Applications In Molecular Biology Research Articles

Viral particles imaging through evanescent wave scattering in a total internal reflection laser microscope

Optical imaging of objects at the nanometric scale is limited by light diffraction, and for this reason several sub-diffractive optical microscopy techniques have been developed in the last decades. In this article we present an optical laser microscope specifically designed for detection of nano-materials exploiting the Mie scattering between the evanescent wave generated by a laser beam travelling in a transparent substrate through total internal reflection and the nano-objects deposited on the substrate itself. The setup, including the laser source, can be fully integrated inside an optical microscope requiring only a geometric alignment with the substrate thus reducing the complexity and the cost. We also report two possible applications: quantitative detection of Au nanoparticles in the 20-100 nm size range and semi-quantitative detection of virions immobilized on the substrate through bioreceptor / antigen binding, including, but not limited to, SARS-CoV-2. A minimum virus amount of ≈105 in the reaction volume is observed. The results presented here open the route towards unprecedent applications in molecular biology or molecular diagnostics.

Improving high-throughput techniques for bacteriophage discovery in multi-well plates

Bacteriophages (also called phages) are viruses of bacteria that have numerous applications in medicine, agriculture, ecology, and molecular biology. With the increasing interest in phages for their many uses, it is now especially important to make phage discovery more efficient and economical.Using the host Mycobacterium smegmatis mc2155, which is a model organism for phage discovery research and is closely related to important pathogens of humans and other animals, we investigated three procedures that are an integral part of phage discovery: enrichment of environmental samples, phage isolation and detection (which can also be used for host range determination), and phage purification. Enrichment in 6-well plates was successful with most environmental samples, and enrichment in 24- and 96-well plates was successful with some environmental samples, demonstrating that larger sample volumes are preferred when possible, but smaller sample volumes may be acceptable if the starting concentration of phages is sufficiently high. Measuring absorbance in multi-well plates was at least as sensitive as the traditional plaque assay for the detection of phages. We also demonstrated a technique for the purification of single phage types from mixed cultures in liquid medium.Multi-well techniques can be used as alternatives or complementary approaches to traditional methods of phage discovery and characterization depending on the needs of the researcher in terms of time, available resources, host species, phage-bacteria matches, and specific goals. In the future, these techniques could be applied to the discovery of phages of aquatic mycobacteria and other hosts for which few phages have currently been isolated.

Application of Novel Molecular Biology in Cancer Therapy

Cancer is a genetic illness that develops for various reasons, including the activation of oncogenes, the failure of tumor suppressor genes, or mutagenesis induced by environmental stimuli. This article was produced using PubMed, Nature, Science Direct, Springer, and Elsevier data. Oncogenes are altered forms of normal proto-oncogenic genes that are important for cell proliferation, development, and regulation. The transformation of a gene to an oncogene is caused by chromosomal translocation or gene mutation due to addition, deletion, duplication, or viral infection. These oncogenes are targeted by medications or the RNAi system to limit malignant cell development. Various molecular biology methods for cancer detection and treatment have been developed, including targeting cancer stem cell pathways for cancer therapy, retroviral therapy, oncogene silencing, and alterations in tumor suppressor genes. Among all the techniques used, RNAi, zinc finger nucleases, and CRISPR have a greater chance of reaching a cancer-free planet.

Graph-based relevancy-redundancy gene selection method for cancer diagnosis

Nowadays, microarray data processing is one of the most important applications in molecular biology for cancer diagnosis. A major task in microarray data processing is gene selection, which aims to find a subset of genes with the least inner similarity and most relevant to the target class. Removing unnecessary, redundant, or noisy data reduces the data dimensionality. This research advocates a graph theoretic-based gene selection method for cancer diagnosis. Both unsupervised and supervised modes use well-known and successful social network approaches such as the maximum weighted clique criterion and edge centrality to rank genes. The suggested technique has two goals: (i) to maximize the relevancy of the chosen genes with the target class and (ii) to reduce their inner redundancy. A maximum weighted clique is chosen in a repetitive way in each iteration of this procedure. The appropriate genes are then chosen from among the existing features in this maximum clique using edge centrality and gene relevance. In the experiment, several datasets consisting of Colon, Leukemia, SRBCT, Prostate Tumor, and Lung Cancer, with different properties, are used to demonstrate the efficacy of the developed model. Our performance is compared to that of renowned filter-based gene selection approaches for cancer diagnosis whose results demonstrate a clear superiority.

