Complex taxonomy of the 'brush tail' peregrine earthworm Pontoscolex corethrurus.

Pontoscolex corethrurus is the most widespread earthworm species in tropical and sub-tropical zones and one of the most studied in soil science. Although, ecological interactions of P. corethrurus with its environment are well documented, the taxonomic status of the species remains unclear. In this study, we investigated phylogenetic relationships within the genus Pontoscolex, in particular focusing on morphologically indistinguishable (i.e., cryptic) lineages. A total of 792 specimens collected from 25 different countries and islands all over the world were analyzed using two mitochondrial (COI and 16S rDNA) and two nuclear (internal transcribed spacers 2 and 28S rDNA) markers, and a total of 11 morphological characters both internal and external were investigated in all genetically characterized lineages. A large-scale multilocus sequence data matrix was also obtained for Pontoscolex spp. specimens using the Anchored Hybrid Enrichment (AHE) method. Multilocus phylogenetic and phylogenomic analyses, combined with species delimitation methods; including single locus (mPTP, ABGD) and multilocus (BPP) approaches, revealed congruent results. Four cryptic species were supported within the P. corethrurus species complex, and four potentially new species within the genus Pontoscolex. One widespread lineage (L1), within P. corethrurus complex was observed in the current population of Fritz Müller's garden where P. corethrurus was first described in 1856. Cryptic lineages were observed in sympatry at several localities. This, in combination with observed heteroplasmy in COI gene in one population raises an important question of reproductive isolation between these species.

Novel polymorphisms in porcine 3'UTR of the leptin gene, including a rare variant within target sequence for MIR-9 gene in Duroc breed, not associated with production traits.

The leptin gene (LEP) is considered as a functional candidate for production traits in livestock due to its crucial role in energy homeostasis. Because polymorphisms in regulatory sequences may affect gene expression, we searched for them in the 3'UTR of LEP and analyzed their association with production traits. Four breeds and a composite line were studied. In the Polish Landrace and Polish Large White breeds, 8 SNPs and 1 indel were observed; whereas, in the Duroc breed, 9 specific SNPs were found. Pietrain and Line 990 were monomorphic. One SNP (g.+168C>T), observed in the Duroc breed only, was located within a target site for microRNA (miR-9). Association studies showed a weak association between one SNP (c.+846C>T) and abdominal fat weight in the Polish Landrace only. Thus, we concluded that contribution of polymorphisms in the 3'UTR to phenotypic variability of pig production traits is marginal. Moreover, we presented an overview of known polymorphisms (128) in the pig leptin gene.

Polymorphism in 3' untranslated region of the pig PPARA gene influences its transcript level and is associated with adipose tissue accumulation.

The PPARA (peroxisome proliferator-activated receptor-α) gene encodes a nuclear receptor that plays an important role in fatty acid catabolism by transcriptional regulation of genes involved in fatty acid oxidation and can be considered as a candidate gene for fatness traits in the pig. The aim of the study was to search for a functional polymorphism in 3' untranslated region (UTR), their association with production traits, and postnatal PPARA transcript level in 2 skeletal muscles (longissimus and semimembranosus) of 5 commercial pig breeds (Polish Landrace [PL], Polish Large White [PLW], Duroc, Pietrain, and Pulawska). Altogether, 9 novel polymorphisms (8 SNP and 1 indel) were found in the 3' UTR. The in silico analysis revealed 6 putative microRNA target sequences in the analyzed region. The c.*636A>G substitution was widely distributed across breeds and located near the putative target sequence for miR-224. The relative PPARA transcript level was higher (P < 0.05) in LM of AA than in those of GG homozygous animals for SNP c.*636A>G. The luciferase assay revealed that miR-224 probably acts as a negative regulator of the PPARA expression in pig adipocytes (P = 2.9 × 10(-7)), but we did not observe the effect of the A or G alleles on the interaction between miR-224 and its putative target sequence. We hypothesize that the 2 predominant haplotypes, differing at 4 sites (including c.*636A>G), present different architecture of its 3' UTR and it could affect the level of the transcript. The c.*636A>G SNP, analyzed in PL and PLW, was significantly associated with backfat thickness at 3 points (P < 0.05) and intramuscular fat content (P < 0.01) in PL. Suggestive associations were found between 4 SNP (c.*321A>C, c.*324G>C, c.*626T>C, and c.*636A>G) and fatty acid contents in LM and subcutaneous and visceral fat tissue of PL, PLW, Duroc and Pietrain pigs. The PPARA mRNA level was higher in semimembranosus muscle than in LM (P = 8.38 × 10(-12)) in a general comparison and the same trend was found in most breeds (except for PL) and at all tested days of age (60, 90, 120, 150, 180, and 210 d). The effect of breed was highly significant in a general comparison (P = 0.48 × 10(-8)), but there was no common expression pattern in both muscles among different age groups. We conclude that the c.*636A>G SNP in the PPARA gene can be considered in PL breed as a useful genetic marker for adipose tissue accumulation.

