These alterations in protein breakdown rates were associated with an inhibition of the ATP-ubiquitin-dependent proteolytic pathway . The cytokine did not modify the plasma levels of corticosterone and insulin in the tumour hosts. The present data give new insights into the mechanisms by which IL-15 exerts its preventive effect on muscle protein wasting and seem to warrant the implementation of experimental protocols involving the use of the cytokine in the treatment of pathological states characterized by TPH, particularly in skeletal muscle, such as in the present model of cancer cachexia. © 2000 Cancer Research CampaignTissue protein hypercatabolism (TPH) is an important feature in cancer cachexia, particularly with regard to the skeletal muscle. The Yoshida AH-130 rat ascites hepatoma is a model system for studying the mechanisms involved in the processes that lead to tissue depletion, since it induces in the host a rapid and progressive muscle wasting, primarily due to TPH. The present study was aimed at investigating if IL-15, which is known to favour muscle fibre hypertrophy, could antagonize the enhanced muscle protein breakdown in this cancer cachexia model. Indeed, IL-15 treatment partly inhibited skeletal muscle wasting in AH-130-bearing rats by decreasing (8-fold) protein degradative rates (as measured by


Ghrelin and one of its functional receptors, GHS-R1a (Growth Hormone Secretagogue Receptor 1a), were firstly studied about 15 years. Ghrelin is a multifunctional peptide hormone that affects several biological functions including food intake, glucose release, cell proliferation… Ghrelin and GHS-R1a are expressed in key cells of both male and female reproductive organs in several species including fishes, birds, and mammals suggesting a well-conserved signal through the evolution and a role in the control of fertility. Ghrelin could be a component of the complex series of nutrient sensors such as adipokines, and nuclear receptors, which regulate reproduction in function of the energy stores. The objective of this paper was to report the available information about the ghrelin system and its role at the level of the hypothalamic-pituitary-gonadal axis in both sexes. Ghrelin was initially discovered as a ligand for the growth hormone secretagogue receptor GHS-R1a) [a [1], anGhrelin is a 28-amino acid peptide derived from preproghrelin . It has Ghrelin is found in mammalian species as well as nonmammalian species. The majority of ghrelin is synthesized by an endocrine cell population, the X/A-like cells, in the stomach mucosa . GhrelinCirculating ghrelin levels increase with fasting and return to basal levels after refeeding in rodents and humans –25. The The ghrelin receptor is a G-protein-coupled receptor (GPCR) firstly identified in pigs and humans in 1996 . It beloThe GHS-R1a receptor mRNA is mainly expressed in the pituitary  and in s2+ increases, due to a transient Ca2+ release from the intracellular store and a Ca2+ influx through voltage-dependent L-type calcium channel, which are observed as the signal transduction. Ghrelin has also been shown to increase AMPK (Adenosine Monophosphate-activated kinase) activity in the hypothalamus  and decrease the expression of markers of apoptosis  , 84. MorIt has also been reported that ghrelin inhibits early embryo development in mice . In porcIn addition, in rats, high levels of ghrelin receptor (GHS-R) mRNA are detected in various peripheral fetal tissues beginning on embryonic day 14 and lasting until birth. Maternal ghrelin regulates fetal development during the late stages of pregnancy .The testis is a complex endocrine organ where different cell types interplay to produce germ cells, under the control of several extragonadal and intragonodal hormones and growth factors. Some evidence suggests that ghrelin participates in such a regulatory network , 89–91 .Expression of ghrelin has been demonstrated in rodents and sheep by immunostaining mainly in Leydig cells . GhrelinExpression of the functional ghrelin receptor, GHS-R1a, has been shown in Sertoli and Leydig cells as well as seminiferous tubules in rats . Some chμg for 2 days) in adult rats inhibited mRNA expression of the gene encoding stem cell factor (SCF), a key signal for germ cells production and a putative regulator of Leydig cell development. Such an inhibitory action of ghrelin on SCF has also been detected in vitro using cultures of staged seminiferous tubules [These latter data suggest that ghrelin could regulate spermatogenesis by an autocrine or/and a paracrine manner. In this sense, intratesticular injection of ghrelin  [In vitro, ghrelin significantly also inhibits in a dose-dependent manner both hCG- and cAMP-stimulated testosterone release by Leydig cells . This inype III) . In vivoype III) . Thus, h It has also been demonstrated by in vivo intratesticular injection of ghrelin that ghrelin is able to inhibit the proliferative rate of immature Leydig cells both during puberty development and after selective ablation of pre-existing mature Leydig cells by administration of EDS (ethylene dimethane sulfonate) . GhrelinGhrelin is able to modulate key testicular functions such as seminiferous tubule gene expression, testosterone secretion, and Leydig cell proliferation. Thus, this peptide could operate as a novel regulator of testicular development.In conclusion, ghrelin is a peptide hormone mainly secreted from the stomach into the circulation, but it can be synthesized by other tissues such as reproductive tissues suggesting local actions (autocrine and/or paracrine). Its functional receptor, GHS-R1a, is also expressed at different levels of the hypothalamic-pituitary-gonadal axis. As described in this paper, ghrelin may participate in the regulation of different aspects of the female and male reproductive functions from germ cell production to embryo development. These actions appear to be species-specific. Ghrelin through its various biological functions including energy metabolism by promoting fat deposition and food intake could be a key signal between energy status and control of fertility (nutrient-gene expression). However, further studies are required to gain insights into the understanding of the fine mechanisms of ghrelin action.


The goal of this study was to understand gene expression signatures of hepatocellular carcinoma (HCC) recurrence in subjects with hepatitis C virus (HCV) infection. Recurrence-free survival (RFS) following curative resection of HCC in subjects with HCV is highly variable. Traditional clinico-pathological endpoints are recognized as weak predictors of RFS. It has been suggested that gene expression profiling of HCC and nontumoral liver tissue may improve prediction of RFS, aid in understanding of the underlying liver disease, and guide individualized patient management. Frozen samples of the tumors and nontumoral liver were obtained from 47 subjects with HCV-associated HCC. Additional nontumoral liver samples were obtained from HCV-free subjects with metastatic liver tumors. Gene expression profiling data was used to determine the molecular signature of HCV-associated HCC and to develop a predictor of RFS.The molecular profile of the HCV-associated HCC confirmed central roles for MYC and TGFβ1 in liver tumor development. Gene expression in tumors was found to have poor predictive power with regards to RFS, but analysis of nontumoral tissues yielded a strong predictor for RFS in late-recurring (>1 year) subjects. Importantly, nontumoral tissue-derived gene expression predictor of RFS was highly significant in both univariable and multivariable Cox proportional hazard model analyses.de novo tumor formation in cirrhotic patients.Microarray analysis of the nontumoral tissues from subjects with HCV-associated HCC delivers novel molecular signatures of RFS, especially among the late-recurrence subjects. The gene expression predictor may hold important insights into the pathobiology of HCC recurrence and  Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide by annual incidence and the third leading cause of cancer death . Wide geOne of the key reasons for poor long-term survival in HCC is high incidence of recurrence, a complication that cannot be prevented effectively by new and existing therapies ,8. Many Several research groups have performed gene expression profiling of both tumor and nontumoral specimens and identified gene signatures of recurrence-free , or overThus, our study focused on a well-defined cohort of HCC patients who were HCV positive (but free of hepatitis B virus) and underwent tumor resection to conduct a comparative microarray-based gene expression profiling of tumor and nontumoral specimens. We report the molecular network signatures of HCV-associated HCC, as well as the outcome of the analysis of the predictive value of gene expression in tumor- and nontumoral tissue-derived samples. This study was not only successful in developing a highly predictive RFS signature for late recurrence from nontumoral samples, but also shows that this signature can be used for biological interpretation of the liver disease leading to recurrence.This study evaluated specimens  collected during liver tumor resection in (i) subjects with solitary tumors in liver that were a result of metastasis from other organs, and (ii) subjects manifesting HCC in combination with chronic HCV infection and absence of known tumors at other sites. Platelet count, alanine aminotransferase and fibrosis score were significantly different between the groups Table . The HCCThe importance of defining liver tumor biology that may aid in the development of new screening and treatment stratification programs to refine diagnosis and improve patient outcome is well recognized . Thus, oTo evaluate the biological features of HCV-associated HCC we compared gene expression profiles of tumors and control  samples. The analysis revealed 155 up- and 1,248 down-regulated genes whose expression levels were significantly different (FDR = 0). Next, functional pathway analysis was performed using significant genes , several top-ranked (based on Cox score) gene sets were found to be predictive of tumor recurrence  recurrence and repeated the cross-validation procedure at both gene and gene set level. Even though a more traditional cut-off for "late" recurrence is 2 years, recent meta-analysis of HCC molecular subclasses suggests that 1 year time point may represent a more biologically-defined cut-off [It has been suggested that distinct gene expression signatures may exist for prediction of early and late recurrence . This is cut-off .When individual transcript-level data was used, very strong predictive signatures could be derived for up to 91 genes  and the whole human genome  Agilent array used in our work, but also the clinico-pathological variables of the patient cohort used by the authors have not been released to the public . Thus, while the cross-study comparisons shall remain as the goal for validation of the findings reported on independent cohorts and array platforms, greater degree of collaboration may be needed in the field of liver tumor biology.Indeed, a comparison of the applicability of the poor-prognosis signatures from this study to the independent HCC cohort is the ultimate test for the 'biological' validation of the findings. There are two published microarray datasets that profiled nontumoral tissues from HCC subjects as potentially suitable for validation ,12 whichWhile the molecular signatures generated in this study may not be clinic-ready without additional meta-analysis once the data becomes publicly available, we posit that late recurrence predictive genes may provide new insights into the oncogenic pathways for HCC development in cirrhotic patients. By examining the biological roles of the predictive genes it may be possible to better understand the process of field cancerization in