Molecular Hybridization Methods Research Articles

Molecular hybridization method for obtaining paeonol-based fibrate derivatives with potent lipid-lowering and hepatoprotective activity

Molecular hybridization method for obtaining paeonol-based fibrate derivatives with potent lipid-lowering and hepatoprotective activity

A Molecular Approach for Detecting Bacteria and Fungi in Healthcare Environment Aerosols: A Systematic Review.

Molecular methods have become integral to microbiological research for microbial identification. This literature review focuses on the application of molecular methods in examining airborne bacteria and fungi in healthcare facilities. In January 2024, a comprehensive electronic search was carried out in esteemed databases including PubMed, Web of Science, and Scopus, employing carefully selected keywords such as ((bacteria) OR (virus) OR (fungi)) AND (aerosol) AND ((hospital) OR (healthcare) OR (dental office)) AND ((molecular) OR (PCR) OR (NGS) OR (RNA) OR (DNA) OR (metagenomic) OR (microarray)), following the PRISMA protocol. The review specifically targets healthcare environments with elevated concentrations of pathogenic bacteria. A total of 487 articles were initially identified, but only 13 met the inclusion criteria and were included in the review. The study disclosed that the prevalent molecular methodology for appraising aerosol quality encompassed the utilization of the PCR method, incorporating either 16S rRNA (bacteria) or 18S rRNA (fungi) amplification techniques. Notably, five diverse molecular techniques, specifically PFGE, DGGE, SBT, LAMP, and DNA hybridization methods, were implemented in five distinct studies. These molecular tests exhibited superior capabilities compared to traditional bacterial and fungal cultures, providing precise strain identification. Additionally, the molecular methods allowed the detection of gene sequences associated with antibiotic resistance. In conclusion, molecular testing offers significant advantages over classical microbiological culture, providing more comprehensive information.

Morpholinodiazenyl chalcone blocks influenza A virus capsid uncoating by perturbing the clathrin-mediated vesicular trafficking pathway.

Influenza A virus (IAV) is a highly contagious respiratory pathogen that significantly threatens global health by causing seasonal epidemics and occasional, unpredictable pandemics. To identify new compounds with therapeutic potential against IAV, we designed and synthesized a series of 4'-morpholinodiazenyl chalcones using the molecular hybridization method, performed a high-content screen against IAV, and found that (E)-1-{4-[(E)-morpholinodiazenyl]phenyl}-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (MC-22) completely neutralized IAV infection. While MC-22 allowed IAV to successfully internalize into the cell and fuse at the acidic late endosomes, it prevented viral capsid uncoating and genome release. Since IAV majorly utilizes clathrin-mediated endocytosis (CME) for cellular entry, we examined whether MC-22 had any effect on CME, using nonviral cargoes that enter cells via clathrin-dependent or -independent pathways. Although MC-22 showed no effect on the uptake of choleratoxin B, a cargo that enters cells majorly via the clathrin-independent pathway, it significantly attenuated the clathrin-dependent internalization of both epidermal growth factor and transferrin. Cell biological analyses revealed a marked increase in the size of early endosomes upon MC-22 treatment, indicating an endosomal trafficking/maturation defect. This study reports the identification of MC-22 as a novel CME-targeting, highly potent IAV entry inhibitor, which is expected to neutralize a broad spectrum of viruses that enter the host cells via CME.

Hybridization-based discovery of novel quinazoline-2-indolinone derivatives as potent and selective PI3Kα inhibitors