Identification, Characterization, and Preliminary X-ray Diffraction Analysis of a Single Stranded DNA Binding Protein (LjSSB) from Psychrophilic Lacinutrix jangbogonensis PAMC 27137

Single-stranded DNA-binding proteins (SSBs) are essential for DNA metabolism, including repair and replication, in all organisms. SSBs have potential applications in molecular biology and in analytical methods. In this study, for the first time, we purified, structurally characterized, and analyzed psychrophilic SSB (LjSSB) from Lacinutrix jangbogonensis PAMC 27137 isolated from the Antarctic region. LjSSB has a relatively short amino acid sequence, consisting of 111 residues, with a molecular mass of 12.6 kDa. LjSSB protein was overexpressed in Escherichia coli BL21 (DE3) and analyzed for binding affinity using 20- and 35-mer deoxythymidine oligonucleotides (dT). In addition, the crystal structure of LjSSB at a resolution 2.6 Å was obtained. The LjSSB protein crystal belongs to the space group C222 with the unit cell parameters of a = 106.58 Å, b = 234.14 Å, c = 66.14 Å. The crystal structure was solved using molecular replacement, and subsequent iterative structure refinements and model building are currently under progress. Further, the complete structural information of LjSSB will provide a novel strategy for protein engineering and for the application on molecular biological techniques.

Preparation and Use of Cellular Reagents: A Low-resource Molecular Biology Reagent Platform.

Protein reagents are indispensable for most molecular and synthetic biology procedures. Most conventional protocols rely on highly purified protein reagents that require considerable expertise, time, and infrastructure to produce. In consequence, most proteins are acquired from commercial sources, reagent expense is often high, and accessibility may be hampered by shipping delays, customs barriers, geopolitical constraints, and the need for a constant cold chain. Such limitations to the widespread availability of protein reagents, in turn, limit the expansion and adoption of molecular biology methods in research, education, and technology development and application. Here, we describe protocols for producing a low-resource and locally sustainable reagent delivery system, termed "cellular reagents," in which bacteria engineered to overexpress proteins of interest are dried and can then be used directly as reagent packets in numerous molecular biology reactions, without the need for protein purification or a constant cold chain. As an example of their application, we describe the execution of polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) using cellular reagents, detailing how to replace pure protein reagents with optimal amounts of rehydrated cellular reagents. We additionally describe a do-it-yourself fluorescence visualization device for using these cellular reagents in common molecular biology applications. The methods presented in this article can be used for low-cost, on-site production of commonly used molecular biology reagents (including DNA and RNA polymerases, reverse transcriptases, and ligases) with minimal instrumentation and expertise, and without the need for protein purification. Consequently, these methods should generally make molecular biology reagents more affordable and accessible. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Preparation of cellular reagents Alternate Protocol 1: Preparation of lyophilized cellular reagents Alternate Protocol 2: Evaluation of bacterial culture growth via comparison to McFarland turbidity standards Support Protocol 1: SDS-PAGE for protein expression analysis of cellular reagents Basic Protocol 2: Using Taq DNA polymerase cellular reagents for PCR Basic Protocol 3: Using Br512 DNA polymerase cellular reagents for loop-mediated isothermal amplification (LAMP) Support Protocol 2: Building a fluorescence visualization device.

Nematode Orthologs of Macrophage Migration Inhibitory Factor (MIF) as Modulators of the Host Immune Response and Potential Therapeutic Targets.

