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The germ cell-less gene is crucial for gonad development in various organisms. Early interventions in its expression suggested a regulatory role at the mitotic stages of spermatogenesis, and its early knockout resulted in complete sterility in Drosophila. Genomic and transcriptomic data available for the catadromous giant prawn Macrobrachium rosenbergii enabled the identification of a germ cell-less homolog for this species, which we termed MroGCL (mRNA accession number OQ533056). An open reading frame containing 494 amino acids and a typical evolutionarily conserved BTB/POZ domain suggests possible protein-protein interaction functions in keeping with the Drosophila germ cell-less protein. Genomic mapping of MroGCL showed a full length of 120 896 bases. Analysis of the temporal expression of MroGCL showed constant expression in early prawn embryonic and larval stages, but a significant increase 10 days after metamorphosis when crucial sexual differentiation processes occur in prawns. In adult animals, high expression was detected in the gonads compared to the somatic tissues. RNAi-based knock-down experiments showed that both the silenced and control groups reached advanced spermatogenic stages, but that there was a significant decrease in the yield of spermatozoa in about half of the silenced animals. This finding supports our hypothesis that MroGCL is crucial for mitosis during early stage spermatogenesis. In conclusion, this study contributes to the understanding of crustacean gonad development and provides a stepping stone in the development of environmentally valuable sterile crustacean populations., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Rapid relaxed-clock dating methods are frequently applied to analyze phylogenomic data sets containing hundreds to thousands of sequences because of their accuracy and computational efficiency. However, the relative performance of different rapid dating methods is yet to be compared on the same data sets, and, thus, the power and pitfalls of selecting among these approaches remain unclear. We compared the accuracy, bias, and coverage probabilities of RelTime, treePL, and least-squares dating time estimates by applying them to analyze computer-simulated data sets in which evolutionary rates varied extensively among branches in the phylogeny. RelTime estimates were consistently more accurate than the other two, particularly when evolutionary rates were autocorrelated or shifted convergently among lineages. The 95% confidence intervals (CIs) around RelTime dates showed appropriate coverage probabilities (95% on average), but other methods produced rather low coverage probabilities because of overly narrow CIs of time estimates. Overall, RelTime appears to be a more efficient method for estimating divergence times for large phylogenies., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Background: Mitochondrial diseases (MD) are genetic metabolic disorders that impair normal mitochondrial structure or function. The aim of this study was to investigate the status of circulating cell-free mitochondrial DNA (ccfmtDNA) in cerebrospinal fluid (CSF), together with other biomarkers (growth differentiation factor-15 [GDF-15], alanine, and lactate), in a cohort of 25 patients with a molecular diagnosis of MD., Methods: Measurement of ccfmtDNA was performed by using droplet digital PCR., Results: The mean copy number of ccfmtDNA was approximately 6 times higher in the MD cohort compared to the control group; patients with mitochondrial deletion and depletion syndromes (MDD) had the higher levels. We also detected the presence of both wild-type mtDNA and mtDNA deletions in CSF samples of patients with single deletions. Patients with MDD with single deletions had significantly higher concentrations of GDF-15 in CSF than controls, whereas patients with point mutations in mitochondrial DNA presented no statistically significant differences. Additionally, we found a significant positive correlation between ccfmtDNA levels and GDF-15 concentrations (r = 0.59, P = 0.016)., Conclusion: CSF ccfmtDNA levels are significantly higher in patients with MD in comparison to controls and, thus, they can be used as a novel biomarker for MD research. Our results could also be valuable to support the clinical outcome assessment of MD patients., (© American Association for Clinical Chemistry 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Motivation: Precise time calibrations needed to estimate ages of species divergence are not always available due to fossil records' incompleteness. Consequently, clock calibrations available for Bayesian dating analyses can be few and diffused, i.e. phylogenies are calibration-poor, impeding reliable inference of the timetree of life. We examined the role of speciation birth-death (BD) tree prior on Bayesian node age estimates in calibration-poor phylogenies and tested the usefulness of an informative, data-driven tree prior to enhancing the accuracy and precision of estimated times., Results: We present a simple method to estimate parameters of the BD tree prior from the molecular phylogeny for use in Bayesian dating analyses. The use of a data-driven birth-death (ddBD) tree prior leads to improvement in Bayesian node age estimates for calibration-poor phylogenies. We show that the ddBD tree prior, along with only a few well-constrained calibrations, can produce excellent node ages and credibility intervals, whereas the use of an uninformative, uniform (flat) tree prior may require more calibrations. Relaxed clock dating with ddBD tree prior also produced better results than a flat tree prior when using diffused node calibrations. We also suggest using ddBD tree priors to improve the detection of outliers and influential calibrations in cross-validation analyses.These results have practical applications because the ddBD tree prior reduces the number of well-constrained calibrations necessary to obtain reliable node age estimates. This would help address key impediments in building the grand timetree of life, revealing the process of speciation and elucidating the dynamics of biological diversification., Availability and Implementation: An R module for computing the ddBD tree prior, simulated datasets and empirical datasets are available at https://github.com/cathyqqtao/ddBD-tree-prior., (© The Author(s) 2021. Published by Oxford University Press.)

