A review of electrophoretic separations in temperature-responsive Pluronic thermal gels.

Gel electrophoresis is a ubiquitous bioanalytical technique used in research laboratories to validate protein and nucleic acid samples. Polyacrylamide and agarose have been the gold standard gel materials for decades, but an alternative class of polymer has emerged with potentially superior performance. Pluronic thermal gels are water-soluble polymers that possess the unique ability to undergo a change in viscosity in response to changing temperature. Thermal gels can reversibly convert between low-viscosity liquids and high-viscosity solid gels using temperature as an adjustable parameter. The properties of thermal gels provide unmatched flexibility as a dynamic separations matrix to measure analytes ranging from small molecules to cells. This review article describes the physical and chemical properties of Pluronic thermal gels to provide a fundamental overview of polymer behavior. The performance of thermal gels is then reviewed to highlight their applications as a gel matrix for electrokinetic separations in capillary, microfluidic, and slab gel formats. The use of dynamic temperature-responsive gels in bioanalytical separations is an underexplored area of research but one that holds exciting potential to achieve performance unattainable with conventional static polymers.

Controlling the separation of native proteins with temperature in thermal gel transient isotachophoresis.

Polyacrylamide gel electrophoresis (PAGE) is a ubiquitous technique used in biochemical research laboratories to characterize protein samples. Despite its popularity, PAGE is relatively slow and provides limited separation resolution, especially for native proteins. This report describes the development of a microfluidic thermal gel transient isotachophoresis (TG-tITP) method to rapidly separate native proteins with high resolution. Thermal gels were employed as a separations matrix because of their unique ability to change viscosity in response to temperature. Proteins were added into thermal gel and loaded into a microfluidic device. Electrolyte optimization was conducted to achieve robust tITP to isotachophoretically preconcentrate proteins and then electrophoretically separate them. Electropherograms were collected through both time and distance to enable both small and large proteins to be measured within a single analysis. The effects of temperature were evaluated and found to exhibit a pronounced effect on the separation. Temperature gradients were then employed to alter thermal gel viscosity over time to maximize separation resolution between proteins. The results herein demonstrate how gradient TG-tITP achieves rapid, high-performance separations of native proteins. This analysis provided a wide mass range (6-464 kDa) with two-fold higher resolution than native PAGE while requiring 15,000-fold less protein loading and providing five-fold faster analysis times.

Simultaneous Preconcentration and Separation of Native Protein Variants Using Thermal Gel Electrophoresis.

Proteins must maintain proper folding conformations and express the correct post-translational modifications (PTMs) to exhibit appropriate biological activity. However, assessing protein folding and PTMs is difficult because routine polyacrylamide gel electrophoresis (PAGE) methods lack the separation resolution necessary to identify variants of a single protein. Additionally, standard PAGE denatures proteins prior to analysis precluding determinations of folding states or PTMs. To overcome these limitations, a microfluidic thermal gel electrophoresis platform was developed to provide high-sensitivity, high-resolution analyses of native protein variants. A thermally reversible gel was utilized as a separation matrix while in its solid state (30 °C). This thermal gel provided sufficient separation resolution to identify three variants of a fluorescently labeled model protein. To increase detection sensitivity, analyte preconcentration was conducted in parallel with the separation. Continuous analyte enrichment afforded detection limits of 500 fg of protein (250 pM) while simultaneous baseline separation resolution was achieved between variants. The effects of temperature on thermal gel electrophoresis were also characterized. The unique temperature-dependent outcomes illustrated how method performance can be tuned through a thermal dimension. Ultimately, the high detection sensitivity and separation resolution provided by thermal gel electrophoresis enabled rapid screening of native protein variants.

Integration of biometric ID for the effective collection and epidemiological evaluation of antibiotic prescription in tuberculosis and other diseases: A medical hypothesis.

