Rapid point-of-care detection of SARS-CoV-2 infection in exhaled breath using ion mobility spectrometry: a pilot study.

BACKGROUND: An effective testing strategy is essential for pandemic control of the novel Coronavirus disease 2019 (COVID-19) caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Breath gas analysis can expand the available toolbox for diagnostic tests by using a rapid, cost-beneficial, high-throughput point-of-care test. We conducted a bi-center clinical pilot study in Germany to evaluate breath gas analysis using multi-capillary column ion mobility spectrometry (MCC-IMS) to detect SARS-CoV-2 infection. METHODS: Between September 23, 2020, and June 11, 2021, breath gas measurements were performed on 380 patients (SARS-CoV-2 real-time polymerase chain reaction (PCR) positive: 186; PCR negative: 194) presenting to the emergency department (ED) with respiratory symptoms. RESULTS: Breath gas analysis using MCC-IMS identified 110 peaks; 54 showed statistically significant differences in peak intensity between the SARS-CoV-2 PCR-negative and PCR-positive groups. A decision tree analysis classification resulted in a sensitivity of 83% and specificity of 86%, but limited robustness to dataset changes. Modest values for the sensitivity (74%) and specificity (52%) were obtained using linear discriminant analysis. A systematic search for peaks led to a sensitivity of 77% and specificity of 67%; however, validation by transferability to other data is questionable. CONCLUSIONS: Despite identifying several peaks by MCC-IMS with significant differences in peak intensity between PCR-negative and PCR-positive samples, finding a classification system that allows reliable differentiation between the two groups proved to be difficult. However, with some modifications to the setup, breath gas analysis using MCC-IMS may be a useful diagnostic toolbox for SARS-CoV-2 infection. TRIAL REGISTRATION: This study was registered at ClinicalTrials.gov on September 21, 2020 (NCT04556318; Study-ID: HC-N-H-2004).

A randomised assessment of the use of a quality of life questionnaire with or without intervention in patients attending a thoracic cancer clinic.

The study examined the impact of using a quality of life (QoL) questionnaire during a clinic to identify QoL issues and to improve QoL. 138 patients were randomised (1:1:1) to either (1) an Intervention group that completed the European Organisation for Research and Treatment of Cancer-Core Quality of Life Questionnaire and Lung Cancer Module (EORTC QLQ-C30 and LC13) at baseline and received feedback during a clinic, (2) an Attention group that completed the questionnaire at baseline without feedback and (3) a Control group that did not complete the questionnaire. All patients completed the same questionnaire 6 weeks later and a contact diary during the study period. There was a significant difference between the Intervention and Control groups for the mean number of QoL issues identified at baseline (4.69 vs. 2.81, P = 0.006) and the mean number of actions taken (4.41 vs. 2.46, P = 0.004). At 6 weeks, there was no difference between the groups in global QoL (Intervention vs. Control group, P = 0.596; Attention vs. Control, P = 0.973). The results suggest that the completion of the EORTC QLQ-C30 LC13 with feedback improves communication and increases the number of QoL issues identified and actions taken. However, the intervention does not impact on QoL per se. Clinicaltrials.gov: NCT01213745.

Behaviour and growth performance of low-birth-weight piglets cross-fostered in multiparous sows with piglets of higher birth weights / Comportamento e desempenho de crescimento de leitões com baixo peso ao nascer uniformizados com leitões de maior peso em fêmeas suínas multíparas

