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PARP inhibitors (PARPis) have changed the treatment paradigm inBRCA-mutant high-grade serous ovarian carcinoma (HGSC). However, most patients eventually develop resistance to PARPis, highlighting an unmet need for novel therapeutic strategies. Using high-throughput drug screens, we identified ATR/CHK1 pathway inhibitors as cytotoxic, and further validated monotherapy activity of the CHK1 inhibitor (CHK1i), prexasertib, in PARPi-resistantBRCA-mutant HGSC cells and animal models. As a proof-of-concept trial, we conducted a phase II study of prexasertib inBRCA-mutant HGSC patients. The treatment was well-tolerated but yielded an objective response rate of 6% (1/17; 1 PR) in patients with prior PARPi treatment. Exploratory biomarker analyses revealed that replication stress and fork stabilization were associated with clinical benefit to CHK1i. In particular, overexpression ofBLM, andCCNE1overexpression or copy number gain/amplification were seen in patients deriving durable benefit from CHK1i. Our findings suggest replication fork–related biomarkers should be further evaluated for CHK1i sensitivity in HGSC.One Sentence SummaryOverexpression of RecQ helicase BLM is a predictive biomarker for CHK1 inhibitor response in PARP inhibitor–resistantBRCA-mutant ovarian cancer.

Summary The PALM trial in the Democratic Republic of the Congo identified a statistically significant survival benefit for two monoclonal antibody-based therapeutics in the treatment of acute Ebola virus disease; however, substantial gaps remain in improving the outcomes of acute Ebola virus disease and for the survivors. Ongoing efforts are needed to develop more effective strategies, particularly for individuals with severe disease, for prevention and treatment of viral persistence in immune-privileged sites, for optimisation of post-exposure prophylaxis, and to increase therapeutic breadth. As antibody-based approaches are identified and advanced, promising small-molecule antivirals currently in clinical stage development should continue to be evaluated for filovirus diseases, with consideration of their added value in combination approaches with bundled supportive care, their penetration in tissues of interest, the absence of interaction with glycoprotein-based vaccines, and filoviral breadth.

Background: Acquired resistance to PARP inhibitors (PARPis) is a pressing problem in high-grade serous ovarian cancer (HGSC), highlighting the development of novel therapeutic options. To address this unmet need, we conducted high-throughput drug combination screens in PARPi-resistant HGSC cells using ATR inhibitor (ATRi), given that ATR/CHK1 signaling activation is one of the main mechanisms of PARPi resistance. In the screens, PI3K/AKT pathway inhibitors were synergistic with ATRi. Mechanistically, ATR signaling plays a major role in R-loop dynamics and replication stress (RS) response pathways, but the role of AKT is unclear. Here, we aim to investigate AKT’s role in R-loop-mediated RS in PARPi-resistant HGSC cells. We also hypothesize that AKT inhibitor (AKTi) would augment ATRi-induced R-loop formation by reducing the expression of helicases. Methods: We used PARPi-resistant variants and parental PEO1 as a negative control. ATRi (ceralasertib) and AKTi (capivasertib) were used for further mechanistic studies. Induction of RS, DNA double-strand break, and R-loop were evaluated using immunofluorescence staining for markers pRPA, γH2AX, and S9.6, respectively. We measured multiple nuclear helicases and in situ DHX9-R-loop interaction by western blot and proximity ligation assay. DHX9-AKT1 interaction was confirmed by co-immunoprecipitation. All data were repeated in triplicate, analyzed using one-way ANOVA test, and shown as mean ± SD. P < 0.05 was considered statistically significant. Results: The ATRi and AKTi combination induced greater RS, as evidenced by a 1.7-12.0-fold increase in pRPA+/γH2AX+ population relative to each drug alone. Adding AKTi to ATRi significantly increased R-loops compared to monotherapy alone (signal intensity 14-19 x 105 vs. 9.9-14.6 x 105, P < 0.001), suggesting that AKT also contributes to R-loop dissolution. Moreover, the combination showed a higher percentage of cells with γH2AX and R-loops relative to monotherapy (54.7-75.4% vs. 33.3-49.0%, P < 0.001) in the presence of pan-caspase inhibitor, indicating the increased DNA damage is unlikely caused by increased apoptosis but by R-loop accumulation. Also, AKTi addition to ATRi significantly reduced the nuclear DHX9 among other helicases. Moreover, we found that AKT1 directly binds with DHX9 via its kinase domain and couples with DHX9 to resolve R-loops. Conclusion: Our data suggest ATRi and AKTi combination could be a new therapeutic combination in HGSC with PARPi resistance. Mechanistically, our data reveal a previously unknown direct role of AKT1 and its interaction with DHX9 in R-loop resolution besides ATR. Citation Format: Tzu-Ting Huang, Chih-Yuan Chiang, Jayakumar R. Nair, Chi-Ting Shih, Kelli Wilson, Ken Chih-Chien Cheng, Jung-Min Lee. AKT1 prevents aberrant R-loops accumulation via regulating DHX9 and suppresses transcription-replication collision in PARP inhibitor-resistant ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6173.

