Modulation of TRPV4 protects against degeneration induced by sustained loading and promotes matrix synthesis in the intervertebral disc.

While it is well known that mechanical signals can either promote or disrupt intervertebral disc (IVD) homeostasis, the molecular mechanisms for transducing mechanical stimuli are not fully understood. The transient receptor potential vanilloid 4 (TRPV4) ion channel activated in isolated IVD cells initiates extracellular matrix (ECM) gene expression, while TRPV4 ablation reduces cytokine production in response to circumferential stretching. However, the role of TRPV4 on ECM maintenance during tissue-level mechanical loading remains unknown. Using an organ culture model, we modulated TRPV4 function over both short- (hours) and long-term (days) and evaluated the IVDs' response. Activating TRPV4 with the agonist GSK101 resulted in a Ca2+ flux propagating across the cells within the IVD. Nuclear factor (NF)-κB signaling in the IVD peaked at 6 h following TRPV4 activation that subsequently resulted in higher interleukin (IL)-6 production at 7 days. These cellular responses were concomitant with the accumulation of glycosaminoglycans and increased hydration in the nucleus pulposus that culminated in higher stiffness of the IVD. Sustained compressive loading of the IVD resulted in elevated NF-κB activity, IL-6 and vascular endothelial growth factor A (VEGFA) production, and degenerative changes to the ECM. TRPV4 inhibition using GSK205 during loading mitigated the changes in inflammatory cytokines, protected against IVD degeneration, but could not prevent ECM disorganization due to mechanical damage in the annulus fibrosus. These results indicate TRPV4 plays an important role in both short- and long-term adaptations of the IVD to mechanical loading. The modulation of TRPV4 may be a possible therapeutic for preventing load-induced IVD degeneration.

embryoTox: Using Graph-Based Signatures to Predict the Teratogenicity of Small Molecules.

Teratogenic drugs can lead to extreme fetal malformation and consequently critically influence the fetus's health, yet the teratogenic risks associated with most approved drugs are unknown. Here, we propose a novel predictive tool, embryoTox, which utilizes a graph-based signature representation of the chemical structure of a small molecule to predict and classify molecules likely to be safe during pregnancy. embryoTox was trained and validated using in vitro bioactivity data of over 700 small molecules with characterized teratogenicity effects. Our final model achieved an area under the receiver operating characteristic curve (AUC) of up to 0.96 on 10-fold cross-validation and 0.82 on nonredundant blind tests, outperforming alternative approaches. We believe that our predictive tool will provide a practical resource for optimizing screening libraries to determine effective and safe molecules to use during pregnancy. To provide a simple and integrated platform to rapidly screen for potential safe molecules and their risk factors, we made embryoTox freely available online at https://biosig.lab.uq.edu.au/embryotox/.

Bullying, harassment, and discrimination of musculoskeletal researchers and the impact of the COVID-19 pandemic: an international study.

PURPOSE: Bullying, harassment, and discrimination (BHD) are prevalent in academic, scientific, and clinical departments, particularly orthopedic surgery, and can have lasting effects on victims. As it is unclear how BHD affects musculoskeletal (MSK) researchers, the following study assessed BHD in the MSK research community and whether the COVID-19 pandemic, which caused hardships in other industries, had an impact. METHODS: A web-based anonymous survey was developed in English by ORS Spine Section members to assess the impact of COVID-19 on MSK researchers in North America, Europe, and Asia, which included questions to evaluate the personal experience of researchers regarding BHD. RESULTS: 116 MSK researchers completed the survey. Of respondents, 34.5% (n = 40) focused on spine, 30.2% (n = 35) had multiple areas of interest, and 35.3% (n = 41) represented other areas of MSK research. BHD was observed by 26.7% (n = 31) of respondents and personally experienced by 11.2% (n = 13), with mid-career faculty both observing and experiencing the most BHD. Most who experienced BHD (53.8%, n = 7) experienced multiple forms. 32.8% (n = 38) of respondents were not able to speak out about BHD without fear of repercussions, with 13.8% (n = 16) being unsure about this. Of those who observed BHD, 54.8% (n = 17) noted that the COVID-19 pandemic had no impact on their observations. CONCLUSIONS: To our knowledge, this is the first study to address the prevalence and determinants of BHD among MSK researchers. MSK researchers experienced and observed BHD, while many were not comfortable reporting and discussing violations to their institution. The COVID-19 pandemic had mixed-effects on BHD. Awareness and proactive policy changes may be warranted to reduce/eliminate the occurrence of BHD in this community.

