The melon Fom-1-Prv resistance gene pair: Correlated spatial expression and interaction with a viral protein.

The head-to-head oriented pair of melon resistance genes, Fom-1 and Prv, control resistance to Fusarium oxysporum races 0 and 2 and papaya ringspot virus (PRSV), respectively. They encode, via several RNA splice variants, TIR-NBS-LRR proteins, and Prv has a C-terminal extra domain with a second NBS homologous sequence. In other systems, paired R-proteins were shown to operate by "labor division," with one protein having an extra integrated domain that directly binds the pathogen's Avr factor, and the second protein executing the defense response. We report that the expression of the two genes in two pairs of near-isogenic lines was higher in the resistant isoline and inducible by F. oxysporum race 2 but not by PRSV. The intergenic DNA region separating the coding sequences of the two genes acted as a bi-directional promoter and drove GUS expression in transgenic melon roots and transgenic tobacco plants. Expression of both genes was strong in melon root tips, around the root vascular cylinder, and the phloem and xylem parenchyma of tobacco stems and petioles. The pattern of GUS expression suggests coordinated expression of the two genes. In agreement with the above model, Prv's extra domain was shown to interact with the cylindrical inclusion protein of PRSV both in yeast cells and in planta.

Failure to mate enhances investment in behaviors that may promote mating reward and impairs the ability to cope with stressors via a subpopulation of Neuropeptide F receptor neurons.

Living in dynamic environments such as the social domain, where interaction with others determines the reproductive success of individuals, requires the ability to recognize opportunities to obtain natural rewards and cope with challenges that are associated with achieving them. As such, actions that promote survival and reproduction are reinforced by the brain reward system, whereas coping with the challenges associated with obtaining these rewards is mediated by stress-response pathways, the activation of which can impair health and shorten lifespan. While much research has been devoted to understanding mechanisms underlying the way by which natural rewards are processed by the reward system, less attention has been given to the consequences of failure to obtain a desirable reward. As a model system to study the impact of failure to obtain a natural reward, we used the well-established courtship suppression paradigm in Drosophila melanogaster as means to induce repeated failures to obtain sexual reward in male flies. We discovered that beyond the known reduction in courtship actions caused by interaction with non-receptive females, repeated failures to mate induce a stress response characterized by persistent motivation to obtain the sexual reward, reduced male-male social interaction, and enhanced aggression. This frustrative-like state caused by the conflict between high motivation to obtain sexual reward and the inability to fulfill their mating drive impairs the capacity of rejected males to tolerate stressors such as starvation and oxidative stress. We further show that sensitivity to starvation and enhanced social arousal is mediated by the disinhibition of a small population of neurons that express receptors for the fly homologue of neuropeptide Y. Our findings demonstrate for the first time the existence of social stress in flies and offers a framework to study mechanisms underlying the crosstalk between reward, stress, and reproduction in a simple nervous system that is highly amenable to genetic manipulation.

Cell specialization in cyanobacterial biofilm development revealed by expression of a cell-surface and extracellular matrix protein.

Cyanobacterial biofilms are ubiquitous and play important roles in diverse environments, yet, understanding of the processes underlying the development of these aggregates is just emerging. Here we report cell specialization in formation of Synechococcus elongatus PCC 7942 biofilms-a hitherto unknown characteristic of cyanobacterial social behavior. We show that only a quarter of the cell population expresses at high levels the four-gene ebfG-operon that is required for biofilm formation. Almost all cells, however, are assembled in the biofilm. Detailed characterization of EbfG4 encoded by this operon revealed cell-surface localization as well as its presence in the biofilm matrix. Moreover, EbfG1-3 were shown to form amyloid structures such as fibrils and are thus likely to contribute to the matrix structure. These data suggest a beneficial 'division of labor' during biofilm formation where only some of the cells allocate resources to produce matrix proteins-'public goods' that support robust biofilm development by the majority of the cells. In addition, previous studies revealed the operation of a self-suppression mechanism that depends on an extracellular inhibitor, which supresses transcription of the ebfG-operon. Here we revealed inhibitor activity at an early growth stage and its gradual accumulation along the exponential growth phase in correlation with cell density. Data, however, do not support a threshold-like phenomenon known for quorum-sensing in heterotrophs. Together, data presented here demonstrate cell specialization and imply density-dependent regulation thereby providing deep insights into cyanobacterial communal behavior.

