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Huge waves caused by typhoons often induce severe disasters along coastal areas, making the effective prediction of typhoon-induced waves a crucial research issue for researchers. In recent years, the development of the Internet of Underwater Things (IoUT) has rapidly increased the prediction of oceanic environmental disasters. Past studies have utilized meteorological data and feedforward neural networks (e.g., BPNN) with static network structures to establish short lead time (e.g., 1 h) typhoon wave prediction models for the coast of Taiwan. However, sufficient lead time for prediction remains essential for preparedness, early warning, and response to minimize the loss of lives and properties during typhoons. The aim of this research is to construct a novel long lead time typhoon-induced wave prediction model using Long Short-Term Memory (LSTM), which incorporates a dynamic network structure. LSTM can capture long-term information through its recurrent structure and selectively retain necessary signals using memory gates. Compared to earlier studies, this method extends the prediction lead time and significantly improves the learning and generalization capability, thereby enhancing prediction accuracy markedly.

Abstract: Hypercatecholaminergic conditions are known to cause heart failure and cardiac fibrosis when severe. Although previous investigations have studied the effects of beta-blockade in experimental models of catecholaminergic states, the detailed benefits of beta-blockade in more realistic models of hyper-adrenergic states were less clear. In this study, we examined acute cardiac changes in rats with hyperacute catecholamine-induced heart failure with and without propranolol treatment. Male Sprague-Dawley rats (n = 12) underwent a 6-hour infusion of epinephrine and norepinephrine alone, with an additional propranolol bolus (1 mg/kg) at hour 1 (n = 6). Cardiac tissues were examined after 6 hours. Cardiac immunohistochemistry revealed significantly decreased expression of phosphorylated p-38 (left ventricle, P = 0.021; right ventricle, P = 0.021), with upregulation of reactive oxidative species and other profibrosis proteins, after catecholamine infusion alone. After 1 propranolol 1 mg/kg bolus, the levels of phosphorylated-p38 returned to levels comparable with sham (left ventricle, P = 0.021; right ventricle, P = 0.043), with additional findings including downregulation of the apoptotic pathway and profibrotic proteins. We conclude that catecholamine-induced heart failure exerts damage through the p-38 mitogen-activated protein kinase pathway and demonstrates profibrotic changes mediated by matrix metalloproteinase 9, alpha-smooth muscle actin, and fibroblast growth factor 23. Changes in these pathways attenuated acute catecholamine-induced heart failure after propranolol bolus 1 mg/kg. We conclude that propranolol bolus at 1 mg/kg is able to mediate the effects of catecholamine excess through the p-38 mitogen-activated protein kinase pathway, profibrosis, and extrinsic apoptosis pathway., Competing Interests: The authors report no conflicts of interest., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Objective: The healthcare challenge driven by an aging population and rising demand is one of the most pressing issues leading to emergency department (ED) overcrowding. An emerging solution lies in machine learning's potential to predict ED dispositions, thus leading to promising substantial benefits. This study's objective is to create a predictive model for ED patient dispositions by employing ensemble learning. It harnesses diverse data types, including structured and unstructured information gathered during ED visits to address the evolving needs of localized healthcare systems., Methods: In this cross-sectional study, 80,073 ED patient records were amassed from a major southern Taiwan hospital in 2018-2019. An ensemble model incorporated structured (demographics, vital signs) and pre-processed unstructured data (chief complaints, preliminary diagnoses) using bag-of-words (BOW) and term frequency-inverse document frequency (TF-IDF). Two random forest base-learners for structured and unstructured data were employed and then complemented by a multi-layer perceptron meta-learner., Results: The ensemble model demonstrates strong predictive performance for ED dispositions, achieving an area under the receiver operating characteristic curve of 0.94. The models based on unstructured data encoded with BOW and TF-IDF yield similar performance results. Among the structured features, the top five most crucial factors are age, pulse rate, systolic blood pressure, temperature, and acuity level. In contrast, the top five most important unstructured features are pneumonia, fracture, failure, suspect, and sepsis., Conclusions: Findings indicate that utilizing ensemble learning with a blend of structured and unstructured data proves to be a predictive method for determining ED dispositions., (© 2024. The Author(s).)

Objectives: The theory of at least 2-week waiting period between tooth extraction and head and neck radiotherapy could reduce osteoradionecrosis remains controversial. Thus, this study examined the theory and associated factors., Materials and Methods: Data were retrieved from the National Health Insurance Research Database, Taiwan Cancer Registry Database, and Cause of Death Statistics. We included 24,353 patients with head and neck cancer who received radiotherapy from 2011 to 2017 and were followed up until 2019. The patients were divided into three groups: those undergoing tooth removal 2-8 weeks before radiotherapy, those undergoing tooth removal within 2 weeks before radiotherapy, and others. Confounding factors were clinical information, physical conditions, and risky habits. We used the Cox regression model to assess osteoradionecrosis risk., Results: No significant difference in osteoradionecrosis risk was observed between those undergoing tooth extraction within 2 weeks before radiotherapy and the other groups. An irradiation dose of ≥60 Gy, chemotherapy, tumor excision, post-radiotherapy tooth extraction, mandibulectomy, hyperlipidemia, and oral cavity as the tumor subsite were significantly positively associated with osteoradionecrosis risk., Conclusion: A waiting period of ≥2 weeks between tooth extraction and radiotherapy did not significantly reduce osteoradionecrosis risk., (© 2022 Wiley Periodicals LLC.)

The impact of different charging currents and surrounding temperatures has always been an important aspect of battery lifetime for various electric vehicles and energy storage equipment. This paper proposes a bidirectional long short-term memory model to quantify these impacts on the aging of gel batteries and calculate their state of health. The training data set of the bidirectional long short-term memory model is collected by charging and discharging the gel battery for 300 cycles in a temperature-controlled box and an automated charge and discharge device under different operating conditions. The testing set is generated by a small energy storage device equipped with small solar panels. Data for 220 cycles at different temperatures and charging currents were collected during the experiment. The results show that the mean absolute error (MAE) and root-mean-square error (RMSE) between the training set and testing set are 0.0133 and 0.0251, respectively. In addition to the proposed model providing high accuracy, the gel battery proved to be stable and long-lasting, which makes the gel battery an ideal energy storage solution for renewable energy.

