Evaluation of reference intervals of haematological and biochemical markers in an Austrian adolescent study cohort.

Background Reference intervals are a prerequisite for the interpretation of laboratory data related to diagnostic issues and treatment strategies. In adolescents, biomarker concentrations change with age, necessitating a continuous age-related definition of the reference intervals. The purpose of this pilot study was to evaluate the reference intervals for a healthy population of adolescents in Salzburg and compare these, when possible, with age- and gender-matched published data. Methods Anthropometrical parameters and blood samples were collected from adolescents (male and female; 14-17 years) in a school setting. Haematological samples were measured using Sysmex XS-1000i, lipid and carbohydrate metabolism markers as well as enzymes and hormones were determined by Cobas c311, Vitros ECiQ® or ELISA. The reference intervals were calculated according to the CLSI guidelines C28-A3c. Results Samples of 102 participants were included. Compared to age- and gender-matched reference intervals, the BMI levels were in the lower normal rage. Most haematological parameters and biomedical makers reveal similar ranges to values published in other studies. Conclusions This data analysis allowed for a partial comparison of reference values with published data and enabled a new determination of paediatric reference intervals for an Austrian cohort.

Development and Characterization of the Neuroregenerative Xanthohumol C/Hydroxypropyl-ß-cyclodextrin Complex Suitable for Parenteral Administration.

The chroman-like chalcone Xanthohumol C, originally found in hops, was demonstrated to be a potent neuroregenerative and neuroprotective natural product and therefore constitutes a strong candidate for further pharmaceutical research. The bottleneck for in vivo experiments is the low water solubility of this chalcone. Consequently, we developed and validated a suitable formulation enabling in vivo administration. Cyclodextrins were used as water-soluble and nontoxic complexing agents, and the complex of Xanthohumol C and 2-hydroxypropyl-ß-cyclodextrin was characterized using HPLC, HPLC-MS, NMR, and differential scanning calorimetry. The water solubility of Xanthohumol C increases with increasing concentrations of cyclodextrin. Using 50 mM 2-hydroxypropyl-ß-cyclodextrin, solubility was increased 650-fold. Furthermore, in vitro bioactivity of Xanthohumol C in free and complexed form did not significantly differ, suggesting the release of Xanthohumol C from 2-hydroxypropyl-ß-cyclodextrin. Finally, a small-scaled in vivo experiment in a rat model showed that after i. p. administration of the complex, Xanthohumol C can be detected in serum, the brain, and the cerebrospinal fluid at 1 and 6 h post-administration. Mean (± SD) Xanthohumol C serum concentrations after 1, 6, and 12 h were determined as 463.5 (± 120.9), 61.9 (± 13.4), and 9.3 (± 0.8) ng/mL upon i. v., and 294.3 (± 22.4), 45.5 (± 0.7), and 13 (± 1.0) ng/mL after i. p. application, respectively. Accordingly, the formulation of Xanthohumol C/2-hydroxypropyl-ß-cyclodextrin is suitable for further in vivo experiments and further pharmaceutical research aiming for the determination of its neuroregenerative potential in animal disease models.

The transcription factors Sox10 and Myrf define an essential regulatory network module in differentiating oligodendrocytes.

Myelin is essential for rapid saltatory conduction and is produced by Schwann cells in the peripheral nervous system and oligodendrocytes in the central nervous system. In both cell types the transcription factor Sox10 is an essential component of the myelin-specific regulatory network. Here we identify Myrf as an oligodendrocyte-specific target of Sox10 and map a Sox10 responsive enhancer to an evolutionarily conserved element in intron 1 of the Myrf gene. Once induced, Myrf cooperates with Sox10 to implement the myelination program as evident from the physical interaction between both proteins and the synergistic activation of several myelin-specific genes. This is strongly reminiscent of the situation in Schwann cells where Sox10 first induces and then cooperates with Krox20 during myelination. Our analyses indicate that the regulatory network for myelination in oligodendrocytes is organized along similar general principles as the one in Schwann cells, but is differentially implemented.

