A few high-performance liquid chromatography (HPLC)[2�C7] and liq

A few high-performance liquid chromatography (HPLC)[2�C7] and liquid chromatography�Cmass spectrometry (LC-MS)[8,9] methods for its determination have been reported. A simultaneous determination of mycophenolic acid and valproic acid in human plasma by HPLC[10] has also been reported. Olaparib molecular weight Figure 1 Structure of mycophenolate mofetil No high-performance thin layer chromatography (HPTLC) method for the quantitative analysis of mycophenolate mofetil as bulk and as formulation or for stability studies was found by a computer-assisted survey of the literature either from chemical abstracts or by using the CAMAG Bibliography Service. Hence, the main objective of the work discussed in this paper was to develop a rapid and reliable HPTLC method for the analysis of mycophenolate mofetil in bulk and its dosage form.

The method was validated in accordance with the guidelines of the International Conference on Harmonization (ICH) International Conference on Harmonization and remove ��o��.[11] Here we describe the investigation in detail. The usage of HPTLC is well appreciated and accepted all over the world. Many methods are being established to standardize the assay methods. HPTLC remains one step ahead when compared with other tools of chromatography. HPTLC is used for the identification of constituents, determination of impurities, and quantitative determination of active substances.

The use of modern apparatus such as video scanners, densitometers, and new chromatographic chambers, more effective elution techniques and high-resolution sorbents with selected particle size or chemically modified surface, the possibility of combining with other instrumental methods, and the development of computer programs for method optimization make HPTLC an important alternative method to HPLC or gas chromatography. Specifically, HPTLC is one of the ideal TLC techniques for analytical purposes because of its increased accuracy, reproducibility, and ability to document the results, compared with standard TLC. Because of this, HPTLC technologies are also the most appropriate TLC techniques for conformity with Good Manufacturing Practices (GMPs). Today, the comprehensive use of TLC in pharmaceutical analysis is demonstrated by the great number of articles published in this field. So the ultimate aim of the present study is to develop and validate the HPTLC method for the determination of mycophenolate mofetil in bulk drug and dosage form.

The optimization of the method separation, validation parameters, and quantification of mycophenolate mofetil as bulk and as formulation are reported in the following sections. MATERIALS AND METHODS Chemicals Carfilzomib and reagents The authentic sample of mycophenolate mofetil was procured from Intas Pharmaceuticals Ltd., Ahmedabad. The pure drug obtained had 99.9% w/w assay value, and was used without further purification.

Detector Further, to develop a suitable and robust LC method

Detector … Further, to develop a suitable and robust LC method for the determination of tobramycin by UV detection, different mobile phases and columns were employed [Table 1] to achieve the best signal response and retention time. Table 1 Employed mobile phases, columns and elution time during the investigation of tobramycin Ganetespib mw Finally, the mobile phase consisting of water: 0.05 M diammonium hydrogen phosphate, pH adjusted to 10.0 �� 0.05 using tetra methyl ammonium hydroxide (25% solution in water) at a constant flow rate of 1.0 ml/min and detector wavelength set at 210 nm, using a Purosphere RP-8e, 250 mm �� 4.6 mm, 5��m column, was found to be appropriate, allowing well signal response of tobramycin. Optimization of HPLC The pH of the mobile phase can affect the analyte retention time as well as the detection sensitivity.

Figure 2 shows the result of detection response (peak area), efficiency (shown as plate number N/column) and capacity factor of tobramycin at different pHs. The optimal pH 10.0 �� 0.05 was chosen for the determination of tobramycin. Figure 2 Effect of pH on (peak area) and effi ciency (shown as plate number N/column) and capacity factor of tobramycin column: Purosphere RP-8e, 250 �� 4.6 mm, 5��m, mobile phase: 0.05 M diammonium hydrogen phosphate, pH adjusted to 10 using tetramethyl … Concentration of the buffer is another factor that can alter the ion-pair formation. Figure 3 shows the capacity factor and detection response (peak area) as the concentration of the buffer varied. Response was minimal when less than 0.05 M diammonium hydrogen phosphate was used.

