An eco-informatics tool for microbial community studies: supervised classification of Amplicon Length Heterogeneity (ALH) profiles of 16S rRNA

Support vector machines (SVM) and K-nearest neighbors (KNN) are two computational machine learning tools that perform supervised classification. This paper presents a novel application of such supervised analytical tools for microbial community profiling and to distinguish patterning among ecosystems. Amplicon length heterogeneity (ALH) profiles from several hypervariable regions of 16S rRNA gene of eubacterial communities from Idaho agricultural soil samples and from Chesapeake Bay marsh sediments were separately analyzed. The profiles from all available hypervariable regions were concatenated to obtain a combined profile, which was then provided to the SVM and KNN classifiers. Each profile was labeled with information about the location or time of its sampling. We hypothesized that after a learning phase using feature vectors from labeled ALH profiles, both these classifiers would have the capacity to predict the labels of previously unseen samples. The resulting classifiers were able to predict the labels of the Idaho soil samples with high accuracy. The classifiers were less accurate for the classification of the Chesapeake Bay sediments suggesting greater similarity within the Bay's microbial community patterns in the sampled sites. The profiles obtained from the V1+V2 region were more informative than that obtained from any other single region. However, combining them with profiles from the V1 region (with or without the profiles from the V3 region) resulted in the most accurate classification of the samples. The addition of profiles from the V 9 region appeared to confound the classifiers. Our results show that SVM and KNN classifiers can be effectively applied to distinguish between eubacterial community patterns from different ecosystems based only on their ALH profiles.     

Persistence of Borrelia burgdorferi in rhesus macaques following antibiotic treatment of disseminated infection

The persistence of symptoms in Lyme disease patients following antibiotic therapy, and their causes, continue to be a matter of intense controversy. The studies presented here explore antibiotic efficacy using nonhuman primates. Rhesus macaques were infected with B. burgdorferi and a portion received aggressive antibiotic therapy 4-6 months later. Multiple methods were utilized for detection of residual organisms, including the feeding of lab-reared ticks on monkeys (xenodiagnosis), culture, immunofluorescence and PCR. Antibody responses to the B. burgdorferi-specific C6 diagnostic peptide were measured longitudinally and declined in all treated animals. B. burgdorferi antigen, DNA and RNA were detected in the tissues of treated animals. Finally, small numbers of intact spirochetes were recovered by xenodiagnosis from treated monkeys. These results demonstrate that B. burgdorferi can withstand antibiotic treatment, administered post-dissemination, in a primate host. Though B. burgdorferi is not known to possess resistance mechanisms and is susceptible to the standard antibiotics (doxycycline, ceftriaxone) in vitro, it appears to become tolerant post-dissemination in the primate host. This finding raises important questions about the pathogenicity of antibiotic-tolerant persisters and whether or not they can contribute to symptoms post-treatment.     

Combinatorial Synthesis of and High-throughput Protein Release from Polymer Film and Nanoparticle Libraries

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with conventional one-sample-at-a-time synthesis techniques, a more recent high-throughput approach has been developed enabling the synthesis and testing of large libraries of polyanhydrides¹. This will facilitate more efficient optimization and design process of these biomaterials for drug and vaccine delivery applications. The method in this work describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries. In this robotically operated method (Figure 1), linear actuators and syringe pumps are controlled by LabVIEW, which enables a hands-free automated protocol, eliminating user error. Furthermore, this method enables the rapid fabrication of micro-scale polymer libraries, reducing the batch size while resulting in the creation of multivariant polymer systems. This combinatorial approach to polymer synthesis facilitates the synthesis of up to 15 different polymers in an equivalent amount of time it would take to synthesize one polymer conventionally. In addition, the combinatorial polymer library can be fabricated into blank or protein-loaded geometries including films or nanoparticles upon dissolution of the polymer library in a solvent and precipitation into a non-solvent (for nanoparticles) or by vacuum drying (for films). Upon loading a fluorochrome-conjugated protein into the polymer libraries, protein release kinetics can be assessed at high-throughput using a fluorescence-based detection method (Figures 2 and 3) as described previously¹. This combinatorial platform has been validated with conventional methods² and the polyanhydride film and nanoparticle libraries have been characterized with ¹H NMR and FTIR. The libraries have been screened for protein release kinetics, stability and antigenicity; in vitro cellular toxicity, cytokine production, surface marker expression, adhesion, proliferation and differentiation; and in vivo biodistribution and mucoadhesion^1-11. The combinatorial method developed herein enables high-throughput polymer synthesis and fabrication of protein-loaded nanoparticle and film libraries, which can, in turn, be screened in vitro and in vivo for optimization of biomaterial performance.     

