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Simple Summary: Ovarian follicular cysts are one of the most common reproductive disturbances in dairy cows. However, the exact mechanism underlying this disorder is not clear enough and remains difficult to identify. Here, we found that there was a disrupted secretion of hormone and abnormal mRNA expression of corresponding receptors in bovine cystic follicles compared with control follicles. Further KEGG enrichment analyses of transcriptome indicated that the differential expressed genes were significantly enriched in the ovarian steroidogenesis pathway. Based on the findings of the study, metabolic and endocrine disorders were associated with bovine ovarian follicular cysts. After estrus, when mature follicles fail to ovulate, they may further develop to form follicular cysts, affecting the normal function of ovaries, reducing the reproductive efficiency of dairy cows and causing economic losses to cattle farms. However, the key points of ovarian follicular cysts pathogenesis remain largely unclear. The purpose of the current research was to analyze the formation mechanism of ovarian follicular cysts from hormone and gene expression profiles. The concentrations of progesterone (P4), estradiol (E2), insulin, insulin-like growth factor 1 (IGF1), leptin, adrenocorticotropic hormone (ACTH) and ghrelin in follicle fluid from bovine follicular cysts and normal follicles were examined using enzyme-linked immunosorbent assay (ELISA) or 125I-labeled radioimmunoassay (RIA); the corresponding receptors' expression of theca interna cells was tested via quantitative reverse transcription polymerase chain reaction (RT-qPCR), and the mRNA expression profiling was analyzed via RNA sequencing (RNA-seq). The results showed that the follicular cysts were characterized by significant lower E2, insulin, IGF1 and leptin levels but elevated ACTH and ghrelin levels compared with normal follicles (p < 0.05). The mRNA expressions of corresponding receptors, PGR, ESR1, ESR2, IGF1R, LEPR, IGFBP6 and GHSR, were similarly altered significantly (p < 0.05). RNA-seq identified 2514 differential expressed genes between normal follicles and follicular cysts. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis linked the ovarian steroidogenesis pathway, especially the STAR, 3β-HSD, CYP11A1 and CYP17A1 genes, to the formation of follicular cysts (p < 0.01). These results indicated that hormone metabolic disorders and abnormal expression levels of hormone synthesis pathway genes are associated with the formation of bovine ovarian follicular cysts. [ABSTRACT FROM AUTHOR]

Background: Vasopressin gene expression has been demonstrated to be a common feature of all small-cell lung cancer (SCLC) and breast cancer. Provasopressin (ProVP) is a component of the cancer cell membrane and a likely target for treatment. However, a measurable fraction of this cancer provasopressin is also normally processed and products are released into the circulation. Vasopressin (VP) and vasopressin-associated human neurophysin (VP-HNP), two of three products of processing, were earlier shown to be reliable plasma markers for determining the presence of SCLC and monitoring response to treatment.Material and Methods: In this study, copeptin, the third product of provasopressin processing, was preliminarily evaluated as a plasma marker for SCLC or breast cancer using radioimmunoassay (RIA). Antibodies directed against the 18 residue C-terminal peptide fragment of copeptin were used to avoid interference from the large-carbohydrate component of this endogenous glycopeptide.Results: The levels of copeptin in 8 male and 6 female patients with SCLC before treatment ranged from 16 to 319 pmol/L, and these levels were elevated (> 2.5 times) in 10 of 14 cases (70%) when compared with healthy volunteers (normal mean, 18 ± 6 pmol/L). Volunteer values for males were smaller than for females (15± 4 pmol/L and 20± 9 pmol/L), but numbers were small. Patients with breast cancer had plasma levels ranging from 12 to 68 pmol/L, with only three of the six elevated.Conclusion: While cancer patients displayed a wide range of plasma copeptin levels over 70% with SCLC and 50% with breast cancer had clearly elevated levels. This finding indicates that for such patients, plasma copeptin, like plasma VP and VP-HNP, could be used to detect disease. The control values found for healthy volunteers using our RIA were in a range predictable from established normal plasma levels of both VP and VP-HNP. [ABSTRACT FROM AUTHOR]

The influences of stocking density on stereotypic behavior, fecal cortisol and the musk secretion of captive Alpine musk deer (Moschus chrysogaster) were studied in the Xinglongshan Musk Deer Farm (XMDF) of Xinglongshan National Nature Reserve in Gansu Province of Northwest China from the 1st November to the 30th December 2020. The behavior of 26 captive male musk deer was recorded using focal animal sampling and all-occurrence recording, while radioimmunoassay was used to determine fecal cortisol and testosterone levels, and the muscone content was determined by gas chromatography-flame ionization detection (GC-FID). The results showed that the stereotypy behavior of individuals in high stocking density group (HDG) was more frequent (88.89%) and lasted longer (8.4 ± 1.0%) than of individuals in low density group (LDG) (50% and 4.2 ± 1.7%, respectively) (p < 0.05). The fecal cortisol level of HDG individuals (60.19 ± 5.83 ng g− 1) was significantly higher than that of LDG musk deer (36.34 ± 4.77 ng g− 1) (p < 0.05). There was no difference in muscone content, musk secretion and testosterone level between HDG and LDG individuals (p > 0.05). In musk deer farming of XMDF, the stocking density significantly influenced the health of captive Alpine musk deer, but had no significant impact on the musk secretion. [ABSTRACT FROM AUTHOR]

Objective: To explore the underlying mechanism of inhibition by Jinkui Shenqi Pills (JKSQP) on glucocorticoid-enhanced axial length elongation in experimental lens-induced myopia (LIM) guinea pigs. Methods: Sixty 2-week old male guinea pigs were randomly divided into 4 groups with 15 guinea pigs in each group, according to the random numbers generated by SPSS software: control, LIM, saline and JKSQP groups. The control group includes animals with no treatment, while the guinea pigs in the other 3 groups received lens-induced myopization on the right eyes throughout the experiment (for 8 weeks). The saline and JKSQP groups were given daily intraperitoneal injections of 10 mg/kg hydrocortisone for 2 consecutive weeks at the same time, and then orally administered either saline or JKSQP [13.5 g/(kg•d) for 6 consecutive weeks. Body weight, anal temperature and animal appearance were observed and recorded to evaluate the GC-associated symptoms. The ocular parameters, including refraction and axial length, were measured by streak retinoscopy and A-scan ultrasonography, respectively. The levels of plasma hormones associated with the hypothalamic-pituitary-adrenal axis (HPAA), including free triiodothyronine, free thyroxine, estradiol and testosterone, were measured by radioimmunoassay, and cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate were measured by enzyme-linked immunosorbent assay. In addition, the mRNA and protein expressions of retinal amphiregulin (AREG) was measured by quantitative real-time polymerase chain reaction and Western blotting, respectively. Results: JKSQP effectively increased body weight and anal temperature, improved animal appearance and suppressed axial length elongation in glucocorticoid-enhanced myopic guinea pigs with normalization of 4 HPAA-associated plasma hormones (all P<0.05). The plasma level of cAMP was significantly increased, whereas the plasma level of cGMP and the mRNA and protein expressions of retinal AREG were decreased after treatment with JKSQP (all P<0.05). Conclusion: JKSQP exhibited a significant inhibitory effect on axial length elongation with decreased expression of AREG in the retina, and normalized 4 HPAA-associated plasma hormones and the expression of cAMP and cGMP in GC-enhanced myopic guinea pigs. [ABSTRACT FROM AUTHOR]

Simple Summary: The development of aquaculture has led to an increased attention towards the welfare of farmed fish. Transportation, a routine procedure in this industry, can be stressful for fish due to handling, air exposure, confinement, and reduced oxygen levels. Therefore, it is crucial to gain insights into optimal practices for organizing transportation, ensuring the well-being of these animals and minimizing excessive stress. The objective of this study was to assess the stress response of meagre juveniles during commercial transport from Monfalcone to Stintino (Italy), which lasted 24 h. Welfare evaluation included measuring cortisol levels in muscle and assessing the potential cellular localization of oxidative stress markers in various organs and tissues. Significant increases in cortisol were observed in the fish during transfer from housing to transport tanks and in subsequent samplings at different phases of transport. This finding suggests that either the animals had insufficient time to recover or the transport conditions were suboptimal. However, in terms of oxidative stress, overall immunohistochemical analysis did not reveal variation in marker localization before and after transport, indicating that the stress experienced by the fish during transportation might have been relatively mild. In aquaculture, the transportation of live fish is a crucial but stress-inducing practice, necessitating a thorough understanding of its impact on fish welfare. This study aimed to assess the physiological stress response of meagre (Argyrosomus regius) juveniles during a 24 h commercial transport by quantifying muscle cortisol levels using a specific radioimmunoassay. Additionally, an immunohistochemical approach was used to detect and localize the cellular distribution of oxidative-stress-related biomarkers within various tissues and organs. The results demonstrated a significant increase in muscle cortisol levels following the loading procedure, remaining elevated above basal levels throughout the 24 h transport period. This effect may be attributed to either insufficient time for recovery from the loading stress or prolonged transportation-related stress. Immunostaining for all the antibodies we examined was observed in multiple tissues and organs, but we found no notable variations among the various transport phases. In conclusion, the observed stress response appears to be mainly linked to loading stress and the transport process itself, emphasizing the importance of implementing appropriate operational procedures to safeguard fish well-being during transport. Nonetheless, the unaltered distribution of oxidative stress markers between the control and transported groups suggests that the experienced stress might be within tolerable limits. [ABSTRACT FROM AUTHOR]

