Higher Dose Noninvasive Transcutaneous Auricular Vagus Nerve Stimulation Increases Feeding Volumes and White Matter Microstructural Complexity in Open-Label Study of Infants Slated for Gastrostomy Tube.

OBJECTIVE: To determine whether transcutaneous auricular vagus nerve stimulation (taVNS) paired with twice daily bottle feeding increases the volume of oral feeds and white matter neuroplasticity in term-age-equivalent infants failing oral feeds and determined to need a gastrostomy tube. STUDY DESIGN: In this prospective, open-label study, 21 infants received taVNS paired with 2 bottle feeds for 2 - 3 weeks (2x). We compared 1) increase oral feeding volumes with 2x taVNS and previously reported once daily taVNS (1x) to determine a dose response, 2) number of infants who attained full oral feeding volumes, and 3) diffusional kurtosis imaging and magnetic resonance spectroscopy before and after treatment by paired t tests. RESULTS: All 2x taVNS treated infants significantly increased their feeding volumes compared with 10 days before treatment. Over 50% of 2x taVNS infants achieved full oral feeds but in a shorter time than 1x cohort (median 7 days [2x], 12.5 days [1x], P < .05). Infants attaining full oral feeds showed greater increase in radial kurtosis in the right corticospinal tract at the cerebellar peduncle and external capsule. Notably, 75% of infants of diabetic mothers failed full oral feeds, and their glutathione concentrations in the basal ganglia, a measure of central nervous system oxidative stress, were significantly associated with feeding outcome. CONCLUSIONS: In infants with feeding difficulty, increasing the number of daily taVNS-paired feeding sessions to twice-daily significantly accelerates response time but not the overall response rate of treatment. taVNS was associated with white matter motor tract plasticity in infants able to attain full oral feeds. TRIAL REGISTRATION: Clinicaltrials.gov (NCT04643808).

Geographic distribution of United States clinical trial sites utilized for guideline changing studies of cholesterol management.

BACKGROUND: This study aimed to evaluate the geographic distribution of United States (US) clinical trial sites utilizedfor guideline changing studies of cholesterol management. METHODS: Randomized trials evaluating pharmacologic interventions for cholesterol treatment and reporting location data (ie, zip code of trial sites) were identified. Location data was abstracted from ClinicalTrials.gov. RESULTS: Half of US counties were over 30 miles from a study site and, social determinants of health were more favorable in counties with versus without clinical trial sites. CONCLUSIONS: Stakeholders such as regulatory bodies andtrial sponsors should incentivize and support infrastructure that would enable a larger number of US counties to be utilized for clinical trial sites. TRIAL REGISTRATION: Not applicable.

Comparison of Pyrrolobenzodiazepine Dimer Bis-imine versus Mono-imine: DNA Interstrand Cross-linking, Cytotoxicity, Antibody-Drug Conjugate Efficacy and Toxicity.

Antibody-drug conjugates (ADC) delivering pyrrolobenzodiazepine (PBD) DNA cross-linkers are currently being evaluated in clinical trials, with encouraging results in Hodgkin and non-Hodgkin lymphomas. The first example of an ADC delivering a PBD DNA cross-linker (loncastuximab tesirine) has been recently approved by the FDA for the treatment of relapsed and refractory diffuse large B-cell lymphoma. There has also been considerable interest in mono-alkylating PBD analogs. We conducted a head-to-head comparison of a conventional PBD bis-imine and a novel PBD mono-imine. Key Mitsunobu chemistry allowed clean and convenient access to the mono-imine class. Extensive DNA-binding studies revealed that the mono-imine mediated a type of DNA interaction that is described as "pseudo cross-linking," as well as alkylation. The PBD mono-imine ADC demonstrated robust antitumor activity in mice bearing human tumor xenografts at doses 3-fold higher than those that were efficacious for the PBD bis-imine ADC. A single-dose toxicology study in rats demonstrated that the MTD of the PBD mono-alkylator ADC was approximately 3-fold higher than that of the ADC bearing a bis-imine payload, suggesting a comparable therapeutic index for this molecule. However, although both ADCs caused myelosuppression, renal toxicity was observed only for the bis-imine, indicating possible differences in toxicologic profiles that could influence tolerability and therapeutic index. These data show that mono-amine PBDs have physicochemical and pharmacotoxicologic properties distinct from their cross-linking analogs and support their potential utility as a novel class of ADC payload.

