Cellular Modifications Research Articles

Obesity and Surgical Wound Healing: A Current Review

Objective. The correlation between obesity and deficient wound healing has long been established. This review examines the current literature on the mechanisms involved in obesity-related perioperative morbidity. Methods. A literature search was performed using Medline, PubMed, Cochrane Library, and Internet searches. Keywords used include obesity, wound healing, adipose healing, and bariatric and surgical complications. Results. Substantial evidence exists demonstrating that obesity is associated with a number of postoperative complications. Specifically in relation to wound healing, explanations include inherent anatomic features of adipose tissue, vascular insufficiencies, cellular and composition modifications, oxidative stress, alterations in immune mediators, and nutritional deficiencies. Most recently, advances made in the field of gene array have allowed researchers to determine a few plausible alterations and deficiencies in obese individuals that contribute to their increased risk of morbidity and mortality, especially wound complications. Conclusion. While the literature discusses how obesity may negatively affect health on various of medical fronts, there is yet to be a comprehensive study detailing all the mechanisms involved in obesity-related morbidities in their entirety. Improved knowledge and understanding of obesity-induced physiological, cellular, molecular, and chemical changes will facilitate better assessments of surgical risks and outcomes and create efficient treatment protocols for improved patient care of the obese patient population.

Sodium channels as gateable non-photonic sensors for membrane-delimited reactive species

Reactive oxygen species (ROS) and reactive oxygen intermediates (ROI) play crucial roles in physiological processes. While excessive ROS damages cells, small fluctuations in ROS levels represent physiological signals important for vital functions. Despite the physiological importance of ROS, many fundamental questions remain unanswered, such as which types of ROS occur in cells, how they distribute inside cells, and how long they remain in an active form. The current study presents a ratiometric sensor of intracellular ROS levels based on genetically engineered voltage-gated sodium channels (roNaV). roNaV can be used for detecting oxidative modification that occurs near the plasma membrane with a sensitivity similar to existing fluorescence-based ROS sensors. Moreover, roNaV has several advantages over traditional sensors because it does not need excitation light for sensing, and thus, can be used to detect phototoxic cellular modifications. In addition, the ROS dynamic range of roNaV is easily manipulated in real time by means of the endogenous channel inactivation mechanism. Measurements on ROS liberated from intracellular Lucifer Yellow and genetically encoded KillerRed have revealed an assessment of ROS lifetime in individual mammalian cells. Flashlight-induced ROS concentration decayed with two major time constants of about 10 and 1000ms.

Differential expression profiling of hypothalamus genes in laying period and ceased period Huoyan geese

The hypothalamus plays a central role in controlling poultry reproductive activity. To increase our understanding of genes involved in egg laying of Huoyan geese, gene profiles in the hypothalamus of laying period and ceased period Huoyan geese were investigated using suppression subtractive hybridization (SSH) method. A total of 95 differentially expressed sequence tags (ESTs), including 46 up-regulated and 49 down-regulated sequences showed homology to known genes of the non-redundant NCBI databases. Bioinformatic analysis demonstrated that these genes were mainly involved in anatomical structure development, signal transduction, cellular nitrogen compound metabolic process, biosynthetic process, cellular protein modification process, cell differentiation, transport, cell adhesion, and reproduction. Ten ESTs were selected for further analyses by quantitative real-time RT-PCR (qRT-PCR). Whose most part of results were consistent with the SSH results. Of note, AdipoR2, Nrg1, and NCAM1, which related with secretion of GnRH and other hormones, were identified to be differentially expressed between laying period and ceased period. These findings provided a new source for mining genes related to higher laying performance of Huoyan geese, which facilitate our understanding of the reproductive biology of the goose.

