Genotype and phenotype in 12 additional individuals with SATB2-associated syndrome.

SATB2-associated syndrome (SAS) is a multisystemic disorder caused by alterations of the SATB2 gene. We describe the phenotype and genotype of 12 individuals with 10 unique (de novo in 11 of 11 tested) pathogenic variants (1 splice site, 5 frameshift, 3 nonsense, and 2 missense) in SATB2 and review all cases reported in the published literature caused by point alterations thus far. In the cohort here described, developmental delay (DD) with severe speech compromise, facial dysmorphism, and dental anomalies were present in all cases. We also present the third case of tibial bowing in an individual who, just as in the previous 2 individuals in the literature, also had a truncating pathogenic variant of SATB2. We explore early genotype-phenotype correlations and reaffirm the main clinical features of this recognizable syndrome: universal DD with severe speech impediment, mild facial dysmorphism, and high frequency of craniofacial anomalies, behavioral issues, and brain neuroradiographic changes. As the recently proposed surveillance guidelines for individuals with SAS are adopted by providers, further delineation of the frequency and impact of other phenotypic traits will become available. Similarly, as new cases of SAS are identified, further exploration of genotype-phenotype correlations will be possible.

RHCE variants inherited with altered RHD alleles in Brazilian blood donors.

BACKGROUND: The high homology and opposite orientation of RH genes promote rearrangements between them and generate a large number of RHD and RHCE variants which can be inherited together. Searching of RHD-CE genotypes predicting partial antigens in donors is of interest in order to find more closely matched donors for African descent patients. This study aimed to evaluate a molecular approach to search for RhCE variants in a cohort of individuals with altered expression of D antigen and determine the association of RH variant alleles in Brazilian blood donors. METHODS: From 80,961 blood samples tested, 421 with atypical D typing results were studied. The samples were phenotyped for C, c, E, e antigens. Rh variants were identified using molecular techniques. RESULTS: All 421 samples had altered RHD alleles, being 56·3% of them partial D. Among them, 94·9% presented variant RHCE*ce and the most common associations were: RHD*weak D type 4.2.2 with RHCE*ceAR; RHD*DAR linked to RHCE*ceVS.02; RHD*weak D type 4.0 linked to RHCE*ceVS.02 and RHCE*ce (c.48C, c.105T, c.733G, c.744C, c.1025T). Among the samples with RhCE variants, 10·6% predict partial c, partial e, hr(B) - and/or hr(S) - and 100% express low prevalence antigens. CONCLUSION: Targeting individuals with altered expression of D antigen can be a good strategy for finding donors with RhCE variants. In our study 94·9% of the partial D samples revealed altered RHCE variant alleles and 5·7% of the samples with altered RHD allele predicted partial c, partial e and the lack of the high prevalence hr(B) and hr(S) antigens.

Active galaxies. A fast and long-lived outflow from the supermassive black hole in NGC 5548.

Supermassive black holes in the nuclei of active galaxies expel large amounts of matter through powerful winds of ionized gas. The archetypal active galaxy NGC 5548 has been studied for decades, and high-resolution x-ray and ultraviolet (UV) observations have previously shown a persistent ionized outflow. An observing campaign in 2013 with six space observatories shows the nucleus to be obscured by a long-lasting, clumpy stream of ionized gas not seen before. It blocks 90% of the soft x-ray emission and causes simultaneous deep, broad UV absorption troughs. The outflow velocities of this gas are up to five times faster than those in the persistent outflow, and, at a distance of only a few light days from the nucleus, it may likely originate from the accretion disk.

Benchmarking value in the pork supply chain: Characterization of US pork in the retail marketplace.

