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Triphenylamine-sensitized 8-dimethylaminoquinoline (TAQ) probes showed fair two-photon absorption and fragmentation cross sections in releasing kainate and GABA ligands. The water-soluble PEG and TEG-analogs allowed cell internalization and efficient light-gated liberation of the rhodamine reporter under UV and two-photon (NIR) irradiation conditions., (© 2024 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Background: Value-based healthcare (VBHC) is an increasingly employed strategy to transform healthcare organizations into economically sustainable systems that deliver high-value care. In dermatology, the need for VBHC is evident as chronic skin diseases require long-term, often expensive treatments. This narrative review aims to introduce dermatologists to the principles and implementation of VBHC., Summary: VBHC emphasizes maximizing outcomes that are directly relevant to patients. Key components of VBHC include a systematic assessment of standardized patient-relevant outcomes by using core outcome sets and measurement of healthcare cost for the individual patient. Systematic reporting and comparing of risk-adjusted outcomes across the full cycle of care for a specific condition provide benchmarked feedback and actionable insights to promote high-value care and reduce low-value care. VBHC aims to organize care around the patient in condition-specific and team-based integrated practice units with multidisciplinary collaboration, utilize information technology platforms to enable digital data monitoring, reduce cost, and eventually reform payment systems to support bundled payments for the overall care cycle. VBHC implementation in practice necessitates the establishment of a systematic framework for outcome-based quality improvement, the incorporation of value and outcomes in shared decision-making practices, and the cultivation of a value-centric culture among healthcare professionals through continuous training., Key Messages: Dermatologists can benefit from implementing VBHC principles in their practice. An essential step toward value-driven dermatological care is to start measuring outcomes relevant for patients for each patient, which is lacking partly due to the absence of core outcome sets developed for clinical practice. By reducing low-value care and emphasizing optimal patient-centered outcomes, VBHC has the potential to improve the quality of care and ensure cost containment. Efforts are needed to enhance the development and uptake of VBHC in dermatological clinical practice to realize these benefits., (© 2024 The Author(s). Published by S. Karger AG, Basel.)

Purinergic receptors and NOD-like receptor protein 3 (NLRP3) inflammasome regulate inflammation and viral infection, but their effects on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection remain poorly understood. Here, we report that the purinergic receptor P2X7 and NLRP3 inflammasome are cellular host factors required for SARS-CoV-2 infection. Lung autopsies from patients with severe coronavirus disease 2019 (COVID-19) reveal that NLRP3 expression is increased in host cellular targets of SARS-CoV-2 including alveolar macrophages, type II pneumocytes and syncytia arising from the fusion of infected macrophages, thus suggesting a potential role of NLRP3 and associated signaling pathways to both inflammation and viral replication. In vitro studies demonstrate that NLRP3-dependent inflammasome activation is detected upon macrophage abortive infection. More importantly, a weak activation of NLRP3 inflammasome is also detected during the early steps of SARS-CoV-2 infection of epithelial cells and promotes the viral replication in these cells. Interestingly, the purinergic receptor P2X7, which is known to control NLRP3 inflammasome activation, also favors the replication of D614G and alpha SARS-CoV-2 variants. Altogether, our results reveal an unexpected relationship between the purinergic receptor P2X7, the NLRP3 inflammasome and the permissiveness to SARS-CoV-2 infection that offers novel opportunities for COVID-19 treatment., Competing Interests: DT and AA were employed by NH TherAguix SAS. Authors DL, DT, AA, FS, OD and J-LP are listed as co-inventors on a patent application related to SARS-CoV-2 therapy. AP and J-LP are founding members of Findimmune SAS, an Immuno-Oncology Biotech company. J-LP disclosed research funding not related to this work from NH TherAguix and Wonna Therapeutics. NC disclosed research funding not related to this work from GlaxoSmithKline, Roche, Cytune pharma and Sanofi. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Lécuyer, Nardacci, Tannous, Gutierrez-Mateyron, Deva Nathan, Subra, Di Primio, Quaranta, Petit, Richetta, Mostefa-Kara, Del Nonno, Falasca, Marlin, Maisonnasse, Delahousse, Pascaud, Deprez, Naigeon, Chaput, Paci, Saada, Ghez, Mariette, Costa, Pistello, Allouch, Delelis, Piacentini, Le Grand and Perfettini.)

