Molecular Biology Tools Research Articles

Pathophysiological basis in the management of myasthenia gravis: a mini review.

Myasthenia gravis is an autoimmune disorder characterized by the presence of autoantibodies against the acetylcholine receptor present in the post-synaptic membrane of the neuromuscular junction impairing the muscle contraction and causing the patient suffering from the disorder to develop a myriad of muscular defects ranging from drooping of eyelids, blurred or double vision, shortness of breath, difficulty in swallowing, as well as weakness of limbs and arms. Myasthenia gravis is known as the disease of old men and young women but in contrast to the global scenario, in India, myasthenia gravis was found to be predominant in males with the ratio of 2.70:1. Though the disorder has been studied for centuries, the true reason for disease and its pathophysiology still eludes us. But recent advancement in molecular biology and diagnostic tools has enabled us to identify many targets for pharmacotherapy as well as for early diagnosis. Thus, improving the patient's morbidity and quality of life. In this article, we are discussing the recent advancements made in diagnosis and therapy of the disease.

Grafting: a potential method to reveal the differential accumulation mechanism of secondary metabolites.

Plant secondary metabolites make a great contribution to the agricultural and pharmaceutical industries. Their accumulation is determined by the integrated transport of target compounds and their biosynthesis-related RNA, protein, or DNA. However, it is hard to track the movement of these biomolecules in vivo. Grafting may be an ideal method to solve this problem. The differences in genetic and metabolic backgrounds between rootstock and scion, coupled with multiple omics approaches and other molecular tools, make it feasible to determine the movement of target compounds, RNAs, proteins, and DNAs. In this review, we will introduce methods of using the grafting technique, together with molecular biological tools, to reveal the differential accumulation mechanism of plant secondary metabolites at different levels. Details of the case of the transport of one diterpene alkaloid, fuziline, will be further illustrated to clarify how the specific accumulation model is shaped with the help of grafting and multiple molecular biological tools.

Genetic identification of bat species for pathogen surveillance across France.

With more than 1400 chiropteran species identified to date, bats comprise one-fifth of all mammalian species worldwide. Many studies have associated viral zoonoses with 45 different species of bats in the EU, which cluster within 5 families of bats. For example, the Serotine bats are infected by European Bat 1 Lyssavirus throughout Europe while Myotis bats are shown infected by coronavirus, herpesvirus and paramyxovirus. Correct host species identification is important to increase our knowledge of the ecology and evolutionary pattern of bat viruses in the EU. Bat species identification is commonly determined using morphological keys. Morphological determination of bat species from bat carcasses can be limited in some cases, due to the state of decomposition or nearly indistinguishable morphological features in juvenile bats and can lead to misidentifications. The overall objective of our study was to identify insectivorous bat species using molecular biology tools with the amplification of the partial cytochrome b gene of mitochondrial DNA. Two types of samples were tested in this study, bat wing punches and bat faeces. A total of 163 bat wing punches representing 22 species, and 31 faecal pellets representing 7 species were included in the study. From the 163 bat wing punches tested, a total of 159 were genetically identified from amplification of the partial cyt b gene. All 31 faecal pellets were genetically identified based on the cyt b gene. A comparison between morphological and genetic determination showed 21 misidentifications from the 163 wing punches, representing ~12.5% of misidentifications of morphological determination compared with the genetic method, across 11 species. In addition, genetic determination allowed the identification of 24 out of 25 morphologically non-determined bat samples. Our findings demonstrate the importance of a genetic approach as an efficient and reliable method to identify bat species precisely.

Using Molecular Visualisation Techniques to Explain the Molecular Biology of SARS-CoV-2 Spike Protein Mutations to a General Audience.

