New students – old sins: elephant in the (lab)room

Laboratory exercises are important in science education as they contributeto general understanding of the topics presented in lectures and enable students to acquire hands-on experience of the scientific research. However, conscious participation in the practical courses allowing for complete achievement of their learning outcomes depends on the proper prelaboratory preparation. Unfortunately, it is a common problem that students show up under-prepared to participate in the laboratory sessions. This study focuses on medical students, and analyses the level of their pre-laboratory preparation, used techniques, resources and motivational force of the activities. The results show that students do not spend enough time for the preparation and do not use online-based materials. The main reason for under-preparation turns out to be the lack of time. This project discussesalso implementation of the possible motivational activities that could enhance the student preparedness and overall performance in the laboratory

Iron Inhibits Respiratory Burst of Peritoneal Phagocytes In Vitro

Objective. This study examines the effects of iron ions Fe3+ on the respiratory burst of phagocytes isolated from peritoneal effluents of continuous ambulatory peritoneal dialysis (CAPD) patients, as an in vitro model of iron overload in end-stage renal disease (ESRD). Material and Methods. Respiratory burst of peritoneal phagocytes was measured by chemiluminescence method. Results. At the highest used concentration of iron ions Fe3+ (100 μM), free radicals production by peritoneal phagocytes was reduced by 90% compared to control. Conclusions. Iron overload may increase the risk of infectious complications in ESRD patients

Peritonitis-induced antitumor activity of peritoneal macrophages from uremic patients

The macrophages belong to the effector cells of both nonspecific and specific immune response. These cells generally express little cytotoxicity unless activated. The present work was intended to determine if peritoneal macrophages collected from patients on Continuous Ambulatory Peritoneal Dialysis (CAPD) during episodes of peritonitis were active against human tumor cell lines without further in vitro stimulation. We also compared macrophage antitumor potential with effectiveness of drugs used in cancer therapy (taxol and suramin). Conditioned medium (CM) of macrophages collected during inflammation-free periods did not exhibit cytostatic and cytotoxic activity against both tumor (A549 and HTB44) and non-transformed (BEAS-2B and CRL2190) cells. Exposure of tumor cells to CM of macrophages harvested during peritonitis resulted in significant suppression of proliferation, impairment of viability and induction of apoptosis, in contrast to non-transformed cells, which remained unaffected. The efficacy of CM of inflammatory macrophages as an antitumor agent appeared to be comparable to cytostatic and cytotoxic potency of taxol and suramin or, in the case of HTB44 cells, even higher. The results obtained suggest that activated human macrophages might represent a useful tool for cancer immunotherapy

Peritonitis-induced antitumor activity of peritoneal macrophages from uremic patients.

The macrophages belong to the effector cells of both nonspecific and specific immune response. These cells generally express little cytotoxicity unless activated. The present work was intended to determine if peritoneal macrophages collected from patients on Continuous Ambulatory Peritoneal Dialysis (CAPD) during episodes of peritonitis were active against human tumor cell lines without further in vitro stimulation. We also compared macrophage antitumor potential with effectiveness of drugs used in cancer therapy (taxol and suramin). Conditioned medium (CM) of macrophages collected during inflammation-free periods did not exhibit cytostatic and cytotoxic activity against both tumor (A549 and HTB44) and non-transformed (BEAS-2B and CRL2190) cells. Exposure of tumor cells to CM of macrophages harvested during peritonitis resulted in significant suppression of proliferation, impairment of viability and induction of apoptosis, in contrast to non-transformed cells, which remained unaffected. The efficacy of CM of inflammatory macrophages as an antitumor agent appeared to be comparable to cytostatic and cytotoxic potency of taxol and suramin or, in the case of HTB44 cells, even higher. The results obtained suggest that activated human macrophages might represent a useful tool for cancer immunotherapy

The Environment Shapes the Inner Vestibule of LeuT

Human neurotransmitter transporters are found in the nervous system terminating synaptic signals by rapid removal of neurotransmitter molecules from the synaptic cleft. The homologous transporter LeuT, found in Aquifex aeolicus, was crystallized in different conformations. Here, we investigated the inward-open state of LeuT. We compared LeuT in membranes and micelles using molecular dynamics simulations and lanthanide-based resonance energy transfer (LRET). Simulations of micelle-solubilized LeuT revealed a stable and widely open inward-facing conformation. However, this conformation was unstable in a membrane environment. The helix dipole and the charged amino acid of the first transmembrane helix (TM1A) partitioned out of the hydrophobic membrane core. Free energy calculations showed that movement of TM1A by 0.30 nm was driven by a free energy difference of similar to 15 kJ/mol. Distance measurements by LRET showed TM1A movements, consistent with the simulations, confirming a substantially different inward-open conformation in lipid bilayer from that inferred from the crystal structure

Purification and functional characterization of nine human Aquaporins produced in Saccharomyces cerevisiae for the purpose of biophysical characterization

The sparse number of high-resolution human membrane protein structures severely restricts our comprehension of molecular physiology and ability to exploit rational drug design. In the search for a standardized, cheap and easily handled human membrane protein production platform, we thoroughly investigated the capacity of S. cerevisiae to deliver high yields of prime quality human AQPs, focusing on poorly characterized members including some previously shown to be difficult to isolate. Exploiting GFP labeled forms we comprehensively optimized production and purification procedures resulting in satisfactory yields of all nine AQP targets. We applied the obtained knowledge to successfully upscale purification of histidine tagged human AQP10 produced in large bioreactors. Glycosylation analysis revealed that AQP7 and 12 were O-glycosylated, AQP10 was N-glycosylated while the other AQPs were not glycosylated. We furthermore performed functional characterization and found that AQP 2, 6 and 8 allowed flux of water whereas AQP3, 7, 9, 10, 11 and 12 also facilitated a glycerol flux. In conclusion, our S. cerevisiae platform emerges as a powerful tool for isolation of functional, difficult-To-express human membrane proteins suitable for biophysical characterization

Artemin and an Artemin-Derived Peptide, Artefin, Induce Neuronal Survival, and Differentiation Through Ret and NCAM

Artemin (ARTN) is a neurotrophic factor from the GDNF family ligands (GFLs) that is involved in development of the nervous system and neuronal differentiation and survival. ARTN signals through a complex receptor system consisting of the RET receptor tyrosine kinase and a glycosylphosphatidylinositol-anchored co-receptor GFL receptor α, GFRα3. We found that ARTN binds directly to neural cell adhesion molecule (NCAM) and that ARTN-induced neuritogenesis requires NCAM expression and activation of NCAM-associated signaling partners, thus corroborating that NCAM is an alternative receptor for ARTN. We designed a small peptide, artefin, that could interact with GFRα3 and demonstrated that this peptide agonist induces RET phosphorylation and mimics the biological functions of ARTN – neuroprotection and neurite outgrowth. Moreover, artefin mimicked the binding of ARTN to NCAM and required NCAM expression and activation for its neurite elongation effect, thereby suggesting that artefin represents a binding site for NCAM within ARTN. We showed that biological effects of ARTN and artefin can be inhibited by abrogation of both NCAM and RET, suggesting a more complex signaling mechanism that previously thought. As NCAM plays a significant role in neurodevelopment, regeneration, and synaptic plasticity we suggest that ARTN and its mimetics are promising candidates for treatment of neurological disorders and warrant further investigations