First Reports of Cladosporium tenuissimum Causing Leaf spots on Hydrangea macrophylla in Anhui province in China.

Forever Summer Hydrangea (Hydrangea macrophylla) is a common flowering plant in the Yangtze River Valley area of China, and it is widely cultivated globally (Chen et al. 2015). In July 2023, H. macrophylla leaves exhibiting visible diseased lesions were reported in a nursery in Wuhu, Anhui Province, China. The incidence reached 40% in a 0.2 ha area. The primary disease symptom was multiple irregular necrotic spots (0.5 to 1 mm in diameter) appearing on the leaves. These spots on the leaves were faded yellow around the perimeter and grayish brown in the center.). 15 leaf samples were sterilized with 75% alcohol and rinsed three times in sterile distilled water, then transferred to antibiotic-added potato dextrose agar (PDA) for incubation at 27°C. The colonies were fluffy, flocculent, or hairy, dark green, gray-green to gray-brown in color, and spreading or protruding punctate with a colorless halo on PDA. The conidiophores were brown to dark brown, smooth or rough surface, mostly unbranched, clearly differentiated, erect or curved. The conidia displayed a light brown to brown hue, lemon shape, fusiform, elongated ellipsoid or others with obvious spore markings and spore umbilicus. Genomic DNA was extracted from fungal colonies on infected leaves of three collections separately (Braun et al. 2003) and the internal transcribed spacer regions (ITS), actin (ACT) genes and partial translation elongation factor-l-alpha (EF) were amplified and sequenced using the primers ITS1/4 (Yin et al. 2012), ACT-512F/ACT-783R and EF 1-728F/986R (Carbone and Kohn 1999), respectively. DNA sequences of isolates were identical and deposited in GenBank (accession no. OR362754 for ITS, OR611929 for ACT and PP209106 for EF). The consensus sequences from ITS, EF and ACT showed 100%, 98.98% and 100% identical to Cladosporium strains (accession no. OQ186140.1, MT154169.1 and OL322092.1), respectively. To confirm the pathogenicity of the isolates, hydrangeas were planted in 15-cm pots containing commercial potting mix (one plant/pot). Three healthy plants were inoculated at the five to eight leaf stage by spraying 50 µL of the isolate conidial suspension (4 × 106 spores/mL) on healthy leaves. Three plants treated with sterile distilled water were used as controls. After inoculation, all plants were placed in a humidity chamber (>95% relative humidity, 26°C) for 48 h and then transferred to a greenhouse at 22/27°C. All inoculated leaves exhibited symptoms similar to those observed in the nursery 10 days after inoculation, while no symptoms were observed for control leaves. The fungus was re-isolated and confirmed to be C. tenuissimum. Based on the above morphological characterization and molecular identification, the causal agent for this leaf spot disease was identified as C. tenuissimum. Although C. tenuissimum has been reported to cause disease on H. paniculata in northern China (Li et al.2021), this is the first time that C. tenuissimum has been found on H. macrophylla in southern China. This new disease of H. macrophylla caused by C. tenuissimum is a threat to urban greening and is worth further investigation.

Joint effects of prenatal exposure to air pollution and pregnancy-related anxiety on birth weight: A prospective birth cohort study in Ma'anshan, China.

BACKGROUND: A growing number of studies have shown that prenatal exposure to chemical and non-chemical stressors has effects on fetal growth. The co-exposure of both better reflects real-life exposure patterns. However, no studies have included air pollutants and pregnancy-related anxiety (PrA) as mixtures in the analysis. METHOD: Using the birth cohort study method, 576 mother-child pairs were included in the Ma'anshan Maternal and Child Health Hospital. Evaluate the exposure levels of six air pollutants during pregnancy using inverse distance weighting (IDW) based on the pregnant woman's residential address and air pollution data from monitoring stations. Prenatal anxiety levels were assessed using the PrA Questionnaire. Generalized linear regression (GLR), quantile g-computation (QgC) and bayesian kernel machine regression (BKMR) were used to assess the independent or combined effects of air pollutants and PrA on birth weight for gestational age z-score (BWz). RESULT: The results of GLR indicate that the correlation between the six air pollutants and PrA with BWz varies depending on the different stages of pregnancy and pollutants. The QgC shows that during trimester 1, when air pollutants and PrA are considered as a whole exposure, an increase of one quartile is significantly negatively correlated with BWz. The BKMR similarly indicates that during trimester 1, the combined exposure of air pollutants and PrA is moderately correlated with a decrease in BWz. CONCLUSION: Using the method of analyzing mixed exposures, we found that during pregnancy, the combined exposure of air pollutants and PrA, particularly during trimester 1, is associated with BWz decrease. This supports the view that prenatal exposure to chemical and non-chemical stressors has an impact on fetal growth.

