A new paradigm emerges from the study of de novo mutations in the context of neurodevelopmental disease.

The study of de novo point mutations (new germline mutations arising from the gametes of the parents) remained largely static until the arrival of next-generation sequencing technologies, which made both whole-exome sequencing (WES) and whole-genome sequencing (WGS) feasible in practical terms. Single nucleotide polymorphism genotyping arrays have been used to identify de novo copy-number variants in a number of common neurodevelopmental conditions such as schizophrenia and autism. By contrast, as point mutations and microlesions occurring de novo are refractory to analysis by these microarray-based methods, little was known about either their frequency or impact upon neurodevelopmental disease, until the advent of WES. De novo point mutations have recently been implicated in schizophrenia, autism and mental retardation through the WES of case-parent trios. Taken together, these findings strengthen the hypothesis that the occurrence of de novo mutations could account for the high prevalence of such diseases that are associated with a marked reduction in fecundity. De novo point mutations are also known to be responsible for many sporadic cases of rare dominant mendelian disorders such as Kabuki syndrome, Schinzel-Giedion syndrome and Bohring-Opitz syndrome. These disorders share a common feature in that they are all characterized by intellectual disability. In summary, recent WES studies of neurodevelopmental and neuropsychiatric disease have provided new insights into the role of de novo mutations in these disorders. Our knowledge of de novo mutations is likely to be further accelerated by WGS. However, the collection of case-parent trios will be a prerequisite for such studies. This review aims to discuss recent developments in the study of de novo mutations made possible by technological advances in DNA sequencing.

Integrating next-generation sequencing into the diagnostic testing of inherited cancer predisposition.

The clinical application of next-generation sequencing (NGS) as a diagnostic tool has become increasingly evident. The coupling of NGS technologies with new genomic sequence enrichment methods has made the sequencing of panels of target genes technically feasible, at the same time as making such an approach cost-effective for diagnostic applications. In this article, we discuss recent studies that have applied NGS in the diagnostic setting in relation to hereditary cancer.

Modulation of fibrin clot formation by human serum amyloid P component (SAP) and heparin.

Serum amyloid P-component (SAP) is a normal plasma constituent in man with a circulating concentration of approximately 40 micrograms/ml. Supraphysiological amounts of SAP (150-300 micrograms/ml) have been reported to affect coagulation. We have investigated this further by studying the effect of SAP upon clot times in both the absence and presence of heparin, a suggested ligand for SAP and itself a modulator of coagulation processes. In the absence of heparin, SAP (5-125 micrograms/ml) had no effect on clot times generated by Activated Thrombofax Reagent, brain thromboplastin, Russell's Viper Venom or thrombin when assessed in normal citrated plasma. However, in the presence of amounts of heparin that had only a minor effect upon clot times, SAP (5-40 micrograms/ml) greatly prolonged clot formation, with the thrombin time the most sensitive to SAP. This suggested that the primary effect of SAP was at this distal level of the coagulation pathway. Evaluation by radioimmunoassay revealed that supraphysiological concentrations of SAP (150-300 micrograms/ml) alone reduced by approximately 25% the release of fibrinopeptide A (FPA) from fibrinogen. In the presence of heparin, substantial synergism was observed with maximal reductions of approximately 70% in FPA production requiring only 25-50 micrograms/ml SAP. This inhibition correlated with increased thrombin clot time but was unrelated to any direct modulation in either the activities of anti-thrombin III or activated Factor XIII, and was independent of an alteration in the rate of fibrinolysis. Further, while SAP itself did not interfere with the process of spontaneous fibrin polymerization, in the presence of heparin a prolonged polymerization time (greater than 145%) was observed. We believe that these data reflect the primary mechanisms by which serum amyloid P component influences blood coagulation.

Optimization of the extraction of anthocyanin from Bokbunja (Rubus coreanus Miq.) marc produced during traditional wine processing and characterization of the extracts.

