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Across biological scales, gene-regulatory networks employ autorepression (negative feedback) to maintain homeostasis and minimize failure from aberrant expression. Here, we present a proof of concept that disrupting transcriptional negative feedback dysregulates viral gene expression to therapeutically inhibit replication and confers a high evolutionary barrier to resistance. We find that nucleic-acid decoys mimicking cis-regulatory sites act as "feedback disruptors," break homeostasis, and increase viral transcription factors to cytotoxic levels (termed "open-loop lethality"). Feedback disruptors against herpesviruses reduced viral replication >2-logs without activating innate immunity, showed sub-nM IC50, synergized with standard-of-care antivirals, and inhibited virus replication in mice. In contrast to approved antivirals where resistance rapidly emerged, no feedback-disruptor escape mutants evolved in long-term cultures. For SARS-CoV-2, disruption of a putative feedback circuit also generated open-loop lethality, reducing viral titers by >1-log. These results demonstrate that generating open-loop lethality, via negative-feedback disruption, may yield a class of antimicrobials with a high genetic barrier to resistance.

Mononuclear phagocytes (MP) have central importance in innate immunity, inflammation, and fibrosis. Recruited MPs, such as macrophages, are plastic cells and can switch from an inflammatory to a restorative phenotype during the healing process. However, the role of the MPs in corneal wound healing is not completely understood. The purpose of this study is to characterize the kinetics of recruited MPs and evaluate the role of macrophage metalloelastase (MMP12) in the healing process, using an in vivo corneal chemical injury model. Unwounded and wounded corneas of wild-type (WT) and Mmp12-/- mice were collected at 1, 3, and 6 days after chemical injury and processed for flow cytometry analysis. Corneal MP phenotype significantly changed over time with recruited Ly6Chigh (proinflammatory) cells being most abundant at 1 day post-injury. Ly6Cint cells were highly expressed at 3 days post-injury and Ly6Cneg (patrolling) cells became the predominant cell type at 6 days post-injury. CD11c+ dendritic cells were abundant in corneas from Mmp12-/- mice at 6 days post-injury. These findings show the temporal phenotypic plasticity of recruited MPs and provide valuable insight into the role of the MPs in the corneal repair response, which may help guide the future development of MP-targeted therapies.

Probabilistic bet hedging, a strategy to maximize fitness in unpredictable environments by matching phenotypic variability to environmental variability, is theorized to account for the evolution of various fate-specification decisions, including viral latency. However, the molecular mechanisms underlying bet hedging remain unclear. Here, we report that large variability in protein abundance within individual herpesvirus virion particles enables probabilistic bet hedging between viral replication and latency. Superresolution imaging of individual virions of the human herpesvirus cytomegalovirus (CMV) showed that virion-to-virion levels of pp71 tegument protein-the major viral transactivator protein-exhibit extreme variability. This super-Poissonian tegument variability promoted alternate replicative strategies: high virion pp71 levels enhance viral replicative fitness but, strikingly, impede silencing, whereas low virion pp71 levels reduce fitness but promote silencing. Overall, the results indicate that stochastic tegument packaging provides a mechanism enabling probabilistic bet hedging between viral replication and latency.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

PurposeMatrix metalloproteinases (MMPs) comprise a family of zinc-dependent endopeptidases involved in wound healing processes, including neovascularization and fibrosis. We assessed MMP protein expression levels in diseased corneas of patients requiring penetrating and deep anterior lamellar keratoplasty. The purpose of this study was to test the hypothesis that upregulation of MMPs in diseased corneas is positively associated with clinical levels of corneal neovascularization and fibrosis.MethodsProtein expression levels of nine individual MMPs were quantified simultaneously in human corneal lysates by using the Bio-Plex Pro Human MMP 9-Plex Panel and the MAGPIX technology. Measurements of MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP12, and MMP13 were performed on diseased specimens from 21 patients undergoing corneal transplantation (17 for penetrating keratoplasty and 4 for deep anterior lamellar keratoplasty) and 6 normal control corneas.ResultsLuminex-based expression analysis revealed a significant overexpression of four of the nine MMPs tested (MMP2, MMP8, MMP12, and MMP13) in patient samples compared to control. Significant overexpression of MMP1, MMP2, MMP8, MMP12, and MMP13 was observed in diseased corneas with neovascularization compared with diseased corneas without neovascularization. Overexpression of MMP1, MMP2, MMP8, MMP12, and MMP13 also corresponded with the levels of corneal fibrosis. Finally, reduced expression of MMP3 was detected in keratoconus patients.ConclusionsMultiple MMPs are expressed in the corneas of patients with chronic disease requiring keratoplasty even when the pathologic process appears to be clinically inactive. In particular, the expression of several MMPs (MMP2, MMP8, MMP12, and MMP13) is positively associated with increased levels corneal fibrosis and neovascularization.