Association of urinary heavy metal combined exposure with periodontitis among US adults from NHANES 2011-2014.

Some heavy metals are associated with periodontitis; whereas most of these associations have focused on individual metal, there are no specific studies on the effects of combined heavy metal exposure on periodontitis. We conducted an analysis on the association between urinary heavy metal exposure and periodontitis in participants aged 30 years and older using multiple logistic regression and Bayesian kernel machine regression (BKMR). This analysis was performed on data from the National Health and Nutrition Examination Survey from 2011 to 2014. The study found that using logistic regression, the 4th quartile of urinary lead and molybdenum and the 3rd quartile of urinary strontium were positively associated with periodontitis compared to the reference quartile after adjusting for covariates. Odds ratio (OR) with 95% confidence interval (CI) was 1.738 (1.069-2.826), 1.515 (1.025-2.239), and 1.498 (1.010-2.222), respectively. The 3rd and 4th quartiles of urinary cobalt were negatively associated with periodontitis, and their ORs and 95% CIs were 0.639 (0.438-0.934) and 0.571 (0.377-0.964), respectively. The BKMR model showed that urinary barium, lead, and molybdenum were positively associated with periodontitis in a range of concentrations and urinary cobalt, manganese, tin, and strontium were negatively correlated with periodontitis. Furthermore, the overall association between urinary heavy metals and periodontitis was positive. Our study provides evidence for an association between exposure to multiple urinary heavy metals and periodontitis. However, further longitudinal studies are needed to explore the specific mechanisms involved.

Elevated HMGB1 and sRAGE levels in cerebrospinal fluid of aneurysmal subarachnoid hemorrhage patients.

BACKGROUND: Neuroinflammation after aneurysmal subarachnoid hemorrhage (aSAH) leads to poor outcome of patients. High mobility group box 1 (HMGB1) contributes to inflammation through binding to receptors for advanced glycation end-products (RAGE) in various diseases. We aimed to determine the production of these two factors after aSAH and their relationship with clinical features. METHODS: HMGB1 and soluble RAGE (sRAGE) levels in cerebrospinal fluid (CSF) of aSAH patients and controls were measured, and their temporal courses were observed. The correlation between early concentrations (days 1-3) and clinical symptoms assessed by disease severity scores, neuroinflammation estimated by CSF IL-6 levels, as well as prognosis evidenced by delayed cerebral ischemia (DCI) and 6-month adverse outcome was investigated. Finally, combined analysis of early levels for predicting prognosis was confirmed. RESULTS: CSF HMGB1 and sRAGE levels were higher in aSAH patients than in controls (P < 0.05), and the levels decreased from higher early to lower over time. Their early concentrations were positively associated with disease severity scores, IL-6 levels, DCI and 6-month poor outcome (P < 0.05). HMGB1 ≥ 6045.5 pg/ml (OR = 14.291, P = 0.046) and sRAGE ≥ 572.0 pg/ml (OR = 13.988, P = 0.043) emerged as independent predictors for DCI, while HMGB1 ≥ 5163.2 pg/ml (OR = 7.483, P = 0.043) and sRAGE ≥ 537.3 pg/ml (OR = 12.653, P = 0.042) were predictors for 6-month poor outcome. Combined analysis of them improved predictive values of adverse prognosis. CONCLUSION: CSF HMGB1 and sRAGE levels of aSAH patients were increased early and then varied dynamically, which might act as potential biomarkers for poor outcome, especially when co-analyzed.

Aerobic Glycolysis Induced by mTOR/HIF-1α Promotes Early Brain Injury After Subarachnoid Hemorrhage via Activating M1 Microglia.

