Biomechanical properties of murine TMJ articular disc and condyle cartilage via AFM-nanoindentation

This study aims to quantify the biomechanical properties of murine temporomandibular joint (TMJ) articular disc and condyle cartilage using AFM-nanoindentation. For skeletally mature, 3-month old mice, the surface of condyle cartilage was found to be significantly stiffer (306 ± 84 kPa, mean ± 95% CI) than those of the superior (85 ± 23 kPa) and inferior (45 ± 12 kPa) sides of the articular disc. On the disc surface, significant heterogeneity was also detected across multiple anatomical sites, with the posterior end being the stiffest and central region being the softest. Using SEM, this study also found that the surfaces of disc are composed of anteroposteriorly oriented collagen fibers, which are sporadically covered by thinner random fibrils. Such fibrous nature results in both an F-D^3/2 indentation response, which is a typical Hertzian response for soft continuum tissue under a spherical tip, and a linear F-D response, which is typical for fibrous tissues, further signifying the high degree of tissue heterogeneity. In comparison, the surface of condyle cartilage is dominated by thinner, randomly oriented collagen fibrils, leading to Hertzian-dominated indentation responses. As the first biomechanical study of murine TMJ, this work will provide a basis for future investigations of TMJ tissue development and osteoarthritis in various murine TMJ models.     

Haematopoietic TLR4 deletion attenuates perivascular brown adipose tissue inflammation in atherosclerotic mice

AimsTo investigate whether haematopoietic TLR4 deletion attenuates perivascular brown adipose tissue inflammation in atherosclerotic mice.Methods and ResultsExperiments were performed using irradiated LDL receptor-deficient (LDLR^−/−) mice with marrow from either TLR4-deficient (TLR4^−/−) or age-matched wild-type (WT) mice. After 12 weeks of being fed a high-cholesterol diet, TLR4^−/− → LDLR^−/− mice developed fewer atherosclerotic lesions in the aorta compared to WT → LDLR^−/− mice. This effect was associated with an increase in multilocular lipid droplets and mitochondria in perivascular adipose tissue (PVAT). Immunofluorescence analysis confirmed that there was an increase in capillary density and M2 macrophage infiltration, accompanied by a decrease in tumour necrosis factor (TNF)-α expression in the localized PVAT of TLR4^−/− → LDLR^−/− mice. In vitro studies indicated that bone marrow-derived macrophages (BMDMs) from WT mice demonstrated an M1-like phenotype and expression of inflammatory cytokines induced by palmitate. These effects were attenuated in BMDMs isolated from TLR4^−/− mice. Furthermore, brown adipocytes incubated with conditioned medium (CM) derived from palmitate-treated BMDMs, exhibited larger and more unilocular lipid droplets, and reduced expression of brown adipocyte-specific markers and perilipin-1 compared to those observed in brown adipocytes exposed to CM from palmitate-treated BMDMs of TLR4^−/− mice. This decreased potency was primarily due to TNF-α, as demonstrated by the capacity of the TNF-α neutralizing antibody to reverse these effects.ConclusionsThese results suggest that haematopoietic-specific deletion of TLR4 promotes PVAT homeostasis, which is involved in reducing macrophage-induced TNF-α secretion and increasing mitochondrial biogenesis in brown adipocytes.     

Braking characteristics during cutting and pivoting in female soccer players

Most biomechanical studies into changing direction focus on final contact (FC), whilst limited research has examined penultimate contact (PEN). The aim of this study was to explore the kinematic and kinetic differences between PEN and FC of cutting and pivoting in 22 female soccer players (mean ± SD; age: 21 ± 3.1 years, height: 1.68 ± 0.07 m, mass: 58.9 ± 7.3 kg). Furthermore, the study investigated whether horizontal force–time characteristics during PEN were related to peak knee abduction moments during FC. Three dimensional motion analyses of cutting and pivoting on the right leg were performed using Qualysis ‘Proreflex’ infrared cameras (240 Hz). Ground reaction forces (GRF) were collected from two AMTI force platforms (1200 Hz) to examine PEN and FC. Both manoeuvres involved significantly (P < 0.05) greater knee joint flexion angles, peak horizontal GRF, but lower average horizontal GRF during PEN compared to FC. Average horizontal GRF during PEN (R = −0.569, R² = 32%, P = 0.006) and average horizontal GRF ratio (R = 0.466, R² = 22%, P = 0.029) were significantly related to peak knee abduction moments during the FC of cutting and pivoting, respectively. The results indicate PEN during pre-planned changing direction helps reduce loading on the turning leg where there is greater risk of injuries to knee ligaments.     

