GRACILE syndrome,a lethal metabolic disorder with iron overload,is caused by a point mutation in BCS1L

GRACILE (growth retardation, aminoaciduria, cholestasis, iron overload, lactacidosis, and early death) syndrome is a recessively inherited lethal disease characterized by fetal growth retardation, lactic acidosis, aminoaciduria, cholestasis, and abnormalities in iron metabolism. We previously localized the causative gene to a 1.5-cM region on chromosome 2q33-37. In the present study, we report the molecular defect causing this metabolic disorder, by identifying a homozygous missense mutation that results in an S78G amino acid change in the BCS1L gene in Finnish patients with GRACILE syndrome, as well as five different mutations in three British infants. BCS1L, a mitochondrial inner-membrane protein, is a chaperone necessary for the assembly of mitochondrial respiratory chain complex III. Pulse-chase experiments performed in COS-1 cells indicated that the S78G amino acid change results in instability of the polypeptide, and yeast complementation studies revealed a functional defect in the mutated BCS1L protein. Four different mutations in the BCS1L gene have been reported elsewhere, in Turkish patients with a distinctly different phenotype. Interestingly, the British and Turkish patients had complex III deficiency, whereas in the Finnish patients with GRACILE syndrome complex III activity was within the normal range, implying that BCS1L has another cellular function that is uncharacterized but essential and is putatively involved in iron metabolism.     

Denaturation of either Manduca sexta aminopeptidase N or Bacillus thuringiensis Cry1A toxins exposes binding epitopes hidden under nondenaturing conditions

The effect of polypeptide denaturation of Bacillus thuringiensis Cry1A toxins or purified Manduca sexta 120-kDa aminopeptidase N on the specificities of their interactions was investigated. Ligand and dot blotting experiments were conducted with (125)I-labeled Cry1Ac, Cry1Ac mutant (509)QNR-AAA(511) (QNR-AAA), or 120-kDa aminopeptidase N as the probe. Mutant QNR-AAA does not bind the N-acetylgalactosamine moiety on the 120-kDa aminopeptidase. Both (125)I-Cry1Ac and (125)I-QNR-AAA bound to 210- and 120-kDa proteins from M. sexta brush border membrane vesicles and purified 120-kDa aminopeptidase N on ligand blots. However, on dot blots (125)I-QNR-AAA bound brush border vesicles but did not bind purified aminopeptidase except when aminopeptidase was denatured. In the reciprocal experiment, (125)I-aminopeptidase bound Cry1Ac but did not bind QNR-AAA. (125)I-aminopeptidase bound Cry1Ab to a limited extent but not the Cry1Ab domain I mutant Y153D or Cry1Ca. However, denatured (125)I-aminopeptidase detected each Cry1A toxin and mutant but not Cry1Ca on dot blots. The same pattern of recognition occurred with native (nondenatured) (125)I-aminopeptidase probe and denatured toxins as the targets. The broader pattern of toxin-binding protein interaction is probably due to peptide sequences being exposed upon denaturation. Putative Cry toxin-binding proteins identified by the ligand blot technique need to be investigated under native conditions early in the process of identifying binding proteins that may serve as functional toxin receptors.     

Oxanthrone esters from the roots of Cassia kleinii

From the roots of Cassia kleinii two new oxanthrone esters, kleinioxanthrone-3 and kleinioxanthrone-4 have been isolated. Their structures were established as 1,8-dihydroxy-3-methyl-9(10H)-anthracenone-10-oxyhexadecanoate 3 and 2,6,7-trihydroxy-1,8-dimethoxy-3-methyl-9(10H)-anthracenone-10-oxydecanoate 4 respectively based on degradative and spectroscopic evidence.     

Vibrio cholerae O139 Bengal: odyssey of a fortuitous variant

Vibrio cholerae O139, the new serogroup associated with epidemic cholera, came into being in the second half of the year 1992 in an explosive fashion and was responsible for several outbreaks in India and other neighbouring countries. This was an unprecedented event in the history of cholera and the genesis of the O139 serogroup was, at that time, thought to be the beginning of the next or the eighth pandemic of cholera. However, with the passage of time, the O1 serogroup of the El Tor biotype again reappeared and displaced the O139 serogroup on the Indian subcontinent, and there was a feeling among cholera workers that the appearance of this new serogroup may have been a one-time event. The resurgence of the O139 serogroup in September 1996 in Calcutta and the coexistence of both the O1 and O139 serogroups in much of the cholera endemic areas in India and elsewhere, suggested that the O139 serogroup has come to stay and is a permanent entity to contend with in the coming years. During the past 10 years, intensive work on all aspects of the O139 serogroup was carried out by cholera researchers around the world. The salient findings on this serogroup over the past 10 years pertinent to its prevalence, clinico-epidemiological features, virulence-associated genes, rapid screening and identification, molecular epidemiology, and vaccine developments have been highlighted.     

