Embryonic gonadal and sexual organ development in a small viviparous skink, Niveoscincus ocellatus

The majority of research into the timing of gonad differentiation (and sex determination) in reptiles has focused on oviparous species. This is largely because: (1) most reptiles are oviparous; (2) it is easier to manipulate embryonic developmental conditions (e.g., temperature) of eggs than oviductal embryos and (3) modes of sex determination in oviparous taxa were thought to be more diverse since viviparity and environmental sex determination (ESD)/temperature-dependent sex determination (TSD) were considered incompatible. However, recent evidence suggests the two may well be compatible biological attributes, opening potential new lines of enquiry into the evolution and maintenance of sex determination. Unfortunately, the baseline information on embryonic development in viviparous species is lacking and information on gonad differentiation and sexual organ development is almost non-existent. Here we present an embryonic morphological development table (10 stages), the sequence of gonad differentiation and sexual organ development for the viviparous spotted snow skink (Niveoscincus ocellatus). Gonad differentiation in this species is similar to other reptilian species. Initially, the gonads are indifferent and both male and female accessory ducts are present. During stage 2, in the middle third of development, differentiation begins as the inner medulla regresses and the cortex thickens signaling ovary development, while the opposite occurs in testis formation. At this point, the Müllerian (female reproductive) duct regresses in males until it is lost (stage 6), while females retain both ducts until after birth. In the later stages of testis development, interstitial tissue forms in the medulla corresponding to maximum development of the hemipenes in males and the corresponding regression in the females.     

Sensitivity of methods for estimating breeding values using genetic markers to the number of QTL and distribution of QTL variance

The objective of this simulation study was to compare the effect of the number of QTL and distribution of QTL variance on the accuracy of breeding values estimated with genomewide markers (MEBV). Three distinct methods were used to calculate MEBV: a Bayesian Method (BM), Least Angle Regression (LARS) and Partial Least Square Regression (PLSR). The accuracy of MEBV calculated with BM and LARS decreased when the number of simulated QTL increased. The accuracy decreased more when QTL had different variance values than when all QTL had an equal variance. The accuracy of MEBV calculated with PLSR was affected neither by the number of QTL nor by the distribution of QTL variance. Additional simulations and analyses showed that these conclusions were not affected by the number of individuals in the training population, by the number of markers and by the heritability of the trait. Results of this study show that the effect of the number of QTL and distribution of QTL variance on the accuracy of MEBV depends on the method that is used to calculate MEBV.     

Purification of an insect derived recombinant human ADAMTS-1 reveals novel gelatin (type I collagen) degrading activities

ADAMTS-1 (A Disintegrin And Metalloprotease with ThromboSpondin repeats) is a member of a family of secreted proteolytic enzymes with a complex modular structure. These enzymes are characterised by an N-terminal metalloproteinase domain, a disintegrin-like domain and a carboxyl terminal region containing variable numbers of a repeat sequence with homology to thrombospondin-1. The expression of the gene for ADAMTS-1 has been associated with inflammation, ovulation, angiogenesis, cellular proliferation and bone formation. ADAMTS-1 can proteolytically process large proteoglycans indicating a potential role in extracellular matrix turnover. In this study, we have tested ADAMTS-1 activity in gelatin zymogram assays. Since previous data demonstrate that ADAMTS-1 is a matrix metalloproteinase (MMP) substrate and is highly unstable in conditioned medium from eukaryotic cell types, we created an insect cell line expressing human ADAMTS-1. We isolated an epitope tagged full-length recombinant ADAMTS-1 from serum free insect cell conditioned medium. The purified protein had aggrecanase activity and appears as two major bands on the silver stained SDS-PAGE corresponding well to a pro-domain on form of 115 kDa and a pro-domain off form of 90 kDa. Using denatured type I collagen in zymographic analysis we demonstrate that ADAMTS-1 has a previously unreported gelatinolytic activity. Also, we notice that processing of its C-terminal region by an apparently autocatalytic process reveals a 27 kDa species with gelatinolytic activity. Furthermore, we show that MMP2 but not MMP13 remove ADAMTS-1 specific gelatin zymopraphic zones.     