Background: Healthcare professionals (HCPs) should strive to create the maximum value for their patients in which value is defined as the patient-relevant health outcomes achieved per costs made. However, currently it remains difficult to determine which outcomes matter to an individual psoriasis patient., Objective: To define outcome profiles, or so called 'patient value profiles', within a cohort of psoriasis patients that can be translated to daily practice to increase value for the individual patient., Methods: Hierarchical clustering on principal components (HCPC) was used to identify groups of patients sharing the same profile within an outcome ranking exercise. Once the clusters were defined, their characterization was provided based on a V-test. In a final step, a multi-class decision tree (MDT) based on relevant socio-demographic and clinical variables was built to allocate patients to a cluster., Results: In the ranking exercise 120 patients participated. The median age was 50.0 (IQR 25.0) years and 36.7% were female. Median PASI score was 2.4 (IQR 5.2) and median duration of psoriasis was 17.0 (IQR 20.0) years. Primary treatment varied from topicals to biologicals. We found three distinct patient value profiles in this cohort (QoL, cost and treatment). A MDT was built which had an accuracy of 64%., Conclusion: We found three distinct patient value profiles in a cohort of psoriasis patients and patients can be easily assigned to one of these profiles based on a MDT. HCPs can use these profiles to steer psoriasis management accordingly allowing for a more goal-orientated approach., (© 2023 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Introduction: Currently, the healthcare sector is under tremendous financial pressure, and many acknowledge that a dramatic shift is required as the current system is not sustainable. Furthermore, the quality of care that is delivered varies strongly. Several solutions have been proposed of which the conceptual framework known as value-based healthcare (VBHC) is further explored in this study for psoriasis. Psoriasis is a chronic inflammatory skin disease, which is associated with a high disease burden and high treatment costs. The objective of this study is to investigate the feasibility of using the VBHC framework for the management of psoriasis., Methods and Analysis: This is a prospective clinical study in which new patients attending the psoriasis clinic (PsoPlus) of the Ghent University Hospital will be followed up during a period of 1 year. The main outcome is to determine the value created for psoriasis patients. The created value will be considered as a reflection of the evolution of the value score (ie, the weighted outputs (outcomes) divided by weighted inputs (costs)) obtained using data envelopment analysis. Secondary outcomes are related to comorbidity control, outcome evolution and treatment costs. In addition, a bundled payment scheme will be determined as well as potential improvements in the treatment process. A total of 350 patients will be included in this trial and the study initiation is foreseen on 1 March 2023., Ethics and Dissemination: This study has been approved by the Ethics Committee of the Ghent University Hospital. The findings of this study will be disseminated by various means: (1) publication in one or more peer-reviewed dermatology and/or management journals, (2) (inter)national congresses, (3) via the psoriasis patient community and (4) through the research team's social media channels., Trial Registration Number: NCT05480917., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

What is this summary about? This summary presents findings from recent research involving people with psoriasis, based on an article originally published in the Journal of the European Academy of Dermatology and Venereology . Psoriasis is a condition that primarily affects the skin. However, it can also influence people's mental health, social activities, work, and relationships too. Current assessment tools used by doctors and nurses do not cover the complete experience of people with psoriasis, which often include other medical conditions and can leave these individuals feeling that treatment has not been successful. Researchers conducted a study in which people with psoriasis, doctors, and nurses were asked in virtual meetings and via questionnaires what freedom from disease in psoriasis means to them. What were the results? In addition to skin symptoms, the areas of mental health, well-being, treatment, and relationships with healthcare teams were found to be important aspects to be addressed. What do the results of the study mean? Focusing on all five aspects of freedom from disease will help people with psoriasis manage their psoriasis with confidence.

Background: With the current trend in healthcare moving towards a more value-based approach, it is essential to understand what value encompasses., Objectives: To develop an actionable value-based outcome set (VOS) for daily practice., Methods: A mixed method approach was used consisting of four phases. Formerly, a systematic review was conducted, providing an overview of all patient-relevant outcomes defined in current literature. These 23 outcomes were then presented to a group of patients, using a modified nominal group technique (NGT), to establish whether these results represented all of their relevant outcomes. Subsequently, these outcomes were ranked according to importance by patients attending our academic specialized psoriasis clinic. A review of the literature was performed to assess which instruments were available and suitable to evaluate the outcomes in this VOS. Finally, a pilot feasibility test was performed amongst patients., Results: Of the 23 outcomes, two were omitted from the ranking exercise after the NGT. In the ranking exercise, 120 patients participated. The median age was 50.0 (IQR 25.0) years and 36.7% were female. Median PASI score was 2.4 (IQR 5.2), and treatments varied from topicals to biologicals. The outcomes scored as most important were symptom control, treatment efficacy, confidence in care and control of disease. The least important outcomes were comorbidity control, productivity and cost of care. A significant difference was shown between the ranking of the outcomes (p < 0.001). In total, 12 instruments were selected, which are reported by both patient and provider, to measure the outcomes in this VOS. Median completion time for the patient part was 30 min (IQR 2.8)., Conclusions: This VOS is a first proposal to evaluate psoriasis care in a value-based manner. Measuring these outcomes can enable us to critically appraise and improve current care processes, within the reality of available resources, thereby increasing value for patients., (© 2022 The Authors. Journal of the European Academy of Dermatology and Venereology published by John Wiley & Sons Ltd on behalf of European Academy of Dermatology and Venereology.)

There is a need to revise the current healthcare organization due to the ever-rising costs and variation in quality of delivered care. Over the past decades there have been several strategic frameworks attempting to tackle this problem. Value-based healthcare (VBHC) is one of those frameworks which has gained increasing popularity the last years. The framework is formulated on the premise that the healthcare sector should deliver integrated care, using integrated practice units (IPUs), and strive to maximize the value created. Value in this context is defined as the health outcomes achieved per costs made. We have designed a lean IPU called PsoPlus in which psoriasis patients are managed by a multidisciplinary team which has all the expertise and skill to manage psoriasis and its associated conditions. In addition, we have developed and implemented guidelines for the management of psoriasis-associated comorbidities, enabling us to deliver integrated care in the Belgian healthcare setting. Finally, we have designed a supporting information technology platform, called PsoSmart, which brings data from patients and healthcare providers together and provides actionable insights for clinical decision making. The created value is documented and captured using a value-based outcome set. Cost assessments at the individual patient level are also performed. To conclude, we describe here a comprehensive IPU setting for psoriasis which incorporates the VBHC principles. This IPU goes further and delivers a higher level of integrated care than other multidisciplinary psoriasis clinics. Monitoring outcomes and costs provides us with further insights to optimize psoriasis care. In addition, a software program designed to enhance psoriasis care is being developed further; however, advances in healthcare technology are needed., (© 2023 S. Karger AG, Basel.)

