Tobacco smoke mediated induction of sinonasal microbial biofilms

Cigarette smokers and those exposed to second hand smoke are more susceptible to life threatening infection than non-smokers. While much is known about the devastating effect tobacco exposure has on the human body, less is known about the effect of tobacco smoke on the commensal and commonly found pathogenic bacteria of the human respiratory tract, or human respiratory tract microbiome. Chronic rhinosinusitis (CRS) is a common medical complaint, affecting 16% of the US population with an estimated aggregated cost of $6 billion annually. Epidemiologic studies demonstrate a correlation between tobacco smoke exposure and rhinosinusitis. Although a common cause of CRS has not been defined, bacterial presence within the nasal and paranasal sinuses is assumed to be contributory. Here we demonstrate that repetitive tobacco smoke exposure induces biofilm formation in a diverse set of bacteria isolated from the sinonasal cavities of patients with CRS. Additionally, bacteria isolated from patients with tobacco smoke exposure demonstrate robust in vitro biofilm formation when challenged with tobacco smoke compared to those isolated from smoke naïve patients. Lastly, bacteria from smoke exposed patients can revert to a non-biofilm phenotype when grown in the absence of tobacco smoke. These observations support the hypothesis that tobacco exposure induces sinonasal biofilm formation, thereby contributing to the conversion of a transient and medically treatable infection to a persistent and therapeutically recalcitrant condition.     

The alanine racemase of Mycobacterium smegmatis is essential for growth in the absence of D-alanine

Alanine racemase, encoded by the gene alr, is an important enzyme in the synthesis of d-alanine for peptidoglycan biosynthesis. Strains of Mycobacterium smegmatis with a deletion mutation of the alr gene were found to require d-alanine for growth in both rich and minimal media. This indicates that alanine racemase is the only source of d-alanine for cell wall biosynthesis in M. smegmatis and confirms alanine racemase as a viable target gene for antimycobacterial drug development.     

Synthetic viability genomic screening defines Sae2 function in DNA repair

DNA double-strand break (DSB) repair by homologous recombination (HR) requires 3′ single-stranded DNA (ssDNA) generation by 5′ DNA-end resection. During meiosis, yeast Sae2 cooperates with the nuclease Mre11 to remove covalently bound Spo11 from DSB termini, allowing resection and HR to ensue. Mitotic roles of Sae2 and Mre11 nuclease have remained enigmatic, however, since cells lacking these display modest resection defects but marked DNA damage hypersensitivities. By combining classic genetic suppressor screening with high-throughput DNA sequencing, we identify Mre11 mutations that strongly suppress DNA damage sensitivities of sae2Δ cells. By assessing the impacts of these mutations at the cellular, biochemical and structural levels, we propose that, in addition to promoting resection, a crucial role for Sae2 and Mre11 nuclease activity in mitotic DSB repair is to facilitate the removal of Mre11 from ssDNA associated with DSB ends. Thus, without Sae2 or Mre11 nuclease activity, Mre11 bound to partly processed DSBs impairs strand invasion and HR.     

Parental contribution and coefficient of coancestry among maize inbreds: pedigree, RFLP, and SSR data

The genetic relationship between inbreds i and j can be estimated from pedigree or from molecular marker data. The objectives of this study were to: (1) determine whether pedigree, restriction fragment length polymorphism (RFLP), and simple sequence repeat (SSR) data give similar estimates of parental contribution and coefficient of coancestry (f _ij ) among a set of maize (Zea mays L.) inbreds, and (2) compare the usefulness of RFLP and SSR markers for estimating genetic relationship. We studied 13 maize inbreds with known pedigrees. The inbreds were genotyped using 124 RFLP and 195 SSR markers. For each type of marker, parental contributions were estimated from marker similarity among an inbred and both of its parents, and were subsequently used to estimate f _ij . Estimates of parental contribution differed significantly (α<0.05) between pedigree data and either type of marker, but not between the marker systems. The RFLP estimates of parental contribution failed to sum to 1.0, reflecting a higher frequency of non-parental bands with RFLP than with SSR markers. The f _ij estimated from pedigree, RFLP, and SSR data were highly correlated (r=0.87–0.97), although significant differences were found among the three sets of f _ij estimates. We concluded that pedigree and marker data often lead to different estimates of parental contribution and f _ij , and that SSR markers are superior to RFLP markers for estimating genetic relationship. A relevant question is whether or not the inbreds previously genotyped with an older marker system (e.g., RFLP) need to be re-analyzed with a newer marker system (e.g., SSR) for the purpose of estimating genetic relationship. Such re-analysis seems unnecessary if data for the same type of marker are available for a given inbred and both of its parents. Received: 2 June 1999 / Accepted: 30 July 1999     

