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Malarmugila Manimaran,1,* Yin Yin Teo,2 James Chen Yong Kah,3 Adilet Beishenaliev,1 Yean Leng Loke,2 Yiing Yee Foo,1 Shiow-Fern Ng,4 Chin Fei Chee,5 Sek Peng Chin,6 Farid Nazer Faruqu,1 Chia-Yu Chang,7 Misni Misran,2 Lip Yong Chung,6 Bey Fen Leo,8,* Shih-Hwa Chiou,9,10 Chia-Ching Chang,7,11– 13 Sun Tee Tay,14 Lik Voon Kiew1,7 1Department of Pharmacology, Faculty of Medicine, Universiti Malaya, Kuala Lumpur, Malaysia; 2Department of Chemistry, Faculty of Science, Universiti Malaya, Kuala Lumpur, Malaysia; 3Department of Biomedical Engineering, College of Design and Engineering, National University of Singapore, Singapore, Singapore; 4Centre for Drug Delivery Technology and Vaccine, Faculty of Pharmacy, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia; 5Nanotechnology Catalysis Research Centre, Universiti Malaya, Kuala Lumpur, Malaysia; 6Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Universiti Malaya, Kuala Lumpur, Malaysia; 7Department of Biological Science and Technology, National Yang Ming Chiao Tung University, Hsinchu, Taiwan, Republic of China; 8Department of Molecular Medicine, Faculty of Medicine, Universiti Malaya, Kuala Lumpur, Malaysia; 9Department of Medical Research, Taipei Veterans General Hospital, Taipei, Taiwan, Republic of China; 10Institute of Pharmacology, National Yang Ming Chiao Tung University, Taipei, Taiwan, Republic of China; 11Department of Electrophysics, National Yang Ming Chiao Tung University, Hsinchu, Taiwan, Republic of China; 12Center for Intelligent Drug Systems and Smart Bio-devices, National Yang Ming Chiao Tung University, Hsinchu, Taiwan, Republic of China; 13Institute of Physics, Academia Sinica, Nankang, Taipei, Taiwan, Republic of China; 14Department of Medical Microbiology, Faculty of Medicine, Universiti Malaya, Kuala Lumpur, Malaysia*These authors contributed equally to this workCorrespondence: Chia-Ching Chang; Lik Voon Kiew, Email ccchang01@nycu.edu.tw; lvkiew@um.edu.myIntroduction: Over 75% of clinical microbiological infections are caused by bacterial biofilms that grow on wounds or implantable medical devices. This work describes the development of a new poly(diallyldimethylammonium chloride) (PDADMAC)/alginate-coated gold nanorod (GNR/Alg/PDADMAC) that effectively disintegrates the biofilms of Staphylococcus aureus (S. aureus), a prominent pathogen responsible for hospital-acquired infections.Methods: GNR was synthesised via seed-mediated growth method, and the resulting nanoparticles were coated first with Alg and then PDADMAC. FTIR, zeta potential, transmission electron microscopy, and UV–Vis spectrophotometry analysis were performed to characterise the nanoparticles. The efficacy and speed of the non-coated GNR and GNR/Alg/PDADMAC in disintegrating S. aureus-preformed biofilms, as well as their in vitro biocompatibility (L929 murine fibroblast) were then studied.Results: The synthesised GNR/Alg/PDADMAC (mean length: 55.71 ± 1.15 nm, mean width: 23.70 ± 1.13 nm, aspect ratio: 2.35) was biocompatible and potent in eradicating preformed biofilms of methicillin-resistant (MRSA) and methicillin-susceptible S. aureus (MSSA) when compared to triclosan, an antiseptic used for disinfecting S. aureus colonisation on abiotic surfaces in the hospital. The minimum biofilm eradication concentrations of GNR/Alg/PDADMAC (MBEC50 for MRSA biofilm = 0.029 nM; MBEC50 for MSSA biofilm = 0.032 nM) were significantly lower than those of triclosan (MBEC50 for MRSA biofilm = 10,784 nM; MBEC50 for MRSA biofilm 5967 nM). Moreover, GNR/Alg/PDADMAC was effective in eradicating 50% of MRSA and MSSA biofilms within 17 min when used at a low concentration (0.15 nM), similar to triclosan at a much higher concentration (50 μM). Disintegration of MRSA and MSSA biofilms was confirmed by field emission scanning electron microscopy and confocal laser scanning microscopy.Conclusion: These findings support the potential application of GNR/Alg/PDADMAC as an alternative agent to conventional antiseptics and antibiotics for the eradication of medically important MRSA and MSSA biofilms.Keywords: biofilm, gold nanorod, S. aureus, PDADMAC, MRSA, MSSA

