Dvl genes have been identified and the developmental roles of Dvl1 and Dvl2 were previously determined. Here, we identify the functions of Dvl3 in development and provide evidence of functional redundancy among the three murine Dvls. Dvl3−/− mice died perinatally with cardiac outflow tract abnormalities, including double outlet right ventricle and persistent truncus arteriosis. These mutants also displayed a misorientated stereocilia in the organ of Corti, a phenotype that was enhanced with the additional loss of a single allele of the PCP component Vangl2/Ltap (LtapLp/+). Although neurulation appeared normal in both Dvl3−/− and LtapLp/+ mutants, Dvl3+/−;LtapLp/+ combined mutants displayed incomplete neural tube closure. Importantly, we show that many of the roles of Dvl3 are also shared by Dvl1 and Dvl2. More severe phenotypes were observed in Dvl3 mutants with the deficiency of another Dvl, and increasing Dvl dosage genetically with Dvl transgenes demonstrated the ability of Dvls to compensate for each other to enable normal development. Interestingly, global canonical Wnt signaling appeared largely unaffected in the double Dvl mutants, suggesting that low Dvl levels are sufficient for functional canonical Wnt signals. In summary, we demonstrate that Dvl3 is required for cardiac outflow tract development and describe its importance in the PCP pathway during neurulation and cochlea development. Finally, we establish several developmental processes in which the three Dvls are functionally redundant.Dishevelled (Dvl) proteins are important signaling components of both the canonical β-catenin/Wnt pathway, which controls cell proliferation and patterning, and the planar cell polarity (PCP) pathway, which coordinates cell polarity within a sheet of cells and also directs convergent extension cell (CE) movements that produce narrowing and elongation of the tissue. Three mammalian  Dishevelled (Dvl) family members and while the roles of Dvl1 and Dvl2 have been described previously, the functions of Dvl3 have remained elusive. Here, we show that the lack of Dvl3 in mice affects the formation of the heart, neural tube, and inner ear. We further show that the defects in these tissues are much more severe when the mice are deficient in more than one Dvl family member, indicating redundant functions for these genes. Congenital heart disease affects approximately 75 in every 1,000 live human births, and approximately 30% of these diseases are due to disruptions in the outflow tract, the region affected in mice lacking Dvl genes. Neural tube defects, similar to those observed in the Dvl mutants, are also common in humans. The animal models described here provide useful tools to elucidate the genetic mechanisms that underlie these abnormalities and may provide novel ways of treating these disorders in the future.Multi-gene families, comprising a set of very similar genes with shared nucleotide sequences, are common in mammals. Individual family members may be expressed in different places and perform separate functions. Alternatively, the genes may have redundant functions, but distinct dosage requirements. Mammals share three  Normal mammalian development is the result of complex signaling networks that regulate and coordinate cell behavior. Wnt signaling controls a broad spectrum of cell fate decisions during embryogenesis and is critical for cell to cell communication in mammalian development. Through the activation of specific target genes, the canonical Wnt pathway tightly regulates cell proliferation, differentiation, adhesion and survival, and controls embryonic patterning Dishevelled (Dvl) proteins, of which three have been identified in humans and mice Islet1 (Isl1), migrate from the pharyngeal mesoderm to contribute to the myocardium of the outflow tract Isl1 expression In the heart, normal development of the cardiac outflow tract requires the addition of cells from the secondary heart field (SHF) dorsal to the primary heart tube Looptail (LtapLp) mice, which carry a missense mutation in Van Gogh-like 2  LtapLp/LtapLp homozygous mutants display the outflow tract abnormality DORV Wnt11Wnt5aWnt5a has been proposed to function in the outflow tract by stimulating intracellular increases in Ca2+ to regulate cells of the CNC.Studies of Vangl2/LtapFz3 and Fz6Dvl1 and Dvl2PCP signaling is also required for the normal development of the auditory sensory organ, the organ of Corti Vangl2/LtapDvl1 and Dvl2Celsr (a homolog of flamingo) Fz3 and Fz6The neural plate undergoes narrowing and lengthening attributable to CE movements during mammalian neurulation. When PCP signaling is disrupted, cells of the neuroepithelium fail to intercalate, preventing the neural tube from fusing at the midline Dvls in mammalian development, we have generated mouse knockouts for each of the Dvl genes. Interestingly distinct phenotypes were revealed, suggesting separate functions for the Dvl proteins. Dvl1 knockout mice are viable and fertile, but display social interaction abnormalities and defects in sensorimotor gating Dvl2 knockout mice Dvl genes is also suggested from their overlapping expression patterns, as well as their high degree of conservation. In support of this, we have previously shown that Dvl1−/−;Dvl2−/− mutants display craniorachischisis, a completely open neural tube, and abnormalities in the organ of Corti, both novel phenotypes not observed in the single Dvl knockouts To elucidate the role of specific Dvl3 and determine its importance in conotruncal, cochlear and neural tube development. Given the overlapping expression patterns of the Dvls, as well as their high degree of conservation, we further addressed the possibility of functional redundancy and demonstrate that many of the roles of Dvl3 are also shared by Dvl1 and Dvl2. We attribute several of the developmental functions of Dvl3 to its role in PCP signaling, enhancing our knowledge of this essential pathway in mammalian development and further defining the specific role of individual Dvl genes.Here we describe the phenotype of mice deficient in Dvl3 knock out mice were successfully generated , as expected from Mendelian ratios. In a mixed genetic background, 121 pups were analyzed, but only 4 out of the expected 30 Dvl3−/− mice (13.3%) survived. In contrast, at E18.5 Dvl3+/+, Dvl3+/− and Dvl3−/− genotypes were recovered in normal Mendelian ratios from heterozygous crosses. These data indicate that 100% of Dvl3 homozygotes in a 129S6 inbred background and approximately 87% in a mixed genetic background die shortly after birth. No gross abnormalities were observed in the few adult Dvl3−/− mice that did survive in a mixed background.enerated . In ordening age . In a 12Dvl3−/− newborn pups had difficulty breathing and were often cyanotic. Examination of hearts from Dvl3−/− mutants at P0 , 31% (37/118) of pups genotyped at weaning age were Dvl3−/− rescued with the transgene, whereas only 1.6% (2/118) Dvl3−/− mutants survived without the transgene. E18.5 hearts collected from Dvl3−/− embryos with the Dvl3 transgene appeared normal and displayed no conotruncal abnormalities . Expression of both Dvl3 and Dvl1 was observed in the developing neural tube at E9.5 , defective neurulation was apparent when we crossed Dvl3 mutants with LtapLp/+ mice. Several Dvl3+/−;LtapLp/+ embryos displayed normal neural tube development  and Dvl3−/−;LtapLp/+ mutants . However, craniorachischisis appears to be a more severe phenotype than exencephaly, indicating a more severe phenotype in Dvl3−/−;LtapLp/+ mutants compared to Dvl3+/−;LtapLp/+ mutants.Homozygous elopment , whereaselopment  and exeng normal  and otheg normal . The freDvl3−/− and LtapLp;LtapLp mutants display cardiac defects but the single heterozygotes do not, we looked for defects in the hearts of the Dvl3+/−;LtapLp/+ double heterozygotes , display misorientation of stereociliary bundles in sensory hair cells due to disrupted planar cell polarity and the cochlear ducts are also shortened and wider due to defects in CE cell movements Dvl3 mutant embryos, with and without an extra LtapLp mutation in Vangl2. No cochlear defects were observed in either Dvl3+/− (data not shown) or LtapLp/+ single heterozygotes in the basal and middle regions . 7/11 Dvl3−/− mutants showed xiphoid bifurication, but this was also observed in several wild type controls.We have previously reported both vertebral and rib malformations in Dvl1 and Dvl2 was evident from our studies of Dvl1−/−;Dvl2−/− double mutants, which displayed novel phenotypes not observed in the single Dvl knockouts Dvl3 and the other two Dvls we examined the phenotypes of Dvl1;Dvl3 and Dvl2;Dvl3 double mutants. Both Dvl1+/−;Dvl3+/− and Dvl1−/−;Dvl3+/− mutants survived to adulthood and were fertile. Normal development was also observed in Dvl1−/−;Dvl3−/− embryos until E12.5  axis, show an abnormal head shape and a truncated snout, as well as a shortened and kinked tail  was able to rescue the lethal Dvl3−/− phenotype. Importantly, western blot analysis revealed that none of the Dvl transgenes were overexpressed compared to protein levels from wild type alleles  cannot survive. Surprisingly, we found that addition of the Dvl1TG rescued the Dvl3−/− phenotype such that Dvl3−/−;Dvl1TG mutants (now Dvl3−/− with 3 copies of Dvl1 and 2 copies of Dvl2) survived. To test whether this was a complete rescue, we crossed these viable Dvl3−/−;Dvl1TG mutants to Dvl3+/− mice and genotyped the progeny at weaning  survived, comparable to 9 expected from normal Mendelian ratios, indicating full rescue. However, no Dvl2+/−;Dvl3−/−;Dvl2TG mutants were recovered, even though 9 were expected from normal Mendelian ratios if the Dvl2TG could still rescue. Thus, correction of Dvl2 dosage from three copies to two copies of Dvl2 in Dvl2+/−;Dvl3−/−;Dvl2TG mutants  eliminated the ability of the Dvl2TG to rescue the Dvl3−/− mutant phenotype, supporting the conclusion that ectopic expression or over-expression of the transgene was not responsible for the rescue