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As the popularity of NAND flash expands in arenas from embedded systems to high-performance computing, a high-fidelity understanding of its specific properties becomes increasingly important. Further, with the increasing trend toward multiple-die, multiple-plane architectures and high-speed interfaces, flash memory systems are expected to continue to scale and cheapen, resulting in their broader proliferation. However, when designing NAND-based devices, making decisions about the optimal system configuration is nontrivial, because flash is sensitive to a number of parameters and suffers from inherent latency variations, and no available tools suffice for studying these nuances. The parameters include the architectures, such as multidie and multiplane, diverse node technologies, bit densities, and cell reliabilities. Therefore, we introduce NANDFlashSim, a high-fidelity, latency-variation-aware, and highly configurable NAND-flash simulator, which implements a detailed timing model for 16 state-of-the-art NAND operations. Using NANDFlashSim, we notably discover the following. First, regardless of the operation, reads fail to leverage internal parallelism. Second, MLC provides lower I/O bus contention than SLC, but contention becomes a serious problem as the number of dies increases. Third, many-die architectures outperform many-plane architectures for disk-friendly workloads. Finally, employing a high-performance I/O bus or an increased page size does not enhance energy savings. Our simulator is available at http://nfs.camelab.org.

Toxoplasma gondii, an intracellular protozoan parasite, is a major cause of opportunistic infectious disease affecting the brain and has been linked to an increased incidence of schizophrenia. In murine hosts, infection with T. gondii stimulates tryptophan degradation along the kynurenine pathway (KP), which contains several neuroactive metabolites, including 3-hydroxykynurenine (3-HK), quinolinic acid (QUIN) and kynurenic acid (KYNA). As these endogenous compounds may provide a mechanistic connection between T. gondii and the pathophysiology of schizophrenia, we measured KP metabolites in both the brain and periphery of T. gondii-treated C57BL/6 mice 8 and 28 days post-infection. Infected mice showed early decreases in the levels of tryptophan in the brain and serum, but not in the liver. These reductions were associated with elevated levels of kynurenine, KYNA, 3-HK and QUIN in the brain. In quantitative terms, the most significant increases in these KP metabolites were observed in the brain at 28 days post-infection. Notably, the anti-parasitic drugs pyrimethamine and sulfadiazine, a standard treatment of toxoplasmosis, significantly reduced 3-HK and KYNA levels in the brain of infected mice when applied between 28 and 56 days post-infection. In summary, T. gondii infection, probably by activating microglia and astrocytes, enhances the production of KP metabolites in the brain. However, during the first two months after infection, the KP changes in these mice do not reliably duplicate abnormalities seen in the brain of individuals with schizophrenia.

During brain disease, astrocytes can reprogram into a reactive state that alters many of their functions. Here, we present a protocol for studying neuroinflammation and reactive astrogliosis in mice using lipopolysaccharide (LPS) from E. coli. We describe steps for employing the Lcn2CreERT2 mouse crossed into a fluorescent Cre reporter line to label a subset of reactive astrocytes during and after inflammation. We then detail procedures for the longitudinal study of reactive astrocytes during the induction, progression, and/or resolution of astrogliosis. For complete details on the use and execution of this protocol, please refer to Agnew-Svoboda et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Excess nitrogen (N) loading to coastal ecosystems impairs estuarine water quality. Land management decisions made within estuarine watersheds have a direct impact on downstream N delivery. Natural features within watersheds can act as landscape sinks for N, such as wetlands, streams and ponds that transform dissolved N into gaseous N, effectively removing it from the aquatic system. Identifying and evaluating these landscape sinks and their spatial relationship to N sources can help managers understand the effects of alternative decisions on downstream resources., Methods: The N-Sink approach uses widely available GIS data to identify landscape sinks within HUC-12 (or larger) catchments, estimate their N removal potential and map the effect of those sinks on N movement through the catchment. Static maps are produced to visualize N removal efficiency, transport and delivery, the latter in the form of an index. The R package nsink was developed to facilitate data acquisition, processing and visualization., Results: nsink creates static maps for a specific HUC-12, or users can visit the University of Connecticut website to explore previously mapped areas. Users can investigate specific flowpaths interactively by clicking on any location within the catchment. A flowpath is generated with a table describing N removal along each segment. We describe the motivation behind developing nsink , discuss implementation in R, and present two use case examples. nsink is available from https://github.com/USEPA/nsink., Conclusions: N-Sink is a decision support tool created for local decision-makers and NGOs to facilitate better understanding of the relationship between land use and downstream N delivery. Local decision-makers that have prioritized N mitigation in their long-term planning can use nsink to better understand the potential impact of proposed development projects, zoning variances, and land acquisition or restoration. nsink also allows resource economists to investigate the tradeoffs among different, often costly, N reduction strategies., Competing Interests: No competing interests were disclosed., (Copyright: © 2024 Kellogg DQ et al.)

SUMMARYExtracellular vesicles (EVs) have been recognized throughout scientific communities as potential vehicles of intercellular communication in both eukaryotes and prokaryotes, thereby influencing various physiological and pathological functions of both parent and recipient cells. This review provides an in-depth exploration of the multifaceted roles of EVs in the context of bacteria and protozoan parasite EVs, shedding light on their contributions to physiological processes and disease pathogenesis. These studies highlight EVs as a conserved mechanism of cellular communication, which may lead us to important breakthroughs in our understanding of infection, mechanisms of pathogenesis, and as indicators of disease. Furthermore, EVs are involved in host-microbe interactions, offering insights into the strategies employed by bacteria and protozoan parasites to modulate host responses, evade the immune system, and establish infections., Competing Interests: The authors assert that this review meets the journal's ethics and integrity policies. The authors have not received any funding or benefits from the industry or elsewhere to conduct this study.

Background: Infection with the protozoan parasite Toxoplasma gondii leads to the formation of lifelong cysts in neurons that can have devastating consequences in the immunocompromised. In the immunocompetent individual, anti-parasitic effector mechanisms and a balanced immune response characterized by pro- and anti-inflammatory cytokine production establishes an asymptomatic infection that rarely leads to neurological symptoms. Several mechanisms are known to play a role in this successful immune response in the brain including T cell production of IFNγ and IL-10 and the involvement of CNS resident cells. This limitation of clinical neuropathology during chronic infection suggests a balance between immune response and neuroprotective mechanisms that collectively prevent clinical manifestations of disease. However, how these two vital mechanisms of protection interact during chronic Toxoplasma infection remains poorly understood., Main Text: This study demonstrates a previously undescribed connection between innate neutrophils found chronically in the brain, termed "chronic brain neutrophils" (CBNeuts), and neuroprotective mechanisms during Toxoplasma infection. Lack of CBNeuts during chronic infection, accomplished via systemic neutrophil depletion, led to enhanced infection and deleterious effects on neuronal regeneration and repair mechanisms in the brain. Phenotypic and transcriptomic analysis of CBNeuts identified them as distinct from peripheral neutrophils and revealed two main subsets of CBNeuts that display heterogeneity towards both classical effector and neuroprotective functions in an age-dependent manner. Further phenotypic profiling defined expression of the neuroprotective molecules NRG-1 andErbB4 by these cells, and the importance of this signaling pathway during chronic infection was demonstrated via NRG-1 treatment studies., Conclusions: In conclusion, this work identifies CBNeuts as a heterogenous population geared towards both classical immune responses and neuroprotection during chronic Toxoplasma infection and provides the foundation for future mechanistic studies of these cells., (© 2024. The Author(s).)

