Spent human embryo culture media contains low-molecular-weight metabolites that can provide a non-invasive readout of preimplantation embryo physiology. However, nuclear magnetic resonance (NMR)-based metabolomic analysis of these samples is technically challenging because routine embryo culture conditions involve microliter volumes, protein supplementation, and paraffin oil overlays that generate background signals and compromise spectral quality. Here, we present an optimized NMR metabolomics workflow for spent embryo culture media incorporating controlled droplet collection to minimize oil contamination, fluorinated ethylene propylene (FEP) tube liners to chemically isolate the sample from the external reference (TMSP) while accommodating microliter volumes, and acquisition using a Carr-Purcell-Meiboom-Gill pulse sequence to suppress macromolecular background signals. The protocol enables consistent detection and quantification of low-molecular-weight metabolites from individually cultured human embryos and supports reproducible analysis under routine laboratory conditions. Due to numerous technical challenges, the method must be properly validated before it can be used for research or clinical purposes.

Ganglioneuromas of the cervical spine are exceptionally rare, well-differentiated neuroblastic tumors. We present the case of a 4-year-old female with a several-month history of progressive fatigue, right upper extremity weakness, and gait instability, in whom MRI demonstrated a homogeneously enhancing, cervical intradural extramedullary mass causing significant ventral spinal cord compression. A posterior midline approach with cervical two to cervical six laminoplasty was performed under continuous neurophysiological monitoring, providing wide dorsal access to the lesion. Microsurgical resection of the ventrolateral mass was accomplished using careful arachnoid dissection and cerebrospinal fluid drainage to achieve gross total resection, with histopathology confirming mature ganglioneuroma without malignant transformation. A postoperative MRI demonstrated gross total resection of the mass. At the 6-month follow-up, the patient has demonstrated marked neurological recovery, with full upper extremity strength and normal gait. This case offers a reproducible procedural framework for ventrolateral intradural cervical tumors in the pediatric population while underscoring the importance of multidisciplinary planning, laminoplasty-based posterior corridor selection, and meticulous microsurgical technique.

Primary adrenal high-grade undifferentiated pleomorphic sarcoma (UPS) is an exceptionally rare malignant mesenchymal neoplasm, and presentation with brain metastasis as the initial manifestation is even more uncommon. This report highlights the diagnostic evaluation and multidisciplinary management of a patient presenting with an undifferentiated brain metastasis accompanied by an adrenal mass. A 75-year-old woman presented with speech disturbance, cognitive slowing, memory decline, gait instability, and right-sided weakness. Brain computed tomography and contrast-enhanced magnetic resonance imaging revealed a left frontal lesion with multiple intracerebral nodules. Positron emission tomography/computed tomography further demonstrated a left adrenal lesion and additional metastatic foci. Owing to symptomatic mass effect, the brain lesion was surgically resected. Initial pathology suggested a poorly differentiated malignant tumor, and the tumor origin remained uncertain after the first immunohistochemical assessment. Subsequent multidisciplinary review, serial adrenal imaging, adrenal core needle biopsy, and comparative pathologic evaluation progressively redirected the diagnosis toward a high-grade undifferentiated sarcoma, morphologically consistent with UPS. The patient subsequently received postoperative brain radiotherapy, anthracycline-based chemotherapy, additional systemic therapy, and local treatment for progressive metastatic disease. Despite multimodal management, the tumor rapidly progressed with thigh metastasis, bilateral adrenal involvement, skeletal dissemination, and bilateral pathologic femoral fractures. The patient ultimately died of intracerebral hemorrhage. This case expands the clinical spectrum of primary adrenal UPS and underscores the importance of dynamic diagnostic reassessment, multisite tissue sampling, extended immunohistochemical evaluation, and multidisciplinary collaboration when evaluating undifferentiated brain metastases associated with an adrenal mass.