Sox10 cooperates with the mediator subunit 12 during terminal differentiation of myelinating glia.

Several transcription factors are essential for terminal differentiation of myelinating glia, among them the high-mobility-group-domain-containing protein Sox10. To better understand how these factors exert their effects and shape glial expression programs, we identified and characterized a physical and functional link between Sox10 and the Med12 subunit of the Mediator complex that serves as a conserved multiprotein interphase between transcription factors and the general transcription machinery. We found that Sox10 bound with two of its conserved domains to the C-terminal region of Med12 and its close relative, Med12-like. In contrast to Med12-like, substantial amounts of Med12 were detected in both Schwann cells and oligodendrocytes. Its conditional glia-specific deletion in mice led to terminal differentiation defects that were highly reminiscent of those obtained after Sox10 deletion. In support of a functional cooperation, both proteins were jointly required for Krox20 induction and were physically associated with the critical regulatory region of the Krox20 gene in myelinating Schwann cells. We conclude that Sox10 functions during terminal differentiation of myelinating glia, at least in part by Med12-dependent recruitment of the Mediator complex.

Human tetherin exerts strong selection pressure on the HIV-1 group N Vpu protein.

HIV-1 groups M and N emerged within the last century following two independent cross-species transmissions of SIVcpz from chimpanzees to humans. In contrast to pandemic group M strains, HIV-1 group N viruses are exceedingly rare, with only about a dozen infections identified, all but one in individuals from Cameroon. Poor adaptation to the human host may be responsible for this limited spread of HIV-1 group N in the human population. Here, we analyzed the function of Vpu proteins from seven group N strains from Cameroon, the place where this zoonosis originally emerged. We found that these N-Vpus acquired four amino acid substitutions (E15A, V19A and IV25/26LL) in their transmembrane domain (TMD) that allow efficient interaction with human tetherin. However, despite these adaptive changes, most N-Vpus still antagonize human tetherin only poorly and fail to down-modulate CD4, the natural killer (NK) cell ligand NTB-A as well as the lipid-antigen presenting protein CD1d. These functional deficiencies were mapped to amino acid changes in the cytoplasmic domain that disrupt putative adaptor protein binding sites and an otherwise highly conserved ßTrCP-binding DSGxxS motif. As a consequence, N-Vpus exhibited aberrant intracellular localization and/or failed to recruit the ubiquitin-ligase complex to induce tetherin degradation. The only exception was the Vpu of a group N strain recently discovered in France, but originally acquired in Togo, which contained intact cytoplasmic motifs and counteracted tetherin as effectively as the Vpus of pandemic HIV-1 M strains. These results indicate that HIV-1 group N Vpu is under strong host-specific selection pressure and that the acquisition of effective tetherin antagonism may lead to the emergence of viral variants with increased transmission fitness.

Transcription factor Sox10 orchestrates activity of a neural crest-specific enhancer in the vicinity of its gene.

The Sox10 transcription factor is a central regulator of vertebrate neural crest and nervous system development. Its expression is likely controlled by multiple enhancer elements, among them U3 (alternatively known as MCS4). Here we analyze U3 activity to obtain deeper insights into Sox10 function and expression in the neural crest and its derivatives. U3 activity strongly depends on the presence of Sox10 that regulates its own expression as commonly observed for important developmental regulators. Sox10 bound directly as monomer to at least three sites in U3, whereas a fourth site preferred dimers. Deletion of these sites efficiently reduced U3 activity in transfected cells and transgenic mice. In stimulating the U3 enhancer, Sox10 synergized with many other transcription factors present in neural crest and developing peripheral nervous system including Pax3, FoxD3, AP2α, Krox20 and Sox2. In case of FoxD3, synergism involved Sox10-dependent recruitment to the U3 enhancer, while Sox10 and AP2α each had to bind to the regulatory region. Our study points to the importance of autoregulatory activity and synergistic interactions for maintenance of Sox10 expression and functional activity of Sox10 in the neural crest regulatory network.

