Emerging Concepts in Vector Development for Glial Gene Therapy: Implications for Leukodystrophies

Central Nervous System (CNS) homeostasis and function rely on intercellular synchronization of metabolic pathways. Developmental and neurochemical imbalances arising from mutations are frequently associated with devastating and often intractable neurological dysfunction. In the absence of pharmacological treatment options, but with knowledge of the genetic cause underlying the pathophysiology, gene therapy holds promise for disease control. Consideration of leukodystrophies provide a case in point; we review cell type – specific expression pattern of the disease – causing genes and reflect on genetic and cellular treatment approaches including ex vivo hematopoietic stem cell gene therapies and in vivo approaches using adeno-associated virus (AAV) vectors. We link recent advances in vectorology to glial targeting directed towards gene therapies for specific leukodystrophies and related developmental or neurometabolic disorders affecting the CNS white matter and frame strategies for therapy development in future

A long-acting formulation of the integrase inhibitor raltegravir protects humanized BLT mice from repeated high-dose vaginal HIV challenges

Pre-exposure prophylaxis (PrEP) using antiretroviral drugs (ARVs) has been shown to reduce HIV transmission in people at high risk of HIV infection. Adherence to PrEP strongly correlates with the level of HIV protection. Long-acting injectable ARVs provide sustained systemic drug exposures over many weeks and can improve adherence due to infrequent parenteral administration. Here, we evaluated a new long-acting formulation of raltegravir for prevention of vaginal HIV transmission

A rapid, simple, and low-blank pumped ion-exchange column chromatography technique for boron purification from carbonate and seawater matrices

Funding: This work is supported by the European Research Council under the European Union's Horizon 2020 research and innovation program (Grant 805246) to J.W.B.R., H.J., a Natural Environmental Research Council (NERC)-IAPETUS2 Doctoral Training Programme (DTP) Studentship to NE/S007431/1 C.X., a NERC-IAPETUS DTP Studentship NE/RO12253/1 to M.T., a Leverhulme Trust Early Career Fellowship ECF-2023-199 to H.J., a NERC UK IODP grant NE/P000878/1 to S.B. and S.N., and a Taiwanese MOST Grant 111-2116M-002-032-MY3 to S.N.Boron isotope ratios (δ11B) are used across the Earth Sciences and are increasing analyzed by Multi-Collector Inductively Coupled Plasma Mass Spectrometry (MC-ICPMS). Accurate δ11B MC-ICPMS analysis requires boron purification from the sample matrix using ion-exchange column chromatography. However, the traditional gravity-drip column method is time-consuming and prone to airborne contamination due to its long duration and open resin surface. To address these issues, we designed a novel, simple, and reliable column chromatography technique called “peri-columns.” This method uses a peristaltic pump to generate vacuum on a commonly used column set up. This method uses sealed collection beakers and does not require solutions to pass through pump tubing, minimizing contamination. The duration is reduced by eight-fold, processing 12 samples in just 1.5 hr. It also yields low and consistent total procedural blanks, averaging 11 pg. The efficiency and efficacy of this method were tested by repeated boron purification from calcium carbonate and high-sodium matrices with international and in-house reference materials. The results matched those obtained using the gravity column method and fell within our laboratory long-term and international certified values. The mean δ11B and 2SD (standard deviation) of repeatedly processed NIST 8301f were 14.57 ± 0.26‰ (n = 31), NIST 8301c was 24.19 ± 0.33‰ (n = 10), STAiG-F1 was 16.20 ± 0.26‰ (n = 13), and seawater was 39.52 ± 0.32‰ (n = 10). All the components of our techniques are commercially available, and it is easily adaptable to other laboratories and isotope systems.Publisher PDFPeer reviewe

A long-acting formulation of the integrase inhibitor raltegravir protects humanized BLT mice from repeated high-dose vaginal HIV challenges