Characterization of the pollen growth transition in self-incompatible <Emphasis Type="Italic">Petunia inflata</Emphasis>

In Petunia inflata, as in other species that shed bicellular pollen, early pollen tube growth in the pistil is slow, then increases 2- to 5-fold depending on the genotype of the female parent. We refer to the time point at which pollen tubes enter the accelerated phase of growth as the pollen growth transition (PGT). Here, we present evidence that pre-PGT and post-PGT growth are quantitatively and qualitatively different, and that the PGT is triggered when pollen tubes reach the transition zone (TZ) below the stigma. The capacity of various pistil zones to precipitate the PGT was tested through 'stump' pollinations: varying lengths of the pistil apex were excised, the cut surface of the remaining pistil (the stump) coated with stigmatic exudates then dusted with compatible pollen. Pollen applied to TZ tissues entered the PGT earlier than pollen growing in intact control pistils; the PGT was delayed in stylar stumps, largely because of delayed germination and reduced pre-PGT growth. In immature pistils, the PGT was delayed by several hours relative to its onset in mature pistils. The PGT fails to occur in pollen cultured in vitro. Collectively, the data suggest that pollen tubes become competent to enter the PGT when they reach a critical size, but the physicochemical environment of the transmitting tissue is necessary for triggering the cellular changes that result in accelerated growth. An analysis of the distribution of pollen tube tips before and after the PGT suggests that pollen competition is most intense during the pre-PGT phase.     

Impedance Spectroscopy in Frost Hardiness Evaluation of Rhododendron Leaves

Impedance spectroscopy was used in studying frost hardinessof leaves of two diploid rhododendron cultivars, RhododendronL. ‘PJM’ and R. ‘Cunningham's White’,and their tetraploid derivatives, R. ‘Northern Starburst’(NSB) and CW4. After the growing season and initial hardeningin a greenhouse, plants were subjected to an acclimation regimein a phytotron: 3 consecutive weeks at +5, +1 and -2°C each.Hardiness was studied with controlled freezing tests beforeeach decrease in temperature and at the end of the experiment,based on data of extracellular resistance r_eand relaxation time of the frost-exposed leaves. The correlation of the two estimateswas 0.92. Generally, the diploid clones had better frost hardinessthan the tetraploid clones. At the end of the experiment, frosthardiness of the diploid ‘PJM’ was -28.7°C andthat of the tetraploid NSB -20.6°C. Leaves of the diploid‘Cunningham's White’ and of the tetraploid CW4 hardenedto -32.0°C and -20.9°C, respectively. Frost hardinessestimated by impedance spectroscopy correlated well with earlierresults based on visual scoring (r = 0.81–0.86) and electrolyteleakage tests (r = 0.84–0.90), but results from impedancespectroscopy indicated weaker hardiness than the other tests.The difference between the results from impedance spectroscopyand the other tests was smaller and more coherent within the‘Cunningham's White’ clones than within ‘PJM’and NSB. Changes in extracellular and intracellular resistanceof non-frozen leaves during the acclimation correlated withthe changes in frost hardiness of ‘Cunningham's White’clones, but not with those of ‘PJM’ and NSB, whichbelong to another subspecies.Copyright 2000 Annals of BotanyCompany Cold resistance, evergreen, frost hardiness, impedance spectroscopy, polyploid, Rhododendron, tetraploid     