Transcriptional responses of neonatal mouse lung to hyperoxia by Nrf2 status

Hyperoxia exposure can inhibit alveolar growth in the neonatal lung through induction of p21/p53 pathways and is a risk factor for the development of bronchopulmonary dysplasia (BPD) in preterm infants. We previously found that activation of nuclear factor erythroid 2 p45-related factor (Nrf2) improved survival in neonatal mice exposed to hyperoxia likely due to increased expression of anti-oxidant response genes. It is not known however, whether hyperoxic induced Nrf2 activation attenuates the growth impairment caused by hyperoxia in neonatal lung. To determine if Nrf2 activation modulates cell cycle regulatory pathway genes associated with growth arrest we examined the gene expression in the lungs of Nrf2^−/− and Nrf2^+/+ neonatal mice at one and 3 days of hyperoxia exposure.MethodsMicroarray analysis was performed in neonatal Nrf2^+/+ and Nrf2^−/− lungs exposed to one and 3 days of hyperoxia. Sulforaphane, an inducer of Nrf2 was given to timed pregnant mice to determine if in utero exposure attenuated p21 and IL-6 gene expression in wildtype neonatal mice exposed to hyperoxia.ResultsCell cycle regulatory genes were induced in Nrf2^−/− lung at 1 day of hyperoxia. At 3 days of hyperoxia, induction of cell cycle regulatory genes was similar in Nrf2^+/+ and Nrf2^−/− lungs, despite higher inflammatory gene expression in Nrf2^−/− lung.Conclusionp21/p53 pathways gene expression was not attenuated by Nrf2 activation in neonatal lung. In utero SUL did not attenuate p21 expression in wildtype neonatal lung exposed to hyperoxia. These findings suggest that although Nrf2 activation induces expression of anti-oxidant genes, it does not attenuate alveolar growth arrest caused by exposure to hyperoxia.     

Target Binding to S100B Reduces Dynamic Properties and Increases Ca2 +-Binding Affinity for Wild Type and EF-Hand Mutant Proteins

Mutations in the second EF-hand (D61N, D63N, D65N, and E72A) of S100B were used to study its Ca^2 + binding and dynamic properties in the absence and presence of a bound target, TRTK-12. With ^D63NS100B as an exception (^D63NK_D = 50 ± 9 μM), Ca^2 + binding to EF2-hand mutants were reduced by more than 8-fold in the absence of TRTK-12 (^D61NK_D = 412 ± 67 μM, ^D65NK_D = 968 ± 171 μM, and ^E72AK_D = 471 ± 133 μM), when compared to wild-type protein (^WTK_D = 56 ± 9 μM). For the TRTK-12 complexes, the Ca^2 +-binding affinity to wild type (^WT + TRTKK_D = 12 ± 10 μM) and the EF2 mutants was increased by 5- to 14-fold versus in the absence of target (^{D61N + TRTK}K_D = 29 ± 1.2 μM, ^{D63N + TRTK}K_D = 10 ± 2.2 μM, ^{D65N + TRTK}K_D = 73 ± 4.4 μM, and ^{E72A + TRTK}K_D = 18 ± 3.7 μM). In addition, R_ex, as measured using relaxation dispersion for side‐chain ¹⁵N resonances of Asn63 (^D63NS100B), was reduced upon TRTK-12 binding when measured by NMR. Likewise, backbone motions on multiple timescales (picoseconds to milliseconds) throughout wild type, ^D61NS100B, ^D63NS100B, and ^D65NS100B were lowered upon binding TRTK-12. However, the X-ray structures of Ca^2 +-bound (2.0 Å) and TRTK-bound (1.2 Å) ^D63NS100B showed no change in Ca^2 + coordination; thus, these and analogous structural data for the wild-type protein could not be used to explain how target binding increased Ca^2 +-binding affinity in solution. Therefore, a model for how S100B–TRTK‐12 complex formation increases Ca^2 + binding is discussed, which considers changes in protein dynamics upon binding the target TRTK-12.     

