Applications In Molecular Biology Research Articles

Gold Nanoparticles as a Biosensor for Cancer Biomarker Determination.

Molecular biology applications based on gold nanotechnology have revolutionary impacts, especially in diagnosing and treating molecular and cellular levels. The combination of plasmonic resonance, biochemistry, and optoelectronic engineering has increased the detection of molecules and the possibility of atoms. These advantages have brought medical research to the cellular level for application potential. Many research groups are working towards this. The superior analytical properties of gold nanoparticles can not only be used as an effective drug screening instrument for gene sequencing in new drug development but also as an essential tool for detecting physiological functions, such as blood glucose, antigen-antibody analysis, etc. The review introduces the principles of biomedical sensing systems, the principles of nanomaterial analysis applied to biomedicine at home and abroad, and the chemical surface modification of various gold nanoparticles.

Application of molecular biology in the detection of human papillomavirus

Application of molecular biology in the detection of human papillomavirus

Comparing Conventional Cytology Smear and Cell Block Techniques for Ovarian Cancer Diagnosis: A Prospective Observational Study

Introduction: The cytological examination of ascitic fluid is widely recognised and well-documented for its importance in staging and prognosis of malignancy, and for providing information about inflammatory lesions. The Cell Block (CB) method offers improved architectural patterns and morphological features, aiding in the differentiation between reactive mesothelial cells and malignant cells, thus enhancing the efficacy of cyst diagnosis. Additionally, the CB technique finds applications in molecular biology and immunocytochemistry, making it advantageous for targeted therapy due to its ability to preserve cytological material. Aim: To compare the accuracy of conventional cytology smear technique and CB from ascitic fluid with histopathology for diagnosing ovarian tumours. Materials and Methods: A cross-sectional study was conducted in the Department of Pathology at Jawaharlal Nehru Medical College, Datta Meghe Institute of Higher Education and Research, Wardha, Maharashtra, India. A total of 45 patients with suspected ovarian tumours or presence of ascites were included. Biopsy samples were sent to the pathology laboratory for histological evaluation, while study samples were collected from the Department of Pathology between January 2021 and December 2022. Sample processing techniques, such as conventional cytology {including cytocentrifugation before Giemsa, Pap, and Haematoxylin and Eosin (H&E) staining} and thromboplastin-plasma technique for CB preparation, were employed. Evaluation parameters included comparing morphological features of frequently stained cytology smears and CB technique of ascitic fluid, along with their concordance with histopathological diagnosis. Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS) program for Windows, version 28.0. Results: Among 45 patients, majority 24 (53.40%) of them belonged to 41-60 years of age group. It was noted that 24 (53.40%) patients had ascites, 11 patients (24.40%) had abdominal pain with ascites, and 10 (22.20%) had ovarian mass with ascites. Conventional cytology smear diagnoses revealed that 22 (48.9%) patients had infiltrates of serous cystadenocarcinoma. A significant correlation was found between the findings of the CB and Conventional Smear (CS) (p=0.0001), with a sensitivity of 94.12%, specificity of 100%, Positive Predictive Value (PPV) of 100%, Negative Predictive Value (NPV) of 85.71%, and a diagnostic accuracy of 95.65% for CB correlating with CS. Conclusion: A combined strategy utilising stained cytology smears and the CB technique of ascitic fluid could be considered in the diagnostic approach for malignant ovarian tumours.

Flow Cytometric Analysis and Sorting of Plant Chromosomes.

Flow cytometry offers a unique way of analyzing and manipulating plant chromosomes. During a rapid movement in a liquid stream, large populations can be classified in a short time according to their fluorescence and light scatter properties. Chromosomes whose optical properties differ from other chromosomes in a karyotype can be purified by flow sorting and used in a range of applications in cytogenetics, molecular biology, genomics, and proteomics. As the samples for flow cytometry must be liquid suspensions of single particles, intact chromosomes must be released from mitotic cells. This protocol describes a procedure for preparation of suspensions of mitotic metaphase chromosomes from meristem root tips and their flow cytometric analysis and sorting for various downstream applications.