Both embryonic-type neuroectodermal tumor and nephroblastoma arising as somatic-type malignancies are rare, particularly nephroblastoma. Concurrent embryonic-type neuroectodermal tumor and nephroblastoma have only been reported once in literature, identified in metastatic pelvic lymph nodes in association with a mixed germ cell tumor. Herein, we report the first concurrent embryonic-type neuroectodermal tumor and nephroblastoma within the primary testicular tumor, arising associated with a mixed germ cell tumor. A 36-year-old man presented with a 4.5 cm right testicular mass. He underwent right orchiectomy. Gross examination revealed a well-circumscribed, solid to cystic mass confined to the testis (pT1). Histological examination demonstrated a mixed germ cell tumor composed of teratoma (40% of the tumor mass), seminoma (10%), embryonal carcinoma (2.5%), and yolk sac tumor (2.5%), with associated germ cell neoplasia in situ. Additionally, two somatic-type malignancy components were identified, including embryonic-type neuroectodermal tumor (40%) and nephroblastoma (5%). The embryonic-type neuroectodermal tumor consisted of primitive neural tubules, rosettes, and blastemal cells. The nephroblastoma was composed of tubules, blastemal cells, and stromal cells. Immunohistochemically, the embryonic-type neuroectodermal tumor showed diffuse positivity for SOX11, with focal expression of synaptophysin and CD99, while the nephroblastoma was positive for PAX8 and WT1. The patient developed metastasis to a retroperitoneal lymph node 1 month after the orchiectomy, subsequently received chemotherapy, and is alive without metastatic disease at 13 months of follow-up.

The assessment of body composition and neuromuscular performance provides valuable information for the clinical management of athletes and diverse patient populations. This protocol describes the combined use of a multifrequency bioelectrical impedance analyzer and a force analyzer to obtain biomarkers of muscle mass and function. The bioelectrical impedance analyzer measures the body's impedance to alternating electrical currents at selected frequencies, enabling the estimation of body composition parameters. The force analyzer detects vertically applied forces during sit-to-stand movements, including single, two-times, or three-times chair stand tests, allowing the evaluation of neuromuscular performance. Both assessments are simple to perform, noninvasive, and provide results quickly through automatically generated reports. The combined analysis of body composition and neuromuscular performance offers a practical approach for characterizing physical performance in athletes and functional and health status in clinical populations. This protocol may facilitate routine monitoring and support individualized training, rehabilitation, and clinical management strategies and outcomes.

Hepatitis E virus (HEV) infection is a major cause of acute viral hepatitis worldwide. HEV is a positive-strand RNA virus encoding three open reading frames (ORFs). The ORF3 protein is a small membrane-associated protein essential for viral particle secretion; however, its precise role in the viral life cycle remains incompletely understood. Here, we performed immunoprecipitation followed by mass spectrometry to identify host proteins interacting with the HEV ORF3 protein. Candidate interactors were validated by co-immunoprecipitation, confirming physical interactions between ORF3 and cysteine-rich and transmembrane domain-containing protein 1 (CYSTM1), Ras-related protein Rab24, and transferrin receptor 1 (TfR1). Confocal microscopy demonstrated colocalization of all three host factors with ORF3 protein. Gene silencing and knockout revealed that each protein contributes to virus production, with TfR1 depletion producing the most pronounced effect. In cells harboring replicating HEV, TfR1 colocalized with the ORF2 (capsid) and ORF3 proteins at Rab11A-positive recycling endosomes. Silencing of TfR1 in primary human hepatocytes, followed by HEV RNA transfection or infection, confirmed its role in virus production, particularly in the assembly of infectious particles, consistent with its colocalization with the HEV ORF2 protein. Collectively, our proteomics-based analysis identifies TfR1, along with CYSTM1 and Rab24, as novel ORF3-interacting host factors required for efficient production of infectious HEV. These findings provide new insights into the role of ORF3 protein in viral assembly and highlight TfR1 as a key host factor in the HEV life cycle.

