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Macrophage metabolic plasticity is central to inflammatory programming, yet mechanisms of coordinating metabolic and inflammatory programs during infection are poorly defined. Here, we show that type I interferon (IFN) temporally guides metabolic control of inflammation during methicillin-resistant Staphylococcus aureus (MRSA) infection. We find that staggered Toll-like receptor and type I IFN signaling in macrophages permit a transient energetic state of combined oxidative phosphorylation (OXPHOS) and aerobic glycolysis followed by inducible nitric oxide synthase (iNOS)-mediated OXPHOS disruption. This disruption promotes type I IFN, suppressing other pro-inflammatory cytokines, notably interleukin-1β. Upon infection, iNOS expression peaks at 24 h, followed by lactate-driven Nos2 repression via histone lactylation. Type I IFN pre-conditioning prolongs infection-induced iNOS expression, amplifying type I IFN. Cutaneous MRSA infection in mice constitutively expressing epidermal type I IFN results in elevated iNOS levels, impaired wound healing, vasculopathy, and lung infection. Thus, kinetically regulated type I IFN signaling coordinates immunometabolic checkpoints that control infection-induced inflammation., Competing Interests: Declaration of interests C.A.L. has received consulting fees from Astellas Pharmaceuticals, Odyssey Therapeutics, and T-Knife Therapeutics and is an inventor on patents pertaining to Kras-regulated metabolic pathways, redox control pathways in pancreatic cancer, and targeting the GOT1 pathway as a therapeutic approach (US patent no. 2015126580-A1, May 7, 2015; US patent no. 20190136238, May 9, 2019; and international patent no. WO2013177426-A2, April 23, 2015). J.M.K. has received grant support from Q32 Bio, Celgene/BMS, Ventus Therapeutics, ROME Therapeutics, and Janssen. J.M.K. has served on advisory boards for AstraZeneca, Eli Lilly, GlaxoSmithKline, Gilead, Bristol Myers Squibb, Avion Pharmaceuticals, Provention Bio, Aurinia Pharmaceuticals, Ventus Therapeutics, and ROME Therapeutics., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The ex vivo endothelialization of small diameter vascular prostheses can prolong their patency. Here, we demonstrate that heterotypic interactions between human adipose tissue-derived endothelial cells and perivascular cells can be exploited to accelerate the endothelialization of an electrospun ionomeric polyurethane scaffold. The scaffold was used to physically separate endothelial cells from perivascular cells to prevent their diffuse neo-intimal hyperplasia and spontaneous tubulogenesis, yet enable their paracrine cross-talk to accelerate the integration of the endothelial cells into a temporally stable endothelial lining of a continuous, elongated, and aligned morphology. Perivascular cells stimulated endothelial basement membrane protein production and suppressed their angiogenic and inflammatory activation to accelerate this biomimetic morphogenesis of the endothelium. These findings demonstrate the feasibility and underscore the value of exploiting heterotypic interactions between endothelial cells and perivascular cells for the fabrication of an endothelial lining intended for small diameter arterial reconstruction. STATEMENT OF SIGNIFICANCE: Adipose tissue is an abundant, accessible, and uniquely dispensable source of endothelial cells and perivascular cells for vascular tissue engineering. While their spontaneous self-assembly into microvascular networks is routinely exploited for the vascularization of engineered tissues, it threatens the temporal stability of an endothelial lining intended for small diameter arterial reconstruction. Here, we demonstrate that an electrospun polyurethane scaffold can be used to physically separate endothelial cells from perivascular cells to prevent their spontaneous capillary morphogenesis, yet enable their cross-talk to promote the formation of a stable endothelium. Our findings demonstrate the feasibility of engineering an endothelial lining from human adipose tissue, poising it for the rapid ex vivo endothelialization of small diameter vascular prostheses in an autologous, patient-specific manner., 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 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Candida albicans is a frequent colonizer of human mucosal surfaces as well as an opportunistic pathogen. C. albicans is remarkably versatile in its ability to colonize diverse host sites with differences in oxygen and nutrient availability, pH, immune responses, and resident microbes, among other cues. It is unclear how the genetic background of a commensal colonizing population can influence the shift to pathogenicity. Therefore, we examined 910 commensal isolates from 35 healthy donors to identify host niche-specific adaptations. We demonstrate that healthy people are reservoirs for genotypically and phenotypically diverse C. albicans strains. Using limited diversity exploitation, we identified a single nucleotide change in the uncharacterized ZMS1 transcription factor that was sufficient to drive hyper invasion into agar. We found that SC5314 was significantly different from the majority of both commensal and bloodstream isolates in its ability to induce host cell death. However, our commensal strains retained the capacity to cause disease in the Galleria model of systemic infection, including outcompeting the SC5314 reference strain during systemic competition assays. This study provides a global view of commensal strain variation and within-host strain diversity of C. albicans and suggests that selection for commensalism in humans does not result in a fitness cost for invasive disease., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Anderson et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Here, we describe a protocol for purifying functional clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) from Staphylococcus aureus within 24 h and over 90% purity. SaCas9 purification begins with immobilized metal affinity chromatography, followed by cation exchange chromatography, and ended with centrifugal concentrators. The simplicity, cost-effectiveness, and reproducibility of such protocols will enable general labs to produce a sizable amount of Cas9 proteins, further accelerating CRISPR research., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Human adipose tissue-resident microvascular endothelial cells are not only garnering attention for their emergent role in the pathogenesis of obesity-related metabolic disorders, but are also of considerable interest for vascular tissue engineering due, in part, to the abundant, accessible, and uniquely dispensable nature of the tissue. Here, we delineate a protocol for the acquisition of microvascular endothelial cells from human fat. A cheaper, smaller, and simpler alternative to fluorescence-assisted cell sorting for the immunoselection of cells, our protocol adapts magnet-assisted cell sorting for the isolation of endothelial cells from enzymatically digested adipose tissue and the subsequent enrichment of their primary cultures. Strategies are employed to mitigate the non-specific uptake of immunomagnetic microparticles, enabling the reproducible acquisition of human adipose tissue-resident microvascular endothelial cells with purities ≥98%. They exhibit morphological, molecular, and functional hallmarks of endothelium, yet retain a unique proteomic signature when compared with endothelial cells derived from different vascular beds. Their cultures can be expanded for >10 population doublings and can be maintained at confluence for at least 28 days without being overgrown by residual stromal cells from the cell sorting procedure. The isolation of human adipose tissue-resident microvascular endothelial cells can be completed within 6 hours and their enrichment within 2 hours, following approximately 7 days in culture. Graphical abstract., Competing Interests: Competing interests The authors have no conflicts of interest to disclose., (Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC.)