One of the adaptations of nematodes, which allows long-term survival in the host, is the production of proteins with immunomodulatory properties. The parasites secrete numerous homologs of human immune mediators, such as macrophage migration inhibitory factor (MIF), which is a substantial regulator of the inflammatory immune response. Homologs of mammalian MIF have been recognized in many species of nematode parasites, but their role has not been fully understood. The application of molecular biology and genetic engineering methods, including the production of recombinant proteins, has enabled better characterization of their structure and properties. This review provides insight into the current state of knowledge on MIF homologs produced by nematodes, as well as their structure, enzymatic activity, tissue expression pattern, impact on the host immune system, and potential use in the treatment of parasitic, inflammatory, and autoimmune diseases.

Unraveling looping efficiency of stochastic Cosserat polymers

Understanding looping probabilities, including the particular case of ring closure or cyclization, of fluctuating polymers (e.g., DNA) is important in many applications in molecular biology and chemistry. In a continuum limit the configuration of a polymer is a curve in the group $\mathit{SE}$(3) of rigid body displacements, whose energy can be modeled via the Cosserat theory of elastic rods. Cosserat rods are a more detailed version of the classic wormlike-chain (WLC) model, which we show to be more appropriate in short-length scale, or stiff, regimes, where the contributions of extension and shear deformations are not negligible and lead to noteworthy high values for the cyclization probabilities (or $J$-factors). We therefore observe that the Cosserat framework is a candidate for gaining a better understanding of the enhanced cyclization of short DNA molecules reported in various experiments, which is not satisfactorily explained by WLC-type models. Characterizing the stochastic fluctuations about minimizers of the energy by means of Laplace expansions in a (real) path integral formulation, we develop efficient analytical approximations for the two cases of full looping, in which both end-to-end relative translation and rotation are prescribed, and of marginal looping probabilities, where only end-to-end translation is prescribed. For isotropic Cosserat rods, certain looping boundary value problems admit nonisolated families of critical points of the energy due to an associated continuous symmetry. For the first time, taking inspiration from (imaginary) path integral techniques, a quantum mechanical probabilistic treatment of Goldstone modes in statistical rod mechanics sheds light on $J$-factor computations for isotropic rods in the semiclassical context. All the results are achieved exploiting appropriate Jacobi fields arising from Gaussian path integrals and show good agreement when compared with intense Monte Carlo simulations for the target examples.

Identification and in-silico analysis of a novel restriction enzyme coding gene from Pseudomonas anguilliseptica

Type II restriction enzymes (REs) which can cleave double stranded DNA in a sequence specific manner have many applications in recombinant DNA technology and are considered the workhorses of molecular biology. This study was carried out to screen bacterial communities for restriction enzymes. Soil and water samples were screened for isolation of bacteria, harboring restriction enzymes. Cell lysates of isolated bacteria were incubated with λ DNA, followed by analysis by agarose gel electrophoresis. The presence of distinct banding patterns indicated the presence of REs. Nine putative isolates harboring REs were morphologically and molecularly characterized using 16S rRNA analysis and belonged to four different genera (Acinetobacter, Lysinibacillus, Pseudomonas, and Brevibacillus). A HindIII like restriction digestion profile was observed in a lysate of a soil bacterium belonging to genus Pseudomonas. Based on 16S rRNA analysis, the bacterial species was identified as P. angulliseptica. The enzyme was partially purified and optimum conditions for enzyme activity and its recognition sequence were determined. The enzyme showed optimum activity at 40 °C and was stable at 40 °C for 20 min without the DNA substrate. The recognition sequence of the enzyme was determined and found to be 5’AAGCTT 3′ indicating it to be an isoschizomer of HindIII. The whole genome of the Pseudomonas species was sequenced and the coding sequence of the gene for the putative HindIII isoschizomer was identified together with other genes encoding putative REs. The gene coding for the HindIII isoschizomer was analyzed in-silico and its homology and evolutionary relationship to other known isoschizomers of HindIII were determined. The enzyme was tentatively designated as PanI.Our study clearly demonstrated that PanI is an isoschizomer of HindIII with very little sequence similarity to the latter. It can be used as an alternative to HindIII in molecular biological applications.

