Hemizygous deletion of COL3A1, COL5A2, and MSTN causes a complex phenotype with aortic dissection: a lesson for and from true haploinsufficiency

Aortic dilatation/dissection (AD) can occur spontaneously or in association with genetic syndromes, such as Marfan syndrome (MFS; caused by FBN1 mutations), MFS type 2 and Loeys-Dietz syndrome (associated with TGFBR1/TGFBR2 mutations), and Ehlers-Danlos syndrome (EDS) vascular type (caused by COL3A1 mutations). Although mutations in FBN1 and TGFBR1/TGFBR2 account for the majority of AD cases referred to us for molecular genetic testing, we have obtained negative results for these genes in a large cohort of AD patients, suggesting the involvement of additional genes or acquired factors. In this study we assessed the effect of COL3A1 deletions/duplications in this cohort. Multiplex ligation-dependent probe amplification (MLPA) analysis of 100 unrelated patients identified one hemizygous deletion of the entire COL3A1 gene. Subsequent microarray analyses and sequencing of breakpoints revealed the deletion size of 3,408,306 bp at 2q32.1q32.3. This deletion affects not only COL3A1 but also 21 other known genes (GULP1, DIRC1, COL5A2, WDR75, SLC40A1, ASNSD1, ANKAR, OSGEPL1, ORMDL1, LOC100129592, PMS1, MSTN, C2orf88, HIBCH, INPP1, MFSD6, TMEM194B, NAB1, GLS, STAT1, and STAT4), mutations in three of which (COL5A2, SLC40A1, and MSTN) have also been associated with an autosomal dominant disorder (EDS classical type, hemochromatosis type 4, and muscle hypertrophy). Physical and laboratory examinations revealed that true haploinsufficiency of COL3A1, COL5A2, and MSTN, but not that of SLC40A1, leads to a clinical phenotype. Our data not only emphasize the impact/role of COL3A1 in AD patients but also extend the molecular etiology of several disorders by providing hitherto unreported evidence for true haploinsufficiency of the underlying gene.

Gene expression of tumour necrosis factor and insulin signalling-related factors in subcutaneous adipose tissue during the dry period and in early lactation in dairy cows

Gene expression of adipose factors, which may be part of the mechanisms that underlie insulin sensitivity, were studied in dairy cows around parturition. Subcutaneous fat biopsies and blood samples were taken from 27 dairy cows in week 8 antepartum (a.p.), on day 1 postpartum (p.p.) and in week 5 p.p. In the adipose tissue samples, mRNA was quantified by real-time reverse transcription polymerase chain reaction for tumour necrosis factor alpha (TNFalpha), insulin-independent glucose transporter (GLUT1), insulin-responsive glucose transporter (GLUT4), insulin receptor, insulin receptor substrate 1 (IRS1), insulin receptor substrate 2 (IRS2), regulatory subunit of phosphatidylinositol-3 kinase (p85) and catalytic subunit of phosphatidylinositol-3 kinase. Blood plasma was assayed for concentrations of glucose, beta-hydroxybutyric acid, non-esterified fatty acids (NEFA) and insulin. Plasma parameters followed a pattern typically observed in dairy cows. Gene expression changes were observed, but there were no changes in TNFalpha concentrations, which may indicate its local involvement in catabolic adaptation of adipose tissue. Changes in GLUT4 and GLUT1 mRNA abundance may reflect their involvement in reduced insulin sensitivity and in sparing glucose for milk synthesis in early lactation. Unchanged gene expression of IRS1, IRS2 and p85 over time may imply a lack of their involvement in terms of insulin sensitivity dynamics. Alternatively, it may indicate that post-transcriptional modifications of these factors came into play and may have concealed an involvement.

