Repair of Annulus fibrosus with Genipin-enhanced Fibrin Hydrogel and silk membrane- fleece

Introduction Low back pain is often caused by a trauma causing disc herniation and /or disc degeneration. Although there are some promising approaches for nucleus pulposus repair, the inner tissue of the intervertebral disc (IVD) so far no treatment or repair is available for annulus fibrosus (AF) injuries. Here we aimed to develop a new method to seal and repair AF injuries by using a silk fleece composite and a genipin enhanced hydrogel. Methods Bovine (b) IVDs were harvested under aseptic conditions and kept in free swelling conditions for 24h in high-glucose DMEM containing 5% bovine serum for equilibration (1). A circular 2mm biopsy punch (Polymed Medical Center, Switzerland) was used to form a reproducible defect in the AF. For filling the defect and keeping the silk composite in place a human-derived fibrin gel (Baxter Tisseel, Switzerland) enhanced with 4.2mg/ml of the cross linker genipin (Wako Chemicals GmbH, Germany) was used. The silk composite consists of a mesh- and a membrane side (Spintec Engineering GmbH, Germany); the membrane is facing outwards to form a seal. bIVDs were cultured in vitro for 14 days either under dynamic load in a custom-built bioreactor under physiological conditions (0.2MPa load and ±2° torsion at 0.2Hz for 8h/day) or static diurnal load of 0.2MPa (2). At the end of culture discs were checked for seal failure, disc height, metabolic activity, cell death by necrosis (LDH assay), DNA content and glycosaminoglycan content. Results Silk composite maintained its position throughout the 14 days of culture under loaded conditions. Although repaired discs performed slightly lower in cell activity, DNA and GAG content were in the range of the control. Also LDH resulted in similar values compared to control discs (Fig 1). Height loss in repaired discs was in the same range as for static diurnal loaded control samples. For dynamically loaded samples the decrease was comparable to the injured, unrepaired discs. Fig 1 LDH of repaired discs compared to control disc after 24h in free swelling conditions for equilibration and first three loading cycles. Conclusions Silk-genipin-fibrin reinforced hydrogel is a promising approach to close AF defects as tested by two degree of freedom loading. In further experiments cytocompatibility of genipin has to be investigated. References 1. Chan SC, Gantenbein-Ritter B. Preparation of intact bovine tail intervertebral discs for organ culture. J Vis Exp 2012, Feb 2;60(60):e3490. 2. Walser J, Ferguson SJ, Gantenbein-Ritter B. Design of a mechanical loading device to culture intact bovine caudal motional segments of the spine under twisting motion. In: Davies J, editors. Replacing animal models: a practical guide to creating and using biomimetic alternatives. Chichester, UK: John Wiley & Sons, Ltd.; 2012. p. 89-105. Acknowledgements This project is funded by the Gerbert Rüf Stiftung project # GRS-028/13 and the Swiss National Science Project SNF #310030_153411.

4'-O-methylhonokiol increases levels of 2-arachidonoyl glycerol in mouse brain via selective inhibition of its COX-2-mediated oxygenation

