Editing Physicians' Responses Using GPT-4 for Academic Research.

The integration of Artificial Intelligence (AI) into digital healthcare, particularly in the anonymisation and processing of health information, holds considerable potential. OBJECTIVES: To develop a methodology using Generative Pre-trained Transformer (GPT) models to preserve the essence of medical advice in doctors' responses, while editing them for use in scientific studies. METHODS: German and English responses from EXABO, a rare respiratory disease platform, were processed using iterative refinement and other prompt engineering techniques, with a focus on removing identifiable and irrelevant content. RESULTS: Of 40 responses tested, 31 were accurately modified according to the developed guidelines. Challenges included misclassification and incomplete removal, with incremental prompting proving more accurate than combined prompting. CONCLUSION: GPT-4 models show promise in medical response editing, but face challenges in accuracy and consistency. Precision in prompt engineering is essential in medical contexts to minimise bias and retain relevant information.

Translation of Ontological Concepts from English into German Using Commercial Translation Software and Expert Evaluation.

Medical ontologies are mostly available in English. This presents a language barrier that is a limitation in research and automated processing of patient data. The manual translation of ontologies is complex and time-consuming. However, there are commercial translation tools that have shown promising results in the field of medical terminology translation. The aim of this study is to translate selected terms of the Human Phenotype Ontology (HPO) from English into German using commercial translators. Six medical experts evaluated the translation candidates in an iterative process. The results show commercial translators, with DeepL in the lead, provide translations that are positively evaluated by experts. With a broader study scope and additional optimization techniques, commercial translators could support and facilitate the process of translating medical ontologies.

Blackbox error management: how do practices deal with critical incidents in everyday practice? A qualitative interview study.

BACKGROUND: Error management plays a key role in patient safety. It is a systematic approach aimed at identifying and learning from critical incidents by reporting, documenting and analyzing them. Almost nothing is known about the incidents physicians in outpatient care consider to be critical and how they deal with them. We carried out an interview study to explore outpatient physicians' views on error management, discover what they regard as critical incidents, and find out how error management is put into practice in ambulatory care. METHODS: We conducted 72 semi-structured interviews with physicians from ambulatory practices. We asked participants what they considered to be a critical incident, how they reacted following an incident, how they discussed incidents with their coworkers, and whether they used critical incident reporting systems. The interviews were transcribed verbatim and analyzed using qualitative content analysis. RESULTS: Interviewed physicians defined the term "critical incident" differently. Most participants reported that they recorded information on incidents and discussed them in their teams. Several physicians reported taking a 'pay better attention next time-approach' to the analysis of incidents. Systematic error management involving incident documentation, analysis, preventive measure development, and follow-up, was the exception. CONCLUSIONS: To promote error management, medical training should include teaching on the topic, so that medical professionals can learn about critical incidents and how to deal with them in an open and structured manner. This would help establish the culture of safety that has long been called for internationally.

Effect of the addition of a mental health specialist for evaluation of undiagnosed patients in centres for rare diseases (ZSE-DUO): a prospective, controlled trial with a two-phase cohort design.