OBJECTIVE: Tuberculosis (TB) poses a huge socioeconomic burden and remains listed among the top ten causes of deaths across the globe, despite enormous efforts of health agencies and researchers. The primary concern in a relatively slower pace of curbing TB is the emergence of multi-drug resistance and difficulty in tracking antibiotic consumption. METHODS: We propose here a biometric-linked digital means of antibiotic prescription data collection and its utility for various epidemiological assessments. RESULTS: In this short communication, we propose here, a visionary workflow to explore the use of biometric identification system to gather the antibiotic prescription information for tuberculosis cases and real-time monitoring over a global scale. Our proposal is mainly aimed at collection of antibiotic prescription data at clinician level and point of sales level using a simple fingerprint-based biometric approach and its linkage to unique biometric IDs such as Aadhar in India and related systems in other countries. CONCLUSION: The online server and web-interface are aimed at facilitating the health organizations and medical councils to monitor the antibiotic prescription and their consumption in real time. This will not only augment the existing policy-making regarding drug-resistance programs but also promote the collection and downstream exploratory analysis of huge amount of data to get a deeper insight into the epidemiology of tuberculosis and related diseases.

Comparative Analysis of the Molecular Adjuvants and Their Binding Efficiency with CR1.

There are so many obstacles in developing a vaccine or vaccine technology for diseases like cancer and human immunodeficiency virus infection. While developing vaccines that target specific infection, molecular adjuvants are indispensable. These molecular adjuvants act as a vaccine delivery vehicle to the immune system to increase the effectiveness of the specific antigens. In the present work, a computational study has been done on molecular adjuvants like IgGFc, GMCSF and C3d to find out how efficiently they are binding to CR1. Sequence, structure and mutational analysis are performed on the molecular adjuvants to understand the features important for their binding with the receptor. Results obtained from our study indicate that the adjuvant IgGFc complexed with the receptor CR1 has the best binding efficiency, which can be used further to develop better vaccine technologies.

Comparative analysis of the molecular adjuvants and their binding efficiency with CR1.

There are so many obstacles in developing a vaccine or vaccine technology for diseases like Cancer and Human Immunodeficiency Virus (HIV) infection. While developing vaccines that targets specific infection, molecular adjuvants are indispensable. These molecular adjuvants act as a vaccine delivery vehicle to the immune system to increase the effectiveness of the specific antigens. In the present work, a computational study has been done on molecular adjuvants like IgGFc, GMCSF and C3d to find out how efficiently they are binding to CR1. Sequence, structure and mutational analysis are performed on the molecular adjuvants to understand the features important for their binding with the receptor. Results obtained from our study indicate that the adjuvant IgGFc complexed with the receptor CR1 has the best binding efficiency, which can be used further to develop better vaccine technologies.

Development of a VIGS vector based on the ß-satellite DNA associated with bhendi yellow vein mosaic virus.

Bhendi yellow vein mosaic virus (BYMV) is a monopartite begomovirus with an associated ß-satellite. ßC1 ORF encoded by the ß-satellite is the symptom determinant and a strong suppressor of post transcriptional gene silencing. To create a virus induced gene silencing vector based upon the ß-satellite associated with BYVMV the ßC1 ORF was replaced with multiple cloning sites. GFP transgene and plant endogenous genes Su, PDS, PCNA and AGO1 were cloned into ß-satellite based VIGS vector. GFP expression was silenced in the GFP expressing transgenic 16c Nicotiana benthamiana plants infiltrated with VIGS vector carrying GFP gene inside. N. benthamiana plants infiltrated with the VIGS vector harboring the endogenous genes Su, PDS, PCNA and AGO1 produced the phenotypic symptoms yellowing of the veins, photobleaching of the veins, stunting of the plant and upward leaf curling, respectively. Real time PCR analyses revealed a reduction in the levels of the corresponding transgene or endogenous target mRNA. The ß-satellite based VIGS vector was able to silence the target genes effectively. Hence, BYVMV ß-satellite based VIGS vector can be used in functional genomics studies.

Expression of mycobacterial cell division protein, FtsZ, and dormancy proteins, DevR and Acr, within lung granulomas throughout guinea pig infection.

