A de novo 2.3 kb structural variant in MITF explains a novel splashed white phenotype in a Thoroughbred family.

Splashed white in horses is characterized by extensive white patterning on the legs, face and abdomen and may be accompanied by deafness. To date, seven variants in microphthalmia-associated transcription factor (MITF) and two variants in Paired Box 3 (PAX3) have been identified to explain this phenotype. A splashed white Thoroughbred stallion, whose sire and dam were not patterned, was hypothesized to have a de novo variant leading to his white coat pattern. A whole-genome sequencing candidate gene approach identified two single nucleotide variants (SNVs) in SOX10, four SNVs in MITF and a 2.3 kb deletion in MITF with the alternative allele present in this stallion but absent in the other 18 horses analyzed. All six SNVs were annotated as modifiers and were not further considered. The deletion in MITF (NC_009159.3:g.21555811_21558139delinsAAAT) encompasses exon 9 encoding a part of the helix-loop-helix domain required for DNA binding. Sanger sequencing and parentage testing confirmed that this deletion was a de novo mutation of maternal origin. Consistent with the published nomenclature, we denote this likely causal variant as SW8. Genotyping three of this stallion's offspring identified SW8 only in the nearly all-white foal that was confirmed deaf by brainstem auditory evoked response testing. This foal was also a compound heterozygote for dominant white variants (W20/W22), but to date, W variants alone have not been connected to deafness. SW8 marks the fourth de novo MITF variant in horses reported to cause white patterning. The link between deafness and all MITF variants with and without other variants impacting melanocyte development and function needs to be further explored.

Bioavailability and bioaccumulation of heavy metals of several soils and sediments (from industrialized urban areas) for Eisenia fetida.

Soils and sediments are susceptible to anthropogenic contamination with Metallic Trace Elements (MTEs) and it can present some risks to ecosystems and human health. The levels of Cd, Cu, Fe, Ni, Pb and Zn were assessed in soils (C, G, K, L) from Estarreja (Portugal) and sediments from a stormwater basin in Lyon (DJG), a harbour (LDB) and a Rhône river site (TRS) (France). An ecotoxicological study was performed with Eisenia fetida (E. fetida) to infer about potential transfer risks to the soil invertebrates. To assess risks associated with MTEs contamination, it is important to know their total concentrations, fractionation and the potential available fractions. CaCl2, DTPA and NaOAc extractions were performed to assess the extractable and available MTEs fractions. The studied sediments were much more contaminated than the soils for all the MTEs analysed. The trace elements fraction linked with DTPA extraction shows higher values when compared with the NaOAc and the CaCl2 pools. Low mortality effects were recorded in the tests with E. fetida. The MTEs levels in soils and sediments and the concentrations bioaccumulated in adult earthworms contributed to a reduction in the number of juveniles produced. E. fetida adults and juveniles accumulated ETMs as follows: Cd > Cu = Zn > Ni > Pb > Fe. Determined BAFs were mostly lower than 1 with some higher values for Cd, Cu and Zn. Calculated SET and ERITME indexes allowed to classify the samples from the most to the less toxic for E. fetida as: LDB > DJG > L > G > C > K > TRS. Despite this order of toxicity, the earthworms exposed to the sediment TRS presented the lowest reproduction rate. The combination of "chemical" measurements with the calculation of BAFs, but especially SET and ERITME indexes can be a useful tool in risk assessment.

Blood pressures in newborns with twin-twin transfusion syndrome.

OBJECTIVE: In addition to unbalanced flow through placental anastomoses, evidence suggests that transfer of circulating vasoactive elements from the donor to the recipient contribute to the pathological process of twin-twin transfusion syndrome (TTTS). The objective of this study was to test the hypothesis that TTTS recipients have higher blood pressure (BP) at birth than donors. STUDY DESIGN: Chart review of all TTTS infants born from 1996 to 2007 with both twins alive 24 h (51 pairs; average gestational age 30±3 weeks). RESULTS: Both systolic and diastolic neonatal BPs were significantly higher in recipients. When expressed relative to predicted BP for birth weight (BW), BP were lower than expected in donors and higher in recipients. CONCLUSIONS: Data indicate that TTTS recipients have BP significantly higher than donors and than BP expected for BW. The long-term impact of these early hemodynamic perturbations remains to be determined.