HCC ,38 and aSecond, gene set analysis was used to evaluate the data from the sub-cohort of late >1 year) recurrence subjects Figures . A numbe year recde novo tumor occurrence in HCV-associated cirrhosis, the biopsy-based gene expression analysis in cirrhotic patients may be a useful method to stratify the risk of tumor occurrence and determine a need for aggressive patient screening. While challenges remain in validating the signatures and translating the findings to clinical practice, we posit that gene expression-based signatures provide important insights into pathobiology of HCC recurrence.Our study demonstrates that microarray analysis of the nontumoral tissues from subjects with HCV-associated HCC delivers novel molecular signatures of RFS, especially among the late-recurrence subjects. Since late recurrence is likely a Forty-seven subjects with HCV-associated HCC who underwent complete removal of the tumor at the University of Yamanashi Hospital  between 2000 and 2007 were enrolled in this study. All of the subjects tested positive for HCV and negative for hepatitis B virus. The presence and identification of the hepatitis virus was determined by one or more of the following techniques: (i) presence of anti-HCV and anti-HBV reactive serum proteins, (ii) reverse transcription-PCR for serum HCV-RNA, or (iii) branched DNA-HCV probe assay. Following the tumor resection procedure and recovery after surgery, each subject visited the ambulatory care clinic for additional tests monthly. Serum α-fetoprotein levels were measured every month. In addition, ultrasonography or computed tomography of the liver was performed every 3 and 6 months, respectively. Eight HCV- and HBV-free subjects who were diagnosed with metastatic liver tumors that required surgical resection were enrolled as the control group and nontumoral tissue samples were obtained. All samples were frozen in liquid nitrogen immediately following surgery and kept as -80°C until processed. Informed consent was received from each subject in the study and the study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in an a priori approval by the Institutional Board on Ethics for Human Science at the University of Yamanashi. All available clinical data for the subjects enrolled in this study are summarized in Table JMP software  was used for statistical analysis of the clinical and molecular (see below) variables. For univariable analysis, two-sample t-test, linear regression, or Logrank test statistical procedures were used to assess what endpoints could be used for predicting prognosis of HCC subjects after surgery. For multivariable analysis, the Cox proportional hazard model was used to calculate hazard ratio and p-value of each parameter. A p value of less than 0.05 was considered significant.Total RNA was isolated from tumor and nontumoral liver samples (~30 mg) with an RNeasy plus mini kit  as detailed by the manufacturer. High quality RNAs was obtained from 43 tumor and 44 nontumoral samples . For microarray analysis, a balanced design was used. Each batch of arrays  was balanced with regards to the tumor stage and RFS and contained both inter-batch and intra-batch replicates.2 ratio of Cy5/Cy3 intensity was normalized using LOWESS smoothing to eliminate intensity bias of features. Transcripts with fewer than 70% available data across samples were excluded from the analysis, reducing the probe list to 25,073 transcripts. Available data are defined as those probes that are neither saturated nor below the limit of quantification. Inter-batch normalization was carried out using Distance Weighted Discrimination procedure [RNA samples were hybridized to arrays individually; none were pooled. RNA amplifications and labeling were performed using Low RNA Input Linear Amplification kits (Agilent Technologies). For hybridization, 1.2 μg of total RNA from each sample, or human reference RNA  was amplified and labeled with fluorescent dye . RNA labeling, microarray hybridization, washing and scanning were performed according to the manufacturer's instructions. Feature extraction, normalization of the raw data and data filtering were performed using the Agilent Feature Extraction software . Raw microarray data was archived in Gene Expression Omnibus (GSE17856) and is available to the public. The logrocedure .Differentially expressed genes between control and tumor sample groups were identified by using Significant Analysis of Microarray analysis . Individhttp://www.broad.mit.edu/cancer/software/genepattern/). The signatures were validated using a leave-one-out cross-validation prediction procedure. In brief, each sample was left out one-by-one and a disease-free survival-correlated signature was deduced using remaining samples (selecting marker genes based on top-ranked highest absolute Cox score). A predicted class label was assigned to the left-out sample based on the closest "template" using the nearest template prediction algorithm. A maximum of 200 top-ranked genes were used to make predictions in each analysis. The outcome of cross-validation based prediction was assessed by Logrank test and the "error rate," defined here as the ratio of the number of subjects predicted incorrectly to that in the model. In parallel, the "gene set" analysis (GSA) was also used to determine the significance of pre-defined sets of genes with respect to an outcome variable, such as a RFS time. GSA package for R http://www-stat.stanford.edu/~tibs/GSA/ was utilized. The output from GSA was used to generate RFS predictors in the same manner as detailed above for gene-level data and validated using a leave-one-out cross-validation procedure.Tumor and adjacent nontumoral liver tissues were considered separately to define an RFS-predictive signature on each type of tissue. In each dataset, genes whose expression was associated with time to recurrence were selected by means of the high absolute Cox score and prediction analysis was performed by evaluating the expression status of the signature using the nearest template prediction method as described elsewhere and implHCC: Hepatocellular Carcinoma; HCV: Hepatitis C Virus; FDR: False Discovery Rate; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; RFS: Recurrence-Free Survival; SAFE: Significance Analysis of Function and Expression.The authors declare that they have no competing interests.MT participated in the design of the study, carried out the molecular and statistical analyses and drafted the manuscript; JP carried out the statistical analyses and participated in writing of the manuscript; HK, MM and HF designed the study, performed sample analyses and assisted with the development of the manuscript; IR designed the study, participated in the analyses of the data and was responsible for writing of the manuscript. All authors read and approved the final manuscript.® pathways analysis) differentially modulated between control  tissue and HCC tumors. table S3. GO categories and KEGG pathways that differ significantly between control and HCC tumor samples were identified using SAFE analysis (see Methods). table S4. Sixty seven top-ranked gene sets used for recurrence prediction analysis in tumor samples (see Figure table S1. Clinical outcomes for each individual patient included in this study. table S2. Molecular networks (IngenuityClick here for fileFigure S1. TGFβ1- and MYC-centered interactomes are most significantly up-regulated molecular networks in HCC tumors (see Additional file Click here for file


Pisum sativum) is a well-established classic model species for genetics studies that has been used since the Mendel era. In addition, the availability of a plethora of developmental mutants makes pea an ideal crop legume for genomics studies. This study aims to utilise genomics tools in isolating genes that play potential roles in the regulation of SAM activity.Despite the importance of the shoot apical meristem (SAM) in plant development and organ formation, our understanding of the molecular mechanisms controlling its function is limited. Genomic tools have the potential to unravel the molecular mysteries of the SAM, and legume systems are increasingly being used in plant-development studies owing to their unique characteristics such as nitrogen fixation, secondary metabolism, and pod development. Garden pea (Pisum sativum cv Torsdag). Custom-designed oligonucleotide arrays were used to compare the transcriptional profiles of pea SAMs and non-meristematic tissues. A total of 184 and 175 transcripts were significantly up- or down-regulated in the pea SAM, respectively. As expected, close to 61% of the transcripts down-regulated in the SAM were found in the public database, whereas sequences from the same source only comprised 12% of the genes that were expressed at higher levels in the SAM. This highlights the under-representation of transcripts from the meristematic tissues in the current public pea protein database, and demonstrates the utility of our SAM EST collection as an essential genetic resource for revealing further information on the regulation of this developmental process. In addition to unknowns, many of the up-regulated transcripts are known to encode products associated with cell division and proliferation, epigenetic regulation, auxin-mediated responses and microRNA regulation.In order to identify genes that are differentially expressed in the SAM, we generated 2735 ESTs from three cDNA libraries derived from freshly micro-dissected SAMs from 10-day-old garden peas (The presented data provide a picture of the transcriptional profile of the pea SAM, and reveal possible roles of differentially expressed transcripts in meristem function and maintenance. Organ formation is not limited to embryonic development, but can occur throughout the lifetime of a plant. The potential to develop new organs post-embryonically is attributed to meristems located at the growing tips of the plants, with the root apical meristem generating the underground part of the plant and the shoot apical meristem (SAM) giving rise to the entire shoot system after seed germination.Like its root counterpart, the SAM contains a pool of pluripotent stem cells that can self-maintain as well as produce the cells that can differentiate into multiple cell and tissue types [reviewed in ]. While Arabidopsis thaliana, has enhanced our understanding of this vital developmental process . This ied state , and theem cells . While tArabidopsis species. On the other hand, garden pea (Pisum sativum) has been a classic model species used in genetics  and theAB176564), while that of 10.7-fold was for a sequence annotated as vegetative lectin (AAA33691). A study of similar genes encoding type 1 metallothionein in cotton revealed their abundant  expression in the root .EX569897), chromatin remodelling factors  and DNA methyltransferases  and FASCIATA2 (FAS2) genes that encode subunits of the chromatin assembly factor leads to dysfunction of the SAM . For in the SAM . This wan factor . Howevern factor .EX570025) annotated as a putative plant-specific transcription factor from a YABBY-family protein – members of this family are reportedly involved in the abaxial cell-fate specification in lateral organs of Arabidopsis [EX570238). Similar members of the auxin response factor group of proteins are known to regulate auxin-mediated transcript activation or repression. For example, the expression of several genes, such as those encoding members of LATERAL ORGAN BOUNDARIES domain proteins and AUXIN-REGULATED GENE INVOLVED IN ORGAN SIZE, are disrupted in the double mutant of ARF7 and ARF19, implicating their roles in auxin-mediated plant development [We also identified a few putative transcription factors among the genes whose expression was higher in the SAM than in the NM. These included an EST , auxin-conjugating protein (DWARF IN LIGHT1) and a ribosomal protein L24B , pathogenesis-related