Manipulation of SSI expression in wheat using RNAi technology leads to the production of wheat grains with novel starch structure and properties. Arabidopsis and rice suggest that manipulation of starch synthase I (SSI) expression in wheat may lead to the production of wheat grains with novel starch structure and properties. This work describes the suppression of SSI expression in wheat grains using RNAi technology, which leads to a low level of enzymatic activity for SSI in the developing endosperm, and a low abundance of SSI protein inside the starch granules of mature grains. The amylopectin fraction of starch from the SSI suppressed lines showed an increased frequency of very short chains , a lower proportion of short chains (dp 8–12), and more intermediate chains (dp 13–20) than in the grain from their negative segregant lines. In the most severely affected line, amylose content was significantly increased, the morphology of starch granules was changed, and the proportion of B starch granules was significantly reduced. The change of the fine structure of the starch in the SSI-RNAi suppression lines alters the gelatinization temperature, swelling power, and viscosity of the starch. This work demonstrates that the roles of SSI in the determination of starch structure and properties are similar among different cereals and Arabidopsis.Studies in  Starch is the major component of wheat grain (approximately 60–70%). Starch granules in higher plants contain two main classes of glucan polymer, amylose and amylopectin. The amylose fraction of wheat starch is a linear or infrequently branched  molecule with a degree of polymerization (dp) of 500–3000. Amylopectin is a large and highly branched polymer  reaching a dp of about 5000–50 000 (see reviews from Starch polymers are produced by a suite of enzymes including ADP-glucose pyrophosphorylases (EC 2.7.7.27), starch synthases (EC 2.4.1.21), starch-branching enzymes (EC 2.4.1.18), disproportionating enzymes (EC 2.4.1.25), and starch-debranching enzymes (EC 3.2.1.41 and 3.2.1.68)   .SSI gene in wheat endosperm. A reduction of SSI protein in the soluble phase and inside starch granules in wheat endosperm was observed to produce several changes in the structure and properties of wheat starch including changes in the proportion of B-granules (<7 μM), the proportion of amylopectin glucan chains of dp 8–12, the content of amylose, an increased starch peak gelatinization temperature, and a reduced starch peak viscosity value.To understand the role of SSI in the complex genetic background of hexaploid bread wheat, we have suppressed the expression of the ccdB  with BamHI site at 5′ site and EcoRI site at 3′ site, and a rice starch branching enzyme I (D10838) intron 4 (467bp from nucleotide 6202 to nucleotide 6668) in reverse orientation to the promoter, located between a HMW glutenin Bx17 promoter and a rice branching enzyme I intron 9 in forward orientation described by ccdB (1435bp) with SpeI site at 5′ site and KpnI at 3′ site between a rice branching enzyme I intron 9 and a NOS 3′ transcription terminator sequence described by A plasmid cloning vector (pBx17IRcasNOT) was first constructed for the suppression of target sequences in wheat endosperm by RNAi technology. The vector was similar to that published by SSI cDNA (GenBank accession No. AF091803) by PCR using the primers SSI-IR-F 5′- AAAAGGATCCGGTA CCAGGGATTGCTGAGGATTCCATCG-3′ (containing BamHI and KpnI sites at 5′ end) and SSI-IR-R 5′- AAAAGAATTCA CTAGTCAGCAAGAAGGACTGGCACAAGG-3′ (containing EcoRI and SpeI sites at the 5′ end). PCR amplification was carried out under standard conditions using Hotstar polymerase  on a Hybaid PCR Express thermal cycler . The thermal profile was 4min at 94 °C followed by 35 cycles of 30 s at 94 °C, 30 s at 58 °C and 1min at 72 °C. The resulting PCR fragments were first cloned in the reverse orientation into SpeI and KpnI sites and then in the forward orientation into BamHI and EcoRI sites to form a SSI-RNAi construct  was amplified from wheat onstruct .Tritium aestivum L. cv. Bob White 26) plants as a source of explant embryos were grown under containment glasshouse conditions (24 °C day and 18 °C night) under natural light. Embryos were transformed by biolistic bombardment as published (–1 geneticin (G418) and maintained at 24 °C in a 16h light (~25 μE m–2 s–1) and 8h dark regime. Calli were transferred onto fresh selection medium every three weeks. Plantlets were grown to 10cm high, transplanted into soil in 10cm pots, hardened on a misting bench for 2 weeks, and then transferred to a containment glasshouse with a temperature regime of 24 °C (day) and 18 °C (night). T2 plants were grown into soil in 10cm pots and T3 plants were grown with random design into soil in 20cm pots in the glasshouse described early.Wheat  was then used to remove contaminating DNA, following the manufacturer’s protocols. Where it was possible, the primers were designed to bind either side of an intron to enable detection of any genomic DNA contamination (–1) (Promega), 0.25 μl of 40 pmol μl–1 of polyT primer and 0.5 μl of SuperScript III reverse transcriptase (200U μl–1) (Invitrogen). For each PCR reaction (20 μl), 25ng of cDNA, 3.5mM MgCl2, 0.2mM of each dNTP, 5 pmol of primers, 1×SYBR Green I (Invitrogen), 1×PCR buffer (no MgCl2), and 0.25U of Platinum Taq DNA polymerase (Invitrogen) was used. The PCRs were conducted and analysed using a RotorGene 6000 . The real-time PCR conditions were one cycle of 95 °C for 10min, 45 cycles of 95 °C for 20 s, 58 °C for 20 s, and 72 °C for 30 s, and melting from 72 °C to 95 °C rising by 1 °C for each step. Comparative quantitation analysis was used to calculate the comparative expression. The comparative expression for each genotype was calculated by mean values of the comparative concentration for mRNAs for SSI individually divided by the mean value of the comparative concentration for mRNAs for α-tubulin (GenBank accession No. DQ435663).Total RNA from 15 days post-anthesis (DPA) developing endosperms was extracted following the procedure described in mination . A total–1 protease inhibitor cocktail for plant cell and tissue extracts  and 20% (v/v) glycerol followed by centrifugation at 13 000×g with microcentrifuge for 10min at 4 °C. The supernatant was decanted and the protein concentration was estimated using Coomassie Plus™ Protein Assay Reagent . PAGE was carried out for the separation of proteins according to –1 oyster glycogen . Starch synthase activity in the gel was revealed by overnight incubation in an assay buffer containing 25mM Tris, 190mM glycine, 133mM (NH4)2SO4, 7.3mM MgCl2, 0.07% w/v BSA, 0.47% v/v β-mercaptoethanol and 1.24mM ADP-glucose, followed by staining of glucan chains with I2–KI solution containing 2g l–1 iodine and 20g l–1 potassium iodine  for the lines containing homozygous transgene from SSI-RNAi construct and their negative segregants which were derived from T1 heterozygous plants. T3 grains used for the analysis were from a single T2 plant of each of two transgenic event-pairs. To conduct the detail analysis, five T3 plants from each of two transgenic event-pairs were grown with random design to produce T4 grain for analysis.Analyses for grain weight , protein content, starch content, starch granules, starch swelling power, starch gelatinization temperature, and starch viscosity were performed on the T4 SSI-RNAi and negative segregant lines were measured. Average grain weight produced was the mean values of triplicates of 100 grains for each of SSI-RNAi lines and their negative segregants.Grains from TProtein content was measured by near infrared analysis instrument NIR 5000 (FOSS) as described previously . Starch content was assayed using AACC method 76.13 using 20mg of wholemeal for each of three replicate samples  overnight at 37 °C. The starch was centrifuged for 20min at 2000×g then washed twice in distilled water, three times in 0.05% (w/v) sodium hydroxide followed by two washes in distilled water. Finally, the starch pellet was freeze dried under vacuum for two days.Starch was progressively eluted into water from a dough made from wholemeal flour of wheat grains. Starch was pelleted at 2000×g for 20min, resuspended in distilled water and pelleted again. Proteins were removed from the granule surface with 50 μL Proteinase K (1mg ml2–KI solution (1:10 w/w in water) and made up to 1ml with water. Sample absorbance of 200 μl solution was read with an Emax Precision Microplate Reader  at 620nm along with standards containing amylose ranging from 0–100%, made from combined potato amylose and corn amylopectin (Sigma). Absorbance values were converted to percentage amylose using a regression equation derived from the standard samples.The amylose content of starch was determined by a modification of the method of Granule size distribution (by volume) of the starch slurries was determined using a laser diffraction particle size analyser . The percentage of small B-type starch granules was determined using a cut-off diameter of 7 μm (refer to the result section). The diameter of A-type starch granules was calculated as the diameter of starch granules at the peak of A-type starch granules.Starch-granule-bound proteins were isolated and separated on SDS–PAGE gel as described previously . The chain length distribution of amylopectin was analysed according to –1 from 20 °C–140 °C with an empty stainless steel pan as a reference. The PYRIS software was used to calculate gelatinization temperatures and enthalpy.Starch gelatinization parameters were measured in a Pyris 1 differential scanning calorimeter . Starch was combined with water in a 1:2 (w/v) ratio. This mixture  was sealed in a stainless steel pan and scanned at 10 °C minSwelling power was measured by the 40mg swelling test . Starch (3.0g) was added to distilled water (25.0ml) in the Rapid Visco Analyzer pan and analysed under the conditions of continuous shear (160rpm). The rapid viscosity analysis (RVA) run profile was: heat at 50 °C for 2min, heat to 95 °C over 6min, hold at 95 °C for 4min, cool to 50 °C over 4min, and hold at 50 °C for 4min. The measured parameters were: peak viscosity (PV) at 95 °C, holding strength (HS) at the end of a 95 °C holding period, breakdown (BD) = PV–HS, final viscosity (FV) at the end of a 50 °C holding period, setback (SB) = FV–HS, pasting temperature (PT) at onset of viscosity, and peak temperature (Pt) at PV. The software Thermocline for Windows version 2.2  was used for collection and analysis of data.Starch granule morphology was examined with a scanning electron microscope (ZEISS EVO LS15). Purified starches were sputter-coated with gold and scanned at 20kV at room temperature at a ×300 or ×1000 magnification.For examination of the crystalline structure, starch granules were mounted in water and observed by light microscopy (Leica-DMR) with crossed polarized filters to reveal birefringence in the starch granules at a ×400 magnification.P<0.05), looking at changes between two transgenic event-pairs.Statistical analyses were performed using Genstat version 13. Analysis of variance was performed for grain weight, starch content, amylose content, protein content, B starch granule, swelling power, RVA data, differential scanning calorimetry (DSC) data, and quantitative RT-PCT data to obtain the least significant difference . SSI and SSIII in these zymograms were identified immunologically as in previous studies  (4 grain)  were unchanged   or Sypro4 grain) . In bothnchanged .SSI measured by quantitative real-time PCR. Relative expression data showed that the level of SSI mRNA in T4 immature endosperms of both SSI-RNAi lines was significantly lower than that from their negative segregants  and starch contents (approximately 56–64% for T3 grains and 57–62% for T4 grains).To determine the effect of the suppression of SSI expression in wheat grain, analysis of grain weight and its composition was undertaken in T3  and T4   . Thus, t4 grains, SSI-RNAi A and SSI-RNAi F contained a significantly lower percentage of B-granules  than that from their negative segregant lines   .4 SSI-RNAi A and SSI-RNAi F lines, purified starches were debranched