Characterizing the metabolome and microbiome at giant panda scent marking sites during the mating season.

Scent marking sites served as a primary means of chemical communication for giant pandas, enabling intraspecific communication. We integrated metabolomics and high-throughput sequencing techniques to examine the non-targeted metabolome and microbial community structure of scent marking sites and feces in the field. Integrative analysis revealed a more comprehensive array of chemical compounds compared to previous investigations, including ketones, acids, heterocycles, alcohols, and aldehydes. Notably, specific compounds such as 2-decenal, (E)-, octanal, decanal, L-α-terpineol, vanillin, and nonanal emerged as potential key players in scent signaling. Intriguingly, our study of the microbial domain identified dominant bacterial species from the Actinobacteria, Bacteroidetes, and Proteobacteria phyla, likely orchestrating metabolic processes at scent marking sites. Comparative analyses showed, for the first time, that feces do not share the same functions as scent markers, indicating distinct functional roles. This research deepens scientific understanding of chemical communication in wild pandas.

Challenges in the diagnosis of the enigmatic primary adrenal leiomyosarcoma: two case reports and review of the literature.

BACKGROUND: Primary adrenal leiomyosarcoma is a rare and aggressive mesenchymal tumor derived from the smooth muscle wall of a central adrenal vein or its tributaries; therefore, tumors tend to invade the inferior vena cava and cause thrombosis. The great majority of tumors grow rapidly, which makes the disease difficult to diagnose in its early clinical stages and needs differentiation from adrenocortical carcinomas for the selection of chemotherapy including mitotane which causes adrenal insufficiency. CASE PRESENTATION: We presented two patients with adrenal leiomyosarcoma who were referred to our hospital with abdominal pain and harboring large adrenal tumors and inferior vena cava thrombosis. The endocrine findings, including serum catecholamine levels, were unremarkable. These two patients were considered clinically inoperable, and CT-guided core needle biopsy was performed to obtain the definitive histopathological diagnosis and determine the modes of therapy. The masses were subsequently diagnosed as primary adrenal leiomyosarcoma based on the histological features and positive immunoreactivity for SMA (smooth muscle actin), desmin, and vimentin. CONCLUSIONS: Adrenal leiomyosarcoma derived from the smooth muscle wall of a central adrenal vein or its tributaries is rare but should be considered a differential diagnosis in the case of nonfunctioning adrenal tumors extending directly to the inferior vena cava. CT-guided biopsy is considered useful for histopathological diagnosis and clinical management of patients with inoperable advanced adrenal tumors without any hormone excess.

Novel SPEG variants in a neonate with severe dilated cardiomyopathy and relatively mild hypotonia.

Striated muscle preferentially expressed protein kinase (SPEG) variants have been reported to cause centronuclear myopathy associated with cardiac diseases. The severity of skeletal muscle symptoms and cardiac symptoms are presumably related to the location of the variant. Here, we report novel SPEG compound heterozygous pathological variants in a neonate with severe dilated cardiomyopathy and relatively mild hypotonia. This report expands the genotype-phenotype correlations of patients with SPEG variants.

Identification of genotype-biochemical phenotype correlations associated with fructose 1,6-bisphosphatase deficiency.

Fructose-1,6-bisphosphatase (FBPase) deficiency, caused by an FBP1 mutation, is an autosomal recessive disorder characterized by hypoglycemic lactic acidosis. Due to the rarity of FBPase deficiency, the mechanism by which the mutations cause enzyme activity loss still remains unclear. Here we identify compound heterozygous missense mutations of FBP1, c.491G>A (p.G164D) and c.581T>C (p.F194S), in an adult patient with hypoglycemic lactic acidosis. The G164D and F194S FBP1 mutants exhibit decreased FBP1 protein expression and a loss of FBPase enzyme activity. The biochemical phenotypes of all previously reported FBP1 missense mutations in addition to G164D and F194S are classified into three functional categories. Type 1 mutations are located at pivotal residues in enzyme activity motifs and have no effects on protein expression. Type 2 mutations structurally cluster around the substrate binding pocket and are associated with decreased protein expression due to protein misfolding. Type 3 mutations are likely nonpathogenic. These findings demonstrate a key role of protein misfolding in mediating the pathogenesis of FBPase deficiency, particularly for Type 2 mutations. This study provides important insights that certain patients with Type 2 mutations may respond to chaperone molecules.

