Decreased postpartum exercise capacity after a diagnosis of pre-eclampsia: Implications for CVD risk prediction.

BACKGROUND: Hypertensive disorders of pregnancy (HDP) are associated with increased long-term risk for cardiometabolic risk factors (chronic hypertension [HTN], obesity, diabetes) and heart failure. Exercise capacity is a known predictor of heart failure in patients with normal resting cardiac filling pressures. In this prospective observational cohort study, we sought to identify predictors of reduced postpartum exercise capacity in participants with normotensive vs. preeclamptic pregnancies. METHODS: Preeclampsia (PreE) and normotensive subjects were enrolled to undergo bedside echocardiography within 48 hours of delivery, and rest/exercise echocardiography 12 weeks postpartum. RESULTS: Recruited subjects (n=68) were grouped according to their blood pressure as: a) normotensive pregnancy n=15; b) PreE with normotensive postpartum (PreE-Resolved, n=36); c) PreE with persistent postpartum HTN (PreE-HTN, n=17). At enrollment, a significantly higher percentage of subjects in the PreE-HTN group were Black. Compared to normotensive and PreE-Resolved subjects, those with PreE-HTN demonstrated higher resting systolic blood pressure (SBP, 112 [normotensive] vs 112 [PreE-Resolved] vs 134 [PreE-HTN], p<0.001) and diastolic blood pressure (DBP, 70.0 vs 72.5 vs 85.0, p<0.001), and significantly less postpartum weight loss (9.6% vs 13.6% vs 3.8%, p<0.001). Following Bruce protocol stress testing, PreE-HTN subjects demonstrated achieved significantly lower exercise duration (10.4 vs 10.2 vs 7.9 minutes, p = 0.001). Subjects with PreE-HTN also demonstrated evidence of exercise-induced diastolic dysfunction as assessed by peak exercise lateral e' (18.0 vs 18.0 vs 13.5, p=0.045) and peak exercise tricuspid regurgitation velocity (TR Vm, 2.4 vs 3.0 vs 3.1, p = 0.045). Exercise duration was negatively associated with gravidity (R=-0.27, p=0.029) and postpartum LV mass index (R=-0.45, p<0.001), resting average E/e' (R=-0.51, p<0.001), BMI (R=-0.6, p<0.001) and resting SBP (R=-0.51, p<0.001). CONCLUSIONS: Postpartum exercise stress testing capacity is related to readily available clinical markers including pregnancy factors, echocardiographic parameters and unresolved cardiometabolic risk factors.

Running Habits and Injury Frequency Following COVID-19 Restrictions in Adolescent Long-Distance Runners.

PURPOSE: A decline in youth running was observed at the start of the COVID-19 pandemic. We investigated whether the resumption of organized running after social distancing restrictions changed running habits or injury frequency in adolescent runners. METHODS: Adolescents (age = 16.1 [2.1] y) who participated in long-distance running activities completed an online survey in the Spring and Fall of 2020. Participants self-reported average weekly running habits and whether they sustained an injury during the Fall 2020 season. Poisson regression models and 1-way analysis of variance compared running habits while Fisher exact test compared differences in frequencies of injuries during Fall 2020 among season statuses (full, delayed, and canceled). RESULTS: All runners, regardless of season status, increased weekly distance during Fall 2020. Only runners with a full Fall 2020 season ran more times per week and more high-intensity runs per week compared with their Spring 2020 running habits. There were no differences in running volume or running-related injury frequency among Fall 2020 season statuses. CONCLUSIONS: There were no significant differences in running-related injury (RRI) frequency among runners, regardless of season status, following the resumption of cross-country. Health care providers may need to prepare for runners to increase running volume and intensity following the resumption of organized team activities.

Early Sport Specialization and Past Injury in Competitive Youth Rock Climbers.

