Parkinson's Disease Genetics and Pathophysiology.

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by degeneration of the substantia nigra pars compacta and by accumulation of α-synuclein in Lewy bodies. PD is caused by a combination of environmental factors and genetic variants. These variants range from highly penetrant Mendelian alleles to alleles that only modestly increase disease risk. Here, we review what is known about the genetics of PD. We also describe how PD genetics have solidified the role of endosomal, lysosomal, and mitochondrial dysfunction in PD pathophysiology. Finally, we highlight how all three pathways are affected by α-synuclein and how this knowledge may be harnessed for the development of disease-modifying therapeutics.

Liquid Biopsy Proteomics in Ophthalmology.

Minimally invasive liquid biopsies from the eye capture locally enriched fluids that contain thousands of proteins from highly specialized ocular cell types, presenting a promising alternative to solid tissue biopsies. The advantages of liquid biopsies include sampling the eye without causing irreversible functional damage, potentially better reflecting tissue heterogeneity, collecting samples in an outpatient setting, monitoring therapeutic response with sequential sampling, and even allowing examination of disease mechanisms at the cell level in living humans, an approach that we refer to as TEMPO (Tracing Expression of Multiple Protein Origins). Liquid biopsy proteomics has the potential to transform molecular diagnostics and prognostics and to assess disease mechanisms and personalized therapeutic strategies in individual patients. This review addresses opportunities, challenges, and future directions of high-resolution liquid biopsy proteomics in ophthalmology, with particular emphasis on the large-scale collection of high-quality samples, cutting edge proteomics technology, and artificial intelligence-supported data analysis.

Lift Every Voice: Engaging Black Adolescents in Social Justice Service-Learning to Promote Mental Health and Educational Equity.

This study reports on the feasibility and acceptability of a social justice infused service-learning (S-L) program to promote Black adolescent mental health and educational equity. We convened a community advisory board to help adapt and pilot test, via open trial mixed method design, an evidence-based service-learning program for Black middle school adolescents (n = 21) attending summer camp at a faith-based setting. We describe a S-L curriculum, with a focus on the achievement gap, and training for church staff and assess staff and youth reports of feasibility, acceptability, and promise to (a) improve/engage psychological engagement targets, and (b) improve academic motivation, and social-emotional and behavioral outcomes. Mixed method findings revealed high feasibility and acceptability of the S-L intervention as indicated by consistent attendance and enthusiastic engagement by staff and youth, high satisfaction, high completion rates of planned sessions, and emergent qualitative themes from staff interviews and adolescent focus groups highlighting that service-learning (1) facilitated skills (e.g., goal-setting, social-emotional and behavioral regulation, and problem-solving), (2) shaped perspectives and inspired openness, and (3) created a space for all to feel valued and included to address the inequities of education that directly impacted them. There was preliminary evidence for efficacy in that youth report of emotional symptoms, peer problems, and staff report of general internalizing symptoms decreased following the intervention, while youth report of prosocial behaviors increased. Implications suggest that S-L programming demonstrates promise to promote mental health outcomes, raise social awareness, and inspire critical consciousness and lift the voices of Black youth by providing tools for working toward systemic changes to reduce inequities in both education and mental health.

The heart of the neural crest: cardiac neural crest cells in development and regeneration.

Cardiac neural crest cells (cNCCs) are a migratory cell population that stem from the cranial portion of the neural tube. They undergo epithelial-to-mesenchymal transition and migrate through the developing embryo to give rise to portions of the outflow tract, the valves and the arteries of the heart. Recent lineage-tracing experiments in chick and zebrafish embryos have shown that cNCCs can also give rise to mature cardiomyocytes. These cNCC-derived cardiomyocytes appear to be required for the successful repair and regeneration of injured zebrafish hearts. In addition, recent work examining the response to cardiac injury in the mammalian heart has suggested that cNCC-derived cardiomyocytes are involved in the repair/regeneration mechanism. However, the molecular signature of the adult cardiomyocytes involved in this repair is unclear. In this Review, we examine the origin, migration and fates of cNCCs. We also review the contribution of cNCCs to mature cardiomyocytes in fish, chick and mice, as well as their role in the regeneration of the adult heart.

Functionalization of Titanium Dioxide by In Situ Surface Crystallization of Bisphosphonate-Based Coordination Complexes.

