Motor skills were significantly associated with caries experience. Regarding the salivary parameters, osmolality presented a stronger association with caries experience than did the salivary flow rate. Children with worse oral motor performance presented a higher rate of caries occurrence. Osmolality exhibited a stronger association with caries occurrence than did salivary flow rate. This parameter, therefore, could be a potential caries risk indicator for spastic cerebral palsy children. “
“Active sports require sufficient energy intake. How do young athletes meet this need? The aim of this study was to investigate self-reported

health and oral behaviors of young athletes and to compare them with a selleck chemicals llc population-based sample of ordinary adolescents. A computer-based questionnaire on oral hygiene habits and dietary habits was conducted Selleckchem PLX4032 in two junior high

schools with special classes for athletes in 2011. Adolescents of similar age (n = 1230) attending ordinary classes had responded the same questionnaire earlier in the city of Oulu (in 2004) and in Kajaani, Finland (in 2006–2007). Answers to individual questions as well as sum scores of the answers were analyzed. The answers of the athletes and ordinary adolescents were analyzed by gender using cross-tabulation and chi-square testing. The mean sum score of the athletes indicated their more favorable health behavior compared with the other adolescents. They also ate more frequently the four daily than the others; in addition, they ate the school lunch as an entity which it was intended. However, the athlete boys consumed more fizzy/soft drinks and ate chocolate more often than the rest. The athletes

also brushed their teeth more frequently than ordinary adolescents. Oral health behavior of the girls was better than that of the boys. Health behavior OSBPL9 of the young athletes is better than that of other adolescents. Continuous oral health education should be targeted to all adolescents; growing boys should be target group of information on healthy sources of energy. “
“This review aims to summarise common paediatric oral and maxillofacial pathology. It will focus on lesions that have a particular predilection for children, lesions that impart significant morbidity or rare and important entities which paediatric specialists may be less familiar with. Although the vast majority of pathology encountered will be benign or require minimal intervention, there are also lesions that may require urgent referral to an appropriate specialist, multidisciplinary team care and significant surgery. Recognition and appreciation of the clinicopathological features should facilitate an appreciation that the growth, anatomy, physiology or relationship of the maxillofacial structures may have been altered by the pathological entity or treatment received. “
“International Journal of Paediatric Dentistry 2010; 20: 125–131 Objective.

Thus, the conditioning

of media with spent culture supernatants or cell-free extracts derived from helper strains has been used for the growth stimulation of species such as Catellibacterium spp., Psychrobacter spp., Sphingomonas spp. and Symbiobacterium spp. (Tanaka et al., 2004; Bae et al., 2005; Kim et al., 2008a, b; Nichols et al., 2008). Signalling molecules may be responsible for such growth promotion. Empirical testing of known signal molecules, cyclic AMP (cAMP) and acyl homoserine lactones was shown to significantly increase the cultivation efficiency of marine bacteria (Bruns et al., 2002) – the addition to liquid media of 10 μM cAMP led to cultivation efficiencies of up to 100%. This remarkable result has not, however, been corroborated by other studies investigating the effect Dabrafenib of cAMP on the growth of individual species. Coppola et al. (1976) observed a growth

inhibition of Escherichia coli in media supplemented with 5 mM cAMP, and in a study by Chen & Brown (1985), the addition of cAMP at levels ranging from 0.01 to 100 μM showed no consistent influence on the growth rates of Legionella pneumophila. A cAMP concentration-dependent effect on growth may explain the differences in the results of the various studies. It is also possible that use of the most-probable-number Epacadostat molecular weight method in the study by Bruns et al. (2002) led to an overestimation Histidine ammonia-lyase of cell numbers.

Another study (Nichols et al., 2008), in this case investigating the growth stimulation of a Psychrobacter strain, successfully characterized the growth-promoting factor responsible and identified this as a 5-amino-acid peptide. An alternative approach for the culture of as-yet-uncultivated organisms is to simulate their natural environment in vitro. Kaeberlein et al. (2002) constructed a diffusion chamber that allowed the passage of substances from the natural environment (intertidal marine sediment) across a membrane and successfully grew bacteria from marine sediment that were previously uncultivated. These bacteria were subsequently cultured on solid media, but grew only in the presence of other bacteria, implying codependency. Similar diffusion chambers have been constructed since, to culture ‘uncultivable’ or rarely cultivated bacteria from marine (Nichols et al., 2008) and freshwater environments (Bollmann et al., 2007). The latter study reported a significantly greater diversity of recovered isolates using the diffusion chamber than on conventional agar plates. Also mimicking the natural environment, sterile fresh- (Stingl et al., 2008; Wang et al., 2009) and marine- (Rappe et al., 2002; Song et al., 2009) waters have been used to culture previously uncultivated bacteria. Ben-Dov et al.

