing an N-terminal methionine, M-proinsulin, was engineered and converted into a perdeuterio derivative, which using an optimized mass spectrometric protocol and manual calculations gave a mass of 9669.6 (+/-1) Da showing the replacement, with deuterium of 146.4 from a total of 149 exchangeable hydrogen atoms (83 from amides and 66 from side-chains). Tryptic digestion of the perdeuterio-M-proinsulin, followed by the transfer of the digest from a deuterio- into a protio-medium showed, at the earliest time of analysis, that of the 27 (+/-1) D atoms retained in M-proinsulin, 24 (+/-1) were found in the insulin nucleus, M-insulin-RR, and 4.2 (+/-1) in the C-peptide-KR. A temporal analysis of the fate of D atoms in these species showed that whereas the C-peptide-KR rapidly exchanged its deuterium, losing all by 6 h, the loss of D atoms from M-proinsulin and M-insulin-RR was gradual and in each case, 12 deuterium atoms survived exchange for 72 h. At all time intervals the loss of D atoms from M-proinsulin mirrored that from M-insulin-RR plus the C-peptide-KR, suggesting that the secondary-structure elements of M-proinsulin are largely conserved in its two component parts.