IL-23 stabilizes an effector Treg cell program in the tumor microenvironment.

Interleukin-23 (IL-23) is a proinflammatory cytokine mainly produced by myeloid cells that promotes tumor growth in various preclinical cancer models and correlates with adverse outcomes. However, as to how IL-23 fuels tumor growth is unclear. Here, we found tumor-associated macrophages to be the main source of IL-23 in mouse and human tumor microenvironments. Among IL-23-sensing cells, we identified a subset of tumor-infiltrating regulatory T (Treg) cells that display a highly suppressive phenotype across mouse and human tumors. The use of three preclinical models of solid cancer in combination with genetic ablation of Il23r in Treg cells revealed that they are responsible for the tumor-promoting effect of IL-23. Mechanistically, we found that IL-23 sensing represents a crucial signal driving the maintenance and stabilization of effector Treg cells involving the transcription factor Foxp3. Our data support that targeting the IL-23/IL-23R axis in cancer may represent a means of eliciting antitumor immunity.

Glutamatergic dysfunction leads to a hyper-dopaminergic phenotype through deficits in short-term habituation: a mechanism for aberrant salience.

Psychosis in disorders like schizophrenia is commonly associated with aberrant salience and elevated striatal dopamine. However, the underlying cause(s) of this hyper-dopaminergic state remain elusive. Various lines of evidence point to glutamatergic dysfunction and impairments in synaptic plasticity in the etiology of schizophrenia, including deficits associated with the GluA1 AMPAR subunit. GluA1 knockout (Gria1-/-) mice provide a model of impaired synaptic plasticity in schizophrenia and exhibit a selective deficit in a form of short-term memory which underlies short-term habituation. As such, these mice are unable to reduce attention to recently presented stimuli. In this study we used fast-scan cyclic voltammetry to measure phasic dopamine responses in the nucleus accumbens of Gria1-/- mice to determine whether this behavioral phenotype might be a key driver of a hyper-dopaminergic state. There was no effect of GluA1 deletion on electrically-evoked dopamine responses in anaesthetized mice, demonstrating normal endogenous release properties of dopamine neurons in Gria1-/- mice. Furthermore, dopamine signals were initially similar in Gria1-/- mice compared to controls in response to both sucrose rewards and neutral light stimuli. They were also equally sensitive to changes in the magnitude of delivered rewards. In contrast, however, these stimulus-evoked dopamine signals failed to habituate with repeated presentations in Gria1-/- mice, resulting in a task-relevant, hyper-dopaminergic phenotype. Thus, here we show that GluA1 dysfunction, resulting in impaired short-term habituation, is a key driver of enhanced striatal dopamine responses, which may be an important contributor to aberrant salience and psychosis in psychiatric disorders like schizophrenia.

Distinct roles for dopamine clearance mechanisms in regulating behavioral flexibility.

Dopamine plays a crucial role in adaptive behavior, and dysfunctional dopamine is implicated in multiple psychiatric conditions characterized by inflexible or inconsistent choices. However, the precise relationship between dopamine and flexible decision making remains unclear. One reason is that, while many studies have focused on the activity of dopamine neurons, efficient dopamine signaling also relies on clearance mechanisms, notably the dopamine transporter (DAT), which predominates in striatum, and catechol-O-methyltransferase (COMT), which predominates in cortex. The exact locus, extent, and timescale of the effects of DAT and COMT are uncertain. Moreover, there is limited data on how acute disruption of either mechanism affects flexible decision making strategies mediated by cortico-striatal networks. To address these issues, we combined pharmacological modulation of DAT and COMT with electrochemistry and behavior in mice. DAT blockade, but not COMT inhibition, regulated sub-second dopamine release in the nucleus accumbens core, but surprisingly neither clearance mechanism affected evoked release in prelimbic cortex. This was not due to a lack of sensitivity, as both amphetamine and atomoxetine changed the kinetics of sub-second release. In a multi-step decision making task where mice had to respond to reversals in either reward probabilities or the choice sequence to reach the goal, DAT blockade selectively impaired, and COMT inhibition improved, performance after reward reversals, but neither manipulation affected the adaptation of choices after action-state transition reversals. Together, our data suggest that DAT and COMT shape specific aspects of behavioral flexibility by regulating different aspects of the kinetics of striatal and cortical dopamine, respectively.