This may be due to highly aqueous environment that is unfavorable for ion pairing. Therefore, pH 10.0 and 0.05 M diammonium hydrogen phosphate was chosen for estimation of tobramycin. Typical chromatogram of the test solution is shown in Figure 3. Figure 3 A typical chromatogram of test sample by proposed methods column: Purosphere RP-8e, 250 �� 4.6 mm, 5��m, mobile phase: 0.05 M diammonium hydrogen phosphate, pH adjusted to 10.0 using tetramethyl ammonium hydroxide, flow rate 1 ml/min, �� … Method validation The test method for the determination of tobramycin was validated to include the essential demands of International Conference on Harmonization (ICH) guidelines.[21] Parameters like specificity, linearity, accuracy, precision, range, robustness, and system suitability were examined.

Specificity No interferences were observed due to the obvious presence of excipients and mobile phase. Linearity Peak areas versus concentration in milligram per milliliter were plotted for tobramycin at the concentration range between 80% and 120% of the target level. Tobramycin showed linearity GSK-3 between 0.47 and 0.71 mg/ml with a correlation coefficient (r2) of 0.9998. Accuracy Accuracy of the proposed HPLC determination was evaluated from the assay results of the components.

But a myoma screw could not be used in cases

But a myoma screw could not be used in cases selleck chemical Ganetespib of endometrial cancer or in uterus with pyometra, for risk of spillage of tumor cells or pus into the peritoneal cavity. To obviate the disadvantages of a uterine manipulator, a myoma screw, or a grasping forceps, we adapted a technique which is simple and easy. We have used laparoscopic uterine hitch technique for more than 6 months in 23 such patients. The main advantage of this technique was that the stitch used for manipulation does not go through the tumor tissue. Moreover the operating surgeon could directly control and adjust the retraction to his/her satisfaction (unlike the vaginal manipulation, in which the operating surgeon had to instruct the assistant off and on). This reduced the operative time and stress.

Ample space for dissection in the posterior compartment of the pelvis was created when the uterus was hitched to the anterior abdominal wall. This hitch resulted in adequate anteversion and anterior displacement. Cranial traction on the sutures facilitated dissection anterior to the uterus. In this position, there was also enough scope for lateral countertraction and dissection on the lateral sides. The left-upper pararectal port that was originally utilized for the myoma screw could now be used for assistance in retraction of the rectum and in suctioning. In some patients we could even avoid the above port. The problems anticipated were tearing of the uterus, inadvertent bleeding from the uterus, breaking of the suture, accidental perforation of the bladder, or abdominal wall vessel.

But apart from some difficulty in passing the suture through the abdominal wall in an obese patient and tearing of the round ligament in one patient, we did not face any other problem due to the uterine hitch. Similar techniques have been used previously for laparoscopy in benign conditions. Tsin and Colombero described the laparoscopic leash technique to prevent specimen loss during laparoscopy [6]. Giesler further extended this concept with a puppet string variation to facilitate traction and manipulation in large broad ligament fibroids [7]. The uterine hitch technique is an excellent alternative to routine manipulation techniques when operating laparoscopically on cancers in the pelvis. 5. Conclusion The hitching of the uterus is an innovative method for manipulating the uterus in several laparoscopic pelvic oncosurgical procedures. It is a simple, effective, easy, and economical technique Brefeldin_A which facilitates dissection posterior to the uterus and in cases where routine uterine manipulators are relatively contraindicated (endometrial carcinoma, pyometra).

Genome properties The genome consist of a 2,147,060 bp long chrom

Genome properties The genome consist of a 2,147,060 bp long chromosome and four large circular plasmids of 315,518 bp, 195,800 bp, 132,270 bp, and 97,188 bp length, and a G+C content reference 2 of 65.6% (Table 3 and Figure 3). Of the 2,799 genes predicted, 2,741 were protein-coding genes, and 58 RNAs; 85 pseudogenes were also identified. The majority of the protein-coding genes (65.0%) were assigned a putative function while the remaining ones were annotated as hypothetical proteins. The distribution of genes into COGs functional categories is presented in Table 4. Table 3 Genome Statistics Figure 3 Graphical circular map of the chromosome (plasmids not shown, but accessible through the img/er pages on the JGI web pages [48]); From outside to the center: Genes on forward strand (color by COG categories), Genes on reverse strand (color by COG categories), .

.. Table 4 Number of genes associated with the general COG functional categories Acknowledgements We would like to gratefully acknowledge the help of Katja Steenblock (DSMZ) for growing D. proteolyticus cultures. This work was performed under the auspices of the US Department of Energy Office of Science, Biological and Environmental Research Program, and by the University of California, Lawrence Berkeley National Laboratory under contract No. DE-AC02-05CH11231, Lawrence Livermore National Laboratory under Contract No. DE-AC52-07NA27344, and Los Alamos National Laboratory under contract No. DE-AC02-06NA25396, UT-Battelle and Oak Ridge National Laboratory under contract DE-AC05-00OR22725, as well as German Research Foundation (DFG) INST 599/1-2.

