Distinct Roles of Ser-764 and Lys-773 at the N Terminus of von Willebrand Factor in Complex Assembly with Coagulation Factor VIII

Complex formation between coagulation factor VIII (FVIII) and von Willebrand factor (VWF) is of critical importance to protect FVIII from rapid in vivo clearance and degradation. We have now employed a chemical footprinting approach to identify regions on VWF involved in FVIII binding. To this end, lysine amino acid residues of VWF were chemically modified in the presence of FVIII or activated FVIII, which does not bind VWF. Nano-LC-MS analysis showed that the lysine residues of almost all identified VWF peptides were not differentially modified upon incubation of VWF with FVIII or activated FVIII. However, Lys-773 of peptide Ser-766–Leu-774 was protected from chemical modification in the presence of FVIII. In addition, peptide Ser-764–Arg-782, which comprises the first 19 amino acid residues of mature VWF, showed a differential modification of both Lys-773 and the α-amino group of Ser-764. To verify the role of Lys-773 and the N-terminal Ser-764 in FVIII binding, we employed VWF variants in which either Lys-773 or Ser-764 was replaced with Ala. Surface plasmon resonance analysis and competition studies revealed that VWF(K773A) exhibited reduced binding to FVIII and the FVIII light chain, which harbors the VWF-binding site. In contrast, VWF(S764A) revealed more effective binding to FVIII and the FVIII light chain compared with WT VWF. The results of our study show that the N terminus of VWF is critical for the interaction with FVIII and that Ser-764 and Lys-773 have opposite roles in the binding mechanism.     

A novel harmony search-K means hybrid algorithm for clustering gene expression data

Recent progress in bioinformatics research has led to the accumulation of huge quantities of biological data at various data sources. The DNA microarray technology makes it possible to simultaneously analyze large number of genes across different samples. Clustering of microarray data can reveal the hidden gene expression patterns from large quantities of expression data that in turn offers tremendous possibilities in functional genomics, comparative genomics, disease diagnosis and drug development. The k- ¬means clustering algorithm is widely used for many practical applications. But the original k-¬means algorithm has several drawbacks. It is computationally expensive and generates locally optimal solutions based on the random choice of the initial centroids. Several methods have been proposed in the literature for improving the performance of the k-¬means algorithm. A meta-heuristic optimization algorithm named harmony search helps find out near-global optimal solutions by searching the entire solution space. Low clustering accuracy of the existing algorithms limits their use in many crucial applications of life sciences. In this paper we propose a novel Harmony Search-K means Hybrid (HSKH) algorithm for clustering the gene expression data. Experimental results show that the proposed algorithm produces clusters with better accuracy in comparison with the existing algorithms.     

Lower vascular tone and larger plasma volume in Parkinson's disease with orthostatic hypotension

The pathophysiology of orthostatic hypotension in Parkinson's disease (PD) is incompletely understood. The primary focus has thus far been on failure of the baroreflex, a central mediated vasoconstrictor mechanism. Here, we test the role of two other possible factors: 1) a reduced peripheral vasoconstriction (which may contribute because PD includes a generalized sympathetic denervation); and 2) an inadequate plasma volume (which may explain why plasma volume expansion can manage orthostatic hypotension in PD). We included 11 PD patients with orthostatic hypotension (PD + OH), 14 PD patients without orthostatic hypotension (PD - OH), and 15 age-matched healthy controls. Leg blood flow was examined using duplex ultrasound during 60° head-up tilt. Leg vascular resistance was calculated as the arterial-venous pressure gradient divided by blood flow. In a subset of 9 PD + OH, 9 PD - OH, and 8 controls, plasma volume was determined by indicator dilution method with radiolabeled albumin ((125)I-HSA). The basal leg vascular resistance was significantly lower in PD + OH (0.7 ± 0.3 mmHg·ml(-1)·min) compared with PD - OH (1.3 ± 0.6 mmHg·ml(-1)·min, P < 0.01) and controls (1.3 ± 0.5 mmHg·ml(-1)·min, P < 0.01). Leg vascular resistance increased significantly during 60° head-up tilt with no significant difference between the groups. Plasma volume was significantly larger in PD + OH (3,869 ± 265 ml) compared with PD - OH (3,123 ± 377 ml, P < 0.01) and controls (3,204 ± 537 ml, P < 0.01). These results indicate that PD + OH have a lower basal leg vascular resistance in combination with a larger plasma volume compared with PD - OH and controls. Despite the increase in leg vascular resistance during 60° head-up tilt, PD + OH are unable to maintain their blood pressure.     

Colony variation in Sinorhizobium meliloti inoculant strain U 45

A culture of Sinorhizobium meliloti strain U 45, maintained on yeast extract-mannitol (YM) agar, produced a mixture of Congo red-absorbing (R1) and non-absorbing (W1) colonies when grown on YM medium containing Congo red. The original freeze-dried (FD) culture formed gummy (G), white (W2) and small red (R2) colony types on the above medium. All colonies were stable except G, which segregated into G and W2-like types. Immune diffusion patterns of all colony types were identical. The W1 colony type dominated R1 when a 1:1 combination was sub-cultured on YM agar. The parent cultures and their variants exhibited a range of N₂-fixing effectiveness and competitiveness when inoculated onto two cultivars of Medicago sativa. Variant R2 from the FD culture was ineffective on both cultivars. Genomic DNA fingerprinting with insertion elements ISRm3 and ISRm2011-2 suggested that transposition of these elements was not a cause of variation, but a DNA band was absent in the profiles of two out of three W2-like colonies. Protein profile comparisons showed high similarity (r = 0.98) between the colony types when grown in YM broth. When grown on Tryptone-Yeast extract medium, variants from the FD and agar-maintained cultures formed separate clusters with r = 0.79. Polymerase chain reaction fingerprinting using repetitive, site-directed and arbitrary primers failed to differentiate the variants. The results emphasize the need to monitor culture variability to maintain the quality of legume inoculants.     

