HIV exposure and its association with paediatric ICU outcomes in children admitted with severe pneumonia at Chris Hani Baragwanath Academic Hospital, South Africa

Background. Pneumonia is one of the leading causes of under-5 death in South Africa and accounts for a substantial burden of paediatric intensive care unit (PICU) admissions. However, little is known about PICU outcomes in HIV-exposed uninfected (HIV-EU) children with pneumonia, despite the growing size of this vulnerable population. Objectives. To determine whether HIV exposure without infection is an independent risk factor for mortality and morbidity in children admitted to PICU with pneumonia. Methods. This retrospective review included all patients with pneumonia admitted to the PICU at Chris Hani Baragwanath Academic Hospital between 1 January 2013 and 31 December 2014. Patients were classified as HIV-unexposed (HIV-U), HIV-EU and HIV-infected. Medical records were reviewed to determine survival to PICU discharge, duration of PICU admission and duration of mechanical ventilation. Survival analysis was used to determine the association between HIV infection/exposure with mortality, and linear regression was used to examine the association with length of stay and duration of mechanical ventilation. This study included 107 patients: 54 were HIV-U; 28 were HIV-EU; 23 HIV-positive; and 2 had an unknown HIV status. Results. Overall, 84% (n=90) survived to PICU discharge, with no difference in survival based on HIV infection or exposure. Both HIV-EU and HIV-U children had significantly shorter PICU admissions and fewer days of mechanical ventilation compared with HIV-infected children (p=0.011 and p=0.004, respectively). Conclusion. HIV-EU children behaved similarly to HIV-U children in terms of mortality, duration of PICU admission and length of mechanical ventilation. HIV infection was associated with prolonged length of mechanical ventilation and ICU stay but not increased mortality.

Gentamicin-mediated suppression of Hurler syndrome stop mutations restores a low level of alpha-L-iduronidase activity and reduces lysosomal glycosaminoglycan accumulation.

Hurler syndrome is the most severe form of a lysosomal storage disease caused by loss of the enzyme alpha-L-iduronidase (encoded by the IDUA gene), which participates in the degradation of glycosaminoglycans (GAGs) within the lysosome. In some populations, premature stop mutations represent roughly two-thirds of the mutations that cause Hurler syndrome. In this study we investigated whether the aminoglycoside gentamicin can suppress stop mutations within the IDUA gene. We found that a Hurler syndrome fibroblast cell line heterozygous for the IDUA stop mutations Q70X and W402X showed a significant increase in alpha-L-iduronidase activity when cultured in the presence of gentamicin, resulting in the restoration of 2.8% of normal alpha-L-iduronidase activity. Determination of alpha-L-iduronidase protein levels by an immunoquantification assay indicated that gentamicin treatment produced a similar increase in alpha-L-iduronidase protein in Hurler cells. Both the alpha-L-iduronidase activity and protein level resulting from this treatment have previously been correlated with mild Hurler phenotypes. Although Hurler fibroblasts contain a much higher level of GAGs than normal, we found that gentamicin treatment reduced GAG accumulation in Hurler cells to a normal level. We also found that a reduced GAG level could be sustained for at least 2 days after gentamicin treatment was discontinued. The reduction in the GAG level was also reflected in a marked reduction in lysosomal vacuolation. Taken together, these results suggest that the suppression of premature stop mutations may provide an effective treatment for Hurler syndrome patients with premature stop mutations in the IDUA gene.

Aminoglycoside antibiotics mediate context-dependent suppression of termination codons in a mammalian translation system.

The translation machinery recognizes codons that enter the ribosomal A site with remarkable accuracy to ensure that polypeptide synthesis proceeds with a minimum of errors. When a termination codon enters the A site of a eukaryotic ribosome, it is recognized by the release factor eRF1. It has been suggested that the recognition of translation termination signals in these organisms is not limited to a simple trinucleotide codon, but is instead recognized by an extended tetranucleotide termination signal comprised of the stop codon and the first nucleotide that follows. Interestingly, pharmacological agents such as aminoglycoside antibiotics can reduce the efficiency of translation termination by a mechanism that alters this ribosomal proofreading process. This leads to the misincorporation of an amino acid through the pairing of a near-cognate aminoacyl tRNA with the stop codon. To determine whether the sequence context surrounding a stop codon can influence aminoglycoside-mediated suppression of translation termination signals, we developed a series of readthrough constructs that contained different tetranucleotide termination signals, as well as differences in the three bases upstream and downstream of the stop codon. Our results demonstrate that the sequences surrounding a stop codon can play an important role in determining its susceptibility to suppression by aminoglycosides. Furthermore, these distal sequences were found to influence the level of suppression in remarkably distinct ways. These results suggest that the mRNA context influences the suppression of stop codons in response to subtle differences in the conformation of the ribosomal decoding site that result from aminoglycoside binding.

Conserved surface-exposed K/R-X-K/R motifs and net positive charge on poxvirus complement control proteins serve as putative heparin binding sites and contribute to inhibition of molecular interactions with human endothelial cells: a novel mechanism for evasion of host defense.

Vaccinia virus complement control protein (VCP) has been shown to possess the ability to inhibit both classical and alternative complement pathway activation. The newly found ability of this protein to bind to heparin has been shown in previous studies to result in uptake by mast cells, possibly promoting tissue persistence. It has also been shown to reduce chemotactic migration of leukocytes by blocking chemokine binding. In addition, this study shows that VCP-through its ability to bind to glycosaminoglycans (heparin-like molecules) on the surface of human endothelial cells-is able to block antibody binding to surface major histocompatibility complex class I molecules. Since heparin binding is critical for many functions of this protein, we have attempted to characterize the molecular basis for this interaction. Segments of this protein, generated by genetic engineering of the DNA encoding VCP into the Pichia pastoris expression system, were used to localize the regions with heparin binding activity. These regions were then analyzed to more specifically define their properties for binding. It was found that the number of putative binding sites (K/R-X-K/R), the overall positive charge, and the percentage of positively charged amino acids within the protein were responsible for this interaction.

