Engineering a DNA polymerase from Pyrobaculum calidifontis for improved activity, processivity and extension rate.

Positively charged amino acids in the DNA polymerase domain are important for interaction with DNA. Two potential residues in the palm domain of Pca-Pol, a DNA polymerase from Pyrobaculum calidifontis, were identified and mutated to arginine in order to improve the properties of this enzyme. The mutant proteins were heterologously produced in Escherichia coli. Biochemical characterization revealed that there was no significant difference in pH, metal ion, buffer preferences, 3' - 5' exonuclease activity and error rate of the wild-type and the mutant enzymes. However, the specific activity, processivity and extension rate of the mutant enzymes increased significantly. Specific activity of one of the mutants (G522R-E555R) was nearly 9-fold higher than that of the wild-type enzyme. These properties make G522R-E555R mutant enzyme a potential candidate for commercial applications.

Gingerol, a Natural Antioxidant, Attenuates Hyperglycemia and Downstream Complications.

Hyperglycemia is seen in approximately 68 percent of patients admitted to a medical intensive care unit (ICU). In many acute circumstances, such as myocardial infarction, brain, injury and stroke, it is an independent predictor of mortality. Hyperglycemia is induced by a mix of genetic, environmental, and immunologic variables in people with type 1 diabetes. These factors cause pancreatic beta cell death and insulin insufficiency. Insulin resistance and irregular insulin production cause hyperglycemia in type 2 diabetes patients. Hyperglycemia activates a number of complicated interconnected metabolic processes. Hyperglycemia is a major contributor to the onset and progression of diabetes' secondary complications such as neuropathy, nephropathy, retinopathy, cataracts, periodontitis, and bone and joint issues. Studies on the health benefits of ginger and its constituent's impact on hyperglycemia and related disorders have been conducted and gingerol proved to be a potential pharmaceutically active constituent of ginger (Zingiber officinale) that has been shown to lower blood sugar levels, because it possesses antioxidant properties and it functions as an antioxidant in the complicated biochemical process that causes hyperglycemia to be activated. Gingerol not only helps in treating hyperglycemia but also shows effectivity against diseases related to it, such as cardiopathy, kidney failure, vision impairments, bone and joint problems, and teeth and gum infections. Moreover, fresh ginger has various gingerol analogues, with 6-gingerol being the most abundant. However, it is necessary to investigate the efficacy of its other analogues against hyperglycemia and associated disorders at various concentrations in order to determine the appropriate dose for treating these conditions.

Nanoparticles in Drug Delivery: From History to Therapeutic Applications.

Current research into the role of engineered nanoparticles in drug delivery systems (DDSs) for medical purposes has developed numerous fascinating nanocarriers. This paper reviews the various conventionally used and current used carriage system to deliver drugs. Due to numerous drawbacks of conventional DDSs, nanocarriers have gained immense interest. Nanocarriers like polymeric nanoparticles, mesoporous nanoparticles, nanomaterials, carbon nanotubes, dendrimers, liposomes, metallic nanoparticles, nanomedicine, and engineered nanomaterials are used as carriage systems for targeted delivery at specific sites of affected areas in the body. Nanomedicine has rapidly grown to treat certain diseases like brain cancer, lung cancer, breast cancer, cardiovascular diseases, and many others. These nanomedicines can improve drug bioavailability and drug absorption time, reduce release time, eliminate drug aggregation, and enhance drug solubility in the blood. Nanomedicine has introduced a new era for drug carriage by refining the therapeutic directories of the energetic pharmaceutical elements engineered within nanoparticles. In this context, the vital information on engineered nanoparticles was reviewed and conferred towards the role in drug carriage systems to treat many ailments. All these nanocarriers were tested in vitro and in vivo. In the coming years, nanomedicines can improve human health more effectively by adding more advanced techniques into the drug delivery system.

Green Tea Catechins Attenuate Neurodegenerative Diseases and Cognitive Deficits.