CHANGES IN SYSTEMIC LEVELS OF VASCULAR ENDOTHELIAL GROWTH FACTOR AFTER INTRAVITREAL INJECTION OF AFLIBERCEPT OR BROLUCIZUMAB FOR NEOVASCULAR AGE-RELATED MACULAR DEGENERATION.

PURPOSE: To analyze and compare the effects of intravitreal brolucizumab versus aflibercept on systemic vascular endothelial growth factor (VEGF)-A levels in patients with neovascular age-related macular degeneration. METHODS: In this prospective interventional case series study, brolucizumab (6.0 mg/50 µL) or aflibercept (2.0 mg/50 µL) was injected intravitreally in 30 patients each. Blood samples were drawn at baseline and 7 days and 28 days after the first injection. Systemic VEGF-A levels were measured using enzyme-linked immunosorbent assay. Thirty healthy individuals served as controls. RESULTS: The median baseline systemic VEGF-A levels in the brolucizumab, aflibercept, and control groups were 10.8 (8.0-13.2), 12.0 (8.0-18.5), and 10.0 (8.0-15.1) pg/mL, respectively (P = 0.315). In the brolucizumab group, VEGF-A levels significantly decreased to 8.0 (8.0-11.5) pg/mL on Day 7 (P = 0.0254) and to 8.0 (8.0-8.0) pg/mL on Day 28 (P < 0.001). In the aflibercept group, VEGF-A levels significantly decreased to 8.0 (8.0-8.0) pg/mL on Day 7 (P < 0.001) but returned to the baseline level, 12.5 (8.5-14.6) pg/mL, on Day 28 (P = 0.120). Vascular endothelial growth factor-A levels were significantly different between the treatment groups after 28 days (P < 0.001). CONCLUSION: Intravitreal brolucizumab resulted in a sustained reduction of systemic VEGF-A levels until 28 days posttreatment, which raises concerns regarding its safety and long-term effects.

Porous assembly of an antifungal protein mediated by zinc and sulfonato-calix[8]arene.

Controlled protein assembly holds great potential in the fabrication of biohybrid materials. However, the structural diversity and complexity of proteins present an obstacle to controlled assembly. Supramolecular chemistry is a possible solution as it offers tools to mediate self-assembly with molecular precision. This paper deals with the calixarene- and zinc-mediated assembly and crystallization of the histidine-rich Penicillium chrysogenum antifungal protein B (PAFB). We report crystal structures of pure PAFB, PAFB in complex with Zn2+, and the ternary complex of PAFB, Zn2+ and sulfonato-calix[8]arene (sclx8). A comparison of the three crystal structures revealed the structural plasticity of PAFB. While the flexible and highly anionic sclx8 resulted in large molecular weight aggregates of PAFB in solution, diffraction-quality crystals of PAFB-sclx8 were not obtained. We report crystals of PAFB-Zn2+-sclx8 in which a trinuclear zinc cluster occurred adjacent to a calixarene binding site. Interestingly, the combination of sclx8 complexation and zinc coordination resulted in a porous framework with channels of circa 2 nm diameter. Detailed analysis of the crystal structure highlighted novel molecular recognition features. This research enriches the set of supramolecular interactions available to promote protein assembly.

Postintervention and follow-up changes in caregiving behavior and representations after individually or group delivered hybrid Circle of Security-intensive intervention with New Zealand caregiver-child dyads.

The Circle of Security Intensive intervention (COS-I) aims to improve child attachment security and reduce disorganisation by improving caregiver capacities, including caregiving behavior and representations. Research on COS-I effectiveness with these goals is limited and none examines if positive changes are sustained. A recently revised hybrid COS-I protocol (COS-I-RH) incorporates Circle of Security-Parenting (COS-P) material and individual or group delivery options. We examined (1) post intervention and follow-up changes in caregiving behavior and representations after COS-I-RH and (2) if individual or group delivery moderated changes. New Zealand parent-child dyads with relationship concerns (n=36; child age M =35 months) referred to a community-based program completed COS-I-RH. Four caregiver capacities (supportive and unsupportive parenting (CTNES), parenting self-efficacy and satisfaction (PSOC)) were measured pre- and post-treatment, and one year later. Regardless of delivery mode, after COS-I-RH, parents showed large improvements on all 4 indices of caregiving behavior and representations, maintained at one-year follow-up.

Parent and practitioner perspectives on Circle of Security Parenting (COS-P): A qualitative study.

