Engineering osteoarthritic cartilage model through differentiating senescent human mesenchymal stem cells for testing disease-modifying drugs

Science China Life Sciences - Significant cellular senescence has been observed in cartilage harvested from patients with osteoarthritis (OA). In this study, we aim to develop a senescence-relevant...     

Adaptation to temperature stress by Vibrio fischeri facilitates this microbe's symbiosis with the Hawaiian bobtail squid (Euprymna scolopes)

For microorganisms cycling between free‐living and host‐associated stages, where reproduction occurs in both of these lifestyles, an interesting inquiry is whether adaptation to stress during the free‐living stage can impact microbial fitness in the host. To address this topic, the mutualism between the Hawaiian bobtail squid (Euprymna scolopes) and the marine bioluminescent bacterium Vibrio fischeri was utilized. Using microbial experimental evolution, V. fischeri was selected to low (8°C), high (34°C), and fluctuating temperature stress (8°C/34°C) for 2000 generations. The temperatures 8°C and 34°C were the lower and upper growth limits, respectively. V. fischeri was also selected to benign temperatures (21°C and 28°C) for 2000 generations, which served as controls. V. fischeri demonstrated significant adaptation to low, high, and fluctuating temperature stress. V. fischeri did not display significant adaptation to the benign temperatures. Adaptation to stressful temperatures facilitated V. fischeri’s ability to colonize the squid host relative to the ancestral lines. Bioluminescence levels also increased. Evolution to benign temperatures did not manifest these results. In summary, microbial adaptation to stress during the free‐living stage can promote coevolution between hosts and microorganisms.     

Phosphate Solubility and the Cyanate-Mediated Synthesis of Pyrophosphate

The justification for a less alkaline primordial ocean (than present) is briefly reviewed, along with constraints on aqueous phosphate under such conditions. Based on the assumption that CaHPO₄ dihydrate determined the availability of phosphorus species, we have carried out laboratory simulations to determine equilibrium concentrations as a function of pH (in PIPES buffer) with added NaCl and CaCl₂. Consistent with expectations, solubility declines with higher pH and [CaCl₂], but increases only slightly with [NaCl]. Significantly, PIPES shows no specific effect on the dissolution beyond its influence on pH and ionic strength. Data are also presented on the synthesis of pyrophosphate from the NaOCN/CaHPO₄·2H₂O system, which could have provided a source of this phosphate anhydride on the early Earth.     

Mitochondrial DNA Variation Dictates Expressivity and Progression of Nuclear DNA Mutations Causing Cardiomyopathy

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FHL1 Reduces Dystrophy in Transgenic Mice Overexpressing FSHD Muscular Dystrophy Region Gene 1 (FRG1)

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal-dominant disease with no effective treatment. The genetic cause of FSHD is complex and the primary pathogenic insult underlying the muscle disease is unknown. Several disease candidate genes have been proposed including DUX4 and FRG1. Expression analysis studies of FSHD report the deregulation of genes which mediate myoblast differentiation and fusion. Transgenic mice overexpressing FRG1 recapitulate the FSHD muscular dystrophy phenotype. Our current study selectively examines how increased expression of FRG1 may contribute to myoblast differentiation defects. We generated stable C2C12 cell lines overexpressing FRG1, which exhibited a myoblast fusion defect upon differentiation. To determine if myoblast fusion defects contribute to the FRG1 mouse dystrophic phenotype, this strain was crossed with skeletal muscle specific FHL1-transgenic mice. We previously reported that FHL1 promotes myoblast fusion in vitro and FHL1-transgenic mice develop skeletal muscle hypertrophy. In the current study, FRG1 mice overexpressing FHL1 showed an improvement in the dystrophic phenotype, including a reduced spinal kyphosis, increased muscle mass and myofiber size, and decreased muscle fibrosis. FHL1 expression in FRG1 mice, did not alter satellite cell number or activation, but enhanced myoblast fusion. Primary myoblasts isolated from FRG1 mice showed a myoblast fusion defect that was rescued by FHL1 expression. Therefore, increased FRG1 expression may contribute to a muscular dystrophy phenotype resembling FSHD by impairing myoblast fusion, a defect that can be rescued by enhanced myoblast fusion via expression of FHL1.     