Hashimoto's thyroiditis (HT) is an autoimmune disorder of the thyroid. While immune cells are implicated in its pathogenesis, their specific roles have yet to be fully clarified. A two-sample Mendelian randomization (MR) analysis was conducted integrating genome-wide association study (GWAS) summary statistics from large public datasets for immune cell traits (ebi-a-GCST90001391 to ebi-a-GCST90002121), plasma metabolites (GCST90199621-9020102), and HT (ebi-a-GCST90018855). Causal effects were estimated using inverse-variance weighted (IVW) methods, with MR-Egger, weighted median, and leave-one-out analyses to assess pleiotropy and robustness. Bidirectional and mediation MR analyses were further applied to test directionality and identify potential metabolite-mediated pathways. CD3⁺CD4⁺CD25⁺CD39⁺Treg cells were quantified in peripheral blood samples using flow cytometry. Isovalerylcarnitine (C5) was measured by liquid chromatography tandem mass spectrometry. IVW analysis identified 32 immune cell phenotypes significantly associated with HT risk (P < 0.05 after FDR correction). Reverse MR analysis demonstrated that HT was positively causally linked with 2 immune characteristics, while 4 immune characteristics (all P < 0.05) were inversely associated with HT. Sensitivity analyses revealed no horizontal pleiotropy or heterogeneity. Additionally, the IVW method preliminarily identified 9 plasma metabolites as causally related to HT, including risk-enhancing C5 (OR = 1.120, 95% CI: 1.032-1.215, P = 0.006) and protective ergothioneine (OR = 0.958, 95% CI: 0.927-0.990, P = 0.010). Two-step MR mediation identified C5 as a candidate mediator connecting CD3⁺ CD39⁺ Treg to HT (mediation proportion 8.89%, 95% CI: 2.34%-15.4%, P = 0.008). Flow cytometry elevated CD39⁺Treg levels and plasma C5 in HT patients, with C5 positively correlated with CD39⁺Treg cells proportion. This study establishes novel causal links between immune cell phenotypes and HT, and highlights plasma metabolites, particularly C5, as potential mediators in HT pathogenesis. These findings deepen mechanistic understanding of autoimmune thyroid disease and may guide future biomarker and therapeutic target discovery.

Postural control relies on the integration of visual, vestibular, proprioceptive, and auditory inputs to maintain stability. While previous studies have explored the effects of individual sensory modalities, the combined influence of multisensory disruptions on postural predictability remains unclear. We preliminary assessed postural behavior during quiet standing under manipulated sensory conditions in healthy participants. Eight adults stood barefoot on a Wii Balance Board, wearing a mixed reality headset with headphones and receiving vibrotactile stimulation to the Achilles tendons. Experimental conditions combined vibrotactile, static, or moving auditory stimuli across three visual states (Eyes Open, Eyes Closed, Blurred Vision). Center of pressure (CoP) in anterior-posterior (AP) and medio-lateral (ML) planes was collected and analyzed for signal predictability using Sample Entropy. The same experimental procedures were simulated by a biologically inspired neural mass model to investigate multisensory integration in postural control at the neural level. Behaviorally, proprioceptive perturbation via Achilles tendon vibration significantly increased Sample Entropy in both spatial planes, indicating reduced system predictability, while blurred vision decreased only in the AP plane, suggesting less flexible postural dynamics. The neural model replicated key behavioral trends and also revealed distinct central mechanisms underlying sensory interactions: while auditory inputs had minimal behavioral effects, at the simulated neural level, they increased Sample Entropy, especially under dynamic conditions. This revealed subtle modulation of predictability from auditory cues, which was not detectable at the behavioral level. These preliminary results highlight the modality-specific contributions to postural control and demonstrate the utility of Sample Entropy as measure of predictability, with our computational modeling uncovering, for the first time to our knowledge, neural integration processes related to multisensory integration and postural control predictability.

The menopausal transition and early postmenopause represent important periods for women's skeletal health, but the genetic relevance of metabolic, behavioral, and hormone-related factors to bone mineral density during midlife remains incompletely understood. This study used publicly available genome-wide association study summary statistics to examine associations between body mass index, 25-hydroxyvitamin D, sex hormone-binding globulin, high-density lipoprotein cholesterol, smoking initiation, and alcohol intake frequency and total body bone mineral density at ages 45-60 years. Exposure genome-wide association study summary statistics were derived from large European-ancestry populations and were not restricted to midlife women, whereas the outcome genome-wide association study captured an age-stratified total body bone mineral density phenotype at age 45-60 years. This age range overlaps with the menopausal transition and early postmenopause in women. Univariable, reverse, and multivariable Mendelian randomization analyses were performed, with inverse-variance weighting as the primary method and complementary sensitivity analyses used to assess heterogeneity, pleiotropy, and result stability. Genetically predicted higher sex hormone-binding globulin was associated with lower total body bone mineral density (β = -0.111, 95% CI: -0.170 to -0.051; P = 0.0003). Reverse Mendelian randomization did not support reverse causation from bone mineral density to sex hormone-binding globulin. Multivariable analyses suggested that this association persisted after adjustment for selected metabolic biomarkers. The other examined exposures did not show consistent evidence of association. These findings provide genetic evidence linking sex hormone-binding globulin to total-body bone mineral density at ages 45-60 years. Further prospective and predictive studies are needed to evaluate its clinical relevance beyond established bone health assessment tools.