Development and cross-national investigation of a model explaining participation in WHO-recommended and placebo behaviours to prevent COVID-19 infection.

To protect themselves from COVID-19, people follow the recommendations of the authorities, but they also resort to placebos. To stop the virus, it is important to understand the factors underlying both types of preventive behaviour. This study examined whether our model (developed based on the Health Belief Model and the Transactional Model of Stress) can explain participation in WHO-recommended and placebo actions during the pandemic. Model was tested on a sample of 3346 participants from Italy, Japan, Poland, Korea, Sweden, and the US. It was broadly supported: objective risk and cues to action showed both direct and indirect (through perceived threat) associations with preventive behaviours. Moreover, locus of control, decision balance, health anxiety and preventive coping moderated these relationships. Numerous differences were also found between countries. We conclude that beliefs about control over health and perceived benefits of actions are critical to the development of interventions to improve adherence to recommendations.

Drug-like molecules with anti-trypanothione synthetase activity identified by high throughput screening.

Trypanothione synthetase (TryS) catalyses the synthesis of N1,N8-bis(glutathionyl)spermidine (trypanothione), which is the main low molecular mass thiol supporting several redox functions in trypanosomatids. TryS attracts attention as molecular target for drug development against pathogens causing severe and fatal diseases in mammals. A drug discovery campaign aimed to identify and characterise new inhibitors of TryS with promising biological activity was conducted. A large compound library (n = 51,624), most of them bearing drug-like properties, was primarily screened against TryS from Trypanosoma brucei (TbTryS). With a true-hit rate of 0.056%, several of the TbTryS hits (IC50 from 1.2 to 36 µM) also targeted the homologue enzyme from Leishmania infantum and Trypanosoma cruzi (IC50 values from 2.6 to 40 µM). Calmidazolium chloride and Ebselen stand out for their multi-species anti-TryS activity at low µM concentrations (IC50 from 2.6 to 13.8 µM). The moieties carboxy piperidine amide and amide methyl thiazole phenyl were identified as novel TbTryS inhibitor scaffolds. Several of the TryS hits presented one-digit µM EC50 against T. cruzi and L. donovani amastigotes but proved cytotoxic against the human osteosarcoma and macrophage host cells (selectivity index ≤ 3). In contrast, seven hits showed a significantly higher selectivity against T. b. brucei (selectivity index from 11 to 182). Non-invasive redox assays confirmed that Ebselen, a multi-TryS inhibitor, induces an intracellular oxidative milieu in bloodstream T. b. brucei. Kinetic and mass spectrometry analysis revealed that Ebselen is a slow-binding inhibitor that modifies irreversible a highly conserved cysteine residue from the TryS's synthetase domain. The most potent TbTryS inhibitor (a singleton containing an adamantine moiety) exerted a non-covalent, non-competitive (with any of the substrates) inhibition of the enzyme. These data feed the drug discovery pipeline for trypanosomatids with novel and valuable information on chemical entities with drug potential.

MG1113, a specific anti-tissue factor pathway inhibitor antibody, rebalances the coagulation system and promotes hemostasis in hemophilia.

BACKGROUND: Replacement therapy is the most common treatment for reduction of bleeding and control of episodic bleeding in individuals with hemophilia. Despite the proven effectiveness of factor replacement therapy, repeated intravenous administration is a heavy burden to individuals with hemophilia. OBJECTIVES: To reduce the burden, therapeutic agents that can be subcutaneously administered need to be developed, and an anti-tissue factor pathway inhibitor (TFPI) antibody may be a suitable candidate for this purpose. METHODS: MG1113 is an IgG4 monoclonal antibody that binds to Kunitz-2 domain (KD2) of TFPI. To confirm the coagulation potential of MG1113, several tests were conducted using factor VIII (FVIII)- or IX (FIX)-deficient plasma. For the ex vivo spiking test, platelet-poor plasma samples from 14 individuals with hemophilia were spiked with MG1113. The in vivo efficacy was determined using blood loss tests, modified prothrombin time (mPT), and free TFPI quantification after intravenous or subcutaneous administration of MG1113 into hemophilia A (HA)-induced rabbits. RESULTS: Radiographic crystallography demonstrated the specific binding site between MG1113 and KD2. In FVIII-deficient plasma and the plasma of individuals with hemophilia, peak thrombin and endogenous thrombin levels were increased by MG1113 in a concentration-dependent manner. Rotational thromboelastometry assay revealed that clotting time, clot formation time, and maximum clot firmness were normalized in MG1113-treated blood of patients. Intravenous or subcutaneous injection of MG1113 into HA-induced rabbits resulted in rebalancing of blood loss, mPT, and free TFPI levels. CONCLUSIONS: These results indicate that subcutaneous administration of MG1113 neutralizes the function of TFPI and regulates bleeding in individuals with hemophilia.