M1 microglial activation is crucial for the pathogenesis of early brain injury (EBI) following subarachnoid hemorrhage (SAH), and there is growing evidence that glucose metabolism is frequently involved in microglial activation. However, the molecular mechanism of glycolysis and its role in M1 microglial activation in the context of EBI are not yet fully understood. In this study, firstly, the relationship between aerobic glycolysis and M1 microglial activation as well as SAH-induced EBI was researched in vivo. Then, intervention on mammalian target of rapamycin (mTOR) was performed to investigate the effects on glycolysis-dependent M1 microglial activation and EBI and its relationship with hypoxia-inducible factor-1α (HIF-1α) in vivo. Next, Hif-1α was inhibited to analyze its role in aerobic glycolysis, M1 microglial activation, and EBI in vivo. Lastly, both in vivo and in vitro, mTOR inhibition and Hif-1α enhancement were administered simultaneously, and the combined effects were further confirmed again. The results showed that aerobic glycolysis and M1 microglial polarization were increased after SAH, and glycolytic inhibition could attenuate M1 microglial activation and EBI. Inhibition of mTOR reduced glycolysis-dependent M1 microglial polarization and EBI severity by down-regulating HIF-1α expression, while enhancement had the opposite effects. Blockading HIF-1α had the similar effects as suppressing mTOR, while HIF-1α agonist worked against mTOR antagonist when administered simultaneously. In conclusion, the present study showed new evidence that aerobic glycolysis induced by mTOR/HIF-1α might promote EBI after SAH by activating M1 microglia. This finding provided new insights for the treatment of EBI.

Association of cerebrospinal fluid advanced oxidation protein products levels with early brain injury and prognosis of aneurysmal subarachnoid hemorrhage: A preliminary prospective study.

OBJECTIVES: In this study, we investigated the time course in the cerebrospinal fluid (CSF) advanced oxidation protein products (AOPPs) levels in patients with aneurysmal subarachnoid hemorrhage (aSAH), and ascertained the relationship between the levels of AOPPs and early brain injury (EBI), hydrocephalus and prognosis of patients with aSAH. METHODS: We measured the CSF AOPPs levels in 50 patients with aSAH at 1-3 d, 4-6 d, 7-9 d, and 10-12 d after hemorrhage. The modified Fisher grades, Hunt-Hess grades, CSF IL-6 levels, peripheral blood count of white blood cells, cerebral edema scores and hydrocephalus were used to assess the severity of brain injury. Modified Rankin Scale (mRS) scores were used to assess the prognosis. Patients with mRS scores greater than 2 were considered to have a poor outcome. RESULTS: CSF AOPPs levels were significantly higher in patients with aSAH with poor prognosis, compared to patients with good prognosis and peaked in the early stage. Among patients with aSAH, the levels of CSF AOPPs on days 1-3 were significantly correlated with modified Fisher grades, Hunt-Hess grades, CSF IL-6 levels, peripheral blood count of white blood cells, and cerebral edema scores. Also, in patients with hydrocephalus, early CSF AOPPs levels were significantly elevated. Levels of CSF AOPPs in aSAH patients on days 1-3, 4-6, and 7-9 were independently associated with poor prognosis at the 90-day follow-up, and the optimal area under the curve (AUC) values for CSF AOPPs levels were found on days 1-3. CONCLUSIONS: AOPPs may serve as the potential biomarker to assess the severity of EBI and prognosis in patients with aSAH.

A Prognostic Model of Differentiated Thyroid Cancer Based on Up-Regulated Glycolysis-Related Genes.