Previously, we reported that a reduction in β-Arrestin1 protein levels in peripheral blood mononuclear leukocytes (PBMC) significantly correlated with the severity of depression symptoms in women with premenstrual dysphoric disorder (PMDD). This study aimed to determine whether the reduced premenstrual β-Arrestin1 protein levels were associated with changes in the regulator for late luteal phase progesterone secretion. The study participants (n = 25) were non-pregnant women between 18 and 42 years of age not taking any antidepressants or receiving therapy and experiencing the luteal phase of menstruation. ELISA determined the β-Arrestin1 protein in PBMC; testosterone and prolactin levels from the plasma were determined by radioimmunoassay. Reduced levels of β-Arrestin1 protein in women with Hamilton Rating Scale for Depression (HAM-D) scores above 19 were observed alongside significantly higher plasma testosterone and prolactin concentrations. Understanding the mechanism underlying the initiation of PMDD will allow for identification of a key perturbed metabolic enzyme that can serve as a target for drug development to ensure the alleviation of PMDD, which has been suggested earlier as a risk factor for developing major depressive disorders. [ABSTRACT FROM AUTHOR]

Purpose: Testosterone analysis in hair allows for retrospective evaluation of endogenous testosterone concentrations, but studies devoted to investigating confounders in hair testosterone analysis have hitherto been scarce. The current study examined the stability of testosterone concentrations between two hair samples collected three months apart and investigated two potential confounding factors: natural hair colour and cosmetic hair treatments. Methods: Testosterone was analysed with an in-house radioimmunoassay with a limit of detection adequate for the purpose. Results: The testosterone concentrations from the two samplings, at baseline and three months later, had an intra-individual correlation of moderate strength (rho = 0.378, p<0.001, n = 146). Hair treatment, such as colouring or bleaching, seemed to increase testosterone concentrations (p = 0.051, n = 191, and in a paired analysis in a subset of the cohort p = 0.005, n = 24), while no effect of natural colour in untreated hair (p = 0.133) could be detected. Conclusion: The current results suggest that cosmetic hair treatments need to be considered in hair testosterone analyses and demonstrate the utility of a radioimmunoassay to reliably measure testosterone concentrations in small hair samples in women. [ABSTRACT FROM AUTHOR]

Background: Timing of reproductive events can be crucial for a species' population growth and stability. Accurate detection of reproductive phenology presents a challenge to scientists studying wild species, including moose (Alces alces). Currently, there are several established methods for monitoring reproductive activity and events in domestic ruminants, including the use of biologging devices. The main objective of this study was to determine whether female moose display a distinct thermal and activity pattern associated with luteal activity during the estrous cycle, which could be used to determine the onset of their breeding season. We deployed biologging devices and collected fecal samples from 12 captive female moose on the Kenai Peninsula, Alaska, USA to explore variation in vaginal temperature and collar activity and the relationship between these variables and fecal progestagen concentrations. Fecal samples were collected from mid-August to mid-October and analyzed using radioimmunoassay to determine the concentration of fecal progestagens to classify luteal activity. Results: Captive female moose displayed an identifiable thermal pattern during the onset of luteal activity from mid-September to mid-October, associated with the initial estrous cycle of their breeding season. In contrast, we did not observe a distinct pattern in activity during this period. Recurring patterns in both vaginal temperature and activity were identified between mid-October and mid-November, however, which were likely associated with subsequent estrous cycles but not included in our fecal sampling period. Conclusions: This study supports that female moose display an identifiable pattern in vaginal temperature which is associated with luteal activity of the initial estrous cycle of the breeding season. An identifiable pattern was observed for both vaginal temperature and activity registrations at the presumed timing of subsequent estrous cycle of the breeding season. [ABSTRACT FROM AUTHOR]

Context Although some studies have examined the connection between circulating 25-hydroxyvitamin D (25(OH)D) concentrations and preeclampsia (PE) risk, the results were inconsistent. Objective A dose-response meta-analysis on epidemiologic investigations was conducted to evaluate the relation of 25(OH)D concentration and PE. Data Source Electronic databases, including Scopus, MEDLINE (PubMed), the Institute for Scientific Information, Embase, and Google Scholar, were comprehensively search until July 2021. Data Extraction A total of 65 observational studies evaluating the link between circulating 25(OH)D concentrations and PE were included. The body of evidence was assessed by the Grading of Recommendations, Assessment, Development, and Evaluations (GRADE) approach. Data Analysis Combining 32 effect sizes from 32 prospective studies with 76 394 participants revealed that highest vs lowest circulating 25(OH)D concentrations was significantly related to a 33% reduced risk of PE (relative risk [RR], 0.67; 95%CI, 0.54–0.83). Subgroup analysis by study design revealed that PE risk has significantly decreased in cohort and case-cohort studies (RR, 0.72; 95%CI, 0.61–0.85), and a slight decline was found in nested case-control studies (RR, 0.62; 95%CI, 0.38–1.02). Dose–response analysis in 27 prospective studies with 73 626 participants illustrated that each 10 ng/mL increment in circulating 25(OH)D concentration led to a 14% reduced incidence of PE (RR, 0.86; 95%CI, 0.83–0.90). A U-shaped significant association in nonlinear dose–response analysis was found between 25(OH)D and PE. A significant inverse association was also found between highest vs lowest circulating 25(OH)D concentration and PE in 32 nonprospective studies with 37 477 participants (odd ratio, 0.37; 95%CI, 0.27–0.52). This inverse association was significant in almost all subgroups, based on different covariates. Conclusion This meta-analysis of observational investigations showed blood 25(OH)D levels were negatively related to PE risk, in a dose-response manner. Systematic Review Registration PROSPERO registration no. CRD42021267486. [ABSTRACT FROM AUTHOR]

The aim of this study was to determine and compare values of carcinoembryonic antigen (CEA) and cancer antigen (CA 15-3) in 50 bitches with mammary tumors and 150 clinically healthy dogs. A modified procedure was used to determine the CEA and CA 15-3 markers with the human kits using the radioimmunoassay method (RIA). Samples collected from extirpated tumors of mammary glands were histologically processed and classified as per WHO guidelines. The mean values of the carcinoembryonic antigen markers ± SD were as follows: control group 0.89 ± 0.79, group with mammary gland tumor 1.53 ± 1.15. The values of cancer antigen markers CA 15-3 ± SD were: 1.52 ± 0.66 and 2.87 ± 1.11, respectively. The statistical significance for the carcinoembryonic antigen marker between groups was P < 0.0001. The cancer antigen CA 15-3 values between groups were also statistically significant with P < 0.0001. The results of the present study show that there are significant differences in both antigens between the control group and groups with mammary gland tumor in dogs. [ABSTRACT FROM AUTHOR]

Background: Nonsmall cell lung carcinoma (NSCLC) is a major cause of cancer-related death worldwide. The resistance of NSCLC to chemical drugs, such as cisplatin (CDDP), poses a heavy burden for NSCLC therapy. Herein, the effects of circular_0008928 (circ_0008928) on the CDDP sensitivity and biological behavior of CDDP-resistant NSCLC cells and underlying mechanism are revealed. Materials and Methods: The expression of circ_0008928 and microRNA-488 (miR-488) was detected by quantitative real-time polymerase chain reaction. The expression of hexokinase 2 (HK2) protein and exosome-specific proteins was determined by Western blot. The half-maximal inhibitory concentration (IC50) value of CDDP was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell proliferation and migratory and invasive abilities were illustrated by cell counting kit-8 and transwell assays. Cell glycolysis metabolism was illustrated by extracellular acidification rate assay, glucose kit and lactate kit assays and Western blot analysis. The binding sites between miR-488 and circ_0008928 or HK2 were predicted by starbase or microT-CDS online database, and identified by dual-luciferase reporter and RNA immunoprecipitation assays. Results: Circ_0008928 expression and HK2 protein expression were significantly upregulated, while miR-488 expression was obviously downregulated in NSCLC cells and CDDP-resistant NSCLC cells. Circ_0008928 expression was increased in serum exosomes of CDDP-resistant NSCLC patients compared with CDDP-sensitive NSCLC patients. In addition, circ_0008928 silencing improved CDDP sensitivity and attenuated CDDP-induced cell proliferation, migration, invasion, and glycolysis metabolism. Circ_0008928 was a sponge of miR-488, and miR-488 bound to HK2 in CDDP-resistant NSCLC cells. Furthermore, both miR-488 inhibitor and HK2 overexpression attenuated circ_0008928 absence-mediated impacts on CDDP sensitivity and tumor process in CDDP-resistant NSCLC. Conclusions: Circ_0008928 knockdown improved CDDP sensitivity and hindered cell proliferation, migration, invasion, and glycolysis metabolism by miR-488/HK2 axis in CDDP-resistant NSCLC. This finding provides a new mechanism for studying CDDP-resistant therapy in NSCLC. [ABSTRACT FROM AUTHOR]