Adipose tissue is an abundant, accessible, and uniquely dispensable source of cells for vascular tissue engineering. Despite its intrinsic endothelial cells, considerable effort is directed at deriving endothelium from its resident stem and progenitor cells. Here, we investigate the composition of human adipose tissue and characterize the phenotypes of its constituent cells in order to help ascertain their potential utility for vascular tissue engineering. Unsupervised clustering based on cell-surface protein signatures failed to detect CD45 - CD31 - VEGFR2 + endothelial progenitor cells within adipose tissue, but supported further investigation of its resident CD45 - CD31 + microvascular endothelial cells (HAMVECs) and CD45 - CD31 - stromal/stem cells (ASCs). The endothelial differentiation of ASCs altered their proteome, but it remained distinct from that of primary endothelial cell controls - as well as HAMVECs - regardless of their arterial-venous specification or macrovascular-microvascular origin. Rather, ASCs retained a proteome indicative of a perivascular phenotype, which was supported by their ability to facilitate the capillary morphogenesis of HAMVECs. This study supports the use of HAMVECs for the generation of endothelium. It suggests that the utility of ASCs for vascular tissue engineering lies in their capacity to remodel the extracellular matrix and to function as mural cells., Competing Interests: 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., (© 2022 The Authors.)

Signaling initiated by type I interferon (IFN) results in the induction of hundreds of IFN-stimulated genes (ISGs). The type I IFN response is important for antiviral restriction, but aberrant activation of this response can lead to inflammation and autoimmunity. Regulation of this response is incompletely understood. We previously reported that the mRNA modification m 6 A and its deposition enzymes, METTL3 and METTL14 (METTL3/14), promote the type I IFN response by directly modifying the mRNA of a subset of ISGs to enhance their translation. Here, we determined the role of the RNA demethylase fat mass and obesity-associated protein (FTO) in the type I IFN response. FTO, which can remove either m 6 A or cap-adjacent m 6 Am RNA modifications, has previously been associated with obesity and body mass index, type 2 diabetes, cardiovascular disease, and inflammation. We found that FTO suppresses the transcription of a distinct set of ISGs, including many known pro-inflammatory genes, and that this regulation requires its catalytic activity but is not through the actions of FTO on m 6 Am. Interestingly, depletion of FTO led to activation of the transcription factor STAT3, whose role in the type I IFN response is not well understood. This activation of STAT3 increased the expression of a subset of ISGs. Importantly, this increased ISG induction resulting from FTO depletion was partially ablated by depletion of STAT3. Together, these results reveal that FTO negatively regulates STAT3-mediated signaling that induces proinflammatory ISGs during the IFN response, highlighting an important role for FTO in suppression of inflammatory genes., 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 © 2021 Elsevier Ltd. All rights reserved.)

Developing safe and effective strategies to deliver biomolecules such as oligonucleotides and proteins into cells has grown in importance over recent years, with an increasing demand for non-viral methods that enable clinical translation. Here, we investigate uniquely configured oligo-urethane nanoparticles based on synthetic chemistries that minimize the release of pro-inflammatory biomarkers from immune cells, show low cytotoxicity in a broad range of cells, and efficiently deliver oligonucleotides and proteins into mammalian cells. The mechanism of cell uptake for the self-assembled oligo-urethane nanoparticles was shown to be directed by caveolae-dependent endocytosis in murine myoblasts (C 2 C 12 ) cells. Inhibiting caveolae functions with genistein and methyl-β-cyclodextrin limited nanoparticle internalization. The nanoparticles showed a very high delivery efficiency for the genetic material (a 47-base oligonucleotide) (∼80% incorporation into cells) as well as the purified protein (full length firefly luciferase, 67 kDa) into human embryonic kidney (HEK293T) cells. Luciferase enzyme activity in HEK293T cells demonstrated that intact and functional proteins could be delivered and showed a significant extension of activity retention up to 24 h, well beyond the 2 h half-life of the free enzyme. This study introduces a novel self-assembled oligo-urethane nanoparticle delivery platform with very low associated production costs, enabled by their scalable chemistry (the benchwork cost is $ 0.152/mg vs $ 974.6/mg for typical lipid carriers) that has potential to deliver both oligonucleotides and proteins for biomedical purposes.