Current therapies for anthrax include the use of antibiotics (i.e., doxycycline, and ciprofloxacin), an anthrax vaccine (BioThrax) and

Poly (ADP-ribose) polymerase inhibitors (PARPis) have changed the treatment paradigm in the management of high-grade serous ovarian cancer (HGSOC). However, most HGSOC ultimately develops resistance to PARPi, leading to unmet medical needs for better therapeutic strategies. ATR-mediated G2/M cell cycle checkpoints are important for cell survival and DNA repair, especially in HGSOC due to its dysfunctional p53-mediated G1/S checkpoint, making them an attractive therapeutic target. However, only modest monotherapy activity of ATR inhibitors (ATRis) has been reported in patients with HGSOC, highlighting the need for combination treatment. We conducted a high-throughput drug combination screen of ATRi (ceralasertib) with 1,912 drugs in two PARPi-resistant BRCA2-mutant PEO1 (PEO1/OlaR and PEO1/OlaJR) HGSOC cell lines. In this screen, 234 drugs showed synergistic cytotoxicity with ATRi, including 31 (13.2%) cell cycle checkpoint inhibitors, 27 (11.5%) chemotherapeutic drugs, and 13 (5.6%) PI3K/AKT pathway inhibitors. We prioritized the PI3K/AKT pathway inhibitors for ATRi combination because the PI3K/AKT pathway is upregulated in >70% of HGSOC and associated with an aggressive phenotype. Moreover, PI3K/AKT signaling is found to be activated in HGSOC cells with PARPi resistance. We separately validated that an ATRi (ceralasertib < 1 μM) in combination with an AKT inhibitor (AKTi; capivasertib < 10 μM) yielded additive/synergistic cytotoxic effects (combination index < 1) in various HGSOC cell lines, including acquired and de novo PARPi-resistant (PEO1/OlaR, PEO1/OlaJR, and PEO4) and platinum-resistant HGSOC cell lines (OVCAR3), by XTT and colony formation assays. With clinically attainable concentrations of ATRi (1 μM) and AKTi (10 μM), the combination treatment increased cell apoptosis compared to ATRi (increased 2.1 to 95.2-fold; P < 0.001) or AKTi alone (increased 3.3 to 92.2-fold; P < 0.001) in both PARPi-sensitive and -resistant HGSOC cells. ATRi+AKTi treatment also showed greater DNA damage as evidenced by increased percentage of cells with ≥ 5 γH2AX foci relative to ATRi (increased 1.1 to 7.6-fold; not significant in PEO1 and P < 0.001 in PARPi-resistant cells) or AKTi (increased 2.3 to 6.2-fold; P < 0.001) in both PARPi-sensitive and -resistant cells. Furthermore, ATRi+AKTi treatment augmented replication stress as measured by increased phospho-RPA+/γH2AX+ populations compared with ATRi (increased 1.7 to 4.7-fold; P < 0.001) or AKTi (increased 3.7 to 12.0-fold; P < 0.001). Overall, our results suggest that dual inhibition of ATR and AKT pathways results in greater cell death by increasing DNA damage and replication stress in HGSOC cells with PARPi resistance. Citation Format: Tzu-Ting Huang, Chih-Yuan Chiang, Nitasha Gupta, Kelli Wilson, Ken Chih-Chien Cheng, Jung-Min Lee. Targeting ATR and AKT pathways promotes cell death in PARP inhibitor-resistant ovarian cancer cells by increasing DNA damage and lethal replication stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5408.