Mapping the KRAS proteoform landscape in colorectal cancer identifies truncated KRAS4B that decreases MAPK signaling.

The KRAS gene is one of the most frequently mutated oncogenes in human cancer and gives rise to two isoforms, KRAS4A and KRAS4B. KRAS post-translational modifications (PTMs) have the potential to influence downstream signaling. However, the relationship between KRAS PTMs and oncogenic mutations remains unclear, and the extent of isoform-specific modification is unknown. Here, we present the first top-down proteomics study evaluating both KRAS4A and KRAS4B, resulting in 39 completely characterized proteoforms across colorectal cancer cell lines and primary tumor samples. We determined which KRAS PTMs are present, along with their relative abundance, and that proteoforms of KRAS4A versus KRAS4B are differentially modified. Moreover, we identified a subset of KRAS4B proteoforms lacking the C185 residue and associated C-terminal PTMs. By confocal microscopy, we confirmed that this truncated GFP-KRAS4BC185∗ proteoform is unable to associate with the plasma membrane, resulting in a decrease in mitogen-activated protein kinase signaling pathway activation. Collectively, our study provides a reference set of functionally distinct KRAS proteoforms and the colorectal cancer contexts in which they are present.

Lateral abdominal muscles of adults with hypermobility may be partially impaired during contraction.

Background: Muscle function may be impaired in people with generalised hypermobility, yet prior studies have primarily focused on muscles within the extremities. We aimed to examine changes in lateral abdominal muscle (transversus abdominis (TrA) and the external (EO) and internal abdominal obliques (IO)) thickness and length during contraction between participants with and without hypermobility. Methods: This cross-sectional study examined 12 participants with hypermobility and 12 age-matched, sex-matched, height-matched and weight-matched participants without hypermobility. The Beighton and Belavy-Owen-Mitchell score assessed systemic hypermobility. Muscle thickness and length were measured via panoramic ultrasound scans at rest and during contraction. Results: When compared with rest across all lumbar levels (L1-L5), contraction produced a lesser increase in TrA thickness (ß=0.03, p=0.034) for participants with hypermobility compared with control. No group-by-condition interaction was observed for TrA length across all lumbar levels (L1-L5; p=0.269). Contraction produced a greater decrease in EO thickness (ß=0.08, p=0.002) at L3 only for participants with hypermobility compared with control. No group-by-condition interactions were observed for IO thickness. Conclusion: Participants with hypermobility had partially impaired lateral abdominal muscle function given a lesser ability to increase TrA muscle thickness during contraction compared with controls.

Efficacy, Tolerability, and Pharmacokinetic Studies of Antibody-Drug Conjugates Containing a Low-Potency Pyrrolobenzodiazepine Dimer.

Antibody-drug conjugate (ADC) research has typically focused on the release of highly potent cytotoxic agents to achieve antitumor efficacy. However, recently approved ADCs trastuzumab deruxtecan and sacituzumab govitecan release lower-potency topoisomerase inhibitors. This has prompted interest in ADCs that release lower-potency cytotoxic drugs to potentially enhance therapeutic index and reduce unwanted toxicity. Pyrrolobenzodiazepine (PBD) dimer ADCs have been widely investigated in human clinical trials, which have focused on high-potency PBDs. In this study, we evaluated five ADCs that release the low-potency PBD dimer SG3650. The relatively low clogD for this agent facilitated higher drug-to-antibody ratio (DAR) conjugation without the need for antibody engineering or functionalization of the drug. The rank order of potency for DAR 2 site-specific ADCs (conjugated at the C239i position) matched the order for the corresponding free drugs in vitro. Despite free drug SG3650 being inactive in vivo, the DAR 2 ADCs derived from the corresponding drug-linker SG3584 showed antitumor efficacy in solid (anti-HER2) and hematologic (anti-CD22) xenograft models. Antitumor activity could be enhanced by conjugating SG3584 to trastuzumab at higher DARs of 4 and 8 and by adjusting dosing and schedule. Higher-DAR conjugates were stable and displayed good rat pharmacokinetic profiles as measured by ELISA and LC/MS-MS. A single intravenous dose of isotype control SG3584 DAR 2 ADC resulted in no mortality in rats or monkeys at doses of up to 25 and 30 mg/kg, respectively. These findings suggest that further investigations of low-potency PBD dimers in ADCs that target hematologic and solid tumors are warranted.