The phylogenetic relationship of the 16S rRNA gene of C. clariflavum DSM 19732 with other cellulolytic clostridia from Cluster III is shown in Figure 1. The sequences shown in here represent mostly cellulolytic and xylanotlytic clostridia sharing over 84.5% sequence identity. The branch comprised by C. clariflavum, C. straminisolvens and C. thermocellum is of particular interest since it includes cellulolytic organisms sharing at least 96.6% sequence homology able to grow at thermophilic temperatures. A few environmental samples have provided sequences with close homology (>99.0% sequence similarity) to the C.

clariflavum 16S rRNA gene, and have been found in thermophilic methanogenic bioreactors [7], enrichment cultures from bioreactors (Accession number “type”:”entrez-nucleotide”,”attrs”:”text”:”AB231801″,”term_id”:”146197920″,”term_text”:”AB231801″AB231801 GSK-3 and “type”:”entrez-nucleotide”,”attrs”:”text”:”AM408567″,”term_id”:”116806963″,”term_text”:”AM408567″AM408567), and enrichments from thermophilic compost [3]. Two pure cultures have been isolated from compost enrichments with >99.7% sequence similarity to C. clariflavum and able to utilize xylan [4]. However, no evidence of this organism has been reported in metagenomic studies from similar environments.

However, some experiments in T thermophilus HB8 suggest NarK1 mi

However, some experiments in T. thermophilus HB8 suggest NarK1 might also function in nitrite extrusion [39]. T. oshimai JL-2 and T. scotoductus SA-01 also contain homologs of NarK2 (annotated as nep in T. scotoductus SA-01 [41]), which likely encodes a nitrate/nitrite antiporter [44,48]. No significant BLASTP selleck chemical U0126 hits for periplasmic nitrate reductase subunits NapB and NapC were found in T. oshimai JL-2 and T. thermophilus JL-18, consistent with the use of the Nar system in the Thermales. Figure 6 Map showing the organization of nar operon and neighboring genes involved in denitrification located on the megaplasmids of T. oshimai JL-2 (pTHEOS01) and T. thermophilusJL-18 (pTTJL1801) and the chromosome of T. scotoductus SA-01. Fe: heme protein-containing …

All three strains contain a dnrST operon adjacent to, but divergently transcribed from, the narGHJIK operon. dnrST encodes transcriptional activators responsible for upregulation of the nitrate respiration pathway in the absence of O2 and the presence of nitrogen oxides or oxyanions [42] (Figure 6). Both the species contain a putative nirK, which encodes the NO-forming, Cu-containing nitrite reductase. In addition, T. oshimai JL-2 and T. scotoductus SA-01 both harbor nirS [41], which encodes the isofunctional tetraheme cytochrome cd1-containing nitrite reductase. Previous studies have suggested that bacteria use either NirK or NirS, but not both, for the reduction of nitrite [49]. The unique presence of NirK and NirS in T. oshimai JL-2 and T. scotoductus SA-01 likely enhances their denitrification abilities since isoenzymes are typically kinetically distinct and/or regulated differently.

This idea is consistent with the distinct denitrification phenotypes of T. oshimai strains as compared to T. thermophilus strains reported previously, including strains T. oshimai JL-2 and T. thermophilus JL-18 [6]. In those studies, nitrite accumulated in the medium at concentrations of <150 ��M in T. thermophilus strains, whereas it was rapidly produced to concentrations >200 ��M but consumed rapidly to below method detection limits in T. oshimai strains. NirK functions as a homo-trimer [50] and contains type 1 (blue) and type 2 (non-blue) copper-binding residues [49]. Comparison of the NirK from T. oshimai JL-2 and T.

scotoductus SA-01 with previously studied NirK amino acid sequences revealed that six of the seven copper-binding residues are conserved, except for a single methionine (M) to glutamine (Q) substitution in both Thermus proteins (Figure Batimastat 7; indicated by an asterisk (*)). Glutamine, not methionine, is the copper-binding ligand in the case of stellacyanin, a blue (type 1) copper-containing protein [52,53]. A M121Q recombinant protein of Alcaligenes denitrificans azurin showed similar electron paramagnetic resonance (EPR), but exhibited a 100-fold lower redox activity when compared to wild-type azurin [54].