Burkholderia mallei, the causative agent of glanders, is a gram-negative intracellular bacterium. Depending on different routes of infection, the disease is manifested by pneumonia, septicemia, and chronic infections of the skin. B. mallei poses a serious biological threat due to its ability to infect via aerosol route, resistance to multiple antibiotics and to date there are no US Food and Drug Administration (FDA) approved vaccines available. Induction of innate immunity, inflammatory cytokines and chemokines following B. mallei infection, have been observed in in vitro and small rodent models; however, a global characterization of host responses has never been systematically investigated using a non-human primate (NHP) model. Here, using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach, we identified alterations in expression levels of host proteins in peripheral blood mononuclear cells (PBMCs) originating from naïve rhesus macaques (Macaca mulatta), African green monkeys (Chlorocebus sabaeus), and cynomolgus macaques (Macaca fascicularis) exposed to aerosolized B. mallei. Gene ontology (GO) analysis identified several statistically significant overrepresented biological annotations including complement and coagulation cascade, nucleoside metabolic process, vesicle-mediated transport, intracellular signal transduction and cytoskeletal protein binding. By integrating an LC-MS/MS derived proteomics dataset with a previously published B. mallei host-pathogen interaction dataset, a statistically significant predictive protein-protein interaction (PPI) network was constructed. Pharmacological perturbation of one component of the PPI network, specifically ezrin, reduced B. mallei mediated interleukin-1β (IL-1β). On the contrary, the expression of IL-1β receptor antagonist (IL-1Ra) was upregulated upon pretreatment with the ezrin inhibitor. Taken together, inflammasome activation as demonstrated by IL-1β production and the homeostasis of inflammatory response is critical during the pathogenesis of glanders. Furthermore, the topology of the network reflects the underlying molecular mechanism of B. mallei infections in the NHP model.

Research to discover and develop antibacterial and antiviral drugs with potent activity against pathogens of biothreat concern presents unique methodological and process-driven challenges. Herein, we review laboratory approaches for finding new antibodies, antibiotics, and antiviral molecules for pathogens of biothreat concern. Using high-throughput screening techniques, molecules that directly inhibit a pathogen’s entry, replication, or growth can be identified. Alternatively, molecules that target host proteins can be interesting targets for development when countering biothreat pathogens, due to the modulation of the host immune response or targeting proteins that interfere with the pathways required by the pathogen for replication. Monoclonal and cocktail antibody therapies approved by the Food and Drug Administration for countering anthrax and under development for treatment of Ebola virus infection are discussed. A comprehensive tabular review of current in vitro, in vivo, pharmacokinetic and efficacy datasets has been presented for biothreat pathogens of greatest concern. Finally, clinical trials and animal rule or traditional drug approval pathways are also reviewed. Opinions; interpretations; conclusions; and recommendations are those of the authors and are not necessarily endorsed by the US Army.

SummaryPathogens commonly utilize endocytic pathways to gain cellular access. The endosomal pattern recognition receptors TLR7 and TLR9 detect pathogen-encoded nucleic acids to initiate MyD88-dependent proinflammatory responses to microbial infection. Using genome-wide RNAi screening and integrative systems-based analysis, we identify 190 cofactors required for TLR7- and TLR9-directed signaling responses. A set of cofactors were crossprofiled for their activities downstream of several immunoreceptors and then functionally mapped based on the known architecture of NF-κB signaling pathways. Protein complexes and pathways involved in ubiquitin-protein ligase activities, sphingolipid metabolism, chromatin modifications, and ancient stress responses were found to modulate innate recognition of endosomal nucleic acids. Additionally, hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) was characterized as necessary for ubiquitin-dependent TLR9 targeting to the endolysosome. Proteins and pathways identified here should prove useful in delineating strategies to manipulate innate responses for treatment of autoimmune disorders and microbial infection.

Mitogen-activated protein kinase (MAPK) pathways form the backbone of signal transduction in the mammalian cell. Here we applied a systematic experimental and computational approach to map 2,269 interactions between human MAPK-related proteins and other cellular machinery and to assemble these data into functional modules. Multiple lines of evidence including conservation with yeast supported a core network of 641 interactions. Using small interfering RNA knockdowns, we observed that approximately one-third of MAPK-interacting proteins modulated MAPK-mediated signaling. We uncovered the Na-H exchanger NHE1 as a potential MAPK scaffold, found links between HSP90 chaperones and MAPK pathways and identified MUC12 as the human analog to the yeast signaling mucin Msb2. This study makes available a large resource of MAPK interactions and clone libraries, and it illustrates a methodology for probing signaling networks based on functional refinement of experimentally derived protein-interaction maps.

