4). The MS/MS ion search was performed by Mascot Daemon (version 2.2.01) to search against the International Protein Index (IPI) rat protein database (version 3.70). Peptide modification settings were: fixed modification, carbamidomethylation on Cys; variable modifications,

oxidation on Met, deamidation on Asn and Gln. The peptide and fragment mass tolerances were set at ±2.5 and 0.7 Da, respectively. AP24534 clinical trial Maximum missed cleavage of 2 was allowed. The “require bold red” option was activated to remove redundancy. The significance threshold was adjusted to give a false-discovery rate (FDR) <1 %, which was calculated on the basis of the number of peptide matches against a decoy database. Proteins identified with matched peptides exceeding the “identity threshold” are reported as identified proteins. Bioinformatics analysis Distributions in subcellular location and molecular function were assigned to each protein based on UniProt/GO (http://​www.​uniprot.​org, http://​www.​geneontolgy.​org) and also by manually searching the literature. Functional enrichment analyses

of cellular components, molecular functions, and biological processes were performed via the FatiGO analytic tool (http://​www.​fatigo.​org). In the enrichment analysis, modified Fisher’s exact tests were used for statistical analysis. The significantly (p value <0.05) enriched GO categories are presented. Each annotated function was assigned a Z score to measure whether a given function or process was significantly overrepresented in our VEC plasma Cell Cycle inhibitor Tryptophan synthase membrane proteome relative to the public databases. Deltex 3-like immunohistochemical and immunofluorescence analysis For immunohistochemical analysis, kidney tissues were fixed

in methyl Carnoy’s solution and embedded in paraffin. The paraffin-embedded tissues were sectioned at thickness of 4 μm, dewaxed, and incubated sequentially with rabbit anti-human Dll3 antibody (Sigma-Aldrich Co., USA) for 1 h and horseradish peroxidase-conjugated goat anti-rabbit immunoglobulins at 37 °C for 1 h. The peroxidase reaction was visualized using 0.5 mg/mL of 3′-diaminobenzidine tetrahydrochloride-0.01 % hydrogen peroxide as substrate. For immunofluorescence, frozen blocks were sectioned at thickness of 3 μm. Rabbit monoclonal anti-Dll3 in combination with mouse monoclonal anti-caveolin-1 antibody were applied as primary antibodies for double-labeled immunostaining. After washing with PBS, the sections were stained with fluorescein isothiocyanate-conjugated goat anti-rabbit IgG, and subsequently with Texas-Red-conjugated anti-mouse immunoglobulins. Immunofluorescence of the stained sections was observed with a microscope (BX50; Olympus, Tokyo, Japan).

In Amacayacu, the

number of species shared by plots in terra firme forests was found to be significantly different from those occurring in the flood forests (várzea) (Table 2) (p = 0.028 when comparing the relatively species rich terra firme plots with the relatively species poor várzea plots, and p < 0.001 for the reciprocal comparison). Thus our sampling efforts revealed significant differences in macrofungal biodiversity between the Araracuara and Amacayacu regions, and between várzea and terra firme forests in the Amacayacu region. Cluster analysis provides for a more detailed illustration of these patterns. ABT-263 solubility dmso Two main clusters for macrofungal species composition appeared (Fig. 6a). One group comprised the Amacayacu plots and the other represented those from

Araracuara. The latter formed two subclusters, namely one comprising plots buy KU-60019 AR-18y, AR-23y, AR-30y, AR-42y and AR-PR, and a second one with the most disturbed plot (AR-1y) and the primary forest plot (AR-MF). In the first subcluster all plots, except AR-PR, corresponded to patches of forests that varied

in age between 18 and 42 years of regeneration Cell Penetrating Peptide after the chagras were abandoned. The analysis of the Amacayacu plots yielded two subclusters with one containing the terra firme forests (AM-MF and AM-RF) occurring on the high terraces, and the other consisting of plots located in flooded areas (AM-FPF and AM-MFIS) (Fig. 6a). Fig. 5 Accumulation graphs of the macrofungal species in Araracuara, Peña Roja and Amacayacu showing the increase of species after the collection trips (left panel). The dots represent the real (overall) data, whereas the lines are based on ‘record based rarefaction’ with 100 randomizations of records in which a record represents all sporocarps of a species at a certain space/time combination (i.e., a sample). The advantage of this method is that information on patchiness is maintained and the ‘record based’ rarefaction provides sufficient resolution leaving small jumps on the x- and y-axis. For comparison the randomization results of a study in a temperate Swiss forest (Straatsma et al.