and the glucan chains were separated by capillary electrophoresis. The relative proportion of the total for each glucan chain length was determined. The mean value (starches from five plants for each of two transgenic lines and their negative segregant lines) for each amylopectin glucan chain of the negative segregant line was subtracted from the corresponding value for the SSI-RNAi line. The resulting differences in chain length distribution between the SSI-RNAi transgenic lines and their negative segregants are shown in The suppression of SSI expression in wheat grain modifies the chain length distribution of grain starch. To reveal the glucan chain length distribution of amylopectin in the SSI-RNAi suppressed T4 grains of both SSI-RNAi lines had significantly higher peak gelatinization temperature and end gelatinization temperature . With the amylose-lipid dissociation peak, there were no significant differences for the dissociation temperature and the DeltaH values between the SSI-RNAi A line or SSI-RNAi F line and their negative segregants (P<0.05).The reduction of SSI proteins in the SSI-RNAi transgenic lines leads to the changes of starch gelatinization. Starch from T P<0.05,  than tha4 grains of SSI-RNAi A and SSI-RNAi F absorb only 11 or 10 times their weight, a statistically significant reduction of 8% or 20% respectively , holding strength (HS), final viscosity (FV), setback (SB), and higher pasting temperature (PT) than that from their negative segregants (P<0.05). The value for HS was lower from the SSI-RNAi F line than that from the SSI-RNAi A line  showed that starch from Ti A line .Changes in starch granule morphology of the SSI-RNAi lines were examined by scanning electron microscopy. There were no obvious changes between starch granules from the SSI-RNAi A line and its negative segregant under ×300  and ×100The suppression of SSI protein in the SSI-RNAi transgenic lines changes the crystalline order in the starch granules. Birefringence provides a visual measure of the level of crystalline order in starch granules. Spherical-shaped granules show a distinct Maltese cross when examined by polarized light . Starch Expressing grain composition data on a percentage basis does not reflect the underlying synthesis rate of starch in wheat grain. To examine the absolute levels of protein and starch synthesis in the SSI-RNAi lines and their negative segregants, contents were calculated on a per caryopsis basis . Such anTwo independent SSI-RNAi transgenic events (SSI-RNAi A and F) showed that insertion of the SSI-RNAi construct into the wheat genome conferred a clearly altered phenotype upon wheat endosperm starch. The RNAi technology has been widely used for the suppression of endogenous genes in cereal and oil grains (Arabidopsis leaves (Arabidopsis) as well as monocot plants (e.g. rice and wheat).The SSI enzymatic activity in the endosperm was clearly reduced as measured by zymogram analysis, and it seems that this limited the extension of the very short chains of dp 6–7 so that these chains increased in abundance while the glucose chains of dp 8–12 were reduced. Thus, we conclude that the very short glucose chains of dp 6–7 residues are the substrates for SSI enzyme, which extends them to a length of 8–12 glucose residues within the amylopectin clusters. The effects of SSI on the chain length distribution are consistent with the role of SSI activities in rice grains  and ArabArabidopsis leaf starch, although in the latter, starch granules were slightly elongated (In the line with the largest decrease in SSI protein (SSI-RNAi F), a large number of abnormal starch granules were detected under high magnification with a scanning electron microscope. The SSI-RNAi F line also had an altered birefringence pattern with an abnormal less bright Maltese cross. Changes in starch granule morphology and crystallinity were not detected in SSI mutants of rice endosperm starch and Arabidopsis leaf, amylose content was also increased and there were more small starch granules , as the SSI-RNAi A line, which has similar amylose content as its negative segregant line, had lower swelling power than that of its negative segregant line. The reduction of starch swelling power was also reported for starch containing increased amylose content through the suppression of SBEIIa in barley . The supBoth SSI-RNAi lines showed decreased starch peak viscosity, decreased starch final viscosity, increased starch peak gelatinization temperature, and increased starch end gelatinization temperature compared with their negative segregants. This may be due to their starch containing less short chains (dp 8–12) and more intermediate chains (dp 13–22). Low peak viscosity and high peak gelatinization temperature were also observed in rice SSI mutant endosperm starch (In conclusion, this work showed that starch synthase I in wheat grains can be effectively suppressed using RNAi technology, that when SSI is sufficiently suppressed the abundance of SSI in the soluble phase and bound to the starch granule is noticeably reduced and that this changes the starch structure, granule morphology, and starch properties. SSI clearly plays an important role in biosynthesis of wheat endosperm storage starch, but more work is required to determine the relative importance of SSI in the granule stroma and granule-bound forms.


Cpecorum.  strains/genotypes that are recognized, either following (a) natural live infection or (b) administration of a recombinant MOMP vaccine. Plasma antibodies from the koalas naturally infected with a Cpecorum.  G genotype strain recognised the epitopes located in the variable domain (VD) four of MOMP G and also VD4 of MOMP H. By comparison, plasma antibodies from an animal infected with a Cpecorum.  F genotype strain recognised epitopes in VD1, 2 and 4 of MOMP F, but not from other genotype MOMPs. When Chlamydia-free koalas were immunised with recombinant MOMP protein they produced antibodies not only against epitopes in the VDs but also in conserved domains of MOMP. Naturally infected koalas immunised with recombinant MOMP protein also produced antibodies against epitopes in the conserved domains. This work paves the way for further refinement of a MOMP-based Chlamydia vaccine that will offer wide cross-protection against the variety of chlamydial infections circulating in wild koala populations.Chlamydial infection in koalas is common across the east coast of Australia and causes significant morbidity, infertility and mortality. An effective vaccine to prevent the adverse consequences of chlamydial infections in koalas  would provide an important management tool to prevent further population decline of this species. An important step towards developing a vaccine in koalas is to understand the host immune response to chlamydial infection. In this study, we used the Pepscan methodology to identify B cell epitopes across the Major Outer Membrane Protein (MOMP) of four  Phascolarctidae and is considered an icon of Australia’s unique biodiversity. Despite this esteem, wild koala populations in geographically diverse regions throughout the country continue to decline. This decline has been attributed to several variables such as (a) habitat loss, resulting in fragmentation of koala colonies  substrate solution was added and incubated for 10 min at room temperature. The optical density was then read at 405 nm . Background for each sample was calculated from the mean plus twice the standard deviation of the negative wells (no sera added). In an attempt to minimise the inter-assay variability and truly reflect the differences in antibody response profiles between samples, (a) the assay was performed on both pre- and post-immunised samples of each animal alongside on the same day, and (b) the background absorbance (green line) used for comparing the response profiles from pre- and post-immunisations was a result of the mean plus two times the standard deviation of the negative controls of both samples.Cpecorum.  MOMP proteins was performed as previously described [Western blots to assess the specificity of the koala plasma to different escribed . In brieChlamydia negative koalas  were evaluated against the 88 peptides in the MOMP peptide library. Chlamydia-free animals. The response profiles for each pre-immunisation sample was used to establish a background response profile for analysis of post-immunisation samples.Plasma from four healthy, pre-immunised, Chlamydia negative koala (Amity) following immunization with recombinant MOMP A. Interestingly, when compared to animals immunised with other MOMPs  was immunized with recombinant MOMP F protein and by 20 weeks post vaccination produced plasma antibodies  with MOMP G protein resulted in antibodies that primarily recognise various linear epitopes in the conserved domains of MOMP   it produced antibodies that recognised the epitopes in conserved domains (peptides 2 and 19) as well as the variable domain one of MOMP F (peptides 13 and 14)  . Finally and 14)  (Table 2 and 14) Cpecorum.  genotype G when compared to the response of four Chlamydia negative animals. Both animals had a similar response profile to the Chlamydia free animals for 84 out of 88 peptides, however, plasma from these two diseased animals reacted strongly with epitopes from variable domain four of MOMP G  and the equivalent epitope in MOMP H , respectively. Interestingly, when this epitope of 15 amino acid length was aligned with all four MOMPs  and NQTLPLPITAGTATD  of MOMP F  did not produce any antibodies to conserved domains, but retained a similar reactivity pattern to the pre-immunization sample  in variable domain four of MOMP G and H and additionally to the conserved domains  of Chlamydia infection, have suggested a significant requirement for antibody-mediated immune responses  along with cellular immune responses (Th1) for an effective vaccine. Morrison et al. [C. trachomatis genital tract reinfection. In a follow up study, Morrison et al. [Studies in various animal models  were located in the variable domains, while the other epitope (peptide 53) was in a conserved domain. These naturally recognised epitopes presumably represent the surface-exposed regions on intact chlamydial EBs/RBs that are recognised during a natural live infection. Of these six naturally recognised MOMP epitopes, none were recognised by all four koalas. One possible explanation for this could be due to major histocompatibility complex restriction in these out bred koalas [Cpecorum.  genotypes .In our current study, we used Pepscan methodology to investigate linear epitopes along the entire MOMP protein to determine the specificity of the antibody responses produced following, (a) natural live infection, (b) recombinant MOMP vaccination, (c) recombinant MOMP vaccination on top of natural live infection. The 15mer peptides (overlapping by 9 amino acids) covered the full length of koala d koalas . We founin silico B cell prediction algorithm has predicted major epitopes in all four variable domains across Cpecorum.  MOMPs [Cpecorum.  G genotype produced antibodies only to the linear epitopes of variable domain four of MOMP G and MOMP H. This cross reactivity to MOMP G and MOMP H was similar to the immunoblot data we observed  produced plasma antibodies to three epitopes at the variable domains of MOMP F  whereas the other animal (Felix Pitt) produced antibodies to epitopes in two variable domains of MOMP F (VD2 and VD4). Both the animals (Nixon and Felix Pitt) shared a common epitope located in variable domain two. Even though there was 73.3% similarity between MOMP F and MOMP A at this linear epitope, and 66.6% between MOMP F and MOMP G/H, both koalas specifically produced antibodies only to MOMP F  or a combination of two recombinant MOMP proteins (A and F), we observed an epitope response that was different to the response seen with naturally infected koalas  in cross neutralization of Cpecorum.  infections. This is very encouraging from a vaccine development perspective, for the fact that seven of the recognised epitopes were in conserved domains and therefore conserved across all 13 Cpecorum.  MOMP genotypes (data not shown) that we currently have sequence information for, bodes well for wider cross neutralisation. It has also become evident from studies in naturally infected koalas that strong, long lasting protection from the neutralising antibodies does not result in natural infections [Early studies by Girjes et al.  reporteda et al.  to produfections . Hence, Cpecorum.  and then administered the recombinant MOMP vaccine. This is critical because, if the vaccine has to be delivered to wild populations, which already have 20-50% Cpecorum.  infections levels , it has to be effective in these groups. In the naturally infected vaccinated animals, a total of eight vaccine induced epitopes were recognised  MOMP linear epitopes responsible for the production of cross recognition antibodies in post-vaccinated healthy and diseased koalas. The findings from this study provides further insights into (a) screening various potential B cell epitopes using the Pepscan methodology in other chlamydial antigens, such as chlamydial heat shock protein (hsp60) [C. trachomatis infected women and demonstrated the potential to develop an epitope-based serological diagnostic assay. Since both humoral and cell mediated responses are critical for a protective immune response against persistent chlamydial infections, our next step is to apply this screening assay to identify several potential T cell epitopes in healthy and diseased koalas.In conclusion, although this study has dealt with the specificity of the polyclonal antibodies against MOMP in wild (outbred) koalas, known to have highly variable MHC Class II genes . We have (hsp60)  and ther (hsp60)  has iden


