Disrupting autorepression circuitry generates "open-loop lethality" to yield escape-resistant antiviral agents.

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.

A molecular mechanism for probabilistic bet hedging and its role in viral latency.

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.

Characterization of Recruited Mononuclear Phagocytes following Corneal Chemical Injury.

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.

Development and nationwide implementation of a postdischarge responsive parenting intervention program for very preterm born children: The TOP program.

A previous randomized controlled trial has suggested the effectiveness of a Dutch postdischarge responsive parenting program for very preterm (VPT) infants, indicating that nationwide implementation was justified. This paper describes the development and nationwide implementation of the intervention, known as the TOP program, which consisted of three phases. In the preparation phase (2006-2010), a theory of change and the structure of the TOP program were developed, and funding for phase two, based on a positive Business Case, was obtained. In the pilot implementation phase (2010-2014), intervention strategies were developed for a real-world setting, capacity and adoption were increased, systematic evaluations were incorporated, and sustained funding was obtained. In the full-implementation phase (2014-2019), all Dutch Healthcare Insurers reimbursed the TOP program, enabling VPT infants to participate in the program without charge. By 2018, the number of interventionists that provided the TOP program had increased from 37 to 91, and all level III hospitals and 65% of regional hospitals in the Netherlands referred VPT infants. Currently, the program reaches 70% of the Dutch target population and parental satisfaction with the TOP program is high. After a 12-year implementation period, the TOP program forms part of routine care in the Netherlands.

Un previo ensayo controlado al azar ha sugerido la eficacia de un programa holandés sobre la crianza sensible para infantes muy prematuros (VPT) posterior al momento en que se les dio de alta, indicando que la implementación a lo largo de toda la nación era justificada. Este artículo describe el desarrollo y la implementación a nivel de toda la nación de la intervención, conocida como el programa ToP, el cual consistía de tres fases. En la fase de preparación (2006-2010), se desarrollaron una teoría de cambio y la estructura del programa ToP, y se obtuvieron los fondos para la fase dos, con base en un Caso de Negocios (BC) positivo. En la fase piloto de implementación (2010-2014), se desarrollaron estrategias de intervención para un escenario del mundo real, se aumentaron la capacidad y la adopción, se incorporaron evaluaciones sistemáticas y se obtuvieron fondos para mantener el programa. En la fase de implementación completa (2014-2019), todas las Aseguradoras Holandesas del Sector Salud reembolsaron el costo del programa ToP, permitiéndoles a los infantes VPT participar en el programa sin costo alguno. Para 2018, el número de practicantes de la intervención que prestaban el servicio del programa ToP había aumentado de 37 a 91, y todos los hospitales del nivel III y 65% de los hospitales regionales en Holanda refirieron los infantes VPT al programa. Actualmente, el programa llega a 70% de la población holandesa para la cual está destinado y la satisfacción de los padres con el programa ToP es alta. Después de un período de implementación de 12 años, el programa ToP forma parte del cuidado de salud rutinario en Holanda.

Un essai contrôlé randomisé précédent a suggéré l'efficacité d'un programme hollandais de sensibilité de parentage après la sortie de l'hôpital pour les nourrissons grands prématurés (GP ici en français), indiquant qu'une mise en œuvre au niveau national était justifiée. Cet article décrit le développement et la mise en œuvre au niveau national de l'intervention, connue en tant que ToP program, qui a consisté en trois phases. Dans la phase de préparation (2006-2010), une théorie du changement et la structure du programme ToP a été développée, et le financement pour la phase deux, basée sur une Etude de Cas positive, a été sécurisé. Dans la phase pilote d'implémentation (2010-2014) des stratégies d'intervention ont été développées pour un contexte réel, la capacité et l'adoption ont été augmentées, les évaluations systématiques ont été incorporées, et un financement durable a été sécurisé. Dans la pleine phase de mise en œuvre (2014-1029), tous les Assurances Santé Hollandaises ont remboursé le programme ToP, permettant aux nourrissons GP de participer au programme sans coût. En 2018 le nombre de prestataires qui offraient le programme ToP a augmenté de 37 à 91, et tous les hôpitaux de niveau III ainsi que 65% des hôpitaux régionaux aux Pays Bas ont envoyé les nourrissons GP au programme. En ce moment le programme atteint 70% de la population cible hollandaise et la satisfaction parentale avec le programme ToP est élevée. Après une période de mise en œuvre de 12 ans le programme ToP fait partie des soins de routine aux Pays Bas.