Antihypertensive Effects of Esaxerenone in Older Patients with Primary Aldosteronism.

Design: Retrospective cohort study. Patients. The data was obtained from a total of 87 PA patients treated with esaxerenone. The treatment group comprised 33 patients who received esaxerenone as first-line therapy and 54 patients that switched from another MRA to esaxerenone. Measurements. Blood pressure (BP), plasma aldosterone concentration (PAC), plasma renin activity (PRA), serum potassium level, estimated glomerular filtration rate (eGFR), urinary albumin-creatinine ratio (UACR), and brain natriuretic peptide (BNP) were assessed before and after treatment with esaxerenone. Patients with overall reductions in their systolic or diastolic BP by 10 mmHg, or more, were considered responders. Unpaired t-tests of the biochemical and personal parameters between responders and nonresponders were run to find the most influencing characteristic for treatment success. Results: BP overall decreased after treatment with esaxerenone (systolic BP: P=0.025, diastolic BP: P=0.096). Serum potassium levels increased, while eGFR decreased (P=0.047 and 0.043, respectively). No patients needed a dose reduction or treatment discontinuation of esaxerenone based on the serum potassium and eGFR criteria. UACR and BNP decreased insignificantly. The responders were significantly older than the nonresponders of the esaxerenone treatment (P=0.0035). Conclusions: Esaxerenone was effective in older patients with primary aldosteronism.

Angelman syndrome with mosaic paternal uniparental disomy suggestive of mitotic nondisjunction.

Angelman syndrome (AS) is caused by the functional absence of the maternal ubiquitin-protein ligase E3A (UBE3A) gene. Approximately 5% of AS is caused by paternal uniparental disomy of chromosome 15 (UPD(15)pat), most of which is considered to result from monosomy rescue. However, little attention has focused on how UPD(15)pat occurs. We suggest the mitotic nondisjunction mechanism as a cause of UPD(15)pat in a six-year-old patient presenting with distinctive characteristics in line with AS. DNA methylation screening of 15q11-q13 showed a paternal band and a faint maternal band, suggestive of mosaic status. By trio-based microsatellite analysis, we confirmed a large proportion of UPD(15)pat cells and a small proportion of cells of biparental origin. Single nucleotide polymorphism (SNP) microarray revealed isodisomy of the entire chromosome 15. These results suggest that the UPD(15)pat of the patient resulted from mitotic nondisjunction, which may also be the cause of other cases of AS with UPD(15)pat.

Liver group 2 innate lymphoid cells regulate blood glucose levels through IL-13 signaling and suppression of gluconeogenesis.

The liver stores glycogen and releases glucose into the blood upon increased energy demand. Group 2 innate lymphoid cells (ILC2) in adipose and pancreatic tissues are known for their involvement in glucose homeostasis, but the metabolic contribution of liver ILC2s has not been studied in detail. Here we show that liver ILC2s are directly involved in the regulation of blood glucose levels. Mechanistically, interleukin (IL)-33 treatment induces IL-13 production in liver ILC2s, while directly suppressing gluconeogenesis in a specific Hnf4a/G6pc-high primary hepatocyte cluster via Stat3. These hepatocytes significantly interact with liver ILC2s via IL-13/IL-13 receptor signaling. The results of transcriptional complex analysis and GATA3-ChIP-seq, ATAC-seq, and scRNA-seq trajectory analyses establish a positive regulatory role for the transcription factor GATA3 in IL-13 production by liver ILC2s, while AP-1 family members are shown to suppress IL-13 release. Thus, we identify a regulatory role and molecular mechanism by which liver ILC2s contribute to glucose homeostasis.