Remarkable induction of UV-signature mutations at the 3′-cytosine of dipyrimidine sites except at 5′-TCG-3′ in the UVB-exposed skin epidermis of xeroderma pigmentosum variant model mice

The human POLH gene is responsible for the variant form of xeroderma pigmentosum (XP-V), a genetic disease highly susceptible to cancer on sun-exposed skin areas, and encodes DNA polymerase η (polη), which is specialized for translesion DNA synthesis (TLS) of UV-induced DNA photolesions. We constructed polη-deficient mice transgenic with lacZ mutational reporter genes to study the effect of Polh null mutation (Polh^−/−) on mutagenesis in the skin after UVB irradiation. UVB induced lacZ mutations with remarkably higher frequency in the Polh^−/− epidermis and dermis than in the wild-type (Polh^+/+) and heterozygote. DNA sequences of a hundred lacZ mutants isolated from the epidermis of four UVB-exposed Polh^−/− mice were determined and compared with mutant sequences from irradiated Polh^+/+ mice. The spectra of the mutations in the two genotypes were both highly UV-specific and dominated by C → T transitions at dipyrimidines, namely UV-signature mutations. However, sequence preferences of the occurrence of UV-signature mutations were quite different between the two genotypes: the mutations occurred at a higher frequency preferentially at the 5′-TCG-3′ sequence context than at the other dipyrimidine contexts in the Polh^+/+ epidermis, whereas the mutations were induced remarkably and exclusively at the 3′-cytosine of almost all dipyrimidine contexts with no preference for 5′-TCG-3′ in the Polh^−/− epidermis. In addition, in Polh^−/− mice, a small but remarkable fraction of G → T transversions was also observed exclusively at the 3′-cytosine of dipyrimidine sites, strongly suggesting that these transversions resulted not from oxidative damage but from UV photolesions. These results would reflect the characteristics of the error-prone TLS functioning in the bypass of UV photolesions in the absence of polη, which would be mediated by mechanisms based on the two-step model of TLS. On the other hand, the deamination model would explain well the mutation spectrum in the Polh^+/+ genotype.     

Truncated amelogenin and LRAP transgenes improve Amelx null mouse enamel

Amelogenin is the most abundant enamel protein involved in enamel mineralization. Our goal was to determine whether all three regions of amelogenin (N-terminus, C-terminus, central core) are required for enamel formation. Amelogenin RNA is alternatively spliced, resulting in at least 16 different amelogenin isoforms in mice, with M180 and LRAP expressed most abundantly. Soon after secretion by ameloblasts, M180 is cleaved by MMP20 resulting in C-terminal truncated (CTRNC) amelogenin. We aimed to determine whether the 2 transgenes (Tg), LRAP and CTRNC together, can improve LRAPTg/Amelx −/− and CTRNCTg/Amelx −/− enamel thickness and prism organization, which were not rescued in Amelx −/− enamel. We generated CTRNCTg/LRAPTg/Amelx −/− mice and analyzed developing and mature incisor and molar enamel histologically, by microCT, SEM and microhardness testing. CTRNCTg and LRAPTg overexpression together significantly improved the enamel phenotype of LRAPTg/Amelx −/− and CTRNCTg/Amelx −/− mouse enamel, however enamel microhardness was recovered only when M180Tg was expressed, alone or with LRAPTg. We determined that both LRAP and CTRNC, which together express all three regions of the amelogenin protein (N-terminus, C-terminus and hydrophobic core) contribute to the final enamel thickness and prism organization in mice.     

