Complete mesocolic excision versus standard resection for colon cancer: a systematic review and meta-analysis of perioperative safety and an evaluation of the use of a robotic approach.

PURPOSE: Complete mesocolic excision (CME) has been associated with improved oncological outcomes in treatment of colon cancer. However, widespread adoption is limited partly because of the technical complexity and perceived risks of the approach. The aim of out study was to evaluate the safety of CME compared to standard resection and to compare robotic versus laparoscopic approaches. METHODS: Two parallel searches were undertaken in MEDLINE, Embase and Web of Science databases 12 December 2021. The first was to evaluate IDEAL stage 3 evidence to compare complication rates as a surrogate marker of perioperative safety between CME and standard resection. The second independent search compared lymph node yield and survival outcomes between minimally invasive approaches. RESULTS: There were four randomized control trials (n = 1422) comparing CME to standard resection, and three studies comparing laparoscopic (n = 164) to robotic (n = 161) approaches. Compared to standard resection, CME was associated with a reduction in Clavien-Dindo grade 3 or higher complication rates (3.56% vs. 7.24%, p = 0.002), reduced blood loss (113.1 ml vs. 137.6 ml, p < 0.0001) and greater mean lymph node harvest (25.6 vs. 20.9 nodes, p = 0.001). Between the robotic and laparoscopic groups, there were no significant differences in complication rates, blood loss, lymph node yield, 5-year disease-free survival (OR 1.05, p = 0.87) and overall survival (OR 0.83, p = 0.54). CONCLUSIONS: Our study demonstrated improved safety with CME. There was no difference in safety or survival outcomes between robotic and laparoscopic CME. The advantage of a robotic approach may lie in the reduced learning curve and an increased penetration of minimally invasive approach to CME. Further studies are required to explore this. PROSPERO ID: CRD42021287065.

Forward and reverse degradomics defines the proteolytic landscape of human knee osteoarthritic cartilage and the role of the serine protease HtrA1.

OBJECTIVES: Proteolytic destruction of articular cartilage, a major pathogenic mechanism in osteoarthritis (OA), was not previously investigated by terminomics strategies. We defined the degradome of human knee OA cartilage and the contribution therein of the protease HtrA1 using Terminal Amine Isotopic Labeling of Substrates (TAILS). DESIGN: Proteins from OA cartilage taken at knee arthroplasty (n = 6) or separately, from healthy cartilage incubated in triplicate with/without active HtrA1, were labeled at natural and proteolytically cleaved N-termini by reductive dimethylation, followed by trypsin digestion, enrichment of N-terminally labeled/blocked peptides, tandem mass spectrometry and positional peptide annotation to identify cleavage sites. Biglycan proteolysis by HtrA1 was validated biochemically and Amino-Terminal Oriented Mass Spectrometry of Substrates (ATOMS) was used to define the HtrA1 cleavage sites. RESULTS: We identified 10,155 unique internal peptides from 2,162 proteins, suggesting at least 10,797 cleavage sites in OA cartilage. 7,635 internal peptides originated in 371 extracellular matrix/secreted components, many undergoing extensive proteolysis. Rampant ragging of protein termini suggested pervasive exopeptidase activity. HtrA1, the most abundant protease in OA cartilage, experimentally generated 323 cleavages in 109 cartilage proteins, accounting for 171 observed cleavages in the OA degradome. ATOMS identified HtrA1 cleavage sites in a selected substrate, biglycan, whose direct cleavage by HtrA1 was thus orthogonally validated. CONCLUSIONS: OA cartilage demonstrates widespread proteolysis by endo- and exopeptidases. HtrA1 contributes broadly to cartilage proteolysis. Forward degradomics of OA cartilage together with reverse degradomics of proteases active in OA, e.g., HtrA1, can potentially fully annotate OA proteolytic pathways and provide new biomarkers.

Clinical Phenotype of Musladin-Lueke Syndrome in 2 Beagles.

Musladin-Lueke syndrome (MLS), previously termed Chinese Beagle syndrome, is an autosomal-recessive connective tissue disorder characterized by extensive fibrosis of the skin and joints that was first identified in Beagles in the 1970s. Recent research identified a founder mutation (c.660C>T; p.R221C) in the ADAMTSL2 gene in Beagles with MLS. Here, we report the detailed clinical phenotype and laboratory findings in 2 Beagles affected with MLS. We discuss these findings in relation to the human disorder geleophysic dysplasia (GD), which also arises from recessive ADAMTSL2 mutations, and recent findings in Adamtsl2-deficient mice.