Ultra-low sample consumption consecutive-detection method for biochemical molecules based on a whispering gallery mode with a liquid crystal microdroplet.

Ultra-low sample consumption detection has many applications in molecular biology, bioanalytical chemistry, and medical science. In this Letter, a novel, to the best of our knowledge, simple type of ultra-low sample consumption detection method based on a whispering gallery mode is proposed as a means to realize consecutive detection with a liquid crystal (LC) microdroplet for biochemical molecule detection, using deoxyribonucleic acid (DNA) as a model biomarker. The sensor consists of a 105-µm-core multimode fiber fused with a hollow capillary tube, with the LC microdroplet suspended stably in the testing solution. Its application to the detection of salmon sperm DNA yielded an adjustable measurement range of 3.75-11.25 μg/ml and a sensitivity of 0.33 nm/µg/ml. The test solution required as little as 3 nl of the sample, and the limit of detection was 1.32 µg/ml, which corresponds to the effective detection of as little as 3.96 pg of DNA. This method has great potential for application in the ultra-low sample consumption detection of biochemical molecules.

Conjugation of IRDye Photosensitizers or Fluorophores to Nanobodies.

Fluorophores have been conjugated to nanobodies for approximately a decade, for several applications in molecular biology. More recently, photosensitizers have been conjugated to nanobodies for targeted photodynamic therapy (PDT). The most common chemistry is the random conjugation in which commercial fluorophores or photosensitizers contain a N-hydroxysuccinimide ester (NHS ester) group that reacts specifically and efficiently with lysines in the amino acid sequence of the nanobody and with the N-terminal amino groups to form a stable amide bond. Alternatively, maleimide-containing fluorophores or photosensitizers can be used for conjugation to thiols, in a site-directed manner through a cysteine incorporated at the C-terminal of the nanobody. This chapter addresses both conjugation strategies, providing details on the reaction conditions, purification, and characterization of the conjugates obtained.

Sounding a New Era in Biomechanics with Acoustic Force Spectroscopy.

The acoustic force spectroscopy (AFS) tool was recently introduced as a novel tool for probing mechanical properties of biomolecules, expanding the application of sound waves to high-throughput quantification of the mechanical properties of single cells. By using controlled acoustic forces in the piconewton to nanonewton range, tens to hundreds of cells functionalized by attached microspheres can simultaneously be stretched and tracked in real-time with sub millisecond time response. Since its first application, several studies have demonstrated the potential and versatility of the AFS for high-throughput measurements of force-induced molecular mechanisms, revealing insight into cellular biomechanics and mechanobiology at the molecular level. In this chapter, we describe the operation of the AFS starting with the underlying physical principles, followed by a run-down of experimental considerations, and finally leading to applications in molecular and cellular biology.

Design and fabrication of microfluidics devices for molecular biology applications.

In the past decade, microfluidics has emerged as a rapidly growing area with potential to reduce cost and reagent consumption. It has been used for detection of nucleic acids and high-throughput screening of cells and metabolites. It is extensively used for extraction of DNA, RNA, proteins, biomolecules, as well as for cloning and transformation of plasmid into cells. Microfluidics is made up of polydimethylsiloxane (PDMS) polymer which is transparent and is used for preparation of a wide range of devices and systems. In this chapter, we discuss advances and challenges of using microfluidics in molecular biology and its biomedical applications.