Circle of Security Parenting (COS-P) is an attachment-theory-informed program for parents of infants and young children. Designed for scalability, COS-P has been widely adopted internationally. Evidence for the program's effectiveness is limited, however, restricting capacity to make informed decisions about program allocation, and threatening ongoing program funding. To help address this evidence gap, this qualitative study explored the experiences and perceptions of 20 COS-P facilitators and 14 parent recipients in Australia, where COS-P uptake has been particularly widespread. Thematic analysis of combined interview and focus group data revealed a perception that COS-P primarily changes the lens through which parents view (a) their child, (b) themselves in the parenting role, and (c) the parent-child relationship, and that this was a pathway to increased empathy, compassion, and parenting confidence. Participants identified four components that underpinned program impact: key content, skills practice, group processes, and facilitator support. Although COS-P was considered suitable for broad application, limitations were noted. Findings can guide clinical application of COS-P and inform empirical research.

El Círculo de Seguridad - Crianza (COS-P) es un programa basado en la teoría de la afectividad para progenitores de infantes y niños pequeños. Diseñado con un enfoque de escala, COS-P ha sido ampliamente adoptado internacionalmente. La evidencia de su eficacia es limitada, sin embargo, lo cual restringe la capacidad de tomar decisiones fundamentadas acerca de la distribución de fondos y amenaza el continuo apoyo económico del programa. Este estudio cualitativo explora las experiencias y percepciones de 20 mediadores que facilitaban el programa COS-P y 14 progenitores que lo recibían en Australia, donde la aceptación de COS-P ha sido particularmente extensa. Los análisis temáticos de entrevistas combinadas y datos de grupos de enfoques revelaron una percepción de que COS-P cambia el lente a través del cual los progenitores ven (a) a su niño, (b) a sí mismos en el papel de crianza, y (c) la relación progenitor-niño, y que este era una trayectoria para incrementar la empatía, la compasión, así como la confianza en la crianza. Los participantes identificaron cuatro componentes que respaldan el impacto del programa: contenido clave, práctica de habilidades, procesos de grupo y apoyo del mediador. Aunque COS-P se consideró apropiado para una aplicación más amplia, se indicaron las limitaciones. Los resultados pueden guiar la aplicación clínica de COS-P y sustentar la investigación empírica.

Le Parentage Cercle de Sécurité (en anglais Circle of Security Parenting, soit COS-P) est un programme basé sur la théorie de l'attachement pour les parents de nourrissons et de jeunes enfants. Conçu pour son extensibilité le COS-P a été largement adopté au niveau international. Les preuves d'efficacité sont cependant limitées, ce qui restreint la capacité à prendre des décisions informées sur les allocations au programme et menaçant le financement continu du programme. Cette étude qualitative a exploré les expériences et les perceptions de 20 facilitateurs COS-P et 14 récipiendaires parents en Australie, ou l'adoption du COS-P est particulièrement répandue. Une analyse thématique consistant en une combinaison d'entretien et de données de groupes de discussion a révélé une perception que le COS-P change principalement le prisme au travers duquel les parents voient (a) leur enfant, (b) eux-mêmes dans le rôle de parentage, et (c) la relation parent-enfant, et que cela crée un chemin d'empathie, de compassion et de confiance de parentage accrue. Les participants ont identifié quatre composantes qui étaient l'impact du programme: un contenu clé, la pratique des compétences, les processus de groupe et le soutien au facilitateur. Bien que le COS-P soit considéré comme étant adapté à une application générale des limites ont été notées. Les résultats peuvent guider l'application clinique du COS-P et éclairer la recherche empirique.

Development of an Improved Peroxidase-Based High-Throughput Screening for the Optimization of D-Glycerate Dehydratase Activity.

Successful directed evolution examples span a broad range of improved enzyme properties. Nevertheless, the most challenging step for each single directed evolution approach is an efficient identification of improved variants from a large genetic library. Thus, the development and choice of a proper high-throughput screening is a central key for the optimization of enzymes. The detection of low enzymatic activities is especially complicated when they lead to products that are present in the metabolism of the utilized genetic host. Coupled enzymatic assays based on colorimetric products have enabled the optimization of many of such enzymes, but are susceptible to problems when applied on cell extract samples. The purpose of this study was the development of a high-throughput screening for D-glycerate dehydratase activity in cell lysates. With the aid of an automated liquid handling system, we developed a high-throughput assay that relied on a pre-treatment step of cell extract prior to performing the enzymatic and assay reactions. We could successfully apply our method, which should also be transferable to other cell extract-based peroxidase assays, to identify an improved enzyme for the dehydration of D-glycerate.