Alteration of calcium homeostasis in primary preeclamptic syncytiotrophoblasts: effect on calcium exchange in placenta.

Preeclampsia (PE) is characterized by maternal hypertension, proteinuria, oedema and, in 30% of cases, by intrauterine growth retardation. Causes are still unknown; however, epidemiological and clinical studies have suggested alterations in maternal calcium metabolism. We suggested that in PE, calcium transport by the syncytiotrophoblast (ST) is disturbed. From total placental tissues, we studied the expression of: calcium channels (TRPV5, TRPV6 [transient receptor potential vanilloid]), calcium binding proteins (CaBP-9K, CaBP-28K), plasma membrane calcium ATPase (PMCA)1,2,3,4 pumps, ATP synthase, genes implicated in Ca(2+) release [inositol-1,4,5-triphosphate receptor (IP3R)1,2,3; Ryanodine receptor (RyR)1,2,3] and replenishment (SERCA1,2,3 [sarcoendoplasmic reticulum Ca(2+) ATPases]) from endoplasmic reticulum, channels implicated in mitochondrial Ca(2+) accumulation (VDAC1,2,3 [voltage-dependent anion channels]) and a marker of oxidative stress (hOGG1 [Human 8-oxoguanine-DNA glycosylase 1]), as well as the influence of these variations on calcium transport in primary ST cultures. The mRNA and protein levels were thereby examined by real-time PCR and Western blot analysis, respectively, in two different groups of pregnant women with similar gestational age: a normal group (n= 16) and a PE group (n= 8), diagnosed by a clinician. Our study showed a significant decrease in calcium transport by the ST cultured from preeclamptic placentas. We found a significant (P < 0.05) decrease in mRNA levels of TRPV5, TRPV6, CaBP-9K, CaBP-28K, PMCA1, PMCA4, ATP synthase, IP3R1, IP3R2, RyR1, RyR2 and RyR3 in PE group compared to normal one. We also noted a significant decrease in protein levels of TRPV5, TRPV6, CaBP-9K, CaBP-28K and PMCA1/4 in PE group. In contrast, SERCA1, SERCA2, SERCA3, VDAC3 and hOGG1 mRNA expressions were significantly increased in PE placentas. Calcium homeostasis and transport through placenta is compromised in preeclamptic pregnancies and it appears to be affected by a lack of ATP and an excess of oxidative stress.

Cytoplasmic membrane response to copper and nickel in Acidithiobacillus ferrooxidans.

Metal tolerance has been found to vary among Acidithiobacillus ferrooxidans strains and this can impact the efficiency of biomining practices. To explain observed strain variability for differences in metal tolerance we examined the effects of Cu(2+) and Ni(2+) concentrations (1-200 mM) on cytoplasmic membrane properties of two A. ferrooxidans type strains (ATCC 23270 and 19859) and four strains isolated from AMD water around Sudbury, Ontario, Canada. Growth rate, membrane fluidity and phase, determined from the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH), and fatty acid profiles indicated that three different modes of adaptation were present and could separate between strains showing moderate, or high metal tolerance from more sensitive strains. To compensate for the membrane ordering effects of the metals, significant remodelling of the membrane was used to either maintain homeoviscous adaptation in the moderately tolerant strains or to increase membrane fluidity in the sensitive strains. Shifts in the gel-to-liquid crystalline transition temperature in the moderately tolerant strains led to multiple phase transitions, increasing the potential for phase separation and compromised membrane integrity. The metal-tolerant strain however, was able to tolerate increases in membrane order without significant compensation via fatty acid composition. Our multivariate analyses show a common adaptive response which involves changes in the abundant 16:0 and 18:1 fatty acids. However, fatty acid composition and membrane properties showed no difference in response to either copper or nickel suggesting that adaptive response was non-specific and tolerance dependent. We demonstrate that strain variation can be evaluated using differences in membrane properties as intrinsic determinants of metal susceptibility.

Cytoplasmic membrane fluidity and fatty acid composition of Acidithiobacillus ferrooxidans in response to pH stress.