Clinical Usefulness of the Growth Hormone-Releasing Peptide-2 Test for Hypothalamic-Pituitary Disorder.

Context: Growth hormone deficiency (GHD) develops early in patients with hypothalamic-pituitary disorder and is frequently accompanied by other anterior pituitary hormone deficiencies, including secondary adrenal insufficiency (AI). A growth hormone-releasing peptide-2 (GHRP2) test, which is widely used for the diagnosis of patients with GHD, is thought to induce release of not only growth hormone (GH) but also ACTH. However, its clinical usefulness in hypothalamic-pituitary disorder is unclear. Objective: We aimed to determine the clinical utility of the GHRP2 test in patients with hypothalamic-pituitary disorders, particularly for AI concomitant with GHD. Methods: The GHRP2 test, a cosyntropin stimulation test, corticotropin-releasing hormone (CRH) tests, and/or insulin tolerance tests (ITTs) were performed on 36 patients with hypothalamic-pituitary disorder. Results: Twenty-two (61%) had severe GHD, and 3 (8%) had moderate GHD by GHRP2. There was no difference in baseline ACTH and cortisol between non-GHD, moderate GHD, and severe GHD participants. However, a cosyntropin stimulation test and subsequent CRH tests and/or ITTs revealed that 17 (47%) had secondary AI and 16/17 (94%) cases of secondary AI were concomitant with severe GHD. ROC curve analysis demonstrated that the ACTH response in the GHRP2 test was useful for screening pituitary-AI, with a cutoff value of 1.55-fold (83% sensitivity and 88% specificity). Notably, the combination of ACTH response and the peak cortisol level in the GHRP2 test using each cutoff value (1.55-fold and 10 µg/dL, respectively) showed high specificity (100%) with high accuracy (0.94) for diagnosis of pituitary-AI. Conclusion: We recommend measuring ACTH as well as GH during the GHRP2 test to avoid overlooking or delaying diagnosis of secondary AI that frequently accompanies GHD.

Long-term follow-up of a patient with autosomal dominant lower extremity-predominant spinal muscular atrophy-2 due to a BICD2 variant.

INTRODUCTION: Bicaudal D homolog 2 (BICD2) is a causative gene of autosomal-dominant lower extremity-predominant spinal muscular atrophy-2 (SMA-LED2). The severity of SMA-LED2 varies widely, ranging from cases in which patients are able to walk to cases in which severe joint contractures lead to respiratory failure. In this study, we report the long-term course of a case of SMA-LED2 in comparison with previous reports. CASE REPORT: The patient was a 19-year-old woman. She had knee and hip dislocations with contractures, femoral fracture, and talipes calcaneovalgus since birth, and was diagnosed with arthrogryposis multiplex congenita. Intense respiratory support was not needed during the neonatal period. She had aspiration pneumonia repeatedly, necessitating NICU admission until 8 months of age. She achieved head control at 9 months of age and was able to sit at 2 years of age; however, she could not walk. Tube feeding was required until 3 years of age. At present, she can eat orally, move around with a wheelchair, and write words by herself. She needs non-invasive positive pressure ventilation during sleep because of a restrictive respiratory disorder during adolescence. Exome analysis identified a de novo heterozygous missense variant (c.2320G>A; p.Glu774Lys) in BICD2. CONCLUSION: Patients with SMA-LED2 may have a relatively better prognosis in terms of social activities in comparison with the dysfunction in the neonatal period. Moreover, it is important to periodically evaluate respiratory function in patients with SMA-LED2 because respiratory dysfunction may occur during adolescence.

Species identification of introduced veronicellid slugs in Japan.