of the Dvl3−/− phenotype.To confirm that the BAC transgenes were actually behaving as wild type alleles and that ectopic or inappropriate expression levels were not responsible for rescuing the mutant phenotypes, we genetically reduced the enotyped . Twelve Dvl1−/−;Dvl2−/−Dvl3−/− mutants , as did those from the Dvl2+/− (Dvl3+/− (not shown) single heterozygote samples. However, several of the sensory hair cells in Dvl2+/−;Dvl3+/− double heterozygotes had rotated stereociliary bundles  results in incomplete neurulation, suggesting a dosage sensitive redundant role for all three Dvls in this process. Interestingly, Dvl1−/−;Dvl3−/− mutants did not display neural tube defects, suggesting that the three Dvls are not functionally equivalent. Neural tube defects appear in single Dvl mutants when crossed with LtapLp mice, , indicating genetic interaction with the Dvls and the PCP component, Vangl2 and therefore that the Dvls signal through the PCP pathway to promote neural tube closure.We have shown here that Dvl1 and Dvl3 colocalize in the developing neural tube with similar expression patterns to that of Dvl2 as we described previously Dvl3;LtapLp mutants. Craniorachischisis has been observed in many mutants with disrupted PCP signaling, whereas exencephaly is often associated with defective ciliogenesis  Wnt3a allele mutant, vestigial tail, which shows loss of caudal vertebrae causing a shortening of the tail Wnt5a are also shortened along the A–P axis with a phenotype similar to Dvl2+/−;Dvl3−/− mutants, and share outgrowth defects in the developing face and lack of tail Dvl mutants using the TOPflash reporter of TCF activity. Interestingly we found that global Wnt signaling was largely unaffected in even the most severely affected mutants, suggesting that only a low level of Dvl is sufficient for functional canonical Wnt signals. However, our data also suggests subtle abnormalities in Wnt signaling. Precise determination of these abnormalities will require careful sectioning of specific tissues and examination of various cell types throughout development.We have previously shown that both The three highly homologous Dvl proteins shared in mammals have very similar broad expression patterns in development. This study completes the initial characterization of the specific, individual roles of each of these proteins and also establishes functional redundancy and overlap in a number of developmental processes. Dvl3 is required for the development of the cardiac outflow tract and signals in the PCP pathway to regulate CE in the developing neural tube and cochlea, as well as cell polarity in the organ of Corti. Dvl1 and Dvl2 are redundant with each other LtapLp mutants were originally acquired from Jackson Laboratory, Wnt1-Cre mice were a kind gift of Dr. Andrew McMahon, Harvard and Isl1-Cre mice were a generous gift from Sylvia Evans, UCSD. We generated the Dvl3−/− and Dvl3-EYFP mouse mutants as described in Dvl3 mutants was performed with the following primers, Dvl3 forward 5′-TCCGATGAGGATGATTCCACC-3′, Dvl3 reverse 5′-TGAGGCACTGCTCTGTTCTGT -3′, Dvl3 knockout 5′-TTGGCCCACAATGGAGATGCCC-3′, NLpgk neo forward 5′-AGGCTTACCCGCTTCCATTGCTCA-3′. The PCR primers used to distinguish between the transgene and wild type Dvl3 allele were Dvl3lnt2 forward 5′-GGACGCAGGAGATCTTTGAA-3′ and Dvl3Int2 reverse 5′-CATAGCTGGGGTTGAAGCTC-3′ which amplify a band of 155 bp for the wild type allele and 189 bp for the transgene, due to the presence of the LoxP site. Genotyping for the following mouse mutants have previously been described; Dvl1Dvl2Dvl2-EYFP transgene Wnt1-CreIsl1-CreRosa-26-lacZ CreBAT-galAll animal care and experiments were performed under protocols approved by the NHGRI/NIH and UCSD Animal Care and Use Committees. After fixing in 3% aldehyde solution  in 0.1 M phosphate buffer pH 7.5, E18.5 hearts were stored in 100% ethanol following dehydration through a graded ethanol series. Hearts were critically point dried, mounted and coated with 300 Angstrom gold-palladium. A Cambridge Instrument Stereoscan 360 scanning electron microscope  was used to view the prepared samples.P0 hearts were dissected into 10% buffered formalin, dehydrated, embedded in paraffin wax, sectioned at 8 µm thickness and stained with hematoxylin and eosin using standard methods.Dvl3−/− hearts, Dvl3+/− mice carrying Wnt1-CreIsl1-CreDvl3+/− mice carrying the Rosa-26-lacZ Cre reporter gene 2, 0.02% NP-40, 0.01% sodium deoxycholate at 37°C overnight. After re-fixing in 4% paraformaldehyde, the slides were counterstained with Nuclear Fast Red (Vector Lab), according to the manufacturer's protocol. Whole embryos from the above crosses were also collected at E10.5 and stained for β-galactosidase activity using the same procedures. All images were captured using a Spot 2 camera mounted on a Leica DMR light microscope.To label the CNC and SHF cell populations in Dvl mutant mice were crossed with mice carrying the BAT-gal reporter gene To visualize canonical Wnt signaling, Dvl3-EYFP embryos or cochleae were fixed in 4% paraformaldehyde for 30 minutes or overnight at 4°C. To examine the stereocilia in the sensory hair cells, organs of Corti were stained with fluorescein- rhodamine-conjugated phalloidin (Molecular Probes). Both native YFP signal and fluorescent staining was observed using an Olympus FV 1000 or a Zeiss LSM 510 confocal scanning microscope.To visualize Dvl3 expression, To measure the severity of the patterning defect, the whole cochlea duct was scanned and the cells numbered for length reference. The number of cells in the defective regions (where there were no longer three outer hair cell rows) was then calculated as a percentage of total cells.Figure S1Dvl3 deficient mice. A Dvl3 genomic clone was isolated from a 129 genomic DNA library in FIX II (Stratagene) as previously described Dvl3 genomic fragments were subcloned into pBluescript KS II (Stratagene) to enable efficient generation of a Dvl3 knock-out construct (A). A 3.6 kb BamH-Bgl2 fragment from within intron 1 to the middle of exon 2 and a 2.5 kb Dra1-Not1 (from vector MCS) fragment from within intron 7 to beyond 13 were cloned into pPNT 2 litters contained wild type and heterozygous offspring. −/−Dvl3 embryos were collected from crosses between heterozygotes and western blot analysis using E13.5 whole embryo lysates confirmed the absence of Dvl3 protein (C). PCR primers (listed in Targeted disruption and generation of (0.5 MB TIF)Click here for additional data file.Figure S2Dvl3 transgene. An EYFP-tagged Dvl3 transgene was generated using homologous recombination of BACs (A), as described previously Dvl3 genomic region, including flanking sequences, were identified using overlapping PCR primers covering the entire region from a BAC library (Genome Systems). BAC modifications were performed as previously described Dvl3 and LoxP sites were introduced within intron 2 and the 3′ UTR flanking region. Western blot analysis of transgenic mouse E13.5 embryo lysates was used to confirm expression of the transgene, which was larger than the wild type Dvl3 protein due to the additional EYFP (3B). PCR primers .Generation of EYFP-tagged (3.8 MB TIF)Click here for additional data file.


Id3, a transcriptional inactivator, was selected for further investigation. Id3 mRNA was expressed at reduced levels in 6 out of 9 ovarian cancer cell lines compared to the HOSE cells while at the protein level, all 7 ovarian cancer cell lines examined expressed the Id3 protein at greatly reduced levels. Expression of Id3 mRNA was also examined in primary ovarian tumours and was found in only 12/38 (32%) cases. A search was conducted for mutations of Id3 in primary ovarian cancers using single stranded conformation polymorphism (SSCP) analysis. Only one nucleotide substitution, present also in the corresponding constitutional DNA, was found in 94 ovarian tumours. Furthermore no association was found between LOH at 1p36 and lack of expression of Id3. These data suggest that Id3 is not the target of LOH at 1p36. © 2001 Cancer Research Campaign http://www.bjcancer.comThe molecular events that drive the initiation and progression of ovarian adenocarcinoma are not well defined. We have investigated changes in gene expression in ovarian cancer cell lines compared to an immortalized human ovarian surface epithelial cell line (HOSE) using a cDNA array. We identified 17 genes that were under-expressed and 10 genes that were over-expressed in the cell lines compared to the HOSE cells. One of the genes under-expressed in the ovarian cancer cell lines,


To evaluate the tolerance of the cirrhotic liver to extended warm ischaemia, 47 patients with cirrhosis who underwent liver,resection over a 4-year period were studied retrospectively. Three groups of patients were identified. In group 1 (14 patients) liver resection was performed under conditions of portal triad occlusion ranging from 50 to 75 (mean 57.1) min. Group 2 (12 patients) was treated with portal occlusion for a period ranging from 30 to 42 (mean 33.1) min. Group 3 comprised 21 patients who underwent  hepatectomy using conventional techniques. Mean blood loss was significantly reduced by portal triad occlusion  compared with the conventional techniques (1652 ml in group 3) . Hospital death occurred in three of the 21 patients in group 3 but in no patient who underwent portal triad occlusion. The morbidity rate was lower in the two occlusion groups (four of 26 patients) than in group 3 (six of 21). Bilirubin metabolism was substantially better after surgery in the occlusion groups . Although the serum levels of transaminases were significantly raised until day 3 in patients undergoing long term occlusion, the cirrhotic liver withstood the ischaemia-reperfusion insult, as assessed by changes in hepatic microcirculation, lipid peroxidation and the morphology of hepatic sinusoids. It is concluded that prolonged ischaemia during liver resection can be sustained in patients with cirrhosis and without high-risk factors.