Strain variation in the acidophile Acidithiobacillus ferrooxidans was examined as a product of membrane adaptation in response to pH stress. We tested the effects of sub and supra-optimal pH in two type strains and four strains isolated from acid mine drainage water around Sudbury, Ontario, Canada. Growth rate, membrane fluidity and phase, determined from the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene, and fatty acid profiles were compared. The effect of pH 1.5 was the most pronounced compared to the other pH values of 1.8, 3.1, and 3.5. Three different types of response to lower pH were observed, the first of which appeared to maintain cellular homeostasis more effectively. This adaptive mode included a decrease in membrane fluidity and concomitant depression of the phase transition in two distinct membrane lipid components. This was explained through the increase in saturated fatty acids (predominantly 16:0 and cyclopropane 19:0 w8c) with a concomitant decrease in 18:1 w7c fatty acid. The other strains also showed common adaptive mechanisms of specific fatty acid remodeling increasing the abundance of short-chain fatty acids. However, we suspect membrane permeability was compromised due to potential phase separation, which may interfere with energy transduction and viability at pH 1.5. We demonstrate that membrane physiology permits differentiating pH tolerance in strains of this extreme acidophile.

Indicators from archaeal secretomes.

Just as in the Eukarya and the Bacteria, members of the Archaea need to export proteins beyond the cell membrane. This would be required to fulfill a variety of essential functions such as nutrient acquisition and biotransformations, maintenance of extracellular structures and more. Apart from the Eukarya and the Bacteria however, members of the Archaea share a number of unique characteristics. Does this uniqueness extend to the protein secretion system? It was the objective of this study to answer this question. To overcome the limited experimental information on secreted proteins in Archaea, this study was carried out by subjecting the available archaeal genomes, which represent halophiles, thermophiles, and extreme thermophiles, to bioinformatics analysis. Specifically, to examine the properties of the secretomes of the Archaea using the ExProt program. A total of 24 genomes were analyzed. Secretomes were found to fall in the range of 6% of total ORFs (Methanopyrus kandleri) to 19% (Halobacterium sp. NRC-1). Methanosarcina acetivorans has the highest fraction of lipoproteins (at 89) and the lowest (at 1) were members of the Thermoplasma, Pyrobaculum aerophilum, and Nanoarchaeum equitans. Based on the Tat consensus sequence, contribution of these secreted proteins to the secretomes were negligible, making up 8 proteins out of a total of 7105 predicted exported proteins. Amino acid composition, an attribute of signal peptides not used as a selection criteria by ExProt, of predicted archaeal signal peptides show that in the haloarchaea secretomes, the frequency of the amino acid Lys is much lower than that seen in bacterial signal peptides, but is compensated for by a higher frequency of Arg. It also showed that higher frequencies for Thr, Val, and Gly contribute to the hydrophobic character in haloarchaeal signal peptides, unlike bacterial signal peptides in which the hydrophobic character is dominated by Leu and Ile.

Dalteparin for the prevention of recurrence of placental-mediated complications of pregnancy in women without thrombophilia: a pilot randomized controlled trial.

BACKGROUND: The role of anticoagulants for the prevention of placental-mediated pregnancy complications is uncertain. OBJECTIVES: Our aim was to investigate the effectiveness of dalteparin, a low-molecular-weight heparin, in preventing the recurrence of these complications in women without thrombophilia. PATIENTS/METHODS: Between August 1 2000 and June 20 2007, 116 pregnant women with: (i)

Relevance of measuring diastolic time intervals in the ductus venosus during the early stages of twin-twin transfusion syndrome.