The ability of Mycobacterium tuberculosis to persist in a dormant state is a hallmark of tuberculosis. An insight into the expression of mycobacterial proteins will contribute to our understanding of bacterial physiology in vivo. To this end, the expression of FtsZ, Acr and DevR was assessed in the lung granulomas of guinea pigs infected with M. tuberculosis. Antigen immunostaining was then compared with the detection of acid-fast bacilli (AFB) and mycobacterial DNA. Surprisingly, immunostaining for all three antigens was observed throughout the course of infection; maximum expression of all antigens was noted at 20 weeks of infection. The intensity of immunostaining correlated well with the presence of intact bacteria, suggesting that mycobacterial antigens in the extracellular fraction have a short half-life; in contrast to protein, extracellular bacterial DNA was found to be more stable. Immunostaining for bacterial division and dormancy markers could not clearly distinguish between replicating and non-replicating organisms during the course of infection. The detection of Acr and DevR from 4 weeks onwards indicates that the dormancy proteins are expressed from early on in infection. Both antigen staining and DNA detection from intact bacilli were useful for detecting intact mycobacteria in the absence of AFB.

Elicitation of efficient, protective immune responses by using DNA vaccines against tuberculosis.

DNA vaccination is an effective method for elicitation of strong humoral as well as cellular immune responses. DNA vaccines expressing mycobacterial antigens ESAT-6 (Rv3875), alpha-crystallin (Rv2031c) and superoxide dismutase A (Rv3846) were evaluated for their immune responses in Balb/c mice and protective efficacy in guinea pigs. Immunization of mice with the DNA vaccines expressing superoxide dismutase A and alpha-crystallin resulted in markedly higher levels of IFN-gamma as compared to the levels of IL-10. The DNA vaccine expressing ESAT-6 elicited a mixed Th1/Th2 response. Immunization of guinea pigs with these DNA vaccines and subsequent challenge of animals with Mycobacterium tuberculosis H(37)Rv, showed that DNA vaccine expressing superoxide dismutase imparted the maximum protection as observed by a 50 and 10 folds reduction in bacillary load in spleens and lungs, respectively, in comparison to immunization with vector control.

Increased expression of Mycobacterium tuberculosis 19 kDa lipoprotein obliterates the protective efficacy of BCG by polarizing host immune responses to the Th2 subtype.

Mycobacterium tuberculosis can not only neutralize immune effector functions, but also has the ability to modulate host-signalling cascades involved in the development of these responses. The 19 kDa antigen (Rv3763), a lipoprotein of M. tuberculosis, elicits high levels of interleukin (IL)-12 from macrophages in addition to its powerful immunomodulatory properties, leading to suppression of antigen-presentation signalling cascades. The present study was aimed at analysing the effect of overexpression of this antigen on the immunostimulatory properties of M. bovis Bacille Calmette-Guerin (BCG). We have constructed a recombinant BCG strain (rBCG19N) producing higher levels of the 19 kDa antigen in both the cytoplasmic (approximately eightfold) and extracellular (approximately fivefold) fractions as compared to the wildtype BCG. Immunization of mice with rBCG19N elicited high levels of interferon-gamma (IFN-gamma) and relatively low levels of IL-10 against the purified 19 kDa antigen. However, in response to total BCG sonicate, mice immunized with rBCG19N produced significantly high levels of IL-10 with relatively very low levels of IFN-gamma. This polarization of the host immune responses towards T-helper 2 subtype resulted in complete abrogation of the protective efficacy of BCG, when rBCG19N was used as a live vaccine against M. tuberculosis challenge in guinea pigs.

Disruption of response regulator gene, devR, leads to attenuation in virulence of Mycobacterium tuberculosis.

The devR-devS two-component system of Mycobacterium tuberculosis was identified earlier and partially characterized in our laboratory. A devR::kan mutant of M. tuberculosis was constructed by allelic exchange. The devR mutant strain showed reduced cell-to-cell adherence in comparison to the parental strain in laboratory culture media. This phenotype was reversed on complementation with a wild-type copy of devR. The devR mutant and parental strains grew at equivalent rates within human monocytes either in the absence or in the presence of lymphocytic cells. The expression of DevR was not modulated upon entry of M. tuberculosis into human monocytes. However, guinea pigs infected with the mutant strain showed a significant decrease in gross lesions in lung, liver and spleen; only mild pathological changes in liver and lung; and a nearly 3 log lower bacterial burden in spleen compared to guinea pigs infected with the parental strain. Our results suggest that DevR is required for virulence in guinea pigs but is not essential for entry, survival and multiplication of M. tuberculosis within human monocytes in vitro. The attenuation in virulence of the devR mutant in guinea pigs together with DevR-DevS being a bona fide signal transduction system indicates that DevR plays a critical and regulatory role in the adaptation and survival of M. tuberculosis within tissues.