Pseudosecure vs. individuated-secure client attachment to therapist: Implications for therapy process and outcome.

OBJECTIVE: We differentiated two hypothesized client subtypes: (a) Pseudosecure clients have high Client Attachment to Therapist Scale (CATS) Secure and high CATS Preoccupied scores, tend to idealize their therapist, and exhibit maladaptive dependency; (b) Individuated-secure clients combine high Secure with low Preoccupied scores and function more autonomously. Clients who, despite insecure attachment to others, "earn" individuated-secure attachment to their therapist benefit most from therapy. METHOD: We examined regression suppressor effects by reanalyzing raw data from four published studies. If pseudosecure attachment is present, when covariance between CATS Secure and Preoccupied scores is removed, residual Secure scores should be significantly better predictors of process/outcome indicators than raw Secure scores. RESULTS: Suppressor effects were observed in eight of nine analyses. Two were statistically significant. Earned individuated-secure attachment predicted improvement in interpersonal relationship symptoms, but only for clients with Avoidant pre-therapy attachment patterns. Finally, significant meta-analytic effect size estimates were obtained for CATS subscales, Secure r = .274 (95% CI = .177, .366), Avoidant, r = -.296 (95% CI = -.392, -193), and Preoccupied, r = -.192 (95% CI = -.289, -.092). CONCLUSIONS: Clients with pre-therapy Avoidant attachment who nevertheless "earn" individuated-secure attachment to their therapist appear to benefit more from therapy.

Antileishmanial Mechanism of Diamidines Involves Targeting Kinetoplasts.

Leishmaniasis is a disease caused by pathogenic Leishmania parasites; current treatments are toxic and expensive, and drug resistance has emerged. While pentamidine, a diamidine-type compound, is one of the treatments, its antileishmanial mechanism of action has not been investigated in depth. Here we tested several diamidines, including pentamidine and its analog DB75, against Leishmania donovani and elucidated their antileishmanial mechanisms. We identified three promising new antileishmanial diamidine compounds with 50% effective concentrations (EC50s) of 3.2, 3.4, and 4.5 µM, while pentamidine and DB75 exhibited EC50s of 1.46 and 20 µM, respectively. The most potent antileishmanial inhibitor, compound 1, showed strong DNA binding properties, with a shift in the melting temperature (ΔTm) of 24.2°C, whereas pentamidine had a ΔTm value of 2.1°C, and DB75 had a ΔTm value of 7.7°C. Additionally, DB75 localized in L. donovani kinetoplast DNA (kDNA) and mitochondria but not in nuclear DNA (nDNA). For 2 new diamidines, strong localization signals were observed in kDNA at 1 µM, and at higher concentrations, the signals also appeared in nuclei. All tested diamidines showed selective and dose-dependent inhibition of kDNA, but not nDNA, replication, likely by inhibiting L. donovani topoisomerase IB. Overall, these results suggest that diamidine antileishmanial compounds exert activity by accumulating toward and blocking replication of parasite kDNA.

Identification of a resveratrol tetramer as a potent inhibitor of hepatitis C virus helicase.