Homeostatic maintenance of corneal endothelial cells is essential for maintenance of corneal deturgescence and transparency. In Fuchs endothelial corneal dystrophy (FECD), an accelerated loss and dysfunction of endothelial cells leads to progressively severe visual impairment. An abnormal accumulation of extracellular matrix (ECM) is a distinctive hallmark of the disease, however the molecular pathogenic mechanisms underlying this phenomenon are not fully understood. Here, we investigate genome-wide and sequence-specific DNA methylation changes of miRNA genes in corneal endothelial samples from FECD patients. We discover that miRNA gene promoters are frequent targets of aberrant DNA methylation in FECD. More specifically, miR-199B is extensively hypermethylated and its mature transcript miR-199b-5p was previously found to be almost completely silenced in FECD. Furthermore, we find that miR-199b-5p directly and negatively regulates Snai1 and ZEB1, two zinc finger transcription factors that lead to increased ECM deposition in FECD. Taken together, these findings suggest a novel epigenetic regulatory mechanism of matrix protein production by corneal endothelial cells in which miR-199B hypermethylation leads to miR-199b-5p downregulation and thereby the increased expression of its target genes, including Snai1 and ZEB1. Our results support miR-199b-5p as a potential therapeutic target to prevent or slow down the progression of FECD disease.

Following corneal injury, coordinated cellular and protein interactions occur at the wound site to restore tissue homeostasis. Regulation of this response is required to prevent the development of chronic inflammation, abnormal neovascularization, and fibrosis. The chemokine CCL2 and its primary receptor CCR2 are key regulators of the inflammatory and neovascular responses to injury. In this study, we investigated the role of macrophage-associated matrix metalloproteinase 12 (MMP12) in the regulation of CCL2 and CCR2 after corneal wounding. Using two corneal injury models, we examined the temporal and spatial expression of CCL2 and CCR2 in Mmp12-/- and wild-type (WT) mice. Our data showed that MMP12 downregulated CCL2 and CCR2 expression in a manner dependent on the timing and mechanism of injury. We also examined the effect of CCL2 on the injury response in Mmp12-/- and WT corneas. We found that macrophage infiltration and neovascularization following CCL2 blockade was significantly reduced in Mmp12-/- corneas as compared with WT corneas. These findings indicate that MMP12 inhibits corneal inflammation and neovascularization after injury through its regulation of CCL2.

Corneal epithelial defects are a common cause of ocular morbidity and can result in corneal scarring if they do not heal properly. Matrix metalloproteinases (MMPs) are extracellular matrix proteinases that regulate multiple aspects of corneal repair. We have previously shown that MMP12 has a protective effect on corneal fibrosis through its regulation of neutrophil and macrophage infiltration and angiogenesis in a chemical injury model involving full thickness damage to the cornea. However, the role of MMP12 in injuries limited to the corneal epithelium is relatively unknown. This study investigates the reparative effects of MMP12 following isolated corneal epithelial injury. Using a corneal epithelial debridement injury model performed on corneas of wild-type (WT) mice, we show that Mmp12 is expressed early following corneal epithelial injury with highest expression levels at 8 h after injury and lower expression levels at 4 and 8 days after injury. We investigated whether MMP12 has an effect on the rate of epithelial repair and cell migration using in vivo and in vitro scratch assays performed on WT and Mmp12-/- mice. We found that loss of MMP12 results in a slower scratch wound repair rate both in vivo and in vitro. We also found that corneas of Mmp12-/- mice have decreased neutrophil infiltration following injury. Loss of MMP12, however, does not affect cell proliferation in the center of the wounds. These data support a role of MMP12 in promoting early repair processes following corneal epithelial injury by enhancing epithelial cell migration and neutrophil infiltration.

Transparency of the human cornea is necessary for vision. Fuchs Endothelial Corneal Dystrophy (FECD) is a bilateral, heritable degeneration of the corneal endothelium, and a leading indication for corneal transplantation in developed countries. While the early onset, and rarer, form of FECD has been linked to COL8A2 mutations, the more common, late onset form of FECD has genetic mutations linked to only a minority of cases. Epigenetic modifications that occur in FECD are unknown. Here, we report on and compare the DNA methylation landscape of normal human corneal endothelial (CE) tissue and CE from FECD patients using the Illumina Infinium HumanMethylation450 (HM450) DNA methylation array. We show that DNA methylation profiles are distinct between control and FECD samples. Differentially methylated probes (10,961) were identified in the FECD samples compared with the control samples, with the majority of probes being hypermethylated in the FECD samples. Genes containing differentially methylated sites were disproportionately annotated to ontological categories involving cytoskeletal organization, ion transport, hematopoetic cell differentiation, and cellular metabolism. Our results suggest that altered DNA methylation patterns may contribute to loss of corneal transparency in FECD through a global accumulation of sporadic DNA methylation changes in genes critical to basic CE biological processes.