protein (AJ586324), disease-resistance-response protein (AF139018), antimicrobial defensin (AF525685) and chitinase (AB037832). This might be attributable to the stems, leaves and roots generally being exposed to greater biotic or abiotic stress than the well-shielded SAM, with the former therefore requiring the constitutive presence of these gene products at a higher level than in the SAM in order to maintain successful defence responses. However, we found at least one other sequence (EX571142) potentially related to stress responses whose expression was higher in the SAM , catalase (EX568770), Cu-Zn superoxide dismutase (AB189165), copper amine oxidase (AB026253) and peroxidase (AB087838). This implies that the level of ROS is lower in the SAM than in the NM, and hence the expression of genes encoding ROS scavengers is lower in the former. The absence of photosynthesis (a source of ROS) in the SAM might explain the lower ROS level therein. However, there is increasing evidence that plants use ROS as signalling molecules for regulating development and various physiological responses, and for mediating abscisic-acid-induced stomatal closure, as well as in auxin signalling and gravitropism in roots [Surprisingly, the down-regulated transcripts included various sequences encoding proteins that scavenge or generate ROS, such as thioredoxin , pectin methylesterase (AF056493), xyloglucan endotransglycosylase (AB042531), were found to be down-regulated in the SAM. This probably reflects the cell-wall structure in the stem and leaf being more complex that the thin primary cell wall in meristematic cells [Some of the sequences predicted to encode products that play roles in cellulose synthesis, such as cellulose synthase EX56963, pectinIn silico analysis of the predicted gene products has implicated several processes in the complex molecular network that regulates this developmental process. Future studies of these genes should attempt to reveal how they interact in the complex molecular network that maintains and regulates the dynamics of the SAM.The development of our EST collection from the pea SAM represents an important advance towards understanding SAM function and maintenance, especially due to the under-representation of SAM-related transcripts in the public database as demonstrated in this study. Subsequent transcriptional profiling experiments using the microarray constructed from ESTs yielded the transcriptional signatures of the pea SAM, and we have reported a repertoire of transcripts with putative or unknown functions that are differentially regulated in the SAM. Pisum sativum) cultivar Torsdag was grown in a greenhouse located at the University of Melbourne, Australia. SAMs were micro-dissected from 10-day-old peas under the dissecting microscope at 40× magnification. Any leaf primordia were excluded in order to create a meristem-enriched tissue collection and the location of tissue sample is indicated in Figure Garden pea , subtracted (S) and normalized (N) libraries were sequenced at Australian Genome Research Facility (AGRF), Australia and subsequently at Macrogen Korea using T7 primer. These sequences have been deposited in GenBank under the accession numbers Sequence data were trimmed off vector, adaptor and low quality sequences using SEQTools . TrimmedAll sequences were then imported into Blast2GO, a web-based Gene Ontology (GO) annotation and analysis tool  for subsTotal RNA was extracted from dissected SAM (approximately 80 SAMs per extraction) or other plant parts  using Qiagen RNeasy Mini Kit. Four independent tissue collections and RNA extractions  were performed for each of the microarray hybridization experiment.in situ synthesis [The Combimatrix arrays are semiconductor-based oligonucleotide microarrays and are generated based on CombiMatrix technology (hyperlink) of ynthesis . The Cus. One microgram of total RNA from SAM or NM was labelled using the Kreatech's ULS™ RNA ampULSe kit to generate Cy5 or Cy3 labelled targets. The Cy5- or Cy3-labelled cDNA was then hybridized to different sector of the chip according to a balanced block design dual label experiment scheme [Target preparation and hybridization were performed in Australian Genome Research Facility Ltd (AGRF) according to the standard CombiMatrix protocol described in detail at t scheme :Sector 1: Cy3-SAM A vs Cy5-NM ASector 2: Cy5-SAM B vs Cy3-NM BSector 3: Cy3-SAM C vs Cy5-NM CSector 4: Cy5-SAM D vs Cy3-NM DGSE9278.The Cy5- and Cy3-hybridized chip was then scanned using Genepix 4000B microarray scanner  according to manufacturer's instructions. The generated tiff image files were then imported into Combimatrix Microarray Imager to produce intensity data. The LimmaGUI software, which is an implementation of the Empirical Bayes linear modelling approach, was used for subsequent statistical analysis of the resulting data . A robusThe one-tube, two enzyme Access RT-PCR system  was used according to manufacturer's instructions in all RT-PCR analysis. Ten ng of RNA isolated from the SAM, mature stem, mature leaf and primary root of 10-day-old pea seedlings were used as a template in a 10 μl reaction volume. The pea actin gene was used as an internal control. The number of cycles used for the transcripts investigated was routinely between 25–28 and 80 % of the PCR reaction was separated on 1% agarose gel containing 0.1 μg/μl ethidium bromide and visualized under UV light.SAM: shoot apical meristem; NM: non-meristem; EST: expressed sequence tag.CEW carried out the EST analysis, participated in the microarray experiment and RT-PCR analysis, and drafted the manuscript. PLB and MBS were responsible for design of the project, standardisation & organization of meristem micro-dissection & making EST libraries as well as overall coordination of the experiments and manuscript editing. HO contributed to EST library characterization and EST sequence analysis. All authors read and approved the final manuscript.Transcripts identified to be up-regulated in the pea shoot apical meristem (SAM) in comparison to the non-meristem (NM) tissues.Click here for fileTranscripts identified to be down-regulated in the pea shoot apical meristem (SAM) in comparison to the non-meristem (NM) tissues.Click here for file


Gastrointestinal stromal tumours (GIST) frequently occur in patients with neurofibromatosis type 1 (NF-1). It has been reported that GIST may co-exist with pancreatic endocrine tumors but this has only been in association with NF-1.A 76 year old woman presented with a 12 month history of hypoglycaemia symptoms. Abdominal CT scan demonstrated a 13 mm insulinoma localized in the tail of her pancreas. She was commenced on diazoxide and later underwent surgery for enucleation of insulinoma when a small (< 1 cm) incidental tumour was discovered on her stomach wall which was identified as GIST.This is the first case report of a pancreatic insulinoma co-existing with a GIST in a patient without NF-1. In addition, we make the first report of rapidly growing cystic GIST recurrence following resection of a primary GIST tumour. Gastro-entero-pancreatic (GEP NE) tumours have an incidence of around 30/million/population/year . PancreaGIST most commonly occur sporadically, but show increased tendency in patients with NF1 ,4. A smaHere we report the first case of insulinoma associated with a GIST in a patient not diagnosed with NF-1 as confirmed by the pre-operative endocrine assessment.A 76 year old woman presented with a 12 month history of hypoglycaemia symptoms. The patient underwent a series of tests including measurement of overnight fasting plasma glucose, measurement of plasma levels of glucose; insulin; and c-peptide during a hypoglycaemic episode, measurement of urinary sulphonylurea, and radiological imaging with a CT scan. Abdominal CT scan demonstrated a 13 mm insulinoma localized in the tail of her pancreas. She was commenced on diazoxide and later underwent surgery for enucleation of insulinoma when a small (< 1 cm) incidental tumour was discovered on her stomach wall. The small incidental tumour was an exophytic gastric wall lesion that had not been detected pre-operatively and revealed no suspicious features suggestive of invasion or spread on intra-operative inspection. For this reason the lesion was excised with diathermy off the surface of the gastric wall, the defect being closed with Polydioxanone suture, and no frozen sections were taken to assess invasion/spread. Macroscopically the lesion was completely excised. The nature of the lesion was unknown at this time. Recovery was uncomplicated and she was discharged home a week later.The pancreatic lesion figure  was compThe gastric lesion was a 6 mm firm whitish nodule in the upper wall of her stomach and its histology revealed an epithelioid cell tumour. Mitotic rate was very low and no necrosis was noted. Immunohistochemistry showed uniform positivity with C-kit figure  and CD34The patient was re-admitted 6 months later with weight loss, anorexia and vague symptoms. She had a CT scan which showed a 3.9 × 3.9 cm fluid attenuation mass superior to the tail of her pancreas (figure She subsequently underwent oesophagogastroduodenoscopy-guided endoscopic ultrasound confirming an ill-defined mass extending into the stomach wall. Cytology revealed groups of spindly and epithelioid cells highly suggestive of recurrent/metastatic GIST but not indicative of a neuroendocrine tumour figure . She wasThe first important message in our case is that a GIST can occur simultaneously with an insulinoma in the absence of NF-1. The GIST tumour was only incidentally discovered by the operating surgeon during enucleation of the insulinoma in our case. The literature contains only 9 published reports of the simultaneous occurrence of GIST and GEP NE tumour in NF-1 patients -12 (TablThe other unique message in our case is the observed rapid recurrence of the GIST following the resection of the primary gastric wall GIST. Small GISTs, like the initial one in our case, are usually an incidental surgical finding, while large tumours are usually symptomatic. In our case, following resection of the insulinoma and the small incidental gastric GIST, the GIST recurred locally with rapid enlargement. The tumour showed marked cystic transformation probably as result of central necrosis and liquefaction. The reason for the rapid recurrence of the GIST is unclear but evidence from animal studies demonstrates that a primary tumour can inhibit its remote metastases via circulating angiostatin such that after primary tumour removal, the inhibition is removed and metastases become vascularized and grow rapidly . We suspIn summary, this is the first case report of a non-NF-1 patient that had a pancreatic insulinoma co-existent with a GIST, and also, the first case report of a GIST that underwent rapid cystic recurrence following resection of the primary GIST lesion. Unfortunately our unique case report raises more questions than provides answers because there are very few cases of NF-1-related concurrent GEP NE tumours and GISTs and also very little understanding of the prognostic implications of such concurrent tumours. Available evidence suggests that mutations in the NF-1 gene might be involved in the pathogenesis of GIST in NF-1 patients . HoweverWritten informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.EA conceived and designed the report, analyzed all the reports and drafted the manuscript. SB performed surgery on the patient and participated in designing the report. BO performed mutation analysis on the GIST tumour and participated in the analysis of histological specimens. BM performed the radiological procedures on the patient and participated in designing the report. PT analyzed and reported on all the histological specimens in the case and participated in designing the report. All authors read and approved the final manuscript.