Retail pork from eight US cities was obtained for quality and palatability evaluations. Boneless pork loin chops were classified into one of three quality categories - "high," "average," or "low" - with higher quality chops possessing more desirable color, marbling, juiciness, and shear force characteristics than lower quality chops. Loin chops that were enhanced (injected with solution to improve juiciness and/or tenderness) had higher (P<0.05) pH, less purge and cook loss, and higher palatability ratings compared to non-enhanced chops. Hams compared by their protein fat free (PFF) classifications showed that ham and water product received the highest (P<0.05) ratings for juiciness and tenderness, and ham with natural juices received the highest (P<0.05) texture, ham flavor intensity, and smoke flavor ratings. Bacon was compared by price/brand categories; however, the highest priced, national branded bacon (US$12.03/kg) was similar (P>0.05) for most quality and all palatability traits to the lowest priced, national branded bacon (US$6.47/kg) and the store branded bacon (US$8.30/kg) even though retail prices differed widely. Overall, there were tremendous ranges in values for these products indicating that retail pork is quite variable and that efforts to improve the quality and consistency of it must continue.

Benchmarking value in the pork supply chain: Processing and consumer characteristics of hams manufactured from different quality raw materials.

Impact of fresh ham quality on finished ham product characteristics was evaluated. Bone-in hams destined for spiral-sliced ham manufacturing were sorted into two pH groups before processing: pH⩽5.5 and pH⩾5.6. For boneless hams, raw materials were sorted into groups with different levels of pale, soft, and exudative (PSE) product before manufacturing into sliced vacuum packaged hams: "Low PSE" (⩽5% PSE muscle), "Intermediate PSE" (20-30% PSE muscle) or "High PSE" (40-60% PSE muscle). Few differences were observed between the pH⩽5.5 and pH⩾5.6 groups in objective color measures and drip loss in bone-in spiral-sliced hams stored under refrigeration, however, after frozen storage, hams from the pH⩽5.5 group had lower L*- and a*-values and had much higher drip loss than those from the pH⩾5.6 group. Processing yields for bone-in spiral-sliced hams were similar through cooking and chilling, however, the pH⩾5.6 group had higher yields after slicing. For boneless hams, defects occurred at a greater frequency in hams formulated with a greater percentage of PSE raw materials than those with lower amounts of PSE. Differences in objective color measures and purge were minimal over the duration of storage time, but hams formulated with greater percentages of PSE raw materials were lighter in appearance and had less redness. Consumers gave lower color responses for hams formulated with "High PSE" amounts, but did not differentiate between hams manufactured with lower quantities of PSE muscle. However, when consumers directly compared packages of ham, there was distinct discrimination against hams manufactured with greater amounts of PSE. Purchase intent showed that consumers favored ham manufactured from fresh ham muscles containing low quantities of PSE tissue. Further research is needed to determine the optimal ratio of allowable PSE product in formulation that enables processors to maximize consumer appeal with the economic realities of sorting out PSE pork.

Benchmarking value in the pork supply chain: Processing characteristics and consumer evaluations of pork bellies of different thicknesses when manufactured into bacon.

Impact of belly thickness on processing yields and consumer evaluations of finished bacon products was measured. Before processing through a commercial facility, pork bellies (n=96 per group) were sorted into three target thickness groups: "thin" (approximately 2.0cm); "average" (approximately 2.5cm); "thick" (approximately 3.0cm). Processing yields at various production points were recorded and samples from each thickness group were evaluated by consumers for palatability characteristics and visual appearance. Bacon manufactured using "thick" bellies had the highest processing yields through the smoking and cooking phase. "Thin" bellies had the lowest slicing yields and generated the highest percentage of less valuable "#2 slices" (slice profile less than 1.9cm at any point) and "ends and pieces." Consumers found that bacon manufactured from "average" thickness bellies did not have deficiencies in palatability characteristics, but bacon manufactured from "thin" bellies lacked crispiness and bacon manufactured from "thick" bellies lacked flavor. Consumers found the lean-to-fat ratio and the visual appearance of bacon from "thick" bellies was less appealing than bacon from "thin" and "thick" bellies. Moreover, consumers showed much stronger purchase intent for bacon from "thin" and "average" bellies. Belly thickness impacted processing yield and consumer palatability evaluations of bacon. Producers need to minimize production of "thin" bellies because of reduced processing yields and "thick" bellies because of reduced consumer appeal.