Integration of the reverse-transcribed genome is a critical step of the retroviral life cycle. Strand-transfer inhibitors (INSTIs) used for antiretroviral therapy inhibit integration but can lead to resistance mutations in the integrase gene, the enzyme involved in this reaction. A significant proportion of INSTI treatment failures, particularly those with second-generation INSTIs, show no mutation in the integrase gene. Here, we show that replication of a selected dolutegravir-resistant virus with mutations in the 3'-PPT (polypurine tract) was effective, although no integrated viral DNA was detected, due to the accumulation of unintegrated viral DNA present as 1-LTR circles. Our results show that mutation in the 3'-PPT leads to 1-LTR circles and not linear DNA as classically reported. In conclusion, our data provide a molecular basis to explain a new mechanism of resistance to INSTIs, without mutation of the integrase gene and highlights the importance of unintegrated viral DNA in HIV-1 replication. IMPORTANCE Our work highlights the role of HIV-1 unintegrated viral DNA in viral replication. A virus, resistant to strand-transfer inhibitors, has been selected in vitro . This virus highlights a mutation in the 3'PPT region and not in the integrase gene. This mutation modifies the reverse transcription step leading to the accumulation of 1-LTR circles and not the linear DNA. This accumulation of 1-LTR circles leads to viral replication without integration of the viral genome.

Importance: There is a need to define which outcomes matter to patients with psoriasis to deliver value for the patient when managing their condition., Objectives: To generate a comprehensive overview of all outcomes relevant in the management of psoriasis as defined by patients., Evidence Review: A systematic review was performed by searching 3 databases (MEDLINE, Embase, and Web of Science) from August 1, 2019, until March 27, 2021, using a comprehensive search strategy consisting of 4 concepts including psoriasis, patients, outcomes, and relevance. A (citing) reference search was also performed of all retrieved articles. Two independent reviewers screened the retrieved records by title/abstract against the eligibility criteria. Studies were eligible for inclusion if they reported on the importance of outcomes for patients with psoriasis. No language restrictions were used. Data extraction and quality assessment were also performed independently. Quality assessment was done using the QUALSYST tool., Findings: In total, 10 365 records were screened for eligibility, of which 24 studies were included for synthesis. A total of 23 317 patients were evaluated, and 273 (154 unique) items were retrieved. These items were aggregated into 23 outcomes: (almost) complete clearance; symptom control; difficult location clearance; time to clearance; treatment efficacy, sustainability, safety, tolerability, and convenience; comorbidity control; daily and social activity; emotional well-being; intimate relationships; productivity; health-related quality of life; confidence in care; control of disease; communication with care professional; information from other sources than care professional; and cost of care (societal and for the patient). These were then further grouped into 4 core areas: physical/clinical, life impact, resource use, and adverse effects. The mean overall quality of the studies was 75.6% (range, 35.7%-100%)., Conclusions and Relevance: This systematic review analyzed patient-relevant outcomes reported in patients with psoriasis to aid in the transition to a value-based treatment approach.

Background: Physician-reported clinical outcome and quality of life (QoL) measures are currently used to assess outcomes and direct treatment of plaque psoriasis. However, people with psoriasis may have different criteria for judging treatment success., Objectives: To build a unified consensus on the definition of 'freedom from disease' from a European stakeholder group, including people with psoriasis, dermatologists and nurses., Methods: The modified Delphi consensus methodology was used to define 'freedom from disease', with a consensus group consisting of people with psoriasis, nurses and dermatologists. This methodology involved people with psoriasis during the entire process and consisted of a 15-member Facilitating Consensus Panel to drive the programme content and a larger Voting Consensus Panel to vote on defining 'freedom from disease'. The Facilitating Panel agreed on disease domains, and aspects of each domain were put forward to the Voting Consensus Panel to establish relative importance. Following two voting rounds, a meeting was held to agree on a final consensus statement., Results: The Facilitating Panel consisted of six patient advocacy group representatives, three specialist nurses and six dermatologists. Voting rounds 1 and 2 were completed by 166 and 130 respondents from the Voting Consensus Panel, respectively. The outputs from both rounds of voting were similar, focusing on normality of living, symptom control, and a relationship of mutual respect and trust between the individual with psoriasis and their healthcare professional. The consensus statement emphasizes that 'freedom from disease' is multifaceted and includes the following domains 'management of clinical symptoms', 'psychosocial elements', 'QoL and well-being', 'treatment' and 'healthcare team support'. 'Freedom from disease' means all aspects are addressed., Conclusions: Freedom from disease in psoriasis is a multicomponent concept including five main domains. This diverse and multifaceted patient perspective will help us to improve understanding of the outcomes of treatment interventions in people with psoriasis., (© 2021 The Authors. Journal of the European Academy of Dermatology and Venereology published by John Wiley & Sons Ltd on behalf of European Academy of Dermatology and Venereology.)

