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Generation of a random transposon mutant library is advantageous in Leptospira as site-directed mutagenesis remains a challenge, especially in pathogenic species. This procedure is typically completed by transformation of Leptospira with a Himar1 containing plasmid via conjugation with Escherichia coli as a donor cell. Here we describe the methodology to generate random transposon mutants in the saprophyte Leptospira biflexa via conjugation of plasmid pSW29T-TKS2 harbored in E. coli β2163. Determination of transposon insertion site by semi-random nested PCR will also be described. A similar methodology may be employed to generate Tn mutants of pathogenic Leptospira species.

Leptospira are emerging zoonotic pathogens transmitted from animals to humans typically through contaminated environmental sources of water and soil. Regulatory pathways of pathogenic Leptospira spp. underlying the adaptive response to different hosts and environmental conditions remains elusive. In this study, we provide the first global Transcriptional Start Site (TSS) map of a Leptospira species. RNA was obtained from the pathogen Leptospira interrogans grown at 30°C (optimal in vitro temperature) and 37°C (host temperature) and selectively enriched for 5' ends of native transcripts. A total of 2865 and 2866 primary TSS (pTSS) were predicted in the genome of L. interrogans at 30 and 37°C, respectively. The majority of the pTSSs were located between 0 and 10 nucleotides from the translational start site, suggesting that leaderless transcripts are a common feature of the leptospiral translational landscape. Comparative differential RNA-sequencing (dRNA-seq) analysis revealed conservation of most pTSS at 30 and 37°C. Promoter prediction algorithms allow the identification of the binding sites of the alternative sigma factor sigma 54. However, other motifs were not identified indicating that Leptospira consensus promoter sequences are inherently different from the Escherichia coli model. RNA sequencing also identified 277 and 226 putative small regulatory RNAs (sRNAs) at 30 and 37°C, respectively, including eight validated sRNAs by Northern blots. These results provide the first global view of TSS and the repertoire of sRNAs in L. interrogans . These data will establish a foundation for future experimental work on gene regulation under various environmental conditions including those in the host.

Leptospira interrogans is the agent of leptospirosis, a reemerging zoonotic disease. It is transmitted to humans through environmental surface waters contaminated by the urine of mammals chronically infected by pathogenic strains able to survive in water for long periods. Little is known about the regulatory pathways underlying environmental sensing and host adaptation of L. interrogans during its enzootic cycle. This study identifies the EbpA-RpoN regulatory pathway in L. interrogans In this pathway, EbpA, a σ 54 activator and putative prokaryotic enhancer-binding protein (EBP), and the alternative sigma factor RpoN (σ 54 ) control expression of at least three genes, encoding AmtB (an ammonium transport protein) and two proteins of unknown function. Electrophoresis mobility shift assay demonstrated that recombinant RpoN and EbpA bind to the promoter region and upstream of these three identified genes, respectively. Genetic disruption of ebpA in L. interrogans serovar Manilae virtually abolished expression of the three genes, including amtB in two independent ebpA mutants. Complementation of the ebpA mutant restored expression of these genes. Intraperitoneal inoculation of gerbils with the ebpA mutant did not affect mortality. However, the ebpA mutant had decreased cell length in vitro and had a significantly lowered cell density at stationary phase when grown with l-alanine as the sole nitrogen source. Furthermore, the ebpA mutant has dramatically reduced long-term survival ability in water. Together, these studies identify a regulatory pathway, the EbpA-RpoN pathway, that plays an important role in the zoonotic cycle of L. interrogans IMPORTANCE: Leptospirosis is a reemerging disease with global importance. However, our understanding of gene regulation of the spirochetal pathogen Leptospira interrogans is still in its infancy, largely due to the lack of robust tools for genetic manipulation of this spirochete. Little is known about how the pathogen achieves its long-term survival in the aquatic environment. By utilizing bioinformatic, genetic, and biochemical methods, we discovered a regulatory pathway in L. interrogans, the EbpA-RpoN pathway, and demonstrated that this pathway plays an important role in environmental survival of this pathogen., (Copyright © 2017 American Society for Microbiology.)

