Comprehensive analysis of CpG island methylator phenotype (CIMP)-high, -low, and -negative colorectal cancers based on protein marker expression and molecular features

CpG island methylator phenotype (CIMP) is being investigated for its role in the molecular and prognostic classification of colorectal cancer patients but is also emerging as a factor with the potential to influence clinical decision-making. We report a comprehensive analysis of clinico-pathological and molecular features (KRAS, BRAF and microsatellite instability, MSI) as well as of selected tumour- and host-related protein markers characterizing CIMP-high (CIMP-H), -low, and -negative colorectal cancers. Immunohistochemical analysis for 48 protein markers and molecular analysis of CIMP (CIMP-H: ? 4/5 methylated genes), MSI (MSI-H: ? 2 instable genes), KRAS, and BRAF were performed on 337 colorectal cancers. Simple and multiple regression analysis and receiver operating characteristic (ROC) curve analysis were performed. CIMP-H was found in 24 cases (7.1%) and linked (p < 0.0001) to more proximal tumour location, BRAF mutation, MSI-H, MGMT methylation (p = 0.022), advanced pT classification (p = 0.03), mucinous histology (p = 0.069), and less frequent KRAS mutation (p = 0.067) compared to CIMP-low or -negative cases. Of the 48 protein markers, decreased levels of RKIP (p = 0.0056), EphB2 (p = 0.0045), CK20 (p = 0.002), and Cdx2 (p < 0.0001) and increased numbers of CD8+ intra-epithelial lymphocytes (p < 0.0001) were related to CIMP-H, independently of MSI status. In addition to the expected clinico-pathological and molecular associations, CIMP-H colorectal cancers are characterized by a loss of protein markers associated with differentiation, and metastasis suppression, and have increased CD8+ T-lymphocytes regardless of MSI status. In particular, Cdx2 loss seems to strongly predict CIMP-H in both microsatellite-stable (MSS) and MSI-H colorectal cancers. Cdx2 is proposed as a surrogate marker for CIMP-H.

Gender-Specific Associations between Circulating T-Cadherin and High Molecular Weight-Adiponectin in Patients with Stable Coronary Artery Disease.

Close relationships exist between presence of adiponectin (APN) within vascular tissue and expression of T-cadherin (T-cad) on vascular cells. APN and T-cad are also present in the circulation but here their relationships are unknown. This study investigates associations between circulating levels of high molecular weight APN (HMW-APN) and T-cad in a population comprising 66 women and 181 men with angiographically proven stable coronary artery disease (CAD). Plasma HMW-APN and T-cad were measured by ELISA and analysed for associations with baseline clinical characteristics and with each other. In multivariable analysis BMI and HDL were independently associated with HMW-APN in both genders, while diabetes and extent of coronary stenosis were independently associated with T-cad in males only. Regression analysis showed no significant association between HMW-APN and T-cad in the overall study population. However, there was a negative association between HMW-APN and T-cad (P=0.037) in a subgroup of young men (age <60 years, had no diabetes and no or 1-vessel CAD) which persisted after multivariable analysis with adjustment for all potentially influential variables (P=0.021). In the corresponding subgroup of women there was a positive association between HMW-APN and T-cad (P=0.013) which disappeared after adjustment for HDL. After exclusion of the young men, a positive association (P=0.008) between HMW-APN and T-cad was found for the remaining participants of the overall population which disappeared after adjustment for HDL and BMI. The existence of opposing correlations between circulating HMW-APN and T-cad in male and female patient populations underscores the necessity to consider gender as a confounding variable when evaluating biomarker potentials of APN and T-cad.

