Absence of sodA Increases the Levels of Oxidation of Key Metabolic Determinants of Borrelia burgdorferi

Borrelia burgdorferi, the causative agent of Lyme disease, alters its gene expression in response to environmental signals unique to its tick vector or vertebrate hosts. B. burgdorferi carries one superoxide dismutase gene (sodA) capable of controlling intracellular superoxide levels. Previously, sodA was shown to be essential for infection of B. burgdorferi in the C3H/HeN model of Lyme disease. We employed two-dimensional electrophoresis (2-DE) and immunoblot analysis with antibodies specific to carbonylated proteins to identify targets that were differentially oxidized in the soluble fractions of the sodA mutant compared to its isogenic parental control strain following treatment with an endogenous superoxide generator, methyl viologen (MV, paraquat). HPLC-ESI-MS/MS analysis of oxidized proteins revealed that several proteins of the glycolytic pathway (BB0057, BB0020, BB0348) exhibited increased carbonylation in the sodA mutant treated with MV. Levels of ATP and NAD/NADH were reduced in the sodA mutant compared with the parental strain following treatment with MV and could be attributed to increased levels of oxidation of proteins of the glycolytic pathway. In addition, a chaperone, HtpG (BB0560), and outer surface protein A (OspA, BBA15) were also observed to be oxidized in the sodA mutant. Immunoblot analysis revealed reduced levels of Outer surface protein C (OspC), Decorin binding protein A (DbpA), fibronectin binding protein (BBK32), RpoS and BosR in the sodA mutant compared to the control strains. Viable sodA mutant spirochetes could not be recovered from both gp91/phox ^−⁄− and iNOS deficient mice while borrelial DNA was detected in multiple tissues samples from infected mice at significantly lower levels compared to the parental strain. Taken together, these observations indicate that the increased oxidation of select borrelial determinants and reduced levels of critical pathogenesis-associated lipoproteins contribute to the in vivo deficit of the sodA mutant in the mouse model of Lyme disease. This study, utilizing the sodA mutant, has provided insights into adaptive capabilities critical for survival of B. burgdorferi in its hosts.     

Low Resistance to First and Second Line Anti-Tuberculosis Drugs among Treatment Naive Pulmonary Tuberculosis Patients in Southwestern Uganda

BackgroundThere are limited data on region-specific drug susceptibility of tuberculosis (TB) in Uganda. We performed resistance testing on specimens collected from treatment-naive patients with pulmonary TB in Southwestern Uganda for first and second line anti-TB drugs. We sought to provide data to guide regional recommendations for empiric TB therapy.MethodsArchived isolates, obtained from patients at Mbarara Regional Referral Hospital from February 2009 to February 2013, were tested for resistance to isoniazid and rifampicin using the MTBDRplus and Xpert MTB/RIF assays. A subset of randomly selected isolates was tested for second line agents, including fluoroquinolones (FQs), aminoglycosides, cyclic peptides, and ethambutol using the MTBDRsl assay. We performed confirmatory testing for FQ resistance using repeated MTBDRsl, the Mycobacteria growth indicator tube (MGIT) assay, and sequencing of the gyrA and gyrB genes.ResultsWe tested isolates from 190 patients. The cohort had a median age of 33 years (IQR 26-43), 69% (131/190) were male, and the HIV prevalence was 42% (80/190). No isolates (0/190) were rifampicin-resistant and only 1/190 (0.5%) was isoniazid-resistant. Among 92 isolates tested for second-line drug resistance, 71 (77%) had interpretable results, of which none were resistant to aminoglycosides, cyclic peptides or ethambutol. Although 7 (10%) initially tested as resistant to FQs by the MTBDRsl assay, they were confirmed as susceptible by repeat MTBDRsl testing as well as by MGIT and gyrase gene sequencingConclusionWe found no MDR-TB and no resistance to ethambutol, FQs, or injectable anti-TB drugs in treatment naïve patients with pulmonary TB in Southwestern Uganda. Standard treatment guidelines for susceptible TB should be adequate for most patients with TB in this population. Where possible, molecular susceptibility testing methods should be routinely validated by culture methods.     