M. tuberculosis infection. At present these assays do not discriminate between disease and latency. Therefore, more promising antigens and diagnostic tools are continuously being searched for tuberculosis immunodiagnostics. The heparin binding hemagglutinin (HBHA) is a surface protein of M. tuberculosis which promotes bacterial aggregation and adhesion to non-phagocytic cells. It has been previously assumed that native, methylated form of this protein would be a promising antigen to discriminate latent from active infection.In recent years T cell based interferon gamma release assays (IGRA) have been developed for immunodiagnosis of M. smegmatis or to synthetic peptides in patients with recent or past tuberculosis, with atypical mycobacteriosis, or in healthy vaccinated individuals. The T cell reactivities to HBHA were compared to the respective reactivities towards Purified Protein Derivative (PPD) and two surface secreted proteins, ie. Early Secretory Antigen Target-6 (ESAT-6) and Culture Filtrate Protein-10 (CFP-10). Our pilot results indicate that methylated recombinant HBHA induced a strong T cell mediated immune response and the production of IgG and IgM-class antibodies in all patient groups, most surprisingly in young Finnish vaccinees, as well. We observed a positive correlation between the reactivities to HBHA and non-specific PPD among all studied subjects. As expected, ESAT-6 and CFP-10 were the most powerful antigens to discriminate disease from immunity caused by vaccination.We performed a pilot investigation to study humoral and T-cell mediated immunological responses to recombinant HBHA produced in On the basis of results of this exploratory investigation we raise concerns that in countries like Finland, where BCG vaccination was routinely used, HBHA utility might not be sufficient for diagnostics because of inability to explicitly discriminate tuberculosis infection from immunoreactivity caused by previous BCG vaccination. Mycobacterium tuberculosisMycobacterium tuberculosis heparin binding hemagglutinin (HBHA) is a virulence factor that promotes bacterial aggregation, adhesion to the heparan sulphate proteoglycans of nonphagocytic cells, and dissemination of tubercle bacilli from the lungs to other tissues in patients suffering from tuberculosis Newly introduced T cell based interferon gamma release assays (IGRA) are of a great diagnostic importance in distinguishing with high specificity the persons who are infected with Mycobacterium smegmatisM. tuberculosis infection ie. ESAT-6 and Culture Filtrate Protein Derivative (CFP-10). For this pilot study we enrolled a few tuberculosis (TB) patients with a variable degree of disease activity and healthy young and middle-age Finnish vaccinees who were practically free from any previous contact with M. tuberculosis and thus served as a valid control group.Because some earlier studies For this study we enrolled the subjects as follows:Those subjects in whom the symptoms of the disease started within one month, who were hospitalized, anti-tuberculosis treatment was commenced within 2–3 weeks and who had a recent positive bacteriological verification. In this group four presented with pulmonary tuberculosis, one with tuberculosis of gastrointestinal tract and one patient had tuberculosis of cervical vertebra. In these subjects the blood samples were obtained within the routine laboratory investigation.This group consisted of outpatients who presented with no or mild symptoms and who were either partially treated for TB with PAS, streptomycin, and INH or thoracotomy in forties and fifties, in whom chest X-ray findings indicated lack of an active process. Patients with vertebral TB had destructive processes of their vertebra at early childhood. At adulthood their TB was confirmed by radiology and the thick needle aspiration biopsy that ruled out other diseases. At the time of investigation these patients did not receive anti-tuberculosis treatment. Culture or nucleic acid amplification analysis (NAA) were either negative or bacteriological analysis was not performed.Four patients with pulmonary manifestations in whom non-tuberculous mycobacterial (NTM) infection was diagnosed by a positive culture isolation. At the time of investigation these subjects were outpatients and their symptoms were mild (mainly cough).Finnish-born University students (mean age 25 years) who on the interview did not admit any previous contact with a TB patient and thus served as an ideal negative control, and Finnish-born laboratory personnel (mean age 50 years), were enrolled. All subjects of this group were born after the routine BCG vaccination campaign was implemented in Finland. Repeated vaccinations were discontunued in Finland in 1990.a) a verbal informed consent was obtained. The verbal informed consent was sufficient because the patient–doctor relationship was earlier established (Dr. Pusa), the patients attended medical care for the routine doctor's check-up and venipuncture was a part of their current medical treatment. From the groups b), c) and d) a written informed consent was obtained before venipuncture. This study was approved by the Ethical Committee of the University Hospital of Helsinki, Internal Disease Department (Drno. 232/E5/07).From the subjects of the group Whole blood was withdrawn and divided into portions. From one portion sera were prepared by conventional methods and frozen at −20°C until use. From another portion peripheral blood mononuclear cells (PBMC) were isolated by Ficoll Paque gradient  and frozen in the CTL  media in a liquid nitrogen until use. The characteristics of the individuals enrolled in this study are presented in M. tuberculosis (H37Rv) HBHA-protein starting from 37th amino acid were synthesized . The average purity of the peptides was ∼73%. Three peptides from the protein C-terminus were chemically methylated at their lysine residues . The pMV3-38 plasmid that contained the full-length HBHA open reading frame was kindly provided by Dr.G. Delogu  Recombinant methylated HBHA was expressed in TB kit, Oxford Immunotec, Oxford, UK), PPD  and rMtb-HBHA was assessed by Enzyme-Linked ImmunoSpot assay . Peripheral blood mononuclear cells (PBMC) were purified from fresh blood samples by density gradient centrifugation and stored with CryoABC Kit Freezing media (Cellular Technology Ltd.) in liquid nitrogen until use. After cells were washed with RPMI  and resuspensed in the CTL Test Media (Cellular Technology Ltd.), the cell count was performed by blood count-analyzer . The cells were diluted at 2.5×106 PBMC/ml in CTL Test Media (Cellular Technology Ltd.). 250 000 PBMCs/well were stimulated in ELISPOT-plates in the presence of synthetic peptide-pool (10 µg/ml each), PPD (10 µg/ml), rMtb-HBHA (25 µg/ml). For viability test 50 000 PBMC/well were stimulated with phytohaemagglutinin (PHA) (Oxford Immunotech). CTL Test Media was used as a negative control. The plates were incubated at +37°C with 5% CO2 for 48 h. Thereafter the plates were washed and the analysis was performed according to the manufacturer's instruction. The spots were counted the ELISPOT-reader  and the net values were calculated by subtracting the readings of the media control. Spot-sizes and the cytokine values were examined with the ELISPOT-reader, AID EliSpot Software Version 4.0 . The viability control test for the HBHA synthetic peptides was performed by exposing PBMC to the CFP-10 peptide mixture with asetonitrile added at concentrations of 15% and 30% . The addition of asetonitrile did not violate the reactivity to the CFP-10 antigen (data not shown).T-cell reactivity to ESAT-6, CFP-10 . IgM rheumatoid factor was controlled for all serum samples by immunonephelometric method at the HUSLAB, Unit of Immunology.The presence of IgG- and IgM-class antibodies to synthetic peptides, rMtb-HBHA and PPD were determined by enzyme-linked immunosorbent assay (ELISA). Synthetic peptides from The correlation was analyzed by the non-parametric Spearman test. The Receiver operating characteristic (ROC) curve analysis and the Area under the curve (AUC) with respective 95% confidence intervals (CI) for each antigen were calculated with the GraphPad Prism version 4.0 .M. tuberculosis and M. bovis were searched with the Entrez Protein search engine and then the sequences were aligned with the Needle program, EBLOSUM62-Matrix .High interindividual variations were observed in serology, ie. 3 out of 9 (Inactive TB) and 2 out of 4 (Active TB) were strong reactors in the IgG and IgM EIA whereas almost all vaccinees showed moderate to high reactivities in IgG and IgM EIAs . NoteworROC curves were constructed for all tested antigens and the AUCs with respective 95% CI were compared for each tested antigen . As expeM. tuberculosis infection and are absent from the majority of non-tuberculous mycobacteria and BCG substrain M. tuberculosis (Active and Inactive TB groups) would be quantitatively and qualitatively different from vaccinated individuals. In other words, we were interested to study the specificity of immune response to HBHA as a nominator of infection. The minor objective was to see whether immunological responses would differ in TB patients with a different degree of disease severity. We did not enrol a group of patients with a proven LTBI which may be considered as a limitation of our study. On the contrary, we enrolled young Finnish vaccinees that were unambiguously interpreted as free from LTBI and some patients with proven NTM infection. In our opinion, these two groups is a strength of our investigation. For analysis we used ELISPOT and EIA techniques.The potential of HBHA for diagnostics have been recently reported M. smegmatis(405nm). In their study, surprisingly, the combined group of vaccinated people and patients with presumed latent tuberculosis infection did not produce IgG reactivities above the cut-off level. Using a larger cohort size than we did, Zanetti et al. The rMtb-HBHA antigen used in this study was prepared as described by Delogu et al Masungi et al. M. tuberculosis H37Ra evoked a much stronger CD8+ and CD4+ mediated immune responses than the recombinant non-methylated HBHA produced in E. coliM. smegmatis expression host was able to produce HBHA with a methylation pattern very similar to that of the native HBHA, as was evidenced by mass spectrometry analysis, amino acid sequencing, electrophoresis and recognition by the same monoclonal antibody M. tuberculosis and of recombinant HBHA produced in M. smegmatis may be different which would cause minor differences in their immunoreactivities. The protein used in our study, rMtb-HBHA, was expressed in M. smegmatis and all attempts were made to avoid potential contamination that might have blurred the results. While it could be argued that minor M. smegmatis genome coded contaminant antigens might have caused the observed T cell responses in our study, the antibody responses (IgM) to the synthetic peptides support the view of weak specificity of the rMtb-HBHA responses in BCG vaccinated persons. In view of a longevity of immunological memory even without boosting Methylation of HBHA is crucial for effective T-cell immunity. Thus, native HBHA purified from BCG or Results of immunological studies with a battery of synthetic peptides comprising mono- and dimethylated peptides of the C- terminus of HBHA were very disappointing. In this study IgM-class reactivities were detectable but in a very unpredictable fashion while IgG-class antibodies did not recognize peptides even methylated ones in any of the patient groups. The peptides did not evoke any T cell mediated response. Noteworthy, our study protocol confirmed that the non-responsiveness to the peptides was not attributed to the toxicity of asetonitrile used in the peptides solution. Temmerman et al Because our cohorts were too small to be able to pick-up minor differences in reactivity between the studied subjects we chose to use ELISPOT, the most sensitive and functional technique to study cell-mediated immunity. We assessed not only the frequencies of reactive lymphocytes  but the i) the presence of closely related antigen in a BCG substrain; ii) observed good correlation with PPD in serology and cell-mediated assays, iii) comparable ROC analysis for HBHA and PPD; iv) evidence of reactivities of vaccinees without any previous risk of contraction of M. tuberculosis infection, HBHA by no means is superior to ESAT-6 and CFP-10. In our environment HBHA practically does not add to tuberculosis immunodiagnostics. In conclusion, our results emphasize that the search for new more promising antigens for TB diagnostics should continue.This pilot study casts some concerns about the possibility to generalize about diagnostic potential of HBHA. We have observed that samples from healthy individuals in a country with almost complete vaccination coverage and even from individuals who have never been vaccinated may exhibit immune reactivities to HBHA, indicating that natural immunization to this protein or to cross-reactive peptides may occur. Therefore, on the basis of