NM was also involved in identification of the isolates. VL did the isolations of anaerobic bacteria and BIOLOGTM BMS-354825 supplier assay. YS and DR designed the study and gave important inputs for preparation of manuscript. All authors have read and approved the manuscript.”
“Background In Gram-positive bacteria, proteins released in the extracellular environment are synthesized as precursor polypeptides with a cleavable N-terminal leader peptide as the sole topogenic signal. Precursors are moved across the plasma membrane by a translocon and signal peptidases act on newly translocated precursors to release

the mature polypeptide from the membrane [1]. The events leading to protein translocation across the plasma membrane have been genetically dissected using the model organism Escherichia coli . Most precursor proteins travel in an unfolded state through the SecYEG translocon INK 128 price [2–5], pushed by the cytoplasmic ATPase SecA [6]. Precursor proteins bearing a leader peptide with the twin-arginine motif are moved across the plasma membrane by the Tat translocon [7, 8]. Recently, it has been observed that some bacteria, in particular Firmicutes and Actinobacteria, can secrete proteins lacking a canonical leader peptide [9]. Many of these proteins share some distinguishing and conserved

features that include small size (approximately 100-amino acid residues), a WXG amino acid motif in the middle of the protein [10] and a conserved three-dimensional structure (helix–turn–helix hairpin) [11, 12]. Together, these proteins form the WXG100 family of proteins [10]. ESAT-6 and CFP-10 of Mycobacterium tuberculosis are the founding members of the WXG100 family of proteins and are identified with the acronym EsxA and EsxB for ESAT-6 extracellular protein A and B[10]. Bioinformatic and genetic approaches have revealed that the esxA and esxB genes cluster with both conserved and non-conserved genes of unknown function that are required for the stability and secretion of WXG100/Esx proteins into

the extracellular milieu [13–16]. These clusters are conserved among several Firmicutes (Figure 1) but not with Mycobacteriaceae who only share EssC-like ATPases [10, 17]. Rebamipide The name ESX has been used to refer to such gene clusters in Mycobacteriaceae and M. tuberculosis for example encodes five ESX clusters (ESX-1 through ESX-5) [17]. In more general term, ESX mediated secretion has been refereed as Type 7 secretion but it was noted that this general designation should not be used for Firmicutes owing to the lack of overall sequence conservation [18]. Clusters bearing esx genes have therefore been referred as ESAT-6 Secretion Systems (ESS) in Staphylococcus aureus and Bacillus anthracis where they have been experimentally examined [16, 19–21] and sometimes as WXG100 Secretion Systems (WSS) [22].

Aside from methodological issues pertaining to beverage composition and protocol design, it has been postulated that participants with a lower performance level may be more responsive to CHO-PRO-PEP supplementation than those individuals who are deemed more superior performers [15]. This notion was based click here on a performance factor calculated from Wmax, VO2max and the mean power output from a familiarisation of a 5 min all-out cycling performance test, and a subsequent correlation analysis [15]. However, as presented previously, we did not observe an ergogenic response in our participant population. In conclusion, the results of the present study suggest that when matching

CHO, CHO-PRO and CHO-PRO-PEP solutions for energetic content, the inclusion of protein hydrolysates produced from salmon may have significant effects upon exercise metabolism during

endurance cycling. However, the translation of these CFTR modulator significant metabolic effects into subsequently meaningful performance benefits remains to be determined. Moreover, in the absence of an empirically supported mechanism, further investigations are warranted to potentially elucidate mechanisms and further determine the efficacy of CHO-PRO-PEP co-ingestion. Acknowledgments The authors would to thank Einar Leid of Nutrimarine Life Science, Bergen, Norway for generously supplying the supplementation for the study. The authors would also like to thank the participants for their time and effort. References 1. Jeukendrup AE: Carbohydrate intake during exercise and performance. Nutrition 2004, 20:669–677.PubMedCrossRef 2. Jeukendrup AE: Carbohydrate feeding during exercise. Eur J Sport Sci 2008 2008,8(2):77–86.CrossRef 3. Ivy JL, Res PT, Sprague Vitamin B12 RC, Widzer MO: Effect

