Microfluidics: From Crystallization to Serial Time-Resolved Crystallography

Capturing protein structural dynamics in real-time has tremendous potential in elucidating biological functions and providing information for structure-based drug design. While time-resolved structure determination has long been considered inaccessible for a vast majority of protein targets, serial methods for crystallography have remarkable potential in facilitating such analyses. Here, we review the impact of microfluidic technologies on protein crystal growth and X-ray diffraction analysis. In particular, we focus on applications of microfluidics for use in serial crystallography experiments for the time-resolved determination of protein structural dynamics

Nitric Oxide Signalling Augments Neuronal Voltage-Gated L-Type (CaV1) and P/Q-Type (CaV2.1) Channels in the Mouse Medial Nucleus of the Trapezoid Body

Nitric Oxide (NO) is a diffusible second messenger that modulates ion channels, intrinsic excitability and mediates synaptic plasticity. In light of its activity-dependent generation in the principal neurons of the medial nucleus of the trapezoid body (MNTB), we have investigated its potential modulatory effects on native voltage-gated calcium channels (CaV) within this nucleus. Whole-cell patch recordings were made from brain slices from P13–15 CBA mice. Slices were incubated with the inhibitor of neuronal nitric oxide synthase (nNOS) 7-nitroindazole (10 µM) and pharmacological blockers used to isolate Ca2+ current subtypes. Unpaired observations in the presence and absence of the NO-donors sodium nitroprusside (SNP, 100 µM) or Diethyl-ammonium-nonoate (DEA, 100 µM) were made to elucidate NO-dependent modulation of the expressed CaV subtypes. A differential effect of NO on the calcium channel subtypes was observed: CaV1 and CaV2.1 (L+R- and P/Q+R-type) conductances were potentiated, whereas N+R-type (CaV2.2) and R-type (CaV2.3) current amplitudes were unaffected. L+R-type currents increased from 0.36±0.04 nA to 0.64±0.11 nA and P/Q+R-type from 0.55±0.09 nA to 0.94±0.05 nA, thereby changing the balance and relative contribution of each subtype to the whole cell calcium current. In addition, N+R-type half-activation voltage was left shifted following NO exposure. NO-dependent modulation of P/Q+R and N+R-type, but not L+R-type, channels was removed by inhibition of soluble guanylyl cyclase (sGC) activity. This data demonstrates a differential effect of NO signalling on voltage-gated calcium entry, by distinct NO-dependent pathways

An Unusual Digestive Foreign Body

Foreign digestive bodies present unusual circumstances because they are associated with various degrees of local trauma and may lead to direct perforation or delayed local injury. Patients with foreign bodies should be evaluated upon admission for signs of impaction and perforation. While all objects impacted in the esophagus require urgent treatment, rectal foreign bodies are usually removable through the anus. The current case illustrates successful endoscopic retrieval of a proximally located foreign body in a particular legal situation where physicians had to work closely with police officers and court members

Rhomboid family member 2 regulates cytoskeletal stress-associated Keratin 16.

Keratin 16 (K16) is a cytoskeletal scaffolding protein highly expressed at pressure-bearing sites of the mammalian footpad. It can be induced in hyperproliferative states such as wound healing, inflammation and cancer. Here we show that the inactive rhomboid protease RHBDF2 (iRHOM2) regulates thickening of the footpad epidermis through its interaction with K16. K16 expression is absent in the thinned footpads of irhom2-/- mice compared with irhom2+/+mice, due to reduced keratinocyte proliferation. Gain-of-function mutations in iRHOM2 underlie Tylosis with oesophageal cancer (TOC), characterized by palmoplantar thickening, upregulate K16 with robust downregulation of its type II keratin binding partner, K6. By orchestrating the remodelling and turnover of K16, and uncoupling it from K6, iRHOM2 regulates the epithelial response to physical stress. These findings contribute to our understanding of the molecular mechanisms underlying hyperproliferation of the palmoplantar epidermis in both physiological and disease states, and how this 'stress' keratin is regulated

Mechanism and consequences of the shift in cardiac arginine metabolism following ischaemia and reperfusion in rats

© Schattauer 2015. Cardiac ischaemia and reperfusion leads to irreversible injury and subsequent tissue remodelling. Initial reperfusion seems to shift arginine metabolism from nitric oxide (NO) to polyamine formation. This may limit functional recovery at reperfusion. The hypothesis was tested whether ischaemia/reperfusion translates such a shift in arginine metabolism in a tumour necrosis factor (TNF)-α-dependent way and renin-angiotensin system (RAS)-dependent way into a sustained effect. Both, the early post-ischaemic recovery and molecular adaptation to ischaemia/reperfusion were analysed in saline perfused rat hearts undergoing global no-flow ischaemia and reperfusion. Local TNF-α activation was blocked by inhibition of TNF-α sheddase ADAM17. To interfere with RAS captopril was administered. Arginase was inhibited by administration of Nor-NOHA. Long-term effects of ischemia/reperfusion on arginine metabolism were analysed in vivo in rats receiving an established ischaemia/reperfusion protocol in the closed chest mode. mRNA expression analysis indicated a shift in the arginine metabolism from NO formation to polyamine metabolism starting within 2 hours (h) of reperfusion and translated into protein expression within 24 h. Inhibition of the TNF-α pathway and captopril attenuated these delayed effects on post-ischaemic recovery. This shift in arginine metabolism was associated with functional impairment of hearts within 24 h. Inhibition of arginase but not that of TNF-α and RAS pathways improved functional recovery immediately. However, no benefit was observed after four months. In conclusion, this study identified TNF-α and RAS to be responsible for depressed cardiac function that occurred a few hours after reperfusion