Construction of Full-Length Infectious cDNA Clones of Citrus Mosaic Virus RNA1 and RNA2 and Infection of Citrus Seedlings by Agrobacterium-Mediated Vacuum-Infiltration

The development of full-length infectious cDNA clones for plant RNA viruses is important for studying their molecular biological characteristics, functional genomics, pathogenesis, and vectorization applications. Citrus mosaic virus (CiMV), a member of the genus Sadwavirus, is of economic importance to the citrus industry and comprises a bipartite, positive-sense, single-stranded RNA genome encapsidated in icosahedral virions. In the present study, full-length cDNA clones of CiMV RNA1 and RNA2 were constructed based on a ternary yeast-Escherichia coli-Agrobacterium tumefaciens shuttle vector, pTY, using transformation-associated recombination (TAR) strategy. Infectivity of cDNA clones of CiMV RNA1 and RNA2 was examined in multiple citrus varieties via Agrobacterium-mediated vacuum-infiltration (AVI) through symptom observation, RT-PCR, and virion detection with an electron microscope. Furthermore, the genome-sized RT-PCR fragments of RNA1 and RNA2 were obtained from symptomatic Jinchengyou (Citrus grandis) plants infected by the cloned virus (CiMV211). In addition, CiMV211 produced typical symptoms of wild-type CiMV in cowpea (Vigna angularis) plants inoculated by Agrobacterium-mediated injection. This is the first report of infectious cDNA clones of CiMV, which may lay the foundation for research on the pathogenesis and vectorization of the virus.

Functional chromopeptide nanoarchitectonics: molecular design, self-assembly and biological applications.

Chromoproteins are a class of delicate natural compounds that elegantly complex photosensitive species with proteins and play a central role in important life processes, such as photosynthesis. Inspired by chromoproteins, researchers integrate simple peptides and photosensitive molecular motifs to generate chromopeptides. Compared with chromoproteins, chromopeptides exhibit a relatively simple molecular structure, flexible and adjustable photophysical properties, and a capability of programmable self-assembly. Chromopeptide self-assembly has attracted great attention as the resultant high-level architectures exhibit an ingenious combination of photofunctions and biofunctions. This review systematically summarizes recent advances in chromopeptide nanoarchitectonics with particular focus on the design strategy, assembly mechanism, and structure-function relationship. Among them, the effect of peptide sequences and the variation in photophysical performance are critically emphasized. On this basis, various applications, including biomedicine and artificial photosynthesis, are discussed together with the future prospects of chromopeptide nanoarchitectonics. This review will provide insights into chromopeptide nanoarchitectonics and corresponding materials with precise designs, flexible nanostructures and versatile functions. In addition, knowledge involving chromopeptide nanoarchitectonics may aid in the development of many other kinds of supramolecular biological materials and bioengineering techniques.

Green immobilization and efficient proliferation of Escherichia coli cells in polyvinyl alcohol hydrogel membranes by unidirectional nanopore dehydration

BackgroundThe immobilized technology for microbial or cells has the advantages of high microbial activity, high microbial density per unit space, good tolerance, strong shock, load resistance, high processing efficiency, and high reuse rate. It is now widely used in environmental remediation, water quality treatment, biodegradation, food industry, chemical analysis, energy development, medicine and pharmaceuticals, and other fields.ResultsA novel Escherichia coli cell-immobilizing polyvinyl alcohol hydrogel membrane (ECI-PVAHM) was prepared by unidirectional nanopore dehydration (UND) from a 10% polyvinyl alcohol (PVA) aqueous solution containing enhanced green fluorescent protein-labeled E. coli. This bacteria-loaded film has high water stability, flexibility, transparency, and mechanical robustness. Its tensile strength, elongation rate, and swelling rate are in the ranges 0.66–0.90 MPa, 300–390%, and 330–800%, respectively. The effective bacterial load of ECI-PVAHM is 2.375 × 109–1010 CFU/g (dry weight), which does not affect the original crystal structure of the PVAHM. This biofilm has a porous network structure with pore sizes between 0.2 and 1.0 μm, and these cells are embedded in the PVAHM network. When the immobilized cells were continuously cultured for 20 days, and the medium was renewed twice daily, their relative proliferation efficiency after 40 cycles could still be maintained at ~ 91%.ConclusionThe above results show that the cell division, proliferation ability, and metabolic activity of immobilized E. coli were not affected by the physical barrier of the porous network structure of the hydrogel. This UND-based ECI-PVAHM has potential applications in molecular biology, biopharmaceutical expression and production, bioreactors, and fuel cells.