Breast cancer is the most common cancer and cause of deaths in women around the world. Oncogene amplification usually occurs late in tumor progression and correlates well with aggressiveness of tumor. In fact the function of the S100A4 protein and its role in metastasis is unclear at present. The purpose of the study was to determine the expression of S100A4 protein in the invasion status and metastatic potential of breast cancer by using tissue microarray and to determine its role in breast cancer based on the expression of S100A4 gene product.S100A4 protein expression was examined by immunohistochemistry (IHC) using commercially available tissue microarray containing malignant and normal breast tissue cores from 216 patients.S100A4 was absent in normal breast tissues while positive in 45.1% of infiltrating ductal carcinoma (IDC) node negative and 48.8% of infiltrating lobular carcinoma node negative. In paired samples, S100A4 protein was expressed in 13.5% of IDC node positive cases and 35.1% of matched lymph node metastasis.S100A4 protein expression appears widely expressed in early and advanced breast cancer stages compared with normal breast. Our study suggests S100A4 may play a role in breast cancer progression and may prove to be an independent marker of breast cancer which appears to be down regulated in more advanced stages of breast cancer. Breast cancer is still the most common public health problem in women worldwide . Cancer The metastasis process is not a random process but consist of a complex series of linked and interrelated steps involving multiple host-tumor interaction . Many prBreast cancer is considered to be a systemic disease this would mean that the most breast carcinoma metastasize before diagnosis of the primary lesion . TherefoThe S100 gene family located on chromosome 1q21, comprises more than 20 members whose protein sequences encompass at least one EF-hand Ca++ binding motif ,31. S100Studies to determine the mechanistic basis for S100A4 function have shown a potential role for S100A4 in several different facts of tumor progression including motility, invasion, and apoptosis ,41, and , and 41,Formalin-fixed paraffin-embedded tissue microarrays from 188 lymph node negative breast cancer patients, 50 breast cancer patients with lymph node metastasis  and 8 normal breast tissue cores were analyzed by immunohistochemistry for the expression S100A4 protein. Included in this study were patients with infiltrating ductal carcinoma, infiltrating lobular carcinoma, normal breast tissue and lymph node metastasis. The final number was 122 tissue cores of node negative infiltrating ductal carcinoma, 41 node negative infiltrating lobular carcinoma, seven normal breast tissue, 40 node positive  and 46 tissue cores of lymph node metastasis. We did not evaluate infiltrating lobular carcinoma node positive because of the low sample size (2 cases). Forty six tissue cores were excluded from statistics because very little cancer cells or no breast cancer tissue were seen. The total number after exclusion was 254 tissue cores (37 matched tissues) of 216 patients.Rabbit Anti-Human polyclonal primary antibody against S100A4 protein  was used on deparaffinized tissue microarray slides . A secondary detection system (DAKO Envision) enhanced with conjugated polymer was used to bind with the primary antibody. DAB chromogen was used for permanent color development and detection under microscope.The percentage of carcinoma cells with cytoplasmic/membranous/nuclear staining was recorded on each specimen at 200× magnification, using light microscope. The expression of S100A4 was scored in all tumors as: positive ≥ 5% and negative < 5% stained cells. Also the intensity of staining was categorized into three groups: weak, moderate and strong. This was ascertained by a single qualified pathologist.A tissue section of breast cancer was used as positive control for S100A4. Rabbit IgG isotype  was used instead of primary antibody in the immunohistochemical technique on a tissue section each of breast cancer and normal breast as negative control  and heated on microwave. Slides were allowed to cool at room temperature and rinsed with Tris Buffered saline (TBS) mixed with tween 20. Slides were covered with peroxidase blocking solution (DAKO), followed by rinsing with TBS buffer mixed with tween 20. Then 200 μl of primary antibody (dilution 1:200) was added on the tissue microarray slides, followed by rinsing with TBS mixed with tween 20. Two drops of DAKO Envision/HRP, Rabbit/Mouse (secondary antibody) were added on the slides, followed by rinsing with TBS mixed with tween 20. After that DAB substrate (DAKO) was added on section slides followed by rinsing, immersion into hematoxylin and 4 different concentrations of ethanol. After that slides were immersed in two changes of xylene and cover slipped. All incubation steps after heat induced epitope retrieval were carried at room temperature.The S100A4 protein was expressed in the cell cytoplasm without evidence of nuclear staining.There were a total of 122 cases of infiltrating ductal carcinoma node negative and 41 cases of infiltrating lobular carcinoma node negative. Infiltrating ductal carcinoma node positive consisted of 38 cases. Thirty seven cases had paired primary infiltrating ductal carcinoma tissue with its matched lymph node core. There were also 9 cases of unrelated lymph node cores containing metastatic deposits.A positive expression of S100A4 was observed in 45.1% 55/122) cases of infiltrating ductal carcinoma node negative  cases in the paired samples (primary breast carcinoma and matched lymph nodes) showed presence of S100A4 protein in the primary site, while 13/37 (35.1%) cases showed S100A4 in lymph node  paired cases (primary breast cancer with matched lymph node) showed expression of S100A4 in the lymph node metastasis with absent expression in the primary site. Four (23.5%) cases showed S100A4 expression in the primary breast carcinoma with absent expression in its matched lymph node metastasis. One case showed S100A4 expression in both sites.Cancer is a disease or disorder characterized by uncontrolled growth (division) of the cells -55 and tIn the present study, S100A4 protein expression was examined by immunohistochemistry in infiltrating ductal, infiltrating lobular carcinoma and lymph node metastasis and their relation to tumor promotion and progression. Positive expression of S100A4 was observed in 45.1% of the infiltrating ductal carcinoma node negative cases, while in infiltrating lobular carcinoma with node negative, the expression of S100A4 protein was observed in 48.8%. This shows that S100A4 has a similar expression level in both infiltrating ductal carcinoma and infiltrating lobular carcinoma with node negative. S100A4 staining was not observed in normal breast tissues. S100A4 was expressed in a higher percentage in breast cancer tissue compared to normal tissue which showed direct correlation of S100A4 protein in infiltrating breast cancer node negative. This suggests that S100A4 is over expressed in infiltrating breast carcinoma compared to normal breast. Positive expression of S100A4 protein was observed only in 13.5 % of cases of infiltrating ductal carcinoma node positive while positive expression of S100A4 protein was observed in 35.1% of matched lymph node metastasis. These results showed there was a decrease in expression of S100A4 in infiltrating ductal carcinoma node positive (13.5%) compared with IDC node negative (45.1% positive staining), but interestingly there is an increase of expression of S100A4 protein at metastatic lymph node site. This suggests S100A4 may play a role in advanced breast cancer especially with lymph node metastasis. It was also interesting to note that the expression was seen in one site i.e. either primary tumor or its metastatic lymph node only.The majority  of paired samples showed that when there was a positive expression of S100A4 protein in metastatic lymph node, there was associated negative expression in the primary tumor  of the same patient. One case showed positive expression in both primary tumor and its metastatic lymph node at the same time. This was comparable with another study which showed that S100A4 over expression directly correlated with tumor progression . This stIn conclusion S100A4 protein expression appears to be expressed widely in early and advanced stages of breast cancer compared with normal breast. This study indicates a complex role of S100A4 in breast cancer of different types and stages. The difference in the expression of S100A4 protein suggests it may be useful as an independent marker of breast cancer which appears to be down regulated in more advanced stages of breast cancer. However a larger study with more ILC and metastatic cases may clarify the role and function of S100A4 in breast cancer progression.IHC: Immunohistochemistry; IDC: Infiltrating ductal carcinoma; ILC: Infiltrating lobular carcinoma.NII carried out Immunohistochemical part of the study and lab work, participated in drafting the manuscript. GK carried out the pathological part of the study, participated in drafting the manuscript. HH performed the statistical analysis. MSH initiated the project, participated in drafting the manuscript. All authors read and approved this manuscript.


Approximately nine out of ten screen-detected prostate cancers were localized  and well or moderately differentiated  grade I and 50% grade II), which suggests a higher proportion of curable cancers compared with cases detected by other means. © 1999 Cancer Research CampaignApproximately 20 000 men 55–67 years of age from two areas in Finland were identified from the Population Registry and randomized either to the screening arm (1/3) or the control arm (2/3) of a prostate cancer screening trial. In the first round, the participation rate in the screening arm was 69%. Of the 5053 screened participants, 428 (8.5%) had a serum prostate-specific antigen (PSA) concentration of 4.0 ng/ml or higher, and diagnostic examinations were performed on 399 of them. A total of 106 cancers were detected among them corresponding to a positive predictive value of 27%, which is comparable with mammography screening for breast cancer. The prostate cancer detection rate based on a serum PSA concentration of 4.0 ng ml


Lymph node and peripheral blood lymphocytes were studied simultaneously for surface markers of T and B cells in 22 patients with lymphoproliferative diseases and 8 patients with non-neoplastic lymphadenopathy. This resulted in the classification of the malignancy from involved lymph nodes into 4 groups. Six patients had B cell lymphomata with normal or strong immunofluorescent staining for surface membrane immunoglobulin; 8 patients had B cell chronic lymphocytic leukaemia with pale staining for surface membrane immunoglobulin; 5 patients had T cell lymphomata and 3 patients were not definitely classifiable. In 6 out of 8 patients with B cell CLL, histopathology of lymph nodes showed infiltration with well differentiated lymphocytes and in all T cell lymphomata, the infiltrating cells were poorly differentiated. By the use of these markers, malignant lymphocytes were identified in the circulation in only 3 out of 6 patients with B cell lymphoma, in all patients with B cell CLL but in none of those with T cell lymphoma or unclassifiable lymphoma. Therefore a more conclusive characterization of the malignant lymphocyte in lymphoproliferative diseases must include an examination of involved lymph nodes.


It was found:C3H/He and CBA/T6T6 mice which share the H2(1) The tumour grows at the same rate with the same median survival time in matched groups of non-immunized mice from both strains after i.p. injection of tumour cells.7 BP8, but this dose, and more intensive treatment with this drug, fails to cure C3H mice.(2) Cyclophosphamide (Cyclo) at 10 mg/kg will cure CBA mice which have received i.p. injections of 10125IUdR-labelled tumour cells and counting 125I loss by whole-mouse counting shows that the cytotoxic effect of Cyclo against BP8 is similar in the 2 mouse strains.(3) Injecting 7 BP8 followed by 10 mg/kg Cyclo.(4) Cyclo itself does not cure CBA mice, for viable tumour cells are recoverable from the peritoneal cavity 10 days after CBA mice have received 10(5) CBA mice cured of BP8 ascites by Cyclo treatment will reject further i.p. inocula of BP8.(6) The strength of immunity induced by irradiated BP8 cells was directly related to the length of exposure to this antigen. An important aspect of Cyclo treatment is that it prolongs the period during which immunity may develop.(7) Immunization of CBA mice with heavily irradiated BP8, with or without Cyclo, failed to show that Cyclo depressed the capacity of CBA mice to develop cytotoxic immunity. There was some indication that animals immunized with irradiated cells plus drug did better than those with irradiated cells alone.(8) A single injection of irradiated BP8 cells into CBA mice induced weak cytotoxic immunity, as assessed by destruction of a subsequent challenge with BP8, but these mice died from tumour more rapidly than non-immunized controls. It is suggested from these data that immunological enhancement may not always be due to blocking of cytotoxic immunity.


