Many biological functions performed by RNAs arise from their in vivo structures. The structure of the same RNA can differ in vitro and in vivo owing in part to the influence of molecules ranging from ...protons to secondary metabolites to proteins. Chemical reagents that modify the Watson-Crick (WC) face of unprotected RNA bases report on the absence of base-pairing and so are of value to determining structures adopted by RNAs. Reagents have thus been sought that can report on the native RNA structures that prevail in living cells. Dimethyl sulfate (DMS) and glyoxal penetrate cell membranes and inform on RNA secondary structure in vivo through modification of adenine (A), cytosine (C), and guanine (G) bases. Uracil (U) bases, however, have thus far eluded characterization in vivo. Herein, we show that the water-soluble carbodiimide 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) is capable of modifying the WC face of U and G in vivo, favoring the former nucleobase by a factor of ∼1.5, and doing so in the eukaryote rice, as well as in the Gram-negative bacterium
While both EDC and glyoxal target Gs, EDC reacts with Gs in their typical neutral state, while glyoxal requires Gs to populate the rare anionic state. EDC may thus be more generally useful; however, comparison of the reactivity of EDC and glyoxal may allow the identification of Gs with perturbed pK
s in vivo and genome-wide. Overall, use of EDC with DMS allows in vivo probing of the base-pairing status of all four RNA bases.
Debugging a genome sequence is imperative for successfully building a synthetic genome. As part of the effort to build a designer eukaryotic genome, yeast synthetic chromosome X (synX), designed as ...707,459 base pairs, was synthesized chemically. SynX exhibited good fitness under a wide variety of conditions. A highly efficient mapping strategy called pooled PCRTag mapping (PoPM), which can be generalized to any watermarked synthetic chromosome, was developed to identify genetic alterations that affect cell fitness ("bugs"). A series of bugs were corrected that included a large region bearing complex amplifications, a growth defect mapping to a recoded sequence in
, and a loxPsym site affecting promoter function of
PoPM is a powerful tool for synthetic yeast genome debugging and an efficient strategy for phenotype-genotype mapping.
Higher dietary protein intake is increasingly recommended for the elderly; however, high protein diets have also been linked to increased cardiovascular disease (CVD) risk. Trimethylamine-N-oxide ...(TMAO) is a bacterial metabolite derived from choline and carnitine abundant from animal protein-rich foods. TMAO may be a novel biomarker for heightened CVD risk. The purpose of this study was to assess the impact of a high protein diet on TMAO. Healthy men (74.2 ± 3.6 years, n = 29) were randomised to consume the recommended dietary allowance of protein (RDA: 0.8 g protein/kg bodyweight/day) or twice the RDA (2RDA) as part of a supplied diet for 10 weeks. Fasting blood samples were collected pre- and post-intervention for measurement of TMAO, blood lipids, glucose tolerance, insulin sensitivity, and inflammatory biomarkers. An oral glucose tolerance test was also performed. In comparison with RDA, the 2RDA diet increased circulatory TMAO (
= 0.002) but unexpectedly decreased renal excretion of TMAO (
= 0.003). LDL cholesterol was increased in 2RDA compared to RDA (
= 0.049), but no differences in other biomarkers of CVD risk and insulin sensitivity were evident between groups. In conclusion, circulatory TMAO is responsive to changes in dietary protein intake in older healthy males.
Common noncoding variants at the human 1p13.3 locus associated with SORT1 expression are among those most strongly associated with low-density lipoprotein cholesterol (LDL-C) in human genome-wide ...association studies. However, validation studies in mice and cell lines have produced variable results regarding the directionality of the effect of SORT1 on LDL-C. This, together with the fact that the 1p13.3 variants are associated with expression of several genes, has raised the question of whether SORT1 is the causal gene at this locus. Using whole exome sequencing in members of an Amish population, we identified coding variants in SORT1 that are associated with increased (rs141749679, K302E) and decreased (rs149456022, Q225H) LDL-C. Further, analysis of plasma lipoprotein particle subclasses by ion mobility in a subset of rs141749679 (K302E) carriers revealed higher levels of large LDL particles compared to noncarriers. In contrast to the effect of these variants in the Amish, the sortilin K302E mutation introduced into a C57BL/6J mouse via CRISPR/Cas9 resulted in decreased non-high-density lipoprotein cholesterol, and the sortilin Q225H mutation did not alter cholesterol levels in mice. This is indicative of different effects of these mutations on cholesterol metabolism in the two species. To our knowledge, this is the first evidence that naturally occurring coding variants in SORT1 are associated with LDL-C, thus supporting SORT1 as the gene responsible for the association of the 1p13.3 locus with LDL-C.
Neoadjuvant systemic therapy (NST) for triple-negative breast cancer and HER2-positive breast cancer yields a pathological complete response in approximately 60% of patients. A pathological complete ...response to NST predicts an excellent prognosis and can be accurately determined by percutaneous image-guided vacuum-assisted core biopsy (VACB). We evaluated radiotherapy alone, without breast surgery, in patients with early-stage triple-negative breast cancer or HER2-positive breast cancer treated with NST who had an image-guided VACB-determined pathological complete response.
