Development of a single-domain antibody to target a G-quadruplex located on the hepatitis B virus covalently closed circular DNA genome.

To achieve a virological cure for hepatitis B virus (HBV), innovative strategies are required to target the covalently closed circular DNA (cccDNA) genome. Guanine-quadruplexes (G4s) are a secondary structure that can be adopted by DNA and play a significant role in regulating viral replication, transcription, and translation. Antibody-based probes and small molecules have been developed to study the role of G4s in the context of the human genome, but none have been specifically made to target G4s in viral infection. Herein, we describe the development of a humanized single-domain antibody (S10) that can target a G4 located in the PreCore (PreC) promoter of the HBV cccDNA genome. MicroScale Thermophoresis demonstrated that S10 has a strong nanomolar affinity to the PreC G4 in its quadruplex form and a structural electron density envelope of the complex was determined using Small-Angle X-ray Scattering. Lentiviral transduction of S10 into HepG2-NTCP cells shows nuclear localization, and chromatin immunoprecipitation coupled with next-generation sequencing demonstrated that S10 can bind to the HBV PreC G4 present on the cccDNA. This research validates the existence of a G4 in HBV cccDNA and demonstrates that this DNA secondary structure can be targeted with high structural and sequence specificity using S10.

nAMD: optimization of patient care and patient-oriented information with the help of an internet-based survey.

PURPOSE: This survey was conducted to identify factors that influence how patients with neovascular age-related macular degeneration (nAMD) deal with their disease and information that are considered useful from a patient's point of view. METHODS: A total of 5035 patients with nAMD living in Germany were interviewed via internet-based cross-sectional survey, where the following information was collected: personal data, disease awareness, and patients' needs. In addition, a Quality of Life questionnaire (SF-12v2) could be completed. RESULTS: Out of the 5035 participants, more males than females participated (55% vs 45%), and most participants were in the age groups 76 to 85 years (37%) and 66 to 75 years (35%). Seventy-three percent of patients rated their understanding of the disease as at least sufficient, and more than two-thirds of the patients (68%) were aware that their disease needs to be controlled on a regular basis and treated on an "as needed" basis. Regarding potential risk factors for AMD, most participants were aware of age (89%), but only 39% of hereditary load and 33% of smoking as evidence-based risk factors, indicating a need for further information. The doctor remains the major source of information (93%), with internet (29%), brochures (14%), opticians (13%), or patient support groups (4%) with only limited contribution. Distance to the treatment center was identified as one of the factors, which had the greatest influence on patients' compliance. A "treat as needed" regime turned out to be the preferred control and treatment schedule in contrast to a "fixed appointment" every 4 weeks. CONCLUSION: This internet-based survey appears to be representative for nAMD patients. To increase patients' compliance, proximity to the treatment center and a "treat as needed" regime turned out to be important factors as well as patients' awareness of their disease. In this regard, the reported desire for more information indicates that patients' knowledge still needs to be improved. Our results will help to further optimize patient care and patient-oriented information.

Targeted Delivery of Soluble Guanylate Cyclase (sGC) Activator Cinaciguat to Renal Mesangial Cells via Virus-Mimetic Nanoparticles Potentiates Anti-Fibrotic Effects by cGMP-Mediated Suppression of the TGF-ß Pathway.

Diabetic nephropathy (DN) ranks among the most detrimental long-term effects of diabetes, affecting more than 30% of all patients. Within the diseased kidney, intraglomerular mesangial cells play a key role in facilitating the pro-fibrotic turnover of extracellular matrix components and a progredient glomerular hyperproliferation. These pathological effects are in part caused by an impaired functionality of soluble guanylate cyclase (sGC) and a consequentially reduced synthesis of anti-fibrotic messenger 3',5'-cyclic guanosine monophosphate (cGMP). Bay 58-2667 (cinaciguat) is able to re-activate defective sGC; however, the drug suffers from poor bioavailability and its systemic administration is linked to adverse events such as severe hypotension, which can hamper the therapeutic effect. In this study, cinaciguat was therefore efficiently encapsulated into virus-mimetic nanoparticles (NPs) that are able to specifically target renal mesangial cells and therefore increase the intracellular drug accumulation. NP-assisted drug delivery thereby increased in vitro potency of cinaciguat-induced sGC stabilization and activation, as well as the related downstream signaling 4- to 5-fold. Additionally, administration of drug-loaded NPs provided a considerable suppression of the non-canonical transforming growth factor ß (TGF-ß) signaling pathway and the resulting pro-fibrotic remodeling by 50-100%, making the system a promising tool for a more refined therapy of DN and other related kidney pathologies.

