Unraveling the Heat- and UV-Induced Degradation of Mixed Halide Perovskite Thin Films via Surface Analysis Techniques.

In recent years, organic-inorganic hybrid perovskite materials have become one of the most promising materials in the new generation of solar cells. These perovskites can provide excellent photoelectric properties after a simple fabrication process. Although perovskite solar cells have achieved high power conversion efficiency, instability concerns regarding material exposure to heat, moisture, air, and UV light present hindrances to commercialization. In this study, three kinds of perovskites (MAPbI3, MAPbI3-xBrx, and MAPbI3-xClx) were used to investigate the crystal stability upon exposure to heat and UV light. SEM, XRD, and FTIR were used to observe the surface morphology, crystal structure, and functional groups of the perovskite thin films. XPS was used to examine the surface composition and chemical state of the perovskite thin films under different conditions. Among these three types of perovskites, it was found that the MAPbI3-xBrx crystal demonstrated the best stability. ToF-SIMS was used to confirm the molecular distribution of the MAPbI3-xBrx films upon exposure to heat and UV light at different depths. ToF-SIMS revealed that [Pb]+ and [PbI]+ aggregated at the interface between the perovskite and ITO substrate after 14 days of thermal treatment. On the other hand, [Pb]+ and [PbI]+ were distributed uniformly after 3 days of UV exposure. This study systematically analyzed and revealed the thermal- and UV-induced degradation process of three perovskite films by using surface analysis techniques. It was concluded that bromine-doped perovskite films had better stability, and UV light caused more severe damage than heat.

Short-term multidisciplinary family-centered workshop for preschool children with global developmental delays.

BACKGROUND: Children with developmental delays have a great impact on their families. Educating families on how to interact with their children is an important task. Therefore, we assessed the short-term effectiveness of the workshop for children with global developmental delays. METHODS: In total, 101 children aged 18-36 months with global developmental delays, all with language delay along with other developmental delays, and their parents participated in six 2-h family-centered workshop sessions for six weeks. Measures were taken before and after the workshop, including the Mandarin-Chinese Communicative Developmental Inventory, Peabody Developmental Motor Scales, Emotional Competency Rating Scales, Pediatric Outcomes Data Collection Instrument, Pediatric Evaluation of Disability Inventory, Pediatric Daily Occupation Scale, Pediatric Quality of Life Inventory (PedsQL), Caregiver Strain Index, and PedsQL-Family Impact Module. RESULTS: Significant improvements with a small or intermediate effect size in emotions, upper extremity and physical functioning and global functioning, daily occupation performance in sensorimotor, communication, cognitive autonomy, and psychosocial domains, and parental quality of life and family impact were noted with high workshop satisfaction. CONCLUSION: Short-term family-centered workshop is effective for children with global developmental delays. However, due to the lack of follow-up after the intervention, it should be careful in inferring the developmental gain effect. IMPACT: The effectiveness of short-term family-centered workshops on children with global developmental delays remains uncertain. Short-term family-centered workshops improved the children's emotions, physical functional performance, and occupational performance in daily life. The short-term family-centered workshop is practical and effective for children with global developmental delays. Further long-term, large-scale, prospective, randomized trials are warranted to confirm these results. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov ID: NCT05418933.

Fisetin attenuates doxorubicin-induced cardiotoxicity by inhibiting the insulin-like growth factor II receptor apoptotic pathway through estrogen receptor-α/-ß activation.

