Developmental and adult phenotyping directly from mutant embryonic stem cells

Tetraploid embryo complementation assay has shown that mouse ES cells alone are capable of supporting embryonic development and adult life of mice. Newly established F(1) hybrid ES cells allow the production of ES cell-derived animals at a high enough efficiency to directly make ES cell-based genetics feasible. Here we report the establishment and characterization of 12 new F(1) hybrid ES cell lines and the use of one of the best (G4) in a gain- and loss-of-function genetic study, where the in vivo phenotypes were assessed directly from ES cell-derived embryos. We found the generation of G4 ES cell-derived animals to be very efficient. Furthermore, even after two consecutive rounds of genetic modifications, the majority of transgenic lines retained the original potential of the parental lines; with 10-40% of chimeras producing ES cell-derived animals/embryos. Using these genetically altered ES cells, this success rate, in most cases, permitted the derivation of a sufficient number of mutants for initial phenotypic analyses only a few weeks after the establishment of the cell lines. Although the experimental design has to take into account a moderate level of uncontrolled damage on ES cell lines, our proof-of-principle experiment provides useful data to assist future designs harnessing the power of this technology to accelerate our understanding of gene function.     

Effect of bisphosphonates on nitric oxide production by inflammatory activated chondrocytes

OBJECTIVE: Bisphosphonates have been reported to possess anti-inflammatory and cartilage protective effects in animal arthritis models but not much is known about their direct effect on chondrocytes. In this study we evaluate the effect of bisphosphonates on nitric oxide (NO) production by activated chondrocytes. METHODS: Isolated bovine chondrocytes and bovine cartilage explants were used. In the second part of the study human cartilage explants (osteoarthritis (OA) and non-OA cartilage) were used. The isolated chondrocytes and cartilage explants were pre-incubated with clodronate, pamidronate or risedronate and stimulated with IL-1 and TNF-alpha (10 ng/mL, 48 h). NO production was quantified using the Griess assay. RESULTS:In bovine cultures, clodronate (10(-4)mol/L) and pamidronate (10(-6)mol/L) showed a small inhibition of NO production (up to 15 % and 25% respectively), whereas risedronate had no effect.In the human cartilage cultures no effect of BPs on the NO production was detected except for the highest concentration of clodronate tested (10(-4)mol/L) which demonstrated a small enhancement (19%) in NO production reaching significance in the non-OA group. CONCLUSION: BPs have a modest effect on NO production by inflammatory activated chondrocytes only in the higher concentrations, indicating that the clinical relevance of these effects is probably negligible.     

Blood pressure before and after electroconvulsive therapy in hypertensive and nonhypertensive patients

The effect of a course of electroconvulsive therapy (ECT) on blood pressure control in hypertensive patients has not been studied. We retrospectively examined pre- and post-ECT blood pressures in hypertensive and nonhypertensive patients. In neither group was there a statistically significant change in blood pressure with a course of ECT. We conclude that a course of ECT does not worsen blood pressure in hypertensive patients beyond the peritreatment period.     

Neuroendocrine responses to an acute bout of eccentric-enhanced resistance exercise

PURPOSE: The purpose of this study was to compare the total testosterone (TT), bioavailable testosterone (BT), growth hormone (GH), lactate, and ratings of perceived exertion (RPE) responses between a single bout of traditional (TRAD) and eccentric-enhanced resistance exercise (ECC+) of matched training volumes. METHODS: Twenty-two previously untrained males (21.9+/-0.8 yr) completed one familiarization and one baseline 1RM testing bout, for the bench press and squat exercises, and then two exercise bouts. During exercise bout 1, all subjects completed a TRAD protocol (four sets of six reps at 52.5% 1RM), and the subsequent exercise bout consisted of either a TRAD or an ECC+ protocol (three sets of six reps at 40% 1RM concentric and 100% 1RM eccentric) for the bench press and squat exercises. Blood samples acquired at rest, immediately after (T1), and 15, 30, 45, and 60 min after exercise were assessed for serum TT, BT, GH, and blood lactate concentrations. RESULTS: Resting and postexercise TT, BT, and GH were not significantly different between groups. Postexercise TT was not elevated during either bout or in either group, whereas BT increased 15-16% at T1 in both groups during bout 2. Postexercise GH concentrations were elevated 500-7000% above baseline after both protocols. Postexercise lactate accumulation and RPE were greater with ECC+ than TRAD. CONCLUSION: TRAD and ECC+ show similar neuroendocrine and differing metabolic responses during the early phase of resistance exercise in untrained, college-age men.     

