Genetic validation of Leishmania genes essential for amastigote survival in vivo using N-myristoyltransferase as a model

BACKGROUND: Proving that specific genes are essential for the intracellular viability of Leishmania parasites within macrophages remains a challenge for the identification of suitable targets for drug development. This is especially evident in the absence of a robust inducible expression system or functioning RNAi machinery that works in all Leishmania species. Currently, if a target gene of interest in extracellular parasites can only be deleted from its genomic locus in the presence of ectopic expression from a wild type copy, it is assumed that this gene will also be essential for viability in disease-promoting intracellular parasites. However, functional essentiality must be proven independently in both life-cycle stages for robust validation of the gene of interest as a putative target for chemical intervention. METHODS: Here, we have used plasmid shuffle methods in vivo to provide supportive genetic evidence that N-myristoyltransferase (NMT) is essential for Leishmania viability throughout the parasite life-cycle. Following confirmation of NMT essentiality in vector-transmitted promastigotes, a range of mutant parasites were used to infect mice prior to negative selection pressure to test the hypothesis that NMT is also essential for parasite viability in an established infection. RESULTS: Ectopically-expressed NMT was only dispensable under negative selection in the presence of another copy. Total parasite burdens in animals subjected to negative selection were comparable to control groups only if an additional NMT copy, not affected by the negative selection, was expressed. CONCLUSIONS: NMT is an essential gene in all parasite life-cycle stages, confirming its role as a genetically-validated target for drug development

Marginal structural models for repeated measures where intercept and slope are correlated: An application exploring the benefit of nutritional supplements on weight gain in HIV-infected children initiating antiretroviral therapy

BackgroundThe impact of nutritional supplements on weight gain in HIV-infected children on antiretroviral treatment (ART) remains uncertain. Starting supplements depends upon current weight-for-age or other acute malnutrition indicators, producing time-dependent confounding. However, weight-for-age at ART initiation may affect subsequent weight gain, independent of supplement use. Implications for marginal structural models (MSMs) with inverse probability of treatment weights (IPTW) are unclear.MethodsIn the ARROW trial, non-randomised supplement use and weight-for-age were recorded monthly from ART initiation. The effect of supplements on weight-for-age over the first year was estimated using generalised estimating equation MSMs with IPTW, both with and without interaction terms between baseline weight-for-age and time. Separately, data were simulated assuming no supplement effect, with use depending on current weight-for-age, and weight-for-age trajectory depending on baseline weight-for-age to investigate potential bias associated with different MSM specifications.ResultsIn simulations, despite correctly specifying IPTW, omitting an interaction in the MSM between baseline weight-for-age and time produced increasingly biased estimates as associations between baseline weight-for-age and subsequent weight trajectory increased. Estimates were unbiased when the interaction between baseline weight-for-age and time was included, even if the data were simulated with no such interaction. In ARROW, without an interaction the estimated effect was +0.09 (95%CI +0.02,+0.16) greater weight-for-age gain per month's supplement use; this reduced to +0.03 (-0.04,+0.10) including the interaction.DiscussionThis study highlights a specific situation in which MSM model misspecification can occur and impact the resulting estimate. Since an interaction in the MSM (outcome) model does not bias the estimate of effect if the interaction does not exist, it may be advisable to include such a term when fitting MSMs for repeated measures

The association between subcortical and cortical fMRI and lifetime noise exposure in listeners with normal hearing thresholds

In animal models, exposure to high noise levels can cause permanent damage to hair-cell synapses (cochlear synaptopathy) for high-threshold auditory nerve fibers without affecting sensitivity to quiet sounds. This has been confirmed in several mammalian species, but the hypothesis that lifetime noise exposure affects auditory function in humans with normal audiometric thresholds remains unconfirmed and current evidence from human electrophysiology is contradictory. Here we report the auditory brainstem response (ABR), and both transient (stimulus onset and offset) and sustained functional magnetic resonance imaging (fMRI) responses throughout the human central auditory pathway across lifetime noise exposure. Healthy young individuals aged 25–40 years were recruited into high (n = 32) and low (n = 30) lifetime noise exposure groups, stratified for age, and balanced for audiometric threshold up to 16 kHz fMRI demonstrated robust broadband noise-related activity throughout the auditory pathway (cochlear nucleus, superior olivary complex, nucleus of the lateral lemniscus, inferior colliculus, medial geniculate body and auditory cortex). fMRI responses in the auditory pathway to broadband noise onset were significantly enhanced in the high noise exposure group relative to the low exposure group, differences in sustained fMRI responses did not reach significance, and no significant group differences were found in the click-evoked ABR. Exploratory analyses found no significant relationships between the neural responses and self-reported tinnitus or reduced sound-level tolerance (symptoms associated with synaptopathy). In summary, although a small effect, these fMRI results suggest that lifetime noise exposure may be associated with central hyperactivity in young adults with normal hearing thresholds