It is every Editor’s dream that articles in ‘their’ journal serve a wide population of interested readers and that some papers are so challenging that they are actually followed up by readers’ reactions.Consequently, the Editors of the current issue of Perspectives on Medical Education are proud of the issue you see on your screen now (or even hold in your hands)!comment on articles in this very issue. Granted: they have been written on invitation, but still…We have six full papers for you, with contributions from all over the globe. Topics ranging from bedside teaching to feedback are waiting here for you. We have a PhD report, a book review and a letter to the Editor. Moreover, two contributions So the Editors of our journal are happy and proud. Apparently the journal attracts authors from diverse backgrounds and it is beginning to serve this community with a wide variation of interesting stuff.When looking at the papers in this issue from a helicopter point of view, directly or indirectly almost all have to do with the issues of professional identity, group and individual norms, self-esteem and vulnerability when acquiring this professional identity.This is coincidental, and it may be indicative of the emerging insight that those in the health professions are a relatively separate community. Apparently we have our own unwritten rules and our initiation rites to make newcomers adapt to the field. During the first years the newcomers report to answer to a ‘calling’. This calling diminishes from the first to the third year, or at least: it is reported less , 2. Do tJuniors in medicine are vulnerable to suffer from mental health problems, burnout or heart rate variability, although no overt relation with stress can be established . The medSeen from these viewpoints, the health care professions bear great resemblance to other important professional groups, such as the military in the army or musicians in an orchestra: initiation rites, insecurity, not a great emphasis on individualism, rules that are not immediately crystal clear to outsiders.similarities between these two types of training. If we could combine the best of these worlds, maybe this would lead to health professionals who would be excellent craftsmen, handling their instruments with great expertise, flexibility and agility, while these instrumentalists would also be great listeners and interpreters and collaborate to achieve harmony.I know too little about the army to illustrate this further, but I do know about music. I was therefore extra thrilled to see the piece that Watling  contribuI’d say: a lot to be gained in medicine!


Welcome to the last issue of the journal for the year 2010, the year that saw the readership base grow as ISAR membership increased from 600 in 2009 to 1015 currently. The journal also has more than 13,000 people accessing the issue online. Scientists, like everyone else, want to publish papers in journals where their work is likely to reach out to as large an audience as possible, thus having the highest impact. The steady increase in our readership base is testimony to the rising popularity of the journal.The current issue starts with a review article by Dr. Korula George on fertility and age, which highlights the importance of the changing social scenario and increasing demand for fertility services in older women. It is a well-known fact that age of the female partner remains the most important factor predicting the success of infertility treatment. As more and more women are choosing to conceive at a later age, it puts pressure not only on the couple but also on the treating physicians because they are left with limited treatment options. Though tests exist to predict ovarian reserve, there is no way that we can predict IVF success.The article on seminal zinc concentration and semen parameters highlights the correlation between seminal zinc and sperm motility, viability and count. As there is no single test to predict male fertility potential, assessment of seminal zinc may be used in conjunction with routine semen parameters.A prospective observational study by M. S. Kamath throws light on predictor factor for pregnancy after COH. Duration of infertility and male factor (TMF) are shown to be negatively correlated with pregnancy outcome. As a sizeable fraction of infertility patients are given IUI as the first-line treatment option, we need to explore further on the factors affecting IUI success rates.Obesity is an epidemic of the modern world and we need to know the effect of obesity on reproductive outcome. The present study shows no adverse effects of obesity on ART outcome, but results published in the literature have been conflicting.P. Saxena’s article shows the effect of metformin therapy in PCOS. An improvement in metabolic profile with metformin may prove helpful in managing these patients. As we all know, managing PCOS is like a double-edged sword requiring a very careful approach to prevent OHSS.Müllerian anomalies are an important cause of repeated pregnancy loss and the article by Priya Selvaraj highlights the importance of Hysteroscopic Septal Resection with Versapoint that is considered to be a safe and an effective approach. New knowledge on post-translational modifications in ER p57 throws light on the molecular process of male infertility. We need to work beyond semen analysis as far as male infertility is concerned, so bio-informatics analysis of ER p57 can be a useful adjunct to semen analysis in the near future.Last but not the least, all of you must join me in congratulating Dr. Dhiraj Gada for his efforts in bringing IFFS to India. It is a matter of great pride to our country and ISAR to get an international conference of the stature of IFFS to India. We have succeeded in our bid to host the IFFS in 2016 and we must join together to ensure that the conference is a huge success. I conclude with best wishes to all for a happy and prosperous new year.


Staphylococcus aureus is a successful human pathogen that has developed several approaches to evade the immune system, including resistance strategies to prevent oxidative killing by immune cells. One mechanism by which this evasion occurs is by production of superoxide dismutase enzymes, which require manganese as a cofactor. Manganese is acquired by the manganese transporter MntABC. One component of this operon, MntC, has been proposed as a potential vaccine candidate due to its early in vivo expression and its ability to provide protection in preclinical models of staphylococcal infection. In the current study, we interrogate the role of this protein in protecting S. aureus from oxidative stress. We demonstrate that mutation of mntC in a number of invasive S. aureus clinical isolates results in increased sensitivity to oxidative stress. In addition, we show that while downregulation of mntC transcription is triggered upon exposure to physiological concentrations of manganese, MntC protein is still present on the bacterial surface at these same concentrations. Taken together, these results provide insight into the role of this antigen for the pathogen. Staphylococcus aureus is the causative agent of diseases ranging from benign skin infections to life-threatening endocarditis, osteomyelitis, toxinoses, and bacteremia. Proliferation and virulence of this pathogen is dependent on its ability to scavenge essential metals which are normally sequestered by the host S. aureus virulence  transporters have been shown to play a central role in manganese transport in several species of bacteria S. aureus vaccine currently under investigation in human clinical trials  which, in addition to MntC, includes antigens designed to address immune evasion (capsular polysaccharide) and adhesion to host tissues (clumping factor A) Staphylococcus species were found to share an average pairwise identity of 72% S. aureus isolates S. aureus and S. epidermidis bacterial burden in a mouse bacteremia model, suggesting that it may be an effective vaccine antigen for the prevention of S. aureus infection MntC is a component of a tetravalent mntABC expression by MntR and corresponding MntC protein levels. We also demonstrate that MntC is surface expressed at physiological concentrations of manganese and that it plays a key role in providing resistance to oxidative stress across diverse disease-causing clinical isolates.Here we present a brief report describing the precise regulation of 2 and Fe(II)SO4 to final concentrations of 50 µM and 1 µM, respectively. Inductively-coupled mass spectrometry analysis  of four independently prepared batches of the TSB medium that had been treated with Chelex 100 beads revealed that the medium contained 50 nM manganese ±17 nM.Primers, strains, and plasmids used in the current study are listed in mntC::neo knockout cassette consisting of two 800 bp fragments (mntC-1 and mntC-2) flanking the neomycin resistance gene, neo, was constructed. The primer sets and DNA templates used to amplify each of the fragments were: (i) mntC-1: oLH81/oLH82, S. aureus COL genomic DNA; (ii) neo: oLH83/oLH84, pUB110; (iii) mntC-2: oLH85/oLH86, S. aureus COL genomic DNA. All PCRs were performed with iProof polymerase , and all cloning and molecular biology techniques were performed as described BamHI site. After electroporation and chromosomal integration into S. aureus RN4220, the final mntC::neo mutant strain was obtained after plasmid excision by secondary recombination. The mutation was introduced into other S. aureus isolates by transduction using protocols described previously mntC was confirmed by PCR. Mutants were shown to be of the expected strain lineage with a Qualicon RiboPrinter .A mntR::ermC knockout cassette, consisting of two ∼1.2 kb fragments (mntR-1 and mntR-2) flanking the erythromycin resistance gene, ermC, was constructed. The primer sets and DNA templates used to amplify each of the fragments were: (i) mntR-1: oLH202/oLH203, S. aureus COL genomic DNA; (ii) ermC: oLH204/oLH205, pE194; (iii) mntR-2: oLH206/oLH207, S. aureus COL genomic DNA. After purification, these three products were spliced together by amplification with oLH202 and oLH207, and the knockout cassette was cloned into pSPT181 at the BamHI and SalI sites. After electroporation and chromosomal integration into S. aureus RN4220, the final mntR::ermC mutant strain was obtained after plasmid excision by secondary recombination. This mutation was transduced into S. aureus 8325-4 geh::pLH76 essentially as described elsewhere mntR was confirmed