Denaturing HPLC-based approach for detection of COL7A1 gene mutations causing dystrophic epidermolysis bullosa

Dystrophic epidermolysis bullosa (DEB) is a rare clinically heterogeneous genodermatosis due to genetic defects in type VII collagen gene (COL7A1). Identification of COL7A1 mutations is a challenge since this gene comprises 118 exons and more than 300 mutations scattered over the gene have been reported. Here, we describe for the first time the use of denaturing high performance liquid chromatography (DHPLC) for COL7A1 mutation detection. To validate the method, exon-specific DHPLC conditions were applied to screen DNA samples from patients carrying known COL7A1 mutations. Abnormal DHPLC profiles were obtained for all known mutations. Subsequent DHPLC analysis of 17 DEB families of unknown genotype allowed the identification of 21 distinct mutations, 9 of which were novel. The DHPLC mutation detection rate was significantly higher compared with our mutation scanning rate with conventional techniques (97% vs 86%), indicating DHPLC as the method of choice for COL7A1 molecular characterization in DEB patients.     

Glucokinase gene mutations: structural and genotype-phenotype analyses in MODY children from South Italy

Background

Maturity onset diabetes of the young type 2 (or GCK MODY) is a genetic form of diabetes mellitus provoked by mutations in the glucokinase gene (GCK).

Methodology/Principal Findings

We screened the GCK gene by direct sequencing in 30 patients from South Italy with suspected MODY. The mutation-induced structural alterations in the protein were analyzed by molecular modeling. The patients'' biochemical, clinical and anamnestic data were obtained. Mutations were detected in 16/30 patients (53%); 9 of the 12 mutations identified were novel (p.Glu70Asp, p.Phe123Leu, p.Asp132Asn, p.His137Asp, p.Gly162Asp, p.Thr168Ala, p.Arg392Ser, p.Glu290X, p.Gln106_Met107delinsLeu) and are in regions involved in structural rearrangements required for catalysis. The prevalence of mutation sites was higher in the small domain (7/12: ∼59%) than in the large (4/12: 33%) domain or in the connection (1/12: 8%) region of the protein. Mild diabetic phenotypes were detected in almost all patients [mean (SD) OGTT = 7.8 mMol/L (1.8)] and mean triglyceride levels were lower in mutated than in unmutated GCK patients (p = 0.04).

Conclusions

The prevalence of GCK MODY is high in southern Italy, and the GCK small domain is a hot spot for MODY mutations. Both the severity of the GCK mutation and the genetic background seem to play a relevant role in the GCK MODY phenotype. Indeed, a partial genotype-phenotype correlation was identified in related patients (3 pairs of siblings) but not in two unrelated children bearing the same mutation. Thus, the molecular approach allows the physician to confirm the diagnosis and to predict severity of the mutation.     

Diffusione pollinica e caratteristiche immunologiche e broncoreattive di soggetti atopici in Pietra Ligure (Savona)

Riassunto Sono state rilevate durante un monitoraggio aerobiologico effettuato a Pietra Ligure (Savona) nel corso del 1987 le concentrazioni polliniche di 50 taxa ed è stata valutata l'influenza relativa dei fattori meteorologici. Le osservazioni palinologiche sono state rapportate alle concentrazioni sieriche delle IgE specifiche, alla reattività bronchiale specifica ed aspecifica valutate in 101 pazienti allergici (rinitici ed asmatici), sensibilizzati a Graminaceae ed Urticaceae (Parietaria) al fine di riconoscere correlazioni tra le caratteristiche aerobiologiche di questi allergeni edi meccanismi patogenetici che sostengono la reattività bronchiale.      

Plasma Free Hemoglobin and Microcirculatory Response to Fresh or Old Blood Transfusions in Sepsis