Effects of noise exposure on young adults with normal audiograms I: electrophysiology

Noise-induced cochlear synaptopathy has been demonstrated in numerous rodent studies. In these animal models, the disorder is characterized by a reduction in amplitude of wave I of the auditory brainstem response (ABR) to high-level stimuli, whereas the response at threshold is unaffected. The aim of the present study was to determine if this disorder is prevalent in young adult humans with normal audiometric hearing. One hundred and twenty six participants (75 females) aged 18-36 were tested. Participants had a wide range of lifetime noise exposures as estimated by a structured interview. Audiometric thresholds did not differ across noise exposures up to 8 kHz, although 16- kHz audiometric thresholds were elevated with increasing noise exposure for females but not for males. ABRs were measured in response to high-pass (1.5 kHz) filtered clicks of 80 and 100 dB peSPL. Frequency-following responses (FFRs) were measured to 80 dB SPL pure tones from 240- 285 Hz, and to 80 dB SPL 4 kHz pure tones amplitude modulated at frequencies from 240-285 Hz (transposed tones). The bandwidth of the ABR stimuli and the carrier frequency of the transposed tones were chosen to target the 3-6 kHz characteristic frequency region which is usually associated with noise damage in humans. The results indicate no relation between noise exposure and the amplitude of the ABR. In particular, wave I of the ABR did not decrease with increasing noise exposure as predicted. ABR wave V latency increased with increasing noise exposure for the 80 dB peSPL click. High carrier-frequency (envelope) FFR amplitudes decreased as a function of noise exposure in males but not females. However, these correlations were not significant after the effects of age were controlled. The results suggest either that noise-induced cochlear synaptopathy is not a significant problem in young, audiometrically normal adults, or that the ABR and FFR are relatively insensitive to this disorder in young humans, although it is possible that the effects become more pronounced with age

Effects of noise exposure on young adults with normal audiograms II: Behavioral measures

An estimate of lifetime noise exposure was used as the primary predictor of performance on a range of behavioral tasks: frequency and intensity difference limens, amplitude modulation detection, interaural phase discrimination, the digit triplet speech test, the co-ordinate response speech measure, an auditory localization task, a musical consonance task and a subjective report of hearing ability. One hundred and thirty-eight participants (81 females) aged 18–36 years were tested, with a wide range of self-reported noise exposure. All had normal pure-tone audiograms up to 8 kHz. It was predicted that increased lifetime noise exposure, which we assume to be concordant with noise-induced cochlear synaptopathy, would elevate behavioral thresholds, in particular for stimuli with high levels in a high spectral region. However, the results showed little effect of noise exposure on performance. There were a number of weak relations with noise exposure across the test battery, although many of these were in the opposite direction to the predictions, and none were statistically significant after correction for multiple comparisons. There were also no strong correlations between electrophysiological measures of synaptopathy published previously and the behavioral measures reported here. Consistent with our previous electrophysiological results, the present results provide no evidence that noise exposure is related to significant perceptual deficits in young listeners with normal audiometric hearing. It is possible that the effects of noise-induced cochlear synaptopathy are only measurable in humans with extreme noise exposures, and that these effects always co-occur with a loss of audiometric sensitivity

SPACA3gene variants in a New Zealand cohort of infertile and fertile couples

SPRASA (also referred to as SLLP1) is a protein identified in the acrosome of human sperm and encoded by the gene SPACA3. SPRASA is associated with sperm-oocyte recognition and binding, and may play a role in fertility. In order to determine whether variants in the SPACA3 gene are associated with human infertility, we undertook a genetic analysis of 102 infertile and 104 fertile couples. Three gene variants were identified using PCR-based DNA sequencing; 1) an insertion of TGC within a quadruple tri-nucleotide (TGC) repeat region in the 5’ untranslated region (UTR) (g.–22TGC(4_5), 2) a guanine to adenosine transition at position 239 (c.239G> A) resulting in a non-synonymous amino acid substitution from cysteine to tyrosine (p.C80Y) at position 80 in the putative transmembrane region, and 3) a novel nucleotide variant (c.691G> C) located in the 3’UTR. A functional effect of the g.–22TGC (4_5) was confirmed by a luciferase expression assay, while the effects of the variants c.239G> A and c.691G> C were predicted using in silico analysis. Although the frequencies of these variants were not significantly different between the infertile and fertile populations, we present evidence that the variants could affect the expression levels or function of SPRASA, thereby affecting a couple's fertility. Larger populations, especially individuals/couples with unexplained infertility, need to be screened for these variants to validate a relationship with fertility

Girls and Boys Born before 28 Weeks Gestation: Risks of Cognitive, Behavioral, and Neurologic Outcomes at Age 10 Years

To compare the prevalence of cognitive, neurological, and behavioral outcomes at 10 years of age in 428 girls and 446 boys who were born extremely preterm (EP)