Chimeric proteins suggest that the catalytic and/or C-terminal domains give CesA1 and CesA3 access to their specific sites in the cellulose synthase of primary walls

CesA1 and CesA3 are thought to occupy noninterchangeable sites in the cellulose synthase making primary wall cellulose in Arabidopsis (Arabidopsis thaliana L. Heynh). With domain swaps and deletions, we show that sites C terminal to transmembrane domain 2 give CesAs access to their individual sites and, from dominance and recessive behavior, deduce that certain CesA alleles exclude others from accessing each site. Constructs that swapped or deleted N-terminal domains were stably transformed into the wild type and into the temperature-sensitive mutants rsw1 (Ala-549Val in CesA1) and rsw5 (Pro-1056Ser in CesA3). Dominant-positive behavior was assayed as root elongation at the restrictive temperature and dominant-negative effects were observed at the permissive temperature. A protein with the catalytic and C-terminal domains of CesA1 and the N-terminal domain of CesA3 promoted growth only in rsw1 consistent with it accessing the CesA1 site even though it contained the CesA3 N-terminal domain. A protein having the CesA3 catalytic and C-terminal domains linked to the CesA1 N-terminal domain dramatically affected growth, but only in the CesA3 mutant. This is consistent with the operation of the same access rule taking this chimeric protein to the CesA3 site. In this case, however, the transgene behaved as a genotype-specific dominant negative, causing a 60% death rate in rsw5, but giving no visible phenotype in wild type or rsw1. We therefore hypothesize that possession of CesA3(WT) protects Columbia and rsw1 from the lethal effects of this chimeric protein, whereas the mutant protein (CesA3(rsw5)) does not.     

Generation and maintenance of diversity in the cattle MHC class I region

Major histocompatibility complex (MHC) class I genes play a crucial role in the immune defence against intracellular pathogens. An important evolutionary strategy is to generate and maintain a high level of diversity in these genes. Humans express three highly polymorphic classical MHC class I genes (HLA-A, HLA-B and HLA-C). In contrast, some species, for example rat and rhesus macaque, maintain diversity by generation of haplotypes that vary considerably with regard to the number and combination of transcribed genes. Cattle appear to use both strategies. We show that various combinations of six apparently classical genes, three of which are highly polymorphic, are transcribed on different haplotypes. Although additional sequences were identified in both cDNA and gDNA, it was not possible to assign them to any of these defined genes. Most were highly divergent or were non-classical class I genes. Thus, we found little evidence for frequent duplication and deletion of classical class I genes as reported in some other species. However, the maintenance of class I diversity in cattle may involve limited gene shuffling and deletion, possibly as a result of unequal crossing-over within the class I region.The first two authors made an equal contribution to this work.     

The intramuscular contribution to the slow oxygen uptake kinetics during exercise in chronic heart failure is related to the severity of the condition

The mechanism for slow pulmonary O(2) uptake (Vo(2)) kinetics in patients with chronic heart failure (CHF) is unclear but may be due to limitations in the intramuscular control of O(2) utilization or O(2) delivery. Recent evidence of a transient overshoot in microvascular deoxygenation supports the latter. Prior (or warm-up) exercise can increase O(2) delivery in healthy individuals. We therefore aimed to determine whether prior exercise could increase muscle oxygenation and speed Vo(2) kinetics during exercise in CHF. Fifteen men with CHF (New York Heart Association I-III) due to left ventricular systolic dysfunction performed two 6-min moderate-intensity exercise transitions (bouts 1 and 2, separated by 6 min of rest) from rest to 90% of lactate threshold on a cycle ergometer. Vo(2) was measured using a turbine and a mass spectrometer, and muscle tissue oxygenation index (TOI) was determined by near-infrared spectroscopy. Prior exercise increased resting TOI by 5.3 ± 2.4% (P = 0.001), attenuated the deoxygenation overshoot (-3.9 ± 3.6 vs. -2.0 ± 1.4%, P = 0.011), and speeded the Vo(2) time constant (τVo(2); 49 ± 19 vs. 41 ± 16 s, P = 0.003). Resting TOI was correlated to τVo(2) before (R(2) = 0.51, P = 0.014) and after (R(2) = 0.36, P = 0.051) warm-up exercise. However, the mean response time of TOI was speeded between bouts in half of the patients (26 ± 8 vs. 20 ± 8 s) and slowed in the remainder (32 ± 11 vs. 44 ± 16 s), the latter group having worse New York Heart Association scores (P = 0.042) and slower Vo(2) kinetics (P = 0.001). These data indicate that prior moderate-intensity exercise improves muscle oxygenation and speeds Vo(2) kinetics in CHF. The most severely limited patients, however, appear to have an intramuscular pathology that limits Vo(2) kinetics during moderate exercise.     