OBJECTIVE: To determine if the discrete myocardial diastolic dysfunction documented previously in the recipient twin during the early stages of twin-twin transfusion syndrome (TTTS) has any repercussion on flow velocities through the ductus venosus (DV) and to investigate if this could allow early differentiation between TTTS and selective intrauterine growth restriction (IUGR). METHODS: Two groups of monochorionic twin pregnancies with growth discordance between twins were reviewed retrospectively. Group I was composed of fetuses in Stages I and II of TTTS; laser or amnioreduction was not performed in any instance. Group II twin pairs each included one fetus with IUGR due to placental circulatory insufficiency. Intertwin differences (smaller minus larger fetus) were analyzed for myocardial performance index of the right ventricle (MPI-RV) and for time variables in the DV. RESULTS: There were 38 pairs of monochorionic twins (24 TTTS and 14 IUGR) in this study. In the TTTS group, the donors had a significantly lower MPI-RV (0.419 +/- 0.18 vs. 0.596 +/- 0.17, F(1,19df) = 24.017, P < 0.001), a significantly longer total ventricular filling time (150.9 +/- 25.6 ms vs. 124.0 +/- 22.6 ms; F(1,21df) = 19.631, P < 0.001) and a significantly longer early filling time (118.9 +/- 22.9 ms vs. 92.6 +/- 18.9 ms, F(1,21df) = 28.419, P < 0.001) than had the recipient. None of these three differences was present in the IUGR group. Probability studies revealed that cut-off values of 12.75 for intertwin differences in total filling time and 8.5 for intertwin differences in early filling time had sensitivities of 71% and 92%, respectively. The false-positive rates were 23% and 15%, respectively, for the early diagnosis of TTTS. CONCLUSION: In monochorionic twin pregnancies, shortening of the ventricular filling time in the recipient twin indicates diastolic myocardial dysfunction occurring early in the pathophysiology of TTTS. This early interwin difference in myocardial function is not found in pregnancies with IUGR in one twin due to placental circulatory insufficiency, allowing early differentiation between TTTS and selective IUGR.

Fluorescence polarization in studies of bacterial cytoplasmic membrane fluidity under environmental stress.

The integrity of the bacterial cytoplasmic membrane is critical in maintaining the viability of cells and their metabolic functions, particularly under stress. Bacteria actively adjust membrane fluidity through changes in lipid composition in response to variations in temperature, pressure, ion concentrations, pH, nutrient availability, and xenobiotics. Fluorescence polarization methods are valuable for measuring bacterial cytoplasmic membrane fluidity. In this review we discuss the mechanisms of bacterial membrane adaptations and present data from research using 1,6-diphenyl-1,3,5-hexatirene (DPH) as a measure of membrane fluidity and phase transitions. We illustrate the range of fluidity in viable cells, extracted membranes, and liposomes under optimal and stressed physiological conditions.

The use of RAPD genomic fingerprinting to study relatedness in strains of Acidithiobacillus ferrooxidans.

Twelve strains of Acidithiobacillus ferrooxidans were recovered from acid mine drainage (AMD) sites from three different geographical locations: Copper Cliff, Ontario, Canada; Mineral City, OH, USA; and Cornwall, England. The spread-plate technique and various culture media were used to isolate and purify all strains. DNA was extracted from each purified culture and amplified using PCR and twenty, 10-mer primers. Amplification products were separated by gel electrophoresis and photographed under UV light. The RAPD (Randomly Amplified Polymorphic DNA) profiles were compared on the basis of the presence or absence of each DNA band and a data matrix was constructed. Strain diversity was calculated using the Jaccard's coefficient and UPGMA (Unweighted Pair-Group Arithmetic Average Clustering) cluster analysis. The variations in the banding patterns indicated genomic variability among the twelve A. ferrooxidans strains tested. The primers used in this study grouped the twelve strains into five major groups. Similarities between the strains ranged from 5.49% to 85.14%. These results show that the strains have a high degree of genomic diversity and that the RAPD procedure is a powerful technique to assess strain variability in this bacterium.

Detection of Acidithiobacillus ferrooxidans in acid mine drainage environments using fluorescent in situ hybridization (FISH).

