Malignant peripheral nerve sheath tumours (MPNST) are rare tumours known to occur at high frequency in neurofibromatosis 1 (NF1), but may also occur in other cancer prone syndromes.The North West Regional Genetic Register covers a population of 4.1 million and was interrogated for incidence of MPNST in 12 cancer prone syndromes. Age, incidence and survival curves were generated for NF1.TP53 mutation carriers. However, there were no cases amongst 5727BRCA1/2 carriers and first degree relatives, 2029 members from Lynch syndrome families, nor amongst 447 Familial Adenomatous Polyposis, 202 Gorlin syndrome, nor 87 vHL cases.Fifty two of 1254 NF1 patients developed MPNST, with MPNST also occurring in 2/181 cases of schwannomatosis and 2/895 NF2 patients. Three cases were also noted in TP53 mutations and is confirmed at high frequency in NF1. It appears to be only increased in NF2 amongst those that have been irradiated. The lifetime risk of MPNST in NF1 is between 9–13%.MPNST is associated with schwannomatosis and  TP53 mutations[Malignant peripheral nerve sheath tumour (MPNST) are uncommon tumours varying substantially in clinicopathologic features. Previouutations and schwutations. We haveBRCA1, BRCA2, MSH2, MLH1 and MSH6 mutations. The register is highly ascertained for NF1, NF2, FAP, Gorlin syndrome von Hippel Lindau disease[TP53 mutation carriers and an international database for schwannomatosis patients referred into the Manchester laboratory for SMARCB1 mutation analysis. All the registries have been the subject of cancer verification using the regional North West Cancer Intelligence Service (NWCIS). We have previously published risks of cancers in Lynch syndrome[BRCA1 and BRCA2 carriers[MSH2, MLH1, MSH6)[BRCA1/2[The North West Regional Genetic Register covers a region of North West England, based around Manchester, with a population of 4.1 million. The genetic register service covers a number of tumour predisposing syndromes in particular NF1, Neurofibromatosis type 2 (NF2), Familial Adenomatous Polyposis (FAP), Gorlin syndrome, von Hippel Lindau disease and non-syndromic families with  disease. In addisyndrome and canccarriers. The NWCcarriers). As a d1, MSH6) and BRCA[BRCA1/2 as 50% ost April 2010. MPNST incidence curves were derived for NF1 for strict regional residents to avoid ascertainment bias. Five-year survival was determined using Kaplan-Meier curves. NF1 MPNST cases who were identified on the periphery of the region during the study period were included for the survival analysis only. The Mann–Whitney U test and Wilcoxon (Gehan) statistic were used to test between-group differences in age at diagnosis and survival.Death details from the registries were used to calculate survival rates, and death certificates were reviewed to determine cause of death. Follow-up was censored on 1BRCA1/2 carriers and first degree relatives, nor among 2029 Mismatch Repair mutation carriers and first degree relatives from Lynch syndrome families. As the annual rate of MPNST is about 1.25 per million[BRCA1/2 carriers but not yet in Lynch syndrome. There were also no cases amongst vHL, Gorlin or FAP patients although numbers were smaller. The presence of two male cases with MPNST aged 32  and 33 years (lower limb) and one female case of triton tumour aged 5 years amongst carriers of a 524 G > A (Arg175His), a 574delC and a 375 G > A TP53 mutation respectively suggests a likely association with Li Fraumeni syndrome. The 375 G > A mutation affects the invariant splice donor site CG/g > CA/g in exon 4. The missense and splicing mutation were inherited, but the framshift deletion de novo. There were also a further three benign nerve sheath tumours in three further unrelated TP53 mutation carriers aged 33 years (Vestibular schwannoma) and two paraspinal/extra-dural at ages 50 and 49 years. There were also three cases with schwannomatosis and MPNST. Two of these occurred in a family with a c.846 C > G, p.N288K SMARCB1 mutation that we have previously reported as probably pathogenic[SMARCB1 mutation[SMARCB1 mutation analysis was not possible. There were two cases occurring amongst 921 NF2 patients. Both of these have been reported as having occurred after radiation treatment for vestibular schwannoma[The presence of MPNST among the study populations is shown in Table million the lifethogenic. It appemutation. Both hahwannoma,17. No MThere were 52 cases of MPNST amongst our NF1 patients including 43/1059 patients within the strict regional.boundaries of the Cancer Register. Fifteen cases of MPNST have occurred in the strict regional population since 1996 maintaining an incidence rate of above 1 per 1000 NF1 patient per year as 1010 NF1 patients were alive at some point after 1996.Within the strict regional population of 1059 individuals the cumulative risk of MPNST was 11.7% (95% CI 9.7–13.7%) by age 70 years (figureThe association between MPNST and NF1 has long been documented-4,18-22.TP53 mutations. The presence of three proven MPNSTs in only 181 schwannomatosis patients is very suggestive of an association. The main difficulty with this association is the previous problems with classifying schwannomatosis[SMARCB1 mutation suggests that this is a real link. It also broadens the tumour spectrum in families with SMARCB1 mutations. There remains a question as to why children with certain SMARCB1 mutations have a very high risk of the highly Malignant atypical teratoid Rhabdoid tumours[In addition to the known link with NF1 this study has confirmed probable links with schwannomatosis and germline omatosis-27. Howe tumours whereas  tumours although tumours,29,30.TP53 mutation carriers with MPNST makes a link with germline TP53 mutations and Li Fraumeni syndrome very likely. It is quite possible that cases of MPNST are buried amongst reports of soft tissue sarcoma[TP53 analysis on MPNST patients. Both the cases of MPNST occurred after the previous report from our group[TP53 mutation was a case of Triton tumour in a three year old[The presence of three confirmed  sarcoma. It is tur group. Nonetheyear old. This reyear old.TP53 mutation, which in addition to an NF1 mutation may cause a very substantial risk of MPNST. Indeed TP53 mutations have been described in the transformation from benign schwannoma to MPNST[TP53 mutation carriers this will need to be confirmed in larger studies.The link between NF2 and MPNST is controversial,17,32-35Up to 50% of malignant peripheral nerve sheath tumours in non NF1 patients harbour NF1 mutations. There iThe poor survival from MPNST particularly in NF1 patients highlights the need for therapies targeted at the main underlying genetic abnormality. Whole genome sequencing is likely to reveal new targets for therapy and the fast reducing cost of such testing may mean that it will be affordable in the clinic within the next 5 years.TP53 mutations as well as those with NF1. Radiation treatment particularly in childhood increases the risk of MPNST in NF1 and may also cause MPNST to occur in NF2.In conclusion MPNST appears to occur at increased frequency in schwannomatosis and in those with germline The authors declare that they have no competing interests.The research and reviews conceived by DGE, initial manuscript and data assessed by DGE, contributions for data on TP53 from JMB and from SMH for NF1. All authors developed the manuscript and approved the final version.


Acute aortic dissection (AAD) is a potentially fatal condition that requires rapid assessment and treatment. The American Heart Association and American College of Cardiology suggested new guidelines for early diagnosis of AAD in 2010, and so we applied retrospectively that system to our known patients with AAD in a community hospital.We reviewed 166 patients with confirmed AAD regardless of types from January 2000 to April 2013. We evaluated 12 newly proposed risks, based on the new guideline.Abrupt onset of pain was the most frequent symptom (67.4%). 6 patients (3.6%) were grouped under the low risk, 88 patients (53.0%) under the intermediate risk, and 72 patients (43.4%) under the high risk. 90 patients (54.2%) demonstrated a widened mediastinum in the chest X-rays. 3 patients showed a mediastinal widening among 6 patients with low risk. In 7 patients (4.2%), were initially diagnosed with acute myocardial infarction, 3 patients were categorized as intermediate risk group (risk score 1) and the others as a high risk of AAD (risk score 2).The risk score system in new guidelines detected ADD with high sensitivity. But, in addition to applying the new guideline, having high suspicion for AAD enables the early diagnosis to be more accurate.


