SARS-CoV-2 testing, positivity, and factors associated with COVID-19 among people with HIV across Europe in the multinational EuroSIDA cohort.

BACKGROUND: Although people with HIV might be at risk of severe outcomes from infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; coronavirus 2019 [COVID-19]), regional and temporal differences in SARS-CoV-2 testing in people with HIV across Europe have not been previously described. METHODS: We described the proportions of testing, positive test results, and hospitalizations due to COVID-19 between 1 January 2020 and 31 December 2021 in the EuroSIDA cohort and the factors associated with being tested for SARS-CoV-2 and with ever testing positive. RESULTS: Of 9012 participants, 2270 (25.2%, 95% confidence interval [CI] 24.3-26.1) had a SARS-CoV-2 polymerase chain reaction test during the study period (range: 38.3% in Northern to 14.6% in Central-Eastern Europe). People from Northern Europe, women, those aged <40 years, those with CD4 cell count <350 cells/mm3 , and those with previous cardiovascular disease or malignancy were significantly more likely to have been tested, as were people with HIV in 2021 compared with those in 2020. Overall, 390 people with HIV (4.3%, 95% CI 3.9-4.8) tested positive (range: 2.6% in Northern to 7.1% in Southern Europe), and the odds of testing positive were higher in all regions than in Northern Europe and in 2021 than in 2020. In total, 64 people with HIV (0.7%, 95% CI 0.6-0.9) were hospitalized, of whom 12 died. Compared with 2020, the odds of positive testing decreased in all regions in 2021, and the associations with cardiovascular disease, malignancy, and use of tenofovir disoproxil fumarate disappeared in 2021. Among study participants, 58.9% received a COVID-19 vaccine (range: 72.0% in Southern to 14.8% in Eastern Europe). CONCLUSIONS: We observed large heterogeneity in SARS-CoV-2 testing and positivity and a low proportion of hospital admissions and deaths across the regions of Europe.

An Adaptive Study to Determine the Optimal Dose of the Tablet Formulation of the PARP Inhibitor Olaparib.

BACKGROUND: Olaparib is poorly soluble, requiring advanced drug delivery technologies for adequate bioavailability. Sixteen capsules/day are required for the approved 400 mg twice-daily dose; a tablet formulation was developed to reduce pill burden. This clinical trial evaluated the optimal dose and administration schedule of the tablet formulation. PATIENTS AND METHODS: Two stages of sequentially enrolled cohorts: stage 1, pharmacokinetic properties of tablet and capsule formulations were compared in patients with advanced solid tumours; stage 2, tablet dose escalation with expansion cohorts at doses/schedules of interest in patients with solid tumours and BRCAm breast/ovarian cancers. RESULTS: Olaparib 200 mg tablets displayed similar Cmax,ss, but lower AUCss and Cmin,ss than 400 mg capsules. Following multiple dosing, steady-state exposure with tablets ≥300 mg matched or exceeded that of 400 mg capsules. After dose escalation, while 400 mg twice daily was the tablet maximum tolerated dose based on haematological toxicity, 65 % of patients in the randomized expansion phase eventually required dose reduction to 300 mg. Intermittent tablet administration did not significantly improve tolerability. Tumour shrinkage was similar for 300 and 400 mg tablet and 400 mg capsule cohorts. CONCLUSIONS: The recommended monotherapy dose of olaparib tablet for Phase III trials was 300 mg twice daily, simplifying drug administration from 16 capsules to four tablets per day. CLINICAL TRIAL NUMBER: NCT00777582 (ClinicalTrials.gov).

Factors associated with HIV-1 virological failure in an outpatient clinic for HIV-infected people in Haiphong, Vietnam.

The objective of our study was to investigate factors associated with virological failure in 100 consecutive HIV-1 infected Vietnamese adults who initiated antiretroviral therapy (ART) from June 2007 to June 2008. Data were collected from medical records, and a structured questionnaire was used in individual interviews to investigate factors associated with adherence to ART. Plasma HIV viral load was measured at the time of the interview. The median age was 35 years, 35% were women and heterosexual intercourse was the most common mode of HIV transmission (61%). After a median of 14 months since starting ART, 23% had detectable HIV-1 viral load (≥ 400 copies/mL). Patients who had developed a World Health Organization (WHO) clinical stage 4 condition at the time of initiation of ART were more likely to experience virological failure than those in stages 1-3, odds ratio (OR): 5.20 (95% confidence interval [CI] 1.34-20.11), P = 0.017. Patients who reported that their health status was evaluated by a physician at each visit were less likely to experience virological failure, OR: 0.02 (95% CI 0.00-0.24), P = 0.002.

