Molecular Biotechnology Research Articles

Breast Cancer Revisited: Stress Induced Conspiracy, Sensitivity and Specificity

Introduction: Cancer and related disorders remain as one of the most devastating ailments worldwide. It signifies the importance to devise particular techniques, which could help in attenuating the effects of breast cancer and related outcomes. Primary aim of the current study is to revisit the role of inflammatory and stress markers in the development and progression of breast cancer and to determine their sensitivity and specificity. Material and Methods: Local sample of premenopausal Pakistani women (age 40 ± 3.6) diagnosed with breast cancer (n = 498) at Jinnah Hospital Lahore, and equal number of controls (age 36 ± 5.2) were selected for the current case control study after informed consent. Serum samples were separated and estimated for MMPs, ILs, HSPs, TNF-α, INOS and Vitamin D on ELISA, whereas, MDA, SOD, NO, CAT, GSH, Vitamin A and D were estimated on Spectrophotometry. Results: Levels of MDA, ILs, MMPs, TNF-α, NO, INOS and HSPs were significantly higher in the breast cancer patients, but the levels of SOD, CAT, GSH, Vitamins A, C and D were significantly lower, determining the relatively positive relationship of oxidants and negative relationship of antioxidants with the increased incidence of pathology among the patients. Furthermore, HSP-27, MMP-9 and IL-1 levels revealed remarkable sensitivity and specificity on ROC analysis. Conclusion: It concludes that HSP-27, MMP-9 and IL-1 may serve as diagnostic and prognostic markers. Furthermore, it can be extrapolated how the increased levels of oxidative and nitrosative stress species along with inflammatory cytokines predispose to the incidence of cell invasion, cell migration and epithelial-mesenchymal transition. Therefore, it infers that increase in growth factors i.e., VEGF, TGF-β and Bcl2 under the influence of said variables play a crucial role in the metastasis of breast cancer. Funding Information: None to declare. Declaration of Interests: Authors declare no conflict of interest. Ethics Approval Statement: All of the patients signed the informed consent and approval was obtained from the Ethical Review Committee, Institute of Molecular Biology and Biotechnology, The University of Lahore, Lahore-Pakistan.

Applications of Charge Detection Mass Spectrometry in Molecular Biology and Biotechnology.

Charge detection mass spectrometry (CDMS) is a single-particle technique where the masses of individual ions are determined from simultaneous measurement of their mass-to-charge ratio (m/z) and charge. Masses are determined for thousands of individual ions, and then the results are binned to give a mass spectrum. Using this approach, accurate mass distributions can be measured for heterogeneous and high-molecular-weight samples that are usually not amenable to analysis by conventional mass spectrometry. Recent applications include heavily glycosylated proteins, protein complexes, protein aggregates such as amyloid fibers, infectious viruses, gene therapies, vaccines, and vesicles such as exosomes.

LIFE MEMBERS WISDOM CLINICAL PERSPECTIVES: FROM MACRO TO MICRO: A REVIEW OF INTERVENTIONS FOR NEUROPSYCHIATRIC DISORDERS: RESTRAINTS, SURGERIES, NEUROMODULATION THERAPIES, PSYCHOTHERAPIES, AND PHARMACEUTICALS TO MOLECULAR BIOTECHNOLOGIES

This Clinical Perspectives will discuss historical macro neuropsychiatric interventions (eg, restraints, surgeries, ECT, and psychotherapies), trace the trajectory of treatments over the decades to the use of pharmaceuticals and neuromodulation, and envision a future with site-specific brain treatments using molecular biotechnologies.

Effects of Soil pH on Gaseous Nitrogen Loss Pathway via Feammox Process

The application of N fertilizer is one of the most critical soil acidification factors in China, and soil acidification significantly alters biogeochemical processes such as N loss. Anaerobic ammonium oxidation coupled with iron reduction (Feammox) is an important biological process for N loss in natural environments, with the end-products of N2, NO2− and NO3−. However, the response of Feammox pathways to soil pH fluctuation has not been thoroughly studied. In the current study, Feammox pathways and microbial communities were explored through a slurry culture experiment with an artificially adjusted pH combined with a 15N isotope tracing technique and molecular biotechnology. Results showed significant differences in the gaseous N loss through Feammox (0.42–0.97 mg N kg−1 d−1) under different pH conditions. The gaseous N loss pathways were significantly affected by the pH, and Feammox to N2 was the predominant pathway in low-pH incubations. The proportion of N loss caused by Feammox coupled with denitrification increased as the soil pH increased. The gaseous N loss through Feammox increased by 43.9% when the soil pH decreased from 6.5 to 5.0. Fe-reducing bacteria, such as Ochrobactrum, Sphingomonas, and Clostridium increased significantly in lower pH incubations. Overall, this study demonstrated the effects of soil pH on Feammox pathways and extended the understanding of the N biogeochemical cycle in acidic soil.