Numerous vaccines have been developed to address the current COVID-19 pandemic, but safety, cross-neutralizing efficacy, and long-term protectivity of currently approved vaccines are still important issues. In this study, we developed a subunit vaccine, ASD254, by using a nanoparticle vaccine platform to encapsulate the SARS-CoV-2 spike receptor-binding domain (RBD) protein. As compared with the aluminum-adjuvant RBD vaccine, ASD254 induced higher titers of RBD-specific antibodies and generated 10- to 30-fold more neutralizing antibodies. Mice vaccinated with ASD254 showed protective immune responses against SARS-CoV-2 challenge, with undetectable infectious viral loads and reduced typical lesions in lung. Besides, neutralizing antibodies in vaccinated mice lasted for at least one year and were effective against various SARS-CoV-2 variants of concern, including B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), B.1.617.2 (Delta), and B.1.1.529 (Omicron). Furthermore, particle size, polydispersity index, and zeta-potential of ASD254 remained stable after 8-month storage at 4°C. Thus, ASD254 is a promising nanoparticle vaccine with good immunogenicity and stability to be developed as an effective vaccine option in controlling upcoming waves of COVID-19.

The ongoing evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), resulting in the emergence of new variants that are resistant to existing vaccines and therapeutic antibodies, has raised the need for novel strategies to combat the persistent global COVID-19 epidemic. In this study, a monoclonal anti-human angiotensin-converting enzyme 2 (hACE2) antibody, ch2H2, was isolated and humanized to block the viral receptor-binding domain (RBD) binding to hACE2, the major entry receptor of SARS-CoV-2. This antibody targets the RBD-binding site on the N terminus of hACE2 and has a high binding affinity to outcompete the RBD. In vitro, ch2H2 antibody showed potent inhibitory activity against multiple SARS-CoV-2 variants, including the most antigenically drifted and immune-evading variant Omicron. In vivo, adeno-associated virus (AAV)-mediated delivery enabled a sustained expression of monoclonal antibody (mAb) ch2H2, generating a high concentration of antibodies in mice. A single administration of AAV-delivered mAb ch2H2 significantly reduced viral RNA load and infectious virions and mitigated pulmonary pathological changes in mice challenged with SARS-CoV-2 Omicron BA.5 subvariant. Collectively, the results suggest that AAV-delivered hACE2-blocking antibody provides a promising approach for developing broad-spectrum antivirals against SARS-CoV-2 and potentially other hACE2-dependent pathogens that may emerge in the future., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Purpose: It is commonly recommended that tooth extraction should be performed prior to radiotherapy (RT) in patients with head neck cancer to prevent osteoradionecrosis (ORN). However, doctors still occasionally encounter patients who require tooth extraction during RT. This study aimed to determine the risk of ORN in patients who undergo tooth extraction during RT., Methods: Data were collected from Taiwan's National Health Insurance Research Database. We retrospectively enrolled 24,412 patients with head and neck cancer treated with radiotherapy between 2011 and 2017. The associations between ORN and demographic characteristics, timing of tooth extraction, and treatments were examined using univariate and multivariable Cox proportional hazards regression models., Results: A total of 24,412 head and neck cancer patients were enrolled; 133 patients underwent tooth extraction during RT and 24,279 patients did not undergo tooth extraction during RT. Tooth extraction during RT was not associated with a significantly higher risk of ORN (hazard ratio [HR] = 1.303, P = 0.4862). Tumor site, RT dose ≥ 60 Gy, age < 55 y/o, mandibulectomy, chronic periodontitis, and chemotherapy were significantly associated with a higher risk of ORN., Conclusion: The risk of ORN in head and neck cancer is not significantly different between patients who undergo tooth extraction during RT and patients who do not undergo tooth extraction during RT., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Exposure to hypoxia during the early postnatal period can have adverse effects on vital organs. Neonatal Sprague-Dawley rats housed in a hypoxic chamber were compared to those in a normoxic chamber from postnatal days 0 to 7. Arterial blood was collected to evaluate renal function and hypoxia. Kidney morphology and fibrosis were evaluated using staining methods and immunoblotting. In the kidneys of the hypoxic group, protein expressions of hypoxia-inducible factor-1 were higher than those in the normoxic group. Hypoxic rats had higher levels of hematocrit, serum creatinine, and lactate than normoxic rats. Body weight was reduced, and protein loss of kidney tissue was observed in hypoxic rats compared to normoxic rats. Histologically, hypoxic rats showed glomerular atrophy and tubular injury. Renal fibrosis with collagen fiber deposition was observed in the hypoxic group. The expression of nicotinamide adenine dinucleotide phosphate oxidases was enhanced in the kidneys of hypoxic rats. Proteins involved in apoptosis were upregulated in the kidneys of hypoxic rats. An increase in the expression of pro-inflammatory cytokines was also observed in the kidneys of hypoxic rats. Hypoxic kidney injury in neonatal rats was associated with oxidative stress, inflammation, apoptosis, and fibrosis.