Complication accrual impacts pediatric extracorporeal life support mortality.

INTRODUCTION: This study tested the hypothesis that complication accrual during pediatric extracorporeal life support (ECLS) increases mortality irrespective of indication for support. METHODS: Prospectively collected Extracorporeal Life Support Organization (ELSO) registry data for all neonatal and pediatric patients cannulated for ECLS at our institution from 1/1/2015 to 12/31/2020 was stratified based on the presence or absence of complications. We excluded renal replacement therapy from complications, as this is frequently and empirically applied within our practice. RESULTS: Of 114 patients, overall survival to discharge was 66%. 62 patients (54%) had 149 total complications: 29% were mechanical (circuit related), and the rest were patient related. Age (neonatal versus pediatric), sex, race/ethnicity, support type, presence of pre-ECLS arrest, pre-ECLS pH and intubation-to-ECLS duration were not significantly associated with the development of complications. Patients with complications required longer ECLS duration (168 versus 86 median hours, p < 0.001) and were more likely to be decannulated due to death or poor prognosis (25% versus 8%, p = 0.022). One or more ECLS complications was associated with significantly decreased survival by Cox proportional hazard regression (p = 0.003). CONCLUSION: Complications on ECLS are associated with longer support duration and predict decreased survival independent of pre-ECLS variables, suggesting a multidisciplinary ECLS team target for improved outcomes.

Ultrasound imaging measures of vertebral bony landmark distances are weakly to moderately correlated with intervertebral disc height as assessed by MRI.

Objectives: To assess the validity and reliability of ultrasound-derived interbony landmark distances as a proxy for MRI-derived intervertebral disc (IVD) height. Methods: This is a cross-sectional criterion validity study. Twelve college-aged participants without current low back pain completed both MRI and ultrasound imaging of the lumbar spine in a prone position. Single-segment and multisegment distances between the spinous and mammillary processes at the lumbar segments (L2/L3, L3/L4, L4/L5) were measured twice using ultrasound and analysed digitally. Sagittal slices of the lumbar spine were taken via T1-weighted MRI and IVD height, and the overall distance between IVDs L2/L3 and L4/L5 was imaged once and measured twice. Results: There was moderate correlation between multilevel-based measurements (overall distance between L2 and L5, r=0.677, p=0.016) and the average across three levels (r=0.596, p=0.041) when using the spinous processes as bony landmarks. Single-segment measures were not significantly correlated (all: p>0.092). Accuracy and precision were better for the overall MRI-derived distance between the three IVDs from L2 and L5 MRI and the distance measured between the spinous processes L2-L5. There was excellent reliability within multiple measurements at each location, with intraclass correlation coefficient, ICC(3,1), ranging from 0.93 to 0.99 (95% CI 0.82 to 0.99) for ultrasound and from 0.98 to 0.99 (95% CI 0.92 to 0.99) for MRI. Conclusion: Findings do not support the use of ultrasound imaging for estimating single-segment IVD height, yet it may be used to measure the change in distance over time with a certain degree of precision based on its excellent reliability.

Internal and External Oblique Muscle Asymmetry in Sprint Hurdlers and Sprinters: A Cross-Sectional Study.