Objective: This study aims to identify reliable prognostic biomarkers for differentiated thyroid cancer (DTC) based on glycolysis-related genes (GRGs), and to construct a glycolysis-related gene model for predicting the prognosis of DTC patients. Methods: We retrospectively analyzed the transcriptomic profiles and clinical parameters of 838 thyroid cancer patients from 6 public datasets. Single factor Cox proportional risk regression analysis and Least Absolute Shrinkage and Selection Operator (LASSO) were applied to screen genes related to prognosis based on 2528 GRGs. Then, an optimal prognostic model was developed as well as evaluated by Kaplan-Meier and ROC curves. In addition, the underlying molecular mechanisms in different risk subgroups were also explored via The Cancer Genome Atlas (TCGA) Pan-Cancer study. Results: The glycolysis risk score (GRS) outperformed conventional clinicopathological features for recurrence-free survival prediction. The GRS model identified four candidate genes (ADM, MKI67, CD44 and TYMS), and an accurate predictive model of relapse in DTC patients was established that was highly correlated with prognosis (AUC of 0.767). In vitro assays revealed that high expression of those genes increased DTC cancer cell viability and invasion. Functional enrichment analysis indicated that these signature GRGs are involved in remodelling the tumour microenvironment, which has been demonstrated in pan-cancers. Finally, we generated an integrated decision tree and nomogram based on the GRS model and clinicopathological features to optimize risk stratification (AUC of the composite model was 0.815). Conclusions: The GRG signature-based predictive model may help clinicians provide a prognosis for DTC patients with a high risk of recurrence after surgery and provide further personalized treatment to decrease the chance of relapse.

Comprehensive Analysis of Prognostic Alternative Splicing Signature Reveals Recurrence Predictor for Papillary Thyroid Cancer.

BACKGROUND: Alternative splicing (AS) plays a key role in the diversity of proteins and is closely associated with tumorigenicity. The aim of this study was to systemically analyze RNA alternative splicing (AS) and identify its prognostic value for papillary thyroid cancer (PTC). METHODS: AS percent-splice-in (PSI) data of 430 patients with PTC were downloaded from the TCGA SpliceSeq database. We successfully identified recurrence-free survival (RFS)-associated AS events through univariate Cox regression, LASSO regression and multivariate regression and then constructed different types of prognostic prediction models. Gene function enrichment analysis revealed the relevant signaling pathways involved in RFS-related AS events. Simultaneously, a regulatory network diagram of AS and splicing factors (SFs) was established. RESULTS: We identified 1397 RFS-related AS events which could be used as the potential prognostic biomarkers for PTC. Based on these RFS-related AS events, we constructed a ten-AS event prognostic prediction signature that could distinguish high-and low-risk patients and was highly capable of predicting PTC patient prognosis. ROC curve analysis revealed the excellent predictive ability of the ten-AS events model, with an area under the curve (AUC) value of 0.889; the highest prediction intensity for one-year RFS was 0.923, indicating that the model could be used as a prognostic biomarker for PTC. In addition, the nomogram constructed by the risk score of the ten-AS model also showed high predictive efficiency for the prognosis of PTC patients. Finally, the constructed SF-AS network diagram revealed the regulatory role of SFs in PTC. CONCLUSION: Through the limited analysis, AS events could be regarded as reliable prognostic biomarkers for PTC. The splicing correlation network also provided new insight into the potential molecular mechanisms of PTC.

Effects of marital status on survival of medullary thyroid cancer stratified by age.

PURPOSE: Marital status has emerged as an important influence on several cancer outcomes, but its role in medullary thyroid cancer (MTC) remains unclear. This study was to explore the effects of marital status on the prognosis of MTC patients and to determine whether its effects vary by age. PATIENTS AND METHODS: We retrospectively extracted 1344 eligible patients diagnosed with MTC between 2004 and 2015 from the Surveillance, Epidemiology, and End Results (SEER) database. Based on the marital status, we divided those patients into married and unmarried groups. We compared the difference in overall survival (OS) and cancer-specific survival (CSS) between married and unmarried via the Kaplan-Meier analysis. Univariate and multivariate Cox proportional models were performed to identify the prognostic factors of OS and CSS. RESULTS: There were 1344 MTC eligible patients in a total of which 883 (65.7%) were married and 461 (34.3%) were unmarried. The comparison observed between married and unmarried patients was as follows: male (45.2% vs. 28.0%), age (≥52 years) (55.9% vs. 44.6%), White (86.7% vs. 78.7%), and undergo surgery (97.7% vs. 93.3%). Multivariate analysis revealed unmarried status as a risk factor independently associated with worse OS (HR: 2.15, 95% CI: 1.59-2.92) rate and CSS (HR: 1.70, 95% CI: 1.17-2.47) rate. In a further analysis stratified by age, there was no significant difference in OS and CSS between married and unmarried patients younger than 52 years. For the remaining group with 52 years old and higher, unmarried patients showed significantly higher risk of OS and CSS than married patients at all stages of the pathology except M1 stage. CONCLUSION: Married patients with MTC have a better prognosis than unmarried ones. Age can affect the association between marital status and the survival of MTC, and married elders may benefit more than youngers.

