[Clinical analysis of retropharyngeal lymph node metastasis in patients with hypopharyngeal squamous cell carcinoma].

Objective: To analyze the incidence and the related risk factors of retropharyngeal lymph node metastasis in patients with hypopharyngeal squamous cell carcinoma, evaluate the accuracy of preoperative enhanced CT in judging retropharyngeal lymph node metastasis, and investigate the impact of retropharyngeal lymph node metastasis on the prognosis. Methods: Retrospective analyses were made on 398 patients with hypopharyngeal squamous cell carcinoma who underwent surgery as the primary therapy and accepted retropharyngeal lymph node exploration and clearance during surgery in Shandong Provincial ENT Hospital from January 2014 to December 2019. Multivariate logistic regression analysis was used to clarify the related risk factors of retropharyngeal lymph node metastasis. Multivariate Cox regression analysis was used to investigate the impact of retropharyngeal lymph node metastasis on prognosis. The retropharyngeal lymph nodes of 218 cases with available preoperative enhanced CT images were evaluated by two experienced radiologists and compared with postoperative pathological results. Results: Retropharyngeal lymph node metastasis were confirmed in 54 of 398 (13.6%) cases according to postoperative pathology. The sensitivity and specificity of preoperative enhanced CT in the diagnosis of retropharyngeal lymph node metastasis were 34.6% and 91.1%, respectively, and the overall accuracy was 84.4%. Multivariate logistic regression analysis showed that the site of the primary lesion and pathological N stage were independent risk factors for retropharyngeal lymph node metastasis in hypopharyngeal squamous cell carcinoma. Patients with primary lesion located in the posterior wall of hypopharynx (OR=4.83, 95% CI: 1.27-18.40), N2 stage (OR=6.30, 95% CI: 2.25-17.67), and N3 stage (OR=26.89, 95% CI: 5.76-125.58) were prone to retropharyngeal lymph node metastasis. The 5-year overall survival rate of the 398 patients was 50.4%, and the 5-year disease-free survival rate was 48.3%. Multivariate Cox regression analysis showed that T stage, N stage, retropharyngeal lymph node metastasis, and radiotherapy were independent influencing factors for overall survival (T stage: HR=1.28, 95% CI: 1.06-1.54; N stage: HR=1.26, 95% CI: 1.14-1.40; retropharyngeal lymph node metastasis: HR=2.13, 95% CI: 1.47-3.08; radiotherapy: HR=0.54, 95% CI: 0.38-0.76) and disease-free survival of patients with hypopharyngeal squamous cell carcinoma (T stage: HR=1.26, 95% CI: 1.06-1.51; N stage: HR=1.25, 95% CI: 1.13-1.37; retropharyngeal lymph node metastasis: HR=2.24, 95% CI: 1.56-3.21; radiotherapy: HR=0.55, 95% CI: 0.40-0.77). Conclusions: Metastasis of retropharyngeal lymph nodes in hypopharyngeal squamous cell carcinoma is not rare. Enhanced CT is of low accuracy and limited value in diagnosing retropharyngeal lymph node metastasis. Primary lesions located in the posterior wall of the hypopharyngx, N2 stage, and N3 stage are independent high-risk factors for retropharyngeal lymph node metastasis. The prognosis of hypopharyngeal cancer patients with retropharyngeal lymph node metastasis is worse, and active surgical exploration and clearance can effectively reduce the mortality caused by retropharyngeal lymph node metastasis.

[Efficacy of surgical comprehensive therapy for 456 cases of hypopharyngeal carcinoma].

