First conjugation directed traverse of gene cassettes harboring α1,3GT from fast-growing Mycobacterium smegmatis mc2 155 to slow-growing pathogen Mycobacterium tuberculosis H37Rv, presumably opening up new scopes in tuberculosis treatment.

Mycobacterium smegmatis strain mc2 155 is a fast-growing and non-pathogenic mycobacteria and widely used in genetic studies of mycobacteria. It has been shown that this species of mycobacterium can transfer its genomic DNA fragments to other species of mycobacteria during the conjugation process. Galα1-3Galß1-4GlcNAc-R (α-gal) glycan epitope is a highly immunogenic epitope exerted by the enzyme α1-3-galactosyltransferase (α1,3GT) in mammalian cells on the glycan skeleton. However, the enzyme is inactive in humans, primates and Old World monkeys as a result of evolutionary mutations. The robust immunogenicity induced by the epitope in human, has attracted much attention to apply the epitope in vaccine research. In this study we proved successful transfer of desired expression cassettes from fast-growing Mycobacterium smegmatis mc2 155, to the slow-growing pathogen Mycobacterium tuberculosis H37Rv. We designed gene cassettes encoding the α1,3GT enzyme under control the potent G13 promoter and the cassette containing hygromycin resistance gene under a Mtb specific promoter, Ptpa in the vector pMV306DIG13 +FflucRT (harboring attP site). The resulting construct was electroporated into mc2 155 strain in combination with pBS-int containing the gene encoding Mycobacteriophage L5 integrase to integrate pMV306DIG13 +FflucRT-cassettes into mc2 155 genome. Following the integration, the recombinant clones were placed in vicinity to the Mycobacterium tuberculosis H37Rv strain to establish conjugation. Conjugated recombinant clones were selected on the medium containing the hygromycin B and transfer of the desired cassettes to Mycobacterium tuberculosis was confirmed. The enzyme α1,3GT in transconjugants were also investigated.

Endogenous stimuli-responsive linkers in nanoliposomal systems for cancer drug targeting.

There are various drug delivery systems (DDSs) among which nanoliposomal formulations are among the most prominent. Despite the superiority of nanoliposomal DDSs compared to conventional drug delivery methods, recent reports have claimed that they can deliver small amounts of the injected dose to target site by passive targeting. However, our understanding of tumor microenvironment features, including dysregulation of pH, the high intracellular concentration of glutathione, change in the amount and expression of some enzymes, reactive oxygen species, hypoxia, and ATP concentrations, has driven the scope of research into the use of these endogenous stimuli for a design of smart linkers. These linkers optimize the release of payloads in favorable target sites and avoid premature releasing in non-favorable off-target sites. In this review, we discuss particular linkers, which are able to respond to the specific endogenous conditions, and could be used in nanoliposomal DDSs, based on pathophysiological changes that occur in tumors. Furthermore, structural and chemical properties of these linkers and other potential linkers, which could be used in nanoliposomal DDSs, have been reviewed.

Electroanalysis of isoniazid and rifampicin: Role of nanomaterial electrode modifiers.

Thanks to operational simplicity, speediness, possibility of miniaturization and real-time nature, electrochemical sensing is a supreme alternative for non-electrochemical methodologies in drug quantification. This review, highlights different nanotech-based sensory designs for electroanalysis of isoniazid and rifampicin, the most important medicines for patients with tuberculosis. We first, concisely mention analyses with bare electrodes, associated impediments and inspected possible strategies and then critically review the last two decades works with focus on different nano-scaled electrode modifiers. We organized and described the materials engaged in several categories: Surfactants modifiers, polymeric modifiers, metallic nanomaterials, carbon based nano-modifiers (reduced graphene oxide, multi-walled carbon nanotubes, ordered mesoporous carbon) and a large class of multifarious nano composites-based sensors and biosensors. The main drawbacks and superiorities associated with each array as well as the current trend in the areas is attempted to discuss. Summary of 79 employed electrochemical approaches for analysis of isoniazid and rifampicin has also been presented.

Electrochemical-based biosensors for detection of Mycobacterium tuberculosis and tuberculosis biomarkers.

Early detection of tuberculosis (TB) reduces the interval between infection and the beginning of treatment. However, commercially available tests cannot discriminate between BCG-vaccinated healthy persons and patients. Also, they are not suitable to be used for immunocompromised persons. In recent years, biosensors have attracted great attention due to their simple utility, accessibility, and real-time outputs. These sensors are increasingly being considered as pioneering tools for point-of-care diagnostics in communities with a high burden of TB and limited accessibility to reference laboratories. Among other types of biosensors, the electrochemical sensors have the advantages of low-cost operation, fast processing, simultaneous multi-analyte analyzing, operating with turbid samples, comparable sensitivity and readily available miniaturization. Electrochemical biosensors are sub-divided into several categories including: amperometric, impedimetric, potentiometric, and conductometric biosensors. The biorecognition element in electrochemical biosensors is usually based on antibodies (immunosensors), DNAs or PNAs (genosensors), and aptamers (aptasensors). In either case, whether an interaction of the antigen-antibody/aptamer or the hybridization of probe with target mycobacterial DNA is detected, a change in the electrical current occurs that is recorded and displayed as a plot. Therefore, impedimetric-based methods evaluate resistance to electron transfer toward an electrode by a Nyquist plot and amperometric/voltammetric-based methods weigh the electrical current by means of cyclic voltammetry, square wave voltammetry, and differential pulse voltammetry. Electrochemical biosensors provide a promising scope for the new era of diagnostics. As a consequence, they can improve detection of Mycobacterium tuberculosis traces even in attomolar scales.