A Prediction Model for Contralateral Central Neck Lymph Node Metastases in Unilateral Papillary Thyroid Cancer.

The health problems caused by the frequent relapse of papillary thyroid carcinoma (PTC) remain a worldwide concern since the morbidity rate of PTC ranks the highest among thyroid cancers. Residues from contralateral central lymph node metastases (con-CLNM) are the key reason for persistence or recurrence of unilateral papillary thyroid carcinoma (uni-PTC); however, the ability to assess the status of con-CLNM in uni-PTC patients is limited. To clarify the risk factors of con-CLNM, a total of 250 patients with uni-PTC who underwent total thyroidectomy and bilateral central lymph node dissection were recruited in this study. We compared the clinical, sonographic, and pathological characteristics of patients with con-CLNM to those without con-CLNM and established a nomogram for con-CLNM in uni-PTC. We found that male sex, without Hashimoto's thyroiditis, present capsular invasion, with ipsilateral lateral lymph node metastases, and the ratio of ipsilateral central lymph node metastases ≥0.16 were independent con-CLNM predictors of uni-PTC (ORs: 2.797, 0.430, 2.538, 2.202, and 26.588; 95% CIs: 1.182-6.617, 0.211-0.876, 1.223-5.267, 1.064-4.557, and 7.596-93.069, respectively). Additionally, a preoperative nomogram for the prediction of con-CLNM based on these risk factors showed good discrimination (C-index 0.881; 95% CI: 0.840-0.923; sensitivity 85.3%; specificity 76.0%) and good agreement via the calibration plot. Our study provided a way to quantitatively and accurately predict whether con-CLNM occurred in patients with uni-PTC, which may guide surgeons to evaluate the nodal status and perform tailored therapeutic central lymph node dissection.

Role of TREM-1 in the development of early brain injury after subarachnoid hemorrhage.

Triggering receptor expressed on myeloid cells-1 (TREM-1) was found to be induced in the context of subarachnoid hemorrhage (SAH) before. This study further investigates its role in the development of SAH-induced early brain injury (EBI). Firstly, rats were randomly divided into Sham and SAH groups for analysis of temporal patterns and cellular localization of TREM-1. Secondly, TREM-1 intervention was administrated to produce Sham, vehicle, antagonist and agonist groups, for analyzing TREM-1, Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88) and NF-κB expressions at 24 h post-modeling, and EBI assessment at 24 h and 72 h. Thirdly, TLR4 inhibitor (TAK-242) was exploited to produce Sham, Sham+TAK-242, SAH, and SAH + TAK-242 groups to analyze the effects of TLR4 inhibition on TREM-1 induction and EBI evaluation at 72 h. Fourthly, the relationship of soluble TREM-1 (sTREM-1) levels in cerebrospinal fluid of SAH patients with Hunt-Hess grades were explored. The results showed that TREM-1 increased in the brain after experimental SAH (eSAH) early at 6 h and peaked at 48 h, which was found to be located in microglia and endothelial cells. TREM-1 inhibition attenuated EBI associated with TLR4/MyD88/NF-κB suppression, while enhancement had the opposite effects. Contrarily, TLR4 inhibition prevented TREM-1 induction and ameliorated EBI. In addition, sTREM-1 levels in SAH patients positively correlated with Hunt-Hess grades. Overall, the present study provides new evidence that TREM-1 increases dynamically in the brain after eSAH and it is located in microglia and endothelial cells, which may aggravate EBI by interacting with TLR4 pathway. And sTREM-1 in patients might act as a monitoring biomarker of EBI, providing new insights for future studies.

Kinase gene fusions: roles and therapeutic value in progressive and refractory papillary thyroid cancer.

