Chondroadherin Fragmentation Mediated by the Protease HTRA1 Distinguishes Human Intervertebral Disc Degeneration from Normal Aging

Chondroadherin, a member of the leucine-rich repeat family, has previously been demonstrated to be fragmented in some juveniles with idiopathic scoliosis. This observation led us to investigate adults with disc degeneration. Immunoblotting analysis demonstrated that non-degenerate discs from three different age groups show no chondroadherin fragmentation. Furthermore, the chondroadherin fragments in adult degenerate disc and the juvenile scoliotic disc were compared via immunoblot analysis and appeared to have a similar size. We then investigated whether or not chondroadherin fragmentation increases with the severity of disc degeneration. Three different samples with different severities were chosen from the same disc, and chondroadherin fragmentation was found to be more abundant with increasing severity of degeneration. This observation led us to the creation of a neoepitope antibody to the cleavage site observed. We then observed that the cleavage site in adult degenerate discs and juvenile scoliotic discs was identical as confirmed by the neoepitope antibody. Consequently, investigation of the protease capable of cleaving chondroadherin at this site was necessary. In vitro digests of disc tissue demonstrated that ADAMTS-4 and -5; cathepsins K, B, and L; and MMP-3, -7, -12, and -13 were incapable of cleavage of chondroadherin at this site and that HTRA1 was indeed the only protease capable. Furthermore, increased protein levels of the processed form of HTRA1 were demonstrated in degenerate disc tissues via immunoblotting. The results suggest that chondroadherin fragmentation can be used as a biomarker to distinguish the processes of disc degeneration from normal aging.     

Inflammatory Cytokines Induce NOTCH Signaling in Nucleus Pulposus Cells: IMPLICATIONS IN INTERVERTEBRAL DISC DEGENERATION*

The objective of the study was to investigate how inflammatory cytokines, IL-1β, and TNF-α control NOTCH signaling activity in nucleus pulposus (NP) cells. An increase in expression of selective NOTCH receptors (NOTCH1 and -2), ligand (JAGGED2), and target genes (HES1, HEY1, and HEY2) was observed in NP cells following cytokine treatment. A concomitant increase in NOTCH signaling as evidenced by induction in activity of target gene HES1 and HEY1 promoters and reporter 12xCSL was seen. Moreover, treatment increased activity of a 2-kb NOTCH2 promoter. Treatment of cells with NF-κB and MAPK inhibitors abolished the inductive effect of cytokines on NOTCH2 promoter and its expression. Gain and loss-of-function studies confirmed the inductive effect of p65 on NOTCH2 promoter activity. In contrast, p50 blocked the cytokine induction of promoter activity. Supporting promoter studies, lentiviral delivery of sh-p65, and sh-IKKβ significantly decreased cytokine dependent change in NOTCH2 expression. Interestingly, MAPK signaling showed an isoform-specific control of NOTCH2 promoter; p38α/β2/δ, ERK1, and ERK2 contributed to cytokine dependent induction, whereas p38γ played no role. Analysis of human NP tissues showed that NOTCH1 and -2 and HEY2 expression correlated with each other. Moreover, expression of NOTCH2 and IL-1β as well as the number of cells immunopositive for NOTCH2 significantly increased in histologically degenerate discs compared with non-degenerate discs. Taken together, these results explain the observed dysregulated expression of NOTCH genes in degenerative disc disease. Thus, controlling IL-1β and TNF-α activities during disc disease may restore NOTCH signaling and nucleus pulposus cell function.     

An improved staining method for intervertebral disc tissue

The objective of this study was to design a new staining procedure for human disc tissue for visualizing both collagen and proteoglycan-matrix components on the same histology section. Weigert's hematoxylin, alcian blue and picrosirius red were combined to produce distinctive staining of collagen (red), proteoglycans (blue) and cellular elements of the intervertebral disc. This novel stain reveals sharp details of collagen composition in the perilacunar, territorial and intraterritorial extracellular matrix, and concomitantly demonstrates the presence of proteoglycan accumulations around cells in the lacunar spaces and in the extracellular matrix. These details reveal variations within the tissue that would not be apparent with routine stains.     

