Molecular Biology Tools Research Articles

Framework for field-scale application of molecular biological tools to support natural and enhanced bioremediation.

Microorganisms naturally present at environmental contaminated sites are capable of biodegrading, biotransforming, or removing contaminants in soil and groundwater through bioremediation processes. Cleanup strategies and goals for site remediation can be effectively achieved by bioremediation leveraging the capabilities of microorganisms to biotransform contaminants into lesser or non-toxic end products; however, reproducible success can be limited by inadequate design or performance monitoring. A group of biological analyses collectively termed molecular biological tools (MBTs) can be used to assess the contaminant-degrading capabilities and activities of microorganisms present in the environment and appropriately implement bioremediation approaches. While successful bioremediation has been demonstrated through previously described lab-scale studies and field-scale implementation for a variety of environmental contaminants, design and performance monitoring of bioremediation has often been limited to inferring biodegradation potential, occurrence, and pathways based on site geochemistry or lab-scale studies. Potential field-scale application of MBTs presents the opportunity to more precisely design and monitor site-specific bioremediation approaches. To promote standardization and successful implementation of bioremediation, a framework for field-scale application of MBTs within a multiple lines of evidence (MLOE) approach is presented. The framework consists of three stages: (i) "Assessment" to evaluate naturally occurring biogeochemical conditions and screen for potential applicability of bioremediation, (ii) "Design" to define a site-specific bioremediation approach and inform amendment selection, and (iii) "Performance Monitoring" to generate data to measure or infer bioremediation progress following implementation. This framework is introduced to synthesize the complexities of environmental microbiology and guide field-scale application of MBTs to assess bioremediation potential and inform site decision-making.

A comprehensive review on advances in storage pest management: Current scenario and future prospects

Modernization of the agricultural production system led to a significant increase in annual food production intended to meet the ever-growing consumer demand. In many countries, most of the food grains produced is stored for contingency and regular supply. These stored grains, in general, are directly or indirectly infested by insects, resulting in severe grain damages and storage losses, thus, causing a threat to food safety and security. Although a variety of insect management options, such as physical, mechanical, biological, and chemical methods, are available, fumigation has been practiced for decades in storage. However, opportunities for fumigation are narrowing after the phase-out of methyl bromide. Besides, safe food and health concerns paved the path for green chemistry and non-chemical management practices. This review includes the list of stored-grain insects and their detection methods. The different management strategies such as the modern storage structures (hermetic and low-pressure storages), modified or controlled storage atmosphere, application of ozone as fumigant, irradiation, and physical options are presented. Further, the details on sustainable biological options, such as semiochemicals, natural enemies, biopesticides, and entomopathogenic nematodes, are supplemented. The use of inert dusts as grain protectant and in combination with the biological entity is included. Studies on alternative fumigants', novel management options, such as molecular biology tools (RNAi and CRISPR) and nanotechnology in stored grain protection, are also highlighted. This review helps the reader to understand the overall factors affecting grain storage and the different options to manage the insects causing storage losses.

Thermally Enhanced Biodegradation of TCE in Groundwater

In situ remediation is usually restricted by temperature, lack of substrate for reductive dechlorination (anaerobic respiration), the presence of dehalogenating microorganisms, and specific bedrock conditions. In this work, trichloroethene (TCE) degradation was studied by a number of methods, from physical–chemical analyses to molecular biological tools. The abundance changes in dechlorinating bacteria were monitored using real-time PCR. The functional genes vcrA and bvcA as well as the 16S rRNA specific for representatives of genera Dehalococcoides, Dehalobacter, and Desulfitobacterium were monitored. Furthermore, the sulfate-reducing bacteria and denitrifying bacteria were observed by amplifying the functional genes apsA and nirK. The elevated temperature and the substrate (whey) addition significantly affected TCE dechlorination. The chlorine index decreased after nine weeks from 2.5 to 0.1 at 22 °C, to 1.1 at 17 °C and 1.7 at 12 °C and complete dechlorination was achieved at 22 °C with whey addition. The achieved results of this work show the feasibility and effectiveness of biological dechlorination of TCE enhanced with elevated temperature and whey addition.

HT-smFISH: a cost-effective and flexible workflow for high-throughput single-molecule RNA imaging.