Motivation: As the number and diversity of species and genes grow in contemporary datasets, two common assumptions made in all molecular dating methods, namely the time-reversibility and stationarity of the substitution process, become untenable. No software tools for molecular dating allow researchers to relax these two assumptions in their data analyses. Frequently the same General Time Reversible (GTR) model across lineages along with a gamma (+Γ) distributed rates across sites is used in relaxed clock analyses, which assumes time-reversibility and stationarity of the substitution process. Many reports have quantified the impact of violations of these underlying assumptions on molecular phylogeny, but none have systematically analyzed their impact on divergence time estimates., Results: We quantified the bias on time estimates that resulted from using the GTR + Γ model for the analysis of computer-simulated nucleotide sequence alignments that were evolved with non-stationary (NS) and non-reversible (NR) substitution models. We tested Bayesian and RelTime approaches that do not require a molecular clock for estimating divergence times. Divergence times obtained using a GTR + Γ model differed only slightly (∼3% on average) from the expected times for NR datasets, but the difference was larger for NS datasets (∼10% on average). The use of only a few calibrations reduced these biases considerably (∼5%). Confidence and credibility intervals from GTR + Γ analysis usually contained correct times. Therefore, the bias introduced by the use of the GTR + Γ model to analyze datasets, in which the time-reversibility and stationarity assumptions are violated, is likely not large and can be reduced by applying multiple calibrations., Availability and Implementation: All datasets are deposited in Figshare: https://doi.org/10.6084/m9.figshare.12594638., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The conventional wisdom in molecular evolution is to apply parameter-rich models of nucleotide and amino acid substitutions for estimating divergence times. However, the actual extent of the difference between time estimates produced by highly complex models compared with those from simple models is yet to be quantified for contemporary data sets that frequently contain sequences from many species and genes. In a reanalysis of many large multispecies alignments from diverse groups of taxa, we found that the use of the simplest models can produce divergence time estimates and credibility intervals similar to those obtained from the complex models applied in the original studies. This result is surprising because the use of simple models underestimates sequence divergence for all the data sets analyzed. We found three fundamental reasons for the observed robustness of time estimates to model complexity in many practical data sets. First, the estimates of branch lengths and node-to-tip distances under the simplest model show an approximately linear relationship with those produced by using the most complex models applied on data sets with many sequences. Second, relaxed clock methods automatically adjust rates on branches that experience considerable underestimation of sequence divergences, resulting in time estimates that are similar to those from complex models. And, third, the inclusion of even a few good calibrations in an analysis can reduce the difference in time estimates from simple and complex models. The robustness of time estimates to model complexity in these empirical data analyses is encouraging, because all phylogenomics studies use statistical models that are oversimplified descriptions of actual evolutionary substitution processes., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Physical stress, including high temperatures, may damage the central metabolic nicotinamide nucleotide cofactors [NAD(P)H], generating toxic derivatives [NAD(P)HX]. The highly conserved enzyme NAD(P)HX dehydratase (NAXD) is essential for intracellular repair of NAD(P)HX. Here we present a series of infants and children who suffered episodes of febrile illness-induced neurodegeneration or cardiac failure and early death. Whole-exome or whole-genome sequencing identified recessive NAXD variants in each case. Variants were predicted to be potentially deleterious through in silico analysis. Reverse-transcription PCR confirmed altered splicing in one case. Subject fibroblasts showed highly elevated concentrations of the damaged cofactors S-NADHX, R-NADHX and cyclic NADHX. NADHX accumulation was abrogated by lentiviral transduction of subject cells with wild-type NAXD. Subject fibroblasts and muscle biopsies showed impaired mitochondrial function, higher sensitivity to metabolic stress in media containing galactose and azide, but not glucose, and decreased mitochondrial reactive oxygen species production. Recombinant NAXD protein harbouring two missense variants leading to the amino acid changes p.(Gly63Ser) and p.(Arg608Cys) were thermolabile and showed a decrease in Vmax and increase in KM for the ATP-dependent NADHX dehydratase activity. This is the first study to identify pathogenic variants in NAXD and to link deficient NADHX repair with mitochondrial dysfunction. The results show that NAXD deficiency can be classified as a metabolite repair disorder in which accumulation of damaged metabolites likely triggers devastating effects in tissues such as the brain and the heart, eventually leading to early childhood death.