Factors that influence primary school teachers’ self-efficacy in online environment are still seldom researched. There are needs to determine these factors to improve online teaching, which is a new norm. The aims of this study were to evaluate the level of teachers’ self-efficacy in online teaching and to identify the factors that influence it. This was a quantitative study with survey method that involved 155 primary school teachers in Lenggong Zone, Hulu Perak District. Simple random sampling technique was used for sampling purposes. Teachers’ self-efficacy was measured by the Teachers’ Self-Effiacay Scale while factors tthat influence teachers’ self-efficacy was gauged by the Sources of Influence on Teachers’ Self-Efficacy Scale. The factors measured include mastery experience, vicarious experience, verbal persuasion, physiological arousal and contextual factor. Data were analysed with descriptive and inferential statistics. Findings show that primary school teachers have got high self-efficacy in online teaching. Multiple linear regression analysis revealed that mastery experience, vicarious experience, verbal persuasion and physiological arousal factors have got significant influence on teachers’ self-efficacy in online teaching. Contextual factor was the only factor that did not have significant influence on teachers’ self-efficacy in online teaching. Findings of this study can be used to develop intervention programmes to improve teachers’ self-efficacy in online teaching and ultimately to enhance the effectiveness of online teaching in primary schools.

Abstract Background Retinoblastoma is caused by compound heterozygosity or homozygosity of retinoblastoma gene (RB1) mutations. In germline retinoblastoma, mutations in the RB1 gene predispose individuals to increased cancer risks during development. These mutations segregate as autosomal dominant traits with high penetrance (90%). Methods We screened 30 family members from one family using high resolution melting assay and DNA direct sequencing for mutations in the RB1 gene. We evaluate the phenotype and penetrance of germline mutations of the RB1 gene in a large Taiwanese family. Results The molecular analysis and clinical details of this family showed phenotypic variability associated with the p.V654L mutation in exon 19 of the RB1 gene in 11 family members. The phenotype varied from asymptomatic to presence of a unilateral tumor. Only four individuals (2 males and 2 females) developed unilateral retinoblastoma, which resulted in calculated low penetrance of 36% (4/11). The four individuals with retinoblastoma were diagnosed before the age of three years. None of their relatives exhibited variable severity or bilateral retinoblastoma. Conclusions The diseased-eye ratio for this family was 0.36, which is lower than current estimates. This suggests that the RB1 p.V654L mutation is a typical mutation associated with low penetrance.

Abstract Background 1-{4-[Bis(2-chloroethyl)amino]phenyl}-3-[2-methyl-5-(4-methylacridin-9-ylamino)phenyl]urea (BO-1051) is an N-mustard DNA alkylating agent reported to exhibit antitumor activity. Here we further investigate the effects of this compound on radiation responses of human gliomas, which are notorious for the high resistance to radiotherapy. Methods The clonogenic assay was used to determine the IC50 and radiosensitivity of human glioma cell lines (U87MG, U251MG and GBM-3) following BO-1051. DNA histogram and propidium iodide-Annexin V staining were used to determine the cell cycle distribution and the apoptosis, respectively. DNA damage and repair state were determined by γ-H2AX foci, and mitotic catastrophe was measure using nuclear fragmentation. Xenograft tumors were measured with a caliper, and the survival rate was determined using Kaplan-Meier method. Results BO-1051 inhibited growth of human gliomas in a dose- and time-dependent manner. Using the dosage at IC50, BO-1051 significantly enhanced radiosensitivity to different extents [The sensitizer enhancement ratio was between 1.24 and 1.50 at 10% of survival fraction]. The radiosensitive G2/M population was raised by BO-1051, whereas apoptosis and mitotic catastrophe were not affected. γ-H2AX foci was greatly increased and sustained by combined BO-1051 and γ-rays, suggested that DNA damage or repair capacity was impaired during treatment. In vivo studies further demonstrated that BO-1051 enhanced the radiotherapeutic effects on GBM-3-beared xenograft tumors, by which the sensitizer enhancement ratio was 1.97. The survival rate of treated mice was also increased accordingly. Conclusions These results indicate that BO-1051 can effectively enhance glioma cell radiosensitivity in vitro and in vivo. It suggests that BO-1051 is a potent radiosensitizer for treating human glioma cells.