DOES TRAINING IN THE CIRCLE OF SECURITY FRAMEWORK INCREASE RELATIONAL UNDERSTANDING IN INFANT/CHILD AND FAMILY WORKERS?

This article evaluated whether attendance at Circle of Security training workshops resulted in attendees showing greater empathy and attachment-related knowledge and understanding, and fewer judgmental responses to viewing a stressful parent-child interaction. Participants were 202 practitioners who attended and completed a 2-day (n = 70), 4-day (n = 105), or 10-day (n = 27) COS training workshop in Australia or New Zealand in 2015. In a pre/post design, participant reactions to a video clip of a challenging parent-child interaction were coded for empathic, judgmental, or attachment-focused language. Attachment understanding was coded in response to questions about the greatest challenge that the dyad faced. In all training conditions, participants provided significantly more attachment-focused descriptors and showed significantly greater attachment understanding after training, but significantly fewer empathic descriptors. While participants at the longer workshops provided significantly fewer judgmental/critical descriptors, there was no change for those attending the 2-day workshop. Irrespective of workshop duration or professional background, participants took a more relational perspective on the vignette after the training workshops. More detailed research is required to establish the extent to which this increased knowledge and understanding is retained and integrated into infant mental health practice with parents and young children.

A Penicillium chrysogenum-based expression system for the production of small, cysteine-rich antifungal proteins for structural and functional analyses.

BACKGROUND: Small, cysteine-rich and cationic antifungal proteins (APs) from filamentous ascomycetes, such as NFAP from Neosartorya fischeri and PAF from Penicillium chrysogenum, are promising candidates for novel drug development. A prerequisite for their application is a detailed knowledge about their structure-function relation and mode of action, which would allow protein modelling to enhance their toxicity and specificity. Technologies for structure analyses, such as electronic circular dichroism (ECD) or NMR spectroscopy, require highly purified samples and in case of NMR milligrams of uniformly 15N-/13C-isotope labelled protein. To meet these requirements, we developed a P. chrysogenum-based expression system that ensures sufficient amount and optimal purity of APs for structural and functional analyses. RESULTS: The APs PAF, PAF mutants and NFAP were expressed in a P. chrysogenum ∆paf mutant strain that served as perfect microbial expression factory. This strain lacks the paf-gene coding for the endogenous antifungal PAF and is resistant towards several APs from other ascomycetes. The expression of the recombinant proteins was under the regulation of the strong paf promoter, and the presence of a paf-specific pre-pro sequence warranted the secretion of processed proteins into the supernatant. The use of defined minimal medium allowed a single-step purification of the recombinant proteins. The expression system could be extended to express PAF in the related fungus Penicillium digitatum, which does not produce detectable amounts of APs, demonstrating the versatility of the approach. The molecular masses, folded structures and antifungal activity of the recombinant proteins were analysed by ESI-MS, ECD and NMR spectroscopy and growth inhibition assays. CONCLUSION: This study demonstrates the implementation of a paf promoter driven expression cassettes for the production of cysteine-rich, cationic, APs in different Penicillium species. The system is a perfect tool for the generation of correctly folded proteins with high quality for structure-function analyses.

Aversive prediction error signals in the amygdala.

Prediction error signals are fundamental to learning. Here, in mice, we show that aversive prediction signals are found in the hemodynamic responses and theta oscillations recorded from the basolateral amygdala. During fear conditioning, amygdala responses evoked by footshock progressively decreased, whereas responses evoked by the auditory cue that predicted footshock concomitantly increased. Unexpected footshock evoked larger amygdala responses than expected footshock. The magnitude of the amygdala response to the footshock predicted behavioral responses the following day. The omission of expected footshock led to a decrease below baseline in the amygdala response suggesting a negative aversive prediction error signal. Thus, in mice, amygdala activity conforms to temporal difference models of aversive learning.

Improved child behavioural and emotional functioning after Circle of Security 20-week intervention.

