The ACADS gene variation spectrum in 114 patients with short-chain acyl-CoA dehydrogenase (SCAD) deficiency is dominated by missense variations leading to protein misfolding at the cellular level

Short-chain acyl-CoA dehydrogenase (SCAD) deficiency is an inherited disorder of mitochondrial fatty acid oxidation associated with variations in the ACADS gene and variable clinical symptoms. In addition to rare ACADS inactivating variations, two common variations, c.511C > T (p.Arg171Trp) and c.625G > A (p.Gly209Ser), have been identified in patients, but these are also present in up to 14% of normal populations leading to questions of their clinical relevance. The common variant alleles encode proteins with nearly normal enzymatic activity at physiological conditions in vitro. SCAD enzyme function, however, is impaired at increased temperature and the tendency to misfold increases under conditions of cellular stress. The present study examines misfolding of variant SCAD proteins identified in patients with SCAD deficiency. Analysis of the ACADS gene in 114 patients revealed 29 variations, 26 missense, one start codon, and two stop codon variations. In vitro import studies of variant SCAD proteins in isolated mitochondria from SCAD deficient (SCAD−/−) mice demonstrated an increased tendency of the abnormal proteins to misfold and aggregate compared to the wild-type, a phenomenon that often leads to gain-of-function cellular phenotypes. However, no correlation was found between the clinical phenotype and the degree of SCAD dysfunction. We propose that SCAD deficiency should be considered as a disorder of protein folding that can lead to clinical disease in combination with other genetic and environmental factors. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Fast in vitro translation system immobilized on a surface via specific biotinylation of the ribosome

The ribosome is the macromolecular machine responsible for translating the genetic code into polypeptide chains. Despite impressive structural and kinetic studies of the translation process, a number of challenges remain with respect to understanding the dynamic properties of the translation apparatus. Single-molecule techniques hold the potential of characterizing the structural and mechanical properties of macromolecules during their functional cycles in real time. These techniques often necessitate the specific coupling of biologically active molecules to a surface. Here, we describe a procedure for such coupling of functionally active ribosomes that permits single-molecule studies of protein synthesis. Oxidation with NaIO4 at the 3' end of 23S rRNA and subsequent reaction with a biotin hydrazide produces biotinylated 70S ribosomes, which can be immobilized on a streptavidin-coated surface. The surface-attached ribosomes are fully active in poly(U) translation in vitro, synthesizing poly(Phe) at a rate of 3-6 peptide bonds/s per active ribosome at 37 degrees C. Specificity of binding of biotinylated ribosomes to a streptavidin-coated quartz surface was confirmed by observation of individual fluorescently labeled, biotinylated 70S ribosomes, using total internal reflection fluorescence microscopy. Functional interactions of the immobilized ribosomes with various components of the protein synthesis apparatus are shown by use of surface plasmon resonance.     

Regeneration from Phylloclade Explants and Callus Cultures of Schlumbergera and Rhipsalidopsis

Phylloclade explants of Schlumbergera and Rhipsalidopsis were cultured in vitro to produce axillary and adventitious shoots. The explants of both species, taken from greenhouse-grown plants, produced only axillary shoots. There was a pronounced improvement in adventitious shoot formation in phylloclade explants of cultivar CB4 of Rhipsalidopsis by increasing numbers of subcultures of axillary shoots used as donor plants. The axillary shoots generated from the explants were either subcultured to produce successive generations of axillary shoot cultures or made into phylloclade explants and tested for adventitious shoot formation at each subculture. The duration of each subculture varied from 6 to 12 weeks. After the first subculture, sporadic adventitious shoot formation began, and after the third subculture 87% explants of cultivar CB4 produced adventitious shoots at a frequency of about 12 shoots per explant. In contrast, there was no improvement in regenerative ability in explants of cultivar Thor-Olga of Schlumbergera up to third subculture. Adventitious shoots could be produced by callus culture too. Cultivar CB4 was highly regenerative, producing as many as 10 adventitious shoots per square cm of callus. In vitro grown plantlets, when transferred to pots continued to show prolific growth.     

Comparison of the spectra of genetic damage in formaldehyde-induced ad-3 mutations between DNA repair-proficient and -deficient heterokaryons of Neurospora crassa.