Biomedical nanoparticle design: What we can learn from viruses.

Viruses are nanomaterials with a number of properties that surpass those of many synthetic nanoparticles (NPs) for biomedical applications. They possess a rigorously ordered structure, come in a variety of shapes, and present unique surface elements, such as spikes. These attributes facilitate propitious biodistribution, the crossing of complex biological barriers and a minutely coordinated interaction with cells. Due to the orchestrated sequence of interactions of their stringently arranged particle corona with cellular surface receptors they effectively identify and infect their host cells with utmost specificity, while evading the immune system at the same time. Furthermore, their efficacy is enhanced by their response to stimuli and the ability to spread from cell to cell. Over the years, great efforts have been made to mimic distinct viral traits to improve biomedical nanomaterial performance. However, a closer look at the literature reveals that no comprehensive evaluation of the benefit of virus-mimetic material design on the targeting efficiency of nanomaterials exists. In this review we, therefore, elucidate the impact that viral properties had on fundamental advances in outfitting nanomaterials with the ability to interact specifically with their target cells. We give a comprehensive overview of the diverse design strategies and identify critical steps on the way to reducing them to practice. More so, we discuss the advantages and future perspectives of a virus-mimetic nanomaterial design and try to elucidate if viral mimicry holds the key for better NP targeting.

Adenovirus-Mimetic Nanoparticles: Sequential Ligand-Receptor Interplay as a Universal Tool for Enhanced In Vitro/In Vivo Cell Identification.

Viral infection patterns often rely on precisely coordinated sequences of distinct ligand-receptor interactions, leading in many cases to an outstanding target cell specificity. A successful mimicry of viral targeting strategies to create more site-specific nanoparticles (NPs) would therefore require particle-cell interactions to also be adequately controllable. In the present study, hetero-multivalent block-copolymer NPs present their attached ligands in a sterically controlled manner to create a sequential NP-cell interaction similar to the cell infiltration strategy of human adenovirus type 2. Targeting renal mesangial cells, particles therefore initially bind angiotensin II receptor type 1 (AT1r) on the cell surface via a structurally flexible AT1r antagonist. After a mandatory spatial approach, particle endocytosis is realized via binding of immobile αVß3 integrins with a previously concealed secondary ligand, thereby creating a stepwise particle-cell interplay of primary NP attachment and subsequent uptake. Manufactured adenovirus-mimetic NPs show great avidity for both target motifs in vitro, leading to a substantial binding as well as subsequent cell uptake into target mesangial cells. Additionally, steric shielding of secondary ligand visibility leads to a highly controllable, sequential ligand-receptor interaction, whereby hetero-functional NPs activate mesangial cell surface integrins only after a successful prior binding to the AT1r. This stepwise cell identification significantly enhances mesangial cell specificity in co-culture assays with different off-target cells. Additionally, described NPs display excellent in vivo robustness by efficiently accumulating in the mesangium upon injection, thereby opening new paths for possible drug delivery applications.

Nanoparticles Mimicking Viral Cell Recognition Strategies Are Superior Transporters into Mesangial Cells.