In pandemic scenarios involving novel human pathogenic viruses, it is highly desirable that vaccines induce strong neutralizing antibodies as quickly as possible. However, current vaccine strategies require multiple immunization doses to produce high titers of neutralizing antibodies and are poorly protective after a single vaccination. We therefore wished to design a vaccine candidate that would induce increased protective immune responses following the first vaccine dose. We hypothesized that antibodies against the receptor-binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein could be increased by drawing upon immunity to a previous infection. We generated a fusion protein containing the influenza H1N1 PR8 virus nucleoprotein (NP) and the SARS-CoV-2 spike RBD. Mice with or without preexisting immunity to PR8 were then vaccinated with NP/RBD. We observed significantly increased SARS-CoV-2 neutralizing antibodies in mice with PR8 immunity compared to mice without preexisting PR8 immunity. Vaccination with NP/RBD protected mice from SARS-CoV-2-induced morbidity and mortality after a single dose. Additionally, we compared SARS-CoV-2 virus titers in the lungs and nasal turbinates 4 days post-challenge of mice vaccinated with NP/RBD. SARS-CoV-2 virus was detectable in the lungs and nasal turbinate of mice without preexisting PR8 immunity, while SARS-CoV-2 virus was completely undetectable in mice with preexisting PR8 immunity. We also found that CD4-positive T cells in mice with preexisting immunity to PR8 play an essential role in producing the increased antibody response against RBD. This vaccine strategy potentially can be modified to target other pathogens of concern and offers extra value in future pandemic scenarios.IMPORTANCEIncreased globalization and changes in human interactions with wild animals has increased the likelihood of the emergence of novel viruses with pandemic potential. Vaccines can be effective in preventing severe disease caused by pandemic viruses. However, it takes time to develop protective immunity via prime-boost vaccination. More effective vaccine designs should quickly induce protective immunity. We propose leveraging preexisting immunity to a different pathogen to boost protection against emerging viruses. We targeted SARS-CoV-2 as a representative pandemic virus and generated a fusion protein vaccine that combines the nucleoprotein from influenza A virus and the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Our vaccine design significantly increased the production of RBD-specific antibodies in mice that had previously been exposed to influenza virus, compared to those without previous exposure. This enhanced immunity reduced SARS-CoV-2 replication in mice. Our results offer a vaccine design that could be valuable in a future pandemic setting., Competing Interests: The authors declare no conflict of interest.

The protozoan parasite Toxoplasma gondii infects approximately 2.5 billion people worldwide. Infection induces a rapid dissemination of parasites throughout the body followed by the formation of lifelong cysts within neurons of the host brain. Both stages require a dynamic immune response comprised of both innate and adaptive cells. Neutrophils are a primary responding cell to acute infection and have been observed in the brain during murine chronic infection. Previous studies investigating human neutrophils found that invasion by Toxoplasma tachyzoites inhibits apoptosis of neutrophils, prolonging their survival under inflammatory conditions. Here, we demonstrate the differentiation of two distinct subsets following exposure of human neutrophil-like-cells (HNLC) to Toxoplasma cysts. In vitro stimulation and imaging studies show cyst-specific induction of cytokines and cyst clearance by HNLCs. Further testing demonstrates that aged HNLCs perform less phagocytosis of cysts compared to non-aged HNLCs. In conclusion, this study identifies a novel response of HNLCs to Toxoplasma cysts and may indicate a role for neutrophils in the clearance of cysts during human infection with Toxoplasma., (© 2023 The Authors. Parasite Immunology published by John Wiley & Sons Ltd.)

Within the brain, a pro-inflammatory response is essential to prevent clinical disease due to Toxoplasma gondii reactivation. Infection in the immunocompromised leads to lethal Toxoplasmic encephalitis while in the immunocompetent, there is persistent low-grade inflammation which is devoid of clinical symptoms. This signifies that there is a well-balanced and regulated inflammatory response to T. gondii in the brain. T cells are the dominant immune cells that prevent clinical disease, and this is mediated through the secretion of effector molecules such as perforins and IFN-γ. The presence of cognate antigen, the expression of survival cytokines, and the alteration of the epigenetic landscape drive the development of memory T cells. However, specific extrinsic signals that promote the formation and maintenance of memory T cells within tissue are poorly understood. During chronic infection, there is an increase in extracellular glutamate that, due to its function as an excitatory neurotransmitter, is normally tightly controlled in the CNS. Here we demonstrate that CD8 + T cells from the T. gondii-infected brain parenchyma are enriched for metabotropic glutamate receptors (mGluR's). Characterization studies determined that mGluR + expression by CD8 + T cells defines a distinct memory population at the transcriptional and protein level. Finally, using receptor antagonists and agonists we demonstrate mGluR signaling is required for optimal CD8 + T cell production of the effector cytokine IFNγ. This work suggests that glutamate is an important environmental signal of inflammation that promotes T cell function. Understanding glutamate's influence on T cells in the brain can provide insights into the mechanisms that govern protective immunity against CNS-infiltrating pathogens and neuroinflammation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Importance: The classical depiction of the Toxoplasma lifecycle is bradyzoite excystation conversion to tachyzoites, cell lysis, and immune control, followed by the reestablishment of bradyzoites and cysts. In contrast, we show that tachyzoite growth slows independent of the host immune response at a predictable time point following excystation. Furthermore, we demonstrate a host cell-dependent pathway of continuous amplification of the cyst-forming bradyzoite population. The developmental plasticity of the excysted bradyzoites further underlines the critical role the cyst plays in the flexibility of the lifecycle of this ubiquitous parasite. This revised model of Toxoplasma recrudescence uncovers previously unknown complexity in the clinically important bradyzoite stage of the parasite, which opens the door to further study these novel developmental features of the Toxoplasma intermediate life cycle., Competing Interests: The authors declare no conflict of interest.

The rapid increase of the potent greenhouse gas methane in the atmosphere creates great urgency to develop and deploy technologies for methane mitigation. One approach to removing methane is to use bacteria for which methane is their carbon and energy source (methanotrophs). Such bacteria naturally convert methane to CO 2 and biomass, a value-added product and a cobenefit of methane removal. Typically, methanotrophs grow best at around 5,000 to 10,000 ppm methane, but methane in the atmosphere is 1.9 ppm. Air above emission sites such as landfills, anaerobic digestor effluents, rice paddy effluents, and oil and gas wells contains elevated methane in the 500 ppm range. If such sites are targeted for methane removal, technology harnessing aerobic methanotroph metabolism has the potential to become economically and environmentally viable. The first step in developing such methane removal technology is to identify methanotrophs with enhanced ability to grow and consume methane at 500 ppm and lower. We report here that some existing methanotrophic strains grow well at 500 ppm methane, and one of them, Methylotuvimicrobium buryatense 5GB1C, consumes such low methane at enhanced rates compared to previously published values. Analyses of bioreactor-based performance and RNAseq-based transcriptomics suggest that this ability to utilize low methane is based at least in part on extremely low non-growth-associated maintenance energy and on high methane specific affinity. This bacterium is a candidate to develop technology for methane removal at emission sites. If appropriately scaled, such technology has the potential to slow global warming by 2050.