of a carbohydrate-protein supplement on endurance performance during exercise of varying intensity. Int J Sport Nutr Exerc Metab 2003, 13:382–395.PubMed 4. Saunders MJ, Kane MD, Todd MK: Effects of a carbohydrate-protein beverage on cycling endurance performance and muscle damage. Med Sci Sports Exerc 2004,36(7):1233–1238.PubMedCrossRef 5. Saunders MJ, Luden ND, Herrick JE: Consumption of an oral carbohydrate-protein gel improves cycling endurance and prevents postexercise muscle damage. J Strength Cond Res 2007,21(3):678–684.PubMed 6. Breen L, Tipton KD, Jeukendrup AE: No effect of carbohydrate-protein on cycling performance and indices of recovery. Med Sci Sports Exerc 2010,42(6):1140–1148.PubMed 7. Osterberg KL, Zachwieja JJ, Smith JW: Carbohydrate and carbohydrate + protein for cycling time trial performance. J Sports Sci 2008,26(3):227–233.PubMedCrossRef 8. Romano-Ely BC, Todd MK, Saunders MJ, St Laurent T: Effect of an isocaloric carbohydrate-protein-antioxidant drink on cycling performance.

Tumor lesions were identified as areas of focally increased FDG uptake exceeding that of surrounding normal tissue. A region of interest was placed over each lesion to include the highest levels of radioactivity. Maximum SUV was calculated with the following formula: SUV = cdc/(di/w), wherein cdc is the decay-corrected tracer tissue concentration (Bq/g), di is the injected dose (Bq), and w is the patient’s body weight (g). Immunohistochemical staining Immunohistochemical staining was performed to determine

GLUT1 and HK2 levels in gastric cancer tumors. Briefly, resected specimens were fixed in 10% buffered formalin solution, embedded in paraffin, and sectioned at a thickness of 4 μm. Slides were then incubated overnight at room temperature with primary rabbit polyclonal antibody against GLUT1 (1:200) or HK2 (1:100). Avidin-biotin-peroxidase complex buy R428 staining Rapamycin datasheet was performed according to the manufacturer’s instructions (Santa Cruz Biotechnology, CA, USA). Finally, nuclei were counterstained with hematoxylin [20]. Real-time PCR Total RNA was isolated from specimens by guanidinium isothiocyanate-acid

phenol extraction and quantified by absorbance at 260 nm. Total RNA (1 μg) was used for reverse transcription, and the resulting cDNA was analyzed by real-time PCR with Power SYBR Green PCR Master Mix and ABI Prism 7000 (Applied Biosystems, Foster, CA, USA). Target-specific oligonucleotide primers and probes were previously described [20, 21]. 18S rRNA was used as an endogenous control. Primers and probes for 18S rRNA were obtained in a Pre-Developed TaqMan Assay Reagent kit (Applied Biosystems, Stockholm, Sweden). Statistical analysis Data are expressed as mean ± SEM. Paired SUV results were compared by student’s t-test. Multiple one-way analysis of variance was used to assess differences in mRNA levels. Correlation analyses were performed with Spearman’s correlation analysis test. P<0.05 was considered statistically significant. Results Relationship between mean SUV and clinicopathological data

in gastric cancer Of the 50 gastric cancer lesions, 45 showed focally increased FDG uptake. The majority of patients had advanced gastric cancer and a mean tumor size of 7.5 ± 0.5 cm, with 16 cases classified as stage 4. The mean SUV of stage 4 patients was 9.0 ± 1.3, while mean SUV of stage 2 Myosin and stage 3 patients combined was 8.3 ± 0.6 (Figure 1a). When tumors were divided into intestinal and non-intestinal tumors, mean SUVs were 7.8 ± 0.7 and 9.2 ± 1.0, respectively (Figure 1b). When divided by median lymph node metastasis, 22 cases had less than three and 28 cases had three or more; mean SUVs were not significant at 9.4 ± 1.0 and 7.8 ± 0.7, respectively. When divided by maximum median tumor diameter, 22 cases were less than 7.0 cm and 28 cases were 7.0 cm or greater; mean SUVs were 7.0 ± 0.6 and 9.7 ± 0.9, respectively (P < 0.05).