The mutagenic effects of formaldehyde (FA) have been compared in DNA repair-proficient (heterokaryon 12) and DNA repair-deficient (heterokaryon 59) two-component heterokaryons of Neurospora crassa. The data from forward-mutation experiments were used to compare the spectra of FA-induced specific-locus mutations at two closely linked loci in the adenine-3 (ad-3) region and on the FA-induced inactivation of heterokaryotic conidia. Previous studies have demonstrated that specific-locus mutations at these two loci result from five major genotypic classes, namely two classes of gene/point mutations (ad-3A(R) and ad-3B(R)), and three classes of multilocus deletion mutations ([ad-3A](IR), [ad-3B](IR), and [ad-3A ad-3B](IR)). Genetic analysis of ad-3 mutants recovered from both heterokaryons after FA treatment demonstrates that predominantly gene/point mutations were found in H-12 (93.2% ad-3(R), 6.8% [ad-3](IR)) and a significantly higher frequency of multilocus deletion mutations in H-59 (62.8% ad-3(R), 37.0% [ad-3](IR)). The data from our experiments with FA on H-12 demonstrate and confirm the data from other assays that FA is a weak mutagen in this DNA repair-proficient strain. However, the data from our experiments with the DNA repair-deficient strain H-59 demonstrate that comparable concentrations of FA cause more pronounced inactivation of heterokaryotic conidia and, at the highest concentration tested, about a 35-fold higher frequency of ad-3 mutations. In addition, FA induced a 5.4-fold higher frequency of ad-3 mutations resulting from multilocus deletion mutation in H-59 than in H-12. Based on our earlier studies with X-ray-induced multilocus deletion mutations, it is this class of FA-induced ad-3 mutations that might be most expected to show deleterious heterozygous effects. The implications of the present data base from our experiments with Neurospora are that the mutagenic (and possibly the carcinogenic) effect of FA exposure might well vary in different human population subgroups.     

The reactivation of demembranated human spermatozoa lacking outer dynein arms is independent of pH

The effect of pH, Mg-ATP, and free calcium on activity of the inner dynein arm was investigated using demembranated human spermatozoa lacking the outer dynein arms (LODA). The results were compared with those obtained for demembranated-reactivated normal spermatozoa to evaluate the functional properties of the inner and outer dynein arms in axonemal motility. The reactivation of Triton X-100–demembranated LODA spermatozoa was analysed at various pHs and concentrations of Mg-ATP and calcium using video recordings. The percentage of reactivated LODA spermatozoa as a function of Mg-ATP concentration was not dependent on pH, whereas reactivation of normal human spermatozoa is pH dependent. This suggests that there may be a pH-dependent regulatory mechanism associated with the outer dynein arms. A delay in the principal bend propagation of normal and LODA reactivated cells was found at pH 7.1. This disappeared at pH 7.8 in normal but not in LODA populations. This suggests a role for outer dynein arms in the initiation of the propagation of flagellar bends at alkaline pH. The level of LODA and normal sperm reactivation both depended on the calcium concentration in the medium. At lower free calcium concentrations, the reactivation level and beat frequency of reactivated cells were higher. Our results suggest a functional difference between outer and inner dynein arms of human spermatozoa based on a differential pH sensitivity. Moreover, calcium seems to exert its regulatory action elsewhere than on the outer dynein arms. Mol. Reprod. Dev. 49:416–425, 1998. © 1998 Wiley-Liss, Inc.     

SARS-CoV-2 seroprevalence in health care workers from 10 hospitals in Quebec,Canada: a cross-sectional study

