Molecular Biology Tools Research Articles

Advanced Fluorescence Imaging to Distinguish Between Intracellular Fractions of Antisense Oligonucleotides.

Antisense oligonucleotides (AON) have been intensively studied as tools in molecular cell biology and as novel therapeutics in various diseases over the past two decades. Especially cellular uptake and endosomal release of AONs are topics of interest, as these are crucial steps in reaching the subcellular AON target sites and achieving biological activity. We used cell-penetrating peptides (CPPs) to enhance uptake and endosomal release of AONs, and monitored these two processes and the subsequent fate of the AONs by advanced fluorescence microscopy in living cells. In this chapter, we discuss the use of automated time-lapse confocal laser scanning microscopy (CLSM) to follow AON uptake and trafficking in time, fluorescence lifetime imaging microscopy (FLIM) to distinguish between free and AON-bound fluorophore, and fluorescence correlation spectroscopy (FCS) to measure subcellular AON concentrations and molecular associations. Additionally, we expand on the analysis of these microscopy data.

Characterisation of utrophin modulator SMT C1100 as a non-competitive inhibitor of firefly luciferase.

Firefly luciferase (FLuc) is a powerful tool for molecular and cellular biology, and popular in high-throughput screening and drug discovery. However, FLuc assays have been plagued with positive and negative artefacts due to stabilisation and inhibition by small molecules from a range of chemical classes. Here we disclose Phase II clinical compound SMT C1100 for the treatment of Duchenne muscular dystrophy as an FLuc inhibitor (KD of 0.40 ± 0.15 µM). Enzyme kinetic studies using SMT C1100 and other non-competitive inhibitors including resveratrol and NFκBAI4 identified previously undescribed modes of inhibition with respect to FLuc's luciferyl adenylate intermediate. Employing a photoaffinity strategy to identify SMT C1100's binding site, a photolabelled SMT C1100 probe instead underwent FLuc-dependent photooxidation. Our findings support novel binding sites on FLuc for non-competitive inhibitors.

Fabrication of flexible microlens arrays for parallel super-resolution imaging

The development of optical microscopy has greatly promoted the progress of biological fields, providing outstanding observation tools for genetics, molecular biology, and bioengineering technology, from the macro- to the micro-scale. Owing to the optical diffraction limit, the imaging resolution of traditional optical microscopy is limited. Recently, the use of microspheres has been demonstrated to aid the capability to realize super-resolution imaging under white light illumination; however, using this approach, the imaging field of view is only a few microns, due to the size of the microspheres. In this paper, we fabricated microlens arrays by embedding microspheres into polydimethylsiloxane (PDMS) films. Using this method, we have successfully achieved parallel imaging under the sub-diffraction-limited resolution using multiple microspheres with a magnification up to ×2.59–×2.99, and the observed results are consistent with finite-difference time-domain (FDTD) simulation results. Furthermore, two imaging modes were developed: the microlens array-based dynamic scanning imaging mode and the stochastic microlens array region imaging overlay reconstruction mode, a surface image of 900 μm2 was presented stitched with 210 images. This study combines the advantages of parallel imaging and dynamic imaging to increase efficiency and observation range.

Computational approaches in cancer multidrug resistance research: Identification of potential biomarkers, drug targets and drug-target interactions.