The ability to visualize RNA in its native subcellular environment by using single-molecule fluorescence in situ hybridization (smFISH) has reshaped our understanding of gene expression and cellular functions. A major hindrance of smFISH is the difficulty to perform systematic experiments in medium- or high-throughput formats, principally because of the high cost of generating the individual fluorescent probe sets. Here, we present high-throughput smFISH (HT-smFISH), a simple and cost-efficient method for imaging hundreds to thousands of single endogenous RNA molecules in 96-well plates. HT-smFISH uses RNA probes transcribed in vitro from a large pool of unlabeled oligonucleotides. This allows the generation of individual probes for many RNA species, replacing commercial DNA probe sets. HT-smFISH thus reduces costs per targeted RNA compared with many smFISH methods and is easily scalable and flexible in design. We provide a protocol that combines oligo pool design, probe set generation, optimized hybridization conditions and guidelines for image acquisition and analysis. The pipeline requires knowledge of standard molecular biology tools, cell culture and fluorescence microscopy. It is achievable in ~20 d. In brief, HT-smFISH is tailored for medium- to high-throughput screens that image RNAs at single-molecule sensitivity.

Approaches for evaluation of novel CPP-based cargo delivery systems.

Cell penetrating peptides (CPPs) can be broadly defined as relatively short synthetic, protein derived or chimeric peptides. Their most remarkable property is their ability to cross cell barriers and facilitate the translocation of cargo, such as drugs, nucleic acids, peptides, small molecules, dyes, and many others across the plasma membrane. Over the years there have been several approaches used, adapted, and developed for the evaluation of CPP efficacies as delivery systems, with the fluorophore attachment as the most widely used approach. It has become progressively evident, that the evaluation method, in order to lead to successful outcome, should concede with the specialties of the delivery. For characterization and assessment of CPP-cargo a combination of research tools of chemistry, physics, molecular biology, engineering, and other fields have been applied. In this review, we summarize the diverse, in silico, in vitro and in vivo approaches used for evaluation and characterization of CPP-based cargo delivery systems.

A novel and robust method for counting components within bio-molecular complexes using fluorescence microscopy and statistical modelling

Cellular biology occurs through myriad interactions between diverse molecular components, many of which assemble in to specific complexes. Various techniques can provide a qualitative survey of which components are found in a given complex. However, quantitative analysis of the absolute number of molecules within a complex (known as stoichiometry) remains challenging. Here we provide a novel method that combines fluorescence microscopy and statistical modelling to derive accurate molecular counts. We have devised a system in which batches of a given biomolecule are differentially labelled with spectrally distinct fluorescent dyes (label A or B), and mixed such that B-labelled molecules are vastly outnumbered by those with label A. Complexes, containing this component, are then simply scored as either being positive or negative for label B. The frequency of positive complexes is directly related to the stoichiometry of interaction and molecular counts can be inferred by statistical modelling. We demonstrate this method using complexes of Adenovirus particles and monoclonal antibodies, achieving counts that are in excellent agreement with previous estimates. Beyond virology, this approach is readily transferable to other experimental systems and, therefore, provides a powerful tool for quantitative molecular biology.

One-Step Conversion of NHS Esters to Reagents for Site-Directed Labeling of IgG Antibodies.

Antibody conjugates are extensively used for diagnostics and therapeutics, and as a tool for molecular biology. To prepare such conjugates N-hydroxysuccinimide (NHS) esters are most often used due to the straightforward experimental procedure and the commercial accessibility of the reagents. Such conjugates are however highly heterogeneous, since only the reactivity of the lysines determines the distribution of labels. This has inspired the development of methods that experimentally are as facile but produce conjugates of higher quality. Herein, we report the development of a reagent that can, in one step, be activated with an NHS ester of choice and subsequently can be directly used for site-directed labeling of antibodies. The reagent can be prepared in three synthetic steps and produces conjugates with similar ease as for NHS esters, however in a site-directed manner. We show that the reagent is quantitatively activated by a variety of NHS esters, and we use these to functionalize IgG1, IgG2, and IgG4 antibodies.

Microfluidic Enrichment and Computational Analysis of Rare Sequences from Mixed Genomic Samples for Metagenomic Mining.