Endothelial cells are among the fundamental building blocks for vascular tissue engineering. However, a clinically viable source of endothelium has continued to elude the field. Here, we demonstrate the feasibility of sourcing autologous endothelium from human fat - an abundant and uniquely dispensable tissue that can be readily harvested with minimally invasive procedures. We investigate the challenges underlying the overgrowth of human adipose tissue-derived microvascular endothelial cells by stromal cells to facilitate the development of a reliable method for their acquisition. Magnet-assisted cell sorting strategies are established to mitigate the non-specific uptake of immunomagnetic microparticles, enabling the enrichment of endothelial cells to purities that prevent their overgrowth by stromal cells. This work delineates a reliable method for acquiring human adipose tissue-derived microvascular endothelial cells in large quantities with high purities that can be readily applied in future vascular tissue engineering applications., (© 2021. The Author(s).)

Heart failure (HF) is associated with pathological remodeling of the myocardium, including the initiation of fibrosis and scar formation by activated cardiac fibroblasts (CFs). Although early CF-dependent scar formation helps prevent cardiac rupture by maintaining the heart's structural integrity, ongoing deposition of the extracellular matrix in the remote and infarct regions can reduce tissue compliance, impair cardiac function, and accelerate progression to HF. In our study, we conducted mass spectrometry (MS) analysis to identify differentially altered proteins and signaling pathways between CFs isolated from 7 day sham and infarcted murine hearts. Surprisingly, CFs from both the remote and infarct regions of injured hearts had a wide number of similarly altered proteins and signaling pathways that were consistent with fibrosis and activation into pathological myofibroblasts. Specifically, proteins enriched in CFs isolated from MI hearts were involved in pathways pertaining to cell-cell and cell-matrix adhesion, chaperone-mediated protein folding, and collagen fibril organization. These results, together with principal component analyses, provided evidence of global CF activation postinjury. Interestingly, however, direct comparisons between CFs from the remote and infarct regions of injured hearts identified 15 differentially expressed proteins between MI remote and MI infarct CFs. Eleven of these proteins ( Gpc1, Cthrc1, Vmac, Nexn, Znf185, Sprr1a, Specc1, Emb, Limd2, Pawr , and Mcam ) were higher in MI infarct CFs, whereas four proteins ( Gstt1, Gstm1, Tceal3 , and Inmt ) were higher in MI remote CFs. Collectively, our study shows that MI injury induced global changes to the CF proteome, with the magnitude of change reflecting their relative proximity to the site of injury.

Recent discoveries have revealed that, during viral infection, the presence of the RNA modification N 6 -methyladenosine (m 6 A) on viral and cellular RNAs has profound impacts on infection outcome. Although m 6 A directly regulates many viral RNA processes, its effects on cellular RNAs and pathways during infection have only recently begun to be elucidated. Disentangling the effects of m 6 A on viral and host RNAs remains a challenge for the field. m 6 A has been found to regulate host responses such as viral RNA sensing, cytokine responses, and immune cell functions. We highlight recent findings describing how m 6 A modulates host responses to viral infection and discuss future directions that will lead to a synergistic understanding of the processes by which m 6 A regulates viral infection., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Type I interferons (IFNs) induce hundreds of IFN-stimulated genes (ISGs) in response to viral infection. Induction of these ISGs must be regulated for an efficient and controlled antiviral response, but post-transcriptional controls of these genes have not been well defined. Here, we identify a role for the RNA base modification N6-methyladenosine (m 6 A) in the regulation of ISGs. Using ribosome profiling and quantitative mass spectrometry, coupled with m 6 A-immunoprecipitation and sequencing, we identify a subset of ISGs, including IFITM1, whose translation is enhanced by m 6 A and the m 6 A methyltransferase proteins METTL3 and METTL14. We further determine that the m 6 A reader YTHDF1 increases the expression of IFITM1 in an m 6 A-binding-dependent manner. Importantly, we find that the m 6 A methyltransferase complex promotes the antiviral activity of type I IFN. Thus, these studies identify m 6 A as having a role in post-transcriptional control of ISG translation during the type I IFN response for antiviral restriction., Competing Interests: Declaration of interests C.E.M. is a cofounder and board member for Biotia and Onegevity Health and an advisor or compensated speaker for Abbvie, Acuamark Diagnostics, ArcBio, Bio-Rad, DNA Genotek, Genialis, Genpro, Illumina, New England Biolabs, QIAGEN, Whole Biome, and Zymo Research., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