by PCR. Mutants were shown to be of the expected strain lineage with a Qualicon RiboPrinter.A mntABC promoter activity, a transcriptional fusion was constructed comprised of the Renilla luciferase (Rluc) gene placed under the control of the mntABC promoter (PmntABC). To prevent non-specific read-through transcription, a transcriptional terminator (TT) from pI258  with primers oLH434 and oLH435. Each of these products was purified  and were spliced together by amplification with primers oLH431 and oLH435 to give TT-PmntABC-Rluc. A promoterless version of this cassette, TT-Rluc, was constructed with PCR primers oLH431 and oLH442 for TT and oLH443 and oLH435 for Rluc. These two PCR products were purified and spliced together as before to yield TT-Rluc. The TT-Rluc and TT-PmntABC-Rluc cassettes were cloned into the L54a attP-encoding integrative vector, pLH71  or PmntABC-Rluc (8325-4 geh::pLH76) were cultured at 37°C in TSB-c medium to mid-exponential phase of growth, then subcultured into the same medium containing increasing concentrations of supplemental MnSO4 to a final optical density at 600 nm (OD600) of 0.01. After one hour incubation with shaking at 37°C, 40 µL of each culture was mixed with 10 µL of 5X Renilla luciferase lysis buffer (Promega) in an opaque white 96-well plate, and the plate was shaken for two minutes at 200 rpm at room temperature. Fifty µL of Renilla luciferase substrate (Promega) was added and the samples were shaken for an additional two minutes before measuring relative luminescence on a Spectramax M5e .4. Cells were harvested in the mid-exponential phase of growth, lysed with lysostaphin, and proteins were denatured in SDS-PAGE loading buffer. Following electrophoretic fractionation and transfer, Western blots were probed with murine anti-MntC 78-7 monoclonal antibody (at 3 µg/mL) as the primary antibody, and goat anti-mouse IgG (H+L)-AP Conjugate  as the secondary antibody. Western blots were processed using standard protocols Cells were grown overnight in TSB-c at 37°C with shaking, and subcultured (1:200 dilution) the following day in fresh TSB-c supplemented with the indicated concentration of MnSOmntC::neo mutant derivatives were grown in 5 mL TSB-c overnight at 37°C with shaking. The next day, cultures were diluted 1∶200 into TSB-c containing 400 nM MnSO4. Cultures were shaken at 37°C until they reached an OD600 = 0.6 – 0.9. An OD600 equivalent of 5 was collected, pelleted by centrifugation, and the supernatant was decanted. Cells were resuspended in 1 mL of 1X DPBS  +20% glycerol and frozen at −70°C until use. Frozen cells were diluted 1∶10 in 1X DPBS, and 100 µL of each strain of S. aureus was added per well to a NUNC Maxisorp plate . Plates were incubated at 4°C for 16 hours. The plate was blocked with 25 µL of porcine serum  per well, and plates were incubated at 4°C for an additional 2 hours. Plates were washed with 300 µL/well three times with TBS containing 0.1% Brij-35 . One hundred µL of 4 mg/mL IgG-purified polyclonal antibodies from either naïve or anti-MntC rabbit sera S. aureus cells.Selected clinical isolates from each clonal complex and their cognate 4 as the growth medium. MIC determinations were made during at least two independent assays.The Minimal Inhibitory Concentration (MIC) of methyl viologen (Sigma-Aldrich) necessary to inhibit bacterial growth was determined by broth microdilution according to standard protocols mntABC transcription occurs via MntR S. aureus would normally encounter during bacteremia. We therefore sought to more clearly define the concentrations of Mn2+ that regulate mntABC at the level of transcript and protein expression. A transcriptional fusion of the mntABC promoter with Rluc was constructed to facilitate precise definition of the manganese concentration required for the regulation of mntABC transcription.The US Department of Health reports that the normal range for manganese levels in humans is ∼4 to 15 µg/L in blood, which is equivalent to 73 nM to 273 nM S. aureus strains with chromosomally integrated reporters bearing either a promoterless Rluc (S. aureus 8325-4 geh::pLH75) or PmntABC-Rluc (8325-4 geh::pLH76) were cultured in TSB-c medium containing various concentrations of supplemental MnSO4. The promoterless Rluc strain had minimal levels of reporter activity at all concentrations of MnSO4 tested . Higher concentrations triggered repression of reporter activity, and maximal repression was observed at concentrations of ≥156 nM MnSO4. Despite this repression, reporter activity was still observed, indicating that, under these growth conditions, MntR only down regulates expression without abolishing it. These results indicate that S. aureus responds to extracellular concentrations of manganese in the range found in human blood (73 nM to 273 nM). The observation that mntABC transcription is not completely repressed at these concentrations correlates well with in vivo expression of MntC 2+ concentration at which promoter repression occurs also correlates with the experimentally determined binding affinity of purified MntC for Mn2+, which is in the nanomolar range To determine the minimum concentration of manganese at which expression of  of Mn2+ . At concmntABC RNA expression impacts MntC protein production, S. aureus 8325-4 wild-type, 8325-4 mntC, and 8325-4 mntR strains were cultured in TSB-c supplemented with increasing concentrations of Mn2+. As shown in S. aureus 8325-4 wild-type strain as a function of increasing levels of Mn2+ in the culture medium. As expected, no MntC protein was detectable in S. aureus 8325-4 mntC at any concentration of Mn2+ tested. For S. aureus 8325-4 mntR, levels of MntC protein were high and not affected by the concentration of manganese in the growth medium. These results indicate that, although manganese-mediated repression of mntABC transcription by MntR lowers the concentration of MntC protein, it is still expressed at the manganese concentrations which were tested under these growth conditions.To determine the degree to which manganese-dependent repression of mntC has been shown to be highly conserved across S. aureus isolates, its role in bacterial fitness, virulence and resistance to neutrophil killing in clinically relevant disease-causing isolates has yet to be demonstrated. In order to show that, (1) the results described above with the laboratory strain 8325-4 could be extended to clinically relevant strains, and (2) the levels of MntC detected by Western Blot correlate with surface expression, we performed whole cell ELISAs using anti-MntC polyclonal antibodies on representative clinical isolates grown in vitro. The isolates were selected from the five S. aureus clonal complexes most often associated with global disease: CC5, CC8, CC22, CC30, and CC45 Although S. aureus. These immune cells are adept at killing engulfed bacteria through the production of a respiratory burst, i.e., a potent cocktail of superoxide radicals, hydrogen peroxide, and hydroxyl radicals that is secreted into the phagosome in vitro to mimic the oxidative stress that the bacteria encounter during the respiratory burst. Increased sensitivity to methyl viologen was observed with a S. aureus mntA mutant made in the laboratory strain background, 8325-4 mntA) likely resulted in polar effects that disrupt expression of the downstream genes mntB and mntC. In addition, it has been shown that S. aureus 8325-4 encodes a truncated copy of the Nramp homolog mntHmntA was the sole cause of the observed sensitivity to methyl viologen . As a result, experiments were performed to look specifically at the contribution of the MntC vaccine antigen to S. aureus sensitivity to oxidative stress. Further, given that the previous observation was made with only a single laboratory strain of S. aureus, it was important to determine whether mntC-inactivation was similarly important for resistance to oxidative stress in diverse S. aureus strains that cause human disease. To test this possibility, a mntC mutation was transduced from S. aureus RN4220 into twelve clinical isolates from nosocomial infections representing different clonal complexes, including the five most often associated with invasive disease. Among the 12 isogenic pairs of clinical isolates were two MRSA strains, including one USA300, a lineage associated mainly with community-acquired infections. In order to quantitate the effect of mntC inactivation, minimum inhibitory concentrations (MICs) of methyl viologen were determined for wild-type and mntC isogenic strains in Mn2+-depleted media . Further, resistance to methyl viologen was restored when the mntC mutant strains were cultured in the presence of manganese  Rluc reporter construct as compared to a Northern blot, or by the potential contribution of additional negative regulators whose binding sites were not present in the 67 bp promoter fragment used in our construct. We also found that MntC was surface expressed at 400 nM MnSO4 in S. aureus clinical isolates representative of the most prevalent disease-causing strains. Finally, mntC mutation correlated with heightened sensitivity to oxidative stress in a wide spectrum of clinical isolates.In the current study, we sought to further define the regulation of MntC and its role in resistance to oxidative stress in S. aureus virulence, antibodies resulting from vaccination with MntC have the potential to prevent MntC-mediated uptake of Mn2+ by S. aureus, thereby rendering the organism more sensitive to oxidative killing by host immune cells. The results presented above, in combination with the high degree of protein sequence conservation and the demonstration of surface expression during murine models of infection S. aureus vaccine antigen.Several antigens have been proposed as potential vaccine components for the prevention of staphylococcal infections


The animal model of the whole-size and reduced-size liver transplantation in both rat and mouse has been successfully established. Because of the difficulties and complexities in microsurgical technology, the animal model of dual liver transplantation was still not established for twelve years since the first human dual liver transplantation has been made a success. There is an essential need to establish this animal model to lay a basic foundation for clinical practice. To study the physiological and histopathological changes of dual liver transplantation, “Y” type vein from the cross part between vena cava and two iliac of donor and “Y’ type prosthesis were employed to recanalize portal vein and the bile duct between dual liver grafts and recipient. The dual right upper lobes about 45–50% of the recipient liver volume were taken as donor, one was orthotopically implanted at its original position, the other was rotated 180° sagitally and heterotopically positioned in the left upper quadrant. Microcirculation parameters, liver function, immunohistochemistry and survival were analyzed to evaluate the function of dual liver grafts. No significant difference in the hepatic microcirculatory flow was found between two grafts in the first 90 minutes after reperfusion. Light and electronic microscope showed the liver architecture was maintained without obvious features of cellular destruction and the continuity of the endothelium was preserved. Only 3 heterotopically positioned graft appeared patchy desquamation of endothelial cell, mitochondrial swelling and hepatocytes cytoplasmic vacuolization. Immunohistochemistry revealed there is no difference in hepatocyte activity and the ability of endothelia to contract and relax after reperfusion between dual grafts. Dual grafts made a rapid amelioration of liver function after reperfusion. 7 rats survived more than 7 days with survival rate of 58.3.