Temporal–spatial variation and source analysis of carbon and nitrogen in a tidal wetland of Luoyuan Bay

Carbon and nitrogen are important elements in biogeochemical studies of tidal wetlands. Three wetland zones in Luoyuan Bay in the Fujian province were chosen for this study; the Spartina alterniflora flat zone with Spartina alterniflora growing, the silt zone with no Spartina alterniflora growing and the Spartina alterniflora-silt flat zone – a transition zone between the two. The spatial and seasonal variations of total organic carbon (TOC), total nitrogen (TN), stable isotopes of organic material (δ¹³C, δ¹⁵N), C/N ratio, average particle size and sediment composition in surface and vertical sediments of different ecological zones were analyzed. Carbon and nitrogen accumulation and particle size effects in the different ecological zones were discussed and the indicators of δ¹³C and C/N ratios were also compared. TOC, TN, δ¹³C contents, C/N ratios, and average particle size varied within the ranges of 0.611–1.133%, 0.053–0.090%, ?22.60 to ?18.92‰, 12.3–15.7, and 6.4–8.7 μm, respectively. Sediments were mainly silt-sized. Besides δ¹⁵N values, the other parameters, such as TOC, TN, δ¹³C contents, C/N ratios, and average particle size showed an obvious zonal distribution in surface sediments. The distribution of TOC and TN contents reflected the distribution of Spartina alterniflora within the bay. The profile and seasonal variations of these parameters in different ecological zones indicated that variations in the Spartina alterniflora flat and transition zones were complex because of the effect of Spartina alterniflora. Vertical and seasonal variations were sampled in the silt flat area. The profile and seasonal variations of TOC, TN and δ¹³C were similar in the transition zone and the Spartina alterniflora flat zone. Seasonal concentrations of TOC, TN and δ¹³C decreased from autumn > spring > winter > summer. The seasonal variation of carbon and nitrogen in the sediments may be influenced by temperature, particle size, plankton and benthos. The particle size effect was significant in the surface sediments and profile sediments of the transition zone. However, other factors had a greater effect on the distributions of TOC and TN in the Spartina alterniflora flat and silt flat zones. C/N ratios in sediments of the Spartina alterniflora flat, transition zone and silt flat were close to or > 12, indicating that the organic material source was dominated by terrestrial inputs. However, δ¹³C values decreased from the Spartina alterniflora flat zone > transition zone > silt flat zone indicating that the organic material source was predominantly from marine inputs. Thus the indications from C/N ratios and δ¹³C were different. There was no clear relationship between C/N ratios and δ¹³C values and a better relationship between δ¹³C values and TOC concentrations suggested that δ¹³C values provided a better indication of the organic source. Limited amounts of organic material came from Spartina alterniflora. This study has provided basic data for researching biogeochemical processes of biogenic elements in tidal wetlands and vegetation restoration, and has also provided a reference for assessing and protecting the environment and ecological systems in wetlands.     

Interlimb symmetry of dynamic knee joint stiffness and co-contraction is maintained in early stage knee osteoarthritis

Individuals with knee OA often exhibit greater co-contraction of antagonistic muscle groups surrounding the affected joint which may lead to increases in dynamic joint stiffness. These detrimental changes in the symptomatic limb may also exist in the contralateral limb, thus contributing to its risk of developing knee osteoarthritis. The purpose of this study is to investigate the interlimb symmetry of dynamic knee joint stiffness and muscular co-contraction in knee osteoarthritis.Muscular co-contraction and dynamic knee joint stiffness were assessed in 17 subjects with mild to moderate unilateral medial compartment knee osteoarthritis and 17 healthy control subjects while walking at a controlled speed (1.0 m/s). Paired and independent t-tests determined whether significant differences exist between groups (p < 0.05).There were no significant differences in dynamic joint stiffness or co-contraction between the OA symptomatic and OA contralateral group (p = 0.247, p = 0.874, respectively) or between the OA contralateral and healthy group (p = 0.635, p = 0.078, respectively). There was no significant difference in stiffness between the OA symptomatic and healthy group (p = 0.600); however, there was a slight trend toward enhanced co-contraction in the symptomatic knees compared to the healthy group (p = 0.051).Subjects with mild to moderate knee osteoarthritis maintain symmetric control strategies during gait.     