Primates have long been a test case for the development of phylogenetic methods for divergence time estimation. Despite a large number of studies, however, the timing of origination of crown Primates relative to the Cretaceous-Paleogene (K-Pg) boundary and the timing of diversification of the main crown groups remain controversial. Here, we analysed a data set of 372 taxa (367 Primates and 5 outgroups, 3.4 million aligned base pairs) that includes nine primate genomes. We systematically explore the effect of different interpretations of fossil calibrations and molecular clock models on primate divergence time estimates. We find that even small differences in the construction of fossil calibrations can have a noticeable impact on estimated divergence times, especially for the oldest nodes in the tree. Notably, choice of molecular rate model (autocorrelated or independently distributed rates) has an especially strong effect on estimated times, with the independent rates model producing considerably more ancient age estimates for the deeper nodes in the phylogeny. We implement thermodynamic integration, combined with Gaussian quadrature, in the program MCMCTree, and use it to calculate Bayes factors for clock models. Bayesian model selection indicates that the autocorrelated rates model fits the primate data substantially better, and we conclude that time estimates under this model should be preferred. We show that for eight core nodes in the phylogeny, uncertainty in time estimates is close to the theoretical limit imposed by fossil uncertainties. Thus, these estimates are unlikely to be improved by collecting additional molecular sequence data. All analyses place the origin of Primates close to the K-Pg boundary, either in the Cretaceous or straddling the boundary into the Palaeogene.

The m.1555A>G mtDNA variant causes maternally inherited deafness, but the reasons for the highly variable clinical penetrance are not known. Exome sequencing identified a heterozygous start loss mutation in SSBP1, encoding the single stranded binding protein 1 (SSBP1), segregating with hearing loss in a multi-generational family transmitting m.1555A>G, associated with mtDNA depletion and multiple deletions in skeletal muscle. The SSBP1 mutation reduced steady state SSBP1 levels leading to a perturbation of mtDNA metabolism, likely compounding the intra-mitochondrial translation defect due to m.1555A>G in a tissue-specific manner. This family demonstrates the importance of rare trans-acting genetic nuclear modifiers in the clinical expression of mtDNA disease., (© The Author (2017). Published by Oxford University Press on behalf of the Guarantors of Brain.)

Both nymphal and adult stages of several species of spittlebugs (Hemiptera: Cercopidae) are key economic pests of brachiariagrasses (Brachiaria spp.) in tropical America. Progress has been made in the characterization and development of antibiosis resistance to nymphs in brachiariagrasses. Essentially no attention has been given to screening germplasm for resistance to adults. To support current breeding programs, a series of experiments was conducted to develop a methodology to screen for adult damage and to study categories of resistance to adult feeding damage. Six host brachiariagrass genotypes were used: two susceptible checks (CIAT 0606 and CIAT 0654) and four nymph-resistant genotypes (CIAT 6294, CIAT 36062, CIAT 36087, and SX01NO/0102). Test insects were Aeneolamia varia (F.) and Zulia carbonaria (Lallemand). None of the nymph-resistant genotypes was antibiotic to adults. All four nymph-resistant genotypes showed tolerance to A. varia and Z. carbonaria feeding damage. The levels of tolerance to adults of Z. carbonaria, a larger, more aggressive species, were lower. Of the four nymph-resistant genotypes, only CIAT 6294 and CIAT 36087 showed some tolerance to Z. carbonaria expressed as lower leaf damage scores, less chlorophyll loss, and lower functional plant loss indices. The fact that a genotype like SX01NO/0102, which is highly antibiotic to nymphs, is susceptible to adult damage suggests that mechanisms of resistance to the two spittlebug life stages may be independent. Results of these studies suggest a need to incorporate routine screening for tolerance to adult feeding damage as an additional selection criterion in the breeding scheme.

Several greenhouse experiments were used to measure how high levels of antibiosis resistance to nymphs in two interspecific Brachiaria (brachiariagrass) hybrids affect life history parameters of the spittlebugs Aeneolamia varia (F.) and Zulia carbonaria (Lallemand), two of the most important spittlebug (Hemiptera: Cercopidae) species affecting Brachiaria production in Colombia. The A. varia-resistant hybrid CIAT 36062, the Z. carbonaria-resistant hybrid SX01NO/0102, and the susceptible accession CIAT 0654 were used to compare the effect of all possible combinations of food sources for nymphs and adults. Calculation of growth indexes showed a significant impact of antibiosis resistance on the biology of immature stages of both species. Median survival times of adults feeding on resistant genotypes did not differ from those recorded on the susceptible genotype, suggesting that factors responsible for high mortality of nymphs in the resistant hybrids did not affect adult survival. Rearing nymphs of A. varia on CIAT 36062 and of Z. carbonaria on SX01NO/0102 had deleterious sublethal effects on the reproductive biology of resulting adult females. It is concluded that high nymphal mortality and subsequent sublethal effects of nymphal antibiosis on adults should have a major impact on the demography of the two spittlebug species studied.

