Pattern and Performance of Understory Bamboos (Chusquea spp.) under Different Canopy Closures in Old-Growth Oak Forests in Costa Rica1

The effect of canopy closure on spatial and morphological patterns of three understory Chusquea species was studied in pristine upper montane oak forests. Plots with either Chusquea talamancenis Widmer & L.G. Clark or C. tomentosa Widmer & L.G. Clark were set out in gaps, under intermediate tree canopy and under closed canopy. The third species Chusquea foliosa L.G. Clark was only found in gap and intermediate canopy sites in the study area. Clumps of all bamboo species tend to be few and large under open conditions and numerous and small under more shady conditions. The larger size of clumps in gaps is reflected in the number of culms per clump, the diameter of the culms and, as a consequence, clump basal area, clump crown area, and biomass (index of plant volume) compared to intermediate and closed canopy. The smaller clump density in gaps compared to closed canopy implies that there is more intraspecific competition and density dependent mortality (self-thinning effect) when a bamboo species is dominant under favourable light conditions. Parameters of performance like culm length, number of nodes per culm, number and length of primary branches, and number of branch nodes seem not to be affected by the light regime, unlike total number of branches, total branch length, and the branching pattern. Species differ in their response to the light environment: C. tomentosa and C. foliosa have a higher degree of morphological plasticity than C. talamancensis, which in turn, appears to be more shade tolerant. No difference between species has been found regarding their contribution to vegetation, parameters reflecting abundance per unit area (site clump area, site crown area and site index of clump volume) were similar for all three species.     

The pilH gene encodes an ABC transporter homologue required for type IV pilus biogenesis and social gliding motility in Myxococcus xanthus

Type IV pilus genes have been shown to be required for social gliding motility in Myxococcus xanthus . We report the discovery of four additional pil genes: pilD , a homologue of type IV prepilin leader peptidases; and pilG , pilH and pilI , which have no known homologues in other type IV pilus systems. pilH encodes an ATP-binding cassette (ABC) transporter homologue, the first such homologue to be required for the biogenesis of any bacterial pilus type. pilG and pilI are co-transcribed with pilH and appear to be functionally related to pilH . Null mutants of pilG , pilH and pilI all lack social motility, are deficient in pilus production, have elevated sporulation efficiencies and display similar developmental abnormalities. In addition, all three mutations reduced the amount of PilA found in the supernatant after cells were sedimented from liquid culture. We suggest that the products of these three genes form a single ABC exporter complex, in which pilI is an integral membrane protein with membrane-spanning domains, and pilG is an accessory factor. The complex may participate in pilus assembly and/or the export of PilA pilin.     

Validation of a NAT-based Mycoplasma assay according European Pharmacopoiea

Eucaryotic expression systems are widely used to produce biologicals for human use, e.g. vaccines, recombinant proteins and monoclonal antibodies. As part of the safety testing the current U.S. Food and Drug Administration (FDA) regulatory guidelines as well as several European Pharmacopoiea monographs requests the demonstration of the absence of Mycoplasma in the cell culture in the bioreactors prior to harvest and further downstream processing. In recent years progress has been made in the development of a sensitive NAT-based method for the detection of Mycoplasma species in CHO cells, e.g. Eldering et al. This method is based on a nucleic acid amplification technique using a very sensitive touch-down PCR-profile. The presence of mollicutes DNA in the test specimens is determined by an approx. 450 bp target sequence which is amplified and this amplicon is finally detected by polyacrylamide gel electrophoresis. Based on this method a ready-to-use test kit was developed. In this report the validation of both method variants according the European Pharmacopoiea monograph 2.6.7 “Mycoplasmas” is described. The validation demonstrated the robustness and precision as well as a sufficient specificity of both assay formats. The validated sensitivity fulfills the requirements of the European Pharmacopoiea for a PCR-based method proposed as an alternative to the time consuming indicator cell culture and the culture method for the detection of Mollicutes (requested sensitivity of at least 10 colony-forming-units/mL).     

The Prognostic Value of the Work Ability Index for Sickness Absence among Office Workers