%. Using “Y” type vein and bile duct prosthesis, we successfully established a novel rat model of dual right upper liver lobe transplantation. Liver transplantation is an effective treatment for end-stage liver disease, but a huge gap remains between the number of people that need a liver transplant and the number of organs available. In order to maximize donor organs, novel surgical techniques such as split liver and living donor transplantation have evolved The hepatic microcirculatory flow in orthotopically implanted graft and heterotopically positioned graft were 10.7±1.01 ml/min/100 g and 11.2±1.21 ml/min/100 g respectively. No significant difference was found between two grafts (p>0.05) .In orthotopically implanted graft, the hepatocytes and portal tracts showed moderate sinusoidal congestion morphological changes. In 3/12 heterotopically positioned graft, obvious sinusoidal congestion and vacuolar change in the cytoplasm of hepatocytes 90 minute after reperfusion was found, scatter necrosis was found 5 days after transplantation in 2/12 heterotopically positioned graft .In orthotopically implanted graft, the liver architecture was maintained without obvious features of cellular destruction. The continuity of the endothelium was preserved except for minimal edema and defenestration. There was no evidence of progressive cell damage. Only 3 heterotopically positioned graft showed the patchy desquamation of endothelial cell 90 mins after reperfusion, mitochondrial swelling and hepatocytes cytoplasmic vacuolization were at 1 day after reperfusion .In order to evaluate the hepatocyte activity to undergo proliferation and apoptosis after reperfusion, proliferation and apoptosis index ratio  was used as the weighing parameter which can reflect priority inclination of cell to proliferation and (or) apoptosis. At 1 and 5 day after reperfusion, PAIR in the orthotopically implanted graft  was higher than that in the heterotopically positioned graft  without significant difference. The PAIR in dual grafts is higher than that in sham group 1 day after reperfusion (p<0.05) .The ability of endothelia to contract and relax after reperfusion was judged through the ET-1 (Endothelin-1) and eNOS  balance ratio (ENBR) which is proportion of immunostaining intensity score timed positive cell number score of ET-1 to that of eNOS. 90 mins and 3 days after reperfusion, grafts displayed an increased immunoreactivity of ET-1, whereas the eNOS protein expression was reduced with an ENBR 2.5±0.56, 2.1±0.51 in the orthotopically implanted graft respectively, 2.6±0.69, 2.3±0.56 in the heterotopically positioned graft respectively. The significant difference in ENBR was not found between the orthotopically implanted graft and the heterotopically positioned graft. Compared to the sham group, the ENBR is higher than those in dual graft 90 minutes and 3 day after reperfusion (p<0.01) .. Bilirubin levels after reperfusion were gradually increased, it was 52.4±19.9 U/l on the 5th day.Preoperatively, all serum parameters were within the normal range . Dual gr12 rats were used to do survival analysis. 7 rats survived more than 7 days with survival rate of 58.3%. Abdominal bleeding, bile leakage, liver abscess in graft was found in 5 dead rats .The shortage of liver donors has put the patients at risk of dying on the waiting list for liver transplantation. Although new surgical techniques such as split liver transplantation and living donor liver transplantation play an important role in extending the donor pool, it can not be routinely carried out, especially when graft weight and (or) volume can not meet the basic needs of recipient and when graft quality is marginal which can lead to small-for-size syndrome or primary non-function The optimal graft volume to provide adequate functional hepatocytes is still a controversial issue. At present, there are two standards worldwide: one is ratio of grafts to recipients’ weight (GRWR) and the other is ratio of grafts volume to recipients’ standard liver volume (GV/SLV). It is generally thought that the former should be more than 0.8% Presently and clinically, dual left lobe or one left and one lateral segment graft or one left and one right lobe graft or two left side  grafts was employed in dual liver transplantation. A left lobe from a relatively small volunteer donor will not meet the metabolic demands of a larger recipient. The possible solutions to this problem are to increase the extent of resection in the donor by harvesting the right lobe of the liver, which accounts for 60% to 70% of the total liver mass, to apply auxiliary partial orthotopic liver transplantation, or to implant dual grafts into one recipient. The right lobe harvest in the donor is not always safe, depending mainly on the volume of the remaining left lobe. Even though the donor has sufficiently large right lobe that is adequate as a graft for an adult recipient, the remaining left lobe may be too small to bring a safety to donor in many occasions. In this instance, the donor cannot be allowed to donate either side of the liver lobe for a large-size adult recipient. As an alternative, dual left lobe or left lateral segment from two living donors can solve the problem of graft-size insufficiency and guarantee donor safety. Furthermore, if the large-size recipient requires a bigger graft liver volume than the volume of the sum from two potential living donors’ left lobes, and if the right lobe harvested from one of two potential donors seems to be safe, one right lobe and one left lobe from two donors can be transplanted into a single recipient to avoid a small-for-size graft problem. In the present animal study, two right upper lobes were used as dual liver grafts. There were two reasons for using two right upper lobes as grafts. Firstly, if the left or middle lobe was used as a graft, in order to preserve the vena cave a large part of the residual right lobe would also need to be taken due to the vena cava going through whole right lobe. Secondly, the total volume of two right upper lobes gives 45–50% of total recipient liver weight which is a more appropriate size for the recipient.Difficulties in dual liver transplantation lie in the anastomosis of suprahepatic inferior vena cava, portal vein and bile duct between the graft and the recipient. Although two-cuff technique has been used in animal models of single liver transplantation, it can not be put into effect in dual liver transplantation due to the double suprahepatic inferior vena cava, double portal vein and double bile duct anastomosis of the grafts to a single surahepatic inferior vena cava, portal vein and bile duct in the recipient. To overcome these problems we use the “Y” type vein from donor and Self-developed “Y” type bile prosthesis. “Y” type vein is the cross part where vena cava is divided into left and right iliac vein. This arrangement that two cuff of left and right iliac were inserted into portal vein of dual graft and the cuff of recipient’ portal vein were inserted into the vena cava of “Y” type vein made the blood flow direction as the original “Y” type vein did without vortex. . The recIt was postulated that excessive portal blood flow relative to small-for-size graft and insufficient arterial perfusion would induce portal hypertension and arterial hypotension, which in turn lead to injury of the graft. Since the maximum additional hepatic blood flow volume that the liver can tolerate is 20% It is interesting to speculate the reasons which might protect the liver architecture from progressive damage. The balance of ET-1/eNOS plays an important role. Under physiological conditions, the sinusoidal diameters are regulated by a balance between ET-1 and NO produced from L-arginine, e.g. by the constitutive eNOS Because one of the dual graft is heterotopically positioned and rotated 180° sagitally, theoretically the change of spacial position potentially lead to the difference of hepatocyte shear force. Although morphological test display that 3 heterotopically positioned graft showed obvious sinusoidal congestion, vascular change in the cytoplasm of hepatocytes, scatter necrosis and patchy desquamation of endothelial cell, no significant differences in microcirculation, cell activity and the ability of endothelia to contract and relax were observed between orthotopically implanted graft and heterotopically positioned graft which means that right upper lobe can be rotated 180° sagitally as a heterotopically positioned donor.In the present study, although using ‘Y’ type vein and ‘Y’ type bile prosthesis we successfully established a rat model of dual liver transplantation, there is still some limitation:1) the dual donor are both right upper lobe which is different form the combination in clinical practice; 2) the operation time, cool ischemia time and anhepatic phase is longer; 3) the requirement for microsurgical skill is higher because of complicated anastomosis of vena cava, portal vein and bile duct, Usually at least two surgeon need to complete this operation; 4) the 7 day accumulative survival is only 58.3%, so still need to try hard to make a improvement.In the future, we will use this animal model to explore mechanism underlying the cooperative regeneration in dual graft, competitive regeneration resulting in one graft atrophy, the immune microenviroment change between dual graft and between recipient and dual donor and study on implication of marginal donor as dual grafts.Male Sprague-Dawley rats were used as donors and recipients. Rats were housed in a standard animal laboratory with free activity and access to water and chow. They were kept under constant environmental conditions with a 12-hour light-dark cycle. The rats were fasted 12 hours before operation. All the operations were performed under clean conditions.The protocol of animal experiments was approved by the Institutional Ethical Committee of Animal Experimentation of Zhejiang University in china, and the experiments were performed strictly according to governmental and international guidelines on animal experimentation. According to requirements for Biosafety and Animal Ethics ‘Units and individuals who are conducting the production and use of experimental animal production, should treat animals humanely and protect animal welfare, should not tease and abuse animals. The use of experimental animals should be in accordance to the scientific, rational and humane requirements. It is advised and encouraged to minimize the use of laboratory animals to reduce suffering of animals to be disposed of, and to explore of alternative methods in replacing animal testing and use’, Every effort was made to minimize any suffering of the animals used in this study. The isogeneic male SD  we used in our study among which 48 rats were taken as donors, 24 as recipients, dual right upper lobes  were planted in the one recipient. 12 recipients were assigned to for specimens harvest and undergo hemodynamic, morphological and serum monitoring, another 12 recipients were kept under observation up to the 7th postoperative day for survival analysis. In sham group (n = 24), the liver was mobilized by dividing the hepatic ligaments. 