The WD40-repeat Proteins WDR-20 and WDR-48 Bind and Activate the Deubiquitinating Enzyme USP-46 to Promote the Abundance of the Glutamate Receptor GLR-1 in the Ventral Nerve Cord of Caenorhabditis elegans

Ubiquitin-mediated endocytosis and degradation of glutamate receptors controls their synaptic abundance and is implicated in modulating synaptic strength. The deubiquitinating enzymes (DUBs) that function in the nervous system are beginning to be defined, but the mechanisms that control DUB activity in vivo are understood poorly. We found previously that the DUB USP-46 deubiquitinates the Caenorhabditis elegans glutamate receptor GLR-1 and prevents its degradation in the lysosome. The WD40-repeat (WDR) proteins WDR20 and WDR48/UAF1 have been shown to bind to USP46 and stimulate its catalytic activity in other systems. Here we identify the C. elegans homologs of these WDR proteins and show that C. elegans WDR-20 and WDR-48 can bind and stimulate USP-46 catalytic activity in vitro. Overexpression of these activator proteins in vivo increases the abundance of GLR-1 in the ventral nerve cord, and this effect is further enhanced by coexpression of USP-46. Biochemical characterization indicates that this increase in GLR-1 abundance correlates with decreased levels of ubiquitin-GLR-1 conjugates, suggesting that WDR-20, WDR-48, and USP-46 function together to deubiquitinate and stabilize GLR-1 in neurons. Overexpression of WDR-20 and WDR-48 results in alterations in locomotion behavior consistent with increased glutamatergic signaling, and this effect is blocked in usp-46 loss-of-function mutants. Conversely, wdr-20 and wdr-48 loss-of-function mutants exhibit changes in locomotion behavior that are consistent with decreased glutamatergic signaling. We propose that WDR-20 and WDR-48 form a complex with USP-46 and stimulate the DUB to deubiquitinate and stabilize GLR-1 in vivo.

Next Generation Sequencing Reveals Regulation of Distinct Aedes microRNAs during Chikungunya Virus Development

BackgroundApplication of genomics and Next Generation sequencing has led to the identification of new class of cellular functional molecules, namely, small RNAs. Of the several classes of ncRNAs (non-coding RNA), microRNAs have been demonstrated to exert determinative influence on various cellular processes. It is becoming abundantly clear that host/vector/pathogen encoded microRNAs impact eventual pathogenesis. In this context, the participation of vector based microRNAs in disease transmission and pathogen development is being investigated intensively. A few studies have highlighted the role of vector encoded microRNAs in pathogen infection. We conducted this study to evaluate the role of host miRNAs upon CHIKV (Chikungunya Virus) infection in an important vector, Aedes albopictus.FindingsWe identified 88 and 79 known miRNAs in uninfected and CHIKV infected Ae. albopictus Singh's cell line respectively. We further identified nine novel miRNAs in Ae. albopictus. Comparison of the two libraries revealed differential expression of 77 common miRNAs between them. CHIKV infection specifically altered the miRNA profile of a specific set of eight miRNAs. Putative targets of these regulated miRNAs were identified and classified into their pathways.ConclusionsIn our study we have identified and described the profiles of various miRNAs upon CHIKV infection in Ae. albopictus. This investigation provides an insight about cellular modification by miRNAs during CHIKV infection and the results provide leads for identifying potential candidates for vector based antiviral strategies.

The role of protein modifications in senescence of freeze-dried Acetobacter senegalensis during storage.