Background: People with complex symptomatology but unclear diagnosis presenting to a centre for rare diseases (CRD) may present with mental (co-)morbidity. We hypothesised that combining an expert in somatic medicine with a mental health specialist working in tandem will improve the diagnostic outcome. Methods: Patients aged 12 years and older who presented to one of the 11 participating German CRDs with an unknown diagnosis were recruited into this prospective cohort trial with a two-phase cohort design. From October 1, 2018 to September 30, 2019, participants were allocated to standard care (SC, N = 684), and from October 1, 2019 to January 31, 2021 to innovative care (IC, N = 695). The cohorts consisted mainly of adult participants with only a minority of children included (N = 67). IC included the involvement of a mental health specialist in all aspects of care (e.g., assessing medical records, clinic visits, telehealth care, and case conferences). Clinicaltrials.gov identifier: NCT03563677. Findings: The proportion of patients with diagnoses established within 12 months after the first visit to the CRD explaining the entire symptomatology (primary outcome) was 19% (N = 131 of 672) in the SC and 42% (N = 286 of 686) in the IC cohort (OR adjusted for centre effects 3.45 [95% CrI: 1.99-5.65]). The difference was mainly due to a higher prevalence of mental disorders and non-rare somatic diseases in the IC cohort. The median time to explaining diagnoses was one month shorter with IC (95% CrI: 1-2), and significantly more patients could be referred to local regular care in the IC (27.5%; N = 181 of 659) compared to the SC (12.3%; N = 81 of 658) cohort (OR adjusted for centre effects 2.70 [95% CrI: 2.02-3.60]). At 12-month follow-up, patient satisfaction with care was significantly higher in the IC compared to the SC cohort, while quality of life was not different between cohorts. Interpretation: Our findings suggested that including a mental health specialist in the entire evaluation process of CRDs for undiagnosed adolescents and adults should become an integral part of the assessment of individuals with a suspected rare disease. Funding: The study was funded by the Global Innovation Fund from the Joint Federal Committee in Germany (Innovationsfonds des Gemeinsamen Bundesausschusses), grant number 01NVF17031.

Engineered murine IL-21-secreting leukemia cells induce granzyme B+ T cells and CD4+CD44+CD62L- effector memory cells while suppressing regulatory T cells, leading to long-term survival.

We have explored the use of an IL-21 cell-based anti-leukemia treatment in a mouse model of acute lymphoblastic leukemia. 70Z/3 leukemia cells, engineered to secrete IL-21 and injected into the peritoneum of syngeneic mice, induced a strong anti-leukemia response resulting in 100% survival. Mice that mounted an IL-21-induced anti-leukemia immune response were immune to the parent cell line (no IL-21) when rechallenged.Above a certain threshold, IL-21 secretion correlated with improved survival compared to mice injected with parent 70Z/3 cells. IL-21 was detected in serum with peak levels on day 7, correlating with the maximum expansion of IL-21-secreting 70Z/3 cells which subsequently were eliminated. Mice injected with IL-21-secreting leukemia cells had elevated numbers of granzyme B+ CD4+ and CD8+ T cells in the peritoneum, compared to mice injected with the parent cell line. Regulatory T cells, which increased greatly in 70Z/3-injected mice, failed to do so in mice injected with IL-21-secreting cells. Upon rechallenge, IL-21-primed mice went through a secondary immune response, primarily requiring CD4+ T cells, triggering a significant increase of CD4+CD44+CD62L- effector memory T cells. Adoptive transfer of T cells from IL21-primed/rechallenged hosts into naïve mice was successful, indicating that IL-21-primed antigen-experienced T cells convey immunity to naïve mice.Our study shows that delivery of IL-21 in a cell-based anti-leukemia protocol has the potential to induce a potent immune response leading to cancer elimination and long-term immunity-properties which make IL-21 an attractive candidate for cancer immunotherapy. Protecting against tumor antigens as well as improving cancer immunity is justified, as current strategies are limited.

Conception, Development and Validation of Classification Methods for Coding Support of Rare Diseases Using Artificial Intelligence.

Automated coding of diseases can support hospitals in the billing of inpatient cases with the health insurance funds. This paper describes the implementation and evaluation of classification methods for two selected Rare Diseases. Different classifiers of an off-the-shelf system and an own application are applied in a supervised learning process and comparatively examined for their suitability and reliability. Using Natural Language Processing and Machine Learning, disease entities are recognized from unstructured historical patient records and new billing cases are coded automatically. The results of the performed classifications show that even with small datasets (≤ 200), high correctness (F1 score ∼0.8) can be achieved in predicting new cases.

Dual guidance structure for evaluation of patients with unclear diagnosis in centers for rare diseases (ZSE-DUO): study protocol for a controlled multi-center cohort study.