BackgroundThe work ability index (WAI) is a frequently used tool in occupational health to identify workers at risk for a reduced work performance and for work-related disability. However, information about the prognostic value of the WAI to identify workers at risk for sickness absence is scarce.ObjectivesTo investigate the prognostic value of the WAI for sickness absence, and whether the discriminative ability differs across demographic subgroups.MethodsAt baseline, the WAI (score 7-49) was assessed among 1,331 office workers from a Dutch financial service company. Sickness absence was registered during 12-months follow-up and categorised as 0 days, 0<days<5, 5≤days<15, and ≥15 days in one year. Associations between WAI and sickness absence were estimated by multinomial regression analyses. Discriminative ability of the WAI was assessed by the Area Under the Curve (AUC) and Ordinal c-index (ORC). Test characteristics were determined for dichotomised outcomes. Additional analyses were performed for separate WAI dimensions, and subgroup analyses for demographic groups.ResultsA lower WAI was associated with sickness absence (≥15 days vs. 0 days: per point lower WAI score OR=1.27; 95%CI 1.21-1.33). The WAI showed reasonable ability to discriminate between categories of sickness absence (ORC=0.65; 95%CI 0.63-0.68). Highest discrimination was found for comparing workers with ≥15 sick days with 0 sick days (AUC=0.77) or with 1-5 sick days (AUC=0.69). At the cut-off for poor work ability (WAI≤27) the sensitivity to identify workers at risk for ≥15 sick days was 7.5%, the specificity 99.6%, and the positive predictive value 82%. The performance was similar across demographic subgroups.ConclusionsThe WAI could be used to identify workers at high risk for prolonged sickness absence. However, due to low sensitivity many workers will be missed. Hence, additional factors are required to better identify workers at highest risk.     

Diversification and Distribution of Ruminant Chlamydia abortus Clones Assessed by MLST and MLVA

Chlamydia abortus, an obligate intracellular bacterium, is the most common infectious cause of abortion in small ruminants worldwide and has zoonotic potential. We applied multilocus sequence typing (MLST) together with multiple-locus variable-number tandem repeat analysis (MLVA) to genotype 94 ruminant C. abortus strains, field isolates and samples collected from 1950 to 2011 in diverse geographic locations, with the aim of delineating C. abortus lineages and clones. MLST revealed the previously identified sequence types (STs) ST19, ST25, ST29 and ST30, plus ST86, a recently-assigned type on the Chlamydiales MLST website and ST87, a novel type harbouring the hemN_21 allele, whereas MLVA recognized seven types (MT1 to MT7). Minimum-spanning-tree analysis suggested that all STs but one (ST30) belonged to a single clonal complex, possibly reflecting the short evolutionary timescale over which the predicted ancestor (ST19) has diversified into three single-locus variants (ST86, ST87 and ST29) and further, through ST86 diversification, into one double-locus variant (ST25). ST descendants have probably arisen through a point mutation evolution mode. Interestingly, MLVA showed that in the ST19 population there was a greater genetic diversity than in other STs, most of which exhibited the same MT over time and geographical distribution. However, the evolutionary pathways of C. abortus STs seem to be diverse across geographic distances with individual STs restricted to particular geographic locations. The ST30 singleton clone displaying geographic specificity and represented by the Greek strains LLG and POS was effectively distinguished from the clonal complex lineage, supporting the notion that possibly two separate host adaptations and hence independent bottlenecks of C. abortus have occurred through time. The combination of MLST and MLVA assays provides an additional level of C. abortus discrimination and may prove useful for the investigation and surveillance of emergent C. abortus clonal populations.     

Personality over ontogeny in zebra finches: long-term repeatable traits but unstable behavioural syndromes

A crucial assumption of animal personality research is that behaviour is consistent over time, showing a high repeatability within individuals. This assumption is often made, sometimes tested using short time intervals between behavioural tests, but rarely thoroughly investigated across long time intervals crossing different stages of ontogeny. We performed such a longitudinal test across three life stages in zebra finches (Taeniopygia guttata), representing about 15-20% of their life span in captivity, and found repeatabilities ranging from 0.03 to 0.67. Fearlessness and exploration were the most repeatable traits both within and across life stages. Activity and aggression were repeatable across, but not or only partly within life stages. Boldness was not repeatable. Furthermore, we found no evidence for a consistent behavioural syndrome structure across ontogeny. Our results indicate that the consistency of behavioural traits and their correlations might be overestimated and suggest that life-long stability of animal personality should not simply be assumed.     

Evaluation of Protein Profiles From Treated Xenograft Tumor Models Identifies an Antibody Panel for Formalin-fixed and Paraffin-embedded (FFPE) Tissue Analysis by Reverse Phase Protein Arrays (RPPA)