Histopathological diagnosis of myocarditis in a dengue outbreak in Sri Lanka, 2009

Background

In 2009, an outbreak of dengue caused high fatality in Sri Lanka. We conducted 5 autopsies of clinically suspected myocarditis cases at the General Hospital, Peradeniya to describe the histopathology of the heart and other organs.

Methods

The diagnosis of dengue was confirmed with specific IgM and IgG ELISA, HAI and RT-PCR techniques. The histology was done in tissue sections stained with hematoxylin and eosin.

Results

Of the 319 cases of dengue fever, 166(52%) had severe infection. Of them, 149 patients (90%) had secondary dengue infection and in 5 patients, DEN-1 was identified as the causative serotype. The clinical diagnosis of myocarditis was considered in 45(27%) patients. The autopsies were done in 5 patients who succumbed to shock (3 females and 2 males) aged 13- 31 years. All had pleural effusions, ascites, bleeding patches in tissue planes and histological evidence of myocarditis. The main histological findings of the heart were interstitial oedema with inflammatory cell infiltration and necrosis of myocardial fibers. One patient had pericarditis. The concurrent pulmonary abnormalities were septal congestion, pulmonary haemorrhage and diffuse alveolar damage; one case showed massive necrosis of liver.

Conclusions

The histology supports occurrence of myocarditis in dengue infection.

     

Infant feeding practices among mildly wasted children: a retrospective study on Nias Island, Indonesia

Background

This study investigated the infant feeding practices of participating mothers who were recruited into a research project aimed at improving the nutritional status of mildly wasted children (< -1.0 to ?? -1.5 Weight-for-Height Z-scores) aged ?? 6 to < 60 months on Nias Island, Indonesia.

Methods

Cross-sectional, questionnaire-based interview of mothers of the index children (n = 215) who were admitted to the community program for mildly wasted children in the study area. Four focus groups and twenty in-depth interviews were conducted to explore further information on infant feeding practices in the study area.

Results

Retrospective results indicated that 6% of the mothers never breastfed. Fifty two percent of mothers initiated breastfeeding within six hours of birth, but 17% discarded colostrum. Exclusive breastfeeding until 6 months of age was practiced by 12%. Seventy-four percent of the mothers offered supplementary liquids besides breast milk within the first 7 days of life, and 14% of infants received these supplementary liquids from 7 days onwards until 6 months of age. Moreover, 79% of the infants were given complementary foods (solid, semi-solid, or soft foods) before 6 months of age. About 9% of the children were breastfed at least two years. Less than one in five of the mildly wasted children (19%) were breastfed on admission to the community program. Qualitative assessments found that inappropriate infant feeding practices were strongly influenced by traditional beliefs of the mothers and paternal grandmothers in the study areas.

Conclusion

Generally, suboptimal infant feeding was widely practiced among mothers of mildly wasted children in the study area on Nias Island, Indonesia. To promote breastfeeding practices among mothers on Nias Island, appropriate nutrition training for community workers and health-nutrition officers is needed to improve relevant counseling skills. In addition, encouraging public nutrition education that promotes breastfeeding, taking into account social-cultural factors such as the influence of paternal grandmothers on infant feeding practice, is needed.     

Virus safety of plasma products using 20 nm instead of 15 nm filtration as virus removing step

During the manufacture of human plasma derivatives, a series of complementary measures are undertaken to prevent transmission of blood-borne viruses. Virus filtration using 15 nm (Planova15N) filters has successfully been implemented in manufacturing processes for various plasma derivatives primarily because virus filtration is a technique, mild for proteins, that can effectively remove even small non-lipid-enveloped viruses, such as HAV and parvovirus B19. However, the use of 15 nm filters has limitations with regard to protein capacity of the filters and the process flow, resulting in an expensive manufacturing step. Therefore, studies were performed to test whether the use of 20 nm (Planova20N) filters, having different characteristics compared to 15 nm filters, can be an alternative for the use of 15 nm filters.It is shown that 20 nm filtration can be an alternative for 15 nm filtration. However, the virus removal capacity of the 20 nm filters depends on the plasma product that is filtered. Therefore, an optimisation study must be performed with regard to process parameters such as pressure, pH and protein concentration for each plasma product. In this study, using optimised conditions, the virus removal capacity of 20 nm filters appears to be comparable or even better when compared to that of 15 nm filters.     

A new late Westphalian fossil marattialean fern from Nova Scotia

Sydneia manleyi gen. et sp. nov. is based on part of a fertile frond from the upper Westphalian D of the Sydney Coalfield, Nova Scotia, Canada. It has small synangia composed of laterally fused sporangia that are elongate and with a circular cross-section. The sporangia yielded variably sized monolete and trilete spores with laevigate and microspinate ornamentation; intermediate forms were also observed. The spores can be correlated with the sporae dispersae species Latosporites minutus , Punctatosporites oculus and Laevigatosporites minimus . Size distribution of the spores is variable and highly skewed, suggesting heterogeneity of the spores within the sporangium. Spore ultrastructure indicates that the fossil is part of a fern, and the morphology of the spores and synangia indicate marattialean affinities.  © 2003 The Linnean Society of London, Botanical Journal of the Linnean Society , 2003, 142 , 199–212.