Can screening items identify surgery patients at risk of limited health literacy?

BACKGROUND: Health literacy skills (HLS) have been shown to have a major impact on patient outcomes. To identify patients with limited or marginal HLS, the accuracy of three established screening items were examined. MATERIALS AND METHODS: We studied English-speaking adults (>or=21 years) attending a university-based vascular surgery clinic. Structured interviews were conducted to assess sociodemographic characteristics, screening items, and HLS. Area under the receiver operating characteristic (AUROC) curves were plotted to assess the discriminatory capacity of each screening item in detecting patients with limited/marginal HLS. RESULTS: One hundred patients agreed to enter the study and met inclusion criteria. The mean age was 62.0 +/- 12.9; 65 were female; 96 were Caucasian; and 32 had not completed high school. The three screening items were effective in detecting patients with limited (n=18) or marginal (n=21) HLS. "How often do you have someone (like a family member, friend, or hospital worker) help you read hospital materials?" (AUROC of 0.83; 95% confidence interval [CI]=0.73, 0.92), "How often do you have problems learning about your medical condition because of difficulty understanding written information?" (AUROC of 0.77; 95% CI=0.67, 0.86), and "How confident are you filling out medical forms by yourself?" (AUROC of 0.76; 95% CI=0.66, 0.86) were effective in detecting those with limited/marginal HLS skills. CONCLUSIONS: Our findings provide further evidence of the clinical usefulness of these screening items for detecting inadequate HLS in this patient population. Surgeons should consider administering these easy screening items to identify patients at greatest risk of limited or marginal HLS.     

A proteomic view into infection of greyback canegrubs (<Emphasis Type="Italic">Dermolepida albohirtum</Emphasis>) by <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis>

Metarhizium anisopliae is a naturally occurring cosmopolitan fungus infecting greyback canegrubs (Dermolepida albohirtum). The main molecular factors involved in the complex interactions occurring between the greyback canegrubs and M. anisopliae (FI-1045) were investigated by comparing the proteomes of healthy canegrubs, canegrubs infected with Metarhizium and fungus only. Differentially expressed proteins from the infected canegrubs were subjected to mass spectrometry to search for pathogenicity related proteins. Immune-related proteins of canegrubs identified in this study include cytoskeletal proteins (actin), cell communication proteins, proteases and peptidases. Fungal proteins identified include metalloproteins, acyl-CoA, cyclin proteins and chorismate mutase. Comparative proteome analysis provided a view into the cellular reactions triggered in the canegrub in response to the fungal infection at the onset of biological control.     

Campylobacter jejuni Glycosylation Island Important in Cell Charge,Legionaminic Acid Biosynthesis,and Colonization of Chickens

