Bovine virus diarrhea (BVD) is an economically important disease in cattle. Switzerland started an eradication program in 2008. After the initial virological examination of all cattle followed by the ...examination of all newborn calves, the BVD prevalence in 2012 was low enough to start serological monitoring. An unusually high number of increased tank milk values were observed during this serological monitoring in autumn 2019. No seropositive animals were found on most farms in the follow-up cattle group testing. The present study was designed in the form of a multiple case report to better assess the BVD situation in a herd after a serological tank milk result above the cut-off value. The tank milk of 13 farms with serological values above the cut-off value from the analyses in autumn 2019 was examined again with two different ELISA tests in spring 2020. In addition, at the same time blood samples were taken to obtain serological values of all adult cattle on the farm. The results of the two tests that were used to examine the tank milk samples correlated well with each another. The results of the tank milk serology showed a low correlation with the seroprevalence in the lactating cows, but no correlation with the seroprevalence of all adult animals in the herd. A single seropositive animal had a major influence on the results of the tank milk serology in some herds. In addition, correct tank milk sampling must be ensured because the representativeness of the tank milk sample is decisive for a meaningful result for the investigated farm. If the result of the tank milk test is above the cut-off value, the examination of a group of cattle is still the best way to identify a current infection in a herd.
Standard treatment for glioblastoma multiforme and other brain tumors consists of surgical resection followed by combined radio-/chemotherapy. However, radiation resistance of tumor cells limits the ...success of this treatment, and the tumors invariably recur. Therefore, the selective inhibition of molecular mediators of radiation resistance may provide therapeutic benefit to the patient. One of these targets is the Rad51 protein, which is a key component of the homologous recombinational repair of DNA double-strand breaks. Here, we investigated whether post-transcriptional silencing of Rad51 by herpes simplex virus-type 1 (HSV-1) amplicon vector-mediated short interfering RNA expression can enhance the antitumor effect of radiation therapy. We demonstrate that these vectors specifically and efficiently inhibited the radiation-induced recruitment of Rad51 into nuclear foci in human glioma cells. The combination of vector-mediated silencing of Rad51 expression and treatment with ionizing radiation resulted in a pronounced reduction of the survival of human glioma cells in culture. In athymyc mice, a single intratumoral injection of Rad51-specific HSV-1 amplicon vector followed by a single radiation treatment resulted in a significant decrease in tumor size. In control animals, including mice that received an intratumoral injection of Rad51-specific amplicon vector but no radiation treatment, the tumor sizes increased.
Up to 7% of term and late-preterm neonates in high-income countries receive antibiotics during the first 3 days of life because of suspected early-onset sepsis. The prevalence of culture-proven ...early-onset sepsis is 0·1% or less in high-income countries, suggesting substantial overtreatment. We assess whether procalcitonin-guided decision making for suspected early-onset sepsis can safely reduce the duration of antibiotic treatment.
We did this randomised controlled intervention trial in Dutch (n=11), Swiss (n=4), Canadian (n=2), and Czech (n=1) hospitals. Neonates of gestational age 34 weeks or older, with suspected early-onset sepsis requiring antibiotic treatment were stratified into four risk categories by their treating physicians and randomly assigned 1:1 using a computer-generated list stratified per centre to procalcitonin-guided decision making or standard care-based antibiotic treatment. Neonates who underwent surgery within the first week of life or had major congenital malformations that would have required hospital admission were excluded. Only principal investigators were masked for group assignment. Co-primary outcomes were non-inferiority for re-infection or death in the first month of life (margin 2·0%) and superiority for duration of antibiotic therapy. Intention-to-treat and per-protocol analyses were done. This trial was registered with ClinicalTrials.gov, number NCT00854932.
Between May 21, 2009, and Feb 14, 2015, we screened 2440 neonates with suspected early-onset sepsis. 622 infants were excluded due to lack of parental consent, 93 were ineligible for reasons unknown (68), congenital malformation (22), or surgery in the first week of life (3). 14 neonates were excluded as 100% data monitoring or retrieval was not feasible, and one neonate was excluded because their procalcitonin measurements could not be taken. 1710 neonates were enrolled and randomly assigned to either procalcitonin-guided therapy (n=866) or standard therapy (n=844). 1408 neonates underwent per-protocol analysis (745 in the procalcitonin group and 663 standard group). For the procalcitonin group, the duration of antibiotic therapy was reduced (intention to treat: 55·1 vs 65·0 h, p<0·0001; per protocol: 51·8 vs 64·0 h; p<0·0001). No sepsis-related deaths occurred, and 9 (<1%) of 1710 neonates had possible re-infection. The risk difference for non-inferiority was 0·1% (95% CI −4·6 to 4·8) in the intention-to-treat analysis (5 0·6% of 866 neonates in the procalcitonin group vs 4 0·5% of 844 neonates in the standard group) and 0·1% (−5·2 to 5·3) in the per-protocol analysis (5 0·7% of 745 neonates in the procalcitonin group vs 4 0·6% of 663 neonates in the standard group).
Procalcitonin-guided decision making was superior to standard care in reducing antibiotic therapy in neonates with suspected early-onset sepsis. Non-inferiority for re-infection or death could not be shown due to the low occurrence of re-infections and absence of study-related death.