The use of exogenous biomolecules (BM) for the purpose of repairing and regenerating damaged cardiac tissue can yield serious side effects if used for prolonged periods. As well, such strategies can be cost prohibitive depending on the regiment and period of time applied. Alternatively, autologous monocytes/monocyte-derived macrophages (MDM) can provide a viable path towards generating an endogenous source of stimulatory BM. Biomaterials are often considered as delivery vehicles to generate unique profiles of such BM in tissues or to deliver autologous cells, that can influence the nature of BM produced by the cells. MDM cultured on a degradable polar hydrophobic ionic (D-PHI) polyurethane has previously demonstrated a propensity to increase select anti-inflammatory cytokines, and therefore there is good rationale to further investigate a broader spectrum of the cells' BM in order to provide a more complete proteomic analysis of human MDM secretions induced by D-PHI. Further, it is of interest to assess the potential of such BM to influence cells involved in the reparative state of vital tissues such as those that affect cardiac cell function. Hence, this current study examines the proteomic profile of MDM secretions using mass spectrometry for the first time, along with ELISA, following their culture on D-PHI, and compares them to two important reference materials, poly(lactic-co-glycolic acid) (PLGA) and tissue culture polystyrene (TCPS). Secretions collected from D-PHI cultured MDM led to higher levels of regenerative BM, AGRN, TGFBI and ANXA5, but lower levels of pro-fibrotic BM, MMP7, IL-1β, IL-6 and TNFα,  when compared to MDM secretions collected from PLGA and TCPS. In the application to cardiac cell function, the secretion collected from D-PHI cultured MDM led to more human cardiac fibroblast (HCFs) migration. A lower collagen gel contraction induced by MDM secretions collected from D-PHI was supported by gene array analysis for human fibrosis-related genes. The implication of these findings is that more tailored biomaterials such as D-PHI, may lead to a lower pro-inflammatory phenotype of macrophages when used in cardiac tissue constructs, thereby enabling the development of vehicles for the delivery of interventional therapies, or be applied as coatings for sensor implants in cardiac tissue that minimize fibrosis. The general approach of using synthetic biomaterials in order to induce MDM secretions in a manner that will guide favorable regeneration will be critical in making the choice of biomaterials for tissue regeneration work in the future. STATEMENT OF SIGNIFICANCE: Immune modulation strategies currently applied in cardiac tissue repair are mainly based on the delivery of defined exogenous biomolecules. However, the use of such biomolecules may pose wide ranging systemic effects, thereby rendering them clinically less practical. The chemistry of biomaterials (used as a potential targeted delivery modality to circumvent the broad systemic effects of biomolecules) can not only affect acute and chronic toxicity but also alters the timeframe of the wound healing cascade. In this context, monocytes/monocyte-derive macrophages (MDM) can be harnessed as an immune modulating strategy to promote wound healing by an appropriate choice of the biomaterial. However, there are limited reports on the complete proteome analysis of MDM and their reaction of biomaterial related interventions on cardiac tissues and cells. No studies to date have demonstrated the complete proteome of MDM secretions when these cells were cultured on a non-traditional immune modulatory ionomeric polyurethane D-PHI film. This study demonstrated that MDM cultured on D-PHI expressed significantly higher levels of AGRN, TGFBI and ANXA5 but lower levels of MMP7, IL-1β, IL-6 and TNFα when compared to MDM cultured on a well-established degradable biomaterials in the medical field, e.g. PLGA and TCPS, which are often used as the relative standards for cell culture work in the biomaterials field. The implications of these findings have relevance to the repair of cardiac tissues. In another aspect of the work, human cardiac fibroblasts showed significantly lower contractility (low collagen gel contraction and low levels of ACTA2) when cultured in the presence of MDM secretions collected after culturing them on D-PHI compared to PLGA and TCPS. The findings place emphasis on the importance of making the choice of biomaterials for tissue engineering and regenerative medicine applied to their use in cardiac tissue repair., 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 © 2020. Published by Elsevier Ltd.)

In the skin, antiviral proteins and other immune molecules serve as the first line of innate antiviral defense. Here, we identify and characterize the induction of cutaneous innate antiviral proteins in response to IL-27 and its functional role during cutaneous defense against Zika virus infection. Transcriptional and phenotypic profiling of epidermal keratinocytes treated with IL-27 demonstrated activation of antiviral proteins OAS1, OAS2, OASL, and MX1 in the skin of both mice and humans. IL-27-mediated antiviral protein induction was found to occur in a STAT1- and IRF3-dependent but STAT2-independent manner. Moreover, using IL27ra mice, we demonstrate a significant role for IL-27 in inhibiting Zika virus morbidity and mortality following cutaneous, but not intravenous, inoculation. Together, our results demonstrate a critical and previously unrecognized role for IL-27 in cutaneous innate antiviral immunity against Zika virus., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Innate immune detection of viral nucleic acids during viral infection activates a signaling cascade that induces type I and type III IFNs as well as other cytokines, to generate an antiviral response. This signaling is initiated by pattern recognition receptors, such as the RNA helicase retinoic acid-inducible gene I (RIG-I), that sense viral RNA. These sensors then interact with the adaptor protein mitochondrial antiviral signaling protein (MAVS), which recruits additional signaling proteins, including TNF receptor-associated factor 3 (TRAF3) and TANK-binding kinase 1 (TBK1), to form a signaling complex that activates IFN regulatory factor 3 (IRF3) for transcriptional induction of type I IFNs. Here, using several immunological and biochemical approaches in multiple human cell types, we show that the GTPase-trafficking protein RAB1B up-regulates RIG-I pathway signaling and thereby promotes IFN-β induction and the antiviral response. We observed that RAB1B overexpression increases RIG-I-mediated signaling to IFN-β and that RAB1B deletion reduces signaling of this pathway. Additionally, loss of RAB1B dampened the antiviral response, indicated by enhanced Zika virus infection of cells depleted of RAB1B. Importantly, we identified the mechanism of RAB1B action in the antiviral response, finding that it forms a protein complex with TRAF3 to facilitate the interaction of TRAF3 with mitochondrial antiviral signaling protein. We conclude that RAB1B regulates TRAF3 and promotes the formation of innate immune signaling complexes in response to nucleic acid sensing during RNA virus infection., (© 2019 Beachboard et al.)

