Molecular epidemiological study of enterovirus D68 in hospitalised children in Hong Kong in 2014-2015 and their complete coding sequences.

Background: Human enterovirus D68 (EV-D68) was first isolated in 1962 and has aroused public concern recently because of a nationwide outbreak among children in 2014-2015 in the USA. The symptoms include fever, runny nose, sneezing, cough and muscle pains. It might be associated with severe respiratory illness in individuals with pre-existing respiratory conditions and its potential association with acute flaccid myelitis is under investigation. In Asia, EV-D68 cases have been reported in several countries. The study: We aimed to understand the EV-D68 prevalence and their genetic diversity in Hong Kong children. Methods: A total of 10 695 nasopharyngeal aspirate (NPA) samples from hospitalised patients aged <18 years were collected from September 2014 to December 2015 in two regional hospitals. NPAs tested positive for enterovirus/rhinovirus (EV/RV) were selected for genotyping. For those identified as EV-D68, their complete coding sequences (CDSs) were obtained by Sanger sequencing. A maximum-likelihood phylogeny was constructed using all EV-D68 complete coding sequences available in GenBank (n=482). Results: 2662/10 695 (24.9%) were tested positive with EV/RV and 882/2662 (33.1%) were selected randomly and subjected to molecular classification. EV-D68 was detected in 15 (1.70%) samples from patients with clinical presentations ranging from wheezing to pneumonia and belonged to subclade B3. Eight CDSs were successfully obtained. A total of 10 amino acid residue polymorphisms were detected in the viral capsid proteins, proteases, ATPase and RNA polymerase. Conclusion: B3 subclade was the only subclade found locally. Surveillance of EV-D68 raises public awareness and provides the information to determine the most relevant genotypes for vaccine development.

Functional identification of an intronic promoter of the human glucose-dependent insulinotropic polypeptide gene.

Glucose-dependent insulinotropic polypeptide (GIP), a physiological incretin and enterogastrone, plays a vital role in regulating glucose-dependent insulin release from the pancreas and gastric acid secretion from the stomach. By using a transgenic mouse approach, we previously reported that the distal 1.2kb promoter region of the human GIP (hGIP) gene (-2545/-346, relative to the ATG) was able to target the transgene expression in the stomach but not in the small intestine where the majority of GIP-producing cells are located. In the present study, in order to identify the cis-acting element(s) that is/are required for intestinal expression, a 1.6kb (-1580/-) DNA fragment within the first intron of the hGIP gene was isolated and characterized in three GIP-expressing cell lines including HuTu80 (duodenal cells), PANC-1 (pancreatic ductal cells) and Hs746T (stomach cells). By 5' and 3' deletion analysis, a proximal promoter element was confined within the nucleotides -102/-1. This promoter element, functions in an orientation-dependent manner, was able to drive 15.1 and 18.3 fold increases in promoter activities in HuTu80 and PANC-1 cells, respectively. Site-directed mutation analysis indicated that the region -54/-23 was essential for promoter function while the region -22/-1 might possess opposite effects in HuTu80 and PANC-1 cells. In competitive and antibody supershift assays, interactions of the progesterone receptor (PR) and some unknown protein factors from HuTu80 and PANC-1 with the motif(s) at -54/-23 were evident. Consistent with this finding, we demonstrated the transcriptional regulation of the hGIP promoter by progesterone via the PR-B isoform and that progesterone treatment in both HuTu80 and PANC-1 cells resulted in an increase in hGIP transcript level. In addition, a sequence motif (ACATGT) residing -48/-43 was found to be responsible for the binding of potential TFII regulator(s). Taken together, our results suggest that the proximal intronic sequences contain essential cis-acting elements for the cell-specific expression of the hGIP gene.

Identification of a proglucagon cDNA from Rana tigrina rugulosa that encodes two GLP-1s and that is alternatively spliced in a tissue-specific manner.