BACKGROUND: In individuals suffering from a rare disease the diagnostic process and the confirmation of a final diagnosis often extends over many years. Factors contributing to delayed diagnosis include health care professionals' limited knowledge of rare diseases and frequent (co-)occurrence of mental disorders that may complicate and delay the diagnostic process. The ZSE-DUO study aims to assess the benefits of a combination of a physician focusing on somatic aspects with a mental health expert working side by side as a tandem in the diagnostic process. STUDY DESIGN: This multi-center, prospective controlled study has a two-phase cohort design. METHODS: Two cohorts of 682 patients each are sequentially recruited from 11 university-based German Centers for Rare Diseases (CRD): the standard care cohort (control, somatic expertise only) and the innovative care cohort (experimental, combined somatic and mental health expertise). Individuals aged 12 years and older presenting with symptoms and signs which are not explained by current diagnoses will be included. Data will be collected prior to the first visit to the CRD's outpatient clinic (T0), at the first visit (T1) and 12 months thereafter (T2). OUTCOMES: Primary outcome is the percentage of patients with one or more confirmed diagnoses covering the symptomatic spectrum presented. Sample size is calculated to detect a 10 percent increase from 30% in standard care to 40% in the innovative dual expert cohort. Secondary outcomes are (a) time to diagnosis/diagnoses explaining the symptomatology; (b) proportion of patients successfully referred from CRD to standard care; (c) costs of diagnosis including incremental cost effectiveness ratios; (d) predictive value of screening instruments administered at T0 to identify patients with mental disorders; (e) patients' quality of life and evaluation of care; and f) physicians' satisfaction with the innovative care approach. CONCLUSIONS: This is the first multi-center study to investigate the effects of a mental health specialist working in tandem with a somatic expert physician in CRDs. If this innovative approach proves successful, it will be made available on a larger scale nationally and promoted internationally. In the best case, ZSE-DUO can significantly shorten the time to diagnosis for a suspected rare disease. Trial registration ClinicalTrials.gov; Identifier: NCT03563677; First posted: June 20, 2018, https://clinicaltrials.gov/ct2/show/NCT03563677 .

The Warburg effect is necessary to promote glycosylation in the blastema during zebrafish tail regeneration.

Throughout their lifetime, fish maintain a high capacity for regenerating complex tissues after injury. We utilized a larval tail regeneration assay in the zebrafish Danio rerio, which serves as an ideal model of appendage regeneration due to its easy manipulation, relatively simple mixture of cell types, and superior imaging properties. Regeneration of the embryonic zebrafish tail requires development of a blastema, a mass of dedifferentiated cells capable of replacing lost tissue, a crucial step in all known examples of appendage regeneration. Using this model, we show that tail amputation triggers an obligate metabolic shift to promote glucose metabolism during early regeneration similar to the Warburg effect observed in tumor forming cells. Inhibition of glucose metabolism did not affect the overall health of the embryo but completely blocked the tail from regenerating after amputation due to the failure to form a functional blastema. We performed a time series of single-cell RNA sequencing on regenerating tails with and without inhibition of glucose metabolism. We demonstrated that metabolic reprogramming is required for sustained TGF-ß signaling and blocking glucose metabolism largely mimicked inhibition of TGF-ß receptors, both resulting in an aberrant blastema. Finally, we showed using genetic ablation of three possible metabolic pathways for glucose, that metabolic reprogramming is required to provide glucose specifically to the hexosamine biosynthetic pathway while neither glycolysis nor the pentose phosphate pathway were necessary for regeneration.

Secondary azoospermia after sleeve gastrectomy: a case report.

OBJECTIVE: To report the first case of secondary azoospermia after sleeve gastrectomy. DESIGN: Case report. SETTING: Academic male infertility clinic. PATIENTS: A 33-year-old man with secondary azoospermia and primary testicular failure with testosterone deficiency after laparoscopic sleeve gastrectomy. INTERVENTIONS: Hormonal therapy with anastrozole for 10 months and diagnostic testicular biopsy. MAIN OUTCOME MEASURES: Semen analyses and testicular histopathology. RESULTS: Non-obstructive azoospermia persisted at 20 months after surgery despite hormonal therapy with anastrozole. Testicular histopathology revealed the presence of Sertoli cells only. CONCLUSIONS: Although further research is need to determine the relationship between sleeve gastrectomy and secondary infertility, men should be informed of the potentially deleterious effects of this surgery on semen parameters.