Like physics in the 19th century, biology and molecular biology in particular, has been fertilized and enhanced like few other scientific fields, by the incorporation of mathematical methods. In the last decades, a whole new scientific field, bioinformatics, has developed with an output of over 30,000 papers a year (Pubmed search using the keyword "bioinformatics"). Huge databases of mass throughput data have been established, with ArrayExpress alone containing more than 2.7 million assays (October 2019). Computational methods have become indispensable tools in molecular biology, particularly in one of the most challenging areas of cancer research, multidrug resistance (MDR). However, confronted with a plethora of different algorithms, approaches, and methods, the average researcher faces key questions: Which methods do exist? Which methods can be used to tackle the aims of a given study? Or, more generally, how do I use computational biology/bioinformatics to bolster my research? The current review is aimed at providing guidance to existing methods with relevance to MDR research. In particular, we provide an overview on: a) the identification of potential biomarkers using expression data; b) the prediction of treatment response by machine learning methods; c) the employment of network approaches to identify gene/protein regulatory networks and potential key players; d) the identification of drug-target interactions; e) the use of bipartite networks to identify multidrug targets; f) the identification of cellular subpopulations with the MDR phenotype; and, finally, g) the use of molecular modeling methods to guide and enhance drug discovery. This review shall serve as a guide through some of the basic concepts useful in MDR research. It shall give the reader some ideas about the possibilities in MDR research by using computational tools, and, finally, it shall provide a short overview of relevant literature.

Natural Product Medicines for Honey Bees: Perspective and Protocols.

The western honey bee remains the most important pollinator for agricultural crops. Disease and stressors threaten honey bee populations and productivity during winter- and summertime, creating costs for beekeepers and negative impacts on agriculture. To combat diseases and improve overall bee health, researchers are constantly developing honey bee medicines using the tools of microbiology, molecular biology and chemistry. Below, we present a manifesto alongside standardized protocols that outline the development and a systematic approach to test natural products as ‘bee medicines’. These will be accomplished in both artificial rearing conditions and in colonies situated in the field. Output will be scored by gene expression data of host immunity, bee survivorship, reduction in pathogen titers, and more subjective merits of the compound in question. Natural products, some of which are already encountered by bees in the form of plant resins and nectar compounds, provide promising low-cost candidates for safe prophylaxis or treatment of bee diseases.

Novel multiplex strategy for DNA methylation-based age prediction from small amounts of DNA via Pyrosequencing

DNA methylation-based age estimation is a promising new tool for forensic molecular biology. There is growing understanding of the best predictive CpG loci and their performance in various sample types. Since forensic samples usually provide only small amounts of DNA, the sensitivity of the method is crucial. Pyrosequencing is one of the most sensitive methods but only capable to analyze different target regions separately. Thus, multiple input DNA samples are required for investigations of different target regions, which is required for all current age estimation models. To overcome this limitation, we developed a novel multiplex strategy for Pyrosequencing, which allows the investigation of different target regions from a single small amount of input DNA. A pre-amplification step was introduced to increase the amount of target-specific template for the subsequent sequencing PCR step. We tested this multiplex strategy for eight target regions including 15 age CpGs associated with the genes of ELOVL2, FHL2, CCDC102B, C1orf132, KLF14, EDARADD, PDE4C and SST. Except for FHL2, all target regions were successfully sequenced with the multiplex strategy and the precision in terms of reproducibility of the measurements was equal to the singleplex strategy. The measured methylation values at the age CpGs displayed borderline significant differences between both analytical strategies for six out of 14 CpG sites whereas both strategies delivered equal methylation values for the remaining eight age CpGs. In total, our results indicate that the multiplex strategy can act as a promising alternative for age estimation studies in cases when only limited amounts of DNA samples are available.

SEROLOGICAL RESULTS OF MVV AND CAEV ANTIBODIES IN SAMPLES COLLECTED FROM SHEEP AND GOAT FLOCKS OF ROMANIA

INTRODUCTION: Maedi-Visna (MV) and Caprine Arthritis Encephalitis (CAE) are specific diseases of small ruminants caused by lentiretroviruses. These diseases are not a public health concern but they are important due to their economic impact.
 OBJECTIVES: The aim of this study is to analyse the results of the serological screening for MVV and CAEV antibodies in samples collected from small ruminants farms located in 14 Romanian counties and the proximity of Bucharest city.
 METHODS: The samples were analysed by indirect ELISA, using a commercial diagnosis kit. We investigated 702 serum samples in 160 iELISA tests: 148 pools (2-5 samples/pool) and 12 individual animals.
 RESULTS: We noticed 27.50% positive samples that confirm the exposure to MVV and CAEV viruses of small ruminants, but the confirmation is requesting investigation using molecular biology tools or other serological techniques.
 CONCLUSION: A reliable picture over the SRLV infections could underlie a national program for monitoring.