SummaryGlioblastoma multiforme (GBM) is a highly aggressive form of brain cancer associated with a very poor prognosis. Recently, the initiation and growth of GBM has been linked to brain tumor-initiating cells (BTICs), which are poorly differentiated and share features with neural stem cells (NSCs). Here we describe a kinome-wide RNA interference screen to identify factors that control the tumorigenicity of BTICs. We identified several genes whose silencing induces differentiation of BTICs derived from multiple GBM patients. In particular, knockdown of the adaptor protein TRRAP significantly increased differentiation of cultured BTICs, sensitized the cells to apoptotic stimuli, and negatively affected cell cycle progression. TRRAP knockdown also significantly suppressed tumor formation upon intracranial BTIC implantation into mice. Together, these findings support a critical role for TRRAP in maintaining a tumorigenic, stem cell-like state.

Whether monoamine oxidase inhibitors (MAOIs) can be used to suppress the reinforcing effect of cocaine remains unknown. This study was undertaken to examine effects of a long-term dosing regimen with selective MAOIs on cocaine and food reward.Since single dose of clorgyline (2 mg/kg), deprenyl (1 mg/kg), and pargyline (10 mg/kg) did not acutely affect mouse locomotor activity, these doses were chosen to treat the male C57BL/6j mice on a daily basis.Fourteen consecutive days of pretreatments with clorgyline, deprenyl, or pargyline (one injection per day) did not affect natural reward-supported operant behavior, since acquisition of the lever pressing responses for food pellets under an FR-1 protocol did not differ among these drug- and saline-treated mice. Likewise, 24 consecutive days of pretreatments with clorgyline did not alter acquisition of the cocaine (0.3 mg/kg per infusion)-supported operant responses under an FR-1 protocol. In contrast, 24 days of pretreatments with deprenyl and pargyline abolished the cocaine-supported operant responses under a similar protocol. Twenty-four days of clorgyline treatment enhanced serotonin contents in striatum, nucleus accumbens, and frontal cortex. Frontal cortical 3,4-dihydroxyphenylacetic acid and 5-hydroxyindoleacidic acid concentrations were decreased following 24 days of pretreatments with deprenyl and pargyline. These changes were not evident in mice pretreated with clorgyline.We suggest that long-term treatments with MAO-B inhibitors may decrease cocaine-supported operant responses in cocaine-naïve mice by selectively decreasing frontal cortical metabolism of dopamine and serotonin.

Background Burkholderia pseudomallei (Bp) and Burkholderia mallei (Bm) are Gram-negative facultative intracellular pathogens, which are the causative agents of melioidosis and glanders, respectively. Depending on the route of exposure, aerosol or transcutaneous, infection by Bp or Bm can result in an extensive range of disease – from acute to chronic, relapsing illness to fatal septicemia. Both diseases are associated with difficult diagnosis and high fatality rates. About ninety five percent of patients succumb to untreated septicemic infections and the fatality rate is 50 % even when standard antibiotic treatments are administered. Results The goal of this study is to profile murine macrophage-mediated phenotypic and molecular responses that are characteristic to a collection of Bp, Bm, Burkholderia thailandensis (Bt) and Burkholderia oklahomensis (Bo) strains obtained from humans, animals, environment and geographically diverse locations. Burkholderia spp. (N = 21) were able to invade and replicate in macrophages, albeit to varying degrees. All Bp (N = 9) and four Bm strains were able to induce actin polymerization on the bacterial surface following infection. Several Bp and Bm strains showed reduced ability to induce multinucleated giant cell (MNGC) formation, while Bo and Bp 776 were unable to induce this phenotype. Measurement of host cytokine responses revealed a statistically significant Bm mediated IL-6 and IL-10 production compared to Bp strains. Hierarchical clustering of transcriptional data from 84 mouse cytokines, chemokines and their corresponding receptors identified 29 host genes as indicators of differential responses between the Burkholderia spp. Further validation confirmed Bm mediated Il-1b, Il-10, Tnfrsf1b and Il-36a mRNA expressions were significantly higher when compared to Bp and Bt. Conclusions These results characterize the phenotypic and immunological differences in the host innate response to pathogenic and avirulent Burkholderia strains and provide insight into the phenotypic alterations and molecular targets underlying host-Burkholderia interactions. Electronic supplementary material The online version of this article (doi:10.1186/s12866-015-0593-3) contains supplementary material, which is available to authorized users.