Dio2 and Dio3) are differentially regulated by the circadian clock and by changes of the ambient light. The expression level of Dio2 in adult rats (2–3 months of age) kept continuously in darkness is modulated by the circadian clock and is up-regulated by 2 fold at midday. When the diurnal ambient light was on, the expression level of Dio2 increased by 4–8 fold and a consequent increase of the related protein was detected around the nuclei of retinal photoreceptors and of neurons in inner and outer nuclear layers. The expression level of Dio3 had a different temporal pattern and was down-regulated by diurnal light. Our results suggest that DIO2 and DIO3 have a role not only in the developing retina but also in the adult retina and are powerfully regulated by light. As the thyroid hormone is a ligand-inducible transcription factor controlling the expression of several target genes, the transcriptional activation of Dio2 could be a novel genomic component of light adaptation.Analysis with DNA-microrrays and real time PCR show that several genes involved in the thyroid hormone cascade, such as deiodinase 2 and 3 ( In photoreceptors, light adaptation has been extensively studied and several mechanisms contribute to it: changes of intracellular calcium concentration In the present manuscript, we analyze changes in gene expression occurring during the circadian rhythm and when the ambient light in the circadian rhythm is modified. A microarray analysis identified the gene coding for the DIO2 enzyme as the gene with the largest changes of expression levels. This observation prompted us to investigate whether the observed DNA microarray results could be confirmed with real time PCR and if the corresponding protein levels are also modified after light exposure. Several reports have described a major role of the thyroid hormone cascade during retinal development We measured changes in gene expression in retinas acutely isolated from adult (3 months old) freely moving rats exposed to controlled ambient steady lights. To determine the amount of light impinging on the retina, we performed a similar analysis also in cultivated intact retinas Dio2 was the maximally up-regulated gene after 3 and 6 hours of light and its up-regulation was consistently observed in all replicas. Since DIO2 regulates the availability of the active thyroid hormone, as a consequence, DIO2 regulates the timing of cellular responses to thyroid hormones We performed an initial screening using the DNA microarray technique. We extracted the mRNA from retinas of rats kept overnight in darkness until 7 am (as control) 0 ZT and subsequently retinas of animals that were exposed either for 3 hours (3 ZT) or 6 hours (6 ZT) to a 1000 lux light. From the 31099 probes present in the microarray, we extracted those whose expression level increased by more than 60% in all three replicas . Up-reguStat3 , Ep300 (E1A binding protein p300) and Pax4 (paired box gene 4) involved in retinal transcription Stat3 and Ep300 are involved in the thyroid hormone cascade as downstream transcription factors Three transcription factors were up-regulated both after 3 and 6 hours of light: http://bioinfo.vanderbilt.edu/webgestalt/). Among up-regulated genes we found 11 genes involved in visual functions and eye development: Psen1 (presenilin 1), Crygb , Crygc , Crygd , Grk1 (G protein-coupled receptor kinase 1), Rpgrip1 (retinitis pigmentosa GTPase regulator interacting protein 1), Myo5a (myosin Va), Stat3 , Pax4 (paired box 4). These genes could be involved in the protection of the retina during exposure to bright lights.We performed a gene ontology analysis of up-regulated genes (Gabrr1 (gamma-aminobutyric acid (GABA) receptor, rho 1) and Cacnb2 . Gabbr1 codes for the GABA receptor subunit rho1, one of the subunits particular of GABAC receptors highly expressed in the retina Cacnb2 corresponds to the beta subunit of a voltage gated calcium channel known to modulate the b-wave of the ERG response in dark There were 18 up-regulated genes involved in cell-to-cell communication, synaptic function and transmission of nerve impulse. Among them we found Atp1a1 , Scarb1 , Crh (corticotropin releasing hormone) and Dio2 .Up-regulated genes involved in general cell functions and metabolism were Adra1b , coding for the alpha 1b adrenergic receptor involved in the control of cyclic AMP (cAMP) when epinephrine is present Adra1b could have an important role in light adaptation.Down-regulated genes were those that had a decreased expression level larger than 0.7 and were 23 and 22 after 3 and 6 hours of light exposure for all three replicas , respectDio2. As a consequence, we decided to confirm changes in the expression of genes involved in the production or reduction of thyroid hormone Dio2 and Dio3 with real-time PCR in retinas extracted from freely moving rats kept in different ambient lights.The gene which was consistently and prominently up-regulated after 3 and 6 hours of light was Dio2 increased by about 2 times at ZT 3, ZT 4, ZT 6, ZT 8 and ZT 12. The expression level of Dio2 in rats exposed to the usual light or to an intense light, from ZT 0 increased with time of light exposure, reaching levels 4–8 times larger than in control conditions  level of related protein, the expression levels of associated proteins were determined by Western blot  in human WERI-Rb1 cell lines Opn1mw and Opn1sw) Opn1sw and Opn1mwOpn1sw and of Opn1mw was respectively about 30% and 40% lower than at 0 ZT in the dark  to locate the existence of a DR4 element. We found that Atp1b2  contains this exact sequence and Ep300 (E1a binding protein), Ccng1 (cyclin g1) and Cpt1a  contained a sequence that had the two half sites but spaced by 5, 6, 6 base pairs, respectively. TR binding and weak transactivation to sequences with 5 and 6 base pairs between the two half sites has been reported Cpt1a is known to be regulated by T3 Atp1b2 and Ep300, but the two genes Ccng1 and Cpt1a, were up-regulated also by light, as shown in Ccng1 could have a protective role and in cellular survival as reported for other cyclins Cpt1a play a role in retina metabolism Thyroid hormone receptors, TRα and TRβ, regulate target gene expression by binding to the T3 response element (TRE) composed of repeated DNA sequences with different configurations. The consensus sequence recognized by nuclear receptors often contains a hexamer AGGTCA known as “the half site”. TR forms heterodimers with members of the retinoid X receptor (RXR) family to mediate T3 action Sag and Gcap1. These biochemical pathways could be novel components of light adaptation.These results support the notion that light could activate the thyroid hormone cascade, regulating therefore the expression level of its target genes, such as the cone opsins, Dio2 and Dio3 genes and of their corresponding proteins in the adult retina. The genomic analysis of changes in gene expression with DNA-microarrays in the adult retina shows that Dio2 is the most up-regulated gene by diurnal light . The relative concentrations of T4 and T3 and their availability to the nuclear thyroid hormone receptor (TR) are controlled by the local conversion of T4 to T3 catalyzed by the enzyme DIO2, while the enzyme DIO3 inactivates T3 Dio2 and Dio3 exhibit a regulation of gene expression similar to the one described here in the retina. Several genes present in the retina and in the pineal gland show a phase shift with respect to each other Dio2 and Dio3. Dio2 has a role in photoperiodic modulation in seasonal reproduction in the mediobasal hypothalamus In the pituitary gland, The thyroid hormone cascade acts in the regulation of neurodevelopment, possibly by activation and repression of complex gene networks http://www.mybioinfo.info/) to locate the existence of a DR4 element. We found that Atp1b2  contains this exact sequence and Ep300 (E1a binding protein), Ccng1 (cyclin g1) and Cpt1a  contained a sequence that had the two half sites but spaced by 5, 6, 6 base pairs, respectively. TR binding and weak transactivation to sequences with 5 and 6 base pairs between the two half sites has been reported Ep300 has a role in TR function TR regulates target gene expression by binding to the T3 response element (TRE) composed of repeated DNA sequences with different configurations. The consensus sequence recognized by nuclear receptors often contains a hexamer AGGTCA known as “the half site”. TR forms heterodimers with members of the retinoid X receptor (RXR) family to mediate T3 action Dio2−/− knock-out mice had significant deficits in thermoregulation and thermogenesis Dio1−/− and Dio2−/− knock-out mice had significant deficits in the Morris water maze test indicating dysfunctions not only in learning and memory development but also, possibly, in visual capability Dio3−/− knock-out mice, almost 80% of cones are lost through neonatal cell death and the amplitude of both the a- and b-wave of the electroretinogram is significantly reduced Dio2 and Dio3 in retinal visual functions.In humans, the majority of patients with dysthyroid eye disease (Graves' disease), an autoimmune disease where the thyroid is overactive, producing an excessive amount of thyroid hormones have developed color vision defects OPN1LW and OPN1MW, i.e. the long (L) and medium (M) cone opsin genes and were identified as transcriptional targets of the thyroid hormone cascade. Also ARR3 , GCAP1, PDE6H (phosphodiesterase 6H) and PDE6C (phosphodiesterase 6C) were found to be similar transcriptional targets. Arrestin, guanylyl cyclase, and phosphodiesterase are proteins involved in the regulation of the cyclic GMP signal transduction pathways in cones and rods Crx, the cone-rod homeobox OPN1LW, Opn1mw, Arr3 and Gcap1 have been identified to be regulated by CrxDio2 and Dio3 have a similar behavior in wild-type mice treated with MMI/perchlorate, treatment causing mice to become hypothyroidic Opn1mw and Opn1sw are down-regulated after 12 hours of light exposure . All rat experiments were carried out according to the Italian and European guidelines for animal care .Dark-adapted Long Evans male adult rats were sacrificed under an infrared light source. The harvested retinas were expelled into 500 µl of TRI Reagent T9424 (Sigma Aldrich) on ice and stored at −80°C. Culture retinas were prepared as described on the protocol used by Reidel et al Immunolabeling was performed by standard protocols for tissue fixation and processing, using as primary antibody anti-Dio2 sc-98716  and DAPI  for nuclear staining.2) in ice. The total amount of protein was determined by using BCA protein assay kit (Pierce Biotechnology). The homogenate was diluted in a sample buffer (20μg), run on SDS-PAGE and western blotted using the following antibodies: anti-Dio2 sc-98716 , anti-Dio3 ab82041 . β-Actin HRP- conjugated A3854 (Sigma-Aldrich) was used as housekeeping control. Signals were detected analyzing the optical density of the spots.Retinas, dissected from light or dark-exposed mice, were homogenized in Lysis buffer . The RNA quality was checked using a bioanalyzer , and the RNA quantity was measured with ND-1000 Nanodrop spectrophotometer. 10 µg of RNA sample was used for microarray analysis on Affymetrix RAT230_2 GeneChip containing 31099 probes, corresponding to 14181 probes with a gene symbol (Affymetrix). Low level analysis was performed using an Robust Multi-array Average (RMA) algorithm  directly on the scanned images. All data is MIAME compliant and that the raw data has been deposited in a MIAME compliant database , as detailed on the MGED Society website Data were organized in matrices ‘‘m×n’’ . Five samples were considered: a control at 7 am in dark , a sample always kept in dark till 10 am (3Dij), a sample with 3 hours of continuous light also at 10 am (3Lij), and two similar samples at 1 pm . Data were analyzed by considering log2 changes in gene expression in each replica against the control condition C, that is, log2(3Dij/Cij), log2(3Lij/Cij) and log2(6Dij/Cij), log2(6Lij/Cij). Up-regulated genes for each replica were obtained by selecting all genes showing an up-regulation higher than 60%. Down-regulated genes were obtained considering genes with a decrease of expression larger than 0.7. Intersection between the three replicas was performed and presented in 2 inhalation, the eyes enucleated, the lenses removed and the retinas collected in TRI Reagent (Sigma Aldrich). Total RNA from retinas was extracted according to the manufacturer's instructions (Sigma Aldrich). After resuspension in DEPC-treated H2O, RNA was further purified using an RNeasy column (Qiagen) and quantified using an ND-1000 Nanodrop spectrophotometer (Nanodrop Technologies). Total RNA (500 ng) was treated with DNAse I (Invitrogen) to remove any genomic DNA contamination and converted to cDNA using Superscript II reverse transcriptase (Invitrogen). Twenty microliter PCR reaction mixtures contained cDNA, SYBR green master mix (BioRad), H2O, and custom primers designed for each gene of interest. The PCR reactions were performed in an iQ5 thermocycler (BioRad). Each reaction was performed at least in duplicate, and threshold cycles (Ct) were calculated using the second derivative of the reaction. The Ct of each gene was normalized against that of the control reference transcript Gapdh. The variation of Gapdh in Ct between samples obtained from rats exposed to darkness and to light showed no significant difference. For the experiments shown in Gapdh and Hprt. Normalization was performed using the geometrical mean as described by Vandesompele et al −ΔΔCt method), using the average of dark control set to one Long Evans rats were bred and maintained under a 12 hour light/dark cycle (7 AM:7 PM). For changes of lighting environment, two groups of overnight dark-adapted animals were maintained in either a darkened or a lighted cage. A 60 W bulb was used as an adjustable light source. For each time point at least six animals were sacrificed by COPrimers shown in Table S1Relative abundance of Dio2 and Dio3. From the data presented in (DOC)Click here for additional data file.