The relationship between non-communicable diseases and eating behaviour has long been attributed to a surplus of food and energy. However, the increase in the prevalence of non-communicable disease and their underlying low-grade inflammatory milieu among people of low socio-economic status has highlighted the existence of a confounding factor. In this work, we aim to study the effect of lysine deficiency on some inflammatory markers in the absence or presence of an inflammatory insult (lipopolysaccharide (LPS)). For this purpose, thirty-two 5-week-old male Sprague Dawley rats were randomly distributed into four groups: (1) control diet, (2) control diet+LPS, (3) lysine-deficient diet and (4) lysine-deficient diet + LPS. Groups were only allowed their experimental diets for 4 weeks, during which LPS (50 µg/kg) or saline injections were administered intraperitoneally three times per week. The study showed that lysine deficiency blunted growth and body compartments development, decreased albumin production and elevated liver C-reactive protein (CRP) expression, independently of IL-6 and IL-1 β , the main precursors of CRP. Also, the insufficient levels of lysine in the diet increased hyperactivity and triggered an anxiety-like behaviour, exacerbated with LPS. This work presents evidence that various physiological changes are associated with the absence of a sufficient amount of lysine in the diet and can potentially increase the risk factor for diseases. Thus, the increment in non-communicable disease among the low socio-economic status populations, who heavily rely on cereals as a main source of protein, can be, at least partially, blamed on low lysine availability in diets. [ABSTRACT FROM AUTHOR]

Objective: Diabetes foot ulcer (DFU) is a serious complication of diabetes, which can lead to significant mortality and amputation rate. Our previous study found circ_072697 was highly expressed in DFU tissues, but the regulatory mechanism of circ_072697 in DFU remains unclear. Methods: The relative expressions of circ_072697, miR-3150a-3p, and KDM2A in DFU patients or advanced glycation end products (AGEs)-treated HaCaT cells (used as DFU cell model) were determined by using qRT-PCR. Cell proliferation and migration abilities were determined by using CCK-8 and Transwell assays. The interaction between miR-3150a-3p with circ_072697 or KDM2A were verified by RNA immunoprecipitation (RIP) and dual-luciferase reporter assays. Furthermore, the protein expression of genes involved in MAPK signaling pathway was detected by western blot. Results: The expression of circ_072697 was significantly upregulated in DFU tissues, while the expression of miR-3150a-3p was downregulated. Circ_072697 knockdown promoted the proliferation and migration of AGEs-treated HaCaT cells. miR-3150a-3p was confirmed as a target of circ_072697 and its inhibitor reversed the promotion effects of circ_072697 knockdown on biological behavior of cells. In addition, KDM2A was considered as a target of miR-3150a-3p and it was highly expressed in DFU samples. Importantly, circ_072697 could regulate KDM2A expression through sponging miR-3150a-3p, and this axis had effect on the MAPK signaling pathway. Conclusions: Overall, circ_072697 regulated the biological behaviors of keratinocytes in DFU via miR-3150a-3p/KDM2A axis and MAPK signaling pathway, revealing a new insight into the pathogenesis and potential therapeutic targets of DFU. [ABSTRACT FROM AUTHOR]

Background: Circulating biomarkers of bone formation and resorption are widely used in exercise metabolism research, but their responses to exercise are not clear. This study aimed to quantify group responses and inter-individual variability of P1NP and β-CTX-1 after prolonged, continuous running (60–120 min at 65–75% V̇O2max) in young healthy adult males using individual participant data (IPD) meta-analysis. Methods: The protocol was designed following PRISMA-IPD guidelines and was pre-registered on the Open Science Framework prior to implementation (https://osf.io/y69nd). Changes in P1NP and β-CTX-1 relative to baseline were measured during, immediately after, and in the hours and days following exercise. Typical hourly and daily variations were estimated from P1NP and β-CTX-1 changes relative to baseline in non-exercise (control) conditions. Group responses and inter-individual variability were quantified with estimates of the mean and standard deviation of the difference, and the proportion of participants exhibiting an increased response. Models were conducted within a Bayesian framework with random intercepts to account for systematic variation across studies. Results: P1NP levels increased during and immediately after running, when the proportion of response was close to 100% (75% CrI: 99 to 100%). P1NP levels returned to baseline levels within 1 h and over the next 4 days, showing comparable mean and standard deviation of the difference with typical hourly (0.1 ± 7.6 ng·mL−1) and daily (− 0.4 ± 5.7 ng·mL−1) variation values. β-CTX-1 levels decreased during and up to 4 h after running with distributions comparable to typical hourly variation (− 0.13 ± 0.11 ng·mL−1). There was no evidence of changes in β-CTX-1 levels during the 4 days after the running bout, when distributions were also similar between the running data and typical daily variation (− 0.03 ± 0.10 ng·mL−1). Conclusion: Transient increases in P1NP were likely biological artefacts (e.g., connective tissue leakage) and not reflective of bone formation. Comparable small decreases in β-CTX-1 identified in both control and running data, suggested that these changes were due to the markers' circadian rhythm and not the running intervention. Hence, prolonged continuous treadmill running did not elicit bone responses, as determined by P1NP and β-CTX-1, in this population. Key Points: It is unclear whether a single running bout produces bone adaptations, but these potential responses were not captured by bone (re)modelling markers P1NP and β-CTX-1. There is a need for studies that investigate the acute responses of bone (re)modelling markers to different types of exercise interventions and across different populations, which include a control (non-exercise) group. [ABSTRACT FROM AUTHOR]

Simple Summary: Hypersomatotropism results from excess growth hormone production by a pituitary tumour and represents an important underlying cause of diabetes mellitus in cats. Diagnosis of hypersomatotropism is mainly based on the measurement of insulin-like growth factor-1 (IGF-1), ideally in combination with imaging of the head. The most well-validated assay (radioimmunoassay), however, requires facilities approved to handle radioactivity and is costly. This study validated an alternative method for IGF-1 measurement in cats, a chemiluminescence assay, and compared it to the radioimmunoassay. It also established a reference interval for IGF-1. This will increase availability of IGF-1 measurement and facilitate diagnosis of hypersomatotropism. Previously, radioimmunoassay (RIA) has been the only assay to measure insulin-like growth factor-1 (IGF-1) to diagnose hypersomatotropism (HS). Due to radiation concerns, availability, and the cost of IGF-1 RIA, validation of assays for automated analysers such as a chemiluminescent immunoassay (CLIA) is needed. The aim of this study was to validate a CLIA for measurement of feline IGF-1 (IMMULITE 2000® XPi, Siemens Medical Solutions Diagnostics, Malvern, PA, USA) compared to IGF1 RIA, establish reference interval (RI), and determine a cut-off value for diagnosis of HS in diabetic cats. Validation of assay performance included precision, linearity, and recovery studies. Right-sided RI was determined using surplus serum of 50 healthy adult cats. Surplus serum samples of diabetic cats with known IGF-1 concentration with (n = 32/68) and without HS (n = 36/68) were used for method comparison with RIA. The cut-off for diagnosis of HS was established using receiver operating characteristic (ROC) analysis. The intra-assay coefficient of variation (CV) was ≤4.7%, and the inter-assay CV was ≤5.6% for samples with low, medium, and high IGF-1 concentration. Linearity was excellent (R2 > 0.99). The correlation between CLIA and RIA was very high (rs = 0.97), with a mean negative bias for CLIA of 24.5%. The upper limit of RI was 670 ng/mL. ROC analysis showed an area under the curve of 0.94, with best cut-off for diagnosis of HS at 746 ng/mL (sensitivity, 84.4%; specificity, 97.2%). The performance of CLIA was good, and the RI and cut-off for HS diagnosis established in this study allow for CLIA to be used in routine work-up of diabetic cats. [ABSTRACT FROM AUTHOR]

Background: Many long noncoding RNAs (lncRNAs) are the key regulators for cancer progression, including breast cancer (BC). RUSC1 antisense 1 (RUSC1‐AS1) has been found to be highly expressed in BC, but its role and potential molecular mechanism in BC remain to be further elucidated. Methods: Quantitative reverse transcription‐polymerase chain reaction (RT‐PCR) was utilized to measure RUSC1‐AS1, microRNA (miR)‐326 and X‐ray repair cross‐complementing group 5 (XRCC5) expression. Cell proliferation, metastasis, cell cycle, apoptosis and angiogenesis were determined by cell counting kit‐8, colony formation, transwell, flow cytometry and tube formation assays. Protein expression was detected by western blot analysis. The targeted relationship between miR‐326 and RUSC1‐AS1 or XRCC5 was validated using dual‐luciferase reporter assay and RIP assay. Xenograft models were constructed to uncover the effect of RUSC1‐AS1 on BC tumorigenesis. Results: RUSC1‐AS1 was upregulated in BC, and its downregulation suppressed BC proliferation, metastasis, cell cycle, angiogenesis, and tumor growth. MiR‐326 was confirmed to be sponged by RUSC1‐AS1, and its inhibitor reversed the regulation of RUSC1‐AS1 silencing on BC progression. XRCC5 could be targeted by miR‐326. Overexpression of XRCC5 reversed the inhibitory impacts of miR‐326 on BC progression. Conclusion: RUSC1‐AS1 could serve as a sponge of miR‐326 to promote BC progression by targeting XRCC5, suggesting that RUSC1‐AS1 might be a target for BC treatment. [ABSTRACT FROM AUTHOR]

Ambulance personnel in emergency medical services are exposed to physical demands and stress during work, and an increased prevalence of ill health has been observed in this group. The aim was to compare hair cortisol concentration (HCC) among Swedish ambulance personnel with HCC in a population-based reference sample, to analyse differences between women and men, and differences due to work-related factors. Samples of hair 1 cm closest to the skin (5–10 mg) were collected and analysed for cortisol by radioimmunoassay. Moreover, the participants responded to a questionnaire regarding their work environment. The HCC among the ambulance personnel did not differ from the HCC in the population-based reference sample (median 19.2 vs. 22.2 pg/mg, p = 0.319), nor were there statistically significant differences between women and men. Furthermore, no associations were found between HCC and physical and psychosocial work demands, work stress, or rest and recovery from work. However, occupational balance was positively correlated with HCC (rp = 0.240; p = 0.044). The association remained statistically significant after adjustment for sex, age, hair bleaching, and corticosteroid treatment in a linear regression model. This study adds knowledge regarding HCC among ambulance personnel, and thus contributes to the overall picture of work environment and health for this group. [ABSTRACT FROM AUTHOR]

