No longer a historical ailment: two cases of childhood scurvy with recommendations for bone health providers.

PURPOSE: Scurvy, due to vitamin C deficiency, is commonly referenced as a "forgotten" or "historical" disease. A growing number of case reports challenge this notion. Bone health providers are often consulted early in the presentation of scurvy to evaluate musculoskeletal complaints resulting from impaired collagen production and disrupted endochondral bone formation. In this report, we describe two cases of childhood scurvy. Our objective is to summarize the key features of scurvy for bone health providers, with the goal of raising awareness and facilitating diagnosis in future cases. CASE DESCRIPTIONS: Case one occurred in a 12-year-old non-verbal, non-ambulatory female on a ketogenic diet for refractory epilepsy. Clinical findings included hemarthrosis, transfusion dependent anemia, elevated inflammatory markers, and epiphysiolysis. Magnetic resonance imaging (MRI) revealed multi-focal bone marrow signal abnormalities and physeal irregularities. Case two occurred in a typically developing 5-year-old male presenting with limp and knee pain. Symptoms progressed despite casting and immobilization. Mild anemia, elevated inflammatory markers, and multi-focal marrow and physeal MRI abnormalities were identified. Subsequent dietary history revealed total absence of fruit or vegetable consumption. The diagnosis of scurvy was confirmed in both cases by undetectable plasma vitamin C concentrations. Treatment with vitamin C led to rapid clinical improvement. CONCLUSION: Scurvy can no longer be considered a historical diagnosis and should not be forgotten when evaluating children with musculoskeletal ailments. Early recognition of the signs, symptoms, and imaging findings of scurvy can reduce the clinical burden of this disease with the timely initiation of vitamin C therapy.

Pathogenesis of Vesicular Stomatitis New Jersey Virus Infection in Deer Mice ( Peromyscus maniculatus) Transmitted by Black Flies ( Simulium vittatum).

The natural transmission of vesicular stomatitis New Jersey virus (VSNJV), an arthropod-borne virus, is not completely understood. Rodents may have a role as reservoir or amplifying hosts. In this study, juvenile and nestling deer mice ( Peromyscus maniculatus) were exposed to VSNJV-infected black fly ( Simulium vittatum) bites followed by a second exposure to naive black flies on the nestling mice. Severe neurological signs were observed in some juvenile mice by 6 to 8 days postinoculation (DPI); viremia was not detected in 25 juvenile deer mice following exposure to VSNJV-infected fly bites. Both juvenile and nestling mice had lesions and viral antigen in the central nervous system (CNS); in juveniles, their distribution suggested that the sensory pathway was the most likely route to the CNS. In contrast, a hematogenous route was probably involved in nestling mice, since all of these mice developed viremia and had widespread antigen distribution in the CNS and other tissues on 2 DPI. VSNJV was recovered from naive flies that fed on viremic nestling mice. This is the first report of viremia in a potential natural host following infection with VSNJV via insect bite and conversely of an insect becoming infected with VSNJV by feeding on a viremic host. These results, along with histopathology and immunohistochemistry, show that nestling mice have widespread dissemination of VSNJV following VSNJV-infected black fly bite and are a potential reservoir or amplifying host for VSNJV.

Host and Potential Vector Susceptibility to an Emerging Orbivirus in the United States: Epizootic Hemorrhagic Disease Virus Serotype 6.