Mycobacterium avium subsp. paratuberculosis (MAP) exist - S and C MAP strains that primarily infect sheep and cattle, respectively. Concentration of iron in the cultivation medium has been suggested as one contributing factor for the observed microbiologic differences. We recently demonstrated that S strains have defective iron storage systems, leading us to propose that these strains might experience iron toxicity when excess iron is provided in the medium. To test this hypothesis, we carried out transcriptional and proteomic profiling of these MAP strains under iron-replete or -deplete conditions.Two genotypically and microbiologically distinct strains of M. smegmatisΔideR with IdeR of C MAP or that derived from S MAP and compared their transcription profiles using M. smegmatis mc2155 microarrays. In the presence of iron, sIdeR repressed expression of bfrA and MAP2073c, a ferritin domain containing protein suggesting that transcriptional control of iron storage may be defective in S strain. We next performed transcriptional and proteomic profiling of the two strain types of MAP under iron-deplete and -replete conditions. Under iron-replete conditions, C strain upregulated iron storage (BfrA), virulence associated (Esx-5 and antigen85 complex), and ribosomal proteins. In striking contrast, S strain downregulated these proteins under iron-replete conditions. iTRAQ (isobaric tag for relative and absolute quantitation) based protein quantitation resulted in the identification of four unannotated proteins. Two of these were upregulated by a C MAP strain in response to iron supplementation. The iron-sparing response to iron limitation was unique to the C strain as evidenced by repression of non-essential iron utilization enzymes (aconitase and succinate dehydrogenase) and upregulation of proteins of essential function .We first complemented Taken together, our study revealed that C and S strains of MAP utilize divergent metabolic pathways to accommodate in vitro iron stress. The knowledge of the metabolic pathways these divergent responses play a role in are important to 1) advance our ability to culture the two different strains of MAP efficiently, 2) aid in diagnosis and control of Johne's disease, and 3) advance our understanding of MAP virulence. Mycobacterium avium subsp. paratuberculosis (MAP), the causative agent of Johne's disease (JD) of ruminants, often requires eight to sixteen weeks to see colonies in culture - a major hurdle in the diagnosis and therefore in implementation of optimal control measures. Unlike other mycobacteria, which mobilize iron via mycobactins, MAP is unable to produce detectable mycobactin in vitro or in vivo  needed by the cell and free iron and sulfide atoms are toxic to cells [Under iron-limiting conditions most other bacteria including F operon ,45. SUF F operon . This isto cells . Our datCattle MAP strain upregulated pyruvate dehydrogenase operon involved in catabolism of propionate a key component of lipid biosynthesis under limiting iron conditions . In contenhanced intracellular survival (eis) interacts with host T cells. Stimulation of recombinant Eis from MTB results in increased production of IL-10 and decreased production of TNF-α thus contributing to mycobacterial survival inside macrophages [MAP2325, a hypothetical protein deleted in the sheep strain was found to be upregulated under iron-limiting conditions by the C strain (Additional file rophages . We haverophages ,25.There is increased protein synthesis and turn over in response to iron in MTB . SimilarOur data revealed striking differences in metabolic pathways used by cattle and sheep strains of MAP to adapt to iron starvation Figure . We haveSS designed the study. HKJ participated in the experimental design with SS and performed most of the experiments. SK and AK helped in some experiments. JBP contributed to new reagents. BAW performed mass spectrometry. PJ and LAH helped in iTRAQ data analysis. HKJ and SS analyzed the data and wrote the manuscript. All authors read and approved the manuscript.Descriptive and pathway analysis of transcriptome and proteome data. This file contains the experimental design, additional microarray, proteomic and Q-RT PCR data along with pathway analysis of iron stress response proteins of C and S MAP strains.Click here for file


Therapeutic hypothermia has been proven to be effective in improving neurological outcome in patients after cardiac arrest due to ventricular fibrillation (VF). Data concerning the effect of hypothermia treatment on long-term survival however is limited.Clinical and outcome data of 107 consecutive patients undergoing therapeutic hypothermia after cardiac arrest due to VF were compared with 98 historical controls. Neurological outcome was assessed at ICU discharge according to the Pittsburgh cerebral performance category (CPC). A Kaplan-Meier analysis of follow-up data concerning mortality after 24 months as well as a Cox-regression to adjust for confounders were calculated.Neurological outcome significantly improved after mild hypothermia treatment . In Kaplan-Meier survival analysis hypothermia treatment was also associated with significantly improved 2-year probability for survival . Cox-regression analysis revealed hypothermia treatment (p = 0.031) and age (p = 0.013) as independent predictors of 24-month survival.Our study demonstrates that the early survival benefit seen with therapeutic hypothermia persists after two years. This strongly supports adherence to current recommendations regarding postresuscitation care for all patients after cardiac arrest due to VF and maybe other rhythms as well. Patients surviving cardiac arrest still have a poor prognosis with regard to both mortality and neurological outcome. Current guidelines recommend mild hypothermia treatment after cardiac arrest due to ventricular fibrillation (VF) as well as for other initial rhythms,2. TheseRecent studies show that neurological performance does not change markedly from the time of ICU discharge to six months after cardiac arrest in the majority of patients . In a fe® Medivance, USA).The study protocol was approved by the local ethics committee on human research. Between 2005 and 2007 a total of 107 patients were admitted consecutively to our MICU after in-hospital (IHCA) or out-of-hospital cardiac arrest (OHCA). Hypothermia treatment was applied to all survivors after cardiac arrest (n = 107) for 24 hours. A historical control group treated prior to the implementation of hypothermia protocols was identified (n = 98 patients admitted to our MICU between 2002 and 2004 after cardiac arrest). Detailed characteristics for the study population are given in Table Neurological outcome was defined at the time of discharge from ICU according to the Pittsburgh cerebral performance category (CPC) . CPC 1 aThe SPSS software (Version 17.0) and Medcalc (Version 11.0) were used for statistical analysis and graphical depiction. Descriptive parameters are given as median and interquartile range (25-75 percentiles). Univariate analysis of differences between hypothermia patients and the control group was performed by using the Mann-Whitney-U test for non-parametric unpaired data. Survival data were analyzed by the Kaplan-Meier method and comparison between groups was performed by the log-rank test. To adjust for confounders a Cox-regression analysis was calculated.During the observation period, 107 consecutive comatose patients after VF cardiac arrest were admitted to our MICU. The baseline characteristics are given in Table Data on neurological outcome of the patient groups at ICU discharge is presented in Table In contrast CPC 5 was almost equally distributed .A follow-up concerning mortality was performed after 24 months. Six patients of the hypothermia group and 11 patients of the control group were lost to follow-up. 101 patients treated with therapeutic hypothermia and 87 control patients were included in this analysis.The Kaplan-Meier analysis showed a significantly higher 2-year probability of survival in the hypothermia group .Univariate analysis showed significant differences between the groups for epinephrine dose, time to ROSC, bystander CPR and APACHE-score. Therefore a Cox-regression analysis was performed to adjust for these possible confounders. Hypothermia treatment p = 0.031) and age (p = 0.013) were the only parameters identified as independent significant predictors for the probability of 24-month survival  was almost identical. Thus the outcome at ICU discharge in both groups was probably not significantly influenced by more early therapy withdrawal in the treatment group. Nevertheless, neurological status may influence the further development and therefore mortality of these patients. It cannot be fully excluded that patients in a good condition are more likely to receive sophisticated medical therapies than patients in persistent coma. This may have also resulted in a higher probability of survival in the treatment group which of course can be only indirectly attributed to hypothermia treatment. Reliable data concerning witnessed arrest and a time delay to defibrillation which could also influence survival are not available unfortunately.In summary, it is known that the prognosis of patients after out-of-hospital cardiac arrest is similar to that of patients with acute myocardial infarction if they survive until hospital discharge . Our stuIn conclusion, our data demonstrate that therapeutic hypothermia may be effective in two ways: First of all the neurological outcome at ICU discharge is significantly improved. Furthermore, there is a long lasting benefit concerning probability of survival when therapeutic hypothermia has been applied.AMI: Acute myocardial infarction; APACHE: Acute Physiology and Chronic Health Evaluation; CPC: Cerebral Performance Category; CPR: Cardiopulmonary resuscitation; HACA: Hypothermia after Cardiac Arrest trial; ICU: Intensive care unit; IQR: Interquartile range; OHCA: Out-of-hospital cardiac arrest; VF: Ventricular fibrillation; ROSC: Return of spontaneous circulation.The authors declare that they have no competing interests.CS, JN and DH designed and supervised the study from data acquisition to data analysis. AK and JCS participated in the design of the study, revised the manuscript for important intellectual content and helped to draft the manuscript. All authors have read and approved the final version of the manuscript.