A Genome-Wide Association Study of a Biomarker of Nicotine Metabolism

Individuals with fast nicotine metabolism typically smoke more and thus have a greater risk for smoking-induced diseases. Further, the efficacy of smoking cessation pharmacotherapy is dependent on the rate of nicotine metabolism. Our objective was to use nicotine metabolite ratio (NMR), an established biomarker of nicotine metabolism rate, in a genome-wide association study (GWAS) to identify novel genetic variants influencing nicotine metabolism. A heritability estimate of 0.81 (95% CI 0.70–0.88) was obtained for NMR using monozygotic and dizygotic twins of the FinnTwin cohort. We performed a GWAS in cotinine-verified current smokers of three Finnish cohorts (FinnTwin, Young Finns Study, FINRISK2007), followed by a meta-analysis of 1518 subjects, and annotated the genome-wide significant SNPs with methylation quantitative loci (meQTL) analyses. We detected association on 19q13 with 719 SNPs exceeding genome-wide significance within a 4.2 Mb region. The strongest evidence for association emerged for CYP2A6 (min p = 5.77E-86, in intron 4), the main metabolic enzyme for nicotine. Other interesting genes with genome-wide significant signals included CYP2B6, CYP2A7, EGLN2, and NUMBL. Conditional analyses revealed three independent signals on 19q13, all located within or in the immediate vicinity of CYP2A6. A genetic risk score constructed using the independent signals showed association with smoking quantity (p = 0.0019) in two independent Finnish samples. Our meQTL results showed that methylation values of 16 CpG sites within the region are affected by genotypes of the genome-wide significant SNPs, and according to causal inference test, for some of the SNPs the effect on NMR is mediated through methylation. To our knowledge, this is the first GWAS on NMR. Our results enclose three independent novel signals on 19q13.2. The detected CYP2A6 variants explain a strikingly large fraction of variance (up to 31%) in NMR in these study samples. Further, we provide evidence for plausible epigenetic mechanisms influencing NMR.     

Vaccination with Human Papillomavirus Pseudovirus-Encapsidated Plasmids Targeted to Skin Using Microneedles

Human papilloma virus-like particles (HPV VLP) serve as the basis of the current licensed vaccines for HPV. We have previously shown that encapsidation of DNA expressing the model antigen M/M2 from respiratory syncytial virus (RSV) in HPV pseudovirions (PsV) is immunogenic when delivered intravaginally. Because the HPV capsids confer tropism for basal epithelium, they represent attractive carriers for vaccination targeted to the skin using microneedles. In this study we asked: 1) whether HPV16 VLP administered by microneedles could induce protective immune responses to HPV16 and 2) whether HPV16 PsV-encapsidated plasmids delivered by microneedles could elicit immune responses to both HPV and the antigen delivered by the transgene. Mice immunized with HPV16 VLP coated microneedles generated robust neutralizing antibody responses and were protected from HPV16 challenge. Microneedle arrays coated with HPV16-M/M2 or HPV16-F protein (genes of RSV) were then tested and dose-dependent HPV and F-specific antibody responses were detected post-immunization, and M/M2-specific T-cell responses were detected post RSV challenge, respectively. HPV16 PsV-F immunized mice were fully protected from challenge with HPV16 PsV and had reduced RSV viral load in lung and nose upon intranasal RSV challenge. In summary, HPV16 PsV-encapsidated DNA delivered by microneedles induced neutralizing antibody responses against HPV and primed for antibody and T-cell responses to RSV antigens encoded by the encapsidated plasmids. Although the immunogenicity of the DNA component was just above the dose response threshold, the HPV-specific immunity was robust. Taken together, these data suggest microneedle delivery of lyophilized HPV PsV could provide a practical, thermostable combined vaccine approach that could be developed for clinical evaluation.     

Micro-Economic Impact of Congenital Heart Surgery: Results of a Prospective Study from a Limited-Resource Setting

Introduction

The microeconomic impact of surgery for congenital heart disease is unexplored, particularly in resource limited environments. We sought to understand the direct and indirect costs related to congenital heart surgery and its impact on Indian households from a family perspective.

Methods

Baseline and first follow-up data of 644 consecutive children admitted for surgery for congenital heart disease (March 2013 – July 2014) in a tertiary referral hospital in Central Kerala, South India was collected prospectivelyfrom parents through questionnaires using a semi-structured interview schedule.

Results

The median age was 8.2 months (IQR: 3.0– 36.0 months). Most families belonged to upper middle (43.0%) and lower middle (35.7%) socioeconomic class. Only 3.9% of families had some form of health insurance. The median expense for the admission and surgery was INR 201898 (IQR: 163287–266139) [I$ 11989 (IQR: 9696–15804)], which was 0.93 (IQR: 0.52–1.49) times the annual family income of affected patients. Median loss of man-days was 35 (IQR: 24–50) and job-days was 15 (IQR: 11–24). Surgical risk category and hospital stay duration significantly predicted higher costs. One in two families reported overwhelming to high financial stress during admission period for surgery. Approximately half of the families borrowed money during the follow up period after surgery.

Conclusion

Surgery for congenital heart disease results in significant financial burden for majority of families studied. Efforts should be directed at further reductions in treatment costs without compromising the quality of care together with generating financial support for affected families.