BACKGROUND AND PURPOSE: Hepatitis C virus (HCV) infection is responsible for various chronic inflammatory liver diseases. Here, we have identified a naturally occurring compound with anti-HCV activity and have elucidated its mode of antiviral action. EXPERIMENTAL APPROACH: Luciferase reporter and real-time RT-PCR assays were used to measure HCV replication. Western blot, fluorescence-labelled HCV replicons and infectious clones were employed to quantitate expression levels of viral proteins. Resistant HCV mutant mapping, in vitro NS3 protease, helicase, NS5B polymerase and drug affinity responsive target stability assays were also used to study the antiviral mechanism. KEY RESULTS: A resveratrol tetramer, vitisin B from grapevine root extract showed high potency against HCV replication (EC50 = 6 nM) with relatively low cytotoxicity (EC50 >10 µM). Combined treatment of vitisin B with an NS5B polymerase inhibitor (sofosbuvir) exhibited a synergistic or at least additive antiviral activity. Analysis of a number of vitisin B-resistant HCV variants suggested an NS3 helicase as its potential target. We confirmed a direct binding between vitisin B and a purified NS3 helicase in vitro. Vitisin B was a potent inhibitor of a HCV NS3 helicase (IC50 = 3 nM). In vivo, Finally, we observed a preferred tissue distribution of vitisin B in the liver after i.p. injection in rats, at clinically attainable concentrations. Conclusion and Implications Vitisin B is one of the most potent HCV helicase inhibitors identified so far. Vitisin B is thus a prime candidate to be developed as the first HCV drug derived from natural products.

In Vitro and In Vivo Investigation of the Inhibition of Trypanosoma brucei Cell Growth by Lipophilic Bisphosphonates.

We report the results of a screen of a library of 925 potential prenyl synthase inhibitors against Trypanosoma brucei farnesyl diphosphate synthase (TbFPPS) and against T. brucei, the causative agent of human African trypanosomiasis. The most potent compounds were lipophilic analogs of the bone resorption drug zoledronate, some of which had submicromolar to low micromolar activity against bloodstream form T. brucei and selectivity indices of up to ∼ 300. We evaluated the effects of two such inhibitors on survival and parasitemia in a T. brucei mouse model of infection and found that survival increased by up to 16 days. We also investigated the binding of three lipophilic bisphosphonates to an expressed TbFPPS using crystallography and investigated the thermodynamics of binding using isothermal titration calorimetry.

In Vitro and in Vivo Activity of Multitarget Inhibitors against Trypanosoma brucei.

We tested a series of amidine and related compounds against Trypanosoma brucei. The most active compound was a biphenyldiamidine that had an EC 50 of 7.7 nM against bloodstream-form parasites. There was little toxicity against two human cell lines with CC 50 > 100 µM. There was also good in vivo activity in a mouse model of infection with 100% survival at 3 mg/kg i.p. The most potent lead blocked replication of kinetoplast DNA (k-DNA), but not nuclear DNA, in the parasite. Some compounds also inhibited the enzyme farnesyl diphosphate synthase (FPPS), and some were uncouplers of oxidative phosphorylation. We developed a computational model for T. brucei cell growth inhibition (R (2) = 0.76) using DNA ΔT m values for inhibitor binding combined with T. brucei FPPS IC 50 values. Overall, the results suggest that it may be possible to develop multitarget drug leads against T. brucei that act by inhibiting both k-DNA replication and isoprenoid biosynthesis.

Pilot test of a measure to assess therapeutic distance and its association with client attachment and corrective experience in therapy.

OBJECTIVES: The Therapeutic Distance Scale (TDS) was developed in this project to assesse clients' experiences of distance versus engagement with their therapist. METHOD: In a survey study of 47 university clients at the "mid-stage" and 34 of these clients at termination, four TDS subscales were identified: Too Close, Too Distant, Growing Autonomy, and Growing Engagement. RESULTS: TDS subscales were correlated as expected with working alliance and Client Attachment to Therapist. As hypothesized, (i) pre-therapy attachment Avoidance was significantly correlated with perceptions of therapists as Too Close (but not Too Distant), (ii) pre-therapy Anxiety was significantly correlated with Too Distant (but not Too Close); furthermore, among clients who developed a secure attachment to their therapist, (iii) pretherapy Avoidance was significantly correlated with Growing Engagement; however, (iv) contrary to expectations, pre-therapy anxiety was not significantly associated with Growing Autonomy. CONCLUSIONS: The TDS is a promising measure for assessing the in-therapy corrective emotional experiences of clients with hyperactivating attachment (i.e. increasing autonomy) and deactivating attachment (i.e. increasing engagement).