Local neutrophil influx following lateral fluid-percussion brain injury in rats is associated with accumulation of complement activation fragments of the third component (C3) of the complement system.

Traumatic brain injury can lead to locally destructive secondary events mediated by several inflammatory components. Following lateral fluid-percussion (FP) brain injury in rats, we examined cortical and hippocampal sections for neutrophil infiltration and accumulation of complement component C3. Neutrophil influx into the brain after injury was detected by an improved myeloperoxidase (MPO) microassay and manual cell counting, while C3 accumulation was detected using immunocytochemistry. MPO levels were elevated in the injured cortical tissue, whereas C3 immunoreactivity was increased in both injured cortical and ipsilateral hippocampal sections. These results show that the FP model of head injury leads to an intense local inflammatory reaction and subsequent tissue destruction.

A Regression Equation for Determining the Dimensionality of Data.

Parallel analysis has received much support and attention as a criterion for using eigenvalues to determine the dimensionality of data. Parallel analysis compares sample eigenvalues to expected eigenvalues of a sample from a correlation matrix generated by independent normally distributed random variables. To make parallel analysis more accessible to researchers, several studies have proposed multiple regression equations for estimating the expected value of the eigenvalues of a sample correlation matrix assuming that the population correlation matrix is the identity matrix. A new regression equation to estimate the mean value of eigenvalues is presented in this article and a comparative study reveals favorable performance of this proposed equation to previously published regression equations. This proposed technique has the advantage that a table of coefficients, listing regression coefficients for each eigenvalue root, is not needed.

Interaction between a Fab fragment against gp41 of human immunodeficiency virus 1 and its peptide epitope: characterization using a peptide epitope library and molecular modeling.

The molecular interaction of the Fab fragment of the human monoclonal antibody 3D6, directed against the transmembrane protein gp41 of human immunodeficiency virus (HIV) 1, with its peptide epitope is characterized by a panel of overlapping peptides, a peptide epitope library and molecular modeling techniques. The sequence CSGKLICTTAVPW, corresponding to amino acids 605-617 of gp41, was identified as the best binding peptide (KD = 1 x 10(-8) mol/l). This peptide served as a starting point to prepare a cellulose-bound peptide epitope library in which each residue of the epitope is substituted by all L- and D-amino acids, resulting in 494 epitope peptide variants which were subsequently analyzed for binding 3D6. The library was synthesized to identify residues critical for binding and to obtain information about the molecular environment of the epitope peptide bound to 3D6. Both cysteine residues, as well as isoleucine 6, threonine 8 and proline 12, of the epitope were highly sensitive to substitution. Using the data obtained from the epitope characterization, as well as a low-resolution electron density map of a 3D6 Fab-peptide complex, a 3-D model of the Fab-peptide complex was generated by molecular modeling. The modeling experiments predict binding of the peptide, which is cyclized via the two cysteine residues, to a pocket formed dominantly by the hypervariable loops complementarity determining regions CDR3L, CDR2H and CDR3H.

Three-dimensional structure of Schistosoma japonicum glutathione S-transferase fused with a six-amino acid conserved neutralizing epitope of gp41 from HIV.

The 3-dimensional crystal structure of glutathione S-transferase (GST) of Schistosoma japonicum (Sj) fused with a conserved neutralizing epitope on gp41 (glycoprotein, 41 kDa) of human immunodeficiency virus type 1 (HIV-1) (Muster T et al., 1993, J Virol 67:6642-6647) was determined at 2.5 A resolution. The structure of the 3-3 isozyme rat GST of the mu gene class (Ji X, Zhang P, Armstrong RN, Gilliland GL, 1992, Biochemistry 31:10169-10184) was used as a molecular replacement model. The structure consists of a 4-stranded beta-sheet and 3 alpha-helices in domain 1 and 5 alpha-helices in domain 2. The space group of the Sj GST crystal is P4(3)2(1)2, with unit cell dimensions of a = b = 94.7 A, and c = 58.1 A. The crystal has 1 GST monomer per asymmetric unit, and 2 monomers that form an active dimer are related by crystallographic 2-fold symmetry. In the binding site, the ordered structure of reduced glutathione is observed. The gp41 peptide (Glu-Leu-Asp-Lys-Trp-Ala) fused to the C-terminus of Sj GST forms a loop stabilized by symmetry-related GSTs. The Sj GST structure is compared with previously determined GST structures of mammalian gene classes mu, alpha, and pi. Conserved amino acid residues among the 4 GSTs that are important for hydrophobic and hydrophilic interactions for dimer association and glutathione binding are discussed.

Preliminary crystallographic studies of four crystal forms of serum albumin.

Several crystal forms of serum albumin suitable for three-dimensional structure determination have been grown. These forms include crystals of recombinant and wild-type human serum albumin, baboon serum albumin, and canine serum albumin. The intrinsic limits of X-ray diffraction for these crystals are in the range 0.28-0.22 nm. Two of the crystal forms produced from human and canine albumin include incorporated long-chain fatty acids. Molecular replacement experiments have been successfully conducted on each crystal form using the previously determined atomic coordinates of human serum albumin illustrating the conserved tertiary structure.