INTRODUCTION: Sport specialization has been shown to have negative effects on athletes but has not been studied within rock climbing. This study seeks to evaluate the proportion and impact of specialization in pediatric climbers. METHODS: Climbers (ages 8-18 y) were recruited from throughout the United States to complete a 1-time survey regarding climbing experience, training patterns, and injury history. The main outcome of proportion of climbers suffering an injury was assessed within the last 12 mo and within their entire climbing experience (defined as "lifetime" injury). Early specialization was defined as exclusive participation in climbing, with training for >8 mo‧y-1, prior to age 12 y (late specialization if after age 12 y). RESULTS: Participants (n=111, 14±3 y [mean±SD], 69 females) were high-level climbers. Fifty-five percent of participants specialized in climbing, and 69% of those specialized early. Hand and ankle injuries occurred most commonly. Seventy-eight percent of late specialized climbers had a lifetime injury. Late specialized climbers were 1.6 times (95% CI: 1.1-2.3) more likely than early specialized climbers to have had a lifetime injury and 1.8 times (95% CI: 1.1-2.8) more likely to have had an injury in the last 12 mo. No difference in overuse injuries was found between specialization groups. CONCLUSIONS: Early specialization is common among youth climbers but was not associated with an increase in injuries. Late specialization was associated with a higher likelihood of having had a climbing injury in the last 12 mo and during an entire climbing career.

The Association of Finger Growth Plate Injury History and Speed Climbing in Youth Competition Climbers.

INTRODUCTION: Finger growth plate injuries are the most common injury among youth climbers, and the association between these injuries and speed climbing, a mandatory discipline in the 2021 Olympics, has not been examined previously. Our primary purpose was to examine the demographic and training characteristics of adolescent competition climbers who reported a history of a finger growth plate injury compared to those who did not report a history of a finger growth plate injury. Our secondary purpose was to determine whether training characteristics differed between adolescent competition climbers who did and did not report speed climbing. METHODS: Our study was a cross-sectional study design. We surveyed adolescent climbers who competed in the 2017 USA Climbing Sport and Speed Youth National Championships. Questions assessed climbing injury history and current rock-climbing training characteristics. RESULTS: Two-hundred sixty-seven adolescent competition climbers, 14±3 (9-18) y of age (mean±SD with range), completed the survey. Those with a history of a finger growth plate injury reported greater approximate time spent speed climbing throughout the year (ß=1.28, 95% CI 0.11-2.46, P=0.032) and training regularly on the International Federation of Sport Climbing speed wall (adjusted odds ratio=3.95, 95% CI 1.14-13.7, P=0.031). CONCLUSIONS: Training regularly at practices on the speed wall was associated with a self-reported history of finger growth plate injuries among elite youth competition climbers. Speed climbing should be limited, especially during periods of rapid growth.

Returning to Climb after Epiphyseal Finger Stress Fracture.

The number of youth participating in rock climbing has increased over the years. Finger stress epiphyseal fractures are the most common injury among youth climbers. These injuries tend to occur around puberty because this is when the physis is most vulnerable to injury. Additionally, it has been found that intensive finger training (campus boarding, a previously known risk factor for epiphyseal fractures) during adolescence can lead to early-onset osteoarthritis of the hand up to a decade later. There is currently a lack of a return-to-climb protocol for youth climbers following a repetitive stress epiphyseal fracture. Because of this gap in the literature, our purpose was to create a structured return-to-play protocol specific to youth climbers who sustained an epiphyseal fracture to the finger. By establishing these guidelines, medical professionals and coaches may be able to guide their athlete to gradually and safely return to sport.

Safety and efficacy of RNAi therapy for transthyretin amyloidosis.