Developing a comprehensive scale to assess college multicultural programming.

A barrier to assessing effectiveness of multicultural programming is lack of a relatively brief instrument to measure the wide range of intended outcomes. A frequent goal of programming is to increase cultural empathy, but this is rarely the only intended outcome. We conducted focus groups of campus administrators, student affairs staff, and undergraduate instructors who identified a full range of racial/ethnic multicultural competencies that undergraduates should possess. An 84-item pool generated from these focus groups was combined with the 31-item Scale of Ethnocultural Empathy (SEE; Wang et al., 2003). These 115 items, together with instruments used to gauge concurrent validity, were administered to White undergraduate students in introductory psychology courses at the midpoint (n = 602) and end (n = 676) of fall semester. Exploratory factor analysis suggested 6 subscales for the Everyday Multicultural Competencies/Revised SEE (EMC/RSEE): (a) Cultural Openness and Desire to Learn; (b) Resentment and Cultural Dominance; (c) Anxiety and Lack of Multicultural Self-Efficacy; (d) Empathic Perspective-Taking; (e) Awareness of Contemporary Racism and Privilege; and (f) Empathic Feeling and Acting as an Ally. Item response theory principles guided final selection of subscale items. Analyses suggested good factor stability, reliability, and discriminant validity of the 48-item EMC/RSEE in these undergraduate samples. EMC/RSEE subscales were not strongly correlated with a measure of impression management and were significantly associated with measures of Openness to Diversity Challenge, and Universal-Diverse Orientation.

Drosophila DJ-1 decreases neural sensitivity to stress by negatively regulating Daxx-like protein through dFOXO.

DJ-1, a Parkinson's disease (PD)-associated gene, has been shown to protect against oxidative stress in Drosophila. However, the molecular mechanism underlying oxidative stress-induced phenotypes, including apoptosis, locomotive defects, and lethality, in DJ-1-deficient flies is not fully understood. Here we showed that Daxx-like protein (DLP), a Drosophila homologue of the mammalian Death domain-associated protein (Daxx), was upregulated under oxidative stress conditions in the loss-of-function mutants of Drosophila DJ-1ß, a Drosophila homologue of DJ-1. DLP overexpression induced apoptosis via the c-Jun N-terminal kinase (JNK)/Drosophila forkhead box subgroup O (dFOXO) pathway, whereas loss of DLP increased resistance to oxidative stress and UV irradiation. Moreover, the oxidative stress-induced phenotypes of DJ-1ß mutants were dramatically rescued by DLP deficiency, suggesting that enhanced expression of DLP contributes to the DJ-1ß mutant phenotypes. Interestingly, we found that dFOXO was required for the increase in DLP expression in DJ-1ß mutants and that dFOXO activity was increased in the heads of DJ-1ß mutants. In addition, subcellular localization of DLP appeared to be influenced by DJ-1 expression so that cytosolic DLP was increased in DJ-1ß mutants. Similarly, in mammalian cells, Daxx translocation from the nucleus to the cytosol was suppressed by overexpressed DJ-1ß under oxidative stress conditions; and, furthermore, targeted expression of DJ-1ß to mitochondria efficiently inhibited the Daxx translocation. Taken together, our findings demonstrate that DJ-1ß protects flies against oxidative stress- and UV-induced apoptosis by regulating the subcellular localization and gene expression of DLP, thus implying that Daxx-induced apoptosis is involved in the pathogenesis of DJ-1-associated PD.

Tsp66E, the Drosophila KAI1 homologue, and Tsp74F function to regulate ovarian follicle cell and wing development by stabilizing integrin localization.

The metastasis suppressor KAI1/CD82 has been implicated in various cellular processes; however, its function in development is not fully understood. Here, we generated and characterized mutants of Tsp66E and Tsp74F, which are Drosophila homologues of KAI1/CD82 and Tspan11, respectively. These mutants exhibited egg elongation defects along with disturbed integrin localization and actin polarity. Moreover, the defects were enhanced by mutation of inflated, an αPS2 integrin gene. Mutant ovaries had elevated αPS2 integrin levels and reduced endocytic trafficking. These results suggest that Drosophila KAI1/CD82 affects the polarized localization and the level of integrin, which may contribute to epithelial cell polarity.