Flexible, water-soluble hosts are capable of selective molecular recognition in cellular environments and can detect neurotransmitters such as choline in cells. Both cationic and anionic water-soluble self-folded deep cavitands can recognize suitable styrylpyridinium dyes in cellular interiors. The dyes selectively accumulate in nucleotide-rich regions of the cell nucleus and cytoplasm. The hosts bind the dyes and promote their relocation to the outer cell membrane: the lipophilic cavitands predominantly reside in membrane environments but are still capable of binding suitable targets in other cellular organelles. Incubating the cells with structurally similar biomarkers such as choline, cholamine, betaine, or butyrylcholine illustrates the selective recognition. Choline and butyrylcholine can be bound by the hosts, but minimal binding is seen with betaine or cholamine. Varying the dye allows control of the optical detection method, and both "turn-on" sensing and "turn-off" sensing are possible.

Astrocytes are vital support cells that ensure proper brain function. In brain disease, astrocytes reprogram into a reactive state that alters many of their cellular roles. A long-standing question in the field is whether downregulation of reactive astrocyte (RA) markers during resolution of inflammation is because these astrocytes revert back to a non-reactive state or die and are replaced. This has proven difficult to answer mainly because existing genetic tools cannot distinguish between healthy versus RAs. Here we describe the generation of an inducible genetic tool that can be used to specifically target and label a subset of RAs. Longitudinal analysis of an acute inflammation model using this tool revealed that the previously observed downregulation of RA markers after inflammation is likely due to changes in gene expression and not because of cell death. Our findings suggest that cellular changes associated with astrogliosis after acute inflammation are largely reversible., (© 2022 The Author(s).)

Introduction: The Accreditation Council for Graduate Medical Education (ACGME) offers descriptions of competencies and milestones but does not provide standardized assessments to track trainee competency. Entrustable professional activities (EPAs) and special assessments (SAs) are emerging methods to assess the level of competency obtained by regional anesthesiology and acute pain medicine (RAAPM) fellows., Methods: A panel of RAAPM physicians with experience in education and competency assessment and one medical student were recruited to participate in a modified Delphi method with iterative rounds to reach consensus on: a list of EPAs, SAs, and procedural skills; detailed definitions for each EPA and SA; a mapping of the EPAs and SAs to the ACGME milestones; and a target level of entrustment for graduating US RAAPM fellows for each EPA and procedural skill. A gap analysis was performed and a heat map was created to cross-check the EPAs and SAs to the ACGME milestones., Results: Participants in EPA and SA development included 19 physicians and 1 medical student from 18 different programs. The Delphi rounds yielded a final list of 23 EPAs, a defined entrustment scale, mapping of the EPAs to ACGME milestones, and graduation targets. A list of 73 procedural skills and 7 SAs were similarly developed., Discussion: A list of 23 RAAPM EPAs, 73 procedural skills, and 7 SAs were created using a rigorous methodology to reach consensus. This framework can be utilized to help assess RAAPM fellows in the USA for competency and allow for meaningful performance feedback., Competing Interests: Competing interests: None declared., (© American Society of Regional Anesthesia & Pain Medicine 2022. No commercial re-use. See rights and permissions. Published by BMJ.)

Maximum tongue strength, mean swallow pressures, and tongue endurance were measured in 324 children ages 6-12 years. The purpose of this study was to measure saliva swallow pressures in absolute terms (i.e., kilopascals) and as a percentage of maximum tongue strength to determine functional reserve in across ages in children and to examine factors that may influence tongue strength and swallow pressures including age, tongue endurance, and tongue-tie. The study results showed that maximum tongue strength and swallow pressures increased with age, while tongue endurance did not. Swallow pressures averaged 44% of maximum tongue strength across ages, indicating that children typically have a functional reserve of 56%. Tongue strength and swallow pressures were not decreased in the 20 children with tongue-tie. A sample clinical case is discussed., (© 2020. Springer Science+Business Media, LLC, part of Springer Nature.)

Engineering microorganisms into biological factories that convert renewable feedstocks into valuable materials is a major goal of synthetic biology; however, for many nonmodel organisms, we do not yet have the genetic tools, such as suites of strong promoters, necessary to effectively engineer them. In this work, we developed a computational framework that can leverage standard RNA-seq data sets to identify sets of constitutive, strongly expressed genes and predict strong promoter signals within their upstream regions. The framework was applied to a diverse collection of RNA-seq data measured for the methanotroph Methylotuvimicrobium buryatense 5GB1 and identified 25 genes that were constitutively, strongly expressed across 12 experimental conditions. For each gene, the framework predicted short (27-30 nucleotide) sequences as candidate promoters and derived -35 and -10 consensus promoter motifs (TTGACA and TATAAT, respectively) for strong expression in M. buryatense . This consensus closely matches the canonical E. coli sigma-70 motif and was found to be enriched in promoter regions of the genome. A subset of promoter predictions was experimentally validated in a XylE reporter assay, including the consensus promoter, which showed high expression. The pmoC , pqqA , and ssrA promoter predictions were additionally screened in an experiment that scrambled the -35 and -10 signal sequences, confirming that transcription initiation was disrupted when these specific regions of the predicted sequence were altered. These results indicate that the computational framework can make biologically meaningful promoter predictions and identify key pieces of regulatory systems that can serve as foundational tools for engineering diverse microorganisms for biomolecule production.

Toxoplasma gondii is a resilient parasite that infects a multitude of warm-blooded hosts and results in a lifelong chronic infection requiring continuous responses by the host. Chronic infection is characterized by a balanced immune response and neuropathology that are driven by changes in gene expression. Previous research pertaining to these processes has been conducted in various mouse models, and much knowledge of infection-induced gene expression changes has been acquired through the use of high throughput sequencing techniques in different mouse strains and post-mortem human studies. However, lack of infection time course data poses a prominent missing link in the understanding of chronic infection, and there is still much that is unknown regarding changes in genes specifically relating to neuropathology and resulting repair mechanisms as infection progresses throughout the different stages of chronicity. In this paper, we present a targeted approach to gene expression analysis during T. gondii infection through the use of NanoString nCounter gene expression assays. Wild type C57BL/6 and BALB/c background mice were infected, and transcriptional changes in the brain were evaluated at 14, 28, and 56 days post infection. Results demonstrate a dramatic shift in both previously demonstrated and novel gene expression relating to neuropathology and resolution in C57BL/6 mice. In addition, comparison between BALB/c and C57BL/6 mice demonstrate initial differences in gene expression that evolve over the course of infection and indicate decreased neuropathology and enhanced repair in BALB/c mice. In conclusion, these studies provide a targeted approach to gene expression analysis in the brain during infection and provide elaboration on previously identified transcriptional changes and also offer insights into further understanding the complexities of chronic T. gondii infection., Competing Interests: CD is an employee of NanoString. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Bergersen, Barnes, Worth, David and Wilson.)

