Biomechanics of single chondrocytes under direct shear

Articular chondrocytes experience a variety of mechanical stimuli during daily activity. One such stimulus, direct shear, is known to affect chondrocyte homeostasis and induce catabolic or anabolic pathways. Understanding how single chondrocytes respond biomechanically and morphologically to various levels of applied shear is an important first step toward elucidating tissue level responses and disease etiology. To this end, a novel videocapture method was developed in this study to examine the effect of direct shear on single chondrocytes, applied via the controlled lateral displacement of a shearing probe. Through this approach, precise force and deformation measurements could be obtained during the shear event, as well as clear pictures of the initial cell-to-probe contact configuration. To further study the non-uniform shear characteristics of single chondrocytes, the probe was positioned in three different placement ranges along the cell height. It was observed that the apparent shear modulus of single chondrocytes decreased as the probe transitioned from being close to the cell base (4.1 ± 1.3 kPa), to the middle of the cell (2.6 ± 1.1 kPa), and then near its top (1.7 ± 0.8 kPa). In addition, cells experienced the greatest peak forward displacement (~30% of their initial diameter) when the probe was placed low, near the base. Forward cell movement during shear, regardless of its magnitude, continued until it reached a plateau at ~35% shear strain for all probe positions, suggesting that focal adhesions become activated at this shear level to firmly adhere the cell to its substrate. Based on intracellular staining, the observed height-specific variation in cell shear stiffness and plateau in forward cell movement appeared to be due to a rearrangement of focal adhesions and actin at higher shear strains. Understanding the fundamental mechanisms at play during shear of single cells will help elucidate potential treatments for chondrocyte pathology and loading regimens related to cartilage health and disease.     

Generation of a tumor vaccine candidate based on conjugation of a MUC1 peptide to polyionic papillomavirus virus-like particles

Virus-like particles (VLPs) are promising vaccine technology due to their safety and ability to elicit strong immune responses. Chimeric VLPs can extend this technology to low immunogenicity foreign antigens. However, insertion of foreign epitopes into the sequence of self-assembling proteins can have unpredictable effects on the assembly process. We aimed to generate chimeric bovine papillomavirus (BPV) VLPs displaying a repetitive array of polyanionic docking sites on their surface. These VLPs can serve as platform for covalent coupling of polycationic fusion proteins. We generated baculoviruses expressing chimeric BPV L1 protein with insertion of a polyglutamic-cysteine residue in the BC, DE, HI loops and the H4 helix. Expression in insect cells yielded assembled VLPs only from insertion in HI loop. Insertion in DE loop and H4 helix resulted in partially formed VLPs and capsomeres, respectively. The polyanionic sites on the surface of VLPs and capsomeres were decorated with a polycationic MUC1 peptide containing a polyarginine-cysteine residue fused to 20 amino acids of the MUC1 tandem repeat through electrostatic interactions and redox-induced disulfide bond formation. MUC1-conjugated fully assembled VLPs induced robust activation of bone marrow-derived dendritic cells, which could then present MUC1 antigen to MUC1-specific T cell hybridomas and primary naïve MUC1-specific T cells obtained from a MUC1-specific TCR transgenic mice. Immunization of human MUC1 transgenic mice, where MUC1 is a self-antigen, with the VLP vaccine induced MUC1-specific CTL, delayed the growth of MUC1 transplanted tumors and elicited complete tumor rejection in some animals.     

Chicoric Acid Is an Antioxidant Molecule That Stimulates AMP Kinase Pathway in L6 Myotubes and Extends Lifespan in Caenorhabditis elegans

Chicoric acid (CA) is a caffeoyl derivative previously described as having potential anti-diabetic properties. As similarities in cellular mechanism similarities between diabetes and aging have been shown, we explored on L6 myotubes the effect of CA on the modulation of intracellular pathways involved in diabetes and aging. We also determined its influence on lifespan of Caenorhabditis elegans worm (C. elegans). In L6 myotubes, CA was a potent reactive oxygen species (ROS) scavenger, reducing ROS accumulation under basal as well as oxidative stress conditions. CA also stimulated the AMP-activated kinase (AMPK) pathway and displayed various features associated with AMPK activation: CA (a) enhanced oxidative enzymatic defences through increase in glutathion peroxidase (GPx) and superoxide dismutase (SOD) activities, (b) favoured mitochondria protection against oxidative damage through up-regulation of MnSOD protein expression, (c) increased mitochondrial biogenesis as suggested by increases in complex II and citrate synthase activities, along with up-regulation of PGC-1α mRNA expression and (d) inhibited the insulin/Akt/mTOR pathway. As AMPK stimulators (e.g. the anti-diabetic agent meformin or polyphenols such as epigallocatechingallate or quercetin) were shown to extend lifespan in C. elegans, we also determined the effect of CA on the same model. A concentration-dependant lifespan extension was observed with CA (5–100 μM). These data indicate that CA is a potent antioxidant compound activating the AMPK pathway in L6 myotubes. Similarly to other AMPK stimulators, CA is able to extend C. elegans lifespan, an effect measurable even at the micromolar range. Future studies will explore CA molecular targets and give new insights about its possible effects on metabolic and aging-related diseases.     