Doxorubicin (DOX), an effective chemotherapeutic drug, has been used to treat various cancers; however, its cardiotoxic side effects restrict its therapeutic efficacy. Fisetin, a flavonoid phytoestrogen derived from a range of fruits and vegetables, has been reported to exert cardioprotective effects against DOX-induced cardiotoxicity; however, the underlying mechanisms remain unclear. This study investigated fisetin's cardioprotective role and mechanism against DOX-induced cardiotoxicity in H9c2 cardiomyoblasts and ovariectomized (OVX) rat models. MTT assay revealed that fisetin treatment noticeably rescued DOX-induced cell death in a dose-dependent manner. Moreover, western blotting and TUNEL-DAPI staining showed that fisetin significantly attenuated DOX-induced cardiotoxicity in vitro and in vivo by inhibiting the insulin-like growth factor II receptor (IGF-IIR) apoptotic pathway through estrogen receptor (ER)-α/-ß activation. The echocardiography, biochemical assay, and H&E staining results demonstrated that fisetin reduced DOX-induced cardiotoxicity by alleviating cardiac dysfunction, myocardial injury, oxidative stress, and histopathological damage. These findings imply that fisetin has a significant therapeutic potential against DOX-induced cardiotoxicity.

A Wearable Assistant Device for the Hearing Impaired to Recognize Emergency Vehicle Sirens with Edge Computing.

Wearable assistant devices play an important role in daily life for people with disabilities. Those who have hearing impairments may face dangers while walking or driving on the road. The major danger is their inability to hear warning sounds from cars or ambulances. Thus, the aim of this study is to develop a wearable assistant device with edge computing, allowing the hearing impaired to recognize the warning sounds from vehicles on the road. An EfficientNet-based, fuzzy rank-based ensemble model was proposed to classify seven audio sounds, and it was embedded in an Arduino Nano 33 BLE Sense development board. The audio files were obtained from the CREMA-D dataset and the Large-Scale Audio dataset of emergency vehicle sirens on the road, with a total number of 8756 files. The seven audio sounds included four vocalizations and three sirens. The audio signal was converted into a spectrogram by using the short-time Fourier transform for feature extraction. When one of the three sirens was detected, the wearable assistant device presented alarms by vibrating and displaying messages on the OLED panel. The performances of the EfficientNet-based, fuzzy rank-based ensemble model in offline computing achieved an accuracy of 97.1%, precision of 97.79%, sensitivity of 96.8%, and specificity of 97.04%. In edge computing, the results comprised an accuracy of 95.2%, precision of 93.2%, sensitivity of 95.3%, and specificity of 95.1%. Thus, the proposed wearable assistant device has the potential benefit of helping the hearing impaired to avoid traffic accidents.

Pheretima aspergillum extract attenuates high-KCl-induced mitochondrial injury and pro-fibrotic events in cardiomyoblast cells.

Hyperkalemia is often associated with cardiac dysfunction. In this study an earthworm extract (dilong) was prepared from dried Pheretima aspergillum powder and its effect against high-KCl challenge was determined in H9c2 cardiomyoblast cells. H9c2 cells pre-treated with dilong (31.25, 62.5, 125, and 250 mg/mL) for 24 hours, where challenged with different doses of KCl treatment for 3 hours to determine the protective mechanisms of dilong against cardiac fibrosis. High-KCl administration induced mitochondrial injury and elevated the levels of pro-apoptotic proteins. The mediators of fibrosis such as ERK, uPA, SP1, and CTGF were also found to be upregulated in high-KCl condition. However, dilong treatment enhanced IGF1R/PI3k/Akt activation which is associated with cell survival. In addition, dilong also reversed high-KCl induced cardiac fibrosis related events in H9c2 cells and displayed a strong cardio-protective effect. Therefore, dilong is a potential agent to overcome cardiac events associated with high-KCl toxicity.

Alterations of Renal Epithelial Glucose and Uric Acid Transporters in Fructose Induced Metabolic Syndrome.