IntroductionPhosphatidylinositol 3-kinases (PI3Ks) overexpression can elicit cellular homeostatic dysregulation, which further contributes to tumorigenesis, with PI3Kα emerging as the most prevalent mutant isoform kinase among PI3Ks. Therefore, selective inhibitors targeting PI3Kα have attracted considerable interest in recent years. Molecular hybridization, with the advantage of simplified pharmacokinetics and drug-drug interactions, emerged as one of the important avenues for discovering potential drugs. ObjectivesThis study aimed to construct PI3Kα inhibitors by hybridization and investigate their antitumor activity and mechanism. Methods26 quinazoline-2-indolinone derivatives were obtained by molecular hybridization, and their structure–activity relationship was analyzed by MTT, in vitro kinase activity and molecular docking. The biological evaluation of compound 8 was performed by transwell, flow cytometry, laser scanning confocal microscopy, Western blot, CTESA and immunohistochemistry. ResultsHere, we employed molecular hybridization methods to construct a series of quinazoline-2-indolinone derivatives as PI3Kα selective inhibitors. Encouragingly, representative compound 8 exhibited a PI3Kα enzymatic IC50 value of 9.11 nM and 10.41/16.99/37.53-fold relative to the biochemical selectivity for PI3Kβ/γ/δ, respectively. Moreover, compound 8 effectively suppressed the viability of B16, HCT116, MCF-7, H22, PC-3, and A549 cells (IC50 values: 0.2 μM ∼ 0.98 μM), and dramatically inhibited the proliferation and migration of NSCLC cells, as well as induced mitochondrial apoptosis through the PI3K/Akt/mTOR pathway. Importantly, compound 8 demonstrated potent in vivo anti-tumor activity in non-small cell lung cancer mouse models without visible toxicity. ConclusionsThis study presented a new avenue for the development of PI3Kα inhibitors and provided a solid foundation for novel QHIDs as potential future therapies for the treatment of NSCLC.

Synthesis and evaluation of linearly fused thiadiazolocoumarins as prospects with broad-spectrum bioactivity

Various pharmacological properties of coumarins brought them under light among the heterocyclics. Also, coumarin derivatives still approve of their importance in the medical field as a valuable building block to elicit further derivatives with diverse, potent, and interesting biological activities. Moreover, the numerous biological actions of thiadiazole-based compounds contribute to the notion of this unique synthesis of linearly fused thiadiazolocoumarins using the molecular hybridization method. This work reported the novel synthesis of seven linearly fused thiadiazolocoumarins, including Precursor N-III and TDA1-TDA6. These derivatives were characterized by their physicochemical properties and through spectrophotometric tools, including FTIR, 1H NMR, and 13C NMR. Also, their pharmacokinetics were predicted using computer-based studies by the SwissADME and PreADMET programs. Furthermore, the derivatives were checked for several biological activities, including anticancer, biosafety, anti-oxidative stress, anti-inflammatory, antimicrobial, normal flora safety, and antidiabetic activities. According to the results, TDA3 showed the greatest anticancer activity against the examined cancer cell lines, with the highest cellular biosafety. TDA3 and TDA4 showed higher anti-oxidative stress activity than the other derivatives. All the prepared derivatives showed significant inflammation-inhibitory activity toward the LOX-5 enzyme while showing poor activity towards COX enzymes. According to antimicrobial activity, TDA4 exhibited the greatest anti-gram-negative activity and also showed the best safety toward the examined normal flora. TDA1 exhibited promising activity against anaerobic bacteria, while Precursor N-III, TDA3, and TDA4 showed significant antifungal activity. Finally, TDA1 demonstrated moderate antidiabetic activity, which was the greatest activity among the other derivatives. The authors concluded from the gathered results that the linearly fused thiadiazolocoumarins represent key structures for developing compounds with versatile bioactivities.

Thiadiazole - A promising structure in design and development of anti-Alzheimer agents.

The design and development of effective multitargeted agents in treating Alzheimer disease (AD) has always been a hot topic in the field of drug discovery. Since AD is a multifactorial disorder, various key hidden players such as deficit of acetylcholine (ACh), tau-protein aggregation, and oxidative stress have been associated with the incidence and progress of AD. In pursuit of improving efficacy and expanding the range of pharmacological activities of current AD drugs, the molecular hybridization method is also used intensively. Five-membered heterocyclic systems such as thiadiazole scaffolds have previously been shown to have therapeutic activity. Thiadiazole analogs as an anti-oxidant compound have been known to include a wide range of biological activity from anti-cancer to anti-Alzheimer properties. The suitable pharmacokinetic and physicochemical properties of the thiadiazole scaffold have introduced it as a therapeutic target in medicinal chemistry. The current review portrays the critical role of the thiadiazole scaffold in the design of various compounds with potential effects in the treatment of Alzheimer's disease. Furthermore, the rationale used behind hybrid-based design strategies and the outcomes achieved through the hybridization of Thiadiazole analogs with various core structures have been discussed. In addition, the data in the present review may help researchers in the design of new multidrug combinations that may provide new options for the treatment of AD.