Transcript level of the porcine ME1 gene is affected by SNP in its 3'UTR, which is also associated with subcutaneous fat thickness.

Pork quality depends on multiple factors, including fatty acid composition in muscle and fat tissues. The ME1 gene is a strong candidate for fat accumulation, as it encodes the malic enzyme, which is required for fatty acid synthesis. We identified seven new polymorphisms in 3'UTR of the ME1 gene and moreover confirmed the presence of 4 polymorphisms detected previously. Interestingly, the studied Duroc pigs were monomorphic at all these polymorphic sites, while in 3 other breeds (Pietrain, Polish Landrace and Polish Large White), the polymorphisms were unevenly distributed. One of the novel SNPs (c.*488A>G) was found in the Polish Large White and the Polish Landrace only, and the association studies revealed that it was significantly associated with backfat thickness and average daily weight gain in the Polish Landrace (N = 207) and the Polish Large White (N = 157). This SNP was differently associated with ME1 transcript level in muscle and backfat. The in silico analysis of another novel SNP (c.*548C>T) indicated that it is located within a binding sequence conserved among vertebrates for the miR-30 family in 3'UTR of the ME1. It was shown that in the longissimus muscle, but not in adipose tissue, CT gilts compared with CC ones had significantly lower levels of the ME1 transcript. This polymorphism, however, was not associated with production traits. Additionally, we observed that transcript level of the ME1 was significantly higher in subcutaneous fat than in the longissimus muscle, as well as both investigated tissues of the Polish Landrace when compared to the other breeds. However, no association was found between this polymorphism and fatty acid profiles. We conclude that the ME1 gene polymorphism (c.*488A>G) is a potential marker for porcine backfat thickness.

Transcript abundance of the pig stearoyl-CoA desaturase gene has no effect on fatty acid composition in muscle and fat tissues, but its polymorphism within the putative microRNA target site is associated with daily body weight gain and feed conversion ratio.

Fatty acid composition in porcine intramuscular fat affects the dietetic value and technological properties of meat. The stearoyl-CoA desaturase (SCD) gene is a strong positional and functional candidate for fatty acid composition. Our sequence analysis in 4 breeds (Duroc, Pietrain, Polish Landrace, and Polish Large White) revealed a novel SNP in the 5'-flanking sequence and 9 novel SNP and 2 novel indels in the 3' untranslated region (UTR). Transcript level of the SCD in subcutaneous fat was significantly greater than in muscle tissue (n=83; P<0.001) and the interbreed comparison revealed a greater transcript level in the fat tissue of Polish Landrace (P<0.01). We found no association between the abundance of the SCD transcript and fatty acid composition in any of the tissues. We performed an association analysis between 4 SNP (c.-353C>T, c.-233T>C, c.*164A>G, and c.*928G>C), 1 indel (c.*2574_2576delGTC), and production traits in Polish Large White (n=185) and synthetic line 990 (n=243). The most pronounced associations were observed for the c.*928G>C polymorphism, which occurs within a predicted target site for 2 microRNA (ssc-miR-185 and ssc-miR-491). In line 990, this polymorphism was significantly associated with daily BW gain (P<0.04 under the general model) and feed conversion ratio (P<0.0004) but not with fatness traits. The same tendency, but not significant, was observed in the Polish Large White breed. When both breeds were analyzed together, these associations were again highly significant (daily BW gain P<0.003; feed conversion ratio P<0.0001). We conclude that c.*928G>C is a promising marker for both porcine traits.