Reliable identification of species is important for protecting native ecosystems against the invasion of non-native species. DNA barcoding using molecular markers, such as the mitochondrial cytochrome oxidase subunit 1 (COI) gene, helps researchers distinguish species. In this study, we focused on introduced veronicellid slugs in the Ryukyu Islands and some greenhouses on mainland Japan. Some veronicellids are medium-to-high risk pest species for humans. Identifying veronicellid species by their external morphology is difficult and unreliable because there is substantial overlap between intraspecific variation and interspecific differentiation. Therefore, internal morphologies such as male genitalia have been the primary traits used to distinguish veronicellids. To identify introduced veronicellid slugs in Japan to the species level, we used morphological assessment of male genitalia and DNA barcoding of the standard COI gene fragment. We also conducted species-delimitation analyses based on the genetic data. The results showed that five evolutionarily significant units, corresponding to four nominal species inhabit the Ryukyu Islands, of which two species were also found in the greenhouses of mainland Japan, including the first record of Sarasinula plebeia in Japan. The presence of non-native slug species could increase the transmission of parasites in Japan.

ICAM1-Negative Intravascular Large B-Cell Lymphoma of the Pituitary Gland: A Case Report and Literature Review.

OBJECTIVE: Intravascular large B-cell lymphoma (IVLBCL) is a rare and aggressive type of B-cell lymphoma with large cells growing within the lumen of blood vessels. Although previous reports revealed highly variable symptoms resulting from small-vessel occlusion by neoplastic cells in a variety of organs, there are few reports of IVLBCL with pituitary involvement. METHOD: We present a case of IVLBCL with pituitary infiltration from our institution together with a literature review of similar cases to better understand this rare case of IVLBCL involving the pituitary gland. RESULTS: Our case and the pertinent literature demonstrated that IVLBCL with pituitary involvement predominantly occurred in women at a mean age of 64 years, and most of them showed panhypopituitarism that was reversible after standard therapy of rituximab-containing chemotherapy with intrathecal methotrexate. Notably, the pituitary biopsy in our case revealed that atypical large B-cells found within blood vessels and the pituitary gland were negative for intercellular adhesion molecule 1. Intercellular adhesion molecule 1-negative lymphoid cells may have contributed to panhypopituitarism by extravasation into the pituitary tissues, which do not have a blood-brain barrier and receive abundant blood flow. CONCLUSION: IVLBCL of the pituitary gland is a rare lymphoma with nonspecific manifestations and a dismal prognosis. Recognition of the clinicopathological features is necessary for early clinical diagnosis and appropriate treatment.

Characteristics of benign adrenocortical adenomas with 18F-FDG PET accumulation.

INTRODUCTION: Although 18F-FDG PET was originally developed to evaluate benign and malignant tumors, the frequency of detection of benign adrenocortical adenomas showing FDG-PET accumulation has increased. However, the details of FDG-PET-accumulated benign adrenocortical adenomas have not been elucidated. METHODS: To elucidate the pathophysiology of FDG-PET-positive cortisol-producing adrenal tumors, we performed clinicopathological and genetic analyses of adrenocortical adenomas examing FDG-PET in 30 operated patients with unilateral cortisol-producing adrenal tumors (26 adrenal adenomas and 4 adrenal cancers). RESULTS: All adrenocortical carcinomas and 17/26 (65%) benign adrenocortical adenomas showed high FDG accumulation (SUVmax ≥ 3). In adrenocortical adenomas with high FDG accumulation (SUVmax ≥ 3), SUVmax showed a positive correlation with the CT Hounsfield units. A higher SUVmax showed a clear black adenoma appearance with predominantly compact cells, which exhibited high T1 and T2 signals, a lack of signal drop on out-of-phase imaging on MRI, and less accumulation on 131-I adsterol scintigraphy. Furthermore, RNA-sequencing analysis revealed significant increases in the lysosomal and autophagy pathways and metabolic pathways, including glycolysis through glucose transporter (GLUT) 1 and 3, in black adenomas with high-level FDG accumulation. DISCUSSION: A black adenoma is blackish due to lipofuscin, which accumulates as a result of damaged mitochondria or proteins that escape lysosomal degradation or autophagy. Since FDG in PET is taken up via GLUTs, alteration of the intracellular metabolic dynamics associated with mitochondrial damage in black adenomas may increase PET accumulation. CONCLUSION: Black adrenal adenomas should be considered with adrenal tumors showing PET accumulation and low lipid contents.