BACKGROUND: Transthyretin amyloidosis is caused by the deposition of hepatocyte-derived transthyretin amyloid in peripheral nerves and the heart. A therapeutic approach mediated by RNA interference (RNAi) could reduce the production of transthyretin. METHODS: We identified a potent antitransthyretin small interfering RNA, which was encapsulated in two distinct first- and second-generation formulations of lipid nanoparticles, generating ALN-TTR01 and ALN-TTR02, respectively. Each formulation was studied in a single-dose, placebo-controlled phase 1 trial to assess safety and effect on transthyretin levels. We first evaluated ALN-TTR01 (at doses of 0.01 to 1.0 mg per kilogram of body weight) in 32 patients with transthyretin amyloidosis and then evaluated ALN-TTR02 (at doses of 0.01 to 0.5 mg per kilogram) in 17 healthy volunteers. RESULTS: Rapid, dose-dependent, and durable lowering of transthyretin levels was observed in the two trials. At a dose of 1.0 mg per kilogram, ALN-TTR01 suppressed transthyretin, with a mean reduction at day 7 of 38%, as compared with placebo (P=0.01); levels of mutant and nonmutant forms of transthyretin were lowered to a similar extent. For ALN-TTR02, the mean reductions in transthyretin levels at doses of 0.15 to 0.3 mg per kilogram ranged from 82.3 to 86.8%, with reductions of 56.6 to 67.1% at 28 days (P<0.001 for all comparisons). These reductions were shown to be RNAi-mediated. Mild-to-moderate infusion-related reactions occurred in 20.8% and 7.7% of participants receiving ALN-TTR01 and ALN-TTR02, respectively. CONCLUSIONS: ALN-TTR01 and ALN-TTR02 suppressed the production of both mutant and nonmutant forms of transthyretin, establishing proof of concept for RNAi therapy targeting messenger RNA transcribed from a disease-causing gene. (Funded by Alnylam Pharmaceuticals; ClinicalTrials.gov numbers, NCT01148953 and NCT01559077.).

5'-(E)-Vinylphosphonate: A Stable Phosphate Mimic Can Improve the RNAi Activity of siRNA-GalNAc Conjugates.

Small interfering RNA (siRNA)-mediated silencing requires siRNA loading into the RNA-induced silencing complex (RISC). Presence of 5'-phosphate (5'-P) is reported to be critical for efficient RISC loading of the antisense strand (AS) by anchoring it to the mid-domain of the Argonaute2 (Ago2) protein. Phosphorylation of exogenous duplex siRNAs is thought to be accomplished by cytosolic Clp1 kinase. However, although extensive chemical modifications are essential for siRNA-GalNAc conjugate activity, they can significantly impair Clp1 kinase activity. Here, we further elucidated the effect of 5'-P on the activity of siRNA-GalNAc conjugates. Our results demonstrate that a subset of sequences benefit from the presence of exogenous 5'-P. For those that do, incorporation of 5'-(E)-vinylphosphonate (5'-VP), a metabolically stable phosphate mimic, results in up to 20-fold improved in vitro potency and up to a threefold benefit in in vivo activity by promoting Ago2 loading and enhancing metabolic stability.

Effect of Preexisting Serum and Mucosal Antibody on Experimental Respiratory Syncytial Virus (RSV) Challenge and Infection of Adults.

We studied preexisting respiratory syncytial virus (RSV)-specific serum and nasal antibodies and their correlation with infectivity, viral dynamics, and disease severity in a human experimental infection model. Higher preinoculation serum neutralizing antibody titers and nasal immunoglobulin (Ig) A predicted lower infectivity and lower measures of viral replication. However, once individuals were infected, no significant protective effect of preexisting antibodies was seen. Lack of correlation between serum and mucosal antibodies was observed, implying that they are independent co-correlates of protection against RSV infection. We suggest that protection from RSV infection is a function of a complex interplay between mucosal and serum humoral immune responses.

Comprehensive evaluation of canonical versus Dicer-substrate siRNA in vitro and in vivo.

Since the discovery of RNA interference (RNAi), researchers have identified a variety of small interfering RNA (siRNA) structures that demonstrate the ability to silence gene expression through the classical RISC-mediated mechanism. One such structure, termed "Dicer-substrate siRNA" (dsiRNA), was proposed to have enhanced potency via RISC-mediated gene silencing, although a comprehensive comparison of canonical siRNAs and dsiRNAs remains to be described. The present study evaluates the in vitro and in vivo activities of siRNAs and dsiRNAs targeting Phosphatase and Tensin Homolog (PTEN) and Factor VII (FVII). More than 250 compounds representing both siRNA and dsiRNA structures were evaluated for silencing efficacy. Lead compounds were assessed for duration of silencing and other key parameters such as cytokine induction. We identified highly active compounds from both canonical siRNAs and 25/27 dsiRNAs. Lead compounds were comparable in potency both in vitro and in vivo as well as duration of silencing in vivo. Duplexes from both structural classes tolerated 2'-OMe chemical modifications well with respect to target silencing, although some modified dsiRNAs demonstrated reduced activity. On the other hand, dsiRNAs were more immunostimulatory as compared with the shorter siRNAs, both in vitro and in vivo. Because the dsiRNA structure does not confer any appreciable benefits in vitro or in vivo while demonstrating specific liabilities, further studies are required to support their applications in RNAi therapeutics.