Despite great efforts to control the infection of poultry with H5N1 viruses, these pathogens continue to evolve and spread in nature, threatening public health. Elucidating the characteristics of H5N1 avian influenza virus will benefit disease control and pandemic preparation. Here, we sequenced the genomes of 15 H5N1 avian influenza viruses isolated in Vietnam in 2006 and 2007 and performed phylogenetic analyses to compare these sequences with those of other viruses available in the public databases. Molecular characterization of the H5N1 viruses revealed that seven genetically distinct clades of H5N1 viruses have appeared in Vietnam. Clade 2.3.4 viruses existed in Vietnam as early as 2005. Fifteen viruses isolated during 2006 and 2007 belonged to clade 1 and clade 2.3.4, and were divided into five genotypes. Reassortants between the clade 1 and clade 2.3.4 viruses were detected in both North and South Vietnam. We also assessed the replication and pathogenicity of these viruses in mice and found that these isolates replicated efficiently and exhibited distinct virulence in mice. Our results provide important information regarding the diversity of H5N1 viruses in nature. Influenza A virus continuously mutates while circulating in nature and overcomes host immunity from previous infections, posing great challenges to disease control Highly pathogenic avian influenza (HPAI) H5N1 viruses have now spread through poultry populations in many countries. These viruses have also crossed species barriers to infect different hosts http://www.who.int]). Nationwide vaccination programs and culling strategies have been performed to control the disease, which has greatly contributed to a reduction in outbreaks. But despite these great efforts to control the disease, HPAI H5N1 viruses continue to evolve and cause outbreaks in poultry and human infections in Vietnam.Vietnam is one of the highest frequencies of HPAI H5N1 outbreaks. HPAI H5N1 virus was first identified in Vietnam in 2001 To better understand the molecular and biological properties of H5N1 avian influenza viruses, we selected 15 H5N1 strains isolated from poultry in Vietnam during 2006 and 2007 and sequenced their entire genomes. We performed phylogenetic analyses combining with the sequence data from the Vietnam influenza viruses and other representative viruses available in public databases, and then genotyped the viruses on the basis of their whole genomes. We also assessed the replication and pathogenicity of these viruses in mice. Understanding the molecular and biological features of avian H5N1 viruses will help reveal the potential evolutionary and transmission features of H5N1 viruses, and benefit disease control and pandemic preparedness.The 15 HPAI H5N1 viruses used in this study were isolated from domestic poultry, including chickens, Muscovy ducks, and ducks on farms in Vietnam. Details of these viruses are given in Viral RNA was extracted from allantoic fluid by using TRIZOL Reagent (Invitrogen), and was then reverse-transcribed. A set of fragment-specific primers (primer sequences available on request) was used for the PCR amplification and sequence analysis. The PCR products were purified with the Watson PCR purification kit (Watson) and sequenced by using the CEQ DTCS-Quick Start Kit on a CEQ 8800 DNA sequencer (Beckman Coulter). Sequence data were compiled with the SEQMAN program . Phylogenetic trees were generated with MEGA 4.0 by neighbor-joining (NJ) methods and bootstrap tests  based on the sequences for the open reading frames (ORFs).The nucleotide sequences obtained in this study are available from GenBank (accession nos. JX420123- JX420242).2 and inoculated intranasally with 106.0 EID50 of H5N1 influenza virus in a volume of 50 µL 1.0 to 106.0 EID50 of virus in a volume of 50 µL and calculated by using the method of Reed and Muench Groups of eight six-week-old female BALB/c mice  were lightly anesthetized with COThis study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Ministry of Science and Technology of the People’s Republic of China. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences .To determine the molecular features of H5N1 avian influenza viruses in Vietnam, all eight gene segments of the 15 viruses were sequenced and those sequences were compared with the sequences in public databases of 25 representative influenza viruses from Vietnam and 4 representative viruses from China. The HA genes of these 44 viruses belonged to seven different clades of the WHO influenza (H5N1) nomenclature system , which could be further divided into five different groups on the basis of their evolutionary relationships .Group 1 contained five clade 0 viruses, three of which were isolated from the eggs of Vietnamese waterfowl in 2005 Analysis of the deduced amino acid sequences of the HA genes showed that all of the 15 isolates sequenced had a series of basic amino acids at the HA cleavage site (-RRRKR/−RRKKR-), a characteristic of highly pathogenic influenza viruses in chickens The NA genes of the 15 strains sequenced and DK/VN/208/05 mainly formed three groups . HomologThe PA genes of the 15 sequenced strains mainly formed two groups . Group 1The PB2 and PB1 genes of the 15 strains also formed two groups, similar to those of the PA genes; however, the PB2 and PB1 genes of MDK/VN/22/07 were included in group 1. Similarly, the same trends were seen in the phylogenetic tree for the NS genes of the viruses. The NP and M genes of all of these viruses shared over 97% homology and were considered to belong to one lineage. Mutations at certain positions in the M2 protein, including I26, A27, and N31, were associated with the amantadine resistance of influenza viruses On the basis of genomic diversity, the 15 strains sequenced in this study were divided into five genotypes . Genotyp6EID50 of virus; three mice were euthanized on day 3 p.i. and their organs, including lung, spleen, kidney, and brain, were collected for virus titration in eggs, while the remaining five mice were observed for two weeks for changes in body weight or signs of death. As shown in 6EID50, and determined their 50% mouse lethal doses (MLD50) as described previously 50 values for these five viruses ranged from 2.5–3.5log10EID50 (To determine the ability of H5N1 avian influenza viruses to replicate and be pathogenic in mammals, we tested 12 viruses in mice. Groups of 8 mice were inoculated intranasally with 10g10EID50 . These r. The clade 0, calde 3, clade 5, and clade 7 viruses have been detected in poultry or poultry products in Vietnam HPAI H5N1 viruses were detected in Vietnam as early as 2001 Analysis of the 15 viruses sequenced in this study further revealed that the dominant viruses circulating in Vietnam in 2006 and 2007 belonged to clade 1 and clade 2.3.4. A previous study reported that HPAI H5N1 viruses were concentrated in specific geographical regions, with clade 1 viruses mainly in Southern Vietnam and clade 2.3.4 viruses mainly in Northern Vietnam Most Eurasian HPAI viruses isolated since 1997 can replicate in mammals The virulence of influenza virus is determined by multiple gene products and amino acid sites. Several determinant sites in the PB2, PA, HA, NS1, and M1 genes are associated with the virulence of avian influenza viruses in mammals In summary, we characterized the genetic and biological diversity of HPAI H5N1 viruses isolated in Vietnam and uncovered important information that contributes to our comprehensive understanding of these viruses. Influenza viruses spread in nature all year around in tropical regions


Yo antibodies are associated with paraneoplastic cerebellar degeneration (PCD). We have characterized Yo sera by measuring CDR2 and CDR2L antibodies and the localization of their antigens.Forty-two Yo sera from patients with paraneoplastic neurological syndromes (PNS), 179 sera from ovarian and 114 sera from breast cancer patients without PNS and 100 blood donors were screened for CDR2 and CDR2L antibodies by radioactive immune assay (RIA). Fluorescence microscopy was also used to determine the presence of CDR2 or CDR2L antibodies by staining of HeLa cells transfected with CDR2 or CDR2L fused to green fluorescent protein (GFP). Confocal microscopy was further used to localize the CDR2 and CDR2L proteins.RIA showed that 36 of the 42 Yo positive sera contained CDR2 and CDR2L antibodies whereas 6 sera contained only CDR2 antibodies. Five of the ovarian cancer patients had CDR2L antibodies and 4 of the breast cancer patients had either CDR2 or CDR2L antibodies. Only patients with both antibodies had PCD. RIA and staining of transfected cells showed similar results. Yo antibodies were not present in the 100 blood donors. Confocal microscopy showed that CDR2 and CDR2L were localized to the cytoplasm, whereas CDR2L was also present on the cell membrane.Yo sera usually contain CDR2 and CDR2L antibodies and both antibodies are associated with PCD. Since only CDR2L is localized to the cell membrane it is likely that CDR2L antibodies may be of primary pathogenic importance for the development of PCD. Patients with paraneoplastic cerebellar degeneration (PCD) often harbour Yo antibodies which cross-react with antigens in tumours (often ovarian or breast cancer) and Purkinje cells in the cerebellum PCD is characterised by rapid development of pancerebellar symptoms and loss of Purkinje cells Yo antibodies react with a 62 kDa protein (454 amino acids), the cerebellar degeneration-related protein 2  www.proteinatlas.org) shows strong staining in Purkinje cells while the CDR2 antibodies HPA018151 and HPA023870 show moderate and weak staining, respectively. We therefore hypothesise that Yo antibodies could be directed against both CDR2 and CDR2L with CDR2L being the primary target on Purkinje cells. This was supported by the Genevestigator gene expression search engine (www.genevestigator.com), indicating low to medium CDR2 expression potential in the nervous system , and medium to high CDR2L mRNA levels , both based on the human genome 47 k array and 47 samples included.Given the high sequence identity between CDR2 and CDR2L, we asked if Yo antibodies could cross-react with both antigens. The CDR2L specific antibody HPA022015  and approved by the Regional Committee for Medical and Health Research Ethics in Western-Norway, Diagnostic markers of cancer (188.05). The retrospective study of patient records was also approved by the Regional Committee for Medical and Health Research Ethics in Western-Norway and the clinical data were part of a larger retrospective study on clinical correlations with onconeural antibodies . For both the bio-bank and the retrospective study, the regional ethics committee as well as the Ministry of Health and Care Services specifically waived the need to obtain consent , due to the large number of included subjects and high number of deceased subjects. All participants were adults.First, we screened 42 Yo positive sera (with antibodies against CDR2) sent to the Neurology Research Laboratory, Haukeland University Hospital, Bergen, Norway for CDR2L antibodies using a transcription-translation and immunoprecipitation (IP) technique. Subsequently, we screened sera from patients with ovarian (n = 179) and breast (n = 114) cancer, as well as 100 blood donors for CDR2L antibodies using the same IP technique. These blood donor sera have previously been examined for CDR2 antibodies www.origene.com, CDR2 sequence transferred from vector RG204900 with C-terminal green fluorescent protein (GFP) to vector PS100001 with C-terminal myc-DDK tag) or CDR2L  were cloned into expression vectors with a T7 promoter. For the IP experiments, two polyclonal