Objectives : Psoriasis is a chronic skin disease requiring a multidimensional approach, given its varying appearance, presence of comorbidities and complex treatment regimens. Psoriasis care is however often performed fragmented and, in case of flares, reactive with little integrated information on and for the patient. Literature suggests a multileveled approach of psoriasis, but the effects of its implementation have not yet been validated. The aim of this study is to analyze the impact of a multileveled psoriasis consultation format, named PsoPlus, which has been implemented since 2012 in the Department of Dermatology at Ghent University Hospital in Belgium. Methods : The patient population was divided into two groups: one following the regular consultation and one following the PsoPlus format. Demographic data, clinical outcome and treatment approach of psoriasis patients were compared. Results : Patients who opted for the specialized PsoPlus consultation were younger and had longer disease duration. Decision parameters such as disease severity and quality of life were reported more often in the PsoPlus group. In the latter, a higher rate of patients were started on systemic therapy compared to the regular consultation group, and reporting on adverse events was done more frequently. Conclusion : The implementation of a specialized consultation with comprehensive guidance facilitates documentation on disease-relevant parameters such as disease severity and quality of life. This format can be seen as a guidance for capturing data in a structured manner, with evidence showing that it significantly impacts treatment decision, treating not only psoriasis but the patient as a whole.

Mitochondria are involved in many cellular pathways and dysfunctional mitochondria are linked to various diseases. Hence efforts have been made to design mitochondria-targeted fluorophores for monitoring the mitochondrial status. However, the factors that govern the mitochondria-targeted potential of dyes are not well-understood. In this context, we synthesized analogues of the TP-2Bzim probe belonging to the vinyltriphenylamine (TPA) class and already described for its capacity to bind nuclear DNA in fixed cells and mitochondria in live cells. These analogues (TP-1Bzim, TP n -2Bzim, TP 1+ -2Bzim, TN-2Bzim) differ in the cationic charge, the number of vinylbenzimidazolium branches and the nature of the triaryl core. Using microscopy, we demonstrated that the cationic derivatives accumulate in mitochondria but do not reach mtDNA. Under depolarisation of the mitochondrial membrane, TP-2Bzim and TP 1+ -2Bzim translocate to the nucleus in direct correlation with their strong DNA affinity. This reversible phenomenon emphasizes that these probes can be used to monitor ΔΨ m variations., (© 2021 Wiley-VCH GmbH.)

Background: The purpose of this paper is to provide an overview of key aspects of specialised dermatology nursing practice in the management of patients with moderate to severe atopic dermatitis. The role of dermatology nurse specialists in supporting patients and promoting disease understanding, education and treatment adherence continues to evolve. As features of specialised nursing care can also inform other nursing staff in a wide range of care settings, an overview of key components is examined. Observations presented are from a pan-European perspective and represent the collected view of a group of dermatology nurse specialists, dermatologists and patient advocates following two round-table discussions., Main Body: Atopic dermatitis is a common, chronic, inflammatory disease characterised by erythematous/scaling skin lesions, with often intense pruritus. Disease course is cyclic with periodic disease flares of varying intensity, presenting management challenges to patients and families. Dermatology nurse specialists play a key role in providing education and substantial patient support to improve treatment outcomes and quality of life to patients and their family, delivered within a multidisciplinary team framework. Nurse-led education and 'eczema schools' are of benefit in reducing disease severity and improving quality of life by enhancing self-management, adherence and patient engagement. eHealth tools, such as patient portals or online training platforms, can provide online learning, individualised education, and help to improve engagement. These and other initiatives, such as written action plans, are all essential to improve or maintain treatment adherence, self-management and quality of life., Conclusions: Dermatology nurse specialists play a central role in the assessment and management of moderate to severe atopic dermatitis patients and families. This places them in an ideal position to build strong and often long-term relationships with patients and parents. Such engagement promotes trust, assists in setting realistic expectations of treatment and outcomes, and enhances self-management and engagement in their own care. Providing emotional support, as well as formal and systematic education (including individualised practical advice) all contribute to improved treatment adherence and can enhance the quality of life of patients and their families throughout the course of this long-term condition.

Triphenylamines (TPAs) were previously shown to trigger cell death under prolonged one- or two-photon illumination. Their initial subcellular localization, before prolonged illumination, is exclusively cytoplasmic and they translocate to the nucleus upon photoactivation. However, depending on their structure, they display significant differences in terms of precise initial localization and subsequent photoinduced cell death mechanism. Here, we investigated the structural features of TPAs that influence cell death by studying a series of molecules differing by the number and chemical nature of vinyl branches. All compounds triggered cell death upon one-photon excitation, however to different extents, the nature of the electron acceptor group being determinant for the overall cell death efficiency. Photobleaching susceptibility was also an important parameter for discriminating efficient/inefficient compounds in two-photon experiments. Furthermore, the number of branches, but not their chemical nature, was crucial for determining the cellular uptake mechanism of TPAs and their intracellular fate. The uptake of all TPAs is an active endocytic process but two- and three-branch compounds are taken up via distinct endocytosis pathways, clathrin-dependent or -independent (predominantly caveolae-dependent), respectively. Two-branch TPAs preferentially target mitochondria and photoinduce both apoptosis and a proper necrotic process, whereas three-branch TPAs preferentially target late endosomes and photoinduce apoptosis only.