Assisted reproductive therapies and imprinting disorders--a preliminary British survey.

BACKGROUND: Recent reports have suggested a higher risk of Beckwith-Wiedemann syndrome (BWS) and Angelman syndrome (AS) after assisted reproductive technologies (ARTs), but it is unclear whether this might also apply to other disorders of genomic imprinting. METHODS: We contacted families of children with BWS, AS, Prader-Willi syndrome (PWS) and transient neonatal diabetes mellitus (TNDM) to determine use of ART. RESULTS: A statistically significant increased frequency of ART in children with BWS was confirmed [2.9%, 95% confidence interval (CI) 1.4-6.3% vs 0.8% expected] but there was no significant association with PWS or TNDM. Consideration of the molecular subgroup of BWS and AS suggested the feasibility of association with ART. CONCLUSIONS: These differences may relate to variations in (i) the molecular mechanisms for disordered imprinting in the different disorders and (ii) the susceptibility of specific imprinting control regions to ART-associated methylation alterations (epimutations).

Epistatic interaction between variations in the angiotensin I converting enzyme and angiotensin II type 1 receptor genes in relation to extent of coronary atherosclerosis.

OBJECTIVE: To test the hypothesis that gene-gene interaction of the renin-angiotensin system is associated with an effect on the extent of coronary atherosclerosis. SETTING AND RESULTS: A cohort of 1162 patients with coronary artery disease were genotyped for genetic polymorphisms in the renin-angiotensin system. Patients carrying the D allele of the angiotensin I converting enzyme (ACE) gene had greater coronary extent scores (defined as the number of coronary segments with 5% to 75% stenosis) than those not carrying this allele (p = 0.006 in non-parametric analysis and p = 0.019 in parametric analysis). This association remained significant after adjusting for age, body mass index, hypertension, and diabetes, which were also significantly associated with coronary extent scores. There was a significant interaction (p = 0.033) between genotypes of ACE and angiotensin II type 1 receptor (AGTR1). The association between the ACE gene D allele and increased coronary extent scores was significant (p = 0.008 in non-parametric and p = 0.027 in parametric analysis) in those carrying the +1166 C allele of the AGTR1 gene, but was absent in those not carrying the AGTR1 gene +1166 C allele. CONCLUSION: These findings suggest that variation in the ACE and AGTR1 genes and their interaction may not only contribute to susceptibility of coronary artery disease as previously found but also modify the disease process, thus contributing to interindividual differences in severity of the disease.

Stimulation of yeast 3-phosphoglycerate kinase gene promoter by paraquat.

Yeast cells exposed to adverse conditions employ a number of defense mechanisms in order to respond effectively to the stress and sustain a high proliferation rate. It has been shown that several glycolytic enzymes are induced upon heat treatment of yeast. In this work, we used a reporter plasmid construct to study the effects of oxidative stress, induced by the O(*-)(2)-generating compound paraquat (PQ), on the yeast 3-phosphoglycerate kinase gene (PGK) promoter. Our results show that (i) moderate, as opposed to excessive, doses of PQ induce increased stimulation of the PGK promoter, at midlogarithmic phase of growth; and (ii) the thiol antioxidant N-acetylcysteine cancels this stimulatory effect. These observations may represent one aspect of a more general role for glycolysis in maintaining the energy pools of yeast cells under stress.

Cloned prokaryotic iron superoxide dismutase protects yeast cells against oxidative stress depending on mitochondrial location.

Superoxide dismutase (SOD) is considered to be the first line of defense against oxygen toxicity. It exists as a family of three metalloproteins with copper,zinc (Cu,ZnSOD), manganese (MnSOD), and iron (FeSOD) forms. In this work, we have targeted Escherichia coli FeSOD to the mitochondrial intermembrane space (IMS) of yeast cells deficient in mitochondrial MnSOD. Our results show that FeSOD in the IMS increases the growth rate of the cells growing in minimal medium in air but does not protect the MnSOD-deficient yeast cells when exposed to induced oxidative stress. Cloned FeSOD must be targeted to the mitochondrial matrix to protect the cells from both physiological and induced oxidative stress. This confirms that the superoxide radical is mainly generated on the matrix side of the inner mitochondrial membrane of yeast cells, without excluding its potential appearance in the mitochondrial IMS where its elimination by SOD is beneficial to the cells.