Leptospirosis is a zoonotic disease that affects ∼1 million people annually, with a mortality rate of >10%. Currently, there is an absence of effective genetic manipulation tools for targeted mutagenesis in pathogenic leptospires. Transcription activator-like effectors (TALEs) are a recently described group of repressors that modify transcriptional activity in prokaryotic and eukaryotic cells by directly binding to a targeted sequence within the host genome. To determine the applicability of TALEs within Leptospira spp., two TALE constructs were designed. First, a constitutively expressed TALE gene specific for the lacO-like region upstream of bgaL was trans inserted in the saprophyte Leptospira biflexa (the TALEβgal strain). Reverse transcriptase PCR (RT-PCR) analysis and enzymatic assays demonstrated that BgaL was not expressed in the TALEβgal strain. Second, to study the role of LigA and LigB in pathogenesis, a constitutively expressed TALE gene with specificity for the homologous promoter regions of ligA and ligB was cis inserted into the pathogen Leptospira interrogans (TALElig). LigA and LigB expression was studied by using three independent clones: TALElig1, TALElig2, and TALElig3. Immunoblot analysis of osmotically induced TALElig clones demonstrated 2- to 9-fold reductions in the expression levels of LigA and LigB, with the highest reductions being noted for TALElig1 and TALElig2, which were avirulent in vivo and nonrecoverable from animal tissues. This study reconfirms galactosidase activity in the saprophyte and suggests a role for LigA and LigB in pathogenesis. Collectively, this study demonstrates that TALEs are effective at reducing the expression of targeted genes within saprophytic and pathogenic strains of Leptospira spp., providing an additional genetic manipulation tool for this genus., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Leptospirosis, an emerging zoonotic disease, remains poorly understood because of a lack of genetic manipulation tools available for pathogenic leptospires. Current genetic manipulation techniques include insertion of DNA by random transposon mutagenesis and homologous recombination via suicide vectors. This study describes the construction of a shuttle vector, pMaORI, that replicates within saprophytic, intermediate, and pathogenic leptospires. The shuttle vector was constructed by the insertion of a 2.9-kb DNA segment including the parA, parB, and rep genes into pMAT, a plasmid that cannot replicate in Leptospira spp. and contains a backbone consisting of an aadA cassette, ori R6K, and oriT RK2/RP4. The inserted DNA segment was isolated from a 52-kb region within Leptospira mayottensis strain 200901116 that is not found in the closely related strain L. mayottensis 200901122. Because of the size of this region and the presence of bacteriophage-like proteins, it is possible that this region is a result of a phage-related genomic island. The stability of the pMaORI plasmid within pathogenic strains was tested by passaging cultures 10 times without selection and confirming the presence of pMaORI. Concordantly, we report the use of trans complementation in the pathogen Leptospira interrogans. Transformation of a pMaORI vector carrying a functional copy of the perR gene in a null mutant background restores the expression of PerR and susceptibility to hydrogen peroxide comparable to that of wild-type cells. In conclusion, we demonstrate the replication of a stable plasmid vector in a large panel of Leptospira strains, including pathogens. The shuttle vector described will expand our ability to perform genetic manipulation of Leptospira spp., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The bacterial stringent response is triggered by deficiencies of available nutrients and other environmental stresses. It is mediated by 5'-triphosphate-guanosine-3'-diphosphate and 5'-diphosphate-guanosine-3'-diphosphate (collectively (p)ppGpp) and generates global changes in gene expression and metabolism that enable bacteria to adapt to and survive these challenges. Borrelia burgdorferi encounters multiple stressors in its cycling between ticks and mammals that could trigger the stringent response. We have previously shown that the B. burgdorferi stringent response is mediated by a single enzyme, RelBbu, with both (p)ppGpp synthase and hydrolase activities, and that a B. burgdorferi 297 relBbu null deletion mutant was defective in adapting to stationary phase, incapable of down-regulating synthesis of rRNA and could not infect mice. We have now used this deletion mutant and microarray analysis to identify genes comprising the rel regulon in B. burgdorferi cultured at 34°C, and found that transcription of genes involved in glycerol metabolism is induced by relBbu. Culture of the wild type parental strain, the relBbu deletion mutant and its complemented derivative at 34°C and 25°C in media containing glucose or glycerol as principal carbon sources revealed a growth defect in the mutant, most evident at the lower temperature. Transcriptional analysis of the glp operon for glycerol uptake and metabolism in these three strains confirmed that relBbu was necessary and sufficient to increase transcription of this operon in the presence of glycerol at both temperatures. These results confirm and extend previous findings regarding the stringent response in B. burgdorferi. They also demonstrate that the stringent response regulates glycerol metabolism in this organism and is likely crucial for its optimal growth in ticks.