Functionalization of highly pure rutile phase titanium dioxide (TiO2) particles with a selected bisphosphonate-based coordination complex (BPCC), ZOLE-Ca form II, was achieved through in situ surface crystallization. The hydrothermal reaction of the selected BPCC was carried out in the presence of photoactivated rutile phase TiO2 by ultraviolet irradiation. The reaction time was varied to control the crystal growth of the BPCC around the TiO2 core, resulting in a functionalized material with different shell thicknesses: TiO2-core:nano-Ca@ZOLE-shell-† (5 min) and TiO2-core:nano-Ca@ZOLE-shell-‡ (10 min). The crystal phase assessment of the BPCC and the polymorphic phase purity of the metal oxide were determined after immobilization through Raman spectroscopy and powder X-ray diffraction. The results initially suggested that the crystallization of a shell comprising the selected BPCC surrounding a highly pure rutile phase TiO2 core was achieved through controlled in situ surface crystallization. Morphological changes, elemental composition and exact atomic distribution in the functionalized materials were addressed employing scanning electron microscopy coupled with energy-dispersive spectroscopy. These analyses unambiguously confirmed that after 5 min, successful incorporation of a thin BPCC shell on the surface of the metal oxide particles was achieved. Particle size distribution measurements revealed an average particle size of 495 d.nm for the functionalized material after the immobilization process. Quantitative determination of the BPCC shell content in TiO2-core:nano-Ca@ZOLE-shell-† was determined through thermogravimetric analysis, estimating a ratio of ∼1:3 (TiO2:BPCC). The cytotoxicity of TiO2-core:nano-Ca@ZOLE-shell-† against MDA-MB-231 (cancer cell model) and hFOB 1.19 (normal osteoblast-like cell model) cell lines was investigated. The results demonstrated significant cell growth inhibition for TiO2-core:nano-Ca@ZOLE-shell-† against MDA-MB-231, specifically at a concentration of 7.5 µM (% RCL = 46 ± 2%, 72 h). Under the same conditions, the functionalized material did not present cytotoxicity against hFOB 1.19 (% RCL ∼ 100%). These important outcomes provide evidence of the surface crystallization of BPCCs onto rutile phase TiO2 for the development of a novel functionalized material with the potential to treat and prevent osteolytic metastases.

Design of Extended Bisphosphonate-Based Coordination Polymers as Bone-Targeted Drug Delivery Systems for Breast Cancer-Induced Osteolytic Metastasis and Other Bone Therapies.

The coordination between benzene 1,4-bis(bisphosphonic acid) (BBPA), the bisphosphonate (BP) analogue of benzene 1,4-dicarboxylic acid (BDC), and bioactive metals led to the formation of extended bisphosphonate-based coordination polymers (BPCPs). Four distinct crystalline phases were obtained, namely, BBPA-Ca forms I and II, BBPA-Zn, and BBPA-Mg. Among these, BBPA-Ca forms I (7 × 9 Å2) and II (8 × 12 Å2) possess channels large enough to encapsulate 5-fluorouracil (5-FU), a drug prescribed in combination with BPs to treat breast cancer-induced osteolytic metastases (OM). Dissolution curves show a 14% release of BBPA from BBPA-Ca form II in phosphate-buffered saline, while ∼90% was released in fasted-state simulated gastric fluid. These results suggest that this material is relatively stable in neutral environments yet collapses in acidic conditions. Moreover, the phase inversion temperature method decreased the particle size of BBPA-Ca form II, resulting in nano-Ca@BBPA (∼134 d.nm). Binding assays showed a higher affinity of nano-Ca@BBPA (∼97%) to hydroxyapatite than BBPA (∼70%) and significantly higher binding than commercial BPs, zolendronic (3.0×), and risedronic (2.4×) acids after 24 h. Furthermore, both BBPA-Ca form II and nano-Ca@BBPA presented comparable drug loading and release (∼30 wt % 5-FU) relative to BDC-based CCs (UiO-66, MIL-53, and BDC-Zr) where other pharmaceutical compounds (caffeine, ibuprofen, aspirin, and α-cyano-4-hydroxycinnamic acid) have been encapsulated. Cell viability assays established that drug-loaded nano-Ca@BBPA increases the cytotoxicity of a triple-negative human breast cancer cell line (MDA-MB-231) when compared to 5-FU (%RCV = 8 ± 5 vs 75 ± 1% at a 100 µM). At the same concentration, no significant decrease in cell viability was observed for normal human osteoblast-like hFOB 1.19 cells (%RCV = 85 ± 1%). Collectively, these results demonstrate the feasibility of nano-Ca@BBPA as a potential drug delivery system (DDS), with high affinity to bone tissue, to treat bone-related diseases such as OM.

Adolescents' Retributive and Restorative Orientations in Response to Intergroup Harms in Schools.

This mixed-methods study examined how adolescents understand and evaluate different ways to address intergroup harms in schools. In individual interviews, 77 adolescents (M age = 16.49 years; 39 girls, 38 boys) in Bogotá, Colombia, responded to hypothetical vignettes wherein a rival group at school engaged in a transgression against their group. Adolescents reported that students who were harmed should and would talk to school authorities, but also noted they would likely retaliate. In terms of teacher-sanctioned responses to harm, youth endorsed compensation most strongly, followed by apologies, and rated suspension least positively. Youths' explanations for their endorsement of different disciplinary practices reflected varied concerns, including their perceptions of how justice is best achieved and how restoration could be attained.