Epizootic hemorrhagic disease viruses (EHDVs) are orbiviruses transmitted by Culicoides biting midges to domestic and wild ruminants. EHDV-1 and EHDV-2 are endemic in the United States, where epizootic hemorrhagic disease is the most significant viral disease of white-tailed deer (WTD;Odocoileus virginianus) and reports of epizootic hemorrhagic disease in cattle are increasing. In 2006, a reassortant EHDV-6 was isolated from dead WTD in Indiana and has been detected each subsequent year over a wide geographic region. Since EHDV-6 is not a historically endemic serotype in the United States, it is important to understand infection outcome in potential hosts. Specifically, we aimed to evaluate the pathogenicity of the virus in 2 primary US ruminant hosts (WTD and cattle) and the susceptibility of a confirmed US vector (Culicoides sonorensis). Five WTD and 4 cattle were inoculated with >10(6)TCID50EHDV-6 by intradermal and subcutaneous injection. All 5 WTD exhibited moderate to severe disease, and 3 died. Viremia was first detected 3 to 5 days postinfection (dpi) with surviving animals seroconverting by 10 dpi. Two of 4 inoculated cattle had detectable viremia, 5 to 10 dpi and 7 to 24 dpi, respectively. No clinical, hematologic, or pathologic abnormalities were observed. Antibodies were detected by 10 dpi in 3 of 4 cows.C. sonorensis were fed on WTD blood spiked with EHDV-6 and held for 4 to 14 days postfeeding at 25°C. From 4 to 14 days postfeeding, 19 of 171 midges were virus isolation positive and 6 of 171 had ≥10(2.7)TCID50EHDV-6. Although outcomes varied, these studies demonstrate the susceptibility of ruminant and vector hosts in the United States for this recently emerged EHDV serotype.

Evolutionary dynamics of West Nile virus in Georgia, 2001-2011.

From 1999-2001, West Nile virus (WNV) spread throughout the eastern United States (US) and was first detected in Georgia in 2001. To date, the virus has been detected in over 2,500 dead wild bird and mosquito samples from across Georgia. We sequenced the premembrane (preM) and envelope gene (E) (2004 bp) from 111 isolates collected from 2001 to 2011. To assess viral gene flow from other geographic regions in the US, we combined our data with WNV sequences available at the National Center for Biotechnology Information (NCBI) and performed phylogenetic analysis. We found evidence that WNV isolates detected in Chatham County Georgia most likely originated from the Northeastern United States. These results highlight the growing importance of adequate genetic surveillance for monitoring and controlling viruses of public health concern.

The sensory shark: high-quality morphological, genomic and transcriptomic data for the small-spotted catshark Scyliorhinus canicula reveal the molecular bases of sensory organ evolution in jawed vertebrates.

Cartilaginous fishes (chimaeras and elasmobranchs -sharks, skates and rays) hold a key phylogenetic position to explore the origin and diversifications of jawed vertebrates. Here, we report and integrate reference genomic, transcriptomic and morphological data in the small-spotted catshark Scyliorhinus canicula to shed light on the evolution of sensory organs. We first characterise general aspects of the catshark genome, confirming the high conservation of genome organisation across cartilaginous fishes, and investigate population genomic signatures. Taking advantage of a dense sampling of transcriptomic data, we also identify gene signatures for all major organs, including chondrichthyan specializations, and evaluate expression diversifications between paralogs within major gene families involved in sensory functions. Finally, we combine these data with 3D synchrotron imaging and in situ gene expression analyses to explore chondrichthyan-specific traits and more general evolutionary trends of sensory systems. This approach brings to light, among others, novel markers of the ampullae of Lorenzini electro-sensory cells, a duplication hotspot for crystallin genes conserved in jawed vertebrates, and a new metazoan clade of the Transient-receptor potential (TRP) family. These resources and results, obtained in an experimentally tractable chondrichthyan model, open new avenues to integrate multiomics analyses for the study of elasmobranchs and jawed vertebrates.

Domestic cattle as a non-conventional amplifying host of vesicular stomatitis New Jersey virus.

The role of vertebrates as amplifying and maintenance hosts for vesicular stomatitis New Jersey virus (VSNJV) remains unclear. Livestock have been considered dead-end hosts because detectable viraemia is absent in VSNJV-infected animals. This study demonstrated two situations in which cattle can represent a source of VSNJV to Simulium vittatum Zetterstedt (Diptera: Simuliidae) by serving: (a) as a substrate for horizontal transmission among co-feeding black flies, and (b) as a source of infection to uninfected black flies feeding on sites where VSNJV-infected black flies have previously fed. Observed co-feeding transmission rates ranged from 0% to 67%. Uninfected flies physically separated from infected flies by a distance of up to 11 cm were able to acquire virus during feeding although the rate of transmission decreased as the distance between infected and uninfected flies increased. Acquisition of VSNJV by uninfected flies feeding on initial inoculation sites at 24 h, 48 h and 72 h post-infection, in both the presence and absence of vesicular lesions, was detected.