Objective: To retrospectively analyse the efficacy of surgerical comprehensive treatment for hypopharyngeal cancer. Methods: Four hundred and fifty-six cases of hypopharyngeal squamous cell carcinoma treated from Jan 2014 to Dec 2019 were analyzed retrospectively, including 432 males and 24 females, aged 37-82 years old. There were 328 cases of pyriform sinus carcinoma, 88 cases of posterior pharyngeal wall carcinoma, and 40 cases of postcricoid carcinoma. According to American Joint Committe on Cancer(AJCC) 2018 criteria, 420 cases were of stage Ⅲ or Ⅳ; 325 cases were of T3 or T4 stage. Treatment methods included surgery alone in 84 cases, preoperative planned radiotherapy plus surgery in 49 cases, surgery plus adjuvant radiotherapy or concurrent chemoradiotherapy in 314 cases, and inductive chemotherapy plus surgery and adjuvant radiotherapy in 9 cases. The primary tumor resection methods included transoral laser surgery in 5 cases, partial laryngopharyngectomy in 74 cases, of them 48 cases (64.9%) presented with supracricoid hemilaryngopharyngectomy, total laryngectomy with patial pharyngectomy in 90 cases, total laryngopharyngectomy or with cervical esophagectomy in 226 cases, and total laryngopharyngectomy with total esophagectomy in 61 cases. Among 456 cases, 226 cases received reconstruction surgery with free jejunum transplantation, 61 cases with gastric pull-up, and 32 cases with pectoralis myocutaneous flaps. All patients underwent retropharyngeal lymph node dissection, and high-definition gastroscopy was performed during admission and follow-up. SPSS 24.0 software was used to analyze the data. Results: The 3-year and 5-year overall survival rates were respectively 59.8%, and 49.5%. The 3-year and 5-year disease specific survival rates were respectively 69.0% and 58.8%. Total metastasis rate of retropharyngeal lymph nodes was 12.7%. A total of 132 patients (28.9%) suffered from simultaneous and metachronous multiple primary carcinoma of the hypopharynx. Multivariate Logistic regression analysis showed that T3-4 disease, cervical lymph node metastasis, retropharyngeal lymph node metastasis and postoperative adjuvant radiotherapy were independent factors affecting the prognosis of patients (all P<0.05). As of April 30, 2022, a total of 221 patients died during follow-up, of 109 (49.3%) with distant metastases, which were the main cause of death. Conclusions: The efficacy of comprehensive treatment for hypopharyngeal cancer can be improved by accurate preoperative evaluation, improved surgical resection, active retropharyngeal lymph node dissection and full process intervention of the second primary cancer.

Filtration efficiency of N95 filtering facepiece respirators during multi-cycles of '8-hour simulated donning + disinfection'.

BACKGROUND: Aerosol-borne diseases such as COVID-19 may outbreak occasionally in various regions of the world, inevitably resulting in short-term shortage and corresponding reuse of disposable respirators. AIM: To investigate the effective disinfection methods, reusable duration and frequency of N95 respirators. METHODS: Based on the self-built respirator simulation test system, and under combinations of experimental conditions of three N95 respirators × 0-200 nm NaCl aerosols × three simulated breathing flow rates (15, 50 and 85 L/min) × two disinfection methods (dry heating and ultraviolet (UV) radiation), this study continuously measured the changes in filtration efficiency of all respirators during multi-cycles of '8-h simulated donning + disinfection' until the penetration reached ≥5%. FINDINGS: Multi-cycles of dry heating and UV radiation treatments on the reused (i.e., multiple 8-h donning) N95 respirators had a minimal effect (<0.5%) on the respirator filtration efficiency, and even at 85 L/min, all tested N95 respirators were able to maintain filtration efficiencies ≥95% for at least 30 h or four reuse cycles of '8-h donning + disinfection', while a lower breathing flow rate (15 L/min) plus the exhalation valve could further extend the N95 respirator's usability duration up to 140 h or 18 reuse cycles of '8-h donning + disinfection'. As the respirator wearing time extended, aerosol penetration slowly increased in a quadratic function with a negative second-order coefficient, and the penetration increment during each cycle of 8-h donning was less than 0.9%. CONCLUSION: Multi-cycles of N95 respirator reuse in combination with dry heating or UV irradiation disinfection are feasible.

The role of metal ions in the behavior of bovine serum albumin molecules under physiological environment.

Metal ions released from metallic implants can affect the conformation and structural stability of proteins in biological fluids, which eventually affects the biocompatibility of implants. The present study aimed at understanding the interactions between the metal ions (Mn2+, Fe2+, Fe3+, Co2+, Cu2+, and Zn2+) and bovine serum albumin (BSA) molecules in physiological context. The structural information of BSA molecules and the microenvironment of functional groups were investigated using UV, Raman, and circular dichroism spectroscopy. The results revealed that addition of Fe3+, Fe2+, and Cu2+ ions alters the tertiary structure of BSA molecules and exposes the aromatic heterocyclic hydrophobic group of BSA amino acid residues. The addition of Fe3+ and Cu2+ ions results in increased viscosity and decreased intensity of the water peak in the BSA solution. Furthermore, Fe3+ and Cu2+ ions evidently promote the α-helix to ß-sheet transformation of BSA molecules due to decreased disulfide bond stability. Tryptophan residues of BSA and metal ions containing BSA (Me+/BSA) solutions were found to be in a hydrophilic environment. Moreover, the addition of metal ions to BSA results in several of tyrosine residues acting as hydrogen-bond donors. Coomassie brilliant blue staining revealed that the addition of Cu2+ ions promotes the aggregation of BSA molecules. The findings of this study will be helpful for evaluating the biocompatibility of metallic implants.