Soluble Guanylate Cyclase α1–Deficient Mice: A Novel Murine Model for Primary Open Angle Glaucoma

Primary open angle glaucoma (POAG) is a leading cause of blindness worldwide. The molecular signaling involved in the pathogenesis of POAG remains unknown. Here, we report that mice lacking the α₁ subunit of the nitric oxide receptor soluble guanylate cyclase represent a novel and translatable animal model of POAG, characterized by thinning of the retinal nerve fiber layer and loss of optic nerve axons in the context of an open iridocorneal angle. The optic neuropathy associated with soluble guanylate cyclase α₁–deficiency was accompanied by modestly increased intraocular pressure and retinal vascular dysfunction. Moreover, data from a candidate gene association study suggests that a variant in the locus containing the genes encoding for the α₁ and β₁ subunits of soluble guanylate cyclase is associated with POAG in patients presenting with initial paracentral vision loss, a disease subtype thought to be associated with vascular dysregulation. These findings provide new insights into the pathogenesis and genetics of POAG and suggest new therapeutic strategies for POAG.     

The spatial scaling of beta diversity

Beta diversity is an important concept used to describe turnover in species composition across a wide range of spatial and temporal scales, and it underpins much of conservation theory and practice. Although substantial progress has been made in the mathematical and terminological treatment of different measures of beta diversity, there has been little conceptual synthesis of potential scale dependence of beta diversity with increasing spatial grain and geographic extent of sampling. Here, we evaluate different conceptual approaches to the spatial scaling of beta diversity, interpreted from ‘fixed’ and ‘varying’ perspectives of spatial grain and extent. We argue that a ‘sliding window’ perspective, in which spatial grain and extent covary, is an informative way to conceptualize community differentiation across scales. This concept more realistically reflects the varying empirical approaches that researchers adopt in field sampling and the varying scales of landscape perception by different organisms. Scale dependence in beta diversity has broad implications for emerging fields in ecology and biogeography, such as the integration of fine‐resolution ecogenomic data with large‐scale macroecological studies, as well as for guiding appropriate management responses to threats to biodiversity operating at different spatial scales.     

Assessing conservation attitudes and behaviors of Congolese children neighboring the world's first bonobo (Pan paniscus) release site

Poaching and habitat destruction in the Congo Basin threaten African great apes including the bonobo (Pan paniscus), chimpanzees (Pan troglodytes), and gorillas (Gorilla spp.) with extinction. One way to combat extinction is to reintroduce rescued and rehabilitated apes and repopulate native habitats. Reintroduction programs are only successful if they are supported by local populations. Ekolo ya Bonobo, located in Equateur province of the Democratic Republic of Congo (DRC), is the world's only reintroduction site for rehabilitated bonobos. Here we assess whether children, of the Ilonga‐Pôo, living adjacent to Ekolo ya Bonobo demonstrate more pro‐ape conservation attitudes than children living in, Kinshasa, the capital city. We examined children's attitudes toward great apes because children are typically the focus of conservation education programs. We used the Great Ape Attitude Questionnaire to test the Contact Hypothesis, which posits that proximity to great ape habitat influences pro‐conservation attitudes toward great apes. Ilonga‐Pôo children who live in closer contact with wild bonobos felt greater responsibility to protect great apes compared to those in Kinshasa who live outside the natural habitat of great apes. These results suggest that among participants in the DRC, spatial proximity to a species fosters a greater sense of responsibility to protect and conserve. These results have implications for the successful implementation of great ape reintroduction programs in the Congo Basin. The data analyzed in this study were collected in 2010 and therefore provide a baseline for longitudinal study of this reintroduction site.     

Experimental calcium-oxalate crystal production and dissolution by selected wood-rot fungi