BACKGROUND/AIMS: Hyperglycemia and hyperuricemia are two major disorders of Metabolic syndrome. Kidney plays a crucial role in maintaining the homeostasis of uric acid and glucose. The aim of the study was to examine the changes of renal glucose and uric acid transporters in animals with metabolic syndrome. METHODS: Sprague-Dawley rats were fed with high fructose diet (60%) for 3 months (FR-3) and 5 months (FR-5). At the end study, serum and urine biochemical data were compared. Gene expression and protein abundance of renal GLUT1, GLUT2, GLUT9, SGLT1, SGLT2, UAT and URAT1 was investigated by using RT-PCR and immunohistochemical staining. RESULTS: Metabolic syndrome was induced by high-fructose diet. Systolic blood pressure and proteinuria was significantly increased in FR-5 animals. In kidney tissue, gene expression of GLUT2 and SGLT2 increased significantly in a time dependent manner. GLUT9, SGLT1 and UAT were also significantly upregulated in FR-5. Immunohistochemical study showed a significant increase of SGLT1 in both FR-3 (413.5 ± 88.3% of control, p< 0.001) and FR-5 (677.6 ± 26.5% of control, p< 0.001). Also, SGLT2 protein was increased in both FR-3 (643.1 ± 41.3% of control, p< 0.001) and FR-5 (563.3 ± 21.7% of control, p< 0.001). Fructose rich food also induced increase of UAT by nearly 5-fold in both FR-3 and FR-5 (both p< 0.05) and more than 3-fold of GLUT-9 in FR-3 and FR-5 (both p< 0.05). CONCLUSION: Long term high fructose diet induced metabolic syndrome with increased blood pressure and proteinuria in rats. Metabolic syndrome was associated with dual increase in renal glucose and uric acid transporters, including SGLT1, SGLT2, GLUT2, GLUT9 and UAT.

Cellular apoptosis and cardiac dysfunction in STZ-induced diabetic rats attenuated by anthocyanins via activation of IGFI-R/PI3K/Akt survival signaling.

Anthocyanins are known cyto-protective agents against various stress conditions. In this study cardio-protective effect of anthocyanins from black rice against diabetic mellitus (DM) was evaluated using a streptozotocin (STZ)-induced DM rat model. Five-week-old male Wistar rats were administered with STZ (55 mg kg-1 , IP) to induce DM; rats in the treatment group received 250 mg oral anthocyanin/kg/day during the 4-week treatment period. DM and the control rats received normal saline through oral gavage. The results reveal that STZ-induced DM elevates myocardial apoptosis and associated proapoptotic proteins but down-regulates the proteins of IGF1R mediated survival signaling mechanism. Furthermore, the functional parameters such as the ejection-fraction and fraction-shortening in the DM rat hearts declined considerably. However, the rats treated with anthocyanins significantly reduced apoptosis and the associated proapoptotic proteins and further increased the survival signals to restore the cardiac functions in DM rats. Anthocyanin supplementation enhances cardiomyocyte survival and restores cardiac function.

Anthocyanin Attenuates Doxorubicin-Induced Cardiomyotoxicity via Estrogen Receptor-α/ß and Stabilizes HSF1 to Inhibit the IGF-IIR Apoptotic Pathway.

Doxorubicin (Dox) is extensively used for chemotherapy in different types of cancer, but its use is limited to because of its cardiotoxicity. Our previous studies found that doxorubicin-induced insulin-like growth factor II receptor (IGF-IIR) accumulation causes cardiomyocytes apoptosis via down-regulation of HSF1 pathway. In these studies, we demonstrated a new mechanism through which anthocyanin protects cardiomyoblast cells against doxorubicin-induced injury. We found that anthocyanin decreased IGF-IIR expression via estrogen receptors and stabilized heat shock factor 1 (HSF1) to inhibit caspase 3 activation and apoptosis of cardiomyocytes. Therefore, the phytoestrogen from plants has been considered as another potential treatment for heart failure. It has been reported that the natural compound anthocyanin (ACN) has the ability to reduce the risk of cardiovascular disease (CVD). Here, we demonstrated that anthocyanin acts as a cardioprotective drug against doxorubicin-induced heart failure by attenuating cardiac apoptosis via estrogen receptors to stabilize HSF1 expression and down-regulated IGF-IIR-induced cardiomyocyte apoptosis.

Renin angiotensin system blockade ameliorates lead nephropathy.