Objective: The aim of the study is to assess the effects of neuromuscular electrical stimulation on the upper limbs of patients with cerebral palsy., Design: We searched PubMed, Cochrane, Embase, and Scopus databases for randomized controlled trials examining the effects of neuromuscular electrical stimulation on the upper limbs of children with cerebral palsy., Results: Eight randomized controlled trials ( N = 294) were included in the meta-analysis. Compared with traditional physical therapy, sensorimotor training and task-oriented training, constraint-induced movement therapy, dynamic bracing, and conventional robot-assisted therapy, neuromuscular electrical stimulation in combination with these therapies resulted in significantly greater functional scale scores (standardized mean difference = 0.80; 95% confidence interval = 0.54 to 1.06), muscle strength of upper limbs (standardized mean difference = 0.57; 95% confidence interval = 0.25 to 0.88), and spasticity of upper limbs (relative risk = 2.53; 95% confidence interval = 1.46 to 4.39; standardized mean difference = -0.18; 95% confidence interval = -0.29 to -0.06) but did not improve the wrist range of motion (standardized mean difference = 0.43; 95% confidence interval = -0.04 to 0.91). In addition, the effect of neuromuscular electrical stimulation on functional scale scores remained after 3-mo follow-up (standardized mean difference = 0.68; 95% confidence interval = 0.16 to 1.2)., Conclusions: Neuromuscular electrical stimulation effectively improved hand function, muscle strength, and spasticity in patients with cerebral palsy., Competing Interests: Financial disclosure statements have been obtained, and no conflicts of interest have been reported by the authors or by any individuals in control of the content of this article., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Background: The diagnosis of head and neck cancer (HNC) may lead to exhaustion and depression. Therefore, the suicide risk of patients with HNC is high. This study aimed to understand the suicide risk of patients with HNC in Taiwan compared with patients with other-cancer and general population during the period from 2010 to 2019., Methods: A total of 74,495 patients with HNC were compared against the other two cohort consisting 148,878 patients with other-cancer and 595,512 individual without cancer by age, sex, and index year from the Taiwan National Health Insurance Research Database. The multivariate Cox proportional hazard regression analysis was performed to estimate the risk of all-cause or suicide mortality., Results: Compared to the non-cancer group, the all-cause mortality risk of the HNC group (the adjusted hazard ratio (aHR), 7.72; 95 % confidence interval (CI), 7.60-7.83) is lower than that of the other-cancer group (aHR, 8.87; 95 % CI, 8.77-8.98). However, the suicide mortality risk compared with non-cancer group in the HNC group (aHR, 3.89; 95 % CI, 3.46-4.37) is much higher than other-cancer group (aHR, 1.86; 95 % CI, 1.64-2.10). HNC only has the seventh highest all-cause mortality risk, but HNC has the second highest suicide mortality risk. Men always have a higher suicide mortality risk than women. Middle-aged patients (age 50-60 years) have the highest suicide mortality risk, whereas younger patients (age < 40 years) have the lowest suicide mortality risk., Limitations: In this study, some factors and information needed were limited to the existing database; thus, preventing recall bias was difficult., Conclusions: This study indicates that patients with HNC did not have a higher risk of all-cause mortality than patients with other cancers. However, the risk of suicide mortality in patients with HNC was higher than that of patients without cancer and patients with other cancers in Taiwan., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Oral squamous cell carcinoma (OSCC) affects tens of thousands of people worldwide. Despite advances in cancer treatment, the 5-year survival rate of patients with late-stage OSCC is low at 50-60%. Therefore, the development of anti-OSCC therapy is necessary. We evaluated the effects of marine-derived triterpene stellettin B in human OC2 and SCC4 cells. Stellettin B dose-dependently decreased the viability of both cell lines, with a significant reduction in OC2 cells at ≥0.1 µM at 24 and 48 h, and in SCC4 cells at ≥1 µM at 24 and 48 h. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive cells were significantly observed at 20 µM of stellettin B at 48 h, with the overexpression of cleaved caspase3 and cleaved poly(ADP-ribose) polymerase (PARP). Moreover, mitochondrial respiratory functions were ablated by stellettin B. Autophagy-related LC3-II/LC3-I ratio and Beclin-1 proteins were increased, whereas p62 was decreased. At 20 µM at 48 h, the expression levels of the endoplasmic reticulum (ER) stress biomarkers calnexin and BiP/GRP78 were significantly increased and mitogen-activated protein kinase (MAPK) signaling pathways were activated. Further investigation using the autophagy inhibitor 3-methyladenine (3-MA) demonstrated that it alleviated stellettin B-induced cell death and autophagy. Overall, our findings show that stellettin B induces the ER stress, mitochondrial stress, apoptosis, and autophagy, causing cell death of OSCC cells.

Titanium nitride (TiN), a transition-metal compound with tight covalent Ti-N bonding, has a high melting temperature and superior mechanical and chemical stabilities compared to noble metals. With a reduction in thickness, the optical transmittance of TiN films can be drastically increased, and in combination with its excellent electrical conductivity, the ultrathin and continuous TiN film can be considered as an ideal alternative of the metal oxide electrodes. However, the deposition of ultrathin and continuous metallic layer with a smooth surface morphology is a major challenge for typical deposition methods such as thermal evaporation or reactive sputtering. In particular, defects mainly related with oxygen contents and surface scattering can significantly limit the performance of ultrathin TiN films. In this work, ultrathin TiN films with 2-10 nm in thickness are grown by using the nitrogen plasma-assisted molecular-beam epitaxy (MBE) method in an ultrahigh vacuum environment. Excellent surface morphology with a root-mean-square roughness of ≤0.12 nm and a high optical transparency of 75% over the whole visible regime are achieved for ultrathin TiN epitaxial films. The dielectric properties determined by the spectroscopic ellipsometry and the electrical properties measured by the terahertz spectroscopy and the Hall effect method reveal that the percolation thickness of the TiN epitaxial film is less than 2.4 nm and its electrical conductivity is higher than 1.1 × 10 4 Ω -1 cm -1 . These features make MBE-grown ultrathin TiN epitaxial films a good candidate for robust, low cost, and large-area transparent conductive electrodes.

Though numerous studies demonstrated the positive effect of rehabilitation on cerebral palsy (CP) children, there was no literature addressing the role of rehabilitation on mortality among children with CP. Therefore, we aimed to evaluate the impact of rehabilitation intensity on mortality among children with moderate to severe CP. This retrospective cohort study was conducted by National Health Insurance Research Database in Taiwan. Children (<12 years) with newly diagnosed moderate to severe CP between 1 January 2000 and 31 December 2013 were included. All patients were followed up for 3 years after CP diagnosis or death or until 31 December 2013. The intensity of rehabilitation therapy within 6 months after CP diagnosis was categorized into <6 times and ≥6 times. The Cox proportional hazard analysis was used to determine the association between rehabilitation intensity and all-cause mortality after adjusting age, sex, other demographic factors and comorbidities. Among 3936 severe CP children, 164 (4.2%) died during the 3-year follow-up period. The mortality rate was higher among patients receiving rehabilitation < 6 times within 6 months than those ≥6 times within 6 months after adjusting demographic profile and comorbidities (adjust HR (aHR): 1.96, 95% CI 1.33-2.89, p < 0.001). We found that patients who were younger (aHR: 0.84, 95% CI 0.76-0.92, p < 0.001), who were receiving inpatient care more than twice in 1 year before their CP diagnosis (aHR: 2.88; 95% CI: 1.96-4.23; p < 0.001), and who have pneumonia (aHR: 1.41, 95% CI 1.00-1.96, p = 0.047), epilepsy (aHR: 1.41, 95% CI: 1.02-1.95, p = 0.039) and dysphagia (aHR: 1.55, 95% CI: 1.06-2.26, p = 0.024) have higher risk of mortality. Rehabilitation ≥ 6 times within 6 months has a potentially positive impact on pediatric CP survival. Besides having a younger age, being hospitalized more than twice within a year before diagnosis and having pneumonia, epilepsy and dysphagia were modifiable risk factors in clinical practice for these children.