The incidence of papillary thyroid cancer (PTC), the major type of thyroid cancer, is increasing rapidly around the world, and its pathogenesis is still unclear. There is poor prognosis for PTC involved in rapidly progressive tumors and resistance to radioiodine therapy. Kinase gene fusions have been discovered to be present in a wide variety of malignant tumors, and an increasing number of novel types have been detected in PTC, especially progressive tumors. As a tumor-driving event, kinase fusions are constitutively activated or overexpress their kinase function, conferring oncogenic potential, and their frequency is second only to BRAFV600E mutation in PTC. Diverse forms of kinase fusions have been observed and are associated with specific pathological features of PTC (usually at an advanced stage), and clinical trials of therapeutic strategies targeting kinase gene fusions are feasible for radioiodine-resistant PTC. This review summarizes the roles of kinase gene fusions in PTC and the value of clinical therapy of targeting fusions in progressive or refractory PTC, and discusses the future perspectives and challenges related to kinase gene fusions in PTC patients.

Predictive Factors for Level V Lymph Node Metastases in Papillary Thyroid Carcinoma with BRAFV600E Mutation and Clinicopathological Features.

BACKGROUND: Therapeutic lateral neck dissection (LND) is recommended in papillary thyroid carcinoma (PTC) patients with clinically lateral lymph node metastasis (LLNM), whether underwent level V LND remains controversial for lacking of sensitive predicting system. BRAFV600E mutation is associated with aggressive tumor behavior, recurrence, and disease-specific mortality of PTC. However, the relationship between BRAFV600E mutation and level V LNM is unclear. METHODS: Univariate and multivariate analyses were retrospectively conducted on the potential predictive factors of 252 PTC patients who underwent initial treatment of neck lymph node dissection from September 2015 to October 2018 in our institute. BRAFV600E mutation and the clinicopathological characteristics of the two groups were compared. RESULTS: LLNM was presented in 208 (82.5%) patients and level II-V LNM was present in 42.8%, 71.2%, 85.1%, 17.8% patients, respectively. BRAFV600E mutation was observed in 188 (74.6%) patients and was significantly associated with patients' age, lymphocytic thyroiditis, capsule invasion, bilateral central lymph node metastasis (CLNM) and level V LNM in PTC. Univariate analysis revealed that lymphocytic thyroiditis, tumor size, number of CLNM, Level II LNM, Level III LNM, simultaneous Level II+III, simultaneous Level III+IV and simultaneous Level II+III+IV were significantly correlated with Level V LNM. In addition, multivariate analysis revealed that tumor size ≥2.5 cm, number of CLNM≥3, level II metastases and BRAFV600E mutation were independent Level V LNM predictors (odds ratio 3.910, 3.660, 8.410, 0.439; 95% CI 1.737-10.135, 1.054-12.713, 1.233-57.355, 0.280-0.827, respectively). CONCLUSION: In summary, we presented several independent predictive factors for level V LNM in PTC patients. We constructed a risk prediction model consisting of tumor size ≥2.5 cm, number of CLNM≥3 and level II metastases and BRAFV600E mutation that may guide surgeons to evaluate the nodal status in PTC and perform tailored therapeutic LND.

Preoperative Sonographic and Clinicopathological Predictors for Solitary Lateral Neck Node Metastasis in Papillary Thyroid Carcinoma: A Retrospective Study.

BACKGROUND: Cervical lymph node metastasis (LNM) is an independent risk factor for poor prognosis of papillary thyroid carcinoma (PTC), but the scope of PTC lateral neck dissection (LND) is controversial. Solitary lateral lymph node metastasis (SLNM) is a special type of PTC with lateral LNM. Currently, study on the preoperative clinical characteristics of SLNM has been seldomly reported. This study evaluated the preoperative characteristics for predicting the SLNM of PTC. METHODS: We included 391 patients diagnosed with PTC between May 2011 and July 2017. Among those patients, 44 had SLNM and 347 had multiple lateral neck node metastasis (MLNM). The clinicopathologic characteristics and other central lymph node metastasis risk factors were retrospectively analyzed. RESULTS: Univariate analysis revealed that age and tumor size (≤1 cm) were significantly correlated with SLNM. In ROC curve analysis, the optimal cutoff age of preoperative predictors for the prediction of SLNM was 46.5 years (AUC=0.623, 0.536-0.710). Besides, the frequency and mean number of CLNM was significantly less in the SLNM than MLNM group. The oval and round tumor shape and well-defined margin of the tumor were more common in the SLNM group (p =0.001; p=0.024, respectively). In addition, multivariate analysis revealed that age ≥47, capsular invasion, no extrathyroidal extension, with central lymph node metastases and irregular shape were independent SLNM predictors of PTCs (odds ratio 2.386, 0.173, 0.284, 0.239, 0.188; 95% CI 1.07-5.140, 0.058-0.840, 0.066-0.926, 0.091-0.437, 0.167-0.864, respectively). CONCLUSION: This study supported that SLNM is more likely to happen in PTC patients with age ≥47 years, capsular invasion, no extrathyroidal extension, with central lymph node metastases and irregular shape. That denotes, selective single level neck dissection can be considered as an alternative to systemic lateral neck dissection in those patients.