A gain-of-function T-DNA insertion mutant of Marchantia polymorpha hyper-accumulates flavonoid riccionidin A.

Marchantia polymorpha is a model liverwort for which many molecular biological techniques are now available. We previously developed the S-AgarTrap method for easy genetic transformation of M. polymorpha using spores. In this study, we report production of a T-DNA insertion mutant library (approx. 10,000 lines) for M. polymorpha using the S-AgarTrap method. We further isolate and characterize a gain-of-function mutant that hyper-accumulates the flavonoid riccionidin A. The present study demonstrates that the S-AgarTrap-mediated production of a T-DNA insertion mutant library is a powerful tool for molecular biology in M. polymorpha.

Advances in Cryopreservation of Bull Sperm.

Cryopreservation of semen and artificial insemination have an important, positive impact on cattle production, and product quality. Through the use of cryopreserved semen and artificial insemination, sperm from the best breeding bulls can be used to inseminate thousands of cows around the world. Although cryopreservation of bull sperm has advanced beyond that of other species, there are still major gaps in the knowledge and technology bases. Post-thaw viability of sperm is still low and differs significantly among the breeding bulls. These weaknesses are important because they are preventing advances both in fundamental science of mammalian gametes and reproductive biotechnology. Various extenders have been developed and supplemented with chemicals to reduce cryodamage or oxidative stress with varying levels of success. More detailed insights on sperm morphology and function have been uncovered through application of advanced tools in modern molecular and cell biology. This article provides a concise review of progress in the cryopreservation of bull sperm, advances in extender development, and frontiers using diverse techniques of the study of sperm viability. This scientific resource is important in animal biotechnology because with the advances in discovery of sperm fertility markers, there is an urgent need to improve post-thaw viability and fertility of sperm through enhanced cryopreservation for precision agriculture to produce food animals to ensure food security on the global scale.

How the 26S Proteasome Degrades Ubiquitinated Proteins in the Cell

The 26S proteasome is the central element of proteostasis regulation in eukaryotic cells, it is required for the degradation of protein factors in multiple cellular pathways and it plays a fundamental role in cell stability. The main aspects of proteasome mediated protein degradation have been highly (but not totally) described during three decades of intense cellular, molecular, structural and chemical biology research and tool development. Contributions accumulated within this time lapse allow researchers today to go beyond classical partial views of the pathway, and start generating almost complete views of how the proteasome acts inside the cell. These views have been recently reinforced by cryo-electron microscopy and mechanistic works that provide from landscapes of proteasomal populations distributed in distinct intracellular contexts, to detailed shots of each step of the process of degradation of a given substrate, of the factors that regulate it, and precise measurements of the speed of degradation. Here, we present an updated digest of the most recent developments that significantly contribute in our understanding of how the 26S proteasome degrades hundreds of ubiquitinated substrates in multiple intracellular environments.

Pattern Analysis of granulomatous inflammatory lesions

Pattern Analysis of granulomatous inflammatory lesions - IJPO- Print ISSN No: - 2394-6784 Online ISSN No:- 2394-6792 Article DOI No:- 10.18231/j.ijpo.2019.069, Indian Journal of Pathology and Oncology-Indian J Pathol Oncol

Liquid Biopsy in Oligometastatic Prostate Cancer-A Biologist's Point of View.