: A specific light trigger for activating endothelial Nitric Oxide-Synthase (eNOS) in real time would be of unique value to decipher cellular events associated with eNOS activation or to generate on demand cytotoxic levels of NO at specific sites for cancer research. We previously developed novel tools called nanotriggers (NT), which recognized constitutive NO-synthase, eNOS or neuronal NOS (nNOS), mainly via their 2' phosphate group which is also present in NADPH in its binding site. Laser excitation of NT1 bound to eNOS triggered recombinant NOS activity and released NO. We recently generated new NTs carrying a 2' or 3' carboxylate group or two 2' and 3' carboxylate moieties replacing the 2' phosphate group of NADPH. Among these new NT, only the 3' carboxylate derivative released NO from endothelial cells upon laser activation. Here, Molecular Dynamics (MD) simulations showed that the 3' carboxylate NT formed a folded structure with a hydrophobic hub, inducing a good stacking on FAD that likely drove efficient activation of nNOS. This NT also carried an additional small charged group which increased binding to e/nNOS; fluorescence measurements determined a 20-fold improved affinity upon binding to nNOS as compared to NT1 affinity. To gain in specificity for eNOS, we augmented a previous NT with a "hook" targeting variable residues in the NADPH site of eNOS. We discuss the potential of exploiting the chemical diversity within the NADPH site of eNOS for reversal of endothelial dysfunction in cells and for controlled generation of cytotoxic NO-derived species in cancer tissues., Competing Interests: the authors declare no conflict of interest.

Restriction factors are antiviral components of intrinsic immunity which constitute a first line of defense by blocking different steps of the human immunodeficiency virus (HIV) replication cycle. In immune cells, HIV infection is also sensed by several pattern recognition receptors (PRRs), leading to type I interferon (IFN-I) and inflammatory cytokines production that upregulate antiviral interferon-stimulated genes (ISGs). Several studies suggest a link between these two types of immunity. Indeed, restriction factors, that are generally interferon-inducible, are able to modulate immune responses. This review highlights recent knowledge of the interplay between restriction factors and immunity inducing antiviral defenses. Counteraction of this intrinsic and innate immunity by HIV viral proteins will also be discussed.

Background: Nitric-oxide synthases (NOS) catalyze the formation of NO using NADPH as electron donor. We have recently designed and synthesized a new series of two-photon absorbing and photoactivatable NADPH analogues (NT). These compounds bear one or two carboxymethyl group(s) on the 2'- or/and 3'-position(s) of the ribose in the adenosine moiety, instead of a 2'-phosphate group, and differ by the nature of the electron donor in their photoactivatable chromophore (replacing the nicotinamide moiety). Here, we addressed the ability of NTs to photoinduce eNOS-dependent NO production in endothelial cells., Methods: The cellular fate of NTs and their photoinduced effects were studied using multiphoton fluorescence imaging, cell viability assays and a BODIPY-derived NO probe for NO measurements. The eNOS dependence of photoinduced NO production was addressed using two NOS inhibitors (NS1 and L-NAME) targeting the reductase and the oxygenase domains, respectively., Results: We found that, two compounds, those bearing a single carboxymethyl group on the 3'-position of the ribose, colocalize with the Golgi apparatus (the main intracellular location of eNOS) and display high intracellular two-photon brightness. Furthermore, a eNOS-dependent photooxidation was observed for these two compounds only, which is accompanied by a substantial intracellular NO production accounting for specific photocytotoxic effects., Conclusions: We show for the first time that NT photoactivation efficiently triggers electron flow at the eNOS level and increases the basal production of NO by endothelial cells., General Significance: Efficient photoactivatable NADPH analogues targeting NOS could have important implications for generating apoptosis in tumor cells or modulating NO-dependent physiological processes., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Integration of the HIV-1 DNA into the host genome is essential for viral replication and is catalyzed by the retroviral integrase. To date, the only substrate described to be involved in this critical reaction is the linear viral DNA produced in reverse transcription. However, during HIV-1 infection, two-long terminal repeat DNA circles (2-LTRcs) are also generated through the ligation of the viral DNA ends by the host cell's nonhomologous DNA end-joining pathway. These DNAs contain all the genetic information required for viral replication, but their role in HIV-1's life cycle remains unknown. We previously showed that both linear and circular DNA fragments containing the 2-LTR palindrome junction can be efficiently cleaved in vitro by recombinant integrases, leading to the formation of linear 3'-processed-like DNA. In this report, using in vitro experiments with purified proteins and DNAs along with DNA endonuclease and in vivo integration assays, we show that this circularized genome can also be efficiently used as a substrate in HIV-1 integrase-mediated integration both in vitro and in eukaryotic cells. Notably, we demonstrate that the palindrome cleavage occurs via a two-step mechanism leading to a blunt-ended DNA product, followed by a classical 3'-processing reaction; this cleavage leads to integrase-dependent integration, highlighted by a 5-bp duplication of the host genome. Our results suggest that 2-LTRc may constitute a reserve supply of HIV-1 genomes for proviral integration., (© 2019 Richetta et al.)

Background Since 1 June 2017, oral pre-exposure prophylaxis (PrEP) could be prescribed and reimbursed in Belgium as prophylactic medication for people who are at increased risk of HIV acquisition. The aim of this study was to determine the uptake of daily and event-driven PrEP in Belgium during the first 9 months of roll-out., Methods: Routine aggregated data on the number of reimbursement requests and the number of boxes of Truvada (Gilead Sciences, Cambridge, UK) delivered for PrEP through the Belgian pharmacies were obtained from the National Institute for Health and Disability Insurance. We also collected aggregated data from seven Aids Reference Centres (ARCs) currently providing most of the PrEP care in Belgium., Results: From 1 June 2017 to 28 February 2018, 1352 requests for reimbursement were approved by the National Institute for Health and Disability Insurance. Almost 98% of those who bought at least one box of 30 tablets of emtricitabine 200mg/tenofovir disoproxil fumarate 300mg (FTC/TDF) in a Belgian pharmacy were male, and most (67%) were between 30 and 50 years of age. According to data obtained from ARCs, the proportion of those choosing event-driven PrEP initially ranged between 29% and 73%., Conclusions: The uptake of PrEP in Belgium since the start of the roll-out in June 2017 has been high, and almost entirely limited to men who have sex with men, of whom 43% initially prefer a non-daily regimen. A better understanding is needed as to why other populations, such as sub-Saharan African migrants, are not accessing PrEP, as well as the development of a more sustainable PrEP delivery model.

