Cytoplasmic RNA sensors and their interplay with RNA-binding partners in innate antiviral response: theme and variations.

Sensing of pathogen-associated molecular patterns including viral RNA by innate immunity represents the first line of defense against viral infection. In addition to RIG-I-like receptors and NOD-like receptors, several other RNA sensors are known to mediate innate antiviral response in the cytoplasm. Double-stranded RNA-binding protein PACT interacts with prototypic RNA sensor RIG-I to facilitate its recognition of viral RNA and induction of host interferon response, but variations of this theme are seen when the functions of RNA sensors are modulated by other RNA-binding proteins to impinge on antiviral defense, proinflammatory cytokine production and cell death programs. Their discrete and coordinated actions are crucial to protect the host from infection. In this review, we will focus on cytoplasmic RNA sensors with an emphasis on their interplay with RNA-binding partners. Classical sensors such as RIG-I will be briefly reviewed. More attention will be brought to new insights on how RNA-binding partners of RNA sensors modulate innate RNA sensing and how viruses perturb the functions of RNA-binding partners.

Peste Des Petits Ruminants Virus Nucleocapsid Protein Interacts with Protein Kinase R-Activating Protein and Induces Stress Granules To Promote Viral Replication.

The pathogenic mechanisms of peste des petits ruminants virus (PPRV) infection remain poorly understood, leaving peste des petits ruminants (PPR) control and eradication especially difficult. Here, we determined that PPRV nucleocapsid (N) protein triggers formation of stress granules (SGs) to benefit viral replication. A mass spectrometry-based profiling of the interactome of PPRV N protein revealed that PPRV N protein interacted with protein kinase R (PKR)-activating protein (PACT), and this interaction was confirmed in the context of PPRV infection. PACT was essential for PPRV replication. Besides, the ectopic expression of N activated the PKR/eIF2α (α subunit of eukaryotic initiation factor 2) pathway through induction of PKR phosphorylation, but it did not induce PKR phosphorylation in PACT-deficient (PACT-/-) cells. PPRV N interacted with PACT, impairing the interaction between PACT and a PKR inhibitor, transactivation response RNA-binding protein (TRBP), which subsequently enhanced the interaction between PACT and PKR and thus promoted the activation of PKR and eIF2α phosphorylation, resulting in formation of stress granules (SGs). Consistently, PPRV infection induced SG formation through activation of the PKR/eIF2α pathway, and knockdown of N impaired PPRV-induced SG formation. PPRV-induced SG formation significantly decreased in PACT-/- cells as well. The role of SG formation in PPRV replication was subsequently investigated, which showed that SG formation plays a positive role in PPRV replication. By using an RNA fluorescence in situ hybridization assay, we found that PPRV-induced SGs hid cellular mRNA rather than viral mRNA. Altogether, our data provide the first evidence that PPRV N protein plays a role in modulating the PKR/eIF2α/SG axis and promotes virus replication through targeting PACT. IMPORTANCE Stress granule (SG) formation is a conserved cellular strategy to reduce stress-related damage regulating cell survival. A mass spectrometry-based profiling of the interactome of PPRV N protein revealed that PPRV N interacted with PACT to regulate the assembly of SGs. N protein inhibited the interaction between PACT and a PKR inhibitor, TRBP, through binding to the M1 domain of PACT, which enhanced the interaction between PACT and PKR and thus promoted PKR activation and subsequent eIF2α phosphorylation as well as SG formation. The regulatory function of N protein was strikingly abrogated in PACT-/- cells. SGs induced by PPRV infection through the PKR/eIF2α pathway are PACT dependent. The loss-of-function assay indicated that PPRV-induced SGs were critical for PPRV replication. We concluded that the PPRV N protein manipulates the host PKR/eIF2α/SG axis to favor virus replication.

Cell-type-specific need of Ddx3 and PACT for interferon induction by RNA viruses.

IMPORTANCE: Interferon-stimulated genes (ISGs) are induced in response to interferon expression due to viral infections. Role of these ISGs can be variable in different cells or organs. Our study highlights such cell-specific role of an ISG, Ddx3, which regulates the translation of mRNAs essential for interferon induction (PACT) and interferon signaling (STAT1) in a cell-specific manner. Our study also highlights the role of PACT in RNA virus-induced RLR signaling. Our study depicts how Ddx3 regulates innate immune signaling pathways in an indirect manner. Such cell-specific behavior of ISGs helps us to better understand viral pathogenesis and highlights the complexities of viral tropism and innate immune responses.