The extraction of anthocyanin from Bokbunja (Rubus coreanus Miq.) marc generated during traditional wine processing was optimized using response surface methodology (RSM). A face-centered cube design (FCD) consisting of 17 experimental runs, including five replicates at the center point, was used to investigate the effects of the three variables (solid-liquid ratio, time, and temperature) on anthocyanin extraction, and the results showed that the relationship between the three variables and the total anthocyanin content followed a quadratic model (R2=0.8853). In addition, the RSM analysis predicted that the optimum conditions for extraction consisted of a solid-liquid ratio of 20, a time of 60min, and a temperature of 60 degrees C. Verification tests performed under these optimum conditions gave 34.7+/-1.4mg/100g of anthocyanin, which was close to predicted value of 37.2mg/100g. Additionally, analysis of water extracts prepared using the predicted optimum conditions revealed that the carbohydrates (sugar and pectin) in Bokbunja marc underwent significant variation toward the formation of by-products (glycerol and uronic acids) during yeast fermentation, and that the amount of anthocyanin produced was reduced 10-fold when compared to the original extraction. Further, the results of HPLC-PDA-MS/MS analysis of the anthocyanins extracted from Bokbunja marc revealed the presence of six anthocyanin components, which were tentatively identified as cyanidin 3-O-sambubioside, cyanidin 3-O-xylosylrutinoside, cyanidin 3-O-rutinoside, pelargonidin 3-O-rutinoside, delphinidin 3-O-rutinoside-?, and delphinidin 3-O-glucuronide.

Characterization of seed oils from fresh Bokbunja (Rubus coreanus Miq.) and wine processing waste.

The physicochemical characteristics, fatty acid (FA) profile, and triacylglyceride (TAG) composition of seed oils from fresh Bokbunja (Rubus coreanus Miq.) fruits and traditional Bokbunja wine processing waste were determined in this study. Oil contents of the fresh seeds and the seeds from wine processing waste were similar, accounting for about 18% of dry weight. The free fatty acid (FFA) content between the two seed oils was significantly different (0.50% for fresh seed oil and 73.14% for wine seed oil). Iodine, conjugated diene, saponification values, and unsaponifiable matter were very similar in the oil samples, but the specific extinction coefficients at 232 and 270 nm of wine seed oil were higher than those of fresh seed oil. Linoleic (C18:2, 50.45-53.18%, L) and linolenic (C18:3, 29.36-33.25%, Ln) acids were the dominant FAs in the two seed oils, whereas oleic (C18:1, 7.32-8.04%, O), palmitic (C16:0, 1.55-1.65%, P), and stearic (C18:0, 0.65-0.68%, S) acids were the minor FAs. LLL, OLL, LLLn, OOL, LLnLn, and OOO were the abundant TAGs, representing >90% of the oils.

Binding affinity of proanthocyanidin from waste Pinus radiata bark onto proline-rich bovine achilles tendon collagen type I.

Binding affinity of proanthocyanidin (PA) purified from Pinus radiata bark waste onto bovine Achilles tendon collagen (type I) was studied. Adsorption of PA onto the collagen was optimized by examining pH, contact time and temperature. The adsorption was pH-dependent. The maximum adsorption capacity (Q(0)) of PA on collagen was found to be 211 mg g(-1) using the Langmuir isotherm. Comparison between two adsorbents also showed that collagen had higher adsorptivity of approximately 20% more than PVPP (polyvinyl polypyrrolidone). The high affinity between PA and collagen was further confirmed in solvent solubility experiments. The observed solvent resistance was thought to be mainly due to a hydrophobic stacking mechanism reinforced by hydrogen bonding. FT-IR spectra clearly indicated the presence of PA adsorbed on collagen. The results have interesting implications that PA can be a good protective agent for collagen against collagenase and other enzymes.

Definition of fibronectin-mediated uptake of gelatinized latex by liver slices and macrophages.