This study examined the efficacy of the attachment-based Circle of Security 20-week intervention in improving child behavioural and emotional functioning. Participants were 83 parents of children (1-7 years) referred to a clinical service with concerns about their young children's behaviour. Parents (and teachers, when available) completed questionnaires assessing child protective factors, behavioural concerns, internalizing and externalizing problems, prior to and immediately after the intervention. The following were considered as potential moderators: child gender and age, parent representations, reflective functioning, child attachment indices and severity of presenting problems, prior to treatment. Results showed significant improvement for parent ratings of child protective factors, behavioural concerns, internalizing and externalizing symptoms, all ps < .05, and children with more severe problems showed most improvement. Teachers also reported improvements, but change was significant only for externalizing problems (p = .030). Findings suggest Circle of Security is effective in improving child behavioural and emotional functioning in clinically referred children aged 1-7 years.

EFFICACY OF THE 20-WEEK CIRCLE OF SECURITY INTERVENTION: CHANGES IN CAREGIVER REFLECTIVE FUNCTIONING, REPRESENTATIONS, AND CHILD ATTACHMENT IN AN AUSTRALIAN CLINICAL SAMPLE.

Circle of Security is an attachment theory based intervention that aims to promote secure parent-child attachment relationships. Despite extensive uptake of the approach, there is limited empirical evidence regarding efficacy. The current study examined whether participation in the 20-week Circle of Security intervention resulted in positive caregiver-child relationship change in four domains: caregiver reflective functioning; caregiver representations of the child and the relationship with the child; child attachment security, and attachment disorganization. Archived pre- and postintervention data were analyzed from 83 clinically referred caregiver-child dyads (child age: 13-88 months) who completed the Circle of Security intervention in sequential cohorts and gave permission for their data to be included in the study. Caregivers completed the Circle of Security Interview, and dyads were filmed in the Strange Situation Procedure before and after the intervention. Results supported all four hypotheses: Caregiver reflective functioning, caregiving representations, and level of child attachment security increased after the intervention, and level of attachment disorganization decreased for those with high baseline levels. Those whose scores were least optimal prior to intervention showed the greatest change in all domains. This study adds to the evidence suggesting that the 20-week Circle of Security intervention results in significant relationship improvements for caregivers and their children.

Artificial Intelligence (AI) Reveals Ethnic Disparities in Cataract Detection and Treatment.

INTRODUCTION: The aim of this work is to identify patients at risk of limited access to healthcare through artificial intelligence using a name-ethnicity classifier (NEC) analyzing the clinical stage of cataract at diagnosis and preoperative visual acuity. METHODS: This retrospective, cross-sectional study includes patients seen in the cataract clinic of a tertiary care hospital between September 2017 and February 2020 with subsequent cataract surgery in at least one eye. We analyzed 4971 patients and 8542 eyes undergoing surgery. RESULTS: The NEC identified 360 patients with names classified as 'non-German' compared to 4611 classified as 'German'. Advanced cataract (7 vs. 5%; p = 0.025) was significantly associated with group 'non-German'. Mean best-corrected visual acuity in group 'non-German' was 0.464 ± 0.406 (LogMAR), and in group 'German' was 0.420 ± 0.334 (p = 0.009). This difference remained significant after exclusion of patients with non-lenticular ocular comorbidities. Surgical time and intraoperative complications did not differ between the groups. Retrobulbar or general anesthesia was chosen significantly more frequently over topical anesthesia in group 'non-German' compared to group 'German' (24 vs. 18% respectively; p < 0.001). CONCLUSIONS: This study shows that artificial intelligence is able to uncover health disparities between people with German compared to non-German names using NECs. Patients with non-German names, possibly facing various social barriers to healthcare access such as language barriers, have more advanced cataracts and worse visual acuity upon presentation. Artificial intelligence may prove useful for healthcare providers to discover and counteract such inequalities and establish tailored preventive measures to decrease morbidity in vulnerable population subgroups.

Three-year clinical outcome of XEN45 Gel Stent implantation versus trabeculectomy in patients with open angle glaucoma.