This multicentre, single-arm, phase 2 trial was done in seven centres in the USA. Women aged 40 years or older who were not pregnant with unicentric cT1–2N0–1M0 triple-negative breast cancer or HER2-positive breast cancer and a residual breast lesion less than 2 cm on imaging after clinically standard NST were eligible for inclusion. Patients had one biopsy (minimum of 12 cores) obtained by 9G image-guided VACB of the tumour bed. If no invasive or in-situ disease was identified, breast surgery was omitted, and patients underwent standard whole-breast radiotherapy (40 Gy in 15 fractions or 50 Gy in 25 fractions) plus a boost (14 Gy in seven fractions). The primary outcome was the biopsy-confirmed ipsilateral breast tumour recurrence rate determined using the Kaplan-Meier method assessed in the per-protocol population. Safety was assessed in all patients who received VACB. This study has completed accrual and is registered with ClinicalTrials.gov, NCT02945579.
Between March 6, 2017, and Nov 9, 2021, 58 patients consented to participate; however, four (7%) did not meet final inclusion criteria and four (7%) withdrew consent. 50 patients were enrolled and underwent VACB following NST. The median age of the enrolled patients was 62 years (IQR 55–77); 21 (42%) patients had triple-negative breast cancer and 29 (58%) had HER2-positive breast cancer. VACB identified a pathological complete response in 31 patients (62% 95% CI 47·2–75·4). At a median follow-up of 26·4 months (IQR 15·2–39·6), no ipsilateral breast tumour recurrences occurred in these 31 patients. No serious biopsy-related adverse events or treatment-related deaths occurred.
Eliminating breast surgery in highly selected patients with an image-guided VACB-determined pathological complete response following NST is feasible with promising early results; however, additional prospective clinical trials evaluating this approach are needed.
US National Cancer Institute (National Institutes of Health).
During the development of heart failure (HF), the capacity for cardiomyocyte (CM) fatty acid oxidation (FAO) and ATP production is progressively diminished, contributing to pathologic cardiac ...hypertrophy and contractile dysfunction. Receptor-interacting protein 140 (RIP140, encoded by Nrip1) has been shown to function as a transcriptional corepressor of oxidative metabolism. We found that mice with striated muscle deficiency of RIP140 (strNrip1-/-) exhibited increased expression of a broad array of genes involved in mitochondrial energy metabolism and contractile function in heart and skeletal muscle. strNrip1-/- mice were resistant to the development of pressure overload-induced cardiac hypertrophy, and CM-specific RIP140-deficient (csNrip1-/-) mice were protected against the development of HF caused by pressure overload combined with myocardial infarction. Genomic enhancers activated by RIP140 deficiency in CMs were enriched in binding motifs for transcriptional regulators of mitochondrial function (estrogen-related receptor) and cardiac contractile proteins (myocyte enhancer factor 2). Consistent with a role in the control of cardiac fatty acid oxidation, loss of RIP140 in heart resulted in augmented triacylglyceride turnover and fatty acid utilization. We conclude that RIP140 functions as a suppressor of a transcriptional regulatory network that controls cardiac fuel metabolism and contractile function, representing a potential therapeutic target for the treatment of HF.
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•Post-weaning isolation reduces SERT fiber density in the IC.•The effect of social isolation occurs in females, not males.•Estrous phase does not influence SERT fiber density.
...Early-life experiences, including maternal deprivation and social isolation during adolescence, have a profound influence on a range of adult social behaviors. Post-weaning social isolation in rodents influences behavior in part through the alteration of neuromodulatory systems, including the serotonergic system. Of significance to social behavior, the serotonergic system richly innervates brain areas involved in vocal communication, including the auditory system. However, the influence of isolation on serotonergic input to the auditory system remains underexplored. Here, we assess whether 4 weeks of post-weaning individual housing alters serotonergic fiber density in the inferior colliculus (IC), an auditory midbrain nucleus in which serotonin alters auditory-evoked activity. Individually housed male and female mice were compared to conspecifics housed socially in groups of three. Serotonergic projections were subsequently visualized with an antibody to the serotonin transporter, which labels serotonergic fibers with relatively high selectivity. Fiber densities were estimated in the three major subregions of the IC using line-scan intensity analysis. Individually housed female mice showed a significantly reduced fiber density relative to socially housed females, which was accompanied by a lower body weight in individually housed females. In contrast, social isolation did not affect serotonergic fiber density in the IC of males. This finding suggests that sensitivity of the serotonergic system to social isolation is sex-dependent, which could be due to a sex difference in the effect of isolation on psychosocial stress. Since serotonin availability depends on social context, this finding further suggests that social isolation can alter the acute social regulation of auditory processing.