Introduction: Thyroid disorders are considered the most common endocrine disorders affecting 5-10% of individuals. Vitamin D is an important bio-molecule that plays a role in the signaling pathways related to immune modulation and oxidative stress. Vitamin D receptor (VDR) gene polymorphisms were found to be associated with the risk for Graves' disease. As thyroid hormones are essential for normal organ growth, development function the basal metabolic rate of all cells, its alteration can affect the entire metabolism and can alter the activity of serum enzymes like LDH. There is negotiation regarding the increased/decreased levels of LDH in thyroid disorders with some studies stating that LDH can be used as a parameter for screening the thyroid disorder. But some contraindicated results prevail in this area of research. To clear some air an attempt was made on one study group to analyse the levels of LDH and Vitamin D in hyperthyroidism disorder and to find out correlation (if any) that exists between LDH and thyroid hormone. Aim and Objectives: To estimate the levels of serum LDH and vitamin D in hyperthyroid patients and to correlate with TSH, T3 and T4 and also to see the mutual correlation. Materials and methods: The study population was serum specimens from 100 patients with hyperthyroidism with ages ranging from 20-40 years. The samples were collected from the Thyroid laboratory, Department of Biochemistry, PGIMS, Rohtak. The levels of LDH and Vitamin D were determined on the autoanalyzer and radioimmunoassay respectively. Results and conclusion: The mean values for LDH and Vitamin D in hyperthyroidism patient were found to be 280.17±52.43 and 13.83±4.71 respectively. Regression analysis of different parameters was also done. Our findings support that hyperthyroid patients have subtle disturbances of LDH and Vitamin D level. [ABSTRACT FROM AUTHOR]

The aim of study was the production of polyclonal antibody of erythropoietin hormone (Anti-EPO) to determine EPO in human serum by liquid-phase radioimmunoassay (RIA). Production of anti-EPO was performed by immunizing Balb/C mice with EPO antigen using primary immunization and four booster doses. EPO tracer (125I-EPO) was prepared using Chloramine-T method. Additionally, EPO standards were mad. Furthermore, the assay was optimized and validated in additional studies. In conclusion, the elaborated EPO-RIA system exhibits a simple, accurate, specific and sensitive method for determining EPO concentration in human serum and might be suitable for clinical diagnosis of myeloproliferative diseases. [ABSTRACT FROM AUTHOR]

Somatostatin (SST) released from capsaicin-sensitive sensory nerves in response to stimulation exerts systemic anti-inflammatory, analgesic actions. Its elevation correlates with the extent of tissue injury. We measured plasma SST alterations during spine operations (scoliosis and herniated disc) to determine whether its release might be a general protective mechanism during painful conditions. Sampling timepoints were baseline (1), after: soft tissue retraction (2), osteotomy (3), skin closure (4), the following morning (5). Plasma SST-like immunoreactivity (SST-LI) determined by radioimmunoassay was correlated with pain intensity and the correction angle (Cobb angle). In scoliosis surgery, postoperative pain intensity (VAS 2.) 1 day after surgery significantly increased (from 1.44 SEM ± 0.68 to 6.77 SEM ± 0.82, p = 0.0028) and positively correlated with the Cobb angle (p = 0.0235). The baseline Cobb degree negatively correlated (p = 0.0459) with the preoperative SST-LI. The plasma SST-LI significantly increased in fraction 3 compared to the baseline (p < 0.05), and significantly decreased thereafter (p < 0.001). In contrast, in herniated disc operations no SST-LI changes were observed in either group. The VAS decreased after surgery both in the traditional (mean 6.83 to 2.29, p = 0.0005) and microdiscectomy groups (mean 7.22 to 2.11, p = 0.0009). More extensive and destructive scoliosis surgery might cause greater tissue damage with greater pain (inflammation), which results in a significant SST release into the plasma from the sensory nerves. SST is suggested to be involved in an endogenous postoperative analgesic (anti-inflammatory) mechanism. [ABSTRACT FROM AUTHOR]

Introduction. Among the plant ingredients, some compounds interfere with the functions of the thyroid gland. However, there is limited research on the effect of curcumin (CMN) on the functions of this gland. The aim of this study was to analyze the effect of CMN on morphology, histochemical reactivity of cytochrome c oxidase (CCO) and secretion functions of the thyroid gland under conditions of hypothyroidism induced by propylthiouracil (PTU). Material and methods. The rats were treated for 30 days by gavage with CMN (100 mg/kg b.w.) and/or PTU (1 mg/kg b.w.). Control rats received vehicle only. Histomorphometric tests were performed on the thyroid glands, cytochrome c oxidase activity was visualized using the histochemical method, and the levels of thyroid hormones were measured using the radioimmunoassay method. Results. Rats receiving PTU showed compensatory changes in their thyroid glands, including a significant increase in thyroid epithelium height, a decrease in colloid volume density, a decrease in the percentage of small follicles, an increase in medium-sized follicles compared to the control group, as well as a significant increase in CCO histochemical reactivity in the columnar epithelium and a decrease in FT4 serum level compared to the control group. The administration of CMN reversed these adverse changes caused by PTU. The PTU + CMN group exhibited a significant decrease in the height of the thyroid follicle epithelium compared to the PTU group. The percentage of small and medium-sized follicles in the CMN + PTU group did not differ from the control group. Furthermore, CCO reactivity in the cubic epithelium and serum FT4 levels increased compared to the PTU group. Administration of CMN alone resulted in a significant increase in FT4 levels compared to the control group. Conclusions. The administration of CMN to rats with induced hypothyroidism resulted in a reduction of hyperplasia, hypertrophy, and and an increase in secretory activity of the thyroid gland. These findings suggest the protective effect of CMN against induced hypothyroidism. (Folia Histochemica et Cytobiologica 2023, Vol. 61, No. 3, 143-152) [ABSTRACT FROM AUTHOR]

Simple Summary: Dog cloning requires in vivo matured recipient oocytes to transfer somatic donor cells for somatic cell cloning. The timing for recovering in vivo matured dog oocytes was determined by predicting the ovulation day based on serum progesterone (P4) concentration in estrus bitches. Radioimmunoassay (RIA) is traditionally used to measure the P4 concentration in dogs. In this study, the P4 concentration for ovulation was measured using next-generation enzyme-linked fluorescence assay (ELFA) and it was compared with that measured using RIA. The oocytes collected in vivo showed a maturation rate of 65.19% after the prediction of ovulation based on the P4 range measured using the ELFA system. These oocytes then produced four cloned puppies. Conclusively, we provide the optimal P4 range for the prediction of ovulation in estrus bitches when ELFA is used to measure the P4 concentration. Thus, we prove the effectiveness of application of ELFA in obtaining in vivo matured oocytes for dog cloning. Successful dog cloning requires a sufficient number of in vivo matured oocytes as recipient oocytes for reconstructing embryos. The accurate prediction of the ovulation day in estrus bitches is critical for collecting mature oocytes. Traditionally, a specific serum progesterone (P4) range in the radioimmunoassay (RIA) system has been used for the prediction of ovulation. In this study, we investigated the use of an enzyme-linked fluorescence assay (ELFA) system for the measurement of P4. Serum samples of estrus bitches were analyzed using both RIA and ELFA, and the measured P4 values of ELFA were sorted into 11 groups based on the standard concentration measured in RIA and compared. In addition, to examine the tendency of changes in the P4 values in each system, the P4 values on ovulation day (from D − 6 to D + 1) in both systems were compared. The ELFA range of 5.0–12.0 ng/mL was derived from the RIA standard range of 4.0–8.0 ng/mL. The rates of acquired matured oocytes in RIA and ELFA were 55.47% and 65.19%, respectively. The ELFA system successfully produced cloned puppies after the transfer of the reconstructed cloned oocytes. Our findings suggest that the ELFA system is suitable for obtaining in vivo matured oocytes for dog cloning. [ABSTRACT FROM AUTHOR]

Background: Steroids play a key role in numerous physiological processes. Steroid determination is a useful tool to explore various endocrine diseases. Because of its specificity, mass spectrometry is considered to be a reference method for the determination of steroids in serum compared to radioimmunoassay. This technology could progress towards more automation for the optimal organization of clinical laboratories and ultimately for the benefit of patients. Methods: A fully automated ultra-high-performance liquid chromatography-tandem mass spectrometry method was developed and fully validated to determine five steroids in serum. Sample preparation was based on protein precipitation with filtration followed by online solid phase extraction. Chromatographic separation was performed using a biphenyl stationary phase. Results: The method was successfully validated according to European Medicine Agency guidelines. Coefficients of variation did not exceed, respectively, 8.4% and 8.1% for intra- and inter-assay precision. Method comparison with radioimmunoassay showed a proportional bias for all compounds, except for testosterone in men. Comparison with another LC-MS/MS method demonstrated acceptable concordance for all steroids, although a small bias was observed for androstenedione. Conclusion: The novelty of this method is that it has been fully automated. Automation provides benefits in traceability and allows significant savings in cost and time.