Radio Frequency-Activated Nanoliposomes for Controlled Combination Drug Delivery

This work was conducted in order to design, characterize, and evaluate stable liposomes containing the hydrophobic drug raloxifene HCl (RAL) and hydrophilic doxycycline HCl (DOX), two potentially synergistic agents for treating osteoporosis and other bone lesions, in conjunction with a radio frequency-induced, hydrophobic magnetic nanoparticle-dependent triggering mechanism for drug release. Both drugs were successfully incorporated into liposomes by lipid film hydration, although combination drug loading compromised liposome stability. Liposome stability was improved by reducing the drug load and by including Pluronics® (PL) in the formulations. DOX did not appear to interact with the phospholipid membranes comprising the liposomes, and its release was maximized in the presence of radio frequency (RF) heating. In contrast, differential scanning calorimetry (DSC) and phosphorus-31 nuclear magnetic resonance (³¹P-NMR) analysis revealed that RAL developed strong interactions with the phospholipid membranes, most notably with lipid phosphate head groups, resulting in significant changes in membrane thermodynamics. Likewise, RAL release from liposomes was minimal, even in the presence of RF heating. These studies may offer useful insights into the design and optimization of multidrug containing liposomes. The effects of RAL on liposome characteristics and drug release performance underscore the importance of appropriate physical-chemical analysis in order to identify and characterize drug-lipid interactions that may profoundly affect liposome properties and performance early in the formulation development process.KEY WORDS: controlled release, drug combination, liposomes, nanoparticles     

Periplasmic Acid Stress Increases Cell Division Asymmetry (Polar Aging) of Escherichia coli

Under certain kinds of cytoplasmic stress, Escherichia coli selectively reproduce by distributing the newer cytoplasmic components to new-pole cells while sequestering older, damaged components in cells inheriting the old pole. This phenomenon is termed polar aging or cell division asymmetry. It is unknown whether cell division asymmetry can arise from a periplasmic stress, such as the stress of extracellular acid, which is mediated by the periplasm. We tested the effect of periplasmic acid stress on growth and division of adherent single cells. We tracked individual cell lineages over five or more generations, using fluorescence microscopy with ratiometric pHluorin to measure cytoplasmic pH. Adherent colonies were perfused continually with LBK medium buffered at pH 6.00 or at pH 7.50; the external pH determines periplasmic pH. In each experiment, cell lineages were mapped to correlate division time, pole age and cell generation number. In colonies perfused at pH 6.0, the cells inheriting the oldest pole divided significantly more slowly than the cells inheriting the newest pole. In colonies perfused at pH 7.50 (near or above cytoplasmic pH), no significant cell division asymmetry was observed. Under both conditions (periplasmic pH 6.0 or pH 7.5) the cells maintained cytoplasmic pH values at 7.2–7.3. No evidence of cytoplasmic protein aggregation was seen. Thus, periplasmic acid stress leads to cell division asymmetry with minimal cytoplasmic stress.     

Maturation of the angiotensin II cardiovascular response in the embryonic White Leghorn chicken (Gallus gallus)

Angiotensin II (Ang II) is an important regulator of cardiovascular function in adult vertebrates. Although its role in regulating the adult system has been extensively investigated, the cardiovascular response to Ang II in embryonic vertebrates is relatively unknown. We investigated the potential of Ang II as a regulator of cardiovascular function in embryonic chickens, which lack central nervous system control of cardiovascular function throughout the majority of incubation. The cardiovascular response to Ang II in embryonic chickens was investigated over the final 50% of their development. Ang II produced a dose-dependent increase in arterial pressure on each day of development studied, and the response increased in intensity as development progressed. The Ang II type-1 receptor nonspecific competitive peptide antagonist [Sar¹ ile⁸] Ang II blocked the cardiovascular response to subsequent injections of Ang II on day 21 only. The embryonic pressure response to Ang II (hypertension only) differed from that of adult chickens, in which initial hypotension is followed by hypertension. The constant level of gene expression for the Ang II receptor, in conjunction with an increasing pressure response to the peptide, suggests that two Ang II receptor subtypes are present during chicken development. Collectively, the data indicate that Ang II plays an important role in the cardiovascular development of chickens; however, its role in maintaining basal function requires further study.     