Interleukin (IL) 11 is produced by human endometrium and endometrial cancer tissue. It has roles in endometrial epithelial cell adhesion and trophoblast cell invasion, two important processes in cancer progression. This study aimed to determine the levels of IL11 in uterine lavage fluid in women with endometrial cancer and postmenopausal women. It further aimed to determine the levels of IL11 protein and its signaling molecules in human endometrial cancer of varying grades, and endometrium from postmenopausal women and IL11 signalling mechanisms in endometrial cancer cell lines.IL11 levels in uterine lavage were measured by ELISA. IL11, IL11 receptor(R) α, phosphorylated (p) STAT3 and SOCS3 were examined by immunohistochemistry in endometrial carcinomas and in control endometrium from postmenopausal women and normal cycling women. The effect of IL11 on pSTAT3/STAT3 and SOCS3 protein abundance in endometrial cancer cell lines and non-cancer endometrial epithelial cells was determined by Western blot.IL11 was present in uterine flushings and was significantly higher in women with Grade 1 carcinomas compared to postmenopausal women (p < 0.05). IL11 immunostaining was significantly elevated in the endometrial tumour epithelial cells from Grade 1 and 3 compared to endometrial epithelium from postmenopausal and cycling women. IL11Rα immunostaining intensity was increased in cancer epithelium in the Grades 1 and 2 tumours compared to epithelium from postmenopausal women. Both IL11 and IL11Rα localized to vascular endothelial and smooth muscle cells while IL11 also localized to subsets of leucocytes in the cancer tissues. pSTAT3 was found in both the tumour epithelial and stromal compartments but was maximal in the tumour epithelial cells, while SOCS3 was predominantly found in the tumour epithelial cells. pSTAT3 staining intensity was significantly higher in Grade 1 and 2 tumour epithelial cells compared to epithelial cells from cycling and postmenopausal women. SOCS3 staining intensity did not differ between between each tumour and postmenopausal endometrial epithelium but SOCS3 in cycling endometrium was significantly higher compared to postmonopausal and Tumour Grades 2 and 3. IL11 increased pSTAT3/STAT3 in all tumour cell lines, while SOCS3 abundance was increased only in one tumour cell line.The present study suggests that IL11 in uterine washings may be useful as a diagnostic marker for early stage endometrial cancer. It indicates that IL11, along with its specific receptor, IL11Rα, and downstream signalling molecules, STAT3 and SOCS3, are likely to play a role in the progression of endometrial carcinoma. The precise role of IL11 in endometrial cancer remains to be elucidated. Endometrial cancer is the most common gynaecological malignancy . Since iInterleukin (IL) 11 belongs to the IL6 family of cytokines and signals via a heterodimeric complex of IL11 receptor (R) α and gp130. The cellular responses of IL11 are induced by the activation of downstream Janus kinases (JAK) that phosphorylate the latent cytoplasmic transcription factors, signal transducer and activator of transcription (STAT) . PhosphoNumerous studies have suggested that IL11 has roles in human gastric, prostate and bone cancer -17. In aIn the current study, we determined the levels of IL11 in uterine lavage in women with endometrial cancer and postmenopausal controls. We compared IL11, IL11Rα, pSTAT3 and SOCS3 protein in human endometrial carcinomas of varying histologic grades with endometrium from postmenopausal and cycling women. We determined the effect of IL11 on its downstream signaling molecules in endometrial cancer and non-cancer endometrial epithelial cells.Endometrial cancer tissue biopsies N = 16) were collected from postmenopausal women undergoing total abdominal hysterectomy for endometrial carcinoma at the Monash Medical Centre Melbourne, Australia. The Human Ethics Committee approved the research project and informed consent was obtained from each patient participating in this study. Details of individual patients are provided in Table 6 were coFor immunohistochemistry studies: There were 16 cancer patients, with an age range of 34-88 years (mean age = 64.4 years with standard deviation = 14.1)  and postmenopausal controls .Uterine lavages (uterine washings) were collected from postmenopausal women (N = 4) and women with endometrial cancer above except for women with Grade 1 carcinoma where washings were collected from 4 women instead of 5 women  as previously described . Uterine2O2 in methanol for 10 min. Non-specific staining was blocked using a blocking solution of 10% normal horse serum  and 2% normal human serum, diluted in 1× Tris-buffered saline (TBS) for 30 min. Primary antibodies were diluted to 4 μg/ml in blocking solution and applied for 18 h at 4°C. A non-immune isotype IgG negative control  diluted to a matching concentration as the primary antibody, was also included for each tissue. Antibody localisation was detected by sequential application of biotinylated horse anti-mouse IgG  diluted 1:200 in blocking solution for 30 min and an avidin-biotin complex conjugated to HRP . The substrate used was diaminobenzidine (DAB)  forming an insoluble brown precipitate. Sections were then counterstained in Harris hematoxylin . Sections from normal endometrium were used as positive controls and included in each immunostaining run to provide quality control.Immunohistochemistry for IL11 and IL11Rα was performed as described previously  using a Immunohistochemistry for pSTAT3 and SOCS3 was conducted using polyclonal rabbit anti-mouse  and monoclonal rabbit anti-human (Clone C204)  antibodies respectively as previously shown , at fina2O2 in methanol for 10 min. Non-specific staining was blocked using blocking solutions consisting of 10% normal swine serum (in-house) and 2% normal human serum for pSTAT3 and 10% normal goat serum (Vector Laboratories) and 2% normal human serum for SOCS3, each diluted in 1×TBS for 30 min. Primary antibodies were diluted in the appropriate blocking solution and applied for 18 h at 4°C. A non-immune isotype IgG negative control (R&D Systems) diluted to a matching concentration as the primary antibody, was also included for each tissue. Antibody localisation was detected by sequential application of biotinylated swine anti rabbit IgG  or biotinylated goat anti-rabbit IgG (Vector Laboratories) diluted 1:200 in blocking solution correspondingly for 30 min and an avidin-biotin complex conjugated to HRP . The substrate used was diaminobenzidine (DAB) (Zymed), which forms an insoluble brown precipitate. Sections were then counterstained in Harris hematoxylin (Sigma Diagnostics). Sections from normal pre-menopausal endometrium were used as positive controls and included in each immunostaining run to provide quality control.Formalin fixed sections were deparaffinized in histosol and rehydrated in a graded series of ethanol. Endogenous activity was blocked by incubation in 3% HThe endometrial carcinoma cells ECC-1, HEC-1A and Ishikawa cells were cultured in DMEM/F12 (1:1), McCoy's 5A and DMEM  respectively supplemented with 10% fetal calf serum , 1% L-glutamine (Sigma-Aldrich Pty. Ltd) and 1% antibiotic-antimycotic . The non-cancer human endometrial epithelial (HES) cell line  was obtaThe endometrial cancer cell lines ECC-1, HEC-1A and Ishikawa and or HES cells were treated with diluents control, IL11  for 15 minutes or 4 hours. Phosphorylated STAT3 and total STAT3 abundance (15 min) and SOCS3 protein abundance  were analysed by Western blot as previously described  and brieAll in vitro cell culture experiments were performed in two independent experiments in duplicate.Positive staining was scored semiquantitatively by two independent observers, blind to the identity of the tissue, with an intensity score assigned as 0 (negative) to 3 . All statistical analyses were performed using GraphPad Prism. Data was analysed by the non-parametric Kruskal-Wallis test followed by the Dunn's post-hoc test. Differences were considered significant at P < 0.05.IL11 was detectable in 3 of 4 postmenopausal controls and in all the flushings from the Grade 1-3 tumours  and the endometrial epithelial cell line HES, secreted very low levels of IL11 under serum free conditions. The cells were subsequently cultured in serum free conditions to examine the effect of IL11 on pSTAT3/STAT3 and SOCS3 protein abundance.Overall, all the human endometrial cancer cell lines  cells were treated with IL-11 for 4 hours and SOCS3 abundance examined at 0 (before treatment) and 4 hrs as previously described . SOCS3 pThis study was the first to show that IL11 protein was increased in uterine fluid and endometrial tumour epithelial cells in women with Grade 1 endometrial carcinoma compared to postmenopausal women. It further demonstrated that IL11's main endometrial signalling molecules, pSTAT3 and SOCS3, were produced by endometrial cancer cells. IL11 was shown to signal via pSTAT3 and SOCS3 in human endometrial cancer cell lines.Endometrial glandular epithelial products are primarily secreted apically into the uterine lumen therefore we investigated the levels of IL11 in uterine flushings. In agreement with our study, a previous study has suggested that factors present in uterine washings may confirm the presence of endometrial cancer . IL11 lePrevious studies have shown that in cycling endometrium, IL11 and IL11Rα predominantly localise to human endometrial glandular epithelium and decidualized human endometrial stromal cells ,28. EndoIn agreement with our study, IL11 localised predominantly to cancer epithelial cells in a recent report . IL11 mRIL11Rα protein was upregulated in endometrial epithelial tumour cells compared to endometrial epithelium from postmenopausal women. Strong staining for both IL11 and IL11Rα was identified in tumour vascular endothelial and smooth muscle cells as recently reported .IL11 localised to leukocytes only in the advanced Grade 3 tumours and not in control postmenopausal endometrium. Numerous studies report that tumour associated macrophages promote angiogenesis and correlate with poor prognosis . In endoIn agreement with the present study, IL11 is significantly upregulated in several non-endometrial cancers. IL11 and IL11Rα transcript levels are linked to breast cancer prognosis - breast tumours with a poor prognostic index show a high level of IL11 . SimilarTumour development and progression depends on cell adherence to extracellular matrix, proliferation, migration and invasion of tumour cells followed by their metastasis into other tissues and on escaping immune detection and destruction. Our previous studies show that IL11 increases the adhesion of human endometrial epithelial cells to various extracellular matrix molecules and to human trophoblast, at least in part by regulating adhesion molecule mRNA expression and protein production . EndometIt remains to be determined whether IL11 similarly regulates tumour cell adhesion, migration and invasion in endometrial cancers. Angiogenesis is also a key determinant of tumour formation  and hencIL11Rα protein is a proposed candidate target for both human osteosarcoma and also bone metastasis . FurtherOverall, SOCS3 imunostaining intensity was low in epithelium from postmenopausal women and all tissues from the cancer patients. There was higher SOCS3 staining in endometrial glandular epithelium from proliferative phase endometrium compared to all other groups. This suggests that SOCS3 has different functions in cycling endometrium compared to endometrium from postmenopausal women and endometrial cancer.IL11 increases pSTAT3 and SOCS3 protein in differentiating human endometrial stromal cells . STAT3 wSeveral studies have shown that SOCS proteins including SOCS3 are expressed in tumours including head and neck cancer ,40, gastIn the present study SOCS3 staining intensity was absent or very minimal in tumour epithelial cells in the Grade 3 cancer specimens perhaps similarly indicating a reduced sensitivity to SOCS3 in endometrial cancers although this remains to be determined. In normal breast epithelial cells SOCS3 is induced, while in several breast cancer cell lines SOCS3 is weakly activated. In breast tumour cells, it has been postulated that the IFNγ induced anti-proliferative effects are reduced due to a lower sensitivity to SOCS3 induction . Our in Our study suggests that IL11 in uterine washings may be useful as an early marker of endometrial cancer. It is also the first study to demonstrate that IL11 protein is upregulated in Grade 1 endometrial cancers compared to postmenopausal endometrial epithelium and suggests that IL11 signalling is active in endometrial cancer cells. The present study suggests that IL11, along with its specific receptor and downstream signalling molecules pSTAT3 and SOCS3, are likely to play a complex role in the progression of endometrial carcinoma. Functional studies are required to elucidate the role of IL11 in tumourigenesis and determine its potential as a prognostic marker and therapeutic target for endometrial cancer. Large scale studies are required to determine whether IL11 in uterine washings may be useful as a diagnostic marker for endometrial cancer.The authors declare that they have no competing interests.JY performed immunohistochemistry, quantitative ELISA, western blots, data analysis and assisted in drafting the manuscript. LAS contributed to tissue grading and collection of biopsies. TJ co-ordinated patient recruitment, tissue cancer grading and collected the biopsies. PKN provided technical assistance with the immunohistochemistry. ED conceived of the study, designed and co-ordinated the study, participated in data analysis and interpretation and drafted the manuscript. All authors read and approved the manuscript.