A Pulse Coupled Neural Network Segmentation Algorithm for Reflectance Confocal Images of Epithelial Tissue

Automatic segmentation of nuclei in reflectance confocal microscopy images is critical for visualization and rapid quantification of nuclear-to-cytoplasmic ratio, a useful indicator of epithelial precancer. Reflectance confocal microscopy can provide three-dimensional imaging of epithelial tissue in vivo with sub-cellular resolution. Changes in nuclear density or nuclear-to-cytoplasmic ratio as a function of depth obtained from confocal images can be used to determine the presence or stage of epithelial cancers. However, low nuclear to background contrast, low resolution at greater imaging depths, and significant variation in reflectance signal of nuclei complicate segmentation required for quantification of nuclear-to-cytoplasmic ratio. Here, we present an automated segmentation method to segment nuclei in reflectance confocal images using a pulse coupled neural network algorithm, specifically a spiking cortical model, and an artificial neural network classifier. The segmentation algorithm was applied to an image model of nuclei with varying nuclear to background contrast. Greater than 90% of simulated nuclei were detected for contrast of 2.0 or greater. Confocal images of porcine and human oral mucosa were used to evaluate application to epithelial tissue. Segmentation accuracy was assessed using manual segmentation of nuclei as the gold standard.     

Neuronal expression of peripherin,a type III intermediate filament protein,in the mouse hindbrain

Peripherin is a 57 kDa Type III intermediate filament protein associated with neurite extension, neuropathies such as amyotrophic lateral sclerosis, and cranial nerve and dorsal root projections. However, knowledge of peripherin expression in the CNS is limited. We have used immunoperoxidase histochemistry to characterise peripherin expression in the mouse hindbrain, including the inferior colliculus, pons, medulla and cerebellum. Peripherin immunolabelling was observed in the nerve fibres and nuclei that are associated with all cranial nerves [(CN) V–XII] in the hindbrain. Peripherin expression was prominent in the cell bodies and axons of the mesenchephalic trigeminal nucleus and the pars compacta region of nucleus ambiguus, and in the fibres that comprise the solitary tract, the descending spinal trigeminal tract and the trigeminal and facial nerves. A small proportion of peripherin positive fibres in CN VIII likely arise from cochlear type II spiral ganglion neurons. Peripherin positive fibres were also observed in the inferior cerebellar peduncle and folia in the intermediate zone of the cerebellum. Antibody specificity was confirmed by absence of labelling in hindbrain tissue from peripherin knockout mice. This study shows that in the adult mouse hindbrain, peripherin is expressed in discrete neuronal subpopulations that have sensory, motor and autonomic functions.     

The murine gammaherpesvirus 68 M2 gene is required for efficient reactivation from latently infected B cells

Murine gammaherpesvirus 68 (gammaHV68) infection of mice provides a tractable small-animal model system for assessing the requirements for the establishment and maintenance of gammaherpesvirus latency within the lymphoid compartment. The M2 gene product of gammaHV68 is a latency-associated antigen with no discernible homology to any known proteins. Here we focus on the requirement for the M2 gene in splenic B-cell latency. Our analyses showed the following. (i) Low-dose (100 PFU) inoculation administered via the intranasal route resulted in a failure to establish splenic B-cell latency at day 16 postinfection. (ii) Increasing the inoculation dose to 4 x 10(5) PFU administered via the intranasal route partially restored the establishment of B-cell latency at day 16, but no virus reactivation was detected upon explant into tissue cultures. (iii) Although previous data failed to detect a phenotype of the M2 mutant upon high-dose intraperitoneal inoculation, decreasing the inoculation dose to 100 PFU administered intraperitoneally revealed a splenic B-cell latency phenotype at day 16 that was very similar to the phenotype observed upon high-dose intranasal inoculation. (iv) After low-dose intraperitoneal inoculation, fractionated B-cell populations showed that the M2 mutant virus was able to establish latency in surface immunoglobulin D-negative (sIgD(-)) B cells; by 6 months postinfection, equivalent frequencies of M2 mutant and marker rescue viral genome-positive sIgD(-) B cells were detected. (v) Like the marker rescue virus, the M2 mutant virus also established latency in splenic naive B cells upon low-dose intraperitoneal inoculation, but there was a significant lag in the decay of this latently infected reservoir compared to that seen with the marker rescue virus. (vi) After low-dose intranasal inoculation, by day 42 postinfection, latency was observed in the spleen, although at a frequency significantly lower than that in the marker rescue virus-infected mice; by 3 months postinfection, nearly equivalent levels of viral genome-positive cells were observed in the spleens of marker rescue virus- and M2 mutant virus-infected mice, and these cells were exclusively sIgD(-) B cells. Taken together, these data convincingly demonstrate a role for the M2 gene product in reactivation from splenic B cells and also suggest that disruption of the M2 gene leads to dose- and route-specific defects in the efficient establishment of splenic B-cell latency.