Background: Dolutegravir, an integrase strand-transfer inhibitor (STI), shows a high genetic barrier to resistance. Dolutegravir is reported to be effective against viruses resistant to raltegravir and elvitegravir. In this study, we report the case of a patient treated with dolutegravir monotherapy. Failure of dolutegravir treatment was observed concomitant with the appearance of N155H-K211R-E212T mutations in the integrase (IN) gene in addition to the polymorphic K156N mutation that was present at baseline in this patient., Methods: The impact of N155H-K156N-K211R-E212T mutations was studied in cell-free, culture-based assays and by molecular modelling., Results: Cell-free and culture-based assays confirm that selected mutations in the patient, in the context of the polymorphic mutation K156N present at the baseline, lead to high resistance to dolutegravir requiring that the analysis be done at timepoints longer than usual to properly reveal the results. Interestingly, the association of only N155H and K156N is sufficient for significant resistance to dolutegravir. Modelling studies showed that dolutegravir is less stable in IN/DNA complexes with respect to the WT sequence., Conclusions: Our results indicate that the stability of STI IN/DNA complexes is an important parameter that must be taken into account when evaluating dolutegravir resistance. This study confirms that a pathway including N155H can be selected in patients treated with dolutegravir with the help of the polymorphic K156N that acts as a secondary mutation that enhances the resistance to dolutegravir.

FDA-approved integrase strand transfer inhibitors (raltegravir, elvitegravir and dolutegravir) efficiently inhibit HIV-1 replication. Here, we present fluorescence properties of these inhibitors. Dolutegravir displays an excitation mode particularly dependent on Mg 2+ chelation, allowing to directly probe its Mg 2+ -dependent binding to the prototype foamy virus (PFV) integrase. Dolutegravir-binding studied by both its fluorescence anisotropy and subsequent emission enhancement, strictly requires a preformed integrase/DNA complex, the ten terminal base pairs from the 3'-end of the DNA reactive strand being crucial to optimize dolutegravir-binding in the context of the ternary complex. From the protein side, mutation of any catalytic residue fully abolishes dolutegravir-binding. We also compared dolutegravir-binding to PFV F190Y, G187R and S217K mutants, corresponding to HIV-1 F121Y, G118R and G140S/Q148K mutations that confer low-to-high resistance levels against raltegravir/dolutegravir. The dolutegravir-binding properties derived from fluorescence-based binding assays and drug susceptibilities in terms of catalytic activity, are well correlated. Indeed, dolutegravir-binding to wild-type and F190Y integrases are comparable while strongly compromised with G187R and S217K. Accordingly, the two latter mutants are highly resistant to dolutegravir while F190Y shows only moderate or no resistance. Intrinsic fluorescence properties of dolutegravir are thus particularly suitable for a thorough characterization of both DNA-binding properties of integrase and resistance mutations.

Integrase strand-transfer inhibitors (INSTIs), such as raltegravir (RAL), elvitegravir, or dolutegravir (DTG), are efficient antiretroviral agents used in HIV treatment in order to inhibit retroviral integration. By contrast to RAL treatments leading to well-identified mutation resistance pathways at the integrase level, recent clinical studies report several cases of patients failing DTG treatment without clearly identified resistance mutation in the integrase gene raising questions for the mechanism behind the resistance. These compounds, by impairing the integration of HIV-1 viral DNA into the host DNA, lead to an accumulation of unintegrated circular viral DNA forms. This viral DNA could be at the origin of the INSTI resistance by two different ways. The first one, sustained by a recent report, involves 2-long terminal repeat circles integration and the second one involves expression of accumulated unintegrated viral DNA leading to a basal production of viral particles maintaining the viral information.

Photodynamic therapy (PDT) is a promising therapeutic method for several diseases, in particular for cancer. This approach uses a photosensitizer, oxygen, and an external light source to produce reactive oxygen species (ROS) at lethal doses to induce cell death. One drawback of current PDT is the use of visible light which has poor penetration in tissues. Such a limitation could be overcome by the use of novel organic compounds compatible with photoactivation under near-infrared light excitation. Triphenylamines (TPAs) are highly fluorescent compounds that are efficient to induce cell death upon visible light excitation (458 nm), but outside the biological spectral window. Interestingly, we recently showed that TPAs target cytoplasmic organelles of living cells, mainly mitochondria, and induce a high ROS production upon 2-photon excitation (in the 760-860 nm range), leading to a fast apoptosis process. However, we observed significant differences among the tested TPA compounds in terms of cell distribution and time courses of cell death-related events (apoptosis vs necrosis). In summary, TPAs represent serious candidates as photosensitizers that are compatible with 2-photon excitation to simultaneously trigger and imaging cell death although the relationship between their subcellular localization and the cell death mechanism involved is still a matter of debate.