Guidelines for maintenance of adult patients with brain death and potential for multiple organ donations: the Task Force of the Brazilian Association of Intensive Medicine the Brazilian Association of Organs Transplantation, and the Transplantation Center of Santa Catarina.

INTRODUCTION: The organ shortage for transplantation, the principal factor that increases waiting lists, has become a serious public health problem. In this scenario, the intensivist occupies a prominent position as one of the professionals that first has a chance to identify brain death and to be responsible for the maintenance of the potential deceased donor. OBJECTIVE: This report attempts to establish guidelines for care and maintenance of adult deceased donor organs guiding and standardizing care provided to patients with brain death. METHOD: These guidelines were composed by intensivists, transplant coordinators, professionals from various transplant teams, and used transplant center. The formulated questions were forwarded to all members and recommendations were constructed after an extensive literature review selecting articles with the highest degree of evidence. RESULTS: Guidelines were developed in the form of questions reflecting frequent experiences in clinical intensive care practices. The main questions were: Is there an optimal interval for keeping organs of deceased donors viable? What actions are considered essential for maintaining deceased donors in this period? What are the limits of body temperature? How should the patient be warmed? Which laboratory tests should be performed? What is the collection interval? What are the limits in the laboratory and the capture scenario? What are the limits of blood pressure? When and how should one use catecholamines? CONCLUSIONS: This pioneer project involved a multidisciplinary team working in organ transplantation seeking to provide treatment guidance to increase the number of viable organs from deceased adult donors.

Hypospadias in a male (78,XY; SRY-positive) dog and sex reversal female (78,XX; SRY-negative) dogs: clinical, histological and genetic studies.

Hypospadias is rarely reported in dogs. In this study we pre-sent 2 novel cases of this disorder of sexual development and, in addition, a case of hereditary sex reversal in a female with an enlarged clitoris. The first case was a male Moscow watchdog with a normal karyotype (78,XY) and the presence of the SRY gene. In this dog, perineal hypospadias, bilateral inguinal cryptorchidism and testes were observed. The second case, representing the Cocker spaniel breed, had a small penis with a hypospadic orifice of the urethra, bilateral cryptorchidism, testis and a rudimentary gonad inside an ovarian bursa, a normal female karyotype (78,XX) and a lack of the SRY gene. This animal was classified as a compound sex reversal (78,XX, SRY-negative) with the hypospadias syndrome. The third case was a Cocker spaniel female with an enlarged clitoris and internally located ovotestes. Cytogenetic and molecular analyses revealed a normal female karyotype (78,XX) and a lack of the SRY gene, while histology of the gonads showed an ovotesticular structure. This case was classified as a typical hereditary sex reversal syndrome (78,XX, SRY-negative). Molecular studies were focused on coding sequences of the SRY gene (case 1) and 2 candidates for monogenic hypospadias, namely MAMLD1 (mastermind-like domain containing 1) and SRD5A2 (steroid-5-alpha-reductase, alpha polypeptide 2). Sequencing of the entire SRY gene, including 5'- and 3'-flanking regions, did not reveal any mutation. The entire coding sequence of MAMLD1 and SRD5A2 was analyzed in all the intersexes, as well as in 4 phenotypically normal control dogs (3 females and 1 male). In MAMLD1 2 SNPs, including 1 missense substitution in exon 1 (c.128A>G, Asp43Ser), were identified, whereas in SRD5A2 7 polymorphisms, including 1 missense SNP (c.358G>A, Ala120Thr), were found. None of the identified polymorphisms cosegregated with the intersexual phenotype, thus, we cannot confirm that hypospadias may be associated with polymorphism in the coding sequence of the studied genes.