Comparative kinetic study between moving bed biofilm reactor-membrane bioreactor and membrane bioreactor systems and their influence on organic matter and nutrients removal

New technologies regarding wastewater treatment have been developed. Among these technologies, the moving bed biofilm reactor combined with membrane bioreactor (MBBR-MBR) is a recent solution alternative to conventional processes. This paper presents the results obtained from three wastewater treatment plants working in parallel. The first wastewater treatment plant consisted of a membrane bioreactor (MBR), the second one was a MBBR-MBR system containing carriers both in anoxic and aerobic zones, and the last one consisted of a MBBR-MBR system which contained carriers only in the aerobic zone. The reactors operated with a hydraulic retention time of 26.47 h. During the study, the difference between the experimental plants was not statistically significant concerning organic matter and nutrients removal. However, different tendencies regarding nutrients removal are shown by the three wastewater treatment plants. In this sense, the performances in terms of nitrogen and phosphorus removal of the MBBR-MBR system which contained carriers only in the aerobic zone (67.34 ± 11.22% and 50.65 ± 11.13%, respectively) were slightly better than those obtained from another experimental plants. As a whole, the pilot plant which consisted of a MBR showed better performance from the point of view of the kinetics of the heterotrophic and autotrophic biomass with values of μ_m,H = 0.00858 h⁻¹, μ_m,A = 0.07646 h⁻¹, K_M = 2.37 mg O₂ L⁻¹ and K_NH = 1.31 mg N L⁻¹.     

Biomechanical characterisation of ovine spinal facet joint cartilage

The spinal facet joints are known to be an important component in the kinematics and the load transmission of the spine. The articular cartilage in the facet joint is prone to degenerative changes which lead to back pain and treatments for the condition have had limited long term success. There is currently a lack of information on the basic biomechanical properties of the facet joint cartilage which is needed to develop tissue substitution or regenerative interventions. In the present study, the thickness and biphasic properties of ovine facet cartilage were determined using a combination of indentation tests and computational modelling. The equilibrium biphasic Young's modulus and permeability were derived to be 0.76±0.35 MPa and 1.61±1.10×10^?15 m⁴/(Ns) respectively, which were within the range of cartilage properties characterised from the human synovial joints. The average thickness of the ovine facet cartilage was 0.52±0.10 mm, which was measured using a needle indentation test. These properties could potentially be used for the development of substitution or tissue engineering interventions and for computational modelling of the facet joint. Furthermore, the developed method to characterise the facet cartilage could be used for other animals or human donors.     

δ15N and δ13C diet–tissue discrimination factors for large sharks under semi-controlled conditions

Stable isotopes (δ¹⁵N and δ¹³C) are being widely applied in ecological research but there has been a call for ecologists to determine species- and tissue-specific diet discrimination factors (?¹³C and ?¹⁵N) for their study animals. For large sharks stable isotopes may provide an important tool to elucidate aspects of their ecological roles in marine systems, but laboratory based controlled feeding experiments are impractical. By utilizing commercial aquaria, we estimated ?¹⁵N and ?¹³C of muscle, liver, vertebral cartilage and a number of organs of three large sand tiger (Carcharias taurus) and one large lemon shark (Negaprion brevirostris) under a controlled feeding regime. For all sharks mean ± SD for ?¹⁵N and ?¹³C in lipid extracted muscle using lipid extracted prey data were 2.29‰ ± 0.22 and 0.90‰ ± 0.33, respectively. The use of non-lipid extracted muscle and prey resulted in very similar ?¹⁵N and ?¹³C values but mixing of lipid and non-lipid extracted data produced variable estimates. Values of ?¹⁵N and ?¹³C in lipid extracted liver and prey were 1.50‰ ± 0.54 and 0.22‰ ± 1.18, respectively. Non-lipid extracted diet discrimination factors in liver were highly influenced by lipid content and studies that examine stable isotopes in shark liver, and likely any high lipid tissue, should strive to remove lipid effects through standardising C:N ratios, prior to isotope analysis. Mean vertebral cartilage ?¹⁵N and ?¹³C values were 1.45‰ ± 0.61 and 3.75‰ ± 0.44, respectively. Organ ?¹⁵N and ?¹³C values were more variable among individual sharks but heart tissue was consistently enriched by ~ 1–2.5‰. Minimal variability in muscle and liver δ¹⁵N and δ¹³C sampled at different intervals along the length of individual sharks and between liver lobes suggests that stable isotope values are consistent within tissues of individual animals. To our knowledge, these are the first reported diet–tissue discrimination factors for large sharks under semi-controlled conditions, and are lower than those reported for teleost fish.     

