Use of air stacking and abdominal compression for cough assistance in people with complete tetraplegia.

STUDY DESIGN: Cross-sectional. OBJECTIVE: To assess cough using air stacking (AS) to assist inspiratory volume with abdominal compression (AC) during expiration in patients with American Spinal Injury Association Impairment Scale (AIS) A. SETTING: Large tertiary hospital in Chile. METHODS: Peak cough flow (PCF) was measured during four different interventions: spontaneous maximal expiratory effort (MEE); MEE while receiving AC (MEE-AC); MEE after AS with a manual resuscitation bag (AS-MEE); and MEE with AS and AC (AS-MEE-AC). RESULTS: Fifteen in-patients with complete tetraplegia (C4-C6) were included. Median age was 33 years (16-56). PCF during the different interventions was PCF for MEE was 183±90 l min(-1); PCF for MEE-AC was 273±119 l min(-1); PCF for AS-MEE was 278±106 l min(-1) and PCF for AS-MEE-AC was 368±129 l min(-1). We observed significant differences in PCF while applying MEE-AC and AS-MEE compared with MEE (P=0.0001). However, the difference in PCF value was greater using the AS-MEE-AC technique (P=0.00001). CONCLUSION: Patients with spinal cord injury (SCI) presented an ineffective cough that constitutes a risk factor for developing respiratory complications. The application of combined techniques (AS-MEE-AC) can reach near normal PCF values. This is a low-cost, simple and easily applied intervention that could be introduced to all patients with tetraplegia.

First Report on the Transmission of 'Candidatus Liberibacter americanus' from Citrus to Nicotiana tabacum cv. Xanthi.

Huanglongbing (HLB), also known as greening, is one of the most important diseases of citrus worldwide. The causal agent is a gram-negative bacterium known to inhabit the phloem of infected plants. Three different candidate species infect citrus: 'Candidatus Liberibacter africanus' found in the African continent; 'Ca. L. asiaticus' found in Asia, Brazil, and the United States; and 'Ca. L. americanus' found in Brazil. (1). Tobacco is an easily transformable plant species that can be used as an experimental host system to quickly screen for candidate genes useful to control plant pathogens. However, no evidence exists on the ability of this plant species to sustain populations of 'Ca. L. americanus'. With the purpose of transmitting 'Ca. L. americanus' from citrus to tobacco, fragments of healthy stems of Cuscuta spp. (dodder) were used to connect an HLB-infected sweet orange plant to each of 10 healthy plants of Nicotiana tabacum L. cv. Xanthi and allowed to remain connected for 30, 45, and 50 days. Three different HLB-infected orange plants and 30 tobacco plants were used in three independent experiments. Most HLB-exposed Xanthi plants exhibited chlorotic leaves after 50 days of exposure probably because of the parasitic effect of dodder; however, an average of 6, 1, and 3 Xanthi plants exhibited a unique blotchy mottle symptom after 30, 45, and 50 days of exposure, respectively. Symptomatic and asymptomatic leaves were collected and analyzed by PCR. The results consistently confirmed the presence of 'Ca. L. americanus' only in symptomatic leaves. Sequencing of the PCR product and comparison to the NCBI database also confirmed the identity of the pathogen as 'Ca. L. americanus'. Electron microscopy analysis of four symptomatic leaves indicated the presence of bacterium-like bodies with round to elongated bacilliform shapes and surrounded by two membranes. These bodies resembled those already described in HLB-infected citrus in Brazil (1). The evidence presented above confirms the successful transmission of 'Ca. L. americanus' from citrus to Xanthi using the parasitic plant Cuscuta spp. Reference: (1) F. A. O. Tanaka et al. Fitopatol. Bras. 31:99, 2006.

First Report of Citrus leprosis virus on Citrus in Santa Cruz, Bolivia.