Burkholderia is a diverse genus of Gram-negative bacteria that cause high mortality rate in humans and cattle. The lack of effective therapeutic treatments poses serious public health threats. Insights toward host-Burkholderia spp. interaction are critical in understanding the pathogenesis of the infection as well as identifying therapeutic targets for drug development. Reverse-phase protein microarray (RPMA) technology was previously proven to characterize novel biomarkers and molecular signatures associated with infectious diseases and cancers. In the present study, this technology was utilized to interrogate changes in host protein expression and post-translational phosphorylation events in macrophages infected with a collection of geographically diverse strains of Burkholderia spp. The expression or phosphorylation state of 25 proteins was altered during Burkholderia spp. infections and of which eight proteins were selected for further validation by immunoblotting. Kinetic expression patterns of phosphorylated AMPK-α1, Src, and GSK3β suggested the importance of their roles in regulating Burkholderia spp. mediated innate immune responses. Modulating inflammatory responses by perturbing AMPK-α1, Src, and GSK3β activities may provide novel therapeutic targets for future treatments.

We have examined the changes in cellular transcription resulting from infection with HIV-based vectors. Previous work suggested that the incoming viral genome may under some circumstances be detected as DNA damage, so to explore this possibility, we compared the transcriptional response to infection with an HIV-based vector to the response to treatment with the DNA-damaging agent etoposide. Expression levels of about 12,000 cellular RNA transcripts were determined in a human B-cell line at different times after either treatment. Statistical analysis revealed that the infection with the lentivirus vector resulted in quite modest changes in gene expression. Treatment with etoposide, in contrast, caused drastic changes in expression of genes known or inferred to be involved in apoptosis. Statistically significant though subtle parallels in the cellular transcriptional responses to etoposide treatment and HIV-vector infection could be detected. Several further data sets analyzing infections with HIV-based vectors or wild-type HIV-1 showed similar modest effects on cellular transcription and very modest parallels among different data sets. These findings establish that HIV-vector or HIV-1 infection has remarkably little effect on cellular transcription. The statistical methods described here may be of wide use in mining microarray data sets. Our observations support the idea that gene therapy with HIV-based vectors should not be particularly toxic to cells due to disruption of cellular transcription.

Chikungunya virus (CHIKV) is a re-emerging alphavirus which causes severe and prolonged arthralgic febrile illness. The recent global spread of the virus and lack of approved therapeutic options makes it imperative to gain greater insight into the molecular mechanisms underlying CHIKV pathogenesis, in particular host factors recruited by the virus. In the current study, we identify sphingosine kinase 2 (SK2) as a CHIKV host factor co-localized with the viral replication complex (VRC) during infection. SK2 was demonstrated to co-localize with viral RNA and nonstructural proteins. Targeted impairment of SK2 expression or function significantly inhibited CHIKV infection. Furthermore, affinity purification-mass spectrometry studies revealed that SK2 associates with a number of proteins involved in cellular gene expression specifically during viral infection, suggesting a role in replication. Collectively these results identify SK2 as a novel CHIKV host factor.

We describe a statistical analysis methodology designed to minimize the impact of off-target activities upon large-scale RNA interference (RNAi) screens in mammalian cells. Application of this approach enhances reconfirmation rates and facilitates the experimental validation of new gene activities through the probability-based identification of multiple distinct and active small interfering RNAs (siRNAs) targeting the same gene. We further extend this approach to establish that the optimal redundancy for efficacious RNAi collections is between 4-6 siRNAs per gene.