Cesarean section is suggested by some authors also in case of fetal death. In such cases, this procedure must be done first and special care taken to avoid contamination of the peritoneum. Indeed, this can itself be a cause of mortality due to a consequent severe puerperal infection [13]. A delay in diagnosis and surgical intervention over 48 h can have a significant impact on the ultimate outcome of the mother and fetus [2]. The management of sigmoid volvulus in pregnancy begins with aggressive hydration and proximal bowel decompression [13]. In the absence of mucosal

ischemia, sigmoidoscopic detorsion and rectal tube insertion is possible. Apoptosis inhibitor In recurrent cases, elective sigmoidectomy can be safely performed in the second trimester

[20]. Otherwise, surgery can be postponed until after delivery. In cases of bowel gangrene or perforation, prompt surgical intervention through a midline laparotomy is essential. Thorough peritoneal Selleck PD-1/PD-L1 inhibitor lavage of the resection of the necrotic bowel segments is mandatory. This is followed by either primary anastomosis or stoma formation (Hartman’s procedure) [28]. The prognosis of sigmoid volvulus in pregnancy is poor. In the last century, the maternal mortality rate was 21–60% and fetal mortality rate was 50% [5]. In recent decades, the maternal mortality has decreased to 6–12% and fetal mortality to 20–26% [29]. The major causes of maternal mortality are toxic and/or hypovolemic shock, whereas impairment of placental blood flow due to increased intraabdominal pressure affects fetal mortality [30]. Conclusion Sigmoid volvulus is a rare and potentially fatal condition in pregnancy that requires a multidisciplinary approach with general surgeons, obstetricians, and neonatologists. Prompt diagnosis is critical for early management, to minimize fetal and maternal morbidity and mortality. Abdominal pain may be the only findings, and sigmoidoscopic detorsion or surgical resection are the treatment options, depending on bowel viability. Consent Written informed

consent was obtained from the patient for publication of this Case report and any accompanying images. References 1. Lord SA, Boswell WC, Hungerpiller JC: Sigmoid volvulus in pregnancy. Am Surg Unoprostone 1996, 62:380–382.PubMed 2. Harer WB Jr, Harer WB Sr: Volvulus complicating pregnancy and puerperium; report of three cases and review of literature. Obstet Gynecol 1958, 12:399–406.PubMed 3. Nascimento EFR, Chechter M, Fonte FP, Puls N, Valenciano JS, Filho CLPF, Nonose R, Bonassa CEG, Martinez CAR: Volvulus of the sigmoid colon during pregnancy: a case report. Case Rep Obstet Gynecol 2012. doi:10.1155/2012/641093 4. Iwamoto I, Miwa K, Fujino T, Douchi T: Perforated colon volvulus coiling around the uterus in a pregnant woman with a history of severe constipation. J Obstet Gynaecol Res 2007, 33:731–733. 10.1111/j.1447-0756.2007.00641.xPubMedCrossRef 5. Perdue PW, Johnson HW Jr, Stafford PW: Intestinal obstruction complicating pregnancy.

Dislocation cores are represented by thin tubes, in which Shockley partial dislocation with 1/6 <112 > Burgers vector and perfect dislocation with 1/2 <110 > Burgers vector are colored gray and red, respectively. It is seen from Figure 4b that the dislocation loop consists of four

partial dislocations and one perfect dislocation. In addition, there is one vacancy formed beneath the probe. Upon further penetration, the other selleck kinase inhibitor three 111 slip planes are activated sequentially, and Figure 4c shows that the defect zone beneath the probe expands greatly. The glide of dislocations on adjacent slip planes leads to the formation of stair-rod dislocations with 1/6 <110 > Burgers vector highlighted by the arrows in Figure 4d. Figure 4e,f presents dislocation network after the completion of scratching and penetration, respectively. It is seen from Figure 4e that there is less dislocations but more

vacancies in the wake of the probe than that in the vicinity of the probe due to the plastic recovery. In addition to the stair-rod dislocations, there are glissile prismatic dislocation loops formed by dislocation reaction and cross-slip events. In particular, the prismatic dislocation half-loops in front of the probe glide parallels to the free surface to transport the materials displaced by the probe without the formation of surface steps [24]. Although small part of the dislocations beneath the probe annihilates at the free surface during the retraction,

Figure 4f shows that the defect structures are stable. Figure AZD2014 4 Close inspections of defect structures in friction with a probe radius of 8 nm. The scratching depth is 0.82 nm. (a,c) Bottom views of defect structures at penetration depths of 0.72 and 0.82 nm, respectively. Atoms are colored according to their BAD values and FCC atoms are not shown. (b,d) Dislocation networks shown in (a) and (c), respectively. (e,f) Dislocation networks after the completion of scratching and retraction, respectively. Effect of probe radius on minimum wear depth To investigate the influence of probe radius on the minimum wear depth, friction simulations CYTH4 with another three probe radiuses of 6, 10, and 12 nm are conducted, in addition to the probe radius of 8 nm. For each probe radius, the penetration stage stops at a penetration depth that is 0.1 nm deeper than the critical penetration depth at which the phenomenon of force drop occurs. Figure 5a,b plots the contact pressure-penetration depth curves and the friction coefficient-scratching length curves during the penetration and scratching stages with the four probe radiuses, respectively. The contact pressure is defined as the ratio of the penetration force to the contact area. A detailed description about the calculation of the contact area during spherical penetration can be found elsewhere [28].