Flavone acetic acid (FAA), 8.6 gm-2 has been administered by 6h intravenous infusion to 19 patients with advanced colorectal carcinoma and 15 patients with advanced malignant melanoma. The drug associated toxicity was generally mild and as predicted from the phase I study. No responses were seen in either disease.


Msx1 in differentiated C2C12 myotubes has been shown to induce their dedifferentiation. While it remains unclear whether dedifferentiation and redifferentiaton occurs endogenously in mammalian muscle, there is considerable interest in induced dedifferentiation as a possible regenerative tool.Adult mammalian muscle retains incredible plasticity. Muscle growth and repair involves the activation of undifferentiated myogenic precursors called satellite cells. In some circumstances, it has been proposed that existing myofibers may also cleave and produce a pool of proliferative cells that can re-differentiate into new fibers. Such myofiber dedifferentiation has been observed in the salamander blastema where it may occur in parallel with satellite cell activation. Moreover, ectopic expression of the homeodomain transcription factor Barx2 at early and late stages of muscle differentiation which may be due to differential recruitment of transcriptional activator or repressor complexes to muscle specific genes by Barx2.We previously showed that the homeobox protein Barx2 promotes myoblast differentiation. Here we report that ectopic expression of Barx2 in young immature myotubes derived from cell lines and primary mouse myoblasts, caused cleavage of the syncytium and downregulation of differentiation markers. Microinjection of Barx2 cDNA into immature myotubes derived from primary cells led to cleavage and formation of mononucleated cells that were able to proliferate. However, injection of Barx2 cDNA into mature myotubes did not cause cleavage. Barx2 expression in C2C12 myotubes increased the expression of cyclin D1, which may promote cell cycle re-entry. We also observed differential muscle gene regulation by Barx2 regulates plasticity of immature myofibers and might act as a molecular switch controlling cell differentiation and proliferation.We show that  Adult mammalian muscle has the potential to regenerate by activation of undifferentiated myogenic precursor cells (satellite cells), which are normally quiescent and situated between the basal membrane and the myofibers It was previously suggested that dedifferentiated muscle cells became multipotent and could contribute to development of not only new muscle but also cartilage and bones msx-1 is induced in the blastema msx1 in mouse C2C12 myotubes induces cleavage of myotubes into proliferating, mononucleated cells Factors controlling dedifferentiation in newt limb are not well understood; however, expression of the muscle segment homeobox gene Barx2 in regulation of myofiber plasticity. During early embryonic stages of mouse development, Barx2 is widely expressed in proliferating and differentiating cartilage and muscle tissue Barx2 expression was found to be restricted to the joint region Barx2 cooperates with other muscle-expressed transcription factors to regulate cytoskeletal remodeling events of early myoblast differentiation In this study we investigated the role of the homeodomain transcription factor Barx2 over-expression, while more mature myotubes appear to have lost this ability. These results also suggest that Barx2, like Msx1, can regulate muscle plasticity and that homeobox factors could be a part of a general mechanism that controls the susceptibility of cells to reprogramming.Here we show that ectopic expression of Barx2 in C2C12 myotubes and MyoD-induced C3H10T1/2 myotubes induced apparent dedifferentiation indicated by myotube cleavage and concomitant down-regulation of muscle differentiation markers. We also extended these studies to differentiated primary mouse muscle cell cultures; we observed two types of myotubes in primary cultures: thin slowly contracting myotubes with nuclei aligned along the middle of the fiber (immature myotubes), and thicker, faster contracting myotubes often with small or large nuclei clusters (mature myotubes). Microinjection of Barx2 cDNA into the immature myotubes induced cleavage and formation of mononucleated cells that were able to proliferate, whereas injection of Barx2 cDNA into mature myotubes induced myotube contraction but not cleavage. Thus our data indicate that only immature myotubes can dedifferentiate in response to in vivo and is upregulated early during differentiation of C2C12 and C3H10T1/2 cells and primary myoblasts in culture. However, we do not observe Barx2 in the nuclei of mature myofibres  into immature (stage 1) or mature (stage 2) myotubes . To visuBarx2 can promote dedifferentiation of single immature myotubes.In contrast to the results in immature myotubes, injections of Barx2-plasmid into mature myotubes did not induce cleavage ; howeverBarx2 could induce re-entry of myotube-derived cells into the cell cycle, we examined whether Barx2 might affect cell cycle genes directly. Quantitative RT-PCR analysis of C2C12 cells stably transfected with a Barx2-expression plasmid showed that the cyclin D1 gene was upregulated approximately 9-fold relative to control-transfected cells  and accelerates differentiation SMA expression is known to be upregulated at an early stage of myoblast differentiation, possibly to facilitate migration and remodeling, and is subsequently downregulated and replaced by skeletal muscle actin SMA gene and can directly bind to the SMA promoter together with MyoD and increase transcriptional activation of the promoter by MyoD in undifferentiated myoblasts The data presented here combined with our previous work SMA promoter by Barx2 and MyoD was examined by co-transfection of the SMA promoter-luciferase construct with Barx2, MyoD, or empty pcDNA3 expression plasmids in C2C12 cells  family of bHLH factors act as transcriptional repressors and have roles in maintaining progenitor cells in an undifferentiated state and regulating cell fate decisions during embryogenesis. For example, lack of certain Barx2 early in myoblast differentiation as previously investigated The homeobox protein Barx2 is expressed during development of skeletal and smooth muscle Msx1Msx1Msx1 is involved in epimorphic digit tip regeneration in both humans and mice Barx2 and Msx1 share similar pathways and even target genes in the regulation of dedifferentiation. However, an important difference between the functions of Barx2 and Msx1 is that Barx2 is also able to promote the early stages of myoblast differentiation in cooperation with MyoDMsx1; on the contrary expression of Msx1 in undifferentiated C2C12 myoblasts downregulates MyoD expression and inhibits differentiation into myotubes These finding were similar to the previously described functions of another homeodomain transcription factor - Msx1 a number of homeobox factors are capable of suppressing myogenic differentiation. These include Mohawk which is a transcriptional repressor that blocks the myogenic conversion of C3H10T1/2 cells Pax3 and Pax7 paired homeodomain transcription factors have also been shown to suppress myogenic differentiation and both the paired domain and homeodomain of Pax3 are required for this anti-myogenic effect Msx1, Mohawk, and Pax3/7 homeobox containing transcription factors appear to be general repressors of myoblast differentiation. In contrast our previous and current studies indicate that Barx2 positively regulates the early steps of myoblast differentiation; however, its subsequent down-regulation may be necessary for maintenance of the differentiated state of myofibers.It is interesting that in addition to Barx2 in C2C12 cells could be an important factor in promoting cell cycle re-entry by myonuclei derived from dedifferentiated myofibres. Re-entry into the cell cycle has been also induced in terminally differentiated cultured cardiomyocytes by expression of G1 cell cycle factors Barx2 regulates a transcriptional program that allows cellular remodeling to be coordinated with cell cycle re-entry.The upregulation of cyclin D1 expression after overexpression of Barx2 at early and late stages of muscle differentiation are unclear, they are very likely to involve differential recruitment of activator or repressor complexes by Barx2 Barx2 function at different stages of myogenic differentiation.Although mechanisms underlying differential gene regulation by Xenopus embryos, Hes6 increased the size of the myotome due to increased proliferation; it also decreased markers of terminal muscle differentiation Enforced expression of Hes6 has been found to inhibit differentiation, leading to a higher proportion of thin, immature myotubes relative to thickened, mature myotubes. Hes6 expression also reduced the proportion of cells undergoing cell cycle withdrawal and allowing more cells to re-enter the cell cycle after differentiation Hes6 expression in C2C12 myotubes relative to myoblasts Barx2 and Hes6, previous work showed that the repression domain of Barx2 binds directly to the transducin-like enhancer of split (TLE) corepressor family Hes6 and other HES family repressors Our corroborating finding of increased Oct, Nanog) have been shown to be important in induction of pluripotent stem cells (iPS cells). Recent work comparing zebrafish regeneration blastema with iPS cells showed that, although blastemal cells are not pluripotent, some of the key iPS reprogramming factors including the Pou5 homeobox protein are also important for regeneration, presumably due to their role in generating a multipotent cell state. This suggests some common mechanisms may be involved in induced cellular reprogramming and in the natural dedifferentiation process observed in the blastema Barx2 to the list of homeobox regulators of adult cellular plasticity. In future work it would be of considerable interest to assess the role of Barx2 in blastemal regeneration of muscle, perhaps in the zebrafish context.Homeodomain transcription factors regulate gene expression in response to a large variety of extracellular stimuli, and act as molecular switches for controlling cell differentiation, proliferation, and apoptosis. Particular homeobox genes (Mouse C3H10T1/2 (clone 8) and C2C12 (ATCC) cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) at 37C in a humidified 5% CO2 atmosphere without antibiotics.Primary myoblast cultures were prepared as described previously The Zeiss LSM 710 laser scanning confocal microscope (LSCM) was used to obtain images. IMARIS software was used for image analysis.7 C2C12 cells were transfected with either 10 µg of Barx2/pcDNA3 expression vector or pcDNA3 control plasmid using Lipofectamine2000 reagent (Invitrogen). The Barx2 expression plasmid contains an in-frame NH2-terminal Myc tag and was described previously 1×10Total protein was prepared from C2C12 and C3H10T1/2 cells using RIPA lysis buffer and sonicated. Equal aliquots of protein were resolved by SDS-PAGE, transferred to polyvinylidene difluoride membrane, and probed with antibodies to MyoD , skeletal fast myosin , myogenin  and a custom-made Barx2 anti-peptide polyclonal antibody (Covance). β-actin, antibody was used as a reference. HRP conjugated secondary antibodies and a chemiluminescent detection system was used to visualize proteins. All experiments were performed in duplicates.Co-immunoprecipitation of Barx2 with Hes6 was performed as essentially as described in  Briefly, COS1 or C2C12 cells were co-transfected with the Barx2/pcDNA3 and Hes6/pCMVSport6 (Open Biosystems) expression plasmids using Lipofectamine-2000 (Invitrogen). Cell lysates were prepared 48 hours after transfection, pre-cleared with Protein A-Sepharose and then incubated overnight at 4°C with 5 µg of anti-Barx2 rabbit antibody or preimmune serum. Complexes were precipitated with Protein A-Sepharose, washed four times and resolved by SDS-PAGE and immunoblotted with anti-Hes6 polyclonal goat antibody (Santa Cruz Biotechnologies) antibody.ctcctgaaccacctgctagaatcc, reverse: ctaaggatgtagacaccaaatccggc, and GAPDH primers: forward: gtgaaggtcggtgtgaacggatttggccg; reverse: ccatggtggtgaagacaccagtagactcc. The Hes6 primer set amplified a 252 base-pair DNA segment of the Hes6 cDNA. The PCR products were resolved by agarose electrophoresis, bands were quantified by densitometry, and the ratio of Hes6/GAPDH PCR products was calculated. Three experiments were performed, and the results were normalized to the values from undifferentiated myoblast cultures. Statistical (t-test) analysis was performed using Microsoft Exel.RNA was isolated from undifferentiated C2C12 cells and C2C12 young and mature myotubes using the RNeasy Plus Mini Kit (Qiagen). The RNA was quantified using a Beckman DU 640 Spectrophotometer. 