Update of contemporary antimicrobial resistance rates across China: reference testing results for 12 medical centers (2011).

Antimicrobial resistance (R) surveillance across Asia and especially in China has documented unique patterns and mechanisms. This 2011 study reports results for 2278 isolates from 12 hospitals in China (94-216 strains/site); most from bacteremia (20.4%), pneumonias (29.1%), or skin and skin structure infections (20.9%). Samples were tested by reference broth microdilution methods, interpreted by published susceptibility (S) breakpoints. The most common species were Staphylococcus aureus (343, 45.8% MRSA), Escherichia coli (EC; 288), Pseudomonas aeruginosa (PSA; 221), Klebsiella spp. (KSP; 208), acinetobacters (ACB; 178), enterobacters (155), Streptococcus pneumoniae (SPN; 154, 46.8% penicillin-S), and enterococci (ENT; 137). Among 849 Gram-positive (GP) cocci, linezolid, tigecycline (TIG), daptomycin, and vancomycin provided best antimicrobial coverage (≥99.7% S). Resistance patterns of concern were 0.3% VISA, 15.4% teicoplanin non-S coagulase-negative staphylococci, 1.5% vancomycin-R ENT (all Enterococcus faecium), 1.9% levofloxacin-R ß-haemolytic streptococci, and 35.1 and 12.7% ceftriaxone-non-S rates for SPN and viridans group streptococci, respectively. For Gram-negative bacilli, R among Enterobacteriaceae was highest against ß-lactams (extended spectrum ß-lactamase-phenotype strains at 73.6 and 42.8% in EC and KSP, respectively; carbapenem-R was only 2.1-4.3% with KPC and IMP type enzymes detected in KSP). The widest spectrum agents were cefoperazone/sulbactam (79.5-86.1%), piperacillin/tazobactam (88.9-92.0%), TIG (98.6-100%), amikacin (AMK; 91.8-93.7%), and meropenem (95.7-97.1% S). PSA was most inhibited by AMK (90.5% S) and colistin (COL; 99.5%), with cefepime (67.9%) best among the tested ß-lactams. Only COL (100% S) and TIG (MIC90, 2 µg/mL) showed significant potencies against ACB. In conclusion, R among pathogens from 12 Chinese hospitals illustrates several agents active against GP pathogens, but more serious R problems were noted among Enterobacteriaceae, PSA, and ACB. Combination treatment for the latter multidrug-R strains appears necessary, guided by local antibiograms and national surveillance results applying reference methods.

Regional resistance surveillance program results for 12 Asia-Pacific nations (2011).

The Regional Resistance Surveillance program monitored susceptibility rates and developing resistance by geographic region, including 12 Asia-Pacific (APAC) countries. Reference broth microdilution methods for susceptibility/interpretations were applied, processing 5,053 strains. Among Staphylococcus aureus isolates (37% methicillin-resistant S. aureus [MRSA], highest in South Korea [73%]), linezolid (LZD), tigecycline (TIG), and vancomycin were 100% active, but 33 and 34% of strains were levofloxacin (LEV) or macrolide resistant, respectively. Streptococcus pneumoniae was most resistant to ß-lactams and macrolides (45%) but was LZD, LEV, and TIG susceptible (>98%). Extended-spectrum ß-lactamase (ESBL) phenotype rates in Escherichia coli and Klebsiella spp. were 48 and 47%, respectively, and were highest in Taiwan, at 75 to 91%. The best anti-ESBL-phenotype agents were amikacin (81 to 96% susceptible), colistin (COL; >98%), TIG (>98%), and carbapenems (81 to 97%). Pseudomonas aeruginosa showed ≥20% resistance to all drugs except COL (99% susceptible). In conclusion, endemic evolving antimicrobial resistances in APAC nations show compromised roles for many commonly used antimicrobials.

Disruption of Kv1.1 N-type inactivation by novel small molecule inhibitors (disinactivators).

Kv1.1 channels are expressed in many regions of the brain and spinal cord [Monaghan, M. M.; Trimmer, J. S.; Rhodes, K. J. J. Neurosci.2001, 21, 5973; Rasband, M. N.; Trimmer, J. S. J. Comp. Neurol.2001, 429, 166; Trimmer, J. S.; Rhodes, K. J. Ann. Rev. Physiol.2004, 66, 477]. When expressed alone, they produce a delayed rectifier slowly inactivating type current that contributes to hyperpolarizing the neuron following depolarization. In the hippocampus Kv1.1 is co-expressed with Kvbeta1 (and other beta subunits), which converts Kv1.1 into a transient, fast inactivating current, reducing its ability to hyperpolarize the cell and thus increasing neuronal excitability. To reduce neuronal excitability, screening for compounds that prevent inactivation of Kv1.1 channels by Kvbeta1 was performed using a yeast two-hybrid screen. A variety of compounds were discovered in this assay and subsequently determined to disrupt inactivation of the ionic currents, and hence were termed 'disinactivators'. Several of these disinactivators also inhibited pentylenetetrazole-induced seizures (PTZ) in mice. Compounds were found to act by several mechanisms to prevent Kvbeta1 inactivation of Kv1.1 channels, including enhancement of Ca(2+) release/influx and by direct mechanisms. Two structural classes were identified that act on a Kvbeta1N70-Kv1.1 chimera where the N-terminal 70 amino acids of Kvbeta1 were attached to the N-terminus of Kv1.1. It is likely that these disinactivators act directly on the Kvbeta1 N-terminus or its receptor site on Kv1.1, thus preventing it from blocking Kv1.1 channels. Compounds acting by this mechanism may be useful for reducing neuronal hyperexcitability in diseases such as epilepsy and neuropathic pain.