10mM glucosamine prevents activation of proADAMTS5 (aggrecanase-2) in transfected cells by interference with post-translational modification of furin.

OBJECTIVE: Glucosamine has been previously shown to suppress cartilage aggrecan catabolism in explant cultures. We determined the effect of glucosamine on ADAMTS5 (a disintegrin-like and metalloprotease domain (reprolysin type) with thrombospondin type-1 motifs 5), a major aggrecanase in osteoarthritis, and investigated a potential mechanism underlying the observed effects. DESIGN: HEK293F and CHO-K1 cells transiently transfected with ADAMTS5 cDNA were treated with glucosamine or the related hexosamine mannosamine. Glucosamine effects on FURIN transcription were determined by quantitative RT-PCR. Effects on furin-mediated processing of ADAMTS5 zymogen, and aggrecan processing by glucosamine-treated cells, were determined by western blotting. Post-translational modification of furin and N-glycan deficient furin mutants generated by site-directed mutagenesis was analyzed by western blotting, and the mutants were evaluated for their ADAMTS5 processing ability in furin-deficient CHO-RPE.40 cells. RESULTS: Ten mM glucosamine and 5-10mM mannosamine reduced excision of the ADAMTS5 propeptide, indicating interference with the propeptide excision mechanism, although mannosamine compromised cell viability at these doses. Although glucosamine had no effect on furin mRNA levels, western blot of furin from glucosamine-treated cells suggested altered post-translational modification. Glucosamine treatment led to decreased glycosylation of cellular furin, with reduced furin autoactivation as the consequence. Recombinant furin treated with peptide N-glycanase F had reduced activity against a synthetic peptide substrate. Indeed, site-directed mutagenesis of two furin N-glycosylation sites, Asn(387) and Asn(440), abrogated furin activation and this mutant was unable to rescue ADAMTS5 processing in furin-deficient cells. CONCLUSIONS: Ten mM glucosamine reduces excision of the ADAMTS5 propeptide via interference with post-translational modification of furin and leads to reduced aggrecanase activity of ADAMTS5.

1-O-alkylglycerol stabilized carbamazepine intravenous o/w nanoemulsions for drug targeting in mice.

Carbamazepine (CBZ) is used in the treatment of generalized tonic clonic and partial seizures. In seizure disorder the focal point of treatment is brain. At present no commercial parenteral formulation of CBZ is available. We developed o/w nanoemulsions of CBZ stabilized by 1-O-alkylglycerol/lecithin for intravenous administration and evaluated the brain targeting potential of these formulations. The nanoemulsions were characterized for globule size, zeta potential (ZP), CBZ content and in vivo tissue distribution in mice. The in vivo data revealed a significant uptake of CBZ in all tissues. Among the nanoemulsions, 1-O-decylglycerol stabilized system showed significantly higher tissue levels and availability of CBZ. Particularly for this system 2.37 times higher brain availability and a brain/serum concentration ratio of 3.0 at 30 min is an important finding. This indicates the brain targeting potential. A systematic formulation development of CBZ nanoemulsions employing 1-O-alkylglycerols might pave way to achieve selective brain delivery of this important antiepileptic drug.

Ascorbyl palmitate vesicles (Aspasomes): formation, characterization and applications.

Vesicles with biological activity or with a targeting function in addition to carrier properties will have an added advantage. Vesicles prepared with amphiphiles having antioxidant property may have potential applications towards disorders implicated with reactive oxygen species. Ascorbyl palmitate (ASP) was explored as bilayer vesicle forming material. It formed vesicles (Aspasomes) in combination with cholesterol and a negatively charged lipid (dicetyl phosphate). Aspasomes were prepared by film hydration method followed by sonication in which aqueous azidothymidine (AZT) solution was encapsulated in aqueous regions of bilayer. Aspasomes were obtained with all compositions containing 18-72 mol% cholesterol. Differential scanning calorimetric data of aspasome dispersion and anhydrous mixtures of ascorbyl palmitate, cholesterol and dicetyl phosphate confirm the formation of bilayered vesicles with ascorbyl palmitate. Cholesterol content in aspasome did not exhibit any relation with vesicle size, zeta potential or percent entrapment. A substantial change in release rate of azidothymidine from aspasome was noticed on varying the proportion of cholesterol. Release rate and cholesterol content in Aspasomes did not exhibit any relation. A preparation with 45 mol% of cholesterol showed maximum retardation in release rate, than other compositions. The change in capture volume with time (latency) was studied for 8 h and with such a short duration study it was difficult to predict long term stability of these vesicles. But release experiments do indicate stability up to 18 h. Percent reducing activity of aspasome was estimated by measuring the absorbance of alpha,alpha-diphenyl-beta-picrylhydrazyl (DPPH) at 517 nm after addition of test antioxidant samples. These studies revealed that the antioxidant potency of ascorbyl moiety is retained even after converting ascorbyl palmitate into vesicles (Aspasomes). The antioxidant potency of Aspasomes was assessed by measuring the protection offered by this preparation against quinolinic acid induced lipoperoxidation of whole human blood in vitro, where in the lipoperoxidation was monitored by measuring thiobarbituric acid reactive substances (TBARS) levels. Aspasome rendered much better antioxidant activity than ascorbic acid. Transdermal permeation of aspasomal AZT, ASP-AZT aqueous dispersion and AZT-solution across excised rat skin was investigated in vitro using Franz diffusion cell. Permeation of aspasomal AZT was much higher than the other two preparations. However, ASP-AZT aqueous dispersion has also enhanced permeation of AZT significantly over the AZT-solution, indicating skin permeation enhancing property of ascorbyl palmitate.