Prokaryotic iron superoxide dismutase replaces cytosolic copper, zinc superoxide dismutase in protecting yeast cells against oxidative stress.

The iron superoxide dismutase (FeSOD) gene of Escherichia coli was cloned in Saccharomyces cerevisiae cells deficient in copper,zinc superoxide dismutase (Cu,ZnSOD). FeSOD replaced Cu,ZnSOD in protecting the yeast cells against oxidative stress. In the recombinant strains the FeSOD gene, which was under the transcriptional control of the yeast phosphoglycerate kinase gene promoter, was functionally expressed at two different levels on episomal and centromeric plasmids. Despite suppression of methionine and lysine auxotrophy, the higher level of FeSOD activity was more beneficial to growth of the mutant yeast cells only when these were exposed to higher levels of oxidative stress induced by paraquat or 100% oxygen. In the presence of paraquat, there was a novel stimulation of FeSOD activity. This was associated with a marked increase in catalase activity, and a decrease in glutathione reductase activity.

Cloning, expression, and characterization of two manganese superoxide dismutases from Caenorhabditis elegans.

Two genes encoding manganese superoxide dismutase (sod-2 and sod-3) have been identified in the nematode Caenorhabditis elegans. Each gene is composed of five exons, and intron positions are identical; however, intron sizes and sequences are not the same. The predicted protein sequences are 86.3% homologous (91.8% conservative), and the cDNAs are only 75.2% homologous. Both deduced protein sequences contain the expected N-terminal mitochondrial transit peptides. Reverse transcriptase polymerase chain reaction analysis shows that both genes are expressed under normal growth conditions and that their RNA transcripts are trans-spliced to the SL-1 leader sequence. The latter result together with Northern blot analysis indicate that both genes have mono-cistronic transcripts. The sod-3 gene was mapped to chromosome X, and the location of sod-2 was confirmed to be chromosome I. Polymerase chain reaction was used to amplify the cDNA regions encoding the predicted mature manganese superoxide dismutase proteins and each was cloned and expressed to high levels in Escherichia coli cells deficient in cytosolic superoxide dismutases. Both proteins were shown to be active in E. coli, providing similar protection against methyl viologen-induced oxidative stress. The expressed enzymes, which were not inhibited by hydrogen peroxide or cyanide, are dimeric, show quite different electrophoretic mobilities and isoelectric points, but exhibit comparable specific activities.

The conserved residue tyrosine 34 is essential for maximal activity of iron-superoxide dismutase from Escherichia coli.

We have expressed, purified, and analyzed the iron-containing superoxide dismutase (FeSOD) of Escherichia coli with mutations directed at tyrosine position 34 to introduce phenylalanine (SODY34F), serine (SODY34S), or cysteine (SODY34C). FeSOD and mutant enzymes were purified from SOD-deficient cells using a GST-FeSOD fusion protein intermediate which was subsequently cleaved with thrombin and repurified. Specific activities were measured using the xanthine-xanthine oxidase method and gave 3148 u/mg for wild-type FeSOD. The SODY34S mutation virtually inactivates the enzyme (42 u/mg); mutation to cysteine greatly reduces activity (563 u/mg), but the SODY34F mutant retains nearly 40% of the activity of wild type (1205 u/mg). Fusion protein intermediates were also shown to be active and were demonstrated to protect SOD-deficient E. coli cells from the induced effects of oxidative stress, with growth rates directly proportional to the specific activities of the expressed mutant enzymes. SODY34F exhibited decreased thermal stability, reduced activity at high pH, and a pronounced increase in sensitivity to the inhibitor sodium azide compared with wild-type FeSOD. These results suggest that tyrosine at position 34 is multifunctional and plays a structural role (probably through hydrogen bonding to glutamine at position 69) in maintaining the integrity of the active site, a stabilizing role at high pH, and a steric role in obstructing access to the active site of both substrate and inhibitor molecules.

Escherichia coli iron superoxide dismutase targeted to the mitochondria of yeast cells protects the cells against oxidative stress.