rabbit antibodies from Eurogentec (www.eurogentech.com) which were produced against CDR2 (two synthetic peptides corresponding to amino acid 123–138 (EELKSSGQGRRSPGKC) and amino acid 429–443 (CDEQRTKYRSLSSHS) of the human CDR2 sequence) and CDR2L (two synthetic peptides corresponding to amino acid 282–297 (APEADDPQPGRGDDLG) and amino acid 392–407+C (CICRDSSWRDLRGGEEG) of the human CDR2L sequence) were used as reference sera. The antibodies were specific and did not cross react in the IP assay. CDR2 and CDR2L patient antibodies were then tested by the IP technique using the recombinant proteins as antigens Full-length CDR2  or CDR2L  were used to transfect HeLa cells following the standard protocol for Lipofectamine2000 . Transfected cells were fixed and immunocytochemically labelled 24 h after transfection.HeLa cells (ATCC-CCL-2) were chosen as a cell-line with a human background that can easily be transfected with a high reproducibility of the results. Cells were grown in DMEM, supplemented with 10% FCS (Sigma), L-Glutamine  and Pen Strep . cDNA for full-length CDR2 (HeLa cells (ATCC-CCL-2) were grown on poly-L-Lysine coated glass-coverslips (10 mm). Immunocytochemical staining of HeLa cells with patient sera and antibodies against CDR2 and CDR2L was performed as described elsewhere Imaging of HeLa cells was performed with a Zeiss Axiovert M200 fluorescence microscope with a 40x objective and TILL photonics monochromator or Leica TCS SP5 with Blue diode 405, Argon laser 488, Helium/Neon I laser 594, objective HCX PL Apo 63x.Patient sera with CDR2 and/or CDR2L antibodies were tested by immunofluorescence using rat cerebellar tissue or immunoblot using recombinant onconeural proteins including CDR2  , CDR2L   and CDR2L  . The immunoperoxidase staining followed the standard procedures at the Department of Pathology, Haukeland University Hospital.www.gelifesciences.com) and then read in a luminicent image analyzer.We subjected extracts of human cerebellum (150 µg) as well as lysates of recombinant CDR2 or CDR2L (from the IP test) to a 10% SDS-PAGE and performed Western blotting Among 42 sera previously tested for paraneoplastic antibodies, 36 were positive for both CDR2L and CDR2 antibodies using the IP technique  and all Twenty eight of the 36 CDR2L and CDR2 antibody positive patients had PNS; 27 had PCD  and one had isolated myelopathy. Three patients had no known PNS and no clinical information was available from five. Of the 36 CDR2L and CDR2 antibody positive patients, 18 had ovarian, 5 uterus , 1 colon and 4 breast cancer; in 8 patients we had no information on a possible underlying tumour .We found 8 patients with only CDR2 antibodies, of which 6 were from the 42 routine sera. Two of the 8 patients had neuropathy, 5 had no known PNS and for one patient we had no clinical information. They all had different cancers; pharyngeal, prostate, uterus, colon, lung cancer or astrocytoma . Two CDRIn 7 patients, we found only CDR2L antibodies; 5 of these were from the ovarian cancer and 2 from breast cancer cohort, and none had PNS. All of the CDR2 or CDR2L positive sera had low antibody avidity and none of them were positive by line blot or with immunofluorescence, including antigen retrieval protocol. None of the 100 blood donors were positive for CDR2L or CDR2 antibodies.Three patient sera that were shown to have different Yo antibody composition in IP experiments were chosen for IC labelling of HeLa cells expressing either CDR2-GFP  A1–3, buTo determine if CDR antigens in intact cells are available to react with Yo antibodies, we performed a surface staining of HeLa cells transfected with CDR2-myc or CDR2L-myc . Cells wPolyclonal CDR2L antibodies gave a granular cytoplasmic staining of human Purkinje cells, but no staining of these cells was seen by the CDR2 antibodies . NeitherThe polyclonal CDR2 and CDR2L antibodies used for the Purkinje cell staining showed no cross reactivity between the recombinant CDR2 and CDR2L antigens . The CDRWe found that 36 of 42 CDR2 positive patient sera also contained antibodies to CDR2L. The majority of the patients with both CDR2 and CDR2L antibodies had PCD, most often associated with ovarian cancer. The co-existence of these antibodies indicated that both antibodies had high avidity for their antigens. It is known that high avidity antibodies are more likely to be of pathogenic importance since they represent a more long-standing immune response When CDR2L or CDR2 antibodies occurred alone they had low avidity. Of the 15 sera that were positive only for one of these antibodies, only two of 8 CDR2 positive patients had possible PNS (neuropathy) and none with only CDR2L antibodies manifested a paraneoplastic syndrome. This suggests that the co-existence of these antibodies may be of relevance for the development of PNS, especially PCD. Low avidity CDR2 or CDR2L antibodies were all associated with cancer, but probably of most importance in the general tumour immune response and less for the development of PNS.Although patient sera containing only CDR2 or CDR2L antibodies gave negative immunohistochemistry, these antibodies did stain HeLa cells transfected with their respective antigens. This is in line with the assumption that when occurring alone these antibodies have low avidity and mirrors findings with acetylcholine receptor (AChR) antibodies in which transfected cell assays are able to detect low avidity antibodies due to cross reactivity of the antigens on the cell surface We have shown previously that the IP test is more sensitive than both immunohistochemistry and line blot for the detection of paraneoplastic antibodies HeLa cells transfected with CDR2L showed a different distribution of positivity than those transfected with CDR2. Localisation of CDR2L to the plasma membrane suggests a possible biological function similar to other membrane associated antibodies such as AChR and P/Q voltage-gated calcium channel antibodies (VGCC) that have been shown to be of pathogenic importance in myasthenia gravis and Lambert-Eaton myasthenic syndrome The polyclonal CDR2L antibodies showed a granular cytoplasmic staining of the human Purkinje cells, similar to that described for Yo positive sera www.genevestigator.com, probeset 213230_at (CDR2L) and 209501_at (CDR2).The polyclonal CDR2 antibody did not react with human or rat Purkinje cells, even after antigen retrieval. Western blot studies of human cerebellar extract showed weak reactivity with the CDR2 antibody, but strong reactivity with the CDR2L antibodies. These results suggest that CDR2L is more highly expressed in human Purkinje cells than CDR2, findings that are in line with data showing that CDR2L mRNA levels are much higher than CDR2 levels in human cerebellum (biogps.org and www.proteinatlas.org). We have shown previously that CDR2 is present in various tumours regardless of CDR2 antibody status According to the human protein atlas, both CDR2L and CDR2 are found in various human tumours  showed that Yo antibodies can be taken up by cultured rat Purkinje cells. Our results suggest that it is likely that these sera contained both CDR2 and CDR2L antibodies, and that the co-expression of these antibodies is necessary for rat Purkinje cell staining. Moreover, based on our results, it is likely that the Yo antibody staining of the Purkinje cells in these experiments was due to CDR2L and not CDR2 antibodies. Furthermore, as we have shown in transfected cells, CDR2L is membrane-bound and probably therefore can mediate intracellular uptake of CDR2L antibodies.The presence of high avidity CDR2L antibodies in patients with PCD and the presence of CDR2L in Purkinje cells make it tempting to speculate as to whether these antibodies are important in the Purkinje cell death that occurs in PCD Figure S1Surface staining of HeLa cells overexpressing CDR2L-myc. HeLa cells transfected with CDR2L-myc (A1–3) were surface-stained with CDR2L antibody  and myc antibody . The surface staining was done without permeabilisation with Triton-x-100 (A1–3). Of the z-stack 15 slices were superimposed to show the localisation of CDR2L to the membrane. The overlay shows a strong co-localisation  of both antibodies, indicating that the myc staining was specific for the transfected CDR2L protein. Scale bar 20 µm.(TIF)Click here for additional data file.


Bovine papillomaviruses (BPVs) induce hyperplastic and tumoral lesions not only in cows but also in other different animal species. The transforming activity of BPVs is due to its major E5 oncogene. Recent studies have highlighted the role of E5 in cancer development but very little is known about E6 and E7 oncogenes. In this letter we argue for the need of investigating E6 as well as E7 to better understand the role of these two oncogenes during carcinogenesis. Bovine papillomavirus (BPV) is considered as causative agent of cutaneous and mucosal tumors in its natural host . HoweverIn the last decade, the role of BPV major oncoprotein E5 in cell transformation has been largely investigated in naturally occurring bovine and equine tumors. Overall, these studies confirmed the pivotal role of E5 in cancer development supporting the role of the virus -18. IntePv’s contribution to tumorigenesis is “paradigmatically” based on the actions of more than one oncogene. Human Pvs (HPVs) E5, E6 and E7 oncoproteins act synergistically disturbing different cellular pathways and in so doing they contribute to initiation and progression of cancer . MoreoveIn this regard, among BPV’s oncogenes the contribution of the other two oncogenes (E6 and E7) to cancer development is less known. It has been suggested that BPV E7 may cooperate with E5 in inducing cell transformation whereas E6 can downregulate p53 transcriptional activity by interacting with CBP/300 -24. HoweThe author declares that he has no competing interests.


We recently characterized several new broadly neutralizing monoclonal antibodies (bnMAbs) remarkable for their in vitro potency. A number of these bnMAbs are almost 10 fold more potent than previously isolated bnMAbs such as PG9 and VRC01 and hundred fold more potent than older prototype bnMAbs such as 2G12 and b12. Of the new antibodies, PGT121 is one of the most broad and potent, and recognizes a glycan-dependent epitope. Based on in vitro neutralization potency, we speculated that PGT121 may be protective in vivo at serum concentrations below those previously determined for other broadly neutralizing anti-HIV antibodies.SF162P3 was evaluated using a TZMbl-based neutralization assay. To evaluate the protective potency of PGT121 in vivo, we then designed a protection study in rhesus macaques. Animals were administered intravenously with 3 different doses of antibody  24 hours before being vaginally challenged with a single high dose of SHIV SF162P3 (300 TCID50). Serum levels of antibody and viral loads were determined throughout the study.The neutralization potency of PGT121 against SHIV SF162P3 with an IC50 of about 0.005 µg/ml. Preliminary data suggest that an administered dose of PGT121 at 1 mg/kg is sufficient to induce sterilizing immunity against a single high dose vaginal challenge of SHIV SF162P3. A lower 0.2 mg/kg dose appears to be partially protective.PGT121 potently neutralizes SHIV PGT121 is one of the most broad and potent neutralizing antibodies isolated to date. Here, we provide evidence that in vivo protection by PGT121 correlates with the high in vitro potency of the antibody. PGT121 can thus mediate sterilizing immunity at concentrations that are significantly lower than previously observed from passive protection studies with bnMAbs and may be readily achievable through vaccination.


