Aerosolisation during tracheal intubation and extubation in an operating theatre setting.

Aerosol-generating procedures such as tracheal intubation and extubation pose a potential risk to healthcare workers because of the possibility of airborne transmission of infection. Detailed characterisation of aerosol quantities, particle size and generating activities has been undertaken in a number of simulations but not in actual clinical practice. The aim of this study was to determine whether the processes of facemask ventilation, tracheal intubation and extubation generate aerosols in clinical practice, and to characterise any aerosols produced. In this observational study, patients scheduled to undergo elective endonasal pituitary surgery without symptoms of COVID-19 were recruited. Airway management including tracheal intubation and extubation was performed in a standard positive pressure operating room with aerosols detected using laser-based particle image velocimetry to detect larger particles, and spectrometry with continuous air sampling to detect smaller particles. A total of 482,960 data points were assessed for complete procedures in three patients. Facemask ventilation, tracheal tube insertion and cuff inflation generated small particles 30-300 times above background noise that remained suspended in airflows and spread from the patient's facial region throughout the confines of the operating theatre. Safe clinical practice of these procedures should reflect these particle profiles. This adds to data that inform decisions regarding the appropriate precautions to take in a real-world setting.

A cross-sectional study to quantify the prevalence of avian influenza viruses in poultry at intervention and non-intervention live bird markets in central Vietnam, 2014.

In Vietnam, live bird markets are found in most populated centres, providing the means by which fresh poultry can be purchased by consumers for immediate consumption. Live bird markets are aggregation points for large numbers of poultry, and therefore, it is common for a range of avian influenza viruses to be mixed within live bird markets as a result of different poultry types and species being brought together from different geographical locations. We conducted a cross-sectional study in seven live bird markets in four districts of Thua Thien Hue Province in August and December, 2014. The aims of this study were to (i) document the prevalence of avian influenza in live bird markets (as measured by virus isolation); and (ii) quantify individual bird-, seller- and market-level characteristics that rendered poultry more likely to be positive for avian influenza virus at the time of sale. A questionnaire soliciting details of knowledge, attitude and avian influenza practices was administered to poultry sellers in study markets. At the same time, swabs and faecal samples were collected from individual poultry and submitted for isolation of avian influenza virus. The final data set comprised samples from 1,629 birds from 83 sellers in the seven live bird markets. A total of 113 birds were positive for virus isolation; a prevalence of 6.9 (95% CI 5.8-8.3) avian influenza virus-positive birds per 100 birds submitted for sale. After adjusting for clustering at the market and individual seller levels, none of the explanatory variables solicited in the questionnaire were significantly associated with avian influenza virus isolation positivity. The proportions of variance at the individual market, seller and individual bird levels were 6%, 48% and 46%, respectively. We conclude that the emphasis of avian influenza control efforts in Vietnam should be at the individual seller level as opposed to the market level.

Two-year outcome of first-line antiretroviral therapy among HIV-1 vertically-infected children in Hanoi, Vietnam.

A retrospective analysis of 86 HIV-1 vertically-infected Vietnamese children with a follow-up period >24 months after initiating antiretroviral therapy (ART) was performed from 2008 to 2012, to assess the outcome of first-line ART in resource-limited settings. Of the 86 children, 68 (79.1%) were treated successfully (plasma HIV-1 viral load [VL] <1000 copies/ml), and 63 (73.3%) had full viral suppression (VL <400 copies/ml) after 24 months of ART. No significant difference between successfully treated patients and failure groups was observed in VL, CD4(+) T-cell count or clinical stage at baseline; age at ART start; or ART regimen. All 14 children with VL >5000 copies/ml, one of four children with VL 1000-5000 copies/ml and none with VL <1000 copies/ml developed reverse transcriptase inhibitor (RTI)-resistance mutations by 24 months of ART. Y181C and M184V/I were the most dominant non-nucleoside and nucleoside RTI-resistance mutations, respectively (13/15, 86.7%). These findings suggest that VL testing after 24 months of ART can be used to efficiently differentiate ART failures among HIV-1 vertically-infected children in resource-limited settings.

