Comparative Genomics of Community-Acquired ST59 Methicillin-Resistant Staphylococcus aureus in Taiwan: Novel Mobile Resistance Structures with IS1216V

Methicillin-resistant Staphylococcus aureus (MRSA) with ST59/SCCmecV and Panton-Valentine leukocidin gene is a major community-acquired MRSA (CA-MRSA) lineage in Taiwan and has been multidrug-resistant since its initial isolation. In this study, we studied the acquisition mechanism of multidrug resistance in an ST59 CA-MRSA strain (PM1) by comparative genomics. PM1’s non-β-lactam resistance was encoded by two unique genetic traits. One was a 21,832-bp composite mobile element structure (MES_PM1), which was flanked by direct repeats of enterococcal IS1216V and was inserted into the chromosomal sasK gene; the target sequence (att) was 8 bp long and was duplicated at both ends of MES_PM1. MES_PM1 consisted of two regions: the 5′-end side 12.4-kb region carrying Tn551 (with ermB) and Tn5405-like (with aph[3′]-IIIa and aadE), similar to an Enterococcus faecalis plasmid, and the 3′-end side 6,587-bp region (MES_cat) that carries cat and is flanked by inverted repeats of IS1216V. MES_cat possessed att duplication at both ends and additional two copies of IS1216V inside. MES_PM1 represents the first enterococcal IS1216V-mediated composite transposon emerged in MRSA. IS1216V-mediated deletion likely occurred in IS1216V-rich MES_PM1, resulting in distinct resistance patterns in PM1-derivative strains. Another structure was a 6,025-bp tet-carrying element (MES_tet) on a 25,961-bp novel mosaic penicillinase plasmid (pPM1); MES_tet was flanked by direct repeats of IS431, but with no target sequence repeats. Moreover, the PM1 genome was deficient in a copy of the restriction and modification genes (hsdM and hsdS), which might have contributed to the acquisition of enterococcal multidrug resistance.     

Analysis of the role of the coat protein N-terminal segment in Potato virus X virion stability and functional activity

Previously, we have reported that intact Potato virus X (PVX) virions cannot be translated in cell-free systems, but acquire this capacity by the binding of PVX-specific triple gene block protein 1 (TGBp1) or after phosphorylation of the exposed N-terminal segment of intravirus coat protein (CP) by protein kinases. With the help of in vitro mutagenesis, a nonphosphorylatable PVX mutant (denoted ST PVX) was prepared in which all 12 S and T residues in the 20-residue-long N-terminal CP segment were substituted by A or G. Contrary to expectations, ST PVX was infectious, produced normal progeny and was translated in vitro in the absence of any additional factors. We suggest that the N-terminal PVX CP segment somehow participates in virion assembly in vivo and that CP subunits in ST virions may differ in structure from those in the wild-type (UK3 strain). In the present work, to test this suggestion, we performed a comparative tritium planigraphy study of CP structure in UK3 and ST virions. It was found that the profile of tritium incorporation into ST mutant virions in some CP segments differed from that of normal UK3 virions and from UK3 complexed with the PVX movement protein TGBp1. It is proposed that amino acid substitutions in ST CP and the TGBp1-driven remodelling of UK3 virions induce structural alterations in intravirus CPs. These alterations affect the predicted RNA recognition motif of PVX CP, but in different ways: for ST PVX, labelling is increased in α-helices 6 and 7, whereas, in remodelled UK3, labelling is increased in the β-sheet strands β3, β4 and β5.     

Human DNA polymerase λ catalyzes lesion bypass across benzo[a]pyrene-derived DNA adduct during base excision repair

The combined action of oxidative stress and genotoxic polycyclic aromatic hydrocarbons derivatives can lead to cluster-type DNA damage that includes both a modified nucleotide and a bulky lesion. As an example, we investigated the possibility of repair of an AP site located opposite a minor groove-positioned (+)-trans-BPDE-dG or a base-displaced intercalated (+)-cis-BPDE-dG adduct (BP lesion) by a BER system. Oligonucleotides with single uracil residues in certain positions were annealed with complementary oligonucleotides bearing either a cis- or trans-BP adduct. The resulting DNA duplexes contained dU either directly opposite the modified dG or shifted to adjacent 5' (-1) or 3' (+1) positions. Digestion with uracil DNA glycosylase was utilized to generate AP sites which were then hydrolyzed by APE1, and the resulting gaps were processed by DNA polymerase β (Polβ) or λ (Polλ). The AP sites in position -1 can be repaired effectively using APE1 and Polβ or Polλ. The AP sites opposite the BP lesions can be repaired using Polλ in the case of cis- but not the trans-isomeric adduct. The AP sites in position +1 are the most difficult to repair. In the case of the AP site located in position +1, the activity of Polλ does not depend on the stereoisomeric properties of the BP lesions and dCTP is the preferred inserted substrate in both cases. The capability of Polλ to introduce the correct dNTP opposite the cis-BP-dG adduct in gap filling reactions suggests that this polymerase may play a specialized role in the process of repair of these kinds of lesions.     