Previously, we identified five genes (Cj1321 to Cj1326, of which Cj1325 and Cj1326 are a single gene) in the O-linked flagellin glycosylation island that are highly prevalent in Campylobacter jejuni isolates from chickens. We report mutagenesis, functional, and structural data to confirm that this locus, and Cj1324 in particular, has a significant contributory role in the colonization of chickens by C. jejuni. A motile ΔCj1324 mutant with intact flagella was considerably less hydrophobic and less able to autoagglutinate and form biofilms than the parent strain, 11168H, suggesting that the surface charge of flagella of Cj1324-deficient strains was altered. The physical and functional attributes of the parent were restored upon complementation. Structural analysis of flagellin protein purified from the ΔCj1324 mutant revealed the absence of two legionaminic acid glycan modifications that were present in the parent strain, 11168H. These glycoform modifications were shown to be prevalent in chicken isolates and confirm that differences in the highly variable flagellin glycosylation locus can relate to the strain source. The discovery of molecular mechanisms influencing the persistence of C. jejuni in poultry aids the rational design of approaches to control this problematic pathogen in the food chain.Campylobacter jejuni is the leading bacterial cause of human gastroenteritis worldwide (7). Infection can cause symptoms including abdominal pain and fever with watery to bloody diarrhea (54). Occasionally, postinfectious sequelae follow C. jejuni infection and include reactive arthritis and Guillain-Barré syndrome (8). Recently, C. jejuni has been associated with immunoproliferative small intestine disease, which is a rare type of mucosa-associated lymphoid tissue lymphoma (31). The main source of transmission through the food chain is the consumption and handling of contaminated poultry, but the underlying reasons why chickens are particularly susceptible to colonization by C. jejuni are unknown (15). C. jejuni has also been recovered from nonavian livestock, unpasteurized milk, and contaminated water (7). The socioeconomic burden of this pathogen means that it is imperative that ways of reducing the levels of C. jejuni in the food chain, particularly poultry, are investigated.The glycosylation of flagellin in a number of gram-negative pathogenic bacteria, including Pseudomonas aeruginosa, Helicobacter pylori, and Aeromonas spp., is increasingly recognized as playing significant roles (2, 24, 32, 43, 49). Glycosylation modifications have been shown to influence the cell''s immunogenicity, interaction with eukaryotic cells, and host cell specificity. Aeromonads are waterborne bacteria that can cause disease in fish, reptiles, and amphibians. Mesophilic aeromonads are important human pathogens causing gastrointestinal infections and, in severe cases, wound disease and septicemia in healthy and immunocompromised patients (63). Flagella of the mesophilic aeromonad Aeromonas caviae have been shown to be glycosylated (43) with a derivative of pseudaminic acid (50). In the plant pathogen Pseudomonas syringae pv. glycinea, the mutation of three genes located in a flagellin glycosylation island results in alterations to host specificity (61). Mutants of P. syringae pv. glycinea fail to cause symptoms in the normal host, soybean plants, but can grow on nonhost tobacco leaves, causing symptom-like changes on leaves. Takeuchi et al. proposed that the posttranslational modification of flagellin may be an adaptation of the bacterium to avoid recognition by host defenses (61). In P. aeruginosa strain PAK, a flagellin glycosylation island comprising 14 genes was discovered and shown to cause glycosylation exclusively for P. aeruginosa isolates expressing a-type flagellin (2). Further studies have shown that there appears to be variation in the glycosylation islands of strains containing the a-type flagellin (4). A glycosylation island comprising four genes in the type b flagellin strain P. aeruginosa PAO has been found. When a mutant unable to glycosylate flagellin was tested in a murine model of burn wound infection, it exhibited a reduction in virulence compared to that of the wild type (3). Thus, it appears that in P. aeruginosa different glycoforms on the flagellin are required for the colonization of different hosts or environments and that these glycoforms may provide the bacterium with a specific survival advantage.We recently examined 111 strains of C. jejuni, including human, chicken, bovine, ovine, and environmental isolates, using comparative phylogenomics (whole-genome comparisons of microbes using DNA microarrays combined with Bayesian-based phylogenies) (10). Isolates fell into two distinct clades, which based on the origins of the isolates were defined as livestock-associated and non-livestock-associated clades. Over 40 genes were identified as being significantly prevalent in either of the clades. Among these was a set of six genes, Cj1321, Cj1322, Cj1323, Cj1324, Cj1325, and Cj1326 (as identified in the initial annotation by Parkhill et al. for the original sequenced C. jejuni strain, NCTC11168 [42]), that lie within a region of the genome encoding the flagellin O-linked glycosylation system. Thus, although genes Cj1321 to Cj1326 are located within a region of the genome which has variability, they are conserved among some C. jejuni strains that are often associated with livestock. Microarray data have shown that the six genes