The citrus crop is rapidly expanding in the Province of Santa Cruz de la Sierra, Bolívia. Citrus, mostly planted by small growers, currently comprises approximately 15,000 ha. Sweet oranges (Citrus sinensis) and mandarins (C. reticulate) are the main citrus-types grown primarily for internal consumption. Recently, there has been an increase in incidence of leprosis-like symptoms (round to elliptic lesions on the leaves, chlorotic to necrotic lesions in young twigs, and depressed small lesions on the fruits). These symptoms were associated with infestations by the tenuipalpid mite Brevipalpus sp. To verify if Citrus leprosis virus was the causal agent of the observed symptoms, leaf and fruit samples (mostly from Valencia sweet orange) were collected from commercial groves in El Torno, 32 km south of Santa Cruz, and Yapacani and Colónia San Juan, 130 km northwest of Santa Cruz. Small fragments of these samples were placed immediately in a mixture of glutaraldehyde and paraformaldehyde in cacodylate buffer and later processed with transmission electron microscopy at ESALQ, Piracicaba, SP, Brazil. Some of the leaf samples were dried at 35°C and used for reverse transcription-polymerase chain reaction (RT-PCR) with primers that specifically amplify portions of the genome of Citrus leprosis virus, cytoplasmic type (CiLV-C) (1) at Centro APTA Citros, Cordeirópolis, SP, Brazil. Brevipalpus sp. mites were also collected and kept in 90% ethanol for further identification at the University of Florida, Gainesville and ESALQ. In the samples from the three surveyed areas, transmission electron microscopy confirmed the presence of short bacilliform particles within endoplasmic reticulum cisternae and electron dense viroplasms in the cytoplasm, typical of infection by CiLV-C (2). CiLV-C specific primers amplified DNA fragments of expected sizes in RT-PCR from dried leaf samples that came from these three localities. Direct sequencing of at least three amplicons of each sample confirmed the identity of the virus. The consensus sequence of the putative movement protein gene in samples from Yapacani and Colónia San Juan (GenBank Accessions Nos. AY960216 and AY960215, respectively) were identical and exhibited 99% nucleotide and 98% amino acid homology with the Brazilian isolate sequence available at GenBank (Accession No. AY289190). The consensus sequence of the putative replicase gene found in the sample from El Torno (GenBank Accession No. AY960214) exhibited 96 and 93% nucleotide and amino acid homology, respectively with the Brazilian isolate (GenBank Accession No. AY289191). Sampled mites were identified as B. phoenicis (Geijskes), the known vector of CiLV-C (2). The symptomatology, particle morphology and cytopathology, detection by molecular methods and the association with infestation by B. phoenicis, together indicate that the foliar, stem, and fruit lesions in sweet orange observed in the Santa Cruz region were caused by CiLV-C. To our knowledge, this is the first report of this virus in Bolivia. References: (1) E. C. Locali et al. Plant Dis. 87:1317, 2003. (2) J. C. V. Rodrigues et al. Exp. Appl. Acarol. 30:161, 2003.

Resistance of tomato and pepper to T3 strains of Xanthomonas campestris pv. vesicatoria is specified by a plant-inducible avirulence gene.

Tomato race 3 (T3) of Xanthomonas campestris pv. vesicatoria (Xcv) elicits a hypersensitive response (HR) in leaves of Lycopersicon esculentum near-isogenic line (NIL) 216 and pepper genotypes. One cosmid clone (35 kb) selected from a genomic library of a T3 strain induced an HR in all resistant plants. A 1.5-kb active subclone containing the putative avirulence (avr) gene, designated avrXv3, was sequenced. The avrXv3 gene encodes a 654-bp open reading frame (ORF) with no homology to any known gene. Expression studies with a fusion of this gene and uidA indicated that avrXv3 is plant inducible and controlled by the hypersensitivity and pathogenicity (hrp) regulatory system. Mutational analysis and transcription activation assays revealed that AvrXv3 has transcription activation activity in yeast, and that the putative domain responsible for that activity is located at the C terminus of the AvrXv3 protein. Agrobacterium tumefaciens-mediated transient expression confirmed the direct role of AvrXv3 in eliciting the HR in tomato NIL 216 and supported the hypothesis that Avr proteins must be present inside the plant host cell to trigger the HR.

Xv4-vrxv4: a new gene-for-gene interaction identified between Xanthomonas campestris pv. vesicatoria race T3 and wild tomato relative Lycopersicon pennellii.