Clerical error, particularly with respect to patients being liste

Clerical error, particularly with respect to patients being listed on afternoon operating lists, resulted in a number of patients suitable for day-case surgery requiring an overnight stay. This issue has been previously selleck chem Ganetespib identified in randomised trials of laparoscopic day-case cholecystectomy versus overnight stay [9]. Following the interim audit in 2008, patients suitable for day-case were predominantly scheduled on a morning list or first on the afternoon list, which resulted in a substantial increase in day-case rates from 30 to over 60 per cent. Increasing the duration of daycase unit opening hours and ensuring patients are discharged according to criteria that do not include set time periods, may enhance this further.

Whilst patient satisfaction and anxiety was not formally assessed in the present study, there is no clear evidence from randomised trials of an increase in anxiety following day-case surgery [5]. Indeed one study found an increased anxiety in those patients randomised to overnight stay [4]. Likewise initial concerns regarding the detection and management of complications in patients discharged on the day of surgery, particularly postoperative bleeding or bile duct injury, have also been unfounded [11]. Major bleeding is uncommon and bile duct injury is predominantly detected at the time of surgery or several days later. The introduction of a telephone follow-up service is therefore proposed at our institution in order to examine patient satisfaction, anxiety, and complication rates as part of a future study.

Readmission rates following day-case cholecystectomy remained relatively unchanged during the study period at around 5 to 7 per cent. This appears higher than the 2 to 3 per cent rate reported in other series [5, 9, 12], however since individual patient data relating to these readmissions was not formally analysed, the reasons for this disparity remain unclear. The overall conversion rates in this study of 6.1 and 14.5 per cent following elective and emergency laparoscopic cholecystectomy, respectively, were comparable to those reported nationally [13, 14]. However since 2008 these rates have fallen further to 3.1 and 10.5 per cent, respectively. This is likely to have arisen as a consequence of more cholecystectomies being performed by the five specialist upper gastrointestinal surgeons.

Whilst cholecystectomy during index admission with cholecystitis is associated with no significant difference in complication rate or conversion rate [15], it is known to reduce costs, in part due to minimising patient readmission whilst awaiting an elective procedure Brefeldin_A [1]. Indeed the estimated cost of a patient admitted with acute cholecystitis and treated conservatively is ��1,875. Despite this, less than 15 per cent of cholecystectomies were performed during an emergency admission in the present study, which is comparable to that reported nationally [14, 16].

We found that flagellin-induced NF��B activation was dramatically

We found that flagellin-induced NF��B activation was dramatically inhibited in TLR5-KD cells, whereas it was markedly induced in WT cells (Fig. 4B). These results indicate STA-9090 that the activated signaling by the flagellin preparation is specifically mediated by TLR5. Third, using a NF��B reporter activity assay, we showed that NCM460 cells are potently responsive to TLR5 ligand (flagellin), but nonresponsive to TLR2 (Pam3Cys) or TLR4 (LPS) stimulation (Fig. 4C). Taken together, our data obtained from these three experiments clearly indicate that flagellin-induced responses are specifically mediated by TLR5, but not compounded by TLR4. Therefore, the remaining signaling in flagellin-treated MyD88-KO cells is not attributed to a potential contaminant such as LPS. FIGURE 4.

Activated intracellular signaling by the flagellin preparation is not affected by the potential LPS contaminant, but rather is specifically mediated by TLR5. A, primary mouse intestinal epithelial cells from C3H/HeJ (TLR4-null) and its control C3H/HeOuJ … TRIF Deficiency Inhibits Flagellin-induced Inflammatory Cytokine Expression Having found that TRIF deficiency negatively regulates some of the TLR5-dependent intracellular signaling, we tested whether inflammatory cytokine expression induced by flagellin/TLR5 engagement is altered in the absence of TRIF expression. For this purpose, we stimulated primary intestinal epithelial cells from TRIF-KO, MyD88-KO, and WT mice with flagellin, followed by measuring TLR5-induced cytokine production.

