Molecular Biotechnology Research Articles

Taxonomy characterization and plumbum bioremediation of novel fungi

The objective of this study was to investigate the screening, taxonomy characterization, Pb biosorption, and application of the high Pb-resistant fungus F1 separated from the heavy metal contaminated soil. Fungus F1 was screened through metal concentration gradient ranging from 25 to 4000 mg L-1 . The internal transcribed spacers (ITS) of the strain was analyzed by molecular biotechnology. The adsorption conditions were also evaluated. The precipitation of fungus F1 was analyzed by Scanning Electron Microscopy (SEM), Fourier transformer infrared spectroscopy (FTIR) and energy dispersive X-ray (EDX) techniques. The Pb speciation was determined by BCR three-step sequential extraction. The highest concentration of fungus F1 resistance to Pb2+ was 3500 mg L-1 . The fungus was identified as Trichoderma asperellum. The optimum condition for the Pb2+ removal rate was discovered as follows: MTL: 3500 mg L-1 ; pH: 7; Pb2+ concentration: 800 mg L-1 ; temperature was 30 °C; initial biosorbent dosage: 6% (v/v). The surface chemical functional groups of fungus F1 were amino, hydroxyl, and carbonyl groups, which may be involved in the biosorption of Pb. Application test showed that the exchangeable, acid-and water soluble Pb were reduced, and the sulfide, organic combination state, and residual Pb were increased. With the preferable absorption capacity, fungus F1 was considered to have good prospects of bioremediation.

A new approach to precise thermodynamic characterization of hybridization properties of modified oligonucleotides: Comparative studies of deoxyribo- and glycine morpholine pentaadenines

The development of new derivatives and analogues of nucleic acids for the purposes of molecular biology, biotechnology, gene diagnostics, and medicine has been a hotspot for the last two decades. Methylenecarboxamide (glycine) morpholine oligomer analogues (gM) seem to be promising therapeutic candidates because of the ability to form sequence specific complexes with DNA and RNA. In this paper we describe new approaches to the determination of thermodynamic parameters for hybridization of tandem oligonucleotide complexes with the complementary template. It makes possible to determine changes in enthalpy and entropy corresponding to the binding of an individual oligomer with the template, and to the formation of cooperative contact at the helix-helix interface of two neighboring duplex fragments (in the nick). We have experimentally analyzed the series of model tandem complexes of different length at various oligomer concentrations, ionic strength, and pH. The analysis of thermodynamic parameters of complex formation for native and modified oligomers revealed higher Gibbs free energy values of hybridization and cooperative interaction of morpholine-containing complexes at the helix-helix interface under standard conditions (1M NaCl, pH7.2). Further comparative analysis of the hybridization properties of modified oligomers at ionic strength and pH allows us to determine the charge state of the morpholine backbone and the thermodynamic origin of the effects observed. It was found that the decrease in pH to 5.5 led to the protonation of internal morpholine nitrogens. The obtained results prove the veracity of the proposed model and the possibility to evaluate thermodynamic parameters of short native and modified oligomers with high accuracy.

Cathepsin B-Mediated NLRP3 Inflammasome Formation and Activation in Angiotensin II -Induced Hypertensive Mice: Role of Macrophage Digestion Dysfunction

Background/Aims: Angiotensin II (Ang II) is an octapeptide hormone that plays a significant role in mediating hypertension. Although hypertension is considered a chronic inflammatory disease, the molecular basis of the sterile inflammatory response involved in hypertension remains unclear. Methods: We investigated the role of macrophage NLRP3 inflammasomes in engulfing and digesting microbes, a key macrophage function, and in early onset of hypertension-associated macrophage injury using biochemical analyses, gene silencing, molecular biotechnology, immunofluorescence, and microbiology. Results: Ang II stimulation decreased nitric oxide (NO) release and macrophage digestion in cultured THP-1 cells and markedly increased NLRP3 inflammasome formation and activation. NO release and macrophage digestion were restored by NLRP3 inflammasome inhibition with isoliquiritigenin and gene silencing. This Ang II-induced upregulation of NLRP3 inflammasomes in macrophages was attributed to lysosomal damage and release of cathepsin B. Mechanistically, losartan, a nonpeptide Ang II receptor antagonist, decreased Ang II-induced NLRP3 inflammasome activation, lysosomal membrane permeability, lysosomal cathepsin B release, and macrophage digestion dysfunction. Similarly, Ang II-induced macrophage microbe digestion and NO production, which were blocked by ATI gene silencing. In addition, in vivo experiments showed that the bacteria scavenging function was clearly decreased in macrophages from Ang II-induced hypertensive mice. Conclusion: Angiotensin II enhances lysosomal membrane permeabilization and the consequent release of lysosomal cathepsin B, resulting in activation of the macrophage NLRP3 inflammasome. This may contribute to NO mediation of dysfunction in digesting microbes.