BackgroundLoss of viability is one of the most important problems during starter culture production. Previous research has mostly focused on the production process of bacterial starters, but there are few studies about cellular protein deterioration causing cell defectiveness during storage. In the present study, we investigated the influence of storage temperature (−21, 4, 35°C) on the cellular protein modifications which may contribute to the senescence of freeze-dried Acetobacter senegalensis.ResultsHeterogeneous populations composed of culturable cells, viable but non-culturable cells (VBNC) and dead cells were generated when freeze-dried cells were kept at −21 and 4°C for 12 months whereas higher storage temperature (35°C) mainly caused death of the cells. The analysis of stored cell proteome by 2D-DiGE demonstrated a modified pattern of protein profile for cell kept at 4 and 35°C due to the formation of protein spot trains and shift of Isoelectric point (pI). Quantification of carbonylated protein by ELISA showed that the cells stored at 4 and 35°C had higher carbonylated protein contents than fresh cells. 2D-DiGE followed by Western blotting also confirmed the carbonylation of cellular proteins involved in translation process and energy generation. The auto-fluorescent feature of cells kept at 35°C increased significantly which may be an indication of protein glycation during storage. In addition, the percentage of cellular unsaturated fatty acid and the solubility of cellular proteins decreased upon storage of cells at higher temperature suggesting that peroxidation of fatty acids and possibly protein lipidation and oxidation occurred.ConclusionsHigh storage temperature induces some deteriorative reactions such as protein oxidation, lipidation and glycation which may cause further protein modifications like pI-shift, and protein insolubility. These modifications can partly account for the changes in cell viability. It can also be deduced that even moderate carbonylation of some critical cellular proteins (like ribosomal proteins) may lead to VBNC formation or death of freeze-dried bacteria. Moreover, it seems that other mechanisms of biomolecule deterioration preceding protein carbonylation lead to VBNC formation under very low storage temperature.

Molecular characterization of a Tax-2C protein variant identified in Brazilian subjects infected by HTLV-2C

Although the etiologic difference in pathogenic properties of HTLV-1 and HTLV-2 still remains unclear, it has been suggested that it could be attributed to the differential structure and activities of their transactivating Tax proteins. Tax-1 and Tax-2, although having 85% amino acid (aa) similarity, present phenotypic differences consistent with a more robust transformation capacity of Tax-1. Interestingly, the HTLV-2C Brazilian variant present in Amerindians and in IDU from urban areas is genotypically close to HTLV-2A but Tax-2C possesses an additional 25aa in the C-terminal region similar to that of Tax-2B. We have already demonstrated that Tax-1 and Tax-2B have several common domains, but present differential cellular distribution. To add some information concerning the structure and site domains present in Tax-2C we conducted the present study. We have obtained the Tax-2C sequence from 25 different HTLV-2C subjects and analyzed the aminoacid homology between Tax-2B and Tax-2C variants. We found that they differ for amino acidic substitutions in eleven different positions that may affect cellular localization or post-translational modification. Studies on phenotypic properties and cellular localization of Tax-2C are in progress. Support: MCT/CNPq # 303545/2012-7, CAPES, IAL # 49D/2010), Brazil.

Chapter 10 - Neural Mechanisms of Odor Rule Learning

Rats that are trained in a particularly difficult olfactory discrimination task demonstrate a dramatic increase in their capability to acquire memories of new odors once they have learned the first discrimination task. Such high-skill learning, termed "rule learning" or "learning set" (see Saar et al., 1998, 2001) is accompanied by a series postsynaptic cellular modifications which have three major traits: a.They are widespread throughout the piriform cortex network. Both physiological and morphological modifications are found in most of the studied neurons.b.The time course in which these modifications appear and disappear is strongly correlated with the time course in which the skill is acquired and decayed. However, memories for specific odors outlast these modifications by far. Thus, the identified modifications are related to rule learning (learning how to learn), rather than to long-term memory for the specific odors for which the rats are trained.c.While the above-mentioned changes act to enhance single-cell excitability, others reduced it; synaptic inhibition is enhanced after learning and the subunit composition of the NMDA receptor is modified in a manner that favors activity-induced synaptic weakening over synaptic enhancement.

N-terminal signal sequence is required for cellular trafficking and hyaluronan-depolymerization of KIAA1199

Recently, we disclosed that KIAA1199-mediated hyaluronan (HA) depolymerization requires an acidic cellular microenvironment (e.g. clathrin-coated vesicles or early endosomes), but no information about the structural basis underlying the cellular targeting and functional modification of KIAA1199 was available. Here, we show that the cleavage of N-terminal 30 amino acids occurs in functionally matured KIAA1199, and the deletion of the N-terminal portion results in altered intracellular trafficking of the molecule and loss of cellular HA depolymerization. These results suggest that the N-terminal portion of KIAA1199 functions as a cleavable signal sequence required for proper KIAA1199 translocation and KIAA1199-mediated HA depolymerization.