Alveolar macrophages (AMs) phagocytose Bacillus anthracis following inhalation and induce the production of pro-inflammatory cytokines and chemokines to mediate the activation of innate immunity. Ames, the virulent strain of B. anthracis, contains two plasmids that encode the antiphagocytic poly-γ-d-glutamic acid capsule and the lethal toxin. The attenuated Sterne strain of B. anthracis, which lacks the plasmid encoding capsule, is widely adapted as a vaccine strain. Although differences in the outcome of infection with the two strains may have originated from the presence or absence of an anti-phagocytic capsule, the disease pathogenesis following infection will be manifested via the host responses, which is not well understood. To gain understanding of the host responses at cellular level, a microarray analysis was performed using primary rhesus macaque AMs infected with either Ames or Sterne spores. Notably, 528 human orthologs were identified to be differentially expressed in AMs infected with either strain of the B. anthracis. Meta-analyses revealed genes differentially expressed in response to B. anthracis infection were also induced upon infections with multiple pathogens such as Francisella Novicida or Staphylococcus aureus. This suggests the existence of a common molecular signature in response to pathogen infections. Importantly, the microarray and protein expression data for certain cytokines, chemokines and host factors provide further insights on how cellular processes such as innate immune sensing pathways, anti-apoptosis versus apoptosis may be differentially modulated in response to the virulent or vaccine strain of B. anthracis. The reported differences may account for the marked difference in pathogenicity between these two strains.

Innate immune sensors such as Toll-like receptors (TLRs) differentially utilize adaptor proteins and additional molecular mediators to ensure robust and precise immune responses to pathogen challenge. Through a gain-of-function genetic screen, we identified the gamma catalytic subunit of protein phosphatase 1 (PP1-γ) as a positive regulator of MyD88-dependent proinflammatory innate immune activation. PP1-γ physically interacts with the E3 ubiquitin ligase TRAF6, and enhances the activity of TRAF6 towards itself and substrates such as IKKγ, whereas enzymatically inactive PP1-γ represses these events. Importantly, these activities were found to be critical for cellular innate responses to pathogen challenge and microbial clearance in both mouse macrophages and human monocyte lines. These data indicate that PP1-γ phosphatase activity regulates overall TRAF6 E3 ubiquitin ligase function and promotes NF-κB-mediated innate signaling responses.

Toll-like receptor 4 (TLR4) is unique among the TLRs in its use of multiple adaptor proteins leading to activation of both the interferon regulatory factor 3 (IRF3) and nuclear factor κB (NF-κB) pathways. Previous work has demonstrated that TLR4 initiates NF-κB activation from the plasma membrane, but that subsequent TLR4 translocation to the endosomes is required for IRF3 activation. Here we have characterized several components of the signaling pathway that governs TLR4 translocation and subsequent IRF3 activation. We find that phospholipase C γ2 (PLCγ2) accounts for LPS-induced inositol 1,4,5-trisphosphate (IP(3)) production and subsequent calcium (Ca(2+)) release. Blockage of PLCγ2 function by inhibitors or knockdown of PLCγ2 expression by siRNAs in RAW 264.7 macrophages lead to reduced IRF3, but enhanced NF-κB activation. In addition, bone marrow-derived macrophages from PLCγ2-deficient mice showed impaired IRF3 phosphorylation and expression of IRF3-regulated genes after LPS stimulation. Using cell fractionation, we show that PLCγ2-IP(3)-Ca(2+) signaling cascade is required for TLR4 endocytosis following LPS stimulation. In conclusion, our results describe a novel role of the PLCγ2-IP(3)-Ca(2+) cascade in the LPS-induced innate immune response pathway where release of intracellular Ca(2+) mediates TLR4 trafficking and subsequent activation of IRF3.

The purpose of the current study was to evaluate the effects of chronic administration of alcohol on the olfactory bulbectomy (OBX)-induced mouse-killing behavior (MKB), an animal model for screening antidepressants. The rats were divided into three groups, which were given alcohol (0, 0.5, or 1 g/kg/day) orally for 28 days. MKB was analyzed before and at the end of each week of the alcohol treatment. The results showed that chronic alcohol treatment produced a significant increase in the latency of MKB, implying that alcohol may have an antidepressant-like activity. This suggests that alcohol dependence or abuse in depressed patients may result from "self-medication". Since it has been reported that OBX causes a decrease in the density of N-methyl-D-aspartate (NMDA) receptors in the brain and that alcohol is a potent and selective inhibitor of NMDA receptors, the possible role of NMDA receptors in this effect is discussed.