Effect of three common SNPs in 5'-flanking region of LEP and ADIPOQ genes on their expression in Polish obese children and adolescents.

Genes encoding adipokines are considered as candidates for human obesity. In this study we analyzed the expression of leptin (LEP) and adiponectin (ADIPOQ) genes in relation to common 5'-flanking or 5'UTR variants: -2548G>A (LEP), 19A>G (LEP) and -11377C>G (ADIPOQ) in Polish obese children and adolescents. Relative transcription levels in the subcutaneous adipose tissue (real time RT-PCR) and serum protein concentrations (RIA) were measured in 48 obese subjects with known genotypes at three polymorphic sites and in five non-obese controls. None of the studied polymorphisms altered significantly the expression. Significantly elevated relative transcription levels of the LEP gene (P < 0.05) and serum leptin concentrations (P < 0.01) were recorded in obese patients, when compared with the non-obese controls, but such differences were not found for the ADIPOQ gene. Interestingly, the leptin to adiponectin protein concentration ratio (L/A) was approximately sevenfold higher in obese children and adolescents when compared with the non-obese controls (P < 0.001). Taking into consideration the observed relationship between the genotypes and the gene expression level we suggest that these SNPs are not conclusive markers for predisposition to obesity in Polish children and adolescents. On the other hand, we confirmed that the leptin to adiponectin gene expression ratio (L/A) is an informative index characterizing obesity.

An unusual location of deep venous thrombosis associated with ischemic stroke and persistent foramen ovale.

Up to 40% of ischemic strokes have no known cause (cryptogenic). The prevalence of persistent foramen ovale (PFO) amongst patients with cryptogenic stroke (CS) is twice as high as that of the normal population, therefore suggesting a causal relationship between the two entities. However, PFO by itself is not sufficient to cause stroke, as an embolic source is needed. This source is often unknown, making the causal relationship between CS and PFO hard to demonstrate. The most frequent, although still seldom, identifiable cause of embolism in an otherwise cryptogenic stroke associated with PFO is a deep venous thrombosis (DVT) of the lower extremities. Here, we present a unique case of brachiocephalic venous DVT associated with PFO and ischemic stroke in a young patient. As the search for DVT in patients with PFO and stroke is often limited to the lower extremities, this case may suggest that an unspecified number of DVTs are overlooked. Our report lends support to paradoxical embolism as a mechanism of stroke in patients with PFO and does, at least in selected cases, suggest a more detailed search for DVT beyond the lower extremities.

Robertsonian translocation in a sex reversal dog (XX, SRY negative) may indicate that the causative mutation for this intersexuality syndrome resides on canine chromosome 23 (CFA23).

A Bernese mountain dog was subjected for clinical evaluation due to the presence of ambiguous external genitalia (enlarged clitoris). Anatomical and histological studies revealed the presence of one testicle, one ovotestis and a uterus. This dog was classified as a female-to-male sex reversal, with 2 normal X chromosomes and a lack of the Y chromosome-linked genes SRY and ZFY. It is the first case of this syndrome in this breed. Apparently a Robertsonian translocation, rob(5;23), was also identified in this dog and it is again the first case of this type of chromosome abnormality in this breed, as well as the first case of co-occurrence of the sex reversal syndrome along with a centric fusion in the dog. Since on the canine chromosome 23 (CFA23) 3 genes (FOXL2,PISRT1 and CTNNB1) involved in the sex determination process are present, further cytogenetic FISH studies were carried out with the use of BAC probes specific for this chromosome. It was found that a pericentromeric fragment of CFA23 was deleted as a result of the centric fusion. We hypothesize that a cis regulatory sequence for the sex determination genes on CFA23 (e.g. proximally located CTNNB1) is present in the deleted fragment. Thus, a causative mutation responsible for this sex reversal syndrome may reside on CFA23.