Genetic variation of 49 human papillomavirus (HPV) 6 and 22 HPV11 isolates from recurrent respiratory papillomatosis (RRP) (n = 17), genital warts (n = 43), anal cancer (n = 6) and cervical neoplasia cells (n = 5), was determined by sequencing the long control region (LCR) and the E6 and E7 genes. Comparative analysis of genetic variability was examined to determine whether different disease states resulting from HPV6 or HPV11 infection cluster into distinct variant groups. Sequence variation analysis of HPV6 revealed that isolates cluster into variants within previously described HPV6 lineages, with the majority (65%) clustering to HPV6 sublineage B1 across the three genomic regions examined. Overall 72 HPV6 and 25 HPV11 single nucleotide variations, insertions and deletions were observed within samples examined. In addition, missense alterations were observed in the E6/E7 genes for 6 HPV6 and 5 HPV11 variants. No nucleotide variations were identified in any isolates at the four E2 binding sites for HPV6 or HPV11, nor were any isolates found to be identical to the HPV6 lineage A or HPV11 sublineage A1 reference genomes. Overall, a high degree of sequence conservation was observed between isolates across each of the regions investigated for both HPV6 and HPV11. Genetic variants identified a slight association with HPV6 and anogenital lesions (p = 0.04). This study provides important information on the genetic diversity of circulating HPV 6 and HPV11 variants within the Australian population and supports the observation that the majority of HPV6 isolates cluster to the HPV6 sublineage B1 with anogenital lesions demonstrating an association with this sublineage (p = 0.02). Comparative analysis of Australian isolates for both HPV6 and HPV11 to those from other geographical regions based on the LCR revealed a high degree of sequence similarity throughout the world, confirming previous observations that there are no geographically specific variants for these HPV types. Human papillomaviruses (HPVs) have been demonstrated to cause a diverse range of epithelial lesions. To date, 120 HPV’s have been identified and each of these types are associated with infection at particular anatomical sites Although the majority of HPV related non-malignant diseases are attributable to HPV 6 and 11 Anal cancer is uncommon in the general population, although incidence in the general Australian population is increasing Cervical cancer and its precursors namely high grade cervical intraepithelial neoplasia (CIN2/3) and adenocarcinoma insitu (AIS) are frequently associated with persistent HPV infection of types HPV16 and HPV18 Several key gene regions and their role in malignant transformation have been extensively studied in high-risk HPV types 16 and 18. For example, viral E6 and E7 oncogenes are key mediators in cell transformation through the disruption of tumour suppressor Rb/E2F and subsequent p53 pathways respectively, which are critical in regulating the initiation of DNA replication. Loss of function or disruption of these pathways due to integration of HPV DNA, leading to increased expression of E6 and E7 proteins, has been demonstrated to result in the development of cervical carcinoma Previous studies investigating genetic variation of these gene regions have used standard reference genomes for comparative purposes. These standard reference genomes for HPV6 are denoted prototypic HPV6b, and non-prototypic HPV6a and HPV6vc, and one standard reference genome for HPV11. A recent study based on phylogenetic analysis of complete genomes derived from published HPV6 and HPV11 variants has proposed a new standard nomenclature for HPV6 and HPV11 This study examines the genetic variability of the LCR, E6 and E7 gene regions in both HPV6 and HPV11 across different lesion types to determine whether different disease states resulting from single HPV6 or HPV11 infection cluster into distinct variant groups. In addition, current data on the genetic diversity of HPV6 and HPV11 variants present within the Australian population are very limited. Only one study has been conducted within the Australian population, sequencing only a portion of the LCR for 47 HPV6 or HPV11 RRP isolates All patients provided written informed consent at time of treatment whereby ethics approval was received from: the human research ethics committee at St Vincent’s Hospital, Sydney, Australia for anal cancer biopsies, the health ethics research committee at the Royal Perth Hospital, Perth, Australia for genital wart biopsies, and the human research ethics committee at the Royal Women’s Hospital, Melbourne, Australia for the collection of cervical cells. In addition, ethics approval was obtained from the ethics in human research committee of the Royal Children’s Hospital, Melbourne, Australia for juvenile RRP biopsies whereby written informed consent was provided by either the parent or guardian at time of treatment.Based on single HPV infection status a total of 71 HPV 6 and HPV11 isolates from four different disease states being: RRP, genital warts, anal cancer, and cervical neoplasia cells were included in this study. Formalin fixed, paraffin embedded (FFPE) biopsies collected at the Royal Children’s Hospital, Melbourne between 1964 and 1994 from 17 juvenile RRP patients described previously For FFPE archival tissue, 5 – 10 µm sections were deparaffinised with 800 µl histolene, then mixed with 400 µl absolute ethanol. The tissue was centrifuged at 14,000 × g for 2 minutes. The resultant pellet was washed with 1 ml of absolute ethanol, centrifuged for 2 minutes and the supernatant was discarded. The pellet was air dried and subsequently incubated for 4 hrs at 55°C with 160 µl tissue lysis buffer  and proteinase K at a final concentration of 1 mg/ml. DNA was then isolated from digested tissue using the MagNA Pure LC DNA Isolation Kit I (Roche Diagnostics) on the automated MagNA Pure LC extraction system, according to the manufacture’s protocol, with the addition of 33.3 µg/ml of poly(A) RNA carrier to the lysis buffer which has been demonstrated to improve extraction efficiency. Nucleic acid was eluted into a final volume of 100 µl.Fresh 1–2 mm biopsies were homogenised using MagNA Lyser (Roche Diagnostic) with 250 µl of Tissue Lysis Buffer (Roche Diagnostics), by two repetitions of 30 seconds at a speed of 7000 rpm in the MagNA Lyser Instrument. Proteinase K was added to a final concentration of 1 mg/ml, and samples were incubated at 55°C until fully digested. DNA was then isolated from the digested tissue as described above. DNA was isolated from the PreservCyt samples of cervical cells using the MagNA Pure LC DNA Isolation Kit I on the automated MagNA Pure LC extraction system, according to a modified protocol HPV genotyping of all archival FFPE tissues were confirmed by using the INNO-LiPA HPV genotyping test  which has demonstrated increased sensitivity and accuracy for archival tissue DNA positive for HPV6 or HPV11 samples were subject to type and gene specific PCRs. All primers used for genetic variation analysis of HPV6 LCR, E6 and E7 , were de2, 1 U AmpliTaq® Gold DNA Polymerase and 1X reaction buffer , and 2.5 to 5 µl of DNA template. Following a denaturation step of 94°C for 9 min, PCR steps at 94°C for 30 sec, 55°C for 20 sec and 72°C for 30 sec for 45 cycles, followed by an extension step at 72°C for 10 min, on a GeneAmp PCR System 9700 thermocycler (Applied Biosystems). Amplicons were run on a 2% agarose gel for visualization, using 1000 base pair HyperLadder IV  for sizing.All PCR reactions were performed in a 50 µl reaction volume containing 0.5 µM of each primer, 2 mM of each deoxynucleoside triphosphate, 2.5 mM MgClAmplicons were sent to the Australian Genome Research Facility for purification and Sanger DNA sequencing using a AB3730xl capillary sequencer (Applied Biosystems). To minimise PCR and sequencing errors, sequencing was performed on both forward and reverse stands with predicted sequencing error of 0.01% based on sequencing of control DNA. For regions which did not meet our quality control criteria  confirmation of HPV sequence was carried out by repeating both the forward and reverse strands in duplicate. Sequences were assembled using SeqMan version 8  and aligned against reference genomes using SeaView version 4 The HPV6 nucleotide sequences for the LCR, E6, E7 gene regions are deposited in the GenBank database under the accession numbers: LCR sequences (KC300093-KC300140), E6 sequences , E7 sequences (KC300189-KC300237).The HPV11 nucleotide sequences for the LCR, E6, E7 gene regions are deposited in the GenBank database under the accession numbers: LCR sequences (KC329850-KC329871), E6 sequences (KC329872-KC329893), E7 sequences (KC329894-KC329915).Data in categories were evaluated using two-tailed Fisher’s exact test.This study used samples with either HPV6 or HPV11 infection. Of the entire sample set for each lesion type a total of 49 specimens were identified as HPV6 isolates, that is 8 from RRP lesions, 33 genital warts, 4 anal cancer biopsy specimens and 4 cervical cell samples. In addition, a total of 22 specimens were identified as HPV11 isolates, that is 9 RRP lesions, 10 genital warts, 2 anal cancer biopsy specimens and 1 cervical cell sample . For theAll 49 HPV6 isolates were successfully amplified and sequenced across the E6 (nt 102–554) and E7 (nt 530–826) ORFs. For HPV6 E6/E7, a total of 18 single nucleotide variations (13 in the E6 and 5 in the E7 ORF) from the lineage A reference genome were identified . No isolA total of 48 HPV6 isolates were successfully amplified and sequenced across the LCR . Single nucleotide variations from the lineage A reference genome were identified at 45 sites across the LCR. In addition, 5 insertions and 4 deletions were identified . Three vA total of 105 HPV6 LCR sequences were used to perform a comparative phylogenetic analysis, which included 48 Australian isolates from this study, 45 Slovenian All 22 HPV11 isolates were successfully amplified and sequenced across the E6 (nt 102–554) and E7 (nt 530–826) ORFs. Three genomic variants were identified across the E6/E7 ORFs for HPV11 . None ofThe LCR from 22 HPV 11 isolates was successfully amplified and sequenced . Comparative analysis with the HPV11 sublineage A1 reference sequence revealed 13 single nucleotide variations, 1 insertion and 3 deletions in the LCR . A totalComparative phylogenetic nucleotide sequence analysis was carried out on a total of 94 HPV11 LCR sequences. These publicly available sequences included 22 Australian isolates from this study, 63 Slovenian We present one of the largest studies of Australian isolates to date which investigates genetic variation for HPV6 or HPV11 in some of the key HPV gene regions thought to be associated with development of lesions and disease progression in epithelial cells. This study compares genetic variation within various lesion types sampled from the Australian population for the two most prevalent types of low risk HPV associated with these diseases. Analysis of genetic variance revealed slight association with HPV6 and anogenital lesion and HPV11 and RRP with genetic variants HPV 6 B1 lineage being more strongly associated with anogenital lesion.The majority of HPV11 isolates from this study clustered to the variant A2–1 for E6 and E7 . This vaThis study identified a series of variations in the E6 and E7 ORFs of the HPV6 genome within the Australian lesions sampled. A total of three amino acid alterations in each of the E6 and E7 ORFs of different HPV6 isolates were found, some of which were unique to a single isolate, while others were found in several or multiple cases. Of these, several unique amino acid alterations were identified: E113D in E6 and E73K in E7. Conversely, amino acid alterations E19K and H50Q in the E6 ORF and F52Y and N88D in the E7 ORF, present in numerous isolates, have been described previously When the prevalence of HPV-type in genital warts and RRP (lesions for which n>10) was considered, a type specific distribution was indicated for genital warts but not RRP. The prevalence of HPV 6 was found to be higher for genital warts (77% HPV6 vs 23% HPV11) in this sample set, as described previously by Variant analysis of the LCR within Australian isolates was completed for both HPV6 and HPV11. In recent years, a growing number of studies have described HPV6 LCR variations in isolates from the nucleotide positions of the lineage A reference genome Genomic regions such as the LCR have been used by previous studies to identify genetic variants as it provides enough genetic variation to differentiate between variants in both HPV6 and HPV11. For this reason, the LCR was selected for comparative phylogenetic sequence analysis between this and previous studies to determine whether genetic lineages previously described This current study also provides unique pre-vaccination data on the genetic variation present in RRP, anal cancer, cervical cells samples for HPV6 and HPV11 within the Australian population. For genital warts, based on age  and sex , at least 88% of these samples would have come from women and men ineligible for publically funded prophylactic HPV vaccination. The remaining 12% of genital warts were derived from women <26 years, whose vaccination status was unknown. The high proportion of genital wart isolates positive for HPV6 or HPV11 suggests that lesions developed either in unvaccinated patients, or in women who were vaccinated with prevalent infection. Australia was the first country to introduce a government-funded, population-based mass vaccination program in 2007 of the female population (12 to 26 years), using the quadrivalent vaccine Garadasil®  targeting HPV types 16, 18, 6 and 11. It has already seen a reduction in the number of cases of genital warts due to a high coverage of vaccine In summary, we found slight association between HPV6 with anogenital lesions with sublineage B1 being predominate. This is consistent with the findings of other studies and suggests that viral and/or host epigenetic and other host factors (such as those associated with immune response) may play significant roles in disease susceptibility and progression. In addition, larger studies investigating whole genome sequences may shed further light on possible associations with pathogenesis. However, this study makes a significant contribution toward the slowly expanding knowledge about HPV6, and in particular HPV11 genomic diversity and pre-vaccination circulating HPV6/11 variants within the Australian population.Table S1Summary of HPV6 and HPV11 lesion types.(DOCX)Click here for additional data file.Table S2Summary of genetic variation identified across all lesion types for HPV6 and HPV11.(DOCX)Click here for additional data file.Table S3Primers used for amplifying and sequencing the LCR, E6 and E7 ORFs.(DOCX)Click here for additional data file.Table S4HPV6 nucleotide and amino acid sequence variation in the ORF of E6 and E7 from 49 clinical isolates representing four different lesion types.(DOCX)Click here for additional data file.Table S5HPV6 nucleotide sequence variation in the LCR from 48 clinical isolates representing four different lesion types.(DOCX)Click here for additional data file.Table S6HPV11 nucleotide and amino acid sequence variation in the ORF of E6 and E7 from 22 clinical isolates representing four different lesion types.(DOCX)Click here for additional data file.Table S7HPV11 nucleotide sequence variation in the LCR from 22 clinical isolates representing four different lesion types.(DOCX)Click here for additional data file.