The aim of the study was to evaluate the behaviour, pre-weaning survival rate and growth performance of low birth weight (BW) piglets cross-fostered with piglets of higher weights. Piglets were transferred to 60 foster sows, and divided in three groups (G; n=20): G1- 12 low BW piglets (0.80 - 1.25kg); G2- six low BW piglets and six intermediate BW piglets (1.40 - 1.60kg), and G3- six low BW piglets and six high BW piglets (>1.70kg). For the analysis, groups G2 and G3 were subdivided in LG2 (six G2 light piglets); IG2 (six G2 intermediate piglets), LG3 (six G3 light piglets), and HG3 (six G3 heavy piglets). Behavioural observations were carried out on days 1, 2, 4 and 6 (visual direct observation) and on days 3 and 5 (video recording) after birth. The percentage of missed nursings was higher in LG3 piglets than in LG1, IG2 and HG3 piglets, on days 1 and 2. On day 4, light piglets (LG1, LG2 and LG3) missed more nursings than IG2 and HG3 piglets. On day 3, video recording showed a higher percentage of missed nursings in LG1, LG2, and LG3 piglets as compared to HG3 piglets. On day 1, the number of fights during nursing was higher in IG2 than in LG1 and LG3 piglets. Also on day 1, number of fights and percentage of piglets engaged in fights, during 15min after nursing, were higher in LG1, LG3 and HG3 than in LG2 piglets. More playful behaviours were observed on day 2 in IG2 and HG3 piglets compared to LG1, LG2 and LG3 piglets. Light piglets (LG1, LG2, and LG3) presented similar body weight on days 4, 8, 12 and 16 after birth, regardless of being mixed with piglets of higher weights or not; however, the survival rate until day 16 was most compromised in LG3 piglets compared to the other groups. Despite the lack of influence of littermates' weight on the growth of low BW piglets, their survival rate indicates that they should not be mixed with high BW piglets...

O objetivo do estudo foi avaliar o comportamento, a taxa de sobrevivência pré-desmame e o desempenho de crescimento de leitões leves ao nascer uniformizados com leitões de maior peso ao nascer (PN). Os leitões foram transferidos para 60 fêmeas e divididos em três grupos (G; n=20): G1 - 12 leitões de baixo PN (0,80-1,25kg); G2 - seis leitões de baixo PN e seis com PN intermediário (1,40-1,60kg); e G3 - seis leitões de baixo PN e seis leitões pesados (>1,70kg). Para a análise, os grupos G2 e G3 foram subdivididos em LG2 (seis G2 leitões leves); IG2 (seis G2 leitões de peso intermediário); LG3 (seis G3 leitões leves) e HG3 (seis G3 leitões pesados). Observações de comportamento foram realizadas nos dias 1, 2, 4 e 6 (observações visuais diretas) e nos dias 3 e 5 (gravações) após o nascimento. A porcentagem de mamadas perdidas foi maior no grupo LG3 quando comparado aos grupos LG1, IG2 e HG3, nos dias 1 e 2. No dia 4, leitões leves (LG1, LG2 e LG3) perderam maior número de mamadas do que os grupos IG2 e HG3. No dia 3, a gravação mostrou maior porcentagem de perda de mamadas nos grupos LG1, LG2 e LG3 do que no grupo HG3. No dia 1, o número de brigas durante a mamada foi maior nos grupos IG2 do que nos grupos LG1 e LG3. Também no dia 1, o número de brigas e porcentagem de leitões envolvidos em brigas, durante 15 minutos após a mamada, foi maior nos grupos LG1, LG3 e HG3 do que no grupo LG2. Brincadeiras foram mais observadas no dia 2 nos grupos IG2 e HG3 quando comparado aos grupos LG1, LG2 e LG3. Leitões leves (LG1, LG2 e LG3) apresentaram peso semelhante nos dias 4, 8, 12 e 16 após o nascimento, independentemente de serem misturados ou não com leitões pesados. No entanto, a taxa de sobrevivência até o dia 16 foi mais comprometida nos leitões do grupo LG3 do que nos outros grupos. Apesar da falta de influência do peso das leitegadas no crescimento de leitões de baixo PN, a taxa de sobrevivência indicou que estes não devem ser misturados com leitões de maior PN...

Effects of breeding at the second oestrus or after post-weaning hormonal treatment with altrenogest on subsequent reproductive performance of primiparous sows.