Glucagon plays a pivotal role in the regulation of metabolism. A glucagon receptor has been previously characterized in the frog, Rana tigrina rugulosa, and the frog and human glucagon receptors have been shown to possess similar binding affinities toward human glucagon. To study the structural evolution of glucagon peptide and its receptor in vertebrates, in the current study, a proglucagon cDNA from the same frog species was cloned. Interestingly, in contrast to the mammalian proglucagons that contain only one GLP-1 peptide, the frog proglucagon cDNA encodes two GLP-1 peptides (GLP-1A and GLP-1B) in addition to a glucagon peptide and a glucagon-like peptide 2 (GLP-2). By reverse transcriptase-PCR (RT-PCR) analysis, the proglucagon gene expression was widely detected in the brain, colon, small intestine, liver, lung, and pancreas, suggesting that the proglucagon-derived peptides have diverse functions in frogs. Moreover, tissue-specific alternative mRNA splicing was observed in the brain, colon, and pancreas. In these tissues, proglucagon transcripts with a 135 bp in frame deletion encoding GLP-1A were found. This splicing event in R. tigrina rugulosa is novel because it deletes a GLP-1 encoding sequence instead of the GLP-2 observed in other vertebrates. These findings should enhance understanding of the proglucagon evolution, structure, and expression in vertebrates.

Total synthesis and antifungal evaluation of cyclic aminohexapeptides.

The need for new therapies to treat systemic fungal infections continues to rise. Naturally occurring hexapeptide echinocandin B (1) has shown potent antifungal activity via its inhibition of the synthesis of beta-1,3 glucan, a key fungal cell wall component. Although this series of agents has been limited thus far based on their physicochemical characteristics, we have found that the synthesis of analogues bearing an aminoproline residue in the 'northwest' position imparts greatly improved water solubility (> 5 mg/mL). The synthesis and structure-activity relationships (SAR) based on whole cell and upon in vivo activity of the series of compounds are reported.

Glucose-dependent insulinotropic polypeptide gene expression in the stomach: revealed by a transgenic mouse study, in situ hybridization and immunohistochemical staining.

Glucose-dependent insulinotropic polypeptide (GIP) plays an important role in stimulating insulin release in the pancreas as well as inhibiting gastric acid secretion in the stomach. GIP has been found in specific endocrine cells located in the mucosal layer of the small intestine and in the submandibular salivary gland. In this study, the tissue-specific expression of GIP guided by 1.2 kb of the human GIP (hGIP) gene 5' flanking region was investigated by a transgenic mouse approach. A chimeric promoter-reporter gene construct linking the 5'-flanking region of the hGIP gene with the thymidine kinase gene of the herpes simplex virus was introduced into the genomes of mice by microinjection. By reverse transcriptase-PCR (RT-PCR) and thymidine kinase assays, transgene expression was found in the stomach and pancreas. The enzyme activity detected in the stomach was about 6-fold higher than that found in the pancreas, suggesting that GIP may be expressed in the stomach. This observation is supported by RT-PCR studies since both human and mouse GIP transcripts are detected in the stomach and small intestine. In addition, distinct GIP-producing cells were identified in both tissues in mouse by in situ hybridization and immunohistochemical staining. Taken together, our data demonstrate for the first time that GIP is expressed in human and mouse stomach.

Antitumor activity of 9(R)-dihydrotaxane analogs.

A novel reduced taxane, 13-acetyl-9(R)-dihydrobaccatin III (1) has been isolated from Taxus canadensis. The selective C-13 deacetylation of this isolate has allowed for the preparation of a wide variety of 9(R)-dihydrotaxane analogs. In general, this series has shown greater stability and water solubility than the 9-carbonyl series while retaining antimicrotubule and tumor cell cytotoxicity activities relative to taxol. Placement of polar functionalities at the C-7 position results in loss of activity whereas alkylation or acylation of either C-7 or C-9 hydroxyl groups ameliorate the activity.