The abdominal muscles are vital in providing core stability for functional movements during most activities. There is a correlation between side asymmetry of these muscles and dysfunction. Thus, the purpose of this study was to evaluate and compare trunk muscle morphology and trunk rotational strength between sprint hurdlers, an asymmetrical sport, and sprinters, a symmetrical sport. Twenty-one trained collegiate sprint hurdlers and sprinters were recruited for the study (Hurdlers: 4M, 7F; Sprinters: 8M, 2F), average age (years) hurdlers: 20 ± 1.2; sprinters: 20.4 ± 1.9, height (cm) hurdlers: 172.6 ± 10.2; sprinters: 181.7 ± 4.5, and weight (kg) hurdlers: 67.6 ± 12.0; sprinters: 73.9 ± 5.6. Using real-time ultrasound, panoramic images of the internal oblique (IO) and external oblique (EO) were obtained at rest and contracted (flexion and rotation) in a seated position for both right and left sides of the trunk. While wearing a specially crafted shoulder harness, participants performed three maximal voluntary trunk rotational contractions (MVC). The three attempts were then averaged to obtain an overall MVC score for trunk rotation strength. Average MVC trunk rotational strength to the right was greater among all participants, p < 0.001. The IO showed greater and significant thickness changes from resting to contracted state than the EO, this was observed in all participants. The IO side asymmetry was significantly different between groups p < 0.01. Hurdlers, involved in a unilaterally demanding sport, exhibited the expected asymmetry in muscle morphology and in trunk rotational strength. Interestingly, sprinters, although involved in a seemingly symmetrical sport, also exhibited asymmetrical trunk morphology and trunk rotational strength.

Emanuel syndrome and congenital diaphragmatic hernia: A systematic review.

BACKGROUND: Emanuel Syndrome (ES), a rare chromosomal disorder caused by a supernumerary chromosome 22 derivative (der(22)t(11;22)), was identified in a fetus with congenital diaphragmatic hernia (CDH) at our fetal center. We aimed to identify a precedent for clinical care and patient outcomes to guide family decision-making. METHODS: This non-funded and non-registered study queried the entire CDH Registry (CDHR) including >10,000 patients since 1995 and conducted a systematic literature review for patients with concomitant ES and CDH. RESULTS: Literature review captured 12 citations and identified 9 patients with CDH+ES from over 400 known ES cases. Given the rarity of the disease and to reduce bias, there were no exclusion criteria aside from non-English language. Of these 9, two underwent surgical CDH repair with neither surviving. The CDHR identified 6 patients with ES, all reported after 2013 and prenatally diagnosed. Median estimated gestational age was 39 weeks (range 37-40) and median birth weight was 2.72 kg (range 2.4-3.4 kg). 3 patients died within the first few postnatal days; surgical repair was not offered due to "anomalies" and "pulmonary hypertension" in two and one family chose comfort measures. The other 3 patients underwent surgical repair, and 2 were supported with ECMO. Two patients survived to discharge, incurring surgical comorbidities associated with severe CDH including gastrostomy dependence, tracheostomy, and CDH recurrence. CONCLUSIONS: ES patients with CDH have potential to tolerate repair and survive to discharge, however with significant additional morbidity combined with severe challenges inherent to ES. This represents the largest series of patients with CDH and ES to date. LEVEL OF EVIDENCE: IV (Case series with no comparison group).

Reassembling protein complexes after controlled disassembly by top-down mass spectrometry in native mode.

The combined use of electrospray ionization run in so-called "native mode" with top-down mass spectrometry (nTDMS) is enhancing both structural biology and discovery proteomics by providing three levels of information in a single experiment: the intact mass of a protein or complex, the masses of its subunits and non-covalent cofactors, and fragment ion masses from direct dissociation of subunits that capture the primary sequence and combinations of diverse post-translational modifications (PTMs). While intact mass data are readily deconvoluted using well-known software options, the analysis of fragmentation data that result from a tandem MS experiment - essential for proteoform characterization - is not yet standardized. In this tutorial, we offer a decision-tree for the analysis of nTDMS experiments on protein complexes and diverse bioassemblies. We include an overview of strategies to navigate this type of analysis, provide example data sets, and highlight software for the hypothesis-driven interrogation of fragment ions for localization of PTMs, metals, and cofactors on native proteoforms. Throughout we have emphasized the key features (deconvolution, search mode, validation, other) that the reader can consider when deciding upon their specific experimental and data processing design using both open-access and commercial software.

Precise Characterization of KRAS4B Proteoforms by Combining Immunoprecipitation with Top-Down Mass Spectrometry.