Twenty-six species of white-rotting Agaricomycotina fungi (Basidiomycota) were screened for their ability to produce calcium-oxalate (CaOx) crystals in vitro. Most were able to produce CaOx crystals in malt agar medium in the absence of additional calcium. In the same medium enriched with Ca²⁺, all the species produced CaOx crystals (weddellite or whewellite). Hyphae of four species (Ganoderma lucidum, Polyporus ciliatus, Pycnoporus cinnabarinus, and Trametes versicolor) were found coated with crystals (weddellite/whewellite). The production of CaOx crystals during the growth phase was confirmed by an investigation of the production kinetics for six of the species considered in the initial screening (Pleurotus citrinopileatus, Pleurotus eryngii, Pleurotus ostreatus, P. cinnabarinus, Trametes suaveolens, and T. versicolor). However, the crystals produced during the growth phase disappeared from the medium over time in four of the six species (P. citrinopileatus, P. eryngii, P. cinnabarinus, and T. suaveolens). For P. cinnabarinus, the disappearance of the crystals was correlated with a decrease in the total oxalate concentration measured in the medium from 0.65 ??g mm⁻² (at the maximum accumulation rate) to 0.30 ??g mm⁻². The decrease in the CaOx concentration was correlated with a change in mycelia morphology. The oxalate dissolution capability of all the species was also tested in a medium containing calcium oxalate as the sole source of carbon (modified Schlegel medium). Three species (Agaricus blazei, Pleurotus tuberregium, and P. ciliatus) presented a dissolution halo around the growth zone. This study shows that CaOx crystal production is a widespread phenomenon in white-rot fungi, and that an excess of Ca²⁺ can enhance CaOx crystal production. In addition, it shows that some white-rot fungal species are capable of dissolving CaOx crystals after growth has ceased. These results highlight a diversity of responses around the production or dissolution of calcium oxalate in white-rot fungi and reveal an unexpected potential importance of fungi on the oxalate cycle in the environment.     

Fully Automated Enhanced Tumor Compartmentalization: Man vs. Machine Reloaded

ObjectiveComparison of a fully-automated segmentation method that uses compartmental volume information to a semi-automatic user-guided and FDA-approved segmentation technique.MethodsNineteen patients with a recently diagnosed and histologically confirmed glioblastoma (GBM) were included and MR images were acquired with a 1.5 T MR scanner. Manual segmentation for volumetric analyses was performed using the open source software 3D Slicer version 4.2.2.3 (www.slicer.org). Semi-automatic segmentation was done by four independent neurosurgeons and neuroradiologists using the computer-assisted segmentation tool SmartBrush® (referred to as SB), a semi-automatic user-guided and FDA-approved tumor-outlining program that uses contour expansion. Fully automatic segmentations were performed with the Brain Tumor Image Analysis (BraTumIA, referred to as BT) software. We compared manual (ground truth, referred to as GT), computer-assisted (SB) and fully-automated (BT) segmentations with regard to: (1) products of two maximum diameters for 2D measurements, (2) the Dice coefficient, (3) the positive predictive value, (4) the sensitivity and (5) the volume error.ResultsSegmentations by the four expert raters resulted in a mean Dice coefficient between 0.72 and 0.77 using SB. BT achieved a mean Dice coefficient of 0.68. Significant differences were found for intermodal (BT vs. SB) and for intramodal (four SB expert raters) performances. The BT and SB segmentations of the contrast-enhancing volumes achieved a high correlation with the GT. Pearson correlation was 0.8 for BT; however, there were a few discrepancies between raters (BT and SB 1 only). Additional non-enhancing tumor tissue extending the SB volumes was found with BT in 16/19 cases. The clinically motivated sum of products of diameters measure (SPD) revealed neither significant intermodal nor intramodal variations. The analysis time for the four expert raters was faster (1 minute and 47 seconds to 3 minutes and 39 seconds) than with BT (5 minutes).ConclusionBT and SB provide comparable segmentation results in a clinical setting. SB provided similar SPD measures to BT and GT, but differed in the volume analysis in one of the four clinical raters. A major strength of BT may its independence from human interactions, it can thus be employed to handle large datasets and to associate tumor volumes with clinical and/or molecular datasets ("-omics") as well as for clinical analyses of brain tumor compartment volumes as baseline outcome parameters. Due to its multi-compartment segmentation it may provide information about GBM subcompartment compositions that may be subjected to clinical studies to investigate the delineation of the target volumes for adjuvant therapies in the future.     

Bat species diversity and distribution in a disturbed regime at the Lake Bogoria National Reserve,Kenya

Human population growth drives intrusion and progressive conversion of natural habitats for agriculture. We evaluated human impacts on bat species diversity and distribution among four vegetation types in and around Lake Bogoria National Reserve between November 2012 and July 2013. Plants were surveyed using the Braun–Blanquet cover/abundance method, whereas bats were sampled using standard mist nets erected on poles at ground level. Floristic similarity analysis revealed three broad vegetation assemblages, namely riverine vegetation, farmland and Acacia woodland/Acacia–Commiphora woodland. Two hundred and 33 bats representing eleven species in eleven genera and seven families were recorded. These were Epomophorus minimus, Rhinolophus landeri, Hipposideros caffer, Cardioderma cor, Lavia frons, Nycteris hispida, Chaerephon pumilus, Mops condylurus, Neoromicia capensis, Scotoecus hirundo and Scotophilus dinganii. Species richness estimators indicated that sampling for bats at ground level was exhaustive. Bat species richness and diversity were highest in the more structurally complex Acacia woodland compared to more homogenous farmlands where we recorded only common and generalist species that often occur in open habitats. The higher bat species richness and diversity in the Acacia woodland as compared to farmland underscore the importance of remnant natural savannah woodlands in the conservation of bats and other elements of biodiversity .