Awareness about pulmonary tuberculosis in post graduate students of Institute of molecular biology & biotechnology Pakistan

Awareness about pulmonary tuberculosis in post graduate students of Institute of molecular biology & biotechnology Pakistan

Third generation antibody discovery methods: in silico rational design.

Owing to their outstanding performances in molecular recognition, antibodies are extensively used in research and applications in molecular biology, biotechnology and medicine. Recent advances in experimental and computational methods are making it possible to complement well-established in vivo (first generation) and in vitro (second generation) methods of antibody discovery with novel in silico (third generation) approaches. Here we describe the principles of computational antibody design and review the state of the art in this field. We then present Modular, a method that implements the rational design of antibodies in a modular manner, and describe the opportunities offered by this approach.

Updates in Diagnostic Techniques for Brucellosis in Livestock

Brucellosis is a zoonotic disease affecting human as well as domestic animals with worldwide distribution. There are several direct and indirect methods are available for diagnosis of brucellosis in animals. Traditionally, isolation of bacteria by culturing on selective media followed by bacteriological characterisation was done for diagnosis, but it is time consuming. Serological tests are having its own importance for laboratory diagnosis of brucellosis as most of the papers published till date use these methods for screening samples. With advancement in molecular biology and biotechnology, different techniques like polymerase chain reaction (PCR), real-time PCR, loop-mediated isothermal amplification assay are gaining importance for diagnosis of brucellosis in livestock now a day's. These methods are more sensitive and give more accurate results than traditional methods.

H2O2 Signaling-Triggered PI3K Mediates Mitochondrial Protection to Participate in Early Cardioprotection by Exercise Preconditioning.

Previous studies have shown that early exercise preconditioning (EEP) imparts a protective effect on acute cardiovascular stress. However, how mitophagy participates in exercise preconditioning- (EP-) induced cardioprotection remains unclear. EEP may involve mitochondrial protection, which presumably crosstalks with predominant H2O2 oxidative stress. Our EEP protocol involves four periods of 10 min running with 10 min recovery intervals. We added a period of exhaustive running and a pretreatment using phosphoinositide 3-kinase (PI3K)/autophagy inhibitor wortmannin to test this protective effect. By using transmission electron microscopy (TEM), laser scanning confocal microscopy, and other molecular biotechnology methods, we detected related markers and specifically analyzed the relationship between mitophagic proteins and mitochondrial translocation. We determined that exhaustive exercise associated with various elevated injuries targeted the myocardium, oxidative stress, hypoxia-ischemia, and mitochondrial ultrastructure. However, exhaustion induced limited mitochondrial protection through a H2O2-independent manner to inhibit voltage-dependent anion channel isoform 1 (VDAC1) instead of mitophagy. EEP was apparently safe to the heart. In EEP-induced cardioprotection, EEP provided suppression to exhaustive exercise (EE) injuries by translocating Bnip3 to the mitochondria by recruiting the autophagosome protein LC3 to induce mitophagy, which is potentially triggered by H2O2 and influenced by Beclin1-dependent autophagy. Pretreatment with the wortmannin further attenuated these effects induced by EEP and resulted in the expression of proapoptotic phenotypes such as oxidative injury, elevated Beclin1/Bcl-2 ratio, cytochrome c leakage, mitochondrial dynamin-1-like protein (Drp-1) expression, and VDAC1 dephosphorylation. These observations suggest that H2O2 generation regulates mitochondrial protection in EEP-induced cardioprotection.