Since the COVID-19 pandemic started in 2019, the virus responsible for the outbreak-SARS-CoV-2-has continued to evolve. Mutations of the virus' spike protein, the main protein driving infectivity and transmissibility, are especially concerning as they may allow the virus to improve its infectivity, transmissibility, and ability to evade the immune system. Understanding how specific molecular changes can alter the behaviour of a virus is challenging for non-experts, but this information helps us to understand the pandemic we are living through and the public health measures and interventions needed to bring it under control. In response to communication challenges arising from the COVID-19 pandemic, we recently developed an online educational application to explain the molecular biology of SARS-CoV-2 spike protein mutations to the general public. We used visualisation techniques such as 3D modelling and animation, which have been shown to be highly effective teaching tools in molecular biology, allowing the viewer to better understand protein structure, function, and dynamics. We also included interactive elements for users to learn actively by engaging with the digital content, and consequently improve information retention.This chapter presents the methodological and technological framework which we used to create this resource, the 'SARS-CoV-2 Spike Protein Mutation Explorer' (SSPME). It explains how molecular visualisation and 3D modelling software were used to develop accurate models of relevant proteins; how 3D animation software was used to accurately visualise the dynamic molecular processes of SARS-CoV-2 infection, transmission, and antibody evasion; and how game development software was used to compile the 3D models and animations into a comprehensive, informative interactive application on SARS-CoV-2 spike protein mutations. This chapter indicates how cutting-edge visualisation techniques and technologies can be used to improve science communication about complex topics in molecular biology and infection biology to the general public, something that is critical to gaining control of the continuing COVID-19 pandemic.

Affinity-Guided Site-Selective Labeling of Nanobodies with Aldehyde Handles.

Antigen-binding proteins such as nanobodies are extensively used in biomedicine, diagnostics, and as tools for molecular biology. Often such applications require modification of the nanobodies with fluorophores or drugs. Here, we describe a robust method for introduction of aldehyde handles into His-tagged nanobodies and further derivatization of these proteins with hydroxylamine functionalized compounds of interest. The method allows for isolation of nanobodies containing one or more labels.

Livestock and Poultry Milieus as Non-Hospital Sources of AmpC and ESBL Producers in Southeast Nigeria

Antimicrobial resistance (AMR) occurs when microorganisms fail to respond to the therapeutic onslaught of antibiotics. Extended-spectrum beta-lactamase (ESBL) and AmpC enzymes are important AMR mechanisms that erode the efficacy of important antibiotics. Here, we report on the detection and susceptibility of ESBL- and AmpC-producing bacteria from livestock and poultry environments. Bacteriological and molecular biology tools were used for the isolation and characterization of bacteria. Combined

Livestock and Poultry Milieus as Non-Hospital Sources of AmpC and Extended-Spectrum Beta-Lactamases Producers in Southeast Nigeria

Antimicrobial resistance (AMR) occurs when microorganisms fail to respond to the therapeutic onslaught of antibiotics. Extended-spectrum beta-lactamase (ESBL) and AmpC enzymes are important AMR mechanisms that erode the efficacy of important antibiotics. Here, we report on the detection and susceptibility of ESBL- and AmpC-producing bacteria from livestock and poultry environments. Bacteriological and molecular biology tools were used for the isolation and characterization of bacteria. Combined

CRISPR Interference as a Tool to Repress Gene Expression in Haloferax volcanii.

To date, a plethora of tools for molecular biology have been developed on the basis of the CRISPR-Cas system. Almost all use the class 2 systems since here the setup is the simplest with only one protein and one guide RNA, allowing for easy transfer to and expression in other organisms. However, the CRISPR-Cas components harnessed for applications are derived from mesophilic bacteria and are not optimal for use in extremophilic archaea.Here, we describe the application of an endogenous CRISPR-Cas system as a tool for silencing gene expression in a halophilic archaeon. Haloferax volcanii has a CRISPR-Cas system of subtype I-B, which can be easily used to repress the transcription of endogenous genes, allowing to study the effects of their depletion. This article gives a step-by-step introduction on how to use the implemented system for any gene of interest in Haloferax volcanii. The concept of CRISPRi described here for Haloferax can be transferred to any other archaeon, that is genetically tractable and has an endogenous CRISPR-Cas I systems.