Neurodegenerative diseases exert an overwhelming socioeconomic burden all around the globe. They are mainly characterized by modified protein accumulation that might trigger various biological responses, including oxidative stress, inflammation, regulation of signaling pathways, and excitotoxicity. These disorders have been widely studied during the last decade in the hopes of developing symptom-oriented therapeutics. However, no definitive cure has yet been discovered. Tea is one of the world's most popular beverages. The same plant, Camellia Sinensis (L.).O. Kuntze, is used to make green, black, and oolong teas. Green tea has been most thoroughly studied because of its anti-cancer, anti-obesity, antidiabetic, anti-inflammatory, and neuroprotective properties. The beneficial effect of consumption of tea on neurodegenerative disorders has been reported in several human interventional and observational studies. The polyphenolic compounds found in green tea, known as catechins, have been demonstrated to have many therapeutic effects. They can help in preventing and, somehow, treating neurodegenerative diseases. Catechins show anti-inflammatory as well as antioxidant effects via blocking cytokines' excessive production and inflammatory pathways, as well as chelating metal ions and free radical scavenging. They may inhibit tau protein phosphorylation, amyloid beta aggregation, and release of apoptotic proteins. They can also lower alpha-synuclein levels and boost dopamine levels. All these factors have the potential to affect neurodegenerative disorders. This review will examine catechins' neuroprotective effects by highlighting their biological, pharmacological, antioxidant, and metal chelation abilities, with a focus on their ability to activate diverse cellular pathways in the brain. This review also points out the mechanisms of catechins in various neurodegenerative and cognitive diseases, including Alzheimer's, Parkinson's, multiple sclerosis, and cognitive deficit.

A novel strategy for developing a tetravalent vaccine (dvac) against dengue utilizing conserved regions from all DENV proteins.

Dengue fever is a global health issue which is infecting millions of people each year and number of reported infections are constantly increasing. Though the only commercialized vaccine i.e. dengvaxia has banned in several countries due to its potential health risk, overall vaccine holds promising potential against viruses. In this study, we have developed a novel formulation of multi-epitope peptide vaccine (dvac), which utilizes peptides from each dengue protein with >80% sequence conservancy within each serotype and their respective genotypes. Simultaneous utilization of all dengue proteins and their conservancy among dengue virus genome is targeted to evoke balanced immunity against dengue serotypes without eliciting antibody-dependent enhancement and antigenic sin like response, which are primarily responsible for severe dengue fever. Immunoinformatic approaches are used to identify the potential of dvac in inducing cytotoxic T-lymphocytes, helper T-lymphocytes, Interleukin-4, Interferon-gamma and B-cell immune responses without inducing allergic responses. Cross-reactivity of dvac with human cellular machinery is also taken into consideration to avoid any cross-reactive pathogenicity. Furthermore, interaction of dvac with immune receptors i.e. toll-like receptors (TLR3 and TLR4) using molecular docking studies revealed favorable interaction between synthetic peptide and immune receptors. Our findings suggest that designed multi-epitope peptide holds great potential to evoke balanced immunity against all dengue serotypes without eliciting any significant harmful side-effects.

Dengue proteins with their role in pathogenesis, and strategies for developing an effective anti-dengue treatment: A review.

Dengue virus is an arbovirus belonging to class Flaviviridae Its clinical manifestation ranges from asymptomatic to extreme conditions (dengue hemorrhagic fever/dengue shock syndrome). A lot of research has been done on this ailment, yet there is no effective treatment available for the disease. This review provides the systematic understanding of all dengue proteins, role of its structural proteins (C-protein, E-protein, prM) in virus entry, assembly, and secretion in host cell, and nonstructural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5) in viral assembly, replication, and immune evasion during dengue progression and pathogenesis. Furthermore, the review has highlighted the controversies related to the only commercially available dengue vaccine, that is, Dengvaxia, and the risk associated with it. Lastly, it provides an insight regarding various approaches for developing an effective anti-dengue treatment.

Variability in the immunogenic preS region of Pakistani hepatitis B virus isolates.