How to design a registry for undiagnosed patients in the framework of rare disease diagnosis: suggestions on software, data set and coding system.

BACKGROUND: About 30 million people in the EU and USA, respectively, suffer from a rare disease. Driven by European legislative requirements, national strategies for the improvement of care in rare diseases are being developed. To improve timely and correct diagnosis for patients with rare diseases, the development of a registry for undiagnosed patients was recommended by the German National Action Plan. In this paper we focus on the question on how such a registry for undiagnosed patients can be built and which information it should contain. RESULTS: To develop a registry for undiagnosed patients, a software for data acquisition and storage, an appropriate data set and an applicable terminology/classification system for the data collected are needed. We have used the open-source software Open-Source Registry System for Rare Diseases (OSSE) to build the registry for undiagnosed patients. Our data set is based on the minimal data set for rare disease patient registries recommended by the European Rare Disease Registries Platform. We extended this Common Data Set to also include symptoms, clinical findings and other diagnoses. In order to ensure findability, comparability and statistical analysis, symptoms, clinical findings and diagnoses have to be encoded. We evaluated three medical ontologies (SNOMED CT, HPO and LOINC) for their usefulness. With exact matches of 98% of tested medical terms, a mean number of five deposited synonyms, SNOMED CT seemed to fit our needs best. HPO and LOINC provided 73% and 31% of exacts matches of clinical terms respectively. Allowing more generic codes for a defined symptom, with SNOMED CT 99%, with HPO 89% and with LOINC 39% of terms could be encoded. CONCLUSIONS: With the use of the OSSE software and a data set, which, in addition to the Common Data Set, focuses on symptoms and clinical findings, a functioning and meaningful registry for undiagnosed patients can be implemented. The next step is the implementation of the registry in centres for rare diseases. With the help of medical informatics and big data analysis, case similarity analyses could be realized and aid as a decision-support tool enabling diagnosis of some undiagnosed patients.

Racial disparities in analgesic use amongst patients presenting to the emergency department for kidney stones in the United States.

INTRODUCTION: We sought quantify racial disparities in use of analgesia amongst patients seen in Emergency Departments for renal colic. METHODS: We identified all individuals presenting to the Emergency Department with urolithiasis from 2003 to 2015 in the nationally representative Premier Hospital Database. We included patients discharged in ≤1 day and excluded those with chronic pain or renal insufficiency. We assessed the relationship between race/ethnicity and opioid dosage in morphine milligram equivalents (MME), and ketorolac, through multivariable regression models adjusting for patient and hospital characteristics. RESULTS: The cohort was 266,210 patients, comprised of White (84%), Black (6%) and Hispanic (10%) individuals. Median opioid dosage was 20 MME and 55.5% received ketorolac. Our adjusted model showed Whites had highest median MME (20 mg) with Blacks (-3.3 mg [95% CI: -4.6 mg to -2.1 mg]) and Hispanics (-6.0 mg [95% CI: -6.9 mg to -5.1 mg]) receiving less. Blacks were less likely to receive ketorolac (OR: 0.72, 95% CI: 0.62-0.84) while there was no difference between Whites and Hispanics. CONCLUSIONS: Black and Hispanic patients in American Emergency Departments with acute renal colic receive less opioid medication than White patients; Black patients are also less likely to receive ketorolac.

Inability to obtain sperm for fresh IVF cycles: analysis and incidence of outcomes using a database from the United States.