Dysregulation of STAT3 signaling is associated with endplate-oriented herniations of the intervertebral disc in Adgrg6 mutant mice.

Degenerative changes of the intervertebral disc (IVD) are a leading cause of disability affecting humans worldwide and has been attributed primarily to trauma and the accumulation of pathology during aging. While genetic defects have also been associated with disc degeneration, the precise mechanisms driving the initiation and progression of disease have remained elusive due to a paucity of genetic animal models. Here, we discuss a novel conditional mouse genetic model of endplate-oriented disc herniations in adult mice. Using conditional mouse genetics, we show increased mechanical stiffness and reveal dysregulation of typical gene expression profiles of the IVD in adhesion G-protein coupled receptor G6 (Adgrg6) mutant mice prior to the onset of endplate-oriented disc herniations in adult mice. We observed increased STAT3 activation prior to IVD defects and go on to demonstrate that treatment of Adgrg6 conditional mutant mice with a small molecule inhibitor of STAT3 activation ameliorates endplate-oriented herniations. These findings establish ADGRG6 and STAT3 as novel regulators of IVD endplate and growth plate integrity in the mouse, and implicate ADGRG6/STAT3 signaling as promising therapeutic targets for endplate-oriented disc degeneration.

Development and validation of the CHIME simulation model to assess lifetime health outcomes of prediabetes and type 2 diabetes in Chinese populations: A modeling study.

BACKGROUND: Existing predictive outcomes models for type 2 diabetes developed and validated in historical European populations may not be applicable for East Asian populations due to differences in the epidemiology and complications. Despite the continuum of risk across the spectrum of risk factor values, existing models are typically limited to diabetes alone and ignore the progression from prediabetes to diabetes. The objective of this study is to develop and externally validate a patient-level simulation model for prediabetes and type 2 diabetes in the East Asian population for predicting lifetime health outcomes. METHODS AND FINDINGS: We developed a health outcomes model from a population-based cohort of individuals with prediabetes or type 2 diabetes: Hong Kong Clinical Management System (CMS, 97,628 participants) from 2006 to 2017. The Chinese Hong Kong Integrated Modeling and Evaluation (CHIME) simulation model comprises of 13 risk equations to predict mortality, micro- and macrovascular complications, and development of diabetes. Risk equations were derived using parametric proportional hazard models. External validation of the CHIME model was assessed in the China Health and Retirement Longitudinal Study (CHARLS, 4,567 participants) from 2011 to 2018 for mortality, ischemic heart disease, cerebrovascular disease, renal failure, cataract, and development of diabetes; and against 80 observed endpoints from 9 published trials using 100,000 simulated individuals per trial. The CHIME model was compared to United Kingdom Prospective Diabetes Study Outcomes Model 2 (UKPDS-OM2) and Risk Equations for Complications Of type 2 Diabetes (RECODe) by assessing model discrimination (C-statistics), calibration slope/intercept, root mean square percentage error (RMSPE), and R2. CHIME risk equations had C-statistics for discrimination from 0.636 to 0.813 internally and 0.702 to 0.770 externally for diabetes participants. Calibration slopes between deciles of expected and observed risk in CMS ranged from 0.680 to 1.333 for mortality, myocardial infarction, ischemic heart disease, retinopathy, neuropathy, ulcer of the skin, cataract, renal failure, and heart failure; 0.591 for peripheral vascular disease; 1.599 for cerebrovascular disease; and 2.247 for amputation; and in CHARLS outcomes from 0.709 to 1.035. CHIME had better discrimination and calibration than UKPDS-OM2 in CMS (C-statistics 0.548 to 0.772, slopes 0.130 to 3.846) and CHARLS (C-statistics 0.514 to 0.750, slopes -0.589 to 11.411); and small improvements in discrimination and better calibration than RECODe in CMS (C-statistics 0.615 to 0.793, slopes 0.138 to 1.514). Predictive error was smaller for CHIME in CMS (RSMPE 3.53% versus 10.82% for UKPDS-OM2 and 11.16% for RECODe) and CHARLS (RSMPE 4.49% versus 14.80% for UKPDS-OM2). Calibration performance of CHIME was generally better for trials with Asian participants (RMSPE 0.48% to 3.66%) than for non-Asian trials (RMPSE 0.81% to 8.50%). Main limitations include the limited number of outcomes recorded in the CHARLS cohort, and the generalizability of simulated cohorts derived from trial participants. CONCLUSIONS: Our study shows that the CHIME model is a new validated tool for predicting progression of diabetes and its outcomes, particularly among Chinese and East Asian populations that has been lacking thus far. The CHIME model can be used by health service planners and policy makers to develop population-level strategies, for example, setting HbA1c and lipid targets, to optimize health outcomes.

