New method for the orthogonal labeling and purification of Toxoplasma gondii proteins while inside the host cell

oxoplasma gondii is an obligate intracellular protozoan parasite that is capable of causing severe disease in immunocompromised humans. How T. gondii is able to modulate the host cell to support itself is still poorly understood. Knowledge pertaining to the host-parasite interaction could be bolstered by developing a system to specifically label parasite proteins while the parasite grows inside the host cell. For this purpose, we have created a strain of T. gondii that expresses a mutant Escherichia coli methionyl-tRNA synthetase (MetRSNLL) that allows methionine tRNA to be loaded with the azide-containing methionine analog azidonorleucine (Anl). Anl-containing proteins are susceptible to a copper-catalyzed “click” reaction to attach affinity tags for purification or fluorescent tags for visualization. The MetRSNLL-Anl system labels nascent T. gondii proteins in an orthogonal fashion, labeling proteins only in MetRSNLL-expressing parasites. This system should be useful for nonradioactive pulse-chase studies and purification of nascently translated proteins. Although this approach allows labeling of a diverse array of parasite proteins, secreted parasite proteins appear to be only minimally labeled in MetRSNLL-expressing T. gondii. The minimal labeling of secreted proteins is likely a consequence of the selective charging of the initiator tRNA (and not the elongator methionine tRNA) by the heterologously expressed bacterial MetRS. IMPORTANCE Studying how T. gondii modifies the host cell to permit its survival is complicated by the complex protein environment of the host cell. The approach presented in this article provides the first method for specific labeling of T. gondii proteins while the parasite grows inside the host cell. We show that this approach is useful for pulse-chase labeling of parasite proteins during in vitro growth. It should also be applicable during in vivo infections and in other apicomplexan parasites, including Plasmodium spp

Primary prevention of diabetes mellitus type 2 and cardiovascular diseases using a cognitive behavior program aimed at lifestyle changes in people at risk: Design of a randomized controlled trial

<p>Abstract</p> <p>Background</p> <p>The number of people with cardiovascular disease (CVD) and diabetes mellitus type 2 (T2DM) is growing rapidly. To a large extend, this increase is due to lifestyle-dependent risk factors, such as overweight, reduced physical activity, and an unhealthy diet. Changing these risk factors has the potential to postpone or prevent the development of T2DM and CVD. It is hypothesized that a cognitive behavioral program (CBP), focused in particular on motivation and self-management in persons who are at high risk for CVD and/or T2DM, will improve their lifestyle behavior and, as a result, will reduce their risk of developing T2DM and CVD.</p> <p>Methods</p> <p>12,000 inhabitants, 30-50 years of age living in several municipalities in the semi-rural region of West-Friesland will receive an invitation from their general practitioner (n = 13) to measure their own waist circumference with a tape measure. People with abdominal obesity (male waist ≥ 102 cm, female waist ≥ 88 cm) will be invited to participate in the second step of the screening which includes blood pressure, a blood sample and anthropometric measurements. T2DM and CVD risk scores will then be calculated according to the ARIC and the SCORE formulae, respectively. People with a score that indicates a high risk of developing T2DM and/or CVD will then be randomly assigned to the intervention group (n = 300) or the control group (n = 300).</p> <p>Participants in the intervention group will follow a CBP aimed at modifying their dietary behavior, physical activity, and smoking behavior. The counseling methods that will be used are <it>motivational interviewing </it>(MI) and <it>problem solving treatment </it>(PST), which focus in particular on intrinsic motivation for change and self-management of problems of the participants. The CBP will be provided by trained nurse practitioners in the participant's general practice, and will consists of a maximum of six individual sessions of 30 minutes, followed by 3-monthly booster sessions by phone. Participants in the control group will receive brochures containing health guidelines regarding physical activity and diet, and how to stop smoking. The primary outcome measures will be changes in T2DM and CVD risk scores. Secondary outcome measures will be changes in lifestyle behavior and cost-effectiveness and cost-utility ratios. All relevant direct and indirect costs will be measured, and there will be a follow-up of 24 months.</p> <p>Discussion</p> <p>Changing behaviors is difficult, requires time, considerable effort and motivation. Combining the two counseling methods MI and PST, followed by booster sessions may result in sustained behavioral change.</p> <p>Trial registration</p> <p>Current Controlled Trials ISRCTN59358434</p