Constraints on haplotype structure and variable gene frequencies suggest a functional hierarchy within cattle MHC class I

Six major histocompatibility complex (MHC) classical class I genes have been identified in cattle, and up to three of these are expressed in variable combinations on different haplotypes. The origin and functional significance of this genetic complexity is unknown. However, an improved assembly of the cattle genome, an expanded database of full-length cDNA sequences and high-resolution frequency data concerning expressed class I genes in an economically important cattle breed combine to provide a new opportunity to study the significance of cattle MHC class I diversity. Analysis of these new data supports assignment of alleles to six discrete genes and further shows that all these classical genes share a common ancestor with a single non-classical gene, NC1. While haplotype structure is variable, with thirteen gene configurations identified, there are nevertheless clear constraints relating to both the number and combination of genes. Haplotypes expressing two classical genes are most frequently observed, and the classical class I gene 2 is almost invariably present. The frequency data support the dominance of gene 2, showing that close to 100?% of individuals carry at least one copy. This indicates a hierarchy in the functional importance of particular genes and haplotype structures. Haplotype frequency in cattle populations is therefore likely to impact on differential disease susceptibilities. This knowledge will be important for development of informed breeding strategies aimed at increasing the ability of cattle to survive in the face of future unpredictable pathogen exposure.     

GPR179 is required for depolarizing bipolar cell function and is mutated in autosomal-recessive complete congenital stationary night blindness

Complete congenital stationary night blindness (cCSNB) is a clinically and genetically heterogeneous group of retinal disorders characterized by nonprogressive impairment of night vision, absence of the electroretinogram (ERG) b-wave, and variable degrees of involvement of other visual functions. We report here that mutations in GPR179, encoding an orphan G protein receptor, underlie a form of autosomal-recessive cCSNB. The Gpr179(nob5/nob5) mouse model was initially discovered by the absence of the ERG b-wave, a component that reflects depolarizing bipolar cell (DBC) function. We performed genetic mapping, followed by next-generation sequencing of the critical region and detected a large transposon-like DNA insertion in Gpr179. The involvement of GPR179 in DBC function was confirmed in zebrafish and humans. Functional knockdown of gpr179 in zebrafish led to a marked reduction in the amplitude of the ERG b-wave. Candidate gene analysis of GPR179 in DNA extracted from patients with cCSNB identified GPR179-inactivating mutations in two patients. We developed an antibody against mouse GPR179, which robustly labeled DBC dendritic terminals in wild-type mice. This labeling colocalized with the expression of GRM6 and was absent in Gpr179(nob5/nob5) mutant mice. Our results demonstrate that GPR179 plays a critical role in DBC signal transduction and expands our understanding of the mechanisms that mediate normal rod vision.     

Valid and reliable instruments for arm-hand assessment at ICF activity level in persons with hemiplegia: a systematic review

Background

Loss of arm-hand performance due to a hemiparesis as a result of stroke or cerebral palsy (CP), leads to large problems in daily life of these patients. Assessment of arm-hand performance is important in both clinical practice and research. To gain more insight in e.g. effectiveness of common therapies for different patient populations with similar clinical characteristics, consensus regarding the choice and use of outcome measures is paramount. To guide this choice, an overview of available instruments is necessary. The aim of this systematic review is to identify, evaluate and categorize instruments, reported to be valid and reliable, assessing arm-hand performance at the ICF activity level in patients with stroke or cerebral palsy.

Methods

A systematic literature search was performed to identify articles containing instruments assessing arm-hand skilled performance in patients with stroke or cerebral palsy. Instruments were identified and divided into the categories capacity, perceived performance and actual performance. A second search was performed to obtain information on their content and psychometrics.

Results

Regarding capacity, perceived performance and actual performance, 18, 9 and 3 instruments were included respectively. Only 3 of all included instruments were used and tested in both patient populations. The content of the instruments differed widely regarding the ICF levels measured, assessment of the amount of use versus the quality of use, the inclusion of unimanual and/or bimanual tasks and the inclusion of basic and/or extended tasks.

Conclusions

Although many instruments assess capacity and perceived performance, a dearth exists of instruments assessing actual performance. In addition, instruments appropriate for more than one patient population are sparse. For actual performance, new instruments have to be developed, with specific focus on the usability in different patient populations and the assessment of quality of use as well as amount of use. Also, consensus about the choice and use of instruments within and across populations is needed.