Endothelialization of implanted cardiovascular biomaterial surfaces: The development from in vitro to in vivo

Restenosis and thrombosis formation after cardiovascular devices implantation continue to be problematic. Although various platforms and parameters of cardiovascular devices have been designed and optimized over the years, postoperative complications are hard to avoid. The native vascular endothelium always provide a nonthrombogenic surface as well as prevent intimal overproliferation, thereby, the presence of a confluent endothelial cell layer on material surfaces have been widely accepted as an ideal approach to improve the biocompatibility of implanted cardiovascular materials. Endothelialization on biomaterial surfaces is initially developed by in vitro cell seeding. However, numerous no-perfect parts of this method are existed for clinical use. The emergency of endothelial progenitor cells may provide a promising way for setting these limitations. Over the last decades, countless researches about EPCs-based in vivo induced self-endothelialization have been reported and mainly focused on cellular therapy, pharmacological therapy, materials designing, or surface biofunctional modification. This review details the development of endothelialization on cardiovascular material surfaces from in vitro to in vivo. Endothelialization progress on the basis of molecular biological level and bioinformatics theory is expected to be the key point in the coming decades.

Anatomical alterations of Phaseolus vulgaris L. mature leaves irradiated with X‐rays

The cultivation of higher plants in Space involves not only the development of new agro-technologies for the design of ecologically closed Space greenhouses, but also understanding of the effects of Space factors on biological systems. Among Space factors, ionising radiation is one of the main constraints to the growth of organisms. In this paper, we analyse the effect of low-LET radiation on leaf histology and cytology in Phaseolus vulgaris L. plants subjected to increasing doses of X-rays (0.3, 10, 50, 100Gy). Leaves irradiated at tissue maturity were compared with not-irradiated controls. Semi-thin sections of leaves were analysed through light and epi-fluorescence microscopy. Digital image analysis was applied to quantify anatomical parameters, with a specific focus on the occurrence of signs of structural damage as well as alterations at subcellular level, such as the accumulation of phenolic compounds and chloroplast size. Results showed that even at high levels of radiation, general anatomical structure was not severely perturbed. Slight changes in mesophyll density and cell enlargement were detected at the highest level of radiation. However, at 100Gy, higher levels of phenolic compounds accumulated along chloroplast membranes: this accompanied an increase in number of chloroplasts. The reduced content of chlorophylls at high levels of radiation was associated with reduced size of the chloroplasts. All data are discussed in terms of the possible role of cellular modifications in the maintenance of high radioresistance and photosynthetic efficiency.

Corneal imaging of intrastromal femtosecond laser treatment for presbyopia (Intracor®)

To evaluate anatomic appearance and corneal cellular modifications after monocular Intracor(®) procedure with two different anterior segment imaging techniques within the first postoperative year. Four patients underwent an Intracor(®) procedure in one eye performed at Clinique de la vision, and corneal imaging was performed in Quinze-Vingts National Ophthalmology Hospital. Slit-lamp photography, confocal microscopy with Heidelberg retinal tomography (HRT) and anterior segment spectral-domain optical coherence tomography (OCT) were performed 2 days, and 1, 6, and 12 months after the procedure. Confocal microscopy showed strong cellular activation of keratocytes within the first postoperative month which diminished over time. The linear femtosecond incisions appeared as hyper-reflective regular lines and reflectivity decreased throughout the follow-up period. After 6 months, a fibrotic process with appearance of corneal scars was visible as small intrastromal hyper-reflective lines and thick hyper-reflective spicules around the incisions and remained stable over time. On spectral-domain OCT, the size and depth of the incisions decreased from the center to the periphery. Visibility of the corneal femtosecond incisions on OCT decreased from day 2 until they almost disappeared at 12 months. The incision angles seemed to curve progressively from the center to the periphery in their deep portion on the peripheral rings. Corneal cellular modifications found on HRT, anatomical features, and the sizes of the intrastromal rings, may provide valuable information on this new refractive technique.

