Our earlier studies showed that the thione tautomer is energetically favored (Wujec

et al., 2007). The IR spectra of compounds 7–9 showed the absorption bands at 3,437–3,411 cm−1 and 1,331–1,328 cm−1, indicating the presence of NH and C=S groups, respectively. In the 1H-NMR spectra, NH proton resonated as a singlet at ~14 ppm. Crystallographic data (unpublished results) also confirm the existence of the mentioned compounds as the C=S tautomers. Scheme 1 Synthetic route to target compounds 10–21. Reagents and conditions: a EtOH, reflux, 5 min; b 2 % LEE011 clinical trial NaOH, reflux, 2 h; c HCHO, amine, EtOH, 30 min The Mannich reaction was carried out in mild conditions; it was quick (30 min) and efficient (yields: 76–87 %). The structure and purity of the products (10–21) was confirmed using 1H-NMR, 13C-NMR (for compound 20), and IR spectra as well as elemental analysis. The 1H-NMR spectra showed characteristic signals which indicated the presence of aminomethyl fragment. Two protons of the N2–CH2– group resonated as a singlet in the range of 5.22–5.34 ppm, while the signals

of the amine residues were visible at 1.20–3.76 ppm. In addition Selleck MK2206 to this, peaks characteristic for para-substituted phenyl rings were visible in the area typical for aromatic protons. The IR spectra also confirmed the suggested structure of the Mannich bases (10–21). Antibacterial screening The antibacterial activity of compounds 10–21 was determined for Gram-positive and Gram-negative bacteria. The growth of Gram-negative bacteria (Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 13883, Proteus mirabilis ATCC 12453, and Pseudomonas aeruginosa ATCC 9027) was not inhibited by any of the compounds. Therefore, Table 1 shows the Mannich bases activity only for five investigated Gram-positive bacterial strains. The activity toward the pathogenic Staphylococcus aureus strains was moderate. Minimum concentrations which inhibited the growth of S. aureus ATCC 25923 ranged to 31.25 μg ml−1 (15, 18, 19), and the most active toward

Oxymatrine methicillin-resistant (MRSA) strain were derivatives with diethylaminomethyl (18) and pyrrolidinylmethyl (19) substituents. In both cases, the MIC values equaled 62.5 μg ml−1. Opportunistic (relatively pathogenic) bacteria was by far more sensitive to the newly obtained compounds. In the case of Bacillus cereus ATCC 10876, the activity of three derivatives (14, 15, 21) was similar to the activity of ampicillin, and the activity of another two derivatives (18, 19) was twice as strong. Moreover, the antibacterial activity of the compound with the N2-pyrrolidinylmethyl fragment (15) toward Bacillus subtilis ATCC 6633 was as strong as cefuroxime’s; as far as Micrococcus luteus ATCC 10240 is concerned, the most active compound was the derivative of 4-(4-bromophenyl)-5-(4-chlorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione with pyrrolidinylmethyl substituent (19, MIC = 7.81 μg ml−1).

The in vitro effects of polyamines on immune functions were first reported over 30 years ago [92]. However, later analysis revealed that the reported immunosuppressive effects are induced not by the direct effect of polyamines but by substances produced by the

interaction between polyamines and serum amine oxidase, present exclusively in ruminants, making these results difficult to extend to humans, which lack this enzyme. Nonetheless, animal experiments have shown that polyamine deprivation prevents the development of tumor-induced immunosuppression [93]. The adhesion characteristics of immune cells are important for eliciting anti-tumor cytotoxic activity, because adhesion is crucial for immune cell recognition of tumor cells [94]. Due to decreased adhesion, immune cells fail to recognize cancer cells or exert tumoricidal activities. Such decreases HM781-36B in immune cell adhesion are

observed not only in cancer patients but also in patients having non-cancerous lesions [82]. These findings suggest the possibility that common factor(s), not specifically produced in cancer patients, can induce immunosuppressive conditions. Polyamines are one such factor, because blood polyamine levels, namely levels in blood cells including immune cells, are often increased in patients with various diseases [36, 95–97]. Immune cells also take up polyamines form their surroundings selleck products [98, 99], and the increase in blood polyamine concentrations often observed Oxymatrine in cancer patients as well as in patients with other diseases reflects the increased polyamine levels in leukocytes [36, 100]. We have shown that increased concentrations of spermine or spermidine in cultured human PBMCs suppress adhesion without sacrificing cell viability and activity. The time- and dose-dependent decrease in adhesion produced by polyamines was accompanied by decreases in the expression of lymphocyte function-associated antigen-1 (LFA-1), which consists of an integrin alpha L (CD11a) and beta