Nuclear replacement of in vitro-matured porcine oocytes by a serial centrifugation and fusion method.

The objective of the present study was to establish a method for nuclear replacement in metaphase-II (M-II) stage porcine oocytes. Karyoplasts containing M-II chromosomes (K) and cytoplasts without chromosomes (C) were produced from in vitro-matured oocytes by a serial centrifugation method. The oocytes were then reconstructed by fusion of one karyoplast with 1, 2, 3 or 4 cytoplasts (K + 1C, K + 2C, K + 3C and K + 4C, respectively). Reconstructed oocytes, karyoplasts without fusion of any cytoplast (K) and zona-free M-II oocytes (control) were used for experiments. The rates of female pronucleus formation after parthenogenetic activation in all groups of reconstructed oocytes (58.2-77.4%) were not different from those of the K and control groups (58.2% and 66.0%, respectively). In vitro fertilization was carried out to assay the fertilization ability and subsequent embryonic development of the reconstructed oocytes. The cytoplast : karyoplast ratio did not affect the fertilization status (penetration and male pronuclear formation rates) of the oocytes. A significantly high monospermy rate was found in K oocytes (p < 0.05, 61.6%) compared with the other groups (18.2-32.8%). Blastocyst formation rates increased significantly as the number of the cytoplasts fused with karyoplasts increased (p < 0.05, 0.0-15.3%). The blastocyst rate in the K + 4C group (15.3%) was comparable with that of the control (17.8%). Total cell numbers in both the K + 3C and K + 4C groups (16.0 and 15.3 cells, respectively) were comparable with that of the control (26.2 cells). Our results demonstrate that a serial centrifugation and fusion (Centri-Fusion) is an effective method for producing M-II chromosome transferred oocytes with normal fertilization ability and in vitro development. It is suggested that the number of cytoplasts fused with a karyoplast plays a critical role in embryonic development.

Immunomodulation of experimental autoimmune encephalomyelitis with ordered peptides based on MHC-TCR binding motifs.

T cell-mediated destruction of the myelin sheath causes inflammatory damage of the CNS in multiple sclerosis (MS). The major T and B cell responses in MS patients who are HLA-DR2 (about two-thirds of MS patients) react to a region between residues 84 and 103 of myelin basic protein (1 ). The crystal structure of HLA-DR2 complexed with myelin basic protein(84-102) confirmed that Lys(91) is the major TCR contact site, whereas Phe(90) is a major anchor to MHC and binds the hydrophobic P4 pocket (2 ). We have tested peptides containing repetitive 4-aa sequences designed to bind critical MHC pockets and to interfere with T cell activation. One such sequence, EYYKEYYKEYYK, ameliorates experimental autoimmune encephalomyelitis in Lewis rats, an animal model of MS.

Transposon tagging of the sulfur gene of tobacco using engineered maize Ac/Ds elements.

The Sulfur gene of tobacco is nuclearly encoded. A Su allele at this locus acts as a dominant semilethal mutation and causes reduced accumulation of chlorophyll, resulting in a yellow color in the plant. An engineered transposon tagging system, based upon the maize element Ac/Ds, was used to mutate the gene. High frequency of transposon excision from the Su locus produced variegated sectors. Plants regenerated from the variegated sector exhibited a similar variegated phenotype. Genetic analyses showed that the variegation was always associated with the transposase construct and the transposon was linked to the Su locus. Sequences surrounding the transposon were isolated, and five revertant sectors possessed typical direct repeats following Ds excisions. These genetic and molecular data are consistent with the tagging of the Su allele by the transposon.

The Pex16p homolog SSE1 and storage organelle formation in Arabidopsis seeds.

Mature Arabidopsis seeds are enriched in storage proteins and lipids, but lack starch. In the shrunken seed 1 (sse1) mutant, however, starch is favored over proteins and lipids as the major storage compound. SSE1 has 26 percent identity with Pex16p in Yarrowia lipolytica and complements pex16 mutants defective in the formation of peroxisomes and the transportation of plasma membrane- and cell wall-associated proteins. In Arabidopsis maturing seeds, SSE1 is required for protein and oil body biogenesis, both of which are endoplasmic reticulum-dependent. Starch accumulation in sse1 suggests that starch formation is a default storage deposition pathway.