Shedding light on the EpCAM: An overview.

The epithelial cell adhesion molecule (EpCAM) is a Type I transmembrane superficial glycoprotein antigen that is expressed on the surface of basolateral membrane of multiple epithelial cells with some exceptions such as epidermal keratinocytes, hepatocytes, thymic cortical epithelial cells, squamous stratified epithelial cells, and myoepithelial cells that do not express the molecule. The molecule plays a pivotal role in the structural integrity, adhesion of the epithelial tissues and their interaction with the underlying layers. EpCAM prevents claudin-7 and claudin-1 molecules from degradation, thereby, decreasing the number of tight junctions and cellular interconnections, and promoting the cells toward carcinogenic transformation. Moreover, the mutations in the EpCAM gene lead to congenital tufting enteropathy, severe intestinal epithelium homeostasis disorders, and Lynch and Lynch syndrome. Overexpression of EpCAM on stem cells of some cancers and the presence of this molecule on circulating tumor cells (CTCs) makes it a promising candidate for cancer diagnosis as well as tracing and isolation of CTCs.

Nano-biosensing approaches on tuberculosis: Defy of aptamers.

Tuberculosis is a major global health problem caused by the bacterium Mycobacterium tuberculosis (Mtb) complex. According to WHO reports, 53 million TB patients died from 2000 to 2016. Therefore, early diagnosis of the disease is of great importance for global health care programs. The restrictions of traditional methods have encouraged the development of innovative methods for rapid, reliable, and cost-effective diagnosis of tuberculosis. In recent years, aptamer-based biosensors or aptasensors have drawn great attention to sensitive and accessible detection of tuberculosis. Aptamers are small short single-stranded molecules of DNA or RNA that fold to a unique form and bind to targets. Once combined with nanomaterials, nano-scale aptasensors provide powerful analytical platforms for diagnosing of tuberculosis. Various groups designed and studied aptamers specific for the whole cells of M. tuberculosis, mycobacterial proteins and IFN-γ for early diagnosis of TB. Advantages such as high specificity and strong affinity, potential for binding to a larger variety of targets, increased stability, lower costs of synthesis and storage requirements, and lower probability of contamination make aptasensors pivotal alternatives for future TB diagnostics. In recent years, the concept of SOMAmer has opened new horizons in high precision detection of tuberculosis biomarkers. This review article provides a description of the research progresses of aptamer-based and SOMAmer-based biosensors and nanobiosensors for the detection of tuberculosis.

Label-free nano-biosensing on the road to tuberculosis detection.

Tuberculosis, an ailment caused by the bacterium Mycobacterium tuberculosis (Mtb) complex, is one of the catastrophic transmittable diseases that affect human. Reports published by WHO indicate that in 2017 about 6.3 million people progressed to TB and 53 million TB patients died from 2000 to 2016. Therefore, early diagnosis of the disease is of great importance for global health care programs. Common diagnostics like the traditional PPD test and antibody-assisted assays suffer the lack of sensitivity, long processing time and cumbersome post-test proceedings. These shortcomings restrict their use and encourage innovations in TB diagnostics. In recent years, the biosensor concept opened up new horizons in sensitive and fast detection of the disease, reducing the interval time between sampling and diagnostic result. Among new diagnostics, label-free nano-biosensors are highly promising for sensitive and accessible detection of tuberculosis. Various specific label-free nano-biosensors have been recently reported detecting the whole cell of M. tuberculosis, mycobacterial proteins and IFN-γ as crucial markers in early diagnosis of TB. This article provides a focused overview on nanomaterial-based label-free biosensors for tuberculosis detection.

Recombinant Protein Expression in Escherichia coli (E.coli): What We Need to Know.

BACKGROUND: Host, vector, and culture conditions (including cultivation media) are considered among the three main elements contributing to a successful production of recombinant proteins. Accordingly, one of the most common hosts to produce recombinant therapeutic proteins is Escherichia coli. METHODOLOGY: A comprehensive literature review was performed to identify important factors affecting production of recombinant proteins in Escherichia coli. RESULTS: Escherichia coli is taken into account as the easiest, quickest, and cheapest host with a fully known genome. Thus, numerous modifications have been carried out on Escherichia coli to optimize it as a good candidate for protein expression and; as a result, several engineered strains of Escherichia coli have been designed. In general; host strain, vector, and cultivation parameters are recognized as crucial ones determining success of recombinant protein expression in Escherichia coli. In this review, the role of host, vector, and culture conditions along with current pros and cons of different types of these factors leading to success of recombinant protein expression in Escherichia coli were discussed. CONCLUSION: Successful protein expression in Escherichia coli necessitates a broad knowledge about physicochemical properties of recombinant proteins, selection among common strains of Escherichia coli and vectors, as well as factors related to media including time, temperature, and inducer.