Point mutations in LRRK2 cause autosomal dominant Parkinson's disease. Despite extensive efforts to determine the mechanism of cell death in patients with LRRK2 mutations, the aetiology of LRRK2 PD is not well understood. To examine possible alterations in gene expression linked to the presence of LRRK2 mutations, we carried out a case versus control analysis of global gene expression in three systems: fibroblasts isolated from LRRK2 mutation carriers and healthy, non-mutation carrying controls; brain tissue from G2019S mutation carriers and controls; and HEK293 inducible LRRK2 wild type and mutant cell lines. No significant alteration in gene expression was found in these systems following correction for multiple testing. These data suggest that any alterations in basal gene expression in fibroblasts or cell lines containing mutations in LRRK2 are likely to be quantitatively small. This work suggests that LRRK2 is unlikely to play a direct role in modulation of gene expression, although it remains possible that this protein can influence mRNA expression under pathogenic cicumstances. Mutations in Leucine Rich Repeat Kinase 2 (LRRK2) are the most common Mendelian genetic cause of Parkinson's disease (PD) currently identified Drosophila model of LRRK2 disease Drosophila data would be that steady state mRNA levels of miRNA targets are altered in the presence of mutant LRRK2. To test whether mutations in LRRK2 cause an alteration in basal gene expression we have examined the impact of mutations in three contexts: fibroblast cells cultured from PD patients carrying mutations in LRRK2 , brain tissue from 5 patients carrying the G2019S mutation compared to idiopathic PD and control brain tissue In addition, a recent study has suggested that pathogenic forms of LRRK2 bind directly to the miRNA processing enzyme Argonaute and thus influence mRNA levels in a 2 sterile Petri dishes at 37°C in 5% CO2 in 2 ml DMEM  until fibroblasts were seen to migrate from the skin explants 2 dishes for culturing. Samples were designated anonymous identifiers following collection. For collection of RNA, cells were plated in 10 cm plates at equivalent passage number (n = 7) and at equal density. Following 24 hours growth, RNA was extracted using the RNeasy kit (Qiagen) and RNA quality ascertained prior to microarray analysis using RIN analysis (Agilent). Western blot analysis of endogenous LRRK2 expression was carried out by immunoblotting following BCA assay , with 10 µg of lysate loaded onto 4–12% Bis Tris gels (Invitrogen) following denaturation in 4x sample buffer (Invitrogen) supplemented with 5% β-Mercaptoethanol (Sigma). Proteins were transferred to PVDF membrane (Millipore) and membranes blocked with 5% milk solution in Phosphate buffered saline for 1 hour prior to probing with primary (90minutes) and secondary (60minutes) antibodies. For LRRK2, rabbit monoclonal MJFF2 (Epitomics – see http://www.pdonlineresearch.org/discussions/lrrk2-antibodies for characterisation of these antibodies) was used at a 1∶1000 dilution, for β Actin mouse monoclonal AC-74 (Sigma) was used at a 1∶5000 dilution. HRP secondary antibodies were used at a dilution of 1∶2000 and 1∶10000 for LRRK2 (rabbit) and β Actin (mouse) respectively. Membranes were washed three times in PBS supplemented with 1% tween, and exposed to Supersignal ECL substrate (Pierce). Membranes were exposed to Kodak biomax film and developed on a Fujifilm developer as per manufacturers instructions.Fibroblast cultures were isolated from PD patients with LRRK2 mutations and healthy controls  and Sun Health brain bank  Illumina HumanWG-6 and HumanHT-12, and Affymetrix Exon 1.0 ST Arrays were used according to manufacturer's instructions as previously described We compared gene expression in three systems: an inducible HEK cell model, patient fibroblasts and patient brain samples for wild type and mutant LRRK2. To confirm expression of LRRK2 in these systems, we carried out western blot analysis using anti-V5 and anti-LRRK2 antibodies for the HEK cells and fibroblasts/brains samples respectively . In the Having confirmed the expression of LRRK2 at the protein level in these systems, we carried out genome wide expression analysis. In the HEK cells we compared each cell line induced for 12 h or 24 h to its uninduced controls; no differences in gene expression were seen in either WT or R1441C expressing cells . SimilarWe also examined gene expression in brains of LRRK2 carriers compared to controls and to idiopathic PD . For thiWe considered that simply looking for genes whose steady state expression might miss more subtle patterns in the data and therefore examined overall gene expression by unsupervised hierarchical clustering . SamplesDrosophila and a HEK cell model This study assessed a series of patient fibroblast cells harbouring mutations in LRRK2, brain samples from G2019S carriers and a cell line system over-expressing LRRK2 wild type and mutant transgenes for alterations in gene expression due to the presence of mutant LRRK2. Overall, our data show that any differences in gene expression are lower than the relatively modest cutoffs  used. In turn, this suggests that mutant LRRK2 does not elicit large changes in steady state mRNA levels within the cell under normal growth conditions. This is in contrast to a recent publication studying a series of mononuclear cells carrying the G2019S mutation via the mTOR pathway, and it is plausible that LRRK2 exerts an influence on gene expression following activation in response to cellular stress. With regard to alterations due to miRNA regulation, our data demonstrates no overall impact on mRNA levels. This does not exclude translational regulation as a mechanism through which a putative LRRK2 miRNA pathway could operate, although the recent publication by Guo et al emphasizes that miRNAs in mammalian cells predominantly function by directly impacting on mRNA levels rather than by premature translation termination or inhibiting ribosome formation or binding Our results do not exclude the possibility that gene expression can be differentially altered by mutations in LRRK2 under conditions of stress, for example under starvation conditions, oxidative challenge or widespread cell loss via its kinase activity or through altered recruitment of other co-factors to an active enzymatic complex, aspects of the biology of LRRK2 that are undergoing investigation in a number of laboratories In summary, our data provides evidence from two independent cellular model systems and brain tissue from patients that LRRK2 mutations do not result in large alterations in basal gene expression under normal growth conditions. These data raise the possibility that mutant LRRK2 exerts its pathogenic affect through an alternative mechanism, for example through post translational modification


Bmal1 fed either a normal or a high fat diet (22% by weight). Bmal1 null mice aged 2 months were killed across 24 hours and plasma adiponectin and leptin, and adipose tissue expression of Adipoq, Lep, Retn and Nampt mRNA measured. Glucose, insulin and pyruvate tolerance tests were conducted and the expression of liver glycolytic and gluconeogenic enzyme mRNA determined. Bmal1 null mice displayed a pattern of increased plasma adiponectin and plasma leptin concentrations on both control and high fat diets. Bmal1 null male and female mice displayed increased adiposity (1.8 fold and 2.3 fold respectively) on the normal diet, but the high fat diet did not exaggerate these differences. Despite normal glucose and insulin tolerance, Bmal1 null mice had increased production of glucose from pyruvate, implying increased liver gluconeogenesis. The Bmal1 null mice had arrhythmic clock gene expression in epigonadal fat and liver, and loss of rhythmic transcription of a range of metabolic genes. Furthermore, the expression of epigonadal fat Adipoq, Retn, Nampt, AdipoR1 and AdipoR2 and liver Pfkfb3 mRNA were down-regulated. These results show for the first time that global loss of Bmal1, and the consequent arrhythmicity, results in compensatory changes in adipokines involved in the cellular control of glucose metabolism.The close relationship between circadian rhythm disruption and poor metabolic status is becoming increasingly evident, but role of adipokines is poorly understood. Here we investigated adipocyte function and the metabolic status of mice with a global loss of the core clock gene  Bmal1 ClockPer1, Per2, Cry1, Cry2, Nr1d1) upon binding to E-box elements in the promoters of the genes. After complexing with Casein kinase 1ε/δ, the PER/CRY/Casein kinase complex returns to the nucleus and inhibits the action of BMAL1/CLOCK, thus reducing the expression of the period and cryptochrome genes. NR1D1 plays an important role in the generation of rhythmicity through its repression of Bmal1 expression. We Clock is associated with alterations in metabolism. The Clock19Δ mutants produce a protein that can bind to BMAL1, but not induce gene expression, resulting in a loss of rhythmic gene expression in peripheral tissues, but not the SCN Clock in certain tissues  are rescued in Clock19Δ mutant mice and Clock null mice by a homologue, Npas2Bmal1 null mouse, originally produced by the Bradfield laboratory Clock19Δ mutant or Clock null mice, Bmal1 null mice have poorly entrained wheel running behaviour under a normal photoperiod and are arrhythmic in continuous darkness Bmal2 rescues the functions of Bmal1 in the Bmal1 null mouse Bmal1 null mice are infertile The links between circadian rhythms of gene expression, hormone secretion and metabolism have emerged over recent years. For example glucose metabolism changes across the day and night in humans Bmal1 expression on plasma adipokine levels (adiponectin and leptin) and adipose tissue expression of adiponectin (Adipoq), leptin (Lep), resistin (Retn) and visfatin (Nampt) mRNA in young animals (2 months of age), before any confounding pathology was likely to emerge. In addition we report the effects of global loss of Bmal1 expression on glucose, insulin and pyruvate tolerance tests and expression of liver glycolytic and gluconeogenic enzyme mRNA. Finally we investigated the metabolic impact of a high fat diet on body and adipose tissue weight, plasma metabolites, insulin and adipokines in Bmal1 null mice.In this study we report for the first time the effects of loss of Bmal1 null mice ad libitum. The genotypes of the offspring were determined as previously described by PCR of tail DNA The founder Bmal1 null male mice and their wild-type litter mate controls (4 mice per time point) were killed by decapitation at 2 months of age every four hours across 24 hours at 0800 h, 1200 h, 1600 h, 2000 h, 2400 h and 0400 h. Blood was collected into heparinised tubes and plasma harvested for metabolite and hormone assays. Liver and epigonadal fat were rapidly dissected and immediately placed in RNAlater®  and then stored at −20°C until processing. An additional group of 6 month old male Bmal1 null and wild-type mice (4–9 mice per time point) were killed at the same times of day and blood collected.Groups of Bmal1 null and wild-type mice were fed a high-fat diet (22% fat (wt/wt), 0.15% cholesterol, 4.6 