Easy and versatile cellulosic support inhibiting broad spectrum strains: synergy between photodynamic antimicrobial therapy and polymyxin B.

Despite advances achieved in the health field over the last decade, infections caused by resistant bacterial strains are an increasingly important societal issue that needs to be addressed. New approaches have already been developed to overcome this problem. Photodynamic antimicrobial chemotherapy (PACT) could provide a promising alternative method to eradicate microbes. This approach has already inspired the development of innovative surfaces. Interesting results were achieved against Gram-positive bacteria, but it also appeared that Gram-negative strains, especially Pseudomonas aeruginosa, were less sensitive to PACT. However, materials coated with cationic porphyrins have already proven their wide-spectrum activity, but these materials were not suitable for industrial-scale production. The main aim of this work was the design of a large-scale evolutionary material based on PACT and antibiotic prophylaxis. Transparent regenerated cellulose has been simply impregnated with a usual cationic porphyrin (N-methylpyridyl) and an antimicrobial peptide (polymyxin B). In addition to its photophysical properties, this film exhibited a wide-spectrum bactericidal activity over 4 days despite daily application of fresh bacterial inoculums. The efficiency of PACT and polymyxin B combination could help to reduce the emergence of bacterial multi-resistant strains and we believe that this kind of material would provide an excellent opportunity to prevent bacterial contamination of bandages or packaging.

Function analysis of fish PACT gene in response to virus infection.

PACT (interferon-inducible double-stranded RNA-dependent protein kinase activator A) is a cellular protein which can activate PKR in dsRNA-independent manner. However, the role of PACT in fish virus infection remains largely unknown. In this study, a PACT homologue from grouper (Epinephelus coioides)(EcPACT) was cloned and characterized. The open reading frame of EcPACT has a full length of 924 bp and encodes a protein of 307 amino acids with a predicted molecular weight of 33.29 kDa. Similar to mammals, EcPACT contains three dsRBD domains. EcPACT shares 99.67 % homology with E. lanceolatus. Real-time fluorescence quantitative PCR results showed that EcPACT mRNA was widely expressed in all tissues and abundantly expressed in brain, blood, head kidney and kidney. In addition, SGIV and RGNNV infection significantly upregulated the transcript levels of EcPACT. Subcellular localization analysis showed that EcPACT was mainly distributed in the nucleus. Overexpression of EcPACT inhibited the replication of SGIV and RGNNV in vitro and positively regulated the expression of interferon (IFN) and pro-inflammatory factors. The results provide a better understanding of the relationship between PACT and viral infection in fish.

The SHREAD gene therapy platform for paracrine delivery improves tumor localization and intratumoral effects of a clinical antibody.

The goal of cancer-drug delivery is to achieve high levels of therapeutics within tumors with minimal systemic exposure that could cause toxicity. Producing biologics directly in situ where they diffuse and act locally is an attractive alternative to direct administration of recombinant therapeutics, as secretion by the tumor itself provides high local concentrations that act in a paracrine fashion continuously over an extended duration (paracrine delivery). We have engineered a SHielded, REtargeted ADenovirus (SHREAD) gene therapy platform that targets specific cells based on chosen surface markers and converts them into biofactories secreting therapeutics. In a proof of concept, a clinically approved antibody is delivered to orthotopic tumors in a model system in which precise biodistribution can be determined using tissue clearing with passive CLARITY technique (PACT) with high-resolution three-dimensional imaging and feature quantification within the tumors made transparent. We demonstrate high levels of tumor cell-specific transduction and significant and durable antibody production. PACT gives a localized quantification of the secreted therapeutic and allows us to directly observe enhanced pore formation in the tumor and destruction of the intact vasculature. In situ production of the antibody led to an 1,800-fold enhanced tumor-to-serum antibody concentration ratio compared to direct administration. Our detailed biochemical and microscopic analyses thus show that paracrine delivery with SHREAD could enable the use of highly potent therapeutic combinations, including those with systemic toxicity, to reach adequate therapeutic windows.

PACT inhibits the replication of SARS-CoV-2 through the blockage of GSK-3ß-N-nsp3 cascade.

