The Rho kinase Rock2b establishes anteroposterior asymmetry of the ciliated Kupffer's vesicle in zebrafish

The vertebrate body plan features a consistent left-right (LR) asymmetry of internal organs. In several vertebrate embryos, motile cilia generate an asymmetric fluid flow that is necessary for normal LR development. However, the mechanisms involved in orienting LR asymmetric flow with previously established anteroposterior (AP) and dorsoventral (DV) axes remain poorly understood. In zebrafish, asymmetric flow is generated in Kupffer's vesicle (KV). The cellular architecture of KV is asymmetric along the AP axis, with more ciliated cells densely packed into the anterior region. Here, we identify a Rho kinase gene, rock2b, which is required for normal AP patterning of KV and subsequent LR development in the embryo. Antisense depletion of rock2b in the whole embryo or specifically in the KV cell lineage perturbed asymmetric gene expression in lateral plate mesoderm and disrupted organ LR asymmetries. Analyses of KV architecture demonstrated that rock2b knockdown altered the AP placement of ciliated cells without affecting cilia number or length. In control embryos, leftward flow across the anterior pole of KV was stronger than rightward flow at the posterior end, correlating with the normal AP asymmetric distribution of ciliated cells. By contrast, rock2b knockdown embryos with AP patterning defects in KV exhibited randomized flow direction and equal flow velocities in the anterior and posterior regions. Live imaging of Tg(dusp6:memGFP)(pt19) transgenic embryos that express GFP in KV cells revealed that rock2b regulates KV cell morphology. Our results suggest a link between AP patterning of the ciliated Kupffer's vesicle and LR patterning of the zebrafish embryo.     

Ultrasonographic assessment of skeletal muscles after experimentally induced neurogenic inflammation (facet injury) in rats

This study set out to examine ultrasonographic attributes of non-neurosegmentally (pectoral-forelimb) and neurosegmentally linked (hindlimb) myotomes in an experimental model that leads to neurogenic inflammation in segmentally linked myotomes, and to evaluate quantitative correlations among ultrasonographic attributes of the muscles, relative content of various inflammatory mediators, and nociceptive thresholds (hot and mechanical) in rats. Twelve male Wistar Kyoto rats were randomly divided into two equinumerous groups: surgery group, in which the left lumbar (L4–L6) facet joints were compressed for 3 min with modified Kelly forceps under general anesthesia, and sham-operated rats. All ultrasonograms were obtained with the Vevo 2100 Visual Sonic scanner connected to a 24-MHz transducer at four different time points: pre-surgery and 7, 14, and 21 days after surgical procedures. Digital ultrasonographic images of quadriceps femoris, hamstring, and pectoral-brachial muscle groups were analyzed using a polygonal meter region of interest placed on the largest cross-sectional area of the muscles displayed in Image ProPlus^® analytical software to compute numerical pixel values and pixel heterogeneity (standard deviation of mean pixel values). On day 21, pain behavior tests (hot plate and von Frey) were performed and then all animals were euthanized. Protein expression of inflammatory mediators in biceps brachii and rectus femoris muscles was measured by Western blot. The most prominent differences in muscle echotextural attributes between the two subsets of rats occurred 14 days post-surgery in pectoral-brachial and quadriceps femoris muscles. The expression of calcitonin-gene-related peptide was directly related to both echotextural variables only in biceps brachii (pixel intensity: r = 0.65, P = 0.02; and heterogeneity: r = 0.66, P = 0.02, respectively). Our findings have revealed the occurrence of echotextural changes in skeletal muscles of rats during myositis; however, the accumulation of inflammatory mediators and the outcomes of sensory tests did not relate to the changes in first-order echotextural characteristics of affected hindlimb muscles.     