Systematic Review of Potential Health Risks Posed by Pharmaceutical, Occupational and Consumer Exposures to Metallic and Nanoscale Aluminum, Aluminum Oxides, Aluminum Hydroxide and Its Soluble Salts

Aluminum (Al) is a ubiquitous substance encountered both naturally (as the third most abundant element) and intentionally (used in water, foods, pharmaceuticals, and vaccines); it is also present in ambient and occupational airborne particulates. Existing data underscore the importance of Al physical and chemical forms in relation to its uptake, accumulation, and systemic bioavailability. The present review represents a systematic examination of the peer-reviewed literature on the adverse health effects of Al materials published since a previous critical evaluation compiled by Krewski et al. (2007). Challenges encountered in carrying out the present review reflected the experimental use of different physical and chemical Al forms, different routes of administration, and different target organs in relation to the magnitude, frequency, and duration of exposure. Wide variations in diet can result in Al intakes that are often higher than the World Health Organization provisional tolerable weekly intake (PTWI), which is based on studies with Al citrate. Comparing daily dietary Al exposures on the basis of “total Al”assumes that gastrointestinal bioavailability for all dietary Al forms is equivalent to that for Al citrate, an approach that requires validation. Current occupational exposure limits (OELs) for identical Al substances vary as much as 15-fold. The toxicity of different Al forms depends in large measure on their physical behavior and relative solubility in water. The toxicity of soluble Al forms depends upon the delivered dose of Al+ 3 to target tissues. Trivalent Al reacts with water to produce bidentate superoxide coordination spheres [Al(O2)(H2O4)+ 2 and Al(H2O)6 + 3] that after complexation with O2•−, generate Al superoxides [Al(O2•)](H2O5)]+ 2. Semireduced AlO2• radicals deplete mitochondrial Fe and promote generation of H2O2, O2 • − and OH•. Thus, it is the Al+ 3-induced formation of oxygen radicals that accounts for the oxidative damage that leads to intrinsic apoptosis. In contrast, the toxicity of the insoluble Al oxides depends primarily on their behavior as particulates. Aluminum has been held responsible for human morbidity and mortality, but there is no consistent and convincing evidence to associate the Al found in food and drinking water at the doses and chemical forms presently consumed by people living in North America and Western Europe with increased risk for Alzheimer\u27s disease (AD). Neither is there clear evidence to show use of Al-containing underarm antiperspirants or cosmetics increases the risk of AD or breast cancer. Metallic Al, its oxides, and common Al salts have not been shown to be either genotoxic or carcinogenic. Aluminum exposures during neonatal and pediatric parenteral nutrition (PN) can impair bone mineralization and delay neurological development. Adverse effects to vaccines with Al adjuvants have occurred; however, recent controlled trials found that the immunologic response to certain vaccines with Al adjuvants was no greater, and in some cases less than, that after identical vaccination without Al adjuvants. The scientific literature on the adverse health effects of Al is extensive. Health risk assessments for Al must take into account individual co-factors (e.g., age, renal function, diet, gastric pH). Conclusions from the current review point to the need for refinement of the PTWI, reduction of Al contamination in PN solutions, justification for routine addition of Al to vaccines, and harmonization of OELs for Al substances

The effectiveness of Robot-Assisted Gait Training versus conventional therapy on mobility in severely disabled progressIve MultiplE sclerosis patients (RAGTIME): Study protocol for a randomized controlled trial