Tailored Quinolines Demonstrate Flexibility to Exert Antitumor Effects through Varied Mechanisms-A Medicinal Perspective.

BACKGROUND: Quinoline is considered to be a privileged heterocyclic ring owing to its presence in diverse scaffolds endowed with promising activity profiles. In particular, quinoline containing compounds have exhibited substantial antiproliferative effects through the diverse mechanism of actions, which indicates that the heteroaryl unit is flexible as well as accessible to subtle structural changes that enable its inclusion in chemically distinct anti-tumor constructs. METHODS: Herein, we describe a medicinal chemistry perspective on quinolines as anticancer agents by digging into the peer-reviewed literature as well as patents published in the past few years. RESULTS: This review will serve as a guiding tool for medicinal chemists and chemical biologists to gain insights about the benefits of quinoline ring installation to tune the chemical architectures for inducing potent anticancer effects. CONCLUSION: Quinoline ring containing anticancer agents presents enough optimism and promise in the field of drug discovery to motivate the researchers towards the continued explorations on such scaffolds. It is highly likely that adequate efforts in this direction might yield some potential cancer therapeutics in the future.

Facilitation of ultrasonic microvesicles on homing and molecular mechanism of bone marrow mesenchymal stem cells in cerebral infarction patients.

OBJECTIVE: Cerebral infarction, or ischemia brain stroke, is a common cerebrovascular disease. Bone marrow mesenchymal stem cells (BMSCs) are widely used to treating ischemia disease such as cardiac infarction. Ultrasonic microvesicles may help the targeting of exogenous factors via localized energy blast. Therefore, this study aims to investigate the effect of ultrasonic microvesicles on the homing of BMSCs on artery thrombosis and the associated molecular mechanisms. MATERIALS AND METHODS: Rat BMSCs were isolated and cultured. Rats were divided into sham, model, BMSCs, and microvesicles groups. Cerebral infarction model was prepared by ligation of cervical artery and middle cerebral artery. 3×106/kg BMSCs were transplanted via tail veins. Microvesicles were used for assisting BMSCs infusion. Sex-determining region Y (SRY) gene expression was measured by Real-time PCR, while 2,3,5-triphenyltetrazolium chloride (TTC) staining was employed for describing the area of cerebral infarction. The activity of caspase 3 was assayed by test kit. Vascular endothelial growth factor (VEGF), nuclear factor kappa B (NF-κB) mRNA/protein levels, were quantified by Real-time PCR, and Western blotting, respectively. RESULTS: Microvesicle group had significantly elevated SRY expression (p<0.05 compared to BMSCs group). Transplantation of BMSCs remarkably decreased cerebral infarction area, caspase 3 activity or NF-κB expression, and increased VEGF expression (p<0.05 compared to model group). Microvesicle induced BMSCs had more potent effects (p<0.01). CONCLUSIONS: Ultrasound microvesicle facilitated homing of BMSCs in cerebral infarction, and improved infarction disease via up-regulating VEGF expression, inhibiting NF-κB expression, and modulating apoptosis.

Frequency of antiseptic resistance genes and reduced susceptibility to biocides in carbapenem-resistant Acinetobacter baumannii.

The aim of this study is to determine the frequency of quaternary ammonium compound-resistant genes, including qacA/B, qacC/D, qacE, qacΔE1, qacG, qacJ, and ß-lactamase genes, including blaCTX-M, blaTEM, blaSHV, blaOXA-23, blaVIM, blaIMP, blaNDM and blaGIM in 51 isolates of carbapenem-resistant Acinetobacter baumannii collected over a period of 2 years and to determine the MIC of chlorhexidine and quaternary ammonium compound-based disinfectant benzalkonium chloride. The qac and bla genes were detected by the PCR method. The MICs were determined using the broth microdilution method. The MIC of benzalkonium chloride was in the range of 4 to 64 µg l-1 and chlorhexidine in the range of 4 to 64 µg l-1. The qacΔE1 gene was detected in 96.07 % (49/51) isolates and qacE in 31.37 % (16/51), qacG in 23.52 % (12/51) and qacA/B in 13.72 % (7/51). The qacC/D genes and qacJ were not found in any of these strains in this study. The frequency of bla genes was as follows: 84.31 % (43/51) for blaOXA-23, 33.33 % (17/51) for blaTEM, 27.45 % (14/51) for blaVIM, 19.61 % (10/51) for blaSHV, 17.65 % (9/51) for blaNDM, 11.76 % (6/51) for blaGIM and 7.8 % (4/51) for the blaIMP. The blaCTX-M genes were negative in all the strains. From the study, we concluded that reduced susceptibility to the two disinfectants, as well as qac and bla genes, was prevalent among A. baumannii isolates from the hospital environment. This study may offer hospitals important information about the control of nosocomial infections caused by A. baumannii.