APLIKASI BIOTEKNOLOGI MOLEKULER DALAM BUDAYA PERAIRAN

MOLECULAR BIOTECHNOLOGY APPLICATION IN MARINE AQUACULTURE. Aquaculture industry, as a promising solution for the decrease of natural broodstock as well as an important part in people's health and economics, has been growing rapidly. Biomolecular technologies are now used as a base for proper management of aquaculture programs. Those technologies include breeding program with genetic maps; mono-sex aquaculture program to maximize production's effectiveness; transgenic program; and pathogen's early detection using bio-molecular methods. it can be said that the modern aquaculture industry nowadays depends on the application of molecular biotechnology with support from better aquaculture-system management for product organization.

Transduction Methods for Cytosolic Delivery of Proteins and Bioconjugates into Living Cells.

The human organism and its constituting cells rely on interplay between multiple proteins exerting specific functions. Progress in molecular biotechnologies has facilitated the production of recombinant proteins. When administrated to patients, recombinant proteins can provide important healthcare benefits. To date, most therapeutic proteins must act from the extracellular environment, with their targets being secreted modulators or extracellular receptors. This is because proteins cannot passively diffuse across the plasma membrane into the cytosol. To expand the scope of action of proteins for cytosolic targets (representing more than 40% of the genome) effective methods assisting protein cytosolic entry are being developed. To date, direct protein delivery is extremely tedious and inefficient in cultured cells, even more so in animal models of pathology. Novel techniques are changing this limitation, as recently developed in vitro methods can robustly convey large amount of proteins into cell cultures. Moreover, advances in protein formulation or protein conjugates are slowly, but surely demonstrating efficiency for targeted cytosolic entry of functional protein in vivo in tumor xenograft models. In this review, various methods and recently developed techniques for protein transport into cells are summarized. They are put into perspective to address the challenges encountered during delivery.

Quantification of immobilized protein in pharmaceutical production by bio-assisted potentiometric multisensor system

Quantification of proteins is a key biochemical assay in molecular biology, biotechnology, medicine and pharmacology. Protein quantification protocols can be based on spectrophotometry, enzyme-linked immunosorbent assay, mass spectrometry or quantitative immunoblotting depending on analyte. In case of immobilized protein these methods require suitable sample preparation. Thus, sophisticated analysis becomes even more complex, expensive and time-consuming. Such drawbacks are highly undesirable in industry. In this study we propose a new approach for evaluation of immobilized protein concentration based on application of bio-assisted potentiometric multisensor system. Surface-immobilized recombinant protein A from Staphylococcus aureus (SpA, expressed in Escherichia coli), which is commonly used as affinity ligand immobilized to stationary phase (сhromatography media) for monoclonal antibody purification was employed as the model object. Chromatography media samples containing different amounts of immobilized SpA were analyzed. Proteinase K from Tritirachium album was employed as a bio-transducer. We demonstrated that the suggested approach provides information about immobilized SpA concentration with 0.8mg/ml accuracy in the range 1–6.7mg/ml and within just 16min. Moreover, the proposed procedure requires no expensive materials and equipment and no bio-transducer immobilization. This method has potential of application for fast monitoring of other immobilized proteins in different tasks.

Contrast Media Research 2017 Durango, Colorado USA, October 22–25, 2017 Convener: Michael F. Tweedle, PhD

Contrast Media Research 2017 Durango, Colorado USA, October 22–25, 2017 Convener: Michael F. Tweedle, PhD

Mapping X-Disease Phytoplasma Resistance in Prunus virginiana.

Phytoplasmas such as “Candidatus Phytoplasma pruni,” the causal agent of X-disease of stone fruits, lack detailed biological analysis. This has limited the understanding of plant resistance mechanisms. Chokecherry (Prunus virginiana L.) is a promising model to be used for the plant-phytoplasma interaction due to its documented ability to resist X-disease infection. A consensus chokecherry genetic map “Cho” was developed with JoinMap 4.0 by joining two parental maps. The new map contains a complete set of 16 linkage groups, spanning a genetic distance of 2,172 cM with an average marker density of 3.97 cM. Three significant quantitative trait loci (QTL) associated with X-disease resistance were identified contributing to a total of 45.9% of the phenotypic variation. This updated genetic linkage map and the identified QTL will provide the framework needed to facilitate molecular genetics, genomics, breeding, and biotechnology research concerning X-disease in chokecherry and other Prunus species.