Strains of tomato race 3 (T3) of Xanthomonas campestris pv. vesicatoria elicit a hypersensitive response (HR) in leaves of Lycopersicon pennellii LA716. Genetic segregation of the resistance exhibited ratios near 3:1 in F2 populations, which confirmed that a single dominant gene controlled the inheritance of this trait. With the aid of a collection of introgression lines, restriction fragment length polymorphism, and cleaved amplified polymorphic sequence markers, the resistance locus was located on chromosome 3 between TG599 and TG134. An avirulence gene named avrXv4 was also isolated by mobilizing a total of 600 clones from a genomic DNA library of the T3 strain 91-118 into the X. campestris pv. vesicatoria strain ME90, virulent on L. pennellii. One cosmid clone, pXcvT3-60 (29-kb insert), induced HR in resistant plants. The avirulent phenotype of pXcvT3-60 was confirmed by comparing growth rates in planta and electrolyte leakages among transconjugants carrying a mutated or intact clone with the wild-type T3 strain 91-118. A 1.9-kb DNA fragment contained within a 6.8-kb active subclone was sequenced and was determined to carry an open reading frame of 1,077 bp. The predicted AvrXv4 protein exhibits high similarity to members of an emerging new family of bacterial proteins from plant and mammalian pathogens comprising AvrRxv, AvrBsT, YopJ, YopP, AvrA, and YL40.

Evidence for a prokaryotic insertion-sequence contamination in eukaryotic sequences registered in different databases.

An insertion-sequence of prokaryotic origin was detected in a genomic clone obtained from a Phaseolus vulgaris bacterial artificial chromosome (BAC) library. This BAC clone, characterized as part of a contig constructed near a virus resistance gene, exhibited restriction fragment length polymorphism with an overlapping clone of the contig. Restriction analysis of DNA obtained from individual colonies of the stock culture indicated the presence of a mixed population of wild-type and insertional mutants. Sequence analysis of both members of the population revealed the presence of IS 10R, an insertion-sequence from Escherichia coli. A BLAST search for IS 10-like sequences detected unexpected homologies with a large number of eukaryotic sequences from Homo sapiens, Arabidopsis thaliana, Drosophila melanogasterand Caenorhabditis elegans. Southern analysis of a random sample of BAC clones failed to detect IS 10 in the BAC DNA. However, prolonged sub-culturing of a set of 15 clones resulted in transposition into the BAC DNA. Eventually, all cultures acquired a 2.3-kb fragment that hybridized strongly with IS 10. Sequence analysis revealed the presence of a preferred site for transposition in the BAC vector. These results indicate that a large number, if not all, of the BAC libraries from different organisms are contaminated with IS 10R. The source of this element has been identified as the DH10B strain of E. coli used as the host for BAC libraries.

Influence of burn-induced lipid-protein complex on IL1 secretion by PBMC in vitro.

The lipid-protein complex (LPC) formed by thermal injury to skin, which has been shown to have a toxic effect on mice, and which suppresses the immune response, was tested for its specific influence on monocytes. Growth of bacterial endotoxin-stimulated peripheral blood mononuclear cells (PBMC) was inhibited in the presence of LPC, however, the inhibition was less at the time of the optimal rate of cell proliferation. Inhibition was proportional to LPC concentration. ConA-stimulated PBMC were also inhibited by LPC in a dose-related manner. PBMC, in the presence of LPC, secreted interleukin 1 (IL1) at an increasing rate as LPC concentration rose from 5 to 40 micrograms/ml, and the levels of IL1 which could be induced by endotoxin were increasingly amplified in the presence of LPC. In comparison to LPC, the native tissue proteins which were isolated from unburned skin by the same techniques which produced LPC from burned skin, were tested for their effect on PBMC. Native proteins had no effect on IL1 secretion, whether on background or endotoxin-stimulated levels. Thus, the thermally induced change in skin proteins has a specific effect on monocyte IL1 secretion which is not matched by the native proteins, indicating that burn injury to skin specifically affects the lymphokine cascade and consequent immune function.

Plasma levels of cutaneous burn toxin and lipid peroxides in thermal injury.

