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Precise binding mode identification and subsequent affinity improvement without structure determination remain a challenge in the development of therapeutic proteins. However, relevant experimental techniques are generally quite costly, and purely computational methods have been unreliable. Here, we show that integrated computational and experimental epitope localization followed by full-atom energy minimization can yield an accurate complex model structure which ultimately enables effective affinity improvement and redesign of binding specificity. As proof-of-concept, we used a leucine-rich repeat (LRR) protein binder, called a repebody (Rb), that specifically recognizes human IgG1 (hIgG1). We performed computationally-guided identification of the Rb:hIgG1 binding mode and leveraged the resulting model to reengineer the Rb so as to significantly increase its binding affinity for hIgG1 as well as redesign its specificity toward multiple IgGs from other species. Experimental structure determination verified that our Rb:hIgG1 model closely matched the co-crystal structure. Using a benchmark of other LRR protein complexes, we further demonstrated that the present approach may be broadly applicable to proteins undergoing relatively small conformational changes upon target binding. Author summary: It is quite challenging for computational methods to determine how proteins interact and to design mutations to alter their binding affinity and specificity. Despite recent advances in computational methods, however, in silico evaluation of binding energies has proven to be extremely difficult. We show that, in the case of protein-protein interactions where only small structural changes occur upon target binding, an integrated computational and experimental approach can identify a binding mode and drive reengineering efforts to improve binding affinity or specificity. Using as a model system a leucine-rich repeat (LRR) protein binder that recognizes human IgG1, our approach yielded a model of the protein complex that was very similar to the subsequently experimentally determined co-crystal structure, and enabled design of variants with significantly improved IgG1 binding affinity and with the ability to recognize IgG1 from other species. [ABSTRACT FROM AUTHOR]

Objective: Magnetic nanoparticles (MNPs) are an emerging platform for targeted diagnostics in cancer. An important component needed for translation of MNPs is the detection and quantification of targeted MNPs bound to tumor cells. Method: This study explores the feasibility of a multifrequency nonlinear magnetic spectroscopic method that uses excitation and pickup coils and is capable of discriminating between quantities of bound and unbound MNPs in 0.5 ml samples of KB and Igrov human cancer cell lines. The method is tested over a range of five concentrations of MNPs from 0 to 80 μg/ml and five concentrations of cells from 50 to 400 000 count per ml. Results: A linear model applied to the magnetic spectroscopy data was able to simultaneously measure bound and unbound MNPs with agreement between the model-fit and lab assay measurements (p < 0.001). The detectable iron of the presented method to bound and unbound MNPs was < 2 μg in a 0.5 ml sample. The linear model parameters used to determine the quantities of bound and unbound nanoparticles in KB cells were also used to measure the bound and unbound MNP in the Igrov cell line and vice versa. Conclusion: Nonlinear spectroscopic measurement of MNPs may be a useful method for studying targeted MNPs in oncology. Significance: Determining the quantity of bound and unbound MNP in an unknown sample using a linear model represents an exciting opportunity to translate multifrequency nonlinear spectroscopy methods to in vivo applications where MNPs could be targeted to cancer cells. [ABSTRACT FROM AUTHOR]

The unique properties of magnetic nanoparticles (MNPs), coupled with versatile surface engineering techniques, have led to a rising class of screening methods that enable separation of specific cell populations from complex biological samples. The growing sophistication and efficiency of these methods have far reaching implications for both fundamental research and clinical applications. In this study, the synthesis and surface engineering of MNPs is reviewed. Here, a model is introduced to illustrate how MNP morphology and particle-particle interactions influence magnetization, which is a key consideration in designing and selecting MNPs for efficient cell separations. Building upon these themes, immunomagnetic assays for capturing, isolating, and characterizing rare cell types from complex biological mixtures are reviewed. Although the focus of this study is on circulating tumor cells, these same techniques can be applied in screening for other rare cells of interest, such as various stem cell populations. In conclusion, current challenges and future directions for magnetic -nanomaterial-based cell screening systems are discussed. [ABSTRACT FROM AUTHOR]

The Dartmouth Center for Cancer Nanotechnology Excellence - one of nine funded by the National Cancer Institute as part of the Alliance for Nanotechnology in Cancer - focuses on the use of magnetic nanoparticles for cancer diagnostics and hyperthermia therapy. It brings together a diverse team of engineers and biomedical researchers with expertise in nanomaterials, molecular targeting, advanced biomedical imaging and translational in vivo studies. The goal of successfully treating cancer is being approached by developing nanoparticles, conjugating them with Fabs, hyperthermia treatment, immunotherapy and sensing treatment response. [ABSTRACT FROM AUTHOR]