Background: The aim of this study was to determine the feasibility, accuracy, and reproducibility of a novel tracking-based echocardiographic ejection fraction (EF) assessment method in comparison with traditional methods based on magnetic resonance imaging and echocardiography., Methods: In a prospective assessment, apical echocardiographic grayscale image loops from 81 patients were read in random order by four experienced readers, blinded to any data of the cases. In three separate sessions, EFs were estimated using biplane tracking-based assessment and according to the modified Simpson's rule, as well as by visual interpretation in three apical views. Data were compared with a reference EF derived from echocardiography and magnetic resonance imaging., Results: On average, no significant difference was found between EF estimates of the different methods. Tracking-based EF assessments were possible in 90% of the patients. Tracking-based EF assessments showed slightly higher deviations from the reference EF than the modified Simpson's rule, while interobserver and intraobserver variability of tracking-based assessment were significantly better. Visual interpretation allowed the fastest EF assessment. Tracking-based EF assessment was approximately twice as fast as the modified Simpson's rule., Conclusions: Tracking-based EF assessment is feasible, has lower interobserver and intraobserver variability, and is faster than traditional echocardiographic EF quantification. Its minimal demand on user interaction makes it a favorable alternative to traditional echocardiographic approaches, with a particular clinical advantage when reliable follow-up measurements are needed.

Borrelia burgdorferi, the spirochetal agent of Lyme disease, is a vector-borne pathogen that cycles between a mammalian host and tick vector. This complex life cycle requires that the spirochete modulate its gene expression program to facilitate growth and maintenance in these diverse milieus. B. burgdorferi contains an operon that is predicted to encode proteins that would mediate the uptake and conversion of glycerol to dihydroxyacetone phosphate. Previous studies indicated that expression of the operon is elevated at 23°C and is repressed in the presence of the alternative sigma factor RpoS, suggesting that glycerol utilization may play an important role during the tick phase. This possibility was further explored in the current study by expression analysis and mutagenesis of glpD, a gene predicted to encode glycerol 3-phosphate dehydrogenase. Transcript levels for glpD were significantly lower in mouse joints relative to their levels in ticks. Expression of GlpD protein was repressed in an RpoS-dependent manner during growth of spirochetes within dialysis membrane chambers implanted in rat peritoneal cavities. In medium supplemented with glycerol as the principal carbohydrate, wild-type B. burgdorferi grew to a significantly higher cell density than glpD mutant spirochetes during growth in vitro at 25°C. glpD mutant spirochetes were fully infectious in mice by either needle or tick inoculation. In contrast, glpD mutants grew to significantly lower densities than wild-type B. burgdorferi in nymphal ticks and displayed a replication defect in feeding nymphs. The findings suggest that B. burgdorferi undergoes a switch in carbohydrate utilization during the mammal to tick transition. Further, the results demonstrate that the ability to utilize glycerol as a carbohydrate source for glycolysis during the tick phase of the infectious cycle is critical for maximal B. burgdorferi fitness.