BACKGROUND AND PURPOSE 4'-O-methylhonokiol (MH) is a natural product showing anti-inflammatory, anti-osteoclastogenic, and neuroprotective effects. MH was reported to modulate cannabinoid CB2 receptors as an inverse agonist for cAMP production and an agonist for intracellular [Ca2+]. It was recently shown that MH inhibits cAMP formation via CB2 receptors. In this study, the exact modulation of MH on CB2 receptor activity was elucidated and its endocannabinoid substrate-specific inhibition (SSI) of cyclooxygenase-2 (COX-2) and CNS bioavailability are described for the first time. METHODS CB2 receptor modulation ([35S]GTPγS, cAMP, and β-arrestin) by MH was measured in hCB2-transfected CHO-K1 cells and native conditions (HL60 cells and mouse spleen). The COX-2 SSI was investigated in RAW264.7 cells and in Swiss albino mice by targeted metabolomics using LC-MS/MS. RESULTS MH is a CB2 receptor agonist and a potent COX-2 SSI. It induced partial agonism in both the [35S]GTPγS binding and β-arrestin recruitment assays while being a full agonist in the cAMP pathway. MH selectively inhibited PGE2 glycerol ester formation (over PGE2) in RAW264.7 cells and significantly increased the levels of 2-AG in mouse brain in a dose-dependent manner (3 to 20 mg kg(-1)) without affecting other metabolites. After 7 h from intraperitoneal (i.p.) injection, MH was quantified in significant amounts in the brain (corresponding to 200 to 300 nM). CONCLUSIONS LC-MS/MS quantification shows that MH is bioavailable to the brain and under condition of inflammation exerts significant indirect effects on 2-AG levels. The biphenyl scaffold might serve as valuable source of dual CB2 receptor modulators and COX-2 SSIs as demonstrated by additional MH analogs that show similar effects. The combination of CB2 agonism and COX-2 SSI offers a yet unexplored polypharmacology with expected synergistic effects in neuroinflammatory diseases, thus providing a rationale for the diverse neuroprotective effects reported for MH in animal models.

TrypanoCyc: a community-led biochemical pathways database for Trypanosoma brucei.

The metabolic network of a cell represents the catabolic and anabolic reactions that interconvert small molecules (metabolites) through the activity of enzymes, transporters and non-catalyzed chemical reactions. Our understanding of individual metabolic networks is increasing as we learn more about the enzymes that are active in particular cells under particular conditions and as technologies advance to allow detailed measurements of the cellular metabolome. Metabolic network databases are of increasing importance in allowing us to contextualise data sets emerging from transcriptomic, proteomic and metabolomic experiments. Here we present a dynamic database, TrypanoCyc (http://www.metexplore.fr/trypanocyc/), which describes the generic and condition-specific metabolic network of Trypanosoma brucei, a parasitic protozoan responsible for human and animal African trypanosomiasis. In addition to enabling navigation through the BioCyc-based TrypanoCyc interface, we have also implemented a network-based representation of the information through MetExplore, yielding a novel environment in which to visualise the metabolism of this important parasite.

Overview of Genetically Engineered Mouse Models of Papillary and Anaplastic Thyroid Cancers: Enabling Translational Biology for Patient Care Improvement.

The prognosis from thyroid cancer subtypes in humans covers a spectrum from "cured at almost 90%" to "100% lethal." Invasive and poorly differentiated forms of thyroid cancer are among the most aggressive human cancers, and there are few effective therapeutic options. Genetically engineered mice, based on mutations observed in patients, can accurately recapitulate the human disease and its progression, providing invaluable tools for the preclinical evaluation of novel therapeutic approaches. This overview details models developed to date as well as their uses for identifying novel anticancer agents. © 2015 by John Wiley & Sons, Inc.

RASSF7 expression and its regulatory roles on apoptosis in human intervertebral disc degeneration

Apoptosis plays an important role in intervertebral disc degeneration (IDD). Overwhelming evidence indicates that RASSF7 is essential for cell growth and apoptosis. Recently, it has been noted that the JNK signaling can be negatively regulated by suppressing phosphorylated-MKK7 activation during pro-apoptosis. We aimed to investigate the RASSF7 expression level in human degenerative nucleus pulposus (NP) cells and non-degenerative NP cells and the link between RASSF7-JNK with NP cells apoptosis. We harvested NP tissues from 20 IDD patients as disease group and 8 cadaveric donors as normal controls. We detected RASSF7 expression by Real-time-PCR and western blotting. Consequently, we found that the expression of RASSF7 was higher in non-degenerative group than in degenerative group (P<0.05). Overexpression of RASSF7 in degenerative NP cells led to decreased apoptosis rate than that in scramble group (P<0.05). Collectively, our findings suggest that RASSF7 plays an important role in human IDD and RASSF7 might be potentially developed as a curative agent.