Poor drug availability in the tissue of interest is a frequent cause of therapy failure. While nanotechnology has developed a plethora of nanocarriers for drug transport, their ability to unequivocally identify cells of interest remains moderate. Viruses are the ideal nanosized carriers as they are able to address their embedded nucleic acids with high specificity to their host cells. Here, it is reported that particles endowed with a virus-like ability to identify cells by three consecutive checks have a superior ability to recognize mesangial cells (MCs) in vivo compared to conventional nanoparticles. Mimicking the initial viral attachment followed by a stepwise target cell recognition process leads to a 5- to 15-fold higher accumulation in the kidney mesangium and extensive cell uptake compared to particles lacking one or both of the viral traits. These results highlight the relevance that the viral cell identification process has on specificity and its application on the targeting strategies of nanomaterials. More so, these findings pave the way for transporting drugs into the mesangium, a tissue that is pivotal in the development of diabetic nephropathy and for which currently no efficient pharmacotherapy exists.

Thermodynamic, Spatial and Methodological Considerations for the Manufacturing of Therapeutic Polymer Nanoparticles.

PURPOSE: Evaluate fundamental parameters that dictate the effectiveness of drug loading. METHODS: A model water-soluble drug lacking ionizable groups, pirfenidone (PFD), was encapsulated through nanoprecipitation in poly(ethylene glycol)-poly(lactic acid) (PEG-PLA)-poly(lactic-co-glycolic acid) (PLGA) NPs. Firstly, the thermodynamic parameters predicting drug-polymer miscibility were determined to assess the system's suitability. Then, the encapsulation was evaluated experimentally by two different techniques, bulk and microfluidic (MF) nanoprecipitation. Additionally, the number of molecules that fit in a particle core were calculated and the loading determined experimentally for different core sizes. Lastly, the effect of co-encapsulation of α-lipoic acid (LA), a drug with complementary therapeutic effects and enhanced lipophilicity, was evaluated. RESULTS: The thermodynamic miscibility parameters predicted a good suitability of the selected system. MF manufacturing enhanced the encapsulation efficiency by 60-90% and achieved a 2-fold higher NP cellular uptake. Considering spatial constrictions for drug encapsulation and increasing the size of the PLGA core the number of PFD molecules per NP was raised from under 500 to up to 2000. More so, the co-encapsulation of LA increased the number of drug molecules per particle by 96%, with no interference with the release profile. CONCLUSIONS: Thermodynamic, spatial and methodological parameters should be considered to optimize drug encapsulation.

The Effect of Ligand Mobility on the Cellular Interaction of Multivalent Nanoparticles.

Multivalent nanoparticle binding to cells can be of picomolar avidity making such interactions almost as intense as those seen with antibodies. However, reducing nanoparticle design exclusively to avidity optimization by the choice of ligand and its surface density does not sufficiently account for controlling and understanding cell-particle interactions. Cell uptake, for example, is of paramount significance for a plethora of biomedical applications and does not exclusively depend on the intensity of multivalency. In this study, it is shown that the mobility of ligands tethered to particle surfaces has a substantial impact on particle fate upon binding. Nanoparticles carrying angiotensin-II tethered to highly mobile 5 kDa long poly(ethylene glycol) (PEG) chains separated by ligand-free 2 kDa short PEG chains show a superior accumulation in angiotensin-II receptor type 1 positive cells. In contrast, when ligand mobility is constrained by densely packing the nanoparticle surface with 5 kDa PEG chains only, cell uptake decreases by 50%. Remarkably, irrespective of ligand mobility and density both particle types have similar EC50 values in the 1-3 × 10-9 m range. These findings demonstrate that ligand mobility on the nanoparticle corona is an indispensable attribute to be considered in particle design to achieve optimal cell uptake via multivalent interactions.

Influenza A virus mimetic nanoparticles trigger selective cell uptake.