Suppressive immunization with DNA encoding a self-peptide prevents autoimmune disease: modulation of T cell costimulation.

Usually we rely on vaccination to promote an immune response to a pathogenic microbe. In this study, we demonstrate a suppressive from of vaccination, with DNA encoding a minigene for residues 139-151 of myelin proteolipid protein (PLP139-151), a pathogenic self-Ag. This suppressive vaccination attenuates a prototypic autoimmune disease, experimental autoimmune encephalomyelitis, which presents clinically with paralysis. Proliferative responses and production of the Th1 cytokines, IL-2 and IFN-gamma, were reduced in T cells responsive to PLP139-151. In the brains of mice that were successfully vaccinated, mRNA for IL-2, IL-15, and IFN-gamma were reduced. A mechanism underlying the reduction in severity and incidence of paralytic autoimmune disease and the reduction in Th1 cytokines involves altered costimulation of T cells; loading of APCs with DNA encoding PLP139-151 reduced the capacity of a T cell line reactive to PLP139-151 to proliferate even in the presence of exogenous CD28 costimulation. DNA immunization with the myelin minigene for PLP-altered expression of B7.1 (CD80), and B7.2 (CD86) on APCs in the spleen. Suppressive immunization against self-Ags encoded by DNA may be exploited to treat autoimmune diseases.

Gene silencing from plant DNA carried by a Geminivirus.

The geminivirus tomato golden mosaic virus (TGMV) replicates in nuclei and expresses genes from high copy number DNA episomes. The authors used TGMV as a vector to determine whether episomal DNA can cause silencing of homologous, chromosomal genes. Two markers were used to asses silencing: (1) the sulfur allele (su) of magnesium chelatase, an enzyme required for chlorophyll formation; and (2) the firefly luciferase gene (luc). Various portions of both marker genes were inserted into TGMV in place of the coat protein open-reading frame and the constructs were introduced into intact plants using particle bombardment. When TGMV vectors carrying fragments of su (TGMV::su) were introduced into leaves of wild type Nicotiana benthamiana, circular, yellow spots with an area of several hundred cells formed after 3-5 days. Systemic movement of TGMV::su subsequently produced varigated leaf and stem tissue. Fragments that caused silencing included a 786 bp 5' fragment of the 1392 bp su cDNA in sense and anti-sense orientation, and a 403 bp 3' fragment. TGMV::su-induced silencing was propogated through tissue culture, along with the viral episome, but was not retained through meiosis. Systemic downregulation of a constitutively expresse luciferase transgene in plants was achieved following infection with TGMV vectors carrying a 623 bp portion of luc in sense or anti-sense orientation. These results establish that homologous DNA sequences localized in nuclear episomes can modulate the expression of active chromosomal genes.

Development and characterization of a generalized gene tagging system for higher plants using an engineered maize transposon Ac.

This report describes a series of transposon tagging vectors for dicotyledonous plants based on the maize transposable element Ac. This binary system includes the transposase (Ts) and the tagging element (Ds) on separate T-DNA vectors. Ts elements include versions in which transcription is driven either by the endogenous Ac promoter or by the cauliflower mosaic virus (CaMV) 35S promoter. Ds tagging element includes a gene conferring methotrexate (Mtx) resistance for selection and a supF gene to facilitate cloning of tagged sequences. The Ds element is flanked by a CaMV 35S promoter and the beta-glucuronidase (GUS) coding sequence so that GUS expression occurs upon excision of the element. We have transformed these Ts and Ds elements into tobacco and demonstrated that the Ts is functional with either promoter, and that the artificial Ds elements are capable of transposition. The amount of excision was found to depend upon both the individual Ts and Ds primary transformants used. Somatic excision of Ds was seen in up to 100% of progeny seedlings containing Ts and Ds. Germinal excision was detected in up to 48% of the progeny of plants containing both elements. Hence, this system can generate a sufficient number of events to be useful in gene tagging.