Purpose: The association between the tumor subsites of the oral cavity and the risk of osteoradionecrosis of the jaw (ORNJ) remains unclear. We study the correlation between oral cavity tumor subsites and the risk of ORNJ in a nationwide population-based database., Methods: We enrolled 16,701 adult patients with oral cavity cancers who were treated with radiotherapy between 2000 and 2013. The subsites of the oral tumor, treatments of oral cavity cancers, and the timing of tooth extraction were examined for their association with ORNJ in oral cancer patients., Results: 903 patients (5.40%) developed ORNJ. Of the relevant variables, pre-RT mandible surgery, tooth extraction either before or after RT, and tumor sites were associated with the risk of ORNJ. The adjusted HRs for ORNJ in the mouth floor, gums, retromolar, and buccal cancer were 2.056 (1.490-2.837), 1.909 (1.552-2.349), 1.683 (1.105-2.562), and 1.303 (1.111-1.528), respectively, compared with the risk of tongue cancer. There was no significant difference in the risk of ORNJ between the pre-RT extraction group, the during-RT extraction group, and the post-RT extraction (less than 6 months) group; the post-RT extraction (more than 6 months) group had a significantly higher risk of ORNJ., Conclusions: This study demonstrated that oral cavity tumor subsite is an independent risk factor of ORNJ after RT. Post-RT extraction (less than 6 months) group did not carry a significantly higher risk of ORNJ compared with pre-RT extraction group or during RT extraction group., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

Since the pandemic of COVID-19 has intensely struck human society, small animal model for this infectious disease is in urgent need for basic and pharmaceutical research. Although several COVID-19 animal models have been identified, many of them show either minimal or inadequate pathophysiology after SARS-CoV-2 challenge. Here, we describe a new and versatile strategy to rapidly establish a mouse model for emerging infectious diseases in one month by multi-route, multi-serotype transduction with recombinant adeno-associated virus (AAV) vectors expressing viral receptor. In this study, the proposed approach enables profound and enduring systemic expression of SARS-CoV-2-receptor hACE2 in wild-type mice and renders them vulnerable to SARS-CoV-2 infection. Upon virus challenge, generated AAV/hACE2 mice showed pathophysiology closely mimicking the patients with severe COVID-19. The efficacy of a novel therapeutic antibody cocktail RBD-chAbs for COVID-19 was tested and confirmed by using this AAV/hACE2 mouse model, further demonstrating its successful application in drug development., Competing Interests: The authors have declared that no competing interests exist.

In order to investigate the thermal and chemical (in)stabilities of MAPbI 3 incorporated with graphene and silver nanowire (AgNW) electrodes, we employed the terahertz (THz) time-domain spectroscopy, which has a unique ability to deliver the information of electrical properties and the intermolecular bonding and crystalline nature of materials. In in situ THz spectroscopy of MAPbI 3 , we observed a slight blue-shift in frequency of the 2 THz phonon mode as temperatures increase across the tetragonal-cubic structural phase transition. For MAPbI 3 with the graphene top electrode, no noticeable frequency shift is observed until the temperature reaches the maximum operating temperature of solar cells (85 °C). Phonon frequency shift is sensitive to the strain-induced tilt of PbI 6 octahedra and our results indicate that graphene forms a stable interface with MAPbI 3 and is also effective in suppression of the undesirable phase transition. Meanwhile, for MAPbI 3 coupled with the AgNW bottom electrode, the THz conductivity was found to be as low as that of the MAPbI 3 single layer, attributed to the chemical reaction between Ag atoms and iodide ions. The THz conductivity is greatly increased when an ultrathin Al 2 O 3 interlayer is introduced to cover the AgNW network via the atomic layer deposition (ALD) method. ALD of Al 2 O 3 on the AgNW surfaces at low temperature guarantees a conformal coating, which strongly affects the ohmic contacts between the NWs. Our results demonstrate the advantage of THz spectroscopy for the comprehensive analysis of thermal and chemical stabilities of perovskites associated with the electrode materials.

Background: To identify the risk factors of strabismus surgery among Taiwanese children with severe cerebral palsy (CP) and strabismus., Methods: This retrospective nationwide population-based cohort study examined a cohort of newly diagnosed pediatric CP patients (age ≤ 10 y) between 1997 and 2013 with strabismus. The primary endpoint was strabismus surgery. A stepwise logistic regression was applied to determine the demographic factors, ophthalmic conditions, and comorbidities associated with strabismus surgery., Results: Out of 808 patients, 115 had received strabismus surgery. The significant factors correlated to strabismus surgery in pediatric patients with severe CP and strabismus were CP diagnosis age < 4 years, residency in a suburban/rural area, low birth weight, and strabismic amblyopia., Conclusion: In CP children with strabismus who have risk factors of younger CP diagnosis age (age < 4 y), residency in a suburban/rural area, a low birthweight, and the presence of strabismic amblyopia, strabismus surgery should be considered.

Objective: Radiotherapy (RT) for head and neck cancer (HNC) within 7 days of tooth extraction is contraindicated because it may increase the risk of osteoradionecrosis of the jaw (ORNJ). However, delayed RT could compromise survival in patients with HNC. By using a national healthcare database, we reviewed the contraindications and analysed other risk factors for ORNJ., Design: A retrospective cohort study., Setting: By using Taiwan's National Health Insurance Research Database, 5,062 HNC patients with at least one tooth extraction 1-21 days before the first RT day (index day) and without any extractions during or after RT from 2000 to 2013 were included. The patients were divided into two groups according to the time of tooth extraction before the index day: 1-7 days and 8-21 days., Participants: Taiwanese patients with head and neck cancer., Main Outcomes Measure: Univariate and multivariate Cox proportional hazard regression models were used to evaluate the risk factors of ORNJ., Results: The overall incidence of ORNJ in the included patients was 1.03% (mean follow-up duration, 4.07 ± 3.01 years; range, 1.00-13.99 years). Tooth extraction within 7 days before RT was not associated with increased ORNJ risk (hazard ratio [HR] =0.734; P = .312). Significant risk factors for ORNJ included oral cancer (adjusted HR = 3.961), tumour excision surgery within 3 months before RT (adjusted HR = 3.488) and mandibulectomy within 3 months before RT (adjusted HR = 5.985; all P < .001)., Conclusion: In a mean follow-up of 4 years, tooth extraction within 7 days before RT for HNC treatment did not increase the ORNJ risk compared with tooth extraction 7-21 days before RT., (© 2020 John Wiley & Sons Ltd.)