Immunohistochemical Fos staining has proven to be a method to identify the neurons that are activated by stimulation. Although methamphetamine (MA)-conditioned place preference (CPP) memory was long-lasting, how this memory was established and retrieved remained unknown. We used the vehicle- and MA-conditioned environment (including cues and context) to reactivate the MA-CPP memory in mice. In the limbic system, Fos-positive neurons were examined following retrieval of the MA-CPP memory. We demonstrated that the current conditioning procedure produced reliable MA-CPP performance. Moreover, enhanced Fos expressions were found in the medial prefrontal cortex and the core of the nucleus accumbens after reactivation of the MA-CPP memory. Furthermore, familiarity with the environmental cues/context was found to significantly enhance Fos expressions in dorsal striatum and dentate gyrus. Nucleus accumbens shell, basolateral or lateral amygdala, in this regard, did not seem to be involved in retrieval of the MA-CPP memory. These results, taken together, suggest that the medial prefrontal cortex and the core of the nucleus accumbens are anatomical substrates responsible for reactivation of the MA-CPP memory.

SummaryHuman Immunodeficiency Viruses (HIV-1 and HIV-2) rely upon host-encoded proteins to facilitate their replication. Here, we combined genome-wide siRNA analyses with interrogation of human interactome databases to assemble a host-pathogen biochemical network containing 213 confirmed host cellular factors and 11 HIV-1-encoded proteins. Protein complexes that regulate ubiquitin conjugation, proteolysis, DNA-damage response, and RNA splicing were identified as important modulators of early-stage HIV-1 infection. Additionally, over 40 new factors were shown to specifically influence the initiation and/or kinetics of HIV-1 DNA synthesis, including cytoskeletal regulatory proteins, modulators of posttranslational modification, and nucleic acid-binding proteins. Finally, 15 proteins with diverse functional roles, including nuclear transport, prostaglandin synthesis, ubiquitination, and transcription, were found to influence nuclear import or viral DNA integration. Taken together, the multiscale approach described here has uncovered multiprotein virus-host interactions that likely act in concert to facilitate the early steps of HIV-1 infection.

Drug-associated cues are critical in reinstating the drug taking behavior even during prolonged abstinence and thus are thought to be a key factor to induce drug craving and to cause relapse. Amygdaloid complex has been known for its physiological function in mediating emotional experience storage and emotional cues-regulated memory retrieval. This study was undertaken to examine the role of basolateral nuclei of amygdala and the intracellular signaling molecule in drug cues-elicited cocaine memory retrieval. Systemic anisomycin treatment prior to the retrieval test abolished the cues-provoked cocaine conditioned place preference (CPP) memory. Likewise, a similar blockade of cues-provoked cocaine CPP performance was achieved by infusion of anisomycin and cycloheximide into the basolateral nuclei of amygdala before the test. Intra-amygdaloid infusion of H89, a protein kinase A inhibitor, or U0126, a MEK inhibitor, did not affect retrieval of the cues-elicited cocaine CPP memory. In contrast, intra-amygdaloid infusion of NPC 15437, a PKC inhibitor, abolished the cues-elicited cocaine CPP expression, while left the memory per se intact. Intra-amygdaloid infusion of NPC 15437 did not seem to affect locomotor activity or exert observable aversive effect. Taken together, our results suggest that activation of PKC signaling pathway and probably downstream de novo protein synthesis in the basolateral nuclei of amygdala is required for the cues-elicited cocaine memory performance. However, temporary inhibition of this signaling pathway does not seem to affect cocaine CPP memory per se.

A major goal of phytoremediation is to transform fast-growing plants with genes from plant species that hyperaccumulate toxic trace elements. We overexpressed the gene encoding selenocysteine methyltransferase (SMT) from the selenium (Se) hyperaccumulator Astragalus bisulcatus in Arabidopsis and Indian mustard (Brassica juncea). SMT detoxifies selenocysteine by methylating it to methylselenocysteine, a nonprotein amino acid, thereby diminishing the toxic misincorporation of Se into protein. Our Indian mustard transgenic plants accumulated more Se in the form of methylselenocysteine than the wild type. SMT transgenic seedlings tolerated Se, particularly selenite, significantly better than the wild type, producing 3- to 7-fold greater biomass and 3-fold longer root lengths. Moreover, SMT plants had significantly increased Se accumulation and volatilization. This is the first study, to our knowledge, in which a fast-growing plant was genetically engineered to overexpress a gene from a hyperaccumulator in order to increase phytoremediation potential.