Prostate cancer (PCa) is the main cause of cancer-related mortality in males and the diagnosis, treatment, and care of these patients places a great burden on healthcare systems globally. Clinically, PCa is highly heterogeneous, ranging from indolent tumors to highly aggressive disease. In many cases treatment—generally either radiotherapy (RT) or surgery—can be curative. Several key genetic and demographic factors such as age, family history, genetic susceptibility, and race are associated with a high incidence of PCa. While our understanding of PCa, which is mainly based on the tools of molecular biology—has improved dramatically in recent years, efforts to better understand this complex disease have led to the identification of a new type of PCa–oligometastatic PCa. Oligometastatic disease should be considered an individual, heterogeneous entity with distinct metastatic phenotypes and, consequently, wide prognostic variability. In general, patients with oligometastatic disease typically present less biologically aggressive tumors whose metastatic potential is more limited and which are slow-growing. These patients are good candidates for more aggressive treatment approaches. The main aim of the presented review was to evaluate the utility of liquid biopsy for diagnostic purposes in PCa and for use in monitoring disease progression and treatment response, particularly in patients with oligometastatic PCa. Liquid biopsies offer a rapid, non-invasive approach whose use t is expected to play an important role in routine clinical practice to benefit patients. However, more research is needed to resolve the many existing discrepancies with regard to the definition and isolation method for specific biomarkers, as well as the need to determine the most appropriate markers. Consequently, the current priority in this field is to standardize liquid biopsy-based techniques. This review will help to improve understanding of the biology of PCa, particularly the recently defined condition known as “oligometastatic PCa”. The presented review of the body of evidence suggests that additional research in molecular biology may help to establish novel treatments for oligometastatic PCa. In the near future, the treatment of PCa will require an interdisciplinary approach involving active cooperation among clinicians, physicians, and biologists.

Abstract 330: A HaloTag-TEV Genetic Cassette for Mechanically Probing Native Titin

Single-molecule methods using recombinant proteins have generated transformative hypotheses on how mechanical forces interact with titin in the sarcomere, enabling muscle contraction. However, testing these mechanical hypotheses on native titin in its natural environment has remained inaccessible to conventional genetics, biophysics and molecular biology tools. To overcome these limitations, here we demonstrate a genetically engineered knock-in mouse model carrying a HaloTag-TEV insertion in titin. Using our system, we have specifically severed the titin filament by digestion with TEV protease, and found that the response of muscle fibers to length changes requires mechanical transduction through titin’s intact polypeptide chain. HaloTag-based covalent tethering has enabled directed examination of the dynamics of native titin under physiological forces using recently developed magnetic tweezers. At physiological pulling forces lower than 10 pN, titin domains are readily recruited to the unfolded state, and produce 41.5 zJ mechanical work during refolding. Our results support an active role of titin in muscle contraction in coordination with actomyosin motors.

Cardiac α-Actin (ACTC1) Gene Mutation Causes Atrial-Septal Defects Associated With Late-Onset Dilated Cardiomyopathy.

Familial atrial septal defect (ASD) has previously been attributed primarily to mutations in cardiac transcription factors. Here, we report a large, multi-generational family (78 members) with ASD combined with a late-onset dilated cardiomyopathy and further characterize the consequences of mutant α-actin. We combined a genome-wide linkage analysis with cell biology, microscopy, and molecular biology tools to characterize a novel ACTC1 (cardiac α-actin) mutation identified in association with ASD and late-onset dilated cardiomyopathy in a large, multi-generational family. Using a genome-wide linkage analysis, the ASD disease locus was mapped to chromosome 15q14 harboring the ACTC1 gene. In 15 affected family members, a heterozygous, nonsynonymous, and fully penetrant mutation (p. Gly247Asp) was identified in exon 5 of ACTC1 that was absent in all healthy family members (n=63). In silico tools predicted deleterious consequences of this variant that was found absent in control databases. Ultrastructural analysis of myocardial tissue of one of the mutation carriers showed sarcomeric disarray, myofibrillar degeneration, and increased apoptosis, while cardiac proteomics revealed a significant increase in extracellular matrix proteins. Consistently, structural defects and increased apoptosis were also observed in neonatal rat ventricular cardiomyocytes overexpressing the mutant, but not native human ACTC1. Molecular dynamics studies and additional mechanistic analyses in cardiomyocytes confirmed actin polymerization/turnover defects, thereby affecting contractility. A combined phenotype of ASD and late-onset heart failure was caused by a heterozygous, nonsynonymous ACTC1 mutation. Mechanistically, we found a shared molecular mechanism of defective actin signaling and polymerization in both cardiac development and contractile function. Detection of ACTC1 mutations in patients with ASD may thus have further clinical implications with regard to monitoring for (late-onset) dilated cardiomyopathy.