Many powerful molecular biology tools have their origins in natural systems, including restriction modification enzymes and the CRISPR effectors, Cas9, Cas12, and Cas13. Heightened interest in these systems has led to mining of genomic and metagenomic data to identify new orthologs of these proteins, new types of CRISPR systems, and uncharacterized natural systems with novel mechanisms. To accelerate metagenomic mining, we developed a high-throughput, low-cost droplet microfluidic-based method for enrichment of rare sequences in a mixed starting population. Using a computational pipeline, we then searched in the enriched data for the presence of CRISPR-Cas systems, identifying a previously unknown CRISPR-Cas system. Our approach enables researchers to efficiently mine metagenomic samples for sequences of interest, greatly accelerating the search for nature's treasures.

Protocols for CRISPR/Cas9 Mutagenesis of the Oriental Fruit Fly Bactrocera dorsalis.

The Oriental fruit fly, Bactrocera dorsalis, is a highly invasive and adaptive pest species that causes damage to citrus and over 150 other fruit crops worldwide. Since adult fruit flies have great flight capacity and females lay their eggs under the skins of fruit, insecticides requiring direct contact with the pest usually perform poorly in the field. With the development of molecular biological tools and high-throughput sequencing technology, many scientists are attempting to develop environmentally friendly pest management strategies. These include RNAi or gene editing-based pesticides that downregulate or silence genes (molecular targets), such as olfactory genes involved in searching behavior, in various insect pests. To adapt these strategies for Oriental fruit fly control, effective methods for functional gene research are needed. Genes with critical functions in the survival and reproduction of B. dorsalis serve as good molecular targets for gene knockdown and/or silencing. The CRISPR/Cas9 system is a reliable technique used for gene editing, especially in insects. This paper presents a systematic method for CRISPR/Cas9 mutagenesis of B. dorsalis, including the design and synthesis of guide RNAs, collecting embryos, embryo injection, insect rearing, and mutant screening. These protocols will serve as a useful guide for generating mutant flies for researchers interested in functional gene studies in B. dorsalis.

Proteomic and Genomic Studies of Micronutrient Deficiency and Toxicity in Plants.

Micronutrients are essential for plants. Their growth, productivity and reproduction are directly influenced by the supply of micronutrients. Currently, there are eight trace elements considered to be essential for higher plants: Fe, Zn, Mn, Cu, Ni, B, Mo, and Cl. Possibly, other essential elements could be discovered because of recent advances in nutrient solution culture techniques and in the commercial availability of highly sensitive analytical instrumentation for elemental analysis. Much remains to be learned about the physiology of micronutrient absorption, translocation and deposition in plants, and about the functions they perform in plant growth and development. With the recent advancements in the proteomic and molecular biology tools, researchers have attempted to explore and address some of these questions. In this review, we summarize the current knowledge of micronutrients in plants and the proteomic/genomic approaches used to study plant nutrient deficiency and toxicity.

Similarities of P1-Like Phage Plasmids and Their Role in the Dissemination of blaCTX-M-55.

ABSTRACTThe P1-like phage plasmid (PP) has been widely used as a molecular biology tool, but its role as an active accessory cargo element is not fully understood. In this study, we provide insights into the structural features and gene content similarities of 77 P1-like PPs in the RefSeq database. We also describe a P1-like PP carrying a blaCTX-M-55 gene, JL22, which was isolated from a clinical strain of Escherichia coli from a duck farm. P1-like PPs were very similar and conserved based on gene content similarities, with only eight highly variable regions. Importantly, two kinds of replicon types, namely, IncY and p0111, were identified and can be used to specifically identify the P1-like phage. JL22 is similar to P1, acquiring an important foreign DNA fragment with two obvious features, namely, the plasmid replication gene repA′ (p0111) replacing the gene repA (IncY) and a 4,200-bp fragment mobilized by IS1380 and IS5 and containing a blaCTX-M-55 gene and a trpB gene encoding tryptophan synthase (indole salvaging). The JL22 phage could be induced but had no lytic capacities. However, a lysogenic recipient and intact structure of JL22 virions were observed, showing that the extended-spectrum β-lactamase blaCTX-M-55 gene was successfully transferred. Overall, conserved genes can be a good complement to improve the identification efficiency and accuracy in future screening for P1-like PPs. Moreover, the highly conserved structures may be important for their prevalence and dissemination.IMPORTANCE As a PP, P1 DNA exists as a low-copy-number plasmid and replicates autonomously with a lysogenization style. This unique mode of P1-like elements probably indicates a stable contribution to antibiotic resistance. After analyzing these elements, we show that P1-like PPs are very similar and conserved, with only eight highly variable regions. Moreover, we observed the occurrence of replicon IncY and p0111 only in the P1-like PP community, implying that these conserved regions, coupled with IncY and p0111, can be an important complement in future screening of P1-like PPs. Identification and characterization of JL22 confirmed our findings that major changes were located in variable regions, including the first detection of blaCTX-M-55 in such a mobile genetic element. This suggests that these variable regions may facilitate foreign DNA mobilization. This study features a comprehensive genetic analysis of P1-like PPs, providing new insights into the dissemination mechanisms of antibiotic resistance through P1 PPs.