BRG1 co-localizes with DNA replication factors and is required for efficient replication fork progression

For DNA replication to occur, chromatin must be remodeled. Yet, we know very little about which proteins alter nucleosome occupancy at origins and replication forks and for what aspects of replication they are required. Here, we demonstrate that the BRG1 catalytic subunit of mammalian SWI/SNF-related complexes co-localizes with origin recognition complexes, GINS complexes, and proliferating cell nuclear antigen at sites of DNA replication on extended chromatin fibers. The specific pattern of BRG1 occupancy suggests it does not participate in origin selection but is involved in the firing of origins and the process of replication elongation. This latter function is confirmed by the fact that Brg1 mutant mouse embryos and RNAi knockdown cells exhibit a 50% reduction in replication fork progression rates, which is associated with decreased cell proliferation. This novel function of BRG1 is consistent with its requirement during embryogenesis and its role as a tumor suppressor to maintain genome stability and prevent cancer.     

Rumen Microbial Population Dynamics during Adaptation to a High-Grain Diet

High-grain adaptation programs are widely used with feedlot cattle to balance enhanced growth performance against the risk of acidosis. This adaptation to a high-grain diet from a high-forage diet is known to change the rumen microbial population structure and help establish a stable microbial population within the rumen. Therefore, to evaluate bacterial population dynamics during adaptation to a high-grain diet, 4 ruminally cannulated beef steers were adapted to a high-grain diet using a step-up diet regimen containing grain and hay at ratios of 20:80, 40:60, 60:40, and 80:20. The rumen bacterial populations were evaluated at each stage of the step-up diet after 1 week of adaptation, before the steers were transitioned to the next stage of the diet, using terminal restriction fragment length polymorphism (T-RFLP) analysis, 16S rRNA gene libraries, and quantitative real-time PCR. The T-RFLP analysis displayed a shift in the rumen microbial population structure during the final two stages of the step-up diet. The 16S rRNA gene libraries demonstrated two distinct rumen microbial populations in hay-fed and high-grain-fed animals and detected only 24 common operational taxonomic units out of 398 and 315, respectively. The 16S rRNA gene libraries of hay-fed animals contained a significantly higher number of bacteria belonging to the phylum Fibrobacteres, whereas the 16S rRNA gene libraries of grain-fed animals contained a significantly higher number of bacteria belonging to the phylum Bacteroidetes. Real-time PCR analysis detected significant fold increases in the Megasphaera elsdenii, Streptococcus bovis, Selenomonas ruminantium, and Prevotella bryantii populations during adaptation to the high-concentrate (high-grain) diet, whereas the Butyrivibrio fibrisolvens and Fibrobacter succinogenes populations gradually decreased as the animals were adapted to the high-concentrate diet. This study evaluates the rumen microbial population using several molecular approaches and presents a broader picture of the rumen microbial population structure during adaptation to a high-grain diet from a forage diet.The rumen is a complex microbial ecosystem that is composed of an immense variety of bacteria, protozoa, fungi, and viruses (5). Among these microorganisms, bacteria are the most investigated population and have a significant effect on the animal''s performance. However, our understanding of how rumen bacteria change and adapt to different ruminal environments is in its infancy.In the feedlot cattle industry, when animals on a forage diet are directly put on a high-grain diet, a decrease in ruminal pH due to lactate production has been observed (23, 31, 42), which leads to the possibility of digestive disorders, which can cause a decrease in the animal''s performance (23, 45). Therefore, feeding programs have been implemented to adapt feedlot cattle from a high-forage diet to a high-concentrate diet by gradually increasing the concentration of grain in the diet and decreasing the fiber content (2, 35). During this adaptation to high-grain diets, significant changes in the ruminal environment and rumen bacterial population structure have been reported (17, 46, 48). However, the microbial changes that occur during this transition phase are poorly understood (17, 21, 26, 46). Studies performed to date have utilized culture-based techniques or have looked at the fluctuation of a few indicator bacteria (48, 47) to evaluate bacterial population changes. Due to limitations in culturing rumen bacteria, the use of culture-based techniques to evaluate bacterial populations substantially underestimates the diversity of microorganisms within the rumen. In this study, we have utilized culture-independent approaches to evaluate bacterial population structure and diversity using terminal restriction fragment length polymorphisms (T-RFLPs) and sequence analysis of 16S rRNA gene libraries to compare the rumen bacterial population structure in animals on prairie hay against that in animals adapting to a high-concentrate (high-grain) diet. We have also quantified the fluctuations in the populations of previously reported indicator bacterial species using quantitative real-time PCR (qRT-PCR) to assess the role of these organisms during adaptation to a high-concentrate diet.