Elasticity in Ionically Cross-Linked Neurofilament Networks

Neurofilaments are found in abundance in the cytoskeleton of neurons, where they act as an intracellular framework protecting the neuron from external stresses. To elucidate the nature of the mechanical properties that provide this protection, we measure the linear and nonlinear viscoelastic properties of networks of neurofilaments. These networks are soft solids that exhibit dramatic strain stiffening above critical strains of 30-70%. Surprisingly, divalent ions such as Mg²⁺, Ca²⁺, and Zn²⁺ act as effective cross-linkers for neurofilament networks, controlling their solidlike elastic response. This behavior is comparable to that of actin-binding proteins in reconstituted filamentous actin. We show that the elasticity of neurofilament networks is entropic in origin and is consistent with a model for cross-linked semiflexible networks, which we use to quantify the cross-linking by divalent ions.     

Building reactive copper centers in human carbonic anhydrase II

Reengineering metalloproteins to generate new biologically relevant metal centers is an effective a way to test our understanding of the structural and mechanistic features that steer chemical transformations in biological systems. Here, we report thermodynamic data characterizing the formation of two type-2 copper sites in carbonic anhydrase and experimental evidence showing one of these new, copper centers has characteristics similar to a variety of well-characterized copper centers in synthetic models and enzymatic systems. Human carbonic anhydrase II is known to bind two Cu²⁺ ions; these binding events were explored using modern isothermal titration calorimetry techniques that have become a proven method to accurately measure metal-binding thermodynamic parameters. The two Cu²⁺-binding events have different affinities (K _a approximately 5 × 10¹² and 1 × 10¹⁰), and both are enthalpically driven processes. Reconstituting these Cu²⁺ sites under a range of conditions has allowed us to assign the Cu²⁺-binding event to the three-histidine, native, metal-binding site. Our initial efforts to characterize these Cu²⁺ sites have yielded data that show distinctive (and noncoupled) EPR signals associated with each copper-binding site and that this reconstituted enzyme can activate hydrogen peroxide to catalyze the oxidation of 2-aminophenol.     

Chemotherapeutic treatment of xenograft Spirocerca lupi-associated sarcoma in a murine model

To date, data are not available concerning the effectiveness of chemotherapy in the treatment of Spirocerca lupi-associated esophageal sarcomas. In the present study, we compared the effectiveness of 4 chemotherapeutic agents against S. lupi-associated osteosarcoma, using a xenograft murine model created in our lab. Samples of xenografted osteosarcoma were inoculated subcutaneously into 5 groups (n = 10 each) of 6-wk-old male and female NOD/SCID mice. Tumor-bearing mice were divided into treatment and control groups. The treatment groups were injected with either pegylated liposomal doxorubicin (6 mg/kg, intravenously, n = 9), doxorubicin (6 mg/kg, intravenously, n = 8), carboplatin (60 mg/kg, intraperitoneally, repeated twice at 1-wk intervals for a total of 2 doses, n = 9), or cisplatin (6 mg/kg, intraperitoneally, n = 8). The control group was injected with buffered saline (n = 9). Tumor size was determined by caliper measurements. Compared with the control group, the pegylated liposomal doxorubicin- and doxorubicin-treated groups, but not the carboplatin or cisplatin groups, showed significant inhibition of tumor growth. Our results indicate that doxorubicin-based drugs are effective against S. lupi-associated sarcomas in a mouse xenograft model. Because it is less toxic than doxorubicin, pegylated liposomal doxorubicin is likely the drug of choice for treatment of S. lupi-associated sarcomas. We suggest that combination of doxorubicin or its pegylated form with surgical excision will improve the prognosis of dogs with this disease.     

A multitrophic model to quantify the effects of marine viruses on microbial food webs and ecosystem processes

Viral lysis of microbial hosts releases organic matter that can then be assimilated by nontargeted microorganisms. Quantitative estimates of virus-mediated recycling of carbon in marine waters, first established in the late 1990s, were originally extrapolated from marine host and virus densities, host carbon content and inferred viral lysis rates. Yet, these estimates did not explicitly incorporate the cascade of complex feedbacks associated with virus-mediated lysis. To evaluate the role of viruses in shaping community structure and ecosystem functioning, we extend dynamic multitrophic ecosystem models to include a virus component, specifically parameterized for processes taking place in the ocean euphotic zone. Crucially, we are able to solve this model analytically, facilitating evaluation of model behavior under many alternative parameterizations. Analyses reveal that the addition of a virus component promotes the emergence of complex communities. In addition, biomass partitioning of the emergent multitrophic community is consistent with well-established empirical norms in the surface oceans. At steady state, ecosystem fluxes can be probed to characterize the effects that viruses have when compared with putative marine surface ecosystems without viruses. The model suggests that ecosystems with viruses will have (1) increased organic matter recycling, (2) reduced transfer to higher trophic levels and (3) increased net primary productivity. These model findings support hypotheses that viruses can have significant stimulatory effects across whole-ecosystem scales. We suggest that existing efforts to predict carbon and nutrient cycling without considering virus effects are likely to miss essential features of marine food webs that regulate global biogeochemical cycles.     

Multiscale model of CRISPR-induced coevolutionary dynamics: diversification at the interface of Lamarck and Darwin

The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system is a recently discovered type of adaptive immune defense in bacteria and archaea that functions via directed incorporation of viral and plasmid DNA into host genomes. Here, we introduce a multiscale model of dynamic coevolution between hosts and viruses in an ecological context that incorporates CRISPR immunity principles. We analyze the model to test whether and how CRISPR immunity induces host and viral diversification and the maintenance of many coexisting strains. We show that hosts and viruses coevolve to form highly diverse communities. We observe the punctuated replacement of existent strains, such that populations have very low similarity compared over the long term. However, in the short term, we observe evolutionary dynamics consistent with both incomplete selective sweeps of novel strains (as single strains and coalitions) and the recurrence of previously rare strains. Coalitions of multiple dominant host strains are predicted to arise because host strains can have nearly identical immune phenotypes mediated by CRISPR defense albeit with different genotypes. We close by discussing how our explicit eco-evolutionary model of CRISPR immunity can help guide efforts to understand the drivers of diversity seen in microbial communities where CRISPR systems are active.     

Osmotic pressure can regulate matrix gene expression in Bacillus subtilis

Many bacteria organize themselves into structurally complex communities known as biofilms in which the cells are held together by an extracellular matrix. In general, the amount of extracellular matrix is related to the robustness of the biofilm. Yet, the specific signals that regulate the synthesis of matrix remain poorly understood. Here we show that the matrix itself can be a cue that regulates the expression of the genes involved in matrix synthesis in Bacillus subtilis. The presence of the exopolysaccharide component of the matrix causes an increase in osmotic pressure that leads to an inhibition of matrix gene expression. We further show that non‐specific changes in osmotic pressure also inhibit matrix gene expression and do so by activating the histidine kinase KinD. KinD, in turn, directs the phosphorylation of the master regulatory protein Spo0A, which at high levels represses matrix gene expression. Sensing a physical cue such as osmotic pressure, in addition to chemical cues, could be a strategy to non‐specifically co‐ordinate the behaviour of cells in communities composed of many different species.