Mori for their technical advice and assistance. Care of experimental animals was within institutional guidelines. Supported by grants from the Ministry of Education, Science, Sports and Culture of Japan, and Circulation Biosystems at the University of Tsukuba. E.T. and J.1. are research fellows of the Japan Society for the Promotion of Science.

SUMMARY Gasdermin D (GSDMD) is considered a proinflammatory factor that mediates pyroptosis in macrophages to protect hosts from intracellular bacteria. Here, we reveal that GSDMD deficiency paradoxically augmented host responses to extracellular Escherichia coli, mainly by delaying neutrophil death, which established GSDMD as a negative regulator of innate immunity. In contrast to its activation in macrophages, in which activated inflammatory caspases cleave GSDMD to produce an N-terminal fragment (GSDMD-cNT) to trigger pyroptosis, GSDMD cleavage and activation in neutrophils was caspase independent. It was mediated by a neutrophil-specific serine protease, neutrophil elastase (ELANE), released from cytoplasmic granules into the cytosol in aging neutrophils. ELANE-mediated GSDMD cleavage was upstream of the caspase cleavage site and produced a fully active ELANE-derived NT fragment (GSDMD-eNT) that induced lytic cell death as efficiently as GSDMD-cNT. Thus, GSDMD is pleiotropic, exerting both pro- and anti-inflammatory effects that make it a potential target for antibacterial and anti-inflammatory therapies.

Disease-associated blood biomarkers exist in exceedingly low concentrations within complex mixtures of high-abundance proteins such as albumin. We have introduced an affinity bait molecule into N-isopropylacrylamide to produce a particle that will perform three independent functions within minutes, in one step, in solution: a) molecular size sieving b) affinity capture of all solution phase target molecules, and c) complete protection of harvested proteins from enzymatic degradation. The captured analytes can be readily electroeluted for analysis.There is an urgent need to discover novel biomarkers that provide sensitive and specific disease detection1 , 2. Cancer is rapidly becoming the leading cause of death for many population groups in the United States, largely due to the fact that the disease is usually diagnosed after the cancer has metastasized and treatment is ineffective. It is widely believed that early detection of cancer prior to metastasis will lead to a dramatic improvement in treatment outcome. Biomarkers are nucleic acids, proteins, protein fragments or metabolites indicative of a specific biological state, that are associated with the risk of contraction or presence of disease3. Biomarker research has revealed that low-abundance circulating proteins and peptides present a rich source of information regarding the state of the organism as a whole 4 . Two major hurdles have prevented these discoveries from reaching clinical benefit: 1) disease-relevant biomarkers in blood or body fluids may exist in exceedingly low concentrations within a complex mixture of biomolecules and could be masked by high-abundance species such as albumin, and 2) degradation of protein biomarkers can occur immediately following the collection of blood or body fluid as a result of endogenous or exogenous proteinases. The goal of this study was to create "smart" nano-particles that allow enrichment and encapsulation of selected classes of proteins and peptides from complex mixtures of biomolecules such as plasma, and protect them from degradation during subsequent sample handling. The captured analytes can be readily extracted from the particles by electrophoresis allowing for subsequent quantitative analysis. This nanotechnology provides a powerful tool that is uniquely suited for the discovery of novel biomarkers for early stage diseases such as cancer.SUPPORTING INFORMATION AVAILABLE: Available in the Supplementary Information are details on particles synthesis protocol, SDS PAGE analysis on molecular sieving properties and enzymatic degradation, and tables (Table S1 and S2) listing proteins (with peptide coverage lists) identified via LC-MS/MS (ESI) on material electroeluted from NIPAm and NIPAm/AAc particles. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public AccessAuthor Manuscript Nano Lett. Author manuscript; available in PMC 2010 May 28. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptThe concentration of proteins and peptides comprising t...