Lytic Reactivation of the Kaposi's Sarcoma-Associated Herpesvirus (KSHV) Is Accompanied by Major Nucleolar Alterations.

The nucleolus is a subnuclear compartment whose primary function is the biogenesis of ribosomal subunits. Certain viral infections affect the morphology and composition of the nucleolar compartment and influence ribosomal RNA (rRNA) transcription and maturation. However, no description of nucleolar morphology and function during infection with Kaposi's sarcoma-associated herpesvirus (KSHV) is available to date. Using immunofluorescence microscopy, we documented extensive destruction of the nuclear and nucleolar architecture during the lytic reactivation of KSHV. This was manifested by the redistribution of key nucleolar proteins, including the rRNA transcription factor UBF. Distinct delocalization patterns were evident; certain nucleolar proteins remained together whereas others dissociated, implying that nucleolar proteins undergo nonrandom programmed dispersion. Significantly, the redistribution of UBF was dependent on viral DNA replication or late viral gene expression. No significant changes in pre-rRNA levels and no accumulation of pre-rRNA intermediates were found by RT-qPCR and Northern blot analysis. Furthermore, fluorescent in situ hybridization (FISH), combined with immunofluorescence, revealed an overlap between Fibrillarin and internal transcribed spacer 1 (ITS1), which represents the primary product of the pre-rRNA, suggesting that the processing of rRNA proceeds during lytic reactivation. Finally, small changes in the levels of pseudouridylation (Ψ) and 2'-O-methylation (Nm) were documented across the rRNA; however, none were localized to the functional domain. Taken together, our results suggest that despite dramatic changes in the nucleolar organization, rRNA transcription and processing persist during lytic reactivation of KSHV. Whether the observed nucleolar alterations favor productive infection or signify cellular anti-viral responses remains to be determined.

An Engineered Nanocomplex with Photodynamic and Photothermal Synergistic Properties for Cancer Treatment.

Photodynamic therapy (PDT) and photothermal therapy (PTT) are promising therapeutic methods for cancer treatment; however, as single modality therapies, either PDT or PTT is still limited in its success rate. A dual application of both PDT and PTT, in a combined protocol, has gained immense interest. In this study, gold nanoparticles (AuNPs) were conjugated with a PDT agent, meso-tetrahydroxyphenylchlorin (mTHPC) photosensitizer, designed as nanotherapeutic agents that can activate a dual photodynamic/photothermal therapy in SH-SY5Y human neuroblastoma cells. The AuNP-mTHPC complex is biocompatible, soluble, and photostable. PDT efficiency is high because of immediate reactive oxygen species (ROS) production upon mTHPC activation by the 650-nm laser, which decreased mitochondrial membrane potential (∆ψm). Likewise, the AuNP-mTHPC complex is used as a photoabsorbing (PTA) agent for PTT, due to efficient plasmon absorption and excellent photothermal conversion characteristics of AuNPs under laser irradiation at 532 nm. Under the laser irradiation of a PDT/PTT combination, a twofold phototoxicity outcome follows, compared to PDT-only or PTT-only treatment. This indicates that PDT and PTT have synergistic effects together as a combined therapeutic method. Our study aimed at applying the AuNP-mTHPC approach as a potential treatment of cancer in the biomedical field.

The Effect of Dehydroepiandrosterone Treatment on Neurogenesis, Astrogliosis and Long-Term Cocaine-Seeking Behavior in a Cocaine Self-Administration Model in Rats.

Cocaine addiction is an acquired behavioral state developed in vulnerable individuals after cocaine exposure. It is characterized by compulsive drug-seeking and high vulnerability to relapse even after prolonged abstinence, associated with decreased neurogenesis in the hippocampus. This addictive state is hypothesized to be a form of "memory disease" in which the drug exploits the physiological neuroplasticity mechanisms that mediate regular learning and memory processes. Therefore, a major focus of the field has been to identify the cocaine-induced neuroadaptations occurring in the usurped brain's reward circuit. The neurosteroid dehydroepiandrosterone (DHEA) affects brain cell morphology, differentiation, neurotransmission, and memory. It also reduces drug-seeking behavior in an animal model of cocaine self-administration. Here, we examined the long-lasting effects of DHEA treatment on the attenuation of cocaine-seeking behavior. We also examined its short- and long-term influence on hippocampal cells architecture (neurons and astrocytes). Using a behavioral examination, immunohistochemical staining, and diffusion tensor imaging, we found an immediate effect on tissue density and activation of astrocytes, which has a continuous beneficial effect on neurogenesis and tissue organization. This research emphasizes the requites concert between astrocytes and neurons in the rehabilitation from addiction behavior. Thus, DHEA may serve as a treatment that corrects brain damage following exposure to and abstinence from cocaine.