kcal/g, SF00-219, Specialty Feeds, Glen Forrest, Western Australia) or the control diet from 3 to 8 weeks of age (n = 10–19 mice of each sex per genotype). Animals were weighed and killed during the mid light period (1400 h), trunk blood collected and the epigonadal/retroperitoneal fat pads, testes/uteri and kidneys were dissected and weighed.To determine the effects of a high-fat diet on plasma hormones and metabolites, male and female Bmal1 null male mice aged 2 and 6 months (n = 5–6) were maintained on the control diet, fasted overnight and injected with glucose  starting 2 h after the lights were turned on as previously described Wild-type and Bmal1 null 2 month old male mice (n = 6) and female mice (n = 5–7) were maintained on the control diet, fasted overnight and injected with sodium pyruvate  starting 2 h after the lights were turned on as previously described Wild-type and Bmal1 null male and female mice (n = 6 per gender) aged 2 months were maintained on the control diet and had food withheld for 2 hours before injection of insulin  2–3 h after lights on as previously described Wild-type and To investigate the expression of clock and clock controlled genes in the liver and adipose tissue total mRNA was extracted, reverse transcribed and amplified by real time PCR. Some of the primers have been used previously by our group Plasma glucose and free fatty acids were measured enzymatically post hoc analysis using the Bonferroni correction for multiple comparisons. For the body composition, hormone and gene expression data collected across 24 h, the Estimated Marginal Means are reported in the text for the various comparisons. To determine whether hormone and gene expression data were rhythmic, i.e., fitted a sine curve, the data were analyzed using CircWaveBatch; http://hutlab.nl/Hormone, body weight, organ weight and gene expression data were analyzed by univariate ANOVA (SPSS v17), using genotype and time of day as the dependent variables followed by Bmal1 null mice, however, plasma insulin was lower (P<0.02) and adiponectin (P<0.001) and leptin (P<0.001) were higher in Bmal1 null compared to wild-type mice , higher adiponectin  and higher leptin  than wild-type mice.Similar differences in plasma insulin and adipokines between Bmal1 null mice compared to wild-type mice while plasma glucose and free fatty acids were unchanged. These differences persisted in 6 month old mice.In summary, across 24 hours, plasma insulin was lower and adiponectin and leptin were both higher in 2 month old male Bmal1 null and wild-type mice had similar fasting blood glucose (data not shown). The peak glucose levels and area under the curve during the glucose tolerance tests in wild-type and Bmal1 null mice at 2 and 6 months of age were not different (data not shown).At 2 and 6 months of age, male Bmal1 null mice had a trend to decreased blood glucose , but not females.At 2 months of age, administration of pyruvate resulted in increased blood glucose levels in both male and female ype mice . The bloBmal1 null mice were 14% lower than wild-type mice (P<0.005). On the high fat diet, male Bmal1 null mice gained weight such that they were no longer lighter than wild type mice . Male Bmal1 null mice had 58% and 52% more epigonadal fat and 182% and 64% more retroperitoneal fat per gram of body weight than wild-type mice maintained on the normal chow and high fat diet respectively (P<0.001). When the epigonadal and retroperitoneal fad pad weights were combined, male Bmal1 null mice had 1.8 fold more fat than the wild-type mice on the chow diet and 1.5 fold more when on the high fat diet. Testes weight in male Bmal1 null mice were reduced compared to the wild type mice only when maintained on the chow diet (P<0.001) and kidney weight in the Bmal1 null males was reduced compared to wild type on both diets (P<0.01).Following five weeks on the control diet, body weight of the male ype mice . Wild tyBmal1 null mice on a high fat diet were increased when compared to chow fed Bmal1 females and wild type females placed on a high fat diet . Bmal1 null mice on a high fat diet had more retroperitoneal fat compared to high fat diet wild type mice (P<0.001) and there was a trend for increased adiposity compared to wild type for Bmal1 null mice on a chow diet and when comparing Bmal1 null females on a high fat vs chow diet (P = 0.016 and P = 0.014 respectively). When the epigonadal and retroperitoneal fad pad weights were combined, female Bmal1 null mice had 2.3 fold more fat than the wild-type mice on the chow diet and 2.3 fold more when on the high fat diet. While showing a trend to smaller uteri in chow fed animals (P = 0.018), female Bmal1 null mice uteri were not lighter than wild-type mice maintained on the normal chow or high fat diet (P>0.05), nor were kidney weights effected.Body weights of female fat diet , P<0.01.Bmal1 null mice weighed less than wild-type mice when maintained on the chow diet but caught up when placed on the high fat diet, whereas female Bmal1 null mice had comparable body weight on a normal chow diet, but weighed more when maintained on a high fat diet compared to chow fed Bmal1 null mice or high fat fed wild type mice. For both control and high fat diets, Bmal1 null male and female mice had a greater degree of adiposity than wild type mice.In summary, male Bmal1 null mice, however, plasma triglyceride (78% and 72%) and adiponectin (91% and 51%) were increased in Bmal1 null mice on chow diet and high fat diet respectively .When the male mice were killed at mid-light (1400 h), there was no difference in plasma glucose, free fatty acids and insulin between wild-type and ectively ; P<0.01.Bmal1 null mice, however adiponectin (by 63%) and leptin (by 56%) were increased in Bmal1 null mice on chow diet, and plasma triglyceride was increased in Bmal1 null mice on both chow (33%) and high fat diets . The high fat diet did not affect plasma glucose, triglyceride or adiponectin, but increased NEFA in the wild type (72%) and Bmal1 null mice (38%), increased circulating insulin by 77% in wild type mice and increased circulating leptin in both wild type and Bmal1 null mice .When the female mice were killed at mid-light (1400 h), there was no difference in plasma glucose, NEFA and insulin between wild-type and Per2 (−30%), Pparγ (24%), Nr1d1 (−98%), Adipoq (−31%), Retn (−29%), Nampt (−52%), Adipor1 (−16%) and Adipor2 (−35%) mRNA was decreased (P<0.05), but Lep mRNA was unchanged (P>0.05) in male Bmal1 null mice compared to the wild-type mice , but Retn and Nampt mRNA expression was arrhythmic. Expression of all genes analysed in male Bmal1 null mice was arrhythmic except for Lep mRNA (P<0.05).In the epigonadal fat, overall expression of ype mice . Expressphosphoenolpyruvate carboxykinase 1), Fbp1 , G6pc (glucose-6-phosphatase ), Gck (glucokinase) and Adipor2 mRNA expression did not vary with genotype, whereas Pfkfb3  mRNA was decreased by 25% in male Bmal1 null mice compared to wild type mice  were also analysed (ANOVA) using only the 0800 h and 1200 h time points (light period), the time around which the glucose and insulin tolerance tests were performed. In contrast to the 24 hour analysis, expression of Gck and G6pc were increased 1.7 fold (P = 0.05) and 1.6 fold (P = 0.09) respectively in Bmal1 null mice at these times.The gene expression levels for the genes involved in the glycolysis and gluconeogenesis pathways . At 2 and 6 months of age, plasma adiponectin and leptin were higher in Bmal1 null mice than wild-type mice. Although tending to weigh less than the wild-type mice, Bmal1 null mice had a greater degree of adiposity . Adipoq, Nampt and Retn mRNA expression was reduced in Bmal1 null compared to the wild-type mice, but their increased adiposity is consistent with the increased secretion of these adipokines. We were not able to measure plasma resistin or visfatin in this study due to the limited volume of plasma available for assay.In this study we show for the first time that global disruption of gene rhythmicity in both male and female Bmal1 null mice and observe initial stages of loss of metabolic control, which may have been obscured with a more extreme and less physiological diet. When the mice were fed the diet for 5 weeks, the differences in adiposity between the Bmal1 null and wild-type mice was maintained but not increased, suggesting the Bmal1 null mice are not more sensitive to this metabolic challenge. There was a pattern towards a small increase in body weight in Bmal1 null mice in response to 5 weeks on the high fat diet, with minimal change in the wild type mice. This was not unexpected given the moderate challenge of the diet, however both Bmal1 null and wild type mice increased their adiposity by approximately 50%. Both lines showed signs of increasing their plasma free fatty acids and leptin to a similar extent on the diet. In previous studies, the body weight of comparable age Bmal1 null mice has been reported as normal The “Western diet” (containing 22% fat and 0.15% cholesterol) used in this study was chosen as we wished to challenge the homeostasis of the Bmal1 null mice at 2 and 6 months of age, which is consistent with the decreased glucose stimulated insulin section reported by 3 independent groups Bmal1 null mice. A possible explanation for the normal glucose despite decreased insulin secretion in Bmal1 null mice may relate to the elevated plasma adiponectin which is known to be an insulin sensitising adipokine Clock19Δ+MEL mutant mice Adipoq mRNA expression, but unlike the Bmal1 null mice, did not show increased adiposity Clock19Δ+MEL mutant and Bmal1 null mice have adapted to a pancreatic insulin secretory defect by altering the levels of adipokines that modulate insulin sensitivity (adiponectin), to maintain glucose homeostasis. Interestingly, Clock19Δ+MEL mutant mice had low plasma free fatty acids Bmal1 null mice did not exhibit this phenotype.In this study, plasma insulin  was decreased in male Bmal1 null mice did not express Per2 mRNA rhythmically in the liver in contrast to the wild-type mice. These results confirm the original observations, that disruption of Bmal1 interferes with circadian rhythm generation Pfkfb3, Pck, Fbp1, and Gck) were rhythmic in wild-type, but not Bmal1 null mice. When analysed across 24 hours, the overall expression of these genes was not altered, except for Pfkfb3 mRNA expression, which was reduced. Expression of Pck and Fbp1 mRNA prior to darkness was clearly reduced in the Bmal1 null mice, perhaps reflecting a change in feeding pattern caused by the loss of rhythmicity.Bmal1 null mice have increased pyruvate tolerance was unexpected because a previous study reported that the loss of Bmal1 resulted in decreased pyruvate tolerance when the test was conducted 7 hours after expected lights on (ZT7) Bmal1. More recently Shimba et al.Bmal1 null mice, as compared to those in control mice following a pyruvate challenge test, similar to that observed here. However, Shimba et al.G6pc and Fbp mRNA in the liver was increased in the Bmal1 null mice and that liver glucose-6 phosphate was significantly higher than that of control mice, whereas we found the overall 24 hour expression of Pfkfb3 mRNA to be lower in the Bmal1 null mice, particularly