Detection and imaging of non-contractile inclusions and sarcomeric anomalies in skeletal muscle by second harmonic generation combined with two-photon excited fluorescence

The large size of the multinucleated muscle fibers of skeletal muscle makes their examination for structural and pathological defects a challenge. Sections and single fibers are accessible to antibodies and other markers but imaging of such samples does not provide a three-dimensional view of the muscle. Regrettably, bundles of fibers cannot be stained or imaged easily. Two-photon microscopy techniques overcome these obstacles. Second harmonic generation (SHG) by myosin filaments and two-photon excited fluorescence (2PEF) of mitochondrial and lysosomal components provides detailed structural information on unstained tissue. Furthermore, the infrared exciting light can penetrate several layers of muscle fibers and the minimal processing is particularly valuable for fragile biopsies. Here we demonstrate the usefulness of SHG, combined with 2PEF, to reveal enlarged lysosomes and accumulations of non-contractile material in muscles from the mouse model for the lysosomal storage disorder Pompe disease (PD), and in biopsies from adult and infant PD patients. SHG and 2PEF also detect sarcomeric defects that may presage the loss of myofibrils in atrophying muscle and signify loss of elasticity. The combination of SHG and 2PEF should be useful in the analysis and diagnosis of a wide range of skeletal muscle pathologies.     

Accuracy of diagnostic registers and management of chronic obstructive pulmonary disease: the Devon primary care audit

Background

Guidelines on COPD diagnosis and management encourage primary care physicians to detect the disease at an early stage and to treat patients according to their condition and needs. Problems in guideline implementation include difficulties in diagnosis, using spirometry and the disputed role of reversibility testing. These lead to inaccurate diagnostic registers and inadequacy of administered treatments. This study represents an audit of COPD diagnosis and management in primary care practices in Devon.

Methods

Six hundred and thirty two patients on COPD registers in primary care practices were seen by a visiting Respiratory Specialist Nurse. Diagnoses were made according to the NICE guidelines. Reversibility testing was carried out either routinely or based on clinical indication in two sub-samples. Dyspnoea was assessed. Data were entered into a novel IT-based software which computed guideline-based treatment recommendations. Current and recommended treatments were compared.

Results

Five hundred and eighty patients had spirometry. Diagnoses of COPD were confirmed in 422 patients (73%). Thirty nine patients were identified as asthma only, 94 had normal spirometry, 23 were restrictive and 2 had a cardiac disorder. Reversibility testing changed diagnosis of 11% of patients with airflow obstruction, and severity grading in 18%. Three quarters of patients with COPD had been offered practical help with smoking cessation. Short and long-acting anticholinergics and long acting beta-2 agonists had been under-prescribed; in 15–18% of patients they were indicated but not received. Inhaled steroids had been over-prescribed (recommended in 17%; taken by 60%), whereas only 4% of patients with a chronic productive cough were receiving mucolytics. Pulmonary rehabilitation was not available in some areas and was under-used in other areas.

Conclusion

Diagnostic registers of COPD in primary care contain mistakes leading to inaccurate prevalence estimates and inappropriate treatment decisions. Use of pre-bronchodilator readings for diagnosis overestimates the prevalence and severity in a significant minority, thus post bronchodilator readings should be used. Management of stable COPD does often not correspond to guidelines. The IT system used in this study has the potential to improve diagnosis and management of COPD in primary care.     

Dramatic down-regulation of oxidoreductases in human hepatocellular carcinoma hepG2 cells: proteomics and gene ontology unveiling new frontiers in cancer enzymology

Background

Oxidoreductases are enzymes that catalyze many redox reactions in normal and neoplastic cells. Their actions include catalysis of the transformation of free, neutral oxygen gas into oxygen free radicals, superoxide, hydroperoxide, singlet oxygen and hydrogen peroxide. These activated forms of oxygen contribute to oxidative stress that modifies lipids, proteins, DNA and carbohydrates. On the other hand, oxidoreductases constitute one of the most important free radical scavenger systems typified by catalase, superoxide dismutase and glutathione peroxidase. In this work, proteomics, Gene Ontology mapping and Directed Acyclic Graphs (DAG) are employed to detect and quantify differential oxidoreductase enzyme expressions between HepG2 cells and normal human liver tissues.