Development and Testing of a Novel Locking Pin Cap to Create a Fixed-Angle K-Wire Plate Construct.

PURPOSE: To test the effectiveness of a novel locking pin cap to attach a K-wire rigidly to a volar locking plate and resist fracture displacement compared with commercially available alternatives. METHODS: Two different methods of fracture fixation were tested on a total of 12 Sawbones models with volar shear distal radius fracture (6/group). The fragments were fixed with either 2 commercially available pin plates (industry standard) or a volar plate with 2 locking screws fixing the scaphoid facet and 2 pins locked to the plate with a novel locking pin cap in the lunate facet. Axial load conditioning was performed followed by sinusoidal loading to 250 N at 50 mm/s. A motion capture system was used to assess the relative movement of the fracture fragments relative to the intact shaft. The strength of the fixation construct was quantified by (1) the force required to achieve a 2-mm gap between the shaft and fracture fragments and (2) ultimate load to failure. RESULTS: One industry standard pin plate demonstrated disassociation of the pin from the plate after fatigue conditioning. This did not occur in the locking pin cap group. The locking pin cap construct group was able to sustain a significantly higher load compared with the industry standard when the construct was displaced to the 2-mm gap. The locking pin cap also significantly increased the ultimate load to failure compared with the industry standard. CONCLUSIONS: The novel locking pin cap creates a fixed-angle attachment of a K-wire to an existing locking screw hole in a plate. This fixed-angle K-wire is significantly stronger in preventing gap formation and resisting ultimate failure than commercially available plates that use bent K-wires. CLINICAL RELEVANCE: The development of novel techniques to secure small articular fragments may ultimately improve clinical outcomes.

The age-dependent effect of high-dose X-ray radiation on NFκB signaling, structure, and mechanical behavior of the intervertebral disc.

Purpose: Ionizing radiation damages tissue and provokes inflammatory responses in multiple organ systems. We investigated the effects of high-dose X-ray radiation on the molecular inflammation and mechanical function of the intervertebral disc (IVD).Methods: Functional spine units (FSUs) containing the vertebrae-IVDs-vertebrae structure extracted from 1-month, 6-month, and 16-month-old NFκB-luciferase reporter mice and from 6-month-old myeloid differentiation factor 88 (MyD88)-null mice. After a preconditioning period in culture, the FSUs were subjected a single dose of ionizing X-ray radiation at 20 Gys, and then NFκB expression was monitored. The IVDs were then subjected to mechanical testing using dynamic compression, glycosaminoglycan (GAG) quantification, and histological analyses.Results: In the 1-month-old FSUs, the NFκB-driven luciferase activity was significantly elevated for 1 day following the exposure to radiation. The 6-month-old FSUs showed increased NFκB activity for 3 days, while the 16-month-old FSUs sustained elevated levels of NFκB activity throughout the 10-day culture period. All irradiated groups showed significant loss of disc height, GAG content, mechanical function and changes in structure. Ablation of MyD88 blunted the radiation-mediated NFκB signaling, and preserved GAG content, and the IVDs' structure and mechanical performance.Conclusions: These results suggest that high-dose radiation affects the IVDs' NFκB-dependent inflammatory processes that subsequently lead to functional deterioration. Blocking the transactivation potential of NFκB via MyD88 ablation preserved the structure and mechanical function of the FSUs. The long-term effects of radiation on IVD homeostasis should be considered in individuals susceptible to occupational and medical exposure.

Cannulated Screw Prominence in Tension Band Wiring of Patella Fractures Increases Fracture Gapping: A Cadaver Study.