Enzymatic characterization of Bacillus licheniformis γ-glutamyltranspeptidase fused with N-terminally truncated forms of Bacillus sp. TS-23 α-amylase.

Bacillus licheniformis γ-glutamyltranspeptidase (BlGGT) was fused at its C-terminal end with N-terminally truncated forms of Bacillus sp. TS-23 α-amylase. BlGGT and six fusion enzymes, BlGGT/SBD, BlGGT/AMYΔN476, BlGGT/AMYΔN443, BlGGT/AMYΔN376, BlGGT/AMYΔN195, and BlGGT/AMYΔN34, were over-expressed in Escherichia coli M15 cells and purified to apparent homogeneity by metal-affinity chromatography. The fusion constructions had no significant effect on the autocatalytic processing of BlGGT. Progressive decrease in the GGT activity of fusion proteins was associated with an increasing level of truncation, and only BlGGT/AMYΔN34 reserved the amylolytic activity. The protein fusions did not alter the optimal temperature and pH of BlGGT. However, as compared with the parental BlGGT, a significant change in circular dichorism and fluorescence spectra was observed in the fusion enzymes. Thermal unfolding of BlGGT, BlGGT/AMYΔN476, BlGGT/AMYΔN443, and BlGGT/AMYΔN376 followed the two-state unfolding process with a transition point (T(m)) of 61.3-63.1 °C, whereas BlGGT/AMYΔN195 and BlGGT/AMYΔN34 displayed two temperature transitions at 40.6 and 46.7 °C as well as at 62.8 and 62.9 °C, respectively. All of the fusion enzymes exhibited the raw-starch-binding ability, and the adsorbed proteins could be eluted from the adsorbent by 50mM Tris-HCl (pH 9.0) containing 2% soluble starch.

Characterization of glycine substitution mutations within the putative NAD+-binding site of Bacillus licheniformis aldehyde dehydrogenase.

The NAD(+)-requiring enzymes of the aldehyde dehydrogenase (ALDH) family contain a glycine motif, GX1- 2GXXG, which is reminiscent of the fingerprint region of the Rossman fold, a conserved structural motif of the classical nicotinamide nucleotide-binding proteins. In this research, the role of three glycine residues situated within the putative NAD(+)-binding motif (211-GPGSSAG) together with Gly233 and Gly238 of Bacillus licheniformis ALDH (BlALDH) were probed by site-directed mutatgenesis. Fifteen mutant BlALDHs were obtained by substitution of the indicated glycine residues with alanine, glutamate and arginine. Except for the Ala replacement at positions 211, 213, 217 and 238, the remaining mutant enzymes lost the dehydrogenase activity completely. Tryptophan fluorescence and far-UV circular dichroism spectra allowed us to discriminate BlALDH and the inactive mutant enzymes, and unfolding analyses further revealed that they had a different sensitivity towards temperature- and guanidine hydrochloride (GdnHCl)-induced denaturation. BlALDH and the functional variants had a comparable T(m) value, but the value was reduced by more than 5.1°C in the rest of mutant enzymes. Acrylamide quenching analysis showed that the inactive mutant enzymes had a dynamic quenching constant greater than that of BlALDH. Native BlALDH started to unfold beyond ~0.21 M GdnHCl and reached an unfolded intermediate, [GdnHCl](0.5, N-U), at 0.92 M equivalent to free energy change (ΔG(N-U)(H2O)) of 12.34 kcal/mol for the N → U process, whereas the denaturation midpoints for mutant enzymes were 0.45-1.61 M equivalent to ΔG(N-U)(H2O) of 0.31-4.35 kcal/mol. Taken together, these results strongly suggest that the explored glycines are indeed important for the catalytic activity and structural stability of BlALDH.