during the 4 hours before lights off. This discrepancy may be explained by the fact that in the Shimba et al. study ad libitum. We found a trend  for higher G6pc mRNA expression in the livers of Bmal1 null mice at 0800 h and 1200 h, the time at which the pyruvate tolerance tests were conducted. Thus these results may explain the increase in glucose conversion in male and female Bmal1 null mice over the wild type mice. The failure of the gene expression data to reach significance in the sub analysis of the 0800 h and 1200 h time points probably reflects the lower power. Future studies on the impact of loss of Bmal1 on gluconeogenesis should carefully control the time of day and feed access that the analyses are conducted and in addition assess possible changes in enzyme activity.The observation in the current study that Bmal1 null mice on normal chow confirms 3 recent reports Clock19Δ (C57Bl/6) mice Clock19Δ+MEL mice Bmal1 null mice is not typical since both adiponectin and leptin are elevated. In the case of Clock19Δ+MEL mice, adiponectin is elevated but not leptin Adipor1 and Adipor2 mRNA expression was decreased in epigonadal fat. This suggests that there are important modifications in adipose tissue that develop in the absence of Bmal1 or disrupted clock function when the defect is present throughout life. Interestingly, the size of the adipocytes in the wild-type and Bmal1 null mice were not different (data not shown), suggesting that the lack of Bmal1 potentiated adipogenesis.The observation of increased adiposity in Nr1d1 mRNA expression in the epigonadal fat pads in Bmal1 null mice. It is well established that CLOCK/BMAL1 heterodimers induce Nr1d1 mRNA through interactions at an E-box on the promoter Clock or Bmal1 will reduce Nr1d1 mRNA expression and eliminate its rhythmicity. Adipogenesis is characterised by high Nr1d1 mRNA expression Nr1d1 mRNA is induced by PPARγ Bmal1 null mice, loss of rhythmic Nr1d1 resulted in arrhythmic Pparγ expression but levels of Pparγ mRNA remained around the circadian nadir levels. The high degree of adiposity in Bmal1 null mice would therefore suggest that low constitutive expression of Nr1d1 may be sufficient to allow adipogenesis and that its rhythmicity is not critical or that the lipolytic pathways in Bmal1 null mice are also significantly downregulated.In the current study we identified a profound decrease in Bmal1 null mice is different from that found in Clock19Δ mutant mice on either C57Bl/6 Clock19Δ mutants is one of obesity, hyper-insulinaemia, hyper-lipidaemia, glucose intolerance and increased insulin sensitivity Bmal1 null mice that were maintained on a mixed background because we did not wish to confound the impact of Bmal1 loss with other metabolically important mutations present in inbred strains like the C57/Bl6 mice Bmal1 null mice with a complete lack of central and peripheral rhythmicity would be exacerbated over and above those observed in Clock19Δ mutants. Bmal1 null mice do not, however, show evidence of a major metabolic disorder. This is not to say that Bmal1 null mice are normal, since they have a reduced life expectancy ad libitum feeding, Bmal1 null mice appear able to maintain normo-glycaemia, despite hypertriglyceridaemia, hyperlipidaemia and hyperleptinaemia. Interestingly despite this potential cardiovascular risk, Bmal1 null mice have recently been shown to be hypotensive and have lower stress induced alterations in blood pressure Bmal1 null mice appear to have compensated for the long term lack of Bmal1 and/or rhythmicity by altering a range of physiological systems including adipose tissue growth, adipokine secretion and feeding behaviour to maintain glucose homeostasis.The metabolic phenotype of Bmal1 null model do not discriminate between that caused by arrhythmicity and any non-circadian functions of this gene. Nevertheless, there is mounting evidence for a role of circadian rhythms in the regulation of metabolic function, as demonstrated through a variety of knock out and mutant mice models. Furthermore, from our results, it is clear that removal of the Bmal1 gene leads to cellular arrhythmicity of not only clock genes, but also genes involved in key metabolic processes including adipogenesis and gluconeogenesis. We believe therefore, that it is this circadian arrhythmicity, at both the cellular and whole organism level, that leads to the metabolic perturbations observed here, rather than any non-clock function of Bmal1.Our analyses of the metabolic parameters of the Bmal1, and the circadian arrhythmicity this creates, leads to increased adiposity when maintained on both control and high fat diets. Furthermore, the Bmal1 null mice displayed increased plasma adiponectin and leptin levels, despite reduced or normal expression of Adipoq and Lep mRNA respectively. Presumably the 1.8–2.3 fold increase in adiposity, and hence the greater number of fat cells available for secretion of these hormones, plays a role in the elevated concentrations in plasma. Bmal1 null mice also displayed an elevated response to pyruvate challenge, which when considered with the normal glucose and insulin tolerance, suggest an increased capacity for gluconeogenesis. Nevertheless, Bmal1 null mice display normal profiles of plasma glucose. Finally the loss of adipocyte and hepatic rhythmic gene expression and down-regulation of key metabolic genes in both tissues emphasises further the regulatory role Bmal1 is able to play. Together these results demonstrate that disrupted circadian rhythmicity leads to changes in adipocyte function and compensatory changes in adipokines involved in the cellular control of glucose metabolism, and provides further support for the role of circadian rhythms in the regulation of metabolic homeostasis.In summary, global loss of Figure S1Blood glucose response to intraperitoneal administration of insulin. Insulin (0.75 IU/kg) was injected into wild-type (open circle) and Bmal1 null (closed circle) mice and blood glucose measured over the subsequent 2 hours. Data are mean ± SEM (n = 6); (a) males, (b) females.(TIF)Click here for additional data file.


An outbreak of oseltamivir-resistant influenza A (H1N1) occurred in a long-term care facility. Eight (47%) of 17 and 1 (6%) of 16 residents in 2 wards had oseltamivir-resistant influenza A virus (H1N1) infections. Initial outbreak response included treatment and prophylaxis with oseltamivir. The outbreak abated, likely because of infection control measures. Outbreaks of influenza virus infection cause illness and death, especially among residents of long-term care facilities (LTCFs). In addition to annual vaccination and infection control measures, antiviral agents for treatment and prophylaxis are useful components for control of influenza outbreaks in LTCFs  and neuraminidase inhibitors (oseltamivir and zanamivir). Circulation of influenza A viruses resistant to both classes of antiviral agents, A (H3N2) to adamantanes and A (H1N1) to oseltamivir, was reported during the 2007–08 influenza season  received the 2007–08 influenza vaccine. Of the 685 LTCF employees involved in direct patient care, 385 (56%) received the 2007–08 influenza vaccine on site.We defined a confirmed case as a positive rapid or reverse transcription–PCR result for influenza virus from January 20 through February 8, 2008, in a resident of the LTCF. Surveillance for new case-patients included obtaining a nasopharyngeal specimen from all residents with new onset of fever or respiratory symptoms or any unusual behavior within 24 hours after illness onset. All specimens were tested by using the QuickVue A and B Influenza Test . A second specimen was obtained from all persons with positive rapid test results and some (57%) from persons with negative results for confirmation of influenza virus infection and virus subtyping by reverse transcription–PCR. Medical records, vaccination records, resident activity, and visitor logs were reviewed.Testing for antiviral drug resistance was conducted directly on clinical specimens by pyrosequencing as described  of 17 residents in ward 1 and 1 (6%) of 16 residents in ward 2 were infected with influenza A viruses (H1N1) that contained the H274Y mutation but did not have markers of resistance to adamantanes or zanamivir.On January 30, high fever developed in a male resident in ward 3 while on the first day of a home visit . He retuCharacteristics of case-patients are shown in the Sequence analysis of the HA1 gene in outbreak influenza A viruses (H1N1) showed identical or nearly identical sequences, differing by only 1 or 2 nt . These vThe attack rate of illness caused by oseltamivir-resistant influenza A viruses (H1N1) in ward 1 was within the range (20%–80%) reported for other facility influenza outbreaks (Although we documented a relatively high attack rate in 1 ward (ward 1), and despite resistance to the antiviral agent initially used, the outbreak abated quickly. High annual vaccination rates among residents and relatively high rates among employees (www.cdc.gov/h1n1flu/recommendations.htm). This outbreak underscores the possibility of 2 influenza A viruses, with different antiviral susceptibilities, in the same facility. During a facility outbreak of influenza, providers should consult antiviral recommendations of the Centers for Disease Control and Prevention and obtain influenza virus typing and subtyping to guide appropriate antiviral drug choices.The proportion of circulating influenza viruses resistant to oseltamivir increased from 12% during the 2007–08 season to 99% during the 2008–09 season in the United States, and new interim guidelines for use of antiviral agents were released in December 2008 (


Lynch syndrome is an autosomal dominant cancer predisposition syndrome which is caused by a germline mutation in one of four genes, MLH1, MSH2, MSH6 or PMS2. Individuals with a germline mutation in one of these genes are at increased lifetime risk of colon, endometrial, ovarian, small intestine, renal pelvis and ureter. Less commonly patients may develop biliary tract cancers, gastric and pancreatic cancers, brain tumours, sebaceous adenomas, carcinomas and skin keratoacanthomas.Immunohistochemical staining for the above four mismatch repair (MMR) proteins is routinely performed for individuals with bowel cancer or a related cancer who are suspected of having Lynch syndrome. This helps target genetic testing to the correct mismatch repair gene. The genes involved in Lynch syndrome work together in a DNA repair complex. The MMR proteins form a heterodimer with MLH1 partnering PMS1, PMS2 or MLH3 and MSH2 partnering MSH3 or MSH6. This means that a mutation in MLH1 leads to loss of staining of both MLH1 and PMS2, while a mutation in MSH2 leads to loss of staining of both MSH2 and MSH6. The same is not true however, for mutations in either PMS2 or MSH6, where only the single protein made by these genes is not expressed in the tumour.However, the pattern of staining using immunohistochemistry is sometimes more complex due to coding microsatellites within the MSH6 gene. We present two unrelated cases with a germline PMS2 mutation which had loss of both MSH6 and PMS2, but normal MLH1 and MSH2 on immunohistochemical staining.