Lead intoxication is usually insidious and may cause a variety of complications such as kidney damage and hypertension. The role of intrarenal renin-angiotensin system (RAS) in lead-induced nephropathy has not been investigated. Adult male Sprague-Dawley rats were fed with water containing 250ppm of lead acetate (lead group) and deionized water (control group) for 4weeks. Another two groups started to receive intraperitoneal captopril (50mg/kg/d) or losartan (10mg/kg/d) after 2weeks of lead feeding and continued for another 2weeks. Immunoblotting was used to analyze the protein amount of intrarenal RAS components and transforming growth factor-beta (TGF-ß). Compared with control group, lead exposure resulted in increased proteinuria after 2-week treatment (4.2±0.9mg/100g vs. 1.8±0.8mg/100g, p<0.05) and 4-week (5.2±1.7mg/100g, p<0.05). Serum creatinine level was increased (0.40±0.2 vs. 0.3 ±.04mg/dL, p<0.05) and calculated glomerular filtration rate (GFR) was decreased (2.68±1.03 vs. 3.37±0.11mL/min, p<0.05). Intrarenal angiotensin converting enzyme (ACE), angiotensin II (ANG II), angiotensin II type 1 receptor (AT1R) and transforming growth factor-beta (TGF-ß) were upregulated in lead group. Captopril and losartan administration reduced proteinuria significantly (3.0±0.50mg/100g of captopril and 2.7±0.4mg/100g of losartan group) and lowered systolic blood pressure when compared with lead group. Furthermore, serum creatinine levels and GFR were improved by RAS blockade. Captopril treatment significantly reduced protein abundance of ACE, ANG II, AT1R and TGF-ß. Losartan treatment also decreased ANG II and TGF-ß. We concluded that lead exposure elicited intrarenal RAS activation with associated proteinuria and impaired renal function. RAS blockade was effective in alleviating lead-associated kidney injury and lowering blood pressure.

Layout Dependence Stress Investigation in through Glass via Interposer Architecture Using a Submodeling Simulation Technique and a Factorial Design Approach.

The multi-chiplet technique is expected to be a promising solution to achieve high-density system integration with low power consumption and high usage ratio. This technique can be integrated with a glass interposer to accomplish a competitive low fabrication cost compared with the silicon-based interposer architecture. In this study, process-oriented stress simulation is performed by the element activation and deactivation technique in finite element analysis architecture. The submodeling technique is also utilized to mostly conquer the scale mismatch and difficulty in mesh gridding design. It is also used to analyze the thermomechanical responses of glass interposers with chiplet arrangements and capped epoxy molding compounds (EMC) during curing. A three-factor, three-level full factorial design is applied using the analysis of variance method to explore the significance of various structural design parameters for stress generation. Analytic results reveal that the maximum first principal stresses of 130.75 and 17.18 MPa are introduced on the sidewall of Cu-filled via and the bottom of the glass interposer, respectively. Moreover, the EMC thickness and through glass via pitch are the dominant factors in the adopted vehicle. They significantly influence the stress magnitude during heating and cooling.

Reliability Assessment of Thermocompressed Epoxy Molding Compound through Glass via Interposer Architecture by the Submodeling Simulation Approach.

In glass interposer architecture and its assembly process, the mechanical responses of interposer structure under thermocompression process-induced thermal loading and generated shrinkage of molding material are regarded as a major reliability issue. Thousands of metal-filled via are involved in glass interposers and are regarded as a potential risk that can lead to cracking and the failure of an entire vehicle. In this study, a finite element-based submodeling approach is demonstrated to overcome the complexity of modeling and the relevant convergence issue of interposer architecture. Convergence analysis results revealed that at least four via pitch-wide regions of a local simulation model were needed to obtain the stable results enabled by the submodeling simulation approach. The stress-generation mechanism during thermocompression, the coefficient of thermal expansion mismatch, and the curing process-induced shrinkage were separately investigated. The critical stress location was explored as the outer corner of the chip, and the maximum first principal stress during the thermocompression process generated on the chip and glass interposer were 34 and 120 MPa, respectively.

Stress Impact of the Annealing Procedure of Cu-Filled TSV Packaging on the Performance of Nano-Scaled MOSFETs Evaluated by an Analytical Solution and FEA-Based Submodeling Technique.