An important microorganism of acid mine drainage (AMD) and bioleaching environments is Acidithiobacillus ferrooxidans which oxidizes ferrous iron and generates ferric iron, an oxidant. Most investigations to understand microbial aspects of sulfide mineral dissolution have focused on understanding physiological, metabolic, and genetic characteristics of A. ferrooxidans. In this study, a 16S rRNA oligonucleotide probe designated S-S-T.ferr-0584-a-A-18, and labeled at the 5'-end with indocarbocyanine dye (CY3), was used in a fluorescent in situ hybridization (FISH) procedure on pure cultures of nine isolates of A. ferrooxidans. These isolates were recovered from acid mine drainage and mining environments. The probe was also used to detect cells of A. ferrooxidans, recovered from AMD samples, growing on FeTSB and FeSo solid media in a FISH procedure. In addition, the presence of cells of A. ferrooxidans in an environmental water sample from an AMD site in Copper Cliff, Ontario, Canada was analyzed using the FISH technique. Probe specificity was first confirmed with A. ferrooxidans ATCC 19859 (positive control) and Acidithiobacillus thiooxidans ATCC 19377, Acidiphilium acidophilum ATCC 27807, and Lactobacillus plantarum ATCC 8014 (negative controls). Positive and negative control cells were also used to determine optimal stringency conditions for hybridizations with the probe. Cells of the nine isolates of A. ferrooxidans stained positive, although the fluorescent signal varied in intensity from isolate to isolate. Colonies of A. ferrooxidans from the environmental water sample of the AMD site were recovered only on FeTSB solid medium after 22 days of incubation. The probe was able to detect cells of A. ferrooxidans in a FISH procedure. However, no cells of A. ferrooxidans were detected in the AMD water sample without cultivation. Thus, probe S-S-T.ferr-0584-a-A-18 hybridized effectively with cells of A. ferrooxidans recovered from pure cultures but failed to directly detect cells of A. ferrooxidans in the AMD site.

Early intertwin differences in myocardial performance during the twin-to-twin transfusion syndrome.

BACKGROUND: In the twin-to-twin transfusion syndrome (TTTS), pressure rather than volume overload is increasingly considered as a key factor in the pathogenesis of the cardiomyopathy of the recipient twin. If this is the case, cardiac dysfunction should be among the first signs observed with TTTS. The objective of this study was to determine whether intertwin differences in myocardial function are modified early in the course of TTTS and whether they can help to differentiate this condition from intrauterine growth restriction (IUGR). METHODS AND RESULTS: Eight variables were analyzed on the first fetal echocardiography on 21 pairs of twins with TTTS and 11 with IUGR. No difference was found between the 2 groups for the cardiothoracic ratio, pulsatility indices in the umbilical and middle cerebral arteries, and peak velocity of the middle cerebral artery. Significant difference was found for ventricular septal thickness, but with no association with the conditions under study. With TTTS, left ventricular shortening fraction was consistently greater in the donor twins, and myocardial performance indices (MPIs) were elevated in the recipient twins. This increase in MPI was caused by a lengthening of the isovolumic periods compared with those of the donor twin: left ventricular and right ventricular isovolumic periods 0.105+/-0.047 and 0.097+/-0.026 seconds, respectively, for the recipient twins versus 0.0561+/-0.46 and 0.065+/-0.03 seconds, respectively, for the donor twins (P<0.001). These changes in the isovolumic periods were mainly due to significant prolongation of isovolumic relaxation times. A change in left ventricular MPI > or =0.09 combined with a change in right ventricular MPI > or =0.05 would identify a TTTS with a sensitivity of 75% and a false-positive rate of 9%. CONCLUSIONS: The observed diastolic function impairment goes along with the pressure-overload pathogenic concept proposed in TTTS. Assessment of intertwin difference in MPI is a valuable tool for early differential diagnosis between TTTS and isolated IUGR.

Monitoring of bacteria in acid mine environments by reverse sample genome probing.

A variety of microorganisms can exist in acid mine drainage (AMD) environments, although their contribution to AMD problems is unclear. Environmental strains of Thiobacillus ferrooxidans and Thiobacillus acidophilus were purified by repeated plating and single-colony isolation on iron salts and tetrathionate media, respectively. Thiobacillus thiooxidans was enriched on sulfur-containing media. For the isolation of Leptospirillum ferrooxidans, iron salts and pyrite media were inoculated with environmental samples. However, L. ferrooxidans was never recovered on solid media. Denatured chromosomal DNAs from type and (or) isolated strains of T. ferrooxidans, T. acidophilus, T. thiooxidans, and L. ferrooxidans were spotted on a master filter for their detection in a variety of samples by reverse sample genome probing (RSGP). Analysis of enrichments of environmental samples by RSGP indicated that ferrous sulfate medium enriched T. ferrooxidans strains, whereas all thiobacilli grew in sulfur medium, T. thiooxidans strains being dominant. Enrichment in glucose medium followed by transfer to tetrathionate medium resulted in the selection of T. acidophilus strains. DNA was also extracted directly (without enrichment) from cells recovered from AMD water or sediments, and was analyzed by RSGP to describe the communities present. Strains showing homology with T. ferrooxidans and T. acidophilus were found to be major community components. Strains showing homology with T. thiooxidans were a minor community component, whereas strains showing homology with L. ferrooxidans were not detected.