Background: Pneumonia is the leading cause of death among children and young people (CYP) with severe cerebral palsy (CP). Only a few studies used nomogram for assessing risk factors and the probability of pneumonia. Therefore, we aimed to identify risk factors and devise a nomogram for identifying the probability of severe pneumonia in CYP with severe CP., Methods: This retrospective nationwide population-based cohort study examined CYP with newly diagnosed severe CP before 18 years old between January 1st, 1997 and December 31st, 2013 and followed them up through December 31st, 2013. The primary endpoint was defined as the occurrence of severe pneumonia with ≥ 5 days of hospitalization. Logistic regression analysis was used for determining demographic factors and comorbidities associated with severe pneumonia. These factors were assigned integer points to create a scoring system to identify children at high risk for severe pneumonia., Results: Among 6,356 CYP with newly diagnosed severe CP, 2,135 (33.59%) had severe pneumonia. Multivariable logistic regression analysis revealed that seven independent predictive factors, namely age <3 years, male sex, and comorbidities of pressure ulcer, gastroesophageal reflux, asthma, seizures, and perinatal complications. A nomogram was devised by employing these seven significant predictive factors. The prediction model presented favorable discrimination performance., Conclusions: The nomogram revealed that age, male sex, history of pressure ulcer, gastroesophageal reflux, asthma, seizures, and perinatal complications were potential risk factors for severe pneumonia among CYP with severe CP., Competing Interests: The authors have declared that no competing interests exist.

Background : Children with cerebral palsy (CP) place a considerable burden on medical costs and add to an increased number of inpatient days in Taiwan. Continuity of care (COC) has not been investigated in this population thus far. Materials and Methods : We designed a retrospective population-based cohort study using Taiwan's National Health Insurance Research Database. Patients aged 0 to 18 years with CP catastrophic illness certificates were enrolled. We investigated the association of COC index (COCI) with medical costs and inpatient days. We also investigated the possible clinical characteristics affecting the outcome. Results : Over five years, children with CP with low COCI levels had higher medical costs and more inpatient days than did those with high COCI levels. Younger age at CP diagnosis, more inpatient visits one year before obtaining a catastrophic illness certificate, pneumonia, and nasogastric tube use increased medical expenses and length of hospital stay. Conclusions : Improving COC reduces medical costs and the number of inpatient days in children with CP. Certain characteristics also influence these outcomes.

Two 11,20-epoxybriaranes, including a known compound, juncenolide K ( 1 ), as well as a new metabolite, fragilide X ( 2 ), have been isolated from gorgonian Junceella fragilis collected off the waters of Taiwan. The absolute configuration of juncenolide K ( 1 ) was determined by single-crystal X-ray diffraction analysis for the first time in this study and the structure, including the absolute configuration of briarane 2 was established on the basis of spectroscopic analysis and compared with that of model compound 1 . One aspect of the stereochemistry of the known compound 1 was revised. Briarane 2 was found to enhance the generation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) release from RAW 264.7 cells.

Deposition of monosodium urate (MSU) crystals in the joint or synovium is the major factor in Gouty arthritis (GA). The clinical features of chronic and recurrent GA include pain and the subsequent development of chronic tophaceous GA with multiple tophi deposits accompanied by osteolysis. The majority of previous animal studies have focused on MSU-induced acute GA without making observations regarding osteolysis. In the study, intra-articular injections of MSU into the knee (2 times/week for 10 weeks) was used to induce chronic and recurrent attacks of GA that in turn induced progressive osteolysis. Moreover, we also evaluated whether the clinical, nonsteroidal anti-inflammatory drug (NSAID) etoricoxib attenuated the osteoclastogenesis of progressive osteolysis. The knee morphometry and the expression of osteoclastogenesis-related proteins (cathepsin K and matrix metalloproteinase-9 and -13) in the knee were examined by micro-CT and immunohistochemistry, respectively. Results showed that oral etoricoxib not only significantly attenuated the nociceptive behaviors of the rats but that it also inhibited the expression of osteoclastogenesis-related proteins in their knee joints in chronic and recurrent attacks of GA. Our findings thus suggest that NSAIDs not only inhibit nociception but also prevent the progression of osteolysis in chronic and repeated attacks of GA., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2020 THE AUTHORS. Published by Elsevier BV on behalf of Cairo University.)

The aim of this study was to clarify the effects of general anesthesia (GA) on joint range of motion (ROM) in children with spastic cerebral palsy (SCP). Eighty-four SCP cases (mean age 8.4 years) admitted for first corrective surgery were retrospectively reviewed. Lower limb ROM were measured 1 day before operation and immediately after GA. Contracture of hip, knee, and ankle joints decreased significantly after GA, with + 11.1° (39.5%) for the hip abduction angle, -3.7° (18.0%) for the Thomas test, -15.0° (19.1%) for the popliteal angle, + 6.6° (39.8%) and 7.0° (109%) for ankle dorsiflexion with knee flexion and extension, respectively (all P < 0.001). These changes were correlated positively to pre-GA contracture and body weight, negatively to age, but independent of preoperative functional level, geographic classification of SCP, or modified Ashworth scale. On the basis of these findings, routine post-GA reassessments of joint ROM before corrective surgeries were recommended for pediatric SCP cases.

Objectives: Cancer of the oral cavity, a well-known global health concern, remains one of most common causes of cancer mortality. Continuity of care (COC), a measurement of the extent to which an individual patient receives care from a given provider over a specified period of time, can help cancer survivors process their experiences of dealing with the illness and recuperation; however, limited research has focused on the survival rate of working-age patients with oral cancer., Methods: A total of 14,240 working-age patients (20 0.38) and non-high COC (COCI ≤ 0.38) groups. After propensity-score matching, the mortality risk in the low and intermediate COC groups was also found to be significantly higher than that in the high COC group (aHR = 1.178, 95% CI = 1.074-1.292, p < 0.001 and aHR = 1.189, 95% CI = 1.107-1.277, p = 0.001, respectively)., Conclusions: In Taiwan, COC and prior dental treatment before RT significantly affected the survival rate of working-age patients with oral cancer. This result merits policymakers' attention., Competing Interests: The authors have declared that no competing interests exist.

Gingival recession (GR) potentially leads to the exposure of tooth root to the oral cavity microenvironment and increases susceptibility to dental caries, dentin hypersensitivity, and other dental diseases. Even though many etiological factors were reported, the specific mechanism of GR is yet to be elucidated. Given the species richness concerning marine biodiversity, it could be a treasure trove for drug discovery. In this study, we demonstrate the effects of a marine compound, (+)-rhodoptilometrin from crinoid, on gingival cell migration, wound healing, and oxidative phosphorylation (OXPHOS). Experimental results showed that (+)-rhodoptilometrin can significantly increase wound healing, migration, and proliferation of human gingival fibroblast cells, and it does not have effects on oral mucosa fibroblast cells. In addition, (+)-rhodoptilometrin increases the gene and protein expression levels of focal adhesion kinase (FAK), fibronectin, and type I collagen, changes the intracellular distribution of FAK and F-actin, and increases OXPHOS and the expression levels of complexes I~V in the mitochondria. Based on our results, we believe that (+)-rhodoptilometrin might increase FAK expression and promote mitochondrial function to affect cell migration and promote gingival regeneration. Therefore, (+)-rhodoptilometrin may be a promising therapeutic agent for GR.

