Proteolytic activities of human ADAMTS-5: comparative studies with ADAMTS-4

Aggrecanases have been characterized as proteinases that cleave the Glu373-Ala374 bond of the aggrecan core protein, and they are multidomain metalloproteinases belonging to the ADAMTS (adamalysin with thrombospondin type 1 motifs) family. The first aggrecanases discovered were ADAMTS-4 (aggrecanase 1) and ADAMTS-5 (aggrecanase 2). They contain a zinc catalytic domain followed by non-catalytic ancillary domains, including a disintegrin domain, a thrombospondin domain, a cysteine-rich domain, and a spacer domain. In the case of ADAMTS-5, a second thrombospondin domain follows the spacer domain. We previously reported that the non-catalytic domains of ADAMTS-4 influence both its extracellular matrix interaction and proteolytic abilities. Here we report the effects of these domains of ADAMTS-5 on the extracellular matrix interaction and proteolytic activities and compare them with those of ADAMTS-4. Although the spacer domain was critical for ADAMTS-4 localization in the matrix, the cysteine-rich domain influenced ADAMTS-5 localization. Similar to previous reports of other ADAMTS family members, very little proteolytic activity was detected with the ADAMTS-5 catalytic domain alone. The sequential inclusion of each carboxyl-terminal domain enhanced its activity against aggrecan, carboxymethylated transferrin, fibromodulin, decorin, biglycan, and fibronectin. Both ADAMTS-4 and -5 had a broad optimal activity at pH 7.0-9.5. Aggrecanolytic activities were sensitive to the NaCl concentration, but activities on non-aggrecan substrates, e.g. carboxymethylated transferrin, were not affected. Although ADAMTS-4 and ADAMTS-5 had similar general proteolytic activities, the aggrecanase activity of ADAMTS-5 was at least 1,000-fold greater than that of ADAMTS-4 under physiological conditions. Our studies suggest that ADAMTS-5 is a major aggrecanase in cartilage metabolism and pathology.     

Biocidal activity of three wood essential oils against Ixodes scapularis (Acari: Ixodidae), Xenopsylla cheopis (Siphonaptera: Pulicidae), and Aedes aegypti (Diptera: Culicidae)

The biocidal activity of three steam distilled wood essential oils-incense cedar, Calocedrus decurrens (Torr.) Florin; Port-Orford-cedar, Chamaecyparis lawsoniana (A. Murr.) Parl.; and western juniper, Juniperus occidentalis (Hook)--were evaluated against adult Aedes aegypti (L.) (Diptera: Culicidae) and Xenopsylla cheopis (Rothchild) (Siphonaptera: Pulicidae) and nymphal Ixodes scapularis Say (Acari: Ixodidae). In vitro laboratory bioassays were conducted to establish baseline dose-mortality data through 24 h. Incense cedar heartwood was the most toxic to all three vector species followed in order of activity by western juniper and Port-Orford-cedar based on LC50 and LC90 values. Ae. aegypti were substantially more susceptible to the oils than either I. scapularis or X. cheopis.     

Differentiating megakaryocytes in myelodysplastic syndromes succumb to mitochondrial derangement without caspase activation

Myelodysplastic syndromes (MDS) constitute a preneoplastic condition in which potentially malignant cancer stem cells continuously die during differentiation. This MDS-associated cell death often involves caspase-3 activation, yet can also occur without caspase activation, for instance in differentiating megakaryocytes (MK). We investigated, the mechanisms through which MK from MDS patients undergo premature cell death. While polyploid, mature MK from healthy subjects or MDS patients manifested caspase-3 activation during terminal differentiation, freshly isolated, immature MK from MDS died without caspase-3 activation. Similarly, purified bone marrow CD34⁺ cells from MDS patients that were driven into MK differentiation in vitro died without caspase-3 activation at an immature stage, before polyploidization. The premature death of MDS MK was accompanied by the mitochondrial release of cytochrome c, Smac/DIABLO and endonuclease G, a caspase-independent death effector, as well loss of the mitochondrial membrane potential and plasma membrane phosphatidylserine exposure before definitive loss of viability. Thus, a stereotyped pattern of mitochondrial alterations accompanies differentiation-associated MK death in MDS. T. Braun and G. Carvalho contributed equally to this paper.     