Dementia caregiving: the role of the primary care physician.

BACKGROUND: Family and friends play an important role in caring for individuals with dementia living in the community. In preparation for the Canadian Consensus Conference on Dementia held in Montreal, Canada in February 1998, the subject of dementia caregiving was reviewed in order to provide primary care physicians with some guidelines for their practice. The review was updated in June 2000 in preparation for this article. METHOD: Pertinent English-language publications and resources from the Alzheimer Society of Canada were reviewed from 1985 onwards. Findings related to the consequences of caregiving, services for caregivers and recommendations regarding the role of the primary care physician were reviewed. FINDINGS: Dementia caregivers experience many positive and negative consequences of caregiving. Some comprehensive services for caregivers have been shown to delay institutionalization and reduce negative consequences of caregiving. The primary care physician has a role to play in working with families and should address the following issues: 1) education about dementia; 2) psychological support for caregivers; 3) assistance mobilizing caregiver social support networks. CONCLUSION: Primary care physicians have an important role to play in acknowledging and supporting the caregiving provided by family and friends to individuals with dementia.

Reference values for differences between cardio-circulatory variables of normal twin fetuses.

BACKGROUND: No reference values exist concerning the differences between cardio-circulatory variables of normal twin fetuses. The normal data could be useful in the identification of conditions causing opposite hemodynamic effects on each twin. OBJECTIVE: To establish the normal differences among cardio-circulatory parameters of twin fetuses during the second and third trimesters of gestation. MATERIALS AND METHODS: Twenty-seven normal twin pregnancies were used in this longitudinal and prospective study. Doppler-echocardiographic investigations were performed every 2-3 weeks starting at an average of 23.11 +/- 3.13 (mean +/- standard deviation) weeks' gestation. At each visit, the following cardio-circulatory variables were evaluated: the cardio-thoracic ratio, the ventricular wall and septal thicknesses, end-diastolic and systolic diameters, ventricular fractional shortenings, velocity of circumferential fibre shortenings and left and right ventricular outputs. In addition the following measurements were made from Doppler recordings: through both aortic and pulmonary valve the acceleration and ejection times, the peak systolic velocities and the velocity time integrals; and through both mitral and tricuspid valves peak velocities of E and A waves and the E/A ratios. Finally the pulsatility index of the umbilical artery was also evaluated. RESULTS: There was no single variable where the intertwin difference changed with gestational age. No systematic difference between the smaller and larger twin could be demonstrated for any variables except for the cardio-thoracic ratio. CONCLUSION: Reference tables should permit a comparative approach between the two twins in the investigation of life threatening complications such as twin-to-twin transfusion syndrome.

Strain variability and the effects of organic compounds on the growth of the chemolithotrophic bacterium Thiobacillus ferrooxidans.

The effects of naturally-occurring organic compounds on ferrous iron oxidation by the bacterium Thiobacillus ferrooxidans were examined with a view to using these compounds to treat or prevent acid mine/rock drainage. The compounds glucose, cellobiose, galacturonic acid, and citric acid were added to the growth medium of five different strains of the bacterium and growth studies were done to determine whether or not strain differences existed with respect to organic compound sensitivity. The effects of these compounds were compared to the effects of sodium dodecyl sulfate (SDS) an anionic detergent. Each of the compounds tested had an inhibitory effect on the strains of the bacterium and sensitivity to these compounds was strain dependent. All strains appeared to be equally susceptible to SDS. Inhibitory concentrations ranged from 70 mM to >280 mM for glucose, 7.5 mM to 150 mM for cellobiose, 20 mM to 230 mM for galacturonic acid, and 50 mM to 130 mM for citric acid. SDS effectively inhibited iron oxidation for all strains at a concentration of 0.3 mM, the lowest concentration tested. Some naturally-occurring organic compounds, therefore, might be candidates for the growth control of T. ferrooxidans.