A Note on “Wiener Index of a Fuzzy Graph and Application to Illegal Immigration Networks”

Connectivity parameters have an important role in the study of communication networks. Wiener index is such a parameter with several applications in networking, facility location, cryptology, chemistry, and molecular biology, etc. In this paper, we show two notes related to the Wiener index of a fuzzy graph. First, we argue that Theorem 3.10 in the paper “Wiener index of a fuzzy graph and application to illegal immigration networks, Fuzzy Sets and Syst. 384 (2020) 132–147” is not correct. We give a correct statement of Theorem 3.10. Second, by using a new operator on matrix, we propose a simple and polynomial-time algorithm to compute the Wiener index of a fuzzy graph.

CRISPR-Powered Microfluidics in Diagnostics: A Review of Main Applications

In the past few years, the CRISPR (clustered regularly interspaced short palindromic repeats) applications in medicine and molecular biology have broadened. CRISPR has also been integrated with microfluidic-based biosensors to enhance the sensitivity and selectivity of medical diagnosis due to its great potentials. The CRISPR-powered microfluidics can help quantify DNAs and RNAs for different diseases such as cancer, and viral or bacterial diseases among others. Here in this review, we discussed the main applications of such tools along with their advantages and limitations.

Bayesian networks as a novel tool to enhance interpretability and predictive power of ecological models

In today's world, it is becoming increasingly important to have the tools to understand, and ultimately to predict, the response of ecosystems to disturbance. However, understanding such dynamics is not simple. Ecosystems are a complex network of species interactions, and therefore any change to a population of one species will have some degree of community level effect. In recent years, the use of Bayesian networks (BNs) has seen successful applications in molecular biology and ecology, where they were able to recover plausible links in the respective systems they were applied to. The recovered network also comes with a quantifiable metric of interaction strength between variables. While the latter is an invaluable piece of information in ecology, an unexplored application of BNs would be using them as a novel variable selection tool in the training of predictive models. To this end, we evaluate the potential usefulness of BNs in two aspects: (1) we apply BN inference on species abundance data from a rocky shore ecosystem, a system with well documented links, to test the ecological validity of the revealed network; and (2) we evaluate BNs as a novel variable selection method to guide the training of an artificial neural network (ANN). Here, we demonstrate that not only was this approach able to recover meaningful species interactions networks from ecological data, but it also served as a meaningful tool to inform the training of predictive models, where there was an improvement in predictive performance in models with BN variable selection. Combining these results, we demonstrate the potential of this novel application of BNs in enhancing the interpretability and predictive power of ecological models; this has general applicability beyond the studied system, to ecosystems where existing relationships between species and other functional components are unknown.

Complementing Cancer Photodynamic Therapy with Ferroptosis through Iron Oxide Loaded Porphyrin-Grafted Lipid Nanoparticles.

Nanomaterials that combine multimodality imaging and therapeutic functions within a single nanoplatform have drawn extensive attention for molecular medicines and biological applications. Herein, we report a theranostic nanoplatform based on a relatively smaller (<20 nm) iron oxide loaded porphyrin-grafted lipid nanoparticles (Fe3O4@PGL NPs). The amphiphilic PGL easily self-assembled on the hydrophobic exterior surface of ultrasmall Fe3O4 NPs, resulting in a final ultrasmall Fe3O4@PGL NPs with diameter of ∼10 nm. The excellent self-assembling nature of the as-synthesized PGL NPs facilitated a higher loading of porphyrins, showed a negligible dark toxicity, and demonstrated an excellent photodynamic effect against HT-29 cancer cells in vitro. The in vivo experimental results further confirmed that Fe3O4@PGL NPs were ideally qualified for both the fluorescence and magnetic resonance (MR) imaging guided nanoplatforms to track the biodistribution and therapeutic responses of NPs as well as to simultaneously trigger the generation of highly cytotoxic reactive oxygen species (ROS) necessary for excellent photodynamic therapy (PDT). After recording convincing therapeutic responses, we further evaluated the ability of Fe3O4@PGL NPs/Fe3O4@Lipid NPs for ferroptosis therapy (FT) via tumor microenvironment (TME) modulation for improved anticancer activity. We hypothesized that tumor-associated macrophages (TAMs) could significantly improve the efficacy of FT by accelerating the Fenton reaction in vitro. In our results, the Fe ions released in vitro directly contributed to the Fenton reaction, whereas the presence of RAW 264.7 macrophages further accelerated the ROS generation as observed by the fluorescence imaging. The significant increase in the ROS during the coincubation of NPs, endocytosed by HT-29 cells and RAW264.7 cells, further induced increased cellular toxicity of cancer cells.