Bioavailability of Lumefantrine Is Significantly Enhanced with a Novel Formulation Approach, an Outcome from a Randomized, Open-Label Pharmacokinetic Study in Healthy Volunteers.

The artemether-lumefantrine combination requires food intake for the optimal absorption of lumefantrine. In an attempt to enhance the bioavailability of lumefantrine, new solid dispersion formulations (SDF) were developed, and the pharmacokinetics of two SDF variants were assessed in a randomized, open-label, sequential two-part study in healthy volunteers. In part 1, the relative bioavailability of the two SDF variants was compared with that of the conventional formulation after administration of a single dose of 480 mg under fasted conditions in three parallel cohorts. In part 2, the pharmacokinetics of lumefantrine from both SDF variants were evaluated after a single dose of 480 mg under fed conditions and a single dose of 960 mg under fasted conditions. The bioavailability of lumefantrine from SDF variant 1 and variant 2 increased up to ∼48-fold and ∼24-fold, respectively, relative to that of the conventional formulation. Both variants demonstrated a positive food effect and a less than proportional increase in exposure between the 480-mg and 960-mg doses. Most adverse events (AEs) were mild to moderate in severity and not suspected to be related to the study drug. All five drug-related AEs occurred in subjects taking SDF variant 2. No clinically significant treatment-emergent changes in vital signs, electrocardiograms, or laboratory blood assessments were noted. The solid dispersion formulation enhances the lumefantrine bioavailability to a significant extent, and SDF variant 1 is superior to SDF variant 2.

Effects of MMP12 on cell motility and inflammation during corneal epithelial repair.

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.

Rethinking preventive post-discharge intervention programmes for very preterm infants and their parents.

Post-discharge preventive intervention programmes with involvement of the parent may support the resilience and developmental outcomes of infants born very preterm. Randomized controlled trials of home-based family-centred intervention programmes in very preterm infants that aimed to improve cognitive outcome, at least at age two, were selected and updated on the basis of a recent systematic review to compare their content and effect over time to form the basis of a narrative review. Six programmes were included in this narrative review. Four of the six programmes led to improved child cognitive and/or motor development. Two programmes, which focused primarily on responsive parenting and development, demonstrated improved cognitive outcome up till 5 years after completion of the programme. The programmes that also focused on maternal anxiety remediation led to improved maternal mental well-being, along with improved child behaviour, in one study - even at 3 years after completion of the programme. The magnitude of the effects was modest. Family-centred preventive intervention programmes that aim at improvement of child development should be continued after discharge home to improve the preterm child's resilience. Programmes may be most effective when they support the evolvement of a responsive parent-infant relationship over time, as well as the parent's well-being.

DR.BEAT: Initial Functional Testing of a BCG Wearable Prototype for Recording Ballistocardiographic Signals.

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.

Rhesus rotavirus trafficking during entry into MA104 cells is restricted to the early endosome compartment.

Endocytosis has recently been implicated in rotavirus (RV) entry. We examined the role of Rabs, which regulate endosomal trafficking, during RV entry. Several structural proteins of neuraminidase-sensitive and -insensitive RVs colocalized with Rab5, an early endosome marker, but not Rab7, a late endosome marker. Dominant-negative and constitutively active mutants demonstrated that Rab5 but not Rab4 or Rab7 affects rhesus RV (RRV) infectivity. These data suggest that early RRV trafficking is confined to the early endosome compartment and requires Rab5.

DR.BEAT: First Insights into a Study to Collect Baseline BCG Data with a Sensor-Based Wearable Prototype in Heart-Healthy Adults.