Artificial Circular RNA Sponges Targeting MicroRNAs as a Novel Tool in Molecular Biology

Artificial Circular RNA Sponges Targeting MicroRNAs as a Novel Tool in Molecular Biology

Community Structures of Bacteria, Archaea, and Eukaryotic Microbes in the Freshwater Glacier Lake Yukidori-Ike in Langhovde, East Antarctica

Since most studies about community structures of microorganisms in Antarctic terrestrial lakes using molecular biological tools are mainly focused on bacteria, limited information is available about archaeal and eukaryotic microbial diversity. In this study, the biodiversity of microorganisms belonging to all three domains in a typical Antarctic freshwater glacier lake (Yukidori-Ike) was revealed using small subunit ribosomal RNA (SSU rRNA) clone library analysis. The bacterial clones were grouped into 102 operational taxonomic units (OTUs) and showed significant biodiversity. Betaproteobacteria were most frequently detected, followed by Cyanobacteria, Bacteroidetes, and Firmicutes as major lineages. In contrast to the bacterial diversity, much lower archaeal diversity, consisting of only two OTUs of methanogens, was observed. In the eukaryotic microbial community consisting of 20 OTUs, Tardigradal DNA was remarkably frequently detected. Genera affiliated with the phyla Ciliophora, Cryptomycota, Chlorophyta, Bacillariophyta, and Apusozoa were also detected. The biodiversity and species compositions of the whole microbial community of Lake Yukidori-Ike are similar to those of freshwater environments in temperate regions but are different from saline lakes in Antarctica, indicating that the salinity seems to affect the microbial composition more than the temperature.

Characterizing the genetic diversity of Atlas cedar and phylogeny of Mediterranean Cedrus species with a new multiplex of 16 SSR markers

Cedar is an emblematic Mediterranean forest tree. Genetic research activities on Mediterranean Cedrus species are progressively developed in relation to conservation, restoration, and exploitation plans of these forest tree species. However, currently available molecular biology tools supporting genetic research in these fields are still scarce and have a limited genetic resolution potential. We developed a new set of 12 nuclear microsatellite markers (nSSRs) on Cedrus atlantica that we combined with four previously developed ones in three multiplexes. We checked their monogenic inheritance in controlled crosses. We used a collection of 131 samples from six populations of C. atlantica to estimate null allele frequencies and probability of identity and to characterize the structure of genetic diversity in the fragmented distribution range of this species. We also tested the transferability of the markers to another set of 36 samples from the other Mediterranean Cedrus species and performed a phylogenetic analysis. The three multiplexes reached a high level of resolution potential that we used to evaluate sampling quality. Null allele frequency estimates showed no specific pattern across populations or across species and did not affect the results of biogeographic and phylogenic analyses. Our results reveal a very clear geographical genetic structure within C. atlantica, and the phylogenic tree matched previous analyses based on other markers. Our results confirm the potential interest of these nSSR multiplexes for genotyping in Cedrus.

An Efficient Approach for Two Distal Point Site-Directed Mutagenesis from Randomly Ligated PCR Products.