2 (CD18) chain [41]. Polyamines in particular decrease the number of cells expressing bright CD11a. Such suppression was exclusively observed for LFA-1 with most other adhesion molecules tested unaffected by polyamines. The suppression of LFA-1 expression by polyamines was further confirmed in human healthy volunteers with polyamines suppressing LFA-1 expression on PBMCs, regardless of the volunteer’s age [41]. In addition to LFA-1 suppression by polyamines, the number of CD56 bright cells was decreased by polyamines in vitro, although the effect was not confirmed in vivo. LFA-1 and CD56 contribute to the induction of tumoricidal cell activities, especially lymphokine activated killer (LAK) activity [101, 102]. LAK cells, which have tumoricidal activities against established (existing) tumors, are induced by co-culture with IL-2 [103, 104].

The bacteria were grown at 37°C or 42°C with rapid shaking (~200 rpm) in flasks with a large headspace and harvested in early stationary phase (~5 × 109 colony forming units [CFU]/ml). Alternatively, the bacteria were grown under low oxygen tension in a bottle filled with medium to minimize the headspace and shaken slowly (75 rpm) to favor biofilm formation [29]. Bacteria were also grown in a strict anaerobic environment on CBA in a BD GasPak system (BD Diagnostic

Systems), or in CTT containing Oxyrase for Broth™ (Oxyrase, Mansfield, OH). For some experiments, the medium was supplemented with 2% NaCl, or the bacteria were harvested during mid- to late-stationary phase (48-72 h post-inoculation). For growth BTK signaling pathway inhibitors supplementation with Neu5Ac, 1 mg (50-μg/ml final concentration)

of Neu5Ac (Sigma Chemical Co.) was added to CBA, TTT, or to a chemically defined medium [31]. Polysaccharide purification H. somni was grown on CBA plates incubated in 5% CO2 or anaerobic conditions for 48-72 h at 37°C. The cells were scraped from the plates and suspended in phosphate buffered saline, pH 7.2, (PBS) to a turbidity of 150 Klett units (about 109 CFU/ml). After vigorous vortexing at room temperature, the cell suspension was incubated at 37°C for 1 h, vortexed again, and the cells removed by centrifugation (10,000 × g for 15 min). Cetavlon (hexadecyltrimethyl ammonium bromide) was added to a final concentration of 0.005 M. Any precipitate that formed was harvested and solubilized in distilled water. Rucaparib chemical structure Tideglusib No further purification was done on this sample. Alternatively, the bacteria were grown to late stationary phase in CTT (48-72 h

post-inoculation), the bacteria harvested as above, and Cetavlon added to the supernatant. Any precipitate that formed following addition of Cetavlon was further purified by enzyme digestion (RNase, DNase, and Proteinase K), phenol extraction, and ultracentrifugation to remove LOS, as described for purification of the capsular polysaccharide of Actinobacillus pleuropneumoniae [32]. The bacteria were also grown at 37°C in filled 1-L bottles containing TTT with shaking at 75 rpm for 4-5 days. The clear supernatant was carefully removed and the sediment was extracted with 45% aqueous phenol at room temperature, digested with DNase, RNase, and Proteinase K, and subjected to ultracentrifugation at 125,000 × g at 4°C, as described for purification of H. somni LOS [33], except that the supernatant from the ultracentrifugation step was retained. Polysaccharide in the supernatant was precipitated by the addition of 30 mM sodium acetate (final) and 5 volumes of cold (-20°C) 95% ethanol, and incubated at -20°C for at least 4 hours. The pellet obtained by centrifugation was suspended in distilled water, and eluted through a Sephacryl S-400 column (2.5 × 50 cm) with distilled water as eluent. The first fractions containing carbohydrate (determined by phenol-sulfuric acid assay) [34] were pooled and lyophilized.

