The World Health Organization (WHO) estimates that 40% of tuberculosis (TB) cases are not diagnosed and treated correctly. Even though there are several diagnostic tests available in the market, ...rapid, easy, inexpensive detection, and drug susceptibility testing (DST) of
is still of critical importance specially in low and middle-income countries with high incidence of the disease. In this work, we have developed a microscopy-based methodology using the reporter mycobacteriophage
ϕ for detection of
and phenotypic determination of rifampicin resistance within just days from sputum sample collection. Fluoromycobacteriophage methodology is compatible with regularly used protocols in clinical laboratories for TB diagnosis and paraformaldehyde fixation after infection reduces biohazard risks with sample analysis by fluorescence microscopy. We have also set up conditions for discrimination between
complex (MTBC) and non-tuberculous mycobacteria (NTM) strains by addition of
-nitrobenzoic acid (PNB) during the assay. Using clinical isolates of pre-XDR and XDR-TB strains from this study, we tested
Φ for extended DST and we compared the antibiotic resistance profile with those predicted by whole genome sequencing. Our results emphasize the utility of a phenotypic test for
extended DST. The many attributes of
Φ suggests this could be a useful component of clinical microbiological laboratories for TB diagnosis and since only viable cells are detected this could be a useful tool for monitoring patient response to treatment.
Computational modeling can provide a mechanistic and quantitative framework for describing intracellular spatial heterogeneity of solutes such as oxygen partial pressure (pO2). This study develops ...and evaluates a finite-element model of oxygen-consuming mitochondrial bioenergetics using the COMSOL Multiphysics program. The model derives steady-state oxygen (O2) distributions from Fickian diffusion and Michaelis–Menten consumption kinetics in the mitochondria and cytoplasm. Intrinsic model parameters such as diffusivity and maximum consumption rate were estimated from previously published values for isolated and intact mitochondria. The model was compared with experimental data collected for the intracellular and mitochondrial pO2 levels in human cervical cancer cells (HeLa) in different respiratory states and under different levels of imposed pO2. Experimental pO2 gradients were measured using lifetime imaging of a Förster resonance energy transfer (FRET)-based O2 sensor, Myoglobin-mCherry, which offers in situ real-time and noninvasive measurements of subcellular pO2 in living cells. On the basis of these results, the model qualitatively predicted (1) the integrated experimental data from mitochondria under diverse experimental conditions, and (2) the impact of changes in one or more mitochondrial processes on overall bioenergetics.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Calmodulin kinase-like vesicle-associated (CaMKv), a pseudokinase belonging to the Ca
2+
/calmodulin-dependent kinase family, is expressed predominantly in brain and neural tissue. It may function in ...synaptic strengthening during spatial learning by promoting the stabilization and enrichment of dendritic spines. At present, almost nothing is known regarding CaMKv structure and regulation. In this study we confirm prior proteomic analyses demonstrating that CaMKv is palmitoylated on Cys5. Wild-type CaMKv is enriched on the plasma membrane, but this enrichment is lost upon mutation of Cys5 to Ser. We further show that CaMKv interacts with another regulator of synaptic plasticity, Arc/Arg3.1, and that the interaction between these two proteins is weakened by mutation of the palmitoylated cysteine in CamKv.
For better understanding the role of dynamic factors in the DNA functioning, it is important to study the internal mobility of DNA and, in particular, the movement of nonlinear conformational ...distortions -kinks along the DNA chains. In this work, we study the behavior of the kinks in the pPF1 plasmid containing two genes of fluorescent proteins (EGFP and mCherry). To simulate the movement, two coupled nonlinear sine-Gordon equations that describe the angular oscillations of nitrogenous bases in the main and complementary chains and take into account the effects of dissipation and the action of a constant torsion field. To solve the equations, approximate methods such as the quasi-homogeneous approximation, the mean field method, and the block method, were used. The obtained solutions indicate that two types of kinks moving along the double strand can be formed in any part of the plasmid. The profiles of the potential fields in which these kinks are moving are calculated. The results of the calculations show that the lowest energy required for the kink formation, corresponds to the region located between the genes of green and red proteins (EGFP and mCherry). It is shown that it is in this region a pit trap is located for both kinks. Trajectories of the kinks in the pit-trap and nearby are constructed. It is shown that there are threshold values of the torsion field, upon reaching which the kinks behavior changes dramatically: there is a transition from cyclic motion inside the pit-trap to translational motion and exit from the potential pit-trap.