A Method for Isolation Bacteriophage Particles-Free Genomic DNA, Exemplified by TP-84, Infecting Thermophilic Geobacillus.

DNA purification methods are indispensable tools of molecular biology, used for many decades. Nevertheless, for certain specialized applications, the currently employed techniques are not sufficiently effective. While examining a number of the existing methods to purify the genomic DNA of the thermophilic bacteriophage TP-84, which infects Geobacillus stearothermophilus (G. stearothermophilus), we have found out that the obtained DNA is contaminated with trace amounts of infectious TP-84 particles. This was detrimental for the bacteriophage genetic manipulation purposes, as finding the recombinant TP-84 clones was essentially impossible due to the appearance of a high background of native bacteriophage plaques. Thus, we have developed a method, which enables the fast and efficient isolation of a bacteriophage genomic DNA from concentrated phage preparations, obtained using CsCl gradient ultracentrifugation, without the need to remove concentrated CsCl solutions. The method employs silica columns and mini-scale isolation of microgram amounts of high quality DNA. It is universal—the silica mini-columns from various manufacturers can be used to conduct the procedure. The purified DNA, free from infectious bacteriophage particles, is ready for further manipulations. This is particularly important for such thermophilic bacteriophages that may partially survive standard isolation procedures and contaminate the final DNA product.

The Electrophoretic Revolution in the 1960s: Historical Epistemology Meets the Global History of Science and Technology.

This paper uses zone electrophoresis, one of the most frequently used tools in molecular biology, to explore two ideas derived from Hans‐Jörg Rheinberger's reflections on experiments. First, the constraining role played by technical objects—instrumentation and material conditions—in the production of knowledge or epistemic things. Second, the production of interconnected experimental systems by such technical objects, which results in the unexpected entanglement of research fields and experimental cultures. By the beginning of the 1960s, the inception of zone electrophoresis in laboratories around the world transformed—some say, revolutionized—the study of proteins. Even today, electrophoresis continues to open research venues and questions in biomedicine, molecular biology, human genetics, and in the field of molecular evolution. In my essay, I seek to look at the interconnected lives of zone electrophoresis and address the broader social, and even global context, in which this apparently humble technique became a salient tool in the production of biological knowledge. In so doing, I aim to take the past and present of the history and historiography of experimental systems to the future, where experiments and technologies are interrogated as they are used in different geographies and contexts, including contexts of poverty.

Quantifying Huntingtin Protein in Human Cerebrospinal Fluid Using a Novel Polyglutamine Length-Independent Assay

Background:The use of biomarkers has become a major component of clinical trial design. In Huntington’s disease (HD), quantifying the amount of huntingtin protein (HTT) in patient cerebrospinal fluid (CSF) has served as a pharmacodynamic readout for HTT-lowering therapeutic approaches and is a potential disease progression biomarker. To date, an ultrasensitive immunoassay to quantify mutant HTT protein (mHTT) has been used, but additional assays are needed to measure other forms of HTT protein.Objective:We aimed to develop an ultrasensitive immunoassay to quantify HTT protein in a polyglutamine length-independent manner (mHTT and non-expanded wild type HTT combined) in control and HD participant CSF samples.Methods:An ultrasensitive, bead-based, single molecule counting (SMC) immunoassay platform was used for the detection of HTT protein in human CSF samples.Results:A novel ultrasensitive SMC immunoassay was developed to quantify HTT protein in a polyglutamine length-independent manner and shown to measure HTT in both control and HD participant CSF samples. We validate the selectivity and specificity of the readout using biochemical and molecular biology tools, and we undertook a preliminary analytical qualification of this assay to enable its clinical use. We also used this novel assay, along with the previously described mHTT assay, to analyze CSF from control and HD participants. The results of this preliminary set suggests that correlation is present between mHTT and the polyglutamine length-independent HTT levels in human CSF.Conclusion:We have developed a novel ultrasensitive immunoassay that is able to quantify HTT protein in a polyglutamine length-independent manner in control and HD participant CSF.

