Molecular basis of RNA recognition by the human alternative splicing factor Fox-1

The Fox-1 protein regulates alternative splicing of tissue-specific exons by binding to GCAUG elements. Here, we report the solution structure of the Fox-1 RNA binding domain (RBD) in complex with UGCAUGU. The last three nucleotides, UGU, are recognized in a canonical way by the four-stranded beta-sheet of the RBD. In contrast, the first four nucleotides, UGCA, are bound by two loops of the protein in an unprecedented manner. Nucleotides U1, G2, and C3 are wrapped around a single phenylalanine, while G2 and A4 form a base-pair. This novel RNA binding site is independent from the beta-sheet binding interface. Surface plasmon resonance analyses were used to quantify the energetic contributions of electrostatic and hydrogen bond interactions to complex formation and support our structural findings. These results demonstrate the unusual molecular mechanism of sequence-specific RNA recognition by Fox-1, which is exceptional in its high affinity for a defined but short sequence element.     

A simple equation for describing the temperature dependent growth of free-floating macrophytes

Temperature is one of the most important factors determining growth rates of free-floating macrophytes in the field. To analyse and predict temperature dependent growth rates of these pleustophytes, modelling may play an important role. Several equations have been published for describing temperature responses of macrophytes and algae. But they are often complex or are only applicable in a limited range of temperatures. In this paper, we present a simple three-parameter equation for describing the temperature dependent growth rates of pleustophytes. The equation that we developed is tested using results from laboratory growth experiments conducted with three different species of pleustophytes (Lemna minor, Salvinia molesta and Azolla filiculoides). The equation is simple and demonstrates reliable fits (adjusted R² reaching from 0.89 to 0.95). Additionally, our equation primarily uses parameters of biological significance, resulting in estimates of useful cardinal temperatures (minimum and maximum).     

Opisthorchis viverrini: identification of a glycine-tyrosine rich eggshell protein and its potential as a diagnostic tool for human opisthorchiasis

A cDNA encoding a novel eggshell protein (OvESP) with high-glycine (49.2%) and -tyrosine (27.8%) content was cloned from the human liver fluke Opisthorchis viverrini. In the adult parasite, the RNA products of the OvESP gene are limited to the vitelline follicles. They have a size of 800 nucleotides and are already present in 2-week-old juveniles. Immune sera of hamsters, experimentally infected, and humans, naturally infected with O. viverrini, detect bacterially expressed recombinant OvESP (rOvESP). A rabbit anti-rOvESP antiserum only reacts with the shells of intrauterine eggs in tissue sections of the parasite. Comparison of rOvESP with the parasite's excretion/secretion products as diagnostic tools for human opisthorchiasis shows a higher sensitivity (0.82-0.48) and specificity (0.97-0.91) of the recombinant protein in the ELISA technique. But the observed weak cross-reactivity of immune sera from mice infected with Schistosoma mansoni, Schistosoma mekongi, and Fasciola gigantica in Western blots of rOvESP indicates that the diagnostic quality of this protein might be compromised if infections by other trematodes are present.     

Protection by sucrose against heat-induced lethal and sublethal injury of Lactococcus lactis: an FT-IR study

The heat inactivation of Lactococcus lactis was studied by determination of cell counts, and by FT-IR spectroscopy recording the average structure of cell proteins. Cell counts were measured after incubation milk buffer or milk buffer with 1. 5 M sucrose, and FT-IR spectra were recorded in (2)H(2)O or (2)H(2)O with 1. 5 M sucrose in the range of 6-75 degrees Celsius. Sucrose protected L. lactis against heat inactivation. The cell counts differed by up to 6-log cycles after treatment in milk buffer as compared to milk buffer with sucrose. The (1)H/(2)H exchange in proteins, and secondary structure elements were detected by the analysis of amide I', amide II and amide II' bands. A reduced (1)H/(2)H exchange as well as a lower content of disordered structural elements was observed when sucrose was present. Conformational fluctuations of native proteins as indicated by the (1)H/(2)H exchange were apparent already at sublethal temperatures. The loss of viability of L. lactis occurred in the same temperature range as the loss of the protein secondary structure. These results demonstrate that sucrose protects L. lactis against heat inactivation, and that the increased heat stability of proteins in the presence of sucrose contributed to this enhanced heat resistance.     