Phylogenetic analysis of receptor FgfrL1 shows divergence of the C-terminal end in rodents

FGFRL1 is a member of the fibroblast growth factor receptor (FGFR) family. Similar to the classical receptors FGFR1-FGFR4, it contains three extracellular Ig-like domains and a single transmembrane domain. However, it lacks the intracellular tyrosine kinase domain that would be required for signal transduction, but instead contains a short intracellular tail with a peculiar histidine-rich motif. This motif has been conserved during evolution from mollusks to echinoderms and vertebrates. Only the sequences of FgfrL1 from a few rodents diverge at the C-terminal region from the canonical sequence, as they appear to have suffered a frameshift mutation within the histidine-rich motif. This mutation is observed in mouse, rat and hamster, but not in the closely related rodents mole rat (Nannospalax) and jerboa (Jaculus), suggesting that it has occurred after branching of the Muridae and Cricetidae from the Dipodidae and Spalacidae. The consequence of the frameshift is a deletion of a few histidine residues and an extension of the C-terminus by about 40 unrelated amino acids. A similar frameshift mutation has also been observed in a human patient with a craniosynostosis syndrome as well as in several patients with colorectal cancer and bladder tumors, suggesting that the histidine-rich motif is prone to mutation. The reason why this motif was conserved during evolution in most species, but not in mice, is not clear.

Gut Microbiota Orchestrates Energy Homeostasis during Cold.

Microbial functions in the host physiology are a result of the microbiota-host co-evolution. We show that cold exposure leads to marked shift of the microbiota composition, referred to as cold microbiota. Transplantation of the cold microbiota to germ-free mice is sufficient to increase insulin sensitivity of the host and enable tolerance to cold partly by promoting the white fat browning, leading to increased energy expenditure and fat loss. During prolonged cold, however, the body weight loss is attenuated, caused by adaptive mechanisms maximizing caloric uptake and increasing intestinal, villi, and microvilli lengths. This increased absorptive surface is transferable with the cold microbiota, leading to altered intestinal gene expression promoting tissue remodeling and suppression of apoptosis-the effect diminished by co-transplanting the most cold-downregulated strain Akkermansia muciniphila during the cold microbiota transfer. Our results demonstrate the microbiota as a key factor orchestrating the overall energy homeostasis during increased demand.

Rapid Communication: Cholesterol deficiency-associated APOB mutation impacts lipid metabolism in Holstein calves and breeding bulls.

During the last months, the number of reports on Holstein calves suffering from incurable idiopathic diarrhea dramatically increased. Affected calves showed severe hypocholesterolemia and mostly died within days up to a few months after birth. This new autosomal monogenic recessive inherited fat metabolism disorder, termed cholesterol deficiency (CD), is caused by a loss of function mutation of the bovine gene. The objective of the present study was to investigate specific components of lipid metabolism in 6 homozygous for the mutation (CDS) and 6 normal Holstein calves with different genotypes. Independent of sex, CDS had significantly lower plasma concentrations of total cholesterol (TC), free cholesterol (FC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), very-low-density lipoprotein cholesterol (VLDL-C), triacylglycerides (TAG), and phospholipids (PL) compared with homozygous wild-type calves ( < 0.05). Furthermore, we studied the effect of the genotype on cholesterol metabolism in adult Holstein breeding bulls of Swissgenetics. Among a total of 254 adult males, the homozygous mutant genotype was absent, 36 bulls were heterozygous carriers (CDC), and 218 bulls were homozygous wild-type (CDF). In CDC bulls, plasma concentrations of TC, FC, HDL-C, LDL-C, VLDL-C, TAG, and PL were lower compared with CDF bulls ( < 0.05). The ratios of FC:cholesteryl esters (CE) and FC:TC were higher in CDC bulls compared with CDF bulls, whereas the ratio of CE:TC was lower in CDC bulls compared with CDF bulls ( < 0.01). In conclusion, the CD-associated mutation was shown to affect lipid metabolism in affected Holstein calves and adult breeding bulls. Besides cholesterol, the concentrations of PL, TAG, and lipoproteins also were distinctly reduced in homozygous and heterozygous carriers of the mutation. Beyond malabsorption of dietary lipids, deleterious effects of apolipoprotein B deficiency on hepatic lipid metabolism, steroid biosynthesis, and cell membrane function can be expected, which may result in unspecific symptoms of reduced fertility, growth, and health.

