Clone library preparation from community DNA

Total community DNA was used for preparing 16S rRNA gene libraries. The 16S rRNA gene was amplified with modified universal primers for bacteria 8FI (5’GGATCCAGACTTTGATYMTGGCTCAI-3’) and 907RI (5’- CCGTCAATTCMTTTGAGTTI-3’) CUDC-907 solubility dmso [27]. The PCR product were purified by gel elution using Gene Elute Gel Extraction Kit (Sigma-aldrich, St Louis USA) and were ligated into pCR4® TOPO vector supplied with the TOPO TA cloning kit (Invitrogen, San Diego, USA) and transformed into One Shot TOPO10 electrocompetent cells of E. coli (Invitrogen, San Diego, USA) following the manufacturer’s instructions. Sterile LB agar with 50 μg/ml of kanamycin were used for selection of the transformed cells which were incubated for 16 h at 37°C. M13F and M13R primers were used for screening and sequencing of the clones. The sequencing was done by ABI 3730 XL DNA analyser (Applied Biosystems Inc, USA) using the ABI Big-Dye terminator version 3.1 sequencing kit as per the manufacturer’s this website instructions. Phylogenetic analysis Sequences from each of the clone libraries

were compared to the current database of 16S RNA gene sequences at Ribosomal Database Project II [28]. The sequences were assembled and contig’s were obtained using ChromasPro software, alignment was done using CLUSTAL X2 and the sequences were edited manually using DAMBE to get unambiguous sequence alignment. All sequences were checked for chimeric artifacts by Mallard program, reference sequence used for this purpose was E. coli U000096 [29] Appropriate subsets of 16S rRNA gene sequences were selected on the basis of initial results and subjected Pregnenolone to further phylogenetic analysis using DNADIST of Phylip (version 3.61). The number of Operational Taxonomic Units (OTU) (clone sequences with > 97% similarity grouped together as one OTU) were obtained

by DOTUR program (version 1.53) using furthest neighbor algorithm [30]. Representative sequences from each of the OTUs were retrieved and checked against the previously determined 16S rRNA gene from the RDPII release 10 version of the database and these sequences were downloaded in FASTA format. Phylogenetic analyses were conducted using MEGA, version 4 [31], and the phylogenetic trees were constructed using neighbor-joining method with Kimura 2 parameter [32, 33]. Normalized heat map was generated using MG-RAST, a modified version of RAST server, using RDP database [34]. Real time PCR The Real Time PCR was done using the 7300 Real time PCR system from Applied Biosystems Inc. (USA) using SYBR green master mix (Applied Biosystems Inc. USA). Primers used for absolute quantification were reported earlier [19]. The primers used are listed in Table  1.

Dis Mon 2007, 53:32–38.PubMedCrossRef 38. Rasheed S, Zinicola R, Watson D, Bajwa A, McDonald PJ: Intra-abdominal and gastrointestinal

tuberculosis. Colorectal Dis 2007, 9:773–783.PubMedCrossRef 39. Baloch NA, Baloch MA, Baloch AF: A study of 86 cases of abdominal tuberculosis. Journal of Surgery Pakistan (International) 2008, 13:30–32. 40. Boukthir S, Murad SM: Abdominal Tuberculosis in children. Acta Gastroenterol Belg 2004, 67:245–249.PubMed 41. Hu ML, Lee CH, Kuo CM, Huang CC, Tai WC, Chang KC, Lee CM, Chuah SK: Abdominal tuberculosis: analysis of clinical features and outcome of adult patients in southern Taiwan. Chang Gung Med J 2009, 32:510–515. 42. Urassa M, Isingo R, Kumogola Y, Mwidunda P, Helelwa M, Changulucha J, Mngara J, Zaba B, Calleja T, Slaymaker E: Effect of PMTCT availability on choice of ANC in Mwanza and Magu districts and its impact on HIV sentinel surveillance in Tanzania. Report of ANC surveillance Mwanza and Magu Districts; 2007. 43. Selonsertib clinical trial TEW-7197 Fee MJ, Oo MM, Gabayan AE, Radin DR, Barnes PF: Abdominal tuberculosis in patients infected with the human immunodeficiency virus. Clin Infect Dis 1995, 20:938–944.PubMedCrossRef 44. Iliyasu Z, Babashani M: Prevalence and predictors of tuberculous co-infection among HIV seropositive patients attending the Aminu Kano Teaching Hospital, northern Nigeria. J Epidemiol 2009, 19:81–87.PubMedCrossRef