BACKGROUND: Transverse patella fractures are often treated with cannulated screws and a figure-of-eight anterior tension band. A common teaching regarding this construct is to recess the screws so that their distal ends do not protrude beyond the patella because doing so may improve biomechanical performance. However, there is a lack of biomechanical or clinical data to support this recommendation. QUESTION: In the treatment of transverse patella fractures, is there a difference between prominent and recessed cannulated screw constructs, supplemented by tension banding, in terms of gap formation from cyclic loading and ultimate load to failure? METHODS: Ten pairs of fresh-frozen cadaver legs (mean donor age, 72 years; range, 64-89 years) were randomized in a pairwise fashion to prominent or standard-length screws. In the prominent screw group, screw length was 15% longer than the measured trajectory, resulting in 4 to 6 mm of additional length. Each patella was transversely osteotomized at its midportion and fixed with screws and an anterior tension band. Gap formation was measured over 40 loaded flexion-extension cycles (90° to 5°). Ultimate load to failure was assessed with a final monotonic test after cyclic loading. Areal bone mineral density (BMD) of each patella was measured with dual energy x-ray absorptiometry (DEXA). There was no difference in BMD between the recessed (1.06 ± 0.262 g/cm) and prominent (1.03 ± 0.197 g/cm) screw groups (p = 0.846). Difference in gap formation was assessed with a Wilcoxon Rank Sum Test. Ultimate load to failure and BMD were assessed with a paired t-test. RESULTS: Patella fractures fixed with prominent cannulated screws demonstrated larger gap formation during cyclic loading. Median gap size at the end of cyclic loading was 0.13 mm (range, 0.00-2.92 mm) for the recessed screw group and 0.77 mm (range, 0.00-7.50 mm) for the prominent screw group (p = 0.039; 95% confidence interval [CI] difference of geometric means, 0.05-2.12 mm). There was no difference in ultimate failure load between the recessed screw (891 ± 258 N) and prominent screw (928 ± 268 N) groups (p = 0.751; 95% CI difference of means, -226 to 301 N). Ultimate failure load was correlated with areal BMD (r = 0.468; p = 0.046). CONCLUSIONS: In this cadaver study, when using cannulated screws and a figure-of-eight tension band to fix transverse patella fractures, prominent screws reduced the construct's ability to resist gap formation during cyclic loading testing. CLINICAL RELEVANCE: This biomechanical cadaver study found that the use of prominent cannulated screws for the fixation of transverse patella fractures increases the likelihood of interfragmentary gap formation, which may potentially increase the risk of fracture nonunion and implant failure. These findings suggest that proximally and distally recessed screws may increase construct stability, which may increase the potential for bony healing. The findings support further laboratory and clinical investigations comparing recessed screws supplemented by anterior tension banding with other repair methods that are in common use, such as transosseous suture repair.

Clinical measurements of bone tissue mechanical behavior using reference point indentation.

Over the last thirty years, it has become increasingly clear the amount of bone (e.g. 'bone quantity') and the quality of the bone matrix (e.g. 'bone quality') both critically contribute to bone's tissue-level mechanical behavior and the subsequent ability of bone to resist fracture. Although determining the tissue-level mechanical behavior of bone through mechanical testing is relatively straightforward in the laboratory, the destructive nature of such testing is unfeasible in humans and in animal models requiring longitudinal observation. Therefore, surrogate measurements are necessary for quantifying tissue-level mechanical behavior for the pre-clinical and clinical evaluation of bone strength and fracture risk in vivo. A specific implementation of indentation known as reference point indentation (RPI) enables the mechanical testing of bone tissue without the need to excise and prepare the bone surface. However, this compromises the ability to carefully control the specimen geometry that is required to define the bone tissue material properties. Yet the versatility of such measurements in clinical populations is provocative, and to date there are a number of promising studies that have utilized this tool to discern bone pathologies and to monitor the effects of therapeutics on bone quality. Concurrently, on-going efforts continue to investigate the aspects of bone material behavior measured by RPI, and the compositional factors that contribute to these measurements. There are currently two variants, cyclic- and impact- RPI, that have been utilized in pre-clinical and clinical studies. This review surveys clinical studies that utilize RPI, with particular emphasis on the clinical instrument, as well as the endeavors to understand the fundamental mechanisms of such measurements. Ultimately, an improved awareness in the tradeoffs and limitations of in vivo RPI is critical towards the effective and successful utilization of this tool for the overall improvement of fragility determination in the clinic.

Protein-induced fluorescence enhancement of two-photon excitable water-soluble diketopyrrolopyrroles.

Fluorescent contrast agents are important tools in cell biology and medical imaging due to their high sensitivity and relative availability. Diketopyrrolopyrrole (DPP) derivatives have been recently studied for applications in bioimaging, but certain drawbacks due to their inherent structure have stifled progress towards their widespread implementation. Aggregation caused quenching (ACQ) associated with π-π stacking in relatively rigid extended conjugation systems as well as hydrophobicity of previously reported DPPs make most unsuitable for biological imaging applications. Addressing these deficiencies, we report the synthesis and photophysical characterization of two new water-soluble diketopyrrolopyrole (DPP) probes that exhibit pronounced protein-induced fluorescence enhancement (PIFE) upon binding serum albumin protein. In vitro studies were also performed showing low cytotoxicity for the new DPP probes. Two-photon fluorescence microscopy (2PFM) images were obtained via excitation at 810 nm and emission in the NIR window of biological transparency, illustrating the potential of these compounds as nonlinear optical bioimaging probes.