Synthetic Biology’s Latest Trends in Antimicrobial Resistance and Biofilm

Recent instances of novel biological circuits that enable cells to gain biosynthetic skills demonstrate synthetic biology’s therapeutic potential. Synthetic biology is a branch of biology whose primary role is to build completely functional biological systems from the smallest basic elements such as DNA, proteins, and other organic molecules to complex bacteria. This review briefly mentions some novel way of synthetic strategies like bacterial modelling, two-component systems, synthetic peptide, and synthetic flavonoids used for targeting biofilm and drug-stable microbial communities. Bacterial modelling was mainly done in Escherichia coli and Mycoplasma using different strategies like introducing quorum sensing devices and CRISPR-mediated editing. Synthetic peptides are also one of the extensively studied ongoing areas which are produced from natural peptides taking as a template and altering amino acid position. Flavonoids are produced by two-step reaction and molecular hybridization methods. This kind of synthetic approach reported significant biofilm dispersion and lethal effects on clinically relevant bacteria like Pseudomonas aeruginosa, Staphylococcus aureus, E. coli, Acinetobacter baumannii, and Streptococcus species and Klebsiella pneumonia.

New Hybrids Derived from the Natural Compound (-)-β-Pinene and Amides or Acylthioureas as Antitumor Agents

Background:Plant-derived natural compounds have a unique molecular structure and rich biological activity, hence, they are treated as important raw materials for the development of drugs.Methods:A natural compound (-)-β-pinene was used as a raw material, and twenty-six novel derivatives with amide or acylthiourea groups were synthesized based on the molecular hybridization method. In vitro antitumor activity of these derivatives on human breast cancer cell line MCF7 and human colon cancer cell line SW1116 were tested by MTT method. The effects of the synthesized derivatives on the morphology of MCF7 and SW1116 were observed by fluorescent inverted microscope.Results:The preliminary structure-activity relationship analysis demonstrates that the position and species of substituents on the aromatic ring of derivatives have an effect on the antitumor activity of derivatives. Observation of the cell morphology reveals that derivatives with antitumor activity can lead to rounding of the cell morphology, a decrease in cell volume and cell density, and ultimately inhibition of the proliferation of MCF7 and SW1116 cells. The antitumor activity evaluation results show that among these derivatives, compounds 5c, 5e, 5h, 7c, 7b and 7e exhibit good antitumor activity against MCF7, and compounds 5c, 5e, 5h and 7j exert moderate antitumor activity against SW1116.Conclusion:This study hopes to promote the high value-added utilization of natural compounds β-pinene and the development of novel antitumor drugs.

(E)-3-[3-(4-Morpholinophenyl)acryloyl]-2H-chromen-2-one

A new compound (E)-3-[3-(4-morpholinophenyl)acryloyl]-2H-chromen-2-one, a coumarin based chalcone derivative, has been successfully synthesized employing a molecular hybridization method through the reaction between 3-acetylcoumarin and 4-morpholinobenzaldehyde using a Claisen–Schmidt reaction using pTSA as a catalyst. The structure of the title compound was established using spectroscopic data FTIR, HRESI-MS, 1H- and 13C-NMR. The anticancer activity against breast cancer cells line T47D and cervix cancer cells line HeLa was determined using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.