Central nervous system (CNS) injury and infection can result in profound tissue remodeling in the brain, the mechanism and purpose of which is poorly understood. Infection with the protozoan parasite Toxoplasma gondii causes chronic infection and inflammation in the brain parenchyma. Control of parasite replication requires the continuous presence of IFNγ-producing T cells to keep T. gondii in its slowly replicating cyst form. During infection, a network of extracellular matrix fibers, revealed using multiphoton microscopy, forms in the brain. The origin and composition of these structures are unknown but the fibers have been observed to act as a substrate for migrating T cells. In this study, we show a critical regulator of extracellular matrix (ECM) remodeling, Secreted Protein, Acidic, Rich in Cysteine (SPARC), is upregulated in the brain during the early phases of infection in the frontal cortex. In the absence of SPARC, a reduced and disordered fibrous network, increased parasite burden, and reduced antigen-specific T cell entry into the brain points to a role for SPARC in T cell recruitment to and migration within the brain. We also report SPARC can directly bind to CCR7 ligands CCL19 and CCL21 but not CXCL10, and enhance migration toward a chemokine gradient. Measurement of T cell behavior points to tissue remodeling being important for access of immune cells to the brain and facilitating cellular locomotion. Together, these data identify SPARC as an important regulatory component of immune cell trafficking and access to the inflamed CNS.

Competing Interests: Disclosure E.H. Wilson and C.M. Burkle: None.

The Accreditation Council for Graduate Medical Education has shifted to competency-based medical education. This educational framework requires the description of educational outcomes based on the knowledge, skills and behaviors expected of competent trainees. It also requires an assessment program to provide formative feedback to trainees as they progress to competency in each outcome. Critical to the success of a curriculum is its practical implementation. This article describes the development of model curricula for anesthesiology residency training in regional anesthesia and acute pain medicine (core and advanced) using a competency-based framework. We further describe how the curricula were distributed through a shared web-based platform and mobile application., Competing Interests: Competing interests: None declared., (© American Society of Regional Anesthesia & Pain Medicine 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

The positioning of chromosomes in the nucleus of a eukaryotic cell is highly organized and has a complex and dynamic relationship with gene expression. In the human malaria parasite Plasmodium falciparum , the clustering of a family of virulence genes correlates with their coordinated silencing and has a strong influence on the overall organization of the genome. To identify conserved and species-specific principles of genome organization, we performed Hi-C experiments and generated 3D genome models for five Plasmodium species and two related apicomplexan parasites. Plasmodium species mainly showed clustering of centromeres, telomeres, and virulence genes. In P. falciparum , the heterochromatic virulence gene cluster had a strong repressive effect on the surrounding nuclear space, while this was less pronounced in Plasmodium vivax and Plasmodium berghei , and absent in Plasmodium yoelii In Plasmodium knowlesi , telomeres and virulence genes were more dispersed throughout the nucleus, but its 3D genome showed a strong correlation with gene expression. The Babesia microti genome showed a classical Rabl organization with colocalization of subtelomeric virulence genes, while the Toxoplasma gondii genome was dominated by clustering of the centromeres and lacked virulence gene clustering. Collectively, our results demonstrate that spatial genome organization in most Plasmodium species is constrained by the colocalization of virulence genes. P. falciparum and P. knowlesi , the only two Plasmodium species with gene families involved in antigenic variation, are unique in the effect of these genes on chromosome folding, indicating a potential link between genome organization and gene expression in more virulent pathogens., Competing Interests: The authors declare no conflict of interest.

Alternaria alternata is a fungal allergen associated with severe asthma and asthma exacerbations. Similarly to other asthma-associated allergens, Alternaria secretes a serine-like trypsin protease(s) that is thought to act through the G protein-coupled receptor protease-activated receptor-2 (PAR 2 ) to induce asthma symptoms. However, specific mechanisms underlying Alternaria-induced PAR 2 activation and signaling remain ill-defined. We sought to determine whether Alternaria-induced PAR 2 signaling contributed to asthma symptoms via a PAR 2 /β-arrestin signaling axis, identify the protease activity responsible for PAR 2 signaling, and determine whether protease activity was sufficient for Alternaria-induced asthma symptoms in animal models. We initially used in vitro models to demonstrate Alternaria-induced PAR 2 /β-arrestin-2 signaling. Alternaria filtrates were then used to sensitize and challenge wild-type, PAR 2 -/- and β-arrestin-2 -/- mice in vivo. Intranasal administration of Alternaria filtrate resulted in a protease-dependent increase of airway inflammation and mucin production in wild-type but not PAR 2 -/- or β-arrestin-2 -/- mice. Protease was isolated from Alternaria preparations, and select in vitro and in vivo experiments were repeated to evaluate sufficiency of the isolated Alternaria protease to induce asthma phenotype. Administration of a single isolated serine protease from Alternaria, Alternaria alkaline serine protease (AASP), was sufficient to fully activate PAR 2 signaling and induce β-arrestin-2 -/- -dependent eosinophil and lymphocyte recruitment in vivo. In conclusion, Alternaria filtrates induce airway inflammation and mucus hyperplasia largely via AASP using the PAR 2 /β-arrestin signaling axis. Thus, β-arrestin-biased PAR 2 antagonists represent novel therapeutic targets for treating aeroallergen-induced asthma.

Extracellular vesicles (EVs) actively participate in intercellular communication and pathological processes. Studying the molecular signatures of EVs is key to reveal their biological functions and clinical values, which, however, is greatly hindered by their sub-100 nm dimensions, the low quantities of biomolecules each EV carries, and the large population heterogeneity. Now, single-EV flow cytometry analysis is introduced to realize single EV counting and phenotyping in a conventional flow cytometer for the first time, enabled by target-initiated engineering (TIE) of DNA nanostructures on each EV. By illuminating multiple markers on single EVs, statistically significant differences are revealed among the molecular signatures of EVs originating from several breast cancer cell lines, and the cancer cell-derived EVs among the heterogeneous EV populations are successfully recognized. Thus, our approach holds great potential for various biological and biomedical applications., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Chemical-genetic interactions-observed when the treatment of mutant cells with chemical compounds reveals unexpected phenotypes-contain rich functional information linking compounds to their cellular modes of action. To systematically identify these interactions, an array of mutants is challenged with a compound and monitored for fitness defects, generating a chemical-genetic interaction profile that provides a quantitative, unbiased description of the cellular function(s) perturbed by the compound. Genetic interactions, obtained from genome-wide double-mutant screens, provide a key for interpreting the functional information contained in chemical-genetic interaction profiles. Despite the utility of this approach, integrative analyses of genetic and chemical-genetic interaction networks have not been systematically evaluated. We developed a method, called CG-TARGET (Chemical Genetic Translation via A Reference Genetic nETwork), that integrates large-scale chemical-genetic interaction screening data with a genetic interaction network to predict the biological processes perturbed by compounds. In a recent publication, we applied CG-TARGET to a screen of nearly 14,000 chemical compounds in Saccharomyces cerevisiae, integrating this dataset with the global S. cerevisiae genetic interaction network to prioritize over 1500 compounds with high-confidence biological process predictions for further study. We present here a formal description and rigorous benchmarking of the CG-TARGET method, showing that, compared to alternative enrichment-based approaches, it achieves similar or better accuracy while substantially improving the ability to control the false discovery rate of biological process predictions. Additional investigation of the compatibility of chemical-genetic and genetic interaction profiles revealed that one-third of observed chemical-genetic interactions contributed to the highest-confidence biological process predictions and that negative chemical-genetic interactions overwhelmingly formed the basis of these predictions. We also present experimental validations of CG-TARGET-predicted tubulin polymerization and cell cycle progression inhibitors. Our approach successfully demonstrates the use of genetic interaction networks in the high-throughput functional annotation of compounds to biological processes., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following potential competing interests: Authors Simpkins, Nelson, and Myers have licensed the CG-TARGET software for commercial use (z.umn.edu/cgtarget).