Background: Geriatric hip fractures are mostly managed by internal fixation (IF) or hemiarthroplasty (HA). Survivorship of dementia patients following these surgeries has not been extensively compared in literature. By analysis of nationwide database, this study aimed to investigate the impact of dementia on two-year mortality after IF and HA in treating geriatric hip fractures., Method: From retrospective review of Taiwan's National Health Insurance Research Database, we enrolled 153,623 subjects aged 65 years and older with hospitalization for first hip fracture operated by IF (93,029 cases) or HA (60,594 cases) between 2000 and 2011. Postoperative mortality was compared between subjects with and without dementia after adjustments of age, gender, Charlson comorbidity index and hospital level., Results: The prevalence of dementia was 5.24% in the IF and 5.29% in the HA group. In the IF group, dementia increased adjusted hazard ratio of one-year (1.06, 95%CI:1.00-1.13) and two-year mortality (1.10, 95%CI:1.05-1.16). However, short and long-term mortality following HA was not significantly impacted by dementia (in-hospital OR:0.79, 95%CI:0.60-1.03; three-month HR:0.99, 95%CI:0.87-1.12; one-year HR:1.01, 95%CI:0.93-1.10; two-year HR:1.03, 95%CI:0.96-1.09). In a subgroup of dementia patients, mortality following IF was 15% higher than HA in one (p = 0.004) and two years (p < 0.001). The negative prognostic factors included female (HR:1.10; 95%CI:1.03-1.18) and aging 65-84 years (HR:1.15; 95%CI:1.00-1.32)., Conclusion: Dementia increased one and two-year mortality following geriatric hip fracture treated by IF, rather than HA. Dementia patients undergoing HA, especially female or 65-84 years old, sustained better one and two-year survival than those receiving IF., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Semiconducting polymer dots (Pdots) have recently emerged as a new type of ultrabright fluorescent probe that has been proved to be very useful for biomedical imaging. However, Pdots often suffer from serious fluorescence aggregation-caused quenching (ACQ) especially for near-infrared (NIR) fluorescent Pdots. This article compared two strategies to overcome the ACQ effect in near-infrared emissive Pdot systems: aggregation-induced emission (AIE) and anti-aggregation-caused quenching (anti-ACQ). The results show that the anti-ACQ platform outperforms the AIE system. The fluorescence quantum yield of anti-ACQ-based Pdots can be over 50% and the average per-particle brightness of the Pdots is about 5 times higher than that of the commercially available quantum dots. To help understand why the monomer conformations could greatly affect the optical properties of Pdots, molecular dynamics simulations were performed for the first time in such complicated Pdot systems. To demonstrate applications for in vivo fluorescence imaging, both microangiography imaging on living zebrafish embryos and specific tumor targeting on mice were performed. We anticipate that these studies will pave the way for the design of new highly fluorescent Pdot systems.

Objectives: The five-year survival rate of head and neck cancer (HNC) after radiotherapy (RT) varies widely from 35% to 89%. Many studies have addressed the effect of socioeconomic status and urban dwelling on the survival of HNC, but a limited number of studies have focused on the survival rate of HNC patients after RT., Materials and Methods: During the period of 2000-2013, 40,985 working age individuals (20 < age < 65 years) with HNC patients treated with RT were included in this study from a registry of patients with catastrophic illnesses maintained by the Taiwan National Health Insurance Research Database (NHIRD)., Results: The cumulative survival rate of HNC following RT in Taiwan was 53.2% (mean follow-up period, 3.75 ± 3.31 years). The combined effects of income and geographic effect on cumulative survival rates were as follows: high income group > medium income group > low income group and northern > central > southern > eastern Taiwan. Patients with moderate income levels had a 36.9% higher risk of mortality as compared with patients with high income levels (hazard ratio (HR) = 1.369; p  < 0.001). Patients with low income levels had a 51.4% greater risk of mortality than patients with high income levels (HR = 1.514, p  < 0.001)., Conclusion: In Taiwan, income and residential area significantly affected the survival rate of HNC patients receiving RT. The highest income level group had the best survival rate, regardless of the geographic area. The difference in survival between the low and high income groups was still pronounced in more deprived areas., Competing Interests: The authors declare there are no competing interests.

Copper is essential for eukaryotic life, and animals must acquire this nutrient through the diet and distribute it to cells and organelles for proper function of biological targets. Indeed, mutations in the central copper exporter ATP7A contribute to a spectrum of diseases, including Menkes disease, with symptoms ranging from neurodegeneration to lax connective tissue. As such, a better understanding of the fundamental impacts of ATP7A mutations on in vivo copper distributions is of relevance to those affected by these diseases. Here we combine metal imaging and optical imaging techniques at a variety of spatial resolutions to identify tissues and structures with altered copper levels in the Calamity gw71 zebrafish model of Menkes disease. Rapid profiling of tissue slices with LA-ICP-MS identified reduced copper levels in the brain, neuroretina, and liver of Menkes fish compared to control specimens. High resolution nanoSIMS imaging of the neuroretina, combined with electron and confocal microscopies, identified the megamitochondria of photoreceptors as loci of copper accumulation in wildtype fish, with lower levels of megamitochondrial copper observed in Calamity gw71 zebrafish. Interestingly, this localized copper decrease does not result in impaired photoreceptor development or altered megamitochondrial morphology, suggesting the prioritization of copper at sufficient levels for maintaining essential mitochondrial functions. Together, these data establish the Calamity gw71 zebrafish as an optically transparent in vivo model for the study of neural copper misregulation, illuminate a role for the ATP7A copper exporter in trafficking copper to the neuroretina, and highlight the utility of combining multiple imaging techniques for studying metals in whole organism settings with spatial resolution.