PubMedCentralPubMedCrossRef 14. Larici AR, Gotway MB, Litt HI, Gautham PR, Reddy GP, Webb WR, Gotway CA, Dawn SK, Marder LY294002 SR, Storto ML: Helical CT with sagittal and coronal reconstructions: Accuracy for detection of diaphragmatic injury. AJR 2002, 179:451–457.PubMedCrossRef 15. Slim K, Bousquet J, Chipponi J: Laparoscopic repair of missed blunt diaphragmatic rupture using a prosthesis. Surg Endosc 1998, 12:1358–1360.PubMedCrossRef 16. Reyad AG, Ahmed I, Bosanac Z, Philips

S: Successful laparoscopic repair of acute intrapericardial diaphragmatic hernia secondary to penetrating trauma. J Trauma 2009, 67:E181-E183.CrossRef 17. Hanna WC, Ferri LE, Fata P, Razek T, Mulder DS: The current status of traumatic diaphragmatic injury: lessons learned from 105 patients over 13 years. Ann Thorac Surg 2008, 85:1044–1048.PubMedCrossRef 18. Amid PK: Classification of biomaterials Daporinad nmr and their related complications in abdominal wall surgery. Hernia 1997, 1:15–21.CrossRef 19. Rashid F, Chakrabarty MM, Singh R, Iftikhar SY: A review on delayed presentation of diaphragmatic rupture. World J Emerg Surg 2009, 4:32.PubMedCentralPubMedCrossRef 20. Paul S, Nasar A, Port JL, Lee PC, Stiles BC, Nguyen AB, Altorki NK, Sedrakyan A: Comparative analysis of diaphragmatic hernia repair outcomes using the

nationwide inpatient sample database. Arch Surg 2012, 147:607–612.PubMedCrossRef Competing interest The authors declare that they have no competing interest. Authors’ contribution RB and DF was involved in the clinical management of the patient. AL and RL contributed conceiving the manuscript. RB, DF and AL performed the operation. RL and RB wrote the manuscript. AL and DF reviewed the literature. All authors read and approved the manuscript. MP and RB answer to the reviewer and all the authors approved the corrections.”
“Background Portal vein aneurysm (PVA) is defined as a focal dilatation of the portal venous system, greater than Ketotifen 2 cm [1]. PVA is a rare vascular anomaly, observed in 0.43% [2] but its incidence was increasing

in recent years with the enlarged use of magnetic resonance (MR) and computed tomography (CT) [3]. Most common sites are the main portal vein and confluence of splenic and superior mesenteric veins, forming extra-hepatic portal vein aneurysm (EPVA). Although risk factors like portal hypertension and liver cirrhosis have been highlighted, the etiology remains to be clarified. PVA may be associated with various complications: thrombosis, aneurismal rupture, inferior vena cava obstruction and duodenal compression. Thrombosis is the most frequent complication with complete thrombosis and non-occlusive thrombus occurring in 13.6% and 6%, respectively [3]. Herein we report the case of a giant EPVA with complete thrombosis, among the largest described so far. A conservative treatment showed satisfying clinical and radiological response. We reviewed the English literature, disclosing 13 cases of thrombosed EPVA in order to assess current treatment [4–13].

Early fluorescence measurements (Murata and Sugahara 1969; Wraight and Crofts 1970) detected the absolute fluorescence from

an illuminated sample and how it changed following different chemical treatments. Because the total fluorescence is proportional to the illumination intensity, comparing the amount of fluorescence across different illumination conditions requires measuring of the fluorescence quantum yield, \(\phi_\rm F.\) $$ \phi_\rm F = \frac\hboxnumber of photons emitted\hboxnumber of photons absorbed. $$ (1) PAM fluorimetry is a widely used tool for measuring changes in the chlorophyll fluorescence yield as plants acclimate to changing light conditions (Schreiber et al. 1986). PAM techniques are reviewed in Brooks and Niyogi (2011) and Schreiber (2004). While absolute fluorescence measurements use a single light source BMS-907351 ic50 to illuminate the sample and induce fluorescence, PAM fluorimeters only detect fluorescence resulting from a low intensity (<0.1 μmol photons m−2 s−1) modulated measuring light that minimally affects the photochemistry or NPQ in the plant.