Stress-induced performance change in electron packaging architecture is a major concern when the keep-out zone (KOZ) and corresponding integration density of interconnect systems and transistor devices are considered. In this study, a finite element analysis (FEA)-based submodeling approach is demonstrated to analyze the stress-affected zone of through-silicon via (TSV) and its influences on a planar metal oxide semiconductor field transistor (MOSFET) device. The feasibility of the widely adopted analytical solution for TSV stress-affected zone estimation, Lamé radial stress solution, is investigated and compared with the FEA-based submodeling approach. Analytic results reveal that the Lamé stress solution overestimates the TSV-induced stress in the concerned device by over 50%, and the difference in the estimated results of device performance between Lamé stress solution and FEA simulation can reach 22%. Moreover, a silicon-germanium-based lattice mismatch stressor is designed in a silicon p-type MOSFET, and its effects are analyzed and compared with those of TSV residual stress. The S/D stressor dominates the stress status of the device channel. The demonstrated FEA-based submodeling approach is effective in analyzing the stress impact from packaging and device-level components and estimating the KOZ issue in advanced electronic packaging.

Auger Electron Spectroscopy Analysis of the Thermally Induced Degradation of MAPbI3 Perovskite Films.

Organometal halide perovskites are highly promising materials for photovoltaic applications due to the rapid growth of power conversion efficiency in recent years. However, thermal stability is still a major hurdle for perovskite solar cells toward commercialization. Herein, we first explore the slow thermal response of the CH3NH3PbI3 perovskite crystal investigated via Auger electron spectroscopy (AES). AES image mapping directly observes the evolution of morphology and elemental distribution over time. The AES small spot analysis demonstrates the precise initial degradation position of perovskite with both information regarding physical changes in crystals and chemical changes in elemental bonding at the nanometer scale. X-ray photoelectron spectroscopy (XPS) was used to confirm the surface chemical bonding and composition of the perovskite crystals. This work provides the first insights into the physical and chemical changes of perovskites investigated by AES upon long-term exposure to heat under ambient conditions.

Comprehensive Stress Effect of Thin Coatings and Silicon-Carbon Lattice Mismatch on Nano-Scaled Transistors with Protruding Poly Gate.

Enhancing the mobility in metal-oxide-semiconductor field-effect transistors (MOSFETs) with narrow channel widths is highly sensitive to the stress effects of Si channel when related advanced strain engineering is introduced and is compatible with semiconductor processes. In practice, layouts have significant effect on the device performance, especially for the protruding gate width on shallow trench isolation structures. The geometric parameter is investigated by systematically analysing an n-channel MOSFET composed of silicon-carbon (SiC) stressors embedded in the source and drain (S/D) regions and a tensile contact etch stop layer (CESL) using three-dimensional finite element simulation. Tensile CESL (1.1 GPa) and a SiC S/D stressor with a carbon mole fraction of 1.65% are loaded on the structure. The difference in the interactive percentages between the mechanical bending effect of the CESL from the top of the poly gate and the downward force of the CESL adjacent to the spacer sidewall of the gate occurs when the protruding gate width is increased. Results indicate that mobility was highly enhanced by approximately 72.5% at a width of approximately 0.2 µm. The mechanical bending effect becomes dominant when the gate width is more than 0.2 µm. Consequently, the mobility gain decays and consequently converges toward a constant.

Energy and nitrogenous waste from glutamate/glutamine catabolism facilitates acute osmotic adjustment in non-neuroectodermal branchial cells.

