Automated error-tolerant macromolecular structure determination from multidimensional nuclear Overhauser enhancement spectra and chemical shift assignments: improved robustness and performance of the PASD algorithm

We report substantial improvements to the previously introduced automated NOE assignment and structure determination protocol known as PASD (Kuszewski et al. (2004) J Am Chem Soc 26:6258-6273). The improved protocol includes extensive analysis of input spectral data to create a low-resolution contact map of residues expected to be close in space. This map is used to obtain reasonable initial guesses of NOE assignment likelihoods which are refined during subsequent structure calculations. Information in the contact map about which residues are predicted to not be close in space is applied via conservative repulsive distance restraints which are used in early phases of the structure calculations. In comparison with the previous protocol, the new protocol requires significantly less computation time. We show results of running the new PASD protocol on six proteins and demonstrate that useful assignment and structural information is extracted on proteins of more than 220 residues. We show that useful assignment information can be obtained even in the case in which a unique structure cannot be determined.     

Use of the EM algorithm to detect QTL affecting multiple-traits in an across half-sib family analysis

QTL detection experiments in livestock species commonly use the half-sib design. Each male is mated to a number of females, each female producing a limited number of progeny. Analysis consists of attempting to detect associations between phenotype and genotype measured on the progeny. When family sizes are limiting experimenters may wish to incorporate as much information as possible into a single analysis. However, combining information across sires is problematic because of incomplete linkage disequilibrium between the markers and the QTL in the population. This study describes formulæ for obtaining MLEs via the expectation maximization (EM) algorithm for use in a multiple-trait, multiple-family analysis. A model specifying a QTL with only two alleles, and a common within sire error variance is assumed. Compared to single-family analyses, power can be improved up to fourfold with multi-family analyses. The accuracy and precision of QTL location estimates are also substantially improved. With small family sizes, the multi-family, multi-trait analyses reduce substantially, but not totally remove, biases in QTL effect estimates. In situations where multiple QTL alleles are segregating the multi-family analysis will average out the effects of the different QTL alleles.     

Design and properties of N(CCG)-gp41, a chimeric gp41 molecule with nanomolar HIV fusion inhibitory activity.

The design and characterization of a chimeric protein, termed N(CCG)-gp41, derived from the ectodomain of human immunodeficiency virus (HIV), type I gp41 is described. N(CCG)-gp41 features an exposed trimeric coiled-coil comprising the N-terminal helices of the gp41 ectodomain. The trimeric coiled-coil is stabilized both by fusion to a minimal thermostable ectodomain of gp41 and by engineered intersubunit disulfide bonds. N(CCG)-gp41 is shown to inhibit HIV envelope-mediated cell fusion at nanomolar concentrations with an IC(50) of 16.1 +/- 2.8 nm. It is proposed that N(CCG)-gp41 targets the exposed C-terminal region of the gp41 ectodomain in its pre-hairpin intermediate state, thereby preventing the formation of the fusogenic form of the gp41 ectodomain, which comprises a highly stable trimer of hairpins arranged in a six-helix bundle. N(CCG)-gp41 has potential as a therapeutic agent for the direct inhibition of HIV cell entry, as an anti-HIV vaccine, and as a component of a rapid throughput assay for screening for small molecule inhibitors of HIV envelope-mediated cell fusion. It is anticipated that antibodies raised against N(CCG)-gp41 may target the trimeric coiled-coil of N-terminal helices of the gp41 ectodomain that is exposed in the pre-hairpin intermediate state in a manner analogous to peptides derived from the C-terminal helix of gp41 that are currently in clinical trials.     

Health and chemical burdens of fish species from polluted and hyper-eutrophic freshwater ecosystems in South Africa

Three aquatic ecosystems in South Africa, the Hartbeespoort, Klipvoor and Bospoort Dams, are classified as hyper-eutrophic, because of high nutrient loads and chemical pollution. Water and two fish species, Clarias gariepinus and Cyprinus carpio, were collected from these dams to assess the impact of eutrophication and chemical pollutants on their health status. Water and muscle samples were analysed for organic and inorganic chemicals. Condition factor was determined and a necropsy performed to note any macroscopic abnormalities. A histology-based fish health assessment was done on the liver, kidney, gills and gonads. A number of fish from the three dams exhibited livers with fatty change and focal discoloration, skin lesions and parasites within the visceral cavity. The prevalence and severity of histopathology in the liver resulted in higher liver index values than the index values for kidneys and gills. Aluminium, silicon and chromium were detected in the water and muscle tissue. The DDT metabolite p,p’-DDE was present in both species, as well as in fish from the reference site, Marico-Bosveld Dam. Only C. gariepinus from Hartbeespoort Dam had p,p’-DDD levels higher than 5 µg g^?1 per edible portion. Water from hyper-eutrophic dams adversely affects the health of freshwater fish.     