COMPRESSION BEHAVIOR AT HIGH STRAIN RATE FOR AN ULTRA HIGH PERFORMANCE CONCRETE

The need for a stronger and more durable building material is becoming more important as the structural engineering field expands and challenges the behavioral limits of current materials. One of the demands for stronger material is rooted in the effects that dynamic loading has on a structure. High strain rates on the order of 101 s-1 to 103 s-1, though a small part of the overall types of loading that occur anywhere between 10-8 s-1 to 104 s-1 and at any point in a structures life, have very important effects when considering dynamic loading on a structure. High strain rates such as these can cause the material and structure to behave differently than at slower strain rates, which necessitates the need for the testing of materials under such loading to understand its behavior. Ultra high performance concrete (UHPC), a relatively new material in the U.S. construction industry, exhibits many enhanced strength and durability properties compared to the standard normal strength concrete. However, the use of this material for high strain rate applications requires an understanding of UHPC’s dynamic properties under corresponding loads. One such dynamic property is the increase in compressive strength under high strain rate load conditions, quantified as the dynamic increase factor (DIF). This factor allows a designer to relate the dynamic compressive strength back to the static compressive strength, which generally is a well-established property. Previous research establishes the relationships for the concept of DIF in design. The generally accepted methodology for obtaining high strain rates to study the enhanced behavior of compressive material strength is the split Hopkinson pressure bar (SHPB). In this research, 83 Cor-Tuf UHPC specimens were tested in dynamic compression using a SHPB at Michigan Technological University. The specimens were separated into two categories: ambient cured and thermally treated, with aspect ratios of 0.5:1, 1:1, and 2:1 within each category. There was statistically no significant difference in mean DIF for the aspect ratios and cure regimes that were considered in this study. DIF’s ranged from 1.85 to 2.09. Failure modes were observed to be mostly Type 2, Type 4, or combinations thereof for all specimen aspect ratios when classified according to ASTM C39 fracture pattern guidelines. The Comite Euro-International du Beton (CEB) model for DIF versus strain rate does not accurately predict the DIF for UHPC data gathered in this study. Additionally, a measurement system analysis was conducted to observe variance within the measurement system and a general linear model analysis was performed to examine the interaction and main effects that aspect ratio, cannon pressure, and cure method have on the maximum dynamic stress.

Clinical and esthetic outcomes of implants placed in postextraction sites

PURPOSE: The aim of this review was to evaluate the clinical outcomes for the different time points of implant placement following tooth extraction. MATERIALS AND METHODS: A PubMed search and a hand search of selected journals were performed to identify clinical studies published in English that reported on outcomes of implants in postextraction sites. Only studies that included 10 or more patients were accepted. For implant success/survival outcomes, only studies with a mean follow-up period of at least 12 months from the time of implant placement were included. The following outcomes were identified: (1) change in peri-implant defect dimension, (2) implant survival and success, and (3) esthetic outcomes. RESULTS AND CONCLUSIONS: Of 1,107 abstracts and 170 full-text articles considered, 91 studies met the inclusion criteria for this review. Bone augmentation procedures are effective in promoting bone fill and defect resolution at implants in postextraction sites, and are more successful with immediate (type 1) and early placement (type 2 and type 3) than with late placement (type 4). The majority of studies reported survival rates of over 95%. Similar survival rates were observed for immediate (type 1) and early (type 2) placement. Recession of the facial mucosal margin is common with immediate (type 1) placement. Risk indicators included a thin tissue biotype, a facial malposition of the implant, and a thin or damaged facial bone wall. Early implant placement (type 2 and type 3) is associated with a lower frequency of mucosal recession compared to immediate placement (type 1).

Long-term estrogen exposure of whitefish Coregonus lavaretus induces intersex but not Lake Thun-typical gonad malformations

A high prevalence of gonad morphological variations has been observed in whitefish Coregonus lavaretus from Lake Thun (Switzerland). To clarify the role of endocrine disruption as a possible cause of the gonad alterations, whitefish were reared in a long-term laboratory experiment under exposure to 17 beta-estradiol (E2). Fish were fed from first-feeding until 3 yr of age at a daily rate of 0 (control), 0.5 or 50 microg E2 kg(-1) fish. E2 exposure resulted in a time- and concentration-dependent increase of prevalence and intensity of intersex gonads, i.e. gonads that macroscopically appeared as either testis or ovary but microscopically contained both male and female germ cells. Four types of intersex could be distinguished: Types 1 and 2 were composed of mainly male tissue, with Type 1 containing single oocytes and Type 2 displaying an ovary-like lamellar structure of the tissue. In Type 3, an increased percentage of the tissue was occupied by female germ cells, while in Type 4, the majority of the gonad tissue consisted of female germ cells. Chronic E2 exposure additionally resulted in a concentration-dependent shift of the sex ratio towards females, a reduced condition factor, retarded gonad growth together with delayed maturation of germ cells, and elevated levels of hepatic vitellogenin mRNA. However, Lake Thun-typical alterations of gonad morphology were not induced by chronic E2 exposure. The results provide evidence that estrogen-active compounds unlikely play a role in the etiology of gonad malformations in Lake Thun whitefish.