Maintenance of homeostasis is one of the most important physiological responses for animals upon osmotic perturbations. Ionocytes of branchial epithelia are the major cell types responsible for active ion transport, which is mediated by energy-consuming ion pumps (e.g., Na+-K+-ATPase, NKA) and secondary active transporters. Consequently, in addition to osmolyte adjustments, sufficient and immediate energy replenishment is essenttableial for acclimation to osmotic changes. In this study, we propose that glutamate/glutamine catabolism and trans-epithelial transport of nitrogenous waste may aid euryhaline teleosts Japanese medaka (Oryzias latipes) during acclimation to osmotic changes. Glutamate family amino acid contents in gills were increased by hyperosmotic challenge along an acclimation period of 72 hours. This change in amino acids was accompanied by a stimulation of putative glutamate/glutamine transporters (Eaats, Sat) and synthesis enzymes (Gls, Glul) that participate in regulating glutamate/glutamine cycling in branchial epithelia during acclimation to hyperosmotic conditions. In situ hybridization of glutaminase and glutamine synthetase in combination with immunocytochemistry demonstrate a partial colocalization of olgls1a and olgls2 but not olglul with Na+/K+-ATPase-rich ionocytes. Also for the glutamate and glutamine transporters colocalization with ionocytes was found for oleaat1, oleaat3, and olslc38a4, but not oleaat2. Morpholino knock-down of Sat decreased Na+ flux from the larval epithelium, demonstrating the importance of glutamate/glutamine transport in osmotic regulation. In addition to its role as an energy substrate, glutamate deamination produces NH4+, which may contribute to osmolyte production; genes encoding components of the urea production cycle, including carbamoyl phosphate synthetase (CPS) and ornithine transcarbamylase (OTC), were upregulated under hyperosmotic challenges. Based on these findings the present work demonstrates that the glutamate/glutamine cycle and subsequent transepithelial transport of nitrogenous waste in branchial epithelia represents an essential component for the maintenance of ionic homeostasis under a hyperosmotic challenge.

The Effects of a Supportive Care Program on the Posttraumatic Stress Symptoms of Patients With Oral Cancer After Surgery.

This study aimed to estimate the effects of a supportive care program on the posttraumatic stress symptoms (PTSSs) of patients with oral cancer after surgery. Participants were divided into two groups. Outcome measurements included the Chinese version of the Davidson trauma scale to examine PTSSs at a clinical follow-up 1 week (T0), 1 month (T1), and 3 months (T2) after hospital discharge. The results indicated that the frequency and severity mean scores of PTSSs for the two groups at T0 were significantly higher than those at T1 and T2. Both the groups and times were significantly different; moreover, the supported group's PTSS score decreased more than that of the nonsupported group. These findings supported the effects of the supportive care program. Health care staff should be aware of the PTSS status of patients with oral cancer who undergo surgery and consider these issues in combination with patients' discharge care plans.

A multisampling reporter system for monitoring microRNA activity in the same population of cells.

MicroRNAs (miRNAs) downregulate gene expression by binding to the partially complementary sites in the 3' untranslated region (UTR) of target mRNAs. Several methods, such as Northern blot analysis, quantitative real-time RT-PCR, microarray, and the luciferase reporter system, are commonly used to quantify the relative level or activity of miRNAs. The disadvantage of these methods is the requirement for cell lysis, which means that several sets of wells/dishes of cells must be prepared to monitor changes in miRNA activity in time-course studies. In this study, we developed a multisampling reporter system in which two secretable bioluminescence-generating enzymes are employed, one as a reporter and the other as an internal control. The reporters consist of a pair of vectors containing the Metridia luciferase gene, one with and one without a duplicated miRNA targeting sequence at their 3'UTR, while the other vector coding for the secreted alkaline phosphatase gene is used as an internal control. This method allows miRNA activity to be monitored within the same population of cells over time by withdrawing aliquots of the culture medium. The practicability and benefits of this system are addressed in this report.

Two C-terminal amino acids, Ser(334) and Ser(335), are required for homologous desensitization and agonist-induced phosphorylation of opioid receptor-like 1 receptors.