Leucine alleviates dexamethasone-induced suppression of muscle protein synthesis via synergy involvement of mTOR and AMPK pathways.

Glucocorticoids (GCs) are negative muscle protein regulators that contribute to the whole-body catabolic state during stress. Mammalian target of rapamycin (mTOR)-signalling pathway, which acts as a central regulator of protein metabolism, can be activated by branched-chain amino acids (BCAA). In the present study, the effect of leucine on the suppression of protein synthesis induced by GCs and the pathway involved were investigated. In vitro experiments were conducted using cultured C2C12 myoblasts to study the effect of GCs on protein synthesis, and the involvement of mTOR pathway was investigated as well. After exposure to dexamethasone (DEX, 100 µmol/l) for 24 h, protein synthesis in muscle cells was significantly suppressed (P<0.05), the phosphorylations of mTOR, ribosomal protein S6 protein kinase 1 (p70s6k1) and eukaryotic initiation factor 4E binding protein 1 (4EBP1) were significantly reduced (P<0.05). Leucine supplementation (5 mmol/l, 10 mmol/l and 15 mmol/l) for 1 h alleviated the suppression of protein synthesis induced by DEX (P<0.05) and was accompanied with the increased phosphorylation of mTOR and decreased phosphorylation of AMPK (P<0.05). Branched-chain amino transferase 2 (BCAT2) mRNA level was not influenced by DEX (P>0.05) but was increased by leucine supplementation at a dose of 5 mmol/l (P<0.05).

[Protective effect of Angelica sinensis polysaccharide against liver injury induced by D-galactose in aging mice and its mechanisms].

OBJECTIVE: To investigate the protective effect of Angelica sinensis polysaccharide (ASP) against liver injury induced by D-galactose in aging mice and its mechanisms. METHODS: Male C57BL/6J were randomly divided into three groups with 10 mice in each group. In the D-galactose model group, the mice were subcutaneously injected with D-galactose (120 mg/kg) qd×42; in the ASP+D-galactose group, from the 8th day of the establishment of D-galactose model, the mice were subcutaneously injected with ASP (120 mg/kg) qd×35. In the normal control group, the mice were subcutaneously injected with isotonic saline of the same volume at the same time point. On the 2nd day after the injection was finished, the ocular blood was collected to prepare serum and measure the content of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin (TBil). The liver tissue homogenate was prepared to measure the content of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), malondialdehyde (MDA), and advanced glycation end products (AGEs). A paraffin section of liver tissue was prepared; HE staining was performed to observe the pathomorphological changes of the liver, periodic acid-Schiff staining (PAS) was used to observe the changes in glycogen in the liver, and a transmission electron microscope was used to observe the hepatocyte ultrastructure. RESULTS: The D-galactose model group had increased content of ALT, AST, and TBil, reduced activities of SOD and GSH-Px, an increased content of MDA, and severe liver injuries; the hepatocytes showed degenerative changes, the amount of glycogen in the liver decreased, and the accumulation of AGEs increased. The ASP+D-galactose group had reduced content of ALT, AST, and TBil, increased activities of SOD and GSH-Px, and reduced content of MDA and AGEs; the amount of glycogen in the liver increased, and liver injury and hepatocyte injury were alleviated. CONCLUSION: ASP can antagonize the liver injury induced by D-galactose in aging mice, and its mechanism may be related to the inhibition of oxidative stress.

Left atrial function as assessed by speckle-tracking echocardiography in hypertension.