Incidence and treatment of developmental hip dysplasia in Mongolia: a prospective cohort study

BACKGROUND In Mongolia, adequate early diagnosis and treatment of developmental hip dysplasia (DDH) have been unavailable and its incidence was unknown. We determined the incidence of ultrasonographic DDH in newborns and established adequate procedures for diagnosis and treatment of DDH at the largest maternity hospital in Ulaanbaatar, Mongolia. METHODOLOGY/PRINCIPAL FINDINGS During one year (Sept 2010 - Aug 2011) we assessed the hips newborns using ultrasound and Graf's classification of DDH. 8,356 newborns were screened; median age at screening was 1 day. We identified 14,873 Type 1 (89.0%), 1715 Type 2a (10.3%), 36 Type 2c (0.2%), 70 Type D (0.4%), 14 Type 3 (0.08%), and 4 Type 4 hips (0.02%). Children with Type 1 hips (normal) were discharged. Children with Type 2a hips (physiologically immature) received follow-up ultrasounds at monthly intervals. Children with Type 2c to 4 (DDH; deformed or misaligned hip joint) hips were treated with a Tubingen hip flexion splint and also followed up. The hip abnormalities resolved to mature hips in all children who were followed up. There was no evidence for severe treatment related complications. CONCLUSION/SIGNIFICANCE This study suggests that the incidence of DDH in Mongolian neonates is comparable to that in neonates in Europe. Early ultrasound-based assessment and splinting treatment of DDH led to mature hips in all children followed up. Procedures are feasible and will be continued.

A model of the roles of essential kinases in the induction and expression of late long-term potentiation.

The induction of late long-term potentiation (L-LTP) involves complex interactions among second-messenger cascades. To gain insights into these interactions, a mathematical model was developed for L-LTP induction in the CA1 region of the hippocampus. The differential equation-based model represents actions of protein kinase A (PKA), MAP kinase (MAPK), and CaM kinase II (CAMKII) in the vicinity of the synapse, and activation of transcription by CaM kinase IV (CAMKIV) and MAPK. L-LTP is represented by increases in a synaptic weight. Simulations suggest that steep, supralinear stimulus-response relationships between stimuli (e.g., elevations in [Ca(2+)]) and kinase activation are essential for translating brief stimuli into long-lasting gene activation and synaptic weight increases. Convergence of multiple kinase activities to induce L-LTP helps to generate a threshold whereby the amount of L-LTP varies steeply with the number of brief (tetanic) electrical stimuli. The model simulates tetanic, -burst, pairing-induced, and chemical L-LTP, as well as L-LTP due to synaptic tagging. The model also simulates inhibition of L-LTP by inhibition of MAPK, CAMKII, PKA, or CAMKIV. The model predicts results of experiments to delineate mechanisms underlying L-LTP induction and expression. For example, the cAMP antagonist RpcAMPs, which inhibits L-LTP induction, is predicted to inhibit ERK activation. The model also appears useful to clarify similarities and differences between hippocampal L-LTP and long-term synaptic strengthening in other systems.

Evolutionarily conserved role of calcineurin in phosphodegron-dependent degradation of phosphodiesterase 4D.

Calcineurin is a widely expressed and highly conserved Ser/Thr phosphatase. Calcineurin is inhibited by the immunosuppressant drug cyclosporine A (CsA) or tacrolimus (FK506). The critical role of CsA/FK506 as an immunosuppressant following transplantation surgery provides a strong incentive to understand the phosphatase calcineurin. Here we uncover a novel regulatory pathway for cyclic AMP (cAMP) signaling by the phosphatase calcineurin which is also evolutionarily conserved in Caenorhabditis elegans. We found that calcineurin binds directly to and inhibits the proteosomal degradation of cAMP-hydrolyzing phosphodiesterase 4D (PDE4D). We show that ubiquitin conjugation and proteosomal degradation of PDE4D are controlled by a cullin 1-containing E(3) ubiquitin ligase complex upon dual phosphorylation by casein kinase 1 (CK1) and glycogen synthase kinase 3beta (GSK3beta) in a phosphodegron motif. Our findings identify a novel signaling process governing G-protein-coupled cAMP signal transduction-opposing actions of the phosphatase calcineurin and the CK1/GSK3beta protein kinases on the phosphodegron-dependent degradation of PDE4D. This novel signaling system also provides unique functional insights into the complications elicited by CsA in transplant patients.