Characterization and Expression Profiles of Galaetinol Synthase and Raffinose Synthase in Rubber Tree

Galactinol synthase (GolS) and Raffinose synthase (RS) are key enzymes for the synthesis of Raffinose family oligosaccharides (RFOs) which are widely involved in the growth development and resistances to stresses. To explore the function of HbGolSs  and HbRSs  during leaf development and tapping period in Hevea brasiliensis, in this study, based on the established library data platform of rubber tree genome, we isolated and identified the family members of HbGolSs  and HbRSs , then we analyzed their expression profiles with emphasis through on-line molecular biological tools and PRISM software as well. The results demonstrated that HbGolS1  and HbRS1 were prime member in their separate family, HbGOlS1  was highly expressed in latex and leaf, and with leaf development, its level gradually increased till the ultimate in mature leave, especially, it was dramatically up-regulated by tapping but markedly inhibited by Ethrel treatment; Different from it, HbRS1  was little influenced by tapping or Ethrel treatment although it was specially expressed in latex. Together, this study demonstrated the expression characteristics of HbGolS1 and HbRS1  with leaf development and tapping, which has laid a foundation for further exploring the metabolism of RFOs and their roles in rubber trees.

Successful Exogenous Expression of ATP8, a Mitochondrial Encoded Protein, From the Nucleus In Vivo

Replicative errors, inefficient repair, and proximity to reactive oxygen species production sites make the mitochondrial DNA (mtDNA) susceptible to damage with time. mtDNA mutations accumulate with age and accompany a progressive decline in organelle function. We lack molecular biology tools to manipulate mtDNA, thus we explore the possibility in vivo of utilizing allotopic expression, or the re-engineering mitochondrial genes and expressing them from the nucleus, as an approach to rescue defects arising from mtDNA mutations. This study uses a mouse model with a mutation in the mitochondrial ATP8 gene that encodes a protein subunit of the ATP synthase. We generated a transgenic mouse with an epitope-tagged recoded and mitochondrial-targeted ATP8 gene expressed from the nucleus. Our results show that the allotopically expressed ATP8 protein in the transgenic mice is robustly expressed across all tested tissues, successfully transported into the mitochondria, and incorporated into ATP synthase. We are currently evaluating if allotopic expression of ATP8 will functionally rescue the behavioral and bioenergetic defects in ATP8 mutant mice. Translating allotopic expression technology into a mammal and demonstrating systemic functional rescue will lend credence to utilizing allotopic expression as a gene therapy in humans to repair physiological consequences of mtDNA defects that may accumulate with age.

Successful Exogenous Expression of ATP8, a Mitochondrial Encoded Protein, From the Nucleus In Vivo.

Replicative errors, inefficient repair, and proximity to reactive oxygen species production sites make the mitochondrial DNA (mtDNA) susceptible to damage with time. mtDNA mutations accumulate with age and accompany a progressive decline in organelle function. We lack molecular biology tools to manipulate mtDNA, thus we explore the possibility in vivo of utilizing allotopic expression, or the re-engineering mitochondrial genes and expressing them from the nucleus, as an approach to rescue defects arising from mtDNA mutations. This study uses a mouse model with a mutation in the mitochondrial ATP8 gene that encodes a protein subunit of the ATP synthase. We generated a transgenic mouse with an epitope-tagged recoded and mitochondrial-targeted ATP8 gene expressed from the nucleus. Our results show that the allotopically expressed ATP8 protein in the transgenic mice is robustly expressed across all tested tissues, successfully transported into the mitochondria, and incorporated into ATP synthase. We are currently evaluating if allotopic expression of ATP8 will functionally rescue the behavioral and bioenergetic defects in ATP8 mutant mice. Translating allotopic expression technology into a mammal and demonstrating systemic functional rescue will lend credence to utilizing allotopic expression as a gene therapy in humans to repair physiological consequences of mtDNA defects that may accumulate with age.

CRISPR-Cas9-mediated Large Cluster Deletion and Multiplex Genome Editing in Paenibacillus polymyxa.

The use of molecular tools based on the clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas) systems has rapidly advanced genetic engineering. These molecular biological tools have been applied for different genetic engineering purposes in multiple organisms, including the quite rarely explored Paenibacillus polymyxa. However, only limited studies on large cluster deletion and multiplex genome editing have been described for this highly interesting and versatile bacterium. Here, we demonstrate the utilization of a Cas9-based system to realize targeted deletions of four biosynthetic gene clusters in the range of 12-41 kb by the use of a single targeting sgRNA. Furthermore, we also harnessed the system for multiplex editing of genes and large genomic regions. Multiplex deletion was achieved with more than 80% efficiency, while simultaneous integration at two distantly located sites was obtained with 58% efficiency. The findings reported in this study are anticipated to accelerate future research in P. polymyxa and related species.