Genetics of fat tissue accumulation in pigs: a comparative approach.

Fatness traits are important in pig production since they influence meat quality and fattening efficiency. On the other hand, excessive fat accumulation in humans has become a serious health problem due to worldwide spread of obesity. Since the pig is also considered as an animal model for numerous human diseases, including obesity and metabolic syndrome, comparative genomic studies may bring new insights into genetics of fatness/obesity. Input of genetic factors into phenotypic variability of these traits is rather high and the heritability coefficient (h(2)) of these traits oscillates around 0.5. Genome scanning revealed the presence of more than 500 QTLs for fatness in the pig genome. In addition to QTL studies, many candidate gene polymorphisms have been analyzed in terms of their associations with pig fatness, including genes encoding leptin (LEP) and its receptor (LEPR), insulin-like growth factor 2 (IGF-2), fatty acid-binding proteins (FABP3 and FABP4), melanocortin receptor type 4 (MC4R), and the FTO (fat mass and obesity-associated) gene. Among them, a confirmed effect on pig fatness was found for a well-known polymorphism of the IGF-2 gene. In humans the strongest association with predisposition to obesity was shown for polymorphism of the FTO gene, while in pigs such an association seems to be doubtful. The development of functional genomics has revealed a large number of genes whose expression is associated with fat accumulation and lipid metabolism, so far not studied extensively in terms of the association of their polymorphism with pig fatness. Recently, epigenomic mechanisms, mainly RNA interference, have been considered as a potential source of information on genetic input into the fat accumulation process. The rather limited progress in studies focused on the identification of gene polymorphism related with fatness traits shows that their genetic background is highly complex.

Stamping of monomeric SAMs as a route to structured crystallization templates: patterned titania films.

Gold-coated glass slides have be patterned by using self-assembled monolayers (SAM) of alkane thiols. Through the use of a special thiol terminated with a styrene monomer, microstructures of 5 to 10 microm width and 70 A height have been formed on the surface by graft polymerization of styrene. These patterned gold slides have then been used to template the precipitation of thin titania films from ethanolic solutions of titanium isopropoxide to create microstructured architectures in the film. Plasmon resonance spectra have established the presence of different steps in the process and have been used to follow the kinetics of the precipitation of titania on the surface. The structured TiO2 films have been characterized by scanning electron microscopy.

Validation of predicted total body water and extracellular water by multi-frequency impedance in young Indonesian adults.

To investigate the validity of the prediction of body water compartments by multi-frequency impedance in Indonesians, 42 adults (19 males and 23 females) were recruited in Jakarta, the capital of Indonesia. Extracellular water (ECW) and total body water (TBW) were measured with dilution techniques and predicted from body impedance at 1 and 100 kHz, respectively, using prediction formulas developed in a Dutch population. Furthermore, body weight, body height and body density were measured and as a measure of body build TBW/height and ECW/height were calculated. Percent ECW of body weight in both males (26 +/- 3%) and females (23 +/- 2%) was higher compared to the Dutch reference population, and also the body water distribution (ECW/TBW) was higher (0.46 +/- 0.04) compared to the reference population. TBW/height and ECW/height appeared to be considerably lower in the Indonesian group compared to the reference group, indicating a more slender body shape. TBW was overestimated by 1.8 +/- 2.4 and 2.5 +/- 1.3 kg in males and females, respectively. ECW was underestimated in males by 0.1 +/- 1.2 kg and slightly overestimated in females (0.1 +/- 1.0 kg). After corrections for differences in body build and body water distribution the bias of the predicted TBW was markedly reduced from 2.2 +/- 1.9 to 1.3 +/- 1.8 kg (males and females combined). For ECW the bias after correction for body water distribution and body build was 0.3 +/- 0.6 kg (males and females combined). It is concluded that multi-frequency impedance is suitable for the prediction of body water compartments at the population level but, due to differences in body build and body water distribution, validation in a larger group of Indonesian subjects is needed to avoid systematic prediction errors.

