Promoter optimisation of lentiviral vectors for efficient insulin gene expression in canine mesenchymal stromal cells: potential surrogate beta cells.

BACKGROUND: The lack of an ideal cell type that can be easily acquired, modified to produce insulin, and re-implanted has been a limitation for ex vivo insulin gene therapy. Canine diabetes is currently treated with human insulin and is a good model for human diabetes. Mesenchymal stromal cells (MSCs) are a promising candidate cell type for gene therapy. In the present study, we optimised insulin production using lentiviral transduced canine MSCs (cMSCs), aiming to evaluate their ability for use as surrogate beta cells. METHODS: Canine MSCs were derived from bone marrow and validated by measuring the expression of MSC lineage specific markers. Lentivirus vectors encoding the proinsulin gene (with or without a Kozak sequence) under the control of spleen focus forming virus, cytomegalovirus, elongation factor 1α and simian virus 40 promotors were generated and used to transduce primary cMSCs and a hepatocyte cell line. The insulin-producing capacity of transduced primary cMSCs was assessed by measuring the concentration of C-peptide produced. RESULTS: Primary cMSC could be readily expanded in culture and efficiently transduced using lentiviral vectors encoding proinsulin. Increasing the multiplicity of infection from 3 to 20 led to an increase in C-peptide secretion (from 1700 to 4000 pmol/l). The spleen focus forming virus promoter conferred the strongest transcriptional ability. CONCLUSIONS: The results of the present study suggest that optimised lentiviral transduction of the insulin gene into primary cMSCs renders these cells capable of secreting insulin over both the short- and long-term, in sufficient quantities in vitro to support their potential use in insulin gene therapy.The study was funded by the Lollipop Trust. Work in the laboratory is supported by the Biomedical Research Centre.This is the final version of the article. It first appeared from Wiley via https://doi.org/10.1002/jgm.290

The Ctf18 RFC-like complex positions yeast telomeres but does not specify their replication time

Peer reviewedPreprin

Serum methylarginines and spirometry-measured lung function in older adults

Rationale: Methylarginines are endogenous nitric oxide synthase inhibitors that have been implicated in animal models of lung disease but have not previously been examined for their association with spirometric measures of lung function in humans. Objectives: This study measured serum concentrations of asymmetric and symmetric dimethylarginine in a representative sample of older community-dwelling adults and determined their association with spirometric lung function measures. Methods: Data on clinical, lifestyle, and demographic characteristics, methylated arginines, and L-arginine (measured using LC-MS/MS) were collected from a population-based sample of older Australian adults from the Hunter Community Study. The five key lung function measures included as outcomes were Forced Expiratory Volume in 1 second, Forced Vital Capacity, Forced Expiratory Volume in 1 second to Forced Vital Capacity ratio, Percent Predicted Forced Expiratory Volume in 1 second, and Percent Predicted Forced Vital Capacity. Measurements and Main Results: In adjusted analyses there were statistically significant independent associations between a) higher asymmetric dimethylarginine, lower Forced Expiratory Volume in 1 second and lower Forced Vital Capacity; and b) lower L-arginine/asymmetric dimethylarginine ratio, lower Forced Expiratory Volume in 1 second, lower Percent Predicted Forced Expiratory Volume in 1 second and lower Percent Predicted Forced Vital Capacity. By contrast, no significant associations were observed between symmetric dimethylarginine and lung function. Conclusions: After adjusting for clinical, demographic, biochemical, and pharmacological confounders, higher serum asymmetric dimethylarginine was independently associated with a reduction in key measures of lung function. Further research is needed to determine if methylarginines predict the decline in lung function

B Cells Regulate Neutrophilia during Mycobacterium tuberculosis Infection and BCG Vaccination by Modulating the Interleukin-17 Response