Preterm delivery may have a strong impact on mothers. In a multicentre randomised controlled trial, including very preterm infants (less than 32 weeks and/or less than 1500 g), the effect of the Infant Behavioral Assessment and Intervention Program (IBAIP) on maternal psychological distress at 6, 12 and 24 (corrected) months after preterm birth was assessed. The programme is designed to assist parents to support and enhance their infants' regulatory competence and development. Eighty-six infants and their parents were randomly assigned to the intervention group and 90 to the control group. Maternal psychological distress was assessed with the General Health Questionnaire. In general, the mothers reported high levels of psychological distress, especially during the first six months after discharge from hospital when 56% had clinical scores. No differences were found in maternal psychological distress between the intervention and control group. Early intervention to decrease maternal distress in mothers of preterm infants is warranted. (Contains 3 tables.)

Background: Prematurity and perinatal insults lead to increased developmental vulnerability. The home-based Infant Behavioral Assessment and Intervention Program (IBAIP) was designed to improve development of preterm infants. In a multicenter randomized controlled trial the effect of IBAIP on mother-infant interaction was studied as a secondary outcome. Method: Mother-infant interaction was assessed during the Still-face procedure at 6 months corrected age. One hundred and twelve mother-infant dyads (57 intervention, 55 control) were studied. Results: Findings partially supported our hypothesis that the intervention would increase maternal sensitivity in interaction with their preterm infants. No effects were found on infant self-regulatory behavior or positive interaction behavior. Conclusion: The family-centered and strength-based approach of IBAIP appears to be a promising intervention method to promote sensitive mother-infant interaction at home after discharge from hospital. However, no positive effects were found on infant interaction behavior.

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DR.BEAT ("Digital Research on Ballistocardiography for Extraterrestrial And Terrestrial use") develops a miniaturized sensor system with signal processing to interpret ballistocardiographic signals and implements an application oriented user interface. Presented is a breadboard prototype's functional tests with regard to data completeness and plausibility. The analysis confirmed a reliability of 99.99995% over the tests and the signals displayed the expected heart-specific characteristics. These results support the ethical justifiability of an initial study. [ABSTRACT FROM AUTHOR]

The ever-increasing flood of information, especially in the medical-clinical field, inter alia due to the increase of data-intensive mobile/wearable devices, is one of the biggest challenges of medical informatics. In this work different possibilities of the integration of wearable devices on the example of the development of a fall prevention system are presented and classified. We started with a commercial off-the-shelf one-on-one system (Shimmer/Arduino) using a Bluetooth connection, integrated via nurse call system as Human-Machine- Interface. During the evaluation of the system we mentioned several new requirements and optimization possibilities. Thus, we adjusted the hardware-software-system; the near-field communication was realized by IEEE802.15.4, we replaced commercial off-the-shelf devices with highly-specialized open hardware in-house developments and we transferred the nurse call integration by setting up our own network and integrating this into an existing (wireless) local area network. With this development, the energy-efficient, simple and intuitive mechanisms of proximity communication via IEEE802.15.4 can be combined with the benefits broadband functionalities, e.g. of Wi-Fi, with both worlds benefits and the compensation of some disadvantages. [ABSTRACT FROM AUTHOR]

This article analyses imaginaries of political decision makers of the European Union in the context of the ‘refugee crisis’ and interprets them according to theories of European integration – neofunctionalism and liberal intergovernmentalism. Speeches, interviews, statements and press releases of the 28 heads of state and government and two Commissioners are analysed through a qualitative content analysis. The aim of the article is to derive prospects for European integration from the imaginaries. We found that the European imaginaries expressed by the largest group of heads of state and government remain blurred without clarification which position is taken on European integration, while the imaginaries expressed by the Commissioners are mainly characterised by support of further integration. Our interpretation of the European imaginaries suggests that the prospects for further integration remain limited according to neofunctionalism, but are higher following liberal intergovernmentalism. In policy fields that are directly related to the management of the crisis further cooperation can be expected. [ABSTRACT FROM PUBLISHER]