Lipid peroxides, formed as a consequence of oxygen free radical formation, are responsible for tissue damage in a great variety of pathological conditions including thermal injury. 'Cutaneous burn toxin', formed by application of heat to skin, is thought to be specific to the burn injury. It causes dose-dependent damage to mitochondrial and red cell membranes, and dose-dependent inhibition of interleukin-2-dependent growth of lymphocytes. The possibility that the toxicity of 'cutaneous burn toxin', a lipid-protein, is exerted through lipid peroxides, was examined by measuring the levels of both agents in plasmas of eight burn patients during the first week after their injury. It was observed that plasma lipid peroxides did not appear in parallel with absorption into the circulation of 'cutaneous burn toxin'. Lipid peroxide levels equally common to very low and very high burn toxin levels, were recorded. The pair of agents correlated negatively (r = -0.26) at a significance of only 0.1. In addition, isolated purified 'cutaneous burn toxin' contained no measurable lipid peroxide. No relationship was therefore demonstrated between plasma levels of 'cutaneous burn toxin' and lipid peroxides.

Complete nucleotide sequence, genomic organization and phylogenetic analysis of Citrus leprosis virus cytoplasmic type.

The complete nucleotide sequence of the genomic RNA 1 (8745 nt) and RNA 2 (4986 nt) of Citrus leprosis virus cytoplasmic type (CiLV-C) was determined using cloned cDNA. RNA 1 contains two open reading frames (ORFs), which correspond to 286 and 29 kDa proteins. The 286 kDa protein is a polyprotein putatively involved in virus replication, which contains four conserved domains: methyltransferase, protease, helicase and polymerase. RNA 2 contains four ORFs corresponding to 15, 61, 32 and 24 kDa proteins, respectively. The 32 kDa protein is apparently involved in cell-to-cell movement of the virus, but none of the other putative proteins exhibit any conserved domain. The 5' regions of the two genomic RNAs contain a 'cap' structure and poly(A) tails were identified in the 3'-terminals. Sequence analyses and searches for structural and non-structural protein similarities revealed conserved domains with members of the genera Furovirus, Bromovirus, Tobravirus and Tobamovirus, although phylogenetic analyses strongly suggest that CiLV-C is a member of a distinct, novel virus genus and family, and definitely demonstrate that it does not belong to the family Rhabdoviridae, as previously proposed. Based on these results it was proposed that Citrus leprosis virus be considered as the type member of a new genus of viruses, Cilevirus.

Characterization of an alpha-amidating activity in a human pancreatic tumour secreting vasoactive intestinal peptide (VIP).

A case of watery diarrhoea hypokalaemia achlorhydria (WDHA) syndrome due to a pancreatic tumour and identified by VIP plasma level, VIP immunocytochemistry, and ultrastructural analysis of tumour sections, is reported. Since VIP is the mediator of the syndrome and is biologically active under its amidated form, the enzymatic alpha-amidating activity was investigated and characterized in tumour extract; using the synthetic substrate D-Tyr-Val-Gly, the enzyme displayed an optimal activity at pH 7.0, under aerobic conditions and with 35 microM CuSO4 and 3 mM ascorbate as co-factors. The Kmax and Vmax values of the enzymatic activity were 133.7 microM and 26.9 pmol/h/micrograms protein respectively. Its molecular weight, determined by molecular sieving, was close to 36 kDa. Other tumours of the human endocrine pancreas were also investigated for the enzymatic activity. The clinical interest of studying the regulation of the alpha-amidating activity in such tumours is discussed.

Selection of Candida glabrata strains with reduced susceptibility to azoles in four liver transplant patients with invasive candidiasis.

The cases of four liver transplant recipients who developed invasive candidiasis (2 cholangitis, 1 perihepatic abscess, 1 candidemia) due to azole-resistant, Candida glabrata are reported. Three patients were receiving azolic compounds (2 itraconazole, 1 fluconazole) when the infection was diagnosed. All four patients received fluconazole as intestinal decontamination during the first three weeks post transplantation. The infections occurred two months after transplantation in all patients, and in one patient Candida infection was the direct cause of death. Infection of the biliary tree was the origin of candidiasis in three patients; the fourth patient developed neutropenic-related candidemia. Fluconazole MICs exceeded 16 micrograms/ml in all cases; itraconazole MICs were 16, 2, 1, and 2 micrograms/ml, respectively. The potential role of Candida species other than albicans in these patients after administration of azole agents is discussed.