We have previously demonstrated that B cells can shape the immune response to Mycobacterium tuberculosis, including the level of neutrophil infiltration and granulomatous inflammation at the site of infection. The present study examined the mechanisms by which B cells regulate the host neutrophilic response upon exposure to mycobacteria and how neutrophilia may influence vaccine efficacy. To address these questions, a murine aerosol infection tuberculosis (TB) model and an intradermal (ID) ear BCG immunization mouse model, involving both the μMT strain and B cell-depleted C57BL/6 mice, were used. IL (interleukin)-17 neutralization and neutrophil depletion experiments using these systems provide evidence that B cells can regulate neutrophilia by modulating the IL-17 response during M. tuberculosis infection and BCG immunization. Exuberant neutrophilia at the site of immunization in B cell-deficient mice adversely affects dendritic cell (DC) migration to the draining lymph nodes and attenuates the development of the vaccine-induced Th1 response. The results suggest that B cells are required for the development of optimal protective anti-TB immunity upon BCG vaccination by regulating the IL-17/neutrophilic response. Administration of sera derived from M. tuberculosis-infected C57BL/6 wild-type mice reverses the lung neutrophilia phenotype in tuberculous μMT mice. Together, these observations provide insight into the mechanisms by which B cells and humoral immunity modulate vaccine-induced Th1 response and regulate neutrophila during M. tuberculosis infection and BCG immunization. © 2013 Kozakiewicz et al

Discrimination of low missing energy look-alikes at the LHC

The problem of discriminating possible scenarios of TeV scale new physics with large missing energy signature at the Large Hadron Collider (LHC) has received some attention in the recent past. We consider the complementary, and yet unexplored, case of theories predicting much softer missing energy spectra. As there is enough scope for such models to fake each other by having similar final states at the LHC, we have outlined a systematic method based on a combination of different kinematic features which can be used to distinguish among different possibilities. These features often trace back to the underlying mass spectrum and the spins of the new particles present in these models. As examples of "low missing energy look-alikes", we consider Supersymmetry with R-parity violation, Universal Extra Dimensions with both KK-parity conserved and KK-parity violated and the Littlest Higgs model with T-parity violated by the Wess-Zumino-Witten anomaly term. Through detailed Monte Carlo analysis of the four and higher lepton final states predicted by these models, we show that the models in their minimal forms may be distinguished at the LHC, while non-minimal variations can always leave scope for further confusion. We find that, for strongly interacting new particle mass-scale ~600 GeV (1 TeV), the simplest versions of the different theories can be discriminated at the LHC running at sqrt{s}=14 TeV within an integrated luminosity of 5 (30) fb^{-1}.Comment: 40 pages, 10 figures; v2: Further discussions, analysis and one figure added, ordering of certain sections changed, minor modifications in the abstract, version as published in JHE

Family-led rehabilitation after stroke in India (ATTEND): a randomised controlled trial

Background: Most people with stroke in India have no access to organised rehabilitation services. The effectiveness of training family members to provide stroke rehabilitation is uncertain. Our primary objective was to determine whether family-led stroke rehabilitation, initiated in hospital and continued at home, would be superior to usual care in a low-resource setting. Methods: The Family-led Rehabilitation after Stroke in India (ATTEND) trial was a prospectively randomised open trial with blinded endpoint done across 14 hospitals in India. Patients aged 18 years or older who had had a stroke within the past month, had residual disability and reasonable expectation of survival, and who had an informal family-nominated caregiver were randomly assigned to intervention or usual care by site coordinators using a secure web-based system with minimisation by site and stroke severity. The family members of participants in the intervention group received additional structured rehabilitation training—including information provision, joint goal setting, carer training, and task-specific training—that was started in hospital and continued at home for up to 2 months. The primary outcome was death or dependency at 6 months, defined by scores 3–6 on the modified Rankin scale (range, 0 [no symptoms] to 6 [death]) as assessed by masked observers. Analyses were by intention to treat. This trial is registered with Clinical Trials Registry-India (CTRI/2013/04/003557), Australian New Zealand Clinical Trials Registry (ACTRN12613000078752), and Universal Trial Number (U1111-1138-6707). Findings: Between Jan 13, 2014, and Feb 12, 2016, 1250 patients were randomly assigned to intervention (n=623) or control (n=627) groups. 33 patients were lost to follow-up (14 intervention, 19 control) and five patients withdrew (two intervention, three control). At 6 months, 285 (47%) of 607 patients in the intervention group and 287 (47%) of 605 controls were dead or dependent (odds ratio 0·98, 95% CI 0·78–1·23, p=0·87). 72 (12%) patients in the intervention group and 86 (14%) in the control group died (p=0·27), and we observed no difference in rehospitalisation (89 [14%]patients in the intervention group vs 82 [13%] in the control group; p=0·56). We also found no difference in total non-fatal events (112 events in 82 [13%] intervention patients vs 110 events in 79 [13%] control patients; p=0·80). Interpretation: Although task shifting is an attractive solution for health-care sustainability, our results do not support investment in new stroke rehabilitation services that shift tasks to family caregivers, unless new evidence emerges. A future avenue of research should be to investigate the effects of task shifting to health-care assistants or team-based community care. Funding: The National Health and Medical Research Council of Australia