This study is the first of its kind in Zambia to describe the molecular typing of M.bovis isolates from indigenous cattle breeds originating from high prevalence settings. Characterization of M. bovis strains based on different geographical locations by districts or region is pivotal in understanding the molecular epidemiology of BTB [21, 23, 27]. It further helps in understanding the dynamics of disease dispersion which are difficult to appreciate through traditional epidemiological investigative tools. However, through the use of modern molecular epidemiological

tools such as spoligotyping, we have been able to demonstrate the presence as well as the specific existing strains of Mycobacterium bovis in Zambian cattle. The technique has shade more light CH5424802 ic50 on the strain diversity, distribution and relatedness within Zambia and globally. Two dominant spoligotypes were identified representing the majority of isolates analyzed. These findings intimate a degree of homogeneity among M. bovis isolates in Zambia. However, when distinguishing between unrelated strains through the application of the Hunter-Gaston Discriminatory Index [28, 29], the spoligotyping technique in this particular case was found Acalabrutinib manufacturer to have a good discrimination power. The index indicated that 98% of the strains had an equal chance of having different spoligo patterns

if randomly sampled. Of the 31 M. bovis isolates that yielded interpretable spoligotypes, 10 different patterns were detected. Based on the global spoligotype patterns diversity provided by the international data base on spoligotyping, http://​www.​mbovis.​org, 83.9% of the isolates have been described. The predominant spoligotype that was widely dispersed geographically was found on the international data base to have a pattern with a spoligotype number SB0120. This spoligotype is similar to the spoligotype of the vaccine strain BCG type, and previously described in France, Belgium, South Africa, The Netherlands, Sri Lanka, Spain, Japan, Portugal, SPTBN5 Russia, Iran, Denmark, China and Brazil http://​www.​mbovis.​org[30]. The

second most predominant spoligotype had a pattern previously numbered SB0871 and has been described from France. These predominant patterns, SB0120 and SB0871, differ only by a single spacer (spacer 10). The most common spoligotype, SB0120, has a considerable degree of geographical dispersion in Zambia, being detected in 5 out of the 6 districts, and has further been shown to be common in other countries including continental Europe [31, 32]. This finding of strains from Europe may suggest the introduction of the disease by early European settlers to Africa, a finding that has been highlighted by different workers [17, 23, 27]. The finding of SB0120 in South Africa strongly infers to this probability, when tracing the early migration routes of colonial settlers to Zambia. In our current study, 16.1% (5/31) of the isolates had spoligotypes that were unique to Zambia.

J Infect Dis 1987, 156:770–776.PubMedCrossRef 28. Katragkou A, Kruhlak MJ, Simitsopoulou M, Chatzimoschou

A, Taparkou A, Cotten CJ, Paliogianni F, Diza-Mataftsi E, Tsantali C, Walsh TJ, et al.: Interactions between human phagocytes and Candida albicans biofilms alone and in combination with antifungal agents. J Infect Dis 2010, 201:(12):1941–1949.PubMedCrossRef 29. Chimento A, Cacciola SO, Garbelotto M: Detection of mRNA by Reverse Transcription PCR as an Indicator of Viability in Phytophthora ramorum . In Proceedings of the Sudden Oak Death Third Torin 1 cost Science Symposium. Santa Rosa, California; 2007. 30. Martinez A, Lahiri R, Pittman TL: Molecular determination of Mycobacterium leprae viability by use of real-time PCR. J Clin