Increasing prevalence in obesity has become a significant public concern. C57BL/6J mice are prone to diet-induced obesity (DIO) when fed high-fat diet (HFD), and develop chronic inflammation and metabolic syndrome, making them a good model to analyze mechanisms whereby obesity elicits pathologies. DIO mice demonstrated profound sex differences in response to HFD with respect to inflammation and hypothalamic function. First, we determined that males are prone to DIO, while females are resistant. Ovariectomized females, on the other hand, are susceptible to DIO, implying protection by ovarian hormones. Males, but not females, exhibit changes in hypothalamic neuropeptide expression. Surprisingly, ovariectomized females remain resistant to neuroendocrine changes, showing that ovarian hormones are not necessary for protection. Second, obese mice exhibit sex differences in DIO-induced inflammation. Microglial activation and peripheral macrophage infiltration is seen in the hypothalami of males, while females are protected from the increase in inflammatory cytokines and do not exhibit microglia morphology changes nor monocyte-derived macrophage infiltration, regardless of the presence of ovarian hormones. Strikingly, the anti-inflammatory cytokine IL-10 is increased in the hypothalami of females but not males. Third, this study posits a potential mechanism of obesity-induced impairment of hypothalamic function whereby obese males exhibit reduced levels of synaptic proteins in the hypothalamus and fewer spines in GnRH neurons, located in the areas exhibiting macrophage infiltration. Our studies suggest that inflammation-induced synaptic remodeling is potentially responsible for hypothalamic impairment that may contribute to diminished levels of gonadotropin hormones, testosterone, and sperm numbers, which we observe and corresponds to the observations in obese humans. Taken together, our data implicate neuro-immune mechanisms underlying sex-specific differences in obesity-induced impairment of the hypothalamic function with potential consequences for reproduction and fertility.

Tachyzoite to bradyzoite development in Toxoplasma is marked by major changes in gene expression resulting in a parasite that expresses a new repertoire of surface antigens hidden inside a modified parasitophorous vacuole called the tissue cyst. The factors that control this important life cycle transition are not well understood. Here we describe an important transcriptional repressor mechanism controlling bradyzoite differentiation that operates in the tachyzoite stage. The ApiAP2 factor, AP2IV-4, is a nuclear factor dynamically expressed in late S phase through mitosis/cytokinesis of the tachyzoite cell cycle. Remarkably, deletion of the AP2IV-4 locus resulted in the expression of a subset of bradyzoite-specific proteins in replicating tachyzoites that included tissue cyst wall components BPK1, MCP4, CST1 and the surface antigen SRS9. In the murine animal model, the mis-timing of bradyzoite antigens in tachyzoites lacking AP2IV-4 caused a potent inflammatory monocyte immune response that effectively eliminated this parasite and prevented tissue cyst formation in mouse brain tissue. Altogether, these results indicate that suppression of bradyzoite antigens by AP2IV-4 during acute infection is required for Toxoplasma to successfully establish a chronic infection in the immune-competent host.

Introduction Intensivist involvement for patients with sepsis is associated with decreased complications and mortality, and lower hospital resource utilization, but few studies have evaluated outcomes for patients exposed to electronic intensive care unit (eICU) telemedicine sepsis management in the emergency department (ED). In this study, we assess whether eICU cart exposure in the ED improved compliance with components of the 2010 Surviving Sepsis Campaign bundles, length of stay (LOS), disposition and hospital costs. Methods An institutional review board-approved, retrospective cohort study was completed on patients with confirmed sepsis who presented to our ED from July 2010 through February 2013. Results Of 711 patient ED encounters, 314 cases met criteria for analysis (95 exposed and 219 non-exposed). Patient cohorts had similar demographics and comorbid International Classification of Diseases, Ninth Edition (ICD-9) diagnoses. The exposed cohort received antibiotics more quickly (122.3 minutes ±83.3 versus 163.4 minutes ±204.4, p = 0.043) and were more likely to have lactic acid levels drawn within six hours (98.9% vs. 90%, p = 0.019). The exposed cohort had a shortened ED LOS (in days) 0.08 ± 0.28 versus 0.16 ± 0.37, p = 0.036. Hospital LOS, disposition and death were similar in both cohorts. Total hospital costs for the exposed cohort were lower and less variable (US$19,713 ± 16,550 vs. US$24,364 ± 25068), but this was not significant ( p = 0.274). Discussion Our findings suggest that in individuals with confirmed sepsis, ED exposure to a telemedicine-based eICU cart impacted adherence to aspects of the Surviving Sepsis Campaign recommended bundle, but did not impact overall survival and medical costs.

High quality DNA design tools are becoming increasingly important as synthetic biology continues to increase the rate and throughput of building and testing genetic constructs. To make effective use of expanded build and test capacity, genotype design tools must not only be efficient enough to allow for many designs to be easily created, but also expressive enough to support the complex design patterns required by scientists on the frontier of genome engineering. Genotype Specification Language (GSL) is a language-based design tool invented at Amyris that enables scientists to quickly create DNA designs using a familiar syntax. This syntax provides a layer of abstraction that moves users away from reading and writing raw DNA sequences toward composing designs in terms of functional parts . GSL increases the speed at which scientists can design DNA constructs, provides a precise and reproducible representation of parts, and achieves these goals while maintaining design flexibility. Finally, the GSL compiler can emit information such as the exact final DNA sequence of the design as well as the reagents (primers and template information) required to physically build the constructs. Since its open-source release in February 2016, the GSL compiler can be freely downloaded and used by genome engineers to efficiently specify genetic designs. This chapter briefly introduces GSL syntax and design principles before examining specific examples of genome engineering tasks with accompanying GSL code.