A gene encoding a fusion protein consisting of Escherichia coli iron superoxide dismutase (FeSOD) with the mitochondrial targeting presequence of yeast manganese superoxide dismutase (MnSOD) was cloned and expressed in E. coli and in Saccharomyces cerevisiae DL1Mn- yeast cells deficient in MnSOD. In the yeast cells the fusion protein was imported into the mitochondrial matrix. However, the presequence was not cleaved. In a control set of experiments, the E. coli FeSOD gene without the yeast MnSOD leader sequence was also cloned and expressed in S. cerevisiae DL1Mn- cells. In this case the FeSOD was located in the cytosol and was not imported into the mitochondrial matrix. E. coli FeSOD, with and without the yeast MnSOD presequence, proved to be active in yeast, but, whereas the FeSOD targeted to the mitochondria of yeast cells deficient in MnSOD protected the cells from the toxic effects of oxidative stress, FeSOD without the yeast MnSOD presequence did not protect the yeast cells deficient in MnSOD against oxidative stress.

Prediction of protein secondary structure from circular dichroism spectra: an attempt to solve the problem of the best-fitting reference protein subsets.

In least-squares fitting of protein circular dichroism (CD) spectra using basis CD spectra for the respective secondary structure components, as given by reference proteins of known structural composition, good fits of the CD spectrum do not necessarily correspond to appropriate fits of the underlying structural composition of a test protein. In an attempt to overcome this problem, CD similarity measures were used to construct a subset of five reference CD spectra which permitted three-component fitting of the CD and prediction of the relative magnitude of total beta-sheet (antiparallel + parallel) and total other structure (beta-turn + remainder), relative to helix, in the test protein. A backpropagation neural network (BPN) was also trained to make this prediction. In subsequent five-component fitting of the CD spectrum, using a Monte Carlo method to generate subsets of reference proteins from the working data set, only those secondary structure fits which conformed to the consensus prediction of the CD similarity measures and the BPN were accepted. The method enhanced the fitting of antiparallel beta-sheet and beta-turn for 16 proteins, compared to the variable selection method of P. Manavalan and W. C. Johnson (1987, Anal. Biochem. 167, 76-85). Some other proteins were less well fitted. Improvement in results is expected with a larger representation of feasible basis CD spectra in the working reference proteins.

Prediction of protein secondary structure from circular dichroism spectra using artificial neural network techniques.

An approach to predict protein secondary structure is presented using circular dichroism (CD) spectra as input to two types of artificial neural networks (ANNs): (i) a three-layer backpropagation network and (ii) a hybrid self-organisation to backpropagation network. The dataset comprised the CD spectra of 22 proteins in the 178-260 nm wavelength range whose secondary structures were known. A total of 22 networks were trained by each method, using the jackknife technique for testing the prediction on each protein in turn. The performance, measured in terms of root mean square residuals and Pearson product-moment correlation coefficients, compares well with that obtained by other statistical and ANN methods, and is likely to improve with the growth of the dataset.

The manganese superoxide dismutase gene of Caenorhabditis elegans.

We have cloned and sequenced a gene (sod-3) encoding manganese superoxide dismutase from the nematode Caenorhabditis elegans. The protein-coding region spans 943 bp including three intron sequences and encodes a protein of 227 amino acids (M(r) = 26,367) of which the first 33 amino acids are the presumed mitochondrial-targeting signal peptide. The deduced mature manganese-superoxide dismutase has 194 amino acids (M(r) = 22,193).

The copper/zinc superoxide dismutase gene of Caenorhabditis elegans.

The nucleotide and deduced amino acid sequences of the copper/zinc superoxide dismutase gene (sod-1) from the nematode Caenorhabditis elegans has been determined. The protein coding region is interrupted by three intron sequences covering 608 bp in total and encodes a protein of 158 amino acids (M(r) = 16,307).

Grading cervical dysplasia with AgNORs using a semiautomated image analysis system.

Colposcopic biopsies were classified according to previously established criteria by a group of three pathologists interested in cervical pathology. Ten cases were identified in each of the following five groups: normal, koilocytosis, low grade squamous intraepithelial lesions (CIN 1), high grade squamous intraepithelial lesions (CIN 2) and high grade squamous intraepithelial lesions (CIN 3). The Crocker technique was used to stain the sections cut 3 microns thick. With ths silver stain the nucleolar organizer regions (NORs) are stained black and referred to as AgNORs. It has been shown that malignant and premalignant changes in cells produce an increase in AgNORs. In each case eight images were captured using a 100x oil-immersion objective and stored in a Datacube Maxvideo system as 512 x 480 pixels in an 8-bit grayscale per image. The images were processed using the NeoPath field-of-view computer to detect the AgNORs and nuclei by using grayscale mathematical morphology algorithms. Color overlays of the AgNORs and nuclei were created using segmentation algorithms. The results show that it is possible to differentiate between low grade squamous intraepithelial lesions (CIN 1) and high grade squamous intraepithelial lesions (CIN 2 and CIN 3) taken together; however, there is no difference between low grade squamous intraepithelial lesions (CIN 1) and koilocytosis. The results support the concept that dysplasia cannot be classified effectively into three grades and that low grade squamous intraepithelial lesions (mild dysplasia [CIN 1]) is indistinguishable from koilocytosis.