Confluences characteristics determine the influence scope of microbial community from confluence hydrodynamic zone on river network

River confluences are crucial parts of the river networks, which connect different rivers and promote material exchange between them. Joining of two rivers would cause sharp shifts of flow dynamics and form confluence hydrodynamic zone (CHZ) with specific flow structure and biogeochemical processes. However, CHZs are small areas compared with river networks, whether and how the biogeochemical processes in CHZs would influence the lower reaches and even the whole river networks remain unclear. To address this gap, the present study focused on the nitrogen dynamics in a river network consisting a series of confluences, and combined molecular biological tools, gene-centric modelling approach and SourceTracker analysis to reveal the microorganisms induced nitrogen transformation processes in CHZs and other normal hydrodynamic zones (NHZs). Results illustrated that the enriched microbial species in CHZs were associated with nitrogen transformation, such as Terrimonas and Sphingobacterium, which were reported to be vital participants in nitrate and nitrite reduction processes. The reactions rates of different nitrogen transformation processes, calculated based on functional genes abundances, further revealed that N2 production rates were significantly higher in CHZs than NHZs (p < 0.05), demonstrating CHZs were the hotspots for nitrogen removal in the researched river network. Besides, the specific microbial communities and stimulated nitrogen removal processes in CHZ would sustain in lower reaches. SourceTracker analysis illustrated that microbial communities would exert significant influences on lower reaches, and the influence scope was controlled by confluences characteristics, such as flow ratio between tributary and main stream. Higher discharge ratio would increase the influence scope of microbial community in CHZ. These results indicate that reasonable design of confluence characteristics can be applied as a critical engineering measure for the improvement of ecological health of river networks.

Acid‐Stable Nucleobase Protection for a Strongly Pairing Pyridone C‐Nucleoside Suitable for Solid‐Phase Synthesis of Oligonucleotides

AbstractOligonucleotides are indispensable tools in diagnostics, therapeutic applications and molecular biology. The low base pairing strength of thymine with adenine complicates their use. EthynylpyridoneC‐nucleosides are analogs of thymidine that pair more strongly and with improved base selectivity, and sequences containing these analogs show improved target affinity and selectivity, but their routine use is hampered by diminished yields of solid‐phase syntheses with the known building blocks. A partial loss of base protecting groups during the acidic deblocking step of chain extension cycles was identified as the cause of lower yields. Here we report the synthesis of an improved phosphoramidite building block featuring a pivaloyloxymethyl (POM) base protecting group. This building block gives oligonucleotides containing the strongly pairing ethynylmethylpyridoneC‐nucleoside in high yield and purity via solid‐phase synthesis.

The burden of T. solium cysticercosis and selected neuropsychiatric disorders in Mocuba district, Zambézia province, Mozambique.