IMPORTANCE: Brain-computer interface (BCI) implants have previously required craniotomy to deliver penetrating or surface electrodes to the brain. Whether a minimally invasive endovascular technique ...to deliver recording electrodes through the jugular vein to superior sagittal sinus is safe and feasible is unknown. OBJECTIVE: To assess the safety of an endovascular BCI and feasibility of using the system to control a computer by thought. DESIGN, SETTING, AND PARTICIPANTS: The Stentrode With Thought-Controlled Digital Switch (SWITCH) study, a single-center, prospective, first in-human study, evaluated 5 patients with severe bilateral upper-limb paralysis, with a follow-up of 12 months. From a referred sample, 4 patients with amyotrophic lateral sclerosis and 1 with primary lateral sclerosis met inclusion criteria and were enrolled in the study. Surgical procedures and follow-up visits were performed at the Royal Melbourne Hospital, Parkville, Australia. Training sessions were performed at patients’ homes and at a university clinic. The study start date was May 27, 2019, and final follow-up was completed January 9, 2022. INTERVENTIONS: Recording devices were delivered via catheter and connected to subcutaneous electronic units. Devices communicated wirelessly to an external device for personal computer control. MAIN OUTCOMES AND MEASURES: The primary safety end point was device-related serious adverse events resulting in death or permanent increased disability. Secondary end points were blood vessel occlusion and device migration. Exploratory end points were signal fidelity and stability over 12 months, number of distinct commands created by neuronal activity, and use of system for digital device control. RESULTS: Of 4 patients included in analyses, all were male, and the mean (SD) age was 61 (17) years. Patients with preserved motor cortex activity and suitable venous anatomy were implanted. Each completed 12-month follow-up with no serious adverse events and no vessel occlusion or device migration. Mean (SD) signal bandwidth was 233 (16) Hz and was stable throughout study in all 4 patients (SD range across all sessions, 7-32 Hz). At least 5 attempted movement types were decoded offline, and each patient successfully controlled a computer with the BCI. CONCLUSIONS AND RELEVANCE: Endovascular access to the sensorimotor cortex is an alternative to placing BCI electrodes in or on the dura by open-brain surgery. These final safety and feasibility data from the first in-human SWITCH study indicate that it is possible to record neural signals from a blood vessel. The favorable safety profile could promote wider and more rapid translation of BCI to people with paralysis. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT03834857
Aim
To investigate associations between clinical factors and the development of autism spectrum disorder (ASD) in children with tuberous sclerosis complex (TSC), specifically seizures, ...electroencephalogram abnormalities, tubers and other neurostructural abnormalities, and genetic factors.
Method
MEDLINE, Embase, PubMed, the Cochrane Library, and Web of Science were searched until January 2019. Studies that considered the predefined factors for development of ASD in children with TSC were included, following PRISMA‐P guidelines. Two authors independently reviewed titles, s, and full texts, extracted data, and assessed risk of bias.
Results
Forty‐two studies with 3542 children with TSC were included. ASD was associated with a history of seizures (odds ratio OR 3.79, 95% confidence interval CI 1.77–8.14), infantile spasms compared with other seizure types (OR 3.04, 95% CI 2.17–4.27), onset of any seizure type during infancy (OR 2.65, 95% CI 1.08–6.54), and male sex (OR 1.62, 95% CI 1.23–2.14). There was no association with tuber number, tuber location, or genotype.
Interpretation
While a causal link between seizures and ASD in children with TSC cannot be inferred, a strong association between seizures and ASD in children with TSC, particularly with seizure onset during infancy and specifically infantile spasms, is present. Children with TSC and infant‐onset seizures should be monitored for emerging features of ASD.
What this paper adds
Seizures and autism spectrum disorder (ASD) strongly associate in children with tuberous sclerosis complex (TSC).
Infant‐onset seizures and infantile spasms are particularly strongly associated with ASD in TSC.
What this paper adds
Seizures and autism spectrum disorder (ASD) strongly associate in children with tuberous sclerosis complex (TSC).
Infant‐onset seizures and infantile spasms are particularly strongly associated with ASD in TSC.
In a single-institution phase II study, we evaluated the safety of a 5-day dose-equivalent neoadjuvant radiotherapy (RT) regimen for high-risk primary soft tissue sarcoma.
Patients received ...neoadjuvant RT alone (30 Gy in five fractions) to the primary tumor with standard margins. The primary endpoint was grade ≥2 late-radiation toxicity. Major wound complications, local recurrences, and distant metastases were also examined. In exploratory analysis, we evaluated germline biomarkers for wound toxicity and the effects of the study on treatment utilization.
Over 2 years, 52 patients were enrolled with median follow-up of 29 months. Seven of 44 evaluable patients (16%) developed grade ≥2 late toxicity. Major wound complications occurred in 16 of 50 patients (32%); a signature defined by 19 germline SNPs in miRNA-binding sites of immune and DNA damage response genes, in addition to lower extremity tumor location, demonstrated strong predictive performance for major wound complications. Compared with the preceding 2-year period, the number of patients treated with neoadjuvant RT alone at our institution increased 3-fold, with a concomitant increase in the catchment area.
A shorter 5-day neoadjuvant RT regimen results in favorable rates of wound complications and grade ≥2 toxicity after 2-year follow-up. Five-day RT significantly increased utilization of neoadjuvant RT at our high-volume sarcoma center. With further validation, a putative germline biomarker for wound complications may guide safer RT utilization.
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