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.

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.

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).

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.

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.

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.

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.

Improved Physical Stability of an Antibody-Drug Conjugate Using Host-Guest Chemistry.

Antibody-drug conjugates (ADCs) are an emerging class of biopharmaceutical products for oncology, with the cytotoxic pyrrolobenzodiazepine (PBD) family of "warheads" well-established in the clinic. While PBDs offer high potency, they are also characterized by their hydrophobicity, which can make formulation of the ADC challenging. Several approaches have been investigated to improve the physicochemical properties of PBD-containing ADCs, and herein a supramolecular approach was explored using cucurbit[8]uril (CB[8]). The ability of CB[8] to simultaneously encapsulate two guests was exploited to incorporate a 12-mer polyethylene glycol harboring a methyl viologen moiety at one terminus (MV-PEG12), together with a PBD harboring an indole moiety at the C2' position (SG3811). This formulation approach successfully introduced a hydrophilic PEG to mask the hydrophobicity of SG3811, improving the physical stability of the ADC while avoiding any loss of potency related to chemical modification.

ADCT-402, a PBD dimer-containing antibody drug conjugate targeting CD19-expressing malignancies.

Human CD19 antigen is a 95-kDa type I membrane glycoprotein in the immunoglobulin superfamily whose expression is limited to the various stages of B-cell development and differentiation and is maintained in the majority of B-cell malignancies, including leukemias and non-Hodgkin lymphomas of B-cell origin. Coupled with its differential and favorable expression profile, CD19 has rapid internalization kinetics and is not shed into the circulation, making it an ideal target for the development of antibody-drug conjugates (ADCs) to treat B-cell malignancies. ADCT-402 (loncastuximab tesirine) is a novel CD19-targeted ADC delivering SG3199, a highly cytotoxic DNA minor groove interstrand crosslinking pyrrolobenzodiazepine (PDB) dimer warhead. It showed potent and highly targeted in vitro cytotoxicity in CD19-expressing human cell lines. ADCT-402 was specifically bound, internalized, and trafficked to lysosomes in CD19-expressing cells and, following release of the PBD warhead, resulted in formation of DNA crosslinks that persisted for 36 hours. Bystander killing of CD19- cells by ADCT-402 was also observed. In vivo, single doses of ADCT-402 resulted in highly potent, dose-dependent antitumor activity in several subcutaneous and disseminated human tumor models with marked superiority to comparator ADCs delivering tubulin inhibitors. Dose-dependent DNA crosslinks and γ-H2AX DNA damage response were measured in tumors by 24 hours after single dose administration, whereas matched peripheral blood mononuclear cells showed no evidence of DNA damage. Pharmacokinetic analysis in rat and cynomolgus monkey showed excellent stability and tolerability of ADCT-402 in vivo. Together, these impressive data were used to support the clinical testing of this novel ADC in patients with CD19-expressing B-cell malignancies.

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.

Crowder-Induced Conformational Ensemble Shift in Escherichia coli Prolyl-tRNA Synthetase.

The effect of molecular crowding on the structure and function of Escherichia coli prolyl-transfer RNA synthetase (Ec ProRS), a member of the aminoacyl-transfer RNA synthetase family, has been investigated using a combined experimental and theoretical method. Ec ProRS is a multidomain enzyme; coupled-domain dynamics are essential for efficient catalysis. To gain insight into the mechanistic detail of the crowding effect, kinetic studies were conducted with varying concentrations and sizes of crowders. In parallel, spectroscopic and quantum chemical studies were employed to probe the "soft interactions" between crowders and protein side chains. Finally, the dynamics of the dimeric protein was examined in the presence of crowders using a long-duration (70 ns) classical molecular dynamic simulations. The results of the simulations revealed a shift in the conformational ensemble, which is consistent with the preferential exclusion of cosolutes. The "soft interactions" model of the crowding effect also explained the alteration in kinetic parameters. In summary, the study found that the effects of molecular crowding on both conformational dynamics and catalytic function are correlated in the multidomain Ec ProRS, an enzyme that is central to protein synthesis in all living cells. This study affirmed that large and small cosolutes have considerable impacts on the structure, dynamics, and function of modular proteins and therefore must be considered for stabilizing protein-based pharmaceuticals and industrial enzymes.

A Reactive Antibody Platform for One-Step Production of Antibody-Drug Conjugates through a Diels-Alder Reaction with Maleimide.

The normal electron-demand Diels-Alder (DA) cycloaddition is a classic transformation routinely used in synthesis; however, applications in biological systems are limited. Here, we report a spiro[2.4]hepta-4,6-diene-containing noncanonical amino acid (SCpHK) capable of efficient incorporation into antibodies and subsequent coupling with maleimide via a DA reaction. SCpHK was stable throughout protein expression in mammalian cells and enabled covalent attachment of maleimide drug-linkers yielding DA antibody-drug conjugates (DA-ADCs) with nearly quantitative conversion in a one-step process. The uncatalyzed DA reaction between SCpHK and maleimide in aqueous buffer was rapid (1.8-5.4 M-1 s-1), and the antibody-drug adduct was stable in rat serum for at least 1 week at 37 °C. Anti-EphA2 DA-ADCs containing AZ1508 or SG3249 maleimide drug-linkers were potent inhibitors of tumor growth in PC3 tumor models in vivo. The DA bioconjugation strategy described here represents a simple method to produce site-specific and stable ADCs with maleimide drug-linkers.