Metastatic basal cell carcinoma: case series and review of the literature.

Metastatic basal cell carcinoma is exceedingly uncommon, with a poorly defined natural history, and its incidence, risk factors, patterns of spread, prognosis and potential treatment options are not well understood. This retrospective single institution case series aims to shed light on these issues. Aggressive local management in patients with locoregional disease may result in long-term survivors.

Novel Augmentation Technique for Patellar Tendon Repair Improves Strength and Decreases Gap Formation: A Cadaveric Study.

BACKGROUND: Patellar tendon ruptures commonly are repaired using transosseous patellar drill tunnels with modified-Krackow sutures in the patellar tendon. This simple suture technique has been associated with failure rates and poor clinical outcomes in a modest proportion of patients. Failure of this repair technique can result from gap formation during loading or a single catastrophic event. Several augmentation techniques have been described to improve the integrity of the repair, but standardized biomechanical evaluation of repair strength among different techniques is lacking. QUESTIONS/PURPOSES: The purpose of this study was to describe a novel figure-of-eight suture technique to augment traditional fixation and evaluate its biomechanical performance. We hypothesized that the augmentation technique would (1) reduce gap formation during cyclic loading and (2) increase the maximum load to failure. METHODS: Ten pairs (two male, eight female) of fresh-frozen cadaveric knees free of overt disorders or patellar tendon damage were used (average donor age, 76 years; range, 65-87 years). For each pair, one specimen underwent the standard transosseous tunnel suture repair with a modified-Krackow suture technique and the second underwent the standard repair with our experimental augmentation method. Nine pairs were suitable for testing. Each specimen underwent cyclic loading while continuously measuring gap formation across the repair. At the completion of cyclic loading, load to failure testing was performed. RESULTS: A difference in gap formation and mean load to failure was seen in favor of the augmentation technique. At 250 cycles, a 68% increase in gap formation was seen for the control group (control: 5.96 ± 0.86 mm [95% CI, 5.30-6.62 mm]; augmentation: 3.55 ± 0.56 mm [95% CI, 3.12-3.98 mm]; p = 0.02). The mean load to failure was 13% greater in the augmentation group (control: 899.57 ± 96.94 N [95% CI, 825.06-974.09 N]; augmentation: 1030.70 ± 122.41 N [95% CI, 936.61-1124.79 N]; p = 0.01). CONCLUSIONS: This biomechanical study showed improved performance of a novel augmentation technique compared with the standard repair, in terms of reduced gap formation during cyclic loading and increased maximum load to failure. CLINICAL RELEVANCE: Decreased gap formation and higher load to failure may improve healing potential and minimize failure risk. This study shows a potential biomechanical advantage of the augmentation technique, providing support for future clinical investigations comparing this technique with other repair methods that are in common use such as transosseous suture repair.

RGD-conjugated two-photon absorbing near-IR emitting fluorescent probes for tumor vasculature imaging.

Observation of the activation and inhibition of angiogenesis processes is important in the progression of cancer. Application of targeting peptides, such as a small peptide that contains adjacent L-arginine (R), glycine (G) and L-aspartic acid (D) residues can afford high selectivity and deep penetration in vessel imaging. To facilitate deep tissue vasculature imaging, probes that can be excited via two-photon absorption (2PA) in the near-infrared (NIR) and subsequently emit in the NIR are essential. In this study, the enhancement of tissue image quality with RGD conjugates was investigated with new NIR-emitting pyranyl fluorophore derivatives in two-photon fluorescence microscopy. Linear and nonlinear photophysical properties of the new probes were comprehensively characterized; significantly the probes exhibited good 2PA over a broad spectral range from 700-1100 nm. Cell and tissue images were then acquired and examined, revealing deep penetration and high contrast with the new pyranyl RGD-conjugates up to 350 µm in tumor tissue.

Age-related changes in the plasticity and toughness of human cortical bone at multiple length scales.