Plasma S-nitrosothiol status in neonatal calves: ontogenetic associations with tissue-specific S-nitrosylation and nitric oxide synthase

Neonatal cattle and in part neonates of other species have manyfold higher plasma concentrations of nitrite plus nitrate than mature cows and subjects of other species, suggesting an enhanced and needed activation of the nitric oxide (NO) axis at birth. While the biological half-life of NO is short (<1 sec), its functionality can be prolonged, and in many regards more discretely modulated, when it reacts with low-molecular-weight and protein-bound thiols to form S-nitrosothiols (RSNO), from which NO subsequently can be rereleased. We used the calf as a model to test the hypothesis that plasma concentrations of RSNO are elevated at birth in mammals, correlate with ascorbate and urate levels, are selectively generated in critical tissue beds, and are generated in a manner temporally coincident with changes in tissue levels of active NO synthases (NOS). Plasma concentrations of RSNO, ascorbate, and urate were highest immediately after birth (Day 0), dropped >50% on Day 1, and gradually decreased over time, reaching a nadir in mature cattle. Albumin and immunoglobulin G were identified as major plasma RSNO. The presence of S-nitrosocysteine (SNC, a validated marker for S-nitrosylated proteins), inducible NOS (iNOS), and activated endothelial NOS (eNOS phosphorylated at Ser1177) in different tissues was analyzed by immunohistochemistry in another group of similar-aged calves. SNC, iNOS, and phosphorylated eNOS were detected in liver and ileum at the earliest timepoint of sampling (4 hrs after birth), increased between 4 and 24 hrs, and then declined to near-nondetectable levels by 2 weeks of life. Our data show that the neonatal period in the bovine species is characterized by highly elevated and coordinated NO-generating and nitrosylation events, with the ontogenetic changes occurring in iNOS and eNOS contents in key tissues as well as RSNO products and associated antioxidant markers.

High-molecular weight adiponectin is independently associated with the extent of coronary artery disease in men

OBJECTIVE: Adiponectin has anti-atherogenic properties and low circulating adiponectin has been linked to coronary atherosclerosis. Yet, there is considerable evidence that the high-molecular weight (HMW) complex of adiponectin is the major active form of this adipokine. We therefore investigated whether HMW adiponectin is associated with the extent of coronary artery disease (CAD) in men. RESEARCH DESIGN AND METHODS: Associations among CAD, HMW adiponectin and the HMW/total-adiponectin ratio were assessed in 240 male patients undergoing elective coronary angiography. Total adiponectin and HMW adiponectin was measured by enzyme-linked immunosorbent assay and serum levels were correlated with defined coronary scores and established cardiovascular risk factors. RESULTS: We found significant inverse correlations between angiographic scores and HMW adiponectin [Extent Score (ES): r=-0.39; Gensini Score (GS): r=-0.35; and Severity Score (SS): r=-0.40, all P<0.001], and the HMW/total-adiponectin ratio (ES: r=-0.49; GS: r=-0.46; SS: r=-0.46; all P<0.001). Multivariable regression analyses revealed that HMW adiponectin and the HMW/total-adiponectin ratio were significantly associated with the extent of CAD (both P<0.001). ROC analyses demonstrated that the predictive value of HMW adiponectin and the HMW/total-adiponectin ratio for the extent of coronary atherosclerosis significantly exceeded that of total adiponectin (P<0.001, P=0.010, respectively). CONCLUSIONS: HMW adiponectin and the HMW/total-adiponectin ratio inversely correlate with the extent of CAD. HMW adiponectin in particular seems to be a better marker for CAD extent than total adiponectin.

M-ficolin in the neonatal period: Associations with need for mechanical ventilation and mortality in premature infants with necrotising enterocolitis

