Cold acclimation and freezing tolerance are the result of complex interaction between low temperature, light, and photosystem II (PSII) excitation pressure. Previous results have shown that expression of the Wcs19 gene is correlated with PSII excitation pressure measured in vivo as the relative reduction state of PSII. Using cDNA library screening and data mining, we have identified three different groups of proteins, late embryogenesis abundant (LEA) 3-L1, LEA3-L2, and LEA3-L3, sharing identities with WCS19. These groups represent a new class of proteins in cereals related to group 3 LEA proteins. They share important characteristics such as a sorting signal that is predicted to target them to either the chloroplast or mitochondria and a C-terminal sequence that may be involved in oligomerization. The results of subcellular fractionation, immunolocalization by electron microscopy and the analyses of target sequences within the Wcs19 gene are consistent with the localization of WCS19 within the chloroplast stroma of wheat (Triticum aestivum) and rye (Secale cereale). Western analysis showed that the accumulation of chloroplastic LEA3-L2 proteins is correlated with the capacity of different wheat and rye cultivars to develop freezing tolerance. Arabidopsis was transformed with the Wcs19 gene and the transgenic plants showed a significant increase in their freezing tolerance. This increase was only evident in cold-acclimated plants. The putative function of this protein in the enhancement of freezing tolerance is discussed.

Cognitive impairment is a common comorbidity in pediatric epilepsy that can severely affect quality of life. In many cases, antiepileptic treatments fail to improve cognition. Therefore, a fundamental question is whether underlying brain abnormalities may contribute to cognitive impairment through mechanisms independent of seizures. Here, we examined the possible effects on cognition of Nav1.1 down-regulation, a sodium channel principally involved in Dravet syndrome but also implicated in other cognitive disorders, including autism and Alzheimer’s disease. Using an siRNA approach to knockdown Nav1.1 selectively in the basal forebrain region, we were able to target a learning and memory network while avoiding the generation of spontaneous seizures. We show that reduction of Nav1.1 expression in the medial septum and diagonal band of Broca leads to a dysregulation of hippocampal oscillations in association with a spatial memory deficit. We propose that the underlying etiology responsible for Dravet syndrome may directly contribute to cognitive impairment in a manner that is independent from seizures.

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.

SUMMARY The enzyme lysostaphin possesses potent anti-staphylococcal activity and represents a promising antibacterial drug candidate; however, its immunogenicity poses a barrier to clinical translation. Here, structure-based biomolecular design enabled widespread depletion of lysostaphin’s DRB1*0401 restricted T cell epitopes, and resulting deimmunized variants exhibited striking reductions in anti-drug antibody responses upon administration to humanized HLA-transgenic mice. This reduced immunogenicity translated into improved efficacy in the form of protection against repeated challenges with methicillin-resistant Staphylococcus aureus, or MRSA. In contrast, while wild type lysostaphin was efficacious against the initial MRSA infection, it failed to clear subsequent bacterial challenges that were coincident with escalating anti-drug antibody titers. These results extend the existing deimmunization literature, in which reduced immunogenicity and retained efficacy are assessed independently of each other. By correlating in vivo efficacy with longitudinal measures of anti-drug antibody development, we provide the first direct evidence that T cell epitope depletion manifests enhanced biotherapeutic efficacy.

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.

BackgroundNeuropathic 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.ResultsWe 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-inflammatory cytokines and chemokines and the phosphorylated form of p38.ConclusionsOur 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.

In plants, O-methylation of phenolic compounds plays an important role in such processes as lignin synthesis, flower pigmentation, chemical defense, and signaling. However, apart from phenylpropanoids and flavonoids, very few enzymes involved in coumarin biosynthesis have been identified. We report here the molecular and biochemical characterization of a gene encoding a novel O-methyltransferase that catalyzes the methylation of 7,8-dihydroxycoumarin, daphnetin. The recombinant protein displayed an exclusive methylation of position 8 of daphnetin. The identity of the methylated product was unambiguously identified as 7-hydroxy-8-methoxycoumarin by co-chromatography on cellulose TLC and coelution from high performance liquid chromatography, with authentic synthetic samples, as well as by UV, mass spectroscopy, 1 H NMR spectral analysis, and NOE correlation signals of the relevant protons. Northern blot analysis and enzyme activity assays revealed that the transcript and corresponding enzyme activity are up-regulated by both low temperature and photosystem II excitation pressure. Using various phenylpropanoid and flavonoid substrates, we demonstrate that cold acclimation of rye leaves increases O-methyltransferase activity not only for daphnetin but also for the lignin precursors, caffeic acid, and 5-hydroxyferulic acid. The significance of this novel enzyme and daphnetin O-methylation is discussed in relation to its putative role in modulating cold acclimation and photosystem II excitation pressure.Low temperature is one of the most important environmental factors limiting the productivity and distribution of plants. Exposure of plants to low, nonfreezing temperatures, a process known as cold acclimation, induces the genetic system required for increased freezing tolerance. Knowledge of the molecular, physiological, and biochemical changes that occur during this process could lead to the improvement of plant productivity.This complex process has been extensively studied and several cold-responsive genes have been isolated from a range of dicotyledonous and monocotyledonous species (1, 2). Although the functions of some of these genes are known (3-6), the details of the processes responsible for their regulation and detection of temperature changes are still incomplete.Previous studies have shown that development of freezing tolerance in winter cereals, such as wheat and rye, is correlated with an increase in their photosynthetic capacity (7). Thus, growth at low temperature not only induces freezing tolerance, but also results in an increased resistance to low temperatureinduced photoinhibition of photosynthesis, and requires adjustment to a combination of light and low temperature. The common photosynthetic response of plants to low temperature and normal light is rationalized in terms of photosystem II (PSII) 1 excitation pressure, which is a measure of the redox state of the first electron acceptor, quinone A (7-9). It has been shown that cold-acclimated rye and wheat grown at 5°C/250 mol m Ϫ2 s Ϫ1

Sensory neuron-specific receptors (SNSRs) belong to a large family of GPCRs, known as Mrgs (Mas-related genes), many of which are preferentially expressed in primary afferent nociceptors. Selective SNSR agonists produce pain-like behaviors in rats, showing that SNSR activation is sufficient to produce pain. However, it is unknown whether SNSR activation is necessary for pain either in the normal condition or in pathological pain states. Here we used small interfering RNA (siRNA) to acutely knockdown rat SNSR1 and test the hypothesis that this receptor mediates pain responses. Administration of siRNA to the lumbar spinal cord in rats dose-dependently knocked down rSNSR1 mRNA and protein and abolished heat hyperalgesia evoked by intradermal administration of specific rSNSR1 agonists. In rats with levels of rSNSR1 knockdown sufficient to block responses to the SNSR1 agonists, there was no effect on normal pain responses, but there was a significant reduction of heat hyperalgesia in an inflammatory pain model (Complete Freund's Adjuvant), supporting a role for rSNSR1 in inflammatory pain. Further in vivo studies revealed that SNSR1 knockdown had no effect on responses to intradermal capsaicin, a selective TRPV1 agonist. In contrast, a selective TRPV1 antagonist abolished heat hyperalgesia produced by an SNSR agonist, suggesting that TRPV1 receptors mediate rSNSR1-evoked responses. We also found that rSNSR1-like immunoreactivity, like TRPV1, is localized in the superficial dorsal horn of the spinal cord. We propose that rSNSR1 represents a new member of the receptors expressed on chemosensitive nociceptors responsible for detecting the "inflammatory soup" of mediators generated by tissue damage.