Typical qE PAM fluorimeter measurements consist of a dark-acclimated sample exposed to actinic light (light that results in productive photosynthesis) until qE reaches a steady state (approximately 10 min), followed by a period of dark reacclimation until qE turns off. To distinguish the effects of photochemical quenching (irreversible charge Afatinib price separation in the RC) and NPQ, fluorescence yield measurements are compared when PSII RCs are open and closed. RCs are considered to be open when the primary plastoquinone electron acceptor in the RC, Q A, is oxidized and is considered closed when Q A is reduced (Baker 2008; Govindjee 2004). During the illumination and dark periods, short (<1 s) pulses of high intensity (up to 20,000 μmol photons m−2 s−1) actinic light are used to close PSII RCs. When RCs are open, excited chlorophyll can relax via photochemical

quenching, NPQ, fluorescence, or ISC. Adenosine When the saturating pulses close the RCs, the only available pathways are NPQ, fluorescence, or ISC. The rates of these processes affect the measured fluorescence quantum yield. To characterize the NPQ response of a plant, it is useful to compare the fluorescence yield when the PSII RCs are closed before and during light acclimation. F m is proportional to the maximum fluorescence yield measured during a saturating pulse of actinic light applied to dark-acclimated leaves. \(F_\rm m^\prime\) is the maximum fluorescence yield following exposure to light, also measured during saturating pulses. A parameter called NPQ can be calculated with these parameters (Schreiber et al. 1994). $$ \hboxNPQ = \fracF_\rm m-F_\rm m^\primeF_\rm m^\prime.

Calibration of the system was performed on suspensions of E. coli ΔmdtM BW25113 cells. Cultures from single bacterial colonies were grown aerobically at 30°C to an OD600 of 3.0 in LB medium supplemented INCB024360 with 30 μg/ml kanamycin. Cultures were then diluted 125-fold into 100 ml of fresh LB medium containing antibiotic and grown aerobically at 37°C to an OD600 of 1.0. Six 10 ml aliquots of cells were pelleted by centrifugation (3000 × g) at 4°C and washed twice in assay buffer (140 mM NaCl, 10 mM HEPES and 1 mM MgCl2) that had pH adjusted with KOH to 7.5, 8.0, 8.5, 9.0, 9.25 or 9.5. To load the cells with fluorescent probe, the washed cells were

pelleted and then resuspended to OD600 of 2.0 in assay buffer that contained 2.5 μM BCECF-AM. To equalize internal and external pH, 10 μM of the protonophore CCCP was added CH5424802 cell line to the buffer and the cells were incubated in the dark at 37°C for 1 h. BCECF-AM-loaded

cells were collected by centrifugation and stored on ice until use. For each pH value investigated, 200 μl of loaded cells were added to 1.3 ml of assay buffer that contained 10 μM CCCP. After incubation at 30°C for 60 s, the fluorescence intensity of the mixture at 530 nm upon excitation at 490 nm and 440 nm was recorded under continuous stirring using a Fluoromax-4 fluorometer with excitation and emission slit widths set to 1.0 nm and 10 nm, respectively. Experiments were performed in triplicate for each pH value investigated and used to construct a calibration plot that correlated the 490 nm/440 nm fluorescence emission ratio to pH. To determine if MdtM functioned in maintenance of a stable intracellular pH under Thalidomide conditions of alkaline stress, fluorescence measurements were performed on pMdtM and pD22A transformants of E. coli ΔmdtM BW25113 cells at six different external alkaline

pH values using the method described above except that carbenicillin (100 μg/ml) and L–arabinose (0.002% w/v) was added to the growth medium, CCCP was omitted from the assay buffer, and D-glucose (1 mM) was added to the assay buffer to energise the cells 60 s prior to recording the fluorescence. Western blot analysis of recombinant MdtM Estimation of expression levels of recombinant wild-type and D22A mutant MdtM by transformed ΔmdtM BW25113 cells grown at different alkaline pH values was performed as described in [25]. Acknowledgements The authors thank Professors Eitan Bibi (Weizmann Institute of Science, Rehovot, Israel) and Hiroshi Kobayashi (Chiba University, Japan) for the kind gifts of E. coli UTL2 and TO114 cells, respectively. This work was supported in part by BBSRC Research Grant BB/K014226/1 (to CJL). SRH was supported by a Northern Ireland Department of Employment and Learning (DEL) postgraduate studentship. Electronic supplementary material Additional file 1: PDF file showing that E.