The structure of human cortical bone evolves over multiple length scales from its basic constituents of collagen and hydroxyapatite at the nanoscale to osteonal structures at near-millimeter dimensions, which all provide the basis for its mechanical properties. To resist fracture, bone's toughness is derived intrinsically through plasticity (e.g., fibrillar sliding) at structural scales typically below a micrometer and extrinsically (i.e., during crack growth) through mechanisms (e.g., crack deflection/bridging) generated at larger structural scales. Biological factors such as aging lead to a markedly increased fracture risk, which is often associated with an age-related loss in bone mass (bone quantity). However, we find that age-related structural changes can significantly degrade the fracture resistance (bone quality) over multiple length scales. Using in situ small-angle X-ray scattering and wide-angle X-ray diffraction to characterize submicrometer structural changes and synchrotron X-ray computed tomography and in situ fracture-toughness measurements in the scanning electron microscope to characterize effects at micrometer scales, we show how these age-related structural changes at differing size scales degrade both the intrinsic and extrinsic toughness of bone. Specifically, we attribute the loss in toughness to increased nonenzymatic collagen cross-linking, which suppresses plasticity at nanoscale dimensions, and to an increased osteonal density, which limits the potency of crack-bridging mechanisms at micrometer scales. The link between these processes is that the increased stiffness of the cross-linked collagen requires energy to be absorbed by "plastic" deformation at higher structural levels, which occurs by the process of microcracking.

Comparison of different intubation techniques performed inside a moving ambulance: a manikin study.

OBJECTIVE. Airway management and endotracheal intubation may be required urgently when a patient deteriorates in an ambulance or aircraft during interhospital transfer or in a prehospital setting. The objectives of this study were: (1) to compare the effectiveness of conventional intubation by Macintosh laryngoscope in a moving ambulance versus that in a static ambulance; and (2) to compare the effectiveness of inverse intubation and GlideScope laryngoscopy with conventional intubation inside a moving ambulance. DESIGN. Comparative experimental study. SETTING. The experiment was conducted in an ambulance provided by the Auxiliary Medical Service in Hong Kong. PARTICIPANTS. A group of 22 doctors performed endotracheal intubation on manikins with Macintosh laryngoscope in a static and moving ambulance. In addition, they performed conventional Macintosh intubation, inverse intubation with Macintosh laryngoscope, and GlideScope intubation in a moving ambulance in both normal and simulated difficult airways. MAIN OUTCOME MEASURES. The primary outcome was the rate of successful intubation. The secondary outcomes were time taken for intubation, subjective glottis visualisation grading, and eventful intubation (oesophageal intubation, intubation time >60 seconds, and incisor breakage) with different techniques or devices. RESULTS. In normal airways, conventional Macintosh intubation in a static ambulance (95.5%), conventional intubation in a moving ambulance (95.5%), as well as GlideScope intubation in a moving ambulance (95.5%) were associated with high success rates; the success rate of inverse intubation was comparatively low (54.5%; P=0.004). In difficult airways, conventional Macintosh intubation in a static ambulance (86.4%), conventional intubation in a moving ambulance (90.9%), and GlideScope intubation in a moving ambulance (100%) were associated with high success rates; the success rate of inverse intubation was comparatively lower (40.9%; P=0.034). CONCLUSIONS. En-route intubation in an ambulance by conventional Macintosh laryngoscopy is superior to inverse intubation unless the cephalad access is impossible. GlideScope laryngoscopy appears to be associated with lower rates of eventful intubation in difficult airways and has better laryngoscopic view versus inverse intubation.

Multi-omics resources for the Australian southern stuttering frog (Mixophyes australis) reveal assorted antimicrobial peptides.

The number of genome-level resources for non-model species continues to rapidly expand. However, frog species remain underrepresented, with up to 90% of frog genera having no genomic or transcriptomic data. Here, we assemble the first genomic and transcriptomic resources for the recently described southern stuttering frog (Mixophyes australis). The southern stuttering frog is ground-dwelling, inhabiting naturally vegetated riverbanks in south-eastern Australia. Using PacBio HiFi long-read sequencing and Hi-C scaffolding, we generated a high-quality genome assembly, with a scaffold N50 of 369.3 Mb and 95.1% of the genome contained in twelve scaffolds. Using this assembly, we identified the mitochondrial genome, and assembled six tissue-specific transcriptomes. We also bioinformatically characterised novel sequences of two families of antimicrobial peptides (AMPs) in the southern stuttering frog, the cathelicidins and ß-defensins. While traditional peptidomic approaches to peptide discovery have typically identified one or two AMPs in a frog species from skin secretions, our bioinformatic approach discovered 12 cathelicidins and two ß-defensins that were expressed in a range of tissues. We investigated the novelty of the peptides and found diverse predicted activities. Our bioinformatic approach highlights the benefits of multi-omics resources in peptide discovery and contributes valuable genomic resources in an under-represented taxon.