Toxoplasma gondii is a widespread parasitic pathogen that infects over a third of the world's population. Following an acute infection, the parasite can persist within its mammalian host as intraneuronal or intramuscular cysts. Cysts will occasionally reactivate, and - depending on the host's immune status and site of reactivation - encephalitis or myositis can develop. Because these diseases have high levels of morbidity and can be lethal, it is important to understand how Toxoplasma traffics to these tissues, how the immune response controls parasite burden and contributes to tissue damage, and what mechanisms underlie neurological and muscular pathologies that toxoplasmosis patients present with. This review aims to summarize recent important developments addressing these critical topics., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Objective: To examine the practices and attitudes of obstetric providers in the state of Michigan on their approach to perinatal oral health., Methods: An online survey was distributed using health care provider databases in the state of Michigan. Descriptive statistics were tabulated and Fisher's Exact Test was used for categorical variables., Results: There were 146 respondents, 80 obstetrician-gynecologist (OB/GYN) and 66 certified nurse midwife (CNM) providers. Eighty percent acknowledged that perinatal oral health is an important consideration for optimal prenatal care, while only 53% reported having questions specific to oral health on their intake documents. Only 35% discussed oral health, 29% performed an oral cavity exam and 42% recommended oral health assessment by a dentist. CNMs were more likely than OB/GYNs to discuss the importance of oral health (48% versus 24%, (p <.05)) and recommend a formal dental assessment (61% versus 26% (p<.05)). These findings demonstrate a dichotomy of care in that obstetric providers recognize the importance of oral health care in pregnancy, but only a minority routinely incorporates these surveillance practices., Conclusion: These data highlight a gap in obstetric services that needs attention through specific educational initiatives and further research endeavors that address health care provider practice and patient barriers.

During chronic infection, memory T cells acquire a unique phenotype and become dependent on different survival signals than those needed for memory T cells generated during an acute infection. The distinction between the role of effector and memory T cells in an environment of persistent antigen remains unclear. Here, in the context of chronic Toxoplasma gondii infection, we demonstrate that a population of CD8 T cells exhibiting a tissue-resident memory (T RM ) phenotype accumulates within the brain. We show that this population is distributed throughout the brain in both parenchymal and extraparenchymal spaces. Furthermore, this population is transcriptionally distinct and exhibits a transcriptional signature consistent with the T RM observed in acute viral infections. Finally, we establish that the CD103 + T RM population has an intrinsic capacity to produce both IFN-γ and TNF-α, cytokines critical for parasite control within the central nervous system (CNS). The contribution of this population to pro-inflammatory cytokine production suggests an important role for T RM in protective and ongoing immune responses in the infected CNS. Accession number:   GSE95105 .

In an effort to find alternatives to the antitumor drug cisplatin, a series of copper (II) complexes possessing alkyl-substituted polypyridyl ligands have been synthesized. Eight new complexes are reported herein: μ-dichloro-bis{2,9-di-sec-butyl-1,10-phenanthrolinechlorocopper(II)} {[( di-sec-butyl phen)ClCu(μ-Cl) 2 CuCl( di-sec-butyl phen)]}(1), 2-sec-butyl-1,10-phenanthrolinedichlorocopper(II) { [mono-sec-butyl phen) CuCl 2 } (2), 2,9-di-n-butyl-1,10-phenanthrolinedichlorocopper(II) {[ di-n-butyl phen) CuCl 2 }(3), 2-n-butyl-1,10-phenanthrolinedichlorocopper(II) {[ mono-n-butyl phen) CuCl 2 } (4), 2,9-di-methyl-1,10-phenanthrolineaquadichlorocopper(II) {[ di-methyl phen) Cu(H 2 O)Cl 2 }(5), μ-dichloro-bis{6-sec-butyl-2,2'-bipyridinedichlorocopper(II)} {( mono-sec-butyl bipy) ClCu(μ-Cl) 2 CuCl( mono-sec-butyl bipy)} (6), 6,6'-di-methyl-2,2'-bipyridinedichlorocopper(II) { 6,6'-di-methyl bipy) CuCl 2 } (7), and 4,4'-dimethyl-2,2'-bipyridinedichlorocopper(II) { 4,4'-di-methyl bipy) CuCl 2 } (8). These complexes have been characterized via elemental analysis, UV-vis spectroscopy, and mass spectrometry. Single crystal X-ray diffraction experiments revealed the complexes synthesized with the di-sec-butyl phen ligand (1) and mono-sec-butyl bipy ligand (6) crystallized as dimers in which two copper(II) centers are bridged by two chloride ligands. Conversely, complexes 2, 7, and 8 were isolated as monomeric species possessing distorted tetrahedral geometries, and the [( di-methyl phen)Cu(H 2 O)Cl 2 ] (5) complex was isolated as a distorted square pyramidal monomer possessing a coordinating aqua ligand. Compounds 1-8 were evaluated for their in vitro antitumor efficacy. Compounds 1, 5, and 7 in particular were found to exhibit remarkable activity against human derived lung cancer cells, yet this class of copper(II) compounds had minimal cytotoxic effect on non-cancerous cells. In vitro control experiments indicate the activity of the copper(II) complexes most likely does not arise from the formation of CuCl 2 and free polypyridyl ligand, and preliminary solution state studies suggest these compounds are generally stable in biological buffer. The results presented herein suggest further development of this class of copper-based drugs as potential anti-cancer therapies should be pursued., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Hematopoietic stem and progenitor cells (HSPCs) function to replenish the immune cell repertoire under steady-state conditions and in response to inflammation due to infection or stress. Whereas the bone marrow serves as the primary niche for hematopoiesis, extramedullary mobilization and differentiation of HSPCs occur in the spleen during acute Plasmodium infection, a critical step in the host immune response. In this study, we identified an atypical HSPC population in the spleen of C57BL/6 mice, with a lineage(-)Sca-1(+)c-Kit(-) (LSK(-)) phenotype that proliferates in response to infection with nonlethal Plasmodium yoelii 17X. Infection-derived LSK(-) cells upon transfer into naive congenic mice were found to differentiate predominantly into mature follicular B cells. However, when transferred into infection-matched hosts, infection-derived LSK(-) cells gave rise to B cells capable of entering into a germinal center reaction, and they developed into memory B cells and Ab-secreting cells that were capable of producing parasite-specific Abs. Differentiation of LSK(-) cells into B cells in vitro was enhanced in the presence of parasitized RBC lysate, suggesting that LSK(-) cells expand and differentiate in direct response to the parasite. However, the ability of LSK(-) cells to differentiate into B cells was not dependent on MyD88, as myd88(-/-) LSK(-) cell expansion and differentiation remained unaffected after Plasmodium infection. Collectively, these data identify a population of atypical lymphoid progenitors that differentiate into B lymphocytes in the spleen and are capable of contributing to the ongoing humoral immune response against Plasmodium infection., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Recent observations of the Martian surface by the Phoenix lander and the Sample Analysis at Mars indicate the presence of perchlorate (ClO 4 - ). The abundance and isotopic composition of these perchlorates suggest that the mechanisms responsible for their formation in the Martian environment may be unique in our solar system. With this in mind, we propose a potential mechanism for the production of Martian perchlorate: the radiolysis of the Martian surface by galactic cosmic rays, followed by the sublimation of chlorine oxides into the atmosphere and their subsequent synthesis to form perchloric acid (HClO 4 ) in the atmosphere, and the surface deposition and subsequent mineralization of HClO 4 in the regolith to form surface perchlorates. To evaluate the viability of this mechanism, we employ a one-dimensional chemical model, examining chlorine chemistry in the context of Martian atmospheric chemistry. Considering the chlorine oxide, OClO, we find that an OClO flux as low as 3.2 × 10 7  molecules cm -2  s -1 sublimated into the atmosphere from the surface could produce sufficient HClO 4 to explain the perchlorate concentration on Mars, assuming an accumulation depth of 30 cm and integrated over the Amazonian period. Radiolysis provides an efficient pathway for the oxidation of chlorine, bypassing the efficient Cl/HCl recycling mechanism that characterizes HClO 4 formation mechanisms proposed for the Earth but not Mars.

