An enzyme cascade fluorescence-based assay for the quantification of phenylalanine in serum.

The quantification of phenylalanine in clinical samples is essential for the diagnosis and treatment of neonatal phenylketonuria. In this report, an enzyme cascade strategy was proposed and a high-efficiency fluorescence assay was established for rapid and convenient phenylalanine quantification. The assay involves phenylalanine dehydrogenase for the quantitative metabolization of phenylalanine and the formation of NADH, as well as nitroreductase combining a nitroaromatic substrate for the fluorescent quantification of NADH and subsequently phenylalanine. The phenylalanine levels in clinical serum determined by this fluorescence assay are consistent with those from HPLC. This straightforward approach provides a versatile strategy for the development of cost-effective and convenient assays for mass screening and metabolite monitoring.

BRAFV600E mutation test on fine-needle aspiration specimens of thyroid nodules: Clinical correlations for 4600 patients.

BACKGROUND: The BRAFV600E mutation is valuable for the diagnosis, prognosis, and therapy of papillary thyroid cancer (PTC). However, studies related to this mutation have involved only a small number of patients. Therefore, we performed a large-scale analysis from a single institute to evaluate the accuracy of combined fine-needle aspiration (FNA) and BRAFV600E mutation tests for PTC diagnosis. METHODS: A total of 4600 patients with thyroid nodules who underwent both FNA cytology and BRAFV600E mutation analysis on FNA specimens were enrolled. The association between the BRAFV600E mutation and clinicopathological features was analyzed. A separate analysis was performed for the 311 patients who underwent repeated FNA for comparison of cytological evaluation and BRAFV600E mutation results. The diagnostic efficacy of the BRAFV600E mutation test and cytologic diagnoses was evaluated for 516 patients who underwent preoperative FNA tests in comparison with conclusive postoperative histopathologic results. RESULTS: The cytology results of all 4600 FNA samples were categorized according to The Bethesda System for Reporting Thyroid Cytology (TBSRTC) stages I-VI, which accounted for 11.76%, 60.02%, 6.46%, 3.61%, 6.71%, and 11.43% of the samples, respectively. The BRAFV600E mutation was detected in 762 (16.57%) FNA samples, with rates of 1.48%, 0.87%, 20.20%, 3.01%, 66.02%, and 87.81% for TBSRTC I-VI lesions, respectively. Among the 311 repeat FNA cases, 81.0% of the BRAFV600E -positive and 4.3% of the BRAFV600E -negative specimens with an initial indication of cytological non-malignancy were ultimately diagnosed as malignant by repeat FNA (p < 0.001). Among the 516 patients who underwent thyroidectomy, the sensitivity and specificity of the BRAFV600E mutation test alone for PTC diagnosis were 76.71% and 100.0%, respectively, which increased to 96.62% and 88.03%, respectively, when combining the BRAFV600E mutation test with cytology. BRAFV600E mutation was significantly associated with lymph node metastasis (p < 0.001), but not with age, gender, or tumor size. CONCLUSIONS: The BRAFV600E mutation test in FNA samples has potential to reduce false negatives in PTC diagnosis, and therefore plays an important role in the diagnosis of thyroid nodules, especially those with an indeterminate or nondiagnostic cytology, which should be considered for repeat FNA.

Flow Assisted Mutation Enrichment (FAME): A highly efficacious and efficient method to enrich Double Knockouts (DKO) after gene editing.

Gene editing has become an essential tool for interrogation of gene function in biomedical research and is also a promising approach for gene therapy. Despite recent progresses, the gene-editing procedure is still a tedious process involving manually isolating large number of single cell colonies to screen for desired mutations. For diploid eukaryotic cells, there is the additional challenge to inactivate both alleles for genes-of-interest, i.e., generating double knockouts (DKOs), for the desired phenotypes or therapeutic effects. In this report, we present a novel method based on Fluorescence Assisted Cell Sorting (FACS) to enrich for DKO cells, using a cell surface marker ß2-microglobulin (B2M) as a basis for negative selection. This method significantly increased percentage of DKOs in isolated cells after gene editing, and in the meantime, significantly improve the efficiency of workflow by automating colony isolation. It would greatly facilitate future biomedical research including potential gene/cell therapies.