Poor target cell specificity is currently a major shortcoming of nanoparticles (NPs) used for biomedical applications. It causes significant material loss to off-target sites and poor availability at the intended delivery site. To overcome this limitation, we designed particles that identify cells in a virus-like manner. As a blueprint, we chose a mechanism typical of influenza A virus particles in which ectoenzymatic hemagglutinin activation by target cells is a mandatory prerequisite for binding to a secondary target structure that finally confirms cell identity and allows for uptake of the virus. We developed NPs that probe mesangial cells for the presence of angiotensin-converting enzyme on their surface using angiotensin I (Ang-I) as a proligand. This initial interaction enzymatically transforms Ang-I to a secondary ligand angiotensin II (Ang-II) that has the potential to bind in a second stage to Ang-II type-1 receptor (AT1R). The presence of the receptor confirms the target cell identity and triggers NP uptake via endocytosis. Our virus-mimetic NPs showed outstanding target-cell affinity with picomolar avidities and were able to selectively identify these cells in the presence of 90% off-target cells that carried only the AT1R. Our results demonstrate that the design of virus-mimetic cell interactive NPs is a valuable strategy to enhance NP specificity for therapeutic and diagnostic applications. Our set of primary and secondary targets is particularly suited for the identification of mesangial cells that play a pivotal role in diabetic nephropathy, one of the leading causes of renal failure, for which currently no treatment exists.

Interaction of functionalized nanoparticles with serum proteins and its impact on colloidal stability and cargo leaching.

The majority of effort in the area of polymeric nanocarriers is aimed at providing controlled drug delivery in vivo. Therefore, it is essential to understand the delicate interplay of polymeric NPs with serum proteins in order to forecast their performance in a biological system. In this study, the interaction of serum proteins with functionalized polymeric colloids as a function of particle charge and hydrophobicity was investigated. Moreover, impact on NP stability and cargo leaching was assessed. The hard protein corona of polymeric NPs with either uncharged methoxy groups (methoxy-NPs), positively charged amine groups (amine-NPs), negatively charged carboxylic acid groups (carboxyl-NPs) or zwitterionic NPs decorated with amine and carboxylic acid groups (zwitterion-NPs) was quantitatively and qualitatively analyzed and correlated with the respective colloidal stability using fluorescence resonance energy transfer. Positively charged amine-NPs displayed an enhanced interaction with serum proteins via electrostatic interactions resulting in a hard corona consisting of diverse protein components. As revealed by FRET and agarose gel electrophoresis, the enhanced adsorption of proteins onto the colloidal surface significantly altered the NP identity and severely impaired the colloidal integrity as the lipophilic cargo was continuously leached out of the hydrophobic NP core. These results highlight the importance of generating a profound knowledge of the bio-nano interface as adherence of biomolecules can severely compromise the performance of a colloidal drug delivery system by changing its identity and integrity.

Perceptions of Supervision in an Outpatient Psychiatry Clinic.

OBJECTIVES: There is little guidance regarding best practices in supervision of psychiatric residents. As a result, expectations for both supervisors and trainees are often unclear. This study explored the experiences of trainees and supervisors in outpatient supervision, in order to identify areas for potential improvement. METHODS: The authors conducted focus groups of residents and faculty members. The sessions were transcribed and analyzed via established methods of qualitative data analysis. RESULTS: A number of themes emerged. In general, residents desire an explicit structure to supervisory sessions, with more specific and concrete instruction. Attendings prefer to let residents lead discussion in supervision and focus on interpersonal aspects, such as the mentor-mentee relationship. Findings were situated within an established model of skill acquisition, the five-stage progression described by Dreyfus and Dreyfus. CONCLUSIONS: The differing experiences of trainees and supervisors reflect their respective stages of skill development as ambulatory psychiatrists. Potential interventions to improve the educational value of supervision include explicit agenda-setting at the beginning of supervision, regular bidirectional feedback, and more frequent opportunities for residents to observe attending interviews with patients.

Helping psychiatry residents cope with patient suicide.