Association of urinary free cortisol with bone formation in patients with mild autonomous cortisol secretion.

CONTEXT: Mild autonomous cortisol secretion (ACS) is associated with an increased risk of vertebral fractures (VFx). However, the influence of this condition on bone turnover or its association with mild ACS is still controversial. OBJECTIVE: This study aimed to evaluate the impact of mild ACS on bone quality among patients living with the disease. DESIGN AND SETTING: A retrospective study was conducted using data from 55 mild ACS and 12 nonfunctioning adrenal tumour (NFT) patients who visited Chiba University Hospital, Japan, from 2006 to 2018. PATIENTS AND MAIN OUTCOME MEASURES: We analysed clinical features and bone-related factors, including bone mineral density (BMD) and VFx, performed blood tests to assess bone metabolism markers in patients with mild ACS and NFT, and assessed the associations between bone-related markers and endocrinological parameters in patients with mild ACS. RESULTS: No significant differences between mild ACS and NFT patients were observed with respect to the presence or absence of VFx and BMD. Urinary free cortisol (UFC) was higher in mild ACS patients with VFx than those without (p = .037). The T-score and young adult mean (YAM) of the BMD of the femoral neck in mild ACS patients with a body mass index <25 were positively correlated with dehydroepiandrosterone sulphate levels (ρ: 0.42, p = .017; ρ: 0.40, p = .024, respectively). Pearson's correlation analysis showed that bone-specific alkaline phosphatase was negatively correlated with UFC in the patients with mild ACS (ρ: -0.37, p = .026). CONCLUSIONS: These results suggest that urinary free cortisol may be useful for predicting bone formation in mild ACS patients.

Steroid metabolites for diagnosing and predicting clinicopathological features in cortisol-producing adrenocortical carcinoma.

BACKGROUND: Approximately 60% of adrenocortical carcinomas (ACC) are functional, and Cushing's syndrome is the most frequent diagnosis that has been revealed to have a particularly poor prognosis. Since 30% of ACC present steroid hormone-producing disorganization, measurement of steroid metabolites in suspected ACC is recommended. Previous reports demonstrated that steroid hormone precursors or their urine metabolites, which can be assessed using liquid chromatography tandem mass spectrometry (LC-MS/MS) or gas chromatography mass spectrometry (GC-MS) respectively, are useful for distinguishing ACC from cortisol-producing adenomas (CPA); however, despite high precision, LC-MS/MS and GC-MS require a highly trained team, are expensive and have limited capacity. METHODS: Here, we examined 12 serum steroid metabolites using an immunoassay, which is a more rapid and less costly method than LC-MS/MS, in cortisol-producing ACC and CPA. Further, the correlation of each steroid metabolite to the classification stage and pathological status in ACC was analyzed. RESULTS: Reflecting disorganized steroidogenesis, the immunoassay revealed that all basal levels of steroid precursors were significantly increased in cortisol-producing ACC compared to CPA; in particular, 17-hydroxypregnenolone (glucocorticoid and androgen precursor) and 11-deoxycorticosterone (mineralocorticoid precursor) showed a large area under the ROC curve with high sensitivity and specificity when setting the cut-off at 1.78 ng/ml and 0.4 mg/ml, respectively. Additionally, a combination of androstenedione and DHEAS also showed high specificity with high accuracy. In cortisol-producing ACC, 11-deoxycortisol (glucocorticoid precursor) showed significant positive correlations with predictive prognostic factors used in ENSAT classification, while testosterone showed significant positive correlations to the Ki67-index in both men and women. CONCLUSION: Less expensive and more widely available RIA and ECLIA may also biochemically distinguish ACC from CPA and may predict the clinicopathological features of ACC.

Aldosterone Reduction Rate After Saline Infusion Test May Be a Novel Prediction in Patients With Primary Aldosteronism.