Disruption of electrostatic contacts in the HNH nuclease from a thermophilic Cas9 rewires allosteric motions and enhances high-temperature DNA cleavage.

Allosteric signaling within multidomain proteins is a driver of communication between spatially distant functional sites. Understanding the mechanism of allosteric coupling in large multidomain proteins is the most promising route to achieving spatial and temporal control of the system. The recent explosion of CRISPR-Cas9 applications in molecular biology and medicine has created a need to understand how the atomic level protein dynamics of Cas9, which are the driving force of its allosteric crosstalk, influence its biophysical characteristics. In this study, we used a synergistic approach of nuclear magnetic resonance (NMR) and computation to pinpoint an allosteric hotspot in the HNH domain of the thermostable GeoCas9. We show that mutation of K597 to alanine disrupts a salt-bridge network, which in turn alters the structure, the timescale of allosteric motions, and the thermostability of the GeoHNH domain. This homologous lysine-to-alanine mutation in the extensively studied mesophilic S. pyogenes Cas9 similarly alters the dynamics of the SpHNH domain. We have previously demonstrated that the alteration of allostery via mutations is a source for the specificity enhancement of SpCas9 (eSpCas9). Hence, this may also be true in GeoCas9.

Multi‐Stimuli Responsive Thiazolothiazole Viologen‐Containing Poly(2‐Isopropyl‐2‐Oxazoline) and Its Multi‐Modal Thermochromism, Photochromism, Electrochromism, and Solvatofluorochromism Applications

AbstractSmart materials are highly desirable over the recent decade due to the growing demand on complicated nature. Smart materials with stable stimuli‐responsive properties are widely applied in sensors, smart windows, displays, and chemical valves, but they are relatively rare. Most of the current chromic materials are triggered by one or two external stimuli, and only a few multi‐stimuli responsive instances still suffer by their restricting functional states, such as solution or polymer. To address these challenges, researchers are focusing their efforts on developing a new generation of multi‐stimulus responsive chromic materials. In this article, our group has adopted a new strategy for the synthesis of the multi‐stimuli responsive single polymeric material, which can be useful for different applications (such as thermochromism, photochromism, electrochromism, and solvatofluorochromism). Thiazolothiazole viologen (TTv)‐containing poly(2‐isopropyl‐2‐oxazoline) (PiPO) was synthesized using a one‐pot reaction where the living cationic polymer chains were reacted with thiazolothiazole viologen to produce multi‐stimuli responsive PiPO‐TTv. PiPO‐TTv‐based device demonstrated multi‐responsive properties due to the thermoresponsive properties of PiPO segments and photo‐, electro‐responsive properties of TTv monomer, respectively. PiPO‐TTv material, additionally exhibits fluorescence characteristics that makes it appealing for environmental and molecular biological sensor applications.

Serial enrichment of heteroduplex DNA using a MutS-magnetic bead system.

Numerous applications in molecular biology and genomics require characterization of mutant DNA molecules present at low levels within a larger sample of non-mutant DNA. This is often achieved either by selectively amplifying mutant DNA, or by sequencing all the DNA followed by computational identification of the mutant DNA. However, selective amplification is challenging for insertions and deletions (indels). Additionally, sequencing all the DNA in a sample may not be cost effective when only the presence of a mutation needs to be ascertained rather than its allelic fraction. The MutS protein evolved to detect DNA heteroduplexes in which the two DNA strands are mismatched. Prior methods have utilized MutS to enrich mutant DNA by hybridizing mutant to non-mutant DNA to create heteroduplexes. However, the purity of heteroduplex DNA these methods achieve is limited because they can only feasibly perform one or two enrichment cycles. We developed a MutS-magnetic bead system that enables rapid serial enrichment cycles. With six cycles, we achieve complete purification of heteroduplex indel DNA originally present at a 5% fraction and over 40-fold enrichment of heteroduplex DNA originally present at a 1% fraction. This system may enable novel approaches for enriching mutant DNA for targeted sequencing.