Considerable effort has been directed toward deriving endothelial cells (ECs) from adipose-derived mesenchymal stem cells (ASCs) since 2004, when it was first suggested that ECs and adipocytes share a common progenitor. While the capacity of ASCs to express endothelial markers has been repeatedly demonstrated, none constitute conclusive evidence of an endothelial phenotype as all reported markers have been detected in other, non-endothelial cell types. In this study, quantitative phenotypic comparisons to representative EC controls were used to determine the extent of endothelial differentiation being achieved with ASCs. ASCs were harvested from human subcutaneous abdominal white adipose tissue, and their endothelial differentiation was induced using well-established biochemical stimuli. Reverse transcription quantitative real-time polymerase chain reaction and parallel reaction monitoring mass spectrometry were used to quantify their expression of endothelial genes and corresponding proteins, respectively. Flow cytometry was used to quantitatively assess their uptake of acetylated low-density lipoprotein (AcLDL). Human umbilical vein, coronary artery, and dermal microvascular ECs were used as positive controls to reflect the phenotypic heterogeneity between ECs derived from different vascular beds. Biochemically conditioned ASCs were found to upregulate their expression of endothelial genes and proteins, as well as AcLDL uptake, but their abundance remained orders of magnitude lower than that observed in the EC controls despite their global proteomic heterogeneity. The findings of this investigation demonstrate the strikingly limited extent of endothelial differentiation being achieved with ASCs using well-established biochemical stimuli, and underscore the importance of quantitative phenotypic comparisons to representative primary cell controls in studies of differentiation. Stem Cells Translational Medicine 2019;8:35-45., (© 2018 The Authors. Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

RNA virus genomes are efficient and compact carriers of biological information, encoding information required for replication both in their primary sequences and in higher-order RNA structures. However, the ubiquity of RNA elements with higher-order folds-in which helices pack together to form complex 3D structures-and the extent to which these elements affect viral fitness are largely unknown. Here we used single-molecule correlated chemical probing to define secondary and tertiary structures across the RNA genome of dengue virus serotype 2 (DENV2). Higher-order RNA structures are pervasive and involve more than one-third of nucleotides in the DENV2 genomic RNA. These 3D structures promote a compact overall architecture and contribute to viral fitness. Disrupting RNA regions with higher-order structures leads to stable, nonreverting mutants and could guide the development of vaccines based on attenuated RNA viruses. The existence of extensive regions of functional RNA elements with tertiary folds in viral RNAs, and likely many other messenger and noncoding RNAs, means that there are significant regions with pocket-containing surfaces that may serve as novel RNA-directed drug targets., Competing Interests: Conflict of interest statement: K.M.W. is an advisor to and holds equity in Ribometrix.

Zika virus (ZIKV) is a re-emerging flavivirus that is transmitted to humans through the bite of an infected mosquito or through sexual contact with an infected partner. ZIKV infection during pregnancy has been associated with numerous fetal abnormalities, including prenatal lethality and microcephaly. However, until recent outbreaks in the Americas, ZIKV has been relatively understudied, and therefore the biology and pathogenesis of ZIKV infection remain incompletely understood. Better methods to study ZIKV infection in live cells could enhance our understanding of the biology of ZIKV and the mechanisms by which ZIKV contributes to fetal abnormalities. To this end, we developed a fluorescent cell-based reporter system allowing for live imaging of ZIKV-infected cells. This system utilizes the protease activity of the ZIKV non-structural proteins 2B and 3 (NS2B-NS3) to specifically mark virus-infected cells. Here, we demonstrate the utility of this fluorescent reporter for identifying cells infected by ZIKV strains of two lineages. Further, we use this system to determine that apoptosis is induced in cells directly infected with ZIKV in a cell-autonomous manner. Ultimately, approaches that can directly track ZIKV-infected cells at the single cell-level have the potential to yield new insights into the host-pathogen interactions that regulate ZIKV infection and pathogenesis., Competing Interests: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