Hyperosmolaric contrast agents in cartilage tomography may expose cartilage to overload-induced cell death

In clinical arthrographic examination, strong hypertonic contrast agents are injected directly into the joint space. This may reduce the stiffness of articular cartilage, which is further hypothesized to lead to overload-induced cell death. We investigated the cell death in articular cartilage while the tissue was compressed in situ in physiological saline solution and in full strength hypertonic X-ray contrast agent Hexabrix^TM. Samples were prepared from bovine patellae and stored in Dulbecco’s Modified Eagle’s Medium overnight. Further, impact tests with or without creep were conducted for the samples with contact stresses and creep times changing from 1 MPa to 10 MPa and from 0 min to 15 min, respectively. Finally, depth-dependent cell viability was assessed with a confocal microscope. In order to characterize changes in the biomechanical properties of cartilage as a result of the use of Hexabrix?, stress-relaxation tests were conducted for the samples immersed in Hexabrix? and phosphate buffered saline (PBS). Both dynamic and equilibrium modulus of the samples immersed in Hexabrix? were significantly (p<0.05) lower than those of the samples immersed in PBS. Cartilage samples immersed in physiological saline solution showed load-induced cell death primarily in the superficial and middle zones. However, under high 8–10 MPa contact stresses, the samples immersed in full strength Hexabrix? showed significantly (p<0.05) higher number of dead cells than the samples compressed in physiological saline, especially in the deep zone of cartilage. In conclusion, excessive loading stresses followed by tissue creep might increase the risk for chondrocyte death in articular cartilage when immersed in hypertonic X-ray contrast agent, especially in the deep zone of cartilage.     

Quantitative analysis of RU38486 (mifepristone) by HPLC triple quadrupole mass spectrometry

A sensitive liquid chromatography–mass spectrometric method was validated for the quantification of RU38486 (mifepristone) in human and murine plasma. The analyte and internal standard (alfaxolone) were extracted by liquid–liquid extraction with diethyl ether, resolved on a C18 column using gradient elution with methanol and ammonium acetate and detected after positive electrospray ionization (m/z 430 → 372; m/z 333 → 297, respectively). Quantification was linear over the range 0.5–500 ng (r² > 0.997), precise and accurate (intra-assay RSD ≤ 7.2%, RME ≤ 8.2%; inter-assay RSD ≤ 15.7% RME ≤ 10.2%). The limit of quantification (LOQ) was 50 pg injected on column, permitting reproducible analysis of RU38486 in small volumes of plasma.     

Mapping riparian condition indicators in a sub-tropical savanna environment from discrete return LiDAR data using object-based image analysis

Mapping, monitoring and managing the environmental condition of riparian zones are major focus areas for local and state governments in Australia. New remotely sensed data techniques that can provide the required mapping accuracies, complete spatial coverage and processing and mapping transferability are currently being developed for use over large spatial extents. The research objective was to develop and apply an approach for mapping riparian condition indicators using object-based image analysis of airborne Light Detection and Ranging (LiDAR) data. The indicators assessed were: streambed width; riparian zone width; plant projective cover (PPC); longitudinal continuity; coverage of large trees; vegetation overhang; and stream bank stability. LiDAR data were captured on 15 July 2007 for two 5 km stretches along Mimosa Creek in Central Queensland, Australia. Field measurements of riparian vegetation structural and landform parameters were obtained between 28 May and 5 June 2007. Object-based approaches were developed for mapping each riparian condition indicator from the LiDAR data. The validation and empirical modelling results showed that the object-based approach could be used to accurately map the riparian condition indicators (R² = 0.99 for streambed width, R² = 0.82 for riparian zone width, R² = 0.89 for PPC, R² = 0.40 for bank stability). These research findings will be used in a 26,000 km mapping project assessing riparian vegetation and physical form indicators from LiDAR data in Victoria, Australia.     