We investigated left atrial (LA) function in relation to hypertension using 2-dimensional speckle-tracking echocardiography (STE) in subjects with preserved left ventricular (LV) ejection fraction, while accounting for LA enlargement and LV mass and diastolic function.We performed standard 2-dimensional and Doppler echocardiography and LA volumetric measurements and STE strain imaging in hypertensive patients (systolic/diastolic blood pressure ≥140/90 mmHg, or use of antihypertensive drugs, n = 124) and age- and sex-matched normotensive subjects (n = 124). We measured the peak LA velocity, strain, and strain rate during systole and early and late diastole, respectively. We investigated the associations of interests in the presence or absence of LA enlargement (LA volume index ≥28 mL/m).Hypertensive and normotensive subjects had similar LV ejection fraction and LA diameter (P ≥ 0.22). However, hypertensive compared with normotensive subjects had enlarged LV and impaired diastolic function, and had increased LA volumetric measurements and decreased LA emptying fractions (P < 0.0001). Hypertensive patients also had impaired LA function, as measured by STE velocity, strain, and strain rate in general and in the absence of LA enlargement (P < 0.0001). The differences in LA STE strain rate during LV systole and LA contraction between hypertension and normotension in the absence of LA enlargement remained statistically significant (P < 0.001), after adjustment for age, sex, and LV mass index and E/E'.Hypertension is associated with impaired LA function, as assessed by STE strain imaging technique, even before LA enlargement develops and after LV remodeling is accounted for.

MiR-873 regulates ERα transcriptional activity and tamoxifen resistance via targeting CDK3 in breast cancer cells.

miRNAs (microRNAs) are frequently and aberrantly expressed in many cancers. MiR-873 has been revealed to be downregulated in colorectal cancer and glioblastoma. However, its function remains unclear. Here we report that miR-873 is downregulated in breast tumor compared with normal tissue. Enforced expression of miR-873 decreases the transcriptional activity of ER (estrogen receptor)-α but not ERß through the modulation of ERα phosphorylation in ER-positive breast cancer cells. We also found that miR-873 inhibits breast cancer cell proliferation and tumor growth in nude mice. Reporter gene assays revealed cyclin-dependent kinase 3 (CDK3) as a direct target of miR-873. CDK3 was shown to be overexpressed in breast cancer and phosphorylate ERα at Ser104/116 and Ser118. Furthermore, we found that Mir-873 inhibits ER activity and cell growth via targeting CDK3. Interestingly, miR-873 was observed to be downregulated in tamoxifen-resistant MCF-7/TamR cells, while CDK3 is overexpressed in these cells. More importantly, re-expression of miR-873 reversed tamoxifen resistance in MCF-7/TamR cells. Our data demonstrate that miR-873 is a novel tumor suppressor in ER-positive breast cancer and a potential therapeutic approach for treatment of tamoxifen-resistant breast cancer.

Microsatellite instability in saliva from patients with hereditary non-polyposis colon cancer and siblings carrying germline mismatch repair gene mutations.

Microsatellites are short tandem repeats of deoxyribonucleic acid (DNA) sequences which are distributed throughout the genome. Tumors in patients with Lynch syndrome tend to accumulate mutations in microsatellites at a much higher rate than other sequences in the genome resulting in microsatellite instability (MSI). This is due to germline mutations in mismatch repair (MMR) genes. Using small pool-polymerase chain reaction (SP-PCR), previous studies have shown that mutant alleles can be detected in microsatellites of DNA from peripheral blood lymphocytes (PBLs) of Lynch syndrome patients at frequencies that were low, but significantly higher than frequencies in PBLs of age-matched non-Lynch syndrome controls. In the present study, SP-PCR detection of frequency of mutant MSI alleles (FMMA) was performed on PBLs and saliva samples from four sets of families. Each family set consisted of a mutation carrying affected proband (initial tumor bearer), a germline mutation-carrier sibling without tumors, and an age-matched normal control, either related (for 3 family sets) without mutation carrier status or unrelated (for 1 family set) without mutation carrier status. FMMAs of saliva and PBL DNA were compared between each proband, sibling and control for each family set, and between family sets. In all five statistically significant saliva comparisons identified between germline mutation carriers (FMMA: 0.080-0.261) and normal controls (FMMA: 0.003-0.087), the measured FMMAs were always higher in the carriers (p < 0.05). A logistic regression model of the data showed a significant increase in FMMAs in saliva DNA from siblings with MMR mutation compared to the normal controls (p < 0.001). These results indicated that the increased FMMAs observed in the saliva DNA as well as PBL DNA of MMR gene mutation carriers compared to normal controls are real and repeatable. Furthermore, the logistic regression also indicated that the FMMAs seen in saliva were nearly double those seen in PBLs (p < 0.001). Saliva testing, a less-invasive procedure than PBL testing, is more sensitive and appears to be a viable alternative for identifying MSI in carriers with MMR mutations.