This study investigated the effects of breeding at the second oestrus after weaning or after feeding an orally active progestagen (altrenogest) on the subsequent reproductive performance of primiparous sows. After 3 weeks of lactation, 663 weaned sows of two genotypes were allocated into three groups: G1--breeding at the first oestrus after weaning; G2--breeding at the second oestrus after weaning and G3--treatment with altrenogest for 5 days after weaning and breeding at the first oestrus after the end of the treatment. Body weight at breeding was lower in G1 and G3 than in G2 sows (p < 0.05). The interval to show oestrus was similar for G1 and G2 groups (p > 0.05) but higher (p < 0.05) than that observed in G3 group. Within genotype A, percentages of females in oestrus within 10 days were not different (p > 0.05) among groups, whereas in genotype B, more G1 and G2 sows (p < 0.05) showed oestrus than G3 sows. In both genotypes, lower farrowing rates were observed in G3 than in G1 and G2 sows (p < 0.05) and a greater litter size (p < 0.05) was observed in G2 sows. In genotype A, the number of total born piglets was similar for G1 and G3 groups (p > 0.05), whereas in genotype B, G1 sows had a greater litter size than G3 sows (p < 0.05). Body weight at weaning and at breeding was similar (p > 0.05) between farrowed and non-farrowed sows in all groups. Reproductive performance is not improved in primiparous sows treated with altrenogest during 5 days after weaning. The reproductive performance of genotype B sows is compromised in Control and Altrenogest-treated sows but not in those bred at the second oestrus after mating. Breeding at the second oestrus after weaning allows primiparous sows to gain weight between weaning and service, and increases their farrowing rate and subsequent litter size.

Palosuran inhibits binding to primate UT receptors in cell membranes but demonstrates differential activity in intact cells and vascular tissues.

BACKGROUND AND PURPOSE: The recent development of the UT ligand palosuran (1-[2-(4-benzyl-4-hydroxy-piperidin-1-yl)-ethyl]-3-(2-methyl-quinolin-4-yl)-urea sulphate salt) has led to the proposition that urotensin-II (U-II) plays a significant pathological role in acute and chronic renal injury in the rat. EXPERIMENTAL APPROACH: In the present study, the pharmacological properties of palosuran were investigated further using a series of radioligand binding and functional bioassays. KEY RESULTS: Palosuran functioned as a 'primate-selective' UT ligand in recombinant cell membranes (monkey and human UT K(i) values of 4 +/- 1 and 5 +/- 1 nM), lacking appreciable affinity at other mammalian UT isoforms (rodent and feline K(i) values >1 microM). Paradoxically, however, palosuran lost significant (10- to 54-fold) affinity for native and recombinant human UT when radioligand binding was performed in intact cells (K(i) values of 50 +/- 3 and 276 +/- 67 nM). In accordance, palosuran also exhibited diminished activity in hUT (human urotensin-II receptor)-CHO (Chinese hamster ovary) cells (IC50 323 +/- 67 nM) and isolated arteries (K(b)>10 microM in rat aorta; K(b)>8.5 microM in cat arteries; K(b)>1.6 microM in monkey arteries; K(b) 2.2 +/- 0.6 microM in hUT transgenic mouse aorta). Relative to recombinant binding K(i) values, palosuran was subjected to a 392- to 690-fold reduction in functional activity in monkey isolated arteries. Such phenomena were peculiar to palosuran and were not apparent with an alternative chemotype, SB-657510 (2-bromo-N-[4-chloro-3-((R)-1-methyl-pyrrolidin-3-yloxy)-phenyl]-4,5-dimethoxybenzenesulphonamide HCl). CONCLUSIONS AND IMPLICATIONS: Collectively, such findings suggest that caution should be taken when interpreting data generated using palosuran. The loss of UT affinity/activity observed in intact cells and tissues cf. membranes offers a potential explanation for the disappointing clinical efficacy reported with palosuran in diabetic nephropathy patients. As such, the (patho)physiological significance of U-II in diabetic renal dysfunction remains uncertain.

Artificial insemination of gilts with 1.5 billion sperms stored in different periods associated with different pre-ovulatory intervals.