We describe here the Genotype Specification Language (GSL), a language that facilitates the rapid design of large and complex DNA constructs used to engineer genomes. The GSL compiler implements a high-level language based on traditional genetic notation, as well as a set of low-level DNA manipulation primitives. The language allows facile incorporation of parts from a library of cloned DNA constructs and from the "natural" library of parts in fully sequenced and annotated genomes. GSL was designed to engage genetic engineers in their native language while providing a framework for higher level abstract tooling. To this end we define four language levels, Level 0 (literal DNA sequence) through Level 3, with increasing abstraction of part selection and construction paths. GSL targets an intermediate language based on DNA slices that translates efficiently into a wide range of final output formats, such as FASTA and GenBank, and includes formats that specify instructions and materials such as oligonucleotide primers to allow the physical construction of the GSL designs by individual strain engineers or an automated DNA assembly core facility.

The immune privileged nature of the CNS can make it vulnerable to chronic and latent infections. Little is known about the effects of lifelong brain infections, and thus inflammation, on the neurological health of the host. Toxoplasma gondii is a parasite that can infect any mammalian nucleated cell with average worldwide seroprevalence rates of 30%. Infection by Toxoplasma is characterized by the lifelong presence of parasitic cysts within neurons in the brain, requiring a competent immune system to prevent parasite reactivation and encephalitis. In the immunocompetent individual, Toxoplasma infection is largely asymptomatic, however many recent studies suggest a strong correlation with certain neurodegenerative and psychiatric disorders. Here, we demonstrate a significant reduction in the primary astrocytic glutamate transporter, GLT-1, following infection with Toxoplasma. Using microdialysis of the murine frontal cortex over the course of infection, a significant increase in extracellular concentrations of glutamate is observed. Consistent with glutamate dysregulation, analysis of neurons reveal changes in morphology including a reduction in dendritic spines, VGlut1 and NeuN immunoreactivity. Furthermore, behavioral testing and EEG recordings point to significant changes in neuronal output. Finally, these changes in neuronal connectivity are dependent on infection-induced downregulation of GLT-1 as treatment with the ß-lactam antibiotic ceftriaxone, rescues extracellular glutamate concentrations, neuronal pathology and function. Altogether, these data demonstrate that following an infection with T. gondii, the delicate regulation of glutamate by astrocytes is disrupted and accounts for a range of deficits observed in chronic infection.

Amphibian populations are declining globally. The potential contribution of glucocorticoid hormones to these declines has received little attention, but chronic elevation of glucocorticoids has been linked to a suite of negative outcomes across vertebrate taxa. Recently, chronic environmental stress has been associated with precipitous declines in sperm count and sperm viability in White's treefrogs (Litoria caerulea), but the mechanism remains unknown. In order to determine whether corticosterone is responsible for suppressing reproductive and immune function in this species, we elevated circulating concentrations of corticosterone in 10 male captive-bred frogs via transdermal application for 7 days. We compared sperm count, sperm viability, splenic cell count and circulating leucocyte counts in corticosterone-treated frogs with those in untreated control frogs. Chronic application of exogenous corticosterone led to supraphysiological circulating concentrations of corticosterone, but had no effect on sperm count or viability. However, corticosterone-treated frogs demonstrated a significant decrease in circulating eosinophils, which are immune cells implicated in fighting a variety of pathogens, including extracellular parasites. These findings suggest that although chronic elevation of circulating corticosterone is not necessarily associated with reproductive suppression in this species, it may cause immunosuppression. Thus, chronic glucocorticoid elevations in amphibians might enhance susceptibility to infection with pathogens and parasites, and their potential contributions to global population declines warrant further study.

The rise of the AIDS epidemic made the requirement for T cells in our continuous protection from pathogens critically apparent. The striking frequency with which AIDS patients exhibited profound neurological pathologies brought attention to many chronic infections that are latent within the immune-privileged CNS. One of the most common lethal opportunistic infections of these patients was with the protozoan parasite, Toxoplasma gondii. Reactivation of Toxoplasma cysts within the brain causes massive tissue destruction evidenced as multiple ring-enhancing lesions on MRI and is called toxoplasmic encephalitis (TE). TE is not limited to AIDS patients, but rather is a risk for all severely immunocompromised patients, including recipients of chemotherapy or transplant recipients. The lessons learned from these patient populations are supported by T cell depletion studies in mice. Such experiments have demonstrated that CD4+ and CD8+ T cells are required for protection against TE. Although it is clear that these T cell subsets work synergistically to fight infection, much evidence has been generated that suggests CD8+ T cells play a dominant role in protection during chronic toxoplasmosis. In other models of CNS inflammation, such as intracerebral infection with LCMV and experimental autoimmune encephalomyelitis (EAE), infiltration of T cells into the brain is harmful and even fatal. In the brain of the immunocompetent host, the well-regulated T cell response to T. gondii is therefore an ideal model to understand a controlled inflammatory response to CNS infection. This review will examine our current understanding of CD8+ T cells in the CNS during T. gondii infection in regards to the (1) mechanisms governing entry into the brain, (2) cues that dictate behavior within the brain, and (3) the functional and phenotypic properties exhibited by these cells.

Urbanization is a major driver of ecological change and comes with a suite of habitat modifications, including alterations to the local temperature, precipitation, light and noise regimes. Although many recent studies have investigated the behavioural and ecological ramifications of urbanization, physiological work in this area has lagged. We tested the hypothesis that anthropogenic noise is a stressor for amphibians and that chronic exposure to such noise leads to reproductive suppression. In the laboratory, we exposed male White's treefrogs, Litoria caerulea, to conspecific chorus noise either alone or coupled with pre-recorded traffic noise nightly for 1 week. Frogs presented with anthropogenic noise had significantly higher circulating concentrations of corticosterone and significantly decreased sperm count and sperm viability than did control frogs. These results suggest that in addition to having behavioural and ecological effects, anthropogenic change might alter physiology and Darwinian fitness. Future work should integrate disparate fields such as behaviour, ecology and physiology to elucidate fully organisms' responses to habitat change.

Glioblastoma multiforme is an extremely aggressive and invasive form of central nervous system tumor commonly treated with the chemotherapeutic drug Temozolomide. Unfortunately, even with treatment, the median survival time is less than 12 months. 2,9-Di-sec-butyl-1,10-phenanthroline (SBP), a phenanthroline-based ligand originally developed to deliver gold-based anticancer drugs, has recently been shown to have significant antitumor activity in its own right. SBP is hypothesized to initiate tumor cell death via interaction with non-DNA targets, and considering most glioblastoma drugs kill tumors through DNA damage processes, SBP was tested as a potential novel drug candidate against glial-based tumors. In vitro studies demonstrated that SBP significantly inhibited the growth of rodent GL-26 and C6 glioma cells, as well as human U-87, and SW1088 glioblastomas/astrocytomas. Furthermore, using a syngeneic glioma model in mice, in vivo administration of SBP significantly reduced tumor volume and increased survival time. There was no significant toxicity toward nontumorigenic primary murine and human astrocytes in vitro, and limited toxicity was observed in ex vivo tissues obtained from noncancerous mice. Terminal deoxynucleotidyl transferase dUTP nick end labeling staining and recovery assays suggest that SBP induces apoptosis in gliomas. This exploratory study suggests SBP is effective in slowing the growth of tumorigenic cells in the brain while exhibiting limited toxicity to normal cells and tissues and should therefore be further investigated for its potential in glioblastoma treatment.