The Past, Present, and Future of Oral Dosage Forms for Children.

The administration of medications to children has been a challenge for parents and caregivers for generations. Pharmaceutical companies have often overcome the difficulties of weight-based dosing and the -inability of most young children to swallow solid dosage forms by creating oral liquids. While oral liquids -offer advantages in terms of dose flexibility, swallowability, and ease of administration for young children and patients with enteral tubes, they have been plagued by issues such as taste, volume, and texture, to name a few. While the recommendations for broader use of oral syringes can help with the issue of measuring accuracy and incremental dosing, the issues of poor taste and frequently unacceptable volumes for doses remain a problem. New oral dosage forms which have begun to enter the United States marketplace have the potential to improve adherence and acceptability of oral medications for children, but come with their own unique challenges.

Ethanol Content of Medications and Its Effect on Blood Alcohol Concentration in Pediatric Patients.

OBJECTIVE: Ethanol is a common excipient used in liquid medications to enhance solubility and inhibit -bacterial growth. While the US Food and Drug Administration (FDA) and European Medicines Agency (EMA) have released guidance for how much ethanol is acceptable in medicines, many medications contain more than the recommended amount. The objective of this study was to determine what effect these medications would have on blood alcohol concentration (BAC) for pediatric patients, defined as those medications that would increase the BAC by ≥2.5 mg/dL. METHODS: A list of medications dispensed to pediatric patients from a single hospital over a period of 4 months was obtained. The package inserts of these medications were reviewed to determine ethanol content. Typical doses were used to determine the amount of ethanol pediatric patients weighing 10, 20, and 40 kg would receive. The theoretical BAC was then calculated for each medication containing ethanol. RESULTS: Seven hundred ninety-six medications were dispensed for pediatric patients during the study period, of which 33 contained ethanol. Seven medications would be projected to increase the BAC above 2.5 mg/dL with a normal pediatric dose. CONCLUSION: While most medications do not contain ethanol, we found 7 that contained enough ethanol to potentially raise the BAC above 2.5 mg/dL. Health care practitioners should consider the ethanol content of medications prior to recommending them in children and when assessing overdoses.

Oral Drug Product Administration via Enteral Feeding Tubes: In Vitro Testing.

Medication administration via enteral feeding tubes (EFT) is a necessary practice for patients unable to swallow oral dosage forms due to a medical condition or treatment that affects the ability to swallow or the function of the gastrointestinal tract. Off-label administration of oral drug products via EFT raises concerns for pharmaceutical sponsors, regulators, and healthcare practitioners (HCPs) because of the potential risks this practice introduces to both the patient and the caregiver. These risks can be mitigated by generating data-supported instructions that patients and HCPs can use to ensure safe and accurate administration of oral drug products via EFT. This commentary presents an industry perspective on the testing that should be conducted to enable development of product-specific instructions in the labeling to support or advise against administration of oral drug products via enteral feeding tube. The proposal outlined in this commentary takes a risk-based approach, addressing recommendations from both regulatory agencies as well as considerations for expanding this testing to address needs specific to neonatal and pediatric populations.

Identification of small molecule inhibitors of G3BP-driven stress granule formation.

Stress granule formation is triggered by the release of mRNAs from polysomes and is promoted by the action of the RNA-binding proteins G3BP1/2. Stress granules have been implicated in several disease states, including cancer and neurodegeneration. Consequently, compounds that limit stress granule formation or promote their dissolution have potential as both experimental tools and novel therapeutics. Herein, we describe two small molecules, G3BP inhibitor a and b (G3Ia and G3Ib), designed to bind to a specific pocket in G3BP1/2 that is targeted by viral inhibitors of G3BP1/2 function. In addition to disrupting the co-condensation of RNA, G3BP1, and caprin 1 in vitro, these compounds inhibit stress granule formation in cells treated prior to or concurrent with stress and dissolve pre-existing stress granules. These effects are consistent across multiple cell types and a variety of initiating stressors. Thus, these compounds represent powerful tools to probe the biology of stress granules and hold promise for therapeutic interventions designed to modulate stress granule formation.