An overview of enzymatic production of biodiesel

Biodiesel production has received considerable attention in the recent past as a biodegradable and nonpolluting fuel. The production of biodiesel by transesterification process employing alkali catalyst has been industrially accepted for its high conversion and reaction rates. Recently, enzymatic transesterification has attracted much attention for biodiesel production as it produces high purity product and enables easy separation from the byproduct, glycerol. But the cost of enzyme remains a barrier for its industrial implementation. In order to increase the cost effectiveness of the process, the enzyme (both intracellular and extracellular) is reused by immobilizing in a suitable biomass support particle and that has resulted in considerable increase in efficiency. But the activity of immobilized enzyme is inhibited by methanol and glycerol which are present in the reacting mixture. The use of tert-butanol as solvent, continuous removal of glycerol, stepwise addition of methanol are found to reduce the inhibitory effects thereby increasing the cost effectiveness of the process. The present review analyzes these methods reported in literature and also suggests a suitable method for commercialization of the enzymatic process.     

Use of a novel pacing mode to achieve biventricular pacing in a patient with recurrent atrial lead dislodgement after CRT-D implantation

Cardiac resynchronization therapy device (CRT-P and CRT-D) implantation has increased tremendously with increasing operator experience, eligible patients and expansion of indications. Refinements in devices and algorithms now aid physicians to improve biventricular pacing and optimize CRT. We report a case in which an interesting device program was used to achieve biventricular pacing after repeated dislodgement of the atrial lead in a patient implanted with CRT-D.     

Recurrent heat shock impairs the proliferation and differentiation of C2C12 myoblasts

Heat-related illness and injury are becoming a growing safety concern for the farmers, construction workers, miners, firefighters, manufacturing workers, and other outdoor workforces who are exposed to heat stress in their routine lives. A primary response by a cell to an acute heat shock (HS) exposure is the induction of heat-shock proteins (HSPs), which chaperone and facilitate cellular protein folding and remodeling processes. While acute HS is well studied, the effect of repeated bouts of hyperthermia and the sustained production of HSPs in the myoblast-myotube model system of C2C12 cells are poorly characterized. In C2C12 myoblasts, we found that robust HS (43 °C, dose/time) significantly decreased the proliferation by 50% as early as on day 1 and maintained at the same level on days 2 and 3 of HS. This was accompanied by an accumulation of cells at G2 phase with reduced cell number in G1 phase indicating cell cycle arrest. FACS analysis indicates that there was no apparent change in apoptosis (markers) and cell death upon repeated HS. Immunoblot analysis and qPCR demonstrated a significant increase in the baseline expression of HSP25, 70, and 90 (among others) in cells after a single HS (43 °C) for 60 min as a typical HS response. Importantly, the repeated HS for 60 min each on days 2 and 3 maintained the elevated levels of HSPs compared to the control cells. Further, the continuous HS exposure resulted in significant inhibition of the differentiation of C2C12 myocytes to myotubes and only 1/10th of the cells underwent differentiation in HS relative to control. This was associated with significantly higher levels of HSPs and reduced expression of myogenin and Myh2 (P < 0.05), the genes involved in the differentiation process. Finally, the cell migration (scratch) assay indicated that the wound closure was significantly delayed in HS cells relative to the control cells. Overall, these results suggest that a repeated HS may perturb the active process of proliferation, motility, and differentiation processes in an in vitro murine myoblast-myotube model.