Reverse phase protein arrays (RPPA) are an established tool for measuring the expression and activation status of multiple proteins in parallel using only very small amounts of tissue. Several studies have demonstrated the value of this technique for signaling pathway analysis using proteins extracted from fresh frozen (FF) tissue in line with validated antibodies for this tissue type; however, formalin fixation and paraffin embedding (FFPE) is the standard method for tissue preservation in the clinical setting. Hence, we performed RPPA to measure profiles for a set of 300 protein markers using matched FF and FFPE tissue specimens to identify which markers performed similarly using the RPPA technique in fixed and unfixed tissues. Protein lysates were prepared from matched FF and FFPE tissue specimens of individual tumors taken from three different xenograft models of human cancer. Materials from both untreated mice and mice treated with either anti-HER3 or bispecific anti-IGF-1R/EGFR monoclonal antibodies were analyzed. Correlations between signals from FF and FFPE tissue samples were investigated. Overall, 60 markers were identified that produced comparable profiles between FF and FFPE tissues, demonstrating significant correlation between the two sample types. The top 25 markers also showed significance after correction for multiple testing. The panel of markers covered several clinically relevant tumor signaling pathways and both phosphorylated and nonphosphorylated proteins were represented. Biologically relevant changes in marker expression were noted when RPPA profiles from treated and untreated xenografts were compared. These data demonstrate that, using appropriately selected antibodies, RPPA analysis from FFPE tissue is well feasible and generates biologically meaningful information. The identified panel of markers that generate similar profiles in matched fixed and unfixed tissue samples may be clinically useful for pharmacodynamic studies of drug effect using FFPE tissues.Many human diseases are characterized by abnormalities in complex signaling pathways (1). The expression and activation status of proteins from these deregulated pathways has traditionally been analyzed using single marker techniques such as immunohistochemistry and Western blotting. Although these techniques have provided valuable information on the molecular abnormalities underlying human disease, they are labor intensive, have a low throughput, and often require high sample volume. Furthermore, techniques such as Western blotting are not applicable in the routine clinical setting. Miniaturized parallel immunoassay techniques have been developed in recent years and have played a pivotal role in biomarker discovery (2). Antibody arrays enable multiple potential disease markers to be investigated in a single sample in parallel (3). Beyond this, Reverse Phase Protein Arrays (RPPA)¹ are sensitive high throughput tools that can quantify protein expression levels and activation status (posttranslational modifications such as phosphorylation) in multiple experimental samples simultaneously. The technique requires only minute amounts of samples, printed as lysate arrays onto slides, and hundreds of markers of interest can be investigated, array by array, in a miniaturized dot blot manner. Numerous reports have demonstrated that RPPA can be applied to various sources of cells and tissues to analyze protein profiles, signaling pathway networks, and for the identification of biomarkers (4–13). A recently published workshop report reviews the full potential and advances of RPPA for use in clinical, translational, and basic research (11).In oncology, the parallel profiling of multiple protein markers is particularly desirable to study tumor initiation and progression, to classify tumor disease states on the molecular level, and to discover and monitor biomarkers that can predict therapeutic response or tumor recurrence (14–16). The study of signaling response and analysis of pharmacodynamic (PD) markers upon treatment using in vitro and in vivo test systems (e.g. cell line or patient derived xenograft tumor models) is an established component of preclinical and early clinical drug development. These techniques can provide evidence of target pathway modulation for new therapeutic lead candidate compounds and provide valuable information on the drug mode of action (17), especially in the translational phase. Multiplex analyses of PD biomarkers by RPPA have been performed in vitro using cancer cell lines (18, 19) as well as in patient-derived tumor tissue and blood samples (20, 21) to assess response to treatment and target inhibition. A combination of RPPA signaling pathway mapping and functional PET imaging has recently been successfully evaluated in xenograft models as an early response PD marker for anti-cancer drug efficacy (13).Translating miniaturized multiple protein analysis platforms-such as RPPA - from preclinical to clinical applicability is highly desirable; however, issues such as the limited amount of available clinical samples and tumor heterogeneity must first be addressed. Furthermore, most studies of RPPA in tumor tissue to date have been conducted using proteins extracted from fresh-frozen (FF) tissue specimens; whereas, formalin fixation and paraffin embedding (FFPE) is the standard method for tissue preservation used in clinical pathology laboratories. FFPE yields excellent tissue architecture for histological assessment and enables analysis of individual proteins in situ by techniques such as immunohistochemistry. However, formalin fixation leads to extensive protein–protein and protein–nucleic acid cross-linking (22), which can hamper protein extraction and reduce both the overall yield of extracted protein and the profile of proteins detectable by proteomic techniques (23, 24). Furthermore, formalin-induced cross-linking induces conformational changes in protein structure that can alter the immunoreactivity of some proteins in situ by hiding or altering peptide epitopes (25, 26). Such artifacts are absent from snap-frozen tissue; therefore, protein profiles obtained from FF tissue are likely to reflect the in vivo biology of the tumor more closely. However, FF tumor tissue is not widely available because it is costly to collect and maintain in the clinical setting. FFPE tissue samples are routinely archived by nearly every hospital and offer a unique opportunity to study thousands of samples retrospectively with extensive clinical records and follow-up information.Several groups have now established protocols for retrieving cross-linked proteins from fixed tissues (27–33). These methods are mainly based on the use of concentrated ionic detergents and high temperature protocols closely related to the antigen retrieval methods developed for immunohistochemistry. These studies show that obtaining nondegraded, full-length proteins from FFPE tissues for multiplex analyses is feasible (27–33). More recently, protein extraction techniques optimized for fixed samples have been used to successfully conduct RPPA using FFPE tissue biopsies from different cancer types (34–40). Guo et al. systematically investigated several protein extraction methods and demonstrated that RPPA of FFPE materials is feasible, reproducible and can generate biologically relevant protein profiles (41). Other studies have confirmed the validity of this approach and shown that data generated from RPPA analyses of FFPE tissue demonstrate good concordance with traditional immunohistochemistry markers such as HER2 protein in breast cancer (34, 40). However, to date, analyses have been performed only for a limited set of protein markers.To evaluate whether analysis of a broader panel of protein markers is feasible and generates meaningful data from FFPE tumor tissue sections, we conducted RPPA on matched samples of FF and FFPE tissues using a set of 300 markers, the largest panel reported to date. Our aim was to identify markers that performed similarly when comparing the protein profiles measured in protein extracts from matched FF and FFPE tissue, using RPPA assays established for use in frozen tissues. Correlating selected markers and assays in such a way should qualify RPPA for further use with FFPE tissues of clinical relevance, e.g. in PD marker studies. In this paper, we have specifically focused on the technical issues relevant for using the RPPA platform in a clinical setting, and did not address the biology of the test systems used in detail. However, the models used have been pre-characterized to identify key signaling parameters in context of targeted drug treatment (42). We conducted a systematic comparison of RPPA protein profiles in matched FF and FFPE tumor tissues resected from three different xenograft models of human cancer, each treated with targeted therapeutic antibodies that have previously been shown to achieve tumor growth inhibition. Furthermore, we investigated the effect of targeted drug treatment on protein expression and activation status, and the concordance of matched FF and FFPE tissue RPPA profiles. Finally, with one of the applied tumor models, we compared a set of protein profiles measured with two different multiple assay platforms - the RPPA and the Luminex Bio-Plex system, and discuss their relevance with respect to the analysis of FFPE tissue.     