The chaperone function of cyclophilin 40 maps to a cleft between the prolyl isomerase and tetratricopeptide repeat domains

Cyclophilin 40 (CyP40), an immunophilin cochaperone present in steroid receptor-Hsp90 complexes, contains an N-terminal peptidylprolyl isomerase (PPIase) domain separated from a C-terminal Hsp90-binding tetratricopeptide repeat (TPR) domain by a 30-residue linker. To map CyP40 chaperone function, CyP40 deletion mutants were prepared and analysed for chaperone activity. CyP40 fragments containing the PPIase domain plus linker or the linker region and the adjoining TPR domain retained chaperone activity, whilst individually, the catalytic and TPR domains were devoid of chaperoning ability. CyP40 chaperone function then, is localized within the linker that forms a binding cleft with potential to accommodate non-native substrates.     

High-Pressure-Mediated Survival of Clostridium botulinum and Bacillus amyloliquefaciens Endospores at High Temperature

Endospores of proteolytic type B Clostridium botulinum TMW 2.357 and Bacillus amyloliquefaciens TMW 2.479 are currently described as the most high-pressure-resistant bacterial spores relevant to food intoxication and spoilage in combined pressure-temperature applications. The effects of combined pressure (0.1 to 1,400 MPa) and temperature (70 to 120°C) treatments were determined for these spores. A process employing isothermal holding times was established to distinguish pressure from temperature effects. An increase in pressure (600 to 1,400 MPa) and an increase in temperature (90 to 110°C) accelerated the inactivation of C. botulinum spores. However, incubation at 100°C, 110°C, or 120°C with ambient pressure resulted in faster spore reduction than treatment with 600 or 800 MPa at the same temperature. This pressure-mediated spore protection was also observed at 120°C and 800, 1,000, or 1,200 MPa with the more heat-tolerant B. amyloliquefaciens TMW 2.479 spores. Inactivation curves for both strains showed a pronounced pressure-dependent tailing, which indicates that a small fraction of the spore populations survives conditions of up to 120°C and 1.4 GPa in isothermal treatments. Because of this tailing and the fact that pressure-temperature combinations stabilizing bacterial endospores vary from strain to strain, food safety must be ensured in case-by-case studies demonstrating inactivation or nongrowth of C. botulinum with realistic contamination rates in the respective pressurized food and equipment.     

Abrupt temporal fluctuations in the chicken fecal microbiota are explained by its gastrointestinal origin

One of the main challenges in understanding the composition of fecal microbiota is that it can consist of microbial mixtures originating from different gastrointestinal (GI) segments. Here, we addressed this challenge for broiler chicken feces using a direct 16S rRNA gene-sequencing approach combined with multivariate statistical analyses. Broiler feces were chosen because of easy sampling and the importance for pathogen transmission to the human food chain. Feces were sampled daily for 16 days from chickens with and without a feed structure-induced stimulation of the gastric barrier function. Overall, we found four dominant microbial phylogroups in the feces. Two of the phylogroups were related to clostridia, one to lactobacilli, and one to Escherichia/Shigella. The relative composition of these phylogroups showed apparent stochastic temporal fluctuations in feces. Analyses of dissected chickens at the end of the experiment, however, showed that the two clostridial phylogroups were correlated to the microbiota in the cecum/colon and the small intestine, while the upper gut (crop and gizzard) microbiota was correlated to the lactobacillus phylogroup. In addition, chickens with a stimulated gizzard also showed less of the proximate GI dominating bacterial group in the feces, supporting the importance of the gastric barrier function. In conclusion, our results suggest that GI origin is a main determinant for the chicken fecal microbiota composition. This knowledge will be important for future understanding of factors affecting shedding of both harmful and beneficial gastrointestinal bacteria through feces.     