1-O-Alkylglycerol vesicles (Algosomes): their formation and characterization.

1-O-alkylglycerols (ALKG) have exhibited several biological activities and a prominent effect on blood-brain barrier permeability. They have markedly improved brain uptake of cancerostatic agents. Since ALKG are amphiphilic, we explored their tendency to assemble into bilayer vesicles, which can be applied as carriers for drugs. Vesicles (Algosomes) were formed by film hydration method using ALKG (tetra-, penta-, hexa-, hepta-, octa- or nona-decylglycerols) in combination with cholesterol (CHOL) and dicetyl phosphate (DCP) (1-O-alkylglycerol:CHOL:DCP in 45:45:10 molar ratio). On microscopic examination, the algosomes were found to be conspicuously spherical and the dispersion was a mixture of multi-lamellar and small-unilamellar vesicles. Phase transition temperatures of 1-O-hexadecylglycerol (HXDG) and CHOL mixtures were tested by differential scanning calorimetry (DSC). The changes in phase transition temperatures indicate the vesicle forming tendency of ALKG in presence of CHOL. Alkyl chain length dependent variations in vesicle size, zeta-potential (ZP) and capture volume (CV) could not be observed. Vesicles of 1-O-tetradecylglycerol (TTDG) showed improvement in CV with increase in CHOL content from 15 to 55 mol%. However the vesicle size decreased. On challenging algosomes with hypertonic salt solution [potassium iodide (KI) in water], vesicle size decreased and thus algosomes were found to be osmotically sensitive. Algosome dispersions on addition of higher concentrations of KI (40-100 mM) brought about increases in vesicle size and at concentrations 60 mM and above showed aggregation. All vesicular dispersions were stable for only a few days.

Release studies on niosomes containing fatty alcohols as bilayer stabilizers instead of cholesterol.

Monomers of some amphiphiles organize into bilayers to form liposomes and niosomes. Such bilayers are unstable or leaky and hence cholesterol is a common ingredient included to stabilize them. Cholesterol stabilizes bilayers, prevents leakiness, and retards permeation of solutes enclosed in the aqueous core of these vesicles. Other than cholesterol a material with good bilayer-stabilizing properties is yet to be identified. We have substituted cholesterol with fatty alcohols in niosomes containing polyglyceryl-3-di-isostearate (PGDS) and polysorbate-80 (PS-80) to explore their membrane-stabilizing property via permeation studies. Niosomes of polyglyceryl-3-di-isostearate, fatty alcohol/cholesterol, and polysorbate were prepared by ether injection method. Aqueous solution of ketorolac tromethamine (KT) was entrapped in them. The effects of alkyl chain length of fatty alcohols (C(12), C(14), C(16), C(18), and C(16+18)), of acyl chain length of polyoxyethylene sorbitan monoester surfactants, and of the molar ratio of lipid mixture on the release rate of ketorolac from niosomes were assessed by employing modified dissolution-dialysis method. Niosomes with cholesterol or fatty alcohols have exhibited a common release pattern. Niosomes containing fatty alcohol showed a considerably slower release rate of KT than those containing cholesterol. Based on the release rate, fatty alcohols can be ranked as stearyl

Procollagen II amino propeptide processing by ADAMTS-3. Insights on dermatosparaxis.