PP2A regulates BCL-2 phosphorylation and proteasome-mediated degradation at the endoplasmic reticulum.

Anti-apoptotic activity of BCL-2 is mediated by phosphorylation at the endoplasmic reticulum (ER), but how this phosphorylation is regulated and the mechanism(s) by which it regulates apoptosis are unknown. We purified macromolecular complexes containing BCL-2 from ER membranes and found that BCL-2 co-purified with the main two subunits of the serine/threonine phosphatase, PP2A. The association of endogenous PP2A and BCL-2 at the ER was verified by co-immunoprecipitation and microcystin affinity purification. Knock down or pharmacological inhibition of PP2A caused degradation of phosphorylated BCL-2 and led to an overall reduction in BCL-2 levels. We found that this degradation was due to the action of the proteasome acting selectively at the ER. Conversely, overexpression of PP2A caused elevation in endogenous BCL-2. Most importantly, we found that PP2A knock down sensitized cells to several classes of death stimuli (including ER stress), but this effect was abolished in a genetic background featuring knock in of a non-phosphorylatable BCL-2 allele. These studies support the hypothesis that PP2A-mediated dephosphorylation of BCL-2 is required to protect BCL-2 from proteasome-dependent degradation, affecting resistance to ER stress.

The control of endoplasmic reticulum-initiated apoptosis by the BCL-2 family of proteins.

Irreversible perturbations in the homeostasis of the endoplasmic reticulum (ER) are thought to lead to apoptosis and cell loss in a number of important human diseases, including Alzheimer disease, Parkinson disease, and type 2 diabetes. However, the exact mechanisms that lead from ER stress to cell death remain incompletely understood. Recent work has shown that the BCL-2 family of proteins plays a central role in regulating this form of cell death, both locally at the ER and from a distance at the mitochondrial membrane.

Sip2, an N-myristoylated beta subunit of Snf1 kinase, regulates aging in Saccharomyces cerevisiae by affecting cellular histone kinase activity, recombination at rDNA loci, and silencing.

Saccharomyces cerevisiae has evolved a number of mechanisms for sensing glucose. In the present study we examine the mechanism by which one of these pathways, involving Snf1, regulates cellular aging. Snf1 is a heterotrimer composed of a catalytic alpha subunit (Snf1p) that phosphorylates target proteins at Ser/Thr residues, an activating gamma subunit (Snf4p), and a beta subunit (Sip1p, Sip2p, or Gal83). We previously showed that forced expression of Snf1p or loss of Sip2p, but not the other beta subunits, causes accelerated aging, while removal of Snf4p extends life span (Ashrafi, K., Lin, S. S., Manchester, J. K., and Gordon, J. I. (2000) Genes Dev. 14, 1872-1885). We now demonstrate that in wild type cells, there is an age-associated shift in Sip2p from the plasma membrane to the cytoplasm, a prominent redistribution of Snf4p from the plasma membrane to the nucleus, a modest increase in nuclear Snf1p, and a concomitant increase in cellular Snf1 histone H3 kinase activity. Covalent attachment of myristate to the N-terminal Gly of Sip2p is essential for normal cellular life span. When plasma membrane association of Sip2p is abolished by a mutation that blocks its N-myristoylation, Snf4p is shifted to the nucleus. Rapidly aging sip2 Delta cells have higher levels of histone H3 kinase activity than their generation-matched isogenic wild type counterparts. Increased Snf1 activity is associated with augmented recombination at rDNA loci, plus desilencing at sites affected by Snf1-catalyzed Ser(10) phosphorylation of histone H3 (the INO1 promoter plus targets of the transcription factor Adr1p). The rapid-aging phenotype of sip2 Delta cells is fully rescued by blocking recombination at rDNA loci with a fob1 Delta allele; rescue is not accompanied by amelioration of an age-associated shift toward gluconeogenesis and glucose storage. Together, these findings suggest that Sip2p acts as a negative regulator of nuclear Snf1 activity in young cells by sequestering its activating gamma subunit at the plasma membrane and that loss of Sip2p from the plasma membrane to the cytoplasm in aging cells facilities Snf4p entry into the nucleus so that Snf1 can modify chromatin structure.

Manipulation of a nuclear NAD+ salvage pathway delays aging without altering steady-state NAD+ levels.

Yeast deprived of nutrients exhibit a marked life span extension that requires the activity of the NAD(+)-dependent histone deacetylase, Sir2p. Here we show that increased dosage of NPT1, encoding a nicotinate phosphoribosyltransferase critical for the NAD(+) salvage pathway, increases Sir2-dependent silencing, stabilizes the rDNA locus, and extends yeast replicative life span by up to 60%. Both NPT1 and SIR2 provide resistance against heat shock, demonstrating that these genes act in a more general manner to promote cell survival. We show that Npt1 and a previously uncharacterized salvage pathway enzyme, Nma2, are both concentrated in the nucleus, indicating that a significant amount of NAD(+) is regenerated in this organelle. Additional copies of the salvage pathway genes, PNC1, NMA1, and NMA2, increase telomeric and rDNA silencing, implying that multiple steps affect the rate of the pathway. Although SIR2-dependent processes are enhanced by additional NPT1, steady-state NAD(+) levels and NAD(+)/NADH ratios remain unaltered. This finding suggests that yeast life span extension may be facilitated by an increase in the availability of NAD(+) to Sir2, although not through a simple increase in steady-state levels. We propose a model in which increased flux through the NAD(+) salvage pathway is responsible for the Sir2-dependent extension of life span.