Background: Gait and mobility impairments affect the quality of life (QoL) of patients with progressive multiple sclerosis (MS). Robot-assisted gait training (RAGT) is an effective rehabilitative treatment but evidence of its superiority compared to other options is lacking. Furthermore, the response to rehabilitation is multidimensional, person-specific and possibly involves functional reorganization processes. The aims of this study are: (1) to test the effectiveness on gait speed, mobility, balance, fatigue and QoL of RAGT compared to conventional therapy (CT) in progressive MS and (2) to explore changes of clinical and circulating biomarkers of neural plasticity. Methods: This will be a parallel-group, randomized controlled trial design with the assessor blinded to the group allocation of participants. Ninety-eight (49 per arm) progressive MS patients (EDSS scale 6-7) will be randomly assigned to receive twelve 2-h training sessions over a 4-week period (three sessions/week) of either: (1) RAGT intervention on a robotic-driven gait orthosis (Lokomat, Hocoma, Switzerland). The training parameters (torque of the knee and hip drives, treadmill speed, body weight support) are set during the first session and progressively adjusted during training progression or (2) individual conventional physiotherapy focusing on over-ground walking training performed with the habitual walking device. The same assessors will perform outcome measurements at four time points: baseline (before the first intervention session); intermediate (after six training sessions); end of treatment (after the completion of 12 sessions); and follow-up (after 3 months from the end of the training program). The primary outcome is gait speed, assessed by the Timed 25-Foot Walk Test. We will also assess walking endurance, balance, depression, fatigue and QoL as well as instrumental laboratory markers (muscle metabolism, cerebral venous hemodynamics, cortical activation) and circulating laboratory markers (rare circulating cell populations pro and anti-inflammatory cytokines/chemokines, growth factors, neurotrophic factors, coagulation factors, other plasma proteins suggested by transcriptomic analysis and metabolic parameters). Discussion: The RAGT training is expected to improve mobility compared to the active control intervention in progressive MS. Unique to this study is the analysis of various potential markers of plasticity in relation with clinical outcomes. Trial registration: ClinicalTrials.gov, identifier: NCT02421731. Registered on 19 January 2015 (retrospectively registered)

Electro-Tactile Stimulation Enhances Cochlear Implant Speech Recognition in Noise

For cochlear implant users, combined electro-acoustic stimulation (EAS) significantly improves the performance. However, there are many more users who do not have any functional residual acoustic hearing at low frequencies. Because tactile sensation also operates in the same low frequencies (&lt;500 Hz) as the acoustic hearing in EAS, we propose electro-tactile stimulation (ETS) to improve cochlear implant performance. In ten cochlear implant users, a tactile aid was applied to the index finger that converted voice fundamental frequency into tactile vibrations. Speech recognition in noise was compared for cochlear implants alone and for the bimodal ETS condition. On average, ETS improved speech reception thresholds by 2.2 dB over cochlear implants alone. Nine of the ten subjects showed a positive ETS effect ranging from 0.3 to 7.0 dB, which was similar to the amount of the previously-reported EAS benefit. The comparable results indicate similar neural mechanisms that underlie both the ETS and EAS effects. The positive results suggest that the complementary auditory and tactile modes also be used to enhance performance for normal hearing listeners and automatic speech recognition for machines

Non-steady-state calcium handling in failing hearts from the spontaneously hypertensive rat

It is generally agreed that changes in Ca(2+) cycling are often associated with heart failure, yet the impact of these changes on a beat-to-beat basis remains unclear. Measurements of isometric force and [Ca(2+)](i) were made at 37°C in left ventricular trabeculae from failing spontaneously hypertensive rat (SHR) hearts, and their normotensive Wistar-Kyoto (WKY) controls. At 1 Hz, peak stress was reduced in SHR (14.5 ± 2.4 mN mm(-2) versus 22.5 ± 6.7 mN mm⁻² for WKY), although the Ca(2+) transients were bigger (peak [Ca(2+)](i) 0.60 ± 0.08 μM versus 0.38 ± 0.03 μM for WKY) with a slower decay of fluorescence (time constant 0.105 ± 0.005 s versus 0.093 ± 0.002 s for WKY). To probe dynamic Ca(2+) cycling, two experimental protocols were used to potentiate force: (1) an interval of 30 s rest, and (2) a 30-s train of paired-pulses, and the recirculation fraction (RF) calculated for recovery to steady-state. No difference was found between rat strains for RF calculated from either peak force or Ca(2+), although the RF was dependent on potentiation protocol. Since SR uptake is slower in SHR, the lack of change in RF must be due to a parallel decrease in trans-sarcolemmal Ca(2+) extrusion. This view was supported by a slower decay of caffeine-induced Ca(2+) transients in SHR trabeculae. Confocal analysis of LV free wall showed t-tubules were distorted in SHR myocytes, with reduced intensity of NCX and SERCA2a labelling in comparison to WKY