A seated saline loading test (SLT) using liquid chromatography-tandem mass spectrometry (LC-MS/MS) is one of the most accepted confirmatory tests of primary aldosteronism. However, LC-MS/MS is time-consuming and is not widely available in diagnostic laboratories compared to immunoassay. With immunoassay, it is unknown whether SLT in the seated position is more accurate than that of the supine position, and a cutoff value of post-seated SLT plasma aldosterone concentration (PAC) must be established in the Korean population. Ninety-eight patients underwent SLT in both positions, and post-SLT PAC was measured by LC-MS/MS and radioimmunoassay. We confirmed primary aldosteronism if post-seated SLT PAC by LC-MS/MS exceeded 5.8 ng/dL. The area under the receiver operating characteristic curve was greater for seated than supine SLT (0.928 vs. 0.834, P=0.003). The optimal cutoff value of post-seated SLT by radioimmunoassay was 6.6 ng/dL (sensitivity 83.3%, specificity 92.2%).

The study aimed to evaluate the usefulness of salivary cortisol (SC) for the assessment of procedural pain intensity in preterm and term newborns. Three groups of neonates (term, 370–416 weeks; moderate to late preterm, 320–366; and very preterm, <320) hospitalized in neonatal intensive care unit were assessed for the study. Response to nappy change, lung ultrasound (LUS), and blood sampling was analyzed. The intensity of pain was evaluated using continuous heart rate and blood oxygen saturation (SpO2) monitoring, Neonatal Infant Pain Scale (NIPS), and SC concentrations. Saliva samples were collected before and 20 min after the procedure's end. Seventy-one infants were examined: 30 term, 21 moderate to late preterm, and 20 very preterm. SC has increased significantly in response to nappy change only in very preterm newborns (2.13 ng/mL [1.55–3.68] vs. 2.84 ng/mL [1.93–9.06], p = 0.01). LUS did not affect concentrations of SC in any group. Significant increase in SC was observed after blood sampling in term and very preterm infants (2.2 ng/mL [1.45–2.92] vs. 4.29 ng/mL [3.88–5.73], p = 0.002, and 1.88 ng/mL [1.47–4.13] vs. 5.3 ng/mL [3.42–8.02], p = 0.002, respectively). A significant correlation between values of SC increase and NIPS scores was found (Spearman's rank correlation coefficient [rs] = 0.31, p = 0.001). We observed the increase in SC concentrations in response to painful stimulus. The presence of a correlation between NIPS scores and SC increase suggests that SC can be used as an objective parameter to assess pain in neonates. [ABSTRACT FROM AUTHOR]

Vitamin D plays an essential role in neonatal skeletal development and maternal weight gain during pregnancy. We aim to study the association between vitamin D status, maternal weight, and materno-neonatal bone metabolism parameters. From January to June 2017, we conducted this cross-sectional study among 103 pregnant women (21–42 years old) and their singletons. The levels of serum 25-(OH)D, PTH, P1NP, OC, and CTX were measured for mothers and neonates (cord blood). Serum vitamin D and OC were measured using chemiluminescence and two-site immunoradiometric assay, respectively. Meanwhile, P1NP, CTX, and PTH were measured by ELISA. The average serum vitamin D levels from mothers were 15.1 ng/mL during pregnancy and 16.2 ng/mL in the umbilical cord. At baseline, vitamin D deficient mothers were more likely to have higher PTH (36.4 vs. 18 pg/mL; p=0.029) and lower P1NP levels (90 vs. 92.5 ng/mL; p=0.026). Also, vitamin D deficient status was associated with lower fetal weight (3,293 vs. 3,358 g; p=0.019). Maternal weight was significantly correlated with P1NP (65.86 vs. 109.35; p=0.001) and OC (14.52 vs. 18.24; p=0.038), as well as cord vitamin D level (13.31 vs. 18.46; p=0.039) among normal vs. overweight women. No significant differences were found for the correlation between maternal weight and fetal parameters except for fetal weight which significantly increased with the increase in maternal weight (overweight vs. obese women=3,280 vs. 3,560; p=0.06). Maternal vitamin D status is associated with maternal and neonatal bone metabolism parameters as well as maternal and neonatal weight. [ABSTRACT FROM AUTHOR]

Monoclonal antibodies (mAbs), such as infliximab, are important treatment options for different diseases. Immunogenicity is a major risk, resulting in anti-drug antibodies (ADAs), being associated with adverse events and loss of response, influencing long-term outcomes. The development of ADAs against infliximab is primarily measured by immunoassays like radioimmunoassay (RIA). Although liquid chromatography-tandem mass spectrometry (LC-MS/MS) is increasingly utilized across different fields, this technique is currently not used for ADAs against infliximab measurements. Therefore, we developed the first LC-MS/MS method. Stable isotopically labeled infliximab antigen-binding fragments (SIL IFX F(ab')2) were used to bind and measure ADAs indirectly. Protein A magnetic beads were used to capture IgG, including ADAs, whereafter SIL IFX F(ab')2 was added for labeling. After washing, internal standard addition, elution, denaturation and digestion samples were measured by LC-MS/MS. Internal validation showed good linearity between 0.1 and 16 mg/L (R2 > 0.998). Sixty samples were used for cross-validation with RIA, and no significant difference between ADA concentrations was found. The methods had high correlation (R = 0.94, p < 0.001) and excellent agreement, intraclass correlation coefficient = 0.912 (95% confidence interval 0.858–0.947, p < 0.001). We present the first ADA against the infliximab LC-MS/MS method. The method is amendable for quantifying other ADAs, making it applicable as a template for future ADA methods. [ABSTRACT FROM AUTHOR]

Casein kinase 1α (CK1α) is present in multiple cellular organelles and plays various roles in regulating neuroendocrine metabolism. Herein, we investigated the underlying function and mechanisms of CK1α-regulated thyrotropin (thyroid-stimulating hormone (TSH)) synthesis in a murine model. Immunohistochemistry and immunofluorescence staining were performed to detect CK1α expression in murine pituitary tissue and its localization to specific cell types. Tshb mRNA expression in anterior pituitary was detected using real-time and radioimmunoassay techniques after CK1α activity was promoted and inhibited in vivo and in vitro. Relationships among TRH/L-T4, CK1α, and TSH were analyzed with TRH and L-T4 treatment, as well as thyroidectomy, in vivo. In mice, CK1α was expressed at higher levels in the pituitary gland tissue than in the thyroid, adrenal gland, or liver. However, inhibiting endogenous CK1α activity in the anterior pituitary and primary pituitary cells significantly increased TSH expression and attenuated the inhibitory effect of L-T4 on TSH. In contrast, CK1α activation weakened TSH stimulation by thyrotropin-releasing hormone (TRH) by suppressing protein kinase C (PKC)/extracellular signal-regulated kinase (ERK)/cAMP response element binding (CREB) signaling. CK1α, as a negative regulator, mediates TRH and L-T4 upstream signaling by targeting PKC, thus affecting TSH expression and downregulating ERK1/2 phosphorylation and CREB transcriptional activity. [ABSTRACT FROM AUTHOR]

Parathyroid hormone (PTH) determination is of paramount importance for the exploration of diseases related with calcium metabolism and for the follow-up of patients suffering from bone and mineral disorders associated with chronic kidney diseases (CKD-MBD). Unfortunately, the biologically active form of PTH, i.e. 1–84 PTH, circulates in the blood stream with many fragments and post-translationally modified forms, which decreases the specificity of immunoassays. The assays used to measure PTH, either from 2nd or 3rd generation, are not standardised, which may lead to interpretation errors and clinical consequences. Reference ranges for PTH have neither been always correctly established and the stability of the peptide is also a matter of concern. Fortunately, these last years, newer techniques using mass spectrometry (either high resolution or triple quadripole) coupled with liquid chromatography have been developed, which will help to standardise the different assays. Indeed, PTH assays standardisation is one of the task of the IFCC Committee for Bone Metabolism. Such standardisation will allow a better consistency in the interpretation of the results and will promote studies aiming at the establishment of correct reference ranges. [ABSTRACT FROM AUTHOR]

Simple Summary: It is essential to study animal models of human disease to understand the progression of disease, the mechanisms involved and their interventional testing on a pre-clinical basis. We proposed to observe a lipopolysaccharide (LPS)-induced effective model of mammary gland inflammation. Additionally, the nutraceutical role of resveratrol, a phenolic compound, against the negative effects of lipopolysaccharides (LPS) in mammary tissues caused by oxidative damage and inflammation has not been fully studied. In our experiments, we found that LPS significantly enhances oxidative stress by repressing the expression of oxidative-related factors and stimulating the secretion of inflammatory cytokines. Moreover, LPS decreases the body weight, water and feed intake and activates NF-κB, Jnk, Erk, and Nrf2. The relative protein intensity of P65 and pP65 were increased by LPS. However, resveratrol administration remarkably decreases the oxidative response as well as inflammatory genes and plays a vital role in protection of the mammary glands from negative alterations in female mice. The aim of this study is to evaluate the defensive role of resveratrol, which is antagonistic to the oxidative stress and inflammation that is prompted by LPS in mammary tissue of female mice. Thirty adult mice were distributed into three groups (n = 10) control (CON), lipopolysaccharides at 2.5 mg/kg (LPS), and lipopolysaccharides at 2.5 mg/kg with 2 mg/kg of resveratrol (RES + LPS). The treatments were applied for 15 consecutive days. Spectrophotometry was used to quantify ROS in the blood, and proinflammatory cytokines concentrations were determined through radioimmunoassay. NF-κB, Jnk, IL-1β, Erk, IL-6, Nrf2 and TNF-α were quantified by RT-qPCR, and Western blots were used to quantifyP65 and pP65 protein intensities. MDA production was considerably increased, and the activity of T-AOC declined in the LPS treatment in comparison with the CON group but was significantly reversed in the RES + LPS group. Proinflammatory cytokines production and the genes responsible for inflammation and oxidative stress also showed higher mRNA and pP65 protein intensity in the LPS group, while Nrf2 showed a remarkable decline in mRNA expression in the LPS versus the CON group. All these mRNA intensities were reversed in the RES + LPS group. There were no remarkable changes in P65 protein intensity observed between the CON, LPS, and RES + LPS groups. In conclusion, resveratrol acts as a protective agent to modulate cellular inflammation and oxidative stress caused by LPS in mammary tissue of female mice. [ABSTRACT FROM AUTHOR]