Background:The COVID-19 pandemic has disproportionately affected health care workers. We sought to estimate SARS-CoV-2 seroprevalence among hospital health care workers in Quebec, Canada, after the first wave of the pandemic and to explore factors associated with SARS-CoV-2 seropositivity.Methods:Between July 6 and Sept. 24, 2020, we enrolled health care workers from 10 hospitals, including 8 from a region with a high incidence of COVID-19 (the Montréal area) and 2 from low-incidence regions of Quebec. Eligible health care workers were physicians, nurses, orderlies and cleaning staff working in 4 types of care units (emergency department, intensive care unit, COVID-19 inpatient unit and non-COVID-19 inpatient unit). Participants completed a questionnaire and underwent SARS-CoV-2 serology testing. We identified factors independently associated with higher seroprevalence.Results:Among 2056 enrolled health care workers, 241 (11.7%) had positive SARS-CoV-2 serology. Of these, 171 (71.0%) had been previously diagnosed with COVID-19. Seroprevalence varied among hospitals, from 2.4% to 3.7% in low-incidence regions to 17.9% to 32.0% in hospitals with outbreaks involving 5 or more health care workers. Higher seroprevalence was associated with working in a hospital where outbreaks occurred (adjusted prevalence ratio 4.16, 95% confidence interval [CI] 2.63–6.57), being a nurse or nursing assistant (adjusted prevalence ratio 1.34, 95% CI 1.03–1.74) or an orderly (adjusted prevalence ratio 1.49, 95% CI 1.12–1.97), and Black or Hispanic ethnicity (adjusted prevalence ratio 1.41, 95% CI 1.13–1.76). Lower seroprevalence was associated with working in the intensive care unit (adjusted prevalence ratio 0.47, 95% CI 0.30–0.71) or the emergency department (adjusted prevalence ratio 0.61, 95% CI 0.39–0.98).Interpretation:Health care workers in Quebec hospitals were at high risk of SARS-CoV-2 infection, particularly in outbreak settings. More work is needed to better understand SARS-CoV-2 transmission dynamics in health care settings.

The COVID-19 pandemic has disproportionately affected health care workers. In France, Spain, Italy, Germany and the United States, at least 10% of cases reported in spring 2020 were in health care workers.¹ In Quebec, 25% (14 177 of 56 565) of all cases declared during the first wave of the pandemic, from March to July 2020, were in health care workers,² about one-third of whom were working in acute care hospitals.¹ The Montréal area was the most affected region in Quebec and Canada during the first wave, reaching a COVID-19 incidence rate of 1336 per 100 000 population.²The number of COVID-19 cases reported among health care workers underestimated the number of those infected with SARS-CoV-2 during that period, given limited testing leading to undiagnosed asymptomatic or paucisymptomatic infections.³ Seroprevalence studies are an important tool to determine the proportion of people infected with SARS-CoV-2, both in the general population and among health care workers.⁴ After the first wave in Quebec, SARS-CoV-2 seroprevalence in adults aged 18–69 years was found to be low (3.1% in Montréal and 1.3% in less affected regions), but this proportion could be much higher among health care workers who had to work despite the general shutdown of social and economic activities, especially if they were exposed to major outbreaks.⁵Only 2 other Canadian studies provide SARS-CoV-2 seroprevalence estimates among health care workers, and both studies were from a single centre.^6,7 Outside Canada, most seroprevalence studies among health care workers include a single site and do not provide a representative estimate for a defined region.⁸ Several studies have reported a higher seroprevalence among health care workers from units treating patients with COVID-19 (COVID-19 units) compared with other units (non–COVID-19 units), emergency departments or intensive care units.^9,10 Other studies have not identified such associations.^11,12 In this study, we aimed to assess the seroprevalence of SARS-CoV-2 antibody among hospital health care workers from a variety of settings after the first pandemic wave in Quebec, and to explore factors associated with SARS-CoV-2 seropositivity.     

X-ray-induced specific-locus mutations in the ad-3 region of two-component heterokaryons of Neurospora crassa. VII. Genetic lesions resulting in gene/point mutations at the ad-3B locus have different dose-response relationships

Genetic characterization of X-ray-induced ad-3 mutants, induced in a two-component heterokaryon (H-12) of Neurospora crassa, has been performed to determine genotype, patterns of allelic complementation, and leakiness, and to distinguish gene/point mutations from multilocus deletions and multiple locus mutations (de Serres, 1989c, 1990a). The array of genotypes in the classes and subclasses in the spectrum of ad-3 mutants induced by a mutagenic agent constitute its mutagenicity profile; for X-rays this profile consists of 29 different genotypes. In the present paper, the data on gene/point mutations induced at the ad-3B locus (designated ad-3BR mutations) have been tabulated as a function of X-ray dose to determine the dose-dependent relationships of complementing and noncomplementing mutants. This analysis has shown that the overall percentages of mutants showing allelic complementation and the percentages of complementing mutants with nonpolarized patterns (both leaky and nonleaky) and noncomplementing mutants were dose-dependent; the former class decreased with increasing X-ray dose, and the latter class increased with increasing X-ray dose. The percentages of complementing mutants with polarized patterns were X-ray dose-independent. In addition, an unexpectedly high frequency of mutants with nonpolarized complementation patterns are discontinuous and probably result from multiple-site mutations.     