CRISPR systems: what’s new, where next?

The genetic signature of natural CRISPR-Cas systems were first noted in a 1989 publication and were characterized in detail from 2002 to 2007, culminating in the first report of a prokaryotic adaptive immune system. Since then, CRISPR-Cas enzymes have been adapted into molecular biology tools that have transformed genetic engineering across domains of life. In this feature article, we describe origins, uses and futures of CRISPR-Cas enzymes in genetic engineering: we highlight advances made in the past 10 years. Central to these advances is appreciation of interplay between CRISPR engineering and DNA repair. We highlight how this relationship has been manipulated to create further advances in the development of gene editing.

Nested Stochastic Block Models applied to the analysis of single cell data

Single cell profiling has been proven to be a powerful tool in molecular biology to understand the complex behaviours of heterogeneous system. The definition of the properties of single cells is the primary endpoint of such analysis, cells are typically clustered to underpin the common determinants that can be used to describe functional properties of the cell mixture under investigation. Several approaches have been proposed to identify cell clusters; while this is matter of active research, one popular approach is based on community detection in neighbourhood graphs by optimisation of modularity. In this paper we propose an alternative and principled solution to this problem, based on Stochastic Block Models. We show that such approach not only is suitable for identification of cell groups, it also provides a solid framework to perform other relevant tasks in single cell analysis, such as label transfer. To encourage the use of Stochastic Block Models, we developed a python library, schist, that is compatible with the popular scanpy framework.

Evolution of ColE1-like plasmids across γ-Proteobacteria: From bacteriocin production to antimicrobial resistance.

Antimicrobial resistance is one of the major threats to Public Health worldwide. Understanding the transfer and maintenance of antimicrobial resistance genes mediated by mobile genetic elements is thus urgent. In this work, we focus on the ColE1-like plasmid family, whose distinctive replication and multicopy nature has given rise to key discoveries and tools in molecular biology. Despite being massively used, the hosts, functions, and evolutionary history of these plasmids remain poorly known. Here, we built specific Hidden Markov Model (HMM) profiles to search ColE1 replicons within genomes. We identified 1,035 ColE1 plasmids in five Orders of γ-Proteobacteria, several of which are described here for the first time. The phylogenetic analysis of these replicons and their characteristic MOBP5/HEN relaxases suggest that ColE1 plasmids have diverged apart, with little transfer across orders, but frequent transfer across families. Additionally, ColE1 plasmids show a functional shift over the last decades, losing their characteristic bacteriocin production while gaining several antimicrobial resistance genes, mainly enzymatic determinants and including several extended-spectrum betalactamases and carbapenemases. Furthermore, ColE1 plasmids facilitate the intragenomic mobilization of these determinants, as various replicons were identified co-integrated with large non-ColE1 plasmids, mostly via transposases. These results illustrate how families of plasmids evolve and adapt their gene repertoires to bacterial adaptive requirements.

Recent Advancements in Microbial Polysaccharides: Synthesis and Applications.

Polysaccharide materials are widely applied in different applications including food, food packaging, drug delivery, tissue engineering, wound dressing, wastewater treatment, and bioremediation sectors. They were used in these domains due to their efficient, cost-effective, non-toxicity, biocompatibility, and biodegradability. As is known, polysaccharides can be synthesized by different simple, facile, and effective methods. Of these polysaccharides are cellulose, Arabic gum, sodium alginate, chitosan, chitin, curdlan, dextran, pectin, xanthan, pullulan, and so on. In this current article review, we focused on discussing the synthesis and potential applications of microbial polysaccharides. The biosynthesis of polysaccharides from microbial sources has been considered. Moreover, the utilization of molecular biology tools to modify the structure of polysaccharides has been covered. Such polysaccharides provide potential characteristics to transfer toxic compounds and decrease their resilience to the soil. Genetically modified microorganisms not only improve yield of polysaccharides, but also allow economically efficient production. With the rapid advancement of science and medicine, biosynthesis of polysaccharides research has become increasingly important. Synthetic biology approaches can play a critical role in developing polysaccharides in simple and facile ways. In addition, potential applications of microbial polysaccharides in different fields with a particular focus on food applications have been assessed.

Spatial and Temporal Constraints on the Composition of Microbial Communities in Subsurface Boreholes of the Edgar Experimental Mine.