[Objectives] This study was conducted to investigate the effects of Polygonatum odoratum polysaccharide (POP) on organ relative weights and reproductive hormone levels in male rats fed a high-fat diet. [Methods] Thirty healthy male Sprague-Dawley (SD) rats were randomly divided into two groups according to their body weight; 10 in normal control group (Group NC, n = 10) and 20 in experimental group (n =20). The rats in experimental group were fed a high-fat diet for eight weeks before they were further randomly divided into two groups; high fat group (Group HF) and high fat +400 mg/(kg - d) POP group (Group HF + POP). In Group HF + POP, the rats were administered with POP for another six weeks, before their blood plasma was collected, and the relative weights of their testis and epididymis were calculated. The plasma levels of testosterone (T), estrogen (E2), follicle-stimulating hormone (FSH), cortisol (C) and luteinizing hormone (LH) were measured by radioimmunoassay, and the plasma levels of sex hormone-binding globulin (SHBG) and insulin-like growth factor-1 (1GF-1) were determined by enzyme-linked immunosorbent assay. [Results] Compared with Group HF, POP could effectively inhibit rat obesity caused by high-fat diets, increase the relative weights of their testis and epididymis, plasma levels of LH, E2, FSH, T, SHBG and IGF-1, and reduce the plasma level of E2. [Conclusions] Polygonatum odoratum polysaccharide (POP) is able to effectively regulate the level of reproductive hormones in high-fat diet fed rats, and helps to protect their reproductive function. [ABSTRACT FROM AUTHOR]

Simple Summary: A three-dimensional spheroid cell culture model resembles the architecture and cellular characteristics of solid tumors much more closely than the conventional monolayer cell cultures often applied in preclinical drug testing. In this study, we validate the efficacy of the RIST treatment protocol, a novel multimodal treatment regimen for high-risk neuroblastoma, in a neuroblastoma spheroid cell culture model characterized by an augmented neoplastic phenotype. The results also indicate the importance of the neuroblastoma spheroid model for preclinical drug testing in a rigorous, robust and efficient high-throughput format, which can be beneficial to identify more effective treatment options for children with high-risk neuroblastoma in the future. Background: The outcome for patients with high-risk neuroblastoma remains poor and novel treatment strategies are urgently needed. The RIST protocol represents a novel metronomic and multimodal treatment strategy for high-risk neuroblastoma combining molecular-targeted drugs as 'pre-treatment' with a conventional chemotherapy backbone, currently evaluated in a phase II clinical trial. For preclinical drug testing, cancer cell growth as spheroid compared to mo-nolayer cultures is of advantage since it reproduces a wide range of tumor characteristics, including the three-dimensional architecture and cancer stem cell (CSC) properties. The objective of this study was to establish a neuroblastoma spheroid model for the rigorous assessment of the RIST treatment protocol. Methods: Evaluation of CSC marker expression was performed by mRNA and protein analysis and spheroid viability by luminescence-based assays. Aberrant expression of RNA-binding protein La in neuroblastoma was assessed by tissue microarray analysis and patients' data mining. Results: Spheroid cultures showed increased expression of a subgroup of CSC-like markers (CXCR4, NANOG and BMI) and higher Thr389 phosphorylation of the neuroblastoma-associated RNA-binding protein La when compared to monolayer cultures. Molecular-targeted 'pre-treatment' of spheroids decreased neoplastic signaling and CSC marker expression. Conclusions: The RIST treatment protocol efficiently reduced the viability of neuroblastoma spheroids characterized by advanced CSC properties. [ABSTRACT FROM AUTHOR]

This study sought to assess the association between vitamin D deficiency (VDD) and chest pain, anxiety, and fatigue in patients with coronary artery disease (CAD). A cross-sectional study was conducted to collect data from a sample of 90 participants with CAD. Serum 25OHD3 was measured using a radioimmunoassay procedure. Chest pain was assessed using the Numeric Pain Rating Scale, anxiety using the Anxiety Subscale, and fatigue by the General Fatigue subscale. The majority of participants (80%) had VDD. Results showed that the level of vitamin D was significantly negatively associated with chest pain (r = −.43, p =.00); anxiety (r = −.466, p =.00); and fatigue (r = −.25, p =.018). Findings suggest the role of VDD in heightened central sensitivity in cardiac-related symptoms such as chest pain, anxiety, and fatigue. Patients with CAD should be monitored for VDD so that the condition can be corrected. [ABSTRACT FROM AUTHOR]

The main objective of this study was the preparation and evaluation of the primary reagents for the liquid phase human chorionic gonadotropin-radioimmunoassay (HCG-RIA) kit. Polyclonal antibody is specific and valid; monoclonal antibodies are more specific. In HCG-RIA technique, polyclonal antibodies for βHCG subunits with high binding and displacement % were used. 125I-HCG radioactive tracer was prepared with high yield 71.58 ± 0.92%, purity 99.2 ± 0.05%, and specific activity 170.42 ± 1.65 µCi/µg using chloramine T method, βHCG polyclonal antisera and HCG standards in assay buffer matrix were locally prepared. In-house HCG-RIA was developed with high sensitivity 0.5 mIU/ml, specificity (99%), precision (CV% < 6.4 and < 8.3 for intra-and inter-assay, respectively), and accuracy (recovery range 96.4–104.3%) could be used for quantitative estimation of HCG in human sera for monitoring pregnancy and diagnosis of gestational trophoblastic diseases. [ABSTRACT FROM AUTHOR]

The radioimmunoassay (RIA) method is widely used to determine the levels of arginine vasopressin (AVP) in studies, especially in cell culture studies. However, there are many difficulties and disadvantages in performing this method. Therefore, this study aimed to assess the comparison between enzyme-linked immunosorbent assay (ELISA) and RIA methods by using the Bland–Altman statistical method and to investigate whether the commonly used RIA can be replaced by the ELISA method for measurement of AVP levels in cell culture medium. For this purpose, wild-type (WT) and three different mutant AVP-NPII vectors were transiently transfected to mouse neuroblastoma (Neuro2A) cells and AVP secretion into the cell culture medium by transfected Neuro2A cells was determined by both RIA and ELISA methods. Following the use of the normalization method, Bland–Altman method, currently the most commonly used statistical method assessing comparison between two methods, was performed to assess agreement between two methods. The order of normalized AVP values obtained using the RIA method was as follows: WT (0.921 ± 0.08), 207_209delGGC mutant (0.801 ± 0.09), G45C mutant (0.508 ± 0.10), and G88V mutant (0.497 ± 0.12). The normalized AVP values obtained using the ELISA method were as follows: WT (0.865 ± 0.12), 207_209delGGC mutant (0.704 ± 0.13), G88V mutant (0.255 ± 0.16), and G45C mutant (0.250 ± 0.15). The order of AVP levels measured from each transfected cell using both methods was quite similar. According to the Bland–Altman method, there is an agreement between RIA and ELISA for measuring the AVP levels in cell culture. It can be recommended to apply ELISA instead of the RIA method for determinating AVP levels. [ABSTRACT FROM AUTHOR]

Background: D4-androstenedione (D4ASD) is an intermediate hormone of androgen biosynthesis by the gonads and the adrenal glands. The interest in D4ASD concentration assessment resides in diagnostics of androgenic hyperproduction pathologies. Currently, many D4ASD quantification methods are available on the market including immunological methods that remain problematic due to the possible cross-reactivity with endogenous or exogenous steroids. Methods: Recently Roche® launched a new fully automated instrument for the measurement of D4ASD concentration. In this paper, the criteria of analytical performance (repeatability and intermediate precision) of the D4ASD Roche® assay were assessed and compared with 2 different methods including a radioimmunoassay (RIA) as well as a liquid chromatography tandem mass spectrometry (LC-MS/MS) method. Results: Repeatability and intermediate precision of the D4ASD Roche® were acceptable according to the predefined RICOS standard (CV ≤ 7.9%) and the assay showed a good correlation with other assays considering the 95% CI obtained for the slope and the y-intercept. Conclusions: This method demonstrates acceptable criteria of analytical performance with an intermediate imprecision and a trueness within the fixed acceptance limits. [ABSTRACT FROM AUTHOR]

BackgroundTrigeminal-mediated headshaking results from a low threshold for firing of the trigeminal nerve. A seasonal component has been implicated in onset of clinical signs, which occur during the spring and summer months. Geldings are overrepresented in the affected population and hormonal differences as compared to a healthy control population of geldings might contribute to headshaking.Objective/hypothesisTo assess concentrations of luteinizing hormone (LH) over an 8-hour period in gelded healthy controls and horses affected with headshaking. Our hypothesis was that geldings with seasonal headshaking would have higher concentrations of LH over an 8-hour period compared to control horses during the summer when affected horses manifested headshaking.AnimalsTwelve geldings (6 controls and 6 affected).MethodsProspective controlled trial. Blood samples were drawn every 15 minutes over an 8-hour time period during summer from all horses to measure circulating LH concentrations by using a radioimmunoassay for equine LH. All affected horses were actively affected by headshaking at the time of sample collection.ResultsNo statistically significant differences in LH concentrations were found throughout the study period in headshakers as compared to control horses. Time had no significant effect, but a slight decrease in LH concentrations was observed for all horses. The main limitation of the study was the low number of horses.Conclusions and clinical importanceHorses affected with headshaking did not have significant differences in circulating LH during the late summer as compared to control horses.