Flatfoot in Africa, the cirripede Chthamalus in the west Indian Ocean.

Barnacles of the genus Chthamalus are commonly encountered rocky intertidal shores. The phylogeography of the different species in the Western Indian Ocean is unclear. Using morphological characteristics as well as the molecular markers mitochondrial cytochrome oxygenase subunit I (COI) and the nuclear sodium-potassium ATPase (NaKA), we identified four clades representing four species in the Western Indian Ocean and its adjacent seas. Among these species, a newly identified species, Chthamalus barilani, which was found in Madagascar, Zanzibar and Tanzania. Chthamalus from the coasts of Tanzania and Zanzibar is identified morphologically as C. malayensis, and clusters with C. malayensis from the Western Pacific and the Indo Malayan regions. C. malayensis is regarded as a group of four genetically differentiated clades representing four cryptic species. The newly identified African clade is genetically different from these clades and the pairwise distances between them justify the conclusion that it is an additional cryptic species of C. malayensis. This type of genetic analyses offers an advantage over morphological characterization and allowed us to reveal that another species, C. barnesi, which is known from the Red Sea, is also distributed in the Arabian Sea and the Persian Gulf. We could also confirm the presence of the South African species C. dentatus in the Mozambique channel. This represents the Northeastern limit of C. dentatus, which is usually distributed along the coast of southern Africa up to the Islands of Cape Verde in West Africa. Altogether, based on a combination of morphology and genetics, we distinct between four clusters of Chthamalus, and designate their distribution in the West Indian Ocean. These distinctions do not agree with the traditional four groups reported previously based merely on morphological data. Furthermore, these findings underline the importance of a combining morphological and genetics tools for constructing barnacle taxonomy.

Reduction of DNMT3a and RORA in the nucleus accumbens plays a causal role in post-traumatic stress disorder-like behavior: reversal by combinatorial epigenetic therapy.

Post-traumatic stress disorder (PTSD) is an incapacitating trauma-related disorder, with no reliable therapy. Although PTSD has been associated with epigenetic alterations in peripheral white blood cells, it is unknown where such changes occur in the brain, and whether they play a causal role in PTSD. Using an animal PTSD model, we show distinct DNA methylation profiles of PTSD susceptibility in the nucleus accumbens (NAc). Data analysis revealed overall hypomethylation of different genomic CG sites in susceptible animals. This was correlated with the reduction in expression levels of the DNA methyltransferase, DNMT3a. Since epigenetic changes in diseases involve different gene pathways, rather than single candidate genes, we next searched for pathways that may be involved in PTSD. Analysis of differentially methylated sites identified enrichment in the RAR activation and LXR/RXR activation pathways that regulate Retinoic Acid Receptor (RAR) Related Orphan Receptor A (RORA) activation. Intra-NAc injection of a lentiviral vector expressing either RORA or DNMT3a reversed PTSD-like behaviors while knockdown of RORA and DNMT3a increased PTSD-like behaviors. To translate our results into a potential pharmacological therapeutic strategy, we tested the effect of systemic treatment with the global methyl donor S-adenosyl methionine (SAM), for supplementing DNA methylation, or retinoic acid, for activating RORA downstream pathways. We found that combined treatment with the methyl donor SAM and retinoic acid reversed PTSD-like behaviors. Thus, our data point to a novel approach to the treatment of PTSD, which is potentially translatable to humans.

Cumulative IVF outcomes after retrieval of testicular spermatozoa: should we use immotile spermatozoa for ICSI?