Non-syndromic progressive hearing loss DFNA38 is caused by heterozygous missense mutation in the Wolfram syndrome gene WFS1.

Dominantly inherited progressive hearing loss DFNA38 is caused by heterozygosity for a novel mutation in WFS1, the gene for recessively inherited Wolfram syndrome. Wolfram syndrome is defined by juvenile diabetes mellitus and optic atrophy and may include progressive hearing loss and other neurological symptoms. Heterozygotes for other Wolfram syndrome mutations generally have normal hearing. Dominant deafness defined by DFNA38 is more severe than deafness of Wolfram syndrome patients and lacks any syndromic features. In a six-generation kindred from Newfoundland, Canada, WFS1 Ala716Thr (2146 G-->A) was shared by all deaf members of the family and was specific to deaf individuals. The causal relationship between this missense mutation and deafness was supported by two observations based on haplotype and mutation analysis of the kindred. First, a relative homozygous for the mutation was diagnosed at age 3 years with insulin-dependent diabetes mellitus, the central feature of Wolfram syndrome. Second, two relatives with normal hearing had an identical haplotype to that defining DFNA38, with the exception of the base pair at position 2146. Other rare variants of WFS1 co-inherited with deafness in the family could be excluded as disease-causing mutations on the basis of this hearing-associated haplotype. The possibility that 'mild' mutations in WFS1 might be a cause of non-syndromic deafness in the general population should be explored.

Mutations in the gene encoding neutrophil elastase in congenital and cyclic neutropenia.

Congenital neutropenia and cyclic neutropenia are disorders of neutrophil production predisposing patients to recurrent bacterial infections. Recently the locus for autosomal dominant cyclic neutropenia was mapped to chromosome 19p13.3, and this disease is now attributable to mutations of the gene encoding neutrophil elastase (the ELA2 gene). The authors hypothesized that congenital neutropenia is also due to mutations of neutrophil elastase. Patients with congenital neutropenia, cyclic neutropenia, or Shwachman-Diamond syndrome were referred to the Severe Chronic Neutropenia International Registry. Referring physicians provided hematologic and clinical data. Mutational analysis was performed by sequencing polymerase chain reaction (PCR)-amplified genomic DNA for each of the 5 exons of the neutrophil ELA2 gene and 20 bases of the flanking regions. RNA from bone marrow mononuclear cells was used to determine if the affected patients expressed both the normal and the abnormal transcript. Twenty-two of 25 patients with congenital neutropenia had 18 different heterozygous mutations. Four of 4 patients with cyclic neutropenia and 0 of 3 patients with Shwachman-Diamond syndrome had mutations. For 5 patients with congenital neutropenia having mutations predicted to alter RNA splicing or transcript structure, reverse transcriptase-PCR showed expression of both normal and abnormal transcripts. In cyclic neutropenia, the mutations appeared to cluster near the active site of the molecule, whereas the opposite face was predominantly affected by the mutations found in congenital neutropenia. This study indicates that mutations of the gene encoding neutrophil elastase are probably the most common cause for severe congenital neutropenia as well as the cause for sporadic and autosomal dominant cyclic neutropenia.

Enzymic catalysis of the accumulation of acetaldehyde from ethanol in human prenatal cephalic tissues: evaluation of the relative contributions of CYP2E1, alcohol dehydrogenase, and catalase/peroxidases.