Non-secretory solitary plasmacytoma of the spleen

A case of primary nonsecretory plasmacytoma of the spleen is reported. On laparotomy and splenectomy a 920 g spleen was removed, measuring 16×14×6 cm. The cut surface of the entire spleen showed that the tumour occupied most of the splenic tissue. A bone marrow aspirate and trephine, skeletal survey showed no signs of myeloma. Biopsy of the liver and regional lymph nodes was normal. Immunocytochemistry of the splenic tumour showed positivity for pan-B and plasma cell markers. After splenectomy the patient was treated with chemotherapy according to protocol VBCMP (M2).     

Association of genetic variants rs641153 (CFB), rs2230199 (C3), and rs1410996 (CFH) with age-related macular degeneration in a Brazilian population

This study aimed to investigate the association among genetic variants of the complement pathway CFB R32Q (rs641153), C3 R102G (rs2230199), and CFH (rs1410996) with age-related macular degeneration (AMD) in a sample of the Brazilian population. In a case-control study, 484 AMD patients were classified according to the clinical age-related maculopathy grading system (CARMS) and compared to 479 unrelated controls. The genetic variants rs1410996 of complement H (CFH), rs641153 of complement factor B (CFB), and rs2230199 of complement 3 (C3) were evaluated through polymerase chain reaction (PCR) and direct sequencing. The associations between single nucleotide polymorphisms (SNPs) and AMD, adjusted by age, were assessed by using logistic regression models. A statistically significant association was observed between AMD risk and rs2230199 variant with an OR of 2.01 (P  = 0.0002) for CG individuals compared to CC individuals. Regarding the comparison of advanced AMD versus the control group, the OR was 2.12 (P = 0.0036) for GG versus AA genotypes for rs1410996 variant. Similarly, the OR for rs2230199 polymorphism was 2.3034 (P  = 5.47^e-05) when comparing CG individuals to CC carriers. In contrast, the rs641153 variant showed a significant protective effect against advanced AMD for GA versus GG genotype (OR = 0.4406; P  = 0.0019). When comparing wet AMD versus controls, a significant association was detected for rs1410996 variant (OR = 2.16; P  = 0.0039) comparing carriers of the homozygous GG versus AA genotype, as well as in the comparisons of GG (OR = 3.0713; P  = 0.0046) and CG genotypes (OR = 2.2249; P  = 0.0002) versus CC genotype for rs2230199 variant, respectively. The rs641153 variant granted a significant protective effect against wet AMD for GA versus GG genotypes (OR = 0.4601; P  = 0.0044). Our study confirmed the risk association between rs2230199 and rs1410996 variants and AMD, and the protective role against AMD for rs641153 variant.     

No difference in between-country variability in use of newly approved orphan and non- orphan medicinal products - a pilot study

Background

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease, which rapidly leads to chronic respiratory failure requiring mechanical ventilation. Currently, forced vital capacity (FVC) < 50% is considered as physiologic marker for admitting patients to Noninvasive Positive Pressure Ventilation (NPPV) intervention, although it has been recently shown the median survival of patients with baseline FVC < 75% much shorter than median survival of patients with baseline FVC > 75%, independently by any treatment.

Aim

To assess the role of NPPV in improving outcome of ALS, a retrospective analysis was performed to investigate 1 year survival of ALS patients with FVC < 75% and nocturnal respiratory insufficiency, treated with NPPV, compared to a well-matched population of ALS patients, who refused or was intolerant to NPPV.

Methods

We investigated seventy-two consecutive ALS patients who underwent pulmonary function test. Forty-four presented a FVC > 75% and served as control group. Twenty-eight patients presented a FVC < 75% and showed, at polysomnography analysis, nocturnal respiratory insufficiency, requiring NPPV; sixteen were treated with NPPV, while twelve refused or were intolerant.

Results

Increased survival rate at 1 year in patients with FVC < 75% treated with NPPV, as compared to those who refused or could not tolerate NPPV (p = 0.02), was observed. The median rate of decline in FVC% was slower in NPPV patients than in patients who did not use NPPV (95% CI: 0.72 to 1.85; p < 0.0001).

Conclusion

This report demonstrates that early treatment with NPPV prolongs survival and reduces decline of FVC% in ALS.