The amino and carboxyl propeptides of procollagens I and II are removed by specific enzymes as a prerequisite for fibril assembly. Null mutations in procollagen I N-propeptidase (ADAMTS-2) cause dermatosparaxis in cattle and the Ehlers-Danlos syndrome (dermatosparactic type) in humans by preventing proteolytic excision of the N-propeptide of procollagen I. We have found that procollagen II is processed normally in dermatosparactic nasal cartilage, suggesting the existence of another N-propeptidase(s). We investigated such a role for ADAMTS-3 in Swarm rat chondrosarcoma RCS-LTC cells, which fail to process the procollagen II N-propeptide. Stable transfection of RCS-LTC cells with bovine ADAMTS-2 or human ADAMTS-3 partially rescued the processing defect, suggesting that ADAMTS-3 has procollagen II N-propeptidase activity. Human skin and skin fibroblasts showed 30-fold higher mRNA levels of ADAMTS-2 than ADAMTS-3, whereas ADAMTS-3 mRNA was 5-fold higher than ADAMTS-2 mRNA in human cartilage. We propose that both ADAMTS-2 and ADAMTS-3 process procollagen II, but ADAMTS-3 is physiologically more relevant, given its preferred expression in cartilage. The findings provide an explanation for the sparing of cartilage in dermatosparaxis and, perhaps, for the relative sparing of some procollagen I-containing tissues.

Chromosomal mapping of Adam9, Adam15 and Adam21.

Adam9, Adam15 and Adam21, genes encoding members of the ADAM or MDC family of metalloproteases, have been mapped to mouse chromosomes 8, 1, and 12, respectively, using an interspecific cross. The mapping of these mouse loci and the extrapolated loci for their human orthologs may facilitate the mapping of diseases involving these genes.

Impaired endochondral ossification and angiogenesis in mice deficient in membrane-type matrix metalloproteinase I.

Membrane-type matrix metalloproteinase I (MT1-MMP)-deficient mice were found to have severe defects in skeletal development and angiogenesis. The craniofacial, axial, and appendicular skeletons were severely affected, leading to a short and domed skull, marked deceleration of postnatal growth, and death by 3 wk of age. Shortening of bones is a consequence of decreased chondrocyte proliferation in the proliferative zone of the growth plates. Defective vascular invasion of cartilage leads to enlargement of hypertrophic zones of growth plates and delayed formation of secondary ossification centers in long bones. In an in vivo corneal angiogenesis assay, null mice did not have angiogenic response to implanted FGF-2, suggesting that the defect in angiogenesis is not restricted to cartilage alone. In tissues from null mice, activation of latent matrix metalloproteinase 2 was deficient, suggesting that MT1-MMP is essential for its activation in vivo.

ADAM-TS8, a novel metalloprotease of the ADAM-TS family located on mouse chromosome 9 and human chromosome 11.

A disintegrin-like and metalloprotease domain with thrombospondin type I modules (ADAM-TS) describes a novel family of zinc metalloendopeptidases. Its members have a common domain organization, which includes, typically, a pre-pro-metalloprotease domain, a disintegrin-like domain, and one or more thrombospondin-like (TS) modules. We describe here the complete primary structure of mouse ADAM-TS8, through cloning of Adamts8 cDNA. This novel member of the family contains two TS modules and is highly similar in sequence and domain organization to three other recently described gene products, ADAM-TS5, ADAM-TS6, and ADAM-TS7. Adamts8 is expressed at low levels throughout development and in adult mouse lung and heart. Through analysis of an interspecific backcross panel, we place the Adamts8 locus on mouse chromosome 9 at a consensus position of 11 cM and its human ortholog, recently reported as the METH2 gene, on human chromosome 11q25.

ADAM-TS5, ADAM-TS6, and ADAM-TS7, novel members of a new family of zinc metalloproteases. General features and genomic distribution of the ADAM-TS family.

We report the primary structure of three novel, putative zinc metalloproteases designated ADAM-TS5, ADAM-TS6, and ADAM-TS7. All have a similar domain organization, comprising a preproregion, a reprolysin-type catalytic domain, a disintegrin-like domain, a thrombospondin type-1 (TS) module, a cysteine-rich domain, a spacer domain without cysteine residues, and a COOH-terminal TS module. These genes are differentially regulated during mouse embryogenesis and in adult tissues, with Adamts5 highly expressed in the peri-implantation period in embryo and trophoblast. These proteins are similar to four other cognate gene products, defining a distinct family of human reprolysin-like metalloproteases, the ADAM-TS family. The other members of the family are ADAM-TS1, an inflammation-induced gene, the procollagen I/II amino-propeptide processing enzyme (PCINP, ADAM-TS2), and proteins predicted by the KIAA0366 and KIAA0688 genes (ADAM-TS3 and ADAM-TS4). Individual ADAM-TS members differ in the number of COOH-terminal TS modules, and some have unique COOH-terminal domains. The ADAM-TS genes are dispersed in human and mouse genomes.