Interaction between host G3BP and viral nucleocapsid protein regulates SARS-CoV-2 replication and pathogenicity.

G3BP1/2 are paralogous proteins that promote stress granule formation in response to cellular stresses, including viral infection. The nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) inhibits stress granule assembly and interacts with G3BP1/2 via an ITFG motif, including residue F17, in the N protein. Prior studies examining the impact of the G3PB1-N interaction on SARS-CoV-2 replication have produced inconsistent findings, and the role of this interaction in pathogenesis is unknown. Here, we use structural and biochemical analyses to define the residues required for G3BP1-N interaction and structure-guided mutagenesis to selectively disrupt this interaction. We find that N-F17A mutation causes highly specific loss of interaction with G3BP1/2. SARS-CoV-2 N-F17A fails to inhibit stress granule assembly in cells, has decreased viral replication, and causes decreased pathology in vivo. Further mechanistic studies indicate that the N-F17-mediated G3BP1-N interaction promotes infection by limiting sequestration of viral genomic RNA (gRNA) into stress granules.

A randomized, double-blind, placebo-controlled study of an RNAi-based therapy directed against respiratory syncytial virus.

RNA interference (RNAi) is a natural mechanism regulating protein expression that is mediated by small interfering RNAs (siRNA). Harnessing RNAi has potential to treat human disease; however, clinical evidence for the effectiveness of this therapeutic approach is lacking. ALN-RSV01 is an siRNA directed against the mRNA of the respiratory syncytial virus (RSV) nucleocapsid (N) protein and has substantial antiviral activity in a murine model of RSV infection. We tested the antiviral activity of ALN-RSV01 in adults experimentally infected with wild-type RSV. Eighty-eight healthy subjects were enrolled into a randomized, double-blind, placebo-controlled trial. A nasal spray of ALN-RSV01 or saline placebo was administered daily for 2 days before and for 3 days after RSV inoculation. RSV was measured serially in nasal washes using several different viral assays. Intranasal ALN-RSV01 was well tolerated, exhibiting a safety profile similar to saline placebo. The proportion of culture-defined RSV infections was 71.4 and 44.2% in placebo and ALN-RSV01 recipients, respectively (P = 0.009), representing a 38% decrease in the number of infected and a 95% increase in the number of uninfected subjects. The acquisition of infection over time was significantly lower in ALN-RSV01 recipients (P = 0.007 and P = 0.03, viral culture and PCR, respectively). Multiple logistic regression analysis showed that the ALN-RSV01 antiviral effect was independent of other factors, including preexisting RSV antibody and intranasal proinflammatory cytokine concentrations. ALN-RSV01 has significant antiviral activity against human RSV infection, thus establishing a unique proof-of-concept for an RNAi therapeutic in humans and providing the basis for further evaluation in naturally infected children and adults.

Patient Weight Should Be Included on All Medication Prescriptions.

Medication prescriptions for both children and adults often require the patient's current weight to determine a safe and effective dose. Medication orders in the inpatient setting typically require a patient weight be recorded prior to order verification. However, in the ambulatory setting a very different standard exists; weights are not required on prescriptions and are rarely provided by practitioners. Without this information, the community pharmacist must either ask the caregiver, who may not know an accurate weight, or simply assume that the prescriber used a current and accurate weight and calculated the dose correctly. Standard doses are prescribed for most adult prescriptions, which makes it possible for the pharmacist to identify a dosing error. Without a current patient weight, the pharmacist is not able to provide the same level of patient care to pediatric patients or adults whose prescriptions require weight-based doses. The Pediatric Pharmacy Association recommends that patient weight, recorded in kilograms, be required on all medication prescriptions in both the inpatient and outpatient settings.