Prevalence of thyroid dysfunction in older Chinese patients with type 2 diabetes-A multicenter cross-sectional observational study across China.

Type 2 diabetes [T2D] and thyroid dysfunction [TD] often co-occur, have overlapping pathologies, and their risk increases with age. Since 1995, universal salt iodization has been implemented in China to prevent disorders caused by iodine deficiency. However, after two decades of implementation of universal salt iodization, the prevalence of TD in elderly Chinese patients with T2D is not well described and may have been underestimated. We conducted a questionnaire-based survey across 24 endocrinology centers in China between December 2015 and July 2016. Demographic and clinical data from 1677 patients with T2D were obtained and analyzed to examine the prevalence of TD along with T2D in these patients. We assessed TD prevalence according to the four TD subtypes [subclinical hypothyroidism, clinical hypothyroidism, subclinical hyperthyroidism, and clinical hyperthyroidism], TD history, gender, and age. The diagnosis rates were calculated for TD and also for the TD subtype. The number of patients reaching treatment goals for T2D [hemoglobin A1c <7%] and TD [normal free thyroxine and thyroid-stimulating hormone [TSH]] and the incidences of complications and comorbidities were recorded. Among the enrolled patients with T2D [N = 1677], TD was diagnosed in 23.79% [399/1677] out of which 61% (245/399) were previously diagnosed and 38.59% (154/399) were newly diagnosed cases. Subclinical hypothyroidism, clinical hypothyroidism, subclinical hyperthyroidism, and clinical hyperthyroidism were reported in 4.89%, 9.3%, 1.13%, and 3.16% of the total population, respectively. Among patients previously diagnosed with TD, the incidence in women [166/795; 20.88%] was higher than in men [79/882; 8.96%]. The treatment goals for TD and T2D were attained in 39.6% [97/245] and 34.41% [577/1677] of the cases, respectively. Diabetic complications and comorbidities were reported in 99.7% of patients, with peripheral neuropathy being the most common [43.46%] followed by cataract [24.73%]. We had found that the incidences of dyslipidemia, elevated LDL levels, and osteoporosis were significantly higher in patients with TD than those without TD. TD is underdiagnosed in elderly Chinese patients with T2D.

Rare malignant insulinoma with multiple liver metastases derived from ectopic pancreas: 3-year follow-up and literature review.

Here, we report the diagnosis and treatment of a very rare case of malignant insulinoma derived from ectopic pancreas. A middle-aged woman presented with a 6-year history of recurrent hypoglycemia with multiple lesions in liver. Admission workup revealed elevated serum insulin and C-peptide, as well as multiple lesions in the liver (largest being 4.3 cm), enlarged lymph nodes around the pancreas, and a lesion (of 3.5 cm) at the proximal jejunum, as shown by contrast computed tomography (CT). Liver biopsy showed the lesions to be well-differentiated neuroendocrine tumors, grade G1. 68Gallium-exendin-4 positron emission tomography/CT confirmed the origin as the lesion located at the jejunum. The combination treatment of everolimus plus long-acting octreotide relieved symptoms and achieved a partial tumor response. Maintenance treatment of the somatostatin analog (ie, octreotide) alone was administered. Three years of follow-up, up to the writing of this report, showed good survival, with the patient remaining asymptomatic and euglycemic without disease progression. This case shows that 68Gallium-exendin-4 positron emission tomography/CT is useful for locating insulinoma, especially for insulinoma derived from ectopic pancreas, and that everolimus plus octreotide with maintenance somatostatin analog alone is an effective drug strategy for treating inoperable malignant insulinoma.

An ultrasensitive bioluminogenic probe of γ-Glutamyltranspeptidase in vivo and in human serum for tumor diagnosis.