Dietary hempseed meal intake increases body growth and shortens the larval stage via the upregulation of cell growth and sterol levels in Drosophila melanogaster.

Hempseed, a rich source of polyunsaturated fatty acids (PUFAs) and phytosterols, has been recognized as a potential therapeutic food used for cardioprotection, preventing platelet aggregation, and improving atopic dermatitis. Although several studies have revealed the physiological benefits of hempseed on a variety of animals, the effects of dietary hempseed intake on animal development are currently unknown. In this study, we evaluated the developmental effects of the addition of hempseed meal (HSM) to the diet of Drosophila. Interestingly, dietary HSM intake was shown to increase the body size of flies by increasing cell numbers, and also truncated the larval period without affecting survival rate or longevity. The oviposition of female flies was also increased by dietary HSM supplementation. Interestingly, the levels of sterols, which are precursors of ecdysone, a molting hormone, were found to be elevated in the larvae fed on HSM. Additionally, the hexane extracts of hempseed mimicked the effects of HSM on growth, developmental timing, and reproduction. Moreover, among the major nonpolar components of HSM, feeding on cholesterol but not PUFA mix or campesterol accelerated pupariation and increased body size. These results indicate that the dietary intake of HSM accelerates both body growth and developmental rates in Drosophila via the stimulation of cell growth and ecdysone synthesis. Additionally, nonpolar components of hempseed, such as cholesterol, might be responsible for the effects of HSM on development and reproduction.

Parkin suppresses c-Jun N-terminal kinase-induced cell death via transcriptional regulation in Drosophila.

Parkin is the most prevalent genetic factor in the onset of autosomal recessive juvenile parkinsonism (AR-JP), and mutations in parkin has been reported to cause motor defects, which result from dopamine deficiency caused by dopaminergic neuronal cell death. Activation of c-Jun N-terminal kinase (JNK) has also been implicated in neuronal cell death in Parkinson's disease (PD). Moreover, Drosophila models for AR-JP, loss of function mutants of Drosophila parkin, also show dopaminergic neural degeneration associated with hyperactivation of JNK, increased apoptosis, and mitochondrial defects. However, the molecular mechanism by which Parkin protects cells from apoptosis remains unclear. In the present study, we tested whether Drosophila Parkin suppressed the JNK signaling pathway in developing tissues. Ectopically expressed parkin strongly suppressed the constitutively active form of Hemipterous (Hep(CA)), a Drosophila JNK kinase that induces an eye degeneration phenotype and apoptosis in the eye imaginal disc. Moreover, parkin also suppressed extra vein formation induced by Basket (Bsk), a Drosophila JNK. Interestingly, the bsk mRNA level was markedly reduced by parkin over-expression, suggesting that the effect of parkin on the phenotype induced by activation of JNK signaling was achieved by transcriptional regulation. Furthermore, we found that the expression level of JNK target genes was reduced by parkin over-expression. Taken together, these results suggest that Drosophila Parkin suppresses JNK signaling by reducing bsk transcription.

dXNP/DATRX increases apoptosis via the JNK and dFOXO pathway in Drosophila neurons.

Mutation of the XNP/ATRX gene, which encodes an SNF2 family ATPase/helicase protein, leads to ATR-X syndrome and several other X-linked mental retardation syndromes. Although XNP/ATRX is a chromatin remodeler, the molecular mechanism by which mental retardation occurs in patients with ATR-X has yet to be determined. To better understand the role of XNP/ATRX in neuronal development, we expressed Drosophila XNP (dXNP/DATRX) ectopically in Drosophila neurons. Neuronal expression of dXNP/DATRX resulted in various developmental defects and induced strong apoptosis. These defects and apoptosis were suppressed by Drosophila inhibitor of apoptosis protein 1. Expression of dXNP/DATRX also increased JNK activity and the levels of reaper and hid transcripts, which are pro-apoptotic factors that activate caspase. Furthermore, dXNP/DATRX-induced rough eye phenotype and apoptosis were suppressed by dFOXO deficiency. These results suggest that dXNP/DATRX is involved in caspase-dependent apoptosis in Drosophila neurons via regulation of the JNK and dFOXO pathway.