Microbiol 2009, 47:2124–2130.PubMedCrossRef 31. Varughese E, Wymer LJ, Haugland RA: An integrated culture and real-time PCR method to assess viability of disinfectant treated Bacillus spores using robotics and the MPN quantification method. J Microbiol Meth 2007, 71:66–70.CrossRef 32. Hao B, Clancy C, Cheng S, Raman S, Iczkowski K, Nguyen M: Candida albicans RFX2 encodes a DNA binding protein involved in DNA damage responses, morphogenesis, and virulence. Nivolumab manufacturer Eukaryot Cell 2009, 8:627–639.PubMedCrossRef 33. Khot P, Suci PA, Miller RL, Nelson RD, Tyler BJ: A small subpopulation of blastospores in Candida albicans biofilms exhibit resistance to amphotericin B associated with differential regulation of ergosterol and β -1,6-glucan pathway genes. Antimicrob Agents Chemother 2006, 50:3708–3716.PubMedCrossRef BCKDHB 34. Taylor B, Hannemann H, Sehnal M, Biesemeier A, Schweizer A, Rollinghoff M, Schroppel K: Induction of SAP7 correlates with virulence in an intravenous infection model of candidiasis but not in a vaginal infection model in mice. Infect Immun 2005, 73:7061–7063.PubMedCrossRef 35. Theiss S, Ishdorj G, Brenot A, Kretschmar M, Lan CY, Nichterlein T, Hacker J, Nigam S, Agabian N, Kohler GA: Inactivation of the phospholipase B gene PLB5 in wild-type Candida albicans reduces cell-associated phospholipase

A2 activity and attenuates virulence. Int J Med Microbiol 2006, 296:405–420.PubMedCrossRef 36. Uppuluri P, Chaturvedi AK, Lopez-Ribot JL: Design of a simplemodel of Candida albicans biofilms formed under conditions of flow: development, architecture, and drug Resistance. Mycopathologia 2009, 168:101–109.PubMedCrossRef 37. Vogel M, Hartmann T, Köberle M, Treiber M, Autenrieth I, Schumacher U: Rifampicin induces MDR1 expression in Candida albicans . J Antimicrob Chemother 2008, 61:541–547.PubMedCrossRef 38. Fonzi WAMI: Isogenic strain construction and gene mapping in Candida albicans . Genetics 1993, 134:717–728.PubMed 39. Dongari-Bagtzoglou A, Kashleva H: Development of a highly reproducible 3D organotypic model of the oral mucosa. Nature Protocols 2006,1(4):2012–2018.PubMedCrossRef 40.

Most of these proteins (61%) had an intact EAL domain, including the EVL motif (Figure 1), and 39% had EAL degenerate domains (Table 1). Some of the EAL degenerate proteins, such as KPK_A0040 and KPN_pKPN3p05966, were found on plasmids. pneumoniae 342, MGH 78578 and NTUH-K2044   K. pneumoniae342 K. pneumoniaeMGH 78578 K. pneumoniaeNTUH-K2044 Total Predicted DGC Total Predicted PDE   GGDEF

GGDEF + EAL EAL GGDEF GGDEF + EAL EAL GGDEF GGDEF + EAL EAL     Total proteins 15 6 15 13 5 15 12 5 10 56 56 Transmembrane segments 11 5 6 9 4 7 8 4 6 41(73%) 32 (57%) Signal peptides 1 1 1 1 0 1 0 0 1 3 (5%) 4 (7%) With GAF domain 3 0 1 4 0 0 3 0 0 10 (18%) 1 (2%) With HAMP domain 2 1 0 2 1 0 1 1 0 8 (14%) 3 (5%) With PAS domain 1 1 0 1 1 0 1 1 0 6 (11%) 3 (5%) With BLUF Doxorubicin mw domain 0 0 3 0 0 2 0 0 2 0 7 (12%) With MASE domain 2 1 1 0 1 1 0 1 1 5 (9%) 6 (11%) With CACHE domain 1 0 0 2 0 0 2