The transmembrane domain of HIV-1 Vpu is sufficient to confer anti-tetherin activity to SIVcpz and SIVgor Vpu proteins: cytoplasmic determinants of Vpu function.

BACKGROUND: The acquisition of effective Vpu-mediated anti-tetherin activity to promote virion release following transmission of SIVcpzPtt from central chimpanzees (Pan troglodytes troglodytes) to humans distinguishes pandemic HIV-1 group M strains from non-pandemic group N, O and P viruses and may have been a prerequisite for their global spread. Some functional motifs in the cytoplasmic region of HIV-1 M Vpus proposed to be important for anti-tetherin activity are more frequently found in the Vpu proteins of SIVcpzPtt than in those of SIVcpzPts infecting eastern chimpanzees (P. t. schweinfurthii), that have not been detected in humans, and SIVgor from gorillas, which is closely related to HIV-1 O and P. Thus, SIVcpzPtt strains may require fewer adaptive changes in Vpu than SIVcpzPts or SIVgor strains to counteract human tetherin. RESULTS: To examine whether SIVcpzPtt may only need changes in the transmembrane domain (TMD) of Vpu to acquire anti-tetherin activity, whereas SIVcpzPts and SIVgor may also require changes in the cytoplasmic region, we analyzed chimeras between the TMD of an HIV-1 M Vpu and the cytoplasmic domains of SIVcpzPtt (n = 2), SIVcpzPts (n = 2) and SIVgor (n = 2) Vpu proteins. Unexpectedly, all of these chimeras were capable of counteracting human tetherin to enhance virion release, irrespective of the presence or absence of the putative adaptor protein binding sites and the DSGxxS ß-TrCP binding motif reported to be critical for effective anti-tetherin activity of M Vpus. It was also surprising that in three of the six chimeras the gain of anti-tetherin function was associated with a loss of the CD4 degradation activity since this function was conserved among all parental HIV-1, SIVcpz and SIVgor Vpu proteins. CONCLUSIONS: Our results show that changes in the TMD of SIVcpzPtt, SIVcpzPts and SIVgor Vpus are sufficient to render them active against human tetherin. Thus, several previously described domains in the extracellular region of Vpu are not absolutely essential for tetherin antagonism but may be required for other Vpu functions.

Candida tenuis xylose reductase catalyzed reduction of aryl ketones for enantioselective synthesis of active oxetine derivatives.

Candida tenuis xylose reductase shows high catalytic efficiencies in carbonyl reduction of acetophenone and 1-phenyl-1-propanone derivatives. The quite low substrate solubility in aqueous buffer systems is circumvented by addition of methanol or by two-phase solvent systems. In the latter, methanol improves the substrate phase transfer as solvent mediator and leads to reasonable space/time yields. Resulting enantiomerically pure chiral alcohols are key intermediates for synthesis of active pharmaceutical ingredients. (R)-Atomoxetine is exemplarily synthesized in four steps, and the further use for generation of other oxetine derivatives and a polo-like kinase 1 inhibitor are discussed.

Light driven magnetic transitions in transition metal dichalcogenide heterobilayers.

Motivated by the recent excitement around the physics of twisted transition metal dichalcogenide (TMD) multilayer systems, we study strongly correlated phases of TMD heterobilayers under the influence of light. We consider both waveguide light and circularly polarized light. The former allows for longitudinally polarized light, which in the high frequency limit can be used to selectively modify interlayer hoppings in a tight-binding model. We argue based on quasi-degenerate perturbation theory that changes to the interlayer hoppings can be captured as a modulation to the strength of the moiré potential in a continuum model. As a consequence, waveguide light can be used to drive transitions between a myriad of different magnetic phases, including a transition from a 120∘Neel phase to a stripe ordered magnetic phase, or from a spin density wave phase to a paramagnetic phase, among others. When the system is subjected to circularly polarized light we find that the effective mass of the active TMD layer is modified by an applied electromagnetic field. By simultaneously applying waveguide light and circularly polarized light to a system, one has a high level of control in moving through the phase diagram in-situ. Lastly, we comment on the experimental feasibility of Floquet state preparation and argue that it is within reach of available techniques when the system is coupled to a judiciously chosen bath.