Targeting Host Store-Operated Ca<sup>2+</sup> Release to Attenuate Viral Infections

Viruses coopt host intracellular Ca(2+) signaling pathways to optimize timing and effectiveness of infection stages against barriers to invasion, pathogenesis, replication, and release. Virus-induced changes in free cytosolic Ca(2+) levels facilitate virus adsorption, uncoating, catalysis, toxin production, structural assembly and stabilization, trafficking, and fusion and budding. Ca(2+)-associated alterations in virus status also selectively precipitate host cytopathologies through, among other events, retardation or induction of apoptosis, elevation of metabolic stress and reactive oxygen species production, and promotion of proinflammatory cytokine and chemokine synthesis and release. Viral particles and proteins tune spatiotemporal dynamics of host free cytosolic Ca(2+) concentrations by modulating Ca(2+) entry from the extracellular environment, upstream first or second messengers, ion- and ATP-dependent Ca2+ pumps that sequester or extrude free cytosolic Ca(2+), store-operated Ca(2+) mobilization and leakage, and viral capsid/envelope and downstream host Ca(2+) binding proteins and sensors. Each of these major viral mechanisms, briefly reviewed in this article, presents a suitable drug target capable of mitigating the severity and incidence of viral infections. Given its pivotal role in cellular response regulation, bioenergetics, posttranslational protein and lipid modification and transport, homeostasis, cell motility and morphogenesis, and T lymphocyte proliferation, targeting virally stimulated inositol 1,4,5-trisphoshate (IP3)-mediated store-operated Ca(2+) release especially offers unique, predictable benefits for augmenting immunoprotection in vertebrate clinical populations. We appraise possibilities of modulating this system with experimental proteins that gate activation kinetics of endoplasmic-reticulum-localized Ca(2+)-conducting IP3 receptors via allosteric protein-protein interactions. Such compounds are expected to be valuable in treating primary disease symptoms and sequelae, including virus-associated dementia.

Effect of the alcohol consumption on osteocyte cell processes: a molecular imaging study

We have previously shown microarchitectural tissue changes with cellular modifications in osteocytes following high chronic alcohol dose. The aim of this study was to assess the dose effect of alcohol consumption on the cytoskeleton activity, the cellular lipid content and modulation of differentiation and apoptosis in osteocyte. Male Wistar rats were divided into three groups: Control (C), Alcohol 25% v/v (A25) or Alcohol 35% v/v (A35) for 17 weeks. Bone mineral density (BMD) was assessed by DXA, osteocyte empty lacunae, lacunae surface, bone marrow fat with bright field microscopy. Osteocyte lipid content was analysed with transmission electron microscopy (TEM) and epifluorescence microscopy. Osteocyte apoptosis was analysed with immunolabelling and TEM. Osteocyte differentiation and cytoskeleton activity were analysed with immunolabelling and real time quantitative PCR. At the end of the protocol, BMD was lower in A25 and A35 compared with C, while the bone marrow lipid content was increased in these groups. More empty osteocyte lacunae and osteocyte containing lipid droplets in A35 were found compared with C and A25. Cleaved caspase-3 staining and chromatin condensation were increased in A25 and A35 versus C. Cleaved caspase-3 was increased in A35 versus A25. CD44 and phosphopaxillin stainings were higher in A35 compared with C and A25. Paxillin mRNA expression was higher in A35 versus A25 and C and sclerostin mRNA expression was higher in A35 versus C. We only observed a dose effect of alcohol consumption on cleaved caspase-3 osteocyte immunostaining levels and on the number of lipid droplets in the bone marrow.