A community-academic partnership to explore informational needs of African American women as a primer for cancer clinical trial recruitment.

Background: African Americans (AAs) are less likely to participate in cancer clinical trials (CCTs) despite experiencing disproportionately higher rates of cancer mortality. As a way to address these ongoing disparities, this study sought to qualitatively explore informational needs regarding CCTs among AA women and identify message considerations for educational information targeting AA women and their community. Methods: Three focus groups were conducted in which AA women viewed a DVD created as a decisional tool for CCT participation and provided feedback regarding content. Results: Results indicated general fear regarding CCTs, which is partially attributable to the impact of historic research abuses, lack of information regarding CCTs, and lack of cultural relevance of the education and outreach materials for AA communities. Recruitment of AAs to CCTs may be enhanced by educational and outreach approaches that increase awareness of CCTs as well as involvement of the AA community in developing such interventions. Conclusion: Interventions should include the perspectives of AA women, as key stakeholders and decision-makers for their family and provide research information in a multimedia format that will facilitate family discussion and decision-making regarding CCTs.

Kawasaki at the Extremes of Age: Thinking Outside the Box.

Kawasaki disease is an acute vasculitis syndrome that typically occurs in children aged 1 to 4 years. Because there is no specific diagnostic test for Kawasaki disease, the diagnosis is made clinically based on specific characteristic signs and symptoms. Cases in which patients fall outside of the typical age range are uncommon and often challenging to diagnose because they have atypical presentations. This is especially true in infants, who rarely meet all the clinical criteria required for diagnosis. Patients at the extremes of ages often have a delayed diagnosis, which can lead to worse cardiac outcomes. We describe the cases of a young infant and an older adolescent who present with Kawasaki disease. These cases illustrate the challenge of diagnosing Kawasaki disease in patients beyond the typical age range. Both patients were return visits to the emergency department after inpatient stays. When fever persists longer than 5 days, clinicians must have a high index of suspicion for Kawasaki disease in all pediatric age groups to prevent treatment delay and disease sequelae.

Synthesis and in vitro evaluation of SG3227, a pyrrolobenzodiazepine dimer antibody-drug conjugate payload based on sibiromycin.

A novel pyrrolobenzodiazepine dimer payload, SG3227, was rationally designed based on the naturally occurring antitumour compound sibiromycin. SG3227 was synthesized from a dimeric core in an efficient fashion. An unexpected room temperature Diels-Alder reaction occurred during the final step of the synthesis and was circumvented by use of an iodoacetamide conjugation moiety in place of a maleimide. The payload was successfully conjugated to trastuzumab and the resulting ADC exhibited potent activity against a HER2-expressing human cancer cell line in vitro.

Transcutaneous auricular vagus nerve stimulation may benefit from the addition of N-acetylcysteine to facilitate motor learning in infants of diabetic mothers failing oral feeds.

Objective: This study aims to determine if pretreating with enteral N-acetylcysteine (NAC) improves CNS oxidative stress and facilitates improvement in oromotor skills during transcutaneous auricular nerve stimulation (taVNS) paired with oral feedings in infants of diabetic mothers (IDMs) who are failing oral feeds. Methods: We treated 10 IDMs who were gastrostomy tube candidates in an open-label trial of NAC and taVNS paired with oral feeding. NAC (75 or 100 mg/kg/dose) was given by nasogastric (NG) administration every 6 h for 4 days, then combined with taVNS paired with 2 daily feeds for another 14 days. NAC pharmacokinetic (PK) parameters were determined from plasma concentrations at baseline and at steady state on day 4 of treatment in conjunction with magnetic resonance spectroscopic (MRS) quantification of CNS glutathione (GSH) as a marker of oxidative stress. We compared increases in oral feeding volumes before and during taVNS treatment and with a prior cohort of 12 IDMs who largely failed to achieve full oral feeds with taVNS alone. Results: NAC 100 mg/kg/dose every 6 h NG resulted in plasma [NAC] that increased [GSH] in the basal ganglia with a mean of 0.13 ± 0.08 mM (p = 0.01, compared to baseline). Mean daily feeding volumes increased over 14 days of NAC + taVNS compared to the 14 days before treatment and compared to the prior cohort of 12 IDMs treated with taVNS alone. Seven IDMs reached full oral feeds sufficient for discharge, while three continued to have inadequate intake. Conclusion: In IDM failing oral feeds, NAC 100 mg/kg/dose every 6 h NG for 4 days before and during taVNS paired with oral feeding increased CNS GSH, potentially mitigating oxidative stress, and was associated with improving functional feeding outcomes compared to taVNS alone in a prior cohort. This represents a novel approach to neuromodulation and supports the concept that mitigation of ongoing oxidative stress may increase response to taVNS paired with a motor task.

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.

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.

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.