Lesion development and replication kinetics during early infection in cattle inoculated with Vesicular stomatitis New Jersey virus via scarification and black fly (Simulium vittatum) bite.

Vesicular stomatitis viruses are the causative agents of vesicular stomatitis, an economically important contagious disease of livestock that occurs in North, Central, and South America. Little is known regarding the early stages of infection in natural hosts. Twelve adult Holstein steers were inoculated with Vesicular stomatitis New Jersey virus (VSNJV) on the coronary bands (CB) of the feet via scarification (SC) or by VSNJV-infected black fly (Simulium vittatum) bite (FB). Three additional animals were inoculated on the neck skin using FB. Clinical disease and lesion development were assessed daily, and animals were euthanatized from 12 hours post inoculation (HPI) through 120 HPI. The animals inoculated in the neck failed to develop any clinical signs or gross lesions, and VSNJV was detected neither by in situ hybridization (ISH) nor by immunohistochemistry (IHC). Lesions on the CB were more severe in the animals infected by FB than by SC. In both groups, peak VSNJV replication occurred between 24 and 48 HPI in keratinocytes of the CB, as evidenced by ISH and IHC. There was evidence of viral replication limited to the first 24 HPI in the local draining lymph nodes, as seen through ISH. Successful infection via FB required logarithmically less virus than with the SC technique, suggesting that components in black fly saliva may facilitate VSNJV transmission and infection in cattle. The lack of lesion development in the neck with the same method of inoculation used in the CB suggests that specific characteristics of the CB epithelium may facilitate VSNJV infection.

Hard choices: a qualitative study of influences on the treatment decisions made by advanced lung cancer patients.

OBJECTIVE: Most patients with non-small cell lung cancer (NSCLC) present with an incurable, advanced disease, and treatment decisions may be hard. This study explored the factors that influence patients' choice of treatment during the oncologist-patient consultation. DESIGN: Semi-structured interviews conducted within 1 month of a consultation with an oncologist. PARTICIPANTS: Five patients newly diagnosed with incurable NSCLC and facing a treatment decision following a consultation with an oncologist. SETTING: A regional oncology unit in the UK. RESULTS: Some of the participants who opted for chemotherapy had made a decision before seeing the oncologist, presented with fewer symptoms, had been more active in seeking information before the consultation, and were willing to accept the risk of side effects. Participants opting for radiotherapy were not willing to accept the risk of side effects for the possibility of a small survival gain and instead focused on symptom relief. CONCLUSION: Some participants sought information before the consultation from various formal and informal sources. This may undermine the oncologist-patient consultation as the information may be incomplete or inaccurate. Patients vary in their willingness to accept risks for small potential gains. More research is required into methods to communicate the extent of the risks of treatment. The Clinical Nurse Specialist performed a valuable role for the patients and was seen as a trusted source of information.

Complete and draft genome sequences of six members of the Aquificales.

The Aquificales are widespread in marine and terrestrial hydrothermal environments. Here, we report the complete and draft genome sequences of six new members of the Aquificales: two marine species, Persephonella marina strain EX-H1 and Hydrogenivirga strain 128-5-R1 (from the East Pacific Rise, 9 degrees 50.3'N, 104 degrees 17.5'W, and the Eastern Lau Spreading Center, 176 degrees 11.5'W, 20 degrees 45.8'S, respectively), and four terrestrial isolates, Sulfurihydrogenibium azorense strain Az-Fu1, Sulfurihydrogenibium yellowstonense strain SS-5, and Sulfurihydrogenibium strain Y03AOP1 (from Furnas, Azores, Portugal, and Calcite Springs and Obsidian Pool in Yellowstone National Park, United States, respectively), and the only thermoacidophilic isolate, Hydrogenobaculum strain Y04AAS1 (from a stream adjacent to Obsidian Pool). Significant differences among the different species exist that include nitrogen metabolism, hydrogen utilization, chemotaxis, and signal transduction, providing insights into their ecological niche adaptations.

Transmission of vesicular stomatitis virus from infected to noninfected black flies co-feeding on nonviremic deer mice.