IntroductionImmunoassays and liquid chromatography-tandem mass spectrometry assays are commonly employed in clinical laboratories for measurement of total testosterone in serum. Results obtained from either of these methodologies compare poorly due to differences in calibration and/or inadvertent detection of interfering substances by the immunoassays. Standardization efforts are underway, but recent studies indicate that accuracy remains an issue.MethodsThis study compares the results from four independently developed and validated LC-MS/MS assays for total testosterone. The calibration for each assay was verified using National Institute of Standards and Technology Standard Reference Material 971.ResultsInitially, one of the four assays had a mean percent difference of +11.44%, compared to the All Method Mean, but following re-verification of all five non-zero calibrator concentrations with the NIST SRM 971, the mean percent difference decreased to -4.88%. Subsequently, the agreement between all four assays showed a mean bias of

We conceptualize and define nanocrinology as the science that studies the nanometric and subnanometric precision that operates in diagnostic and therapeutic endocrinology. It includes advanced generation assays, which can detect low concentrations of hormones, and modern drug delivery systems that allow more efficient delivery of endocrinotropic agents. Nanocrinology is a rapidly growing field of endocrinology, and we call for greater research and adoption of this science. Keywords: Diagnosis, endocrinology, hormone, radioimmunoassay, nanoformulation.

Cortisol is a steroid hormone that is involved in a broad range of physiological processes in human/animal organisms. Cortisol levels in biological samples are a valuable biomarker, e.g., of stress and stress-related diseases; thus, cortisol determination in biological fluids, such as serum, saliva and urine, is of great clinical value. Although cortisol analysis can be performed with chromatography-based analytical techniques, such as liquid chromatography–tandem mass spectrometry (LC-MS/MS), conventional immunoassays (radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs), etc.) are considered the "gold standard" analytical methodology for cortisol, due to their high sensitivity along with a series of practical advantages, such as low-cost instrumentation, an assay protocol that is fast and easy to perform, and high sample throughput. Especially in recent decades, research efforts have focused on the replacement of conventional immunoassays by cortisol immunosensors, which may offer further improvements in the field, such as real-time analysis at the point of care (e.g., continuous cortisol monitoring in sweat through wearable electrochemical sensors). In this review, most of the reported cortisol immunosensors, mainly electrochemical and also optical ones, are presented, focusing on their immunosensing/detection principles. Future prospects are also briefly discussed. [ABSTRACT FROM AUTHOR]

Context: The 250µg-cosyntropin stimulation test (CST) is used to diagnose non-classic congenital adrenal hyperplasia (NCCAH). The current recommendation is to perform CST when follicular 17-hydroxyprogesterone (17OHP) is 6-30 nmol/L, a cutoff derived from radioimmunoassay (RIA). Recently, enzyme-linked immunosorbent assay (ELISA) has replaced RIA. Objectives: We aimed to (1) determine the RIA and ELISA-based 17OHP cutoffs at which CST should be performed, (2) identify predictors of NCCAH. Methods: A retrospective study at an Israeli Health Maintenance Organization. Data were retrieved from women with suspected NCCAH, referred for CST during 2001-2020. NCCAH was defined as a stimulated 17OHP >30 nmol/L. Serum 17OHP levels were assayed by RIA from 1/2000-3/2015, and by ELISA from 4/2015-12/2020. ROC curves were generated and optimal 17OHP thresholds were determined. Multivariate analysis was performed. Results: CST was performed in 2409 women (1564 in RIA, 845 in ELISA). NCCAH was diagnosed in 4.7% of the RIA group and 7.5% of the ELISA group. The optimal basal 17OHP cutoff values predicting NCCAH were 6.1 nmol/L in RIA (sensitivity=93.2%, specificity=91.7%) and 8.2 nmol/L in ELISA (sensitivity=93.7%, specificity=92.3%). In multivariate analysis, higher basal 17OHP, lower LH: FSH ratio, and oligomenorrhea were predictors of NCCAH in RIA. Higher basal 17OHP, androstenedione, and total testosterone were predictors of NCCAH in ELISA. A lower LH: FSH ratio showed similar trend in ELISA. Conclusions: Optimal RIA-based basal 17OHP cutoff was comparable with that recommended in guidelines. The results suggest adopting a higher 17OHP cutoff when using ELISA. LH: FSH ratio improves the negative predictive value of basal 17OHP. [ABSTRACT FROM AUTHOR]

Background: Measurement of pregnancy-associated glycoproteins (PAGs) by radioimmunoassay or enzyme-linked immunosorbent assay has been commonly used for early pregnancy diagnosis in ruminants. However, an accurately pregnancy detection depends on test antibody, breed and number of embryos. Only few studies have conducted to detect or predict animals at risk of late embrionic mortality (LEM) and to use hormonal interventions for embryo losses reducing, but this area is still open in sheep. The current study aimed to evaluate the effect of gonadotropin releasing hormone (GnRH) administration on Day 4 after artificial insemination on the pregnancy-associated glycoproteins in dairy sheep with different reproductive status in relation to pregnancy and late embrionic mortality detection. Materials, Methods & Results: Sixty-five East Friesian sheep were divided in 2 groups - I (Control group, n = 35) and II (GnRH group, n = 30) and sublected to estrus synchronization and artificial insemination (AI). Group I was not treated and Group II received 50 µg GnRH on Day 4 after AI. PAGs in blood serum were measured by Alertys Ruminant Pregnancy test on Days 4, 12, 20, 25 and 35 after AI and ultrasound pregnancy test was conducted on Days 20, 25, 35 and 60. Reproductive status (non-pregnant, pregnant and animals with LEM) was determined by ultrasound and the results between different groups were compared. The PAGs mean values according to reproductive status and Day after AI were analysed. Accuracy, sensitivity and specificity of the PAGs test for pregnancy diagnosis on Day 35 were calculated. On Day 20 after AI the pregnat sheep (83.3%) in group I tended to be higher than those (77.1%) in group II with decrease of 25.7% and 20% for the same groups on Day 25. On day 25 LEM was recorded in 33.3% and 24% in group I and II, respectively. The ultrasound exams on Day 60 confirmed the results from Day 25 after AI. A total value of non-pegnant, pregnant animals and LEM was 20%, 56.9% and 28.8%, respectively. The mean values of PAGs in animals with the same reproductive status in group I and II no differed statistically between Days 4 and 35 after AI. On Day 25 the PAGs values in LEM groups (0.126 ± 0.072 and 0.179 ± 0.029) were higher than those (0.062 ± 0.038 and 0.083 ± 0.023) in the non-pregnant groups, but no significan difference was deternined. On Day 35 after AI the mean values of PAGs (0.414 ± 0.125 and 0.421 ± 0.121) for the pregnant groups were significantly (P < 0.05) higher than those (0.078 ± 0.053 and 0.093 ± 0.034) for the non-pregnant groups. The values of PAGs in LEM groups on Day 25 (0.099 ± 0.062 and 0.113 ± 0.058) were decreased and close to the mean values in non-pregnant sheep. A significant effect of the day after AI on the PAGs values was evidenced in pregnant animals only (control r = 0.97 and GnRH r = 0.98; P < 0.05). The PAGs started to rise rapidly between Days 25 and 35 after AI. On Day 35 the accuracy (98%), the sensitivity (97.3%) and the specificity (100%) of the PAG test for pregnancy diagnosis were similar to the values (100%) for ultrasound method. Discussion: The gonadotropin treatment on Day 4 after AI tended to improve the reproductive performance in the sheep, but no significant effect of GnRH on the PAGs values in the animals with different reproductive status was found. The PAGs profiles in non-pregnant and LEM sheep were close and distinguishing between non-pregnant and LEM ewes on Day 35 was impossible. The PAGs values in pregnant sheep showed significantly (P < 0.05) increase between Days 25 and 35 after AI with higher mean values in pregnant than non-pregnant groups on Day 35 (P < 0.05). Alertys Ruminant Pregnancy test was a reliable for pregnancy detection in sheep on Day 35 after artificial insemination. [ABSTRACT FROM AUTHOR]