are all transcribed in the same orientation, but it is unknown if they are an operon (N. Dorrell and B. W. Wren, unpublished data). NCTC11168 has since been reannotated, and as a result, Cj1325 and Cj1326 are now considered to be one gene, hereinafter referred to as Cj1325/6 (22). Previous BLAST analyses have shown that the Cj1321 protein has amino acid similarity to many bacterial acetyl transferases, both Cj1322 and Cj1323 proteins are similar to hydroxyacyl dehydrogenases, and the product of Cj1324 is similar to WbpG, a protein involved in lipopolysaccharide synthesis in many bacteria.In C. jejuni NCTC11168 (the original sequenced strain, found in the livestock clade), the O-linked flagellar glycosylation system is thought to consist of a cluster of approximately 50 genes (Cj1293 to Cj1342) adjacent to flaA and flaB which encode the structural flagellin proteins (42). The full glycan structure(s) in NCTC11168 (and most other strains associated with livestock) is unknown, but given the considerably larger size of the O-linked glycosylation loci in the livestock-associated strains than in the non-livestock-associated strains, it is likely that the livestock-associated strains may have additional modifications to the pseudaminic acid basic structure, as well as other unique glycan moieties, compared to those of the non-livestock-associated strains. The flagellin O-linked glycosylation locus in C. jejuni 81-176 (a frequently studied human strain found in the non-livestock-associated clade) is far simpler than that in C. jejuni NCTC11168, comprising just 26 genes (21). Two modifications predominantly decorate FlaA and FlaB of strain 81-176, the nine-carbon sugar pseudaminic acid (5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-α-l-manno-nonulosonic acid [Pse5Ac7Ac]) and an acetamidino form of pseudaminic acid, 5-acetamido-7-acetamidino-3,5,7,9-tetradeoxy-l-glycero-α-l-manno-nonulosonic acid (Pse5Ac7Am). Derivatives of Pse5Ac7Am also decorate the flagellin of 81-176 in minor quantities (34, 37, 62). Genetic analysis of 81-176 showed that pse genes are involved in the biosynthesis of pseudaminic acid and its derivatives (21, 37, 62). More recently, the full biosynthetic pathway for pseudaminic acid was determined; in a six-step reaction, UDP-N-acetylglucosamine (UDP-GlcNAc) is converted to pseudaminic acid through the actions of PseB/Cj1293, PseC/Cj1294, PseH/Cj1313, PseG/Cj1312, PseI/Cj1317, and PseF/Cj1311 proteins (The Cj designations refer to predicted coding sequences in C. jejuni NCTC11168) (11, 19, 21, 35, 52, 62).The most detailed analysis of the flagellin O-linked glycosylation locus has been undertaken with C. jejuni strain 81-176 and the related species Campylobacter coli (strain VC167) (34). Structural studies of the flagellum modifications of C. coli VC167 revealed that in addition to Pse5Ac7Ac, acetamidino and N-methylacetimidoyl derivatives of legionaminic acid [5-acetamidino-7-acetamido-3,5,7,9-tetradeoxy-d-glycero-d-galacto-nonulosonic acid (Leg5Am7Ac) and 5-E/Z-N-(N-methlyacetimidoyl)-7-acetamidino-3,5,7,9-tetradeoxy-d-galacto-nonulosonic acid (Leg5AmNMe7Ac), respectively] decorate the C. coli flagellin, the first demonstration of a legionaminic acid derivative modification of bacterial flagellin (36). Biosynthesis of these legionaminic acid derivatives involves a distinct pathway encoded by the posttranslational modification (ptm) genes (34, 36). Although the precise pathway for the production of legionaminic acid has yet to be determined, tentative functions have been assigned which have identified PtmA to PtmH to be required for biosynthesis (PtmA, PtmB, PtmC, PtmD, PtmE, PtmF, PtmG, and PtmH are equivalent to the Cj1332, Cj1331, Cj1327, Cj1328, Cj1329, Cj1330, Cj1324, and Cj1325/6 proteins, respectively) (36). This ptm pathway is absent in C. jejuni strain 81-176. PtmG and PtmH from C. coli VC167 show 86 and 76% amino acid sequence similarity, respectively, to two hypothetical proteins, the Cj1324 and Cj1325/6 proteins of C. jejuni NCTC11168. The enzyme(s) involved in the attachment of glycan(s) to the flagellin protein of Campylobacter strains and the consensus sequence for the O-linked glycosylation process have yet to be identified.In C. jejuni strain 81-176, glycosylation of flagellin has been shown to be necessary for the assembly of flagella and subsequent motility (19). There is extensive polymorphism in the C. jejuni O-linked glycosylation cluster, suggesting that selective pressure may cause the bacterium to alter surface antigens in attempts to evade the host immune defenses (59). Evidence supporting this possibility is demonstrated by comparing the glycan moieties of the flagella of C. jejuni 81-176 and C. coli VC167, as these strains produce unique modifications on their flagella which affect serospecificity (34).Given the diversity of the O-linked glycosylation system in C. jejuni and the prevalence of the locus of Cj1321 to Cj1325/6 in chicken isolates, we hypothesized that these genes may be important for the abilities of some C. jejuni strains to colonize poultry and that colonization may be mediated through structural and surface charge changes in the glycan that modifies the flagellin. In this study, we demonstrate that Cj1324 is involved in the biosynthesis of two novel legionaminic acid modifications found on the flagellin of strain 11168H. The presence of these modifications affects autoagglutination, cell charge, and the efficiency with which C. jejuni 11168H colonizes chickens.