Objectives: Osteoradionecrosis of the jaws (ORNJ) is painful for patients and relatively difficult to treat clinically. The high risk of ORNJ for post radiotherapy R/T dental extraction is known; however, many patients still have to have teeth extracted after head and neck R/T. The objective of the present study is to review post R/T dental extraction and determine the ORNJ risk., Materials and Methods: We preformed a retrospective cohort study of 1759 patients with head and neck cancer s/p R/T from a random sample of 1,000,000 insurants in the National Health Insurance Research Database during 2000-2013 in Taiwan. Statistical methods included two-proportion Z-test., Results: We evaluated two cohorts: 522 patients with post R/T dental extraction and 1237 patients without post R/T extraction. Overall moderate-to-severe ORNJ after R/T was 2.22% (39/1759), and a total of 39 ORNJ cases were noted during an average of 3.02years (range: 0.62-8.89years, ±2.07). ORNJ prevalence in the overall post R/T extraction-exposed cohort (5.17%, 27/522) was significantly greater than that in the unexposed cohort (0.97%, 12/1237). In a group of patients with ⩽5 post R/T dental extractions (n=373), the ORNJ risk was 2.4% (ORNJ case n=9); in a group of patients with >5 dental extractions (n=149), the ORNJ risk was 12.1% (ORNJ case n=18) (Z-score=4.5062; p-value<0.0001). In the extraction-exposed cohort, the ORNJ risk is higher if the index day to first extraction day was ⩽0.5year (n=103) compared with the group with the index day to first extraction day >0.5year (n=419) (Z-score=-2.1506; p-value=0.0315)., Conclusion: A tooth extraction time less than half a year after R/T or during the head and neck R/T period, and extraction tooth number ⩽5 would significant lower the ORNJ prevalence., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Coronavirus (CoV) nucleocapsid (N) protein is a highly phosphorylated protein required for viral replication, but whether its phosphorylation and the related kinases are involved in the viral life cycle is unknown. We found the severe acute respiratory syndrome CoV N protein to be an appropriate system to address this issue. Using high resolution PAGE analysis, this protein could be separated into phosphorylated and unphosphorylated isoforms. Mass spectrometric analysis and deletion mapping showed that the major phosphorylation sites were located at the central serine-arginine (SR)-rich motif that contains several glycogen synthase kinase (GSK)-3 substrate consensus sequences. GSK-3-specific inhibitor treatment dephosphorylated the N protein, and this could be recovered by the constitutively active GSK-3 kinase. Immunoprecipitation brought down both N and GSK-3 proteins in the same complex, and the N protein could be phosphorylated directly at its SR-rich motif by GSK-3 using an in vitro kinase assay. Mutation of the two priming sites critical for GSK-3 phosphorylation in the SR-rich motif abolished N protein phosphorylation. Finally, GSK-3 inhibitor was found to reduce N phosphorylation in the severe acute respiratory syndrome CoV-infected VeroE6 cells and decrease the viral titer and cytopathic effects. The effect of GSK-3 inhibitor was reproduced in another coronavirus, the neurotropic JHM strain of mouse hepatitis virus. Our results indicate that GSK-3 is critical for CoV N protein phosphorylation and suggest that it plays a role in regulating the viral life cycle. This study, thus, provides new avenues to further investigate the specific role of N protein phosphorylation in CoV replication.

Persistent hepatitis B virus (HBV) infection is a major risk of hepatocellular carcinoma (HCC). One intriguing feature of HBV-related HCC is the male predominance, with a male to female ratio of 5-7:1. This dominance has been attributed to the elevated androgen level and the enhanced androgen receptor (AR)-mediated activity in the host. How HBV infection and AR signaling modulate HCC is unknown. We investigated whether the HBV nonstructural protein, X protein (HBx) could cooperate with the AR signaling pathway to enhance carcinogenesis. We found that HBx increased the anchorage-independent colony-formation potency of AR in a nontransformed mouse hepatocyte cell line. We also found that HBx functioned as a positive transcriptional coregulator to increase AR-mediated transcriptional activity. This transcription enhancement was increased in the presence of androgen in a concentration-responsive manner, thus explaining a more prominent effect in males. HBx did not physically associate with ligand-bound AR in the nucleus, and it likely augmented AR activity by increasing the phosphorylation of AR through HBx-mediated activation of the c-Src kinase signaling pathway. Our study documents HBx as a previously undescribed class of noncellular positive coregulators for AR. The results reveal a mechanism for the vulnerability of males to microbial infections and the subsequent development of cancer.

Ultralong cadmium oxide nanowires were synthesized in high yield on gold-coated silicon substrates by using a vapor transport process. Cadmium vapor generated by the carbothermal reduction of CdO powder in a tube furnace heated to 500 degrees C was carried to the substrate zone by an argon flow with a trace amount of oxygen. The CdO nanowires grew via a vapor-liquid-solid growth mechanism. The diameters of the nanowires are approximately 40-80 nm, and can reach lengths of 30-50 mum. Because the nanowire formation was gold particle catalyzed, patterned nanowire growth on substrates can be achieved. These nanowires grew along the [111] direction and have slightly rough surfaces due to the presence of crystalline CdO shells formed via a physical vapor deposition process. Interesting CdO nanowires with a necklace-like morphology were also observed in a small region of the substrate, where the oxygen supply may be ample to facilitate the lateral growth of rhombohedron-shaped crystals over the straight wires. Electron diffraction and high-resolution TEM results suggest that these side crystals should grow epitaxially on the wire surfaces. The band gap of the CdO nanowires with smoother surfaces was determined to be approximately 2.53 eV. These nanowires exhibit a relatively weak emission band centered at approximately 550 nm.

Enolase (2-phospho-D-glycerate hydrolase) is an enzymatic component of the glycolytic pathway and is conserved through evolution. The TR-CaENO1/Caeno1 stain, of which the expression of CaENO1 is under control of the tetracycline-regulatable (TR) expression system, is utilized for elucidating the functions of CaENO1 in Candida albicans. As expected, there was no detectable CaENO1 mRNA when the TR-CaENO1/Caeno1 cells grew on media containing doxycycline repressing the expression of TR-CaENO1.The TR-CaENO1/Caeno1 cells were arrested in media containing doxycycline in the presence of glucose but not in non-fermentable carbon sources, such as glycerol. Furthermore, the TR-CaENO1/Caeno1 cells were also arrested in media containing 4% serum. In this study, we have showed that CaENO1 is required for the cell growth of C. albicans in the presence of glucose. Our findings may help us to design new and more effective antifungal agents for preventing and treating bloodstream fungal infections by blocking the function(s) of enolases.