Detection of Methicillin-Resistant Staphylococcus aureus Using mecA/nuc Genes and Antibiotic Susceptibility Profile of Malaysian Clinical Isolates

The aim of this study is to compare methicillin-resistant Staphylococcus aureus (MRSA) detection methods and to generate antibiogram profile of S. aureus clinical isolates from two teaching hospitals in Malaysia including three reference isolates from American Type Culture Collection (ATCC). The mecA/nuc gene PCR amplification, spot inoculation test and oxacillin disc diffusion test were applied to compare its MRSA detection abilities. No disagreement between the three methods was observed. From 29 bacterial isolates (including the ATCC strains) tested, 19 isolates were confirmed as S. aureus with 14 isolates exhibiting multidrug-resistance. All isolates are still susceptible to vancomycin as indicated by the E-test result. Current biochemical tests are comparable with the molecular detection method for MRSA used in this study while multidrug-resistance traits are present in both MRSA and MSSA clinical isolates. Presently, mupirocin seems to be the best alternative for vancomycin against multidrug-resistant S. aureus infections in Malaysia. Susceptibility profile of 19 S. aureus isolates acquired from two teaching hospitals and ATCC towards 16 selected antibiotics was analyzed and an antibiogram was generated. Findings also indicated resistance against many of the available antibiotics and thus an urgent need to search for alternative antibiotics.     

Quantitative proteomic analysis of eight cartilaginous tissues reveals characteristic differences as well as similarities between subgroups

Human synovial joints display a characteristic anatomic distribution of arthritis, e.g. rheumatoid arthritis primarily affects the metacarpophalangeal and proximal finger joints, but rarely the distal finger joints, whereas osteoarthritis occurs in the distal and proximal finger joints. Pelvospondylitis has a selective localization to the spine and sacroiliac joints. Is this tropism due to differences between the cartilages at the molecular level? To substantiate this concept the present study provides a background detailed compositional analysis by relative quantification of extracellular matrix proteins in articular cartilages, meniscus, intervertebral disc, rib, and tracheal cartilages on samples from 5–6 different individuals using an optimized approach for proteomics. Tissue extraction followed by trypsin digestion and two-dimensional LC separations coupled to tandem mass spectrometry, relative quantification with isobaric labeling, iTRAQ^TM, was used to compare the relative abundance of about 150 proteins. There were clear differences in protein patterns between different kinds of cartilages. Matrilin-1 and epiphycan were specific for rib and trachea, whereas asporin was particularly abundant in the meniscus. Interestingly, lubricin was prominent in the intervertebral disc, especially in the nucleus pulposus. Fibromodulin and lumican showed distributions that were mirror images of one other. Analyses of the insoluble residues from guanidine extraction revealed that a fraction of several proteins remained unextracted, e.g. asporin, CILP, and COMP, indicating cross-linking. Distinct differences in protein patterns may relate to different tissue mechanical properties, and to the intriguing tropism in different patterns of joint pathology.     

Measurement of local strains in intervertebral disc anulus fibrosus tissue under dynamic shear: Contributions of matrix fiber orientation and elastin content

Shear strain has been implicated as an initiator of intervertebral disc anulus failure, however a clear, multi-scale picture of how shear strain affects the tissue microstructure has been lacking. The purposes of this study were to measure microscale deformations in anulus tissue under dynamic shear in two orie ntations, and to determine the role of elastin in regulating these deformations. Bovine AF tissue was simultaneously shear loaded and imaged using confocal microscopy following either a buffer or elastase treatment. Digital image analysis was used to track through time local shear strains in specimens sheared transversely, and stretch and rotation of collagen fiber bundles in specimens sheared circumferentially. The results of this study suggest that sliding does not occur between AF plies under shear, and that interlamellar connections are governed by collagen and fibrilin rather than elastin. The transverse shear modulus was found to be approximately 1.6 times as high in plies the direction of the collagen fibers as in plies across them. Under physiological levels of in-plane shear, fiber bundles stretched and re-oriented linearly. Elastin was found to primarily stiffen plies transversely. We conclude that alterations in the elastic fiber network, as found with IVD herniation and degeneration, can therefore be expected to significantly influence the AF response to shear making it more susceptible to micro failure under bending or torsion loading.     