Taenia solium (neuro-)cysticercosis, a neglected tropical disease, can be associated with epileptic seizures and other neuropsychiatric (= neurological and psychiatric) disorders. This study aimed to evaluate the association of T. solium cysticercosis with selected neuropsychiatric disorders and/or symptoms (chronic headache, epileptic seizures/epilepsy and psychosis) in Mocuba district, Mozambique. Between March and May 2018, a cross-sectional study was conducted among 1,086 participants aged 2 years or above in Mocuba district, Zambézia province, central Mozambique, to assess the seroprevalence of human cysticercosis and risk factors for infection, as well as to explore its relation to selected neuropsychiatric disorders. Socio-demographic and clinical data were collected from each participant using a modified questionnaire designed by the Cysticercosis Working Group for Eastern and Southern Africa. Additionally, neuropsychiatric disorders, such as chronic headache, epileptic seizures/epilepsy and psychosis were assessed using four vignettes. T. solium antigen and cysticercosis IgG in serum were detected using both T. solium antigen B158/B60 enzyme linked immunosorbent assay (ELISA) and LDBIO Cysticercosis Western Blot, respectively. Overall, 112/1,086 participants (10.3%) were sero-positive for T. solium antigen or antibodies. Prevalence of antibodies (6.6%; n = 72) was higher than of antigens (4.9%; n = 54). In the questionnaires, 530 (49.5%) of participants reported chronic headache, 293 (27%) had generalized epileptic seizures, 188 (18%) focal seizures and 183 (18.3%) psychosis. We found a statistically significant association between seropositivity for T. solium and chronic headache (p = 0.013). Additionally, increasing age (p = 0.03) was associated with Ag-ELISA seropositivity. Our study revealed that in Mocuba, T. solium cysticercosis is prevalent and associated with self-reported chronic headache. Additionally, in the study setting, the seroprevalence of cysticercosis increased with age. However, it is not associated with other neuropsychiatric disorders such epileptic seizures/epilepsy and psychosis. Future studies are needed to confirm the high burden of neuropsychiatric disorders and their possible etiology, including neurocysticercosis, using additional serological, molecular biological and radiological diagnostic tools, as well as in-depth clinical examinations.

Antibody-Antigen Binding Interface Analysis in the Big Data Era.

Antibodies have become the Swiss Army tool for molecular biology and nanotechnology. Their outstanding ability to specifically recognise molecular antigens allows their use in many different applications from medicine to the industry. Moreover, the improvement of conventional structural biology techniques (e.g., X-ray, NMR) as well as the emergence of new ones (e.g., Cryo-EM), have permitted in the last years a notable increase of resolved antibody-antigen structures. This offers a unique opportunity to perform an exhaustive structural analysis of antibody-antigen interfaces by employing the large amount of data available nowadays. To leverage this factor, different geometric as well as chemical descriptors were evaluated to perform a comprehensive characterization.

Entanglement of polymer chains in hypertonic medium enhances the delivery of DNA and other biomacromolecules into cells

HypothesisMost experimental procedures applied in modern biology involve cargo delivering into cells. One of the ways to cargo introduction is osmotic-mediated intracellular vesicle swelling. However, its widespread use was hindered due to cargo size (<10 nm) and cell-type-related restrictions. We addressed the issue of the composition of colloidal loading solution to enhance the efficiency of cellular delivery. ExperimentsWe examined the effectiveness of colloidal loading solutions of varied compositions, including various types and sizes of polymers building osmotic pressure. We used confocal imaging coupled with fluorescence correlation spectroscopy to evaluate the introduction of polymers, proteins, nanoparticles, and DNA plasmids (cargos of sizes 1–175 nm) to cells representing eight cell lines: cancer, normal, epithelial, and mesenchymal ones. FindingsWe found that cellular delivery effectiveness strongly correlates with the size and concentration of osmotic pressure building polymers and not with the high value of the osmotic pressure itself. We show that polymer solutions at the entangled regime of concentrations enhance the delivery of large biomacromolecules even of size 200 nm (DNA plasmids) into cells, including MDA-MB-231 cells – so far resistant to the osmotic procedure. We show that the colloid loading medium based on entangled polymer chains is a versatile cargo delivery tool for molecular biology.

Paradigm Shift in Phytochemicals Research: Evolution from Antioxidant Capacity to Anti-Inflammatory Effect and to Roles in Gut Health and Metabolic Syndrome.

Food bioactive components, particularly phytochemicals with antioxidant capacity, have been extensively studied over the past two decades. However, as new analytical and molecular biological tools advance, antioxidants related research has undergone significant paradigm shifts. This review is a high-level overview of the evolution of phytochemical antioxidants research. Early research used chemical models to assess the antioxidant capacity of different phytochemicals, which provided important information about the health potential, but the results were overused and misinterpreted despite the lack of biological relevance (Antioxidants v1.0). This led to findings in the anti-inflammatory properties and modulatory effects of cell signaling of phytochemicals (Antioxidants v2.0). Recent advances in the role of diet in modulating gut microbiota have suggested a new phase of food bioactives research along the phytochemicals-gut microbiota-intestinal metabolites-low-grade inflammation-metabolic syndrome axis (Antioxidants v3.0). Polyphenols and carotenoids were discussed in-depth, and future research directions were also provided.