BACKGROUND: The human prenatal brain is very sensitive to the toxic effects of ethanol, but very little information is available concerning the conversion of ethanol to the highly cytotoxic metabolite, acetaldehyde, in that organ. Thus, experiments were designed to investigate rates of accumulation of acetaldehyde from ethanol in the prenatal human brain. METHODS: Prenatal human cephalic tissue homogenates were used as enzyme sources and were compared with analogous preparations of adult rat livers. Generated acetaldehyde was derivatized with cyclohexane-1,3-dione to yield fluorescent decahydroacrizine-1,8-dione, which was readily separated, detected, and quantitated with HPLC. RESULTS: Detected rates of accumulation were unexpectedly high, even in the absence of added NADPH, NAD+, or H2O2, which are cofactors/cosubstrates for cytochrome P-450-, alcohol dehydrogenase- and catalase/peroxidase-catalyzed reactions, respectively. Without added cofactors/cosubstrates or other components and under linear reaction conditions, rates in human prenatal cephalic preparations were approximately 20% of those observed with analogous preparations of adult rat livers. Cofactor/cosubstrate-independent reactions were localized in the cytosolic (soluble) fraction and were strongly dependent on molecular oxygen (O2). They were not inhibited substantially by carbon monoxide (CO:O2 = 80:20 vs N2:O2 = 80:20) or by pyrazole in concentrations up to 10 mM and were only weakly inhibited by azide. Preincubations with excess catalase did not result in decreased activity. Reactions exhibited substrate saturation and heat inactivation indicating enzymic catalysis. CONCLUSIONS: Experiments indicated a relatively rapid accumulation of acetaldehyde from ethanol in human prenatal brain tissues and suggested that the observed cofactor/cosubstrate-independent reactions were largely independent of P-450 cytochromes, alcohol dehydrogenases, or catalase/peroxidases. Results were consistent with catalysis by an as yet unidentified cytosolic oxidase(s).

Hepatic lesion differentiated from accessory spleen by a heat-damaged red blood cell scan.

A 34-year-old woman referred for evaluation of an abdominal mass underwent a computed tomographic portagram that showed a large mass in the region of the left hepatic lobe and adjacent to the spleen. A liver-spleen scan with sulfur colloid did not clearly show whether the mass originated in the liver or the spleen. To evaluate the possibility of an accessory spleen, a selective spleen scan using Tc-99m-labeled heat-damaged red blood cells was performed and showed intense uptake in the region of the spleen only. The patient underwent surgical exploration and excision of the lesion, which proved to be focal nodular hyperplasia of the liver. Thus, the heat-damaged red blood cell scan ruled out an accessory spleen as a cause for the mass.

Mutations in ELA2, encoding neutrophil elastase, define a 21-day biological clock in cyclic haematopoiesis.

Human cyclic haematopoiesis (cyclic neutropenia, MIM 162800) is an autosomal dominant disease in which blood-cell production from the bone marrow oscillates with 21-day periodicity. Circulating neutrophils vary between almost normal numbers and zero. During intervals of neutropenia, affected individuals are at risk for opportunistic infection. Monocytes, platelets, lymphocytes and reticulocytes also cycle with the same frequency. Here we use a genome-wide screen and positional cloning to map the locus to chromosome 19p13.3. We identified 7 different single-base substitutions in the gene (ELA2) encoding neutrophil elastase (EC 3. 4.21.37, also known as leukocyte elastase, elastase 2 and medullasin), a serine protease of neutrophil and monocyte granules, on unique haplotypes in 13 of 13 families as well as a new mutation in a sporadic case. Neutrophil elastase (a 240-aa mature protein predominantly found in neutrophil granules) is the target for protease inhibition by alpha1-antitrypsin, and its unopposed release destroys tissue at sites of inflammation. We hypothesize that a perturbed interaction between neutrophil elastase and serpins or other substrates may regulate mechanisms governing the clock-like timing of haematopoiesis.

Role of free radicals in the limb teratogenicity of L-NAME (N(G)-nitro-(L)-arginine methyl ester): a new mechanistic model of vascular disruption.