New drug developments of snake venom polypeptides and progress

The value of snake venom polypeptides in clinical application has drawn extensive attention, and the development of snake polypeptides into new drugs with anti-tumor, anti-inflammatory, antithrombotic, analgesic or antihypertensive properties has become the recent research hotspot. With the rapid development of molecular biology and biotechnology, the mechanisms of snake venom polypeptides are also gradually clarified. Numerous studies have demonstrated that snake venom polypeptides exert their pharmacological effects by regulating ion channels, cell proliferation, apoptosis, intracellular signaling pathway, and expression of cytokine as well as binding to relevant active sites or receptors.

Construction of Plasmonic Nano‐Biosensor‐Based Devices for Point‐of‐Care Testing

AbstractNext‐generation healthcare equipment and devices are in great demand for point‐of‐care testing (POCT), in view of their applications in rapid disease diagnosis, personalized medicine, portable or mobile health care, nontechnician assays, etc. Localized surface plasmon resonance (LSPR) biosensors, which are based on noble‐metal nanostructures and which provide fast, real‐time, nonlabeled, and sensitive biochemical detections, have become one of the leading candidates for POCT. The advances in nanofabrication, molecular biotechnology, and optoelectronics integration technology have promoted LSPR biosensing to low‐cost, easy‐to‐use, and on‐site POCT. Here, a synopsis of LSPR nano‐biosensors and LSPR‐based POCT devices is provided by delineating the fundamental principles of LSPR sensing, various metal nanostructures, the modification layers on LSPR nanostructures, and the integration of LSPR biosensors into devices. Examples of applications of existing LSPR‐based microfluidic platforms and POCT devices in healthcare, public security, environmental monitoring, and food safety, for example, are given. Finally, the challenges and perspectives of developing LSPR‐based POCT techniques are highlighted. A relatively complete and instructive summary is provided for the development of plasmonic nano‐biosensor‐based portable, mobile, and wearable POCT devices.

Thermostable proteins bioprocesses: The activity of restriction endonuclease-methyltransferase from Thermus thermophilus (RM.TthHB27I) cloned in Escherichia coli is critically affected by the codon composition of the synthetic gene.

Obtaining thermostable enzymes (thermozymes) is an important aspect of biotechnology. As thermophiles have adapted their genomes to high temperatures, their cloned genes’ expression in mesophiles is problematic. This is mainly due to their high GC content, which leads to the formation of unfavorable secondary mRNA structures and codon usage in Escherichia coli (E. coli). RM.TthHB27I is a member of a family of bifunctional thermozymes, containing a restriction endonuclease (REase) and a methyltransferase (MTase) in a single polypeptide. Thermus thermophilus HB27 (T. thermophilus) produces low amounts of RM.TthHB27I with a unique DNA cleavage specificity. We have previously cloned the wild type (wt) gene into E. coli, which increased the production of RM.TthHB27I over 100-fold. However, its enzymatic activities were extremely low for an ORF expressed under a T7 promoter. We have designed and cloned a fully synthetic tthHB27IRM gene, using a modified ‘codon randomization’ strategy. Codons with a high GC content and of low occurrence in E. coli were eliminated. We incorporated a stem-loop circuit, devised to negatively control the expression of this highly toxic gene by partially hiding the ribosome-binding site (RBS) and START codon in mRNA secondary structures. Despite having optimized 59% of codons, the amount of produced RM.TthHB27I protein was similar for both recombinant tthHB27IRM gene variants. Moreover, the recombinant wt RM.TthHB27I is very unstable, while the RM.TthHB27I resulting from the expression of the synthetic gene exhibited enzymatic activities and stability equal to the native thermozyme isolated from T. thermophilus. Thus, we have developed an efficient purification protocol using the synthetic tthHB27IRM gene variant only. This suggests the effect of co-translational folding kinetics, possibly affected by the frequency of translational errors. The availability of active RM.TthHB27I is of practical importance in molecular biotechnology, extending the palette of available REase specificities.