The DR.BEAT project aims at the further development of a measurement system for recording ballistocardiographic signals into a body-worn sensor system combined with extensive signal processing, data evaluation and visualization. With a first breadboard prototype, an explorative feasibility study for acquiring initial signals of healthy cardiac activity in adults was performed. This paper briefly presents the DR.BEAT project, the breadboard prototype, the study conducted, and initial insights into the study results. The signals obtained in the study exhibit the seismocardiographic characteristics as reported in the literature and form the basis for further development of the hardware as well as the pre-processing and automated analysis algorithms in the DR.BEAT project.Clinical Relevance- The characteristics of ballisto- and seismocardiographic signals allow to infer about the mechanical work of the heart. The development of a body-worn sensor system to record ballisto- and seismocardiographic signals, compact enough for everyday wear, enables the acquisition of heart-specific parameters in terrestrial as well as extraterrestrial application scenarios. Combined with extensive signal analysis and visualization, it holds the potential to monitor heart health in a variety of contexts and support its maintenance and improvement.

Rhesus rotavirus entry into a polarized epithelium is endocytosis dependent and involves sequential VP4 conformational changes.

Rotavirus (RV) cell entry is an incompletely understood process, involving VP4 and VP7, the viral proteins composing the outermost layer of the nonenveloped RV triple-layered icosahedral particle (TLP), encasing VP6. VP4 can exist in three conformational states: soluble, cleaved spike, and folded back. In order to better understand the events leading to RV entry, we established a detection system to image input virus by monitoring the rhesus RV (RRV) antigens VP4, VP6, and VP7 at very early times postinfection. We provide evidence that decapsidation occurs directly after cell membrane penetration. We also demonstrate that several VP4 and VP7 conformational changes take place during entry. In particular, we detected, for the first time, the generation of folded-back VP5 in the context of the initiation of infection. Folded-back VP5 appears to be limited to the entry step. We furthermore demonstrate that RRV enters the cell cytoplasm through an endocytosis pathway. The endocytosis hypothesis is supported by the colocalization of RRV antigens with the early endosome markers Rab4 and Rab5. Finally, we provide evidence that the entry process is likely dependent on the endocytic Ca(2+) concentration, as bafilomycin A1 treatment as well as an augmentation of the extracellular calcium reservoir using CaEGTA, which both lead to an elevated intraendosomal calcium concentration, resulted in the accumulation of intact virions in the actin network. Together, these findings suggest that internalization, decapsidation, and cell membrane penetration involve endocytosis, calcium-dependent uncoating, and VP4 conformational changes, including a fold-back.

Roles of VP4 and NSP1 in determining the distinctive replication capacities of simian rotavirus RRV and bovine rotavirus UK in the mouse biliary tract.

Rotavirus replication and virulence are strongly influenced by virus strain and host species. The rotavirus proteins VP3, VP4, VP7, NSP1, and NSP4 have all been implicated in strain and species restriction of replication; however, the mechanisms have not been fully determined. Simian (RRV) and bovine (UK) rotaviruses have distinctive replication capacities in mouse extraintestinal organs such as the biliary tract. Using reassortants between UK and RRV, we previously demonstrated that the differential replication of these viruses in mouse embryonic fibroblasts is determined by the respective NSP1 proteins, which differ substantially in their abilities to degrade interferon (IFN) regulatory factor 3 (IRF3) and suppress the type I IFN response. In this study, we used an in vivo model of rotavirus infection of mouse gallbladder with UK × RRV reassortants to study the genetic and mechanistic basis of systemic rotavirus replication. We found that the low-replication phenotype of UK in biliary tissues was conferred by UK VP4 and that the high-replication phenotype of RRV was conferred by RRV VP4 and NSP1. Viruses with RRV VP4 entered cultured mouse cholangiocytes more efficiently than did those with UK VP4. Reassortants with RRV VP4 and UK NSP1 genes induced high levels of expression of IRF3-dependent p54 in biliary tissues, and their replication was increased 3-fold in IFN-α/ß and -γ receptor or STAT1 knockout (KO) mice compared to wild-type mice. Our data indicate that systemic rotavirus strain-specific replication in the murine biliary tract is determined by both viral entry mediated by VP4 and viral antagonism of the host innate immune response mediated by NSP1.

A neurobehavioral intervention and assessment program in very low birth weight infants: outcome at 24 months.