This study evaluated the reproductive performance of gilts inseminated at three intervals before ovulation (0-12, 13-23, 24-30 h) with sperm doses (SD) stored for 0-48 and 96-120 h. A total of 218 PIC Camborough 22 gilts were inseminated once with SD of 1.5 x 10(9) sperms. Pregnant gilts (n = 166) were slaughtered 30.8 +/- 3.7 days after artificial insemination. The number of corpora lutea (CL) and total embryos (TE) was counted. Pregnancy rates (PR) were analysed by chi-square test. TE and embryonic survival (ES), obtained as the ratio between viable embryos and CL, were analysed by GLM procedure (SAS) and mean values were compared by Tukey's test. Pregnancy rate was similar among artificial insemination-ovulation (AIOV) intervals when semen was stored for 0-48 h. However, the lowest PR was observed in the 24-30 h AIOV interval with storage time (ST) of 96-120 h (p < 0.05). There was a significant effect of the interaction between ST and AIOV (p < 0.05) on TE and ES variables. Total embryos and ES did not differ (p > 0.05) among AIOV intervals in ST of 0-48 h. However, gilts inseminated at 24-30 h AIOV interval with ST of 96-120 h showed a reduction of 6.7 embryos (p < 0.05) compared with gilts in the same interval inseminated with semen stored for 0-48 h. ES for the 24-30 h AIOV interval and ST of 96-120 h was lower than that observed in the other groups (p < 0.05).

Maturation of dendritic cells leads to up-regulation of cellular FLICE-inhibitory protein and concomitant down-regulation of death ligand-mediated apoptosis.

Dendritic cells (DCs) disappear from lymph nodes 1 to 2 days after antigen presentation, presumably by apoptosis. To evaluate the role of death ligands in elimination of DCs, we analyzed the sensitivity of human DCs to CD95 ligand (CD95L) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). We found mature DCs to be resistant to killing via CD95L or TRAIL, whereas only immature DCs were partially sensitive. However, all DC populations expressed CD95, TRAIL-R2, and TRAIL-R3 at comparable levels, suggesting that sensitivity to death ligand-induced DC apoptosis is not regulated at the receptor level. Interestingly, mature DCs highly expressed the caspase 8 inhibitory protein cFLIP, whereas only low levels were detected in immature DCs. Thus, death ligand sensitivity proved to be dependent on DC maturation and inversely correlated with expression levels of cFLIP. Induction of apoptosis by TRAIL or CD95L does not seem to play a role in the elimination of mature DCs, but instead might serve to regulate immature DC populations.

Listeria monocytogenes-infected human dendritic cells: uptake and host cell response.

Dendritic cells (DCs) are potent antigen-presenting cells and play a crucial role in initiation and modulation of specific immune responses. Various pathogens are able to persist inside DCs. However, internalization of the gram-positive bacterium Listeria monocytogenes into human DCs has not yet been shown. In the present study, we demonstrate that human monocyte-derived immature DCs can efficiently phagocytose L. monocytogenes. This uptake is independent of listerial adhesion factors internalin A and internalin B but requires cytoskeletal motion and factors present in human plasma. A major portion of internalized bacteria is found in membrane-bound phagosomes and is rarely free in the cytosol, as shown by transmission electron microscopy and by using an L. monocytogenes strain expressing green fluorescent protein when in the host cell cytosol. The infection caused maturation of the immature DCs into mature DCs displaying high levels of CD83, CD25, major histocompatibility complex class II, and the CD86 costimulator molecule. This effect appeared to be largely mediated by listerial lipoteichoic acid. Although L. monocytogenes infection is known to induce death in other cell types, infection of human DCs was found to induce necrotic but not apoptotic death in fewer than 20% of DCs. Therefore, the ability of DCs to act as effective antigen-presenting cells for listerial immunity is probably enhanced by their resistance to cell death, as well as their ability to rapidly differentiate into mature, immunostimulatory DCs upon encountering bacteria.

Differential expression of Rel/NF-kappaB and octamer factors is a hallmark of the generation and maturation of dendritic cells.