Ancient origin of a deletion in human BST2/Tetherin that confers protection against viral zoonoses.

Bone marrow stromal antigen 2 (BST2)/Tetherin is an antiviral factor that blocks the release of enveloped virions from infected cells. Recent data suggest that efficient BST2 antagonism was a prerequisite for the global spread of HIV/AIDS. Most simian immunodeficiency viruses (SIVs), including the direct precursors of HIV, use their Nef protein to antagonize BST2 of their respective host species. Human BST2, however, contains a five amino acid deletion in its cytoplasmic domain that confers resistance to Nef. Thus, this antiviral factor constitutes a major barrier to effective zoonotic transmissions of SIVs to humans. Here, we show that this protective deletion has already been present in Neanderthal and Denisovan BST2 and thus evolved at least 800,000 years ago. This ancient origin helps to explain why effectively spreading zoonotic transmissions of SIVs to humans have been rare, although SIVs are widespread in African nonhuman primates and humans must have been exposed to these viruses many times.

Enzyme identification and development of a whole-cell biotransformation for asymmetric reduction of o-chloroacetophenone.

Chiral 1-(o-chlorophenyl)-ethanols are key intermediates in the synthesis of chemotherapeutic substances. Enantioselective reduction of o-chloroacetophenone is a preferred method of production but well investigated chemo- and biocatalysts for this transformation are currently lacking. Based on the discovery that Candida tenuis xylose reductase converts o-chloroacetophenone with useful specificity (kcat/Km=340 M(-1) s(-1)) and perfect S-stereoselectivity, we developed whole-cell catalysts from Escherichia coli and Saccharomyces cerevisiae co-expressing recombinant reductase and a suitable system for recycling of NADH. E. coli surpassed S. cerevisiae sixfold concerning catalytic productivity (3 mmol/g dry cells/h) and total turnover number (1.5 mmol substrate/g dry cells). o-Chloroacetophenone was unexpectedly "toxic," and catalyst half-life times of only 20 min (E. coli) and 30 min (S. cerevisiae) in the presence of 100 mM substrate restricted the time of batch processing to maximally ∼5 h. Systematic reaction optimization was used to enhance the product yield (≤60%) of E. coli catalyzed conversion of 100 mM o-chloroacetophenone which was clearly limited by catalyst instability. Supplementation of external NAD+ (0.5 mM) to cells permeabilized with polymyxin B sulfate (0.14 mM) resulted in complete conversion providing 98 mM S-1-(o-chlorophenyl)-ethanol. The strategies considered for optimization of reduction rate should be generally useful, however, especially under process conditions that promote fast loss of catalyst activity.

Candida tenuis xylose reductase catalysed reduction of acetophenones: the effect of ring-substituents on catalytic efficiency.

The catalytic efficiencies of Candida tenuis xylose reductase catalysed reductions of mono-substituted acetophenones are in reasonable correlation with the σ-Hammett coefficients of the substituted phenyl groups. Variations of the substrate transformation rates are hence mainly caused by mesomeric and inductive effects of the substituents, while differences in substrate binding have a secondary relevance. Some substrate (1)H NMR chemical shifts and carbonyl IR absorption bands are in reasonable accordance with the catalytic activities and allow the estimation of the transformation rates with good accuracy. The resulting substituted (S)-1-phenyl ethanols are generated in very high enantiomeric excess.

The Prenylflavonoid ENDF1 Overrules Central Nervous System Growth Inhibitors and Facilitates Regeneration of DRG Neurons.