Gossypol detoxification in the rumen and Helicoverpa armigera larvae: A review

Gossypol, a phenolic compound found in the cotton plant, is widely distributed in cottonseed by-products. Although ruminant animals are believed to be more tolerant of gossypol toxicity than monogastric animals due to rumen microbial fermentation, the actual mechanisms of detoxification remain unclear. In contrast, the metabolic detoxification of gossypol by Helicoverpa armigera (Lepidoptera: Noctuidae) larvae has achieved great advances. The present review discusses the clinical signs of gossypol in ruminant animals, as well as summarizing advances in the study of gossypol detoxification in the rumen. It also examines the regulatory roles of several key enzymes in gossypol detoxification and transformation known in H. armigera. With the rapid development of modern molecular biotechnology and -omics technology strategies, evidence increasingly indicates that research into the biological degradation of gossypol in H. armigera larvae and some microbes, in terms of these key enzymes, could provide scientific insights that would underpin future work on microbial gossypol detoxification in the rumen, with the ultimate aim of further alleviating gossypol toxicity in ruminant animals.

Immune-mediated inflammatory disease therapeutics: past, present and future.

Immune-mediated inflammatory diseases are common and clinically diverse. Although they are currently incurable, the therapeutic armamentarium for immune-mediated inflammatory diseases has been transformed in the past two decades. We have moved from the wide application of broad-spectrum immune modulators to the routine use of agents with exquisite specificity, arising from monoclonal and molecular biotechnology and more recently from highly targeted medicinal chemistry. Here we describe key advances and lessons that drove this remarkable progress and thereafter reflect on the next steps in this ongoing journey.

Prokaryotic expression of chimeric GFP-hFc protein as a potential immune-based tool.

GFP is an old-yet-powerful protein marker, which has been widely used in molecular biotechnology due to its capacity of exhibiting bright green fluorescence when exposed to ultraviolet light. The hFc region of IgG antibodies is a specific binding ligand of expressed receptors on immune cells with well-known cellular-associated functions like opsonization and phagocytosis. In this present study, we proceeded to fuse gfp-hfc gene into pET-28a to create a recombinant pET-28a-gfp-hfc vector. The expression of GPF-hFc was induced by IPTG and confirmed using SDS-PAGE and followed by Western blot probed with 6xHis antibodies. This chimeric protein was utilized in specific binding experiments with protein A/G-coated magnetic beads using a fluorescence microscope. Due to its fluorescence and binding ability, GFP-hFc could be used as a model molecule for monitoring molecule detection studies, tracking nanoparticle migration and distribution, or stimulating immune responses.

Mass spectrometry imaging-based multi-modal technique: Next-generation of biochemical analysis strategy

Mass spectrometry imaging-based multi-modal technique: Next-generation of biochemical analysis strategy

Molecular Genetics, Genomics and Biotechnology in Crop Plant Breeding, Series II

This second volume of this series of Special Issues provides research papers and reviews on the use of molecular marker technologies, genomics selection, site-directed mutagenesis, gene-discovery by genome-wide association studies and biotechnology in important grain crops, tubers, fruit bearing shrub and small fruit crops [...]

Model genetic profiles of pigs of mother breeds on genes - productivity markers

Currently, development of molecular genetics and biology allows for genomic analysis and selection directly at the DNA level (marker-dependent selection). The aim of the research was to develop model genetic profiles for marker genes of quantitative traits of productivity of pigs of planned maternal breeds used in pig breeding. Such breeds in the Republic of Belarus are as follows: Belarusian large white, Belarusian black and white and Yorkshire. The studies were carried out at the agricultural branch of Zadneprovsky SGC, Orshansky Plant of Bread Products, JSC, Zarechye SGC, Zapadny SGC with populations of purebred animals of Belarusian Large White, Belarusian Black and White and Belarusian plant type of Yorkshire pigs during 2002-2018. Genetic testing was carried out with sows, boars and finishing pigs of maternal breeds. As a starting material, tissue samples from the auricle of pigs were used with DNA isolated and optimized for the analysis of gene polymorphism using PCR - RFLP method in the laboratories of molecular biotechnology and DNA testing (Research and Practical Center of the National Academy of Sciences of Belarus for Animal Breeding) and animal genetics (Institute of Genetics and Cytology of the National Academy of Sciences of Belarus). As a result of studies based on the established polymorphism, model genetic profiles of maternal breeds of pigs were developed for marker genes of quantitative traits of productivity. The maximum achieved level of the preferred genotype for each marker gene among the three maternal breeds served as a model profile for the evaluated breeds (breeding young animals). For breeds with an established high level of marker gene polymorphism and productivity, a model profile has been developed that exceeds the achieved indicator by 8-10 p.p.