Synthesis and biological evaluation of C-3'-modified analogs of 9(R)-dihydrotaxol.

Taxol (1) is considered a most exciting new drug in cancer chemotherapy. The promising antitumor activity of 9(R)-dihydrotaxol (3) encouraged us to further explore the structure-activity relationship of this new member of the taxane family. Studies indicated that the C-13 side chain of taxol is indispensable for antitumor activity and that the natural substitution pattern of a 2'(R)-hydroxy and a 3'(S)-acylamino group might be optimal. However, relatively little is known about the effects of the 3'-phenyl ring on activity. The synthesis and biological evaluation of analogs of 3 modified at the C-3' position are described. This study revealed that the 3'-phenyl ring was not required for activity and identified several compounds which had equal or greater in vitro and in vivo activity than taxol.

Synthesis and antifungal activity of 1,3,2-benzodithiazole S-oxides.

The preparation of 1,3,2-benzodithiazole S-oxide analogs exhibiting in vitro antifungal activity against several strains of Candida is described. For the preparation of derivatives bearing aromatic substituents, a novel electrophilic aromatic thiolation reaction was utilized which produced substituted aromatic 1,2-dithiol intermediates. The reactions of nucleophiles with the parent heterocyclic system have led to an efficient transamidation process which allows for the direct production of these analogs. The S-oxide bond exhibits poor stereochemical stability and has been found to epimerize under ambient conditions. The structure-activity data report that a side chain of greater than 10 carbons effects a loss in activity as does the placement of polar groups in this chain.

Synthesis and antitumor activity of structural analogues of the epipodophyllotoxins.

Several ring-contracted analogues of the antitumor agent etoposide have been prepared. The synthesis of the simple indanyl system 3 is described along with two bicyclic systems of general structure 4 prepared through a stereoselective allylation of the keto-ester 6. A cis-fused lactone analogue 5, which is isomeric with the etoposide aglycone, has been synthesized via a dialkylation of the indene-2-carboxylate anion. Regiochemical and stereochemical results of these alkylations are described. The cytotoxicity of these derivatives toward several tumor cell lines is described and generally follows the structure-activity relationships known for the agent podophyllotoxin (2).

Synthesis and activity of nonhydrolyzable pseudomonic acid analogues.

Several series of pseudomonic acid analogues have been prepared that incorporate modified functionalities in place of the C1-C3 alpha,beta-unsaturated ester group. The inhibition of isoleucyl-tRNA synthetase and the in vitro activity of these compounds against various Gram-positive and Gram-negative strains are described. Several derivatives showed enzyme inhibition equivalent to or better than that of methyl pseudomonate (3), while lacking the hydrolyzable ester group at C1. These analogues include the corresponding phenyl ketone and the ether 12. The long-chain ketone 24 exhibited similar in vitro activity as the parent ester.

Stabilization of gas cavitation nuclei by surface-active compounds.

Gas bubbles are the primary agent in producing the pathogenic effects of decompression sickness. Numerous experiments indicate that bubbles originate in water, and probably also in man, as pre-existing gas nuclei. This is surprising considering that gas phases larger than 1 micron should rise to the surface of a standing liquid, whereas smaller ones should dissolve rapidly due to surface tension. Several stabilizing mechanisms have been suggested, and each has been refuted on experimental grounds. In this article, we propose a new model that arises out of a systematic study of the earlier theories. We review these theories and conclude that gas cavitation nuclei must be held intact by surface-active skins that are initially permeable. The first quantitative analysis of bubble formation data from supersaturated gelatin is summarized and leads to the further conclusion that skins can become impermeable if the ambient pressure is increased rapidly by a sufficient amount. Our model owes much to Sirotyuk, who "demonstrated experimentally that stabilization of gas bubbles acting as cavitation nuclei in water is always attributable to the presence of surface-active substances in the water".