OBJECTIVE: To determine whether the Infant Behavioral Assessment and Intervention Program (IBAIP) improves development and behavior in very low birth weight (VLBW) infants at 24-month corrected age. STUDY DESIGN: In a multicenter, randomized, controlled trial 86 infants received postdischarge intervention until 6-month corrected age. The intervention consisted of supporting infants' self-regulation and development, and facilitating sensitive parent-infant interactions; 90 control infants received regular care. At 6 months, positive intervention effects were found. At 24 months, development and behavior were evaluated with the Bayley Scales of Infant Development-II (BSID-II) and the Child Behavior Check List (CBCL). RESULTS: Eighty-three intervention and 78 control infants were available for follow-up. After adjustment for differences in perinatal characteristics, an intervention effect of 6.4 points (+/- standard error, 2.4) on the Psychomotor Developmental Index favored the intervention infants. Groups did not differ on the Mental Developmental Index, the Behavioral Rating Scale of the BSID-II, or on the CBCL. Subgroup analyses revealed improved motor as well as improved mental outcomes in intervention infants with bronchopulmonary dysplasia and with combined biological and social risk factors. CONCLUSIONS: The IBAIP shows sustained motor improvement in VLBW infants until 2-year corrected age.

The effect of the Infant Behavioral Assessment and Intervention Program on mother-infant interaction after very preterm birth.

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.

The major form of hepatitis C virus alternate reading frame protein is suppressed by core protein expression.

Hepatitis C virus (HCV) is a human RNA virus encoding 10 proteins in a single open reading frame. In the +1 frame, an 'alternate reading frame' (ARF) overlaps with the core protein-encoding sequence and encodes the ARF protein (ARFP). Here, we investigated the molecular regulatory mechanisms of ARFP expression in HCV target cells. Chimeric HCV-luciferase reporter constructs derived from the infectious HCV prototype isolate H77 were transfected into hepatocyte-derived cell lines. Translation initiation was most efficient at the internal AUG codon at position 86/88, resulting in the synthesis of a truncated ARFP named (86/88)ARFP. Interestingly, (86/88)ARFP synthesis was markedly enhanced in constructs containing an inactivated core protein reading frame. This enhancement was reversed by co-expression of core protein in trans, demonstrating suppression of ARFP synthesis by HCV core protein. In conclusion, our results indicate that translation of ARFP occurs mainly by alternative internal initiation at position 86/88 and is regulated by HCV core protein expression. The suppression of ARFP translation by HCV core protein suggests that ARFP expression is inversely linked to the level of viral replication. These findings define key mechanisms regulating ARFP expression and set the stage for further studies addressing the function of ARFP within the viral life cycle.

Development of a Presentation Interface for Seismo- and Ballistocardiographic Data.

Ballistocardiography is a method to gain detailed information about body movements imparted by the ballistic forces associated with cardiac contraction. We measured using different setups and sensor positions to gain information of a reference signal of healthy adults. We used two resting state recordings and two recordings under physical stress (ergometer and treadmill) with stepwise increasing load. Data was gathered from 34 subjects, which results in 72 data sets, overall, more than 18h of signal data. With these data we have created a first database for BCG reference. We started data analysis and created a first naive data representation prototype. Using this naive attempt, we created a user interface for the intuitive representation of live data as well as retrospective data.

A Structured Measurement of Highly Synchronous Real-Time Ballistocardiography Signal Data of Heart Failure Patients.

Ballistocardiography (BCG) has gained more attention due to the fundamental goal of medical intervention in diagnostics and follow-up. BCG is particularly suitable for the study of heart failure, which a recent study has shown. The results of this working group shall be validated and reproduced with another study trial. Therefore, acceleration sensor prototypes will be placed on various parts of the patient's body and be connected to a computer unit, which allows a high data quality and high signal resolution. A temporal shift of only 20 ns ensures real-time measurement of BCG parameters. The reference measurement will be done with a 12-channel ECG. The study will include patients with heart failure. All conducted tests take place as part of the diagnostic-therapeutic routine. The only change in the procedure concerns the additional equipment with the measuring sensors. The results will be the validation of the data from the other working group, as well as the information about the choice of sensors and clock frequency, the measuring points and the needed features for early detection of heart failure in BCG signals.

Author Correction: Aberrant DNA methylation of miRNAs in Fuchs endothelial corneal dystrophy.

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