Possible response of molecular biotechnology to call SARS-CoV-2

COVID-19 pandemic caused by coronavirus SARS-CoV-2 affected virtually all life aspects in most countries and nations. During 2020 around 100 mln of Earth inhabitants contracted this morbid infection and 2 mln people died thereafter. It is natural therefore that many pharmaceutical companies around the globe have joined into the race aimed at elaboration of prophylactic antiviral vaccines. However, the option can not be ruled out that parenteral vaccines to counter rapidly mutating pandemic coronavirus may be designed hastily, neglecting due tests of remote side effects, so that in the long run they risk to be expelled from the marked due to doubtful safety and efficiency.As a real alternative to traditional vaccination techniques the present mini-review has proposed application of intranasal adjuvant nano-vaccine. If necessary, this approach may be complemented with pharmacological inactivation of coronavirus under the impact of ribo-favipiravir, recombinant arginine deiminase, RNAse L or lactoferrin, as well as biogenic silver or copper nanoparticles - the preparations derived from up-to-date molecular biotechnological processes.

Biosynthesis of <i>Escherichia coli</i> adenosine deaminase using cell-free protein synthesis

One of the recent perspective trends of molecular biotechnology is cell-free protein synthesis (CFPS). The procedure of CFPS is based on in vitro reconstruction of all stages of a biosynthesis of protein in a whole cell, including a transcription, an aminoacylation of tRNA and translation of mRNA by ribosomes.Previously, we constructed a strain Escherichia coli that produces homologous adenosine deaminase (ADase). In the present study, as an alternative to canonical submerged cultivation in a fermenter, the possibility of the ADase synthesis in the system of CFPS was studied. For synthesis of this enzyme we used the E. coli-30 cell extract, T7 bacteriophage RNA polymerase, and high-copy plasmid vector pET42mut with gene ADase inserted into it.As a result of the work we have demonstrated for the first time the possibility of synthesis of ADase E. coli in the CFPS system. In a partially optimized process conditions, an experimental sample of recombinant AD with an activity of 530 U/ml of enzyme preparation was obtained.

Advances in Agricultural Biotechnology

Agricultural biotechnology is becoming the major sector in crop improvement through the use of scientific techniques for the modification of genes conferring resistance to biotic, abiotic stress and improving the quality of crops. With the evolvement from Mendelian genetics to molecular biotechnology, there have been several developments in the field of crop improvement. Recent biotechnological advances have aimed towards removing the physiological constraints of the crops and increasing crop yield potential. With the use of different tools of agricultural biotechnologies like genetic engineering, tissue culture, embryo rescue, somatic hybridization, molecular marker-assisted selection, genome doubling, and omics technologies, various transgenic crops have been developed over the decades and have been approved for commercialization. This development and adoption of transgenic technology have been shown to increase crop yields, reduce CO2 emission, reduce pesticide and insecticide use and decrease the costs of crop production. Even though the biotechnological approach and transgenic organisms have immense potential to contribute to the world’s food security, several concerns of genetically modified crops being a threat to the environment and human health have developed. This review will address applications and concerns of biotechnology in crop improvement considering health hazards and ecological risks.

Challenges for Nanotechnology

The term “Nanotechnology” describes a large field of scientific and technical activities dealing with objects and technical components with small dimensions. Typically, bodies that are in–at least–two dimensions smaller than 0.1 µm are regarded as “nanobjects”. By this definition, a lot of advanced materials, as well as the advanced electronic devices, are objects of nanotechnology. In addition, many aspects of molecular biotechnology as well as macromolecular and supermolecular chemistry and nanoparticle techniques are summarized under “nanotechnology”. Despite this size-oriented definition, nanotechnology is dealing with physics and chemistry as well as with the realization of technical functions in the area between very small bodies and single particles and molecules. This includes the shift from classical physics into the quantum world of small molecules and low numbers or single elementary particles. Besides the already established fields of nanotechnology, there is a big expectation about technical progress and solution to essential economic, medical, and ecological problems by means of nanotechnology. Nanotechnology can only meet these expectations if fundamental progress behind the recent state of the art can be achieved. Therefore, very important challenges for nanotechnology are discussed here.