Purpose: Allelic loss at chromosome 4q21-23 occurs frequently in human hepatocellular carcinoma, and the putative tumor suppressor gene (TSG) has not yet been identified. We studied the Fas-associated phosphatase-1 (FAP-1) gene as a potential candidate TSG in this region., Experimental Design: The expression level of FAP-1 RNA in hepatocellular carcinomas was evaluated by RNase protection and quantitative PCR. Sodium bisulfite modification and subsequent single-strand conformational polymorphism and sequence analyses were used to assay the methylation of CpGs at FAP-1 promoter. Direct sequencing of the FAP-1 coding region was conducted for detecting the genetic mutations. Two common single nucleotide polymorphisms of FAP-1 were selected for evaluating their association with the hepatocellular carcinoma trait in sporadic and familial hepatocellular carcinomas. Moreover, the functional effect of FAP-1 on cellular proliferation has been evaluated by small interfering RNA approach., Results: Around 50% of hepatocellular carcinomas showed significantly decreased expression of FAP-1 compared with the corresponding nontumorous liver tissues. In most cases, the RNA level was well correlated with the methylation status of promoter CpGs, suggesting that the promoter methylation may contribute to the down-regulation. Several genetic mutations of FAP-1 have been identified in hepatocellular carcinomas. The G/G genotype of FAP-1 cSNP6304 was significantly associated with the increased risk of multiplex familial hepatocellular carcinomas (odds ratio, 2.44; 95% confidence interval, 1.19-5.01). Finally, knockdown expression of FAP-1 was shown to enhance the cellular proliferation in PLC5 cells., Conclusions: FAP-1 could be inactivated during hepatocarcinogenesis, mainly attributed by allelic loss and promoter methylation. The genetic mutations and polymorphisms may also confront with the higher hepatocellular carcinoma risk. These results first suggested FAP-1 as a putative TSG in hepatocarcinogenesis.

Unusual ZnO microspheres constructed of interconnected sheetlike nanostructures were prepared by the hydrothermal synthesis approach. These microspheres possess high surface areas (28.9 m(2)/g) and are amorphous. Trisodium citrate plays a key role in directing the formation of these microstructures. By increasing the reaction time, these microspheres gradually dissolved to form short hexagonal microrods with stacked nanoplate or nanosheet structure. The microrods were also formed under the influence of trisodium citrate. They are crystalline and show a strong (002) X-ray diffraction peak of wurtzite ZnO structure. Both microsphere and microrod samples show near-band-edge emission at approximately 385 nm, but only the microrod sample exhibits yellow luminescence at approximately 560 nm. Due to their high surface areas, these ZnO microstructures were examined for their ability to photodecompose phenol. The as-prepared samples did not display photocatalytic activity due to possible surface adsorption of solution species. However, microspheres with heat treatment to 300 degrees C can substantially enhance the photodecomposition of phenol under direct sunlight irradiation and still maintain their high surface area nanosheet structure.

Background/aims: Both viral titer and genotype of hepatitis B virus (HBV) play critical roles in determining clinical outcome and response to antiviral treatment in hepatitis B patients. In this study, a method was developed to determine both parameters in a single-tube reaction., Methods: The method contains two consecutive steps, the first step used real-time PCR for quantification and second step used melting curve analysis for genotyping. For accurate quantification, the PCR primers and hybridization probes were selected from highly conserved regions to ensure the equivalent amplification and hybridization of all genotypes of HBVs. Within the sensor probe there exists signature single nucleotide polymorphisms (SNPs), which could effectively differentiate different HBV genotypes by showing different melting temperatures., Results: The quantification results showed great consistency with the commercial assays in linear range from 10(2) to 10(11) copies/ml. By comparison with the traditional restriction fragment length polymorphism (RFLP) methods, 99% of samples were accurately genotyped by current assay, and with a higher detection rate. In addition, this method can detect mixed HBV infections., Conclusions: Currently, this methodology can be applied to areas prevalent with HBV genotypes B and C, providing an efficient alternative for clinical diagnosis and large-scaled longitudinal studies.

The coding region of the copper/zinc superoxide dismutase (Cu/Zn SOD) cDNA from papaya fruit, Carica papaya L. cv. Tainong 2, was cloned into an expression vector, pET-20b(+). The Cu/Zn SOD was expressed in Escherichia coli and purified by His-tag technique. Two active forms of the enzyme (30% dimer and 70% monomer) in equilibrium were observed. The activity of the dimeric enzyme was higher than that of the monomeric form. The thermal inactivation rate constant K(d) values calculated for the dimer and monomer at 90 degrees C were -0.0203 and -0.0216 min(-1), and the half-lives for inactivation were 41.9 and 31.8 min, respectively. This indicated that the dimeric enzyme was more stable than its monomeric form. The dimerization of the enzyme was inhibited under acidic pH (below 3.0) or imidazole buffer (above 0.5 M), whereas it was not affected under alkaline pH (above 9.0). Both activity and forms of the enzyme were not affected by 1-4% SDS. Furthermore, the dimeric enzyme was much more resistant to proteolytic attack after 3 h of incubation at 37 degrees C with trypsin or chymotrypsin. In addition, mutation of the papaya Cu/Zn SOD at position 48 from Leu to Phe (L48F) affected the association of monomer, whereas a mutant with Lys substitution (L48K) at the same position tended to dissociate into monomeric form.

A full-length complementary DNA (cDNA) clone encoding a putative copper/zinc superoxide dismutase (Cu/Zn SOD) of papaya fruit, Carica papaya L. cv. Tainong 2, was amplified according to the polymerase chain reaction technique from cDNA synthesized from fruit messenger RNA. Nucleotide sequence analysis of this cDNA clone revealed that it comprised a complete open reading frame coding for 152 amino acid residues. The deduced amino acid sequence showed higher identity (81.6-84.9%) with the sequences of the cytosolic Cu/Zn SODs than those of the chloroplast from other plant species, and no recognizable plastid targeting peptide was found. These suggest that the papaya fruit cDNA clone encodes a cytosolic Cu/Zn SOD. The residues required for coordinating copper (His-45, -47, -62, and -119) and zinc (His-62, -70, and -79 and Asp-82), as well as the two cysteines (56 and 145) that form a single disulfide bond, are conserved as they are among all reported Cu/Zn SOD sequences. In addition, the coding region of Cu/Zn SOD cDNA from papaya was introduced into an expression vetor, pET-20b(+), and transformed into Escherichia coli AD494(DE3)pLysS. A predominant protein band was detected by Coomassie blue staining of native PAGE, and activity staining confirmed the result of Coomassie blue staining. These indicate that this Cu/Zn SOD cDNA clone can overexpress active Cu/Zn SOD enzyme in E. coli.