OBJECTIVE: To reliably compare the three-year clinical outcome and safety of XEN45 Gel Stent implantation (XEN) vs. trabeculectomy (TRAB) in patients with glaucoma. SUBJECT/METHODS: We conducted a retrospective cohort study with patients with primary open angle or pseudoexfoliation glaucoma with uncontrolled intraocular pressure (IOP) undergoing XEN or TRAB at the Innsbruck University Clinic of Ophthalmology and Optometry, Austria and analysed changes in IOP, numbers of IOP-lowering medications, and complete surgical success (i.e., IOP ≤ 18 mmHg, ≥20% IOP reduction and not requiring IOP-lowering medication) up to 36 months postoperatively. RESULTS: Between 2013 and 2019, we performed XEN Gel Stent implantation in 58 eyes and trabeculectomy in 84 eyes. From baseline to 36 months, mean IOP decreased from 23.4 to 13.8 mmHg (mean reduction 35%, 95% confidence interval 23-48%, p < 0.001) in the XEN group and from 25.1 to 11.2 mmHg (mean reduction 50%, 41-60%, p < 0.001) in the TRAB group. TRAB provided higher IOP reduction than XEN Gel Stent implantation at 12, 24, and 36 months (all p < 0.05). In XEN versus TRAB, IOP-lowering medication was required by 98.3% vs. 97.6% before surgery (p = 0.781), differed significantly at month 12 (43.2% vs. 2.0%, p < 0.001)but not at month 24 or 36. Complete surgical success was achieved in 40.0% vs. 62.8% at month 24 (adjusted odds ratio 2.70; 1.04-7.00, p = 0.040) and 27.3% vs. 56.8% at month 36 (4.36; 1.25-15.18, p = 0.021). CONCLUSION: Compared to XEN, TRAB was associated with lower intraocular pressure, less IOP-lowering medication, and higher probability of achieving complete surgical success over a 36-month follow-up period.

A Ketone Monoester with Carbohydrate Improves Cognitive Measures Postexercise, but Not Performance in Trained Females.

PURPOSE: The acute ingestion of a ketone monoester with the coingestion of a carbohydrate (KME + CHO) compared with carbohydrate (CHO) was investigated on cycling performance and cognitive performance in trained females. METHODS: Using a two condition, placebo-controlled, double-blinded and crossover design, 12 trained females (mean ± SD: age, 23 ± 3 yr; height, 1.64 ± 0.08 m; mass, 65.2 ± 12.7 kg) completed a baseline assessment of cognitive performance (psychomotor vigilance testing (PVT), task switching, and incongruent flanker), followed by 6 × 5-min intervals at 40%, 45%, 50%, 55%, 60%, and 65% of their maximal power output (W max ) and then a 10-km time trial, concluding with the same assessments of cognitive performance. Participants consumed either 375 mg·kg -1 body mass of KME with a 6% CHO solution (1 g·min -1 of exercise) or CHO alone, across three boluses (50:25:25). RESULTS: Blood ß-hydroxybutyrate concentrations averaged 1.80 ± 0.07 and 0.13 ± 0.01 mM during exercise in KME + CHO and CHO, respectively. Blood glucose decreased after drink 1 of KME + CHO (~15%; P = 0.01) but not CHO, and lactate concentrations were lower in KME + CHO at 50%, 55%, 60%, and 65% W max (all P < 0.05) compared with CHO. Despite these changes, no differences were found between conditions for time trial finishing times (KME + CHO, 29.7 ± 5.7 min; CHO, 29.6 ± 5.7 min; P = 0.92). However, only KME + CHO resulted in increases in psychomotor vigilance testing speed (~4%; P = 0.01) and faster reaction times (~14%; P < 0.01), speed (~15%; P < 0.01), and correct responses (~13%; P = 0.03) in the incongruent flanker during posttesting compared with CHO. CONCLUSIONS: The acute ingestion of a KME + CHO elevated blood ß-hydroxybutyrate and lowered glucose and lactate across multiple time points during exercise compared with CHO. Although these changes did not affect physical performance, several markers of cognitive performance were improved by the addition of a KME in trained females.

Generation and Characterization of a Human Neuronal In Vitro Model for Rett Syndrome Using a Direct Reprogramming Method.

Rett Syndrome (RTT) is a severe neurodevelopmental disorder, afflicting 1 in 10,000 female births. It is caused by mutations in the X-linked methyl-CpG-binding protein gene (MECP2), which encodes for the global transcriptional regulator methyl CpG binding protein 2 (MeCP2). As human brain samples of RTT patients are scarce and cannot be used for downstream studies, there is a pressing need for in vitro modeling of pathological neuronal changes. In this study, we use a direct reprogramming method for the generation of neuronal cells from MeCP2-deficient and wild-type human dermal fibroblasts using two episomal plasmids encoding the transcription factors SOX2 and PAX6. We demonstrated that the obtained neurons exhibit a typical neuronal morphology and express the appropriate marker proteins. RNA-sequencing confirmed neuronal identity of the obtained MeCP2-deficient and wild-type neurons. Furthermore, these MeCP2-deficient neurons reflect the pathophysiology of RTT in vitro, with diminished dendritic arborization and hyperacetylation of histone H3 and H4. Treatment with MeCP2, tethered to the cell penetrating peptide TAT, ameliorated hyperacetylation of H4K16 in MeCP2-deficient neurons, which strengthens the RTT relevance of this cell model. We generated a neuronal model based on direct reprogramming derived from patient fibroblasts, providing a powerful tool to study disease mechanisms and investigating novel treatment options for RTT.