45. Mawalla BM, Mshana SE, Chalya PL, Imirzalioglu C, Mahalu W: Predictors of surgical site infections among patients undergoing major surgery at Bugando Medical Centre in Northwestern Tanzania. BMC Surg 2011, 11:21.PubMedCrossRef 46. Balthazar EJ, Gordon R, Hulnick D: Ileocecal tuberculosis: CT and radiographic evaluation. AJR Am J Roentgenol 1990, 154:499–503.PubMedCrossRef HAS1 47. Watters DAK: Surgery for tuberculosis before and after HIV infection: a tropical perspective. Br J Surg 1997, 84:8–14.PubMedCrossRef 48. Iseman MD: Treatment of multidrug resistant tuberculosis. N Engl Jt Med 1993, 329:784–791.CrossRef 49. Wadhwa N, Agarwal S, Mishra K: Reappraisal of abdominal tuberculosis. J Ind

Med Assoc 2004, 102:31–42. 50. Paustian FF: Tuberculosis of the intestine. In Bockus HL, editor. Gastroenterology Volume 11 . 2nd edition. Philadelphia: W.B. Saunders Co; 1964:p. 311. 51. Nguyen VH: Intestinal obstruction due to tuberculosis. Asian J Surg 2002, 25:145–148.PubMedCrossRef 52. Sial K, Baloch Q: Presentation and surgical management of Abdominal Tuberculosis. Med Channel 2004, 10:20–22. 53. Tariq N: Abdominal Tuberculosis. The surgical audit of its presentation. Pak J Surg 1993, 13:82–88. 54. Katariya RN, Sood S, Rao PG, Rao PLNG: Stricturoplasty for tubercular stricture of the gastrointestinal tract. Br J Surg 1977, 64:494–496.CrossRef 55. Prichard TJ, Schoetz DJ, Caushaj FP, Roberts LP, Marry JJ: Strictroplasty of small bowel in patients with Crohn’s Disease. Arch Surg 1990, 125:715–717.CrossRef 56. Pujari BD: Modified surgical procedures in intestinal tuberculosis.

In the occupational health setting, more attention for illness perceptions by health professionals seems therefore sensible. Many health professionals are unaware of the relevance of discussing patient’s illness representations or strategies patients adopt to deal with their illness. At the same time, patients do not often spontaneously articulate these issues if they are not encouraged to do so. Discussing illness perceptions

is appreciated by patients and create a feeling of support (Theunissen et al. 2003). Preventative actions can be taken by an occupational professional for a worker who is at risk of dropping out with an illness. This could include offering more positive https://www.selleckchem.com/products/nepicastat-hydrochloride.html views about the illness and possibilities to work, provide ability to vent emotions, encourage social support and communication with the supervisor, and train problem-focused coping at work. Identifying

which patients develop maladaptive illness representations would be helpful for health professionals. It seems sensible to target interventions by (occupational) health professionals to (patterns of) maladaptive illness representations. For example, if patients have unhelpful perceptions regarding the consequences of their illness than the aim could be to help the patient understand these and filter out any unrealistic scenario’s. The same applies when patients have unrealistic perceptions of the chronic or recurrent timeline of their illness, or work participation is unnecessarily postponed as only the negative

consequences of work are considered by the patient. selleck compound Also, providing information on occupational interventions Metalloexopeptidase or job accommodations could empower a patient to keep working with a chronic disease and boost the patient’s perception to control the negative effects of the illness while at work. The above would require the health professional to have an adequate knowledge of the effects that different illnesses have on functioning or more specific work participation, and more importantly, how any of these cognitive or emotional representations can be accommodated for or trained by the worker. This would require skills in cognitive and behavioral therapy, which may be feasible as shown in the Theunissen et al. (2003) who provided GP trainees with a short (6 h) training in these principles. Other promising vocational rehabilitation strategies are increasingly used in the occupational health field (Hoving et al. 2009; Verbeek 2006) and would benefit from including the concept of illness perceptions. The use of illness perception measures by health professionals would also target specific interventions to those who need it, in contrast to offering the same treatment to everyone, and would be a potential cost-effective option.