Older adults can maximally activate the biceps brachii muscle by voluntary command

De Serres, Sophie J., and Roger M. Enoka. Older adultscan maximally activate the biceps brachii muscle by voluntary command.J. Appl. Physiol. 84(1): 284-291, 1998.Because some of the decline in strength with age may beexplained by an impairment of muscle activation, the purpose of thisstudy was to determine the activation level achieved in biceps brachiiby older adults during a maximum voluntary contraction (MVC). Thiscapability was assessed with two superimposition techniques: onecalculated the activation level that was achieved during an MVC, andthe other provided an estimate of the expected MVC force based on extrapolation with submaximal forces. The activation level in bicepsbrachii was incomplete (<100%) for the young(n = 16) and elderly(n = 16) subjects, with the elderlysubjects exhibiting the greater deficit. In contrast, there was nodifference between the measured and expected MVC forces for eithergroup of subjects, whether the extrapolation involved a third-orderpolynomial or linearization of the data. Because of the lowersignal-to-noise ratio associated with the measurement of activationlevel and the greater number of measurements that contributed to theestimate of the expected MVC force, we conclude that the older adultswere able to achieve complete activation of the biceps brachii muscle during an MVC.

     

Kinetics of lactate,acetate and propionate in unadapted and lactate-adapted thermophilic,anaerobic sewage sludge: the influence of sludge adaptation for start-up of thermophilic UASB-reactors

Summary A thermophilic anaerobic sludge digestor was adapted to lactate metabolism. The adapted sludge showed an improved capacity for lactate degradation when tested by a batch activity test, compared to the performance of unadapted sludge. Acetate was the major intermediate produced during the degradation. When adapted sludge was used as the inoculum for a lactate-fed, upflow anaerobic sludge blanket (UASB) reactor, the chemical oxygen demand reduction rate was higher than with unadapted sludge. After 39 days, however, the difference vanished due to an extensive wash-out of sludge from the reactor inoculated with adapted sludge.Offprint requests to: B. K. Ahring     

X-ray-induced specific-locus mutations in the ad-3 region of two-component heterokaryons of Neurospora crassa. VIII. Dose-dependence of the overall spectrum

There is considerable controversy in the literature concerning the nature of X-ray-induced specific-locus mutations in various experimental organisms. To investigate this problem in Neurospora crassa a series of experiments (Webber and de Serres, 1965) was performed to study the induction-kinetics of X-ray-induced mutation in the adenine-3 (ad-3) region of a two-component heterokaryon (H-12). Subsequent genetic analyses (de Serres, 1989a,b,c, 1990a), on a series of 832 mutants recovered in these experiments, have shown that 3 different classes of ad-3 mutants were recovered, namely gene/point mutations, multilocus deletions and multiple-site mutations. Complementation studies with a series of genetic markers that define 21 genetic loci in the ad-3 and immediately adjacent genetic regions have shown that ad-3 mutants classified as multilocus deletions result from the inactivation of a series of loci in the ad-3 and immediately adjacent regions of Linkage Group I, whereas multiple-locus mutations result from combinations of gene/point mutations and multilocus deletions. Analysis of the induction kinetics of these 3 different classes, after completion of the genetic characterization of all mutants (de Serres, 1990b) demonstrated that gene/point mutations increase linearly with X-ray dose, whereas multilocus deletions and multiple-site mutations increase as the square of X-ray dose. Further analysis of allelic complementation among the gene/point mutations at the ad-3B locus (de Serres, 1990c), demonstrated that the spectrum of complementation patterns was dose-dependent: complementing mutants with nonpolarized patterns decreased and noncomplementing mutations increased with increasing X-ray dose. There was little or no change with dose in the frequency of mutants with polarized patterns. In the present report, data from studies published previously have been utilized, along with additional data from the original X-ray experiments (12-5, 12-6, 12-7, and 12-10; see Webber and de Serres, 1965) to develop composite complementation maps of the X-ray-induced specific-locus mutations in the ad-3 and immediately adjacent regions as a function of X-ray dose. This analysis of the overall spectrum of X-ray-induced specific-locus mutations in the ad-3 region demonstrated marked dose-dependence and provides an explanation for the discrepancies in the literature with regard to specific-locus studies in different experimental organisms.