BackgroundFree hemoglobin (fHb) may induce vasoconstriction by scavenging nitric oxide. It may increase in older blood units due to storage lesions. This study evaluated whether old red blood cell transfusion increases plasma fHb in sepsis and how the microvascular response may be affected.MethodsThis is a secondary analysis of a randomized study. Twenty adult septic patients received either fresh or old (<10 or >15 days storage, respectively) RBC transfusions. fHb was measured in RBC units and in the plasma before and 1 hour after transfusion. Simultaneously, the sublingual microcirculation was assessed with sidestream-dark field imaging. The perfused boundary region was calculated as an index of glycocalyx damage. Tissue oxygen saturation (StO₂) and Hb index (THI) were measured with near-infrared spectroscopy and a vascular occlusion test was performed.ResultsSimilar fHb levels were found in the supernatant of fresh and old RBC units. Despite this, plasma fHb increased in the old RBC group after transfusion (from 0.125 [0.098–0.219] mg/mL to 0.238 [0.163–0.369] mg/mL, p = 0.006). The sublingual microcirculation was unaltered in both groups, while THI increased. The change in plasma fHb was inversely correlated with the changes in total vessel density (r = -0.57 [95% confidence interval -0.82, -0.16], p = 0.008), De Backer score (r = -0.63 [95% confidence interval -0.84, -0.25], p = 0.003) and THI (r = -0.72 [95% confidence interval -0.88, -0.39], p = 0.0003).ConclusionsOld RBC transfusion was associated with an increase in plasma fHb in septic patients. Increasing plasma fHb levels were associated with decreased microvascular density.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01584999","term_id":"NCT01584999"}}NCT01584999     

Effects of tunable excitation in carotenoids explained by the vibrational energy relaxation approach

Carotenoids are fundamental building blocks of natural light harvesters with convoluted and ultrafast energy deactivation networks. In order to disentangle such complex relaxation dynamics, several studies focused on transient absorption measurements and their dependence on the pump wavelength. However, such findings are inconclusive and sometimes contradictory. In this study, we compare internal conversion dynamics in \(\beta\)-carotene, pumped at the first, second, and third vibronic progression peak. Instead of employing data fitting algorithms based on global analysis of the transient absorption spectra, we apply a fully quantum mechanical model to treat the high-frequency symmetric carbon–carbon (C=C and C–C) stretching modes explicitly. This model successfully describes observed population dynamics as well as spectral line shapes in their time-dependence and allows us to reach two conclusions: Firstly, the broadening of the induced absorption upon excess excitation is an effect of vibrational cooling in the first excited state (\(S_{1}\)). Secondly, the internal conversion rate between the second excited state (\(S_{2}\)) and \(S_{1}\) crucially depends on the relative curve displacement. The latter point serves as a new perspective on solvent- and excitation wavelength-dependent experiments and lifts contradictions between several studies found in literature.     

Persistence of experimental Rocio virus infection in the golden hamster (Mesocricetus auratus)

Rocio virus (ROCV) is an encephalitic flavivirus endemic to Brazil. Experimental flavivirus infections have previously demonstrated a persistent infection and, in this study, we investigated the persistence of ROCV infection in golden hamsters (Mesocricetus auratus). The hamsters were infected intraperitoneally with 9.8 LD50/0.02 mL of ROCV and later anaesthetised and sacrificed at various time points over a 120-day period to collect of blood, urine and organ samples. The viral titres were quantified by real-time-polymerase chain reaction (qRT-PCR). The specimens were used to infect Vero cells and ROCV antigens in the cells were detected by immunefluorescence assay. The levels of antibodies were determined by the haemagglutination inhibition technique. A histopathological examination was performed on the tissues by staining with haematoxylin-eosin and detecting viral antigens by immunohistochemistry (IHC). ROCV induced a strong immune response and was pathogenic in hamsters through neuroinvasion. ROCV was recovered from Vero cells exposed to samples from the viscera, brain, blood, serum and urine and was detected by qRT-PCR in the brain, liver and blood for three months after infection. ROCV induced histopathological changes and the expression of viral antigens, which were detected by IHC in the liver, kidney, lung and brain up to four months after infection. These findings show that ROCV is pathogenic to golden hamsters and has the capacity to cause persistent infection in animals after intraperitoneal infection.     

Proteomic analysis of mouse growth plate cartilage

Cartilage is a highly specialized load-bearing tissue with a small number of cells and a high proportion of extracellular matrix (ECM). The abundance of heavily sulfated proteoglycans and a poorly soluble collagenous ECM presents a major technical challenge to 2-DE. Here we report proteomic analysis of mouse growth plate cartilage using novel methodology for tissue dissection and sample prefractionation. We have successfully resolved cartilage tissue extracts by 2-DE for the first time and identified cartilage ECM proteins by Western blotting and MS/MS.     