A novel synthesized sulfonamido-based gallic acid – LDQN-C: Effects on chondrocytes growth and phenotype maintenance

Chondrocyte based therapy is promising to treat symptomatic chondral and osteochondral lesions. Growth factors to accelerate the proliferation and retain the phenotype of chondrocytes in vitro are imperative. However, the high cost and rapid degradation of growth factors limited their further application. Therefore, it is significant to find substitutes that can preserve chondrocytes phenotype and ensure sufficient cells for cytotherapy. Antioxidant and anti-inflammatory agents or their derivatives that have effect on arthritis may be an alternative. In this study, we synthesized sulfonamido-based gallate – LDQN-C and investigated its effect on rat articular chondrocytes through examination of the cell proliferation, morphology, viability, glycosaminoglycans (GAGs) synthesis and cartilage specific gene expression. Results showed that LDQN-C could enhance secretion and synthesis of cartilage extracellular matrix (ECM) by up-regulating expression levels of aggrecan, collagen II and Sox9 genes compared to the GA treated group and control group. Expression of collagen type II was effectively up-regulated while collagen I was down-regulated, which demonstrated that the inhibition of chondrocytes dedifferentiation by LDQN-C. Range of 1.36 × 10⁻⁹ M to 1.36 × 10⁻⁷ M is recommended dose of LDQN-C, among which the most profound response was observed with 1.36 × 10⁻⁸ M. GA at concentration of 0.125 μg/mL was compared. This study might provide a basis for the development of a novel agent for the treatment of articular cartilage defect.     

Faunal utilization of constructed intertidal oyster (Crassostrea rivularis) reef in the Yangtze River estuary,China

An intertidal oyster reef (～260 ha) was created by planting hatchery-reared seed oysters (Crassostrea rivularis) on an artificial concrete modular reef in the Deepwater Navigation Channel Regulation Project of the Yangtze River estuary. We examined the development of reef communities (oyster, barnacle and motile epibenthic macrofauna), characterized nekton use and assessed the habitat value of the constructed reef. The C. rivularis oyster population showed a rapid exponential increase with time and reached maximum density (3410 ± 241 ind./m²) and biomass (3175 ± 532 g/m²) after one year of restoration. The barnacle Balanus albicostatus was the most abundant sessile macrofauna and had a significantly greater density in the high intertidal zone than in the low intertidal zone (P < 0.05). The reef also supported diverse motile epibenthic macrofauna (11 mollusks, 11 crustaceans, 4 annelids and 2 fishes), and the reef-associated communities were numerically dominated by Neanthes japonica, Perinereis aibuhitensis, Nerita yoldi and Littorinopsis intermedia. A total of 50 nekton species (31 fishes, 9 shrimps and 10 crabs) utilized the constructed intertidal oyster reef, and grass shrimp Palaemon spp. dominated the nekton communities in term of abundance. Since the constructed intertidal oyster reef supports a variety of reef communities and abundant nektons, it should be recognized as an important and protective fish habitat in the Yangtze River estuary.     

Hepatic stellate cells retain the capacity to synthesize retinyl esters and to store neutral lipids in small lipid droplets in the absence of LRAT