D-Peptide and D-Protein Technology: Recent Advances, Challenges, and Opportunities.

Total chemical protein synthesis provides access to entire D-protein enantiomers enabling unique applications in molecular biology, structural biology, and bioactive compound discovery. Key enzymes involved in the central dogma of molecular biology have been prepared in their D-enantiomeric forms facilitating the development of mirror-image life. Crystallization of a racemic mixture of L- and D-protein enantiomers provides access to high-resolution X-ray structures of polypeptides. Additionally, D-enantiomers of protein drug targets can be used in mirror-image phage display allowing discovery of non-proteolytic D-peptide ligands as lead candidates. This review discusses the unique applications of D-proteins including the synthetic challenges and opportunities.

Easily-controllable, helper phage-free single-stranded phagemid production system

BackgroundSingle-stranded (ss) DNAs are utilized in various molecular biological and biotechnological applications including the construction of double-stranded DNAs with a DNA lesion, and are commonly prepared by using chimeric phage-plasmids (phagemids) plus M13-derived helper phages. However, the yields of ss DNA with these methods are poorly reproducible, and multiple factors must be optimized.ResultsIn this report, we describe a new arabinose-inducible ss phagemid production method without helper phage infection. The newly exploited DNA derived from VCSM13 expresses the pII protein, which initiates ss DNA synthesis, under the control of the araBAD promoter. In addition, the packaging signal is deleted in the DNA to reduce the contamination of the phage-derived ss DNA. The phagemid DNA of interest, carrying the M13 origin of replication and the packaging signal, was introduced into bacterial cells maintaining the modified VCSM13 DNA as a plasmid, and the ss phagemid DNA production was induced by arabinose. The DNA recovered from the phage particles had less contamination from VCSM13 DNA, as compared to the conventional method. Moreover, we extended the method to purify the ss DNAs by using an anion-exchange column, to avoid the use of hazardous chemicals.ConclusionUsing this combination of methods, large quantities of phagemid ss DNAs of interest can be consistently obtained.

The DAMA Protocol, an Introduction: Finding Pathogens before They Find Us

Globally, humanity is coming to recognize the magnitude of the interactive crisis for emerging infectious disease (EID). Strategies for coping with EID have been largely in the form of reactive measures for crisis response. The DAMA protocol (Document, Assess, Monitor, Act), the operational policy extension of the Stockholm paradigm, constitutes a preventive/proactive dimension to those efforts. DAMA is aimed at focusing and extending human and material resources devoted to coping with the accelerating wave of EID. DAMA is integrative, combining efforts to strategically document the distribution of complex pathogen and host assemblages in the biosphere in the context of dynamic environmental interfaces that provide the opportunities for pathogen exchange and emergence. Movement of habitats and animals (a breakdown in ecological isolation) catalyzed by climate change and broader anthropogenic trajectories of environmental disruption provide the landscape of opportunity for emergence. Evolutionarily and ecologically conserved capacities for exploitation of host-based resources allow pathogens to persist in one place or among a particular spectrum of hosts and provide insights to predict outcomes of persistence and emergence in novel conditions and across changing ecological interfaces. DAMA trajectories combine “boots on the ground” contributions of citizen scientists working with field biologists in development and application of sophisticated archival repositories, bioinformatics, molecular biology, and satellite surveillance. DAMA is a focus for anticipation, mitigation, and prevention of EID through knowledge of pathogens present in the environment and actions necessary to diminish risk space for their emergence. DAMA can be an effective strategy for buying time in the arena of accelerating environmental and socioeconomic disturbance and expanding EID linked to a future of climate change. Information + action = prediction and lives saved in a realm of EID. This article has been produced in support of and with appreciation for the efforts by Gábor Földvári of the Institute of Evolution, Centre for Ecological Research, and the Centre for Eco-Epidemiology, National Laboratory for Health Security (both located at 1121 Budapest, Konkoly-Thege Miklós út 29-33, Hungary). Through his untiring efforts, team building, and leadership, he has secured the first EU-wide team research grant. This work was supported by the National Research, Development and Innovation Office in Hungary (RRF-2.3.1-21-2022-00006) and the COST Action CA21170 “Prevention, anticipation and mitigation of tick-borne disease risk applying the DAMA protocol (PRAGMATICK),” which represent the first funded efforts to apply the principles of the DAMA protocol.