Delivering extinction trials minutes after fear conditioning yields only a short-term fear suppression that fully recovers the following day. Because extinction has been reported to increase CS-evoked spike firing and spontaneous bursting in the infralimbic (IL) division of the medial prefrontal cortex (mPFC), we explored the possibility that this immediate extinction deficit is related to altered mPFC function. Single-units were simultaneously recorded in rats from neurons in IL and the prelimbic (PrL) division of the mPFC during an extinction session conducted 10 minutes (immediate) or 24 hours (delayed) after auditory fear conditioning. In contrast to previous reports, IL neurons exhibited CS-evoked responses early in extinction training in both immediate and delayed conditions and these responses decreased in magnitude over the course of extinction training. During the retention test, CS-evoked firing in IL was significantly greater in animals that failed to acquire extinction. Spontaneous bursting during the extinction and test sessions was also different in the immediate and delayed groups. There were no group differences in PrL activity during extinction or retention testing. Alterations in both spontaneous and CS-evoked neuronal activity in the IL may contribute to the immediate extinction deficit. Failure to extinguish fear memory is a core feature of several anxiety disorders Recently, two regions of the mPFC have been implicated in the expression of fear. The infralimbic division (IL) projects to inhibitory neurons in the amygdala involved in suppressing fear after extinction In the light of this circuitry, we hypothesize that neuronal activity in the IL that is involved in the acquisition of long-term extinction memory may be disrupted under immediate extinction conditions Unit recording sites in IL and PrL are illustrated in Rats were submitted to four phases of training: baseline (BL), fear conditioning, extinction (EXT), and a retention test (TEST). For the baseline session, all rats were presented with ten tone conditional stimuli  in a novel context. They next received fear conditioning with five tone-shock  pairings delivered in another novel context. Depending on their group assignments, the rats were then extinguished with 50 tones either 15 minutes  or 24 hours  in the baseline context. Twenty-four hours after extinction all rats received a retention test consisting of 10 CS-alone presentations in the baseline context. Unit activity was recorded during both the 2-s tone CS period and the inter-trial intervals during BL, EXT, and TEST.Post hoc comparisons revealed that freezing behavior between the two groups was equivalently low during the BL session. As we have previously observed Freezing behavior during each session is shown in Studies have shown that IL, but not PrL, neurons exhibit increases in CS-evoked firing after extinction training that correlate with fear suppression As shown in z>0 within 200 ms of CS onset). Among all the neurons recorded, only about 30% exhibited tone-responsivity in each of the behavioral phase . Average peri-event time histograms of spike firing in these neurons are shown in We characterized CS-evoked single-unit activity in both IL and PrL neurons during each behavioral session. Overall, the number of CS-responsive neurons in both the IL and PrL was low. Only 13 out of 245 neurons in the IL and 13 out of 327 neurons in the PrL met our standard criterion (z>3 in any post-CS bin within 200 ms of CS onset) for tone responsivity lower levels of CS-evoked firing than animals in the IMMED group that did not exhibit extinction. Indeed, CS-evoked spike firing in the IL appeared to correlate more strongly with the expression of fear than extinction. This outcome was not expected based on previously published work For IL, there was no significant difference in CS-evoked spike firing among the groups [F<1] during the BL session . During In the prelimbic division of the mPFC, CS-evoked responses were generally more robust in amplitude than in the IL, and PrL neurons exhibited CS-evoked firing in all of the behavioral sessions . HoweverPrevious studies have shown that spike bursting in the IL is a neural correlate of extinction in rats To elucidate whether IL bursting is linked to CS onset, we quantified trial-related bursting during both the 2-s CS and the 1-min inter-trial interval (ITI) following the CS in each behavioral phase. Consistent with an earlier report In the present study, we used electrophysiological recordings in the medial prefrontal cortex to examine the neural correlates of the immediate extinction deficit in rats. Our results reveal that rats undergoing immediate extinction exhibited minimal suppression of CS-evoked firing in the IL during extinction training, and maintain CS-responsiveness during a retention test twenty-four hours later. Despite elevated CS-evoked responses, IL neurons in rats undergoing immediate extinction exhibited similar levels of bursting to the CS during extinction training, although these burst responses were significantly attenuated during the inter-trial intervals. Altered CS-evoked neuronal firing and intertrial bursting in the IL may contribute to the failure of rats undergoing immediate extinction to maintain fear suppression the following day.In previous studies, increases in IL bursting have been reported during the consolidation of extinction memory after extinction training was complete In addition to the bursting, we found that CS-evoked activity in the IL (at least among those neurons that exhibited an excitatory response to the CS) did not dissipate during the course of immediate extinction training, unlike that in rats undergoing delayed extinction. Furthermore, CS-evoked responses in IL were significantly greater during the retention test in animals that failed to extinguish. This result was unexpected insofar as an earlier report found that CS-evoked responses in the IL were minimal before extinction training, and increased in magnitude after extinction What might account for changes in mPFC activity in rats undergoing immediate extinction? One possibility is that the stress engendered by a recent traumatic event, i.e., fear conditioning, might yield the immediate extinction deficit. Stress-induced arousal appears to compromise the function of mPFC circuits involved in extinction learning. Several studies have found that stress impairs IL function and impairs extinction Ultimately, there is considerable debate in the clinical literature about when therapeutic interventions should be attempted after psychological trauma The subjects were eight male Long-Evans rats  obtained from a commercial supplier . They were singly housed in individual cages on a 14-h light/10-h dark cycle (lights on at 7:00 am) and allowed food and water ad libitum. During the first 5 days, they were handled for 10 sec each day to habituate them to the experimenter. All experiments were carried out in accordance with guidelines approved by the University of Michigan University Committee on Use and Care of Animals.Each rat was implanted with a head assembly containing 18 individually drivable tetrodes Neuronal and behavioral data acquisition was performed using a 96-channel amplifier system (Boston University Electronics Design Facility) and acquired on-line using SciWorks software . Single-unit activity was amplified , filtered (300 Hz to 6 kHz), and digitized (31.25 kHz) and written to disk. Tetrodes were progressively lowered into target area across several days and were left in place at least three days prior to the starting of behavioral procedures (roughly ten days after surgery). Tetrode placement was performed on a swivel stool outside of the recording chamber that was to be used for the behavioral experiments. Neuronal and behavioral data were continuously acquired during each behavioral session and stored for further analyses.Single units with  were detected off-line using SciWorks, and then manually discriminated and clustered using Offline Sorter . Data were then imported into NeuroExplorer  for analyses of firing rate, inter-spike intervals, peri-event time histograms, and bursting.One standard rodent conditioning chamber  was modified to accommodate electrophysiological recording. It rested on a load-cell platform that was used to record chamber displacement in response to each rat's motor activity. The load cell amplifier output was digitized at 5 Hz and acquired on-line using SciWorks .Two distinct contexts were used in this experiment. For the first context (context A), a 15 W houselight mounted opposite the speaker was turned on, and room lights remained on. The chamber was cleaned with a 1% acetic acid solution. To provide a distinct odor, a stainless steel pan containing a thin layer of this solution was placed underneath the grid floors before the rat was placed inside. The ventilation fan in chest supplied background noise (65 dB). For the second context (context B), the room lights were turned dim and the chamber houselight was turned off. Additionally, the door on the sound-attenuating cabinet was closed, the ventilation fan was turned off, and the chamber was cleaned with 1% ammonium hydroxide solution. Also, stainless steel pans containing a thin layer of the same solution was placed underneath the grid floors before the rat was placed inside to provide a distinct odor.Rats were submitted to four phases of training: baseline (BL), fear conditioning, extinction (EXT), and a retention test (TEST). Unit activities were acquired during the BL, EXT, and TEST sessionsOn Day 1, rats received 10 tone-alone  presentations during the BL session after placement in the recording chamber (context A). Rats then received five tone-footshock  trials ) either 10 min after the BL session  or 10 min before the EXT session  on Day 2 in another context (context B). On Day 2, all rats received 50 tone-alone presentations during the extinction session . On Day 3, all rats were returned to the extinction context (context A) again and exposed to another 10 CS-alone presentations for the retention test (TEST). Freezing was assessed during the BL, COND, EXT, and TEST session.At the end of experiments, anodal current  was passed through the tetrode tips to create small marking lesions. Rats were then perfused across the heart with 0.9% saline followed by a 10% formalin solution. After extraction from the skull, brains were post-fixed in 10% formalin solution for two days, at which time the solution was replaced with a 10% formalin and 30% sucrose solution until sectioning. Sections (45 µm thick) were cut on a cryostat (−20°C), and wet-mounted on glass microscope slides with 70% ethanol. After drying, the sections were stained with 0.25% thionin for visualization of lesions.Post hoc comparisons in the form of Fisher's PLSD tests were performed after a significant F ration.All behavioral data are expressed as means and standard error of the means (SEM) and were submitted to analysis of variance (ANOVA) in 10-trial blocks during BL, early/late EXT , and TEST sessions unless specified otherwise. z-score) during the 2-sec CS period. A burst was defined as three or more consecutive spikes with an interval of less than 30 ms between the first two spikes and less than 50 ms in subsequent spikes.Tone-evoked responses for each single unit were summed across 10 CS trials in different behavioral phases and post-CS activity was normalized to the 2s pre-CS baseline (200ms bin-size) to a generate standard score (