ABSTRACTThe deep biosphere hosts uniquely adapted microorganisms overcoming geochemical extremes at significant depths within the crust of the Earth. Attention is required to understand the near subsurface and its continuity with surface systems, where numerous novel microbial members with unique physiological modifications remain to be identified. This surface-subsurface relationship raises key questions about networking of surface hydrology, geochemistry affecting near-subsurface microbial composition, and resiliency of subsurface ecosystems. Here, we apply molecular and geochemical approaches to determine temporal microbial composition and environmental conditions of filtered borehole fluid from the Edgar Experimental Mine (∼150 m below the surface) in Idaho Springs, CO. Samples were collected over a 4-year collection period from expandable packers deployed to accumulate fluid in previously drilled boreholes located centimeters to meters apart, revealing temporal evolution of borehole microbiology. Meteoric groundwater feeding boreholes demonstrated variable recharge rates likely due to a complex and undefined fracture system within the host rock. 16S rRNA gene analysis determined that unique microbial communities occupy the four boreholes examined. Two boreholes yielded sequences revealing the presence of Desulfosporosinus, Candidatus Nitrotoga, and Chelatococcus associated with endemic subsurface communities. Two other boreholes presented sequences related to nonsubsurface-originating microbiota. High concentration of sulfate along with detected sulfur reducing and oxidizing microorganisms suggests that sulfur related metabolic strategies are prominent within these near-subsurface boreholes. Overall, results indicate that microbial community composition in the near-subsurface is highly dynamic at very fine spatial scales (<20 cm) within fluid-rock equilibrated boreholes, which additionally supports the role of a relationship for surface geochemical processes infiltrating and influencing subsurface environments.IMPORTANCE The Edgar Experimental Mine, Idaho Springs, CO, provides inexpensive and open access to borehole investigations for subsurface microbiology studies. Understanding how microbial processes in the near subsurface are connected to surface hydrological influences is lacking. Investigating microbial communities of subsurface mine boreholes provides evidence of how geochemical processes are linked to biogeochemical processes within each borehole and the geochemical connectedness and mobility of surface influences. This study details microbial community composition and fluid geochemistry over spatial and temporal scales from boreholes within the Edgar Mine. These findings are relevant to biogeochemistry of near-surface mines, caves, and other voids across planetary terrestrial systems. In addition, this work can lead to understanding how microbial communities relate to both fluid-rock equilibration, and geochemical influences may enhance our understanding of subsurface molecular biological tools that aid mining economic practices to reflect biological signals for lucrative veins in the near subsurface.

Molecular and MALDI-TOF MS characterisation of Hyalomma aegyptium ticks collected from turtles and their associated microorganisms in Algeria

The identification of ticks and their associated pathogens is important for knowledge on tick-borne diseases. The objective of this study was to use morphological, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and/or molecular biology tools to identify ticks collected from turtles in north-eastern Algeria, as well as to investigate the microorganisms associated with these ticks. A total of 471 adult ticks were collected and identified morphologically as Hyalomma aegyptium, of which 248 (52.7%) were female and 223 (47.3%) were male. amongst them, 230 specimens were randomly selected for molecular and MALDI-TOF MS analysis. Molecular biology confirmed that our ticks were Hy. aegyptium. MALDI-TOF MS analysis revealed that 100% of the spectra were of excellent quality. Four spectra were selected to update our own database MALDI-TOF MS arthropod. The blind test of the 226 remaining spectra showed that all ticks were correctly identified, with scores ranging from 1.774 to 2.655 with a mean of 2.271 ± 0.16 of which, 223 (98.6%) had log score value (LSV)>1.8. Molecular biology screening showed that the ticks carried the DNA of Borrelia turcica, Rickettsia africae, Rickettsia aeschlimannii, Rickettsia sibirica mongolitimonae and with the Anaplasmataceae were close to a potentially new, undescribed Ehrlichia sp. This study confirms that MALDI-TOF MS is a reliable tool for the identification of ticks and that ticks collected from turtles in Algeria are carriers of several species of microorganisms which may be responsible for diseases in humans and animals.

Molecular Cloning, Functional and Biophysical Characterization of an Antimicrobial Peptide from Rhizosphere Soil.