LEUKEMIA;

Background: Leukemia is a hematological malignancy i.e. that arises in theblood progenitor cells. It is a tumor of white blood cells which is characterized by uncontrolledproliferation of immature hematopoietic WBCs in bone marrow. Leukemia is of two types myeloidand lymphoid and two subtypes acute and chronic. Acute lymphoid leukemia is most commonin children. There are certain risk factors associated with leukemia like ionizing radiations,genetic polymorphisms. Drug resistance in leukemia is linked with polymorphisms for genecoding enzymatic antioxidants. Multiple circulating biochemical markers depict pathogenesisof leukemia. Elevated reactive oxygen species and decreased antioxidant elements revealoccurrence of various malignancies. Study Design: Case Control Study. Setting: Study wasconducted at Institute of Molecular Biology and Biotechnology (IMBB), University of Lahore.Period of Study: One year. Materials and Methods: Blood samples of 270 leukemic patientsbetween the age of 30-55 were collected from PINUM and Allied Hospital, Faisalabad. Whilethe samples of 100 individuals were served as a control. Levels of both enzymatic and nonenzymaticantioxidant such as CAT, GSH, SOD, GPx and GRx, vitamin A, C and E and levels ofMDA were determined spectrophotometrically. While the concentration of IL6, AOPPS, AGEs,TNF-α, MMP9 and Isoprostanes were measured by using commercially available Elisa kits.Results: This research work was conducted on a total of 100 control subjects and 270 leukemicpatients and the results were described on mean values. Many circulating biochemical stressmarkers, antioxidants and vitamins were investigated. All enzymatic and non-enzymaticantioxidants, vitamins were decreased in leukemic individuals while markers which showedhigh oxidative stress like MDA, IL6, AOPPS, TNF-α and MMP9 were elevated in leukemicpatients. Conclusion: It is concluded from this study that low levels of antioxidants and highlevels of oxidative stress depict the pathological state of leukemia and defected cell proliferationand growth. Continuous evaluation of antioxidant levels during leukemia can serve as a way ofbetter prognosis and treatment of leukemia.

English

Background: Leukemia is a hematological malignancy i.e. that arises in theblood progenitor cells. It is a tumor of white blood cells which is characterized by uncontrolledproliferation of immature hematopoietic WBCs in bone marrow. Leukemia is of two types myeloidand lymphoid and two subtypes acute and chronic. Acute lymphoid leukemia is most commonin children. There are certain risk factors associated with leukemia like ionizing radiations,genetic polymorphisms. Drug resistance in leukemia is linked with polymorphisms for genecoding enzymatic antioxidants. Multiple circulating biochemical markers depict pathogenesisof leukemia. Elevated reactive oxygen species and decreased antioxidant elements revealoccurrence of various malignancies. Study Design: Case Control Study. Setting: Study wasconducted at Institute of Molecular Biology and Biotechnology (IMBB), University of Lahore.Period of Study: One year. Materials and Methods: Blood samples of 270 leukemic patientsbetween the age of 30-55 were collected from PINUM and Allied Hospital, Faisalabad. Whilethe samples of 100 individuals were served as a control. Levels of both enzymatic and nonenzymaticantioxidant such as CAT, GSH, SOD, GPx and GRx, vitamin A, C and E and levels ofMDA were determined spectrophotometrically. While the concentration of IL6, AOPPS, AGEs,TNF-α, MMP9 and Isoprostanes were measured by using commercially available Elisa kits.Results: This research work was conducted on a total of 100 control subjects and 270 leukemicpatients and the results were described on mean values. Many circulating biochemical stressmarkers, antioxidants and vitamins were investigated. All enzymatic and non-enzymaticantioxidants, vitamins were decreased in leukemic individuals while markers which showedhigh oxidative stress like MDA, IL6, AOPPS, TNF-α and MMP9 were elevated in leukemicpatients. Conclusion: It is concluded from this study that low levels of antioxidants and highlevels of oxidative stress depict the pathological state of leukemia and defected cell proliferationand growth. Continuous evaluation of antioxidant levels during leukemia can serve as a way ofbetter prognosis and treatment of leukemia.