A new series of photoactivable NADPH mimics bearing one or two O-carboxymethyl groups on the adenosine moiety have been readily synthesized using click chemistry. These compounds display interesting one- or two-photon absorption properties. Their fluorescence emission wavelength and quantum yields (Φ) are dependent on the solvent polarity, with a red-shift in a more polar environment (λ max,em = 460-467 nm, Φ > 0.53 in DMSO, and λ max,em = 475-491 nm, Φ < 0.17 in Tris). These compounds show good binding affinity towards the constitutive nNOS and eNOS, confirming for the first time that the carboxymethyl group can be used as a surrogate of phosphate. Two-photon fluorescence imaging of nanotriggers in living cells showed that the presence of one carboxymethyl group (especially on the 3' position of the ribose) strongly favors the addressing of nanotriggers to eNOS in the cell context.

Cadmium is a toxic metal that inactivates DNA-repair proteins via multiple mechanisms, including zinc substitution. In this study, we investigated the effect of Cd(2+) on the Bloom protein (BLM), a DNA-repair helicase carrying a zinc-binding domain (ZBD) and playing a critical role to ensure genomic stability. One characteristics of BLM-deficient cells is the elevated rate of sister chromatid exchanges, a phenomenon that is also induced by Cd(2+). Here, we show that Cd(2+) strongly inhibits both ATPase and helicase activities of BLM. Cd(2+) primarily prevents BLM-DNA interaction via its binding to sulfhydryl groups of solvent-exposed cysteine residues and, concomitantly, promotes the formation of large BLM multimers/aggregates. In contrast to previously described Cd(2+) effects on other zinc-containing DNA-repair proteins, the ZBD appears to play a minor role in the Cd(2+)-mediated inhibition. While the Cd(2+)-dependent formation of inactive multimers and the defect of DNA-binding were fully reversible upon addition of EDTA, the inhibition of the DNA unwinding activity was not counteracted by EDTA, indicating another mechanism of inhibition by Cd(2+) relative to the targeting of a catalytic residue. Altogether, our results provide new clues for understanding the mechanism behind the ZBD-independent inactivation of BLM by Cd(2+) leading to accumulation of DNA double-strand breaks.

Integration of HIV-1 linear DNA into host chromatin is required for high levels of viral expression, and constitutes a key therapeutic target. Unintegrated viral DNA (uDNA) can support only limited transcription but may contribute to viral propagation, persistence and/or treatment escape under specific situations. The molecular mechanisms involved in the differential expression of HIV uDNA vs integrated genome (iDNA) remain to be elucidated. Here, we demonstrate, for the first time, that the expression of HIV uDNA is mainly supported by 1-LTR circles, and regulated in the opposite way, relatively to iDNA, following NF-κB pathway modulation. Upon treatment activating the NF-κB pathway, NF-κB p65 and AP-1 (cFos/cJun) binding to HIV LTR iDNA correlates with increased iDNA expression, while uDNA expression decreases. On the contrary, inhibition of the NF-κB pathway promotes the expression of circular uDNA, and correlates with Bcl-3 and AP-1 binding to its LTR region. Finally, this study identifies NF-κB subunits and Bcl-3 as transcription factors binding the HIV promoter differently depending on viral genome topology, and opens new insights on the potential roles of episomal genomes during the HIV-1 latency and persistence.

Photodynamic therapy (PDT) leads to cell death by using a combination of a photosensitizer and an external light source for the production of lethal doses of reactive oxygen species (ROS). Since a major limitation of PDT is the poor penetration of UV-visible light in tissues, there is a strong need for organic compounds whose activation is compatible with near-infrared excitation. Triphenylamines (TPAs) are fluorescent compounds, recently shown to efficiently trigger cell death upon visible light irradiation (458 nm), however outside the so-called optical/therapeutic window. Here, we report that TPAs target cytosolic organelles of living cells, mainly mitochondria, triggering a fast apoptosis upon two-photon excitation, thanks to their large two-photon absorption cross-sections in the 760-860 nm range. Direct ROS imaging in the cell context upon multiphoton excitation of TPA and three-color flow cytometric analysis showing phosphatidylserine externalization indicate that TPA photoactivation is primarily related to the mitochondrial apoptotic pathway via ROS production, although significant differences in the time courses of cell death-related events were observed, depending on the compound. TPAs represent a new class of water-soluble organic photosensitizers compatible with direct two-photon excitation, enabling simultaneous multiphoton fluorescence imaging of cell death since a concomitant subcellular TPA re-distribution occurs in apoptotic cells.

Myostatin, also known as growth differentiation factor 8, is a member of the transforming growth factor-beta superfamily that has been shown to play a key role in the regulation of the skeletal muscle mass. Indeed, while myostatin deletion or loss of function induces muscle hypertrophy, its overexpression or systemic administration causes muscle atrophy. Since myostatin blockade is effective in increasing skeletal muscle mass, myostatin inhibitors have been actively sought after. Decorin, a member of the small leucine-rich proteoglycan family is a metalloprotein that was previously shown to bind and inactivate myostatin in a zinc-dependent manner. Furthermore, the myostatin-binding site has been shown to be located in the decorin N-terminal domain. In the present study, we investigated the anti-myostatin activity of short and soluble fragments of decorin. Our results indicate that the murine decorin peptides DCN48-71 and 42-65 are sufficient for inactivating myostatin in vitro. Moreover, we show that the interaction of mDCN48-71 to myostatin is strictly zinc-dependent. Binding of myostatin to activin type II receptor results in the phosphorylation of Smad2/3. Addition of the decorin peptide 48-71 decreased in a dose-dependent manner the myostatin-induced phosphorylation of Smad2 demonstrating thereby that the peptide inhibits the activation of the Smad signaling pathway. Finally, we found that mDCN48-71 displays a specificity towards myostatin, since it does not inhibit other members of the transforming growth factor-beta family., (Copyright © 2016 Elsevier Inc. All rights reserved.)

