A Modular Cloning Toolkit for the production of recombinant proteins in Leishmania tarentolae.

Modular Cloning (MoClo) is based on libraries of standardized genetic parts that can be directionally assembled via Golden Gate cloning in one-pot reactions into transcription units and multigene constructs. Here, a team of bachelor students established a MoClo toolkit for the protist Leishmania tarentolae in the frame of the international Genetically Engineered Machine (iGEM) competition. Our modular toolkit is based on a domesticated version of a commercial LEXSY expression vector and comprises 34 genetic parts encoding various affinity tags, targeting signals as well as fluorescent and luminescent proteins. We demonstrated the utility of our kit by the successful production of 16 different tagged versions of the receptor binding domain (RBD) of the SARS-CoV-2 spike protein in L. tarentolae liquid cultures. While highest yields of secreted recombinant RBD were obtained for GST-tagged fusion proteins 48 h post induction, C-terminal peptide tags were often degraded and resulted in lower yields of secreted RBD. Fusing secreted RBD to a synthetic O-glycosylation SP20 module resulted in an apparent molecular mass shift around 10 kDa. No disadvantage regarding the production of RBD was detected when the three antibiotics of the LEXSY system were omitted during the 48-h induction phase. Furthermore, the successful purification of secreted RBD from the supernatant of L. tarentolae liquid cultures was demonstrated in pilot experiments. In summary, we established a MoClo toolkit and exemplified its application for the production of recombinant proteins in L. tarentolae.

Deciphering the mechanism of glutaredoxin-catalyzed roGFP2 redox sensing reveals a ternary complex with glutathione for protein disulfide reduction.

Glutaredoxins catalyze the reduction of disulfides and are key players in redox metabolism and regulation. While important insights were gained regarding the reduction of glutathione disulfide substrates, the mechanism of non-glutathione disulfide reduction remains highly debated. Here we determined the rate constants for the individual redox reactions between PfGrx, a model glutaredoxin from Plasmodium falciparum, and redox-sensitive green fluorescent protein 2 (roGFP2), a model substrate and versatile tool for intracellular redox measurements. We show that the PfGrx-catalyzed oxidation of roGFP2 occurs via a monothiol mechanism and is up to three orders of magnitude faster when roGFP2 and PfGrx are fused. The oxidation kinetics of roGFP2-PfGrx fusion constructs reflect at physiological GSSG concentrations the glutathionylation kinetics of the glutaredoxin moiety, thus allowing intracellular structure-function analysis. Reduction of the roGFP2 disulfide occurs via a monothiol mechanism and involves a ternary complex with GSH and PfGrx. Our study provides the mechanistic basis for understanding roGFP2 redox sensing and challenges previous mechanisms for protein disulfide reduction.

Commuting accidents in the German chemical industry.

Due to accident severity and the extent of claim payments commuting accidents are a significant expense factor in the German industry. Therefore the aim of the present study was the identification of risk factors for commuting accidents in a German chemical company. A retrospective analysis of commuting accidents recorded between 1990 and 2003 was conducted in a major chemical company in Germany. A logistic regression-model was calculated in order to determine factors influencing the duration of work inability as a result of commuting accidents. The analysed data included 5,484 employees with commuting accidents. Cars (33.1%) and bicycles (30.5%) were the most common types of vehicles used by commuters who had an accident. The highest number of commuting accidents was observed in the age group under 26 yr. Accidents on the route from the work site to the worker's residence were less frequently observed, but they caused longer periods of work inability than accidents on the way to the work site. The longest periods of work inability were found in the groups of motorcyclists and older employees. The present study identifies specific groups at risk for commuting accidents. The data of the present investigation also underline the need for developing group specific prevention strategies.

Comparisons of musculoskeletal complaints and data entry between a sitting and a sit-stand workstation paradigm.