The protein activator of protein kinase R (PKR) (PACT) has been shown to play a crucial role in stimulating the host antiviral response through the activation of PKR, retinoic acid-inducible gene I, and melanoma differentiation-associated protein 5. Whether PACT can inhibit viral replication independent of known mechanisms is still unrevealed. In this study, we show that, like many viruses, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) hijacks GSK-3ß to facilitate its replication. GSK-3ß-induced phosphorylation on N protein increased the interaction between N protein and nsp3. Thus, GSK-3ß-N-nsp3 cascade promotes viral replication. Although SARS-CoV-2 can sabotage the activation of AKT, the upstream proteins suppressing the activation of GSK-3ß, we found that the host can use PACT, another protein kinase, instead of AKT to decrease the activity of GSK-3ß and the interaction between PACT and GSK-3ß is enhanced upon viral infection. Moreover, PACT inhibited the activity of GSK-3ß independent of its well-studied double-stranded RNA binding and PKR activating ability. In summary, this study identified an unknown function of PACT in inhibiting SARS-CoV-2 replication through the blockage of GSK-3ß-N-nsp3 cascade.

Sex differences in the severity and natural recovery of child PTSD symptoms: a longitudinal analysis of children exposed to acute trauma.

BACKGROUND: Higher levels of PTSD symptoms are present among trauma-exposed females v. males in adulthood; however, much less is known about the emergence of this sex difference during development. METHODS: In a multi-study sample of 7-18-year-olds (n = 3397), we examined the effect of sex and age on the severity of PTSD symptoms after a single incident trauma at 1 month (T1), and on symptom change after a natural recovery period of 3 (T2) and 6 months (T3). PTSD scores were harmonised across measurement types, and linear regressions were used to determine sex and age effects, adjusting for study level variance and trauma type. RESULTS: A sex × age interaction was observed at T1 (p < 0.001) demonstrating that older age was associated with greater PTSD symptom severity in females (ß = 0.008, p = 0.047), but less severe symptoms in males (ß = -0.011, p = 0.014). The same pattern was observed at T2 and T3, with sex differences beginning to emerge by age 12 years. PTSD symptoms decreased naturally by ~25% at T2 with little further improvement by T3. Further, females showed a greater reduction in symptoms at T3 than males, although the same effect was not observed at T2. CONCLUSIONS: Sex differences in PTSD symptoms become apparent during adolescence, due to opposing changes in susceptibility occurring in females and males with age. Understanding the factors contributing to these findings is likely to provide wider insight into sex-specific psychological vulnerability to trauma-related psychopathology.

Impact of Glasgow Climate Pact and Updated Nationally Determined Contribution on Mercury Mitigation Abiding by the Minamata Convention in India.

India is one of the largest emitters of atmospheric anthropogenic mercury (Hg) and the third-largest emitter of greenhouse gases in the world. In the past decade, India has been committed to the Minamata Convention (2017) in addition to the Paris Climate Change Agreement (2015) and the Glasgow Pact (2021). More than 70% to 80% of India's mercury and carbon dioxide emissions occur because of anthropogenic activities from coal usage. This study explores nine policy scenarios, the nationally determined contribution (NDC) scenario, and two deep decarbonization pathways (DDP) with and without mercury control technologies in the energy and carbon-intensive sectors using a bottom-up, techno-economic model, AIM/Enduse India. It is estimated that NDC scenarios reduce mercury emissions by 4%-10% by 2070; while coal intensive (DDP-CCS) pathways and focus on renewables (DDP-R) reduce emissions by 10%-54% and 15%-59%, respectively. Increase in the renewables share (power sector) can result in a significant reduction in the costs of additional pollution-abating technologies in the DDP-R scenario when compared with the coal intensive DDP-CCS scenario. However, the industry sector, especially iron and steel and metal production, will require stringent policies to encourage installation of pollution-abating technologies to mitigate mercury emissions under all the scenarios.

Performance comparison of photodynamic antimicrobial chemotherapy with visible-light-activated organic dyes: Rose bengal, crystal violet, methylene blue, and toluidine blue O.