BACKGROUND: Azoospermia is present in 10% of men presenting with infertility and surgical sperm retrieval rates for men with azoospermia due to spermatogenic dysfunction remain low. We investigated the incidence of failed fresh IVF cycles due to inability to obtain sperm and describe predictors for subsequent IVF. METHODS: A national IVF database was used to identify fresh IVF cycles in which there was failure to obtain sperm. Patient linkage was utilized to determine outcomes of subsequent IVF. RESULTS: 243,291 fresh IVF cycles were identified; 719 (0.3%) listed "inability to obtain sperm" as reason for embryo non-transfer. Male infertility was a factor in 537 (75%) and ejaculation was the most common anticipated sperm source (414, 57%). 713 (99.2%) cycles resulted in retrieved oocytes, but only 627 (87.2%) cryopreserved oocytes. 265 (37%) of couples underwent subsequent IVF. On multivariable analysis, lack of initial oocyte cryopreservation (OR 0.34, p = 0.01) and male infertility (OR 0.14, p = 0.01) were associated with having no subsequent cycles. Partner sperm was used in 213 (80%) second cycles and sperm retrieval method was largely conserved (181/213, 85%). Embryos were transferred in 186 (70%) second cycles. Failed embryo transfers were due to repeat inability to obtain sperm in 5 (6%) cycles. CONCLUSIONS: Failure to obtain sperm during fresh IVF is rare, but most affected couples will not pursue further cycles of IVF after their initial failed attempt.

Neurokinin-1 Receptor Signaling Is Required for Efficient Ca2+ Flux in T-Cell-Receptor-Activated T Cells.

Efficient Ca2+ flux induced during cognate T cell activation requires signaling the T cell receptor (TCR) and unidentified G-protein-coupled receptors (GPCRs). T cells express the neurokinin-1 receptor (NK1R), a GPCR that mediates Ca2+ flux in excitable and non-excitable cells. However, the role of the NK1R in TCR signaling remains unknown. We show that the NK1R and its agonists, the neuropeptides substance P and hemokinin-1, co-localize within the immune synapse during cognate activation of T cells. Simultaneous TCR and NK1R stimulation is necessary for efficient Ca2+ flux and Ca2+-dependent signaling that sustains the survival of activated T cells and helper 1 (Th1) and Th17 bias. In a model of contact dermatitis, mice with T cells deficient in NK1R or its agonists exhibit impaired cellular immunity, due to high mortality of activated T cells. We demonstrate an effect of the NK1R in T cells that is relevant for immunotherapies based on pro-inflammatory neuropeptides and its receptors.

Hemokinin-1 as a Mediator of Arthritis-Related Pain via Direct Activation of Primary Sensory Neurons.

The tachykinin hemokinin-1 (HK-1) is involved in immune cell development and inflammation, but little is known about its function in pain. It acts through the NK1 tachykinin receptor, but several effects are mediated by a yet unidentified target. Therefore, we investigated the role and mechanism of action of HK-1 in arthritis models of distinct mechanisms with special emphasis on pain. Arthritis was induced by i.p. K/BxN serum (passive transfer of inflammatory cytokines, autoantibodies), intra-articular mast cell tryptase or Complete Freund's Adjuvant (CFA, active immunization) in wild type, HK-1- and NK1-deficient mice. Mechanical- and heat hyperalgesia determined by dynamic plantar esthesiometry and increasing temperature hot plate, respectively, swelling measured by plethysmometry or micrometry were significantly reduced in HK-1-deleted, but not NK1-deficient mice in all models. K/BxN serum-induced histopathological changes (day 14) were also decreased, but early myeloperoxidase activity detected by luminescent in vivo imaging increased in HK-1-deleted mice similarly to the CFA model. However, vasodilation and plasma protein extravasation determined by laser Speckle and fluorescent imaging, respectively, were not altered by HK-1 deficiency in any models. HK-1 induced Ca2+-influx in primary sensory neurons, which was also seen in NK1-deficient cells and after pertussis toxin-pretreatment, but not in extracellular Ca2+-free medium. These are the first results showing that HK-1 mediates arthritic pain and cellular, but not vascular inflammatory mechanisms, independently of NK1 activation. HK-1 activates primary sensory neurons presumably via Ca2+ channel-linked receptor. Identifying its target opens new directions to understand joint pain leading to novel therapeutic opportunities.

Interleukin-15 in cancer immunotherapy: IL-15 receptor complex versus soluble IL-15 in a cancer cell-delivered murine leukemia model.