Syndromic immunodeficiencies: a pediatrician's perspective on selected diseases.

Syndromic immunodeficiencies are a genetically and pathophysiologically heterogeneous group of inborn errors of immunity. These are characterized by multiple extra immune clinical symptoms and a wide range of immunological phenotypes with increased susceptibility to infections, autoimmune phenomena, immune dysregulation, organ-specific pathology, and malignancy. To increase the pediatricians' awareness of this multifaceted group of primary immunodeficiencies in children. A comprehensive review of genetic background and clinical symptomatology of syndromic immunodeficiencies as well as current diagnostic approach and treatment modalities. From the pediatrician's perspective, an early-life diagnosis of syndromic immunodeficiencies, which is frequently indispensable for successful life-saving immunocorrection, poses a diagnostic challenge. Increased pediatricians' awareness to recognize signs and symptoms of these diseases in affected children is of paramount importance. Current advances in molecular biotechnology and immunogenetics, resulting in the implementation of newborn screening and new-generation sequencing, provide informative tools for definitive diagnosis and, in many new disease entities, for their definition and genotype-phenotype delineation and correlation. A broad spectrum of clinical phenotypes in children with syndromic primary immunodeficiencies requires pediatrician's special attention, that is, individualized multidisciplinary approach under the supervision of a clinical immunologist.

An approach for quantifying western blots: the case of signal intensity and the statistical analysis

Article Details: Received: 2020-04-07 | Accepted: 2021-01-25 | Available online: 2021-06-30 https://doi.org/10.15414/afz.2021.24.02.141-146 In biological sciences, western blotting technique is widely used to quantify the expression of proteins in a given sample. However, there is no unified method for quantifying the expression of proteins. As a consequence, quantitative analysis of expression of protein through western blotting often suffers from data inconsistency. At the same time, extraction of the poor sample size (n=3/5/7) turns such analysis non-Gaussian and less robust to statistical errors. In present study, we attempt  a noble approach while analyzing an image from western blotting using Gaussian blur as filter and thereby generating data in order to perform meaningful statistical analysis. The differences among various blots that correspond to the expressed target proteins are tested viably using appropriate statistical tools. This procedure of quantifying western blotting is comprehensive, simple and can be applied to collect data in compliance with statistical norms.  Furthermore, repeating western blotting on a set of particular proteins may improve the analysis part as well. Keywords:  blotting, statistical analysis, non-parametric, densitometry References Aldridge, G.M. et al. (2008). The use of total protein stains as loading controls: An alternative to high-abundance singleprotein controls in semiquantitative immunoblotting. Journal of Neuroscience Methods, 172(2), 250–254. Butler, T.A.J. et al. (2019). Misleading westerns: common quantification mistakes in western blot densitometry and proposed corrective measures. BioMedical Research, ID 5214821. https://doi.org/10.1155/2019/5214821 Eaton, S.L. et al. (2013). Total Protein Analysis as a Reliable Loading Control for Quantitative Fluorescent Western Blotting. PLoS ONE, 8(8), e72457. https://doi.org/10.1371/journal.pone.0072457 Ghosh, R., Gilda, J.E. & Gomes, A.V. (2014). The necessity of and strategies for improving confidence in the accuracy of western blots. Expert Review of Proteomics, 11, 549–560. Graham, B. (2016). Quantifying western blots: none more black. BMC Biology, 14, 116. https://doi.org/10.1186/s12915-016-0339-1 Kreutz, C. et al. (2007). An error model for protein quantification. Bioinformatics, 23 (20), 2747–2753, https://doi.org/10.1093/bioinformatics/btm397 Maleki, F. et al. (2019). Size matters: how sample size affects the reproducibility and specificity of gene set analysis. Hum Genomics, 13, 42. https://doi.org/10.1186/s40246-019-0226-2 Taylor, S.C. et al. (2013). A defined methodology for reliable quantification of Western blot data. Molecular Biotechnology, 55(3), 217–226. https://doi.org/10.1007/s12033-013-9672-6 Towbin, H., Staehelin, T. & Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences, 76(9), 4350–4354. Welinder, C. & Ekblad, L. (2011). Coomassie staining as loading control in Western blot analysis. Journal of Proteome Research, 10(3), 1416–1419

Synthetic counter-selection markers and their application in genetic modification of Synechococcus elongatus UTEX2973.