Improving Stability and Specificity of CRISPR/Cas9 System by Selective Modification of Guide RNAs with 2'-fluoro and Locked Nucleic Acid Nucleotides.

The genome editing approach using the components of the CRISPR/Cas system has found wide application in molecular biology, fundamental medicine and genetic engineering. A promising method is to increase the efficacy and specificity of CRISPR/Cas-based genome editing systems by modifying their components. Here, we designed and chemically synthesized guide RNAs (crRNA, tracrRNA and sgRNA) containing modified nucleotides (2'-O-methyl, 2'-fluoro, LNA-locked nucleic acid) or deoxyribonucleotides in certain positions. We compared their resistance to nuclease digestion and examined the DNA cleavage efficacy of the CRISPR/Cas9 system guided by these modified guide RNAs. The replacement of ribonucleotides with 2'-fluoro modified or LNA nucleotides increased the lifetime of the crRNAs, while other types of modification did not change their nuclease resistance. Modification of crRNA or tracrRNA preserved the efficacy of the CRISPR/Cas9 system. Otherwise, the CRISPR/Cas9 systems with modified sgRNA showed a remarkable loss of DNA cleavage efficacy. The kinetic constant of DNA cleavage was higher for the system with 2'-fluoro modified crRNA. The 2'-modification of crRNA also decreased the off-target effect upon in vitro dsDNA cleavage.

RiboXYZ: a comprehensive database for visualizing and analyzing ribosome structures.

Recent advances in Cryo-EM led to a surge of ribosome structures deposited over the past years, including structures from different species, conformational states, or bound with different ligands. Yet, multiple conflicts of nomenclature make the identification and comparison of structures and ortholog components challenging. We present RiboXYZ (available at https://ribosome.xyz), a database that provides organized access to ribosome structures, with several tools for visualisation and study. The database is up-to-date with the Protein Data Bank (PDB) but provides a standardized nomenclature that allows for searching and comparing ribosomal components (proteins, RNA, ligands) across all the available structures. In addition to structured and simplified access to the data, the application has several specialized visualization tools, including the identification and prediction of ligand binding sites, and 3D superimposition of ribosomal components. Overall, RiboXYZ provides a useful toolkit that complements the PDB database, by implementing the current conventions and providing a set of auxiliary tools that have been developed explicitly for analyzing ribosome structures. This toolkit can be easily accessed by both experts and non-experts in structural biology so that they can search, visualize and compare structures, with various potential applications in molecular biology, evolution, and biochemistry.

Scaled Torus Principal Component Analysis

A particularly challenging context for dimensionality reduction is multivariate circular data, that is, data supported on a torus. Such kind of data appears, for example, in the analysis of various phenomena in environmental sciences and astronomy, as well as in molecular structures. This article introduces Scaled Torus Principal Component Analysis (ST-PCA), a novel approach to perform dimensionality reduction with toroidal data. ST-PCA finds a data-driven map from a torus to a sphere of the same dimension and a certain radius. The map is constructed with multidimensional scaling to minimize the discrepancy between pairwise geodesic distances in both spaces. ST-PCA then resorts to principal nested spheres to obtain a nested sequence of subspheres that best fits the data, which can afterwards be inverted back to the torus. Numerical experiments illustrate how ST-PCA can be used to achieve meaningful dimensionality reduction on low-dimensional torii, particularly with the purpose of clusters separation, while two data applications in astronomy (on a three-dimensional torus) and molecular biology (seven-dimensional torus) show that ST-PCA outperforms existing methods for the investigated datasets. Supplementary materials for this article are available online.