e. identification of bacteria and microorganismal pathogens within the peritoneal fluid, the presence of yeasts (if applicable), and the antibiotic susceptibilities of bacterial isolates. Selleckchem Eltanexor This observational study does not attempt to change or modify the laboratory or clinical practices of the participating physicians or their respective institutions, and neither informed

consent nor formal approval by an Ethics Committee is required. The study will continue to meet and abide by the standards outlined in the Declaration of Helsinki and Good Epidemiological Practices. A Scientific Committee was established to impartially assess the objectives, methodology, and overall scientific quality of the project. The study is monitored by the Coordination Center, which investigates and verifies missing

or unclear data submited to the central database. Statistical analyses were performed using MedCalc® statistical software. Results PD0332991 price Patients 912 patients with a mean age of 54.4 years (range 4–98) were enrolled in the study during the first three-month period. 432 patients (47.7%) were women and 480 (52.3%) were men. Among these patients, 753 (83.3%) were affected by community-acquired IAIs while the remaining 159 (16.7%) suffered from healthcare-associated infections. Intraperitoneal specimens were collected from 586 (64.2%) of the enrolled patients. 338 patients (37%) were affected by generalized peritonitis while 574 (63%) suffered from localized Oxymatrine peritonitis or abscesses. 123 patients (13.5%) were admitted in critical condition (severe sepsis, septic shock). Tables 1 and 2 contain the clinical findings and radiological assessments

recorded upon patient admission. Table 1 Clinical findings Clinical findings Patients n° (%) Abdominal pain 102 (11,2%) Abdominal pain, abdominal rigidity 87 (9,5%) Abdominal pain, abdominal rigidity, T > 38°C or <36°C, WBC >12000 or < 4000 38 (4,2%) Abdominal pain, abdominal rigidity, T > 38°C or <36°C, 184 (20,2) Abdominal pain, abdominal rigidity, WBC >12000 or < 4000 182 (20%) Abdominal pain, T > 38°C or <36°C, 28 (3%) Abdominal pain, T > 38°C or <36°C, WBC >12000 or < 4000 100 (11%) Abdominal pain, WBC >12000 or < 4000 138 (15,1) T > 38°C or <36°C 5 (0,5%) T > 38°C or <36°C, WBC >12000 or < 4000 22 (2,4%) WBC >12000 or < 4000 15 (1,7) Not reported 11 (1,2%) Table 2 Radiological procedures Radiological procedures Patients n° (%) Abdomen X ray 91 (10%) Abdomen X ray, CT 73 (8%) Abdomen X ray, ultrasound 167 (18,3%) Abdomen X ray, ultrasound, CT 88 (9,6%) Abdomen X ray, ultrasound, MRI 2 (0,2%) CT 208 (22,8%) Ultrasound 153 (16,8%) Ultrasound, CT 74 (8,1%) Ultrasound, CT, MRI 1 (0,1%) Ultrasound, MRI 2 (0,2%) Not reported 53 (5,8%) Source control The various sources of infection are outlined in Table 3. The most frequent source of infection was acute appendicitis. 350 cases (38.

, Chiyoda, Tokyo, Japan) was used to characterize the morphology of the samples. The crystal structure of the TiO2 nano-branched arrays was examined by X-ray diffraction (XRD; XD-3, PG Instruments Ltd., Beijing, China) with Cu Kα radiation (λ = 0.154 nm) at a scan rate of 4° per min. X-ray tube voltage and current were set to 36 kV and 20 mA, eFT-508 respectively. The optical absorption spectrum was obtained using a UV-visible spectrometer (TU-1900, PG Instruments, Ltd., Beijing, China). Solar cell assembly and performance measurement Solar cells were assembled

using nano-branched TiO2/CdS nanostructures as photoanodes. Pt counter electrodes were prepared by depositing a 20-nm-thick Pt film on FTO glass https://www.selleckchem.com/products/empagliflozin-bi10773.html using magnetron

sputtering. A 60-μm-thick sealing material (SX-1170-60, Solaronix SA, Aubonne, Switzerland) with a 5 × 5 mm2 aperture was pasted onto the Pt counter electrodes. The Pt counter electrode and the nano-branched TiO2/CdS photoelectrode were sandwiched and sealed with the conductive sides facing inward. A polysulfide electrolyte was injected into the space between the two electrodes. The polysulfide electrolyte was composed of 0.5 M sulfur, 1 M Na2S, and 0.1 M NaOH, all of which were dissolved in methanol/water (7:3, v/v) and stirred at 80°C for 2 h. A solar simulator (Model 94022A, Newport, OH, USA) with an AM1.5 filter was used to illuminate the working solar cell at a light intensity of 1 sun illumination (100 mW/cm2). A sourcemeter (2400, Keithley Instruments Inc., Cleveland, OH, USA) provided electrical characterization during the measurements. Measurements were calibrated using an OSI standard silicon solar photodiode. Results and discussion Figure 1 shows the typical FESEM images of TiO2 nanorod arrays on Buspirone HCl FTO-coated glass substrates, at both (a)