OBJECTIVE: Necrotising enterocolitis (NEC) causes significant mortality in premature infants. The involvement of the innate immune system in the pathogenesis of NEC remains unclear. M-, L- and H-ficolins recognize microorganisms and activate the complement system, but their role in host defense is largely unknown. This study investigated whether ficolin concentrations are associated with NEC. STUDY DESIGN: Case-control study including 30 premature infants with NEC and 60 controls. M-, L- and H-ficolins were measured in cord blood using time-resolved immunofluorometric assays. Multivariate logistic regression was performed. RESULTS: Of the 30 NEC cases (median gestational age, 29.5 weeks), 12 (40%) were operated and 4 (13%) died. No difference regarding ficolin concentration was found when comparing NEC cases versus controls (p>0.05). However, infants who died of NEC had significantly lower M-ficolin cord blood concentrations than NEC survivors (for M-ficolin <300ng/ml; multivariate OR 12.35, CI 1.03-148.59, p=0.048). In the entire study population, M-, L- and H-ficolins were positively correlated with gestational age (p<0.001) and birth weight (p<0.001). Infants with low M-ficolin required significantly more often mechanical ventilation after birth multivariate (OR 10.55, CI 2.01-55.34, p=0.005). CONCLUSIONS: M-, L- and H-ficolins are already present in cord blood and increase with gestational age. Low cord blood concentration of M-ficolin was associated with higher NEC-associated fatality and with increased need for mechanical ventilation. Future studies need to assess whether M-ficolin is involved in multiorgan failure and pulmonary disease.

The evolution of host associations in the parasitic wasp genus Ichneumon (Hymenoptera: Ichneumonidae): convergent adaptations to host pupation sites

Background: The diversification of organisms with a parasitic lifestyle is often tightly linked to the evolution of their host associations. If a tight host association exists, closely related species tend to attack closely related hosts; host associations are less stable if associations are determined by more plastic traits like parasitoid searching and oviposition behaviour. The pupal-parasitoids of the genus Ichneumon attack a variety of macrolepidopteran hosts.They are either monophagous or polyphagous, and therefore offer a promissing system to investigate the evolution of host associations. Ichneumon was previously divided into two groups based on general body shape; however, a stout shape has been suggested as an adaptation to buried host pupation sites, and might thus not represent a reliable phylogenetic character. Results: We here reconstruct the first molecular phylogeny of the genus Ichneumon using two mitochondrial (CO1 and NADH1) and one nuclear marker (28S). The resulting phylogeny only supports monophyly of Ichneumon when Ichneumon lugens Gravenhorst, 1829 (formerly in Chasmias, stat. rev.) and Ichneumon deliratorius Linnaeus, 1758 (formerly Coelichneumon) are included. Neither parasitoid species that attack hosts belonging to one family nor those attacking butterflies (Rhopalocera) form monophyletic clades. Ancestral state reconstructions suggest multiple transitions between searching for hosts above versus below ground and between a stout versus elongated body shape. A model assuming correlated evolution between the two characters was preferred over independent evolution of host-searching niche and body shape. Conclusions: Host relations, both in terms of phylogeny and ecology, evolved at a high pace in the genus Ichneumon. Numerous switches between hosts of different lepidopteran families have occurred, a pattern that seems to be the rule among idiobiont parasitoids. A stout body and antennal shape in the parasitoid female is confirmed as an ecological adaptation to host pupation sites below ground and has evolved convergently several times. Morphological characters that might be involved in adaptation to hosts should be avoided as diagnostic characters for phylogeny and classification, as they can be expected to show high levels of homoplasy.