Restoration of neuronal connectivity after lesion of the central nervous system, such as spinal cord injury, is one of the biggest challenges in modern medicine. In particular, the accumulation of axon growth inhibitory factors at the site of injury constitutes a major obstacle to structural and thus functional repair. We previously investigated a group of prenylflavonoids derived from hops for their capacity to promote neuroregeneration. We identified a molecule called ENDF1 that was very potent to enhance regrowth and branching of neurites from dorsal root ganglion neurons in culture on growth promoting substrates. In the present study, we investigated ENDF1's capacity to promote regeneration of rat dorsal root ganglion neurons in vitro in the presence of three main components of the extracellular matrix acting as axon growth inhibitors: Semaphorin 3A, Ephrin A4 and mixed chondroitin sulfate proteoglycans. We report that ENDF1 application significantly promoted the percentages of sensory neurons able to regrow their neurites regardless of the presence of those inhibitors, and this to an extent similar to the one obtained after NGF treatment. Moreover, ENDF1 strongly enhanced the total neurite length and the complexity of neurites extending from neurons challenged with axon growth inhibitors. Although the impact of NGF and ENDF1 on the regeneration of neurons was similar, the activity of ENDF1 was not mediated by signaling through the TrkA receptor, indicating that each molecule act through different signaling pathways. In addition, ENDF1 did not decrease the phosphorylation of cofilin, a downstream effector of the regeneration-associated RhoA/ROCK signaling pathway. Hence, ENDF1 is a potent pro-neuroregenerative factors that could help in identifying new efficient targets for regenerative therapies of the nervous system.

Corticosteroid-binding globulin: structure-function implications from species differences.

Corticosteroid-binding globulin (CBG) transports glucocorticoids and progesterone in the blood and thereby modulates the tissue availability of these hormones. As a member of the serine protease inhibitor (SERPIN) family, CBG displays a reactive center loop (RCL) that is targeted by proteinases. Cleavage of the RCL is thought to trigger a SERPIN-typical stressed-to-relaxed (S-to-R) transition that leads to marked structural rearrangements and a reduced steroid-binding affinity. To characterize structure-function relationships in CBG we studied various conformational states of E. coli-produced rat and human CBG. In the 2.5 Å crystal structure of human CBG in complex with progesterone, the RCL is cleaved at a novel site that differs from the known human neutrophil elastase recognition site. Although the cleaved RCL segment is five residues longer than anticipated, it becomes an integral part of ß-sheet A as a result of the S-to-R transition. The atomic interactions observed between progesterone and CBG explain the lower affinity of progesterone in comparison to corticosteroids. Surprisingly, CD measurements in combination with thermal unfolding experiments show that rat CBG fails to undergo an S-to-R transition upon proteolytic cleavage of the RCL hinting that the S-to-R transition observed in human CBG is not a prerequisite for CBG function in rat. This observation cautions against drawing general conclusions about molecular mechanisms by comparing and merging structural data from different species.

Chromatin-remodeling factor Brg1 is required for Schwann cell differentiation and myelination.

Schwann cells produce myelin sheaths and thereby permit rapid saltatory conductance in the vertebrate peripheral nervous system. Their stepwise differentiation from neural crest cells is driven by a defined set of transcription factors. How this is linked to chromatin changes is not well understood. Here we show that the glial transcription factor Sox10 functions in Schwann cells by recruiting Brg1-containing chromatin-remodeling complexes via Baf60a to regulatory regions of Oct6 and Krox20 genes. It thereby stimulates expression of these transcriptional regulators that then cooperate with Sox10 to convert immature into myelinating Schwann cells. The functional interaction between Sox10 and Brg1 is evident from gain- and loss-of-function studies, similar neuropathies in the corresponding mouse mutants, and an aggravated neuropathy in compound mutants. Our results demonstrate that the transcription factor-mediated recruitment of the chromatin-remodeling machinery to specific genomic loci is an essential driving force for Schwann cell differentiation and myelination.