low magnification and (b) high magnification. It can be observed that the FTO-coated glass substrate was uniformly covered with ordered TiO2 nanorods. The density of the nanorods was 20 nanorods/μm2, which allows suitable space for growth of TiO2 nanobranches. After immersion in an aqueous TiCl4 solution for a period of time ranging from 6 to 24 h, nanobranches appeared along the trunks of the TiO2 nanorods. The morphology of the branches, shown in Figure 2, is strongly dependent on the amount of time the nanorods remain immersed in the TiCl4 solution. As the immersion time increases, the branches become greater in number and longer in length. These branches coated on TiO2 nanorod would greatly improve the specific surface area and roughness, which is urgent for solar cell applications. However, when immersed for 24 h or more, the branches form continuous networks that greatly suppress the effective surface area, preventing the CdS quantum dots from fully contracting with the TiO2 and therefore decreasing the overall photovoltaic performance.

SCCHN is the 5th most common cancer worldwide [9] with high mortality ratios among all malignancies accounting for 12% of all cancers in men and 8% of all cancers among women [10]. SCCHN are the commonest forms of cancers of the head and neck that start in the cells forming the lining of the mouth, nose, throat and ear or the surface covering the tongue. The major head and neck Natural Product Library screening sites include the oral cavity, the pharynx (nasopharynx, oropharynx and hypopharynx),

the tongue (anterior 2/3rd and posterior 1/3rd or base of tongue), the larynx and the paranasal sinuses. Breast cancer is the primary subtype of cancer leading to death among women in developing countries.

13% out of the 58 million deaths worldwide in the year 2005 were caused due to cancer which included 502,000 deaths per year due to breast cancer. Well-established risk factors ascribed to breast cancer include early menarche, late menopause, age of first child’s birth, nulliparity and family history (FH) [11]. DNA repair is considered to play a key role in cancer susceptibility whereby some individuals are at very high risk of cancer due to SNPs in crucial DNA repair genes [12–15]. Inactivation or defect in DNA selleck chemical repair genes may be associated with increased cancer risk [16]. Genetic polymorphisms in DNA repair genes are very common events [17–19], and some studies have shown a significant

effect of some of these polymorphisms in DNA repair capacity [20–22]. Evidence of inherited abnormalities in DNA repair genes and genes controlling carcinogen metabolism has been found to underline increase in risk of cancers [23]. The gene ERCC2 (located in the chromosomal location 19q13.3; OMIM ID 126340; Gene ID 2068; Gene length 18984) encodes the ERCC2/Xeroderma pigmentosum Type D (XPD) protein, which is one of the seven genetic complementation groups that forms an essential component of the Nucleotide excision repair (NER) pathway, a major DNA repair pathway that Clomifene removes photoproducts from UV radiation and bulky adducts from a huge number of chemicals, cross-links and oxidative damage through the action of 20 proteins and several multiprotein complexes [13, 24]. XPD is a highly polymorphic gene and correlation of its polymorphisms and cancer risk have been extensively studied [20, 25]. Among the genetic polymorphisms in ERCC2, the SNP causing amino acid change in codon 751 (Lys to Gln) (SNP ID rs13181) have been considered very important and there is evidence that subjects homozygous for the variant genotypes of XPD have suboptimal DNA repair capacity for benzo(a)pyrene adducts and UV DNA damage [26, 27].

Methods Operating principle A near-midgap state in the zigzag graphene nanoribbon (zzGNR) [7] with periodic edge roughness is extensively studied in [8]. In this work, we study novel device characteristics where the channel consists of a 1-nm wide zzGNR as shown in Figure 1a. The device structure is shown in Figure 1b, where the channel is gated by two side gates to create an electric field in the width direction. For such a side-gated nanoribbon, we show the electronic structure in Figure 1c

using extended Hückel AZD0156 mw theory (see [8–12] for the detailed model). The two interesting electronic structure features are a significant band gap opening of about 2 eV, which is not very sensitive to the external electric field, and secondly a near-midgap state with a finite bandwidth, the bandwidth and dispersion of which can be manipulated by the gate-induced electric field. In Figure 1d, we show the dependence of the bandwidth on the gate voltage in the limit of relative permittivity