Egr-1 mediates extracellular matrix-driven transcription of membrane type 1 matrix metalloproteinase in endothelium.

Matrix metalloproteinase activity is instrumental in processes of cellular invasion. The interstitial invasion of endothelial cells during angiogenesis is accompanied by up-regulation of several matrix metalloproteinases, including membrane type 1 matrix metalloproteinase (MT1-MMP). In this study, we show that endothelial cells stimulated to undergo angiogenesis by a three-dimensional extracellular matrix environment increase production of the transcription factor Egr-1. Increased binding of Egr-1 to the MT1-MMP promoter correlates with enhanced transcriptional activity, whereas mutations in the Egr-1 binding site abrogate the increased transcription of MT1-MMP in the stimulated cells. These data identify Egr-1-mediated transcription of MT1-MMP as a mechanism by which endothelial cells can initiate an invasive phenotype in response to an alteration in extracellular matrix environment, thus functionally associating MT1-MMP with a growing number of proteins known to be up-regulated by Egr-1 in response to tissue injury or mechanical stress.

Human tissue inhibitor of metalloproteinases 3 interacts with both the N- and C-terminal domains of gelatinases A and B. Regulation by polyanions.

We compared the association constants of tissue inhibitor of metalloproteinases (TIMP)-3 with various matrix metalloproteinases with those for TIMP-1 and TIMP-2 using a continuous assay. TIMP-3 behaved more like TIMP-2 than TIMP-1, showing rapid association with gelatinases A and B. Experiments with the N-terminal domain of gelatinase A, the isolated C-terminal domain, or an inactive progelatinase A mutant showed that the hemopexin domain of gelatinase A makes an important contribution to the interaction with TIMP-3. The exchange of portions of the gelatinase A hemopexin domain with that of stromelysin revealed that residues 568-631 of gelatinase A were required for rapid association with TIMP-3. The N-terminal domain of gelatinase B alone also showed slower association with TIMP-3, again implying significant C-domain interactions. The isolation of complexes between TIMP-3 and progelatinases A and B on gelatin-agarose demonstrated that TIMP-3 binds to both proenzymes. We analyzed the effect of various polyanions on the inhibitory activity of TIMP-3 in our soluble assay. The association rate was increased by dextran sulfate, heparin, and heparan sulfate, but not by dermatan sulfate or hyaluronic acid. Because TIMP-3 is sequestered in the extracellular matrix, the presence of certain heparan sulfate proteoglycans could enhance its inhibitory capacity.

Time dependent influence of diazepam on the pharmacokinetics of ibuprofen in man.

Circadian variation in the disease activity of rheumatoid arthritis has been established. Several nonsteroidal anti-inflammatory drugs have been studied for their chronokinetic behaviour. Time dependent influence of diazepam on the pharmacokinetics of diclofenac and naproxen has been reported. We report the time dependent influence of diazepam on the pharmacokinetics of ibuprofen in healthy subjects. Either ibuprofen or ibuprofen with diazepam was administered at 10.00 or 22.00 hours to eight healthy volunteers in a randomized crossover study. Serum ibuprofen levels were estimated by high performance liquid chromatography. There was a significant (p < 0.05) increase in mean elimination half life (2.39 +/- 0.42 to 3.59 +/- 0.35 h) following ibuprofen and diazepam administration compared to ibuprofen alone administered at 22.00 hours. The mean clearance of ibuprofen was therefore lowered from 62.7 +/- 8.9 to 41.7 +/- 2.6 ml/h/kg under the influence of diazepam during the night. Such a time dependent influence of diazepam on the pharmacokinetics of ibuprofen may be due to circadian variation in the pattern of protein production in the liver and/or competitive protein binding of the two drugs during the dark period.

Cloning of the human tissue inhibitor of metalloproteinase-4 gene (TIMP4) and localization of the TIMP4 and Timp4 genes to human chromosome 3p25 and mouse chromosome 6, respectively.

We have isolated genomic DNA containing the human tissue inhibitor of metalloproteinases-4 gene (TIMP4) and determined the structure of the exons comprising the gene. Like other members of the TIMP family, the TIMP-4 protein is encoded by five exons. These span 6 kb of genomic DNA, so that TIMP4 is similar in size to Timp1 but considerably smaller than TIMP2 and TIMP3. The exon-intron boundaries of TIMP4 are at locations very similar to those of the other TIMP genes, demonstrating the high degree of conservation of gene structure in this family. The human and mouse TIMP-4 genes map to comparable locations in the respective genomes, localizing to human chromosome 3p25 and mouse chromosome 6.