This paper reports the genetic variability of hepatitis B virus in Pakistan population. The worldwide prevalence of hepatitis B virus is estimated to be around 350 million that causes significant mortality especially in developing countries like Pakistan. In this study, genetic diversity of HBV was checked by using preS domain of HBV. About seventy-five samples were selected for study. Among these samples nine samples showed positive results after PCR and gel analysis. These nine samples were named SBS001-SBS008. After gel purification these samples were ligated in T/A cloning vector and transformed with E. coli DH5α. After successful cloning and positive restriction analysis these samples were subjected to DNA sequencing. Sequencing results showed that eight samples (SBS002-SBS008) have a deletion of 33 nucleotides at N-terminal that is characteristics of genotype D while SBS001 belongs to genotype C. Silent mutations and amino acid changes were also searched in this highly variable region of genome. Based upon this study it was concluded that genotype D is the most common genotype in Pakistan.

Construction and recombinant expression of Pseudomonas aeruginosa truncated exotoxin A in Escherichia coli.

Pseudomonas aeruginosa exotoxin A (PE) is a bacterial toxin composed of three domains namely: cell binding, translocation and enzymatic domain. The cytotoxic activity of PE is attributed to the enzymatic domain, which inhibits protein synthesis through ADP-ribosylation of EF-2. PE can be genetically modified to fight cancer. In this regard, a truncated and modified form of PE was produced that could be used for more potent immunotoxins. This modified form termed PE38KDEL was completely devoid of cell binding domain and parts of translocation domain II and Ib which are reported to be inessential for cytotoxicity of the toxin. The resultant expressed protein consisted of the essential translocation domain II and catalytic subunit (domain Ib, III). The deletions in the exotoxin A gene for truncated protein production were made via overlapping PCR extension. The amplicon was cloned in pTZ57r-T vector for DNA works and sub cloned in pET22b expression vector.  It is demonstrated here that PE38KDEL can be expressed in huge quantities in Escherichia coli by using the recombinant vector PE38KDEL/pET under control of T7 promoter and E. coli host strain BL21 (DE3) CodonPlus. The protein expression was optimized at 0.5 mM IPTG concentration for induction as soon as the OD600 nm reached 0.6 with 6 hours of post induction culturing at 37°C.  The recombinant protein was expressed both as soluble and inclusion body forms however the expression of the soluble form was more pronounced.

Structural and quantum mechanical computations to elucidate the altered binding mechanism of metal and drug with pyrazinamidase from Mycobacterium tuberculosis due to mutagenicity.

Pyrazinamide is known to be the most effective treatment against tuberculosis disease and is known to have bacteriostatic action. By targeting the bacterial spores, this drug reduces the chances for the progression of the infection in organisms. In recent years, increased instances of the drug resistance of bacterial strains are reported. Pyrazinamidase, activator for pyrazinamide, leads to resistance against the drug due to mutagenicity across the world. The present study aimed at the quantum mechanistic analysis of mutations in pyrazinamidase to gain insights into the mechanism of this enzyme. Quantum mechanical calculations were performed to analyse the effect of mutations at the metal coordination site using ORCA software program. Moreover, conformational changes in PZase binding cavity has also been analysed due to mutations of binding pocket residues using CASTp server. In order to elucidate the behaviour of the mutant pyrazinamidase, docking of PZA in the binding pocket of PZase was performed using AutoDock Vina. Analysis of results revealed that iron showed weak binding with the metal coordination site of the mutant proteins due to alteration in electron transfer mechanism. The binding cavity of the mutant PZase has undergone major conformational changes as the volume of pocket increased due to bulky R-chains of mutated amino acids. These conformational changes lead to weak binding of the drug at binding cavity of PZase and reduce the drug activation mechanism leading to increased drug resistance in the bacterial strains.

N-terminal deletion of Tk1689, a subtilisin-like serine protease from Thermococcus kodakaraensis, copes with its cytotoxicity in Escherichia coli.

Tk1689, a subtilisin-like serine protease from Thermococcus kodakaraensis, was found to be toxic to the host cells when produced in the pro-protein form (Pro-Tk1689) in Eschericia coli. Cytotoxic effect of Pro-Tk1689 was reduced when signal and pro-peptide both were removed and the protein was produced in the mature form (MP-Tk1689). The mature protein was produced in E. coli in the soluble form. Recombinant MP-Tk1689 was catalytically active and exhibited optimum activity at 55°C and pH 7. Specific activity of the enzyme was 700 U/mg. The enzyme displayed a half life of 80 min at 60°C.