The presence of anti-HBc IgG in the absence of HBsAg is usually indicative of a past self-limiting HBV infection. But it is frequently associated with co-infection with HCV which can worsen the existing status of chronic liver disease (CLD).The present study was planned to evaluate the significance of isolated HBc IgG positivity in patients of CLD and look for the presence of HCV co-infection in such patients.Clinical profiles and biochemical tests were done for all the 77 CLD cases included in the study. Blood samples were taken from these patients and tested by the commercially available EIA for the presence of HBsAg, anti-HBc IgG, anti-HBs and anti-HCV. HBV DNA was detected by amplifying the surface region in all the cases.Isolated anti-HBc IgG positivity defined as the presence of anti-HBc IgG in absence of any other serological markers of HBV infection was detected in 28 patients. Out of 64 patients positive for anti-HBc IgG 36 had the markers of HBV, either HBsAg, HBV DNA or anti-HBs alone or in combination. There was a significant association between isolated anti-HBc IgG positivity and HCV co-infection.Anti-HBc IgG should be tested in all patients with CLD as it is frequently the only marker of HBV infection in such patients and they should be monitored closely as such patients can develop CLD. Presence of co-infection with HCV should be actively searched for in such patients. Both Hepatitis B virus (HBV) and Hepatitis C virus (HCV) are the major causes of chronic liver disease (CLD) worldwide.2 The dia3In cases of co-infection with both HBV and HCV serious consequences are seen with more severe and aggressive liver disease. PrevalenEpidemiologic results have also shown that prior exposure to HBV infection is a risk factor for the development of HCC. Hence inThe present study was conducted in the Departments of Microbiology and Medicine, Maulana Azad Medical College and associated Lok Nayak and GB Pant hospitals in New Delhi from January 2003 - March 2004.The study group included a total of 77 cases of chronic liver disease selected randomly from the Medical Outpatient Department or Medical Wards of Lok Nayak Hospital. All the three types of CLD i.e. chronic hepatitis, cirrhosis and hepatocellular carcinoma were included and were diagnosed on the basis of clinical, biochemical, serological and/ or histopathological study. Informed consent was taken from these patients before their inclusion in the study. The patients were evaluated on the basis of a detailed history and detailed physical and systemic examination. Autoimmune hepatitis, Wilson's disease, primary hemachromatosis and other infective causes besides viral hepatitis could not be ruled out.Seven ml of blood was collected aseptically from all patients and it was subjected to a battery of biochemical and hematological investigations. Serum for viral serology was stored at −20°C until further testing was done. Serological tests were performed using commercially available ELISA kits according to the manufacturer's instructions. The various serological tests performed in all the patients included HBsAg provided by BIORAD, antibody to the core antigen, (HBc IgG) using commercially available ELISA kits by Biochem Immunosystems and antibody to Hepatitis C Virus (anti HCV) by Dia Sorin. Anti HBS was tested in all the patients using ELISA kit by SRL.III digest) on 3% agarose gel stained with ethidium bromide and the 321 base pair product was compared with the known DNA marker (f × 174 Hae III digest) under UV illumination. The results were calculated according to manufacturer's instruction. The results were statistically analyzed using Chi square test and Student's t-test. P value of < 0.05 was considered as statistically significant.Polymerase chain reaction was performed in all the 77 samples for HBV DNA. HBV DNA was extracted from the serum sample using the standard phenol-chloroform-isoamylalcohol method. PCR was A total of 77 cases of chronic liver disease were included in the study. The cases were divided as having either chronic hepatitis, cirrhosis or hepatocellular carcinoma. Since other etiology of chronic liver disease besides hepatitis markers was not specifically looked for, hepatitis B virus was the most common cause which could be assigned to 83.1% based on the presence of HBc IgG. Presence of various serological markers in each group is given in P value being < 0.05. The patients showing isolated anti-HBc IgG were further grouped into those having HCV co-infection (8 cases) and those who did not have HCV co-infection (20 cases). Clinical parameters and the biochemical profile among both the groups was similar and there was no statistically significant difference among the groups [Tables Out of 28 cases of isolated anti-HBc IgG positive patients as many as eight also had the presence of HCV co-infection (28.57%). On comparing the prevalence of HCV co-infection in HBsAg positive and isolated anti-HBc IgG positive patients, a statistically significant association was seen between isolated anti-HBc IgG seropositivity and HCV co-infection with s Tables .Surface antigen of HBV (HBsAg) by convention is used as the only marker to diagnose the prevalence of HBV infection, but this can miss a substantial number of cases. Additional tests like anti-HBc IgG and HBV DNA can help identify further cases as is seen in the present study too.11 The prThe prevalence of HCV infection in CLD patients has been reported to be between 10–40%13 whereaAll the 12 patients in our study who tested positive for anti-HCV were HBsAg negative. This is in accordance with other studies having reported the presence of HCV in serologically silent HBV infection with the prevalence ranging between 50–87%. However The clinical significance of the silent HBV infection in HCV associated CLD has not been fully understood. A few studies have reported that the clinical course is generally more severe in patients with HBV / HCV co-infection,–8 wherea17The patients having isolated HBc IgG and those having HCV co-infection did not have significantly different liver function profile but the deranged liver function test in patients having isolated HBc IgG positivity confirms the presence of liver disease in such patients.Thus it can be concluded from the present study that anti-HBc IgG could frequently be the only marker for past HBV infection and its presence should be tested in all patients of CLD as patients with isolated anti-HBc IgG positivity can develop liver disease. Furthermore patients showing isolated anti-HBc IgG should be monitored closely for liver function. These patients are also more likely to have presence of co-infection with HCV which should be actively searched for in them.


Several unicellular organisms (prokaryotes and protozoa) can live under permanently anoxic conditions. Although a few metazoans can survive temporarily in the absence of oxygen, it is believed that multi-cellular organisms cannot spend their entire life cycle without free oxygen. Deep seas include some of the most extreme ecosystems on Earth, such as the deep hypersaline anoxic basins of the Mediterranean Sea. These are permanently anoxic systems inhabited by a huge and partly unexplored microbial biodiversity.Spinoloricus nov. sp., Rugiloricus nov. sp. and Pliciloricus nov. sp.) new to science. Using radioactive tracers, biochemical analyses, quantitative X-ray microanalysis and infrared spectroscopy, scanning and transmission electron microscopy observations on ultra-sections, we provide evidence that these organisms are metabolically active and show specific adaptations to the extreme conditions of the deep basin, such as the lack of mitochondria, and a large number of hydrogenosome-like organelles, associated with endosymbiotic prokaryotes.During the last ten years three oceanographic expeditions were conducted to search for the presence of living fauna in the sediments of the deep anoxic hypersaline L'Atalante basin (Mediterranean Sea). We report here that the sediments of the L'Atalante basin are inhabited by three species of the animal phylum Loricifera (This is the first evidence of a metazoan life cycle that is spent entirely in permanently anoxic sediments. Our findings allow us also to conclude that these metazoans live under anoxic conditions through an obligate anaerobic metabolism that is similar to that demonstrated so far only for unicellular eukaryotes. The discovery of these life forms opens new perspectives for the study of metazoan life in habitats lacking molecular oxygen. These are characterised by very low oxygen availability (O2 < 0.5 mM) and high sulphide concentrations in the bottom sediments (>0.1 mM in the surface centimetre) ..rain of -2). This finding is per se surprising, as only two individuals of the phylum Loricifera have been found in the deep Mediterranean Sea over the last 40 years [-3, whereas the density of the brines above the sediment is significantly higher (1.23 g cm-3). Moreover, the presence of laminated sediment layers along with the lack of turbidites in the L'Atalante basin [The permanent reducing conditions of anoxic sediments can preserve dead organisms and their protein for a long time, so that microscopic analyses do not provide proof of the viability of an organism. However, the abundance of these loriciferans was the highest reported so far world-wide per unit of surface sediment investigated , in the dark and at the in-situ temperature (ca 14°C) immediately after sample retrieval. In the first investigations, intact and undisturbed sediment cores were injected with (3H)-leucine (Table 3H)-leucine of both living and killed nematodes. Thanks to this experiment we demonstrated the presence of significant differences in the incorporation of radio-labelled compounds and proved the linearity between the number of nematodes and the incorporated radioactivity  which has been previously used to identify living unicellular eukaryotes in anoxic sediments [A second expedition was dedicated to the demonstration of the viability of these loriciferans of the L'Atalante basin, through independent experimental approaches. All of the experiments were conducted on deck 101,325 Pa), under anoxic conditions  tolerating an enormous osmotic pressure ; (ii) detoxifying highly toxic compounds (due to the high hydrogen sulphide concentrations); and (iii) living without oxygen. Quantitative X-ray micro-analysis and Fourier-transformed infrared spectroscopy on the body composition of the loriciferans collected from the anoxic sediments revealed significant differences with the loriciferans collected in the oxygenated deep Atlantic Ocean  and high contents of Na+ , Cl-  and Mg+ (410 mM) [2), in 1998, 2005, 2006 and 2008. The samples from the DHAB sediment were collected in December 1998 , August 2005 , and June 2008 . In 1998 and 2008 additional sediment samples were collected outside the L'Atalante basin  at ca. 3,250 m depth, for investigation of the characteristics of meiofaunal metazoans from the oxygenated adjacent systems (three sampling sites per period with three to five replicated deployments per site). In the northern-eastern Atlantic Ocean, oxygenated deep-sea sediment samples  were collected during the 2006 expedition. Loriciferans retrieved from these sediments were used for the comparison of their body composition with loriciferan specimens collected in the anoxic sediments of the L'Atalante basin. Sediments retrieved from the deep anoxic basin were immediately processed under strict anaerobic conditions.The L'Atalante deep hypersaline anoxic basin (DHAB) was discovered in the Mediterranean Sea in 1993 during an expedition that was a part of the European funded project "Mediterranean Ridge Fluid Flow". The bottom of the L'Atalante basin is a relatively flat area bounded to the southwest by the Cleft Basin and it is characterised by a morphological escarpment that is several hundreds of metres high, which is the sea-bottom expression of the main back thrust of the accretionary ridge. These characteristics originated from the dissolution of buried salt deposits (evaporitic deposits), which remained from the hypersaline waters of the Miocene period (5.5 My before present). The L'Atalante basin is characterised by the presence of a thick brine layer (ca. 40 m) with high density (1.23 g cm(410 mM)  and is c-3) [-1), a stain commonly used to highlight the body structures under light microscopy. On average of all collected samples metazoan abundance was 2,075 ind. m-2 in the L'Atalante sediments vs 21,548 ind. m-2 in the oxygenated sediments surrounding the basin. In the anoxic sediments of the L'Atalante basin, Loricifera accounted for 16.1% of the total metazoan abundance. No Loricifera were encountered in the oxygenated sediments