Objective: To assess the utility of liquefaction degeneration as a marker of apoptosis in oral lichen planus (OLP)., Methods: TdT-mediated dUTP-biotin nick-end labelling (TUNEL) assay and immunohistochemical methods were used to detect p21 proteins and the active form of caspase 3 in 32 tissue samples of oral mucosa with OLP and 20 samples of normal oral mucosa., Results: Liquefaction degeneration was moderate or intense in 27.5% (n=8) and slight in 72.4% (n=21) of OLP samples. There was low expression of apoptosis markers (TUNEL, active caspase 3 form), which was not significantly associated with liquefaction degeneration of the basal cell layer. Basal and suprabasal expression of p21 was significantly more frequent in samples with more intense liquefaction degeneration of basal cells (P<0.01)., Conclusions: Our results demonstrate that liquefaction degeneration, as a morphological expression of T lymphocyte attack, does not unequivocally indicate apoptosis. Attacked basal cells more frequently respond with cell-cycle arrest or senescence than with apoptosis.

Background: Adult film production is a legal, multibillion dollar industry in California. In response to reports of human immunodeficiency virus (HIV) transmission by an adult film worker, we sought to determine the extent of HIV infection among exposed workers and to identify means of improving worker safety., Methods: The Los Angeles County Department of Health Services initiated an outbreak investigation that included interviews of infected workers to elicit information about recent sex partners, review of the testing agency's medical records and laboratory results, molecular analysis of HIV isolates from the 4 infected workers, and a risk assessment of HIV transmission in the adult film industry., Results: Many adult film workers participate in a monthly program of screening for HIV infection by means of polymerase chain reaction-based technology to detect HIV DNA in blood. A male performer tested negative for HIV on 12 February 2004 and 17 March 2004, then tested positive for HIV on 9 April 2004. During the period between the negative test results, he experienced a flulike illness after performing unprotected vaginal and anal intercourse for an adult film produced outside the United States by a US company. After returning to California, he performed unprotected sex acts for adult films with 13 female partners who had all tested negative for HIV in the preceding 30 days; 3 subsequently tested positive for HIV (a 23% attack rate). Contact tracing identified no reasonable sources of infection other than the male index patient., Conclusion: Although current testing methods may shorten the window period to diagnosis of new HIV infection, they fail to prevent occupational acquisition of HIV in this setting. A California Occupational Safety and Health Administration-approved written health and safety program that emphasizes primary prevention is needed for this industry.

Aims: Mycobacteria are a serious cause of infections in humans, with limited treatment options, as no new antibiotics have been developed against mycobacteria since the 1960s. In this study, the antimycobacterial activity of a small library of acetophenone (AP) compounds was analysed., Methods and Results: Twenty-three AP derivatives were examined for activity against mycobacteria using a microbroth assay. The compounds were bacteriostatic, with the most effective (cyclohexylacetophenone and piperidinoacetophenone) having minimal inhibitory concentrations of 246 microM. Active compounds tended to be more hydrophobic, and may work by alkylation of as yet undetermined intracellular target protein(s). Cytotoxicity against eukaryotic cells was also determined and appears to be unrelated to the bacteriostatic activity., Significance and Impact of the Study: AP may serve as a novel group of useful therapeutics against the mycobacteria.

The molecular mechanisms involved in the regulation of the balance between rRNA and mRNA in mitochondria are poorly understood. The mitochondrial transcription termination factor (mTERF) was highlighted as a potential transcription-control point. In this study, rat mTERF has been expressed in vitro and in Escherichia coli. The mature protein, in addition to the expected specific DNA-binding capacity for the sequence required for termination, has a new DNA-binding activity, and is able to bind to rat mitochondrial promoter region. This finding suggests communication between transcription initiation and termination regions. However, the results of a competition experiment argue against the formation of a complex between rat mTERF and the termination probe and promoter probe simultaneously, although it remains to be investigated whether another factor(s) might be involved in this interaction. In addition, recombinant human mTERF is also able to bind to human mitochondrial promoter region.

Mitochondrial transcription factor A (TFAM) stimulates transcription from mitochondrial DNA (mtDNA) promoters in vitro and in organello. To investigate whether changes of TFAM levels also modulate transcription and replication in situ, the protein was transiently overexpressed in cultured cells. Mitochondrial mRNAs were significantly elevated at early time points, when no expansion of the TFAM pool was yet observed, but were decreased when TFAM levels had doubled, resemb-ling in vitro results. HEK cells contain about 35 molecules of TFAM per mtDNA. High levels of TFAM were not associated with increases of full-length mtDNA, but nucleic acid species sensitive to RNAse H increased. Stimulation of transcription was more evident when TFAM was transiently overexpressed in cells pre-treated with ethidium bromide (EBr) having lowered mtDNA, TFAM and mitochondrial transcript levels. EBr rapidly inhibited mtDNA transcription, while decay of mtDNA was delayed and preferentially slowly migrating, relaxed mtDNA species were depleted. In conclusion, we show that transcription of mtDNA is submaximal in cultured cells and that a subtle increase of TFAM within the matrix is sufficient to stimulate mitochondrial transcription. Thus, this protein meets all criteria for being a key factor regulating mitochondrial transcription in vivo, but other factors are necessary for increasing mtDNA copy number, at least in cultured cells.