Clinical Streptococcus pneumoniae isolates induce differing CXCL8 responses from human nasopharyngeal epithelial cells which are reduced by liposomes.

BACKGROUND: Streptococcus pneumoniae causes several human diseases, including pneumonia and meningitis, in which pathology is associated with an excessive inflammatory response. A major inducer of this response is the cholesterol dependent pneumococcal toxin, pneumolysin. Here, we measured the amount of inflammatory cytokine CXCL8 (interleukin (IL)-8) by ELISA released by human nasopharyngeal epithelial (Detroit 562) cells as inflammatory response to a 24 h exposure to different pneumococcal strains. RESULTS: We found pneumolysin to be the major factor influencing the CXCL8 response. Cholesterol and sphingomyelin-containing liposomes designed to sequester pneumolysin were highly effective at reducing CXCL8 levels from epithelial cells exposed to different clinical pneumococcal isolates. These liposomes also reduced CXCL8 response from epithelial cells exposed to pneumolysin knock-out mutants of S. pneumoniae indicating that they also reduce the CXCL8-inducing effect of an unidentified pneumococcal virulence factor, in addition to pneumolysin. CONCLUSION: The results indicate the potential of liposomes in attenuating excessive inflammation as a future adjunctive treatment of pneumococcal diseases.

CCK2 receptor splice variant with intron 4 retention in human gastrointestinal and lung tumours

The wild-type cholecystokinin type 2 (CCK(2)) receptor is expressed in many gastrointestinal and lung tumours. A splice variant of the CCK(2) receptor with retention of intron 4 (CCK(2)Ri4sv) showing constitutive activity associated with increased tumour growth was described in few colorectal, pancreatic and gastric cancers. Given the potential functional and clinical importance of this spliceoform, its occurrence was quantitatively characterized in a broad collection of 81 gastrointestinal and lung tumours, including insulinomas, ileal carcinoids, gastrointestinal stromal tumours (GIST), gastric, colorectal and pancreatic ductal adenocarcinomas, cholangiocellular and hepatocellular carcinomas, small cell lung cancers (SCLC), non-SCLC (nSCLC) and bronchopulmonary carcinoids, as well as 21 samples of corresponding normal tissues. These samples were assessed for transcript expression of total CCK(2) receptor, wild-type CCK(2) receptor and CCK(2)Ri4sv with end-point and real-time RT-PCR, and for total CCK(2) receptor protein expression on the basis of receptor binding with in vitro receptor autoradiography. Wild-type CCK(2) receptor transcripts were found in the vast majority of tumours and normal tissues. CCK(2)Ri4sv mRNA expression was present predominantly in insulinomas (incidence 100%), GIST (100%) and SCLC (67%), but rarely in pancreatic, colorectal and gastric carcinomas and nSCLC. It was not found in wild-type CCK(2) receptor negative tumours or any normal tissues tested. CCK(2)Ri4sv transcript levels in individual tumours were low, ranging from 0.02% to 0.14% of total CCK(2) receptor transcripts. In conclusion, the CCK(2)Ri4sv is a marker of specific gastrointestinal and lung tumours. With its high selectivity for and high incidence in SCLC and GIST, it may represent an attractive clinical target.