In continuing studies of limb effects resulting from fetal exposure to N(G)-nitro-(L)-arginine methyl ester (L-NAME), we examined the early time course of vascular changes and the effectiveness of fetal intraamniotic injection. Vascular engorgement and hemorrhage occurred within 4 hr of L-NAME treatment on gestational day (gd) 17, and direct injection appeared to be as effective as maternal intraperitoneal injection in inducing limb hemorrhage. Further studies examined protein nitration and electron transport inhibition in tissues of exposed fetuses. L-NAME caused significant increases in nitrotyrosine (NT) formation in limb but not in heart or brain, and reduced electron transport rates in limb. Three agents, alpha-phenyl-N-t-butylnitrone (PBN), a radical trap and inhibitor of inducible nitric oxide synthase (iNOS), allopurinol, an inhibitor of xanthine oxidase, and aminoguanidine, a relatively specific inhibitor of iNOS, significantly moderated limb hemorrhage and protein nitration in distal limb. These results suggest that L-NAME works directly on the fetal limb vasculature and indicate a cytotoxic role for peroxynitrite, a potent oxidant and nitrating agent that is the reaction product of nitric oxide and superoxide anion radical. We propose that L-NAME and other vasoactive toxicants disrupt the fetal limb in a sequential process. Initially, nitric oxide (NO) is depleted, causing hemorrhage and edema in the limb. Within hours, iNOS is induced, resulting in cytotoxic tissue concentrations of NO and reactive nitrogen species that induce apoptosis and/or necrosis in the limb. We suggest that L-NAME exposure may serve as a model of vascular disruptive limb malformations.

Hematopoietic dynamics in grey collies.

Using Lomb periodogram analysis we have quantified variations in the peripheral neutrophil and platelet counts of the cyclical neutropenia animal model-the grey collie. We found that the amplitudes of the oscillations in these two cell lineages vary concomitantly. Further, the power spectrum and the shape of the oscillations in the absolute neutrophil counts vary together with the amplitude of the oscillations. As the amplitude of the oscillations increases, the height of the second subharmonic increases, giving rise to a distorted oscillation with two peaks per cycle. The particular dynamics of the absolute neutrophil counts can be reproduced by a combination of a delayed peripheral feedback, representing the peripheral control of granulopoiesis through granulocyte colony stimulating factor, together with a sinusoidal input representing an oscillatory input from the pluripotential stem cells to the granulocytic lineage. The same pluripotential stem cell input is probably responsible for the sinusoidal oscillations observed in the other cell lineages.

Catalytic activity and quantitation of cytochrome P-450 2E1 in prenatal human brain.

Cytochrome P-450 2E1 (CYP2E1) is a readily inducible hemoprotein that catalyzes the oxidation of endogenous compounds and many low molecular weight xenobiotics. As the major component of the microsomal ethanol oxidizing system, it contributes significantly to ethanol metabolism and the formation of the highly reactive metabolite acetaldehyde. The leaky property of this enzyme results in the generation of reactive oxygen species that can induce oxidative stress and cytotoxic conditions deleterious to development. To further investigate the proposed role of CYP2E1 in the etiology of alcohol teratogenesis, the current study focused on the quantification of CYP2E1 in prenatal human brain, a tissue that is highly vulnerable to the damaging effects of ethanol throughout gestation. In microsomal samples prepared from pools of brain tissues, immunoreactive protein was detected by Western blot analysis using enhanced chemiluminescence, whereas functional protein was estimated with an enzymatic assay using p-nitrophenol and an electrochemical detection system. CYP2E1 transcript was consistently detected in RNA samples prepared from individual brain tissues using the ribonuclease protection assay. Quantitative data were collected by scanning densitometry and phosphorimaging technology. There was a dramatic increase in human brain CYP2E1 content around gestational day 50 and a fairly constant level was maintained throughout the early fetal period, until at least day 113. The relatively low levels of the P-450 isoform present in conceptal brain may be sufficient to generate reactive intermediates that elicit neuroembryotoxicity following maternal alcohol consumption.

Reactive oxygen species and DNA oxidation in fetal rat tissues.