1 µg of each RNA sample was used to synthesize cDNA using a First-Strand cDNA Synthesis kit and SuperScript III/RNaseOUT Enzyme mix and 50 ng/ul random hexamer primers. The RT PCR was performed using Perkin Elmer 9600 PCR machine. Each sample was amplified with Hes6 primers: forward: gtatttgtctaccccagacaggtt, R: tcatcctgcgattctgatacc; mouse Cyclin D1 F: tctttccagagtcatcaagtgtg, R: gactccagaagggcttcaatc. Mouse GAPDH (NM_008084) (QIAGEN SaBiosciences) and mouse ribosomal protein S26 (RPS26) F: aggtgcagaaggctgagg, R: ggttctcccgagtgatgaag, were used as controls.RNA was prepared from primary myoblast or C2C12 cell cultures using Trizol reagent (Gibco). Cells were lysed directly in Trizol and RNA was prepared according to manufacturer protocol (Invitrogen). RNA was treated with DNase using the DNA-free kit (Ambion) and reverse transcribed using random primers and MMluV reverse transcriptase (New England Biolabs). Quantitative RT-PCR reactions were performed on an ABI 7300 machine using Superarray Biosciences RT2 SYBR green reagent and the following primers. Mouse Barx2 F: 4 1.25, NaHCO3 26, MgCl2 1, Glucose 25, CaCl2 2.Injections into myofibers were performed using traditional patch-clamp electrophysiology equipment In experiments to assess the proliferation of cells derived from microinjected myotubes, the cultures were maintained for an additional 4–5 days and then incubated with 5-bromo-2-deoxyuridine (BrdU)  for one hour. Cultures were then fixed and BrdU detection was performed using an Alexa Fluor® 594-conjugated secondary antibody according to the manufacturers protocol. Proliferating BrdU-labeled cells were detected as fluorescein-positive cells by confocal microscopy.


To identify and understand physiological processes and behavior that depends on the InsP3 signaling pathway at a systemic level, we are studying Drosophila mutants for the InsP3R (itpr) gene. Here, we show that growth defects precede larval lethality and both are a consequence of the inability to feed normally. Moreover, restoring InsP3R function in insulin producing cells (IPCs) in the larval brain rescues the feeding deficit, growth and lethality in the itpr mutants to a significant extent. We have previously demonstrated a critical requirement for InsP3R activity in neuronal cells, specifically in aminergic interneurons, for larval viability. Processes from the IPCs and aminergic domain are closely apposed in the third instar larval brain with no visible cellular overlap. Ubiquitous depletion of itpr by dsRNA results in feeding deficits leading to larval lethality similar to the itpr mutant phenotype. However, when itpr is depleted specifically in IPCs or aminergic neurons, the larvae are viable. These data support a model where InsP3R activity in non-overlapping neuronal domains independently rescues larval itpr phenotypes by non-cell autonomous mechanisms.The Inositol 1,4,5- trisphosphate receptor (InsP It is known that InsP3R is widely expressed and its role in various cellular processes has been identified using in vitro studies 3R function in the context of whole organism physiology is not well understood.Calcium is a versatile signaling molecule that has been found to regulate a multitude of processes, from fertilization to cell death. The regulation of such diverse processes depends on the intricate regulation of calcium levels by an extensive toolkit that consists of calcium channels and pumps on the plasma membrane and the membrane of intracellular stores that help in assembling signaling systems with very different temporal and spatial dynamics Drosophila melanogaster, a model system amenable to genetic and physiological manipulations, has therefore been utilized to understand both systemic and cellular requirements for the InsP3R 3R activity could underlie a physiological output. By this process, we have previously demonstrated that InsP3R expression in the neuronal domain and specifically the aminergic interneurons (with the DdcGAL4) rescues larval viability itpr mutant phenotypes can be significantly rescued by restoring InsP3R activity in insulin producing cells (IPCs) with use of the Dilp2GAL4itpr mutants arise as a consequence of disrupted feeding behavior. An independent requirement of InsP3R activity in the prothoracic gland cells that synthesize and secrete the insect molting hormone ecdysone also exists. The Dilp2GAL4 and DdcGAL4 expression domains do not exhibit any obvious overlap suggesting that the Dilp2GAL4 rescue is mediated by a non-cell autonomous mechanism.In this study, we show that larval Drosophila itpr gene exhibit larval and adult phenotypes based on the strength of the heteroallelic combination. Stronger mutant combinations are larval lethal while adult viable combinations exhibit defective wing posture with reduced flight ability and altered flight physiology sv35/ug3itpr has been well characterized; a majority of these larvae die as second instars with a slightly extended lethality profile as compared with itpr null organisms ug3itpr is a hypomorph in which the single point mutation lies in the N-terminal ligand binding domain while sv35itpr is a null allele with a stop codon in the modulatory domain sv35/ug3itpr larvae are smaller in size as compared to wild-type controls  in the background of sv35/ug3itpr. A significant rescue of larval size was observed  or due to a feeding defect in these mutants or a combination of both. The feeding ability of itpr mutants was determined quantitatively by measuring ingestion of colored food  analysis  (which stimulates ecdysone synthesis in the prothoraic gland of the ring gland) producing neurons are ablated  animals . While 5 animals . Delays ng pupae  and pupailp2GAL4 . Pupal ailp2GAL4 . The timecdysone . Ecdysonitpr mutant phenotypes by restoring itpr function in the IPCs and aminergic neurons is that an overlap exists between the two domains. In order to determine this, a membrane bound GFP (UASmCD8GFP) was expressed with Dilp2GAL4 and larval brains of these animals were stained with an anti-Ddc antibody DdcGAL4 expresses in both serotonergic and dopaminergic neurons, itpr mutant phenotypes are not rescued by expression of +UASitpr in the dopaminergic domain  lines for the itpr gene and measured their effect on larval viability by ubiquitous expression with an Actin5cGAL4. Amongst the dsitpr lines tested, one line referred to as 1063UASdsitpr, does not survive beyond the larval stages on expression with the Actin5cGAL4 line  or with either Dilp2GAL4 or DdcGAL4 had no significant effect on larval viability or size (data not shown) as judged by the number and size of pupae formed  mRNA are present in the larval and adult nervous system Drosophila serotonin receptors 5-HT1BDro (d5-HT1B) and 5-HT2Dro have been observed in larval and adult brains 3-coupled 5-HT2CR is a key mediator of the serotonergic suppression of feeding and agonists of this receptor were found to significantly improve glucose tolerance and reduce plasma insulin in murine models of obesity and type 2 diabetes + channels that respond directly to glucose levels and signal insulin release are not present on Drosophila IPCs 3 pathway could be one such mechanism The absence of any cellular overlap between aminergic and DILP producing neurons suggests that these domains regulate feeding and growth through secreted serotonin and DILPs and thus communicate with each other or influence a common subset of downstream cells by binding of serotonin and DILP to their cognate receptors. High levels of sv35/ug3itpr is a heteroallelic combination of single point mutants in the itpr gene that were generated in an EMS (ethyl methanesulfonate) screen. Detailed molecular information on these alleles has been published itpr cDNA (+UASitpr) itpr RNAi experiments were done with the UASdsitpr (1063R-2) line from the National Institute of Genetics Fly Stock Center, Japan. The Dilp2GAL4 strain was from Dr. E. Rulifson DdcGAL4P0163GAL4Actin5cGAL4 (4414), C155GAL4Elav and UASdicer(III) (24651) were obtained from the Bloomington Stock Centre. The other fly strains used were generated by standard genetic methods using individual mutant and transgenic fly lines described above.To obtain molting profiles, staging experiments were performed with minor modifications as described previously g for 5 minutes and the supernatant was transferred to a fresh tube. The supernatant was mixed with PBS and the Abs520 read.Yeast paste containing red dye  was placed centrally on 90mm petri dishes plated with 2% agar in Phosphate Buffered Saline (PBS). Larvae of the appropriate age and genotype were placed on red yeast paste and allowed to feed for 4 hrs (at 60 hrs AEL) or 2 hrs (at 108 hrs AEL). After feeding, each group of larvae were washed in distilled water, dried on blotting paper and placed in 1.5 ml tubes and immediately frozen in liquid nitrogen. Larvae were then homogenized in PBS, centrifuged at 14 nd instar larvae of the indicated genotypes were selected at 56–64 h AEL and snap frozen in liquid nitrogen. Total RNA was extracted with Trizol Reagent (Invitrogen) according to the manufacturer's protocol. Approximately 1 µg of purified total RNA was used for reverse transcription reactions. cDNA was generated using gene specific primers and MMLV reverse transcriptase (Invitrogen) at 42°C for 1 hour. Polymerase chain reactions (PCRs) were performed using cDNA as template in a 25 µl reaction. rp49 gene primers were used for internal normalization of every batch of RNA. The same sense and antisense primers were used for RT-PCR and Realtime PCR. Quantitative