A key feature of maturation of dendritic cells is the down-regulation of antigen-processing and up-regulation of immunostimulatory capacities. To study the differential expression of transcription factors in this process, we investigated the nuclear translocation and DNA binding of Rel/NF-kappaB and octamer factors during in vitro generation and maturation of dendritic cells compared with macrophage development. RelB was the only factor strongly up-regulated during the generation of both immature dendritic cells and macrophages. Cytokine-induced maturation of dendritic cells resulted in an increase in nuclear RelB, p50, p52, and especially c-Rel, whereas cytokine-treated macrophages responded poorly. This up-regulation of NF-kappaB factors did not correlate with lower levels of cytosolic NF-kappaB inhibitors, the IkappaBs. One IkappaB, Bcl-3, was strongly expressed only in mature dendritic cells. Furthermore, generation and maturation of dendritic cells led to a continuous down-regulation of the octamer factor Oct-2, whereas monocytes and macrophages displayed high Oct-2 levels. A similar pattern of maturation-induced changes in transcription factor levels was found in cultured murine epidermal Langerhans cells, suggesting a general physiological significance of these findings. Finally, this pattern of differential activation of Rel and octamer factors appears to be suitable in determining the maturation stage of dendritic cells generated by treatment with different cytokine combinations in vitro. (Blood. 2000;95:277-285)

Urinary excretion of platinum from platinum industry workers.

OBJECTIVES: Assessment of level of exposure to platinum and platinum concentration in urine from platinum industry workers to evaluate internal exposures and excretion kinetics. METHODS: Platinum concentrations in urine samples from 34 workers were measured by adsorptive voltammetry after UV-photolysis. Morning and evening samples were taken two to six times during six months. Individual exposures were assessed by personal air sampling. Also, two male volunteers were exposed to platinum dust for four hours at a typical platinum refinery workplace. RESULTS: Urinary platinum excretion after a shift in platinum industry workers was found to be up to 6270 ng/g creatinine--that is, 1000 times above the median value of unexposed people. Urinary excretion reached the maximum nearly 10 hours after inhalative exposure to dust containing platinum. Elimination corresponded to a first half life of about 50 (95% confidence interval (95% CI) 36 to 66) hours, but there were indications that a part of the incorporated platinum is stored longer. The amount of urinary platinum excretion showed a close correlation with the exposure level monitored by personal air sampling. CONCLUSIONS: A newly developed analytical method enabled the detection of even natural background concentrations of platinum. Thus, increased urinary platinum concentrations could be detected early, which is important to avoid damaging health of exposed workers.

The ATPase activity of purified CDC48p from Saccharomyces cerevisiae shows complex dependence on ATP-, ADP-, and NADH-concentrations and is completely inhibited by NEM.

The cell cycle protein CDC48p from Saccharomyces cerevisiae is a member of a protein superfamily (AAA superfamily) characterized by a common region of approximately 200 amino-acid residues including an ATP binding consensus. CDC48p purified to homogeneity showed considerable ATPase activity which could be completely abolished by preincubation with NEM in the absence of ATP. ATP protects the protein from NEM and stabilizes the otherwise labile enzyme. The ATPase activity is reversibly inhibited by NADH and shows cooperativity with its substrate ATP. The application of the in vitro ATPase activity to the identification of physiologically interacting molecules is discussed. By electron microscopy, the enzyme was shown to consist of hexameric ring structures similar to its vertebrate homologue.

The ins and outs of Raf kinases.

Raf kinases are signal-integrating enzymes that have the ability to switch tyrosine kinase signalling to serine/threonine phosphorylation and connect growth factor receptors with transcription factors. The connection involves a cascade of protein kinases that is essential for cellular proliferation and differentiation of species ranging from worms to humans. This cascade also mediates transformation by most oncogenes.

Localisation of hyaluronate (HA) in primary tumors and nude mouse xenografts of human pancreatic carcinomas using a biotinylated HA-binding protein.

A biotinylated hyaluronate (HA)-binding protein isolated from bovine cartilage was used to analyze the distribution of HA in nude mouse xenografts derived from human pancreatic adenocarcinoma cell lines as well as in primary human pancreatic adenocarcinomas. The most reproducible results for the localisation of HA were obtained using cryostat sections. When the biotinylated HA-binding protein was applied to histological sections of nude mouse xenografts, the specific staining found could be inhibited by preincubating the HA-binding protein with an excess of HA or by hyaluronidase treatment of the tissue before staining. The highest HA concentration was found at the tumor boundaries, while in the central part of the tumor staining was slight or absent. In cryostat sections of primary tumors HA was found predominantly in the connective tissue immediately around tumor cells or at the border between the tumor and normal pancreatic tissue.

Yeast cell cycle protein CDC48p shows full-length homology to the mammalian protein VCP and is a member of a protein family involved in secretion, peroxisome formation, and gene expression.