RESEARCH QUESTION: What are the cumulative clinical pregnancy rates (CPR) and live births rates (LBR) in intracytoplasmic sperm injection (ICSI) cycles using testicular motile compared with immotile spermatozoa, obtained from testicular sperm aspiration (TESA) or extraction (TESE)? DESIGN: A retrospective analysis of ICSI cycles using TESA or TESE over a period of 7 years. Cycles were divided into two groups according to the motility of the retrieved spermatozoa: Group A consisted of couples with motile spermatozoa; Group B of couples with immotile spermatozoa. Group B was subdivided into two groups: B1 consisted of couples with motile spermatozoa and B2 with immotile spermatozoa after the addition of pentoxifylline. RESULTS: No differences in CPR and LBR per transfer was found between the study groups after fresh embryo transfer. No pregnancies were achieved by vitrified-warmed embryo transfer in group B2. Fertilization rates decreased when using immotile spermatozoa (64.4%, 56%, 37.9%, for groups A, B1 and B2, respectively, P < 0.001). Top-quality embryo rates were higher in groups A and B1 compared with B2 (40.7% and 40.1% versus 19.1%, respectively, P = 0.015). Cumulative CPR (53%, 41.7%, 13.6% for groups A, B1 and B2, respectively, P = 0.005) and LBR (42.4%, 30%, 13.6% for groups A, B1 and B2, respectively P = 0.03) per oocyte retrieval was significantly higher when using motile spermatozoa compared with motile or immotile spermatozoa after adding pentoxifylline. CONCLUSIONS: Although fertilization, top-quality embryo rates, cumulative CPR and LBR decreased when using immotile spermatozoa, ICSI is still valid; therefore, it should be considered and offered to couples before embarking on a donor sperm insemination cycle, or cryopreserving oocytes for future additional testicular sperm retrieval.

TMF1 is upregulated by insulin and is required for a sustained glucose homeostasis.

Insulin-regulated glucose homeostasis is a critical and intricate physiological process, of which not all regulatory components have been deciphered. One of the key players in modulating glucose uptake by cells is the glucose transporter-GLUT4. In this study, we aimed to explore the regulatory role of the trans-Golgi-associated protein-TATA Element Modulatory Factor (TMF1) in the GLUT4 mediated, insulin-directed glucose uptake. By establishing and using TMF1-/- myoblasts and mice, we examined the effect of TMF1 absence on the insulin driven functioning of GLUT4. We show that TMF1 is upregulated by insulin in myoblasts, and is essential for the formation of insulin responsive, glucose transporter GLUT4-containing vesicles. Absence of TMF1 leads to the retention of GLUT4 in perinuclear compartments, and to severe impairment of insulin-stimulated GLUT4 trafficking throughout the cytoplasm and to the cell plasma membrane. Accordingly, glucose uptake is impaired in TMF1-/- cells, and TMF1-/- mice are hyperglycemic. This is reflected by the mice impaired blood glucose clearance and increased blood glucose level. Correspondingly, TMF1-/- animals are leaner than their normal littermates. Thus, TMF1 is a novel effector of insulin-regulated glucose homeostasis, and dys-functioning of this protein may contribute to the onset of a diabetes-like disorder.

Entrapment and release kinetics study of dyes from BSA microspheres forming a matrix and a reservoir system.

Two kinds of Bovine Serum Albumin (BSA)-loaded microspheres were prepared in water-organic bilayer systems using ultrasonic irradiation. The first method included an aqueous solution of BSA and water-soluble dye together, mixed with dodecane, that upon sonication formed a matrix system where the dye is concentrated in the protein shell. The other system included an aqueous solution of BSA mixed with octanol-soluble dye that, upon sonication, formed a reservoir system in which the dye filled the inner volume of the microspheres. Each of these microspheres was prepared with two different dyes and their leaching profiles into pure solvents were studied using UV-vis spectrometry. Fast leaching was observed at the beginning for both systems, which levelled-off after a certain time. For the matrix system, an equilibrium state was obtained after 100-200 hours, whereas for the reservoir system, leaching occurred much faster, within 1-3 hours. Such systems can serve as models for drug delivery agents.

A single nucleotide variant of human PARP1 determines response to PARP inhibitors.