Site-directed mutagenesis is one of the most important tools in molecular biology. The majority of the mutagenesis methods have been developed to mutate one region of target DNA in each cycle of mutagenesis, while in some cases there is a need to mutate several distal points. We used a new method to simultaneously mutate two distal points in the target DNA. Different regions of the target DNA were amplified in three separate PCR reactions. The PCR products were back-to-back and together they made the complete length of the template DNA. Mutations were introduced to PCR products by middle mutagenic primers. PCR products were mixed and ligated with random blunt ligation, and then the desired mutated DNA fragments were selected in two steps by flanking restriction enzyme digestion and size selection. Selected fragments were amplified in another PCR reaction using flanking primers and finally cloned into the plasmid vector. This mutagenesis process is simple, there is no need to use modified primers and long or difficult PCR reactions.

Abstract 3048: Targeting protein deglycosylation as a newly identified vulnerable point in melanoma

Abstract As an enzyme that removes N-glycans from glycopeptides, N-glycanase 1 (NGLY1) deglycosylates denatured glycoproteins and allows proteasome-mediated protein degradation to efficiently occur. Although NGLY1 is known for this pivotal function, information regarding the responses of human cancer and normal cells to NGLY1 suppression is limited. Here, we examined how NGLY1 expression affects viability, tumor growth, and responses to therapeutic agents in melanoma cells and an animal model. Molecular mechanisms contributing to NGLY1 suppression-induced anticancer responses were revealed by systems biology and chemical biology studies. Using computational and medicinal chemistry-assisted approaches, we established novel NGLY1-inhibitory small molecules. Compared with normal cells, NGLY1 was upregulated in melanoma cell lines and patient tumors. NGLY1 knockdown caused melanoma cell death and tumor growth retardation. Targeting NGLY1 induced pleiotropic responses, predominantly stress signaling-associated apoptosis and cytokine surges, which synergize with the anti-melanoma activity of chemotherapy and targeted therapy agents. Pharmacological and molecular biology tools that inactivate NGLY1 elicited highly similar responses in melanoma cells. Unlike normal cells, melanoma cells presented distinct responses and high vulnerability to NGLY1 suppression. Our work demonstrated the significance of NGLY1 in melanoma cells, provided mechanistic insights into how NGLY1 inactivation leads to eradication of melanoma with limited impact on normal cells, and suggested that targeting NGLY1 represents a novel anti-melanoma strategy with the opportunity for a broad therapeutic window. Citation Format: Victor J.T. Lin, Ashwini Zolekar, Nigam M. Mishra, Yin Ying Ho, Hamed S. Hayatshahi, Abhishek Parab, Rohit Sampat, Xiaoyan Liao, Peter Hoffmann, Jin Liu, Kyle A. Emmitte, Yu-Chieh Wang. Targeting protein deglycosylation as a newly identified vulnerable point in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3048.

MicroRNAs meet with quantitative trait loci: Small powerful players in regulating quantitative yield traits in rice.

MicroRNAs (miRNAs) are small noncoding RNAs which regulate various functions related to growth, development, and stress responses in plants and animals. Rice, Oryza sativa, is one of the most important food crops of the world. In rice, a number of quantitative trait loci (QTL) controlling yield-related traits have been identified. Some of them are actually controlled by miRNAs, which control various yield-related quantitative traits in rice. On one hand, many of these miRNAs are found to regulate more than one yield-related traits, such as tillering, grain size, and branch number of a panicle. On the other hand, a rice yield-related trait is usually controlled by multiple miRNAs, for example, grain size being controlled by miR156, miR167, miR396, miR397, and miR1432. In rare case, a single miRNA may specifically regulate only one yield-related trait, such as, miR444 regulating rice tillering. In this review, we focus on the functions of miRNAs in controlling yield-related quantitative traits in rice, including panicle grain number, grain weight/size, panicle length and branching, tiller number per plant, spikelet number, seed setting rate, and leaf inclination, and discuss how to modulate the expression of these miRNAs using modern molecular biology tools to promote grain yield. This article is categorized under: RNA in Disease and Development > RNA in Development.