As expected, the levels of KC (mouse ortholog of human IL-8), macrophage inflammatory protein 3��, and IL-6 expression were inhibited in MyD88-KO cells compared with the levels in WT cells (Fig. 5, A�CC). Notably, the cytokine expression was also dramatically suppressed in TRIF-KO cells compared with the expression level in WT cells. These results are in agreement with our findings demonstrating that NF��B and MAPK activation by flagellin is reduced in TRIF-KD and TRIF-KO cells (Figs. 2B and and33F). FIGURE 5. TRIF deficiency inhibits flagellin-induced cytokine expression. A�CC, primary mouse intestinal epithelial cells from WT, MyD88-KO, and TRIF-KO mice were stimulated with flagellin (100 ng/ml, 8 h). KC (A), macrophage inflammatory protein 3�� …

When the primary intestinal epithelial cells are stimulated with IL-1�� mediating its responses via IL-1R in a MyD88-dependent manner, IL-1�� stimulation failed to induce KC cytokine expression in MyD88-KO cells, whereas it strongly induced the cytokine expression in both TRIF-KO cells and WT cells (Fig. 5D). Moreover, TNF��, utilizing non�CTLR-associated signaling pathways, potently elicited the cytokine expression in the primary intestinal epithelial GSK-3 cells from MyD88-KO, TRIF-KO, and WT mice (Fig. 5E).

The vlogs are a good

The vlogs are a good Lenalidomide TNF-alpha inhibitor example of what appear to be authentic user-generated promotional videos. Even without tobacco industry involvement, all the vlogs in this sample mention (and often heavily promote) a specific ST product, essentially providing free marketing to various ST brands. Analysis of the vlogs reveals that there is a community of users who create what they commonly refer to as ��dip videos�� on YouTube in which they review and promote various products and comment on each other��s dip videos. These vlogs are particularly interesting because unlike most other ST videos, they create a real sense of community and interpersonal interaction between people who make and/or regularly watch these videos.

In this respect, YouTube��s influence on people��s attitudes and behaviors may be unique compared with other traditional media because it integrates elements of interpersonal interaction with a medium that allows a broad audience to have constant easy access to information. This hybrid interpersonal media platform and other social media websites could potentially be a highly influential source of ��information.�� These social networks transcend space and time, making it easy for this dipping community to spread their protobacco message to a vast audience. Further research should track these pro-ST vlogs over time and assess who is watching them and how they affect viewers�� attitudes and behaviors regarding ST. Since traditional regulations and restrictions on tobacco marketing are likely to have a limited effect on protobacco messages on YouTube and other user-generated Internet websites, it is important to consider other means of tobacco control.

Although YouTube bans videos that violate their ��community guidelines�� (such as underage smoking and copywritten materials; YouTube, 2010b), these policies are subjectively enforced and rely on users voluntarily flagging videos (YouTube, 2010b; Zeller, 2006). In addition, these guidelines do not apply to most of these ST videos because tobacco use in general (when not underage) is not banned from YouTube. When videos do not meet the standards for banning, YouTube can put age restrictions on videos that do not ��violate our community guidelines but may not be appropriate for everyone�� (YouTube, 2010b). To view these restricted videos, users must be signed into a YouTube account and be registered as 18 years or older.

Protobacco messaging could easily fall into this ��age restriction�� category, yet none of the protobacco videos in this sample, including tobacco advertisements, were restricted to 18+ users. Interestingly, the one video in the sample that was restricted to 18+ was the most highly viewed anti-ST PSA and was restricted because it contained graphic images of facial deformities Anacetrapib from ST use (Narconon PSA, 2008).

Thus, these drugs show promise in the treatment of liver cancer 1

Thus, these drugs show promise in the treatment of liver cancer.15 Wang et al used tanshinone IIA and tanshinone IIA nanoparticles selleck in the treatment of tumor-bearing mice with transplanted liver cancer H22 cells. Compared with the control group, tanshinone IIA and tanshinone IIA nanoparticles significantly inhibited tumor growth, and at the same level of drug concentration, tanshinone IIA nanoparticles inhibited tumor growth significantly better than tanshinone IIA, as demonstrated by significantly increased tumor necrosis and apoptosis, and lower cyclin E expression. This indicated that tanshinone IIA nanoparticles were superior to tanshinone IIA.16 The uptake of nanoparticles was affected by the size, shape, surface characteristics, medium concentration, incubation time, temperature, and many other factors.