We used resting-state functional MRI to investigate spatial patterns of spontaneous brain activity in 22 healthy elderly subjects, as well as 16 mild cognitive impairment (MCI) and 16 Alzheimer's disease (AD) patients. The pattern of intrinsic brain activity was measured by examining the amplitude of low-frequency fluctuations (ALFF) of blood oxygen level dependent signal during rest. There were widespread ALFF differences among the three groups throughout the frontal, temporal, and parietal cortices. Both AD and MCI patients showed decreased activity mainly in the medial parietal lobe region and lentiform nucleus, while there was increased activity in the lateral temporal regions and superior frontal and parietal regions as compared with controls. Compared with MCI, the AD patients showed decreased activity in the medial prefrontal cortex and increased activity in the superior frontal gyrus and inferior and superior temporal gyri. Specifically, the most significant ALFF differences among the groups appeared in the posterior cingulate cortex, with a reduced pattern of activity when comparing healthy controls, MCI, and AD patients. Additionally, we also showed that the regions with ALFF changes had significant correlations with the cognitive performance of patients as measured by mini-mental state examination scores. Finally, while taking gray matter volume as covariates, the ALFF results were approximately consistent with those without gray matter correction, implying that the functional analysis could not be explained by regional atrophy. Together, our results demonstrate that there is a specific pattern of ALFF in AD and MCI, thus providing insights into biological mechanisms of the diseases.

We have undertaken a global analysis of sumoylated proteins in Saccharomyces cerevisiae by tandem mass spectrometry. Exposure of cells to oxidative and ethanol stresses caused large increases in protein sumoylation. A large number of new sumoylated proteins were identified in untreated, hydrogen peroxide-treated, and ethanol-treated cells. These proteins are known to be involved in diverse cellular processes, including gene transcription, protein translation, DNA replication, chromosome segregation, metabolic processes, and stress responses. Additionally, the known enzymes, including E1, E2, and E3 of the sumoylation cascade were found to be auto-sumoylated. Taken together, these results show that protein sumoylation is broadly involved in many cellular functions and this mass spectrometry-based proteomic approach is useful in studying the regulation of protein sumoylation in the cells.Protein ubiquitination is well known to be involved in protein degradation through the proteasome and intracellular protein sorting (1-3). A family of ubiquitin-like proteins has also been identified to be attached to substrate proteins through enzymatic processes similar to that of ubiquitination (4-6). However, the function of these ubiquitin-like proteins is unclear. Among them, SUMO, 1 a small ubiquitin-like modifier, is the most widely studied to date. In mammals, SUMO was first identified to be conjugated to substrate protein such as 8). The Smt3 gene, in the budding yeast Saccharomyces cerevisiae, is essential for cell viability and is the only budding yeast homolog of mammalian SUMO. Smt3 was originally identified as a suppressor of Mif2 mutation, and its function in the nucleus has been suggested (9). With an approach similar to that used in the identification of ubiquitination enzymes, the enzymes of protein sumoylation in budding yeast were identified (10,11).In the yeast S. cerevisiae, Smt3 is first proteolytically processed to expose its Gly-Gly repeat at the C terminus by a SUMO-specific protease, Ulp1 (12). In an ATP-dependent reaction, the C terminus of mature Smt3 forms a thioester bond with the cysteine residue at the active site of the E1 enzyme, which is a heterodimeric Aos1/Uba2 complex (10). Through a trans-* This work was supported in part by the Ludwig Institute for Cancer Research.To whom correspondence should be addressed: Ludwig Institute for Cancer Research, University of California at San Diego, 9500 Gilman Dr., CMM-East, Rm. 3050, La Jolla, CA 92093-0660. Tel.: 858-534-7808; Fax: 858-534-7750; huzhou@ucsd.edu EXPERIMENTAL PROCEDURES MaterialsExpression plasmid pYES2/NT was from Invitrogen. Plasmid pFA6a-3HA-kanMX6 was obtained from Mark Longtine (21). Yeast was grown in YPD (1% yeast extract, 2% peptone, and 2% dextrose) or in synthetic minimal defined medium with appropriate sugar at 30 °C. Ni + -NTA resin was from Qiagen; anti-FLAG antibody and conjugated-agarose were from Sigma. Anti-HA antibody and anti-HA affinity matrix were from Roche Applied Science. Protease inhibitor mixture for pur...

