Bone-targeted ultrasound-responsive nanobubbles for siRNA delivery to treat osteoporosis in mice.

This project aimed to study the efficacy of a bone-targeted ultrasound-responsive nanobubble (NB) platform to deliver gene-silencing cathepsin K (CTSK) siRNA into the bone for osteoporosis treatment using in vitro and in vivo studies. To this end, characterization of CTSK siRNA loaded NB functionalized with alendronate (NB-CTSK siRNA-AL) was performed using transmission electron microscopy (TEM) imaging, and a release profile was obtained through fluorescent spectroscopy. In vitro studies were conducted by culturing NB-CTSK siRNA-AL with osteoclasts to evaluate siRNA uptake, CTSK expression, and the expression of tartrate-resistant acid phosphatase (TRAP). A control group and an NB-CTSK siRNA-AL treated group of ovariectomized (OVX) mice (n = 4) were tested. The OVX group that received treatment underwent weekly sessions for 4 weeks, during which they were exposed to low-intensity pulsed ultrasound (LIPUS) stimulation following administration of NB-CTSK siRNA-AL, prior to being sacrificed. Both groups underwent a series of tests to evaluate the bone targeting, safety, and efficacy of the nanoplatform. These tests included biodistribution studies conducted at 4 h and 24 h post-injection, a 3-point bending test of the femurs, nano-computed tomography analysis, as well as Hematoxylin & Eosin histological staining, Masson's Trichrome staining, and CTSK staining. The biodistribution showed the accumulation of NB-CTSK siRNA-AL in the bone and liver. Results showed that the OVX mice treated with NB-CTSK siRNA-AL had increased distal cortical bone thickness (174.4 ± 5.28 µm vs. 144.3 ± 10.66 µm, p > 0.05)) and bone volume fraction (16.5 ± 3.96 % vs. 6.55 ± 0.13 % (p > 0.05)). A reduced collagen degradation and downregulated CTSK expression were evident in the staining procedures. No adverse effects were recorded within histological assessments on the liver, kidney, and heart post-treatment. Morphology was shown to be normal and healthy within muscle cells post-LIPUS stimulation of NB-CTSK siRNA-AL. From these results, it can be concluded that an ultrasound-mediated NB-CTSK siRNA-AL can serve as a reliable, safe CTSK siRNA carrier to bone-specific targets for in vivo osteoporosis treatment.

Level Up Your Residency: Can a Novel Two-Week Simulation Rotation Really Make a Difference?

INTRODUCTION: This study describes a unique two-week simulation-based medical education (SBME) rotation for transitional year (TY) residents. During the rotation, residents are fully integrated into the simulation team, actively participating in clinically based interprofessional scenarios, procedural techniques, and mixed reality experiences. Residents also created and ran their own simulations while receiving content expert feedback. We evaluated the rotation's effectiveness in preparing TY graduates for their specific advanced residency track. METHODS: A retrospective survey evaluated the experiences of 11 TY residents who participated in a unique two-week simulation rotation. The survey assessed residents' perceptions of the program's value and skill development, course design, scenario relevance to future practice, and preparedness to develop future scenarios. RESULTS: Residents (11 out of 12 residents, 92% response rate) overwhelmingly endorsed the simulation rotation (100% positive, 45.45% extremely valuable). The program demonstrably improved core clinical skills (100% reported improvement) and fostered self-efficacy for future practice. Scenario relevance was high (81.82% highly relevant). Collaboration and communication skills showed promise (72.73% positive) while highlighting a potential area for future refinement. Residents unanimously agreed on effective time allocation and the program's value for debriefing skills. Notably, 91% strongly supported residency-specific simulation training. DISCUSSION: The two-week simulation was perceived by prior TY residents as valuable, with a majority finding the experience highly valuable across multiple survey questions. Residents overwhelmingly expressed a preference for residency-specific training, suggesting future development of specialty-tailored modules and enhanced debriefing sessions. The findings highlight the program's effectiveness and successful implementation into a TY residency curriculum.

The road to virtual control groups and the importance of proper body-weight selection.