The introduction of novel cancer drugs and innovative treatments brings great hope for cancer patients, but also an urgent need to match drugs to suitable patients, since certain drugs that benefit one patient may actually harm others. The newly developed poly-ADP ribose polymerase (PARP) inhibitors (PARPis) are a group of pharmacological enzyme inhibitors used clinically for multiple indications. Several forms of cancer tend to be PARP dependent, making PARP an attractive target for cancer therapy. Specifically, PARPis are commonly used in BRCA-associated breast cancers patients, since unrepaired single-strand breaks are converted into double-strand breaks and BRCA-associated tumors cannot repair them by homologous recombination so that PARPi leads to tumor cell death, by a mechanism called "Synthetic Lethality". Unfortunately, not all patients respond to PARPi, and it is not currently possible to predict who will or will not respond. Here, we present a specific genomic marker, which reflects a single-nucleotide polymorphism of human PARP1 and correlates in vitro with response to PARPi, throughout all indications. In addition, we report that this SNP is associated with re-shaping mRNA, and mRNA levels, and influences the final protein structure to expose new binding sites while hiding others. The status of the SNP is therefore critical to patients' care, as it relates responses to PARPi to the PARP1-SNP carried.

A Lipophilic 4-Phenylbutyric Acid Derivative That Prevents Aggregation and Retention of Misfolded Proteins.

Chemical chaperones prevent protein aggregation. However, the use of chemical chaperones as drugs against diseases due to protein aggregation is limited by the very high active concentrations (mm range) required to mediate their effect. One of the most common chemical chaperones is 4-phenylbutyric acid (4-PBA). Despite its unfavorable pharmacokinetic properties, 4-PBA was approved as a drug to treat ornithine cycle diseases. Here, we report that 2-isopropyl-4-phenylbutanoic acid (5) has been found to be 2-10-fold more effective than 4-PBA in several in vitro models of protein aggregation. Importantly, compound 5 reduced the secretion rate of autism-linked Arg451Cys Neuroligin3 (R451C NLGN3).

Proliferation of Inhibitory Input to the Substantia Nigra in Experimental Parkinsonism.

The substantia nigra pars reticulata (SNr) is one of the output nuclei of the basal ganglia (BG) and plays a vital role in movement execution. Death of dopaminergic neurons in the neighboring nucleus, the substantia nigra pars compacta (SNc), leads to Parkinson's disease. The ensuing dopamine depletion affects all BG nuclei. However, the long-term effects of dopamine depletion on BG output are less characterized. In this in vitro study, we applied electrophysiological and immunohistochemical techniques to investigate the long-term effects of dopamine depletion on GABAergic transmission to the SNr. The findings showed a reduction in firing rate and regularity in SNr neurons after unilateral dopamine depletion with 6-OHDA, which we associate with homeostatic mechanisms. The strength of the GABAergic synapses between the globus pallidus (GP) and the SNr increased but not their short-term dynamics. Consistent with this observation, there was an increase in the frequency and amplitude of spontaneous inhibitory synaptic events to SNr neurons. Immunohistochemistry revealed an increase in the density of vGAT-labeled puncta in dopamine depleted animals. Overall, these results may suggest that synaptic proliferation can explain how dopamine depletion augments GABAergic transmission in the SNr.

Imaging within single NPCs reveals NXF1's role in mRNA export on the cytoplasmic side of the pore.

Translocation of mRNA through the nuclear pore complex (NPC) requires interactions with different NPC regions. To determine the interactions that are crucial for effective mRNA export in living cells, we examined mRNA export within individual pores by applying various types of mRNA export blocks that stalled mRNPs at different stages of transition. Focusing on the major mRNA export factor NXF1, we found that initial mRNP binding to the NPC did not require NXF1 in the NPC, whereas release into the cytoplasm did. NXF1 localization in the NPC did not require RNA or RNA binding. Superresolution microscopy showed that NXF1 consistently occupied positions on the cytoplasmic side of the NPC. Interactions with specific nucleoporins were pinpointed using FLIM-FRET for measuring protein-protein interactions inside single NPCs, showing that Dbp5 helicase activity of mRNA release is conserved in yeast and humans. Altogether, we find that specific interactions on the cytoplasmic side of the NPC are fundamental for the directional flow of mRNA export.

Nanostructures, Faceting, and Splitting in Nanoliter to Yoctoliter Liquid Droplets.

