Extracellular Vesicles Secreted from Cancer Cell Lines Stimulate Secretion of MMP-9, IL-6, TGF-β1 and EMMPRIN

Extracellular vesicles (EVs) are key contributors to cancer where they play an integral role in cell-cell communication and transfer pro-oncogenic molecules to recipient cells thereby conferring a cancerous phenotype. Here, we purified EVs using straightforward biochemical approaches from multiple cancer cell lines and subsequently characterized these EVs via multiple biochemical and biophysical methods. In addition, we used fluorescence microscopy to directly show internalization of EVs into the recipient cells within a few minutes upon addition of EVs to recipient cells. We confirmed that the transmembrane protein EMMPRIN, postulated to be a marker of EVs, was indeed secreted from all cell lines studied here. We evaluated the response to EV stimulation in several different types of recipient cells lines and measured the ability of these purified EVs to induce secretion of several factors highly upregulated in human cancers. Our data indicate that purified EVs preferentially stimulate secretion of several proteins implicated in driving cancer in monocytic cells but only harbor limited activity in epithelial cells. Specifically, we show that EVs are potent stimulators of MMP-9, IL-6, TGF-β1 and induce the secretion of extracellular EMMPRIN, which all play a role in driving immune evasion, invasion and inflammation in the tumor microenvironment. Thus, by using a comprehensive approach that includes biochemical, biological, and spectroscopic methods, we have begun to elucidate the stimulatory roles.     

Streptococcus pneumoniae G5 domains bind different ligands

Many bacterial pathogens express small G5 domains that exist in the context of various membrane‐anchored proteins and these G5 domains have been associated with colonization, cellular adhesion, and biofilm formation. However, despite over a decade since the computational prediction of these G5 domains, many remain uncharacterized, particularly those from Streptococcus pneumoniae. Of five previously predicted G5 domains we found that four of these, all derived from S. pneumoniae, are independently folded modules. As one of these exhibits extreme line broadening due to self‐association, we were able to use NMR solution studies to probe the potential ligand interactions of the remaining three G5 domains. None of these G5 domains engage N‐acetylglucosamine (NAG) as previously predicted but do interact with other small molecules that may modulate adherence to both bacteria and host cells. Specifically, while all G5 domains tested engage Zn, only one of these G5 domains engage heparin. NMR solution structural studies of the IgA1 Protease G5 (IgA1P‐G5) and endo‐beta‐N‐acetylglucosaminidase‐D G5 (ENDD‐G5) also facilitated identification of the ligand binding sites and confirm the typical G5 fold that comprises two connected β‐sheets with no canonical core. NMR relaxation experiments indicate flexibility on both ends and within the connecting regions between the β‐sheets. Our studies thus establish a basis for future biological experiments to test whether the ligands presented here are involved in bacterial adherence, either to bacteria or to host cells.     

Characterizing and controlling the inherent dynamics of cyclophilin-A

With the recent advances in NMR relaxation techniques, protein motions on functionally important timescales can be studied at atomic resolution. Here, we have used NMR-based relaxation experiments at several temperatures and both 600 and 900 MHz to characterize the inherent dynamics of the enzyme cyclophilin-A (CypA). We have discovered multiple chemical exchange processes within the enzyme that form a “dynamic continuum” that spans 20–30 Å comprising active site residues and residues proximal to the active site. By combining mutagenesis with these NMR relaxation techniques, a simple method of counting the dynamically sampled conformations has been developed. Surprisingly, a combination of point mutations has allowed for the specific regulation of many of the exchange processes that occur within CypA, suggesting that the dynamics of an enzyme may be engineered.     

Influence of decorin and biglycan on mechanical properties of multiple tendons in knockout mice

Evaluations of tendon mechanical behavior based on biochemical and structural arrangement have implications for designing tendon specific treatment modalities or replacement strategies. In addition to the well studied type I collagen, other important constituents of tendon are the small proteoglycans (PGs). PGs have been shown to vary in concentration within differently loaded areas of tendon, implicating them in specific tendon function. This study measured the mechanical properties of multiple tendon tissues from normal mice and from mice with knock-outs of the PGs decorin or biglycan. Tail tendon fascicles, patellar tendons (PT), and flexor digitorum longus tendons (FDL), three tissues representing different in vivo loading environments, were characterized from the three groups of mice. It was hypothesized that the absence of decorin or biglycan would have individual effects on each type of tendon tissue. Surprisingly, no change in mechanical properties was observed for the tail tendon fascicles due to the PG knockouts. The loss of decorin affected the PT causing an increase in modulus and stress relaxation, but had little effect on the FDL. Conversely, the loss of biglycan did not significantly affect the PT, but caused a reduction in both the maximum stress and modulus of the FDL. These results give mechanical support to previous biochemical data that tendons likely are uniquely tailored to their specific location and function. Variances such as those presented here need to be further characterized and taken into account when designing therapies or replacements for any one particular tendon.     