The genome of Borrelia burgdorferi, the causative agent of Lyme disease, is comprised of a large linear chromosome and numerous smaller linear and circular plasmids. B. burgdorferi exhibits substantial genomic variation, and previous studies revealed genotype-specific variation at the right chromosomal telomere. A correlation has also been established between genotype and invasiveness. The correlation between chromosome length and genotype and between genotype and invasiveness suggested that a gene(s) at the right chromosome telomere may be required for virulence. Of particular interest was bb0844, an RpoS-regulated gene at the right telomere, the expression of which is induced when the spirochete undergoes adaptation to the mammalian host. The structure of the right chromosomal telomere was examined in 53 B. burgdorferi clinical isolates of various genotypes. Four distinct patterns were observed for bb0844: (i) chromosomal localization, (ii) plasmid localization, (iii) presence on both chromosome and plasmid, and (iv) complete absence. These patterns correlated with the B. burgdorferi genotype. On the basis of available sequence data, we propose a mechanism for the genomic rearrangements that accounts for the variability in bb0844 genomic localization. To further explore the role of BB0844 in the spirochete life cycle, a bb0844 deletion mutant was constructed by allelic exchange, and the viability of wild-type and bb0844 deletion mutants was examined in an experimental mouse-tick infection model. The bb0844 mutant was fully infectious in C3H/HeJ mice by either needle inoculation or tick transmission with B. burgdorferi-infected Ixodes scapularis larvae. Naïve larval ticks acquired both wild-type and mutant spirochetes with equal efficiency from B. burgdorferi-infected mice. The results demonstrate that BB0844 is not required for spirochete viability, pathogenicity, or maintenance in the tick vector or the mammalian host. At present, a defined role for BB0844 in B. burgdorferi cannot be ascertained.

Objective: Velocity vector imaging (VVI) is a new echocardiographic technique of measuring regional myocardial velocities and deformation. Our aim was to evaluate the feasibility and accuracy of VVI in defining regional functional abnormalities in patients with an acute myocardial infarction., Methods: Standard echocardiography and delayed enhancement (DE) magnetic resonance imaging were performed in 32 patients (29 men, mean age 61.2 +/- 8.1 years) within 36 hours of primary angioplasty. Twenty healthy volunteers (16 men, mean age 34.6 +/- 6.3 years) served as control subjects. Using VVI for offline analysis, segmental longitudinal deformation indices were measured. Infarcted, adjacent, and remote left ventricular segments were defined according to DE magnetic resonance imaging and coronary angiography. Infarct transmurality was also graded based on the DE extent within each segment on DE magnetic resonance imaging (0%-25%, 26%-50%, 51%-75%, and >76% of wall thickness)., Results: As compared with remote segments, myocardial infarction segments had significantly lower longitudinal systolic strain (S) (-9.6% vs -14.6%, P < .0001), lower S rate (-0.75 vs -1.08 s(-1), P < .0001), and a higher postsystolic S index (21% vs 8.3%, P < .001). By receiver operating characteristic curve analysis, a myocardial peak systolic longitudinal S lower than -6.5% in at least one ventricular segment showed best predictive value (94%) for detecting an infarcted left ventricle. Peak systolic S and S rate were useful predictors of the presence of regional dysfunction, and for the localization and transmural extent of the infarct., Conclusions: VVI is a promising new tool for studying myocardial motion and deformation with good feasibility in the clinical setting. The assessment of myocardial longitudinal systolic S and S rate with VVI can be used to identify the presence, location, and the transmural extent of myocardial infarction.

Lyme borreliosis in North America is caused by the tick-borne spirochete Borrelia burgdorferi, a zoonotic bacterium that is able to persistently infect a wide range of vertebrate species. Given the pronounced strain structure of B. burgdorferi in the northeastern United States, we asked whether the fitness of the different genotypes varies among susceptible vertebrate hosts. The transmission dynamics of two genetically divergent human isolates of B. burgdorferi, BL206 and B348, were analyzed experimentally in white-footed mice and in C3H/HeNCrl mice over a time period of almost 3 months. We found that the initially high transmission efficiency from white-footed mice to ticks declined sharply for isolate B348 but remained considerably high for isolate BL206. In contrast, in C3H/HeNCrl mice, high transmission efficiency persisted for both isolates. Our findings provide proof-of-principle evidence for intrinsic fitness variation of B. burgdorferi strains in vertebrate host species, perhaps indicating the beginnings of adaptive radiation.