17�C22 Combining drug-loaded nanoparticles with monoclonal antibodies against human hepatocellular carcinoma produces a drug-nanoparticle-monoclonal antibody immune complex. McAb can carry drug-loaded nanoparticles to specific target sites, thus enhancing specific cancer cell-drug combinations, increasing the drug concentration, and improving efficacy. Wu et al combined the highly specific anti-human liver acid ferritin monoclonal antibody with doxorubicin-poly butyl cyanoacrylate nanoparticles and prepared liver-specific doxorubicin immuno-nanoparticles. Experiments showed that the nanoparticles had a significantly longer half-life and high targeting in nude mice tumor inhibition experiments in vivo and cytotoxicity experiments in vitro.

The drug accumulated in the liver tumor, greatly increasing the drug��s concentration, extending the time during which it is effective, enhancing the efficacy, and reducing toxicity to other organs.23 Liu et al connected anti-tumor monoclonal antibody HAb18 with mitoxantrone-bovine serum albumin nanoparticles and prepared liver-specific immuno-nanoparticles, which could effectively combine with human liver cancer cells. In vitro studies indicated that the nano-drug could significantly enhance the inhibition of human hepatoma SMMC-7721 cell growth.24 Chen et al wrapped doxorubicin in a phospholipid bilayer and prepared liver-specific immuno-phospholipid nanoparticles, which showed greatly increased inhibition of human liver cancer cell growth compared with doxorubicin or an ordinary plasmid.

Compared with liposomes, lipid nanoparticles significantly improved the inhibition rate in mice bearing nude human liver cancer. Nanoparticles significantly increased the targeting of tumors, elevated drug concentration, extended the time during which it was effective, and reduced doxorubicin toxicity to other organs.25 Alpha-fetoprotein (AFP) is a AV-951 major plasma protein produced by the yolk sac and the liver during fetal development.

Fig 5 Immunofluorescence staining

Fig 5 Immunofluorescence staining selleck inhibitor of HBsAg in cells transfected with wild-type and mutated M88-cl4 sequences. The position and nature of the OBI-specific amino acid substitutions introduced into the genotype B control M88-cl4 are indicated. Several modeling programs predicted very similar arrangements of the S protein in the bilayer lipid membrane of the endoplasmic reticulum (ER). Persson and Argos’ model as well as SOSUI’s and DAS’s identified the same four transmembrane (TM) domains for wild-type S. However, SOSUI predicted a fifth TM domain (amino acids 149 to 171) that we considered doubtful. Nevertheless, the three amino acid substitutions identified as critical to HBsAg excretion had identical locations predicted by the three models.

M75T was located in the cytosolic loop separating TM1 and TM2, Y100S was at the end of TM2, and P178R was in TM3. HBsAg excretion deficit as potential contributor to OBI phenotype. Amino acid substitutions impairing HBsAg excretion reflected by HBsAg quantification in vitro have been identified in individual OBI clones. However, HBVs in HBsAg-positive as well as OBI strains circulate as quasispecies of related variants not necessarily represented by a single clone tested in vitro. The genetic diversity and the relevance of specific mutations as potential explanation for the OBI phenotype were therefore examined by sequencing multiple clones from several donor samples containing pattern 2 clones (Fig. 6). Fig 6 Phylogenetic analysis of the S amino acid sequences of multiple clones from OBI and non-OBI strains.

Phylogenetic analysis was performed as previously described (10). HBV reference sequences of genotypes/subgenotypes A1-3 and B-H are identified by their … The diversity of patterns was shown in one control (M92; two clones with pattern 2 and one clone with pattern 3) and three OBIs: TW0498 (one pattern 2 and one pattern 3), TW8964 (one pattern 1 and one pattern 3) and HK6794 (3 pattern 1, one pattern 2, and one pattern 3) (Fig. 6). In OBI HK6794, the average amino acid diversity between clones was 20 residues (range, 11 to 32 residues) without a significant relationship with the HBsAg phenotype (data not shown). The Y100S mutations responsible for pattern 2 of clone 2 were present in a subcluster of five clones associated with clone 2, but Y100F was present in all 12 other clones.

In contrast, in OBIs HK01556, HK3110, Brefeldin_A and HK3475, genetic diversity was very limited (Fig. 6), and each of the 8 to 12 clones contained Y100S (HK01556) or P178R (HK3110 and HK3475). For these three OBI strains, it is likely that these two mutations shown to prevent HBsAg excretion contribute to the OBI phenotype. DISCUSSION High mutation rates in the HBsAg in OBI strains from various geographical origins have been reported (4, 5, 8, 10).