Virtual control groups (VCGs) created from historical control data (HCD) can reduce the number of concurrent control group animals needed in regulatory toxicity studies by up to 25%. This study investigates the performance of VCGs on statistical outcomes of body weight development between treatment and control groups in legacy studies. The objective is to reproduce the statistical outcomes of 28-day sub-chronic studies (legacy studies) after replacing the concurrent control group with virtual ones. In rodent toxicity studies initial body weight is used as surrogate for the age of animals. For the assessment of VCG-sampling methods, three different approaches were explored: (i) sam­pling VCGs from the entire HCD, ignoring initial body weight information of the legacy study, (ii) sampling from HCD by matching the legacy study's initial body weights, and (iii) sampling from HCD with assigned statistical weights derived from legacy study initial body weight information. The ability to reproduce statistical outcomes using virtual controls was determined by the congruence between the legacy study and the HCD weight distribution: regardless of the chosen approach, the ability to reproduce statistical outcomes was high for VCGs when the legacy study's initial body weight distribution was similar to the HCD's. When the initial body weight range of the legacy study was at the extreme ends of the HCD's distribution, the weighted sampling approach was superior. This article demonstrates the importance of proper HCD matching by the legacy study's initial body weight and discusses conditions to accurately reproduce body weight development.

Animal control data from past studies performed in a standardized manner can be used to create virtual control groups (VCGs) to use in new studies instead of control animals. This approach can reduce the number of study animals needed by up to 25%. This study assessed the performance of VCGs selected by body weight in rat studies. The objective was to reproduce the original study results as closely as possible after replacing the original control group values with VCGs from a pool of historical control values. Several methods for selecting control animal data to create VCGs were compared. Among these, assigning statistical weights to the sampling pool yielded the best performance. Ideally, the body weight distributions on day 1 of the study should be similar between the VCG and the original study animals. This article shows that proper selection of VCGs can yield reliable study data with fewer animals.

Eye movements track prioritized auditory features in selective attention to natural speech.

Over the last decades, cognitive neuroscience has identified a distributed set of brain regions that are critical for attention. Strong anatomical overlap with brain regions critical for oculomotor processes suggests a joint network for attention and eye movements. However, the role of this shared network in complex, naturalistic environments remains understudied. Here, we investigated eye movements in relation to (un)attended sentences of natural speech. Combining simultaneously recorded eye tracking and magnetoencephalographic data with temporal response functions, we show that gaze tracks attended speech, a phenomenon we termed ocular speech tracking. Ocular speech tracking even differentiates a target from a distractor in a multi-speaker context and is further related to intelligibility. Moreover, we provide evidence for its contribution to neural differences in speech processing, emphasizing the necessity to consider oculomotor activity in future research and in the interpretation of neural differences in auditory cognition.

Carrier-frequency specific omission-related neural activity in ordered sound sequences is independent of omission-predictability.

Regularities in our surroundings lead to predictions about upcoming events. Previous research has shown that omitted sounds during otherwise regular tone sequences elicit frequency-specific neural activity related to the upcoming but omitted tone. We tested whether this neural response is depending on the unpredictability of the omission. Therefore, we recorded magnetencephalography (MEG) data while participants listened to ordered or random tone sequences with omissions occurring either ordered or randomly. Using multivariate pattern analysis shows that the frequency-specific neural pattern during omission within ordered tone sequences occurs independent of the regularity of the omissions. These results suggest that the auditory predictions based on sensory experiences are not immediately updated by violations of those expectations.

Points to consider regarding the use and implementation of virtual controls in nonclinical general toxicology studies.

The replacement of a proportion of concurrent controls by virtual controls in nonclinical safety studies has gained traction over the last few years. This is supported by foundational work, encouraged by regulators, and aligned with societal expectations regarding the use of animals in research. This paper provides an overview of the points to consider for any institution on the verge of implementing this concept, with emphasis given on database creation, risks, and discipline-specific perspectives.

Replacing concurrent controls with virtual control groups in rat toxicity studies.

Virtual control groups (VCGs) in nonclinical toxicity represent the concept of using appropriate historical control data for replacing concurrent control group animals. Historical control data collected from standardized studies can serve as base for constructing VCGs and legacy study reports can be used as a benchmark to evaluate the VCG performance. Replacing concurrent controls of legacy studies with VCGs should ideally reproduce the results of these studies. Based on three four-week rat oral toxicity legacy studies with varying degrees of toxicity findings we developed a concept to evaluate VCG performance on different levels: the ability of VCGs to (i) reproduce statistically significant deviations from the concurrent control, (ii) reproduce test substance-related effects, and (iii) reproduce the conclusion of the toxicity study in terms of threshold dose, target organs, toxicological biomarkers (clinical pathology) and reversibility. Although VCGs have shown a low to moderate ability to reproduce statistical results, the general study conclusions remained unchanged. Our results provide a first indication that carefully selected historical control data can be used to replace concurrent control without impairing the general study conclusion. Additionally, the developed procedures and workflows lay the foundation for the future validation of virtual controls for a use in regulatory toxicology.

Comprehensive evaluation of recombinant lactate dehydrogenase production from inclusion bodies.