Abstract Background The root of Boehmeria spp (ramie) is a hepatoprotective Chinese herbal medicine. Medicinal properties vary between Boehmeria nivea var. nivea and Boehmeria nivea var. tenacissima, which are local species found in Taiwan. As commercial preparations may use either species, there is a need for a rapid and simple assay to identify variants for quality control. Methods Four methods were developed and tested for their applicability in differentiating the two species. These methods were random amplified polymorphic DNA (RAPD); sequence characterized amplified regions (SCAR); single nucleotide polymorphisms (SNP) and cleaved amplified polymorphic sequences (CAPS). Results Three RAPD markers were developed that produced unique bands in B. nivea var. tenacissima and B. nivea var. nivea. Based on sequenced RAPD bands, one SCAR marker was developed that produced a single DNA band in B. nivea var. nivea. Two SNP markers differentiated between B. nivea var. nivea and B. nivea var. tenacissima based on single nucleotide substitutions. A pair of CAPS oligonucleotides was developed by amplifying a 0.55-kb DNA fragment that exhibited species-specific digestion patterns with restriction enzymes Alf III and Nde I. Consistent results were obtained with all the four markers on all tested Boehmeria lines. Conclusion The present study demonstrates the use of the RAPD, SCAR, SNP and CAPS markers for rapid identification of two closely related Boehmeria species.

Abstract Background Islet transplantation provides a promising cure for Type 1 diabetes; however it is limited by a shortage of pancreas donors. Bone marrow-derived multipotent mesenchymal stem cells (MSCs) offer renewable cells for generating insulin-producing cells (IPCs). Methods We used a four-stage differentiation protocol, containing neuronal differentiation and IPC-conversion stages, and combined with pellet suspension culture to induce IPC differentiation. Results Here, we report adding extracellular matrix proteins (ECM) such as fibronectin (FN) or laminin (LAM) enhances pancreatic differentiation with increases in insulin and Glut2 gene expressions, proinsulin and insulin protein levels, and insulin release in response to elevated glucose concentration. Adding FN or LAM induced activation of Akt and ERK. Blocking Akt or ERK by adding LY294002 (PI3K specific inhibitor), PD98059 (MEK specific inhibitor) or knocking down Akt or ERK failed to abrogate FN or LAM-induced enhancement of IPC differentiation. Only blocking both of Akt and ERK or knocking down Akt and ERK inhibited the enhancement of IPC differentiation by adding ECM. Conclusions These data prove IPC differentiation by MSCs can be modulated by adding ECM, and these stimulatory effects were mediated through activation of Akt and ERK pathways.

Abstract Background Canine distemper virus (CDV) is present worldwide and produces a lethal systemic infection of wild and domestic Canidae. Pre-existing antibodies acquired from vaccination or previous CDV infection might interfere the interpretation of a serologic diagnosis method. In addition, due to the high similarity of nucleic acid sequences between wild-type CDV and the new vaccine strain, current PCR derived methods cannot be applied for the definite confirmation of CD infection. Hence, it is worthy of developing a simple and rapid nucleotide-based assay for differentiation of wild-type CDV which is a cause of disease from attenuated CDVs after vaccination. High frequency variations have been found in the region spanning from the 3'-untranslated region (UTR) of the matrix (M) gene to the fusion (F) gene (designated M-F UTR) in a few CDV strains. To establish a differential diagnosis assay, an amplification refractory mutation analysis was established based on the highly variable region on M-F UTR and F regions. Results Sequences of frequent polymorphisms were found scattered throughout the M-F UTR region; the identity of nucleic acid between local strains and vaccine strains ranged from 82.5% to 93.8%. A track of AAA residue located 35 nucleotides downstream from F gene start codon highly conserved in three vaccine strains were replaced with TGC in the local strains; that severed as target sequences for deign of discrimination primers. The method established in the present study successfully differentiated seven Taiwanese CDV field isolates, all belonging to the Asia-1 lineage, from vaccine strains. Conclusions The method described herein would be useful for several clinical applications, such as confirmation of nature CDV infection, evaluation of vaccination status and verification of the circulating viral genotypes.