realtime PCRs were performed on the Rotor-Gene 3000  operated with Rotor Gene software version 6.0.34 using SYBR® Green JumpStart™ Taq ReadyMix (Sigma). Experiments were performed with rp49 and the gene of interest, using serial dilutions  of the cDNA preparation. The experiment was repeated three times with independently isolated RNA samples. Cycling parameters were 95°C for 10 min, 45 cycles of 95°C for 20 s and 53°C (for rp49) and 55°C (for 4E-BP and Lipase-3) for 30 s, 72°C for 30 s, then 1 cycle of 72°C for 5 min and hold at 50°C for 1 min. The fluorescent signal produced from the amplicon was acquired at the end of the polymerization step at 72°C. A melt curve was also performed after the assay to check for specificity of the reaction. Amplification primers were as follows:2rp49, 5′ATGACCATCCGCCCAGCATAC; 3′TTACCTCGTTCTTCTTGAGAC; 4E-BP, 5′CATGCAGCAACTGCCAAATC;3′CCGAGAGAACAAACAAGGTGG ; Lipase-3, 5′TGAGTACGGCAGCTACTTCCCT; 3′TCAACTTGCGGACATCGCTDrosophila (Canton S) was determined by the comparative ▵▵Ct method −▵▵Ct where ▵▵Ct = (Ct(target gene)−Ct(rp49))mutant −(Ct(target gene)−Ct(rp49))Wild type.The fold change in the mutant's target gene cDNA relative to wild-type rp49 were as follows: 5′CGGATCGATATGCTAAGCTGT; 3′ATGCCTAGCTTGTTCGCG.Amplification primers used for the experiment in Drosophila larval brains expressing a membrane bound GFP (UASmCD8GFP) with the Dilp2GAL4 or DdcGAL4 that were fixed in 4% paraformaldehyde for 30 minutes. The following primary antibodies were used - rat anti-Ddc , rabbit anti-GFP antibody  and monoclonal anti-5-HT antibody . The following fluorescent secondary antibodies were used at a dilution of 1∶400 - anti-rabbit Alexa Fluor 488 and anti-rat Alexa Fluor 633  and anti-mouse Rhodamine Red X (Jackson Laboratories). Confocal analysis was performed on a Zeiss LSM 510 Meta microscope  or an Olympus Confocal FV1000 microscope using 20X 0.9 N.A. or 63X 1.4 N.A. objectives. Confocal data were acquired as image stacks of separate channels and combined and visualized as three- dimensional projections using the LSM5 version 3.2/SP2 software or FV10-ASW 1.3 viewer.Immunohistochemistry was performed on Salivary glands derived from 60 hr AEL larvae were dissected in PBS, fixed in 4% paraformaldehyde, and stained with DAPI to visualize nuclei. Images were acquired at different focal planes and the total number of nuclei per salivary gland was counted.rd instar larvae of the indicated genotype were run on a 5% SDS-polyacrylamide gel and transferred to nitrocellulose membrane by standard western blotting protocols. The affinity purified anti-DInsP3R rabbit polyclonal antibody (IB-9075) raised against KLH-conjugated peptide CEQRKQKQRLGLLNTTANSLLPFQ derived from the DInsP3R sequence Protein extracts from 3t-tests was performed using Origin software  in all experiments.Computation of means, SEM, and Student


A series of plasma globulin studies was carried out on 108 patients who were operated on for splenictrauma during the last 3 years. The reasons for splenectomy or spleen salvage were; gunshot woundsin 22 patients (20.3%); stab injuries in 10 patients (9.2%) and blunt abdominal trauma in 76 patients(70.3%). Plasma gamma globulin determinations were made on the 8th postoperative day and at 3months. In the splenectomy group; plasma gamma globulin determinations demonstrated a significantreduction in serum IgM levels (p < 0.001) but no significant changes in IgA and IgG levels (p > 0.05).No changes were detected in IgA, IgG and IgM levels in the spleen salvage group (p > 0.05).        We believe that the preservation of the traumatized spleen should be the prime aim of surgeons.


Changes in cardiac output may occur during insufflation for laparoscopic procedures. However, there are limited data regarding its potential effects on cerebral oxygenation.2), end tidal CO2, heart rate, blood pressure and oxygen saturation by pulse oximetry were recorded every 5 minutes prior to insufflation, during insufflation and after desufflation. Minute ventilation was increased to maintain normocapnia and the depth of anesthesia was adjusted or fluids/phenylephrine administered to maintain the blood pressure within 20% of the baseline.Cerebral oxygenation . Eighty-two (8.2%) of the values were less than 80% of the baseline value, while 25 values (2.5%) were less than 75% of the baseline value. Twelve patients had at least one ScO2 value that was less than 80% of the baseline and 6 had at least one ScO2 value that was less than 75% of the baseline. Four patients of the cohort had ScO2 values less than 80% of the baseline for more than 50% of the laparoscopic procedure.The cohort for the study included 70 adults for laparoscopic herniorrhaphy, gastric bypass or cholecystectomy. A total of 1004 ScOAlthough relatively uncommon, significant changes in cerebral oxygenation do occur in some patients during insufflation for laparoscopic surgery. Although CO2 is generally accepted as the optimal gas for insufflation, increases in PaCO2 and resultant alterations in cerebral blood flow may occur.[Laparoscopic surgery has become a popular surgical tool due to its less invasive nature, thereby providing a more rapid recovery and improved cosmetic outcome when compared to traditional open surgery. However, a requirement of the procedure is the insufflation of the peritoneal cavity with an inert gas, most commonly carbon dioxide (COay occur.–3 Additiay occur.–6 These 2), end tidal CO2 (ETCO2), heart rate (HR), blood pressure (BP) and oxygen saturation by pulse oximetry (SaO2) were recorded every five minutes prior to insufflation, during insufflation and after desufflation. Peritoneal insufflation pressures were maintained at 8–12 mmHg. The surgical procedures were performed with the patient in the supine or at a 20–30° head-up position. During insufflation, minute ventilation was increased as needed, to maintain normocarbia. The blood pressure was maintained within 20% of baseline values by either increasing the concentration of the inhalational agent for increases in BP or the administration of a fluid bolus and/or phenylephrine for decreases in BP. The ScO2 values obtained prior to insufflation were averaged for each patient and used as that patient's baseline rSO2 for subsequent evaluations. The values obtained during insufflation were then compared to the baseline values and categorized as an increase in ScO2 from the baseline or as an absolute decrease in rSO2 of 0–9, 10–19, 20–29 or ≤ 30 or greater, from baseline. The number of values representing an ScO2 decrease to less than 75% and less than 80% of the baseline, were also determined. Patients who had any ScO2 value less than 80% of baseline, were compared to those without cerebral desaturation using a non-paired t-test and a contingency table, with a Fisher's exact test. All data are presented as the mean ± SD.This study was approved by the Institutional Review Board of the University of Missouri and informed consent was obtained from the patients. Patients scheduled for laparoscopic procedures of the abdomen requiring peritoneal insufflation were considered eligible for inclusion. The choice of anesthetic agents was not standardized and was at the discretion of the attending anesthesiologist. Cerebral oxygen saturation was continuously monitored using near infrared spectroscopy . After the induction of anesthesia, a single NIRS sensor was placed on the right side of the patient's forehead, with the caudad border approximately 1 cm above the eyebrow, with the medial edge at the midline according to the manufacturer's guidelines. Cerebral oxygen saturation (ScOThe cohort for the study included 70 adult patients ranging in age from 27 to 80 years (50.3 ± 13.9 years) and in weight from 40 to 186 kgs (104.9 ± 31.6 kgs). Twelve patients were ASA II and 58 were ASA III. The surgical procedures included laparoscopic herniorrhaphy or cholecystectomy in 34 patients and laparoscopic gastric bypass in 36 patients. The anesthetic technique included premedication with midazolam (1–2 mg), followed by intravenous induction with thiopental or propofol. Maintenance anesthesia consisted of 1–1.5 MAC  of a potent inhalational anesthetic agent, fentanyl (3–5 μg/kg) and neuromuscular blockade with intermittent doses of either cis-atracurium or rocuronium. The duration of insufflation and laparoscopy varied from 25 to 175 minutes (71.7 ± 33.3 minutes).2 varied from 47 to 95 (78 ± 11). A total of 1004 ScO2 values were obtained during laparoscopy. The ScO2 increased from the baseline during 246 of the 1004 data points and decreased from the baseline in 758 of the1004 data points. For the 758 of the 1004 ScO2 values that decreased from baseline during insufflation, the ScO2 decrease was 0–9 less than the baseline in 480 values , 10–19 less than the baseline in 252 values (24.9%) and 20–29 less than the baseline in 26 values (2.6%) [2 value was greater than 30 less than the baseline value. Eighty-two (8.2%) of the values were less than 80% of the baseline value, while 25 values (2.5%) were less than 75% of the baseline value. Twelve of the 70 patients (17%) had at least one ScO2 value that was less than 80% of the baseline value and 6 (0.09%) had at least one ScO2 value that was less than 75% of the baseline value. Four of the 70 patients of the cohort had ScO2 values less than 80% of the baseline, for more than 50% of the laparoscopic procedure. The demographics of patients with an ScO2 value less than 80% of the baseline during insufflation compared to patients without cerebral oxygen desaturation, are outlined in P =0.1). However, these patients weighed more , had longer durations of insufflation  and were more likely to be undergoing a laparoscopic gastric bypass than a laparoscopic herniorrhaphy or cholecystectomy.The baseline ScOs (2.6%) . No ScO23).[The potential use of NIRS, otherwise known as cerebral oximetry to monitor cerebral oxygenatio was first suggested by Jobsis in 1977.[3). Unlike s3).–11 Unlik3).–14et al[et al studied the response of the INVOS 3100A to arterial hypoxemia, induced by breathing a hypoxic mixture in 22 healthy adults and demonstrated that the cerebral oximeter reading correlated well with the calculated cerebral saturation.[The ability of NIRS to detect sudden changes in cerebral oxygenation when cerebral blood flow is altered by extreme maneuvers, has been demonstrated by Levy et al NIRS haset al16 Additiet al18 Pollaret al. compared changes in cerebral oxygenation using NIRS monitoring with neurologic damage assessed with biochemical markers, including neuron specific enolase (NSE) and S-100, a protein released during neuronal damage.[2 showed a significant correlation with the postoperative increase in NSE (r2 = 0.57) and S-100 (r2 = 0.52).Clinical and biochemical studies have validated the correlation of NIRS monitoring with eventual neurologic outcome. Although the majority of the literature using NIRS has compared periods of cerebral desaturation with outcome assessed by clinical assessment of neurologic status, Plachky l damage. The cohoet al have demonstrated that monitoring and acting on decreased ScO2 values may actually decrease the incidence of postoperative neurocognitive dysfunction in elderly patients undergoing major abdominal surgery under general anesthesia.