A host's immune system plays a central role in shaping the composition of the microbiota and, in return, resident microbes influence immune responses. Symbiotic associations of the maternally transmitted bacterium Wolbachia occur with a wide range of arthropods. It is, however, absent from the dengue and Zika vector mosquito Aedes aegypti in nature. When Wolbachia is artificially forced to form symbiosis with this new mosquito host, it boosts the basal immune response and enhances the mosquito's resistance to pathogens, including dengue, Zika virus and malaria parasites. The mechanisms involved in establishing a symbiotic relationship between Wolbachia and A. aegypti, and the long-term outcomes of this interaction, are not well understood. Here, we have demonstrated that both the immune deficiency (IMD) and Toll pathways are activated by the Wolbachia strain wAlbB upon its introduction into A. aegypti. Silencing the Toll and IMD pathways via RNA interference reduces the wAlbB load. Notably, wAlbB induces peptidoglycan recognition protein (PGRP)-LE expression in the carcass of A. aegypti, and its silencing results in a reduction of symbiont load. Using transgenic mosquitoes with stage-specific induction of the IMD and Toll pathways, we have shown that elevated wAlbB infection in these mosquitoes is maintained via maternal transmission. These results indicate that host innate immunity is utilized to establish and promote host-microbial symbiosis. Our results will facilitate a long-term projection of the stability of the Wolbachia-A. aegypti mosquito system that is being developed to control dengue and Zika virus transmission to humans.

The endosymbiont Wolbachia w AlbB induces refractoriness to Plasmodium falciparum in Anopheles stephensi , the primary mosquito vector of human malaria in the Middle East and South Asia. However, it remains unknown whether such refractoriness can be extended to other malaria species. In particular, it was reported that under very specific conditions, w AlbB can enhance Plasmodium infection in some hosts. Here, we measured the impact of w AlbB on the rodent malaria parasite Plasmodium berghei in A. stephensi by comparing the load of oocysts and sporozoites in midguts and salivary glands, respectively, between w AlbB-infected and -uninfected mosquitoes. To investigate whether w AlbB modulated mosquito immune defense against parasites, we compared the expression of the immune genes, which were previously reported to involve in antimalarial response, in both midguts and the remaining carcass tissues of mosquitoes. The stable association of w AlbB with A. stephensi resulted in reduction of parasites by more than half at the oocyst stage, and up to 91.8% at the sporzoite stage. The anti- plasmodium immune genes, including TEP1 , LRIM1 , Toll pathway gene Rel1 and the effector Defensin 1 , were induced by w AlbB in different mosquito body tissues. These findings suggest that immune priming is a potential cause of w AlbB-mediated antimalarial response in A. stephensi . More importantly, no evidence was found for any enhancement of Plasmodium infection in A. stephensi stably infected with w AlbB. We discuss these findings with possible implementations of Wolbachia for malaria control in disease endemic areas.

The ability to recognize invading viral pathogens and to distinguish their components from those of the host cell is critical to initiate the innate immune response. The efficiency of this detection is an important factor in determining the susceptibility of the cell to viral infection. Innate sensing of viruses is, therefore, an indispensable step in the line of defense for cells and organisms. Recent discoveries have uncovered novel sensors of viral components and hallmarks of infection, as well as mechanisms by which cells discriminate between self and non-self. This review highlights the mechanisms used by cells to detect viral pathogens in the cytosol, and recent advances in the field of cytosolic sensing of viruses., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The RNA modification N6-methyladenosine (m 6 A) post-transcriptionally regulates RNA function. The cellular machinery that controls m 6 A includes methyltransferases and demethylases that add or remove this modification, as well as m 6 A-binding YTHDF proteins that promote the translation or degradation of m 6 A-modified mRNA. We demonstrate that m 6 A modulates infection by hepatitis C virus (HCV). Depletion of m 6 A methyltransferases or an m 6 A demethylase, respectively, increases or decreases infectious HCV particle production. During HCV infection, YTHDF proteins relocalize to lipid droplets, sites of viral assembly, and their depletion increases infectious viral particles. We further mapped m 6 A sites across the HCV genome and determined that inactivating m 6 A in one viral genomic region increases viral titer without affecting RNA replication. Additional mapping of m 6 A on the RNA genomes of other Flaviviridae, including dengue, Zika, yellow fever, and West Nile virus, identifies conserved regions modified by m 6 A. Altogether, this work identifies m 6 A as a conserved regulatory mark across Flaviviridae genomes., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Disruption of calmodulin (CaM)-based protein interactions has been touted as a potential means for modulating several disease pathways. Among these is SOX9, which is a DNA binding protein that is involved in chrondrocyte differentiation and regulation of the hormones that control sexual development. In this work, we employed a "magnetic fishing"/mass spectrometry assay in conjunction with intrinsic fluorescence to examine the interaction of CaM with the CaM-binding domain of SOX9 (SOX-CAL), and to assess the modulation of this interaction by known anti-CaM compounds. Our data show that there is a high affinity interaction between CaM and SOX-CAL (27±9 nM), and that SOX-CAL bound to the same location as the well-known CaM antagonist melittin; unexpectedly, we also found that addition of CaM-binding small molecules initially produced increased SOX-CAL binding, indicative of binding to both the well-known high-affinity CaM binding site and a second, lower-affinity binding site., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Wolbachia is a maternally transmitted intracellular bacterium that is estimated to infect up to 65% of insect species, but it is not naturally present in Anopheles malaria vectors. Wolbachia-based strategies for malaria vector control can be developed either through population replacement to reduce vectorial capacity or through population suppression to reduce the mosquito population. We have previously generated An. stephensi mosquitoes carrying a stable wAlbB Wolbachia infection and have demonstrated their ability to invade wild-type laboratory populations and confer resistance to Plasmodium on these populations., Methods: We assessed wAlbB-associated fitness by comparing the female fecundity, immature development and survivorship, body size, male mating competiveness, and adult longevity of the infected An. stephensi to that of wild-type mosquitoes., Results: We found that wAlbB reduced female fecundity and caused a minor decrease in male mating competiveness. We also observed that wAlbB increased the life span of both male and female mosquitoes when they were maintained solely on sugar meals; however, there was no impact on the life span of blood-fed females. In addition, wAlbB did not influence either immature development and survivorship or adult body sizes., Conclusions: These results provide significant support for developing Wolbachia-based strategies for malaria vector control.