The present study evaluated the secretions of interleukin (IL)-1beta and tumor necrosis factor (TNF) alpha by fetal membranes stimulated with group B streptococci (GBS) and lipopolysaccharide (LPS). The aim was to evaluate the initial response of full-thickness membranes to the microbial insult using an in vitro experimental model that allowed testing of the individual contributions of amnion and choriodecidua to stimulation. Full-thickness membranes were obtained after delivery by elective cesarean section from women at 37-40 wk of gestation without evidence of active labor. The membranes were mounted in Transwell devices, physically separating the upper and lower chambers. The LPS (500 ng/ml) or GBS (1 x 10(6) colony-forming units/ml) was added to either the amniotic or choriodecidual surface, and accumulation of IL-1beta and TNFalpha were measured in both compartments using a specific ELISA. Fetal membranes followed different patterns of secretion of proinflammatory cytokines that depended on the side to which the stimulus was added or the nature of the stimulus itself. The TNFalpha was secreted by amnion and choriodecidua in the presence of LPS or GBS, and stimulation with GBS induced a greater synthesis of IL-1beta than did stimulation with LPS. Choriodecidual tissue was more responsive than amniotic tissue, and this response tended to be higher even when the stimulation was only on the amniotic side. However, the amnion plays an active role in recognizing LPS or GBS, contributing a significant amount of TNFalpha. Thus, cooperative and bidirectional communications occur between amnion and choriodecidua in response to bacterial products, which include intermembranous cytokine traffic and signaling between tissues.

Despite the crucial importance of mitochondrial transcription, knowledge of its regulation is poor. Therefore, characterization of mammalian mitochondrial transcription termination factor (mTERF) functionality and regulation is of fundamental biological interest in order to understand the regulation of mitochondrial transcription. Here we report that mTERF is the first protein having a role in mammalian mitochondrial gene expression that appears to be controlled by phosphorylation. Recombinant mature rat mTERF protein has specific DNA-binding capacity for the sequence required for transcription termination. Furthermore, unlike recombinant human mTERF, the rat protein bound to its mitochondrial DNA binding site promotes the termination of transcription initiated with heterologous RNA polymerase. Interestingly, mTERF is a phosphoprotein with four phosphate groups, and while the DNA-binding activity of mTERF is unaffected by the phosphorylation/dephosphorylation state, only the phosphorylated form of the protein is active for termination activity. Moreover, natural human mTERF is also a phosphoprotein and its termination activity is inhibited by dephosphorylation. These data suggest that mTERF functioning in vivo is regulated by phosphorylation.

Among patients undergoing heart transplantation, Aspergillus is the opportunistic pathogen with the highest attributable mortality. The median time of onset from transplantation for invasive pulmonary aspergillosis (IPA) was 46 days, but the median time to first positive culture result was 104 days among patients with Aspergillus colonization but no invasive disease. Most patients with IPA presented with fever and cough within the first 90 days of transplantation and with single or multiple pulmonary nodules. None of the heart transplant recipients with either IPA or invasive extrapulmonary aspergillosis (IEPA) had associated neutropenia. Human leukocyte antigen A1 locus was found significantly more frequently among patients colonized with Aspergillus than among patients with IPA (P<.006) or IEPA (P<.001). Even in the absence of neutropenia, IPA should be suspected for heart transplant recipients who have fever and respiratory symptoms within the first 3 months of transplantation, have a positive result of culture of respiratory secretions, and have abnormal radiological findings (particularly nodules).

Mitochondrial transcription factor A (TFAM) has been shown to stimulate transcription from mitochondrial DNA promoters in vitro. In order to determine whether changes in TFAM levels also regulate RNA synthesis in situ, recombinant human precursor proteins were imported into the matrix of rat liver mitochondria. After uptake of wt-TFAM, incorporation of [alpha-32P]UTP into mitochondrial mRNAs as well as rRNAs was increased 2-fold (P < 0.05), whereas import of truncated TFAM lacking 25 amino acids at the C-terminus had no effect. Import of wt-TFAM into liver mitochondria from hypothyroid rats stimulated RNA synthesis up to 4-fold. We conclude that the rate of transcription is submaximal in freshly isolated rat liver mitochondria and that increasing intra-mitochondrial TFAM levels is sufficient for stimulation. The low transcription rate associated with the hypothyroid state observed in vivo as well as in organello seems to be a result of low TFAM levels, which can be recovered by treating animals with T3 in vivo or by importing TFAM in organello. Thus, this protein meets the criteria for being a key factor in regulating mitochondrial gene expression in vivo.

Sperm mitochondria play an important role in spermatozoa because of the high ATP demand of these cells. Different mitochondrial DNA (mtDNA) mutations and haplogroups influence sperm function. The mtDNA dose also contributes to genetic variability and pathology in different tissues and organs, but nothing is known about its relevance in the performance of spermatozoa. We estimated the variability in mtDNA content within a population of men. Different mtDNA:nuclear DNA ratios were characteristic of progressive and nonprogressive spermatozoa, confirming the influence of mtDNA content on sperm functionality. We also estimated that the absolute content of mtDNA was 700 and 1200 mtDNA copies per cell in progressive and nonprogressive human spermatozoa, respectively. These results suggest that a marked increase of mtDNA copy number per cell volume takes place during spermatogenesis.