Analysis of Infiltrating Immune Cells Following Intervertebral Disc Injury Reveals Recruitment of Gamma-Delta (γδ) T cells in Female Mice.

Inadequate repair of injured intervertebral discs (IVD) leads to degeneration and contributes to low back pain. Infiltrating immune cells into damaged musculoskeletal tissues are critical mediators of repair, yet little is known about their identities, roles, and temporal regulation following IVD injury. By analyzing longitudinal changes in gene expression, tissue morphology, and the dynamics of infiltrating immune cells following injury, we characterize sex-specific differences in immune cell populations and identify the involvement of previously unreported immune cell types, γδ and NKT cells. Cd3+Cd4-Cd8- T cells are the largest infiltrating lymphocyte population with injury, and we identified the presence of γδ T cells in this population in female mice specifically, and NKT cells in males. Injury-mediated IVD degeneration was prevalent in both sexes, but more severe in males. Sex-specific degeneration may be associated with the differential immune response since γδ T cells have potent anti-inflammatory roles and may mediate IVD repair.

Pain, disability, and quality of life in participants after concurrent onabotulinumtoxinA treatment of upper and lower limb spasticity: Observational results from the ASPIRE study.

INTRODUCTION: Upper and lower limb spasticity is commonly associated with central nervous system disorders including stroke, traumatic brain injury, multiple sclerosis, cerebral palsy, and spinal cord injury, but little is known about the concurrent treatment of upper and lower limb spasticity with botulinum toxins. OBJECTIVE: To evaluate onabotulinumtoxinA (onabotA) utilization and to determine if concurrent onabotA treatment of the upper and lower limbs has supported improvements in participants with spasticity. DESIGN: Sub-analysis of a 2-year, international, prospective, observational registry (ASPIRE, NCT01930786). SETTING: International clinic sites (54). PARTICIPANTS: Adult spasticity participants across etiologies, who received ≥1 concurrent onabotA treatment of the upper and lower limbs during the study. INTERVENTION: Participants were treated with onabotA at the clinician's discretion. OUTCOMES: Baseline characteristics and outcomes of disability (Disability Assessment Scale [DAS]), pain (Numeric Pain Rating Scale [NPRS]), participant satisfaction, physician satisfaction, and quality of life (QoL; Spasticity Impact Assessment [SIA]) were evaluated. Adverse events were monitored throughout the study. RESULTS: Of 744 participants enrolled, 730 received ≥1 dose of onabotA; 275 participants received treatment with onabotA in both upper and lower limbs during ≥1 session; 39.3% of participants were naïve to onabotA for spasticity. The mean (SD) total dose per treatment session ranged from 421.2 (195.3) to 499.6 (188.6) U. The most common baseline upper limb presentation was clenched fist (n = 194, 70.5%); lower limb was equinovarus foot (n = 219, 66.9%). High physician and participant satisfaction and improvements in pain, disability and QoL were reported after most treatments. Nine participants (3.3%) reported nine treatment-related adverse events; two participants (0.7%) reported three serious treatment-related severe adverse events. No new safety signals were identified. CONCLUSION: More than a third of enrolled participants received at least one concurrent onabotA treatment of the upper and lower limbs, with reduced pain, disability, and improved QoL after treatment, consistent with the established safety profile of onabotA for the treatment of spasticity.

Assessment of bovine cortical bone fracture behavior using impact microindentation as a surrogate of fracture toughness.

The fracture behavior of bone is critically important for evaluating its mechanical competence and ability to resist fractures. Fracture toughness is an intrinsic material property that quantifies a material's ability to withstand crack propagation under controlled conditions. However, properly conducting fracture toughness testing requires the access to calibrated mechanical load frames and the destructive testing of bone samples, and therefore fracture toughness tests are clinically impractical. Impact microindentation mimicks certain aspects of fracture toughness measurements, but its relationship with fracture toughness remains unknown. In this study, we aimed to compare measurements of notched fracture toughness and impact microindentation in fresh and boiled bovine bone. Skeletally mature bovine bone specimens (n = 48) were prepared, and half of them were boiled to denature the organic matrix, while the other half remained preserved in frozen conditions. All samples underwent a notched fracture toughness test to determine their resistance to crack initiation (KIC) and an impact microindentation test using the OsteoProbe to obtain the Bone Material Strength index (BMSi). Boiling the bone samples increased the denatured collagen content, while mineral density and porosity remained unaffected. The boiled bones also showed significant reduction in both KIC (P < .0001) and the average BMSi (P < .0001), leading to impaired resistance of bone to crack propagation. Remarkably, the average BMSi exhibited a high correlation with KIC (r = 0.86; P < .001). A ranked order difference analysis confirmed the excellent agreement between the 2 measures. This study provides the first evidence that impact microindentation could serve as a surrogate measure for bone fracture behavior. The potential of impact microindentation to assess bone fracture resistance with minimal sample disruption could offer valuable insights into bone health without the need for cumbersome testing equipment and sample destruction.