Admixture mapping of an allele affecting interleukin 6 soluble receptor and interleukin 6 levels

Circulating levels of inflammatory markers can predict cardiovascular disease risk. To identify genes influencing the levels of these markers, we genotyped 1,343 single-nucleotide polymorphisms (SNPs) in 1,184 African Americans from the Health, Aging and Body Composition (Health ABC) Study. Using admixture mapping, we found a significant association of interleukin 6 soluble receptor (IL-6 SR) with European ancestry on chromosome 1 (LOD 4.59), in a region that includes the gene for this receptor (IL-6R). Genotyping 19 SNPs showed that the effect is largely explained by an allele at 4% frequency in West Africans and at 35% frequency in European Americans, first described as associated with IL-6 SR in a Japanese cohort. We replicate this association (P<1.0x10-12) and also demonstrate a new association with circulating levels of a different molecule, IL-6 (P<3.4x10-5). After replication in 1,674 European Americans from Health ABC, the combined result is even more significant: P<1.0x10-12 for IL-6 SR, and P<2.0x10-9 for IL-6. These results also serve as an important proof of principle, showing that admixture mapping can not only coarsely localize but can also fine map a phenotypically important variant.     

Acute beta-adrenergic overload produces myocyte damage through calcium leakage from the ryanodine receptor 2 but spares cardiac stem cells

A hyperadrenergic state is a seminal aspect of chronic heart failure. Also, "Takotsubo stress cardiomyopathy," is associated with increased plasma catecholamine levels. The mechanisms of myocyte damage secondary to excess catecholamine exposure as well as the consequence of this neurohumoral burst on cardiac stem cells (CSCs) are unknown. Cardiomyocytes and CSCs were exposed to high doses of isoproterenol (ISO), in vivo and in vitro. Male Wistar rats received a single injection of ISO (5 mg kg-1) and were sacrificed 1, 3, and 6 days later. In comparison with controls, LV function was impaired in rats 1 day after ISO and started to improve at 3 days. The fraction of dead myocytes peaked 1 day after ISO and decreased thereafter. ISO administration resulted in significant ryanodine receptor 2 (RyR2) hyperphosphorylation and RyR2-calstabin dissociation. JTV519, a RyR2 stabilizer, prevented the ISO-induced death of adult myocytes in vitro. In contrast, CSCs were resistant to the acute neurohumoral overload. Indeed, CSCs expressed a decreased and inverted complement of beta1/beta2-adrenoreceptors and absence of RyR2, which may explain their survival to ISO insult. Thus, a single injection of ISO causes diffuse myocyte death through Ca2+ leakage secondary to the acutely dysfunctional RyR2. CSCs are resistant to the noxious effects of an acute hyperadrenergic state and through their activation participate in the response to the ISO-induced myocardial injury. The latter could contribute to the ability of the myocardium to rapidly recover from acute hyperadrenergic damage.     

Isolation and characterization of heterotepalins, type 1 ribosome-inactivating proteins from Phytolacca heterotepala leaves

Leaves from Phytolacca heterotepala H. Walter (Mexican pokeweed) contain at least 10 type 1 RIP isoforms, named heterotepalins. Their Mr values are included in the range 28,000-36,000, as shown by SDS-PAGE performed under reduced conditions and the pI values in the pH range 8.50-9.50. Some heterotepalins are glycosylated. ESI-QTOF mass spectrometry provides the accurate Mr of heterotepalin 4 (29,326.00) and heterotepalin 5b (30,477.00), two isoforms purified to homogeneity by conventional chromatographic techniques. The N-terminal sequences up to residue 35, show that heterotepalins exhibit an high percentage identity with other type 1 RIPs isolated from Phytolaccaceae. Some heterotepalins cross-react with antisera raised against RIPs isolated from Phytolacca dioica leaves. The complete amino acid sequence of heterotepalin 4 matches that of Phytolacca heterotepala anti-viral protein PAP (RIP1), deduced from the cDNA sequence of PhRIP1 gene (AC: AY327475), with one exception concerning residue 245 which, in the native protein, is Ile instead of Met. This substitution, found by mass spectrometry mapping, has been directly confirmed by Edman degradation sequencing of the C-terminal tryptic peptide 242-262. The results show the high potential of mass spectrometry and Edman degradation to verify and to uncover possible amino acid substitutions between native proteins and their cDNA deduced sequences.