Efficient DNA repair mechanisms frequently limit the effectiveness of chemotherapeutic agents that act through DNA damaging mechanisms. Consequently, proteins involved in DNA repair have increasingly become attractive targets of high-throughput screening initiatives to identify modulators of these pathways. Disruption of the XRCC4-Ligase IV interaction provides a novel means to efficiently halt repair of mammalian DNA double strand break repair; however; the extreme affinity of these proteins presents a major obstacle for drug discovery. A better understanding of the interaction surfaces is needed to provide a more specific target for inhibitor studies. To clearly define key interface(s) of Ligase IV necessary for interaction with XRCC4, we developed a competitive displacement assay using ESI-MS/MS and determined the minimal inhibitory fragment of the XRCC4-interacting region (XIR) capable of disrupting a complex of XRCC4/XIR. Disruption of a single helix (helix 2) within the helix-loop-helix clamp of Ligase IV was sufficient to displace XIR from a preformed complex. Dose-dependent response curves for the disruption of the complex by either helix 2 or helix-loop-helix fragments revealed that potency of inhibition was greater for the larger helix-loop-helix peptide. Our results suggest a susceptibility to inhibition at the interface of helix 2 and future studies would benefit from targeting this surface of Ligase IV to identify modulators that disrupt its interaction with XRCC4. Furthermore, helix 1 and loop regions of the helix-loop-helix clamp provide secondary target surfaces to identify adjuvant compounds that could be used in combination to more efficiently inhibit XRCC4/Ligase IV complex formation and DNA repair., (Copyright © 2013 Wiley Periodicals, Inc.)

Protein-protein interactions are an intricate part of biological pathways and have become important targets for drug discovery. Here we present a two-stage magnetic bead assay to functionally screen small-molecule mixtures for modulators of protein-based interactions, with simultaneous affinity-based isolation of active compounds and identification by mass spectrometry. Proteins of interest interact in solution prior to the addition of Ni(II)-functionalized magnetic beads to recover an intact protein-protein complex through affinity capture of a polyhistidine-tagged primary target ("protein-complex fishing"). Protein-complex fishing, utilizing His(6)-tagged calmodulin (CaM) as the primary (bait) protein and melittin (Mel) as the target, was used to screen a mass-encoded library of 1000 bioactive compounds (50 mixtures, 20 compounds each) and successfully identified three known antagonists, three naturally occurring phenolic compounds previously reported to disrupt CaM-activated phosphodiesterase activity, and two newly identified modulators of the CaM-Mel interaction, methylbenzethonium and pempidine tartrate. The ability to produce quantitative inhibition data is also shown through the development of dose-dependent response curves and the determination of inhibition constants (K(I)) for the novel compound methylbenzethonium (K(I) = 14-49 nM) and two known antagonists, calmidazolium (K(I) = 1.7-7.5 nM) and trifluoperazine (K(I) = 1.2-3.0 μM), with the latter two values being in close agreement with literature values.

A reagentless bioactive paper-based solid-phase biosensor was developed for detection of acetylcholinesterase (AChE) inhibitors, including organophosphate pesticides. The assay strip is composed of a paper support (1 x 10 cm), onto which AChE and a chromogenic substrate, indophenyl acetate (IPA), were entrapped using biocompatible sol-gel derived silica inks in two different zones (e.g., sensing and substrate zones). The assay protocol involves first introducing the sample to the sensing zone via lateral flow of a pesticide-containing solution. Following an incubation period, the opposite end of the paper support is placed into distilled deionized water (ddH(2)O) to allow lateral flow in the opposite direction to move paper-bound IPA to the sensing area to initiate enzyme catalyzed hydrolysis of the substrate, causing a yellow-to-blue color change. The modified sensor is able to detect pesticides without the use of any external reagents with excellent detection limits (bendiocarb approximately 1 nM; carbaryl approximately 10 nM; paraoxon approximately 1 nM; malathion approximately 10 nM) and rapid response times (approximately 5 min). The sensor strip showed negligible matrix effects in detection of pesticides in spiked milk and apple juice samples. Bioactive paper-based assays on pesticide residues collected from food samples showed good agreement with a conventional mass spectrometric assay method. The bioactive paper assay should, therefore, be suitable for rapid screening of trace levels of organophosphate and carbamate pesticides in environmental and food samples.