Inoculation of cucumber, melon and zucchini varieties with Tomato leaf curl New Delhi virus and evaluation of infection using different detection methods

AbstractThe disease caused by Tomato leaf curl New Delhi virus (ToLCNDV), which is naturally transmitted by the whitefly Bemisia tabaci, causes important economic losses in cucurbit crops. The availability of simple and efficient inoculation protocols and detection methods is necessary for screening varieties and germplasm collections as well as for breeding populations. We evaluated the infectivity of ToLCNDV inocula prepared using three different buffers for mechanical sap inoculation in a susceptible variety of zucchini. We found that inoculum prepared with buffer III, which contains polyvinylpyrrolidone, is highly efficient for mechanical inoculation, with 100% of plants displaying severe symptoms 21 days post‐inoculation. Using this buffer, we mechanically inoculated 19 commercial varieties of cucurbit crops (six of cucumber, six of melon and seven of zucchini), evaluated the evolution of symptoms and diagnosed infection using nine different ToLCNDV detection methods (four based on serology, four based on molecular hybridization and one based on PCR detection). The results revealed that all varieties are susceptible, although cucumber varieties display less severe symptoms than those of melon or zucchini. All detection methods were highly efficient (more than 85% of plants testing positive) in melon and zucchini, but in cucumber, the percentage of positive plants detected with serology and molecular hybridization methods ranged from 20.4% with Squash leaf curl virus (SLCV) antiserum, to 78.5% with DNA extract hybridization. Overall, the best detection results were obtained with PCR, with 92.6%, 92.4% and 98.4% cucumber, melon and zucchini plants, respectively, testing positive. When considering the overall results in the three crops, the best serology and molecular hybridization methods were those using Watermelon chlorotic stunt virus (WmCSV) antiserum and DNA extract, respectively. The inoculation methodology developed and the information on detection methods are of great relevance for the selection and breeding of varieties of cucurbit crops that are tolerant or resistant to ToLCNDV.

High diagnostic yield of line probe assay as a point-of care test in a TB rural setting

Background Tuberculosis is the second leading cause of death due to infectious disease worldwide. Delay in bacilli isolation in culture, cumbersome susceptibility testing methods, lack of methods for differentiation of Mycobacterium tuberculosis (MTB) from non tuberculous mycobacteria, are important issues which impinge on the WHO’s millennium goal of disease reduction by 2015. Traditional methods of TB detection and anti tB drug susceptibility assays are time consuming and with a steep rise in number of MDR tB cases the need of the hour is rapid diagnostic methods like direct drug susceptibility testing in liquid medium, molecular hybridization methods and RealTime PCR.

Distinct biphasic histomorphological pattern in gastrointestinal stromal tumours (GISTs) with common primary mutations but divergent molecular cytogenetic progression

The morphological diversity of gastrointestinal stromal tumours (GISTs) is well appreciated. The aim of this study was to shed light on the molecular pathogenesis of GISTs displaying a distinct biphasic histomorphological pattern, which is poorly understood. Six biphasic gastric GISTs (four high, one intermediate and one low risk for aggressive behaviour) were studied by histological, immunohistochemical, molecular and comparative genomic hybridization methods. The different tumour components were designated as primary and secondary compartments, based on cellularity and mitotic index. In addition, metastases from two patients were analysed separately. According to the classification of Miettinen et al., four biphasic histomorphological patterns were seen: (i) sclerosing spindle cell/dyscohesive or paraganglioma-like epithelioid (n = 2); (ii) sarcomatous spindle cell/pleomorphic sarcomatous spindle cell (n = 1); (iii) sarcomatous spindle cell/sarcomatous epithelioid (n = 2); and (iv) sclerosing epithelioid/hypercellular epithelioid/sarcomatous epithelioid (n = 1) morphology. In each case, both tumour compartments revealed the same KIT (n = 5) or platelet-derived growth factor receptor-alpha (n = 1) mutation, as well as common chromosomal imbalances reflecting their common clonal origin. Additional chromosomal imbalances were detected in the secondary tumour compartments and their respective metastases. Our results indicate that the intratumoral phenotypic diversity in GIST reflects histomorphological progression, which is associated with higher chromosomal instability, irrespective of the primary mutation.