OBJECTIVE: Accurate assessment and localization of aldosterone-producing adenomas (APAs) are essential for the treatment of primary aldosteronism (PA). Although adrenal venous sampling (AVS) is the standard method of reference for subtype diagnosis in PA, controversy exists concerning the criteria for its interpretation. This study aims to determine better indicators that can reliably predict subtypes of PA. METHOD: Retrospective, single-cohort analysis including 209 patients with PA who were subjected to AVS. Eighty-two patients whose plasma aldosterone concentrations (PAC) were normalized after surgery were histopathologically or genetically diagnosed with APA. The accuracy of image findings was compared to AVS results. Receiver operating characteristic (ROC) curve analysis between the operated and the no-apparent laterality groups was performed using AVS parameters and loading test for diagnosis of PA. RESULT: Agreement between image findings and AVS results was 56.3%. ROC curve analysis revealed that the lateralization index (LI) after adrenocorticotropin stimulation cutoff was 2.40, with 98.8% sensitivity and 97.1% specificity. The contralateral suppression index (CSI) cutoff value was 1.19, with 98.0% sensitivity and 93.9% specificity. All patients over the LI and CSI cutoff values exhibited unilateral subtypes. Among the loading test, the best classification accuracy was achieved using the PAC reduction rate after a saline infusion test (SIT) >33.8%, which yielded 87.2% sensitivity or a PAC after a SIT <87.9 pg/mL with 86.2% specificity for predicting bilateral PA. CONCLUSION: The combined criteria of the PAC reduction rate and PAC after the SIT can determine which subset of patients with APA who should be performed AVS for validation.

R3hdml regulates satellite cell proliferation and differentiation.

In this study, we identified a previously uncharacterized skeletal satellite cell-secreted protein, R3h domain containing-like (R3hdml). Expression of R3hdml increases during skeletal muscle development and differentiation in mice. Body weight and skeletal muscle mass of R3hdml knockout (KO) mice are lower compared to control mice. Expression levels of cell cycle-related markers, phosphorylation of Akt, and expression of insulin-like growth factor within the skeletal muscle are reduced in R3hdml KO mice compared to control mice. Expression of R3hdml increases during muscle regeneration in response to cardiotoxin (CTX)-induced muscle injury. Recovery of handgrip strength after CTX injection was significantly impaired in R3hdml KO mice, which is rescued by R3hdml. Our results indicate that R3hdml is required for skeletal muscle development, regeneration, and, in particular, satellite cell proliferation and differentiation.

Syntroph diversity and abundance in anaerobic digestion revealed through a comparative core microbiome approach.

Anaerobic digestion is an important biotechnology treatment process for conversion of waste to energy. In this study, a comparative core microbiome approach, i.e., determining taxa that are shared in functioning digesters but not shared in non-functioning digesters, was used to determine microbial taxa that could play key roles for effective anaerobic digestion. Anaerobic digester functions were impaired by adding the broad-spectrum antimicrobial triclosan (TCS) or triclocarban (TCC) at different concentrations, and the core microbiomes in both functioning and non-functioning anaerobic digesters were compared. Digesters treated with high (2500 mg/kg) or medium (450 mg/kg) TCS and high (850 mg/kg) TCC concentrations lost their function, i.e., methane production decreased, effluent volatile fatty acid concentrations increased, and pH decreased. Changes in microbial community diversity and compositions were assessed using 16S rRNA gene amplicon sequencing. Microbial richness decreased significantly in non-functioning digesters (p < 0.001). Microbial community compositions in non-functioning digesters significantly differed from those in functioning digesters (p = 0.001, ANOSIM). Microbes identified as potentially key taxa included previously known fatty acid-degrading syntrophs and amino acid-degrading syntrophs. A diverse group of syntrophs detected in this study had low relative abundance in functioning digesters, suggesting the importance of rare microbes in anaerobic digester operation. The comparative microbiome approach used in this study can be applied to other microbial systems where a community-driven biological phenomena can be observed directly.