The characterization of biologically relevant post-translational modifications (PTMs) on KRAS4B has historically been carried out through methodologies such as immunoblotting with PTM-specific antibodies or peptide-based proteomic methods. While these methods have the potential to identify a given PTM on KRAS4B, they are incapable of characterizing or distinguishing the different molecular forms or proteoforms of KRAS4B from those of related RAS isoforms. We present a method that combines immunoprecipitation of KRAS4B with top-down mass spectrometry (IP-TDMS), thus enabling the precise characterization of intact KRAS4B proteoforms. We provide detailed protocols for the IP, LC-MS/MS, and data analysis comprising a successful IP-TDMS assay in the contexts of cancer cell lines and tissue samples.

NAC and Vitamin D Restore CNS Glutathione in Endotoxin-Sensitized Neonatal Hypoxic-Ischemic Rats.

Therapeutic hypothermia does not improve outcomes in neonatal hypoxia ischemia (HI) complicated by perinatal infection, due to well-described, pre-existing oxidative stress and neuroinflammation that shorten the therapeutic window. For effective neuroprotection post-injury, we must first define and then target CNS metabolomic changes immediately after endotoxin-sensitized HI (LPS-HI). We hypothesized that LPS-HI would acutely deplete reduced glutathione (GSH), indicating overwhelming oxidative stress in spite of hypothermia treatment in neonatal rats. Post-natal day 7 rats were randomized to sham ligation, or severe LPS-HI (0.5 mg/kg 4 h before right carotid artery ligation, 90 min 8% O2), followed by hypothermia alone or with N-acetylcysteine (25 mg/kg) and vitamin D (1,25(OH)2D3, 0.05 µg/kg) (NVD). We quantified in vivo CNS metabolites by serial 7T MR Spectroscopy before, immediately after LPS-HI, and after treatment, along with terminal plasma drug concentrations. GSH was significantly decreased in all LPS-HI rats compared with baseline and sham controls. Two hours of hypothermia alone did not improve GSH and allowed glutamate + glutamine (GLX) to increase. Within 1 h of administration, NVD increased GSH close to baseline and suppressed GLX. The combination of NVD with hypothermia rapidly improved cellular redox status after LPS-HI, potentially inhibiting important secondary injury cascades and allowing more time for hypothermic neuroprotection.

Extended field-of-view ultrasound imaging is reliable for measuring Transversus Abdominis muscle size at rest and during contraction.

BACKGROUND: The strength and size of core muscles, including the abdominal muscles, are crucial to proper function in most activities. Therefore, it is important to reliably assess these characteristics. Our primary objective was to determine if the length, thickness and cross-sectional area of the transversus abdominis (TrA) can be visualized independently from the internal and external abdominal oblique muscles using extended field of view ultrasound imaging at rest and with contraction and to establish its intra- and inter-tester reliability. METHODS: Twenty-six individuals were recruited to participate in the study (20 F, 6 M), average age 24.0 years (SD 9.4), height 170.7 cm (SD 8.6) and weight 63.9 kg (SD 9.0). From this total number of participants, two groups of 16 randomly selected participants were assessed to determine intra- and inter-tester reliability respectively. Extended field of view ultrasound images were obtained at three vertebral levels during rest and contraction in the side lying position for both the right and left sides of the trunk. RESULTS: Excellent intra-tester and inter-tester reliability was seen (ICC range of 0.972 to 0.984). The overall average percent standard error of the measurement for all measurements and locations was approximately 4%. The overall average minimal difference for the thickness measurement for the resting and contraction conditions combined were as follows: intratester 0.056 (0.014) cm and intertester 0.054 (0.017) cm, for area intratester 0.287 (0.086) cm2 and intertester 0.289 (0.101) cm2 and for length intratester 0.519 (0.097) cm and intertester 0.507 (0.085) cm. CONCLUSIONS: Extended field of view ultrasound imaging is an effective method of reliably capturing clear images of the TrA during rest and contraction. It provides an efficient mechanism for the analysis of muscle morphology by being able to measure the cross-sectional area, thickness, and length on one image. This methodology is recommended for studies investigating TrA function and training.

Effects of Distal Mutations on Prolyl-Adenylate Formation of Escherichia coli Prolyl-tRNA Synthetase.

