Toll/Interleukin-1 Receptor Domain Dimers as the Platform for Activation and Enhanced Inhibition of Toll-like Receptor Signaling

TIR (Toll/IL-1 receptor) domains mediate interactions between TLR (Toll-like) or IL-1 family receptors and signaling adapters. While homotypic TIR domain interactions mediate receptor activation they are also usurped by microbial TIR domain containing proteins for immunosuppression. Here we show the role of a dimerized TIR domain platform for the suppression as well as for the activation of MyD88 signaling pathway. Coiled-coil dimerization domain, present in many bacterial TCPs, potently augments suppression of TLR/IL-1R signaling. The addition of a strong coiled-coil dimerization domain conferred the superior inhibition against the wide spectrum of TLRs and prevented the constitutive activation by a dimeric TIR platform. We propose a molecular model of MyD88-mediated signaling based on the dimerization of TIR domains as the limiting step.     

Metabolic engineering of Saccharomyces cerevisiae for bioconversion of D-xylose to D-xylonate

An NAD(+)-dependent D-xylose dehydrogenase, XylB, from Caulobacter crescentus was expressed in Saccharomyces cerevisiae, resulting in production of 17 ± 2 g D-xylonate l(-1) at 0.23 gl(-1)h(-1) from 23 g D-xylose l(-1) (with glucose and ethanol as co-substrates). D-Xylonate titre and production rate were increased and xylitol production decreased, compared to strains expressing genes encoding T. reesei or pig liver NADP(+)-dependent D-xylose dehydrogenases. D-Xylonate accumulated intracellularly to ～70 mgg(-1); xylitol to ～18 mgg(-1). The aldose reductase encoding gene GRE3 was deleted to reduce xylitol production. Cells expressing D-xylonolactone lactonase xylC from C. crescentus with xylB initially produced more extracellular D-xylonate than cells lacking xylC at both pH 5.5 and pH 3, and sustained higher production at pH 3. Cell vitality and viability decreased during D-xylonate production at pH 3.0. An industrial S. cerevisiae strain expressing xylB efficiently produced 43 g D-xylonate l(-1) from 49 g D-xylose l(-1).     

Accelerated Return to Play in Professional Basketball Players With Surgically-Treated Metacarpal Shaft Fractures

BackgroundTraditional rehabilitation protocols for surgically treated metacarpal shaft fracture allow for return to play at 6-8 weeks post-operative. This may be devastating for the elite athlete. We outline a protocol that may allow for professional basketball players to successfully return to sport within four weeks following surgery.MethodsProfessional basketball players who sustained non-thumb metacarpal shaft fractures were included. All athletes underwent open reduction and internal fixation of the injured metacarpal. Patients were subsequently enrolled into an accelerated rehabilitation protocol.ResultsThe five athletes in our case series successfully passed return to sport testing within four weeks of surgery.ConclusionA plate and screw construct can potentially allow for professional basketball players to return to play in half the time. Future research studies should include a larger pool of athletes to further investigate accelerated rehabilitation following surgical fixation of metacarpal fractures. Level of Evidence: IV     

Noninvasive High-Throughput Single-Cell Analysis of the Intracellular pH of Saccharomyces cerevisiae by Ratiometric Flow Cytometry

The ability of cells to maintain pH homeostasis in response to environmental changes has elicited interest in basic and applied research and has prompted the development of methods for intracellular pH measurements. Many traditional methods provide information at population level and thus the average values of the studied cell physiological phenomena, excluding the fact that cell cultures are very heterogeneous. Single-cell analysis, on the other hand, offers more detailed insight into population variability, thereby facilitating a considerably deeper understanding of cell physiology. Although microscopy methods can address this issue, they suffer from limitations in terms of the small number of individual cells that can be studied and complicated image processing. We developed a noninvasive high-throughput method that employs flow cytometry to analyze large populations of cells that express pHluorin, a genetically encoded ratiometric fluorescent probe that is sensitive to pH. The method described here enables measurement of the intracellular pH of single cells with high sensitivity and speed, which is a clear improvement compared to previously published methods that either require pretreatment of the cells, measure cell populations, or require complex data analysis. The ratios of fluorescence intensities, which correlate to the intracellular pH, are independent of the expression levels of the pH probe, making the use of transiently or extrachromosomally expressed probes possible. We conducted an experiment on the kinetics of the pH homeostasis of Saccharomyces cerevisiae cultures grown to a stationary phase after ethanol or glucose addition and after exposure to weak acid stress and glucose pulse. Minor populations with pH homeostasis behaving differently upon treatments were identified.     