Study Objectives: The assay currently used worldwide to measure cerebrospinal fluid hypocretin-1 (CSF-hcrt-1) for diagnosing narcolepsy uses a competitive radioimmunoassay with polyclonal anti-hcrt-1 antibodies. This assay detects multiple hypocretin-1 immunoreactive species in the CSF that are all derived from full-length hcrt-1. We aimed to revalidate CSF-hcrt-1 cut-offs for narcolepsy type 1 (NT1) diagnosis and to evaluate temporal changes in CSF-hcrt-1 levels in patients suspected of having central hypersomnia.Method: We carried out a repeat lumbar puncture with a mean follow-up of 4.0 years, to measure CSF-hcrt-1 in patients suspected of having central hypersomnia in a follow-up study. Data from CSF samples of patients with NT1 and of controls without known hypersomnia, from the Italian-Stanford and Danish populations, were examined using a receiver-operating characteristic analysis.Results: The optimal CSF-hcrt-1 cut-offs for identifying NT1 were 129 pg/ml and 179 pg/ml for the Italian-Stanford and Danish populations, respectively. The sensitivity was 0.93-0.99 and the specificity was 1. Follow-up lumbar puncture measurements of CSF-hcrt-1 were obtained from 73 patients. 30 of 32 patients with low CSF-hcrt-1 levels continued to be categorized as low, with an unaltered diagnosis; two patients showed a marked increase in CSF-hcrt-1, attaining normal values at follow-up. One of these patients relapsed to low CSF-hcrt-1 after follow-up. All 41 patients with normal CSF-hcrt-1 at baseline had normal CSF-hcrt-1 at follow-up.Conclusion: CSF-hcrt-1 measurement can provide an accurate test for diagnosing NT1, although it is important to validate the CSF-hcrt-1 cut-off for specific testing locations. Stable CSF-hcrt-1 levels support the already established prognosis of narcolepsy as permanent once the disorder has fully developed. [ABSTRACT FROM AUTHOR]

Previous studies of cortisol concentrations in hair have concluded that it is not possible to measure more than 6 months retrospectively. This study shows for the first time that it is possible to analyze hair cortisol concentrations month-by-month for a retrospective period of 24 months. In addition, we have determined whether cortisol concentration decreases with time. The study population was 48 women in the age range 20-51 years, all with hair of length of 24 cm or longer. The participants completed a questionnaire that examined exposure to life stressors and potential confounders. Competitive radioimmunoassay was used to extract and analyze cortisol levels in hair. The overall intraclass correlation for the participants was substantial (ICC = 0.38, 95% CI 0.29, 0.49), indicating a strong within-person correlation during the growth period. The median levels of cortisol were reasonably stable. Wash-out effects were small, even for those who reported that they washed their hair every day. We conclude that it is possible to detect hair cortisol concentrations every month at least two years back in time. Changes in hair cortisol concentration are more likely to be related to life stressors than changes due to time since growth. [ABSTRACT FROM AUTHOR]

Background: Ghrelin is widely distributed in an animal's body, playing important biological roles. In mammals, Ghrelin can stimulate food intake and weight gain, but in poultry, it inhibits food intake and drinking. We aimed to determine the effect of Ghrelin on feeding, drinking, body weight and growth hormone (GH) content of African ostrich chicks. Methods: Sixteen 40-day-old African ostrich chicks were randomly divided into four groups and injected intravenously with saline (control) or Ghrelin (10, 50 and 100 µg/kg) for 7 days. The feeding, drinking and growth hormone (GH) levels using measurement method and radioimmunoassay were determined. Result: The results show that exogenous Ghrelin increased the feed intake rate, the drinking rate and the secretion of GH in the ostriches. [ABSTRACT FROM AUTHOR]

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Aims. Abnormal changes in cardiac function have been reported in menopausal women, but there are few clinical studies on this topic. Erxian decoction (EXD) is a classic prescription that is widely used in the treatment of female menopausal diseases. The purpose of this study was to investigate the dynamic evolution of cardiac function and glucose and lipid metabolism in ovariectomized (OVX) rats and the intervention effect of EXD. Materials and Methods. The OVX climacteric rat model was established by bilateral ovariectomy. After successful modeling, the rats were randomly divided into four groups: the sham operation (SHAM) group (equal volumes of purified water), OVX group (equal volumes of purified water), estradiol (E2) group (1.8 × 10−4 g/kg), and EXD group (9 g/kg). Each group of rats was treated for 16 weeks. At the 4th, 8th, 12th, and 16th weeks after treatment, the cardiac function of the rats in each group was evaluated by ultrasound. The coaxial method was used to measure blood pressure (BP). Serum endothelin-1 (ET-1) and angiotensin-2 (Ang II) levels were determined by the enzyme-linked immunosorbent assay (ELISA). The strip method was used to measure fasting blood glucose (FBG). The serum total cholesterol (TC) and triglyceride (TG) levels of rats were measured with the oxidase method. Direct methods were used to measure serum high-density lipoprotein (HDL-C) and low-density lipoprotein (LDL-C) levels. At week 16 of dosing, serum E2 levels were determined by E2 radioimmunoassay. The myocardium and uterus of the rats in each group were stained with HE (hematoxylin-eosin). The ultrastructure of the rat myocardium was observed by electron microscopy. Results. After the 16th week of treatment, the serum E2 level decreased (P < 0.05), and the uterus was atrophied in OVX rats. The cardiac ejection fraction (EF%) decreased at 4 weeks after treatment, and systolic and diastolic function decreased after 12 weeks. After the 16th week, the EF%, which reflects the "pump" function of the heart, decreased significantly (P < 0.05 or P < 0.01). At the 4th, 8th, 12th, and 16th weeks of treatment, the systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean pressure (MBP) of the rats in the OVX group increased with time (P < 0.05 or P < 0.01). The serum ET-1 and Ang II levels of rats in the OVX group increased (P < 0.05 or P < 0.01). In the OVX group, FBG was increased (P < 0.05 or P < 0.01), and blood lipids, especially LDL-C, were significantly increased (P < 0.05 or P < 0.01). After the 16th week of treatment, the myocardial tissue of OVX rats showed obvious pathological changes. EXD significantly increased serum E2 levels (P < 0.01), decreased ET-1 and Ang II levels (P < 0.01), reduced the cardiac function risk factors BP, FBG, and blood lipids, and significantly improved cardiac function and structural changes in OVX rats (P < 0.05 or P < 0.01). Conclusions. EXD can improve abnormal cardiac structure and function in OVX rats. [ABSTRACT FROM AUTHOR]

One animal-based method to evaluate welfare is the presence of stress. In dairy ruminants, the responses to stressors include the activation of both neuroendocrine and autonomous nervous system that can be evaluated through an endocrine assessment. The present study aimed to validate a radioimmunoassay method for cortisol in buffalo milk. Three formulations (whole and skimmed milk and whey) and three solvents (methanol diethyl ether and dichloromethane) were tested: methanol was characterised by the best extraction efficiency (69.88%), whey cortisol concentrations showed a significant correlation with whole extracted milk and were not affected by fat content variation during the milking session. The RIA used in the present study showed good precision, sensitivity and specificity: the dilutions test indicated the high reproducibility of the results, overlapping of the dilution curve and standard curve highlighted high specificity and the lack of interfering factors by buffalo whey matrix. It is concluded that the present assay suits the cortisol measurement in buffalo milk and the ranges described could be employed in the calibration of a biosensing technologies directly integrated in milking parlour systems. Buffalo milk whey revealed to be a matrix of great interest because of its high stability in terms of storage, transportation and processing. RIA method suits the cortisol measurement in buffalo milk Ranges described can be employed in the calibration of biosensors for non-invasive assessment of cortisol [ABSTRACT FROM AUTHOR]

A novel Radioimmunoassay (RIA) method was developed for measuring C-reactive protein (CRP) in human serum. The development of CRP-RIA includes CRP radiolabelling with Na125I using the Chloramine -T method to prepare 125I-CRP tracer and purify it using Sephadex G-25 Column. Polyclonal anti-CRP was coupled with magnetizable cellulose particles with an average particle size of < 2 µm. Two types of assays were developed: (1) An assay with an extended standard range (0–6400 ng/mL), which can measure CRP levels up to 160,000 ng/mL, and (2) A sensitive assay (0–200 ng/mL), which can measure the CRP level in human serum as low as 3 ng/mL. The developed assay procedures were validated after studying standard assay parameters such as sensitivity, assay variations, linearity of dilution, etc. This assay is user-friendly, with only three pipetting steps and a convenient incubation time of 1 h at room temperature. The developed CRP-RIA method covers the entire physiological and clinical useful range for routine estimation of CRP in human serum. [ABSTRACT FROM AUTHOR]

Previously, radioimmunoassay (RIA) has been the only assay to measure insulin-like growth factor-1 (IGF-1) to diagnose hypersomatotropism (HS). Due to radiation concerns, availability, and the cost of IGF-1 RIA, validation of assays for automated analysers such as a chemiluminescent immunoassay (CLIA) is needed. The aim of this study was to validate a CLIA for measurement of feline IGF-1 (IMMULITE 2000® XPi, Siemens Medical Solutions Diagnostics, Malvern, PA, USA) compared to IGF1 RIA, establish reference interval (RI), and determine a cut-off value for diagnosis of HS in diabetic cats. Validation of assay performance included precision, linearity, and recovery studies. Right-sided RI was determined using surplus serum of 50 healthy adult cats. Surplus serum samples of diabetic cats with known IGF-1 concentration with (n = 32/68) and without HS (n = 36/68) were used for method comparison with RIA. The cut-off for diagnosis of HS was established using receiver operating characteristic (ROC) analysis. The intra-assay coefficient of variation (CV) was ≤4.7%, and the inter-assay CV was ≤5.6% for samples with low, medium, and high IGF-1 concentration. Linearity was excellent (R2 > 0.99). The correlation between CLIA and RIA was very high (rs = 0.97), with a mean negative bias for CLIA of 24.5%. The upper limit of RI was 670 ng/mL. ROC analysis showed an area under the curve of 0.94, with best cut-off for diagnosis of HS at 746 ng/mL (sensitivity, 84.4%; specificity, 97.2%). The performance of CLIA was good, and the RI and cut-off for HS diagnosis established in this study allow for CLIA to be used in routine work-up of diabetic cats.