Purpose: To investigate the functional status and quality of life of patients at a low-vision clinic and to evaluate the impact of low-vision services., Methods: Interviews, including the Medical Outcomes Study 36-Item Short Form (SF-36), the Visual Function-14 (VF-14), and the 51-item Field Test Version of the National Eye Institute Visual Functioning Questionnaire (NEI-VFQ), were administered to 156 consecutive patients 1 week before and 3 months after their low-vision clinic visit., Results: Low-vision patients scored lower (P < .001) in physical functioning and role limitations caused by physical and emotional health problems than published SF-36 scores of the age-appropriate United States normal population, patients with congestive heart failure, and clinically depressed patients. Low-vision services were associated with improvement in the subjective functional status of 150 patients (98.7%) and were rated "very useful" by 82 (53.9%) patients. The SF-36 scores did not change significantly after low-vision services. The VF-14 mean score improved from 35.8 to 41.2 (P < .001). Four NEI-VFQ subscale scores improved significantly (P < .001): general vision, near activities, distance activities, and peripheral vision., Conclusions: The SF-36, VF-14, and NEI-VFQ demonstrate that low-vision clinic patients perceive marked impairment of functional status and quality of life. Low-vision services are associated with high patient satisfaction. Vision-targeted questionnaires are more sensitive than general health-related quality of life questionnaires to changes in functional status and quality of life after low-vision services, and they may help elucidate the outcomes of low-vision services.

Residue levels of parathion methyl on field-sprayed Granny-Smith apples were studied. The pesticide was applied according to producer recommendations. Apples received a single spraying at a rate of 40 g active ingredient/100 L. Residues were determined with a simple gas chromatographic method. Recovery of parathion methyl was 88-108%, and the limit of determination was 0.002 mg/kg. Decomposition of parathion methyl was studied in apples remaining on trees after spraying and in apples harvested and stored under ambient-temperature, refrigerated-room, and controlled-atmosphere conditions. During post harvest storage, parathion methyl degrades more slowly than on apples remaining on the trees. Best-fit curves were determined, and kinetic equations, rate constants, and half-lives were calculated. Half-lives found were 8 days for apples on trees, 45 days for apples stored at ambient conditions, 68 days for apples stored in controlled-atmosphere room, and 62 days for apples stored in a refrigerated room. Under storage conditions, levels of parathion methyl residues need a long time to become lower than the legal limit (0.2 mg/kg).

Although numerous operations have been devised for lymphedema, most surgeons manage this vexing condition by nonsurgical means. Previous studies by us showed that high-pressure (90 to 100 torr) sequential external pneumatic compression (SEP) reduced both limb girth and volume in a lymphedematous extremity. To assess the long-term effects of a program entailing (1) SEP, (2) elastic compression stockings to maintain the post-SEP girth, and (3) daily skin care, we reviewed the long-term courses of 49 patients managed by one surgeon. Limb girths measured at nine levels on the limb were obtained serially in follow-up (mean 25 months) by an independent observer to provide an objective response to therapy. The relative reduction in lymphedematous tissue was determined by the difference between the pretreatment, postacute treatment, and long-term treatment girths at nine points in the limb. In long-term follow-up, 26 of the patients maintained a full response (reduction at > 3 levels), whereas 10 maintained a partial response (reduction at < or = 3 levels). At late follow-up, calf and ankle girths were reduced by an absolute value of 5.37 +/- 1.01 and 4.63 +/- 0.88 cm in the full-response group and 5.43 +/- 1.58 and 3.98 +/- 1.18 cm in the partial-response group over pretreatment measurements. The degree of subcutaneous fibrosis in relationship to the duration of the edema appeared to influence results greatly. The treatment of lymphedema with SEP and compression stockings is associated with long-term maintenance of reduced limb girth in 90% of patients.

In a study of 56 patients, the old glasses of a previously refracted individual were as close to the final subjective refraction as the reading obtained with a 6600 Autorefractor. Therefore, the previous spectacle correction might logically be used for the starting point of a subjective refraction, with retinoscopy or automatic refractor results being used for this purpose in patients whose previous spectacle refraction is unknown or in the first refraction of an aphakic patients.

The 6600 Autorefractor was evaluated on 200 patients undergoing refraction. The automatic refractor showed a high degree of accuracy in determining the needed refractive correction, but the errors are sufficient that the automatic refractor alone without subjective refinement cannot be substituted for conventional complete refracting methods with subjective refinement. However, for aphakic patients and for patients with clear media and cylindric corrections over 0.50 diopters, the automatic refractor can be used as a substitute for retinoscopy in determining the starting point for a subjective refraction.