Regulatory T cells and the immune pathogenesis of prenatal infection

Pregnancy in placental mammals offers exceptional comprehensive benefits of in utero protection, nutrition, and metabolic waste elimination for the developing fetus. However, these benefits also require durable strategies to mitigate maternal rejection of fetal tissues expressing foreign paternal antigens. Since the initial postulate of expanded maternal immune tolerance by Sir Peter Medawar 60 years ago, an amazingly elaborate assortment of molecular and cellular modifications acting both locally at the maternal-placental interface and systemically have been shown to silence potentially detrimental maternal immune responses. In turn, simultaneously maintaining host defense against the infinite array of potential pathogens during pregnancy is equally important. Fortunately, resistance against most infections is preserved seamlessly throughout gestation. On the other hand, recent studies on pathogens with unique predisposition for prenatal infections have uncovered distinctive holes in host defense associated with the reproductive process. Using these infections to probe the response during pregnancy, the immune suppressive regulatory subset of maternal CD4 T cells has been increasingly shown to dictate the inter-workings between prenatal infection susceptibility and pathogenesis of ensuing pregnancy complications. Herein, the recent literature suggesting a necessity for maternal regulatory T cells (Tregs) in pregnancy-induced immunological shifts that sustain fetal tolerance is reviewed. Additional discussion is focused on how expansion of maternal Treg suppression may become exploited by pathogens that cause prenatal infections and the perilous potential of infection-induced immune activation that may mitigate fetal tolerance and inadvertently inject hostility into the protective in utero environment.

Dysregulation of KSHV replication by extracts from Carthamus tinctorius L.

Carthamus tinctorius L. (CT) is traditionally used to reduce ailments from diseases of the musculoskeletal system and connective tissue and diseases of blood circulation and the cardiovascular system. Flower extracts from CT are known to have antibacterial activity, anti-inflammatory activity, and to inhibit tumor promotion in mouse skin carcinogenesis. In order to discover new antiviral agents from CT extracts, we tested whether CT extracts contain antiviral activity against gammaherpesvirus infection. This study demonstrated that treatment with CT extracts disrupted KSHV latency in the viral-infected host cells, iSLK-BAC16. n-Hexane and EtOH fractions of CT extracts critically affected at least two stages of the KHSV life-cycle by abnormally inducing KSHV lytic reactivation and by severely preventing KSHV virion release from the viral host cells. In addition to the effects on KSHV itself, CT extract treatments induced cellular modifications by dysregulating cell-cycle and producing strong cytotoxicity. This study demonstrated for the first time that CT extracts have antiviral activities that could be applied to development of new anti-gammaherpesviral agents.

Gene expression profiling in the pituitary gland of laying period and ceased period huoyan geese.

Huoyan goose is a Chinese local breed famous for its higher laying performance, but the problems of variety degeneration have emerged recently, especially a decrease in the number of eggs laid. In order to better understand the molecular mechanism that underlies egg laying in Huoyan geese, gene profiles in the pituitary gland of Huoyan geese taken during the laying period and ceased period were investigated using the suppression subtractive hybridization (SSH) method. Total RNA was extracted from pituitary glands of ceased period and laying period geese. The cDNA in the pituitary glands of ceased geese was subtracted from the cDNA in the pituitary glands of laying geese (forward subtraction); the reverse subtraction was also performed. After sequencing and annotation, a total of 30 and 24 up and down-regulated genes were obtained from the forward and reverse SSH libraries, respectively. These genes mostly related to biosynthetic process, cellular nitrogen compound metabolic process, transport, cell differentiation, cellular protein modification process, signal transduction, small molecule metabolic process. Furthermore, eleven genes were selected for further analyses by quantitative real-time PCR (qRT-PCR). The qRT-PCR results for the most part were consistent with the SSH results. Among these genes, Synaptotagmin-1 (SYT1) and Stathmin-2 (STMN2) were substantially over-expressed in laying period compared to ceased period. These results could serve as an important reference for elucidating the molecular mechanism of higher laying performance in Huoyan geese.

