Second, several publically available methanotroph genomes are not

Second, several publically available methanotroph genomes are not yet completely assembled, and absence of evidence does not provide Androgen Receptor signaling Antagonists evidence of absence. Third, the required pathway reactions could be performed by proteins whose sequence bears little or no resemblance to experimentally characterized enzymes. Clearly, more research is needed to elucidate how facultative

methanotrophs assimilate carbon from multicarbon compounds into biomass, and the increasing availability of genome sequences represents as much a great asset as a sobering reminder of our ignorance. It has been confirmed that facultative methanotrophy does indeed exist, but corresponding isolates can only utilize a small number of organic acids and ethanol to support growth, i.e., sugars cannot be used, possibly due to lack of sugar transporters and/or lack of key steps of the glycolytic pathway. Also, to date, no methanotrophs of the gammaproteobacterial phylum have conclusively been shown to be facultative. These methanotrophs present

several key differences to Alphaproteobacteria methanotrophs including, as noted above, the lack of a complete TCA cycle, as well as their utilization of the RuMP pathway for growth. One Gammaproteobacteria methanotroph, M. capsulatus Bath, has been found to have genes for the E1 and E2 subunits learn more of the 2-ketoglutarate dehydrogenase (Ward et al., 2004). At this Cepharanthine time, it is

unclear under what conditions, if any, these genes are transcribed, and active enzyme synthesized. The absence of 2-ketoglutarate dehydrogenase activity may limit growth of Gammaproteobacteria methanotrophs with alternative multicarbon compounds, as well as the fact that isocitrate lyase and malate synthase are apparently missing in these microorganisms (Trotsenko & Murrell, 2008). Further, the acetate assimilation pathways described above do not lead to the production of intermediates of the RuMP pathway. Accordingly, and unlike Alphaproteobacteria methanotrophs that utilize the serine cycle, Gammaproteobacteria methanotrophs appear to be unable to use these pathways for carbon assimilation from multicarbon compounds. This may help explain why all known facultative methanotrophs utilize the serine cycle and not the RuMP pathway for carbon assimilation. We suggest that more effort be invested to isolate Gammaproteobacteria methanotrophs from environments with high acetate concentrations, for example, peat bogs and acidic forest soils, to determine if such conditions promote facultative growth in a broader phylogenetic range of methanotrophs. Molecular evidence indicates that such methanotrophs exist in these environments, particular peat bogs, but that they do not represent a significant fraction of the overall methanotrophic population (Dedysh, 2009).

We recommend all patients should have the option of treatment, an

We recommend all patients should have the option of treatment, and have the pros and cons of opting for initiation of treatment

and of deferring treatment discussed with them. We suggest for patients with non-cirrhotic disease there is the option to defer treatment until newer therapies or a suitable trial become available. We recommend those deferring treatment are monitored by non-invasive tests at least annually and if they have confirmed progression of fibrosis are reconsidered for initiation of therapy. The response rates of genotypes 2 and 3 infection to pegylated interferon and ribavirin regimens are much higher than in genotype 1 infection in both monoinfected and coinfected individuals. In a recent meta-analysis, treatment response rates of genotype EPZ015666 purchase 2 and 3 did not differ between HIV-infected and -uninfected populations [95]. Neither telaprevir nor boceprevir has substantial activity against genotypes 2 and 3, although second-generation protease inhibitors and other DAA classes as well as several interferon-sparing strategies have reported high rates of SVR in monoinfected populations [77,96–98]. Because of differential activity of the newer DAAs on GT2 and GT3 virus, there may be a requirement to separate recommendations in future guidelines [99–100].

Therefore the only available therapy for CX-5461 ic50 genotype 2 and 3 hepatitis C in the context of HIV infection remains pegylated interferon and ribavirin. Ribavirin should be

prescribed as weight-based due to higher response rates when this method is employed. In individuals who are naïve to hepatitis C therapy, do not have cirrhosis (Metavir F4) and achieve an RVR, treatment duration should be 24 weeks, as longer courses of therapy have not translated into higher rates of SVR. Individuals not achieving an RVR but reaching an EVR should receive 48 weeks of therapy. All individuals receiving treatment after failing a previous interferon-based regimen should receive 48 weeks of therapy. Erythropoietin and granulocyte colony stimulating factors should be used as required and should be given in Florfenicol preference to interferon and ribavirin dose reduction. We suggest for patients with genotype 4 infection without cirrhosis, there is the option to defer treatment until newer therapies or a suitable clinical trial become available. We recommend if treatment is given now, this should be with pegylated interferon and ribavirin. The duration of therapy should be 48 weeks if RVR is achieved. If the RNA is still detectable at 12 weeks, consideration should be given to discontinuing treatment. For those with previous treatment failure, we recommend waiting for the availability of interferon-sparing regimens with active DAAs.