Cytokines of the common γ-chain receptor family such as IL-15 are vital with respect to activating immune cells, sustaining healthy immune functions, and augmenting the anti-tumor activity of effector cells, making them ideal candidates for cancer immunotherapy. IL-15, either in its soluble form (IL-15sol) or complexed with IL-15Rα (IL-15Rc), has been shown to exhibit potent anti-tumor activities in various experimental cancer studies. Here we describe the impact of intraperitoneal IL-15 in a cancer cell-delivered IL-15 immunotherapy approach using the 70Z/3-L leukemia mouse model. Whereas both forms of IL-15 led to significantly improved survival rates compared to the parent cell line, there were striking differences in the extent of the improved survival: mice receiving cancer cells secreting IL-15sol showed significantly longer survival and protective long-term immunity compared to those producing IL-15Rc. Interestingly, injection of leukemia cells secreting IL-15sol lead to heightened expansion of CD4+ and CD8+ T-cell populations in the peritoneum compared to IL-15Rc. Cell-secreted IL-15Rc resulted in an influx and/or expansion of NK1.1+ cells in the peritoneum which was much less pronounced in the IL-15sol model. Furthermore, IL-15Rc but not IL-15sol lead to T-cell exhaustion and disease progression. To our knowledge, this is the first study detailing a significantly different biological effect of cell-delivered IL-15sol versus IL-15Rc in a mouse cancer immunotherapy study.

Impact of Index Surgical Care Setting on Perioperative Outcomes and Cost Following Penile Prosthesis Surgery.

BACKGROUND: Penile prosthesis surgery has witnessed a migration from the inpatient to ambulatory surgical care setting. However, little is known about the cost savings afforded by this change in care setting and whether or not these savings come at the expense of worse perioperative outcomes. AIM: The aim of this study was to identify predictors of index penile prosthesis (PP) surgery care setting, and whether ambulatory vs inpatient surgery is associated with comparable perioperative outcomes and costs. METHODS: This was a retrospective cohort study using all-payer claims data from the 2014 Healthcare Cost and Utilization Project State Databases from Florida and New York. Patient demographics, regional data, total charges (converted to costs), and 30-day revisit rates were abstracted for all patients undergoing index placement of an inflatable or malleable PP. Multivariable logistic and linear regression adjusted for facility clustering was utilized. OUTCOMES: The outcomes were index surgical and 30-day postoperative costs, as well as 30-day revisit rates. RESULTS: Of the 1,790 patients undergoing an index surgery, 394 (22.0%) received care in the inpatient setting compared to 1,396 (78.0%) in the ambulatory setting. Adjusted index procedural ($9,319.66 vs $ 10,191.35; P < .001) and 30-day acute care costs ($9,461.74 vs $10,159.42; P < .001) were lower in the ambulatory setting. The underinsured experienced lower odds of receiving surgery in the ambulatory setting (Medicaid vs private: odds ratio [OR] 0.19; 95% CI 0.06-0.55; P < .001). There was no difference in risk-adjusted odds of experiencing a 30-day revisit between patients undergoing surgery in the ambulatory vs inpatient settings (OR 1.31; 95% CI 0.78-2.21; P = .3). CLINICAL TRANSLATION: Ambulatory PP surgery confers significant cost savings and is associated with comparable perioperative outcomes relative to inpatient-based surgery. CONCLUSIONS: Both clinical and nonclinical factors predict the care setting of index PP surgery. Notably, underinsured patients experienced lower odds of undergoing ambulatory surgery. Ambulatory surgery was less costly with similar 30-day revisit rates relative to inpatient-based care. Berger A, Friedlander DF, Herzog P, et al. Impact of Index Surgical Care Setting on Perioperative Outcomes and Cost Following Penile Prosthesis Surgery. J Sex Med 2019;16:1451-1458.

Differential regulation of IgA+ B cells in vitro by stromal cells from distinctive anatomical compartments.

B cell development is regulated by stromal cells (SCs) that form a supportive microenvironment. These SCs along with other cell types produce cytokines, chemokines, and adhesion molecules that guide B cell commitment and differentiation. BM, spleen (Sp), and the gut lamina propria (LP) constitute distinctive anatomical compartments that support B cell differentiation. In order to characterize and compare the signals necessary to generate IgA+ B cells, we developed an in vitro system to co-culture gut LP, BM, or Sp-derived SCs with B lineage cells. Using this co-culture system, we found that gut LP SCs promote IgA+ B cell accumulation through the production of soluble stimulatory factors. In contrast to gut LP SCs, BM and splenic SCs were found to impair IgA+ B cell accumulation in vitro. Taken together, these observations provide new insights into how SCs derived from different anatomical locations shape IgA+ B cell responses.