This study was designed to screen and identify an antimicrobial peptide from rhizosphere soil. The study was further focused towards overexpression, purification and characterization of this antimicrobial peptide, and to functionally validate its efficiency and efficacy as an antimicrobial agent. Yet, the study was further aimed at corroborating structural and functional studies using biophysical tools. Antimicrobial resistance is emerging as one of the top 10 global health crisis, it is multifaceted and the second largest cause of mortality. According to the World Health Organization (WHO), around the world, an estimated 700,000 people die each year from infection caused by antibiotic-resistant microbes. Antimicrobial peptides offer the best alternative to combat and overcome this crisis. In this manuscript, we report cloning, expression, purification and characterization of an antimicrobial peptide discovered from rhizosphere soil. Objectives of this study include construction, screening and identification of antimicrobial peptide from metagenome followed by its expression, purification and functional and biophysical investigation. Yet another objective of the study was to determine antimicrobial efficacy and efficiency as an antimicrobial peptide against MRSA strains. In this study, we used an array of molecular biology tools that include genetic engineering, PCR amplification, construction of an expression construct and NI-NTA based purification of the recombinant peptide. We have also carried out antimicrobial activity assay to determine MIC (minimum inhibitory concentration) and IC50 values of antimicrobial peptide. To establish the structural and functional relationship, circular dichroism, and both extrinsic and intrinsic fluorescence spectroscopy studies were carried out. Screening of metagenomic library resulted in the identification of gene (~500bp) harbouring an open reading frame (ORF) consisting of 282 bp. Open reading frame identified in gene encodes an antimicrobial peptide which had shared ~95% sequence similarity with the antimicrobial peptide of Bacillus origin. Purification of recombinant protein using Ni-NTA column chromatography demonstrated a purified protein band of ~11 kDa on 14% SDS-PAGE, which is well corroborated to theoretical deduced molecular weight of peptide from its amino acids sequence. Interestingly, the peptide exhibited antimicrobial activity in a broad range of pH and temperature. MIC determined against gram positive Bacillus sp. was found to be 0.015mg/ml, whereas, in the case of gram negative E. coli, it was calculated to be 0.062mg/ml. The peptide exhibited IC50 values corresponding to ~0.25mg/ml against Bacillus and ~0.5 mg/ml against E. coli. Antimicrobial susceptibility assay performed against methicillin resistant Staphylococcus aureus strain ATCC 3412 and standard strain of Staphylococcus aureus ATCC 9144 revealed its strong inhibitory activity against MRSA, whereby we observed a ~16mm clearance zone at higher peptide concentrations ~2mg/ml (~181.8μM). Biophysical investigation carried out using Trp fluorescence, ANS fluorescence and circular dichroism spectroscopy further revealed conformational stability in its secondary and tertiary structure at a wide range of temperature and pH. Altogether, the peptide discovered from rhizosphere metagenome holds potential in inhibiting the growth of both gram positive and gram negative bacteria, and was equally effective in inhibiting the multidrug resistant pathogenic strains (MRSA).

Plant Gravitropism: From Mechanistic Insights into Plant Function on Earth to Plants Colonizing Other Worlds.

Gravitropism, the growth of roots and shoots toward or away from the direction of gravity, has been studied for centuries. Such studies have not only led to a better understanding of the gravitropic process itself, but also paved new paths leading to deeper mechanistic insights into a wide range of research areas. These include hormone biology, cell signal transduction, regulation of gene expression, plant evolution, and plant interactions with a variety of environmental stimuli. In addition to contributions to basic knowledge about how plants function, there is accumulating evidence that gravitropism confers adaptive advantages to crops, particularly under marginal agricultural soils. Therefore, gravitropism is emerging as a breeding target for enhancing agricultural productivity. Moreover, research on gravitropism has spawned several studies on plant growth in microgravity that have enabled researchers to uncouple the effects of gravity from other tropisms. Although rapid progress on understanding gravitropism witnessed during the past decade continues to be driven by traditional molecular, physiological, and cell biological tools, these tools have been enriched by technological innovations in next-generation omics platforms and microgravity analog facilities. In this chapter, we review the field of gravitropism by highlighting recent landmark studies that have provided unique insights into this classic research topic while also discussing potential contributions to agriculture on Earth and beyond.