Bone canonical Wnt signaling is downregulated in type 2 diabetes and associates with higher advanced glycation end-products (AGEs) content and reduced bone strength.

Type 2 diabetes (T2D) is associated with higher fracture risk, despite normal or high bone mineral density. We reported that bone formation genes (SOST and RUNX2) and advanced glycation end-products (AGEs) were impaired in T2D. We investigated Wnt signaling regulation and its association with AGEs accumulation and bone strength in T2D from bone tissue of 15 T2D and 21 non-diabetic postmenopausal women undergoing hip arthroplasty. Bone histomorphometry revealed a trend of low mineralized volume in T2D (T2D 0.249% [0.156-0.366]) vs non-diabetic subjects 0.352% [0.269-0.454]; p=0.053, as well as reduced bone strength (T2D 21.60 MPa [13.46-30.10] vs non-diabetic subjects 76.24 MPa [26.81-132.9]; p=0.002). We also showed that gene expression of Wnt agonists LEF-1 (p=0.0136) and WNT10B (p=0.0302) were lower in T2D. Conversely, gene expression of WNT5A (p=0.0232), SOST (p<0.0001), and GSK3B (p=0.0456) were higher, while collagen (COL1A1) was lower in T2D (p=0.0482). AGEs content was associated with SOST and WNT5A (r=0.9231, p<0.0001; r=0.6751, p=0.0322), but inversely correlated with LEF-1 and COL1A1 (r=-0.7500, p=0.0255; r=-0.9762, p=0.0004). SOST was associated with glycemic control and disease duration (r=0.4846, p=0.0043; r=0.7107, p=0.00174), whereas WNT5A and GSK3B were only correlated with glycemic control (r=0.5589, p=0.0037; r=0.4901, p=0.0051). Finally, Young's modulus was negatively correlated with SOST (r=-0.5675, p=0.0011), AXIN2 (r=-0.5523, p=0.0042), and SFRP5 (r=-0.4442, p=0.0437), while positively correlated with LEF-1 (r=0.4116, p=0.0295) and WNT10B (r=0.6697, p=0.0001). These findings suggest that Wnt signaling and AGEs could be the main determinants of bone fragility in T2D.

Type 2 diabetes is a long-term metabolic disease characterised by chronic high blood sugar levels. This in turn has a negative impact on the health of other tissues and organs, including bones. Type 2 diabetes patients have an increased risk of fracturing bones compared to non-diabetics. This is particularly true for fragility fractures, which are fractures caused by falls from a short height (i.e., standing height or less), often affecting hips or wrists. Usually, a lower bone density is associated with higher risk of fractures. However, patients with type 2 diabetes have increased bone fragility despite normal or higher bone density. One reason for this could be the chronically high levels of blood sugar in type 2 diabetes, which alter the properties of proteins in the body. It has been shown that the excess sugar molecules effectively 'react' with many different proteins, producing harmful compounds in the process, called Advanced Glycation End-products, or AGEs. AGEs are ­ in turn ­thought to affect the structure of collagen proteins, which help hold our tissues together and decrease bone strength. However, the signalling pathways underlying this process are still unclear. To find out more, Leanza et al. studied a signalling molecule, called sclerostin, which inhibits a signalling pathway that regulates bone formation, known as Wnt signaling. The researchers compared bone samples from both diabetic and non-diabetic patients, who had undergone hip replacement surgery. Analyses of the samples, using a technique called real-time-PCR, revealed that gene expression of sclerostin was increased in samples of type 2 diabetes patients, which led to a downregulation of Wnt signaling related genes. Moreover, the downregulation of Wnt genes was correlated with lower bone strength (which was measured by compressing the bone tissue). Further biochemical analysis of the samples revealed that higher sclerostin activity was also associated with higher levels of AGEs. These results provide a clearer understanding of the biological mechanisms behind compromised bone strength in diabetes. In the future, Leanza et al. hope that this knowledge will help us develop treatments to reduce the risk of bone complications for type 2 diabetes patients.