of the gate dielectric to be much larger than that of the nanoribbon. Figure 1 Device structure and operating principle of an electronic structure modulation transistor. (a) The channel consists of a 1-nm wide hydrogenated zigzag graphene nanoribbon with edge roughness. (b) The channel is side-gated to create an electric field in the width direction. Gate dielectric

surrounds the channel and is not shown for clarity. (c) For such a ribbon, a near-midgap state with Apoptosis Compound Library a small bandwidth is observed which can be modulated by the gate-induced electric field (left = 0 V/nm electric field, middle = 0.2 V/nm electric field, right = zoomed bandwidth comparison for the two electric fields). (d) The bandwidth of the near-midgap state is linearly dependent on the gate voltage [8]. Such a bandwidth modulation can be understood in terms of the real-space localization of the wavefunction for various momentum values. At the Γ point, the wavefunction of the near-midgap state is distributed throughout the nanoribbon width, whereas at the X point is localized on the pristine edge. Additionally, the wavefunctions are localized on Sucrase only one sublattice of graphene [8]. By applying a positive gate voltage at this edge, the energy values shift downward, thereby increasing the bandwidth as shown in Figure 1c. One should note that such modulation may happen due to intrinsic or extrinsic electric fields. In case of gate-voltage-induced modulation (extrinsic electric field) as shown in Figure 1d, the BW is given as follows: (1) where α is a dimensionless parameter, called the modulation factor. BWo is the residual BW at zero gate voltage (Mag ≡ absolute magnitude) and V g is the applied gate voltage. In Figure 1d, α = 0.47 and BWo = 0.12 eV.

5 days and 4 days post inoculation, respectively. The expression of bacterial DnaK was used as the internal control. Protein samples were reacted with antibodies against the FLAG sequence (top panel) and DnaK (low panel). Each lane was loaded with material from

5 × 107 CFU bacteria. (C-D). Level of tagged proteins from the bacterial EPZ015938 strains recovered from the macrophages and spleens of infected mice as determined in (A) and (B). The values, which are the means of triplicate experiments, represent the relative percentage of the levels of the tagged proteins in the bacteria recovered from macrophages (C) at 5 hours postinfection and from the spleen at 5 days postinoculation (D), as compared to those in the bacteria recovered from macrophages at 0.2 hours postinfection and from spleen at

0.5 days post inoculation, respectively. In cultured macrophages, SipA, SipC, and SopB were all expressed at the early phase (e.g. 0.2 h) of infections. However, by 5 hr post infection, the levels of the three SPI-1 proteins diverged, with the SipC level increased, the SopB level decreased while SipA level remained unchanged (Figure 6A and 6C). To determine the relative abundance of these proteins in the spleen during systemic infection, BALB/c mice were infected intraperitoneally. Salmonella was recovered from the spleen at different time points postinfection, and CBL0137 concentration the expression levels of the tagged proteins were determined. Similar to the results of macrophage infection, all three proteins were

detected during the early stage of infection (i.e. 0.5 days). However, at a later stage of systemic infection (i.e. 5 days), the level of SipC increased and the level of SopB decreased while the level of SipA remained unchanged (Figure 6B and 6D). These results correlated with those observed in the proteomic analyses and in the macrophage experiments. Furthermore, these data strongly suggest that different SPI-1 factors are specifically expressed at late stage of Salmonella infection, and highlight a possible role of SipC in late phase of macrophage and in vivo infections of Salmonella. Discussion Stable isotope labeling procedure coupled with MS-based analysis for quantitative Immune system proteomic study of bacterial protein expression In the postgenomic era, new methodologies are needed that can quantitatively, globally, and accurately measure protein expression in cells and tissues [37]. In this study, we have modified the SILAC method to develop a stable isotope labeling procedure coupled with MS analysis to carry out quantitative proteomic analysis of Salmonella. As a “”proof of principle”" pilot study, a total of 103 SE2472 proteins were monitored for their expression profiles upon exposure to H2O2. At least seventy six proteins have been found to be modulated in the presence of H2O2.