Hepatic stellate cells (HSCs) play an important role in liver physiology and under healthy conditions they have a quiescent and lipid-storing phenotype. Upon liver injury, HSCs are activated and rapidly lose their retinyl ester-containing lipid droplets. To investigate the role of lecithin:retinol acyltransferase (LRAT) and acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) in retinyl ester synthesis and lipid droplet dynamics, we modified LC–MS/MS procedures by including multiple reaction monitoring allowing unambiguous identification and quantification of all major retinyl ester species. Quiescent primary HSCs contain predominantly retinyl palmitate. Exogenous fatty acids are a major determinant in the retinyl ester species synthesized by activated HSCs and LX-2 cells, indicating that HSCs shift their retinyl ester synthesizing capacity from LRAT to DGAT1 during activation. Quiescent LRAT^−/− HSCs retain the capacity to synthesize retinyl esters and to store neutral lipids in lipid droplets ex vivo. The median lipid droplet size in LRAT^−/− HSCs (1080 nm) is significantly smaller than in wild type HSCs (1618 nm). This is a consequence of an altered lipid droplet size distribution with 50.5 ± 9.0% small (≤ 700 nm) lipid droplets in LRAT^−/− HSCs and 25.6 ± 1.4% large (1400–2100 nm) lipid droplets in wild type HSC cells. Upon prolonged (24 h) incubation, the amounts of small (≤ 700 nm) lipid droplets strongly increased both in wild type and in LRAT^−/− HSCs, indicating a dynamic behavior in both cell types. The absence of retinyl esters and reduced number of lipid droplets in LRAT-deficient HSCs in vivo will be discussed.     

Distal skeletal tibia assessed by HR-pQCT is highly correlated with femoral and lumbar vertebra failure loads

Dual energy X-ray absorptiometry (DXA) is the standard for assessing fragility fracture risk using areal bone mineral density (aBMD), but only explains 60–70% of the variation in bone strength. High-resolution peripheral quantitative computed tomography (HR-pQCT) provides 3D-measures of bone microarchitecture and volumetric bone mineral density (vBMD), but only at the wrist and ankle. Finite element (FE) models can estimate bone strength with 86–95% precision. The purpose of this study is to determine how well vBMD and FE bone strength at the wrist and ankle relate to fracture strength at the hip and spine, and to compare these relationships with DXA measured directly at those axial sites. Cadaveric samples (radius, tibia, femur and L4 vertebra) were compared within the same body. The radius and tibia specimens were assessed using HR-pQCT to determine vBMD and FE failure load. aBMD from DXA was measured at the femur and L4 vertebra. The femur and L4 vertebra specimens were biomechanically tested to determine failure load. aBMD measures of the axial skeletal sites strongly correlated with the biomechanical strength for the L4 vertebra (r = 0.77) and proximal femur (r = 0.89). The radius correlated significantly with biomechanical strength of the L4 vertebra for vBMD (r = 0.85) and FE-derived strength (r = 0.72), but not with femur strength. vBMD at the tibia correlated significantly with femoral biomechanical strength (r = 0.74) and FE-estimated strength (r = 0.83), and vertebral biomechanical strength for vBMD (r = 0.97) and FE-estimated strength (r = 0.91). The higher correlations at the tibia compared to radius are likely due to the tibia’s weight-bearing function.     

Browning deficiency and low mobilization of fatty acids in gonadal white adipose tissue leads to decreased cold-tolerance of transglutaminase 2 knock-out mice

During cold-exposure ‘beige’ adipocytes with increased mitochondrial content are activated in white adipose tissue (WAT). These cells, similarly to brown adipose tissue (BAT), dissipate stored chemical energy in the form of heat with the help of uncoupling protein 1 (UCP1). We investigated the effect of tissue transglutaminase (TG2) ablation on the function of ATs in mice. Although TG2^+/+ and TG2^−/− mice had the same amount of WAT and BAT, we found that TG2^+/+ animals could tolerate acute cold exposure for 4 h, whereas TG2^−/− mice only for 3 h. Both TG2^−/− and TG2^+/+ animals used up half of the triacylglycerol content of subcutaneous WAT (SCAT) after 3 h treatment; however, TG2^−/− mice still possessed markedly whiter and higher amount of gonadal WAT (GONAT) as reflected in the larger size of adipocytes and lower free fatty acid levels in serum. Furthermore, lower expression of ‘beige’ marker genes such as UCP1, TBX1 and TNFRFS9 was observed after cold exposure in GONAT of TG2^−/− mice, paralleled with a lower level of UCP1 protein and a decreased mitochondrial content. The detected changes in gene expression of Resistin and Adiponectin did not provoke glucose intolerance in the investigated TG2^−/− mice, and TG2 deletion did not influence adrenaline, noradrenaline, glucagon and insulin production. Our data suggest that TG2 has a tissue-specific role in GONAT function and browning, which becomes apparent under acute cold exposure.