Immunology and HIV expression in skin, in vitro, and in response to ultraviolet light

HIV can enter the body through Langerhans cells, dendritic cells, and macrophages in skin mucosa, and spreads by lysis or by syncytia. Since UVL induces of HIV-LTR in transgenic mice mid in cell lines in vitro, we hypothesized that UVB may affect HIV in people and may affect HIV in T cells in relation to dose, apoptosis, and cytokine expression. To determine whether HIV is induced by UVL in humans, a clinical study of HIV+ patients with psoriasis or pruritus was conducted during six weeks of UVB phototherapy, Controls were HIV-psoriasis patients receiving UVB and HIV+ KS subjects without UVB.Blood and skin biopsy specimens were collected at baseline, weeks 2 and 6, and 4 weeks after UVL. AIDS-related skin diseases showed unique cytokine profiles in skin and serum at baseline. In patients and controls on phototherapy, we observed the following: (1) CD4+ and CD8+ T cell numbers are not significantly altered during phototherapy, (2) p24 antigen levels, and also HIV plasma levels increase in patients not on antiviral therapy, (3) HIV-RNA levels in serum or plasma. (viral load) can either increase or decrease depending on the patient's initial viral load, presence of antivirals, and skin type, (4) HIV-RNA levels in the periphery are inversely correlated to serum IL-10 and (5) HIV+ cell in skin increase after UVL at 2 weeks by RT-PCR in situ hybridization mid we negatively correlated with peripheral load. To understand the mechanisms of UVB mediated HIV transcription, we treated Jurkat T cell lines stably transfected with an HIV-LTR-luciferase plasmid only or additionally with tat-SV-40 early promoter with UVB (2 J/m2 to 200 J/m2), 50 to 200 ng/ml rhIL-10, and 10 μg/ml PHA as control. HIV promoter activity was measured by luciferase normalized to protein. Time points up to 72 hours were analyzed for HIV-LTR activation. HIV-LTR activation had the following properties: (1) requires the presence of Tat, (2) occurs at 24 hours, and (3) is UVB dose dependent. Changes in viability by MTS (3-(4,5-dimethyhhiazol-2-y1)-5-(3-carboxymethoxyphonyl)-2-(4-sulfophenyl)-2H-tetrazolium) mixed with PMS (phenazine methosulfate) solution and apoptosis by propidium iodide and annexin V using flow cytometry (FC) were seen in irradiated Jurkat cells. We determined that (1) rhIL-10 moderately decreased HIV-LTR activation if given before radiation and greatly decreases it when given after UVB, (2) HIV-LTR activation was low at doses of greater than 70 J/m2, compared to activation at 50 J/m2. (Abstract shortened by UMI.)^

Discusiones sobre el realismo en la narrativa argentina contemporánea

A partir de las recientes lecturas sobre el realismo argentino de, centralmente, Martín Kohan, Graciela Speranza, Nora Avaro y Analía Capdevila, se piensan y discuten hipótesis en torno de los siguientes ejes: 1. los límites del alcance y de la vigencia de la categoría de realismo en su relación con la narrativa argentina actual; 2. alcances y límites de una relectura de la teoría lukacsiana hoy; 3. el problema de la definición del tipo realista; 4. variaciones realistas en la narrativa argentina contemporánea.

Postglacial deposition of sand in the Eckernfoerder Bay, western Baltic Sea

At the NW-slope of Eckernforder Bay (Western Baltic) between 14 and 21 m water depth 7 sand cores were taken with a vibrocorer. The cores were between 85 and 250 cm long. The sand was analysed for grain size distribution, proportions of organic carbon and carbonate, and contents of microfossils. The radiometric age and stable carbon isotope ratios were determined on organic material from 14 sample. With regard to benthic foraminifera and other microorganisms four different types of depositional conditions could be distinguished: Types 1 and 2: two types of offshore sand areas. Type 3: lagoon and nearshore. Type 4: subaerial or limnic. Using sedimentological and geochemical parameters two formation areas could be distinguished with the aid of a discriminant analysis: offshore (types 1 and 2) and nearshore (types 3 and 4). A juxtaposition of core sections indicated two distinct profiles. Their ages fit into the picture of the assumed postglacial sea-level rise. The lagoon- and nearshore sands are interpreted as the result of sea-level stagnation at 17-18 m below present sea-level. The accumulation rates of the sand in the offshore areas are, with a maximum of 0.15 mm/yr., an order of magnitude smaller than in the mud areas, located several hundred metres away.