A broad application spectrum ranging from clinical diagnostics to biosensors in a variety of sectors, makes the enzyme Lactate dehydrogenase (LDH) highly interesting for recombinant protein production. Expression of recombinant LDH is currently mainly carried out in uncontrolled shake-flask cultivations leading to protein that is mostly produced in its soluble form, however in rather low yields. Inclusion body (IB) processes have gathered a lot of attention due to several benefits like increased space-time yields and high purity of the target product. Thus, to investigate the suitability of this processing strategy for ldhL1 production, a fed-batch fermentation steering the production of IBs rather than soluble product formation was developed. It was shown that the space-time-yield of the fermentation could be increased almost 3-fold by increasing qs to 0.25 g g-1 h-1 which corresponds to 21% of qs,max, and keeping the temperature at 37°C after induction. Solubilization and refolding unit operations were developed to regain full bioactivity of the ldhL1. The systematic approach in screening for solubilization and refolding conditions revealed buffer compositions and processing strategies that ultimately resulted in 50% product recovery in the refolding step, revealing major optimization potential in the downstream processing chain. The recovered ldhL1 showed an optimal activity at pH 5.5 and 30∘C with a high catalytic activity and KM values of 0.46 mM and 0.18 mM for pyruvate and NADH, respectively. These features, show that the here produced LDH is a valuable source for various commercial applications, especially considering low pH-environments.

Toward implementing virtual control groups in nonclinical safety studies.

Historical data from control groups in animal toxicity studies is currently mainly used for comparative purposes to assess validity and robustness of study results. Due to the highly controlled environment in which the studies are performed and the homogeneity of the animal collectives it has been proposed to use the historical data for building so-called virtual control groups, which could replace partly or entirely the concurrent control. This would constitute a substantial contribution to the reduction of animal use in safety studies. Before the concept can be implemented, the prerequisites regarding data collection, curation and statistical evaluation together with a validation strategy need to be identified to avoid any impairment of the study outcome and subsequent consequences for human risk assessment. To further assess and develop the concept of virtual control groups the transatlantic think tank for toxicology (t4) sponsored a workshop with stakeholders from the pharmaceutical and chemical industry, academia, FDA, pharmaceutical, contract research organizations (CROs), and non-governmental organizations in Washington, which took place in March 2023. This report summarizes the current efforts of a European initiative to share, collect and curate animal control data in a centralized database and the first approaches to identify optimal matching criteria between virtual controls and the treatment arms of a study as well as first reflections about strategies for a qualification procedure and potential pitfalls of the concept.

Animal safety studies are usually performed with three groups of animals where increasing amounts of the test chemical are given to the animals and one control group where the animals do not receive the test chemical. The design of such studies, the characteristics of the animals, and the measured parameters are often very similar from study to study. Therefore, it has been suggested that measurement data from the control groups could be reused from study to study to lower the total number of animals per study. This could reduce animal use by up to 25% for such standardized studies. A workshop was held to discuss the pros and cons of such a concept and what would have to be done to implement it without threatening the reliability of the study outcome or the resulting human risk assessment.

Rosai-Dorfman Disease in a Pediatric Patient: Imaging Findings and Pathology with a brief review of the Literature.

Rosai-Dorfman Disease, otherwise known as sinus histiocytosis with massive lymphadenopathy, is a rare form of non-Langerhans cell histiocytosis with an estimated incidence of 100 cases per year in the United States. Due to its variable presentation and nonspecific clinical findings, it is particularly difficult to diagnose in pediatric patients. We report a case of an 11-month-old male who presented with a 4-day history of a right groin mass. Ultrasound of the groin and pelvis demonstrated, and MRI of the abdomen and pelvis confirmed an inguinal mass with surrounding lymphadenopathy. Pathology confirmed Rosai-Dorfman Disease and the patient improved after starting oral steroid therapy. To the best of our knowledge, this is the first case of Rosai-Dorfman Disease involving the inguinal region in an infant under 1 year of age reported in the literature. In this case report, we discuss the imaging and histology findings as well as provide a brief literature review for this diagnosis.

Can Historical Control Group Data Be Used to Replace Concurrent Controls in Animal Studies?

The availability of large amounts of high-quality control data from tightly controlled regulated animal safety data has created the idea to re-use these data beyond its classical applications of quality control, identification of treatment-related effects and assessing effect-size relevance for building virtual control groups (VCGs). While the ethical and cost-saving aspects of such a concept are immediately evident, the potential challenges need to be carefully considered to avoid any effect which could lower the sensitivity of an animal study to detect adverse events, safety thresholds, target organs, or biomarkers. In our brief communication, we summarize the current discussion regarding VCGs and propose a path forward how the replacement of concurrent control with VCGs resulting from historical data could be systematically assessed and to come to conclusions regarding the scientific value of the concept.