BACKGROUND: Seated working positions are often regarded as a cause for discomfort in the musculoskeletal system. Performing work in different working positions--that is, alternating between sitting and standing (sit-stand workstation paradigm)--could help reduce physical complaints. OBJECTIVE: The questions were whether performing office work partly in a standing position leads to reduced complaints and whether standing would change the efficiency of data entry office work. METHOD: We investigated the effect of a sit-stand workstation paradigmd during experimental data entry office work on physical and psychological complaints and data entry efficiency by conducting a randomized controlled trial with 60 male participants ages 18 to 35 years. RESULTS: In this experiment, musculoskeletal complaints were reduced by a sit-stand workstation paradigm. A trend could be identified indicating a small but nonsignificant loss of efficiency in data entry while standing. CONCLUSION: A sit-stand workstation paradigm reduces musculoskeletal complaints without considerably affecting data entry efficiency under the presented study conditions (young male participants, short duration, fixed and controlled sit-stand workstation paradigm, simulated experimental working condition). APPLICATION: According to the present data, implementing a sit-stand workstation paradigm can be an effective workplace health intervention to reduce musculoskeletal complaints. This experiment encourages further studies on the effectiveness of a sit-stand workstation paradigm. Experimental research and field studies that prove the reduction of complaints when introducing a sit-stand workstation paradigm in the workplace could be the basis for evidence-based recommendations regarding such interventions.

Effects of locomotion training with assistance of a robot-driven gait orthosis in hemiparetic patients after stroke: a randomized controlled pilot study.

BACKGROUND AND PURPOSE: The success of gait rehabilitation after stroke depends on active walking exercises. However, the disabling after-effects of stroke often make such exercises impossible at the onset of therapy. To facilitate treadmill training of paraparetic patients, a robot-driven gait orthosis (Lokomat) was developed. We investigated the effects of the Lokomat when used with hemiparetic patients. METHODS: The authors conducted a randomized, controlled pilot study of 30 acute stroke survivors. The treatment group received 30 minutes of robotic training daily and the control group 30 minutes of conventional physiotherapy daily in addition to 30 minutes of conventional physiotherapy for each group. Outcome measures were independence of gait, gait speed, gait parameters, and body tissue composition. RESULTS: After 4 weeks of therapy, the walking ability of the Lokomat group and the control group expressed as the functional ambulation classification was significantly improved. The functional ambulation category (median+/- interquartile range) was at baseline 0+/-0 in control and 0+/-1 in the therapy group and increased after therapy to 1+/-3 in both groups significantly (P=0.01). There was no significant difference in gain of these parameters between the groups. The Lokomat group had a significantly longer single stance phase (sec; mean+/-SEM) on the paretic leg when walking on the floor. At baseline, it was 0.19+/-0.17 and after therapy 0.49+/-0.07 (P=0.014). The control group had increased their body weight approximately 1.33+/-1.40 kg (mean+/-SEM; P=0.046), mostly as fat mass, whereas the Lokomat group had lost fat mass approximately -2.9+/-1.0 kg (mean+/-SEM; P=0.016) and increased their muscle mass approximately 3.36+/-1.4 kg (mean+/- SEM; P=0.031). CONCLUSIONS: This pilot study indicates that Lokomat therapy is a promising intervention for gait rehabilitation. Although there was no difference between groups in gain of functional scores, the Lokomat group showed an advantage of robotic training over conventional physiotherapy in improvement of gait abnormality and body tissue composition.

The influence of different Lokomat walking conditions on the energy expenditure of hemiparetic patients and healthy subjects.

To determine the strenuousness and efficacy of therapy, the energy expenditure of 10 healthy and 10 hemiparetic subjects were measured while they walked on a treadmill that was combined with a robot-driven gait orthosis, the Lokomat, which physiologically exercises the legs of a patient on a moving treadmill. Subjects performed different Lokomat conditions after measurement of the baseline, i.e., standing in the Lokomat with 30% body weight support (BWS). Robotic strategies with a position control scheme used fixed gait patterns to produce the following conditions: walking with 100% BWS at a speed of 1 km/h versus 2 km/h and walking with 30% BWS at a speed of 1 km/h versus 2 km/h. Another robotic control option with a force control scheme allowed the force to be reduced on only one leg of the orthosis. In this option a reduction to 60% and to 0% assistance was tested. Oxygen consumption and heart rate were measured by a breath-by-breath respiratory gas analyzing system using standard open circuit methodology. The results for O(2) rate [ml/kg/min] indicate that: (1) walking in the Lokomat is not passive; (2) oxygen uptake is significantly increased due to an effect of loading during active stance phase; (3) speed is not a factor leading to increased oxygen consumption; (4) patients do not significantly increase their oxygen uptake due to the advanced force control scheme.