0 0 5 (9%) 0 With CHASE domain 0 1 0 this website 0 0 0 0 0 0 1 (2%) 1 (2%) With CSS-motif domain 0 0 5 0 0 6 0 0 5 0 16 (28%) With sensor domains 9 4 10 9 3 9 7 3 8 35 (62%) 37 (66%) With allosteric I site 7 0 0 7 0 0 5 0 0 19 (34%) 0 With degenerate GGDEF 1 3 0 1 3 0 0 3 0 11 (20%) 9 (56%)* With degenerate EAL 0 2 6 0 2 6 0 2 4 6 (38%)* 22 (39%) *Average calculated based only on the number of hybrid proteins (16). Numbers in parenthesis indicate % of total for either DGCs or PDEs. Figure 1 Logo sequences for DCG and PDE domains. Logos are shown for the active DGC domain and the I site (A) and the PDE domain (B). Red rectangles show the conserved A site (GGEEF or GGDEF), the I site (RxxD), and the EAL domain. The error bars indicate an approximate, Bayesian 95% confidence interval. To further characterize these proteins, signal peptides, sensor and conserved domains were identified. Only 5% of GGDEF and 7% of EAL proteins in Bacterial neuraminidase K. pneumoniae included signal peptides (Table 1), indicating that they could be transported across or anchored

in membranes [20, 21]. A larger proportion of the proteins contained transmembrane segments, 73% of the GDDEF and 57% of EAL-containing proteins (Table 1), suggesting that regulation and/or enzyme activity is most likely occurring at the membrane, as has been suggested [22, 23]. Sensor domains found in GGDEF and EAL containing proteins One of the most intriguing aspects of the enzymes involved in modulating intracellular levels of c-di-GMP is their modular structure characterized by the presence of additional input sensory domains [24]. Therefore, a search was carried out for the diverse periplasmic, cytoplasmic, and integral membrane domains that have been described [23, 25]. Most of the GGDEF and EAL-containing proteins in K. pneumoniae contained sensor domains, 62% and 66%, respectively (Table 1).

Scand J Work Environ Health 22:251–259CrossRef Vingard E, Alfredsson L, Goldie I, Hogstedt C (1991) Occupation and osteoarthrosis of the hip and knee, a register-based cohort study. Int J Epidemiol 20:1025–1031CrossRef Wickström G, Hänningen K, Mattison T, Niskanen T, Riihimäki H, Waris P, Zitting A (1983) Knee degeneration in concrete reinforcement workers. Br J Ind Med 40:216–219 Zelle J, Barink M, De Malefjit Waal M, Verdonschot N (2009) Thigh-calf contact: does it affect the loading of the knee in the high-flexion range? J Biomech 42(5):87–93CrossRef”
“Background Stress-related mental disorders and musculoskeletal disorders are the

two most important factors behind long-term sick leave in Sweden and account for a considerable amount of the total economic burden on society, companies and organizations (Statistics Sweden 2010). Regarding human STI571 manufacturer service organizations in Sweden, structural changes during the 1990s led to a decrease in the total number check details of employees from 1.6 million in 1992 to 1.3 million in 2001 (Statistics Sweden 2008). This influenced not only the governing of human service organizations, but also daily tasks and performances within the organizations (Hertting et al. 2004). Along with the decrease in the number of employees, long-term sick

leave due to mental disorders started to increase, and psychosocial stress at work was identified as a predominant factor behind this increase (Stefansson 2006). This rise in sick leave continued until 2003. Since then, the total amount of sick leave has gone down considerably,

but still both mental disorders and musculoskeletal disorders constitutes a major reason for long-term sick leave and productivity loss within the Swedish workforce (Statistics Sweden 2011). Results from previously conducted studies have also indicated that these disorders are especially common among women working in human service organizations (Leijon et al. 2004; Fronteira and Ferrinho 2011). Several studies have shown that reduced working capacity is a predictor of long-lasting sickness, absence and that persons at risk often scored high on instruments measuring different Osimertinib mw aspects of work-related stress (Ahola et al. 2008; Borritz et al. 2010). Moreover, it is well known that loss in productivity caused by a decreased working capacity due to medical conditions increases the so-called “hidden costs” among companies and organizations both in the long- and short-time perspectives (Stewart et al. 2003b). Thus, it is therefore of vital importance to investigate antecedents of decreased work performance and work ability in order to implement preventive strategies. The term work performance could be defined as a combination of both quantitative and qualitative aspects of performing a work task by a worker or a work group. To objectively measure these dimensions of work are difficult, hence, most studies in this field use self-reports (de Vries et al. 2012; Waghorn and Chant 2011).