Vesicular stomatitis is an economically important arboviral disease of livestock. Viremia is absent in infected mammalian hosts, and the mechanism by which insects become infected with the causative agents, vesicular stomatitis viruses, remains unknown. Because infected and noninfected insects potentially feed on the same host in nature, infected and noninfected black flies were allowed to feed on the same host. Viremia was not detected in the host after infection by a black fly bite, but because noninfected black flies acquired the virus while co-feeding on the same host with infected black flies, it is concluded that a viremic host is not necessary for an insect to be infected with the virus. Thus co-feeding is a mechanism of infection for an insect-transmitted virus.

Inhibition of a Mycobacterium tuberculosis beta-ketoacyl ACP synthase by isoniazid.

Although isoniazid (isonicotinic acid hydrazide, INH) is widely used for the treatment of tuberculosis, its molecular target has remained elusive. In response to INH treatment, saturated hexacosanoic acid (C26:0) accumulated on a 12-kilodalton acyl carrier protein (AcpM) that normally carried mycolic acid precursors as long as C50. A protein species purified from INH-treated Mycobacterium tuberculosis was shown to consist of a covalent complex of INH, AcpM, and a beta-ketoacyl acyl carrier protein synthase, KasA. Amino acid-altering mutations in the KasA protein were identified in INH-resistant patient isolates that lacked other mutations associated with resistance to this drug.

Experimental transmission of vesicular stomatitis New Jersey virus from Simulium vittatum to cattle: clinical outcome is influenced by site of insect feeding.

Vesicular stomatitis New Jersey virus (VSNJV) is an insect-transmitted Rhabdovirus causing vesicular disease in domestic livestock including cattle, horses, and pigs. Natural transmission during epidemics remains poorly understood, particularly in cattle, one of the most affected species during outbreaks. This study reports the first successful transmission of VSNJV to cattle by insect bite resulting in clinical disease. When infected black flies (Simulium vittatum Zetterstedt) fed at sites where VS lesions are usually observed (mouth, nostrils, and foot coronary band), infection occurred, characterized by local viral replication, vesicular lesions, and high neutralizing antibody titers (> 1: 256). Viral RNA was detected up to 9 d postinfection in tissues collected during necropsy from lesion sites and lymph nodes draining those sites. Interestingly, when flies were allowed to feed on flank or neck skin, viral replication was poor, lesions were not observed, and low levels of neutralizing antibodies (range, 1:8-1:32) developed. Viremia was never observed in any of the animals and infectious virus was not recovered from tissues on necropsies performed between 8 and 27 d postinfection. Demonstration that VSNJV transmission to cattle by infected black flies can result in clinical disease contributes to a better understanding of the epidemiology and potential prevention and control methods for this important disease.

Modulation of chaperone gene expression in mutagenized Saccharomyces cerevisiae strains developed for recombinant human albumin production results in increased production of multiple heterologous proteins.

The yeast Saccharomyces cerevisiae has been successfully established as a commercially viable system for the production of recombinant proteins. Manipulation of chaperone gene expression has been utilized extensively to increase recombinant protein production from S. cerevisiae, focusing predominantly on the products of the protein disulfide isomerase gene PDI1 and the hsp70 gene KAR2. Here we show that the expression of the genes SIL1, LHS1, JEM1, and SCJ1, all of which are involved in regulating the ATPase cycle of Kar2p, is increased in a proprietary yeast strain, developed by several rounds of random mutagenesis and screening for increased production of recombinant human albumin (rHA). To establish whether this expression contributes to the enhanced-production phenotype, these genes were overexpressed both individually and in combination. The resultant strains showed significantly increased shake-flask production levels of rHA, granulocyte-macrophage colony-stimulating factor, and recombinant human transferrin.

Phospholipid methylation in brain membrane preparations: kinetic mechanism.