Mutational analysis of splicing activities of ribonucleotide reductase α subunit protein from lytic bacteriophage P1201.

A CP1201 RIR1 intein is found in the ribonucleotide reductase alpha subunit (RNR α subunit) protein of lytic bacteriophage P1201 from Corynebacterium glutamicum NCHU 87078. This intein can be over-expressed and spliced in Escherichia coli NovaBlue cells. Mutations of C539, the N-terminal residue of the C-extein in the CP1201 RIR1 protein, led to the changes of pattern and level of protein-splicing activities. A G392S variant was found to be a temperature-sensitive protein with complete splicing activity at 17 and 28°C but not at 37°C or higher. We also found that the cleavage at the CP1201 RIR1 intein C-terminus of the double mutant G392S/C539G was blocked, but other cleavage activities could be efficiently performed at 17°C. G392S/C539G variant possessed the properties of low-temperature-induced cleavage at the intein N-terminus.

Biochemical characterization of two thymidylate synthases in Corynebacterium glutamicum NCHU 87078.

The genome of Corynebacterium glutamicum NCHU 87078 contains two putative thymidylate synthase genes, designated CgthyA and CgthyX. These two genes were expressed in Escherichia coli NovaBlue and the expressed His(6)-tagged enzymes were purified by nickel-chelate chromatography. The purified CgThyA had a specific activity of 414 mU mg(-)(1) protein, whereas thymidylate synthase activity for CgThyX could not be detected in a functional complementation assay using a 10-day incubation period. Gel filtration chromatography and chemical cross-linking experiments showed that CgThyX may exist as a dimer in solution, unlike a typical ThyX protein with homotetrameric structure for catalytic activity. Spectroscopic analysis indicated that purified CgThyX lacked the cofactor FAD. The 2.3A resolution crystal structure of CgThyX-FAD demonstrated a loose tetramer, in which FAD is chelated between the subunits via a manner distinct from that of other flavin-dependent thymidylate synthases. Structure-based mutational studies have identified a non-conserved segment (residues 70-73) of CgThyX protein with crucial role in binding to FAD. Taken together, our biochemical and structural analyses highlight unique features of the C. glutamicum ThyX that distinguish this enzyme from ThyX proteins from other organisms. Our results also suggest that thymidylate synthesis in C. glutamicum requires ThyA but not ThyX.

Deletion analysis of the C-terminal region of a molecular chaperone DnaK from Bacillus licheniformis.

Bacillus licheniformis DnaK (BlDnaK) is predicted to consist of a 45-kDa N-terminal ATPase domain and a 25-kDa C-terminal substrate-binding domain. In this study, the full-length BlDnaK and its T86W and three C-terminally truncated mutants were constructed to evaluate the role of up to C-terminal 255 amino acids of the protein. The steady-state ATPase activity for BlDnaK, T86W, T86W/DeltaC120, T86W/DeltaC249, and T86W/DeltaC255 was 65.68, 53.21, 116.04, 321.38, and 90.59 nmol Pi/min per mg, respectively. In vivo, BldnaK, T86W and T86W/DeltaC120 genes allowed an E. coli dnaK756-ts mutant to grow at 44 degrees C. Except for T86W/DeltaC255, simultaneous addition of B. licheniformis DnaJ and GrpE, and NR-peptide synergistically stimulated the ATPase activity of BlDnaK, T86W, T86W/DeltaC120, and T86W/DeltaC249 by 16.9-, 13.9-, 33.9-, 9.9-fold, respectively. Measurement of intrinsic tryptophan fluorescence revealed significant alterations of microenvironment of aromatic amino acids in the C-terminally truncated mutants. The temperature-dependent signal in the far-UV region for T86W was consistent with that of BlDnaK, but the C-terminally truncated mutant proteins showed a higher sensitivity toward temperature-induced denaturation. These results suggest that C-terminal truncations alter the ATPase activity and thermal stability of BlDnaK and induce the conformation change of the ATPase domain.