This study evaluated the photobiocidal performance of four widely distributed visible-light-activated (VLA) dyes against two bacteria (Staphylococcus epidermidis and Escherichia coli) and two bacteriophages (phages MS2 and phi 6): rose bengal (RB), crystal violet, methylene blue, and toluidine blue O (TBO). The photobiocidal performance of each dye depended on the relationship between the type of dye and microorganism. Gram-negative E. coli and the non-enveloped structure of phage MS2 showed more resistance to the photobiocidal reaction than Gram-positive S. epidermidis and the enveloped structure of phage phi 6. RB had the highest potential to yield reactive oxygen species. However, the photobiocidal performance of RB was dependent on the magnitude of the surface charge of the microorganisms; for example, anionic RB induced a negative surface charge and thus electrical repulsion. On the other hand, the photobiocidal performance of TBO was observed to be less affected by the microorganism type. The comparative results presented in our study have significant implications for selecting photodynamic antimicrobial chemotherapy (PACT) dyes suitable for specific situations and purposes. Furthermore, they contribute to the advancement of PACT-related technologies by enhancing their applicability and scalability.

A comparative analysis of solitary suicides, suicides following homicide, and suicide pacts using the National Violent Death Reporting System.

BACKGROUND: Incidents of suicide can be categorized into three main types: solitary suicides, suicides following homicide, and suicide pacts. Although these three suicide incidents vary by definition, no studies to-date have simultaneously examined and compared them for potential differences. The objective of the current study was to empirically and descriptively compare solitary suicides, suicides following homicide, and suicide pacts in the United States. METHODS: Restricted-access data from the National Violent Death Report System for 2003-2019 for 262,679 solitary suicides, 4,352 suicides following homicide, and 450 suicide pacts were used. Pairwise comparisons of the three suicide incident types were made for demographic factors, method of suicide, preceding circumstances, mental health status, and toxicology findings. RESULTS: Solitary suicides, suicides following homicide, and suicide pacts have distinct profiles, with statistically significant (p < 0.05) differences across all pairwise comparisons of sex, race, ethnicity, marital status, education, method of suicide, financial problems, interpersonal relationship problems, physical health problems, mental health problems, mood disorders, suicide attempt history, and opiate use at the time of death. CONCLUSION: Despite sharing a few commonalities, solitary suicides, suicides following homicide, and suicide pacts represent distinct phenomena. Each of these suicide incident types likely have their own unique prevention pathways.

Steering clear of Akrasia: An integrative review of self-binding Ulysses Contracts in clinical practice.

In many jurisdictions, legal frameworks afford patients the opportunity to make prospective medical decisions or to create directives that contain a special provision forfeiting their own ability to object to those decisions at a future time point, should they lose decision-making capacity. These agreements have been described with widely varying nomenclatures, including Ulysses Contracts, Odysseus Transfers, Psychiatric Advance Directives with Ulysses Clauses, and Powers of Attorney with Special Provisions. As a consequence of this terminological heterogeneity, it is challenging for healthcare providers to understand the terms and uses of these agreements and for ethicists to engage with the nuances of clinical decision-making with such unique provisions surrounding patient autonomy. In theory, prospective self-binding agreements may safeguard patient's "authentic" wishes from future "inauthentic" changes of mind. In practice, it is unclear what may be comprised within these agreements or how-and to what effect-they are used. The primary focus of this integrative review is to curate the existing literature describing Ulysses Contracts (and analogous decisions) used in the clinical arena, in order to empirically synthesize their shared essence and provide insights into the traditional components of these agreements when used in practice, the requirements of their consent processes, and the outcomes of their utilization.

Novel Antiviral Molecules against Ebola Virus Infection.

Infection with Ebola virus (EBOV) is responsible for hemorrhagic fever in humans with a high mortality rate. Combined efforts of prevention and therapeutic intervention are required to tackle highly variable RNA viruses, whose infections often lead to outbreaks. Here, we have screened the 2P2I3D chemical library using a nanoluciferase-based protein complementation assay (NPCA) and isolated two compounds that disrupt the interaction of the EBOV protein fragment VP35IID with the N-terminus of the dsRNA-binding proteins PKR and PACT, involved in IFN response and/or intrinsic immunity, respectively. The two compounds inhibited EBOV infection in cell culture as well as infection by measles virus (MV) independently of IFN induction. Consequently, we propose that the compounds are antiviral by restoring intrinsic immunity driven by PACT. Given that PACT is highly conserved across mammals, our data support further testing of the compounds in other species, as well as against other negative-sense RNA viruses.