The sea urchin mitochondrial displacement (D)-loop binding protein mtDBP has been previously identified and cloned. The polypeptide (348 amino acids) displays a significant homology with the human mitochondrial transcription termination factor mTERF. This similarity, and the observation that the 3' ends of mitochondrial RNAs coded by opposite strands mapped in correspondence of mtDBP-binding sites, suggested that mtDBP could function as transcription termination factor in sea urchin mitochondria. To investigate such a role we tested the capability of mtDBP bound to its target sequence in the main non-coding region to affect RNA elongation by mitochondrial and bacteriophage T3 and T7 RNA polymerases. We show that mtDBP was able to terminate transcription bidirectionally when initiated by human mitochondrial RNA polymerase but only unidirectionally when initiated by T3 or T7 RNA polymerases. Time-course experiments indicated that mtDBP promotes true transcription termination rather than transcription pausing. These results indicate that mtDBP is able to function as a bipolar transcription termination factor in sea urchin mitochondria. The functional significance of such an activity could be linked to the previously proposed dual role of the protein in modulating mitochondrial DNA transcription and replication.

The light strand promoter of mammalian mitochondrial DNA gives rise to a primary transcript, but also to the RNA primer necessary for initiation of replication and 7S DNA synthesis as well as 7S RNA. Here we have studied the turnover of 7S DNA in isolated rat liver mitochondria and whether import of mitochondrial transcription factor A (mtTFA), which is necessary for transcription initiation, increases its rate of synthesis. 7S DNA was present as two species, probably due to two different sites of RNA-DNA transition. Time course and pulse-chase experiments showed that the half-life of this DNA is approximately 45 min. Import of mtTFA, produced in vitro, into the mitochondrial matrix in stoichiometric amounts significantly increased the rate of 7S DNA formation. We conclude that isolated rat liver mitochondria faithfully synthesize and degrade 7S DNA and that increased matrix levels of mtTFA are sufficient to increase its rate of synthesis, strongly supporting the hypothesis that this process is transcription primed.

A total of 1073 infectious episodes (IEs) that occurred in 620 consecutive heart transplantation patients at Stanford Medical Center between 16 December 1980 and 30 June 1996 were reviewed. Infectious complications were a major cause of morbidity and mortality, second only to rejection as the cause of early deaths and the most common cause of late deaths. Of the IEs, 468 (43.6%) were caused by bacteria, 447 (41.7%) by viruses, 109 (10.2%) by fungi, 43 (4.0%) by Pneumocystis carinii, and 6 (0.6%) by protozoa. The largest number of IEs occurred in the lungs (301 [28.1%]). A significant reduction in the incidence of IEs and a delay in presentation after transplantation were observed; these were most likely related to the introduction of new chemoprophylactic regimens during the study period and prevention of significant disease caused by cytomegalovirus.

The usefulness of testing for IgG avidity in association with Toxoplasma gondii was evaluated in a US reference laboratory. European investigators have reported that high-avidity IgG toxoplasma antibodies exclude acute infection in the preceding 3 months. In this US study, 125 serum samples taken from 125 pregnant women in the first trimester were chosen retrospectively, because either the IgM or differential agglutination (AC/HS) test in the Toxoplasma serologic profile suggested or was equivocal for a recently acquired infection. Of 93 (74.4%) serum samples with either positive or equivocal results in the IgM ELISA, 52 (55.9%) had high-avidity antibodies, which suggests that the infection probably was acquired before gestation. Of 87 (69.6%) serum samples with an acute or equivocal result in the AC/HS test, 35 (40.2%) had high-avidity antibodies. Forty women were given spiramycin, to prevent congenital transmission, and 7 (17.5%) had high-avidity antibodies. These findings highlight the value of testing a single serum sample obtained in the first trimester of pregnancy for IgG avidity.

Meningitis caused by Pseudallescheria boydii is an uncommon infection of the CNS that usually has a poor prognosis and a difficult treatment. We describe a case of chronic meningitis caused by P. boydii in an immunocompetent host that was successfully treated with voriconazole, a new antifungal agent.

The efficacy of sequential protease inhibitor therapy was studied in 16 human immunodeficiency virus (HIV) 1-infected persons in whom saquinavir with multiple nucleoside reverse transcriptase (RT) inhibitors (NRTI) had failed. Nelfinavir plus two NRTIs (new or continued) resulted in minimal (0.59 log RNA copies/mL) and transient (8 weeks) suppression of plasma HIV RNA levels. Rapid failure was surprisingly associated with baseline presence of protease gene mutation L90M (P=.04) in the absence of D30N and with RT mutations D67N (P<.01), K70R/S (P=.02), and K219Q/W/R/E (P<.01). Ten patients were subsequently switched to indinavir plus nevirapine and 2 NRTIs, resulting in a median 1.62 log reduction in plasma HIV RNA, with 3 patients maintaining 400 copies/mL for 24 weeks. These results suggest that nelfinavir may have limited utility after saquinavir failure, particularly without potent concomitant therapy. Combining an NRTI with a new protease inhibitor for rescue may improve response.