Hepatotoxicity of AKR1C3 Inhibitor BAY1128688: Findings from an Early Terminated Phase IIa Trial for the Treatment of Endometriosis.

INTRODUCTION: BAY1128688 is a selective inhibitor of aldo-keto reductase family 1 member C3 (AKR1C3), an enzyme implicated in the pathology of endometriosis and other disorders. In vivo animal studies suggested a potential therapeutic application of BAY1128688 in treating endometriosis. Early clinical studies in healthy volunteers supported the start of phase IIa. OBJECTIVE: This manuscript reports the results of a clinical trial (AKRENDO1) assessing the effects of BAY1128688 in adult premenopausal women with endometriosis-related pain symptoms over a 12-week treatment period. METHODS: Participants in this placebo-controlled, multicenter phase IIa clinical trial (NCT03373422) were randomized into one of five BAY1128688 treatment groups: 3 mg once daily (OD), 10 mg OD, 30 mg OD, 30 mg twice daily (BID), 60 mg BID; or a placebo group. The efficacy, safety, and tolerability of BAY1128688 were investigated. RESULTS: Dose-/exposure-dependent hepatotoxicity was observed following BAY1128688 treatment, characterized by elevations in serum alanine transferase (ALT) occurring at around 12 weeks of treatment and prompting premature trial termination. The reduced number of valid trial completers precludes conclusions regarding treatment efficacy. The pharmacokinetics and pharmacodynamics of BAY1128688 among participants with endometriosis were comparable with those previously found in healthy volunteers and were not predictive of the subsequent ALT elevations observed. CONCLUSIONS: The hepatotoxicity of BAY1128688 observed in AKRENDO1 was not predicted by animal studies nor by studies in healthy volunteers. However, in vitro interactions of BAY1128688 with bile salt transporters indicated a potential risk factor for hepatotoxicity at higher doses. This highlights the importance of in vitro mechanistic and transporter interaction studies in the assessment of hepatoxicity risk and suggests further mechanistic understanding is required. CLINICAL TRIAL REGISTRATION: NCT03373422 (date registered: November 23, 2017).

Perspectives of data science in preclinical safety assessment.

The data landscape in preclinical safety assessment is fundamentally changing because of not only emerging new data types, such as human systems biology, or real-world data (RWD) from clinical trials, but also technological advancements in data-processing software and analytical tools based on deep learning approaches. The recent developments of data science are illustrated with use cases for the three factors: predictive safety (new in silico tools), insight generation (new data for outstanding questions); and reverse translation (extrapolating from clinical experience to resolve preclinical questions). Further advances in this field can be expected if companies focus on overcoming identified challenges related to a lack of platforms and data silos and assuring appropriate training of data scientists within the preclinical safety teams.

Involuntary shifts of spatial attention contribute to distraction-Evidence from oscillatory alpha power and reaction time data.

Imagine you are focusing on the traffic on a busy street to ride your bike safely when suddenly you hear the siren of an ambulance. This unexpected sound involuntarily captures your attention and interferes with ongoing performance. We tested whether this type of distraction involves a spatial shift of attention. We measured behavioral data and magnetoencephalographic alpha power during a cross-modal paradigm that combined an exogenous cueing task and a distraction task. In each trial, a task-irrelevant sound preceded a visual target (left or right). The sound was usually the same animal sound (i.e., standard sound). Rarely, it was replaced by an unexpected environmental sound (i.e., deviant sound). Fifty percent of the deviants occurred on the same side as the target, and 50% occurred on the opposite side. Participants responded to the location of the target. As expected, responses were slower to targets that followed a deviant compared to a standard. Crucially, this distraction effect was mitigated by the spatial relationship between the targets and the deviants: responses were faster when targets followed deviants on the same versus different side, indexing a spatial shift of attention. This was further corroborated by a posterior alpha power modulation that was higher in the hemisphere ipsilateral (vs. contralateral) to the location of the attention-capturing deviant. We suggest that this alpha power lateralization reflects a spatial attention bias. Overall, our data support the contention that spatial shifts of attention contribute to deviant distraction.

The evolving role of investigative toxicology in the pharmaceutical industry.