HIV-1 transcription and latency: an update

Combination antiretroviral therapy, despite being potent and life-prolonging, is not curative and does not eradicate HIV-1 infection since interruption of treatment inevitably results in a rapid rebound of viremia. Reactivation of latently infected cells harboring transcriptionally silent but replication-competent proviruses is a potential source of persistent residual viremia in cART-treated patients. Although multiple reservoirs may exist, the persistence of resting CD4+ T cells carrying a latent infection represents a major barrier to eradication. In this review, we will discuss the latest reports on the molecular mechanisms that may regulate HIV-1 latency at the transcriptional level, including transcriptional interference, the role of cellular factors, chromatin organization and epigenetic modifications, the viral Tat trans-activator and its cellular cofactors. Since latency mechanisms may also operate at the post-transcriptional level, we will consider inhibition of nuclear RNA export and inhibition of translation by microRNAs as potential barriers to HIV-1 gene expression. Finally, we will review the therapeutic approaches and clinical studies aimed at achieving either a sterilizing cure or a functional cure of HIV-1 infection, with a special emphasis on the most recent pharmacological strategies to reactivate the latent viruses and decrease the pool of viral reservoirs.

Intermolecular interactions of the malate synthase of Paracoccidioides spp

BackgroundThe fungus Paracoccidioides spp is the agent of paracoccidioidomycosis (PCM), a pulmonary mycosis acquired by the inhalation of fungal propagules. Paracoccidioides malate synthase (PbMLS) is important in the infectious process of Paracoccidioides spp because the transcript is up-regulated during the transition from mycelium to yeast and in yeast cells during phagocytosis by murine macrophages. In addition, PbMLS acts as an adhesin in Paracoccidioides spp. The evidence for the multifunctionality of PbMLS indicates that it could interact with other proteins from the fungus and host. The objective of this study was to identify and analyze proteins that possibly bind to PbMLS (PbMLS-interacting proteins) because protein interactions are intrinsic to cell processes, and it might be possible to infer the function of a protein through the identification of its ligands.ResultsThe search for interactions was performed using an in vivo assay with a two-hybrid library constructed in S. cerevisiae; the transcripts were sequenced and identified. In addition, an in vitro assay using pull-down GST methodology with different protein extracts (yeast, mycelium, yeast-secreted proteins and macrophage) was performed, and the resulting interactions were identified by mass spectrometry (MS). Some of the protein interactions were confirmed by Far-Western blotting using specific antibodies, and the interaction of PbMLS with macrophages was validated by indirect immunofluorescence and confocal microscopy. In silico analysis using molecular modeling, dynamics and docking identified the amino acids that were involved in the interactions between PbMLS and PbMLS-interacting proteins. Finally, the interactions were visualized graphically using Osprey software.ConclusionThese observations indicate that PbMLS interacts with proteins that are in different functional categories, such as cellular transport, protein biosynthesis, modification and degradation of proteins and signal transduction. These data suggest that PbMLS could play different roles in the fungal cell.

Development of an in vitro analog concomitantly expressing the cellular correlates of sensitization and suppression of feeding in Aplysia californica

When presented with aversive stimuli, the marine mollusk Aplysia californica shows increased defensive responses (sensitization) and suppression of feeding. The cellular correlates of sensitization and suppression of feeding include increased excitability of the tail sensory neurons (TSNs, which mediate defensive responses) and decreased excitability in neuron B51 (a cell critical to the Aplysia feeding circuit), respectively. The goal of this project was to develop a reduced preparation of the isolated Aplysia nervous system (buccal, pleural‐pedal, and cerebral ganglia) in which the effects of aversive stimuli within the defense and feeding neural circuits could be mimicked in vitro by electrical stimulation of afferent nerves. We successfully observed significant concomitant increase in excitability of the TSNs (measured by spikes evoked) and decrease in excitability of B51 (increase in the burst threshold) 15 min post stimulation. Currently, we are examining whether this in vitro analog expresses long term (24 h) correlates. This in vitro analog will facilitate further analyses of cellular and molecular modifications resulting from aversive stimulation, as it would allow manipulations not feasible in vivo. Supported by NSF Grant IOS 1120304 to RM.