It is well recognized that reactive oxygen species (ROS) are formed during the reperfusion of ischemic tissues and ROS may be pathogenic in adult tissues. Although there is little information on the formation and toxicity of ROS during prenatal life, a strong association has been made between limb and possibly brain malformations and uteroplacental ischemia during fetal stages of gestation. It has been proposed that these malformations result from attack by ROS formed during the resumption of placental perfusion. Studies reported here examined formation of ROS in teratogenically sensitive limb and brain and insensitive heart before and during the period of teratogenic sensitivity. Also examined was the formation of ROS following hypoxia and reoxygenation in fetal culture and DNA hydroxylation in sensitive and insensitive fetal tissues during uteroplacental ischemia and reperfusion in vivo. Rates of formation of superoxide anion radical and hydrogen peroxide declined with increasing gestational age whereas those for hydroxyl radical increased. Hydrogen peroxide generation was greatest in insensitive heart whereas hydroxyl radical formation was significantly lower in brain than in limb or heart, which had comparable rates. Hydrogen peroxide generation, which declined significantly in fetuses, but not in membranes with gestation, failed to respond to reoxygenation in vitro. Finally, there were significant increases in DNA hydroxylation in fetal hearts and limbs, but not in brains during uteroplacental ischemia but no further significant change could be detected after reperfusion.

Studies of the cellular distribution of superoxide dismutases in adult and fetal rat tissues.

Activities of three types of superoxide dismutase in tissue fractions were significantly lower in fetal and adult brain and fetal limb preparations than in fetal and adult heart preparations. An exception was the cytoplasmic fraction of adult brain that had levels of Cu, Zn-superoxide dismutase activity comparable to those in cytoplasmic fractions of heart. In addition, Mn superoxide dismutase activity appeared to be very low in all fetal mitochondrial matrix fractions and cytoplasmic fractions as well as in adult brain. Finally, the results of these studies emphasize the importance of two antioxidant defense systems in the tissues studied, one associated with the mitochondrial electron transport system and the other, the cytosolic Cu, Zn enzyme.

Motor unit discharge during muscular after-contraction.

Discharges of single motor units (MUs) in human triceps brachii and deltoid muscle were recorded using needle electromyography during after-contraction and voluntary contraction performed either against a small elastic load or under isometry. The steady-state firing rate of the MUs was lower under after-contraction than during voluntary movement of comparable amplitude and time course (or isometric force level), whereas variability of interspike intervals was similar under the two conditions. In the tibialis anterior muscle (where after-contraction was lacking), a weak voluntary contraction preceded by sustained strong voluntary effort also showed lower firing rate of MUs as compared to similar voluntary movement performed after a rest period. We concluded that sustained contraction gave rise to peripheral potentiation of contractile properties of the muscle, irrespective of whether it was proximal or distal, whereas after-contraction was due to a central tonic drive that differed for proximal and distal muscles.

Studies of mitochondria in oxidative embryotoxicity.

While the limb bud and brain of the rat develop abnormally in response to transient uteroplacental hypoperfusion during late gestation, the heart appears to be protected. These malformations have been associated with the generation of reactive oxygen species (ROS). Studies were designed to examine superoxide generation by mitochondrial electron transport particles (ETP) from adult and conceptal tissues and to investigate characteristics that could be responsible for heightened concentrations of ROS in sensitive tissues. Parameters investigated included NADH oxidase and cytochrome c oxidase activities, cytochrome content, and superoxide dismutase activity. NADH oxidase activities were significantly lower in sensitive tissues that also developed the highest concentrations of superoxide. Because ETP from adult CNS also had low NADH oxidase activity but did not show increased concentrations of superoxide, inhibition of electron transport did not adequately account for increased ROS concentrations. The reduced NADH oxidase activity of sensitive tissues could not be caused by inhibition at the cytochrome c oxidase region since this latter activity equaled or exceeded the former in all instances. No significant differences were found in the cytochrome contents of different tissues. There was significantly less superoxide dismutase activity in homogenates prepared from either of the two sensitive conceptual tissues compared with those from insensitive conceptual or adult tissues. These studies confirm the presence of heightened concentrations of superoxide anion radical in ETP from teratogenically sensitive tissues and suggest that these concentrations may result primarily from decreased activity of superoxide dismutase(s) in those tissues. Superoxide anion radical could therefore be available to participate in the generation of the more toxic oxidant species such as the hydroxyl radical.