Abnormal expression of γ-Glutamyltranspeptidase (GGT) in living organisms is closely implicated in the development of several human tumors. The GGT levels in tissue and serum have emerged as a potential criterion for tumor diagnosis. However, precise "light up" GGT activity in vivo is still challenging due to the signal interferes of background. Bioluminescence based on the firefly luciferase-catalyzed reaction for light production provides a feasible strategy for GGT detection in vivo. In this report, a bioluminogenic probe, Glu-Luc, was designed and synthesized by connecting D-luciferin with γ-glutamyl group. The cleavage of γ-glutamyl group is triggered by GGT, resulting in the release of D-luciferin, which generates a bright bioluminescence emission in the present of luciferase and ATP. The probe exhibits very high selectivity and sensitivity toward GGT activity from in vitro to in vivo and in clinical samples, which offers a promising tool for investigations of the GGT-overexpressing related biological process including tumor diagnosis and prognosis in living organisms.

[Distribution of IgG subclasses of TgAb and TPOAb in sera from patients with Graves' disease, Graves' disease plus Hashimoto's thyroiditis and Hashimoto's thyrotoxicosis].

OBJECTIVE: To evaluate the distribution of IgG subclasses of TgAb and TPOAb in sera from patients with Graves' disease (GD), Graves' disease plus Hashimoto's thyroiditis (GH) and Hashimoto's thyrotoxicosis. METHODS: Patients with GD (n = 33), GH (n = 31) or Hashimoto's thyrotoxicosis (n = 18) diagnosed by fine needle aspiration cytology at Department of Endocrinology of Peking University First Hospital, Beijing Haidian Hospital, China-Japan Friendship Hospital and Civil Aviation General Hospital during the period from January 2010 to May 2013 were enrolled. All of them had TgAb and TPOAb. The total serum IgG and IgG subclasses of TgAb and TPOAb were detected by antigen-specific enzyme-linked immunosorbent assay (ELISA). The prevalence and relative amount of IgG subclasses were calculated and compared among three groups. RESULTS: The levels of TRAb in GD group (21.80(7.53, 40) U/L) were significantly higher than those in GH (7.30(3.10, 25.40) U/L) (P = 0.000) and Hashimoto's thyrotoxicosis groups (4.90(1.69, 16.43) U/L) (P = 0.003). And no significant differences were found in the levels of TgAb and TPOAb. The prevalence of TgAb IgG3 subclass in Hashimoto's thyrotoxicosis group (66.7%) was higher than GD group (35.5%) and GH group (36.4%) and the difference was close to significance (P = 0.066). There were significant differences of relative amount of TgAb IgG2 and TgAb IgG4 among three groups (P = 0.039 and 0.013), and GD patients had higher relative amounts of TgAb IgG2 (0.59(0.34, 0.94)) and TgAb IgG4 (0.57(0.28, 0.97)) than GH patients (TgAb IgG2, 0.31(0.23, 0.34); TgAb IgG4, 0.26(0.09, 0.48)) or patients with Hashimoto's thyrotoxicosis (TgAb IgG2, 0.32(0.24, 0.83); TgAb IgG4, 0.33(0.10, 0.65)) (for TgAb IgG2, P = 0.009 and 0.167; for TgAb IgG4, P = 0.005 and 0.041 respectively). No significant difference was found in the prevalence of each TPOAb IgG subclass. The difference of relative amount of TPOAb IgG2 among three groups was close to significance (P = 0.069). And the relative amount was higher in sera from GD patients (0.39 ± 0.04) than that in GH patients (0.29 ± 0.13) or patients with Hashimoto's thyrotoxicosis (0.26 ± 0.02) (P = 0.104 and 0.002 respectively). CONCLUSION: The patients with high levels of TgAb IgG2, TgAb IgG4 and TPOAb IgG2 subclasses have a greater risk of GD. The IgG subclass distribution of TgAb and TPOAb might help to differentiate the causes of thyrotoxicosis in autoimmune thyroid diseases.

A case of Klinefelter's syndrome with type 1 diabetes mellitus.

Klinefelter's syndrome (KS) is the most common sex chromosome disease in men. Classical features of the syndrome include a eunuchoidal body habitus, small testes and hypergonadotrophic hypogonadism. There has been an increased risk of diabetes mellitus and autoimmune disease for KS patients. This paper reports a case of KS in association with type 1 diabetes mellitus. The patient was a 21-year-old man, who has been confirmed by absolute insulin deficiency and positive IA-2 autoantibody. The hyperinsulinemic euglycemic clamp test indicated his insulin sensitivity in normal range, and his blood glucose was controlled well by the insulin therapy.