It is well-recognized that in the past, GSI-IX processing of lead–zinc and zinc–lead ores in smelters has resulted in widespread contamination of the environment and has severely affected the health of the community. Studies have reported significantly higher BPb levels12–15 and TPb levels13 in children residing near lead factories/mines compared to those of children residing away from the lead source. Thus, the present study comprised of

five villages located in the vicinity of a zinc–lead smelter in Dariba, Rajasthan, India. Paediatric lead poisoning is associated with an increased risk of adverse effects in a variety of target organs, with the central nervous, haematopoietic, and renal systems receiving the greatest attention16,17. Exposure to lead is estimated by measuring levels of lead in the blood (μg/dL). The US Center for Disease Control and Prevention (CDC) has set a ‘level of concern’ for children at 10 μg/dL. However, studies have provided evidence of the possibility of very harmful effects at even levels of exposure as low as 5 μg/dL. Hence, no level of lead exposure can be considered safe enough3,16. Blood-lead levels primarily reflect recent exposure (i.e., Galunisertib over the last 3–5 weeks) and correlate poorly with lead levels in shed primary teeth17. Shed primary teeth can be

used as indicators of long-term lead exposure during early life because much of lead deposited in teeth during mineralization is retained. The metabolism of lead is affected by the same factors

that affect calcium metabolism, SDHB with a tendency to ‘follow the calcium stream’. Mineralized tissues are thus long-term storage sites for lead2,3. Mean dentine lead levels increase with age and duration of exposure to high levels of lead17. Primary teeth provide a readily accessible bone biopsy, hence the concentrations of lead in the whole primary teeth, the enamel, or the dentin (particularly circumpulpal) have served as proxy measures for skeletal lead, and thus for total body lead burden, in epidemiologic studies of childhood lead toxicity. Also, the lead burden of children is more pronounced than that of adults and higher lead levels have been reported in primary teeth than permanent teeth18–21. Hence, in the present study, primary teeth that were either shed or nearing exfoliation were analysed for lead levels. Considering the advantages of using teeth to assess lead exposure, the relation between TPb and BPb levels deserves more attention, and several studies7,22 have already attempted to determine the same. However, in the face of a severe paucity of such data pertaining to the Indian population, it is vital that data be collected, correlated, and compared with that of different populations.

The assembled sequence was manually examined for errors. Potential genes were identified using blast and the annotation of significant hits was accepted. ORFs larger than 100 codons were identified using ORFfinder (http://www.ncbi.nlm.nih.gov/projects/gorf/) and codon usage table 4 (Mold, Protozoan, and Coelenterate Mitochondrial Code). The predicted exon–intron boundaries for three buy Palbociclib selected genes, cytochrome oxidase subunits 1 (cox1) and 2 (cox2) and the small ribosomal subunit (rns) gene were confirmed by sequencing reverse transcriptase (RT)-PCR products. Total RNA from fungal hyphae growing in 2% malt extract was obtained using TRIzol reagent (Invitrogen Corp.,

CA) and RT-PCR was performed using the Omniscript RT Kit (Qiagen Inc., CA) following the manufacturer’s recommended protocol. The primers selleck chemical used are shown in Table 1. Intronic sequences were analyzed using RNAweasel (Lang et al., 2007). The mitochondrial genomes and annotation of P. ostreatus, M. perniciosa, S. commune, C. neoformans and U. maydis are available at GenBank (http://www.ncbi.nlm.nih.gov/sites/entrez?db=nucleotide) under accession numbers EF204913, AY376688, AF402141, AY101381 and DQ157700, respectively. The accession number for the T. cingulata mitochondrial genome is GU723273. The T. cingulata mitochondrial genome was assembled into a

single 91 500 bp circular molecule with a coverage depth of about 140-fold. blast comparison with other fungal mitochondrial genomes identified genes encoding 15 proteins and the small and large rRNAs (Fig. 1). tRNAscan-SE (Lowe & Eddy, 1997) identified 25 tRNAs in the genome corresponding to all 20 amino acids. We also found five ORFs not overlapping any other gene on either strand and larger than 100 codons (Fig. 1). However, these ORFs showed little similarity to sequences found in the mitochondrial genomes of P. ostreatus, M. perniciosa, S. commune, C. neoformans and U. maydis (Fig. 1, rings v–ix). Additionally, tblastx and blastn comparison of