ScPif1 DNA helicase is the prototypical member of a 5'-to-3' helicase superfamily conserved from bacteria to human and plays various roles in the maintenance of genomic homeostasis. While many studies have been performed with eukaryotic Pif1 helicases, including yeast and human Pif1 proteins, the potential functions and biochemical properties of prokaryotic Pif1 helicases remain largely unknown. Here, we report the expression, purification and biochemical analysis of Pif1 helicase from Bacteroides sp. 3&#95;1&#95;23 (BsPif1). BsPif1 binds to a large panel of DNA substrates and, in particular, efficiently unwinds partial duplex DNAs with 5'-overhang, fork-like substrates, D-loop and flap-like substrates, suggesting that BsPif1 may act at stalled DNA replication forks and enhance Okazaki fragment maturation. Like its eukaryotic homologues, BsPif1 resolves R-loop structures and unwinds DNA-RNA hybrids. Furthermore, BsPif1 efficiently unfolds G-quadruplexes and disrupts nucleoprotein complexes. Altogether, these results highlight that prokaryotic Pif1 helicases may resolve common issues that arise during DNA transactions. Interestingly, we found that BsPif1 is different from yeast Pif1, but resembles more human Pif1 with regard to substrate specificity, helicase activity and mode of action. These findings are discussed in the context of the possible functions of prokaryotic Pif1 helicases in vivo., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Triphenylamines are on/off fluorescent DNA minor groove binders, allowing nuclear staining of fixed cells. By contrast, they accumulate in the cytoplasm of living cells and efficiently trigger cell apoptosis upon prolonged visible light irradiation. This process occurs concomitantly with their subcellular re-localization to the nucleus, enabling fluorescence imaging of apoptosis.

Objectives: HIV-1 integration can be efficiently inhibited by strand-transfer inhibitors such as raltegravir, elvitegravir or dolutegravir. Three pathways conferring raltegravir/elvitegravir cross-resistance (involving integrase residues Q148, N155 and Y143) were identified. Dolutegravir, belonging to the second generation of strand-transfer compounds, inhibits the Y143 and N155 pathways, but is less efficient at inhibiting the Q148 pathway. The aim of this study was to characterize the combination of two pathways involved in raltegravir resistance described in one patient failing a dolutegravir regimen for their propensity to confer dolutegravir resistance., Methods: In this study, a patient first failing a regimen including raltegravir was treated with dolutegravir and showed an increase in viruses carrying a combination of two pathways (N155 and Q148). Impacts of these mutations on integrase activity and resistance to strand-transfer inhibitors were characterized using both in vitro and virological assays., Results: Our data showed that the combination of N155H, G140S and Q148H mutations led to strong resistance to dolutegravir., Conclusions: Combination of N155H, G140S and Q148H mutations originating from two distinct resistance pathways to raltegravir or elvitegravir led to a high level of dolutegravir resistance. Due to its high genetic barrier of resistance, it would be reasonable to use dolutegravir in first-line therapy before emergence of raltegravir or elvitegravir resistance., (© The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Background: Genomic integration, an obligate step in the HIV-1 replication cycle, is blocked by the integrase inhibitor raltegravir. A consequence is an excess of unintegrated viral DNA genomes, which undergo intramolecular ligation and accumulate as 2-LTR circles. These circularized genomes are also reliably observed in vivo in the absence of antiviral therapy and they persist in non-dividing cells. However, they have long been considered as dead-end products that are not precursors to integration and further viral propagation., Results: Here, we show that raltegravir action is reversible and that unintegrated viral DNA is integrated in the host cell genome after raltegravir removal leading to HIV-1 replication. Using quantitative PCR approach, we analyzed the consequences of reversing prolonged raltegravir-induced integration blocks. We observed, after RAL removal, a decrease of 2-LTR circles and a transient increase of linear DNA that is subsequently integrated in the host cell genome and fuel new cycles of viral replication., Conclusions: Our data highly suggest that 2-LTR circles can be used as a reserve supply of genomes for proviral integration highlighting their potential role in the overall HIV-1 replication cycle.

Objectives: Strand transfer inhibitors (raltegravir, elvitegravir and dolutegravir) are now commonly used to inhibit HIV-1 integration. To date, three main pathways conferring raltegravir/elvitegravir resistance, involving residues Y143, Q148 and N155, have been described. However, no pathway has been clearly described for dolutegravir resistance. The aim of this study was to characterize the susceptibility of two mutations, F121Y and G118R, originally described in patients failing raltegravir-containing regimens, to dolutegravir and raltegravir, and then to compare the resistance of these mutations with that of other well-known mutations involved in raltegravir resistance., Methods: Both the F121Y and G118R mutations were introduced by site-directed mutagenesis into the pNL4.3 backbone and studied in cell-based and in vitro assays. The effects of the mutations were characterized at the different steps of infection by quantitative PCR., Results: Results obtained with in vitro and ex vivo assays consistently showed that both mutations impaired the catalytic properties of integrase, especially at the integration step. Moreover, both mutations conferred an intermediate level of resistance to dolutegravir. Interestingly, the F121Y mutation, but not the G118R mutation, displayed differential resistance to raltegravir and dolutegravir. Indeed, the F121Y mutation was more resistant to raltegravir than to dolutegravir., Conclusions: Mutations at G118 and F121, which have been described in patients failing raltegravir-containing regimens, must be included in drug-resistance-testing algorithms., (© The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)