Inherent dynamics within the Crimean-Congo Hemorrhagic fever virus protease are localized to the same region as substrate interactions

Crimean-Congo Hemorrhagic fever virus (CCHFV) is one of several lethal viruses that encodes for a viral ovarian tumor domain (vOTU), which serves to cleave and remove ubiquitin (Ub) and interferon stimulated gene product 15 (ISG15) from numerous proteins involved in cellular signaling. Such manipulation of the host cell machinery serves to downregulate the host response and, therefore, complete characterization of these proteases is important. While several structures of the CCHFV vOTU protease have been solved, both free and bound to Ub and ISG15, few structural differences have been found and little insight has been gained as to the structural plasticity of this protease. Therefore, we have used NMR relaxation experiments to probe the dynamics of CCHFV vOTU, both alone and in complex with Ub, discovering a highly dynamic protease that exhibits conformational exchange within the same regions found to engage its Ub substrate. These experiments reveal a structural plasticity around the N-terminal regions of CCHFV vOTU, which are unique to vOTUs, and provide a rationale for engaging multiple substrates with the same binding site.     

Changes in Socioeconomic Inequality in Indonesian Children’s Cognitive Function from 2000 to 2007: A Decomposition Analysis

Background

Measuring social inequalities in health is common; however, research examining inequalities in child cognitive function is more limited. We investigated household expenditure-related inequality in children’s cognitive function in Indonesia in 2000 and 2007, the contributors to inequality in both time periods, and changes in the contributors to cognitive function inequalities between the periods.

Methods

Data from the 2000 and 2007 round of the Indonesian Family Life Survey (IFLS) were used. Study participants were children aged 7–14 years (n = 6179 and n = 6680 in 2000 and 2007, respectively). The relative concentration index (RCI) was used to measure the magnitude of inequality. Contribution of various contributors to inequality was estimated by decomposing the concentration index in 2000 and 2007. Oaxaca-type decomposition was used to estimate changes in contributors to inequality between 2000 and 2007.

Results

Expenditure inequality decreased by 45% from an RCI = 0.29 (95% CI 0.22 to 0.36) in 2000 to 0.16 (95% CI 0.13 to 0.20) in 2007 but the burden of poorer cognitive function was higher among the disadvantaged in both years. The largest contributors to inequality in child cognitive function were inequalities in per capita expenditure, use of improved sanitation and maternal high school attendance. Changes in maternal high school participation (27%), use of improved sanitation (25%) and per capita expenditures (18%) were largely responsible for the decreasing inequality in children’s cognitive function between 2000 and 2007.

Conclusions

Government policy to increase basic education coverage for women along with economic growth may have influenced gains in children’s cognitive function and reductions in inequalities in Indonesia.     

Structure and dynamics of pin1 during catalysis by NMR

The link between internal enzyme motions and catalysis is poorly understood. Correlated motions in the microsecond-to-millisecond timescale may be critical for enzyme function. We have characterized the backbone dynamics of the peptidylprolyl isomerase (Pin1) catalytic domain in the free state and during catalysis. Pin1 is a prolyl isomerase of the parvulin family and specifically catalyzes the isomerization of phosphorylated Ser/Thr-Pro peptide bonds. Pin1 has been shown to be essential for cell-cycle progression and to interact with the neuronal tau protein inhibiting its aggregation into fibrillar tangles as found in Alzheimer's disease. (15)N relaxation dispersion measurements performed on Pin1 during catalysis reveal conformational exchange processes in the microsecond timescale. A subset of active site residues undergo kinetically similar exchange processes even in the absence of a substrate, suggesting that this area is already "primed" for catalysis. Furthermore, structural data of the turning-over enzyme were obtained through inter- and intramolecular nuclear Overhauser enhancements. This analysis together with a characterization of the substrate concentration dependence of the conformational exchange allowed the distinguishing of regions of the enzyme active site that are affected primarily by substrate binding versus substrate isomerization. Together these data suggest a model for the reaction trajectory of Pin1 catalysis.     

Multilocus evidence for globally distributed cryptic species and distinct populations across ocean gyres in a mesopelagic copepod

Zooplanktonic taxa have a greater number of distinct populations and species than might be predicted based on their large population sizes and open‐ocean habitat, which lacks obvious physical barriers to dispersal and gene flow. To gain insight into the evolutionary mechanisms driving genetic diversification in zooplankton, we developed eight microsatellite markers to examine the population structure of an abundant, globally distributed mesopelagic copepod, Haloptilus longicornis, at 18 sample sites across the Atlantic and Pacific Oceans (n = 761). When comparing our microsatellite results with those of a prior study that used a mtDNA marker (mtCOII, n = 1059, 43 sample sites), we unexpectedly found evidence for the presence of a cryptic species pair. These species were globally distributed and apparently sympatric, and were separated by relatively weak genetic divergence (reciprocally monophyletic mtCOII lineages 1.6% divergent; microsatellite F_ST ranging from 0.28 to 0.88 across loci, P < 0.00001). Using both mtDNA and microsatellite data for the most common of the two species (n = 669 for microsatellites, n = 572 for mtDNA), we also found evidence for allopatric barriers to gene flow within species, with distinct populations separated by continental landmasses and equatorial waters in both the Atlantic and Pacific Ocean basins. Our study shows that oceanic barriers to gene flow can act as a mechanism promoting allopatric diversification in holoplanktonic taxa, despite the high potential dispersal abilities and pelagic habitat for these species.