Tyrosine phosphatase PTP1B interacts with TRPV6 in vivo and plays a role in TRPV6-mediated calcium influx in HEK293 cells

This study investigates the role of tyrosine phosphorylation and dephosphorylation in the regulation of the Ca(2+) permeant TRPV6 channel. HEK293 cells co-transfected with TRPV6 and the tyrosine phosphatase PTP1B show a constitutive Ca(2+) entry which was independent of tyrosine phosphorylation under resting conditions. Following depletion of intracellular Ca(2+) stores, TRPV6-mediated Ca(2+) entry could be increased in the presence of a tyrosine phosphatase inhibitor (bis-(N,N-dimethyl-hydroxamido) hydroxo-vanadate; DMHV). Inhibition of Src-kinases completely abolished DMHV-induced increase in TRPV6-mediated Ca(2+) influx. Co-transfection with Src led to tyrosine phosphorylation of TRPV6 which could be dephosphorylated by PTP1B. In vivo interaction of TRPV6 with PTP1B was visualized using the bimolecular fluorescence complementation (BiFC) method and proved by co-immunoprecipitation of both proteins. These data indicate that tyrosine phosphorylation is involved in the regulation of the TRPV6 channel protein.     

Epitope-specific human influenza antibody repertoires diversify by B cell intraclonal sequence divergence and interclonal convergence

We generated from a single blood sample five independent human mAbs that recognized the Sa antigenic site on the head of influenza hemagglutinin and exhibited inhibitory activity against a broad panel of H1N1 strains. All five Abs used the V(H)3-7 and J(H)6 gene segments, but at least four independent clones were identified by junctional analysis. High-throughput sequence analysis of circulating B cells revealed that each of the independent clones were members of complex phylogenetic lineages that had diversified widely using a pattern of progressive diversification through somatic mutation. Unexpectedly, B cells encoding multiple diverging lineages of these clones, including many containing very few mutations in the Ab genes, persisted in the circulation. Conversely, we noted frequent instances of amino acid sequence convergence in the Ag combining sites exhibited by members of independent clones, suggesting a strong selection for optimal binding sites. We suggest that maintenance in circulation of a wide diversity of somatic variants of dominant clones may facilitate recognition of drift variant virus epitopes that occur in rapidly mutating virus Ags, such as influenza hemagglutinin. In fact, these Ab clones recognize an epitope that acquired three glycosylation sites mediating escape from previously isolated human Abs.     

The role of temporal sensing in bioelectromagnetic effects

Experiments were conducted to see whether the cellular response to electromagnetic (EM) fields occurs through a detection process involving temporal sensing. L929 cells were exposed to 60 Hz magnetic fields and the enhancement of ornithine decarboxylase (ODC) activity was measured to determine cellular response to the field. In one set of experiments, the field was turned alternately off and on at intervals of 0.1 to 50 s. For these experiments, field coherence was maintained by eliminating the insertion of random time intervals upon switching. Intervals ≥ 1 s produced no enhancement of ODC activity, but fields switched at intervals ≥ 10 s showed ODC activities that were enhanced by a factor of approximately 1.7. These data indicate that it is the interval over which field parameters (e.g., amplitude or frequency) remain constant, rather than the interval over which the field is coherent, that is critical to cellular response to an EMF. In a second set of experiments, designed to determine how long it would take for cells to detect a change in field parameters, the field was interrupted for brief intervals (25–200 ms) once each second throughout exposure. In this situation, the extent of EMF-induced ODC activity depended upon the duration of the interruption. Interruptions ≥ 100 ms were detected by the cell as shown by elimination of field-induced enhancement of ODC. That two time constants (0.1 and 10 s) are involved in cellular EMF detection is consistent with the temporal sensing process associated with bacterial chemotaxis. By analogy with bacterial temporal sensing, cells would continuously sample and average an EM field over intervals of about 0.1 s (the “averaging” time), storing the averaged value in memory. The cell would compare the stored value with the current average, and respond to the EM field only when field parameters remain constant over intervals of approximately 10 s (the “memory” time). Bioelectromagnetics 18:388–395, 1997. © 1997 Wiley-Liss, Inc.