OBJECTIVE: Every clinical specialty has its own high risk patient challenges that threaten to undermine their trainees' professional identity, evolving sense of competence. In psychiatric training, it is patient suicide, an all-too frequently encountered consequence of severe mental illness that may leave the treating resident perplexed, guilt-ridden, and uncertain of their suitability for the profession. This study evaluates a patient suicide training program aimed at educating residents about patient suicide, common reactions, and steps to attenuate emotional distress while facilitating learning. METHODS: The intervention was selected aspects of a patient suicide educational program, "Collateral Damages,"-video vignettes, focused discussions, and a patient-based learning exercise. Pre- and post-survey results were compared to assess both knowledge and attitudes resulting from this educational program. Eight psychiatry residency training programs participated in the study, and 167 of a possible 240 trainees (response rate = 69.58 %) completed pre- and post-surveys. RESULTS: Knowledge of issues related to patient suicide increased after the program. Participants reported increased awareness of the common feelings physicians and trainees often experience after a patient suicide, of recommended "next" steps, available support systems, required documentation, and the role played by risk management. CONCLUSIONS: This patient suicide educational program increased awareness of issues related to patient suicide and shows promise as a useful and long overdue educational program in residency training. It will be useful to learn whether this program enhances patient care or coping with actual patient suicide. Similar programs might be useful for other specialties.

Exploring the impact of suicide on clinicians: a multidisciplinary retreat model.

OBJECTIVE: We developed an every other year, scheduled retreat model for clinicians and trainees to explore emotional and professional reactions that they may face after a patient's suicide. METHODS: Psychiatry ambulatory clinical staff, residents, and faculty participated in a halfday retreat, which consisted of an opening panel discussion, in which panel members related their experiences of patient suicide, break out groups, and a final panel discussion. Unlinked preand post-retreat surveys were electronically sent to all potential participants. RESULTS: The pre-retreat survey was completed by 103 clinicians; 20% of the respondents were trainees or fellows, and 47% reported that they had experienced a patient suicide. Text responses to the pre-retreat survey reflected the wish to obtain a better understanding of the impact of patient suicide on caregivers, to cope with the event from a personal and professional standpoint, and to get a clearer understanding of what supports are available within the department. The post-retreat survey was completed by 45 clinicians. Comments after the retreat reflected an increased awareness of both shortand long-term effects on clinicians after a patient suicide and the extensive impact that a patient suicide can have on providers within their ambulatory care department. CONCLUSION: An alldepartment ambulatory retreat model has value in providing clinicians with support and information in a structured, educational setting to help reduce the sense of stigma and provide an increased awareness of the magnitude of the impact on clinicians who experience the death of a patient by suicide.

Fomitiporia punctata (Basidiomycota, Hymenochaetales) and its presumed taxonomic synonyms in America: taxonomy and phylogeny of some species from tropical/subtropical areas.

The status of the F. punctata complex in tropical/subtropical America, and more specifically in Cuba and the surrounding area, was partially assessed. Fomitiporia langloisii, Fomitiporia dryophila and Fomitiporia maxonii, three names long considered taxonomic synonyms of F. punctata, are recognized as representing three distinct species that are distinguished from the latter on the basis of morphological, molecular and ecological (biogeographical) data. The three species are described again and their preliminary phylogenetic relationships within Fomitiporia are discussed. Fomitiporia tabaquilio comb. nov. is proposed. The status of Fomitiporia in America and its present circumscription are discussed.

Phellinus caribaeo-quercicolus sp. nov., parasitic on Quercus cubana: taxonomy and preliminary phylogenetic relationships.

Phellinus caribaeo-quercicolus sp. nov. is described from several collections made in western Cuba, so far exclusively on Quercus cubana. The species is characterized by a perennial, resupinate basidiomes, cushion-shaped to nodulose and multi-layered when old, apically hooked to hamate hymenial setae, and ellipsoid to broadly ellipsoid, thin- to thick-walled, hyaline to faintly yellowish basidiospores, 4.5-5.5 x 3.5-4.5 microm. The species is compared to other Phellinus species with hooked setae, especially Phellinus undulatus, also recorded in Cuba. The preliminary phylogenetic relationships of Ph. caribaeo-quercicolus within the poroid Hymenochaetales complex of genera is presented and discussed here.