Enzymes play important roles in many biological processes. Amino acid residues in the active site pocket of an enzyme, which are in direct contact with the substrate(s), are generally believed to be critical for substrate recognition and catalysis. Identifying and understanding how these "catalytic" residues help enzymes achieve enormous rate enhancement has been the focus of many structural and biochemical studies over the past several decades. Recent studies have shown that enzymes are intrinsically dynamic and dynamic coupling between distant structural elements is essential for effective catalysis in modular enzymes. Therefore, distal residues are expected to have impact on enzyme function. However, few studies have investigated the role of distal residues on enzymatic catalysis. In the present study, the effects of distal residue mutations on the catalytic function of an aminoacyl-tRNA synthetase, namely, prolyl-tRNA synthase, were investigated. The present study demonstrates that distal residues significantly contribute to catalysis of the modular Escherichia coli prolyl-tRNA synthetase by maintaining intrinsic protein flexibility.

Comparison of melphalan- And busulfan-based myeloablative conditioning in children undergoing allogeneic transplantation for acute myeloid leukemia or myelodysplasia.

BACKGROUND: The optimal conditioning regimen for alloHCT in children with myeloid malignancies remains undefined. PROCEDURE: We performed a retrospective review of children undergoing alloHCT for AML and MDS over a 10-year period (2008-2018) at our institution, comparing the outcomes of recipients of either a myeloablative busulfan- or reduced toxicity mel/thio-based conditioning regimen. RESULTS: A total of 49 patients underwent alloHCT for AML/MDS (mel/thio, N = 21; busulfan, N = 28). Mel/thio recipients were selected due to pretransplant comorbidities. Recipients of mel/thio were more likely to have t-AML, and less likely to have MRD <0.1% at the time of alloHCT (57.1% vs 82.1%). Graft failure was more common in busulfan recipients; engraftment kinetics were similar between groups. Sinusoidal obstructive syndrome was diagnosed in 21% of busulfan and no mel/thio recipients (P = .03). One patient in each group died from TRM. Relapse incidence was comparable (mel/thio-29% vs busulfan-32%); however, relapse occurred significantly later in recipients of mel/thio conditioning (median d + 396 vs d + 137; P = .01). As a result, there was a trend toward improved OS at 1 and 3 years in mel/thio recipients (95% vs 74%, P = .06; and 75% vs 50%, P = .11; respectively). CONCLUSION: In our single institution, when compared to myeloablative busulfan-based conditioning, use of a mel/thio-based reduced toxicity regimen resulted in comparable outcomes, despite higher risk patient and disease characteristics. Mel/thio recipients had both more comorbidities and higher risk disease profile, which did not translate into higher rates of either TRM or relapse.

Editing Domain Motions Preorganize the Synthetic Active Site of Prolyl-tRNA Synthetase.

Prolyl-tRNA synthetases (ProRSs) catalyze the covalent attachment of proline onto cognate tRNAs, an indispensable step for protein synthesis in all living organisms. ProRSs are modular enzymes and the "prokaryotic-like" ProRSs are distinguished from "eukaryotic-like" ProRSs by the presence of an editing domain (INS) inserted between motifs 2 and 3 of the main catalytic domain. Earlier studies suggested the presence of coupled-domain dynamics could contribute to catalysis; however, the role that the distal, highly mobile INS domain plays in catalysis at the synthetic active site is not completely understood. In the present study, a combination of theoretical and experimental approaches has been used to elucidate the precise role of INS domain dynamics. Quantum mechanical/molecular mechanical simulations were carried out to model catalytic Pro-AMP formation by Enterococcus faecalis ProRS. The energetics of the adenylate formation by the wild-type enzyme was computed and contrasted with variants containing active site mutations, as well as a deletion mutant lacking the INS domain. The combined results revealed that two distinct types of dynamics contribute to the enzyme's catalytic power. One set of motions is intrinsic to the INS domain and leads to conformational preorganization that is essential for catalysis. A second type of motion, stemming from the electrostatic reorganization of active site residues, impacts the height and width of the energy profile and has a critical role in fine tuning the substrate orientation to facilitate reactive collisions. Thus, motions in a distal domain can preorganize the active site of an enzyme to optimize catalysis.