For decades, preclinical toxicology was essentially a descriptive discipline in which treatment-related effects were carefully reported and used as a basis to calculate safety margins for drug candidates. In recent years, however, technological advances have increasingly enabled researchers to gain insights into toxicity mechanisms, supporting greater understanding of species relevance and translatability to humans, prediction of safety events, mitigation of side effects and development of safety biomarkers. Consequently, investigative (or mechanistic) toxicology has been gaining momentum and is now a key capability in the pharmaceutical industry. Here, we provide an overview of the current status of the field using case studies and discuss the potential impact of ongoing technological developments, based on a survey of investigative toxicologists from 14 European-based medium-sized to large pharmaceutical companies.

A rodent thyroid-liver chip to capture thyroid toxicity on organ function level.

Endocrine disruption by environmental chemicals continues to be a concern for human safety. The rat, a widely used model organism in toxicology, is very sensitive to chemical-induced thyroid perturbation, e.g., histopathological alterations in thyroid tissue. Species differences in the susceptibility to thyroid perturbation lead to uncertainty in human safety risk assessments. Hazard identification and characterization of chemically induced thyroid perturbation would therefore benefit from in vitro models addressing different mechanisms of action in a single functional assay, ideally across species. We here introduce a rat thyroid-liver chip that enables simultaneous identification of direct and indirect (liver-mediated) thyroid perturbation on organ-level functions in vitro. A second manuscript describes our work toward a human thyroid-liver chip (Kühnlenz et al., 2022). The presented microfluidic model consisting of primary rat thyroid follicles and liver 3D spheroids maintains a tissue-specific phenotype for up to 21 days. More precisely, the thyroid model exhibits a follicular architecture expressing basolateral and apical markers and secretes T4. Likewise, liver spheroids retain hepatocellular characteristics, e.g., a stable release of albumin and urea, the presence of bile canalicular networks, and the formation of T4-glucuronide. Experiments with reference chemicals demonstrated proficiency to detect direct and indirect mechanisms of thyroid perturbation through decreased thyroid hormone secretion and increased gT4 formation, respectively. Prospectively this rat thyroid-liver chip model, together with its human counterpart, may support a species-specific quantitative in vitro to in vivo extrapolation to improve a data-driven and evidence-based human safety risk assessment with significant contributions to the 3R principles.

Radiologic image sharing among U.S. children's hospitals.

BACKGROUND: There is no streamlined approach for sharing radiologic images among medical institutions. Common methods to transfer imaging between facilities include electronic image-sharing platforms and physical media, such as compact discs (CDs). The prompt and secure transfer of imaging is vital for patient safety as demand for imaging increases. OBJECTIVE: Use a survey-based study to outline the methods and difficulties of image sharing among U.S. children's hospitals. MATERIALS AND METHODS: A multi-question survey regarding radiologic image sharing was distributed to children's hospital department chairs in the United States in August 2021. Descriptive statistical analyses of the results were performed. RESULTS: Our results reveal 78% of responding U.S. children's hospitals have an electronic image-sharing platform. Twenty-seven percent of surveyed institutions experience daily difficulties with radiologic image sharing. Most of the difficulties are with CDs (67%) and a lack of interoperability among electronic image-sharing platforms (51%). CONCLUSION: Our study identified the various methods used by U.S. children's hospitals for radiologic image sharing and quantified the ongoing challenges with these systems.

A microfluidic thyroid-liver platform to assess chemical safety in humans.

Thyroid hormones (THs) are crucial regulators of human metabolism and early development. During the safety assessment of plant protection products, the human relevance of chemically induced TH perturbations observed in test animals remains uncertain. European regulatory authorities request follow-up in vitro studies to elucidate human-relevant interferences on thyroid gland function or TH catabolism through hepatic enzyme induction. However, human in vitro assays based on single molecular initiating events poorly reflect the complex TH biology and related liver-thyroid axis. To address this complexity, we present human three-dimensional thyroid and liver organoids with key functions of TH metabolism. The thyroid model resembles in vivo-like follicular architecture and a TSH-dependent triiodothyronine synthesis over 21 days, which is inhibited by methimazole. The HepaRG-based liver model, secreting the critical TH-binding proteins albumin and thyroxine-binding globulin, emulates an active TH catabolism via the formation of glucuronidated and sulfated thyroxine (gT4/sT4). Activation of the nuclear receptors PXR and AHR was demonstrated via the induction of specific CYP isoenzymes by rifampicin, pregnenolone-16α-carbonitrile, and ß-naphthoflavone. However, this nuclear receptor activation, assumed to regulate UDP-glucuronosyltransferases and sulfotransferases, appeared to have no effect on gT4 and sT4 formation in this human-derived hepatic cell line model. Finally, established single-tissue models were successfully co-cultured in a perfused two-organ chip for 21 days. In conclusion, this model presents a first step towards a complex multimodular human platform that will help to identify both direct and indirect thyroid disruptors that are relevant from a human safety perspective.