Structural and dynamic insights into the HNH nuclease of divergent Cas9 species

CRISPR-Cas9 is a widely used biochemical tool with applications in molecular biology and precision medicine. The RNA-guided Cas9 protein uses its HNH endonuclease domain to cleave the DNA strand complementary to its endogenous guide RNA. In this study, novel constructs of HNH from two divergent organisms, G. stearothermophilus (GeoHNH) and S. pyogenes (SpHNH) were engineered from their respective full-length Cas9 proteins. Despite low sequence similarity, the X-ray crystal structures of these constructs reveal that the core of HNH surrounding the active site is conserved. Structure prediction of the full-length GeoCas9 protein using Phyre2 and AlphaFold2 also showed that the crystallographic construct of GeoHNH represents the structure of the domain within the full-length GeoCas9 protein. However, significant differences are observed in the solution dynamics of structurally conserved regions of GeoHNH and SpHNH, the latter of which was shown to use such molecular motions to propagate the DNA cleavage signal. Indeed, molecular simulations show that the intradomain signaling pathways, which drive SpHNH function, are non-specific and poorly formed in GeoHNH. Taken together, these outcomes suggest mechanistic differences between mesophilic and thermophilic Cas9 species.

Athermal, Chemically Triggered Release of RNA from Thioester Nucleic Acids

AbstractAn athermal approach to mRNA enrichment from total RNA using a self‐immolative thioester linked nucleic acids (TENA) is described. Oligo(thymine) (oT) TENA has a six‐atom spacing between bases which allowed TENA to selectively base‐pair with polyadenine RNA. As a result of the neutral backbone of TENA and the hydrophobicity of the octanethiol end group, oT TENA is water insoluble and efficiently pulled down 93±2 % of EGFP mRNA at a concentration of 10 ng μL−1. Self‐immolative degradation of TENA upon ambient temperature exposure to nucleophilic buffer components (Tris, DTT) allowed recovery of 55±27 ng of mRNA from 3.1 μg of total RNA, which was not statistically different from the amount recovered using Dynabeads® mRNA DIRECT Kit (89±24 ng). Gene expression as measured by RT‐qPCR was comparable for both enrichment methods, suggesting that the mild conditions required for enrichment of mRNA using oT TENA are compatible with RT‐qPCR and other downstream molecular biology applications.

Athermal, Chemically Triggered Release of RNA from Thioester Nucleic Acids.

An athermal approach to mRNA enrichment from total RNA using a self-immolative thioester linked nucleic acids (TENA) is described. Oligo(thymine) (oT) TENA has a six-atom spacing between bases which allowed TENA to selectively base-pair with polyadenine RNA. As a result of the neutral backbone of TENA and the hydrophobicity of the octanethiol end group, oT TENA is water insoluble and efficiently pulled down 93±2 % of EGFP mRNA at a concentration of 10 ng μL-1 . Self-immolative degradation of TENA upon ambient temperature exposure to nucleophilic buffer components (Tris, DTT) allowed recovery of 55±27 ng of mRNA from 3.1 μg of total RNA, which was not statistically different from the amount recovered using Dynabeads® mRNA DIRECT Kit (89±24 ng). Gene expression as measured by RT-qPCR was comparable for both enrichment methods, suggesting that the mild conditions required for enrichment of mRNA using oT TENA are compatible with RT-qPCR and other downstream molecular biology applications.