Discusiones sobre el realismo en la narrativa argentina contemporánea

A partir de las recientes lecturas sobre el realismo argentino de, centralmente, Martín Kohan, Graciela Speranza, Nora Avaro y Analía Capdevila, se piensan y discuten hipótesis en torno de los siguientes ejes: 1. los límites del alcance y de la vigencia de la categoría de realismo en su relación con la narrativa argentina actual; 2. alcances y límites de una relectura de la teoría lukacsiana hoy; 3. el problema de la definición del tipo realista; 4. variaciones realistas en la narrativa argentina contemporánea.

Discusiones sobre el realismo en la narrativa argentina contemporánea

A partir de las recientes lecturas sobre el realismo argentino de, centralmente, Martín Kohan, Graciela Speranza, Nora Avaro y Analía Capdevila, se piensan y discuten hipótesis en torno de los siguientes ejes: 1. los límites del alcance y de la vigencia de la categoría de realismo en su relación con la narrativa argentina actual; 2. alcances y límites de una relectura de la teoría lukacsiana hoy; 3. el problema de la definición del tipo realista; 4. variaciones realistas en la narrativa argentina contemporánea.

Sucrose is a signal molecule in assimilate partitioning

The proton–sucrose symporter mediates the key transport step in the resource distribution system that allows many plants to function as multicellular organisms. In the results reported here, we identify sucrose as a signaling molecule in a previously undescribed signal-transduction pathway that regulates the symporter. Sucrose symporter activity declined in plasma membrane vesicles isolated from leaves fed exogenous sucrose via the xylem transpiration stream. Symporter activity dropped to 35–50% of water controls when the leaves were fed 100 mM sucrose and to 20–25% of controls with 250 mM sucrose. In contrast, alanine symporter and glucose transporter activities did not change in response to sucrose treatments. Decreased sucrose symporter activity was detectable after 8 h and reached a maximum by 24 h. Kinetic analysis of transport activity showed a decrease in Vmax. RNA gel blot analysis revealed a decrease in symporter message levels, suggesting a drop in transcriptional activity or a decrease in mRNA stability. Control experiments showed that these responses were not the result of changing osmotic conditions. Equal molar concentrations of hexoses did not elicit the response, and mannoheptulose, a hexokinase inhibitor, did not block the sucrose effect. These data are consistent with a sucrose-specific response pathway that is not mediated by hexokinase as the sugar sensor. Sucrose-dependent changes in the sucrose symporter were reversible, suggesting this sucrose-sensing pathway can modulate transport activity as a function of changing sucrose concentrations in the leaf. These results demonstrate the existence of a signaling pathway that can control assimilate partitioning at the level of phloem translocation.

The Formation of an Insulin-responsive Vesicular Cargo Compartment Is an Early Event in 3T3-L1 Adipocyte Differentiation

Differentiating 3T3-L1 cells exhibit a dramatic increase in the rate of insulin-stimulated glucose transport during their conversion from proliferating fibroblasts to nonproliferating adipocytes. On day 3 of 3T3-L1 cell differentiation, basal glucose transport and cell surface transferrin binding are markedly diminished. This occurs concomitant with the formation of a distinct insulin-responsive vesicular pool of intracellular glucose transporter 1 (GLUT1) and transferrin receptors as assessed by sucrose velocity gradients. The intracellular distribution of the insulin-responsive aminopeptidase is first readily detectable on day 3, and its gradient profile and response to insulin at this time are identical to that of GLUT1. With further time of differentiation, GLUT4 is expressed and targeted to the same insulin-responsive vesicles as the other three proteins. Our data are consistent with the notion that a distinct insulin-sensitive vesicular cargo compartment forms early during fat call differentiation and its formation precedes GLUT4 expression. The development of this compartment may result from the differentiation-dependent inhibition of constitutive GLUT1 and transferrin receptor trafficking such that there is a large increase in, or the new formation of, a population of postendosomal, insulin-responsive vesicles.