12 rats in sham group were assigned for specimen harvest and the remaining 12 rats for survival analysis .The graft weight relative to the liver weight of recipient was determined after the recipient liver was harvested. Whole operation time, cold ischemic time, warm ischemic time and anhepatic phase were calculated .In order to recanalize portal vein and the bile duct between dual liver grafts and recipient, “Y” type vein and bile duct from donor were used and designed in this study. The branch of vena cava and iliac vein were ligated and then “Y” type part where vena cava is divided into left and right iliac vein was harvest. Two cuffs were made in left and right iliac vein in vitro. The vena cava of “Y” type vein was left to be inserted into by the cuff of recipient vena cava. The recanalization of the bile duct was carried out with an interposion of the “Y” type prosthesis between the donor and the recipient grafts. Two catheters inserted into bile duct of the dual grafts were inserted into the two upper leg of “Y’ type prosthesis respectively in vitro, the catheter inserted into the lower leg of the “Y” type was inserted into the recipient’ bile duct in vivo .2O/O2 = 2∶1+1.5% isoflurane). Liver transplantation was performed on the basis of two cuffs technique described by Kamada N et al All procedures were carried out under nitrous oxide/isoflurane anaesthesia . Then the second liver graft is heterotopically positioned in the left upper quadrant and rotated 180° sagitally (left graft). The rotation of the heterotopic second liver graft through 180° sagitally brings the hilar structures into a reversed position. Thus the bile duct comes to lie behind the portal vein. The anastomosis of the suprahepatic vena cava was performed using continuous #8-0 polypropylene sutures between the recipient’s suprahepatic and the reconstructed suprahepatic vena cava of the dual liver donors. The portal anastomosis was performed using the ‘Y’ type vein, two cuffs of two iliac vein of “Y” type vein were already inserted into the recipient’s portal vein when repairing the graft in vitro. The portal vein of recipient was inserted into a cuff which was inserted into the vena cava of “Y” type vein in vivo. The recanalization of the bile duct was carried out using the ‘Y’ type prosthesis. Two catheters inserted into bile duct of the dual grafts were already inserted into the two upper leg of “Y’ type prosthesis respectively in vitro. The catheter attached the lower leg of the “Y” type was inserted into the lumen of recipient’ bile duct. Anastomosis of infrahepatic vena cava of the first donor was completed by the cuff method and the infrahepatic vena cava of the left graft was ligated .After the completion of the surgical procedure, recipient animals were cared for under an intensive post operative recovery protocol.2 Blood Perfusion Monitor, Laserflo BPM2; Vasamedics, St Paul.Min). A laser Doppler probe  was placed on the surface of the right upper lobe of the graft. Satisfactory contact of the probe on graft surface was maintained during the blood flow detection. Hemodynamic data were analyzed using the PowerLab software system .Microcirculation of the liver graft was measured with laser Doppler . Photographs were taken at a magnification of 12,000.Ultrastructural parameters were recorded by scanning electron microscopy. Liver specimens were cut into 2×2×5 mmFor immunohistochemical staining a HRP/DAB detection IHC kit  was used per the manufacturer’s protocol. Deparaffinize and rehydrate formalin-fixed paraffin-embedded tissue section. Add Hydrogen Peroxide Block to cover the sections and incubate for 10 minutes. After antigen retrieval  for 20 min in a domestic pressure cooker and after blocking non-specific binding sites with protein block, the sections were immunoreacted with primary antibodies  overnight at 4°C. Apply Rabbit specific HRP conjugate and incubate for 15 minutes at room temperature. DAB was applied to the tissue and sections counterstained with haematoxylin.Sections were stained for apoptotic cells with a DeadEnd™ Colorimetric TUNEL System from Promega Corporation. Images shown are representative of at least three independent experiments which gave similar results. The proliferation and apoptosis index ratio (PAIR), i.e. the ratio of the proportion of TUNEL and PCNA decorated nuclei per 100 hepatocellular nuclei, was semi-quantitated by counting approximately 1,000 hepatocytes. The immunostaining intensity of eNOS and ET-1 was evaluated by a four-grade score: 0 = negative, 1 = partially (<25%) weak positive-stained cells, 2 = partially (<25%) moderate or diffuse (>75%) weak positive cells, 3 = diffuse (>75%) moderate or strong positive cells, 4 = diffuse (>75%) strong positive cells. The number of eNOS and ET-1 labeled cells was scored semi-quantitatively from 0 to 4 . Calculate the ET-1 and eNOS balance ratio (ENBR), i.e. the ratio of staining intensity multiplied positive semi-quantitative number of ET-1 and eNOS.Preoperatively, 90 min after reperfusion and 1, 3 and 5 days after operation, alanine-aminotransferase (ALT), albumin and bilirubin were determined at 25°C by standard enzymatic techniques .12 recipients in each group were used for the survival study.P<0.05 was considered as sufficient in all experimental groups.All data are presented as means±SD. Statistical analysis was performed by the t test and Kruskal–Wallis test using SPSS 13.0 software. Survival rates were assessed by the Kaplan–Meier method. The log-rank test was used to compare significance. A level of significance of


Journal of Trauma Management and Outcomes and BioMed Central would like to show our appreciation for the following reviewers for their time, hard work and support by reviewing manuscripts for the journal in 2013.The editors of the  Mazhar AbbasIndiaIbrahim AlhabdanSaudi ArabiaAbdulbari BenerQatarAna CarlottiBrazilCharles GerardoUnited States of AmericaMorad HameedCanadaManu MalbrainBelgiumGerrit MatthesGermanyJeffrey IndesUnited States of AmericaLynne MooreCanadaMichael PasqualeUnited States of AmericaJoao Rezende-NetoCanadaUli SchmuckerGermanyVance SohnUnited States of AmericaSandy WidderCanadaHiroshi YatsushigeJapanNiels ZorgerGermany


Clostridium thermocellum is an anaerobic, thermophilic, cellulolytic bacterium that produces cellulosomes capable of efficiently degrading plant cell walls. The end-product cellobiose, however, inhibits degradation. To maximize the cellulolytic ability of C. thermocellum, it is important to eliminate this end-product inhibition.Cellulases continue to be one of the major costs associated with the lignocellulose hydrolysis process. C. thermocellum cultures supplemented with thermostable beta-glucosidases make up this system. This approach does not require any supplementation with cellulases and hemicellulases. When C. thermocellum strain S14 was cultured with a Thermoanaerobacter brockii beta-glucosidase (CglT with activity 30 U/g cellulose) in medium containing 100 g/L cellulose , we observed not only high degradation of cellulose, but also accumulation of 426 mM glucose in the culture broth. In contrast, cultures without CglT, or with less thermostable beta-glucosidases, did not efficiently hydrolyze cellulose and accumulated high levels of glucose. Glucose production required a cellulose load of over 10 g/L. When alkali-pretreated rice straw containing 100 g/L glucan was used as the lignocellulosic biomass, approximately 72% of the glucan was saccharified, and glucose accumulated to 446 mM in the culture broth. The hydrolysate slurry containing glucose was directly fermented to 694 mM ethanol by addition of Saccharomyces cerevisiae, giving an 85% theoretical yield without any inhibition.This work describes a system for biological saccharification that leads to glucose production following hydrolysis of lignocellulosic biomass. C. thermocellum supplemented with a thermostable beta-glucosidase and cultured under a high cellulose load. We named this approach biological simultaneous enzyme production and saccharification (BSES). BSES may resolve a significant barrier to economical production by providing a platform for production of fermentable sugars with reduced enzyme amounts.Our process is the first instance of biological saccharification with exclusive production and accumulation of glucose from lignocellulosic biomass. The key to its success was the use of  Clostridium thermocellum, an anaerobic, thermophilic, spore-forming bacterium, is the most potent cellulose-degrading bacterium known to produce cellulosomes [C. thermocellum contain a surprisingly large variety of enzymes and show attractive enzymatic properties for the degradation of complex plant biomass. In promising attempts to enhance the hydrolytic ability of C. thermocellum cellulosomes, designer cellulosomes [C. thermocellum produces low levels of cellulosomes (approximately 0.1 g/L) compared to the high level of cellulase secretion  of Trichoderma reesei, which can produce several functionally distinct cellulase components [C. thermocellum exhibits one of the highest observed microbial growth rates on cellulose [C. thermocellum to ferment substrate to desired products  in one step without adding externally produced enzymes. This approach is widely recognized as the ultimate configuration for low-cost hydrolysis and fermentation of cellulosic biomass. However, intrinsic features of C. thermocellum limit its immediate and direct application to industrial ethanol fermentation from cellulose because it produces only low levels of ethanol and shows weak tolerance to ethanol [C. thermocellum S14 strain [Thermoanaerobacter brockii (CglT) [C. thermocellum cultures supplemented with thermostable β-glucosidases, which we named biological simultaneous enzyme production and saccharification (BSES). BSES required no addition of cellulolytic enzymes. It can directly produce glucose from cellulosic materials due to supplementation of cellulose-degrading cultures with β-glucosidase. Exclusive glucose accumulation of glucose occurred when C. thermocellum was cultured with a thermostable β-glucosidase under a high cellulose load. This approach may resolve a significant barrier to economical production of bio-based chemicals and fuels from lignocellulosic biomass.Many microorganisms capable of producing cellulose and hemicellulose-degrading enzymes have been reported and characterized . Clostriulosomes . The celulosomes -5 and reulosomes  have beemponents . Althoug ethanol . In a pr4 strain  in combiT. brockii was characterized as having not only high thermostability, but also relatively high glucose tolerance [C. thermocellum cellulosomes. We found that when C. thermocellum was cultured with CglT under a high cellulose load, cellulose was effectively converted to glucose, and a high concentration of glucose accumulated in the culture broth. Figure C. thermocellum was cultured alone on the medium for 10 days, the maximum consumption of cellulose was approximately 234 mM, and most cellulose remained in the broth. The glucose and cellobiose concentrations in the broth were less than 68 mM and 37 mM, respectively, indicating that C. thermocellum cultivated on cellulose does not usually produce glucose and cellobiose in the culture broth. In contrast, when C. thermocellum S14 was cultured with 30 U/g CglT, it degraded all of the cellulose within 10 days. Furthermore, glucose accumulated to as high as 426 mM in the broth. Based on the cellulose consumption, the majority (70%) was apparently converted to glucose, implying that approximately 30% of the input cellulose was utilized in maintaining cellular homeostasis, such as for energy and enzyme production. The cellobiose content was less than 5.9 mM during cultivation. Cultures both with and without CglT had a pH range of 6.1 to 7.8, with no pH control during cultivation  in the medium  [Thermoanaerobacterium thermosaccharolyticum) [http://www.cazy.org/Glycoside-Hydrolases.html), have been reported as other promising β-glucosidases to enhance cellulose saccharification in combination with cellulosomes. In particular, Bgl was characterized as possessing higher glucose tolerance, which may be an important property for compatibility with C. thermocellum cellulosomes under a high cellulose load [C. thermocellum cultures grown with 100 g/L cellulose were supplemented with recombinant BglA or Bgl instead of CglT. Supplementation with BglA or Bgl led to lower cellulose degradation ability and glucose production than with CglT, and a higher accumulation of cellobiose was observed for both   and Bgl lyticum) ,16, whicC. thermocellum S14 strain [C. thermocellum such as ATCC 27405 and DSM 1313 also show high cellulose degradation and glucose accumulation, C. thermocellum ATCC 27405 and DSM 1313 supplemented with CglT were cultured with 100 g/L cellulose  after 10% (w/v) NaOH pretreatment at 70°C for 5 days was 85.5 ± 0.4% glucan, 5.2 ± 0.5% xylan, 0.7 ± 0.03% arabinan, and 0.3 ± 0.09% galactan (w/w). The Klason lignin content was 6.9 ± 0.3% (w/w), indicating a delignification ratio of 62.3% of the original lignin content (18.3% w/w).C. thermocellum S14 supplemented with CglT was cultured under a high solid load of 120 g/L alkali-pretreated rice straw (3% moisture content) containing 100 g glucan and 6.1 g xylan. Although the biomass slurry at the beginning had a high viscosity, after supplementation with CglT, viscosity was dramatically reduced. Cellobiose was maintained at a very low concentration, less than 3.9 mM throughout cultivation. Glucose accumulated to 