Ezetimibe: A biomarker for efficacy of liver directed UGT1A1 gene therapy for inherited hyperbilirubinemia

As recently demonstrated in patients with factor IX deficiency, adeno-associated virus (AAV)-mediated liver-directed therapy is a viable option for inherited metabolic liver disorders. Our aim is to treat Crigler-Najjar syndrome type I (CN I), an inherited severe unconjugated hyperbilirubinemia, as a rare recessive inherited disorder. Because the number of patients eligible for this approach is small, the efficacy can only be demonstrated by a beneficial effect on the pathophysiology in individual patients. Serum bilirubin levels in potential candidates have been monitored since birth, providing an indication of their pathophysiology. Adjuvant phototherapy to prevent brain damage reduces serum unconjugated bilirubin (UCB) levels in CN I patients to the level seen in the milder form of the disease, CN type II. This therapy increases the excretion of UCB, thereby complicating the use of UCB and conjugated bilirubin levels in serum as biomarkers for the gene therapy we try to develop. Therefore, a suitable biomarker that is not affected by phototherapy is currently needed. To this end, we have investigated whether estradiol, ethinylestradiol or ezetimibe could be used as markers for uridine 5'-di-phospho-glucuronosyltransferase isoform 1A1 (UGT1A1) activity restored by AAV gene therapy in Gunn rats, a relevant animal model for CN I. Of these compounds, ezetimibe appeared most suitable because its glucuronidation rate in untreated control Gunn rats is low. Subsequently, ezetimibe glucuronidation was studied in both untreated and AAV-treated Gunn rats and the results suggest that it may serve as a useful serum marker for restored hepatic UGT1A1 activity.     

Functional markers in wheat: current status and future prospects

Functional markers (FM) are developed from sequence polymorphisms present in allelic variants of a functional gene at a locus. FMs accurately discriminate alleles of a targeted gene, and are ideal molecular markers for marker-assisted selection in wheat breeding. In this paper, we summarize FMs developed and used in common wheat. To date, more than 30 wheat loci associated with processing quality, agronomic traits, and disease resistance, have been cloned, and 97 FMs were developed to identify 93 alleles based on the sequences of those genes. A general approach is described for isolation of wheat genes and development of FMs based on in silico cloning and comparative genomics. The divergence of DNA sequences of different alleles that affect gene function is summarized. In addition, 14 molecular markers specific for alien genes introduced from common wheat relatives were also described. This paper provides updated information on all FMs and gene-specific STS markers developed so far in wheat and should facilitate their application in wheat breeding programs.     

Revolutionizing medicine in the 21(st) century through systems approaches

Personalized medicine is a term for a revolution in medicine that envisions the individual patient as the central focus of healthcare in the future. The term "personalized medicine", however, fails to reflect the enormous dimensionality of this new medicine that will be predictive, preventive, personalized, and participatory-a vision of medicine we have termed P4 medicine. This reflects a paradigm change in how medicine will be practiced that is revolutionary rather than evolutionary. P4 medicine arises from the confluence of a systems approach to medicine and from the digitalization of medicine that creates the large data sets necessary to deal with the complexities of disease. We predict that systems approaches will empower the transition from conventional reactive medical practice to a more proactive P4 medicine focused on wellness, and will reverse the escalating costs of drug development an will have enormous social and economic benefits. Our vision for P4 medicine in 10 years is that each patient will be associated with a virtual data cloud of billions of data points and that we will have the information technology for healthcare to reduce this enormous data dimensionality to simple hypotheses about health and/or disease for each individual. These data will be multi-scale across all levels of biological organization and extremely heterogeneous in type - this enormous amount of data represents a striking signal-to-noise (S/N) challenge. The key to dealing with this S/N challenge is to take a "holistic systems approach" to disease as we will discuss in this article.