Crystal structure analysis of Flavivirus methyltransferases uncovered a flavivirus-conserved cavity located next to the binding site for its cofactor, S-adenosyl-methionine (SAM). Chemical derivatization of S-adenosyl-homocysteine (SAH), the product inhibitor of the methylation reaction, with substituents that extend into the identified cavity, generated inhibitors that showed improved and selective activity against dengue virus methyltransferase (MTase), but not related human enzymes. Crystal structure of dengue virus MTase with a bound SAH derivative revealed that its N6-substituent bound in this cavity and induced conformation changes in residues lining the pocket. These findings demonstrate that one of the major hurdles for the development of methyltransferase-based therapeutics, namely selectivity for disease-related methyltransferases, can be overcome.Methyltransferases (MTases) 3 play key roles in normal physiology and human diseases through methylating DNA, RNA, and proteins. Almost all MTases use S-adenosyl-L-methionine (SAM) as a methyl donor and generate S-adenosyl-Lhomocysteine (SAH) as a by-product. Pharmacological modulation of MTases by small molecules represents a novel approach to therapeutic intervention in cancer and other diseases (1). However, because the core domains of various MTases are conserved, designing inhibitors that specifically block the disease-related MTase without affecting other MTases, has been challenging. The ability to rationally design and generate selective inhibitors would have profound implications for development of new medicines for many methyltransferase-mediated diseases.Dengue virus (DENV), from genus Flavivirus in the family Flaviviridae, is the most prevalent mosquito-borne viral pathogen that infects humans. The four serotypes of DENV (DENV-1 to -4) pose a public health threat to 2.5 billion people worldwide, and cause 50 -100 million human infections each year. Neither vaccine nor antiviral therapy is currently available for DENV. The flavivirus MTase methylates the guanine N7 and ribose 2Ј-O positions of the viral RNA cap in a sequential manner (i.e. GpppA-RNA 3 m7GpppA-RNA 3 m7GpppAm-RNA) (2, 3). Recent studies have shown that flavivirus MTase is critical for viral replication and, therefore, represents a valid target for antiviral therapeutics (4 -6). We therefore examined the feasibility to design inhibitors that specifically modulate flavivirus MTase. EXPERIMENTAL PROCEDURESPreparation of DENV-3 MTases-The DNA fragment representing the MTase domain of DENV-3 was cloned into expression vector pGEX4T1 (Amersham Biosciences). Ala-substitution mutant MTases were prepared using a standard overlapping PCR procedure. Recombinant MTases, containing an N-terminal GST, were expressed in Escherichia coli. BL21 cells and purified through a GSTPrep TM FF 16/10 column (GE Healthcare). The GST tag was then cleaved by thrombin and removed from the MTases using the GST column. The MTases were further purified through gel filtration to ensure protein purity was Ͼ95%. The p...

In order to provide an effective whistle-blowing system, it is expected that companies would provide employees with a high level of disclosure regarding the whistle-blowing process. This study investigates variation in the extent of whistle-blowing disclosures. As a measure of whistle-blowing implementation, this study further examines the provision of a hotline channel. The results suggest that the extent of whistle-blowing disclosures is positively associated with the permissibility of anonymous reporting and organisational support for whistle-blowing, the number of external directors on the audit committee, and the existence of concentrated shareholdings. The mere existence of whistle-blowing disclosures could simply be symbolic. The findings also indicate a greater likelihood of the provision of hotlines when companies are larger in size, have a higher level of current inventory, are cross-listed in the US, and permit anonymous reporting.

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Whistleblowers have been credited for uncovering financial scandals in companies globally, including Enron, Olympus Corporation, and WorldCom. Despite increasing support and incentives for whistleblowing, there generally remains reluctance to blow the whistle. Thus, the purpose of this study is to review: (1) the determinants of internal and external whistleblowing on accounting-related misconduct, (2) U.S. whistleblowing legislation on accounting-related misconduct and related research, and (3) the effects of whistleblowing on firms and whistleblowers. Within each area, suggestions for future research are offered.

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