Identification of small molecule inhibitors of G3BP-driven stress granule formation.

Stress granule formation is triggered by the release of mRNAs from polysomes and is promoted by the action of the paralogs G3BP1 and G3BP2. G3BP1/2 proteins bind mRNAs and thereby promote the condensation of mRNPs into stress granules. Stress granules have been implicated in several disease states, including cancer and neurodegeneration. Consequently, compounds that limit stress granule formation or promote their dissolution have potential as both experimental tools and novel therapeutics. Herein, we describe two small molecules, referred to as G3BP inhibitor a and b (G3Ia and G3Ib), designed to bind to a specific pocket in G3BP1/2 that is known to be targeted by viral inhibitors of G3BP1/2 function. In addition to disrupting co-condensation of RNA, G3BP1, and caprin 1 in vitro, these compounds inhibit stress granule formation in cells treated prior to or concurrent with stress, and dissolve pre-existing stress granules when added to cells after stress granule formation. These effects are consistent across multiple cell types and a variety of initiating stressors. Thus, these compounds represent ideal tools to probe the biology of stress granules and hold promise for therapeutic interventions designed to modulate stress granule formation.

Hepatocyte Smoothened Activity Controls Susceptibility to Insulin Resistance and Nonalcoholic Fatty Liver Disease.

BACKGROUND & AIMS: Nonalcoholic steatohepatitis (NASH), a leading cause of cirrhosis, strongly associates with the metabolic syndrome, an insulin-resistant proinflammatory state that disrupts energy balance and promotes progressive liver degeneration. We aimed to define the role of Smoothened (Smo), an obligatory component of the Hedgehog signaling pathway, in controlling hepatocyte metabolic homeostasis and, thereby, susceptibility to NASH. METHODS: We conditionally deleted Smo in hepatocytes of healthy chow-fed mice and performed metabolic phenotyping, coupled with single-cell RNA sequencing (RNA-seq), to characterize the role of hepatocyte Smo in regulating basal hepatic and systemic metabolic homeostasis. Liver RNA-seq datasets from 2 large human cohorts were also analyzed to define the relationship between Smo and NASH susceptibility in people. RESULTS: Hepatocyte Smo deletion inhibited the Hedgehog pathway and promoted fatty liver, hyperinsulinemia, and insulin resistance. We identified a plausible mechanism whereby inactivation of Smo stimulated the mTORC1-SREBP1c signaling axis, which promoted lipogenesis while inhibiting the hepatic insulin cascade. Transcriptomics of bulk and single Smo-deficient hepatocytes supported suppression of insulin signaling and also revealed molecular abnormalities associated with oxidative stress and mitochondrial dysfunction. Analysis of human bulk RNA-seq data revealed that Smo expression was (1) highest in healthy livers, (2) lower in livers with NASH than in those with simple steatosis, (3) negatively correlated with markers of insulin resistance and liver injury, and (4) declined progressively as fibrosis severity worsened. CONCLUSIONS: The Hedgehog pathway controls insulin sensitivity and energy homeostasis in adult livers. Loss of hepatocyte Hedgehog activity induces hepatic and systemic metabolic stress and enhances susceptibility to NASH by promoting hepatic lipoxicity and insulin resistance.

Interaction between host G3BP and viral nucleocapsid protein regulates SARS-CoV-2 replication.

G3BP1/2 are paralogous proteins that promote stress granule formation in response to cellular stresses, including viral infection. G3BP1/2 are prominent interactors of the nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the functional consequences of the G3BP1-N interaction in the context of viral infection remain unclear. Here we used structural and biochemical analyses to define the residues required for G3BP1-N interaction, followed by structure-guided mutagenesis of G3BP1 and N to selectively and reciprocally disrupt their interaction. We found that mutation of F17 within the N protein led to selective loss of interaction with G3BP1 and consequent failure of the N protein to disrupt stress granule assembly. Introduction of SARS-CoV-2 bearing an F17A mutation resulted in a significant decrease in viral replication and pathogenesis in vivo, indicating that the G3BP1-N interaction promotes infection by suppressing the ability of G3BP1 to form stress granules.