446 mM, corresponding to approximately 72% saccharification of input glucan, which was comparable to tests using pure cellulose  contained 446 mM of glucose, C. thermocellum cells, and CglT, as well as fermentation products such as lactic acid (2.15 ± 0.02 mg/mL), acetic acid (4.18 ± 0.07 mg/mL), and ethanol (0.13 ± 0.02 mg/mL). Fermentation profiles of ethanol production and glucose consumption were obtained from the slurry  reactions into a single step [Here we describe the first example of biological saccharification with glucose production from lignocellulosic biomass. To achieve this, we used gle step .C. thermocellum cellulase complex is substantially more effective during microbial hydrolysis than purified cellulase preparations from this microorganism [It is known that a combination of cellulosomes and a thermostable β-glucosidase leads to efficient saccharification due to elimination of end-product inhibition under high cellulose loading ,15,21. Aorganism . This phorganism . Thus, tC. thermocellum has an uptake system capable of assimilating cellodextrins, which are released through simultaneous multiple hydrolysis steps mediated by adjacent cellulosomal subunits [C. thermocellum ATCC 27405 on glucose was obtained only after an exceptionally long lag of over 100 hours [C. thermocellum for utilization of monosaccharides such as glucose are understood as the lack of any beneficial availability of ATP derived from phosphorolytic cleavage by intracellular cellobiose phosphorylase (CbP) and cellodextrin phosphorylase (CdP) during growth on glucose [C. thermocellum. The phenomenon of high glucose accumulation may also be found in BSES using mesophilic cellulolytic anaerobes possessing similar uptake and phosphorylation (CbP and CdP) systems that are known to be widely distributed in cellulose-utilizing anaerobes, such as C. cellulovorans[C. cellulolyticum[Ruminococcus flavefaciens[C. thermocellum may be better suited for BSES than the mesophilic anaerobes for the following reasons: (1) C. thermocellum exhibits substantially higher enzyme production levels and growth rates on crystalline cellulose than any mesophiles [C. thermocellum, are considered to be robust microorganisms and the cellulolytic enzymes are stable in thermophilic conditions [C. thermocellum[C. thermocellum can degrade hemicellulose as well as mesophiles [subunits . Glucosesubunits . Growth 00 hours , and the00 hours . The phy glucose , and the glucose . Thus, tlolyticum, and Rumvefaciens. On the sophiles ; (2) thenditions ; (3) thermocellum; and (4)sophiles -34, but T. reesei cellulases [C. thermocellum cellulosomes and chimeric CBM-CglT created by fusing the carbohydrate-binding module CBM3 from the scaffolding protein CipA into the N-terminal region of CglT [C. thermocellum and CglT. If CBM-CglT instead of CglT is used during biological saccharification, hydrolysis and glucose production would be carried out for several days, after which cellulosomes, CBM-CglT, and insoluble solid residues bound to C. thermocellum cells may be recovered by adding fresh pretreated rice straw to the broth. C. thermocellum and CBM-CglT would only be expected to be added during the first round, making additional β-glucosidase or fresh inoculum unnecessary  and simply prepared by direct heat treatment (65°C) and cell lysis [C. thermocellum expressible BglA was created in shuttle vector pIBglA in an attempt to increase cellulase activity [One method that may reduce the amount of cellulase used and increase enzymatic productivity is recycling of the cellulases. Many studies have already been carried out on recycling strategies using llulases . Cellulallulases  and intrllulases . Recentl of CglT . It seemy Figure . CglT anll lysis ,40. Alteactivity . When thC. thermocellum is the most potent cellulose-degrading bacterium known to produce cellulosomes. Biological saccharification using C. thermocellum cultures supplemented with thermostable β-glucosidases provide a platform for production of fermentable sugars with drastically reduced requirements for enzyme amounts. This advance is expected to immediately benefit for biomass refinery. To take full advantage of these prospects, future studies will address improvement of C. thermocellum, the cellulase system, and thermostable β-glucosidases. The goals are much higher tolerance to glucose, cellobiose, and fermentation products such as ethanol and acetate.Economical production of bio-based chemicals and fuels from lignocellulosic biomass by enzymatic means continues to require considerable research in terms of both technical and economic aspects. In particular, cellulolytic enzymes continue to be one of the major problems associated with the hydrolysis process. C. thermocellum S14 has been deposited with the National Institute of Technology and Evaluation Patent Microorganisms Depositary  as NITE P-627. C. thermocellum ATCC 27405 and T. brockii ATCC 33075 were obtained from the American Type Culture Collection . C. thermocellum DSM 1313 was obtained from Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures . T. thermosaccharolyticum NOI-1 was originally isolated from soil and stored in our laboratory [C. thermocellum was grown on BM7CO medium [T. brockii was grown in modified DSMZ 122 medium supplemented with 5 g/L cellobiose as the carbon source [E. coli DH5α , E. coli BL21(DE3), and plasmid pET19b  served as the cloning host, expression host, and vector, respectively. E. coli cells were grown at 37°C in Luria-Bertani medium containing ampicillin (50 μg/mL). The sake-brewing yeast S. cerevisiae Kyokai no. 3 (K3) was obtained from the National Research Institute of Brewing . In preculture, the yeast was grown aerobically in static culture at 30°C on complete medium (YPD) containing 20 g/L peptone, 10 g/L yeast extract , and 20 g/L glucose.The hypercellulolytic strain boratory . C. therO medium ,43 suppln source . All BM7bglA from C. thermocellum and bgl from T. thermosaccharolyticum NOI-1. Primer pairs containing artificial restriction enzyme recognition sites  were used to amplify fragments of bglA  from C. thermocellum ATCC 27405 and bgl  from T. thermosaccharolyticum NOI-1 by PCR using genomic DNA as template. PCR was performed with PrimeSTAR HS DNA polymerase (Takara Bio) under standard conditions according to the manufacturer’s instructions. The amplified fragments for bglA and bgl were respectively inserted between the XhoI and Bpu1102 sites and the NdeI and XhoI sites of pET19b to generate pET19BglA and pET19Bgl. Expressed CglT, BglA, and Bgl were purified by nickel affinity column chromatography using Ni-NTA agarose resins and desalted using a desalting column.Preparation of chromosomal and plasmid DNA, and transformation were carried out by standard procedures or according to supplier protocols . Constructed plasmid pET19CglT was used to prepare recombinant CglT . We desi2 under static conditions for 10 minutes [p-nitrophenol from p-nitrophenyl β-D-glucoside. One unit of enzyme releases 1 μmol equivalent of p-nitrophenol per minute. All protein concentrations were determined using the Pierce BCA assay kit  with BSA as the standard.Assays for crude and recombinant β-glucosidase were performed at 60°C in 0.1 M sodium acetate buffer (pH 6.0) with 5 mM CaClg for 5 minutes), and the protein concentration in the supernatant was estimated using the Pierce BCA assay kit. Cellulosomes in cell-free broth were isolated from fermenting cultures using the affinity digestion method [Cell growth in BM7CO supplemented with cellulose was monitored based on the increase in the pellet protein concentration. Briefly, cells were lysed in a NaOH/SDS solution of 0.2 N NaOH  containing 0.2% (w/v) sodium dodecyl sulfate . Cell debris and residual solids were pelleted from the NaOH/SDS solution and removed by centrifugation  alkali-pretreated rice straw as the sole carbon sources. After incubation at 60°C for 6 hours with rotary shaking, the cultures were directly supplemented with 30 U/g cellulose or glucan of β-glucosidase  in an anaerobic chamber  with a CO2-based mixed gas atmosphere. Cultures containing β-glucosidase were incubated again at 60°C for 10 days with rotary shaking at 130 rpm. The concentration of the released sugar was measured by HPLC using culture supernatants obtained by centrifugation . To measure cellulose and glucan concentration in the hydrolyzed residues, aliquots of the separated pellets were dried overnight at 70°C to calculate the percentage of remaining cellulose and the glucan concentration by measuring the weight or following the National Renewable Energy Laboratory  chemical analysis procedure described below.For subculturing, part of a http://www.nrel.gov/biomass/analytical_procedures.html). Each sample was hydrolyzed with 72% sulfuric acid at 30°C for 60 minutes, followed by 3% sulfuric acid at 121°C for 60 minutes. The autoclaved hydrolyzed solution was neutralized to pH 6.0 with calcium carbonate and vacuum-filtered through a filtering crucible. Mono- and oligosaccharide components were separated on an Aminex HPX-87H column  by HPLC with 1 mM sulfuric acid at a flow rate of 0.6 mL/min on a Prominence instrument  operated at 60°C and equipped with a Shimadzu RID-10A refractive index detector. Acid insoluble lignin (Klason lignin) content was defined as the weight of the filter cake (oven-dried at 70°C to constant weight).The chemical composition of oven-dried natural and alkali-treated rice straw was analyzed following the NREL chemical analysis and testing standard procedure  was purchased from Miyahara . Rice straw was ground through a 0.5 mm mesh screen  and 30 g of the substrate was added to a 300 mL autoclavable bottle with 150 mL of 10% (w/v) NaOH solution. The bottle was incubated for 5 days at 70°C. The slurry was filtered through a disposable non-woven filter to recover the insoluble solids. The solids were washed with distilled water until the pH of the solid became neutral. The washed solids were dried in an oven at 60°C for 96 hours. After drying, the moisture content of the samples was measured.S. cerevisiae strain K3 precultured on YPD medium was washed twice with sterilized water and inoculated into each medium at 5% (v/v) and incubated at 30°C. Samples were analyzed for ethanol using a model GC-2014 gas chromatograph (Shimadzu) with a flame ionization detector (FID).The cultures were directly fermented by addition of yeast without any supplements. BM7CO medium adjusted to pH 7.0 with an HCl solution (containing the same concentration of glucose) was inoculated with yeast to obtain the fermentation profile as a reference fermentation test. ARS: alkaline-treated rice straw; ATCC: American Type Culture Collection; BSA: bovine serum albumin; BSES: biological simultaneous enzyme production and saccharification; CBM: carbohydrate-binding module; CBM-CglT: Chimera β-glucosidase fused CBM3 from the scaffolding protein CipA into the N-terminal region of CglT; CBP: consolidated bioprocessing; CbP: cellobiose phosphorylase; CdP: cellodextrin phosphorylase; FID: flame ionization detector; HPLC: high-performance liquid chromatography; NPMD: National Institute of Technology and Evaluation Patent Microorganisms Depositary; NREL: National Renewable Energy Laboratory; NRIB: National Research Institute of Brewing; PASC: phosphoric acid-swollen cellulose; PCR: polymerase chain reaction; SD: standard deviation; SDS: sodium dodecyl sulfate; SHF: separate hydrolysis and fermentation; YPD: yeast extract peptone dextrose.The authors declare that they have no competing interests.PPra carried out the cultivation, collected HPLC data, and helped in the design and interpretation of the experiments. RW helped carry out enzyme assays and participated in the design and interpretation of experiments, and in design and construction of the vectors. CT, PPa, KR, LD, JS, and KS assisted in the design, interpretation, and coordination of the experiments and edited the manuscript. YM helped to draft the manuscript. AK conceived of the project, assisted in the design and interpretation of experiments, and helped to draft and edit the manuscript. All authors read and approved the final manuscript.