Results

For the set of bioinformatics calculations whose BLAST searches are performed using the BLAST program BLASTP 2.2.13 [Nov-27-2005], DAG of the Gene Ontology's Molecular Function annotations show that oxidoreductase activity parent node of the liver proteome contains 331 annotated protein sequences, 7 child nodes and an annotation score of 188.9, whereas that of HepG2 cells has 188 annotated protein sequences, 3 child nodes and an annotation score of only 91.9. Overwhelming preponderance of oxidoreductases in the liver is additionally supported by the isomerase DAGs: nearly all the reactions described in the normal liver isomerase DAG are oxidoreductase isomerization reactions, whereas only one of the three child nodes in the HepG2 isomerase DAG is oxidoreductase. Upon normalization of the annotation scores to the parent Molecular Function nodes, oxidoreductases are down-regulated in HepG2 cells by 58%. Similarly, for the set of bioinformatics calculations whose BLAST searches are carried out using BLASTP 2.2.15 [Oct-15-2006], oxidoreductases are down-regulated in HepG2 cells by 56%.

Conclusion

Proteomics and Gene Ontology reveal, for the first time, differential enzyme activities between HepG2 cells and normal human liver tissues, which may be a promising new prognostic marker of Hepatocellular carcinoma. Two independent sets of bioinformatics calculations that employ two BLAST program versions, and searched different databases, arrived at essentially the same conclusion: oxidoreductases are down-regulated in HepG2 cells by approximately 57%, when compared to normal human liver tissues. Down-regulation of oxidoreductases in hepatoma is additionally supported by Gene Ontology analysis of isomerises.     

Sample prep for proteomics of breast cancer: proteomics and gene ontology reveal dramatic differences in protein solubilization preferences of radioimmunoprecipitation assay and urea lysis buffers

Background

An important step in the proteomics of solid tumors, including breast cancer, consists of efficiently extracting most of proteins in the tumor specimen. For this purpose, Radio-Immunoprecipitation Assay (RIPA) buffer is widely employed. RIPA buffer's rapid and highly efficient cell lysis and good solubilization of a wide range of proteins is further augmented by its compatibility with protease and phosphatase inhibitors, ability to minimize non-specific protein binding leading to a lower background in immunoprecipitation, and its suitability for protein quantitation.

Results

In this work, the insoluble matter left after RIPA buffer extraction of proteins from breast tumors are subjected to another extraction step, using a urea-based buffer. It is shown that RIPA and urea lysis buffers fractionate breast tissue proteins primarily on the basis of molecular weights. The average molecular weight of proteins that dissolve exclusively in urea buffer is up to 60% higher than in RIPA. Gene Ontology (GO) and Directed Acyclic Graphs (DAG) are used to map the collective biological and biophysical attributes of the RIPA and urea proteomes. The Cellular Component and Molecular Function annotations reveal protein solubilization preferences of the buffers, especially the compartmentalization and functional distributions. It is shown that nearly all extracellular matrix proteins (ECM) in the breast tumors and matched normal tissues are found, nearly exclusively, in the urea fraction, while they are mostly insoluble in RIPA buffer. Additionally, it is demonstrated that cytoskeletal and extracellular region proteins are more soluble in urea than in RIPA, whereas for nuclear, cytoplasmic and mitochondrial proteins, RIPA buffer is preferred. Extracellular matrix proteins are highly implicated in cancer, including their proteinase-mediated degradation and remodelling, tumor development, progression, adhesion and metastasis. Thus, if they are not efficiently extracted by RIPA buffer, important information may be missed in cancer research.

Conclusion

For proteomics of solid tumors, a two-step extraction process is recommended. First, proteins in the tumor specimen should be extracted with RIPA buffer. Second, the RIPA-insoluble material should be extracted with the urea-based buffer employed in this work.     