An attempted "suicide pact" in Covid-19 era - psychiatric perspectives.

BACKGROUND: A "suicide pact" is a joint and actively induced death of two individuals with the essential and unavoidable characteristic of a mutual consent. One of the partners (dominant in the relationship, commonly male) usually induces the action and in most cases, it is the one who actively carries it out. Undiagnosed psychopathological dimension or pathological subthreshold traits are found in those who enter into suicide agreements, the presence of cluster B personality traits such as narcissistic or borderline is of particular relevance in the dominant partner, while in the submissive one dependent personality traits are more frequent. As in the case of other similar health emergencies, COVID-19 pandemic seems to lead to greater suicidality, including the "suicide pacts" of couples whose motivation varies including firstly financial problems, strictly followed by fear of infection and not being able to return home from abroad. CASE PRESENTATION: We reported a case of a couple who entered a suicide agreement consequently to the economic difficulties caused by COVID-19 pandemic, hospitalized in our department. Both partners were assessed with Adult Autism Subthreshold Spectrum (AdAS Spectrum) and both crossed the threshold for clinically relevant autistic traits (M = 67; F = 49). CONCLUSION: This case further confirms the link between COVID-19 pandemics and suicidality. The role of autism spectrum traits as a vulnerability factor towards the development of severe psychopathological consequences after traumatic events is also stressed.

Opposite actions of two dsRNA-binding proteins PACT and TRBP on RIG-I mediated signaling.

An integral aspect of innate immunity is the ability to detect foreign molecules of viral origin to initiate antiviral signaling via pattern recognition receptors (PRRs). One such receptor is the RNA helicase retinoic acid inducible gene 1 (RIG-I), which detects and is activated by 5'triphosphate uncapped double stranded RNA (dsRNA) as well as the cytoplasmic viral mimic dsRNA polyI:C. Once activated, RIG-I's CARD domains oligomerize and initiate downstream signaling via mitochondrial antiviral signaling protein (MAVS), ultimately inducing interferon (IFN) production. Another dsRNA binding protein PACT, originally identified as the cellular protein activator of dsRNA-activated protein kinase (PKR), is known to enhance RIG-I signaling in response to polyI:C treatment, in part by stimulating RIG-I's ATPase and helicase activities. TAR-RNA-binding protein (TRBP), which is ∼45% homologous to PACT, inhibits PKR signaling by binding to PKR as well as by sequestration of its' activators, dsRNA and PACT. Despite the extensive homology and similar structure of PACT and TRBP, the role of TRBP has not been explored much in RIG-I signaling. This work focuses on the effect of TRBP on RIG-I signaling and IFN production. Our results indicate that TRBP acts as an inhibitor of RIG-I signaling in a PACT- and PKR-independent manner. Surprisingly, this inhibition is independent of TRBP's post-translational modifications that are important for other signaling functions of TRBP, but TRBP's dsRNA-binding ability is essential. Our work has major implications on viral susceptibility, disease progression, and antiviral immunity as it demonstrates the regulatory interplay between PACT and TRBP IFN production.

New Metallophthalocyanines Bearing 2-Methylimidazole Moieties-Potential Photosensitizers against Staphylococcus aureus.

Newly developed tetra- and octasubstituted methimazole-phthalocyanine conjugates as potential photosensitizers have been obtained. Synthesized intermediates and final products were characterized by the MALD-TOF technique and various NMR techniques, including 2D methods. Single-crystal X-ray diffraction was used to determine the crystal structures of dinitriles. The studied phthalocyanines revealed two typical absorption bands-the Soret band and the Q band. The most intense fluorescence was observed for octasubstituted magnesium(II) phthalocyanine in DMF (ΦFL = 0.022). The best singlet oxygen generators were octasubstituted magnesium(II) and zinc(II) phthalocyanines (Φ∆ 0.56 and 0.81, respectively). The studied compounds presented quantum yields of photodegradation at the level between 10-5 and 10-6. Due to their low solubility in a water environment, the liposomal formulations were prepared. Within the studied group, octasubstituted zinc(II) phthalocyanine at the concentration of 100 µM activated with red light showed the highest antibacterial activity against S. aureus equal to a 5.68 log reduction of bacterial growth.