1999; Johnson et al. 2000; Konermann et al. 2008). In the first step, the gas is adsorbed onto the surface

of the membrane; in the second step the analyte molecules enter the membrane (permeation); and the third step is desorption of the molecules into the vacuum on the other side of the membrane. The gas transmission rate (k trans) across the membrane is given by Fick’s law of diffusion (Hoch and Kok 1963) $$ k_\texttrans = (P \, A \, \Updelta p)/l $$ (3)and is selleck products defined by the gas permeability (P) constant,2 the area of the membrane inlet (A), the partial pressure difference across the membrane (∆p), and the membrane thickness (l). As the partial pressure of gases on the low pressure (vacuum) side of the membrane is very small, the transmission rate is proportional to the gas concentration in the liquid phase. The overall sensitivity (gain factor) of detection is greater for thinner membranes and membrane types with high permeability. There are also effects due to relative diffusion of different molecular weight gases and “stickiness” of gas such as CO2. Therefore, for

quantitative measurements, calibrations need to be performed for each different analyte using a volume of a liquid or calibration gas. The choice of membrane depends on the experiment. If high sensitivity is required then a highly permeable membrane and a large inlet area are advantageous to facilitate a higher rate of gas sampling.

It may also be possible in some circumstances to operate with a higher vacuum to influence greater gas GSK126 mw transmission. In contrast, if long term sampling is required with near constant background gas concentrations, then a low consumption (i.e., thicker) membrane is required and/or use of a small sampling area. Most membranes have a good chemical resistance and if measurements are undertaken at elevated pressure (e.g., 20 bar) a supported membrane with an embedded metal grid can be used. A range of membranes suitable for MIMS applications are the following: silicone Carnitine palmitoyltransferase II membranes (MEM-213, Mem Pro); Teflon films such as FET or AF (DuPont); silicone rubber; oxygen electrode membranes3; HDPE plastic films (various sources); silicon membranes with embedded metal grid (Franatech GmbH, Germany). Thus, the choice of MIMS sensitivity versus gas concentration stability is an important factor in the experimental design. Isotopic enrichment Isotopes are defined as atoms with the same number of protons, but a different number of neutrons and thus differ in atomic weight. There are 80 elements with stable isotopes (26 with only one isotope) and 94 elements that occur naturally on earth. The MIMS approach makes use of the stable isotopes which can be found at natural abundance or purchased from many suppliers in varying enrichments. Table 1 lists many elements that are useful to study with photosynthesis and respiration in plants.

J Cell Sci 1967, 2:617–640.PubMed Competing interests The authors declare that they have no competing interests. Authors’ contributions HA and AI collected animals and made histological studies. HA conceived of the study, and participated in its design and draft the manuscript. AI carried out the histological staining and performed the morphometric analysis. All authors read and approved the final manuscript.”
“Background The prevalence of obesity and metabolic syndrome has increased at an alarming rate. By the year 2030, the number of adults with either type-1 or

type-2 diabetes is estimated to be greater U0126 order than 350 million [1]. Adult onset type-2 diabetes (T2DM) constitutes over 90% of all diabetes cases and is characterized by insulin resistance, abnormal insulin secretion, or both. Of these cases, it is estimated that 16% of people have undiagnosed or poorly managed diabetes (NIDDK National Health Interview survey, 2007–2009). It is well documented that Type-2 diabetes and hepatic steatosis are co-present [2]. The incidence of non-alcoholic fatty liver disease (NAFLD) is prevalent in 40 to 70% of patients with T2DM [3, 4]. This type of liver disease originates as hepatic steatosis, and can progress to non-alcoholic steatohepatitis (NASH), cirrhosis,

and end stage liver failure [5]. T2DM-related NAFLD is not fully understood, but it is known that leptin and insulin are important mediators in the progression of NAFLD [6]. Leptin is a hormone secreted by adipocytes, which binds to the leptin receptor and CH5424802 cell line increases partitioning of fatty