these five ORFs with Calpain the nonredundant database did not identify any sequence with an expected value of <0.1, further indicating that they may not be authentic. GC skew analysis has been used to identify the origin of replication in bacterial genomes (Grigoriev, 1998) and a similar technique has been proposed in fungal mitochondrial genomes (Formighieri et al., 2008). We were unable to detect any obvious origin of replication based on the GC content or GC skew analysis. Like the mitochondrial genes of the other Agaricomycotina, most of the T. cingulata mitochondrial genes are located on one strand. The only identifiable gene on the anticlockwise strand is the one encoding tRNATrp, which is found nowhere else in this genome. While gene order is not conserved among the mitochondrial genomes of T. cingulata, P. ostreatus, M. perniciosa, S. commune, C. neoformans and U. maydis, they share a similar set of genes (Fig. 2).

The model predicts that the apparently fast circuit of the cerebellar cortex may control the timing of slow processes without having

to rely on sensory feedback. Thus, the cerebellar cortex may contain an adaptive temporal integrator, with the sensitivity of integration to the baseline spike rate offering a potential mechanism of plasticity of the response time-constant. “
“Area V3A was identified in five human subjects on both a functional and retinotopic basis using functional magnetic resonance imaging techniques. V3A, along with other visual areas responsive to motion, was then targeted for disruption by repetitive transcranial magnetic stimulation (rTMS) whilst the participants performed a delayed speed matching task. The stimuli used for this task included chromatic, isoluminant motion stimuli that activated either the L−M or S−(L+M) Epigenetics inhibitor cone-opponent mechanisms, in addition to moving stimuli that contained only luminance contrast (L+M). The speed matching task was performed for chromatic and luminance stimuli that moved at slow (2°/s) or faster (8°/s) speeds. The application of rTMS to area V3A produced a perceived slowing of all chromatic and luminance stimuli at both slow and fast speeds. Similar deficits

were found when selleck screening library rTMS was applied to V5/MT+. No deficits in performance were found when areas V3B and V3d were targeted by rTMS. These results provide evidence of a causal link between neural activity in human area V3A and the perception of chromatic isoluminant motion. They establish area V3A, alongside V5/MT+, as a key area in a cortical network that underpins the analysis of not only luminance but also chromatically-defined motion. “
“Nerve axons and the apical Palmatine epidermal cap (AEC) are both essential for the formation of an accumulation blastema by amputated limbs of urodele salamanders. The AEC forms in the absence of axons, but is not maintained, and blastema formation fails. Growth stages of the blastema become

nerve-independent for morphogenesis, but remain dependent on the nerve for blastema growth. Denervated growth stage blastemas form smaller than normal skeletal parts, owing to diminished mitosis, but form the full proximodistal array of skeletal elements. This difference in nerve dependency of morphogenesis and proliferation is hypothesized to be the result of a dependence of the AEC on nerves for blastema cell proliferation but not for blastema morphogenesis. Regenerating axons induce the synthesis and secretion of the anterior gradient protein (AGP) by distal Schwann cells during dedifferentiation and by the gland cells of the AEC during blastema growth stages. AGP promotes the regeneration of a denervated limb to digit stages when electroporated into the limb during dedifferentiation.

4%) had a significant (fourfold or greater) increase in titre after vaccination (Table 2). In contrast, only eight patients (6.3%) had an antibody titre ≤ 1:10. These differences were found to be statistically significant (χ2 = 61.09; P < 0.0001). No correlation was found between age www.selleckchem.com/hydroxysteroid-dehydrogenase-hsd.html and pre-vaccination HI titre. In the χ2 analysis, a nonsignificant trend for a higher proportion of patients with HI titres ≥ 1:40 (P = 0.083) in the > 60 years old group was found; 13 of 19 patients (68.4%) in the > 60 years old group had HI titres