Dynamic 3D culture: Models of chondrogenesis and endochondral ossification

The formation of cartilage from stem cells during development is a complex process which is regulated by both local growth factors and biomechanical cues, and results in the differentiation of chondrocytes into a range of subtypes in specific regions of the tissue. In fetal development cartilage also acts as a precursor scaffold for many bones, and mineralization of this cartilaginous bone precursor occurs through the process of endochondral ossification. In the endochondral formation of bones during fetal development the interplay between cell signalling, growth factors, and biomechanics regulates the formation of load bearing bone, in addition to the joint capsule containing articular cartilage and synovium, generating complex, functional joints from a single precursor anlagen. These joint tissues are subsequently prone to degeneration in adult life and have poor regenerative capabilities, and so understanding how they are created during development may provide useful insights into therapies for diseases, such as osteoarthritis, and restoring bone and cartilage lost in adulthood. Of particular interest is how these tissues regenerate in the mechanically dynamic environment of a living joint, and so experiments performed using 3D models of cartilage development and endochondral ossification are proving insightful. In this review, we discuss some of the interesting models of cartilage development, such as the chick femur which can be observed in ovo, or isolated at a specific developmental stage and cultured organotypically in vitro. Biomaterial and hydrogel‐based strategies which have emerged from regenerative medicine are also covered, allowing researchers to make informed choices on the characteristics of the materials used for both original research and clinical translation. In all of these models, we illustrate the essential importance of mechanical forces and mechanotransduction as a regulator of cell behavior and ultimate structural function in cartilage. Birth Defects Research (Part C) 105:19–33, 2015. © 2015 Wiley Periodicals, Inc.     

A Novel Virus Causes Scale Drop Disease in Lates calcarifer

From 1992 onwards, outbreaks of a previously unknown illness have been reported in Asian seabass (Lates calcarifer) kept in maricultures in Southeast Asia. The most striking symptom of this emerging disease is the loss of scales. It was referred to as scale drop syndrome, but the etiology remained enigmatic. By using a next-generation virus discovery technique, VIDISCA-454, sequences of an unknown virus were detected in serum of diseased fish. The near complete genome sequence of the virus was determined, which shows a unique genome organization, and low levels of identity to known members of the Iridoviridae. Based on homology of a series of putatively encoded proteins, the virus is a novel member of the Megalocytivirus genus of the Iridoviridae family. The virus was isolated and propagated in cell culture, where it caused a cytopathogenic effect in infected Asian seabass kidney and brain cells. Electron microscopy revealed icosahedral virions of about 140 nm, characteristic for the Iridoviridae. In vitro cultured virus induced scale drop syndrome in Asian seabass in vivo and the virus could be reisolated from these infected fish. These findings show that the virus is the causative agent for the scale drop syndrome, as each of Koch’s postulates is fulfilled. We have named the virus Scale Drop Disease Virus. Vaccines prepared from BEI- and formalin inactivated virus, as well as from E. coli produced major capsid protein provide efficacious protection against scale drop disease.