Professor Bin Xu, a Legendary Pharmacologist in People’s Republic of China

Abstract: Professor Bin Xu was a pioneer pharmacologist of People’s Republic of China, especially anti-cancer drug pharmacology. His expertise ranged from drug efficacy screening, molecular biology, drug targeting and clinical applications. As one of the legendary figures (highest-ranking scientist of pharmacology in China), his contributions to the Chinese pharmacology and pharmaceutical industry were enormous and far-reaching. His long scientific career has greatly contributed and has a strong impact on People’s Republic of China. This article describes the engagement and major contributions of Prof Xu (including childhood history, medical experience, experimental work, pharmacology investigations, graduate education, clinical studies, major scientific contributions, social engagements, and global influence). It is necessary to review his invaluable contributions to China and find new light for the Chinese pharmaceutical industry.

Biohydrogen production and purification: Focusing on bioelectrochemical systems

Innovative technologies on green hydrogen production become significant as the hydrogen economy has grown globally. Biohydrogen is one of green hydrogen production methods, and microbial electrochemical cells (MECs) can be key to biohydrogen provision. However, MECs are immature for biohydrogen technology due to several limitations including extracellular electron transfer (EET) engineering. Fundamental understanding of EET also needs more works to accelerate MEC commercialization. Interestingly, studies on biohydrogen gas purification are limited although biohydrogen gas mixture requires complex purification for use. To facilitate an MEC-based biohydrogen technology as the green hydrogen supply this review discussed EET kinetics, engineering of EET and direct interspecies electron transfer associated with hydrogen yield and the application of advanced molecular biology for improving EET kinetics. Finally, this article reviewed biohydrogen purification technologies to better understand purification and use appropriate for biohydrogen, focusing on membrane separation.

Plant Melatonin: Regulatory and Protective Role

Melatonin is an antioxidant that is widely distributed in plants and animals. It is a conservative molecule. In early studies, scientists often used isolation and identification techniques to observe whether the endogenous melatonin cycle in plants was related to the external photoperiod, plant growth, and development cycles, including seed germination, plant rooting, and floral induction. With the development of isolation and identification technology, there is ample evidence that plants possess a variety of melatonin-synthesis pathways. The comprehensive application of molecular biology, genomics, and computational biology has also led to a comprehensive understanding of the physiological functions of plant melatonin. In this paper, we not only highlight the candidate genes from Arabidopsis thaliana and Oryza sativa that might be contributing to increasing plant endogenous melatonin but also elucidate and characterize the role of melatonin in plant growth and development in response to biotic and abiotic stresses.

Insight into achirality and chirality effects in interactions of an racemic ruthenium(II) polypyridyl complex and its Δ- and Λ-enantiomers with an RNA triplex

RNA triplexes have a variety of potential applications in molecular biology, diagnostics and therapeutics, while low stabilization of the third strand hinders their practical utilities under physiological conditions. In this regard, achieving the third-strand stabilization by binding small molecules is a promising strategy. Chirality is one of the basic properties of nature. To clarify achirality and chirality effects on the binding and stabilizing effects of RNA triplexes by small molecules, we report for the first time the RNA interactions of an racemic ruthenium(II) polypyridyl complex [Ru(bpy)2(11-CN-dppz)]2+ (rac-Ru1) and its two enantiomers Δ/Λ-[Ru(bpy)2(11-CN-dppz)]2+ (Δ/Λ-Ru1) with an RNA triplex poly(U-A*U) (where “-” represents Watson-Crick base pairing, and “*” denotes Hoogsteen base pairing, respectively) in this work. Research shows that although rac-Ru1 and its two enantiomers Δ/Λ-Ru1 bind to the RNA triplex through the same mode of intercalation, the binding affinity for enantiomer Δ-Ru1 is much higher than that for rac-Ru1 and enantiomer Λ-Ru1. However, compared to enantiomer Λ-Ru1, the binding affinity for rac-Ru1 does not show much of an advantage, which is slightly greater than that for the former. Thermal denaturation measurements reveal both rac-Ru1 and Δ-Ru1 to have a preference for stabilizing the third strand rather than the template duplex of the RNA triplex, while Λ-Ru1 stabilizes the RNA triplex without significant selectivity. Besides, the third-strand stabilizing effects by rac-Ru1 and Δ-Ru1 are not markedly different from each other, but more marked than that by Λ-Ru1. This work shows that the binding properties of the racemic Ru(II) polypyridyl complex with the RNA triplex are not simply an average of its two enantiomers, indicating potentially complicated binding events.