Prevalence and Risk Factors of HPV Infection among High-Risk Rural and Urban Lithuanian Women

Aims: To investigate the prevalence, persistence and risk factors of high oncogenic risk human papillomavirus (HPV) among urban and rural women of reproductive age coming to consult a gynaecologist. Methods: A prospective cohort study in urban (Kaunas) and rural (Marijampole) regions of Lithuania. The data were collected in 8 healthcare institutions from women seeking consultation of gynaecologists using a questionnaire for finding out demographic, social, behavioural and biomedical factors. HPV DNA was determined by molecular hybridization method (hybrid capture version II) determining HPV of high oncogenic risk. Result: 1,120 women participated in the study. The prevalence of high-risk HPV among the studied women was 25.1%. It was higher among the urban women than among the rural women. The prevalence of high-risk HPV was increased if the subjects had 2 or more sexual partners during the last 12 months (OR 2.81; 95% CI 1.83–4.32), were 19 years of age or younger (OR 2.68; 95% CI 1.47–4.91), were smoking (OR 1.81; 95% CI 1.16–2.81), and had secondary or lower education level (OR 1.43; 95% CI 1.01–2.04). This infection was obviously associated with high- and low-grade squamous intraepithelial changes of the cervix (OR 1.66, 95% CI = 1.08–2.53). Conclusion: The incidence rate for cervical cancer in Lithuania is one of the highest in comparison with other European countries. HPV infection was also particularly common in the studied population. About one-fourth of the women were infected with high-risk HPV infection. Young and less educated women were found to be the group that was most exposed to HPV, and therefore public health interventions and education seem to be essential in programs aimed at reducing the incidence of cervical cancer.

Estimating the Growth Rate of a Bacterial Species in a Complex Mixture by Hybridization of Genomic DNA

> Abstract Advances in molecular techniques have enabled new approaches to identifying bacteria. However, once identified, there is no quantitative information on the in situ growth rate of the species, mainly because the technology has not been available. The quantitative incorporation of [methyl-3H]thymidine into dividing bacteria is coupled with a molecular (hybridization) method, to determine the growth rate of bacterial species in situ. The basis of this molecular method is a reverse gene probe-natural populations are labeled in situ with [methyl-3H]thymidine. The probe (3H-Tdr-DNA) is captured, using a hybridization procedure, on a positively charged nylon membrane on which is attached non-labeled target DNA. Two bacterial species, Bacillus cereus and Zoogloea ramigera, were used to demonstrate the principle in laboratory cultures and in a municipal activate sludge treatment process. The DNA of the dividing bacteria in activated sludge was radioactively labeled with [methyl-3H]thymidine, and the DNA of Z. ramigera was recovered using a DNA hybridization method. The recovered radioactively-labeled DNA was used to estimate the growth rate (0.03 x 10(9) cells . ml-1 . h-1) of Z. ramigera in situ. Simultaneously applying these two powerful molecular-based methods could potentially be used to study bacterial population dynamics in situ.

Non‐radioactive molecular hybridization detection of carnation mottle virus in infected carnations and its comparison to serological and biological techniques

A non‐radioactive molecular hybridization method was developed to detect carnation mottle virus (CarMV) in carnation plants. This method was compared to the standard ELISA test and biological assays. For crude plant extracts, the molecular hybridization technique proved to be at least 125 times more sensitive than double‐antibody sandwich enzyme‐linked immunosorbent assay (DAS‐ELISA). The method was adapted to avoid the necessity to clarify the samples by centrifugation and to use pipettes to load them. By introducing this modification, the hybridization assay was found to be a suitable method for routine virus detection. The relative benefits of using this procedure for large‐scale indexing are discussed.

Quantitative PCR and RT-PCR in virology.