surrounding the basin, where nematodes and copepods accounted for 95% and 4%, respectively, of the total metazoan abundance.For the extraction of metazoan fauna from the sediments, the samples (top 15 to 20 cm of the sediment cores) were pre-filtered through a 1,000-μm mesh (to remove larger debris), and a 20-μm mesh was used to retain all of the multi-cellular organisms. The fraction remaining on this latter sieve was re-suspended and centrifuged three times with Ludox HS40 (density 1.31 g cm-3) . All of -3) ,35. OnlyThe extracted specimens were mounted on microslides in a drop of distilled water. The water was progressively replaced by increasing glycerol concentrations . Then the specimens were sealed with Glyceel. The microslides were analyzed using a light microscope with phase contrast and Nomarski DIC optics. Micrographs of the specimens were taken on an Olympus BX51 microscope equipped with a digital Olympus C-3030 zoom camera and on a Leica DMRXA microscope with a digital Leica DC200 camera . Morphological details of the loriciferans were obtained by scanning electron microscopy. Loriciferans extracted from sediments were carefully rinsed in distilled water and then dehydrated through a graded series of ethanol and acetone prior to critical-point drying. The dried specimens were mounted on aluminium stubs and coated with gold prior to observation under scanning electron microscopy .3H)-leucine [3 of sediment per replicate sample) were kept in the dark at in-situ temperature and under anoxic conditions (a N2 atmosphere); these were injected with 10 mL (3H)-leucine dissolved in 0.2 μm filtered, autoclaved and degassed deep-sea water . Controls for the incubation experiments were obtained as follows: additional sediment cores were frozen immediately after collection at -80°C, to kill all metazoans within the samples. After thawing, when the samples reached the in-situ temperature, the sediments were incubated with an aqueous solution of (3H)-leucine and then processed as described above. We used deep freezing to kill animals, as previous studies have demonstrated that meiofauna fixed using chemical compounds  show a significant loss in the incorporated radioactivity [2 atmosphere) for four hours in the dark, and at the in-situ temperature (about 14°C). At the end of the incubations, the samples were deep-frozen in liquid N2 to stop any additional substrate uptake. In the laboratory, the organisms were extracted from the sediment as previously described. Due to the relatively low numbers of loriciferans in the sediment cores (n = 3 both in the control and treated samples) the organisms were analyzed individually. Meiofaunal organisms were rinsed with 0.2-μm pre-filtered seawater  [For investigating the vitality of the meiofaunal metazoans, the top 5 cm of intact sediment cores were incubated with (-leucine . Replicaactivity . All samsurface)  and tranTo test the accuracy and consistency of the radiotracer experiments carried out on sediments collected in the L'Atalante basin, additional experiments were performed on coastal sediments of the Mediterranean Sea. Loriferans were not present in these samples; therefore nematodes were used as model organisms. After incubation with the radiolabelled substrate, the nematodes (diameter: 20 to 30 μm and length: 200 to 900 μm) were extracted from the sediments and analyzed individually or pooled together . These experiments demonstrated that the radioactivity incorporated into the nematodes is significantly higher than that found in organisms used as controls, even when a single individual is analyzed (Table 2 atmosphere) and incubated on deck  in the dark and at the in-situ temperature (ca 14°C). The samples were used for incorporation experiments with Cell-Tracker™ Green CMFDA, fluorescent probe . The Cell-Tracker™ Green fluorescent CMFDA probe penetrates the cells and reacts with the intracellular enzymes, generating fluorescence [in-situ temperature, the sediments were incubated with an aqueous solution Cell-Tracker™ Green CMFDA, and then processed as described above. At the end of the incubation, the samples were deep-frozen in liquid N2 to stop any metabolic reactions, and the recovered loriciferans were placed on concave slides containing a drop of 0.9% NaCl solution (previously autoclaved). The fluorescence of the organisms was examined using a confocal microscope equipped with Kr/Ar mixed gas laser  using excitation wavelengths 488 nm and the emission has been detected after passing a bandpass filter of 522/35 nm. The confocal laser images were acquired  in the Bio-Rad PIC format using the Bio-Rad Lasersharp Acquisition software (Release 2.1). The organisms were investigated using exactly the same magnification (× 40) in order to allow data comparison. Images were taken at depths of 3 μm for a total of 21 sections per animal and analyzed using the Bio-Rad Lasersharp processing tool. This enabled merging all of the sections (without any contrast manipulation) and measuring the mean scale colour (0 to 255) of the entire animal body. Images were sequentially acquired and stored as TIFF files. The reliability of the control used in the experiment was previously tested by means of repeated (n = 5) incubation experiments with Cell-Tracker™ Green CMFDA performed on two nematode species cultured in the laboratory (Diplolamelloides myily and Diplolaimella diewgatentis). All of the specimens were analyzed by confocal laser microscopy, as described above.After sediment retrieval from the anoxic basin, the top 5 cm of the sediment cores and its anoxic overlying water were maintained under strict anaerobic conditions  and the deep NE Atlantic Ocean (Rugiloricus cauliculus cfr) underwent quantitative X-ray micro-analysis, after coating with graphite. Specimens collected in the oxygenated sediments were used as a reference. The selected parts were: abdomen, the posterior lorica and the whole organism  was performed in all cases while Second Derivative, Fourier Self Deconvolution and Curve Fitting (Gaussian character) procedures were used to determine the absorbance ratio between the bands of interest. All spectra were scaled for equal intensity in the Amide I band. For data handling, the Spectrum v.303 (Perkin-Elmer) software package was used.Fourier transformed Infra-Red (FT-IR) spectroscopic determinations were carried out on loriciferans collected both from the anoxic sediments of the L'Atalante basin and from oxic sediments of the NE Atlantic Ocean. Spectral data were obtained with a Perkin-Elmer Spectrum One FT-IR equipped with a Perkin-Elmer Autoimage microscope . Spectra were measured from 4,000 to 400 cmRugiloricus) extracted from sediments were carefully rinsed in distilled water and then stored in glutaraldehyde  for transmission electron microscopy examinations. After treatment with osmium (one hour incubation) and acetone dehydration , loriciferans were embedded in epoxy resin. Ultrathin sections (78 nm) were obtained using a microtome  equipped with a diamond knife. Sections were collected on carbon-coated formvar supports, stained with lead citrate, and examined by transmission electron microscopy (Philips EM 208).For ultrastructural studies, loriciferans ; DPM: disintegration per minute; TIFF: tagged image file format; FT-IR: Fourier transformed infra-red.RD performed the project planning. AD, CG, KH and RM performed the experimental work, while RD, AD, AP and CG performed the data analysis. RD, AD, AP, CG, KH and RM wrote the manuscript.The study area. Location of the sampling areas in the central Mediterranean Sea, showing: (a), area (rectangle) including the deep hypersaline anoxic basin ; and (b), detailed contour map of the L'Atalante basin.Click here for fileThe effect of Rose Bengal on living and dead specimens. (a and b) Light microscopy (LM) images of living deep-sea nematodes collected from oxygenated sediments adjacent to the anoxic basin and stained with Rose Bengal; (c) LM image of dead deep-sea nematode stained with Rose Bengal; (d) LM image of living deep-sea copepods collected from oxygenated sediments adjacent to the anoxic basin and stained with Rose Bengal; (e) LM image of deep-sea copepod exuviae stained with Rose Bengal.Click here for fileMeiofaunal abundance and community structure in the L'Atalante basin and the adjacent oxygenated deep-sea sediments. Comparison of: (a), total abundance of benthic metazoans  in the anoxic sediments of the L'Atalante deep hypersaline anoxic basin (DHAB) and oxygenated deep-sea sediments surrounding the anoxic basin; and (b), contribution of the different taxa encountered in the anoxic sediments of the L'Atalante DHAB and oxygenated deep-sea sediments surrounding the anoxic basin (expressed as percentages).Click here for fileElemental composition of loriciferans from the L'Atalante basin and oxygenated NE Atlantic deep-sea sediments. Reported are the relative contents (expressed as percentage) of Na, Mg, Si, P, S, Ca, Fe, Cu, Zn, Br in the abdomen, posterior lorica and whole body of loriciferans.Click here for fileDifferences in body composition of loriciferans from the L'Atalante basin and the NE Atlantic Ocean. Output of the principal component analysis carried out on the elemental composition data of different parts of the loriciferans bodies, collected in the L'Atalante basin and in the NE Atlantic Ocean. Vectors in the left part of the panel are proportional to the importance of the investigated chemical elements in distinguishing the Loricifera of the L'Atalante basin from those of the deep-Atlantic oxygenated sediments.Click here for fileFourier-transformed infra-red spectroscopy of loriciferans from the L'Atalante basin and the NE Atlantic Ocean. Comparison of: (a) Fourier-transformed infra-red spectra of lorificerans collected from the L'Atalante basin (blue line) and from the oxygenated sediments of the NE Atlantic Ocean (red line); (b) spectra of chitosan (black line) and the lorica of the loriciferans collected in the L'Atalante basin (blue line); (c) spectra of chitin (green line) and the lorica of the loriciferans collected from the oxygenated sediments of the NE Atlantic Ocean (red line).Click here for file


Inhibin is a polypeptide hormone produced by the granulosa cells of the ovary, and is present in body fluids as dimers of various sizes each comprising an alpha- and beta-subunit. Free forms of the alpha-subunit also circulate, and the presently available radioimmunoassay (Monash assay) cannot distinguish these from biologically active dimeric inhibin. Recently we described a new two-site enzyme immunoassay able for the first time to measure the levels of dimeric inhibin throughout the human menstrual cycle. The sensitivity limit of this assay is 2 pg ml-1 in human serum with cross-reactivity against activin of 0.05%. The normal range of inhibin in post-menopausal women is < 5 pg ml-1, in pre-menopausal women 2-80 pg ml-1 . This assay was used to determine inhibin levels in sera from 15  patients with granulosa cell tumours of the ovary. It was raised in a pre-menopausal patient preoperatively (261 pg ml-1), in six post-menopausal patients  and one pre-menopausal patient with recurrent tumour, (237 pg ml-1), all confirmed clinically. Inhibin was normal in six patients in remission. Oestradiol levels were normal in all patients. Serial levels of inhibin predicted recurrence before overt clinical relapse in two patients. In 29 patients with malignant epithelial ovarian tumours inhibin levels were modestly elevated in nine and normal in the rest. Three patients with endometrioid histology, two with undifferentiated tumours, three with mucinous adenocarcinoma and one with clear cell carcinoma had elevated inhibin levels. Functional inhibin is secreted by all granulosa cell tumours of the ovary studied and can be used as a tumour marker to determine response to therapy and predict recurrence and is superior to oestradiol. A more detailed analysis of the levels of inhibin, and its subunits in epithelial ovarian cancer is needed to identify the molecular forms of the immunoreactive material before optimised assays can be applied to this more common tumour.