≥ 1:40 compared with 88 of 199 patients (48.9%) in the ≤ 60 years old group. The SPR, SCR and mean increase in GMT in the ≤ 60 years old group were 91.2%, 68.1% and 2.43 ± 0.51, respectively. In the > 60 years old group, the SPR, SCR and mean increase in GMT were 92.3%, 61.5% and 2.32 ± 0.52, respectively. In the univariate analysis, ART status, VL and baseline H1N1 antibody titres were found to be significantly associated with H1N1 antibody titres of ≥ 1:40. Nine of 16 patients (56%) not on treatment did not achieve antibody titres required for seroprotectivity. In contrast, 79 of 110 patients

(72%) on treatment achieved HI antibody levels ≥ 1:40. Lower click here HIV VL and higher baseline HI H1N1 antibody titres were also associated with higher post-vaccination HI titres. Further analysis found that only 12 of 26 patients (46%) with detectable VL, but 74 of 100 patients (74%) with undetectable VL (< 50 copies/mL), achieved antibody titres ≥ 1:40 (χ2 = 7.384; P = 0.007). VL and baseline HI H1N1 antibody titre were found to be the predictors of

a response to vaccination (≥ 1:40) in the BLR model (χ2 = 15.71; d.f. = 2; P < 0.0001) (Table 3). The model correctly predicted 71.4% of cases. The Hosmer–Lemeshow goodness of Amoxicillin fit statistic showed that the model was good (P > 0.05). During the Southern Hemisphere winter of 2009 there was considerable concern about the impact of the H1N1 influenza virus as it started spreading within the general population, particularly in those considered at increased risk for complications. Clinics dealing with high-risk patients were disseminated free vaccines via the State’s Public Health Units for mass immunization. HIV-infected patients were considered to be at greater risk of complications from H1N1 infection compared with the general population, although subsequent audits from a number of large HIV centres have suggested that the opposite was actually true. Patients with HIV-1 infection have been shown to have weaker responses to seasonal influenza vaccination, with lower antibody response rates being associated with lower CD4 T-cell count, not being on ART and previous AIDS, with an impaired response being anticipated to H1N1 09 vaccination in this population [8].

For assessing growth characteristics, overnight cultures were inoculated GW-572016 mw into fresh media at an OD595 nm of 0.02. Cultures were divided into twelve 1-ml aliquots and incubated at 27 °C with shaking for 24 h. Samples were taken for 24 h at indicated intervals, and OD595 nm of 100 μL of the cell suspension was measured in a microtiter plate. Growth rates were measured using

the slopes of the trend lines fitted to data at exponential phase as described before using the formula: rate constant (k) = slope/0.301 (Slonczewski & Foster, 2011). Biofilm assays were performed as described in the study of (Karatan et al., 2005). To determine vpsL promoter activity, 200 μL of stationary or one ml of exponential phase cultures grown at 27 °C were pelleted, washed once with Z buffer (Miller, 1992), and resuspended in 200 μL

of Z-buffer. Protease inhibitors and Ortho-nitrophenyl-β-D-galactopyranoside were added to each lysate and incubated Panobinostat research buy at 37 °C for 2 h. β-galactosidase activity was determined by measuring the A415 nm. To assess motility, three isolated colonies were stabbed on semi-soft LB-agar plates (0.3% agar). The swarm diameters were measured after incubation at 27 °C for 24 h. All assays were repeated multiple times to confirm reproducibility of the results. Extraction of polyamines from shaking cultures were performed as previously described (McGinnis et al., 2009). To extract cellular polyamines from biofilm cultures, biofilms were formed in glass bottles in 20 mL LB for 24 h, and planktonic cells were removed and pelleted. Biofilms were washed once with PBS, biofilm-associated cells were dispersed in 10 mL PBS by vortexing with 1-mm glass beads (Bartlesville,

OK), and the cells were pelleted by centrifugation. Planktonic and biofilm-associated cells were resuspended in 10 μL of water per mg wet weight. Cells were lysed by sonication, cell debris was removed by centrifugation, and cellular protein was precipitated by the addition of trichloroacetic acid. The supernatant containing isothipendyl the polyamines were used for benzoylation. In addition, 500 μL of the conditioned media was set aside for benzoylation for all culture conditions. A standard mix containing 0.1 mM each of putrescine, diaminopropane, cadaverine, norspermidine, and spermidine was also prepared every time polyamines were quantified. Benzoylation procedure was performed as described previously (Morgan, 1998). Benzoylated polyamines were extracted with chloroform, evaporated to dryness, and dissolved in 100 μL of a 60% methanol and 40% water solution. HPLC was conducted using a Waters 1525 Binary Pump with a 2487 Dual Wavelength Absorbance Detector and a Waters Spherisorb ODS2 column (5 μm, 250 × 4.6 mm), fitted with a 50 × 4.6 mm guard cartridge (Waters Corporation, Milford, MA).