Calculation of crystal optical properties from molecular electron density

We have recently developed a method to obtain distributed atomic polarizabilities adopting a partitioning of the molecular electron density (for example, the Quantum Theory of Atoms in Molecules, [1]), calculated with or without an applied electric field. The procedure [2] allows to obtained atomic polarizability tensors, which are perfectly exportable, because quite representative of an atom in a given functional group. Among the many applications of this idea, the calculation of crystal susceptibility is easily available, either from a rough estimation (the polarizability of the isolated molecule is used) or from a more precise estimation (the polarizability of a molecule embedded in a cluster representing the first coordination sphere is used). Lorentz factor is applied to include the long range effect of packing, which is enhancing the molecular polarizability. Simple properties like linear refractive index or the gyration tensor can be calculated at relatively low costs and with good precision. This approach is particularly useful within the field of crystal engineering of organic/organometallic materials, because it would allow a relatively easy prediction of a property as a function of the packing, thus allowing "reverse crystal engineering". Examples of some amino acid crystals and salts of amino acids [3] will be illustrated, together with other crystallographic or non-crystallographic applications. For example, the induction and dispersion energies of intermolecular interactions could be calculated with superior precision (allowing anisotropic van der Waals interactions). This could allow revision of some commonly misunderstood intermolecular interactions, like the halogen bonding (see for example the recent remarks by Stone or Gilli [4]). Moreover, the chemical reactivity of coordination complexes could be reinvestigated, by coupling the conventional analysis of the electrostatic potential (useful only in the circumstances of hard nucleophilic/electrophilic interaction) with the distributed atomic polarizability. The enhanced reactivity of coordinated organic ligands would be better appreciated. [1] R. F. W. Bader, Atoms in Molecules: A Quantum Theory. Oxford Univ. Press, 1990. [2] A. Krawczuk-Pantula, D. Pérez, K. Stadnicka, P. Macchi, Trans. Amer. Cryst. Ass. 2011, 1-25 [3] A. S. Chimpri1, M. Gryl, L. H.R. Dos Santos1, A. Krawczuk, P. Macchi Crystal Growth & Design, in the press. [4] a) A. J. Stone, J. Am. Chem. Soc. 2013, 135, 7005−7009; b) V. Bertolasi, P. Gilli, G. Gilli Crystal Growth & Design, 2013, 12, 4758-4770.

Molecular Classification of Ependymal Tumors across All CNS Compartments, Histopathological Grades, and Age Groups

Ependymal tumors across age groups are currently classified and graded solely by histopathology. It is, however, commonly accepted that this classification scheme has limited clinical utility based on its lack of reproducibility in predicting patients' outcome. We aimed at establishing a uniform molecular classification using DNA methylation profiling. Nine molecular subgroups were identified in a large cohort of 500 tumors, 3 in each anatomical compartment of the CNS, spine, posterior fossa, supratentorial. Two supratentorial subgroups are characterized by prototypic fusion genes involving RELA and YAP1, respectively. Regarding clinical associations, the molecular classification proposed herein outperforms the current histopathological classification and thus might serve as a basis for the next World Health Organization classification of CNS tumors.

Molecular Characterization of Three Canine Models of Human Rare Bone Diseases: Caffey, van den Ende-Gupta, and Raine Syndromes.

One to two percent of all children are born with a developmental disorder requiring pediatric hospital admissions. For many such syndromes, the molecular pathogenesis remains poorly characterized. Parallel developmental disorders in other species could provide complementary models for human rare diseases by uncovering new candidate genes, improving the understanding of the molecular mechanisms and opening possibilities for therapeutic trials. We performed various experiments, e.g. combined genome-wide association and next generation sequencing, to investigate the clinico-pathological features and genetic causes of three developmental syndromes in dogs, including craniomandibular osteopathy (CMO), a previously undescribed skeletal syndrome, and dental hypomineralization, for which we identified pathogenic variants in the canine SLC37A2 (truncating splicing enhancer variant), SCARF2 (truncating 2-bp deletion) and FAM20C (missense variant) genes, respectively. CMO is a clinical equivalent to an infantile cortical hyperostosis (Caffey disease), for which SLC37A2 is a new candidate gene. SLC37A2 is a poorly characterized member of a glucose-phosphate transporter family without previous disease associations. It is expressed in many tissues, including cells of the macrophage lineage, e.g. osteoclasts, and suggests a disease mechanism, in which an impaired glucose homeostasis in osteoclasts compromises their function in the developing bone, leading to hyperostosis. Mutations in SCARF2 and FAM20C have been associated with the human van den Ende-Gupta and Raine syndromes that include numerous features similar to the affected dogs. Given the growing interest in the molecular characterization and treatment of human rare diseases, our study presents three novel physiologically relevant models for further research and therapy approaches, while providing the molecular identity for the canine conditions.