2.19 was obtained from the NCBI BLAST website [45]. Using default parameters, blastp was used to align the wBm protein sequences against the protein sequences contained in DEG. To produce the multi-hit score, the check details negative log 10 of the e-values of the highest scoring alignments to each of the DEG organisms were normalized between 0 and 1, squared, then averaged for all DEG organisms. E-values greater than 1 were truncated at 1. Where N = the number of DEG organisms and 1 × 10-200 is the smallest e-value reported by BLAST. Jackknife Analysis Complete Refseq protein sequences for the 15 organisms contained within DEG were downloaded from the NCBI Refseq

ftp site ftp://​ftp.​ncbi.​nlm.​nih.​gov/​genomes/​Bacteria. For each organism, a filtered version of DEG was prepared, removing just the

proteins from that organism. The full protein complement of that organism was then subjected to MHS analysis using the filtered version of DEG, and ranked based on MHS. Moving through the ranked genome from highest prediction of essentiality to lowest, the cumulative sum of DEG genes encountered was calculated. The area under the curve (AUC) of the cumulative sum describes the effectiveness of the ranking. The upper bound of the AUC is defined by an ideal sorting which places all GSK126 chemical structure DEG genes at the top of the list. The mean and standard deviation of the AUC for the null hypothesis of no sorting was determined by randomly permuting the genome sorting 1000 times. The AUCs for the random assortments MTMR9 was assumed to represent a normal distribution with the observed mean and standard deviation. The p-value of the MHS sorting versus the null hypothesis was calculated using the probability density for a normal distribution. For the calculation of percent sorting, the AUC for the unsorted diagonal was one-half of the total area of the graph, calculated

as the total number genes in the genome multiplied by the number of DEG genes, divided by two. Gene Conservation Across Rickettsiales Refseq protein sequences were downloaded from the NCBI Refseq ftp site for the 27 sequenced organisms in the order Rickettsiales (Table 3). The standalone version 1.4 of the OrthoMCL ortholog prediction program was downloaded http://​www.​orthomcl.​org/​common/​downloads/​software/​[38]. OrthoMCL was used with default settings and an inflation value of 1.5 to predict orthologs among the protein sequences of the 27 genomes. Briefly, OrthoMCL begins by using an all-versus-all BLAST search to identify reciprocal best BLAST hits among the genomes as putative orthologs, and reciprocal best BLAST hits within genomes as putative in-paralogs. These interconnections are used to form a similarity graph that is used by the MCL clustering algorithm to break mega-clusters into suitable sub-clusters of orthologs [46]. For each cluster of orthologous genes the minimum spanning tree (MST) distance was calculated based on the phylogenetic distances among the member genomes.

HupF contributes to HupL stability under elevated oxygen tensions The existence of hupF in hydrogenase systems from bacteria synthesizing this enzyme in the presence of oxygen prompted us to study the potential role of this protein in protection against oxygen. To this aim, we analyzed the possible effect of HupF on the status of hydrogenase large subunit in cultures maintained under different

oxygen tensions (1% and 3%). The higher oxygen tension (3%) still allowed the expression of hydrogenase in R. leguminosarum wild-type strain, although at a reduced level (40% of the level induced under 1% O2, Table  2). The presence

and processing status of the hydrogenase large subunit (HupL) were analyzed in crude cell extracts from microaerobic cultures through immunoblot (Figure  2). In these experiments click here Tipifarnib clinical trial we found that the wild-type cells contained a clear band associated to the mature form of HupL, irrespective of whether cells were induced under 1% or 3% oxygen (Figure  2A and 2B, upper panel). This band was absent in a ΔhupL mutant used as negative control (Figure  2A). Analysis of the cell extracts from the ΔhupF strain grown at 1% oxygen revealed the presence of HupL, although in the unprocessed form (Figure  2A, upper panel). Interestingly, HupL was not detected when cultures from the same mutant

strain were incubated under 3% O2 (Figure  2B). In contrast, extracts from a R. leguminosarum mutant lacking HypC, used as a hydrogenase non-processing control, showed a clear band of unprocessed HupL after exposure to both 1% and 3% oxygen tension (Figure  2A and 2B). Similar levels of an immunoreactive band corresponding to HypB were detected in all the extracts (Figure  2, lower panels), indicating that the microaerobic induction of Hup expression was equally effective for all strains in each treatment. These data suggest that, below in the presence of 3% oxygen, HupL is either unstable or not synthesized in the absence of HupF. In order to further evaluate these possibilities, we analyzed the in vivo stability of HupL as a function of the presence/absence of HupF. To address this question, we first induced R. leguminosarum cultures for hydrogenase expression under 1% oxygen, and then the induced cells, carrying either processed HupL (wild-type strain) or unprocessed HupL (ΔhupF and ΔhypC mutants), were exposed to atmospheres containing either 1% O2 or 21% O2 for up to 3 hours. After such treatments, the amount and processing status of HupL was determined through immunoblot assay in cell extracts (Figure  3A).