, 2008) A significant finding from our model was that top-down a

, 2008). A significant finding from our model was that top-down attentional signals and simulated mAChRs decreased correlations between excitatory–inhibitory and inhibitory–inhibitory neurons in the cortex; however, excitatory–excitatory correlations remained unchanged (Figs 8 and 9). Several experimental studies have shown that attention and neuromodulation decrease interneuronal noise correlations (Cohen & Maunsell, 2009; Goard & Dan, 2009; Mitchell et al., 2009). In fact, Cohen and Maunsell showed that

decorrelation caused more than 80% of the attentional improvement in the population signal. This suggested that decreasing noise ITF2357 price correlations was more important than firing rate-related

biases. These studies, however, did not identify the types of neurons they were recording from, which may be difficult using conventional ICG-001 chemical structure recording techniques. Our model predicts that the decorrelations seen in these studies may be excitatory–inhibitory pairs of neurons rather than excitatory–excitatory pairs. In our model, we found no change in excitatory–excitatory correlations when applying top-down attention and stimulating the BF, but saw a significant decrease in excitatory–inhibitory and inhibitory–inhibitory correlations. In this view, excitatory–excitatory pairs are able to maintain a constant, low correlation state regardless of the amount of excitatory drive (which should STK38 increase correlations) due to fast-spiking inhibitory neurons (Fig. 13B). Because muscarinic receptors caused a further decrease in excitatory–inhibitory correlations, we suggest that they may act as a buffer, absorbing increases in excitation that

occur with attention and BF stimulation by changing either the inhibitory spike waveform (i.e. inhibitory speed) or the inhibitory strength. A recently published study further substantiates our finding that excitatory–inhibitory pairs of neurons have stronger decorrelation than excitatory–excitatory pairs. Middleton et al. (2012) were able to distinguish between excitatory and inhibitory neurons and looked at the correlations between these pairs in layer 2/3 of the rat’s whisker barrel cortex. They compared correlations during spontaneous and sensory stimulated states and found that excitatory–inhibitory pairs of neurons became decorrelated when sensory stimuli were presented to the animal, whereas excitatory–excitatory pairs of neurons remained at low levels of correlations. Our model suggests that the spiking pattern of the inhibitory neuron is important for maintaining neuronal decorrelation when further excitatory drive is applied (Fig. 10). Given excitatory–inhibitory decorrelation and minimal excitatory–excitatory correlations both in our model and in Middleton et al.

522, 2270 and 1868 is tentatively denoted as LS1 and the other

522, 2.270 and 1.868 is tentatively denoted as LS1 and the other species with g values of 2.430, 2.250 and 1.910 as LS2. No signals of ferric high-spin heme were observed. When dithionite was added to the protein solution, the EPR signals of LS1 and LS2 decreased in intensity but the decrease, which was larger in LS1 than in LS2, was <30% even after several hours (Fig. 3b). This indicates that both the low-spin hemes in NaxLS, LS1 and LS2, are quite resistant to the dithionite reduction learn more and this is consistent with the optical results that showed only a small shift of the Soret-band maxima upon reduction with dithionite (Fig. 2a). Only the broad signal at g=2.09 completely disappeared

by the reduction. Reduction with dithionite also shifted the g-values of LS1 drastically to 2.570, 2.260 and 1.844 (LS1′). At the same time, the line width of the LS1 signals broadened with an apparent decrease in the intensity. In contrast, the LS2 signals were only slightly affected by reduction and showed very small g-shifts (Fig. 3). The two LS species found in NaxLS are attributable to each redox center of NaxL and NaxS. The EPR results Selleckchem CX 5461 suggest that the two heme sites have not only different redox potentials but also different protein milieus: one (LS1) is