[2 value was monitored and maintained at ≥ 75% of preinduction values or a monitor only group, in which cerebral oximetry was monitored, but not visible to the anesthesiologist. Cerebral oxygen desaturation (≤ 75% of baseline) was observed in 11 patients of the treatment group (20%) and 15 patients in the control group (23%). The treatment protocol to treat cerebral oxygen desaturation, included ensuring adequate ventilation, checking head positioning, increasing the delivered oxygen concentration, allowing the arterial CO2 to increase and increasing the BP with fluids or vasoconstricting medications. Postoperative neurocognitive decline was evaluated using a mini-mental status examination (MMSE). Control patients with cerebral oxygen desaturation had a lower MMSE on postoperative day 7, longer PACU stays (median time of 47 minutes) and longer hospital stays (median of 24 days), when compared with patients in the treatment group whose cerebral oxygen desaturation was reversed by interventions. The latter group had a median PACU discharge time of 25 minutes (P =0.01) and a median hospital stay of 10 days (P =0.007).More recently, Casati esthesia. The 122 et al,[2 decrease to less than 75% of baseline correlates with neurocognitive changes assessed using the MMSE, 6 or approximately 1% of our patients would be at risk. Four of our patients had ScO2 values less than 80% of their baseline during more than 50% of the insufflation time. Patients who experienced an ScO2 value less than 80% of the baseline, weighed more and had a longer duration of peritoneal insufflation. As the majority of the patients were ASA III, aside from body weight, we cannot comment as to whether other co-morbid features increased the likelihood of cerebral oxygen desaturation.In the current study, we noted that the majority of patients experienced a decrease in cerebral oxygenation during insufflation. Although the modest decreases are unlikely to be clinically significant, if we use the criteria of Casati et al, who sugget al evaluated changes in cerebral oxygenation during laparoscopy using another type of cerebral oxygenation monitor , in a study that included 12 adult patients (ASA I or II) undergoing laparoscopic cholecystectomy with a pneumoperitoneum of 10–12 mmHg.[2 increased from a baseline of 33.9 ± 1.3 to a maximum of 52.8 ± 3.3 mmHg during laparoscopy. Both HbO2  and HbR (reduced hemoglobin) increased from the baseline following insufflation, which they related to changes in cerebral blood volume induced by the hypercapnia. No change in the oxygenation status of cytochrome aa3 was noted. The maximum increase of HbO2 (7.3 ± 2.8 μmol/L) occurred 90 minutes after the start of laparoscopy.Previous studies have evaluated changes in cerebral oxygenation using NIRS during insufflation for laparoscopy. Kitajima –12 mmHg. Minute v2 from the baseline with insufflation (−1.4 ± 1.4 from baseline), with a more significant effect, 30 minutes after insufflation and assumption of a head-up positioning for the surgery (−5.1 ± 1.9 from baseline). No change in cytochrome aa3 was noted during the study.A follow-up study by the same group of investigators used the same technology to evaluate changes in cerebral oxygenation and cerebral blood volume during laparoscopic cholecystectomy in 12 adult ASA I and II patients, in whom minute ventilation was increased to maintain normocapnia. They notet al evaluated changes in cerebral oxygenation and cerebral blood volume in 15 ASA I-III children during laparoscopic fundoplication, with an insufflation pressure of 5 to 8 mmHg.[2 increased from 30.0 ± 2.8 to 38.3 ± 5.1 mmHg and PaCO2 increased from 32.0 ± 4.7 to 40.4 ± 5.9 mmHg. Cerebral oxygenation increased by 15.7 ± 8.8% and cerebral blood volume increased by 4.6 ± 8.8%.Using the INVOS cerebral oximeter, de Waal o 8 mmHg. During i2 values that we noted, it is possible that smaller studies including only 10–15 patients may miss such changes. Additionally, we collected ScO2 values at 5 minute intervals with 1000 values more, as compared with the other studies where cerebral oxygenation was assessed at 30 minutes intervals. We did not average our values, whereby a low value might be lost when averaged with other values, rather we compared each ScO2 value to the baseline, to assess its relative change. Our cohort mostly included ASA III patients, as opposed to the ASA I-II patients of Kitajima et al. Minute ventilation was increased to maintain normocapnia. As demonstrated by the studies of Kitajima et al,[2 values, although the greatest difference they noted was when assuming the head-up position for the surgical procedure.The data in our current study are somewhat different that what has been reported in the 3 previous studies that have been mentioned.–23 We woma et al,22 this met al and therefore we cannot comment as to whether treatment of decreases in ScO2 values would have an impact on the outcome. Recommendations for the treatment of decreases in ScO2 values include checking ventilator function and head positioning, increasing the FiO2, increasing the PaCO2 for values less than 35 mmHg, increasing mean arterial pressure with fluid or vasoconstrictors, followed by decreasing the cerebral metabolic rate of oxygen by the administration of propofol or a barbiturate if the above measures fail.The current study did not separate randomized patients into treatment and non-treatment groups as was done by Casati 2, which have been shown to correlate with neurocognitive changes in other studies. The limitations of the current study are, that we did not evaluate our patients postoperatively to see if changes in ScO2 correlated with postoperative neurocognitive changes and therefore we cannot determine if monitoring and treating changes in ScO2 can prevent postoperative neurocognitive changes, as has been previously demonstrated by the study of Casati et al.[2 values, an analysis to determine additional “at risk” features other than body weight and duration of insufflation, such as co-morbid features or intraoperative conditions , was not feasible. However, given that the deleterious physiologic effects of increased intraabdominal pressure during insufflation on stroke volume, cardiac output and other hemodynamic variables have been well documented,[In summary, we noted that although most patients tolerated insufflation without significant effects on cerebral oxygenation, a subset of patients developed significant decreases in ScOti et al. As all tcumented, it is li


Sir,Improved self-management following participation in a clinical trial with better glycemic control has been recently reported in the literature.2 ParticiRetrospective analysis of glycemic control in type 2 diabetic patients who participated in clinical trials was done by comparing the fasting glucose and HbA1C values at screening and randomization visits. We selected the patients who participated in a randomized controlled clinical trial on new therapy for type 2 diabetes mellitus. A total of three such studies were selected. All these studies were approved by the Institutional Ethics Committee. Eligible patients for the evaluation were those who undertook both the screening as well as randomization visits. Those who had undergone only screening were excluded. The interval between the two visits varied between studies depending upon the study design. Antidiabetic medication remained unchanged between the two visits. Fasting glucose, HbA1C, and body weight were measured at both the visits. All these trials included adult, males or females.2) who were inadequately controlled with insulin alone or in combination with oral hypoglycemic agents. Patients were screened at visit 1 and those found eligible continued with stable dose of insulin and oral hypoglycemic agents along with placebo medication for 2 months. At the end of 2 months patients were randomized to receive active study medication. Trial 2 included type 2 diabetic patients with inadequate glycemic control. Patients were drug naïve at the time of inclusion. Patients were screened at visit 1 and received placebo for 1 month after which the eligible patients were randomized. Trial 3 included patients with history of type 2 diabetes mellitus and body mass index between 23 and 45 kg/m2. These were inadequately controlled (HbA1C above 8.0%) and received a combination therapy including metformin and another oral hypoglycemic agent. After screening, all eligible patients were put on maximum tolerated dose of metformin along with pioglitazone for 3 months after which the eligible patients were randomized to receive the study medication.Trial 1 included obese patients with features of metabolic syndrome (body mass index above 25 kg/mP < 0.05 was considered significant. The main outcome was short-term glycemic control measured by glycated hemoglobin (%) and fasting glucose level.Fasting glucose and HbA1C were measured at screening as well as randomization visit in all the three studies. Glucometer was dispensed to all patients for self-monitoring of blood glucose. Patients were given adequate education and were taught to use the glucometer. They were also advised to maintain a diary for daily record of glucometer readings. Data from all three studies were combined as well as analyzed individually. The Student ‘t’-test was used for statistical analysis and a A total of 61 patients  were studied . Patients in trial 3 had maximum duration of diabetes (17.65±8.02 years) and trial 2 patients had shortest. The median interval between screening (visit 1) and randomization (visit 2) was 60 days in trial 1, 30 days in trial 2, and 90 days in trial 3.P=0.074). Overall analysis has shown a decrease in HbA1C between the two visits. Baseline fasting blood glucose was also highest in trial 1 patients and was lowest in trial 2 patients. Fasting blood glucose increased from visit 1 to visit 2 in trial 1 patients and decreased in trial 2 and 3 patients. The drop in fasting glucose level in trial 3 patients was statistically significant (P=0.01). Overall analysis also showed a decrease in fasting glucose level between the two visits [Baseline HbA1C was highest in trial 1 patients. It increased further in trial 1 patients from baseline to randomization visit, whereas it decreased in trial 2 and 3 patients. Drop in HbA1C was much more in trial 3 patients though statistically insignificant (o visits . Body weet al.[th percentile of HbA1C. Improved glycemic control following participation in a clinical trial can be best understood as a result of enhanced awareness in patients, increased care given by health professionals, frequent monitoring and motivation of patients for better adherence to therapy and lifestyle modification.[The present analysis has shown that study participation itself causes sufficient changes in the glycemic control. Combined analysis of all the three studies has shown that there was sufficient reduction in blood glucose and HbA1c level in patients who participated in clinical trials. Similar observations have also been made before.3 In the fication.5 It is o