Histamine (re)uptake by astrocytes: an experimental and computational study

Astrocytes participate in the clearance of neurotransmitters by their uptake and subsequent enzymatic degradation. Histamine as a polar and/or protonated molecule must use a carrier to be transported across the cell membrane, although a specific histamine transporter has not been elucidated, yet. In this work we upgraded the kinetic studies of histamine uptake into neonatal rat cultured type 1 astrocytes with quantum chemical calculations of histamine pKa values in conjunction with Langevin dipoles solvation model as the first step toward microscopic simulation of transport. Our results indicate that astrocytes transport histamine by at least two carrier mediated processes, a concentration gradient dependent passive and a sodium-dependent and ATP-driven active transport. We also demonstrated that histamine protonation states depend on the polarity of the environment. In conclusion we suggest that histamine, a polar molecule at physiological pH uses at least two different mechanisms for its uptake into astrocytes –an electrodiffusion and Na⁺-dependent and ouabain sensitive active process. We emphasize relevance of knowledge of histamines protonation states at the rate limiting step of its transport for microscopic simulation that will be possible when structure of histamine transporter is known.     

Pharmacokinetics of Drugs in Cachectic Patients: A Systematic Review

Cachexia is a weight-loss process caused by an underlying chronic disease such as cancer, chronic heart failure, chronic obstructive pulmonary disease, or rheumatoid arthritis. It leads to changes in body structure and function that may influence the pharmacokinetics of drugs. Changes in gut function and decreased subcutaneous tissue may influence the absorption of orally and transdermally applied drugs. Altered body composition and plasma protein concentration may affect drug distribution. Changes in the expression and function of metabolic enzymes could influence the metabolism of drugs, and their renal excretion could be affected by possible reduction in kidney function. Because no general guidelines exist for drug dose adjustments in cachectic patients, we conducted a systematic search to identify articles that investigated the pharmacokinetics of drugs in cachectic patients.     

Mechanical stability of membrane nanotubular protrusions influenced by attachment of flexible rod-like proteins

It is indicated that nonhomogeneous lateral distribution of membrane attached and flexible rod-like proteins (MRPs) may stabilize nanotubular membrane protrusions. We have shown that curvature induced accumulation of MRPs in the nanotubular membrane protrusion and the corresponding reduction of the membrane free energy are possible if the decrease of the deviatoric free energy of MRPs in the nanotubular protrusions is large enough to overcome the increase of the free energy due to decrease of configurational entropy in the process of lateral sorting of MRPs. The decrease of isotropic curvature energy of MRPs in the region of membrane protrusion is usually not sufficient for substantial MRPs sorting and consequent stabilization of the nanotubular membrane protrusions.     

Polymorphisms of interleukin-8 and -18 genes and diabetic retinopathy

We evaluated possible roles of interleukin-8 gene polymorphisms (1633T/C-rs2227543, 251A/T-rs4073) and interleukin-18 gene polymorphisms (-607C/A-rs1946518, -137G/C-rs187238) in the development of diabetic retinopathy (DR) in Caucasians with type 2 diabetes. 271 patients with DR and 113 without diabetic retinopathy were enrolled in this cross-sectional study. We did not observe an association between either interleukin-8 gene polymorphisms (1633T/C, 251A/T) or interleukin-18 gene polymorphisms (-607C/A, -137G/C) and diabetic retinopathy in Caucasians with type 2 diabetes. We did not find statistically significant differences in interleukin-8 serum levels between diabetics with the TT and AA genotype and those with other genotypes. The interleukin-18 serum levels between diabetics with the CC genotype of the -607C/A polymorphism and those with other genotypes (AA, AC) were not significantly different. Moreover, we did not observe a statistically significant effect of the tested polymorphisms of either interleukin-8 or interleukin-18 genes on serum levels in diabetics. In conclusion, our study indicates that the examined polymorphisms of interleukin-8 (1633T/C, 251A/T) and interleukin-18 (-607C/A or the -137G/C) genes are not genetic risk factors for diabetic retinopathy. Therefore, they may not be used as genetic markers for diabetic retinopathy in Caucasians with type 2 diabetes.     

Kinetics of cholesterol extraction from lipid membranes by methyl-beta-cyclodextrin--a surface plasmon resonance approach

The kinetics of cholesterol extraction from cellular membranes is complex and not yet completely understood. In this paper we have developed an experimental approach to directly monitor the extraction of cholesterol from lipid membranes by using surface plasmon resonance and model lipid systems. Methyl-beta-cyclodextrin was used to selectively remove cholesterol from large unilamellar vesicles of various compositions. The amount of extracted cholesterol is highly dependent on the composition of lipid membrane, i.e. the presence of sphingomyelin drastically reduced and slowed down cholesterol extraction by methyl-beta-cyclodextrin. This was confirmed also in the erythrocyte ghosts system, where more cholesterol was extracted after erythrocytes were treated with sphingomyelinase. We further show that the kinetics of the extraction is mono-exponential for mixtures of 1,2-dioleoyl-sn-glycero-3-phosphocholine and cholesterol. The kinetics is complex for ternary lipid mixtures composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine, bovine brain sphingomyelin and cholesterol. Our results indicate that the complex kinetics observed in experiments with cells may be the consequence of lateral segregation of lipids in cell plasma membrane.