flexible and the other (LS2) is fixed. Because the broad signal at g=2.09 is indicative of some spin–spin interaction, the g-shifts of LS1 might be caused, in part, by the decoupling of such an interaction. To assign LS1/LS2 to NaxL/NaxS, the independent expression of each gene in Escherichia coli cell is under way. The g-values of LS1 and LS2 of NaxLS are remarkably similar to those of the LS species of SoxA of the SoxAX complex, involved in sulfur oxidation of Paracoccus pantotrophus (Cheesman et al., 2001; Reijerse et al., 2007): LS1, g=2.54, 2.30, 1.87 and LS2, g=2.43, 2.26, 1.90. The axial

heme ligands of the SoxA LS1 and LS2 are determined to be His-Cys− and His-Cys-S−, respectively, by X-ray crystallography. Then, the axial heme ligands of LS1 and LS2 of NaxLS are strongly suggested to be His-thiolate. An alignment of the amino acid sequences of NaxS and four other proteins having c-type heme was performed using ClustalW program (Fig. 4). The amino-acid sequences of NaxS and four Florfenicol related heme c proteins (obtained by clustalw program). The heme c of Arthrospira cytochrome c6 has Met/His coordination [Kerfeld et al., 2002; PDB ID, 1KIB; (5) in Fig. 4] and the axial Met is conserved in two other proteins, a heme protein (EES51901) from Leptospirillum [(3) in Fig. 4] and cytochrome c552 (AAY86372) from C. Kuenenia stuttgartiensis [(4) in Fig. 4]. On the other hand, in NaxS [(1) in Fig. 4] and a deduced protein (CAJ70833) from C. Kuenenia stuttgartiensis [(2) in Fig. 4], Cys occupies the Met position. Similar g-values to those of LS1 and LS2 of the NaxLS complex are generally obtained for the b-type hemes with the Cys axial ligand: for example CO-sensor CooA (Cys-Pro, Aono et al.

psychrophilum isolates The DNA sequence revealed a genome

psychrophilum isolates. The DNA sequence revealed a genome 5-FU in vitro of 46 978 bp containing 63 predicted ORFs, of which 13% was assigned a putative function, including an integrase. Sequence analysis showed > 80% amino acid similarity to a specific region found in the virulent F. psychrophilum

strain JIP02/86 (ATCC 49511), suggesting that a prophage similar to phage 6H was present in this strain. Screening for a collection of 49 F. psychrophilum strains isolated in Chile, Denmark, and USA for the presence of four phage 6H genes (integrase, tail tape protein and two hypothetical proteins) by PCR showed the presence of these prophage genes in 80% of the isolates. In conclusion, we hypothesize that bacteriophage 6H belongs to an abundant group of temperate phages which has lysogenized a large fraction of the global F. psychrophilum community. “
“Swainsonine is a polyhydroxy indolizidine alkaloid with various research and potential therapeutic applications. In this work, swainsonine was partially purified (2.5-folds) with acetone–methanol solvent system from Metarhizium anisopliae fermentation broth. The partially purified broth was further subjected to mass-directed preparative-cum-quantitative

analysis. Swainsonine was eluted as MS1 fraction [M + H]+ this website 174.36 ± 0.21 at 4.91 ± 0.04 min with calculated yield of 7.85 ± 1.59 μg mL−1 corresponding to 3.74 × 105 counts. In situ antiproliferative activity of standard and purified swainsonine fractions was tested against Spodoptera frugiperda, Sf-21 cell line with IC50 values of 2.96 μM

and 3.28 μM, respectively, at 36 h. This analytical procedure for purification and quantitative analysis of swainsonine may ensure its suitability for routine laboratory studies and research. “
“The rumen bacterium Butyrivibrio proteoclasticus B316T has a 4.4-Mb genome composed of four replicons (approximately 3.55 Mb, 361, 302 and 186 kb). Mutagenesis of B316T was performed with the broad host-range conjugative transposon Tn916 Sitaxentan to screen for functionally important characteristics. The insertion sites of 123 mutants containing a single copy of Tn916 were identified and corresponded to 53 different insertion points, of which 18 (34.0%), representing 39 mutants (31.7%), were in ORFs and 12 were where transposition occurred in both directions (top and bottom DNA strand). Up to eight mutants from several independent conjugation experiments were found to have the same integration site. Although transposition occurred in all four replicons, the number of specific insertion sites, transposition frequency and the average intertransposon distance between insertions varied between the four replicons. In silico analysis of the 53 insertion sites was used to model a target consensus sequence for Tn916 integration into B316T.