Hantavirus pulmonary syndrome (HPS) is characterized by the rapid onset of pulmonary edema and a high case-fatality rate. Hantavirus antigens have been demonstrated in pulmonary capillary endothelial cells, but the mechanisms causing capillary leakage remain unclear. Immunohistochemical staining was used to enumerate cytokine-producing cells (monokines: interleukin [IL]-1alpha, IL-1beta, IL-6, and tumor necrosis factor [TNF]-alpha; lymphokines: interferon-gamma, IL-2, IL-4, and TNF-beta) in tissues obtained at autopsy from subjects with HPS. High numbers of cytokine-producing cells were seen in the lung and spleen tissues of HPS patients, but only low numbers in the livers and kidneys. A modest increase in the numbers of cytokine-producing cells was detected in the lungs of patients who died with non-HPS acute respiratory distress syndrome (ARDS), and very few (or no) cytokine-producing cells were detected in the lungs of patients who died of causes other than ARDS. These results suggest that local cytokine production may play an important role in the pathogenesis of HPS.

Until now, little attention has been paid to the contribution of mitochondrial dysfunction to germinal tissue disorders. The target of this study was to investigate the relationship between sperm motility and mitochondrial respiratory chain enzyme activities. The results obtained showed that semen samples of control individuals (n = 33) have substantially higher activities of complexes I, II, and IV compared with those of asthenozoospermic subjects (n = 86). Moreover, a direct and positive correlation was found in the whole population studied between spermatozoa motility and all the mitochondrial respiratory complex activities assayed (I, II, I+III, II+III, and IV). The ratio of these enzymes to citrate synthase (a reliable enzymatic marker of mitochondrial volume) activities did not correlate with sperm motility. This suggests that motility depends largely on the mitochondrial volume within the sperm midpiece. These observations could be of physiopathological relevance because they suggest that factors affecting the mitochondrial energy production could be then responsible for particular cases of idiopathic asthenozoospermia.

The presence of both toxoplasmic myocarditis and myositis in the same individual has been reported only at autopsy. We report the first case of biopsy-proven toxoplasmic myocarditis and polymyositis simultaneously occurring in the same individual that was diagnosed during life. Results of her toxoplasmic serology were consistent with acute toxoplasmosis. She subsequently developed visual symptoms consistent with toxoplasmic chorioretinitis. She had a positive clinical response to therapeutic agents specific against Toxoplasma gondii. Her toxoplasmic serological profile established the diagnosis of acute toxoplasmosis. A toxoplasmic serological profile should be obtained for patients with myocarditis and/or polymyositis of unclear etiology. Endomyocardial or skeletal muscle tissue biopsies may establish the definitive diagnosis of toxoplasmic myocarditis or polymyositis, respectively. Examination of blood by polymerase chain reaction analysis before antitoxoplasmic treatment and early in the course of primary infection with T. gondii may prove useful.

Ocular toxoplasmosis is considered to be the most commonly recognized cause of chorioretinitis in the United States. It is commonly believed that the majority of cases of acute toxoplasmic chorioretinitis involving adults in the United States are late sequelae of congenital infection and that the condition is rarely associated with acute postnatally acquired infection. We report here the clinical and serological test findings for 22 adults with acute toxoplasmic chorioretinitis that occurred in the setting of acute postnatally acquired toxoplasmosis. The initial serum specimen from each adult yielded an acute toxoplasmic serological profile, on the basis of the following positive results: 95.5%, Sabin-Feldman dye test [titer of > or = 1:1,024]; 95.5%, IgM ELISA; 90.9%, IgA ELISA; 77.3%, IgE ELISA; 95.5%, IgE immunosorbent agglutination assay; and 86.4%, differential agglutination (AC/HS) test (acute pattern). Detection of IgA or IgE antibodies or an acute pattern in the AC/HS test was particularly helpful in diagnosis for those patients whose ELISA IgM titers at presentation were negative or lowly positive. Thus, acute toxoplasmic chorioretinitis occurring with a recently acquired Toxoplasma gondii infection would appear to be more common in the United States than previously recognized, and a toxoplasmic serological profile is useful in diagnosing this entity.