The Thrasher Foundation, the NutsOhra Foundation, the Sophia Foundation for Scientific research.
Herpesviruses consistently transmit from immunocompetent carriers, implying that their neutralization is hard to achieve. Murid herpesvirus-4 (MuHV-4) exploits host IgG Fc receptors to bypass blocks ...to cell binding, and pH-dependent protein conformation changes to unveil its fusion machinery only after endocytosis. Nevertheless, neutralization remains possible by targeting the virion glycoprotein H (gH)-gL heterodimer, and the neutralizing antibody responses of MuHV-4 carriers are improved by boosting with recombinant gH-gL. We analysed here how gH-gL-directed neutralization works. The MuHV-4 gH-gL binds to heparan sulfate. However, most gH-gL-specific neutralizing antibodies did not block this interaction; neither did they act directly on fusion. Instead, they blocked virion endocytosis and transport to the late endosomes, where membrane fusion normally occurs. The poor endocytosis of gH-gL-neutralized virions was recapitulated precisely by virions genetically lacking gL. Therefore, driving virion uptake appears to be an important function of gH-gL that provides a major target for antibody-mediated neutralization.
In primary glioblastomas and other tumor types, the epidermal growth factor receptor (EGFR) is frequently observed with alterations, such as amplification, structural rearrangements, or ...overexpression of the gene, suggesting an important role in glial tumorigenesis and progression. In this study, we investigated whether posttranscriptional gene silencing by vector-mediated RNAi to inhibit EGFR expression can reduce the growth of cultured human gli36 glioma cells. To "knock down" EGFR expression, we have created HSV-1-based amplicons that contain the RNA polymerase III-dependent H1 promoter to express double-stranded hairpin RNA directed against EGFR at two different locations (pHSVsiEGFR I and pHSVsiEGFR II). We demonstrate that both pHSVsiEGFR I and pHSVsiEGFR II mediated knock-down of transiently transfected full-length EGFR or endogenous EGFR in a dose-dependent manner. The knock-down of EGFR resulted in the growth inhibition of human glioblastoma (gli36-luc) cells both in culture and in athymic mice in vivo. Cell cycle analysis and annexin V staining revealed that siRNA-mediated suppression of EGFR induced apoptosis. Overall HSV-1 amplicons can mediate efficient and specific posttranscriptional gene silencing.
Light regulation of enzyme activities in oxygenic photosynthesis is mediated by ferredoxin:thioredoxin reductase (FTR), a novel class of disulfide reductase with an active site comprising a Fe4S42+ ...cluster and an adjacent disulfide, that catalyzes reduction of the thioredoxin disulfide in two sequential one-electron steps using a Fe2S22+/+ ferredoxin as the electron donor. In this work, we report on spectroscopic (EPR, VTMCD, resonance Raman, and Mössbauer) and redox characterization of the active site of FTR in various forms of the enzyme, including wild-type FTR, point-mutation variants at each of the active-site cysteine residues, and stable analogues of the one-electron-reduced FTR-Trx heterodisulfide intermediate. The results reveal novel site-specific Fe4S4-cluster chemistry in oxidized, one-electron-reduced, and two-electron-reduced forms of FTR. In the resting enzyme, a weak interaction between the Fe4S4 cluster and the active-site disulfide promotes charge buildup at a unique Fe site and primes the active site to accept an electron from ferredoxin to break the disulfide bond. In one-electron-reduced analogues, cleavage of the active-site disulfide is accompanied by coordination of one of the cysteine residues that form the active-site disulfide to yield a Fe4S43+ cluster with two cysteinate ligands at a unique Fe site. The most intriguing result is that two-electron-reduced FTR in which the disulfide is reduced to a dithiol contains an unprecedented electron-rich Fe4S42+ cluster comprising both valence-delocalized and valence-localized Fe2+Fe3+ pairs. These results provide molecular level insights into the catalytic mechanism of FTR, and two viable mechanisms are proposed.
The AP-1 transcription factor composed of @@ifos@ and @@ijun@ gene products mediates transcriptional responses to hormonal and metabolic stimulations of pancreatic beta cells. Here, we investigated ...the mechanisms that dynamically control expression of AP-1 subunit proteins. In MIN6 cells, glucose and GLP-1 raised c-FOS protein with biphasic kinetics, an initial peak being followed by a plateau that persisted as long as stimuli were maintained. ERK1/2 activation paralleled c-FOS expression. Whereas initial induction of c-FOS protein required ERK1/2-dependent activation of c-@@ifos@ transcription and @@ide novo@ protein synthesis, persistent accumulation of c-FOS under sustained stimulation did not. Indeed, dependent on ERK1/2 activation, c-FOS accumulated in its hyperphosphorylated form protected from degradation through the proteasome pathway. The implication of ERK.1/2 in the accumulation of c-FOS protein was confirmed in rat primary ss cells, and the functional consequences of this mechanism were demonstrated with DNA-binding and reporter assays. Altogether these findings reveal a sequential regulation of AP-1 by ERK1/2, which initially increases transcription of c-@@ifos@ and, if stimulation persists, stabilizes freshly synthesized c-FOS protein to efficiently activate the transcription of AP-1-regulated genes. This ERK1/2-AP-1 module can function as a temporal integrator converting metabolic stimuli of different durations into differential transcriptional outputs.
PDF