Disruption of mptpB impairs the ability of Mycobacterium tuberculosis to survive in guinea pigs.

Protein tyrosine kinases and tyrosine phosphatases from several bacterial pathogens have been shown to act as virulence factors by modulating the phosphorylation and dephosphorylation of host proteins. The identification and characterization of two tyrosine phosphatases namely MptpA and MptpB from Mycobacterium tuberculosis has been reported earlier. MptpB is secreted by M. tuberculosis into extracellular mileu and exhibits a pH optimum of 5.6, similar to the pH of the lysosomal compartment of the cell. To determine the role of MptpB in the pathogenesis of M. tuberculosis, we constructed a mptpB mutant strain by homologous recombination and compared the ability of parent and the mutant strain to survive intracellularly. We show that disruption of the mptpB gene impairs the ability of the mutant strain to survive in activated macrophages and guinea pigs but not in resting macrophages suggesting the importance of its role in the host-pathogen interaction. Infection of guinea pigs with the mutant strain resulted in a 70-fold reduction in the bacillary load of spleens in infected animals as compared with the bacillary load in animals infected with the parental strain. Upon reintroduction of the mptpB gene into the mutant strain, the complemented strain was able to establish infection and survive in guinea pigs at rates comparable to the parental strain. These observations demonstrate a role of MptpB in the pathogenesis of M. tuberculosis.

Biochemical and histochemical changes pertaining to active and healed cutaneous tuberculosis.

BACKGROUND: Fibrosis is one of the major causes of post-treatment morbidity in tuberculosis. The molecular basis of fibrosis in active and healed tuberculous lesions is yet to be fully characterized. OBJECTIVES: To measure the tissue levels of collagen, elastin, fibronectin, transforming growth factor-beta (TGF-beta) and zinc in active and healed lesions of cutaneous tuberculosis. PATIENTS AND METHODS: Biopsy lesions obtained from 17 patients with active cutaneous tuberculosis and 12 patients after a 6-month regimen of antituberculous chemotherapy were examined. Collagen, elastin and zinc were estimated biochemically. In addition, the presence of collagen IV, TGF-beta and fibronectin were determined immunohistochemically. RESULTS: It was found that collagen, elastin, fibronectin and TGF-beta levels were higher in the active lesions. The levels of zinc were similar in both active and healed lesions. Clinically, scar tissue or keloid formation was not seen in any of the healed lesions. CONCLUSIONS: Effective antituberculous chemotherapy will lead to a substantial reduction of fibrosis and the consequent disability that can arise in patients with tuberculosis.

The clearance of tubercle bacilli & mycobacterial antigen vis a vis the granuloma in different organs of guinea pigs.

In the present study, an attempt was made to define the relationship of intact tubercle bacilli and/or their antigenic fragments to a granuloma in the guinea pig in order to distinguish an active from a resolving granuloma. In one set of animals, granuloma was induced in the skin by injecting heat-killed Mycobacterium tuberculosis intradermally and in another set, granuloma was produced in the lung and spleen by injecting live M. tuberculosis intramuscularly. The animals were sacrificed at various time points and skin, lung and spleen from the two groups were subjected to histological examination for the presence of granuloma, bacilli and antigenic fragments. In the dermal lesion, intact acid fast bacilli were cleared first by day 42 followed by the removal of their antigenic fragments by day 63 and finally by day 84, the granuloma had resolved completely. In the guinea pigs infected with live M. tuberculosis, removal of the bacilli followed by the clearance of antigen was observed. Though the granuloma itself did not subside completely in these animals, it was found that there was a reduction in congestion and oedema of the granulomatous area. It is concluded from the results that the demonstration of antigen at the site of lesion may be potentially useful to discriminate between a persisting and a resolving tuberculous granuloma.