Sucrose utilization during ovule and seed development ofGasteria verrucosa (Mill.) H. Duval as monitored by sucrose synthase and invertase localization

Summary The development ofGasteria verrucosa ovules and seeds seems to follow a pattern of growth in which the majority of carbohydrates is first used in the sporophytic tissue (nucellus, integuments, and arillus) around the gametophyte-derived cells. After fertilization the carbohydrates are used for further development of the arillus and seed coat. During the next stage carbohydrates are directed to develop the endosperm, followed by carbohydrate investment in the developing embryo and in storage products. This utilization pattern is deducted from a localization study on sucrose synthase and invertase. These two enzymes break down imported sucrose and are in that perspective used as markers for carbohydrate transport since diffusion is expected to be induced towards cells and tissues with high sucrose-hydrolyzing activities.     

MICROPYLAR EXUDATE IN GASTERIA (ALOACEAE) AND ITS POSSIBLE FUNCTION IN POLLEN TUBE GROWTH

The micropylar exudate of Gasteria verrucosa (Mill.) H. Duval was studied using light and electron microscopic techniques. Ovules may contain micropylar exudate before stigma receptivity. During successive phases of stigma receptivity, the number of ovules with micropylar exudate and the amount of micropylar exudate per ovule increases. At the late phase of stigma receptivity, large amounts of micropylar exudate with a smooth to cauliflowerlike appearance were observed. Micropylar exudate is viscous and contains, among other components, proteins and carbohydrates. At all stages of the stigma investigated, ovules situated at the base of the ovary contain a larger quantity of micropylar exudate than those at the top. The appearance of micropylar exudate is related to the degree of development of the embryo sac and it originates primarily from the filiform apparatus. It is assumed that an uptake of water by the ovule initiates the outflow of micropylar exudate from the filiform apparatus into the micropyle. Both ovular pollen tube ingrowth and seed set mark the optimum pollination stage of the stigma, which for both events lies around the onset of stigma receptivity. When pollen tubes have reached the ovary, young micropylar exudate stimulates their growth rate. The presence of micropylar exudate seems to be a requirement for pollen tube penetration, and an interaction between the pollen tube and the micropylar exudate has been proposed. Possibly, the micropylar exudate serves as a nutritional source and, in an optimum condition, as an attractant for approaching pollen tubes.     

Validation and Application of a Custom-Designed Targeted Next-Generation Sequencing Panel for the Diagnostic Mutational Profiling of Solid Tumors

The inevitable switch from standard molecular methods to next-generation sequencing for the molecular profiling of tumors is challenging for most diagnostic laboratories. However, fixed validation criteria for diagnostic accreditation are not in place because of the great variability in methods and aims. Here, we describe the validation of a custom panel of hotspots in 24 genes for the detection of somatic mutations in non-small cell lung carcinoma, colorectal carcinoma and malignant melanoma starting from FFPE sections, using 14, 36 and 5 cases, respectively. The targeted hotspots were selected for their present or future clinical relevance in solid tumor types. The target regions were enriched with the TruSeq approach starting from limited amounts of DNA. Cost effective sequencing of 12 pooled libraries was done using a micro flow cell on the MiSeq and subsequent data analysis with MiSeqReporter and VariantStudio. The entire workflow was diagnostically validated showing a robust performance with maximal sensitivity and specificity using as thresholds a variant allele frequency >5% and a minimal amplicon coverage of 300. We implemented this method through the analysis of 150 routine diagnostic samples and identified clinically relevant mutations in 16 genes including KRAS (32%), TP53 (32%), BRAF (12%), APC (11%), EGFR (8%) and NRAS (5%). Importantly, the highest success rate was obtained when using also the low quality DNA samples. In conclusion, we provide a workflow for the validation of targeted NGS by a custom-designed pan-solid tumor panel in a molecular diagnostic lab and demonstrate its robustness in a clinical setting.     

Dynamic Localization of Tat Protein Transport Machinery Components in Streptomyces coelicolor

The Tat pathway transports folded proteins across the bacterial cytoplasmic membrane and is a major route of protein export in the Streptomyces genus of bacteria. In this study, we have examined the localization of Tat components in the model organism Streptomyces coelicolor by constructing enhanced green fluorescent protein (eGFP) and mCherry fusions with the TatA, TatB, and TatC proteins. All three components colocalized dynamically in the vegetative hyphae, with foci of each tagged protein being prominent at the tips of emerging germ tubes and of the vegetative hyphae, suggesting that this may be a primary site of Tat secretion. Time-lapse imaging revealed that localization of the Tat components was highly dynamic during tip growth and again demonstrated a strong preference for apical sites in growing hyphae. During aerial hypha formation, TatA-eGFP and TatB-eGFP fusions relocalized to prespore compartments, indicating repositioning of Tat components during the Streptomyces life cycle.     

Anthropometric Characteristics,Physical Fitness and Motor Coordination of 9 to 11 Year Old Children Participating in a Wide Range of Sports

ObjectivesThe aim of this study was to investigate to what extent 9 to 11 year old children participating in a specific sport already exhibit a specific anthropometric, physical fitness and motor coordination profile, in line with the requirements of that particular sport. In addition, the profiles in children with a different training volume were compared and possible differences in training hours per week between children from a low, moderate, and high level of physical fitness and motor coordination were investigated.DiscussionThe study showed that in general, children at a young age do not exhibit sport-specific characteristics, except in children with a high training volume. It is possible that on the one hand, children have not spent enough time yet in their sport to develop sport-specific qualities. On the other hand, it could be possible that they do not take individual qualities into account when choosing a sport.     

Molecular architecture of the ribosome‐bound Hepatitis C Virus internal ribosomal entry site RNA

Internal ribosomal entry sites (IRESs) are structured cis‐acting RNAs that drive an alternative, cap‐independent translation initiation pathway. They are used by many viruses to hijack the translational machinery of the host cell. IRESs facilitate translation initiation by recruiting and actively manipulating the eukaryotic ribosome using only a subset of canonical initiation factor and IRES transacting factors. Here we present cryo‐EM reconstructions of the ribosome 80S‐ and 40S‐bound Hepatitis C Virus (HCV) IRES. The presence of four subpopulations for the 80S•HCV IRES complex reveals dynamic conformational modes of the complex. At a global resolution of 3.9 Å for the most stable complex, a derived atomic model reveals a complex fold of the IRES RNA and molecular details of its interaction with the ribosome. The comparison of obtained structures explains how a modular architecture facilitates mRNA loading and tRNA binding to the P‐site. This information provides the structural foundation for understanding the mechanism of HCV IRES RNA‐driven translation initiation.     

Hormone-regulated expression and distribution of versican in mouse uterine tissues

Background  

Remodeling of the extracellular matrix is one of the most striking features observed in the uterus during the estrous cycle and after hormone replacement. Versican (VER) is a hyaluronan-binding proteoglycan that undergoes RNA alternative splicing, generating four distinct isoforms. This study analyzed the synthesis and distribution of VER in mouse uterine tissues during the estrous cycle, in ovariectomized (OVX) animals and after 17beta-estradiol (E2) and medroxyprogesterone (MPA) treatments, either alone or in combination.