These studies show that both liver slices and macrophages carried out fibronectin concentration-dependent uptake of 125I-labeled gelatin-coated latex (test latex). Lack of phagocytosis of test latex by liver slices was shown directly by electron microscopy and indirectly by trypsin treatment, which caused the release of all test latex taken up in response to fibronectin. Inhibitors of phagocytosis did not alter this uptake. On the other hand, trypsin released only a portion of test latex from macrophages. Inhibitors of phagocytosis did not effect the released radioactive particles from macrophages but greatly reduced the trypsin-resistant radioactivity, taken as representing phagocytized particles. Opsonization of test latex with fibronectin did not require heparin but its association with liver slices occurred only in the presence of heparin. Macrophages, however, readily bound and internalized the opsonized test latex and heparin only potentiated these reactions. Gelatin competed with test latex for fibronectin for opsonization, but did not inhibit binding and phagocytosis of fibronectin-test latex complexes. Finally, soluble fibronectin-gelatin complexes did not compete for binding and phagocytosis of fibronectin-test latex complexes. Thus, fibronectin concentrated on the surface of latex is preferred for interaction with the fibronectin receptor of macrophages. Gelatin, however, was not essential for this reaction, because fibronectin directly coupled to latex was also readily taken up.

Further studies on the modulation of blood coagulation by human serum amyloid P component and its acute phase homologue C-reactive protein.

Serum amyloid P component (SAP), and its acute phase homologue C-reactive protein (CRP), prolonged activated partial thromboplastin times (APTT) in cell free plasma when assayed at physiological concentrations in the presence of heparin. SAP also inhibited clot formation initiated through the extrinsic and terminal phases of coagulation in heparinized cell free plasma, an activity not shared with CRP. When CRP and SAP were similarly evaluated in whole blood using the thromboelastograph (TEG), CRP delayed the onset of coagulation and the initial rate of fibrin formation/polymerization; final clot patency was unaltered. SAP suppressed the anticoagulant activity of heparin in the TEG assay, unlike results obtained in heparinized cell free plasma, by facilitating a more rapid onset of coagulation, increasing the rate of fibrin formation/polymerization, and correcting clot patency. The data provided offer further evidence that these homologues can intercede in blood coagulation.

Spontaneous contrast in the pericardial sac caused by gas-forming organisms.

Purulent pericarditis is a life-threatening disease associated with a variety of microorganisms. The case presented herein has unusual manifestations of contrast echoes on two-dimensional echocardiography. To our knowledge, this is the first reported case of pericarditis caused by gas-forming group D Streptococcus and Klebsiella pneumoniae with pyopericardium, resulting in contrast echoes on the two-dimensional echocardiographic image.

Meningoencephalitis, myocarditis and disseminated intravascular coagulation in a patient with scrub typhus.

A 21-year-old male soldier was admitted due to a sore throat, headache, generalized lymphadenopathy and persistent fever for 12 days. Despite empirical antibiotic treatment for four days at a clinic prior to admission, he continued to have persistent abdominal pain over his right upper quadrant region and progressive jaundice was followed by shock. After admission, he developed an episode of clonic seizures and became delirious and agitated. An electrocardiogram showed first degree atrioventricular (AV) block and non-specific ST-T wave changes. Hematological studies revealed thrombocytopenia, hypofibrinogenemia, abnormal partial thromboplastin time (PTT) and a positive test for D-dimer. The cerebrospinal fluid analysis showed pleocytosis with white cells of 84/mm3 with a lymphocyte predominance, protein of 97 mg/dL and glucose of 79 mg/dL. Indirect immunofluorescence assay showed a fourfold rise in antibodies to Orientia tsutsugamushi in paired serum with IgM antibody titer of 1:640. The patient had a favorable response after parenteral chloramphenicol in addition to oral tetracycline. Early ricognition of scrub typhus and early prescription of anti-rickettsial agents prevent complications of central nervous system involvement and further deterioration of cardiac and hematological function.