The nqr operon from Vibrio cholerae, encoding the entire six-subunit, membrane-associated, Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR), was cloned under the regulation of the P(BAD) promoter. The enzyme was successfully expressed in V. cholerae. To facilitate molecular genetics studies of this sodium-pumping enzyme, a host strain of V. cholerae was constructed in which the genomic copy of the nqr operon was deleted. By using a vector containing a six-histidine tag on the carboxy terminus of the NqrF subunit, the last subunit in the operon, the recombinant enzyme was readily purified by affinity chromatography in a highly active form from detergent-solubilized membranes of V. cholerae. The recombinant enzyme has a high specific activity in the presence of sodium. NADH consumption was assessed at a turnover number of 720 electrons per second. When purified using dodecyl maltoside (DM), the isolated enzyme contains approximately one bound ubiquinone, whereas if the detergent LDAO is used instead, the quinone content of the isolated enzyme is negligible. Furthermore, the recombinant enzyme, purified with DM, has a relatively low rate of reaction with O(2) (10-20 s(-1)). In steady state turnover, the isolated, recombinant enzyme exhibits up to 5-fold stimulation by sodium and functions as a primary sodium pump, as reported previously for Na(+)()-NQR from other bacterial sources. When reconstituted into liposomes, the recombinant Na(+)-NQR generates a sodium gradient and a Delta Psi across the membrane. SDS-PAGE resolves all six subunits, two of which, NqrB and NqrC, contain covalently bound flavin. A redox titration of the enzyme, monitored by UV-visible spectroscopy, reveals three n = 2 redox centers and one n = 1 redox center, for which the presence of three flavins and a 2Fe-2S center can account. The V. cholerae Na(+)-NQR is well-suited for structural studies and for the use of molecular genetics techniques in addressing the mechanism by which NADH oxidation is coupled to the pumping of Na(+) across the membrane.

Domestic chickens are valuable sources of protein associated with producing meat and eggs for humans. The gastrointestinal tract (GIT) houses a large microbial community, and these microbiota play an important role in growth and health of chickens, contributing to the enhancement of nutrient absorption and improvement of the birds' immune systems. To improve our understanding of the chicken intestinal microbial composition, microbiota inhabiting 5 different intestinal locations (duodenum, jejunum, ileum, cecum, and colon) of 42-day-old broiler chickens were detected based on 16S rRNA gene sequence analysis. As a result, 1,502,554 sequences were clustered into 796 operational taxonomic units (OTUs) at the 97% sequence similarity value and identified into 15 phyla and 288 genera. Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, and Cyanobacteria were the major microbial groups and Firmicutes was the dominant phylum in duodenum, jejunum, ileum and colon accounting for > 60% of sequences, while Bacteroidetes was the dominant phylum in cecum (>50% of sequences), but little in the other four gut sections. At the genus level, the major microbial genera across all gut sections were Lactobacillus, Enterococcus, Bacteroides, and Corynebacterium. Lactobacillus was the predominant genus in duodenum, jejunum, and ileum (>35%), but was rarely present in cecum, and Bacteroides was the most dominant group in cecum (about 40%), but rarely present in the other 4 intestinal sections. Differences of microbial composition between the 5 intestinal locations might be a cause and consequence of gut functional differences and may also reflect host selection mediated by innate or adaptive immune responses. All these results could offer some information for the future study on the relationship between intestinal microbiota and broiler chicken growth performance as well as health.

There is conflicting evidence regarding a possible association between the apolipoprotein E4 (APOE4) allele and the consequences of traumatic brain injury (TBI). Our aim was to carry out a meta-analysis of cohort studies of sufficient rigor to determine whether the presence of the APOE4 allele contributes to initial injury severity and/or poor outcome following TBI. MEDLINE, EMBase, CBMdisc, and CNKI databases were searched for literature published from January 1993 to October 2007. Of the 100 identified studies, 14 cohort studies were selected for analysis based on comprehensive quality assessment using a standardized scale. Data from the 14 eligible cohort studies included a total of 2527 participants, 736 with and 1791 without the APOE4 allele. The APOE4 allele was not associated with initial injury severity of TBI. The pooled RR were 1.11 (95% confidence interval [CI], 0.91 to 1.35) for severe injury, 1.06 (95% CI, 0.86-1.31) for moderate injury and 0.93 (95% CI, 0.81-1.06) for mild injury. However, the APOE4 allele was significantly associated with a poor outcome of TBI at 6 months after injury (RR = 1.36; 95% CI, 1.04-1.78). The association remained significant in sensitivity tests. This meta-analysis indicates that the presence of the APOE4 allele is not associated with the initial severity of brain injury following TBI but is associated with increased risk of poor long-term outcome at 6 months after injury.