Rapamycin‐mediated lifespan increase in mice is dose and sex dependent and metabolically distinct from dietary restriction

Rapamycin, an inhibitor of mTOR kinase, increased median lifespan of genetically heterogeneous mice by 23% (males) to 26% (females) when tested at a dose threefold higher than that used in our previous studies; maximal longevity was also increased in both sexes. Rapamycin increased lifespan more in females than in males at each dose evaluated, perhaps reflecting sexual dimorphism in blood levels of this drug. Some of the endocrine and metabolic changes seen in diet‐restricted mice are not seen in mice exposed to rapamycin, and the pattern of expression of hepatic genes involved in xenobiotic metabolism is also quite distinct in rapamycin‐treated and diet‐restricted mice, suggesting that these two interventions for extending mouse lifespan differ in many respects.     

A 52-week,open-label study evaluating the safety and efficacy of tabalumab,an anti-B-cell–activating factor monoclonal antibody,for rheumatoid arthritis

Introduction

The objective of this study was to evaluate the long-term safety and efficacy of tabalumab, a monoclonal antibody that neutralizes membrane-bound and soluble B-cell-activating factor, in rheumatoid arthritis (RA) patients.

Methods

Patients with RA who completed one of two 24-week randomized controlled trials (RCTs) participated in this 52-week, flexible-dose, open-label extension study. Patients in RCT1 received intravenous placebo, 30-mg tabalumab or 80-mg tabalumab every 3 weeks, and patients in RCT2 received subcutaneous placebo or 1-, 3-, 10-, 30-, 60- or 120-mg tabalumab every 4 weeks (Q4W). Regardless of prior treatment, all patients in this study received subcutaneous 60-mg tabalumab Q4W for the first 3 months, then a one-time increase to 120-mg tabalumab Q4W (60-mg/120-mg group) and a one-time decrease to 60-mg tabalumab Q4W per patient was allowed (60-mg/120-mg/60-mg group).

Results

There were 182 patients enrolled: 60 mg (n = 60), 60/120 mg (n = 121) and 60/120/60 mg (n = 1). Pretabalumab baseline disease activity was generally higher in the 60-mg/120-mg group. There was a higher frequency of serious adverse events and treatment-emergent adverse events, as well as infections and injection-site reactions, in the 60-mg/120-mg group. One death unrelated to the study drug occurred (60-mg/120-mg group). In both groups, total B-cell counts decreased by approximately 40% from the baseline level in the RCT originating study. Both groups demonstrated efficacy through 52 weeks of treatment relative to baseline pretabalumab disease activity based on American College of Rheumatology criteria improvement ≥20%, ≥50% and ≥70%; European League against Rheumatism Responder Index in 28 joints; Disease Activity Score in 28 joints–C-reactive protein; and Health Assessment Questionnaire–Disability Index.

Conclusions

With long-term, open-label tabalumab treatment, no unexpected safety signals were observed, and B-cell reductions were consistent with previous findings. Despite differences in RCT originating studies, both groups demonstrated an efficacy response through the 52-week extension.

Trial registration

ClinicalTrials.gov Identifier: {"type":"clinical-trial","attrs":{"text":"NCT00837811","term_id":"NCT00837811"}}NCT00837811 (registered 3 February 2009).

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-014-0415-2) contains supplementary material, which is available to authorized users.     

Applying Circuit Theory for Corridor Expansion and Management at Regional Scales: Tiling,Pinch Points,and Omnidirectional Connectivity

Connectivity models are useful tools that improve the ability of researchers and managers to plan land use for conservation and preservation. Most connectivity models function in a point-to-point or patch-to-patch fashion, limiting their use for assessing connectivity over very large areas. In large or highly fragmented systems, there may be so many habitat patches of interest that assessing connectivity among all possible combinations is prohibitive. To overcome these conceptual and practical limitations, we hypothesized that minor adaptation of the Circuitscape model can allow the creation of omnidirectional connectivity maps illustrating flow paths and variations in the ease of travel across a large study area. We tested this hypothesis in a 24,300 km² study area centered on the Montérégie region near Montréal, Québec. We executed the circuit model in overlapping tiles covering the study region. Current was passed across the surface of each tile in orthogonal directions, and then the tiles were reassembled to create directional and omnidirectional maps of connectivity. The resulting mosaics provide a continuous view of connectivity in the entire study area at the full original resolution. We quantified differences between mosaics created using different tile and buffer sizes and developed a measure of the prominence of seams in mosaics formed with this approach. The mosaics clearly show variations in current flow driven by subtle aspects of landscape composition and configuration. Shown prominently in mosaics are pinch points, narrow corridors where organisms appear to be required to traverse when moving through the landscape. Using modest computational resources, these continuous, fine-scale maps of nearly unlimited size allow the identification of movement paths and barriers that affect connectivity. This effort develops a powerful new application of circuit models by pinpointing areas of importance for conservation, broadening the potential for addressing intriguing questions about resource use, animal distribution, and movement.     