Investigators, study coordinators,

and subjects were blinded to treatment assignment. After initiating the study drug, subjects were see more asked to maintain a daily diary to record details regarding medication compliance, geographic location, and number of loose stools, symptoms, and daily eating habits. Subjects were asked to grade their symptoms (Appendix, Table A1). The study coordinator contacted the patient within 7 days of their return from the trip to monitor for toxicity, study outcomes, and reminded subjects to submit a fresh stool sample within 5–7 days of the last study dose. Adverse event (AE) monitoring was done via the daily diary and the final phone interview. An AE was defined as any untoward medical occurrence in a study subject exposed to AKSB or placebo. An AE could be any unfavorable and unintended effect (including an abnormal laboratory finding), symptom,

or disease temporally associated with the use of AKSB or placebo. Serious adverse events (SAEs) were defined as those that were life-threatening, resulted in hospitalizations of >24-hour duration, or were disabling or resulted in death. All AEs were assessed whether they were possibly, probably, or definitely related to the study drug or not related at all. All SAEs were to be reported to the IRB within 24 hours and all other AEs were summarized in annual reports to the IRB. Unused capsules http://www.selleckchem.com/products/gsk1120212-jtp-74057.html from subjects on AKSB were returned to Agri-King, Inc. for probiotic viability studies. Subjects

received a $50 honorarium for the inconvenience of participating in the study. All subjects were asked to submit a fresh stool specimen in a Para-Pak culture Parvulin and sensitivity vial within 5–7 days of returning home from their trip. The specimens were submitted for culture of enteric pathogens (Campylobacter species, Salmonella, Shigella, Aeromonas, and Yersinia), enterotoxigenic E coli toxin assay, and ova and parasite examination at the Mayo Clinic Microbiology Laboratory. The fecal specimen was inoculated onto selective media designed to inhibit growth of normal bowel flora while allowing growth of the enteric pathogens. The following media were used: sheep blood agar, Hektoen enteric agar, eosin-methylene blue agar, Campylobacter agar, cefsulodin-irgasan-novobiocin agar, and the enrichment broth, selenite F. Suspect colonies were identified using conventional biochemical and serologic methods. These tests were performed per standards set by the Clinical and Laboratory Standards Institute. Returned capsules were analyzed for AKSB organisms’ post-travel viability (Analab Laboratories, Fulton, IL, USA). The primary endpoint was the development of diarrhea. Assuming that the frequency of TD is 25% in those receiving placebo, 348 volunteers (174 placebo and 174 AKSB) were required to have an 85% power to detect a 50% reduction in the frequency of TD for the AKSB group (based on a comparison of 25% vs 12.5%, using a two-sided, α = 0.05 level test).

coli TOP10. The resulting pCR4-16S rDNA vectors were analyzed by DNA sequencing (GATC Biotech, Germany) and the blast program was used to compare the sequences with the data in GenBank. All the colonies formed on 2YT or YPD agar were scraped from the plates into 5 mL 2YT (2YT plates) or YPD medium (YPD plates)

and centrifuged (3381 g for 15 min). DNA was extracted according to Matson et al. (2007), with some modifications. The pellet of bacteria was suspended in 1 × TE buffer (1 mM Tris-HCL, 0.1 mM EDTA pH 8.0, Sigma-Aldrich) containing 1% w/v polyvinylpolypyrrolidone (PVPP), 2% sodium dodecyl sulfate, 30% phenol, and 500 mg Zirconia/Silica Beads Selleckchem Dabrafenib (Stratech Scientif Unit). The cells were broken by three cycles of homogenization at 376 g for 30 s (Mikro-Dismembrator U, Sartorius) and chilling on ice for 30 s. The lysate was purified with the Blood and Tissue Kit (Qiagen) according to the manufacturer’s instructions (for the method described for crude lysate purification). Genomic DNA was partially digested with Sau3AI check details and fragments ranging

in size from 5 to 10 kb were purified from the agarose gel with the QIAEX II Gel Extraction Kit (Qiagen). The digested DNA fragments were ligated into the BamHI-linearized vector YEp356 (Myers et al., 1986) and the ligation products introduced into E. coli Max Efficiency DH5α competent cells (Invitrogen). Each colony was collected into a well of a 96-well plate containing 2YT