Proteins from food-grade expression systems can be used in food products and medical applications. Herein, we describe a one-step method of constructing an expression vector in Kluyveromyces lactis ...by combining a URA3-deficient strain and a plasmid vector with no drug-resistant selection. Adjacent DNA elements of the vector were assembled in a targeted manner through a reaction with a special recombinase to form a plasmid vector using a one-step reaction. The unnecessary fragments containing the pUC origin and the ampicillin resistance gene were removed, and the vector was isolated and purified before transformation. A single transformation of the vector can produce a URA3-deficient strain. PCR assay, sequencing, and western blot analysis all indicated that the method of vector construction and target protein expression (mCherry and human serum albumin) were successful. This method may potentially be applied to any species containing the URA3 gene; this system has the potential to become a safe and powerful tool for promoting protein expression in food-safe species.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The insulin/insulin-like growth factor-like signaling (IIS) pathway is highly conserved across metazoans and regulates numerous physiological functions, including development, metabolism, fecundity, ...and lifespan. The insulin receptor (InR), a crucial membrane receptor in the IIS pathway, is known to be ubiquitously expressed in various tissues, albeit at generally low levels, and its subcellular localization remains incompletely characterized. In this study, we employed CRISPR-mediated mutagenesis in the fruit fly
to create knock-in alleles of InR tagged with fluorescent proteins (InR::mCherry or InR::EYFP). By inserting the coding sequence of the fluorescent proteins mCherry or EYFP near the end of the coding sequence of the endogenous InR gene, we could trace the natural InR protein through their fluorescence. As an example, we investigated epithelial cells of the male accessory gland (AG), an internal reproductive organ, and identified two distinct patterns of InR::mCherry localization. In young AG, InR::mCherry accumulated on the basal plasma membrane between cells, whereas in mature AG, it exhibited intracellular localization as multiple puncta, indicating endocytic recycling of InR during cell growth. In the AG senescence accelerated by the mutation of Diuretic hormone 31 (Dh31), the presence of InR::mCherry puncta was more pronounced compared to the wild type. These findings raise expectations for the utility of the newly created InR::mCherry/EYFP alleles for studying the precise expression levels and subcellular localization of InR. Furthermore, this fluorescently tagged allele approach can be extended to investigate other membrane receptors with low abundance, facilitating the direct examination of their true expression and localization.
Abstract Like most animal viruses, studying influenza A in model systems requires secondary methodologies to identify infected cells. To circumvent this requirement, we describe the generation of ...replication-competent influenza A red fluorescent viruses. These influenza A viruses encode mCherry fused to the viral non-structural 1 (NS1) protein and display comparable growth kinetics to wild-type viruses in vitro . Infection of cells with influenza A mCherry viruses was neutralized with monoclonal antibodies and inhibited with antivirals to levels similar to wild-type virus. Influenza A mCherry viruses were also able to lethally infect mice, and strikingly, dose- and time-dependent kinetics of viral replication were monitored in whole excised mouse lungs using an in vivo imaging system (IVIS). By eliminating the need for secondary labeling of infected cells, influenza A mCherry viruses provide an ideal tool in the ongoing struggle to better characterize the virus and identify new therapeutics against influenza A viral infections.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract
The total community genomic DNA (gDNA) from permafrost was extracted using four commercial DNA extraction kits. The gDNAs were compared using quantitative real-time PCR (qPCR) targeting 16S ...rRNA genes and bacterial diversity analyses obtained via 454 pyrosequencing of the 16S rRNA (V3 region) amplified in single or nested PCR. The FastDNA® SPIN (FDS) Kit provided the highest gDNA yields and 16S rRNA gene concentrations, followed by MoBio PowerSoil® (PS) and MoBio PowerLyzer™ (PL) kits. The lowest gDNA yields and 16S rRNA gene concentrations were from the Meta-G-Nome™ (MGN) DNA Isolation Kit. Bacterial phyla identified in all DNA extracts were similar to that found in other soils and were dominated by Actinobacteria,Firmicutes,Gemmatimonadetes,Proteobacteria, and Acidobacteria. Weighted UniFrac and statistical analyses indicated that bacterial community compositions derived from FDS, PS, and PL extracts were similar to each other. However, the bacterial community structure from the MGN extracts differed from other kits exhibiting higher proportions of easily lysed β- and γ-Proteobacteria and lower proportions of Actinobacteria and Methylocystaceae important in carbon cycling. These results indicate that gDNA yields differ between the extraction kits, but reproducible bacterial community structure analysis may be accomplished using gDNAs from the three bead-beating lysis extraction kits.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Secondary experimental procedures such as immunostaining have been utilized to study wild-type influenza A viruses (IAV) but are inadequate to rapidly determine the virus in infected cells or for the ...high-throughput screening (HTS) of antivirals or neutralizing antibodies. Reverse genetics approaches have allowed the generation of recombinant IAV expressing bioluminescent (BL) reporters or fluorescent proteins (FPs). These approaches can easily track viral infections in cultured cells and in validated animal models of infection using in vivo imaging systems (IVIS). Here, we describe the experimental procedures to generate recombinant monomeric (m)Cherry-expressing influenza A/Puerto Rico/8/34 (PR8-mCherry) H1N1 by altering the non-structural (NS) vRNA segment and its use in mCherry-based microneutralization assays to assess antivirals and neutralizing antibodies. The experimental procedures could be used for the generation of other recombinant influenza virus types (e.g., influenza B) or IAV subtypes (e.g., H3N2) expressing mCherry or other BL reporters or FPs from the NS or other vRNA segment. These recombinant reporter-expressing viruses represent an excellent toolbox for the identification of prophylactics or therapeutics for the treatment of influenza viral infections in HTS settings as well as to study different aspects related with the biology of influenza viruses and/or its interaction with the host.
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