Objectives: Pre-exposure prophylaxis (PrEP) using antiretroviral drugs (ARVs) has been shown to reduce HIV transmission in people at high risk of HIV infection. Adherence to PrEP strongly correlates with the level of HIV protection. Long-acting injectable ARVs provide sustained systemic drug exposures over many weeks and can improve adherence due to infrequent parenteral administration. Here, we evaluated a new long-acting formulation of raltegravir for prevention of vaginal HIV transmission. Methods: Long-acting raltegravir was administered subcutaneously to BALB/c, NSG (NOD -scid -gamma) and humanized BLT (bone marrow -liver -thymus) mice and rhesus macaques. Raltegravir concentration in peripheral blood and tissue was analysed. Suppression of HIV replication was assessed in infected BLT mice. Two high-dose HIV vaginal challenges were used to evaluate protection from HIV transmission in BLT mice. Results: Two weeks after a single subcutaneous injection of long-acting raltegravir in BLT mice (7.5 mg) and rhesus macaques (160 mg), the plasma concentration of raltegravir was comparable to 400 mg orally, twice daily in humans. Serum collected from mice 3 weeks post-administration of long-acting raltegravir efficiently blocked HIV infection of TZM-bl indicator cells in vitro. Administration of long-acting raltegravir suppressed viral RNA in plasma and cervico-vaginal fluids of infected BLT mice, demonstrating penetration of active raltegravir into the female reproductive tract. Using transmitted/founder HIV we observed that BLT mice administered a single subcutaneous dose of long-acting raltegravir were protected from two high-dose HIV vaginal challenges 1 week and 4 weeks after drug administration. Conclusions: These preclinical results demonstrated the efficacy of long-acting raltegravir in preventing vaginal HIV transmission

Motor cortical excitability and pre-supplementary motor area neurochemistry in healthy adults with substantia nigra hyperechogenicity

Substantia nigra (SN) hyperechogenicity, viewed with transcranial ultrasound, is a risk marker for Parkinson\u27s disease. We hypothesized that SN hyperechogenicity in healthy adults aged 50 – 70 years is associated with reduced short-interval intracortical inhibition in primary motor cortex, and that the reduced intracortical inhibition is associated with neurochemical markers of activity in the pre-supplementary motor area (pre-SMA). Short-interval intracortical inhibition and intracortical facilitation in primary motor cortex was assessed with paired-pulse transcranial magnetic stimulation in 23 healthy adults with normal (n = 14; 61 ± 7 yrs) or abnormally enlarged (hyperechogenic; n = 9; 60 ± 6 yrs) area of SN echogenicity. Thirteen of these participants (7 SN − and 6 SN+) also underwent brain magnetic resonance spectroscopy to investigate pre-SMA neurochemistry. There was no relationship between area of SN echogenicity and short-interval intracortical inhibition in the ipsilateral primary motor cortex. There was a significant positive relationship, however, between area of echogenicity in the right SN and the magnitude of intracortical facilitation in the right (ipsilateral) primary motor cortex (p = .005; multivariate regression), evidenced by the amplitude of the conditioned motor evoked potential (MEP) at the 10 – 12 ms interstimulus interval. This relationship was not present on the left side. Pre-SMA glutamate did not predict primary motor cortex inhibition or facilitation. The results suggest that SN hyperechogenicity in healthy older adults may be associated with changes in excitability of motor cortical circuitry. The results advance understanding of brain changes in healthy older adults at risk of Parkinson\u27s disease

Association between the timing of childhood adversity and epigenetic patterns across childhood and adolescence:findings from the Avon Longitudinal Study of Parents and Children (ALSPAC) prospective cohort