The use of optical interconnects for communication between points on a microchip is motivated by system-level interconnect modeling showing the saturation of metal wire capacity at the global layer. Free-space optical solutions are analyzed for intrachip communication at the global layer. A multiscale solution comprising microlenses, etched compound slope microprisms, and a curved mirror is shown to outperform a single-scale alternative. Microprisms are designed and fabricated and inserted into an optical setup apparatus to experimentally validate the concept. The multiscale free-space system is shown to have the potential to provide the bandwidth density and configuration flexibility required for global communication in future generations of microchips.

A dual-rate (2 Gbit/s and 100 Mbit/s) optical transceiver designed for power-efficient connections within and between modern high-speed digital systems is described. The transceiver can dynamically adjust its data rate according to performance requirements, allowing for power-on-demand operation. Dynamic power management permits energy saving and lowers device operating temperatures, improving the reliability and lifetime of optoelectronic-devices such as vertical-cavity surface-emitting lasers (VCSELs). To implement dual-rate functionality, we include in the transmitter and receiver circuits separate high-speed and low-power data path modules. The high-speed module is designed for gigabit operation to achieve high bandwidth. A simpler low-power module is designed for megabit data transmission with low power consumption. The transceiver is fabricated in a 0.5 microm silicon-on-sapphire complementary metal-oxide semiconductor. The VCSEL and photodetector devices are attached to the transceiver's integrated circuit by flip-chip bonding. A free-space optical link system is constructed to demonstrate correct dual-rate functionality. Experimental results show reliable link operation at 2 Gbit/s and 100 Mbit/s data transfer rates with approximately 104 and approximately 9 mW power consumption, respectively. The transceiver's switching time between these two data rates is demonstrated as 10 micros, which is limited by on-chip register reconfiguration time. Improvement of this switching time can be obtained by use of dedicated input-output pads for dual-rate control signals.

Optical links are traditionally set to transmit maximum power for worst-case loss and consequently to dissipate more power than is required. We describe a technique to minimize power consumption based on the measured bit-error rate (BER) of the link. This technique uses a novel power-negotiation algorithm that optimizes the link power setting to achieve minimum power dissipation for a target BER. A 0.5 microm complementary metal-oxide semiconductor optical transceiver chip was fabricated, and a free-space optical interconnect system was built for validation. The results showed that the algorithm was able to find the optimum power settings for the VCSELs for a target BER and to account for dynamic changes such as variation in the optical loss in the system.

Experimental validation of a distortion removal technique for multi-chip free-space optical shuffle interconnections is presented. The free-space fabric links dense two-dimensional arrays of vertical cavity surface emitting laser(s) (VCSEL)(s) and detectors and must achieve full field registration on the order of 10 microns across the entire array. The new hybrid micro-macro optical concept realizes the required high-registration accuracy by simultaneously eliminating distortion in each of the interleaved off-axis imaging systems that comprise the complete fabric. This is achieved by exploiting the typically low numerical aperture of VCSELs. Individually tailored beam-deflecting micro-optical elements were used to create symmetry about a central aperture for VCSEL beams in the optical system. Experiments were developed to quantify the registration accuracy, the VCSEL images, and the associated spot sizes. The experimental results show that beam steering can be implemented to remove distortion in off-axis free-space optical-interconnection systems.

The ACTIVE-EYES (adaptive control for thermal imagers via electro-optic elements to yield an enhanced sensor) architecture, an adaptive image-segmentation and processing architecture, based on digital micromirror (DMD) array technology, is described. The concept provides efficient front-end processing of multispectral image data by adaptively segmenting and routing portions of the scene data concurrently to an imager and a spectrometer. The goal is to provide a large reduction in the amount of data required to be sensed in a multispectral imager by means of preprocessing the data to extract the most useful spatial and spectral information during detection. The DMD array provides the flexibility to perform a wide range of spatial and spectral analyses on the scene data. The spatial and spectral processing for different portions of the input scene can be tailored in real time to achieve a variety of preprocessing functions. Since the detected intensity of individual pixels may be controlled, the spatial image can be analyzed with gain varied on a pixel-by-pixel basis to enhance dynamic range. Coarse or fine spectral resolution can be achieved in the spectrometer by use of dynamically controllable or addressable dispersion elements. An experimental prototype, which demonstrated the segmentation between an imager and a grating spectrometer, was demonstrated and shown to achieve programmable pixelated intensity control. An information theoretic analysis of the dynamic-range control aspect was conducted to predict the performance enhancements that might be achieved with this architecture. The results indicate that, with a properly configured algorithm, the concept achieves the greatest relative information recovery from a detected image when the scene is made up of a relatively large area of moderate-dynamic-range pixels and a relatively smaller area of strong pixels that would tend to saturate a conventional sensor.

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