The Relationship of Maternal Prepregnancy Body Mass Index and Pregnancy Weight Gain to Neurocognitive Function at Age 10 Years among Children Born Extremely Preterm

OBJECTIVE: To assess the association between maternal prepregnancy body mass index and adequacy of pregnancy weight gain in relation to neurocognitive function in school-aged children born extremely preterm. STUDY DESIGN: Study participants were 535 ten-year-old children enrolled previously in the prospective multicenter Extremely Low Gestational Age Newborns cohort study who were products of singleton pregnancies. Soon after delivery, mothers provided information about prepregnancy weight. Prepregnancy body mass index and adequacy of weight gain were characterized based on this information. Children underwent a neurocognitive evaluation at 10 years of age. RESULTS: Maternal prepregnancy obesity was associated with increased odds of a lower score for Differential Ability Scales-II Verbal IQ, for Developmental Neuropsychological Assessment-II measures of processing speed and visual fine motor control, and for Wechsler Individual Achievement Test-III Spelling. Children born to mothers who gained an excessive amount of weight were at increased odds of a low score on the Oral and Written Language Scales Oral Expression assessment. Conversely, children whose mother did not gain an adequate amount of weight were at increased odds of a lower score on the Oral and Written Language Scales Oral Expression and Wechsler Individual Achievement Test-III Word Reading assessments. CONCLUSION: In this cohort of infants born extremely preterm, maternal obesity was associated with poorer performance on some assessments of neurocognitive function. Our findings are consistent with the observational and experimental literature and suggest that opportunities may exist to mitigate risk through education and behavioral intervention before pregnancy

Dolutegravir twice-daily dosing in children with HIV-associated tuberculosis: a pharmacokinetic and safety study within the open-label, multicentre, randomised, non-inferiority ODYSSEY trial

Background: Children with HIV-associated tuberculosis (TB) have few antiretroviral therapy (ART) options. We aimed to evaluate the safety and pharmacokinetics of dolutegravir twice-daily dosing in children receiving rifampicin for HIV-associated TB. Methods: We nested a two-period, fixed-order pharmacokinetic substudy within the open-label, multicentre, randomised, controlled, non-inferiority ODYSSEY trial at research centres in South Africa, Uganda, and Zimbabwe. Children (aged 4 weeks to <18 years) with HIV-associated TB who were receiving rifampicin and twice-daily dolutegravir were eligible for inclusion. We did a 12-h pharmacokinetic profile on rifampicin and twice-daily dolutegravir and a 24-h profile on once-daily dolutegravir. Geometric mean ratios for trough plasma concentration (Ctrough), area under the plasma concentration time curve from 0 h to 24 h after dosing (AUC0–24 h), and maximum plasma concentration (Cmax) were used to compare dolutegravir concentrations between substudy days. We assessed rifampicin Cmax on the first substudy day. All children within ODYSSEY with HIV-associated TB who received rifampicin and twice-daily dolutegravir were included in the safety analysis. We described adverse events reported from starting twice-daily dolutegravir to 30 days after returning to once-daily dolutegravir. This trial is registered with ClinicalTrials.gov (NCT02259127), EudraCT (2014–002632-14), and the ISRCTN registry (ISRCTN91737921). Findings: Between Sept 20, 2016, and June 28, 2021, 37 children with HIV-associated TB (median age 11·9 years [range 0·4–17·6], 19 [51%] were female and 18 [49%] were male, 36 [97%] in Africa and one [3%] in Thailand) received rifampicin with twice-daily dolutegravir and were included in the safety analysis. 20 (54%) of 37 children enrolled in the pharmacokinetic substudy, 14 of whom contributed at least one evaluable pharmacokinetic curve for dolutegravir, including 12 who had within-participant comparisons. Geometric mean ratios for rifampicin and twice-daily dolutegravir versus once-daily dolutegravir were 1·51 (90% CI 1·08–2·11) for Ctrough, 1·23 (0·99–1·53) for AUC0–24 h, and 0·94 (0·76–1·16) for Cmax. Individual dolutegravir Ctrough concentrations were higher than the 90% effective concentration (ie, 0·32 mg/L) in all children receiving rifampicin and twice-daily dolutegravir. Of 18 children with evaluable rifampicin concentrations, 15 (83%) had a Cmax of less than the optimal target concentration of 8 mg/L. Rifampicin geometric mean Cmax was 5·1 mg/L (coefficient of variation 71%). During a median follow-up of 31 weeks (IQR 30–40), 15 grade 3 or higher adverse events occurred among 11 (30%) of 37 children, ten serious adverse events occurred among eight (22%) children, including two deaths (one tuberculosis-related death, one death due to traumatic injury); no adverse events, including deaths, were considered related to dolutegravir. Interpretation: Twice-daily dolutegravir was shown to be safe and sufficient to overcome the rifampicin enzyme-inducing effect in children, and could provide a practical ART option for children with HIV-associated TB