The feeding behavior, seasonality, and natural infection rate of Lutzomyia evansi (Nuñez-Tovar) with Leishmania chagasi (Cuna & Chagas) was studied during a 12-mo period at 2 hamlets, El Contento and Vidales. Sand fly abundance in extra-, peri-, and intradomestic habitats was evaluated with sticky traps and CDC light traps, whereas human bait and Shannon trap collections were made only in peridomestic habitats. All trapping methods showed a clear predominance of L. evansi throughout the year. Sand flies were present during most of the year, with the exception of the driest months (February and March). Although the total number of sand flies was higher in El Contento than in Vidales, a larger proportion of L. evansi was found in intradomestic habitat than in the peri- and extradomestic habitats at Vidales. Also, sand flies from Vidales had a higher infection rate with L. chagasi than did those from El Contento. Although 2 of 9 promastigote infections detected in L. evansi were identified as L. chagasi, the difficulty of isolating and propagating leishmania strains from this visceral leishmaniasis focus precluded characterization of most parasite samples. Parous and infected sand flies were most abundant toward the end of the rainy season (October-December). For this reason, control strategies based on reducing sand fly populations or avoiding human-vector contact should be concentrated during the October-December period.

The frequency of HLA-DQ antigens in AIDS patients with toxoplasmic encephalitis (TE) were examined. HLA-DQ3 was significantly more frequent in white North American AIDS patients with TE (85.0%) than in the general white population (51.8%; P = .007, corrected P = .028) or randomly selected control AIDS patients who had not developed TE (40.0%; P = .016). In contrast, the frequency of HLA-DQ1 was lower in TE patients than in healthy controls (40.0% vs. 66.5%, P = .027), but this difference did not reach statistical significance when corrected for the number of variables tested (corrected P = .108 for the general white population). HLA-DQ3 thus appears to be a genetic marker of susceptibility to development of TE in AIDS patients, and DQ1 may be a resistance marker. These HLA associations with disease indicate that development of TE in AIDS patients is affected by a gene or genes in the HLA complex and that HLA-DQ typing may help in decisions regarding TE prophylaxis.

The purpose of this study was to determine the value of conventional and newer serological tests (toxoplasmic serological profile) in the diagnosis of toxoplasmic lymphadenitis (TL). We studied 40 consecutive patients with biopsy-proven TL. Cervical, axillary, or occipital adenopathy was present in 72.5%, 20%, and 7.5% of the patients, respectively. Low-grade fever, fatigue, general malaise, or sore throat were present in only 6 (15%) of the 40 patients. A positive result for all serological tests was time dependent from the clinical onset of lymphadenopathy. The initial serum samples were positive for antibody for each patient, as shown by a Sabin-Feldman dye test. Between 3 and 6 months after clinical onset of TL, all of the patients had antibody titers of > or = 1:1,024. The ELISA was positive for IgM antibodies in all of the patients in the first 3 months. Detection of IgA or IgE antibodies or an acute pattern in the differential agglutination test was helpful in diagnosing TL in those patients who had negative, low-positive, or equivocal titers of IgM antibodies (as measured by ELISA) after 3 months. A toxoplasmic serological profile on the first serum specimen drawn after clinical onset of TL had a sensitivity of 100%. It is advisable to obtain such a serological profile in cases of asymptomatic lymphadenopathy before biopsy is carried out, especially for those individuals who have negative or equivocal IgM antibody titers.

We have developed a highly efficient DNA-synthesizing system with isolated intact rat liver mitochondria. The ATP requirements for this in organello DNA synthesis are provided by endogenous synthesis in the presence of exogenous ADP and an oxidizable substrate. In this system, mitochondrial DNA synthesis strikingly proceeds at a constant rate for about 5 h at 37 degrees C. Gel electrophoresis, hybridization and restriction enzyme analyses show that intact mitochondria synthesize nucleic acids with a size of 16.5 kb, that correspond to mitochondrial DNA, and that both DNA strands are replicated. This in organello DNA synthesis requires the supply of dNTPs and decreases at high ADP concentration in the incubation medium.

The safety and efficacy of combined therapy with polyethylene glycolated (PEG) interleukin (IL)-2 and zidovudine was assessed in 19 human immunodeficiency virus type 1 (HIV-1)-seropositive subjects in a phase I/II open-label dose-ranging study. During courses of three weekly infusions of PEG IL-2, dose-limiting side effects were seen at 5 x 10(6) IU/m2 and reversible encephalopathy in 1 subject at 3 x 10(6) IU/m2. Significant increases were seen in CD4 cell counts (P < .01), NK cell activity (P < .05), and HIV-specific cytotoxicity (P < .01). Virologic monitoring (quantitative DNA polymerase chain reaction and p24 antigen assay) showed no evidence of increased HIV activation. Patients with CD4 cells < 200/mm3 were entered into a chronic dosing phase. PEG IL-2 was given at 14-day intervals at doses of 10(6) IU/m2 for 8 weeks and 3 x 10(6) IU/m2 for up to 16 weeks, resulting in mean CD4 cell count elevations of 16% and 33%, respectively. PEG IL-2 appears to warrant further investigation, especially in subjects with CD4 cell counts < 200/mm3, to determine whether increased lymphocyte numbers will translate into improved clinical outcome.