Simple procedure for production of short DNA size markers of 100 to 2000 bp

DNA size markers (ladder) are essential tools in molecular biology, genetics and biotechnology. In this study, a simple and cost-effective method for laboratory production of DNA ladders is introduced. For this purpose, different sizes of 100 to 2000 bp DNA segments were designed using PCR technique. For producing 14 different gene fragments as DNA molecular weight markers, recombinant plasmid pET28a containing α-amylase gene as a DNA source and one forward and 14 reverse primers were used. The gene fragments containing 100 to 400bp segments with a distance of 50 bp and 400 to 1600 bp segments with a distance of 200 bp as well as 1600 to 2000 bp segments with the distance of 400 bp were generated in a single run of PCR. The present technique could prove to be simple, time saving, inexpensive and good quality approach as compared to the usual DNA ladder preparation procedures. Also, according to the same conditions for designed primers there is a possibility of producing other marker sizes by choosing different types of forward and reverse primers. The PCR product mixture could be directly loaded onto the agarose gel and used as a molecular weight marker without further purification because that was as reliable and uniform as markers from commercial sources. Finally, this marker can be useful for most of molecular biology laboratory techniques.

VITAMIN-D DEFICIENCY;

Background: Deficiency of vitamin D is characterized by the low bone masswhich leads to the bone fragility and high risk of fractures. Bone fractures causes the formationof free radicals, generated by the tissue damaged. Uncontrolled production of free radicalsaccelerates the oxidative stress and increased the bone remodeling process ultimatelycauses osteoporosis. One of the most damaging effects of free radicals is lipid peroxidation;end product of which is MDA, it also act as major factor in osteoblastic activity. Low level ofantioxidative defense system found in osteoporotic patients due to the deficiency of vitamin D.Many important mineral ions removed from bones and risk of bone fragility increases. Currentstudy is aim to check the antioxidative effect produced from excess reactive oxygen speciescompared with low level of vitamin D which is held responsible for higher or lower activityof bone cells. Study Design: Case Control Study. Setting: Study was conducted at Instituteof Molecular Biology and Biotechnology (IMBB), University of Lahore. Period of Study: Oneyear. Materials and Methods: Blood samples of 272 post-menopausal osteoporotic womenbetween the age 49-57 were collected from Jinnah hospital Lahore. While the samples of 92individuals were served as a control. Concentration of both enzymatic and non-enzymaticantioxidant such as CAT, GSH, SOD, GPx and GR, vitamin A, C and E and levels of MDAwere estimated spectrophotometrically. While the concentration of IL6, AOPPS, AGEs, TNF-α,MMP9, Isoprostanes, LDH, cholesterol, triglycerides, free fatty acids and phospholipid weremeasured by using commercially available Elisa kits. Results: Blood plasma levels of vitaminD were significantly lower in osteoporosis patients than in normal subjects. In addition, levelof stress biomarker such as MDA was found to be higher in patients as compared to control.Due to oxidative stress, level of antioxidants (GSH, CAT, and SOD) was found to be reduced.Blood cells and many other important minerals are also reduces in patient group from theirnormal amount. Conclusion: It concludes that excess production of free radicals over whelmsthe antioxidative system, thus it may leads to osteoporosis. Further more antioxidant speciessubjected to body might protect bone loss and also help in acceleration of healing of fracturedbones.

Role of Biocatalysis in Sustainable Chemistry.

Based on the principles and metrics of green chemistry and sustainable development, biocatalysis is both a green and sustainable technology. This is largely a result of the spectacular advances in molecular biology and biotechnology achieved in the past two decades. Protein engineering has enabled the optimization of existing enzymes and the invention of entirely new biocatalytic reactions that were previously unknown in Nature. It is now eminently feasible to develop enzymatic transformations to fit predefined parameters, resulting in processes that are truly sustainable by design. This approach has successfully been applied, for example, in the industrial synthesis of active pharmaceutical ingredients. In addition to the use of protein engineering, other aspects of biocatalysis engineering, such as substrate, medium, and reactor engineering, can be utilized to improve the efficiency and cost-effectiveness and, hence, the sustainability of biocatalytic reactions. Furthermore, immobilization of an enzyme can improve its stability and enable its reuse multiple times, resulting in better performance and commercial viability. Consequently, biocatalysis is being widely applied in the production of pharmaceuticals and some commodity chemicals. Moreover, its broader application will be further stimulated in the future by the emerging biobased economy.