In the last few years, molecular hybridization methods have been used extensively in basic and applied virology because of their technical flexibility and high specificity. Using these techniques, the detection of DNA and RNA viruses directly from clinical specimens, the analysis of the specific transcriptional activity of viral genes in vitro and in vivo, and the study of virus-host relationships have all been carried out at the molecular level. However, although these methods are efficient for many purposes, only development (~) and optimization (z) of PCR amplification have dramatically improved sensitivity. Currently, PCR is the method of choice for the detection of viral nucleic acids present at very low amounts in biological samples, and it allows the molecular study of most acute and persistent viral infections. Impressive achievements that are rap

Interrupted replication of hepatitis B virus in liver tissue of HBsAg carriers with hepatocellular carcinoma

To search for events underlying reduction of peripheral viremia and integration of hepatitis B virus (HBV) DNA into the liver cell genome in long-term virus carriers with hepatocellular carcinoma, paired samples of liver and tumor tissue were analyzed by molecular hybridization and immunological methods. Most tumor tissues contained integrated viral DNA; in none was extrachromosomal HBV DNA detected. Integrated HBV DNA was also found in peritumor liver tissue in the majority of patients. However, liver of patients either with or without peripheral viremia also contained free HBV DNA and replicative intermediates. In three nonviremic patients with replicative HBV DNA in liver, viral core antigen expression was markedly reduced or absent, whereas viral envelope protein (surface antigen) expression was normal. In one case, replicative intermediates in liver were sensitive to DNase I digestion, indicating that viral DNA was not encapsidated in normal viral core particles. These results suggest that decreased or defective core antigen production can lead to reduced viremia associated with blocked virus assembly/secretion and accumulation of unencapsidated HBV DNA replicative intermediates in the liver cell. Accumulation of such HBV DNA molecular forms in the liver may lead to an increased propensity for HBV DNA to integrate into the host genome, which has been found with high frequency in hepatic neoplasms from patients infected with hepatitis B virus.

Comparison of bi‐directional electrophoresis and molecular hybridization methods to detect potato spindle tuber viroid and chrysanthemum stunt viroid1

A comparison was made of methods for viroid detection. Molecular hybridization using cDNA is a very sensitive method that can handle large quantities of samples at the same time but it has the disadvantage that only small amounts of the sample can be applied to the nitrocellulose filter. The method therefore can only detect viroid in plants when its concentration is 10–20 ng g‐1 of leaves, using 32P as a marker system. Bi‐directional electrophoresis can detect viroid in plants when its concentration is 10 ng g‐1 of leaves, because it uses larger samples. It does not need hazardous chemicals like 32P and formamide, and the reading of the results of the test is less liable to failures because it is based on two criteria (position and intensity of RNA band). The Dutch Plant Protection Service and the Dutch General Inspection Service for Ornamentals therefore use a modified bi‐directional electrophoresis method to detect potato spindle tuber viroid and chrysanthemum stunt viroid, respectively.

Ethanol metabolism

Alcohol is metabolized by two pathways in humans: the ADH pathway which accounts for the bulk of the metabolism, and the MEOS pathway which contributes to the increased rate of ethanol elimination at high blood alcohol levels. The increased rate of elimination which results from chronic alcohol consumption is due to an increase in MEOS activity. The activities of these pathways are influenced by environmental factors such as smoking, diet, and endocrine factors. In addition, individuals inherit different types of ADH isoenzymes which have different kinetic properties. Individuals with different phenotypic variants, e.g. the beta 1 vs beta 2 isoenzymes, appear to have different rates of ethanol elimination. The cloning of the ADH genes and the availability of molecular hybridization methods now make it possible to genotype individuals and to correlate the genotype with both alcohol elimination rates and with the risk of developing medical complications of alcoholism or even of developing alcoholism itself.

Molecular hybridization methods for determination of serum HBV-DNA

Molecular hybridization methods for determination of hepatitis B virus DNA (HBV-DNA) in serum were studied. A simple method by which serum was treated with sodium hydroxide, followed by dot hybridization procedure on filter sheets provides a sensitive and direct result for detecting HBV-DNA. Another method in which DNAs extracted from Dane particle fraction were subjected to the molecular hybridization method on a filter membrane, provided similar results although this method is time consuming. The third method in which serum was directly spotted on filter sheets, followed by alkaline-treatment seems to be less sensitive. Three filter papers, NC filter, Zeta-Probe and Biodyne, on which molecular hybridization was performed, gave similar sensitivity.