The PCR reactions were carried out in a final volume of 50 μL con

The PCR reactions were carried out in a final volume of 50 μL containing 1 × PCR buffer, 2.5 mM MgCl2, 0.2 mM of each dNTP, 0.2 μM of each primer, 1.25 U of BIBW2992 manufacturer Taq DNA Polymerase and 5 μL of DNA template and distilled water. Initial denaturation was performed at 94 °C

for 5 min, followed by amplification comprising 35 cycles of denaturation at 94 °C for 30 s, annealing at 52 °C for 30 s and extension at 72 °C for 45 s. A further 2-min final extension at 72 °C was carried out following the final cycle. The amplified PCR products were analyzed using 1.5% agarose gel (Promega) electrophoresis in 1 × TBE buffer at 90 V for 1 h and visualized using ethidium bromide staining under UV illumination. The positive PCR products were purified using Wizard PCR Purification Kit (Promega) and confirmed by sequencing (Research Biolabs

Sdn. Bhd, Singapore). The limit of dilution was determined by subjecting the DNA of the targeted organisms to PCR after 10-fold serial dilutions to produce a DNA concentration ranging from 10 μg mL−1 to 10 fg mL−1. Real-time duplex PCR amplification and melt curve analysis were carried out in an iQ5 real-time PCR detection system (BioRad Laboratories, Hercules, CA). QuantiTect SYBR green PCR Tacrolimus kit (Qiagen) was used for amplification with 0.3 μM of mprA and 0.2 μM of zmpA primers. The PCR was performed with the following cycling protocol. Initial denaturation for 15 min at 95 °C was followed by 30 cycles with 15 s at 94 °C, 30 s at 52 °C and 30 s at 72 °C. Fluorescence data were captured at the elongation step of each cycle. Following amplification, melt curves were acquired by increasing the temperature from 65 to 95 °C at the rate of 0.5 °C 10 s−1, with continuous measurement O-methylated flavonoid of the fluorescence. In general, all three query gene sequences retrieved from the GenBank

and analyzed by blast were correct with an exact match of 100% identity. clustalw alignment revealed that the groEL gene sequence of B. pseudomallei was highly homologous to B. mallei, B. thailandensis and B. cepacia, with a score of 99%, 97% and 95%, respectively. The alignment scores of other organisms such as the Pseudomonads, Xanthomonas campestris, Bordetella pertussis and Ralstonia picketti displayed a distant relation to Burkholderia spp. Therefore, the regions of groEL appropriate for primer design were targeted at the part where there was 100% identity of bases among Burkholderia spp. and vast variation with other organisms. The mprA gene sequenced was not aligned with any other organisms as no database was found for a similar gene in other organisms. The zmpA of B. cepacia was aligned with that of B. pseudomallei. Alignment results revealed an identity of 86% between these two sequences. Thus, the regions that displayed significant nucleotide variation within zmpA sequences of these two organisms were chosen for primer design.

3) Ralstonia eutropha is an industrially important bacterium, bu

3). Ralstonia eutropha is an industrially important bacterium, but its metabolic engineering is somewhat limited due to the lack of an efficient gene knockout system, with the exception of the system utilizing suicide vectors. Thus, here, we developed a gene CP-868596 knockout strategy based on the mobile group II intron system. After the integration of the intron into the target DNA site, the

Ll.LtrB intron cannot be mobilized and spliced in the absence of IEP and is less sensitive to the degradation by nuclease than the full-length intron (Cousineau et al., 1998). These properties result in high integration frequencies of up to ∼22% (Karberg et al., 2001). For the strong induction of the knockout system, an IPTG-inducible tac promoter was used (Baek et al., 2007). Plasmid pBBR1MCS2 was used as a backbone plasmid because it is a broad-host-range vector. It has been reported to replicate in at least 16 different types of bacteria, including Acetobacter xylinum, Bartonella bacilliformis, Bordetella spp., Brucella spp., Caulobacter crescentus, E. coli,