Technical note: Evaluation of a triaxial accelerometer for monitoring selected behaviors in dairy calves.

The objectives of this study were (1) to develop an algorithm for the acceleration sensor of the Smartbow Eartag (Smartbow GmbH, Weibern, Austria) to distinguish between postures (lying and standing or locomotion) and to detect 6 kinds of activities (milk intake, water intake, solid feed intake, ruminating, licking or sucking without milk intake, and other activities) in dairy calves and (2) to evaluate this sensor for identifying these behaviors in dairy calves compared with observations from video. Accelerometers were applied to the left ears of 15 preweaned Holstein dairy calves. Calves were kept in a group pen and received milk replacer from an automatic calf feeder. Based on 38 h of acceleration data and video observation, an algorithm was established to detect the predefined behaviors. Using cross-validation, video recordings were used to analyze whether a behavior was detected correctly by the developed algorithm. For posture, sensitivity (94.4%), specificity (94.3%), precision (95.8%), and accuracy (94.3%) were high. Cohen's kappa was calculated as 0.88. For the 6 defined activities, overall (i.e., aggregated for all activities) accuracy was 70.8% and kappa was calculated as 0.58. Some activities (e.g., ruminating, feed intake, other activities) were identified better than others. In conclusion, the developed algorithm based on the acceleration data of the Smartbow Eartag was successful in detecting lying behavior, rumination, feed intake, and other activities in calves, but further development of the underlying algorithm will be necessary to produce reliable results for milk and water intake.

Functional Consequences of the Postnatal Switch From Neonatal to Mutant Adult Glycine Receptor α1 Subunits in the Shaky Mouse Model of Startle Disease.

Mutations in GlyR α1 or ß subunit genes in humans and rodents lead to severe startle disease characterized by rigidity, massive stiffness and excessive startle responses upon unexpected tactile or acoustic stimuli. The recently characterized startle disease mouse mutant shaky carries a missense mutation (Q177K) in the ß8-ß9 loop within the large extracellular N-terminal domain of the GlyR α1 subunit. This results in a disrupted hydrogen bond network around K177 and faster GlyR decay times. Symptoms in mice start at postnatal day 14 and increase until premature death of homozygous shaky mice around 4-6 weeks after birth. Here we investigate the in vivo functional effects of the Q177K mutation using behavioral analysis coupled to protein biochemistry and functional assays. Western blot analysis revealed GlyR α1 subunit expression in wild-type and shaky animals around postnatal day 7, a week before symptoms in mutant mice become obvious. Before 2 weeks of age, homozygous shaky mice appeared healthy and showed no changes in body weight. However, analysis of gait and hind-limb clasping revealed that motor coordination was already impaired. Motor coordination and the activity pattern at P28 improved significantly upon diazepam treatment, a pharmacotherapy used in human startle disease. To investigate whether functional deficits in glycinergic neurotransmission are present prior to phenotypic onset, we performed whole-cell recordings from hypoglossal motoneurons (HMs) in brain stem slices from wild-type and shaky mice at different postnatal stages. Shaky homozygotes showed a decline in mIPSC amplitude and frequency at P9-P13, progressing to significant reductions in mIPSC amplitude and decay time at P18-24 compared to wild-type littermates. Extrasynaptic GlyRs recorded by bath-application of glycine also revealed reduced current amplitudes in shaky mice compared to wild-type neurons, suggesting that presynaptic GlyR function is also impaired. Thus, a distinct, but behaviorally ineffective impairment of glycinergic synapses precedes the symptoms onset in shaky mice. These findings extend our current knowledge on startle disease in the shaky mouse model in that they demonstrate how the progression of GlyR dysfunction causes, with a delay of about 1 week, the appearance of disease symptoms.