Effects of Free Ca2+ on Kinetic Characteristics of Holotransketolase

Catalytic activity has been demonstrated for holotransketolase in the absence of free bivalent cations in the medium. The two active centers of the enzyme are equivalent in both the catalytic activity and the affinity for the substrates. In the presence of free Ca²⁺ (added to the medium from an external source), this equivalence is lost: negative cooperativity is induced on binding of either xylulose 5-phosphate (donor substrate) or ribose 5-phosphate (acceptor substrate), whereupon the catalytic conversion of the bound substrates causes the interaction between the centers to become positively cooperative. Moreover, the enzyme total activity increase is observed.     

Thyroid hormone induces sprouting angiogenesis in adult heart of hypothyroid mice through the PDGF‐Akt pathway

Study of physiological angiogenesis and associated signalling mechanisms in adult heart has been limited by the lack of a robust animal model. We investigated thyroid hormone‐induced sprouting angiogenesis and the underlying mechanism. Hypothyroidism was induced in C57BL/6J mice by feeding with propylthiouracil (PTU). One year of PTU treatment induced heart failure. Both 12 weeks‐ (young) and 1 year‐PTU (middle age) treatment caused a remarkable capillary rarefaction observed in capillary density. Three‐day Triiodothyronine (T3) treatment significantly induced cardiac capillary growth in hypothyroid mice. In cultured left ventricle (LV) tissues from PTU‐treated mice, T3 also induced robust sprouting angiogenesis where pericyte‐wrapped endothelial cells formed tubes. The in vitro T3 angiogenic response was similar in mice pre‐treated with PTU for periods ranging from 1.5 to 12 months. Besides bFGF and VEGF₁₆₄, PDGF‐BB was the most robust angiogenic growth factor, which stimulated notable sprouting angiogenesis in cultured hypothyroid LV tissues with increasing potency, but had little effect on tissues from euthyroid mice. T3 treatment significantly increased PDGF receptor beta (PDGFR‐β) protein levels in hypothyroid heart. PDGFR inhibitors blocked the action of T3 both on sprouting angiogenesis in cultured LV tissue and on capillary growth in vivo. In addition, activation of Akt signalling mediated in T3‐induced angiogenesis was blocked by PDGFR inhibitor and neutralizing antibody. Our results suggest that hypothyroidism leads to cardiac microvascular impairment and rarefaction with increased sensitivity to angiogenic growth factors. T3‐induced cardiac sprouting angiogenesis in adult hypothyroid mice was associated with PDGF‐BB, PDGFR‐β and downstream activation of Akt.     

Antihyperglycaemic activity of 2,4:3,5-dibenzylidene-D-xylose-diethyl dithioacetal in diabetic mice

We have recently generated lipophilic D-xylose derivatives that increase the rate of glucose uptake in cultured skeletal muscle cells in an AMP-activated protein kinase (AMPK)-dependent manner. The derivative 2,4:3,5-dibenzylidene-D-xylose-diethyl dithioacetal (EH-36) stimulated the rate of glucose transport by increasing the abundance of glucose transporter-4 in the plasma membrane of cultured myotubes. The present study aimed at investigating potential antihyperglycaemic effects of EH-36 in animal models of diabetes. Two animal models were treated subcutaneously with EH-36: streptozotocin-induced diabetes in C57BL/6 mice (a model of insulin-deficient type 1 diabetes), and spontaneously diabetic KKAy mice (Kuo Kondo rats carrying the A(y) yellow obese gene; insulin-resistant type 2 diabetes). The in vivo biodistribution of glucose in control and treated mice was followed with the glucose analogue 2-deoxy-2-[(18) F]-D-glucose; the rate of glucose uptake in excised soleus muscles was measured with [(3) H]-2-deoxy-D-glucose. Pharmacokinetic parameters were determined by non-compartmental analysis of the in vivo data. The effective blood EH-36 concentration in treated animals was 2 μM. It reduced significantly the blood glucose levels in both types of diabetic mice and also corrected the typical compensatory hyperinsulinaemia of KKAy mice. EH-36 markedly increased glucose transport in vivo into skeletal muscle and heart, but not to adipose tissue. This stimulatory effect was mediated by Thr(172) -phosphorylation in AMPK. Biochemical tests in treated animals and acute toxicological examinations showed that EH-36 was well tolerated and not toxic to the mice. These findings indicate that EH-36 is a promising prototype molecule for the development of novel antidiabetic drugs.     

Hit clustering can improve virtual fragment screening: CDK2 and PARP1 case studies

Virtual fragment screening could be a promising alternative to existing experimental screening techniques. However, reliable methods of in silico fragment screening are yet to be established and validated. In order to develop such an approach we first checked how successful the existing molecular docking methods can be in predicting fragment binding affinities and poses. Using our Lead Finder docking software the RMSD of the binding energy prediction was observed to be 1.35 kcal/mol(-1) on a set of 26 experimentally characterized fragment inhibitors, and the RMSD of the predicted binding pose from the experimental one was <1.5 ?. Then, we explored docking of 68 fragments obtained from 39 drug molecules for which co-crystal structures were available from the PDB. It appeared that fragments that participate in oriented non-covalent interactions, such as hydrogen bonds and metal coordination, could be correctly docked in 70-80% of cases suggesting the potential success of rediscovering of corresponding drugs by in silico fragment approach. Based on these findings we've developed a virtual fragment screening technique which involved structural filtration of protein-ligand complexes for specific interactions and subsequent clustering in order to minimize the number of preferable starting fragment candidates. Application of this method led to 2 millimolar-scale fragment PARP1 inhibitors with a new scaffold.