Abstract Background This study analyzed the likelihood of less-urgent emergency department (ED) visits among type 2 diabetic patients receiving care under a diabetes disease management (DM) program offered by the Louisiana State University Health Care Services Division (LSU HCSD). Methods All ED and outpatient clinic visits made by 6,412 type 2 diabetic patients from 1999 to 2006 were extracted from the LSU HCSD Disease Management (DM) Evaluation Database. Patient ED visits were classified as either urgent or less-urgent, and the likelihood of a less-urgent ED visit was compared with outpatient clinic visits using the Generalized Estimating Equation methodology for binary response to time-dependent variables. Results Patients who adhered to regular clinic visit schedules dictated by the DM program were less likely to use the ED for less urgent care with odds ratio of 0.1585. Insured patients had 1.13 to 1.70 greater odds of a less-urgent ED visit than those who were uninsured. Patients with better-managed glycated hemoglobin (A1c or HbA1c) levels were 82 times less likely to use less-urgent ED visits. Furthermore, being older, Caucasian, or a longer participant in the DM program had a modestly lower likelihood of less-urgent ED visits. The patient's Charlson Comorbidity Index (CCI), gender, prior hospitalization, and the admitting facility showed no effect. Conclusion Patients adhering to the DM visit guidelines were less likely to use the ED for less-urgent problems. Maintaining normal A1c levels for their diabetes also has the positive impact to reduce less-urgent ED usages. It suggests that successful DM programs may reduce inappropriate ED use. In contrast to expectations, uninsured patients were less likely to use the ED for less-urgent care. Patients in the DM program with Medicaid coverage were 1.3 times more likely to seek care in the ED for non-emergencies while commercially insured patients were nearly 1.7 times more likely to do so. Further research to understand inappropriate ED use among insured patients is needed. We suggest providing visit reminders, a call centre, or case managers to reduce the likelihood of less-urgent ED visit use among DM patients. By reducing the likelihood of unnecessary ED visits, successful DM programs can improve patient care.

Abstract The progression and metastasis of solid tumors, including head and neck squamous cell carcinoma (HNSCC), have been related to the behavior of a small subpopulation of cancer stem cells. Here, we have established a highly malignant HNSCC cell line, SASVO3, from primary tumors using three sequential rounds of xenotransplantation. SASVO3 possesses enhanced tumorigenic ability both in vitro and in vivo. Moreover, SASVO3 exhibits properties of cancer stem cells, including that increased the abilities of sphere-forming, the number of side population cells, the potential of transplanted tumor growth and elevated expression of the stem cell marker Bmi1. Injection of SASVO3 into the tail vein of nude mice resulted in lung metastases. These results are consistent with the postulate that the malignant and/or metastasis potential of HNSCC cells may reside in a stem-like subpopulation.

Abstract Metastatic renal cell carcinoma (RCC) is highly resistant to conventional systemic treatments, including chemotherapy, radiotherapy and hormonal therapies. Previous studies have shown over-expression of EGFR is associated with high grade tumors and a worse prognosis. Recent studies suggest anticancer therapies targeting the EGFR pathway have shown promising results in clinical trials of RCC patients. Therefore, characterization of the level and localization of EGFR expression in RCC is important for target-dependent therapy. In this study, we investigated the clinical significance of cellular localization of EGFR in human normal renal cortex and RCC. RCC and adjacent normal kidney tissues of 63 patients were obtained for characterization of EGFR expression. EGFR protein expression was assessed by immunohistochemistry on a scale from 0 to 300 (percentage of positive cells × staining intensity) and Western blotting. EGFR membranous staining was significantly stronger in RCC tumors than in normal tissues (P < 0.001). In contrast, EGFR cytoplasmic staining was significantly higher in normal than in tumor tissues (P < 0.001). The levels of membranous or cytoplasmic EGFR expression in RCC tissues were not correlated with sex, tumor grade, TNM stage or overall survival (P > 0.05). These results showed abundant expression of membranous EGFR in RCC, and abundant expression of cytoplasmic EGFR in normal tissues. EGFR expression in RCC was mostly located in the cell membrane, whereas the EGFR expression in normal renal tissues was chiefly seen in cytoplasm. Our results suggest different locations of EGFR expression may be associated with human renal tumorigenesis.

Abstract Background Hemophilia A represents the most common and severe inherited hemorrhagic disorder. It is caused by mutations in the F8 gene, which leads to a deficiency or dysfunctional factor VIII protein, an essential cofactor in the factor X activation complex. Methods We used long-distance polymerase chain reaction and denaturing high performance liquid chromatography for mutation scanning of the F8 gene. We designed the competitive multiplex PCR to identify the carrier with exonal deletions. In order to facilitate throughput and minimize the cost of mutation scanning, we also evaluated a new mutation scanning technique, high resolution melting analysis (HRM), as an alternative screening method. Results We presented the results of detailed screening of 122 Taiwanese families with hemophilia A and reported twenty-nine novel mutations. There was one family identified with whole exons deletion, and the carriers were successfully recognized by multiplex PCR. By HRM, the different melting curve patterns were easily identified in 25 out of 28 cases (89%) and 15 out of 15 (100%) carriers. The sensitivity was 93 % (40/43). The overall mutation detection rate of hemophilia A was 100% in this study. Conclusion We proposed a diagnostic strategy for hemophilia A genetic diagnosis. We consider HRM as a powerful screening tool that would provide us with a more cost-effective protocol for hemophilia A mutation identification.