Paracoccous AC220 supplier denitrificans, Pseudomonas fluorescens, Pseudomonas putida., R. eutropha, Rhizobium meliloti, Rhizobium leguminosarum bv. viciae, Rhodobacter sphaeroides, Salmonella typhimurium, Vibrio cholerae, and Xanthomonas campestris (Kovach et al., 1995). Thus, the gene knockout system developed for R. eutropha in this study could be useful for knocking out genes of interest in these bacteria. As an example, the phaC1 gene encoding polyhydroxyalkanoate synthase was successfully knocked out in R. eutropha. For the construction of multiple knockout strains, the procedure described in this study can be repeated for the knockout of other genes after curing

the intron donor plasmid. For this knockout system to yield high intron integration efficiency, the base pairing interactions with the intron RNA and the reliable intron integration site predicted by the computer algorithm are important. The sequences of the primers for the overlapping PCR of the retargeted intron and the region of the retargeted intron in pBBR1RInt should be confirmed thoroughly by sequencing analysis for the successful base pairing with the of intron RNA. During primer synthesis, some error sequences can be introduced. If the error sequences are present, it can cause a change in the RNA structure and a mismatch in the complementary regions between the exon-binding sites (EBS2 and EBS1 sequences) in the intron RNA and the intron-binding sites (IBS2 and IBS1 sequences) in the DNA target site. For the knockout of the phaC1 gene in this study, computer simulation provided us with the best target site, which worked successfully. However, the best intron insertion site predicted by the computer algorithm might not always yield good results (Yao & Lambowitz, 2007). Thus, one should test the other predicted sites as well.

In the years since our earlier studies in Nepal, empiric self-tre

In the years since our earlier studies in Nepal, empiric self-treatment of diarrhea is routinely recommended to travelers.21–23 It is possible that the clinic sees a higher percentage of patients with more severe diarrhea, or those who have failed empiric treatment.

Indeed, in our study, 14% of patients with diarrhea who came to the clinic had already taken an FQ antibiotic. Among those patients who had taken an FQ and were later proved to have Campylobacter, all of the isolates were resistant to ciprofloxacin. FQ resistance among Campylobacter has been documented BIBW2992 order in Thailand.24,25 Most travelers to the developing world have enjoyed a “golden age” of empiric treatment with ciprofloxacin for suspected bacterial diarrhea, in which virtually 100% of pathogens were sensitive to one drug. This article adds to the concern that ciprofloxacin may no longer be able to cover 100% of pathogens in regions in which resistance to Campylobacter is common. It is important to note that antibiotic resistance has mainly occurred in Campylobacter species and not in the other bacterial pathogens. Some authorities have implicated agricultural use of FQs as increasing the likelihood of resistant Campylobacter.26,27 It has also been noted in prior studies that in vitro FQ resistance in Campylobacter has not always predicted a failed clinical

outcome.28 In one study from Thailand, 58% of patients treated with ciprofloxacin either for ciprofloxacin-resistant Campylobacter achieved a cure.28 Anecdotal experience at the CIWEC clinic suggests that ciprofloxacin works rapidly and is well tolerated, though its use has been associated with BTK inhibitor a low, but noticeable rate of clinical failures. Azithromycin is used as a backup medication if there is a lack of response to ciprofloxacin within 24 to 48 hours. Similarly if azithromycin is used as the first-line drug for treatment, ciprofloxacin is employed for treatment failures. This study was not designed to record clinical outcomes, so we were unable to match

antibiotic failure rates to microbiologic findings in specific patients. Such a study would obviously be very valuable. The data show that when all isolates were taken into account, overall resistance to either ciprofloxacin or azithromycin was the same, and no isolates were resistant to both drugs. Based on this information, the standard of care for pretravel advice should be for travelers to carry both drugs for empiric TD treatment, use one first and reserve the other one for treatment failures. Bacterial pathogens were more often isolated among younger patients, tourists, and those with recent onset of symptoms (fever, watery diarrhea, or WBCs in the stool; Table 1). Viral pathogens presented with similar symptoms, but were still much less common than bacterial pathogens in this population. Of concern is the documentation for the first time of norovirus in 3.