Expression, Purification, Characterization and Cellular Uptake of MeCP2 Variants.

The transcriptional regulator Methyl-CpG-binding protein 2 (MeCP2) is an intrinsically disordered protein, mutations in which, are implicated in the onset of Rett Syndrome, a severe and debilitating neurodevelopmental disorder. Delivery of this protein fused to the cell-penetrating peptide TAT could allow for the intracellular replenishment of functional MeCP2 and hence potentially serve as a prospective Rett Syndrome therapy. This work outlines the expression, purification and characterization of various TAT-MeCP2 constructs as well as their full-length and shortened eGFP fusion variants. The latter two constructs were used for intracellular uptake studies with subsequent analysis via western blotting and live-cell imaging. All purified MeCP2 samples exhibited high degree of stability and very little aggregation propensity. Full length and minimal TAT-MeCP2-eGFP were found to efficiently transduce into human dermal and murine fibroblasts and localize to cell nuclei. These findings clearly support the utility of MeCP2-based protein replacement therapy as a potential Rett Syndrome treatment option.

The impact of compliance among patients with diabetic macular oedema treated with intravitreal aflibercept: a 48-month follow-up study.

PURPOSE: This study aimed to compare anatomical and functional outcomes between patients with non-proliferative diabetic retinopathy (NPDR) with diabetic macular oedema (DME) who adhered to intravitreal aflibercept therapy and patients lost to follow-up (LTFU). METHODS: We enrolled 200 patients and recorded the interval between each procedure and the subsequent follow-up visit. Moreover, visual acuity (VA) and anatomical outcomes were measured at each follow-up examination. RESULTS: Among the patients, 103 (51%) patients adhered to intravitreal aflibercept therapy and follow-up examination while 97 (49%) patients were LTFU. Forty-six (47%) patients LTFU who returned for further treatment showed a significant decrease in VA from 0.51 (±0.46) to 0.89 (±0.38) logarithm of the minimum angle of resolution (logMAR) after 48 months (p = 0.004). Compared with the adherent group, the return group showed a worse VA at 48 months (p = 0.036). Further, 1 (1%) patient in the adherent group and 8 (17%) patients in the return group developed a proliferative DR. Patients who were LTFU had a 13.0 times greater chance to develop a proliferative DR (p = 0.022). CONCLUSIONS: Patients who did not adhere to intravitreal aflibercept therapy for DME showed significantly worse visual outcomes compared to patients with good therapy adherence. Moreover, patients with LTFU had a 13 times higher risk of developing a proliferative DR. Considering the potential disease progress, better strategies should be applied to optimize the functional outcome of patients at risk of reduced adherence.

TAT-MeCP2 protein variants rescue disease phenotypes in human and mouse models of Rett syndrome.

Rett syndrome (RTT) is a neurodevelopmental disorder caused by pathogenic variants leading to functional impairment of the MeCP2 protein. Here, we used purified recombinant MeCP2e1 and MeCP2e2 protein variants fused to a TAT protein transduction domain (PTD) to evaluate their transduction ability into RTT patient-derived fibroblasts and the ability to carry out their cellular function. We then assessed their transduction ability and therapeutic effects in a RTT mouse model. In vitro, TAT-MeCP2e2-eGFP reversed the pathological hyperacetylation of histones H3K9 and H4K16, a hallmark of abolition of MeCP2 function. In vivo, intraperitoneal administration of TAT-MeCP2e1 and TAT-MeCP2e2 extended the lifespan of Mecp2-/y mice by >50%. This was accompanied by rescue of hippocampal CA2 neuron size in animals treated with TAT-MeCP2e1. Taken together, these findings provide a strong indication that recombinant TAT-MeCP2 can reach mouse brains following peripheral injection and can ameliorate the phenotype of RTT mouse models. Thus, our study serves as a first step in the development of a potentially novel RTT therapy.