Evaluation of Schistosome Promoter Expression for Transgenesis and Genetic Analysis

Schistosome worms of the genus Schistosoma are the causative agents of schistosomiasis, a devastating parasitic disease affecting more than 240 million people worldwide. Schistosomes have complex life cycles, and have been challenging to manipulate genetically due to the dearth of molecular tools. Although the use of gene overexpression, gene knockouts or knockdowns are straight-forward genetic tools applied in many model systems, gene misexpression and genetic manipulation of schistosome genes in vivo has been exceptionally challenging, and plasmid based transfection inducing gene expression is limited. We recently reported the use of polyethyleneimine (PEI) as a simple and effective method for schistosome transfection and gene expression. Here, we use PEI-mediated schistosome plasmid transgenesis to define and compare gene expression profiles from endogenous and nonendogenous promoters in the schistosomula stage of schistosomes that are potentially useful to misexpress (underexpress or overexpress) gene product levels. In addition, we overexpress schistosome genes in vivo using a strong promoter and show plasmid-based misregulation of genes in schistosomes, producing a clear and distinct phenotype- death. These data focus on the schistosomula stage, but they foreshadow strong potential for genetic characterization of schistosome molecular pathways, and potential for use in overexpression screens and drug resistance studies in schistosomes using plasmid-based gene expression.     

Polymorphisms in K13 and Falcipain-2 Associated with Artemisinin Resistance Are Not Prevalent in Plasmodium falciparum Isolated from Ugandan Children

The emergence of resistance to artemisinin derivatives in Southeast Asia, manifested as delayed clearance of Plasmodium falciparum following treatment with artemisinins, is a major concern. Recently, the artemisinin resistance phenotype was attributed to mutations in portions of a P. falciparum gene (PF3D7_1343700) encoding kelch (K13) propeller domains, providing a molecular marker to monitor the spread of resistance. The P. falciparum cysteine protease falcipain-2 (FP2; PF3D7_1115700) has been shown to contribute to artemisinin action, as hemoglobin degradation is required for potent drug activity, and a stop mutation in the FP2 gene was identified in parasites selected for artemisinin resistance. Although delayed parasite clearance after artemisinin-based combination therapy (ACT) has not yet been noted in Uganda and ACTs remain highly efficacious, characterizing the diversity of these genes is important to assess the potential for resistance selection and to provide a baseline for future surveillance. We therefore sequenced the K13-propeller domain and FP2 gene in P. falciparum isolates from children previously treated with ACT in Uganda, including samples from 2006–7 (n = 49) and from 2010–12 (n = 175). Using 3D7 as the reference genome, we identified 5 non-synonymous polymorphisms in the K13-propeller domain (133 isolates) and 35 in FP2 (160 isolates); these did not include the polymorphisms recently associated with resistance after in vitro selection or identified in isolates from Asia. The prevalence of K13-propeller and FP2 polymorphisms did not increase over time, and was not associated with either time since prior receipt of an ACT or the persistence of parasites ≥2 days following treatment with an ACT. Thus, the K13-propeller and FP2 polymorphisms associated with artemisinin resistance are not prevalent in Uganda, and we did not see evidence for selection of polymorphisms in these genes.     

Initial Characterization of the FlgE Hook High Molecular Weight Complex of Borrelia burgdorferi

The spirochete periplasmic flagellum has many unique attributes. One unusual characteristic is the flagellar hook. This structure serves as a universal joint coupling rotation of the membrane-bound motor to the flagellar filament. The hook is comprised of about 120 FlgE monomers, and in most bacteria these structures readily dissociate to monomers (∼ 50 kDa) when treated with heat and detergent. However, in spirochetes the FlgE monomers form a large mass of over 250 kDa [referred to as a high molecular weight complex (HMWC)] that is stable to these and other denaturing conditions. In this communication, we examined specific aspects with respect to the formation and structure of this complex. We found that the Lyme disease spirochete Borrelia burgdorferi synthesized the HMWC throughout the in vitro growth cycle, and also in vivo when implanted in dialysis membrane chambers in rats. The HMWC was stable to formic acid, which supports the concept that the stability of the HMWC is dependent on covalent cross-linking of individual FlgE subunits. Mass spectrometry analysis of the HMWC from both wild type periplasmic flagella and polyhooks from a newly constructed ΔfliK mutant indicated that other proteins besides FlgE were not covalently joined to the complex, and that FlgE was the sole component of the complex. In addition, mass spectrometry analysis also indicated that the HMWC was composed of a polymer of the FlgE protein with both the N- and C-terminal regions remaining intact. These initial studies set the stage for a detailed characterization of the HMWC. Covalent cross-linking of FlgE with the accompanying formation of the HMWC we propose strengthens the hook structure for optimal spirochete motility.     