Gold(III) complexes bearing bidentate ligands based on the 1,10-phenanthroline and 2,2'-bipyridine scaffolds have shown promising anticancer activity against a variety of tumor cell lines. In particular, our laboratory has previously found that a pseudo five coordinate gold(III) complex possessing the 2,9-di-sec-butyl-1,10-phenanthroline ligand {[((di-sec-butyl)phen)AuCl3]} exhibits antitumor activity against a panel of five different lung and head-neck tumor cell lines. However, the [((di-sec-butyl)phen)AuCl3] complex was determined to be less active than the free 2,9-di-sec-butyl-1,10-phenanthroline ligand. In order to determine if this class of gold(III) complexes has a distinct mechanism of initiating tumor cell death or if these gold complexes simply release the polypyridyl ligand in the intracellular environment, structural analogues of the [((di-sec-butyl)phen)AuCl3] complex have been synthesized and structurally characterized. These structural congeners were prepared by using mono-alkyl and di-phenyl substituted 1,10-phenanthroline ligands, di-alkyl and di-phenyl substituted 4-methyl-1,10-phenanthroline ligands, and mono-alkyl 2,2'-bipyridine ligands. The redox stability of this library of distorted square pyramidal gold(III) complexes has been studied and the in vitro antitumor activity of gold(III) complexes and corresponding polypyridyl ligands has been determined. The [((di-n-butyl)phen)AuCl3] and [((mono-n-butyl)phen)AuCl3] complexes have been found to be significantly more potent at inhibiting the growth of A549 lung tumor cells than the clinically used drug cisplatin. More importantly, these two gold(III) complexes are significantly more active than their respective free ligands, providing evidence that this class of pseudo five coordinate gold(III) complexes has a mechanism of initiating tumor cell death that is independent of the free ligand., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Chitinases are chitin-degrading enzymes. Chitinases play essential roles in combating chitin-containing pathogens as well as established roles in asthmatic inflammation. This assay is designed to detect chitinase activity in macrophage cell lysates. The chitin substrate is labeled with 4-methylumbelliferone. Hydrolysis of chitin releases 4-methylumbelliferone, and is measured fluorometrically to determine chitinase activity.

In the more than 100 years since its discovery, our knowledge of Toxoplasma biology has improved enormously. The evolution of molecular biology, immunology and genomics has had profound influences on our understanding of this ubiquitous bug. However, it could be argued that in science today the adage "seeing is believing" has never been truer. Images are highly influential and in the time since the first description of T. gondii, advances in microscopy and imaging technology have been and continue to be dramatic. In this review we recount the discovery of T. gondii and the contribution of imaging techniques to elucidating its life cycle, biology and the immune response of its host.

A water-soluble synthetic receptor molecule is capable of selective, controlled endocytosis of a specifically tagged target molecule in different types of living human cells. The presence of suitable choline-derived binding handles is essential for the molecular recognition and transport process, allowing selective guest transport and imaging of cancer cells.

Parasites are diverse eukaryotic pathogens that can have complex life cycles. Their clearance, or control within a mammalian host requires the coordinated effort of the immune system. The cell types recruited to areas of infection can combat the disease, promote parasite replication and survival, or contribute to disease pathology. Location and timing of cell recruitment can be crucial. In this review, we explore the role chemokines play in orchestrating and balancing the immune response to achieve optimal control of parasite replication without promoting pathology.

In response to central nervous system (CNS) injury and infection, astrocytes, neurons, and CNS vasculature express several chemokines, including CCL21. Quantitative polymerase chain reaction (qPCR), western blot, and immunohistochemical methods can quantify mRNA and protein expression. However, these methods do not quantify chemokine bioavailability and bioactivity, variables modified by many environmental factors including composition of the extracellular matrix (ECM). Here we illustrate how two-photon microscopy and carboxyfluorescein succinimidyl ester (CFSE or CFDA SE) labeling of T cells coupled with flow cytometry can be used as tools to assess chemokine-mediated regulation of T cell proliferation, activation, and migration.

Glioblastoma multiforme is a very aggressive and common form of brain tumor. Current therapies consist of a combination of surgical removal, chemotherapy and radiation therapy. These drastic treatments still leave a current prognosis of median survival of less than 1 year. Lack of effectiveness of these treatments has left researchers looking for alternative forms of treatment. A significant alternative currently being investigated is the use of the immune system to potentially target and eliminate tumor cells directly. Stabilin-1, a scavenger receptor expressed by macrophages, has the potential in inhibiting tumor growth by binding and internalizing secreted protein acidic and rich in cysteine (SPARC). SPARC is known to be upregulated in the tumor microenvironment and is involved in extracellular matrix remodeling, cell proliferation and migration. Decreasing SPARC expression using siRNA has been shown to decrease tumor invasiveness and survival. We investigated the phenotype of stabilin-1 expressing immune cells in the tumor environment and demonstrated a transient population of alternatively activated macrophages expressing stabilin-1 in the tumor environment and the disappearance of that population as the tumor progresses., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Proteinase-Activated receptor-2 (PAR(2)), a G-protein-coupled Receptor, activated by serine proteinases, is reported to have both protective and proinflammatory effects in the airway. Given these opposing actions, both inhibitors and activators of PAR(2) have been proposed for treating asthma. PAR(2) can signal through two independent pathways: a β-arrestin-dependent one that promotes leukocyte migration, and a G-protein/Ca(2+) one that is required for prostaglandin E(2) (PGE(2)) production and bronchiolar smooth muscle relaxation. We hypothesized that the proinflammatory responses to PAR(2) activation are mediated by β-arrestins, whereas the protective effects are not. Using a mouse ovalbumin model for PAR(2)-modulated airway inflammation, we observed decreased leukocyte recruitment, cytokine production, and mucin production in β-arrestin-2(-/-) mice. In contrast, PAR(2)-mediated PGE(2) production, smooth muscle relaxation, and decreased baseline airway resistance (measures of putative PAR(2) "protective" effects) were independent of β-arrestin-2. Flow cytometry and cytospins reveal that lung eosinophil and CD4 T-cell infiltration, and production of IL-4, IL-6, IL-13, and TNFα, were enhanced in wild-type but not β-arrestin-2(-/-) mice. Using the forced oscillation technique to measure airway resistance reveals that PAR(2) activation protects against airway hyperresponsiveness by an unknown mechanism, possibly involving smooth muscle relaxation. Our data suggest that the PAR(2)-enhanced inflammatory process is β-arrestin-2 dependent, whereas the protective anticonstrictor effect of bronchial epithelial PAR(2) may be β-arrestin independent.