medium/50 μg mL−1 ampicillin and incubated at 37 °C for 16 h before replication and long-term storage at −80 °C. The quality of the library was determined on 96 randomly selected clones. The plasmids were isolated and then digested with EcoRI and HindIII. The digestion products were analyzed by agarose gel electrophoresis. Olopatadine The colonies in 96-well plates were replicated on the following media for enzyme detection. α-Amylase activity was detected after incubation for 24 h at 37 °C on 2YT agar containing 0.5% soluble starch. The medium was then covered with Gram’s iodine solution (Sigma-Aldrich), and α-amylase-producing colonies were identified by the absence of dark blue stain (due to the starch–iodine complex) in the zone surrounding them. Xylanase and endoglucanase were detected on 2YT agar containing an appropriate chromogenic substrate (respectively, AZCL-xylan or AZCL-HE-cellulose, Megazyme). Colonies producing these activities were identified by the blue color produced. β-Glucosidase was detected on 2YT agar containing 0.5% esculin (Sigma-Aldrich) and 0.1% ammonium iron (III) citrate (Sigma-Aldrich) after incubation at 37 °C for 24 h. Bacteria with β-glucosidase activity hydrolyze the substrate esculin to glucose and esculetin, which combines with ferric ions to yield a dark-brown color in the agar around the bacteria. The plasmid of the positive clone (named P11-6B) was isolated and analyzed by DNA sequencing (GATC Biotech).

coli. However, hydrophobicity selleck products profile analysis revealed that the N-terminus of the A domain has a putative transmembrane segment. The N-terminus of the A domain might act as an integral membrane anchor, indispensable for FtsY membrane association (Bibi et al., 2001). When this putative transmembrane segment was fused to the E. coli NG domain, the chimera construct was capable

of rescuing wild-type EcFtsY depletion in a conditional FtsY-deletion mutant of E. coli. In contrast, the E. coli NG domain alone could not fully rescue wild-type FtsY depletion (Maeda et al., 2008). These results suggest that the N-terminus of S. coelicolor FtsY (ScFtsY) has a functional role. The ScFtsY N-terminus may contribute to the membrane targeting of FtsY, but there is no direct evidence. In this study, the membrane-targeting ability of the N-terminal hydrophobic segment of the ScFtsY A domain was assessed by membrane protein extraction and Mal-PEG

labeling experiments. Results show that this part of the ScFtsY A domain might form a membrane insertion structure that can anchor ScFtsY to the membrane. The S. coelicolor strains used in this study are listed in Table 1. The E. coli strain ET12567 (MacNeil et al., 1992), which contains the plasmid pUZ8002, was used for plasmid introduction by conjugation into S. coelicolor M145 (Kieser et al., 2000). selleck kinase inhibitor All S. coelicolor strains were grown at 30 °C, 220 r.p.m. min−1 in TSB liquid media for protein expression. Apramycin (50 μg mL−1) was added when necessary. All the plasmids used in this study are listed in Table 2. All primers are listed in Supporting Information, Appendix S1, and the detailed protocol for plasmid construction and protein expression is provided in Appendix S2. Subcellular fractions were isolated as described in the study by de Leeuw et al. (1997). Cells were suspended in lysis buffer (Mao et al., 2009) and lysed by freezing and short ultrasonic treatment. The cellular debris was removed from

the lysate by sedimentation (12 000 g for 15 min); the supernatant was then subjected to ultracentrifugation (356 000 g for 45 min), and the membrane pellet fraction [precipitant (‘P’)] was separated from the soluble fraction aminophylline [supernatant (‘S’)]. The supernatant was precipitated with 1 vol 10% TCA and resuspended in SDS-loading buffer, whereas the pellet fraction was directly dissolved in the same amount of SDS-loading buffer. The same amount of ‘P’ and ‘S’ samples was loaded onto an SDS-PAGE gel. The EGFP mutants in the samples were detected using the EGFP antibody. The protein content in ‘P’ and ‘S’ was calculated using the Quantity One software (Bio-Rad™). For carbonate extraction, the membrane pellet fraction, ‘P’, was incubated with 0.2 M Na2CO3 for 30 min at 4 °C and subsequently ultracentrifuged for 45 min at 356 000 g; the precipitant was the membrane pellet fraction (‘P′’), and the supernatant was the soluble fraction (‘S′’).