Identification of high-confidence somatic mutations in whole genome sequence of formalin-fixed breast cancer specimens

The utilization of archived, formalin-fixed paraffin-embedded (FFPE) tumor samples for massive parallel sequencing has been challenging due to DNA damage and contamination with normal stroma. Here, we perform whole genome sequencing of DNA isolated from two triple-negative breast cancer tumors archived for >11 years as 5 μm FFPE sections and matched germline DNA. The tumor samples show differing amounts of FFPE damaged DNA sequencing reads revealed as relatively high alignment mismatch rates enriched for C · G > T · A substitutions compared to germline samples. This increase in mismatch rate is observable with as few as one million reads, allowing for an upfront evaluation of the sample integrity before whole genome sequencing. By applying innovative quality filters incorporating global nucleotide mismatch rates and local mismatch rates, we present a method to identify high-confidence somatic mutations even in the presence of FFPE induced DNA damage. This results in a breast cancer mutational profile consistent with previous studies and revealing potentially important functional mutations. Our study demonstrates the feasibility of performing genome-wide deep sequencing analysis of FFPE archived tumors of limited sample size such as residual cancer after treatment or metastatic biopsies.     

Photoreceptors for biosynthesis, energy storage and vision

Abstract Living organisms use light as a source of energy and as a means of obtaining information about their environment. Photoreactivating enzyme, provitamins D, retinal (rhodopsins and bacteriorhodopsin), porphyrins (chlorophyll, protochlorophyll and heme), photosynthetic accessory pigments (carotenoids and bilins), phytochrome and riboflavin: these are the molecules which life has settled upon to play the role of light receptor. For some of these photoreceptor molecules a great deal is now known about the chemistry which they perform upon absorbing light; for others virtually nothing is known. Riboflavin, the molecule believed to be functioning in a variety of organisms as the receptor for physiological responses to blue light, is an especially interesting case. Its widespread occurrence in cellular roles other than photoreception make it difficult to separate out the particular flavin which functions as the photoreceptor. It represents a case of a photoreceptor which is at once ubiquitous and elusive.     

Non-pertussis components of combination vaccines: problems with potency testing.

Vaccines comprising combinations of diphtheria, tetanus and pertussis (DTP) with Haemophilus influenzae type b polysaccharide-protein conjugate (Hib), inactivated poliomyelitis virus (IPV) and hepatitis B virus (HBV) are already available, and new combinations using acellular pertussis components in a triple vaccine (DTaP) are under development. Evidence to date has shown that control of the efficacy, safety and stability of combination vaccines cannot be based on information already available on the individual components or existing licensed formulations. Several examples of immunological interference between components of a combination vaccine have been observed both in clinical trials and in laboratory tests. Examples of these for D, T and Hib components in DTP and DTaP combinations have been investigated.     

Topsoil Thickness Influences Nitrogen Management of Switchgrass

Switchgrass (Panicum virgatum L.) is an attractive bioenergy crop option for eroded portions of claypan landscapes where grain crop production is marginally profitable. Topsoil thickness above the claypan, or depth to claypan (DTC), can vary widely within fields, and little information exists on its impacts on N management of switchgrass. Therefore, a study was conducted at the University of Missouri South Farm near Columbia, Missouri, to determine whether topsoil thickness influenced fertilizer N requirements of switchgrass. Switchgrass was planted in 2009 on main plots with a range of DTC classified as exposed (<8 cm), shallow (8–15 cm), moderate (16–30 cm), and deep (>30 cm) and was harvested annually at postdormancy during 2011 to 2015. Three split-plot treatments were 0, 67, or 101 kg N ha^?1 applied annually in May, and a fourth was three intercropped native legumes as the N source. Across years, the legume treatment apparently supplied no N because it produced the same or less switchgrass yield than the nonfertilized treatment. Topsoil proved valuable as switchgrass yield, nutrient removal, and profit usually increased as DTC increased. Fertilization with 101 kg N ha^?1 on exposed, shallow, or moderate DTC and 67 kg N ha^?1 on deep DTC was required to obtain the highest biomass yield, but it also increased nutrient removal. Strikingly, profit across years was negative for the legume treatment and highest with no fertilizer on all DTC classes. Therefore, improvements are needed before intercropped legumes are profitable, and N fertilization may be needed only periodically to maximize switchgrass profit on claypan soils.