BACKGROUND: Childhood adversity is a potent determinant of health across development and is associated with altered DNA methylation signatures, which might be more common in children exposed during sensitive periods in development. However, it remains unclear whether adversity has persistent epigenetic associations across childhood and adolescence. We aimed to examine the relationship between time-varying adversity (defined through sensitive period, accumulation of risk, and recency life course hypotheses) and genome-wide DNA methylation, measured three times from birth to adolescence, using data from a prospective, longitudinal cohort study.METHODS: We first investigated the relationship between the timing of exposure to childhood adversity between birth and 11 years and blood DNA methylation at age 15 years in the Avon Longitudinal Study of Parents and Children (ALSPAC) prospective cohort study. Our analytic sample included ALSPAC participants with DNA methylation data and complete childhood adversity data between birth and 11 years. We analysed seven types of adversity (caregiver physical or emotional abuse, sexual or physical abuse [by anyone], maternal psychopathology, one-adult households, family instability, financial hardship, and neighbourhood disadvantage) reported by mothers five to eight times between birth and 11 years. We used the structured life course modelling approach (SLCMA) to identify time-varying associations between childhood adversity and adolescent DNA methylation. Top loci were identified using an R 2 threshold of 0·035 (ie, ≥3·5% of DNA methylation variance explained by adversity). We attempted to replicate these associations using data from the Raine Study and Future of Families and Child Wellbeing Study (FFCWS). We also assessed the persistence of adversity-DNA methylation associations we previously identified from age 7 blood DNA methylation into adolescence and the influence of adversity on DNA methylation trajectories from ages 0-15 years. FINDINGS: Of 13 988 children in the ALSPAC cohort, 609-665 children (311-337 [50-51%] boys and 298-332 [49-50%] girls) had complete data available for at least one of the seven childhood adversities and DNA methylation at 15 years. Exposure to adversity was associated with differences in DNA methylation at 15 years for 41 loci (R 2 ≥0·035). Sensitive periods were the most often selected life course hypothesis by the SLCMA. 20 (49%) of 41 loci were associated with adversities occurring between age 3 and 5 years. Exposure to one-adult households was associated with differences in DNA methylation at 20 [49%] of 41 loci, exposure to financial hardship was associated with changes at nine (22%) loci, and physical or sexual abuse was associated with changes at four (10%) loci. We replicated the direction of associations for 18 (90%) of 20 loci associated with exposure to one-adult household using adolescent blood DNA methylation from the Raine Study and 18 (64%) of 28 loci using saliva DNA methylation from the FFCWS. The directions of effects for 11 one-adult household loci were replicated in both cohorts. Differences in DNA methylation at 15 years were not present at 7 years and differences identified at 7 years were no longer apparent by 15 years. We also identified six distinct DNA methylation trajectories from these patterns of stability and persistence. INTERPRETATION: These findings highlight the time-varying effect of childhood adversity on DNA methylation profiles across development, which might link exposure to adversity to potential adverse health outcomes in children and adolescents. If replicated, these epigenetic signatures could ultimately serve as biological indicators or early warning signs of initiated disease processes, helping identify people at greater risk for the adverse health consequences of childhood adversity.FUNDING: Canadian Institutes of Health Research, Cohort and Longitudinal Studies Enhancement Resources, EU's Horizon 2020, US National Institute of Mental Health.</p

Challenges to access and provision of palliative care for people who are homeless : a systematic review of qualitative research

Background: People who are homeless or vulnerably housed are a marginalized group who often experience high rates of morbidity and die young as a result of complex problems. Access to health care and support can be challenging, with access to palliative care even more so. This review presents a synthesis of published qualitative research exploring from the perspective of homeless people and those working to support them, current challenges to palliative care access and provision, in addition to suggestions for what may improve palliative care for this population. Methods: Systematic review of qualitative research analysed using thematic synthesis. PsycINFO, Medline, Sociological Abstracts, Social Services Abstracts, Science citations index and CINAHL were searched up to September 2016. Thematic synthesis involved a three-step inductive process to develop a deeper understanding of the challenges to and suggestions for the access and provision of palliative care for homeless people. Results: Thirteen qualitative articles, reporting nine studies were identified. The challenges to access and provision to palliative care were drawn from the data covering three broad areas, namely “the chaotic lifestyles sometimes associated with being homeless”, “the delivery of palliative care within a hostel for homeless people” and provision within “mainstream health care systems”. Obstacles were related to homeless persons competing day-to-day priorities, their experience of stigma in mainstream settings, the high burden on hostel staff in supporting residents at the end of life and inflexibility in mainstream health care systems. Suggestions for improving access to palliative care include building trust between homeless persons and health professionals, increasing collaboration between and flexibility within services, and providing more training and support for all professionals. Conclusions: The provision of palliative care can be complicated for all populations, however delivering palliative care for people who are homeless is influenced by a potentially greater and more varied range of factors, on both individual and systemic levels, than providing palliative care for the housed population. Careful consideration and potentially great changes will be needed within health care systems to ensure homeless populations have equitable access to palliative care

A Multiparent Advanced Generation Inter-Cross to Fine-Map Quantitative Traits in Arabidopsis thaliana