Due to its robustness to environmental stresses and fast growth, Synechococcus elongatus UTEX2973 is developed as a new model for researches on cyanobacterial molecular biology and biotechnology. However, systematic genetic modifications of S. elongatus UTEX2973 were hindered by the lack of effective genetic manipulation tools, especially available counter-selection markers. Here, six synthetic counter-selection markers (SCOMs) were assembled by fusing six toxin genes from either Escherichia coli or cyanobacteria with a theophylline-inducible promoter. The SCOMs containing SYNPCC7002_G0085 from Synechococcus sp. PCC7002 or mazF from E. coli were proved to be inducible by theophylline in S. elongatus UTEX2973. By using the mazF-based SCOM, the neutral locus 1 and 23 small regulatory RNAs were completely deleted from the genome of S. elongatusUTEX2973 after one round of selection with both kanamycin and theophylline. The genetic tools developed in this work will facilitate future researches on molecular genetics and synthetic biology in S. elongatus UTEX2973. KEY POINTS: • Two inducible counter-selection markers are lethal to S. elongatus UTEX2973. • The counter-selection marker benefits the gene targeting in S. elongatus UTEX2973. • Twentry-three small regulatory RNAs were fully deleted via the novel gene targeting method.

Cell scientists to watch – Zdeněk Lánský and Marcus Braun

ABSTRACT Zdeněk Lánský received his PhD in physics from the Charles University in Prague in 2006. He then moved to The Netherlands for his postdoc, where he studied the biophysics of molecular motors – first at the Vrije Universiteit in Amsterdam with Erwin Peterman, and then at Wageningen University with Marcel Janson. In 2011 he joined the lab of Stefan Diez at the B CUBE Center for Molecular Bioengineering in Dresden, where his research focused on crosslinker-mediated force generation in the cytoskeleton. Zdeněk set up his lab at the Institute of Biotechnology, Czech Academy of Sciences, BIOCEV in 2015, which he has run together with Marcus Braun since 2017. Marcus Braun received his PhD in biochemistry from King's College London in 2009 for his work on microtubule-based molecular motors in the labs of Andrew McAinsh and Rob Cross. He then moved to Dresden for his postdoc to study cytoskeletal transport, with a particular focus on microtubule crosslinkers, in Stefan Diez's lab – first at the Max Planck Institute of Molecular Cell Biology and Genetics, then later at B CUBE. In 2017, he joined Zdeněk Lánský at the Institute of Biotechnology, Czech Academy of Sciences, BIOCEV, as a principal investigator. The Lánský and Braun lab investigates ensemble dynamics of cytoskeletal proteins.

Molecular diversity of arbuscular mycorrhizal fungi associated with two alpine plant species in the Tibetan Plateau

Alpine ecosystem in the Tibetan Plateau has a special ecological environment, which may harbor a high richness of arbuscular mycorrhizal fungi (AMF) communities. To explore the molecular diversity of AMF community associated with plants in alpine grasslands, root and rhizosphere soil of two dominant plant species, Kobresia sp. and Leontopodium pusillum in Qinghai province, were studied by molecular biotechnology (clone library and sequencing) at five sites for each plant species. A total of 30 operational taxonomic units (OTUs) were identified, including seven families of Glomeromycota. Glomeraceae and Glo1 and Glo2 were most abundant at family and OUT level in roots respectively, while both Glomeraceae and Diversisporaceae were frequently present and Glo1 and Div1 were the most dominant OTUs in the soil. The AMF community was different in the roots and soil. Nineteen OTUs were identified in the roots of Kobresia sp., and 21 OTUs were identified in the rhizosphere soil. As to L. pusillum, fourteen OTUs were identified in roots and 17 OTUs in the soil. Specifically, three OTUs were only found in L. pusillum, and eight OTUs were only found in Kobresia sp. The root biomass and soil C/N ratio were the main factors affecting the AMF community in roots, whereas in soil, the important factors were root carbon content and shoot nitrogen content. These results showed a high diversity of AMF associated with the typical alpine plants, and distinct AMF community exist in roots and soil. Our results confirmed the importance of plant and soil factors in driving AMF assemblages in the fragile ecosystem of Tibetan Plateau.

Recent Advances in Cellular and Molecular Bioengineering for Building and Translation of Biological Systems.

In January of 2020, the Biomedical Engineering Society (BMES)- Cellular and Molecular Bioengineering (CMBE) conference was held in Puerto Rico and themed “Vision 2020: Emerging Technologies to Elucidate the Rule of Life.” The annual BME-CMBE conference gathered worldwide leaders and discussed successes and challenges in engineering biological systems and their translation. The goal of this report is to present the research frontiers in this field and provide perspectives on successful engineering and translation towards the clinic. We hope that this report serves as a constructive guide in shaping the future of research and translation of engineered biological systems.