Dispelling the shadow of fiscal dominance? Fiscal and monetary announcement effects for euro area sovereign spreads in the corona pandemic.

We use event study regressions to compare the impact of monetary versus fiscal policy announcements on euro area government bond spreads in the unfolding Covid-19 pandemic. Throughout our specifications and robustness checks, we detect larger effects for monetary than for fiscal announcements. Among monetary policy instruments, the PEPP has the largest spread compressing effects. Comparing the announcement effects for fiscal crisis tools, Next Generation EU shows significant results in contrast to news on pure loan instruments. The relaxation of European fiscal rules through the activation of the emergency-escape clause under the Stability and Growth Pact is associated with rising spreads. We conclude that the stability of euro area bond markets in the presence of a severe solvency shock depends to a large extent on the Eurosystem's unconstrained sovereign bond purchases. Our results suggest that fiscal support can play a stabilizing role if it includes, like Next Generation EU, a significant transfer component.

Synthesis of novel nicotinamide susbstituted phthalocyanine and photodynamic antomicrobial chemotherapy evaluation potentiated by potassium iodide against the gram positive S. aureus and gram negative E. coli.

In the present work, we propose the synthesis of novel nicotinamide subsituted phthlocyanine photosensitizer (PS) and characterized by FTIR, UV-visible, H-NMR and MALDI Toff spectroscopy. Nicotinamide plays a vital rule in the central nervous system and its potential as a therapeutic for neurodegenerative disease. Nicotinamide substituted PS (3) efficiently produced ROS via type-1 process as measured by DCF assay. We observed that our PS after red light illumination (22 J/cm2) killed gram positive S. aureus upto 3 log reduction. Furher the addition of Potassium Iodide (100 mM) significantly potentiated PS at lower concentrations and enhanced the bacterial killing upto 6 log reduction against the S. aureus. We further found that the synergistic effect of PS and KI also eradicated the gram negative E. coli strain at lower concentraion of PS and found to killed E. coli upto 5 log reduction under the red light illumination at 22 J/cm2 of light dose. The conjugation of such biologically important form of vitamin B3 with PS would be a great addition and could pav the way for the novel photodynamic agent in the treatement of cancer and infectious diseases. A new symmetrical Nicotinamide tetrasubstituted zinc phthalocyanine (3) was synthesized. Upon addition of potassium Iodide with PS, the PS exhibited significant photodynamic activity with 5-6 logs reduction in bacterial load was achieved.

Efficacy of antimicrobial photodynamic therapy (aPDT) for nonsurgical treatment of periodontal disease: a systematic review.

Although the standard treatment for periodontal disease is based on scaling and root planing (SRP), the use of antimicrobial photodynamic therapy (aPDT) has been studied as a complement to obtain better clinical results. The purpose of this study was to evaluate the effect of aPDT as adjuncts to SRP, compared with SRP alone, on clinical parameters of chronic periodontal patients. Only randomized controlled trials with at least 3-month follow-ups, of SRP alone and in association with aPDT, were included. The MEDLINE (PubMed), Google Scholar, and LILACS databases were searched for articles published up to July 2020. Random-effects meta-analyses were conducted for clinical attachment level (CAL) and probing pocket depth (PPD) change after treatment. Of 141 potentially relevant papers, 22 were included. The association between SRP and aPDT promoted a significant CAL gain and PPD reduction. Periodontal treatment was partially improved by aPDT, and a favorable effect of indocyanine green-mediated aPDT was observed, and high concentrations of phenothiazine chloride presented clinical improvement as well.

The Development of Ru(II)-Based Photoactivated Chemotherapy Agents.

Photoactivated chemotherapy (PACT) is a novel cancer treatment method that has drawn increasing attention due to its high selectivity and low side effects by spatio-temporal control of irradiation. Compared with photodynamic therapy (PDT), oxygen-independent PACT is more suitable for treating hypoxic tumors. By finely tuning ligand structures and coordination configurations, many Ru(II) complexes can undergo photoinduced ligand dissociation, and the resulting Ru(II) aqua species and/or free ligands may have anticancer activity, showing their potential as PACT agents. In this mini-review, we summarized the progress in Ru(II)-based PACT agents, as well as challenges that researchers in this field still face.