Identifying natural allelic variation that underlies quantitative trait variation remains a fundamental problem in genetics. Most studies have employed either simple synthetic populations with restricted allelic variation or performed association mapping on a sample of naturally occurring haplotypes. Both of these approaches have some limitations, therefore alternative resources for the genetic dissection of complex traits continue to be sought. Here we describe one such alternative, the Multiparent Advanced Generation Inter-Cross (MAGIC). This approach is expected to improve the precision with which QTL can be mapped, improving the outlook for QTL cloning. Here, we present the first panel of MAGIC lines developed: a set of 527 recombinant inbred lines (RILs) descended from a heterogeneous stock of 19 intermated accessions of the plant Arabidopsis thaliana. These lines and the 19 founders were genotyped with 1,260 single nucleotide polymorphisms and phenotyped for development-related traits. Analytical methods were developed to fine-map quantitative trait loci (QTL) in the MAGIC lines by reconstructing the genome of each line as a mosaic of the founders. We show by simulation that QTL explaining 10% of the phenotypic variance will be detected in most situations with an average mapping error of about 300 kb, and that if the number of lines were doubled the mapping error would be under 200 kb. We also show how the power to detect a QTL and the mapping accuracy vary, depending on QTL location. We demonstrate the utility of this new mapping population by mapping several known QTL with high precision and by finding novel QTL for germination data and bolting time. Our results provide strong support for similar ongoing efforts to produce MAGIC lines in other organisms

Frequency drift in MR spectroscopy at 3T

Purpose: Heating of gradient coils and passive shim components is a common cause of instability in the B-0 field, especially when gradient intensive sequences are used. The aim of the study was to set a benchmark for typical drift encountered during MR spectroscopy (MRS) to assess the need for real-time field-frequency locking on MRI scanners by comparing field drift data from a large number of sites.Method: A standardized protocol was developed for 80 participating sites using 99 3T MR scanners from 3 major vendors. Phantom water signals were acquired before and after an EPI sequence. The protocol consisted of: minimal preparatory imaging; a short pre-fMRI PRESS; a ten-minute fMRI acquisition; and a long post-fMRI PRESS acquisition. Both pre- and post-fMRI PRESS were non-water suppressed. Real-time frequency stabilization/adjustment was switched off when appropriate. Sixty scanners repeated the protocol for a second dataset. In addition, a three-hour post-fMRI MRS acquisition was performed at one site to observe change of gradient temperature and drift rate. Spectral analysis was performed using MATLAB. Frequency drift in pre-fMRI PRESS data were compared with the first 5:20 minutes and the full 30:00 minutes of data after fMRI. Median (interquartile range) drifts were measured and showed in violin plot. Paired t-tests were performed to compare frequency drift pre- and post-fMRI. A simulated in vivo spectrum was generated using FID-A to visualize the effect of the observed frequency drifts. The simulated spectrum was convolved with the frequency trace for the most extreme cases. Impacts of frequency drifts on NAA and GABA were also simulated as a function of linear drift. Data from the repeated protocol were compared with the corresponding first dataset using Pearson's and intraclass correlation coefficients (ICC).Results: Of the data collected from 99 scanners, 4 were excluded due to various reasons. Thus, data from 95 scanners were ultimately analyzed. For the first 5:20 min (64 transients), median (interquartile range) drift was 0.44 (1.29) Hz before fMRI and 0.83 (1.29) Hz after. This increased to 3.15 (4.02) Hz for the full 30 min (360 transients) run. Average drift rates were 0.29 Hz/min before fMRI and 0.43 Hz/min after. Paired t-tests indicated that drift increased after fMRI, as expected (p &lt; 0.05). Simulated spectra convolved with the frequency drift showed that the intensity of the NAA singlet was reduced by up to 26%, 44 % and 18% for GE, Philips and Siemens scanners after fMRI, respectively. ICCs indicated good agreement between datasets acquired on separate days. The single site long acquisition showed drift rate was reduced to 0.03 Hz/min approximately three hours after fMRI.Discussion: This study analyzed frequency drift data from 95 3T MRI scanners. Median levels of drift were relatively low (5-min average under 1 Hz), but the most extreme cases suffered from higher levels of drift. The extent of drift varied across scanners which both linear and nonlinear drifts were observed.</p