Microbial Informatics and Experimentation (MIE) is a journal about computers and microbes. We created a new journal to fill a gap for which there is no publication avenue that is particularly geared to computationally-oriented, strongly biologically motivated, pragmatic articles focused on microbes. On the one hand, the bioinformatics journals are generally very computer technical and unlikely to be read by the diverse community of microbiologists. There is also a strong emphasis in bioinformatics literature on human/mammalian systems, though this is a secondary issue. On the other hand, microbial informatics work has appeared in a variety of microbiological publications, but it is seldom a good fit there either, and methods that span diverse microbes have no obvious home.We envisage the readership being practising microbiologists who are interested in computational insights into their data, and bioinformaticists interested in the microbial species and systems that sustain life on our fragile planet, but that also can make us very ill. The 1918 Influenza (Spanish flu) had an estimated death toll of 50-100 million [We see our topics of interest being as broad as the diversity of species we cover. A starting list of topics would include: microbial comparative genomics, microbial protein structure and function, microbial systems and metagenomics, microbial systems biology and the mathematical modelling of microbial systems. The list is not exhaustive, and will certainly evolve over time. If you have work at the interface between computation and microbiology (both broadly defined), we would be happy to consider it. Our aim for the journal, embodied in the refereeing processes, is that it be collegial and constructive. In a phrase, a community. Please join us.Microbial Informatics and Experimentation TeamMichael J Wise and Barry L Wanner, on behalf of The


The composite graft from the conchal cartilage is a graft that is often used, especially in surgery on the nose, due to its capacity to resolve problems of cover and tissue deficit, arising from the removal of neoplasms or as the result of trauma, burns or following over-aggressive rhinoplasty. We have started to use skin-perichondrium-cartilage graft from the ear to cover large areas of the nose with very satisfying results as well as we describe in the reported clinical case.The operation consisted of reconstruction of the cartilaginous nasal septum, which had previously been removed, using two vestibular labial mucosa flaps to reconstruct the mucosa, and cartilage from the ear conch for the cartilaginous septum. After this, the skin edges of the fistula were turned to recreate the inner lining of the nose and form a vascular base of wide area to accept the composite graft. The case concerns a female 74-year old patient who had undergone several oncological surgery for a relapsing basal cell carcinoma on the dorsum of the nose. The operation consisted of reconstruction of the cartilaginous nasal septum using two vestibular labial mucosa flaps to reconstruct the mucosa, and cartilage from the ear conch for the cartilaginous septum.The perichondrial cutaneous graft has shown in this surgical case very favorable peculiarities that make it usable even in facial plastic surgery.We believe that the positive experience that we achieved in the use of composite grafts for the reconstruction of large areas of the nose could be interesting for others surgeons. The composite graft from the conchal cartilage is a graft that is often used, especially in surgery on the nose, due to its capacity to resolve problems of cover and tissue deficit, arising from the removal of neoplasms or as the result of trauma, burns or following over-aggressive rhinoplasty -3.Complex defects of the nose are aesthetically difficult to repair, however, the colour, the quality and thickness of the composite-skin graft harvested from the preauricular site compare favourably with the skin of the nose region, even after extensive oncological surgery such as in patients affected by basal cell carcinoma of the nose .The technique for removal of the graft is very simple, leaves no trace and does not cause any residual functional deficit ,6. InserWorking from this point of view, in the last 5 years, we have started to use skin-perichondrium-cartilage graft from the ear to cover large areas of the nose with very satisfying results.We would like to describe a clinical case-study which we believe can be useful to illustrate our technique.The case concerns a female 74-year old patient who had undergone several oncological surgery for a relapsing basal cell carcinoma on the dorsum of the nose. It was three years after the last operation and the patient was very keen on a definitive covering of the affected area Figure , which hThe operation consisted of reconstruction of the cartilaginous nasal septum, which had previously been removed, using two vestibular labial mucosa flaps to reconstruct the mucosa, and cartilage from the ear conch for the cartilaginous septum Figure . After tThe perichondrial cutaneous graft is a performant and versatile composite graft that shows useful properties for maxillofacial surgery. The graft consists of epidermis, dermis and subcutaneous tissue, and the perichondrial layer.The perichondrial cutaneous graft has very favorable peculiarities that make it usable in different types of plastic surgery, in some cases we can certainly use it even in facial plastic surgery. The auricular concha is easily accessible, has a good tissue consistency and adaptability. Useful characteristics are that it does not contract and we can see a low donor site morbidity . HoweverOne limitation of the technique is determined by the area that can be considered to be sure of proper establishment and revascularisation of the composite graft.This constraint is however more a subjective limitation than an objective one, since there are no rules that can indicate it to us. What is considered to be important is that the graft should have the maximum possible contact with the vascularised tissue, which can be considered to be the bed upon which the graft is placed; postoperative gentle scarification of the graft, in combination with a constantly applied heparin solution decongests venous stasis normally seen in such grafts helps to establish a stable and early blood supply enhancing graft take ,9. ImporWe believe that the positive experience that we achieved in the use of composite grafts for the reconstruction of large areas of the nose could be interesting for others surgeons and will stimulate wider application of the technique described in this report.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.FI: participated in the surgical treatment and in the follow-up of this patient, MT: drafted the manuscript and reviewed the literature, FMA: participated in the surgical treatment and in the follow-up of this patient, MM: participated in the design of this case study and in the follow-up of this patient, ADI: revised the literature sources, RC: participated in the surgical treatment and in the follow-up of this patient, AMI: documented this case report with digital pictures, GD: participated in the follow-up of this patient. All the authors read and approved the final manuscript.


It is my great privilege to take over as Editor-in-Chief of the Journal of Epidemiology after Dr. Tomotaka Sobue, who served in that role between 2008 and 2010. Under the outstanding leadership of Dr. Sobue and his team, an online submission system was started in 2008, and the number of manuscripts from abroad has substantially increased. In 2010, 40% of submitted articles were from Japan, 12% from Europe, 11% from China, 9% each from Taiwan and countries in the Middle East, 5% from Korea, 6% from other Asian countries, 6% from the United States, and 2% from South America. The average impact factor from 2007 through 2009 was 1.73.The journal will continue to cover a broad range of high-quality epidemiological research throughout the world and to contribute to the basic, clinical, public health, and political sciences. The editorial team seeks to maintain our high international standard by publishing original and review articles on a variety of research topics in order to eventually establish us as one of the leading journals in epidemiology.Hiroyasu Iso, MD, PhD, MPHEditor-in-Chief, Journal of EpidemiologyProfessor of Public HealthDepartment of Environment and Social MedicineOsaka University Graduate School of Medicine


Cell & Bioscience welcomes the submission of your best work for rapid open access publication. This is the official journal of the Society of Chinese Bioscientists in America (SCBA). The SCBCell & Bioscience aims to publish the best works of broad general interest. We have an impeccable collection of dedicated editors and Editorial Board members who will guarantee you fast and fair peer review. We want to publish your articles, and we welcome your submission. Please remember that while Cell & Bioscience is the official journal of the SCBA, you do not need to be a society member to submit to and publish in Cell & Bioscience.Cell & Bioscience from others? In 2011, one appreciates that perhaps the fastest growing region for scientific research is Asia. This is where a new group of scientists and readers are poised to emerge in large numbers and come online. All journals will strive to target the new demographics, but Cell & Bioscience with our roots firmly based in both the United States and China will offer the premier platform for your work to reach both the Western and Asian audience. No other journal can provide you the best of both worlds like Cell & Bioscience. Give us a try and see if you don't become a believer!What distinguishes Cell & Bioscience Editorial Board pledge to work tirelessly to achieve a high visibility journal with excellent impact. But we cannot succeed without your support. We look forward to receiving your manuscript submissions and your advice about Cell & Bioscience. I offer you my best wishes for a joyous and successful 2011. Let this be a good year for you and a great year for Cell & Bioscience.I want to close with some personal reflections. I am extremely honored to be the founding Editor-in-chief and to lead our outstanding Editorial Board. This journal is the product of long and patient planning by many whose hard work has led finally to its fruition. I would like to thank Chuxia Deng and CC Wang as well as the other journal planning committee members for charting the path for the journal and for their trust in me, Kuan-Teh Jeang for his tireless effort and leadership throughout this process, and the BioMed Central team, especially Elizabeth Bal and Ciaran O'Neill, for their excellent support for the successful, timely launch of the journal. I, the editors, and the