The role of glutathione in protection against DNA damage induced by rifamycin SV and copper(II) ions.

Incubation of calf thymus DNA in the presence of rifamycin SV induces a decrease in the absorbance of DNA at 260 nm. The effect, was found to be proportional to the antibiotic concentration and enhanced by copper(II) ions. In the presence of rifamycin SV and copper(II), a significant increase in thiobarbituric acid-reactive (TBA-reactive) material is also observed. This effect is inhibited to different degrees by the following antioxidants: catalase 77%; thiourea 72%; glutathione (GSH) 62%; ethanol 52%; and DMSO 34%, suggesting that both hydrogen peroxide (H2O2) and hydroxyl radicals (OH.) are involved in DNA damage. Rifamycin SV-copper(II) mixtures were also found to induce the production of peroxidation material from deoxyribose and, in this case, glutathione and ethanol were the most effective antioxidant substrates with inhibition rates of 91% and 88% respectively. Electrophoretic studies show that calf thymus DNA becomes damaged after 20 min. incubation in the presence of both agents together and that the damaged fragments run with migration rates similar to those obtained by the metal chelating agent 1,10-phenanthroline. Normal DNA electrophoretic pattern was found to be preserved by catalase, and GSH at physiological concentrations and by thiourea. No protection is observed in the presence of ethanol or DMSO. The results obtained indicate the involvement of different reactive species in the degradation process of DNA due to rifamycin SV-copper(II) complex and emphasize the role of reduced glutathione as an oxygen free radical scavenger.

A processing strategy for automated Papanicolaou smear screening.

A multilayer processing strategy was developed for the automatic screening of conventionally prepared Papanicolaou smears. The processing stages include image segmentation, feature extraction, object classification and slide classification. Mathematical morphology functions were implemented in hardware with custom-built gate array processors for image segmentation. There were 68 features used for classifier training. In object classification we combined the evidential supports of a binary decision tree classifier and a multilayer perceptron classifier to achieve an integrated decision. In this feasibility study, 449 conventionally prepared cervical Papanicolaou smears were tested in a prototype research system between January and May 1991. The 95% confidence interval for the slide false-negative rate was 1-9%, and the 95% confidence interval for the slide sort rate was 45-55%. The estimated sort rate for clearly normal slides is within the range required for a cost-efficient screening system, and the estimated false-negative rate for premalignant and malignant smears is an improvement over published false-negative rates for human performance. Several performance improvement efforts are still under way. We expect that they will result in a vastly reduced slide false-negative rate.

Effect of metal ion catalyzed oxidation of rifamycin SV on cell viability and metabolic performance of isolated rat hepatocytes.

The effect of rifamycin SV on metabolic performance and cell viability was studied using isolated hepatocytes from fed, starved and glutathione (GSH) depleted rats. The relationships between GSH depletion, nutritional status of the cells, glucose metabolism, lactate dehydrogenase (LDH) leakage and malondialdehyde (MDA) production in the presence of rifamycin SV and transition metal ions was investigated. Glucose metabolism was impaired in isolated hepatocytes from both fed and starved animals, the effect is dependent on the rifamycin SV concentration and is enhanced by copper (II). Oxygen consumption by isolated hepatocytes from starved rats was also increased by copper (II) and a partial inhibition due to catalase was observed. Cellular GSH levels which decrease with increasing the rifamycin SV concentration were almost depleted in the presence of copper (II). A correlation between GSH depletion and LDH leakage was observed in fed and starved cells. Catalase induced a slight inhibition of the impairment of gluconeogenesis, GSH depletion and LDH leakage in starved hepatocytes incubated with rifamycin SV, iron (II) and copper (II) salts. Lipid peroxidation measured as MDA production by isolated hepatocytes was also augmented by rifamycin SV and copper (II), especially in hepatic cells isolated from starved and GSH depleted rats. Higher cytotoxicity was observed in isolated hepatocytes from fasted animals when compared with fed or GSH depleted animals. It seems likely that in addition to GSH level, there are other factors which may have an influence on the susceptibility of hepatic cells towards xenobiotic induced cytotoxicity.