Sellar collision tumor involving pituitary gonadotroph adenoma and chondroma: a potential clinical diagnosis

We report on a 74-year-old male patient who presented with progressive neuroophthalmologic symptoms soon after the administration of a long-acting gonadotropin-releasing hormone agonist for treatment of a prostate cancer. Imaging revealed a destructively growing and extensively calcified sellar mass inconsistent with a pituitary adenoma. A transseptal transsphenoidal tumor mass reduction yielded a histological diagnosis of a collision tumor comprised of a gonadotroph adenoma intermingled with osteochondroma. We discuss a potential causal relationship between the administration of the long-acting gonadotropin-releasing hormone agonist and the sudden appearance of the previously unsuspected sellar lesion. Although the association of these two tumors is very likely coincidental, the possibility of causal relationship is addressed.

A Hybrid Multimodal Non-Rigid Registration of MR Images Based on Diffeomorphic Demons

n this paper we present a novel hybrid approach for multimodal medical image registration based on diffeomorphic demons. Diffeomorphic demons have proven to be a robust and efficient way for intensity-based image registration. A very recent extension even allows to use mutual information (MI) as a similarity measure to registration multimodal images. However, due to the intensity correspondence uncertainty existing in some anatomical parts, it is difficult for a purely intensity-based algorithm to solve the registration problem. Therefore, we propose to combine the resulting transformations from both intensity-based and landmark-based methods for multimodal non-rigid registration based on diffeomorphic demons. Several experiments on different types of MR images were conducted, for which we show that a better anatomical correspondence between the images can be obtained using the hybrid approach than using either intensity information or landmarks alone.

Atlas-Based Segmentation of Brain Tumor Images Using a Markov Random Field-Based Tumor Growth Model and Non-Rigid Registration

We propose a new and clinically oriented approach to perform atlas-based segmentation of brain tumor images. A mesh-free method is used to model tumor-induced soft tissue deformations in a healthy brain atlas image with subsequent registration of the modified atlas to a pathologic patient image. The atlas is seeded with a tumor position prior and tumor growth simulating the tumor mass effect is performed with the aim of improving the registration accuracy in case of patients with space-occupying lesions. We perform tests on 2D axial slices of five different patient data sets and show that the approach gives good results for the segmentation of white matter, grey matter, cerebrospinal fluid and the tumor.

Validity of an automatic measure protocol in distal femur for allograft selection from a three-dimensional virtual bone bank system

Osteoarticular allograft is one possible treatment in wide surgical resections with large defects. Performing best osteoarticular allograft selection is of great relevance for optimal exploitation of the bone databank, good surgery outcome and patient’s recovery. Current approaches are, however, very time consuming hindering these points in practice. We present a validation study of a software able to perform automatic bone measurements used to automatically assess the distal femur sizes across a databank. 170 distal femur surfaces were reconstructed from CT data and measured manually using a size measure protocol taking into account the transepicondyler distance (A), anterior-posterior distance in medial condyle (B) and anterior-posterior distance in lateral condyle (C). Intra- and inter-observer studies were conducted and regarded as ground truth measurements. Manual and automatic measures were compared. For the automatic measurements, the correlation coefficients between observer one and automatic method, were of 0.99 for A measure and 0.96 for B and C measures. The average time needed to perform the measurements was of 16 h for both manual measurements, and of 3 min for the automatic method. Results demonstrate the high reliability and, most importantly, high repeatability of the proposed approach, and considerable speed-up on the planning.

Anatomically-Driven Soft-Tissue Simulation Strategy for Cranio-Maxillofacial Surgery Using Facial Muscle Template Model

We propose a computationally efficient and biomechanically relevant soft-tissue simulation method for cranio-maxillofacial (CMF) surgery. A template-based facial muscle reconstruction was introduced to minimize the efforts on preparing a patient-specific model. A transversely isotropic mass-tensor model (MTM) was adopted to realize the effect of directional property of facial muscles in reasonable computation time. Additionally, sliding contact around teeth and mucosa was considered for more realistic simulation. Retrospective validation study with postoperative scan of a real patient showed that there were considerable improvements in simulation accuracy by incorporating template-based facial muscle anatomy and sliding contact.