Realizing the full potential of iron oxide nanoparticles (IONP) for cancer diagnosis and therapy requires selective tumor cell accumulation. Here, we report a systematic analysis of two key determinants for IONP homing to human breast cancers: (i) particle size and (ii) active vs passive targeting. In vitro, molecular targeting to the HER2 receptor was the dominant factor driving cancer cell association. In contrast, size was found to be the key determinant of tumor accumulation in vivo, where molecular targeting increased tumor tissue concentrations for 30 nm but not 100 nm IONP. Similar to the in vitro results, PEGylation did not influence in vivo IONP biodistribution. Thus, the results reported here indicate that the in vitro advantages of molecular targeting may not consistently extend to pre-clinical in vivo settings. These observations may have important implications for the design and clinical translation of advanced, multifunctional, IONP platforms. [ABSTRACT FROM AUTHOR]

Background: Neuropathic pain due to nerve injury is one of the most difficult types of pain to treat. Following peripheral nerve injury, neuronal and glial plastic changes contribute to central sensitization and perpetuation of mechanical hypersensitivity in rodents. The mitogen activated protein kinase (MAPK) family is pivotal in this spinal cord plasticity. MAPK phosphatases (MKPs) limit inflammatory processes by dephosphorylating MAPKs. For example, MKP-1 preferentially dephosphorylates p-p38. Since spinal p-p38 is pivotal for the development of chronic hypersensitivity in rodent models of pain, and p-p38 inhibitors have shown clinical potential in acute and chronic pain patients, we hypothesize that induction of spinal MKP-1 will prevent the development of peripheral nerve-injury-induced hypersensitivity and p-p38 overexpression. Results: We cloned rat spinal cord MKP-1 and optimize MKP-1 cDNA in vitro using transfections to BV-2 cells. We observed that in vitro overexpression of MKP-1 blocked lipopolysaccharide-induced phosphorylation of p38 (and other MAPKs) as well as release of pro-algesic effectors (i.e., cytokines, chemokines, nitric oxide). Using this cDNA MKP-1 and a non-viral, in vivo nanoparticle transfection approach, we found that spinal cord overexpression of MKP-1 prevented development of peripheral nerve-injury-induced tactile hypersensitivity and reduced pro- inflammatorycytokines and chemokines and the phosphorylated form of p38. Conclusions: Our results indicate that MKP-1, the natural regulator of p-p38, mediates resolution of the spinal cord pro-inflammatory milieu induced by peripheral nerve injury, resulting in prevention of chronic mechanical hypersensitivity. We propose that MKP-1 is a potential therapeutic target for pain treatment or prevention [ABSTRACT FROM AUTHOR]

Previous comparisons of winter rye plants ( Secale cereale L. cv. Musketeer) grown in a combination of specific temperature (°C)/irradiance (μmol m−2 s−1) regimes (20/50; 20/250; 20/800; 5/50; 5/250) revealed (1) that photosynthetic acclimation to low temperature mimics photosynthetic acclimation to high light because both conditions result in comparable reduction states of photosystem II (PSII), that is, comparable PSII excitation pressure; (2) that the relative redox state of PSII also appears to regulate a specific cold acclimation gene, Wcs19. In order to identify additional genes regulated differentially by either low temperature, irradiance or excitation pressure, we initiated a detailed analysis of gene expression. We identified and characterized 42 differentially expressed genes from wheat and rye. Based on their patterns of regulation under the five growth conditions employed, 37 of the cDNAs could be classified into four groups: genes regulated by PSII excitation pressure, low temperature, growth irradiance and interaction between growth temperature and irradiance. Partial sequence analyses revealed that several of these genes encode known chloroplastic proteins such as ELIPs, transketolase, carbonic anhydrase and Mg-chelatase. However, five of the genes could not be classified unambiguously into any one of these four categories. The implications of these results and the limitations of the experimental design are discussed in terms of larger-scale genomic studies designed to understand the interactions of multiple abiotic stresses to which a plant may be exposed when examining regulation of gene expression. [ABSTRACT FROM AUTHOR]

To elucidate the molecular basis of cold acclimation in strawberry (Fragaria × anannassa), we have begun studies to identify genes associated with low temperature (LT) acclimation. Differential screening of a cDNA library prepared from cold-acclimated strawberry plants allowed us to isolate several cDNAs showing differential expression at LT. Northern analysis showed that the transcript level of Fcorl (Fragaria cold-regulated) peaked after 2 days of LT exposure while that of Fcor2 peaked after 2 weeks. On the other hand, the level of Fcor3 transcript decreased within 24 hours of LT exposure and remained low during the 8 weeks acclimation period. Fcorl and Fcor2 are expressed in all tissues while Fcor3 is specific to leaves. The Fcorl-encoded protein has a compositional bias for leucine, isoleucine, glycine, proline and serine. This protein shares homology with the proteins encoded by blt101, a LT-re-sponsive gene from barley, and ESI3, a gene induced by salt stress in Lophopyrum. The FCOR2 protein is rich in lysine, leucine, valine, alanine and arginine, and shows no homology with any known gene products. The partial Fcor3 cDNA clone encodes a polypeptide that shows a very high identity with the spinach PSI subunit V and with the PSI PsaG polypeptide from barley. The level of Fcor1 transcript accumulation is correlated with the freezing tolerance of the strawberry cultivars used in our study. This suggests that Fcorl may be useful as a molecular marker to select for this trait in related species of the Rosaceae family. [ABSTRACT FROM AUTHOR]