Measurements of body fat in Indonesian adults: Comparison between a three-compartment model and widely used methods.

Body composition was assessed in Indonesian male (n = 18) and female (n = 23) students using densitometry (underwater weighing), deuterium oxide dilution, skinfold thickness measurements, bioelectrical impedance analysis (BIA) and a prediction equation based on the body mass index. From body density and total body water percentage body fat (BF%) was calculated using a three-compartment body composition model. Percentage body fat obtained by this three-compartment model was regarded as the reference value and BF% obtained by the single methods were compared with this value. Mean differences (± SD) in BF% from the threecompartment model minus the single methods were -1.1 ± 2.1 for densitometry, +1.1 ± 1.6 for deuterium oxide dilution, +1.3 ± 2.8 for skinfold thickness measurement, +2.8 ± 4.3 for BIA and +3.4 ± 4.8 for body mass index in males. In females these values were +0.1 ± 1.7, +0.2 ± 1.4, +3.6 ± 3.3, +3.6 ± 2.4 and +8.7 ± 2.0 BF%, respectively. Correlation coefficients between different methods were high and significant (P < 0.05 in males, P < 0.001 in females). This study shows that the single predictive methods have considerable mean and individual biases compared with the three-compartment model and all predictive methods underestimated body fat in the studied subjects. It is concluded that the development of population-specific prediction formulas may be necessary.

Short stature and failure of pubertal development in thalassaemia major: evidence for hypothalamic neurosecretory dysfunction of growth hormone secretion and defective pituitary gonadotropin secretion.

UNLABELLED: In patients with beta-thalassaemia major, frequent blood transfusions combined with desferrioxamine chelation therapy lead to an improved rate of survival. Endocrine disorders related to secondary haemosiderosis such as short stature, delayed puberty and hypogonadism are major problems in both adolescent and adult patients. A total of 32 patients with beta-thalassaemia major undergoing treatment at the Children's Hospital, University of Göttingen were examined. Fourteen of these were short in stature. Growth hormone (GH) secretion was investigated in 13 patients exhibiting either a short stature or reduced growth rate. The stimulated GH secretion of 10 patients in this subgroup lay within the normal range. Studies of their spontaneous GH secretion during the night revealed that these patients had a markedly reduced mean GH and reduced amplitudes in their GH peaks. Low insulin-like growth factor (IGF)-I levels were seen in the growth-retarded thalassaemic patients. Eight were subjected to an IGF generation test and showed a strong increase in both IGF-I and insulin-like growth factor binding protein (IGFBP)-3 levels indicating intact IGF-I generation by the liver. Hypogonadotropic hypogonadism was found to be present in both the male and female patients with impaired sexual development. After priming with LH-releasing hormone (GnRH) per pump in 2 female and 5 male patients, no change in either their serum oestradiol or testosterone levels or in LH/FSH response to GnRH was observed suggesting that they were suffering from a severe pituitary gonadotropin insufficiency. Three male patients at the age of puberty but exhibiting short stature. low GH, low IGF-I and hypogonadism received low dose long-acting testosterone. After 3 12 months of therapy there was a marked growth spurt, higher nocturnal GH levels and an increase in both IGF-I and IGFBP-3. CONCLUSION: Reduced GH secretion and low IGF-I in thalassaemic patients are related to a neurosecretory dysfunction due to iron overload rather than to liver damage. Hypogonadotropic hypogonadism is caused by the selective loss of pituitary gonadotropin function. In patients with both GH deficiency and hypogonadism, low dose sexual steroid treatment should be considered either as an alternative or an additional treatment before starting GH therapy.