A MIV-150/Zinc Acetate Gel Inhibits SHIV-RT Infection in Macaque Vaginal Explants

To extend our observations that single or repeated application of a gel containing the NNRTI MIV-150 (M) and zinc acetate dihydrate (ZA) in carrageenan (CG) (MZC) inhibits vaginal transmission of simian/human immunodeficiency virus (SHIV)-RT in macaques, we evaluated safety and anti-SHIV-RT activity of MZC and related gel formulations ex vivo in macaque mucosal explants. In addition, safety was further evaluated in human ectocervical explants. The gels did not induce mucosal toxicity. A single ex vivo exposure to diluted MZC (1∶30, 1∶100) and MC (1∶30, the only dilution tested), but not to ZC gel, up to 4 days prior to viral challenge, significantly inhibited SHIV-RT infection in macaque vaginal mucosa. MZC''s activity was not affected by seminal plasma. The antiviral activity of unformulated MIV-150 was not enhanced in the presence of ZA, suggesting that the antiviral activity of MZC was mediated predominantly by MIV-150. In vivo administration of MZC and CG significantly inhibited ex vivo SHIV-RT infection (51–62% inhibition relative to baselines) of vaginal (but not cervical) mucosa collected 24 h post last gel exposure, indicating barrier effect of CG. Although the inhibitory effect of MZC (65–74%) did not significantly differ from CG (32–45%), it was within the range of protection (∼75%) against vaginal SHIV-RT challenge 24 h after gel dosing. Overall, the data suggest that evaluation of candidate microbicides in macaque explants can inform macaque efficacy and clinical studies design. The data support advancing MZC gel for clinical evaluation.     

Unbiased Identification of Patients with Disorders of Sex Development

Disorders of sex development (DSD) represent a collection of rare diseases that generate substantial controversy regarding best practices for diagnosis and treatment. A significant barrier preventing a better understanding of how patients with these conditions should be evaluated and treated, especially from a psychological standpoint, is the lack of systematic and standardized approaches to identify cases for study inclusion. Common approaches include “hand-picked” subjects already known to the practice, which could introduce bias. We implemented an informatics-based approach to identify patients with DSD from electronic health records (EHRs) at three large, academic children’s hospitals. The informatics approach involved comprehensively searching EHRs at each hospital using a combination of structured billing codes as an initial filtering strategy followed by keywords applied to the free text clinical documentation. The informatics approach was implemented to replicate the functionality of an EHR search engine (EMERSE) available at one of the hospitals. At the two hospitals that did not have EMERSE, we compared case ascertainment using the informatics method to traditional approaches employed for identifying subjects. Potential cases identified using all approaches were manually reviewed by experts in DSD to verify eligibility criteria. At the two institutions where both the informatics and traditional approaches were applied, the informatics approach identified substantially higher numbers of potential study subjects. The traditional approaches yielded 14 and 28 patients with DSD, respectively; the informatics approach yielded 226 and 77 patients, respectively. The informatics approach missed only a few cases that the traditional approaches identified, largely because those cases were known to the study team, but patient data were not in the particular children’s hospital EHR. The use of informatics approaches to search electronic documentation can result in substantially larger numbers of subjects identified for studies of rare diseases such as DSD, and these approaches can be applied across hospitals.     

Large Scale Spatial Risk and Comparative Prevalence of Borrelia miyamotoi and Borrelia burgdorferi Sensu Lato in Ixodes pacificus

Borrelia miyamotoi is a newly described emerging pathogen transmitted to people by Ixodes species ticks and found in temperate regions of North America, Europe, and Asia. There is limited understanding of large scale entomological risk patterns of B. miyamotoi and of Borreila burgdorferi sensu stricto (ss), the agent of Lyme disease, in western North America. In this study, B. miyamotoi, a relapsing fever spirochete, was detected in adult (n = 70) and nymphal (n = 36) Ixodes pacificus ticks collected from 24 of 48 California counties that were surveyed over a 13 year period. Statewide prevalence of B. burgdorferi sensu lato (sl), which includes B. burgdorferi ss, and B. miyamotoi were similar in adult I. pacificus (0.6% and 0.8%, respectively). In contrast, the prevalence of B. burgdorferi sl was almost 2.5 times higher than B. miyamotoi in nymphal I. pacificus (3.2% versus 1.4%). These results suggest similar risk of exposure to B. burgdorferi sl and B. miyamotoi from adult I. pacificus tick bites in California, but a higher risk of contracting B. burgdorferi sl than B. miyamotoi from nymphal tick bites. While regional risk of exposure to these two spirochetes varies, the highest risk for both species is found in north and central coastal California and the Sierra Nevada foothill region, and the lowest risk is in southern California; nevertheless, tick-bite avoidance measures should be implemented in all regions of California. This is the first study to comprehensively evaluate entomologic risk for B. miyamotoi and B. burgdorferi for both adult and nymphal I. pacificus, an important human biting tick in western North America.