Performance of a three-stage aerobic RBC reactor in food canning wastewater treatment

Biological treatment using attached growth in a three-stage lab-scale rotating biological contactor (RBC) was implemented for wastewater from food cannery industries. The wastewater contained high level of organic compounds due to fish and fruit cleaning, cooking and filling processes. Nutrients available in the wastewater enhanced the growth of microorganisms and allowed the biological treatment to be effective. The RBC consisted of 54 parallel discs rotating in a reservoir and was arranged in three stages, i.e. 18 discs oriented in each stage. Effect of major operating and physical variables such as hydraulic retention time (HRT), disc submergence and disc rotational speed were examined in COD removal. For duration of 5 days, 96.4% BOD removal was achieved in batch experiment. BOD constant rate (k) and ultimate BOD were determined respectively, 0.8198 day⁻¹ and 6349 mg/l by Thomas graphical method. COD removal efficiency was increased from 85.3 to 97.4% while the HRT was increased from 24 to 48 h. The COD removal efficiency increased from 74.9 to 87.5% as the disc submergence was increased from 31 to 36%. At submergence level of 23.7%, removal efficiency was increased due to activation of second and third compartments. When the rotational speed was increased from 3 to 11 rpm, the COD removal efficiency was also increased from 62.7 to 93.7%, respectively. The stage COD removal efficiency was gradually decreased with an increase number of stage and about 88% of organic compounds were removed in the first stage of aerobic RBC, indicating that the single stage reactor may be sufficient in practical application.     

Cell shape and gene expression in human intervertebral disc cells: in vitro tissue engineering studies

The objective of the present study was to examine the relation between gene expression and the shape of human intervertebral disc cells cultured in vitro in three-dimensional (3D) scaffolds. Disc cells from 19 subjects were seeded into either a collagen sponge or collagen gel and cultured for 10 days. In situ hybridization was performed on serial sections of paraffin embedded specimens and assessed for expression of selected genes important for extracellular matrix formation: Types I and II collagen, aggrecan and chondroitin-6 sulfotransferase. Rounded cells grown in collagen gel showed expression of Types I and II collagen, aggrecan and chondroitin-6 sulfotransferase; expression of these genes was absent in spindle shaped cells. Cells in the collagen sponge that lay on the sponge margin were frequently spindle shaped; these cells expressed type I collagen, but not type II collagen, aggrecan or chondroitin-6 sulfotransferase. Results presented here provide novel data concerning disc cell gene expression with collagen 3D constructs. This information is useful for future tissue engineering studies that have the challenging goal of selectively modulating gene expression.     

澜沧荛花(瑞香科)的花部形态发生 及其系统学意义

通过扫描电镜对澜沧荛花Wikstroemia delavayi花部的形态发生过程进行了观察和分析,旨在为该属的系统学研究提供花部发育形态学资料。澜沧尧花花部的发生和早期发育呈远轴面向近轴面的顺序,但这一式样由于近轴面的器官在早期发育之后生长加速发生了转变。因此,花开放时所表现的所谓辐射对称,显然是由同一轮器官的异率生长所导致的次生现象。花盘发生于花萼筒基部的远轴面上,与花萼、雄蕊的发生间隔时间较长。花盘原基在下轮雄蕊着生处凹陷或间断,与之相对应,花盘裂片与下轮雄蕊呈互生。由此,花盘显然不是花托的一部分,也不是象花萼、雄蕊和心皮一样的独立结构,将其解释为雄蕊群的一部分更合理。花盘的发生和早期发育及其着生位置同其他花部器官的发生和发育式样具有明显的相关性,这种相关性对进一步阐明瑞香属Daphne和荛花属Wikstroemia的系统发育关系具有—定意义。根据对雌蕊群的发生和发育过程观察,该种的子房是由一个近轴面的可育心皮和一个远轴面的不育心皮融合而成的单室子房,为假单心皮雌蕊。尽管荛花属和瑞香属均属于单室产房,但澜沧荛花的子房维管束中的腹束排列于中轴位置,而目前资料显示瑞香属植物的腹束接近于侧膜位置,这方面仍需进一步研究。