The methylation reactions which convert phosphatidylethanolamine (PE) to phosphatidylcholine (PC) have been studied kinetically using exogenously added intermediates and crude membrane preparations from brain. The addition of exogenous PE resulted in no change in the methylation rates compared to that of endogenous PE. The addition of the two intermediates, monomethylphosphatidylethanolamine (PMME) and dimethylphosphatidylethanolamine (PDME), resulted in significantly increased rates of methylation and allowed the kinetic analysis of these latter two methylation reactions. The mechanism for this enzyme appears to be similar to human RBC (Reitz et al. (1989) J. Biol. Chem. 264, 8097-8106) which was a rapid-equilibrium random Bi-Bi sequential mechanism. There were some slight differences between the brain enzyme and that from the RBC, but there is little reason to suggest a fundamentally different mechanism. It is more likely that the differences may relate to an additional dead-end complex for the enzyme from brain such that saturation with AdoMet cannot eliminate AdoHcy inhibition. The KM values for the two phospholipid substrates were 41-44 microM and 39 microM for the methylation of PMME and PDME, respectively. The KM for S-adenosylmethionine (AdoMet) was 7-9 microM with PMME and 4 microM with PDME as the other substrates. The Ki(lipid) varied from 54 microM with PMME to 225 microM with PDME, and the Ki(AdoMet) was 11 microM with PMME and 21 microM with PDME. The product from the use of AdoMet, S-adenosylhomocysteine (AdoHcy), was shown to be a noncompetitive inhibitor of both lipid substrates as well as AdoMet. The methylation of PMME was somewhat higher in cerebellum and brain stem compared to cortex and striatum, but the methylation of PDME was similar in cerebellum, brain stem and cortex.

Cloning and expression of the hup encoding a histone-like protein of Campylobacter jejuni.

A Campylobacter jejuni gene, designated hup, that appears to encode a homolog of the histone-like DNA-binding protein, HU, has been cloned, sequenced and expressed in Escherichia coli. Immunoblotting and in vitro transcription/translation analyses revealed a 11-kDa protein that was produced by recombinant plasmids containing hup. The gene contains an open reading frame (ORF) sufficient to encode a protein of 98 amino acids (aa) with a calculated molecular mass of 10,267 Da and a predicted isoelectric point of 10.1. The deduced aa sequence of the protein, designated HCj, exhibits considerable sequence identity with members of the HU family of proteins from other eubacterial species. The transcription start point was identified by primer extension analysis and appropriately spaced promoter sequences were found which exhibit considerable similarity to E. coli and Bacillus promoters. Southern hybridization analyses indicate that C. jejuni has a single copy of hup.

Cloning and applications of the two/three-base restriction endonuclease R.CviJI from IL-3A virus-infected Chlorella.

The gene (cviJIR) encoding the two/three-base R.CviJI eukaryotic restriction endonuclease (ENase) from IL-3A virus-infected Chlorella was cloned into Escherichia coli. A high frequency of DNA cleavage by R.CviJI required overexpression of the gene encoding the M.CviJI methyltransferase prior to cloning the gene for the ENase. Both genes were sequenced and their organization was determined to be in head-to-tail order. The open reading frame coding for R.CviJI can potentially translate a 41.4-kDa protein; however, in the E. coli host, a truncated version of the enzyme is produced (32.5 kDa). The recombinant ENase does not exhibit ATP-induced 'star' activity (R.CviJI cleaves at RGCY, while R.CviJI* also cleaves at RGCR and YGCY, but not at YGCR), as is characteristic for native R.CviJI. The very high frequency of DNA cleavage by R.CviJI* was exploited in the development of a quasi-random shotgun library method. R.CviJI*-generated oligodeoxyribonucleotides were applied to improve certain molecular biology applications, i.e., DNA labeling, detection, high-resolution restriction mapping, amplification and epitope mapping.

Isolation and complete nucleotide sequence of the gene for bovine parathyroid hormone.