Phenomenology of Light Sneutrino Dark Matter in cMSSM/mSUGRA with Inverse Seesaw

We study the possibility of a light Dark Matter (DM) within a constrained Minimal Supersymmetric Standard Model (cMSSM) framework augmented by a SM singlet-pair sector to account for the non-zero neutrino masses by inverse seesaw mechanism. Working within a 'hybrid' scenario with the MSSM sector fixed at high scale and the singlet neutrino sector at low scale, we find that, contrary to the case of the usual cMSSM where the neutralino DM cannot be very light, we can have a light sneutrino DM with mass below 100 GeV satisfying all the current experimental constraints from cosmology, collider as well as low-energy experiments. We also note that the supersymmetric inverse seesaw mechanism with sneutrino as the lightest supersymmetric partner can have enhanced same-sign dilepton final states with large missing transverse energy (mET) coming from the gluino- and squark-pair as well as the squark-gluino associated productions and their cascade decay through charginos. We present a collider study for the same-sign dilepton+jets+mET signal in this scenario and propose some distinctions with the usual cMSSM. We also comment on the implications of such a light DM scenario on the invisible decay width of an 125 GeV Higgs boson.Comment: 24 pages, 4 figures, 7 tables; matches published versio

Study of the Decays B0 --> D(*)+D(*)-

The decays B0 --> D*+D*-, B0 --> D*+D- and B0 --> D+D- are studied in 9.7 million Y(4S) --> BBbar decays accumulated with the CLEO detector. We determine Br(B0 --> D*+D*-) = (9.9+4.2-3.3+-1.2)e-4 and limit Br(B0 --> D*+D-) < 6.3e-4 and Br(B0 --> D+D-) < 9.4e-4 at 90% confidence level (CL). We also perform the first angular analysis of the B0 --> D*+D*- decay and determine that the CP-even fraction of the final state is greater than 0.11 at 90% CL. Future measurements of the time dependence of these decays may be useful for the investigation of CP violation in neutral B meson decays.Comment: 21 pages, 5 figures, submitted to Phys. Rev.

Improved Measurement of the Pseudoscalar Decay Constant fDsf_{D_{s}}

We present a new determination of the Ds decay constant, f_{Ds} using 5 million continuum charm events obtained with the CLEO II detector. Our value is derived from our new measured ratio of widths for Ds -> mu nu/Ds -> phi pi of 0.173+/- 0.021 +/- 0.031. Taking the branching ratio for Ds -> phi pi as (3.6 +/- 0.9)% from the PDG, we extract f_{Ds} = (280 +/- 17 +/- 25 +/- 34){MeV}. We compare this result with various model calculations.Comment: 23 page postscript file, postscript file also available through http://w4.lns.cornell.edu/public/CLN