A comparison of alternative methods to compute conditional genotype probabilities for genetic evaluation with finite locus models

An increased availability of genotypes at marker loci has prompted the development of models that include the effect of individual genes. Selection based on these models is known as marker-assisted selection (MAS). MAS is known to be efficient especially for traits that have low heritability and non-additive gene action. BLUP methodology under non-additive gene action is not feasible for large inbred or crossbred pedigrees. It is easy to incorporate non-additive gene action in a finite locus model. Under such a model, the unobservable genotypic values can be predicted using the conditional mean of the genotypic values given the data. To compute this conditional mean, conditional genotype probabilities must be computed. In this study these probabilities were computed using iterative peeling, and three Markov chain Monte Carlo (MCMC) methods – scalar Gibbs, blocking Gibbs, and a sampler that combines the Elston Stewart algorithm with iterative peeling (ESIP). The performance of these four methods was assessed using simulated data. For pedigrees with loops, iterative peeling fails to provide accurate genotype probability estimates for some pedigree members. Also, computing time is exponentially related to the number of loci in the model. For MCMC methods, a linear relationship can be maintained by sampling genotypes one locus at a time. Out of the three MCMC methods considered, ESIP, performed the best while scalar Gibbs performed the worst.     

Haplotype inference in crossbred populations without pedigree information

Background

Current methods for haplotype inference without pedigree information assume random mating populations. In animal and plant breeding, however, mating is often not random. A particular form of nonrandom mating occurs when parental individuals of opposite sex originate from distinct populations. In animal breeding this is called crossbreeding and hybridization in plant breeding. In these situations, association between marker and putative gene alleles might differ between the founding populations and origin of alleles should be accounted for in studies which estimate breeding values with marker data. The sequence of alleles from one parent constitutes one haplotype of an individual. Haplotypes thus reveal allele origin in data of crossbred individuals.

Results

We introduce a new method for haplotype inference without pedigree that allows nonrandom mating and that can use genotype data of the parental populations and of a crossbred population. The aim of the method is to estimate line origin of alleles. The method has a Bayesian set up with a Dirichlet Process as prior for the haplotypes in the two parental populations. The basic idea is that only a subset of the complete set of possible haplotypes is present in the population.

Conclusion

Line origin of approximately 95% of the alleles at heterozygous sites was assessed correctly in both simulated and real data. Comparing accuracy of haplotype frequencies inferred with the new algorithm to the accuracy of haplotype frequencies inferred with PHASE, an existing algorithm for haplotype inference, showed that the DP algorithm outperformed PHASE in situations of crossbreeding and that PHASE performed better in situations of random mating.     

Clustering by neurocognition for fine mapping of the schizophrenia susceptibility loci on chromosome 6p

Chromosome 6p is one of the most commonly implicated regions in the genome-wide linkage scans of schizophrenia, whereas further association studies for markers in this region were inconsistent likely due to heterogeneity. This study aimed to identify more homogeneous subgroups of families for fine mapping on regions around markers D6S296 and D6S309 (both in 6p24.3) as well as D6S274 (in 6p22.3) by means of similarity in neurocognitive functioning. A total of 160 families of patients with schizophrenia comprising at least two affected siblings who had data for eight neurocognitive test variables of the continuous performance test (CPT) and the Wisconsin card sorting test (WCST) were subjected to cluster analysis with data visualization using the test scores of both affected siblings. Family clusters derived were then used separately in family-based association tests for 64 single nucleotide polymorphisms (SNPs) covering the region of 6p24.3 and 6p22.3. Three clusters were derived from the family-based clustering, with deficit cluster 1 representing deficit on the CPT, deficit cluster 2 representing deficit on both the CPT and the WCST, and a third cluster of nondeficit. After adjustment using false discovery rate for multiple testing, SNP rs13873 and haplotype rs1225934-rs13873 on BMP6-TXNDC5 genes were significantly associated with schizophrenia for the deficit cluster 1 but not for the deficit cluster 2 or nondeficit cluster. Our results provide further evidence that the BMP6-TXNDC5 locus on 6p24.3 may play a role in the selective impairments on sustained attention of schizophrenia.     