ZBP-89 enhances Bak expression and causes apoptosis in hepatocellular carcinoma cells.

ZBP-89 can enhance tumor cells to death stimuli. However, the molecular mechanism leading to the inhibitory effect of ZBP-89 is unknown. In this study, 4 liver cell lines were used to screen for the target of ZBP-89 on cell death pathway. The identified Bak was further analyzed for its role in ZBP-89-mediated apoptosis. The result showed that ZBP-89 significantly and time-dependently induced apoptosis. It significantly upregulated the level of pro-apoptotic Bak. ZBP-89 targeted a region between -457 and -407 of human Bak promoter to stimulate Bak expression based on the findings of Bak promoter luciferase report gene assay and electrophoretic mobility shift assay. ZBP-89-induced Bak increase and ZBP-89-mediated apoptosis were markedly suppressed by Bak siRNA, confirming that Bak was specifically targeted by ZBP-89 to facilitate apoptosis. In conclusion, this study demonstrated that ZBP-89 significantly induced apoptosis of HCC cells via promoting Bak level.

Effects of extraction conditions on improving the yield and quality of an anthocyanin-rich purple corn (Zea mays L.) color extract.

Purple corn (Zea mays L.) is a rich and economic source of anthocyanin colorants and functional ingredients. However, high levels of anthocyanin-rich waste are generated during processing, reducing the yields and increasing the costs of the final product. This waste has been associated with anthocyanin complexation with tannins and proteins. Our objective was to evaluate anthocyanin extraction methods to reduce purple corn waste. Different solvents (water, 0.01%-HCl-acidified water, and 0.01%-HCl-acidified ethanol), temperatures (room temperature, 50, 75, and 100 degrees C), and times of exposure to the solvents were investigated. Acetone (70% acetone in water) extraction was used as control. Anthocyanins, total phenolics, tannins, and proteins in extracts were measured by the pH differential, Folin-Ciocalteu, protein precipitation, and BCA assay methods. Qualitative analyses were done by HPLC coupled to a PDA detector and SDS-PAGE analysis. Water at 50 degrees C achieved the highest yield of anthocyanins (0.94 +/- 0.03 g per 100 g dry corncob) with relatively low tannins and proteins, comparable to the anthocyanin yield obtained by 70% acetone (0.98 +/- 0.08 g per 100 g dry corncob). Extending the extraction time from 20 to 60 min and using consecutive reextraction procedures reduced anthocyanin purity, increasing the yields of other phenolics. A neutral protease was applied to the extracts and effectively decomposed the major protein that was believed to contribute to the development of anthocyanin complexation and waste generation. Extraction time, consecutive reextraction procedures, and enzyme hydrolysis should be considered for high yield of anthocyanins and waste reduction.

Islet autoimmunity and genetic mutations in Chinese subjects initially thought to have Type 1B diabetes.

AIMS: To explore the contribution of islet autoimmunity and genetic mutations in Chinese patients initially thought to have Type 1B diabetes. METHODS: A group of 33 Chinese patients with newly diagnosed Type 1B diabetes, were identified by the absence of autoantibodies to glutamic acid decarboxylase (GAD), IA-2, insulin, thyroid globulin or thyroid peroxidase, or high-risk HLA-DQ haplotypes. The cohort was further characterized by measurement of autoantibodies to carboxypeptidase H (CPH) and SOX13 using radioligand assays, and testing for genetic mutations associated with MODY3/MODY6 and mitochondrial diabetes. Mutations of HNF-1alpha (MODY3) and neuroD1/beta2 (MODY6) genes were screened using the single-strand conformation polymorphism (SSCP) technique and sequencing. Mitochondrial DNA mutations were analysed with polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). RESULTS: Within the cohort, we found one patient with a novel mutation, R321H (CGC-->CAC) in exon 5 of the HNF-1alpha gene, one with ND1 mt3316 G-->A mutation in mitochondrial DNA, five with Ala45Thr polymorphisms in the neuroD1/beta2 gene, and two patients with autoantibodies to SOX13. CONCLUSIONS: Some of the Chinese patients originally thought to have Type 1B diabetes do have other evidence of islet autoimmunity and genetic mutations involved in the underlying aetiology. This suggests that more rigorous screening for these conditions is needed before classifying subjects as having Type 1B diabetes.