Yeast mutants of cell cycle gene cdc48-1 arrest as large budded cells with microtubules spreading aberrantly throughout the cytoplasm from a single spindle plaque. The gene was cloned and disruption proved it to be essential. The CDC48 sequence encodes a protein of 92 kD that has an internal duplication of 200 amino acids and includes a nucleotide binding consensus sequence. Vertebrate VCP has a 70% identity over the entire length of the protein. Yeast Sec18p and mammalian N-ethylmaleimide-sensitive fusion protein, which are involved in intracellular transport, yeast Pas1p, which is essential for peroxisome assembly, and mammalian TBP-1, which influences HIV gene expression, are 40% identical in the duplicated region. Antibodies against CDC48 recognize a yeast protein of apparently 115 kD and a mammalian protein of 100 kD. Both proteins are bound loosely to components of the microsomal fraction as described for Sec18p and N-ethylmaleimide-sensitive fusion protein. This similarity suggests that CDC48p participates in a cell cycle function related to that of N-ethylmaleimide-sensitive fusion protein/Sec18p in Golgi transport.

Biosynthesis of the 25-kDa protein in the macrogametes/zygotes of Plasmodium falciparum.

Synthesis of the 25-kDa protein in the early midgut stages of Plasmodium falciparum was studied, using metabolic inhibitors (colchicine and actinomycin D) and pulse-labeling experiments. Experiments with colchicine showed that, immediately after induction of macrogametogenesis, 25-kDa protein synthesis occurs in both fertilized and nonfertilized macrogametes. The amount of 25-kDa protein synthesized increased slowly during time. Experiments with actinomycin D revealed that the slow increase of synthesis may be dependent on de novo messenger RNA synthesis.

Characterization of epitopes on the 25 kD protein of the macrogametes/zygotes of Plasmodium falciparum.

A sexual stage-specific protein of Plasmodium falciparum with a Mr of 25,000 is one of the target antigens of transmission-blocking antibodies. The contributions of tertiary structure and post-translational modifications (glycosylation and acylation) to the structure of the epitopes on this protein were the subject of detailed investigations. After modification of the three-dimensional structure and modification or cleavage of carbohydrate groups and linked fatty acids, the immunological reactivity was investigated by three different techniques: (i) immunoprecipitation of radiolabelled proteins, (ii) enzyme-linked immunosorbent assay (ELISA), and (iii) Western blotting. The results of the experiments indicate that the immunological reactivity of the major epitopes on the 25 kD protein, including the epitope involved in transmission-blocking immunity, are dependent on the tertiary structure of the protein and on the presence of linked fatty acids, but not on the presence or absence of carbohydrate groups.

The effect of long-term inhibition of mitochondrial protein synthesis on the oxidation capacity of mitochondria for NADH-linked substrates.

Experiments are presented showing that specific inhibition of mitochondrial protein synthesis by tetracyclines decreases the activity of the NADH-dehydrogenase complex in liver mitochondria, if rats are treated for long periods with these antibiotics. The corresponding inhibition of this complex in tumor cells (Zajdela hepatoma) and tumor mitochondria (Leydig cell tumor) is even more pronounced. It is concluded that the mitochondrial genetic system is involved in the assembly of the NADH-dehydrogenase complex, most likely by coding for one or more subunits. It is argued that this information, contrary to the situation for cytochrome c oxidase, the cytochrome bc1 complex and ATPsynthase, has been missed in previous experiments employing differential inhibition of mitochondrial protein synthesis, because of the circumstance that the inhibition did not reach the level at which it became rate-limiting.

Choice of peptide and peptide length for the generation of antibodies reactive with the intact protein.

N-terminal peptides of bovine ribonuclease (RNase) of 20, 13 and 7 amino acid residues were isolated by reversed-phase high-performance liquid chromatography (HPLC). Antibodies were raised in mice against these peptides coupled to bovine serum albumin (BSA). It was shown that antibodies against the peptides reacted with the intact protein and that the immune response decreased with decreasing size of peptide. In order to obtain a satisfactory reaction with the intact protein, the peptide immunogen should be longer than 7 amino acids.