Multicellular redox regulation in an early-evolving animal treated with glutathione

Redox signaling has emerged as a unifying theme in many seemingly disparate disciplines. Such signaling has been widely studied in bacteria and eukaryotic organelles and is often mediated by reactive oxygen species (ROS). In this context, reduced glutathione (GSH) acts as an important intracellular antioxidant, diminishing ROS and potentially affecting redox signaling. Complementing this cell-level perspective, colonial hydroids can be a useful model for understanding organism-level redox signaling. These simple, early-evolving animals consist of feeding polyps connected by tubelike stolons. Colonies treated exogenously with GSH or reduced glutathione ethyl ester (GEE) were expected to show a morphological change to sheetlike growth typical of low levels of ROS. Contrary to expectations, diminished stolon branching and polyp initiation was observed. Such runnerlike growth is associated with higher levels of ROS, and surprisingly, such higher levels were found in GSH- and GEE-treated colonies. Further investigations show that GSH triggered a feeding response in hydroid polyps, increasing oxygen uptake but at the same time relaxing mitochondrion-rich contractile regions at the base of polyps. Diminished gastrovascular flow and increased emissions of mitochondrial ROS also correlated with the observed runnerlike growth. In contrast to cell-level, "bottom-up" views of redox signaling, here the phenotype may arise from a "top-down" interaction of mitochondrion-rich regions and organism-level physiology. Such multicellular redox regulation may commonly occur in other animals as well.     

Hierarchical Bayesian integrated model for estimating migratory bird harvest in Canada

The Canadian Wildlife Service (CWS) requires reliable estimates of the harvest of migratory game birds, including waterfowl, to effectively manage populations of these hunted species. The National Harvest Survey is an annual survey of hunters who purchase Canada's mandatory migratory game bird hunting permit, integrating information from a survey of hunting activity with information from a separate survey of species composition in the harvest. We used these survey data to estimate the number of birds harvested for each species and hunting activity metrics (e.g., number of active hunters, days spent hunting). The analytical methods used to generate these estimates have not changed since the survey was first designed in the early 1970s. We describe a new hierarchical Bayesian integrated model, which replaces the series of ratio estimators that comprised the old model. We are using this new model to generate estimates for migratory bird harvests as of the 2019–2020 hunting season, and to generate updated estimates for all earlier years. The hierarchical Bayesian model uses over-dispersed Poisson distributions to model mean hunter activity and harvest (zero-inflated Poisson and zero-truncated Poisson, respectively). It also includes multinomial distributions to model some key components (e.g., variation in harvest across periods of the hunting season, the species composition of the harvest within each of those periods, the age and sex composition in the harvests of a given species). We estimated the parameters of the Poisson and the multinomial distributions for each year as random effects using first-difference time-series. This time-series component allows the model to share information across years and reduces the sensitivity of the estimates to annual sampling noise. The new model estimates are generally very similar to those from the old model, particularly for the species that occur most commonly in the harvest, so the results do not suggest any major changes to harvest management decisions and regulations. Estimates for all species from the new model are more precise and less susceptible to annual sampling error, particularly for species that occur less commonly in the harvest (e.g., sea ducks, other species of conservation concern). This new model, with its hierarchical Bayesian framework, will also facilitate future improvements and elaborations, allowing the incorporation of prior information from the rich literature and knowledge in game bird management and biology.     

The Impact of Collagen Fibril Polarity on Second Harmonic Generation Microscopy

In this work, we report the implementation of interferometric second harmonic generation (SHG) microscopy with femtosecond pulses. As a proof of concept, we imaged the phase distribution of SHG signal from the complex collagen architecture of juvenile equine growth cartilage. The results are analyzed in respect to numerical simulations to extract the relative orientation of collagen fibrils within the tissue. Our results reveal large domains of constant phase together with regions of quasi-random phase, which are correlated to respectively high- and low-intensity regions in the standard SHG images. A comparison with polarization-resolved SHG highlights the crucial role of relative fibril polarity in determining the SHG signal intensity. Indeed, it appears that even a well-organized noncentrosymmetric structure emits low SHG signal intensity if it has no predominant local polarity. This work illustrates how the complex architecture of noncentrosymmetric scatterers at the nanoscale governs the coherent building of SHG signal within the focal volume and is a key advance toward a complete understanding of the structural origin of SHG signals from tissues.