The structure of the bovine parathyroid hormone (PTH) gene has been analyzed by Southern blot hybridization of genomic DNA and by nucleotide sequence analysis of a cloned PTH gene. In the Southern analysis, several restriction enzymes produced single fragments that hybridized to PTH cDNA suggesting that there is a single bovine PTH gene. The restriction map of the cloned gene is the same as that determined by Southern blot analysis of bovine DNA. The sequence of 3154 bp of the cloned gene has been determined including 510 bp and 139 bp in the 5' and 3' flanking regions, respectively. The gene contains two introns which separate three exons that code primarily for: (i) the 5' untranslated region, (ii) the pre-sequence of preProPTH, and (iii) PTH and the 3' untranslated region. The gene contains 68% A + T and unusually long stretches of 100- to 150-bp sequences containing alternating A and T nucleotides in the 5' flanking region and intron A. The 5' flanking region contains two TATA sequences, both of which appear to be functional as determined by S1 nuclease mapping. Compared to the rat and human genes, the locations of the introns are identical but the sizes differ. Comparable human and bovine sequences in the flanking regions and introns are about 80% homologous.

Phage lambda cDNA cloning vectors for subtractive hybridization, fusion-protein synthesis and Cre-loxP automatic plasmid subcloning.

We describe the construction and use of two classes of cDNA cloning vectors. The first class comprises the lambda EXLX(+) and lambda EXLX(-) vectors that can be used for the expression in Escherichia coli of proteins encoded by cDNA inserts. This is achieved by the fusion of cDNA open reading frames to the T7 gene 10 promoter and protein-coding sequences. The second class, the lambda SHLX vectors, allows the generation of large amounts of single-stranded DNA or synthetic cRNA that can be used in subtractive hybridization procedures. Both classes of vectors are designed to allow directional cDNA cloning with non-enzymatic protection of internal restriction sites. In addition, they are designed to facilitate conversion from phage lambda to plasmid clones using a genetic method based on the bacteriophage P1 site-specific recombination system; we refer to this as automatic Cre-loxP plasmid subcloning. The phage lambda arms, lambda LOX, used in the construction of these vectors have unique restriction sites positioned between the two loxP sites. Insertion of a specialized plasmid between these sites will convert it into a phage lambda cDNA cloning vector with automatic plasmid subcloning capability.

Bst DNA polymerase permits rapid sequence analysis from nanogram amounts of template.

A simple, rapid method is presented for the enzymatic sequence analysis of nanogram amounts of single-stranded or double-stranded DNA. This approach employs the thermostable DNA polymerase from Bacillus sterothermophilus and exploits its ability to efficiently extend all of the template-primer complex, even at low substrate concentrations. The procedure requires few pipetting steps, no preannealing step and very short reaction time. This method can significantly reduce the cost associated with DNA polymerase and the amount of template and time required to perform the enzymatic sequencing reactions. As little as a 10-ng aliquot of such sequencing reactions can be analyzed on a fluorescence-based capillary gel electrophoresis instrument recently developed in our laboratory. This highly sensitive detection, in conjunction with the ability to efficiently sequence nanogram amounts of template, strongly suggests the feasibility of direct DNA sequencing of single bacteriophage M13 plaques without prior amplification.

Expression of drug resistance-associated mdr-1, GST pi, and topoisomerase II genes during cell cycle traverse.

The expression of drug resistance-associated mdr-1, GST pi, and topoisomerase II genes was analyzed in cell cycle phase enriched populations of doxorubicin-resistant murine leukemic P388/R-84 cells. Flow cytometric analysis of bromodeoxyuridine (BrdU) incorporation and staining with anti-BrdU antibodies was used to confirm the purity of cell cycle phase enriched populations obtained by centrifugal elutriation. Doxorubicin (DOX) and daunorubicin (DNR) accumulation was significantly lower in S-phase cells, and coincubation with verapamil (VPL) or chlorpromazine (CPZ) enhanced DOX and DNR accumulation more in S-phase than in G1- and G2/M-phase cells. While the cellular content of mdr-1 and topoisomerase II mRNAs changed, GST pi mRNA content remained constant during the cell cycle. S-phase cells had about 3-fold higher mdr-1 mRNA content than G1- and G2/M-phase cells. In G1 cells, P-glycoprotein expression, as determined by C219 monoclonal antibody, was 12% less than that of S and G2/M cells. Topoisomerase II mRNA content increased with the progression of cell cycle and peaked in G2/M cells. These observations suggest that cell cycle stage related changes in expression of drug resistance markers may have a major bearing on chemosensitivity of drug-resistant cells.