acids towards oxidation instead of triacylglycerol formation [7]. In mice and rats, leptin deficiency causes hyperphagia and obesity [8]. Moreover, the lack of leptin action causes increased insulin secretion, which is hypothesized to cause insulin resistance in rodents and humans [9]. Insulin resistance syndrome is hypothesized to cause NAFLD and augment progression to NASH [10]. T2DM and hepatic steatosis are modeled by a variety of diet and genetically modified rodent models. Db/db mice (BKS.Cg-m +/+ Leprdb/J) mice possess a spontaneous diabetes (Db) mutation in the leptin receptor. Db/db mice are insulin resistant, hyperinsulinemic, hyperglycemic, glucose intolerant, and possess abnormal islet cell morphology [11–13]. They become hyperinsulinemic filipin from 10–14 days after birth; and exhibit significant weight gain with abnormally high triglycerides and low- and very low-density lipoproteins at 3 to 4 weeks of age. Hyperglycemia appears after 4–6 weeks of age. Other mouse models of obesity, diabetes, and NAFLD exhibit altered transporter expression in liver and kidney [14]. Transporters are membrane proteins, which facilitate chemical transport into and out of cells [15]. Organic anion transporting polypeptides, organic anion transporters and organic cation transporters are often referred to as “uptake transporters”.

flexneri strains and serotype-converting bacteriophages used in this study were listed in Table 2. S. flexneri strain 036 (serotype Y) was Obeticholic Acid supplier used as host for phage infection and large propagation. S. flexneri strains 014 (serotype X) and 019 (serotype 1a) were used as positive controls in the serological assays for group specific antigen 7;8 and type specific antigen I respectively. Twenty four S. flexneri serotype X and 17 of S. flexneri serotype 1a strains isolated

from patients and stored at National Institute for Communicable Disease Control and Prevention, China CDC (ICDC) were used for infection with serotype-converting phages SfI and SfX respectively. Table 2 Strains and serotype-converting bacteriophages used in this study Strains or phages Relevant characteristic Reference or source S. flexneri strains 036 Serotype Y ICDC 014 Serotype X ICDC 019 Serotype 1a ICDC 036_1a 036 infected by SfI, serotype 1a This study 036_X 036 infected by SfX, serotype X This study 036_1d 036 infected by SfI and SfX, serotype Procaspase activation 1d This study Phages SfI Phage SfI, induced from S. flexneri strain 019 This study SfX Phage SfX, induced from S. flexneri strain 2002017 This study ICDC, National

Institute for Communicable Disease Control and Prevention, China CDC Serotype-converting bacteriophages SfI and SfX were induced from S. flexneri serotype 1a strain 019 and serotype Xv strain 2002017 respectively, following the methods described by Mavris et al. [12]. Phage infection and lysogen isolation We used the procedures described for lambda phage (Φλ) for phage infection [22]. S. flexneri cells were inoculated into LB broth and incubated for 3 h at 37°C with aeration. Cells were harvested by centrifugation at 4000 rpm and the cell density was adjusted to 2.0 OD (A595 nm) with MgSO4 buffer (10 mmol/L). A proportion of cells (200 μl) were most infected with purified phages with phage to bacterial cell ratio of about 1:1000 and incubated for 20 min at 37°C. The infected cells were mixed

with 3 ml semisolid agar (Luria Broth (LB) with 0.7% agar) and immediately spread on LB solid agar plates. After incubation at 37°C for 20 h, the lysogens were detected from turbid single colonies. Slide agglutination and LPS analysis Serological identification was performed using two commercial slide agglutination serotyping kits: monovalent anti-sera (Denka Seiken, Japan) and monoclonal antibody reagents (Reagensia AB, Sweden) according to manufacturer’s instructions. The new serotype was further confirmed by Western-blot assay. Briefly, LPS was prepared using the method of Hitchcock & Brown [23] and transferred onto a PVDF membrane in a Tris/glycine/methanol buffer. The membrane was blocked with phosphate buffered saline (PBS) containing 5% (w/v) skimmed milk and 0.