The number of ipsilateral

and contralateral retrievals ma

The number of ipsilateral

and contralateral retrievals made by each mouse was counted until the mouse made a total of 20 retrievals, or a maximum time of 5 min elapsed. A ‘retrieval’ is defined as an exploration into a pot, whether or not a pellet is eaten, and a new retrieval can only be made by investigating a new pot (Dowd et al. 2005a). Data are expressed as percentage contralateral retrievals, calculated as the number of contralateral retrievals expressed as a percentage of the total retrievals made from both sides relative to the lesion. Forelimb akinesia was assessed using the stepping test (Olsson et al., 1995), as adapted for mice. Briefly, the mouse was held by the experimenter with one forelimb restrained and the free forepaw placed on a table surface. The number of adjusting steps made by the mouse, using the free forelimb, was counted as it was moved sideways along a table surface over a distance of 30 cm, in both

Buparlisib datasheet forehand and backhand directions. Data are Androgen Receptor antagonist expressed as the sum of forehand and backhand steps made by each paw. Forelimb use was assessed using the cylinder test, as previously described by (Schallert & Tillerson, 2000). Mice were placed in a glass cylinder (diameter 19 cm, height 20 cm), with mirrors placed behind to allow for a 360° view of all touches, until at least 30 weight-bearing paw touches were made by the forelimbs against the side of the cylinder. The session was videotaped and later scored. Paw touches were analysed using freeze-frame analysis of the recording and, in Org 27569 cases where both paws were used simultaneously, these touches were

not counted. Data are expressed as percentage contralateral touches, calculated as the number of contralateral touches expressed as a percentage of the total touches made using both paws. Once behavioural analysis was complete, mice were terminally anaesthetised with sodium pentobarbitone i.p. (Apoteket, Sweden). Mice were then transcardially perfused with 15 mL of room-temperature (21°C) 0.9% saline, followed by 100 mL of ice-cold 4% paraformaldehyde in phosphate-buffered saline (PBS). Brains were post-fixed for 2 h at 4°C and then transferred to 25% sucrose in PBS at 4°C for cryoprotection overnight. The brains were then sectioned in the coronal plane using a freezing microtome at a thickness of 35 μm. Sections were collected in six series and stored at −20°C in an antifreeze solution (phosphate buffer containing 30% glycerol and 30% ethylene glycol) until free-floating immunohistochemistry was performed. Briefly, sections were rinsed three times in potassium phosphate-buffered saline (KPBS) and then endogenous peroxidase activity was quenched in 3% H2O2 and 10% methanol in KPBS for 20 min. After three rinsing steps in KPBS, the sections were incubated in a blocking solution consisting of 5% normal goat serum in KPBS and 0.25% Triton X-100, to block nonspecific binding sites.

The date of data freezing of the database for this analysis was 1

The date of data freezing of the database for this analysis was 1 June 2008. We investigated the time to discontinuation of at least one drug in the first HAART regimen within 1 year for any reason, and for reasons grouped according to the categories listed in the coded form described

above: intolerance/toxicity, low compliance, OSI-744 mouse clinical and immunovirological failure, or simplification. Changes in international guidelines, therapy discontinuation following the clinician’s decision and therapy discontinuation following the patient’s decision were included in the group ‘other reasons for discontinuation’ and they were not studied in detail. Changes of drug formulation and lamivudine/emtricitabine (FTC) switch were not counted as discontinuation. Similarly, adding a new drug to a regimen without stopping one of the original ones did not count as an event. Standard survival analysis employing Kaplan–Meier estimates was used to estimate BTK inhibitor ic50 the probability of discontinuing at least one drug of the HAART regimen by a certain

time after starting therapy. Time zero for the analysis was the date of initiating HAART; the date of discontinuation was defined as the first time one of the drugs in the specific combination was terminated; the reason for discontinuing this drug was defined as the reason associated with discontinuing the prescribed treatment combination. The objective was to compare the incidence of discontinuation according to calendar period of HAART initiation, so the follow-up time of patients who did not discontinue ≥1 drug after the first year of observation Cediranib (AZD2171) was censored at 1 year after starting

HAART in order to minimize potential bias related to different lengths of follow-up time in patients starting in different calendar years. The follow-up time of patients who discontinued in the first year for reasons other than those under evaluation was censored at the time of discontinuation, under the assumption that the probability of discontinuing for one reason was totally unrelated to that of discontinuing for another. In order to evaluate whether ignoring the informative censoring mechanism could have substantially influenced the estimates of rate of discontinuation, we performed a competing-risk analysis where follow-up of patients who discontinued in the first year for reasons other than those under evaluation was censored at 1 year. In both the analyses, the follow-up time of patients who were followed up for less than 1 year was censored at the date of the last visit.