You can't always get what you want: size assortative mating by mutual mate choice as a resolution of sexual conflict

Background  

Assortative mating patterns for mate quality traits like body size are often observed in nature. However, the underlying mechanisms that cause assortative mating patterns are less well known. Sexual selection is one important explanation for assortment, suggesting that i) one (usually the female) or both sexes could show preferences for mates of similar size or ii) mutual mate choice could resolve sexual conflict over quality traits into assortment. We tested these hypotheses experimentally in the socially monogamous cichlid fish Pelvicachromis taeniatus, in which mate choice is mutual.     

SERPINE2, a serine protease inhibitor extensively expressed in adult male mouse reproductive tissues, may serve as a murine sperm decapacitation factor

SERPINE2, one of the potent serine protease inhibitors that modulates the activity of plasminogen activator and thrombin, is implicated in many biological processes. In the present study, we purified SERPINE2 from mouse seminal vesicle secretion (SVS), using liquid chromatography and identified it by liquid chromatography/tandem mass spectrometry, and it showed potent inhibitory activity against the urokinase-type plasminogen activator. SERPINE2 was expressed predominantly in seminal vesicles among murine male reproductive tissues. It was immunolocalized to the SVS and mucosal epithelium of the seminal vesicle, epididymis, coagulating gland, and vas deferens. In the testes, SERPINE2 was immunostained in spermatogonia, spermatocytes, spermatids, Leydig cells, and spermatozoa. SERPINE2 was also detected on the acrosomal cap of testicular and epididymal sperm and was suggested to be an intrinsic sperm surface protein. The purified SERPINE2 protein could bind to epididymal sperm. A prominent amount of SERPINE2 was detected on ejaculated and oviductal spermatozoa. Nevertheless, SERPINE2 was detected predominantly on uncapacitated sperm, indicating that SERPINE2 is lost before initiation of the capacitation process. Moreover, SERPINE2 could inhibit in vitro bovine serum albumin-induced sperm capacitation and prevent sperm binding to the egg, thus blocking fertilization. It acts through preventing cholesterol efflux, one of the initiation events of capacitation, from the sperm. These findings suggest that the SERPINE2 protein may play a role as a sperm decapacitation factor.     

Genome-wide association and genomic prediction for host response to porcine reproductive and respiratory syndrome virus infection

Background

Host genetics has been shown to play a role in porcine reproductive and respiratory syndrome (PRRS), which is the most economically important disease in the swine industry. A region on Sus scrofa chromosome (SSC) 4 has been previously reported to have a strong association with serum viremia and weight gain in pigs experimentally infected with the PRRS virus (PRRSV). The objective here was to identify haplotypes associated with the favorable phenotype, investigate additional genomic regions associated with host response to PRRSV, and to determine the predictive ability of genomic estimated breeding values (GEBV) based on the SSC4 region and based on the rest of the genome. Phenotypic data and 60 K SNP genotypes from eight trials of ~200 pigs from different commercial crosses were used to address these objectives.

Results

Across the eight trials, heritability estimates were 0.44 and 0.29 for viral load (VL, area under the curve of log-transformed serum viremia from 0 to 21 days post infection) and weight gain to 42 days post infection (WG), respectively. Genomic regions associated with VL were identified on chromosomes 4, X, and 1. Genomic regions associated with WG were identified on chromosomes 4, 5, and 7. Apart from the SSC4 region, the regions associated with these two traits each explained less than 3% of the genetic variance. Due to the strong linkage disequilibrium in the SSC4 region, only 19 unique haplotypes were identified across all populations, of which four were associated with the favorable phenotype. Through cross-validation, accuracies of EBV based on the SSC4 region were high (0.55), while the rest of the genome had little predictive ability across populations (0.09).

Conclusions

Traits associated with response to PRRSV infection in growing pigs are largely controlled by genomic regions with relatively small effects, with the exception of SSC4. Accuracies of EBV based on the SSC4 region were high compared to the rest of the genome. These results show that selection for the SSC4 region could potentially reduce the effects of PRRS in growing pigs, ultimately reducing the economic impact of this disease.