Various cellular signaling pathways induced by nociceptin activation of ORL1 (opioid receptor-like 1 receptor) develop homologous desensitization. Multiple lines of evidence suggest that agonist-induced phosphorylation of serine (Ser)/threonine (Thr) residues at intracellular carboxyl tail leads to homologous desensitization of G protein-coupled receptors. In the present study, we investigated the functional role played by C-terminal Ser/Thr residues in agonist-induced desensitization and phosphorylation of ORL1. In HEK 293 cells expressing wild-type ORL1 and ORL1(CDelta21), which lacks C-terminal 21 amino acids, nociceptin inhibition of adenylate cyclase activity exhibited homologous desensitization after 1 h pretreatment of nociceptin. In contrast, ORL1(CDelta34), which differs with ORL1(CDelta21) by lacking C-terminal Ser(334), Ser(335) and Ser(343) residues, failed to develop agonist-induced desensitization. Point mutant (S343A) ORL1 underwent homologous desensitization after nociceptin pretreatment. Substituting Ser(334) or Ser(335) with alanine greatly impaired nociceptin-induced ORL1 desensitization. In HEK 293 cells expressing double mutant (S334A/S335A) ORL1, nociceptin pretreatment failed to significantly affect the efficacy and potency by which nociceptin inhibits forskolin-stimulated cAMP formation. Mutation of Ser(334) and Ser(335) also greatly reduced nociceptin-induced ORL1 phosphorylation. These results suggest that two C-terminal serine residues, Ser(334) and Ser(335), are required for homologous desensitization and agonist-induced phosphorylation of ORL1.

Polyglutamine-expanded ataxin-7 activates mitochondrial apoptotic pathway of cerebellar neurons by upregulating Bax and downregulating Bcl-x(L).

Spinocerebellar ataxia type 7 (SCA7) is an autosomal dominant neurodegenerative disorder caused by polyglutamine-expanded ataxin-7. In the present investigation, we expressed disease-causing mutant ataxin-7-Q75 in the primary neuronal culture of cerebellum with the aid of recombinant adenoviruses. Subsequently, this in vitro cellular model of SCA7 was used to study the molecular mechanism by which mutant ataxin-7-Q75 induces neuronal death. TUNEL staining studies indicated that polyglutamine-expanded ataxin-7-Q75 caused apoptotic cell death of cultured cerebellar neurons. Mutant ataxin-7-Q75 induced the formation of active caspase-3 and caspase-9 without activating caspase-8. Polyglutamine-expanded ataxin-7-Q75 promoted the release of apoptogenic cytochrome-c and Smac from mitochondria, which was preceded by the downregulation of Bcl-x(L) protein and upregulation of Bax protein expression in cultured cerebellar neurons. Further real-time TaqMan RT-PCR assays showed that mutant ataxin-7-Q75 upregulated Bax mRNA level and downregulated Bcl-x(L) mRNA expression in the primary neuronal culture of cerebellum. The present study provides the evidence that polyglutamine-expanded ataxin-7-Q75 activates mitochondria-mediated apoptotic cascade and induces neuronal death by upregulating Bax expression and downregulating Bcl-x(L) expression of cerebellar neurons.

Gel electrophoresis of polyphenol oxidase with instant identification by in situ blotting.

Polyphenol oxidase (PPO) or tyrosinase is an important and ubiquitous enzyme responsible for browning in plants and melanization in animals. The molecular size of the plant PPO is varied among the species and its activity can be enhanced by a variety of anionic detergents. In the present study, we developed a simple method for the first-step identification of PPO in fruit and vegetable extracts. First, 3mm chromatographic paper was immersed in 0.5% (w/v) catechol solution as an immobilized PPO substrate. After running the extract with 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), one side of the glass plate was removed. The plate was immediately laid on top of the dried catechol-paper. A dark-brown band corresponding to PPO was visualized within 1 min and was further confirmed by a conventional Western blot using an antibody prepared against mushroom PPO. It also reveals that some vegetation (such as tomato, radish, and oriental melon) with low or no detectable activity in a conventional enzyme assay actually possessed marked levels of PPO activity when assessed by PAGE-blot. We propose that an inhibitor is associated with PPO in some plants; the inhibitor, however, is dissociated during the electrophoresis. Therefore, in addition to identify the molecular form of PPO, the present technique may explore the existence of PPO inhibitor(s) in plants. The detail of the method with respect to its relevance for searching a natural PPO inhibitor is described and discussed.