Contrary to everyday experience, where all liquid droplets assume rounded, near-spherical shapes, the temperature-tuning of liquid droplets to faceted polyhedral shapes and to spontaneous splitting has been recently demonstrated in oil-in-water emulsions. However, the elucidation of the mechanism driving these surprising effects, as well as their many potential applications, ranging from faceted nanoparticle synthesis through new industrial emulsification routes to controlled-release drug delivery within the human body, have been severely hampered by the micron-scale resolution of the light microscopy employed to date in all in situ studies. Thus, the thickness of the interfacially frozen crystalline monolayer, suggested to drive these effects, could not be directly measured, and the low limit on the droplet size still showing these effects remained unknown. In this study, we employ a combination of super-resolution stimulated emission depletion microscopy, cryogenic transmission and freeze-fracture electron microscopy, to study these effects well into the nanometer length scale. We demonstrate the occurrence of the faceting transition in droplets spanning an incredible 12 decades in volume from nanoliters to yoctoliters and directly visualize the interfacially frozen, few nanometer thick, crystalline monolayer suggested to drive these effects. Furthermore, our measurements allow placing an upper-limit estimate on the two-dimensional Young modulus of the interfacial nanometer-thick surface crystal in the smallest droplets, providing insights into the virtually unexplored domain of nanoelasticity.

ARTS mediates apoptosis and regeneration of the intestinal stem cell niche.

Stem cells (SCs) play a pivotal role in fueling homeostasis and regeneration. While much focus has been given to self-renewal and differentiation pathways regulating SC fate, little is known regarding the specific mechanisms utilized for their elimination. Here, we report that the pro-apoptotic protein ARTS (a Septin4 isoform) is highly expressed in cells comprising the intestinal SC niche and that its deletion protects Lgr5+ and Paneth cells from undergoing apoptotic cell death. As a result, the Sept4/ARTS-/- crypt displays augmented proliferation and, in culture, generates massive cystic-like organoids due to enhanced Wnt/ß-catenin signaling. Importantly, Sept4/ARTS-/- mice exhibit resistance against intestinal damage in a manner dependent upon Lgr5+ SCs. Finally, we show that ARTS interacts with XIAP in intestinal crypt cells and that deletion of XIAP can abrogate Sept4/ARTS-/--dependent phenotypes. Our results indicate that intestinal SCs utilize specific apoptotic proteins for their elimination, representing a unique target for regenerative medicine.

Uptake mechanism of metabolic-targeted gold nanoparticles.

AIM: To elucidate the interactions, uptake mechanisms and cytotoxicity profile of glucose-functionalized gold nanoparticles (2GF-GNPs), for expanding and advancing the recently proposed technology of metabolic-based cancer detection to a variety of cancer diseases. METHODS: Several cell types with different metabolic features were used to assess the involvement of GLUT-1 and different endocytosis pathways in 2GF-GNP uptake, and the cytotoxicity profile of 2GF-GNPs. RESULTS: Cellular uptake of 2GF-GNP strongly correlated with GLUT-1 surface expression, and occurred mainly through clathrin-mediated endocytosis. 2GF-GNPs showed no toxic effect on cell cycle and proliferation. CONCLUSION: These findings promote development of metabolic-based cancer detection technologies, and suggest that 2GF-GNPs may enable specific cancer detection in a wide range of tumors characterized by high GLUT-1 expression.

Quantifying mRNA targeting to P-bodies in living human cells reveals their dual role in mRNA decay and storage.

The 5'-to-3' mRNA degradation machinery localizes to cytoplasmic processing bodies (P-bodies), which are non-membranous structures found in all eukaryotes. Although P-body function has been intensively studied in yeast, less is known about their role in mammalian cells, such as whether P-body enzymes are actively engaged in mRNA degradation or whether P-bodies serve as mRNA storage depots, particularly during cellular stress. We examined the fate of mammalian mRNAs in P-bodies during translational stress, and show that mRNAs accumulate within P-bodies during amino acid starvation. The 5' and 3' ends of the transcripts residing in P-bodies could be identified, but poly(A) tails were not detected. Using the MS2 mRNA-tagging system for mRNA visualization in living cells, we found that a stationary mRNA population formed in P-bodies during translational stress, which cleared gradually after the stress was relieved. Dcp2-knockdown experiments showed that there is constant degradation of part of the P-body-associated mRNA population. This analysis demonstrates the dual role of P-bodies as decay sites and storage areas under regular and stress conditions.