Impact of Age and School Instruction Mode on Children's Occupations Early in the COVID-19 Pandemic: A Longitudinal Survey Study.

IMPORTANCE: The coronavirus disease 2019 (COVID-19) pandemic disrupted participation in routines and daily activities. It is unclear how children reengaged in activities during the pandemic, particularly as a new school year began. Differences in school instruction mode (in person, hybrid, or remote) during the pandemic could further affect activity participation. OBJECTIVE: To examine changes in sedentary and nonsedentary activity participation in children and to determine whether school instruction mode was associated with participation. SETTING: Home-based survey of U.S. residents. PARTICIPANTS: Parents of 208 children (46.2% female) between ages 5 and 18 yr were surveyed at two waves: March through April 2020 and October 2020. OUTCOMES AND MEASURES: Frequency of sedentary (indoor play, electronic device usage) and nonsedentary (outdoor play, leisure and extracurriculars) activity participation was observed. Descriptive and inferential statistics of the changes from Wave 1 to Wave 2 and linear regression were used to determine statistically significant variables associated with activity participation. RESULTS: Nonsedentary activity participation increased and sedentary activity decreased from Wave 1 to Wave 2. Instruction mode was statistically associated with nonsedentary, but not sedentary, participation. Negative mood, local COVID-19 severity, and household income were also associated with nonsedentary and sedentary participation. CONCLUSIONS AND RELEVANCE: Children's participation in sedentary and nonsedentary activities normalized during the new school year; however, many factors likely contributed to these changes. What This Article Adds: Despite differences in school instruction mode and the influence of mental health early in the pandemic, children's nonsedentary activity participation increased. Occupational therapy practitioners can reinforce the importance of reengaging in activities and regular routines to promote health and well-being during challenging situations.

Gain-of-function mutations in a member of the Src family kinases cause autoinflammatory bone disease in mice and humans.

Autoinflammatory syndromes are characterized by dysregulation of the innate immune response with subsequent episodes of acute spontaneous inflammation. Chronic recurrent multifocal osteomyelitis (CRMO) is an autoinflammatory bone disorder that presents with bone pain and localized swelling. Ali18 mice, isolated from a mutagenesis screen, exhibit a spontaneous inflammatory paw phenotype that includes sterile osteomyelitis and systemic reduced bone mineral density. To elucidate the molecular basis of the disease, positional cloning of the causative gene for Ali18 was attempted. Using a candidate gene approach, a missense mutation in the C-terminal region of Fgr, a member of Src family tyrosine kinases (SFKs), was identified. For functional confirmation, additional mutations at the N terminus of Fgr were introduced in Ali18 mice by CRISPR/Cas9-mediated genome editing. N-terminal deleterious mutations of Fgr abolished the inflammatory phenotype in Ali18 mice, but in-frame and missense mutations in the same region continue to exhibit the phenotype. The fact that Fgr null mutant mice are morphologically normal suggests that the inflammation in this model depends on Fgr products. Furthermore, the levels of C-terminal negative regulatory phosphorylation of Fgr Ali18 are distinctly reduced compared with that of wild-type Fgr. In addition, whole-exome sequencing of 99 CRMO patients including 88 trios (proband and parents) identified 13 patients with heterozygous coding sequence variants in FGR, including two missense mutant proteins that affect kinase activity. Our results strongly indicate that gain-of-function mutations in Fgr are involved in sterile osteomyelitis, and thus targeting SFKs using specific inhibitors may allow for efficient treatment of the disease.

Doublecortin-like Kinase 1 Regulates α-Synuclein Levels and Toxicity.

α-Synuclein (α-Syn) accumulation is a pathological hallmark of Parkinson's disease. Duplications and triplications of SNCA, the gene coding for α-Syn, cause genetic forms of the disease, which suggests that increased α-Syn dosage can drive PD. To identify the proteins that regulate α-Syn, we previously performed a screen of potentially druggable genes that led to the identification of 60 modifiers. Among them, Doublecortin-like kinase 1 (DCLK1), a microtubule binding serine threonine kinase, emerged as a promising target due to its potent effect on α-Syn and potential druggability as a neuron-expressed kinase. In this study, we explore the relationship between DCLK1 and α-Syn in human cellular and mouse models of PD. First, we show that DCLK1 regulates α-Syn levels post-transcriptionally. Second, we demonstrate that knockdown of Dclk1 reduces phosphorylated species of α-Syn and α-Syn-induced neurotoxicity in the SNc in two distinct mouse models of synucleinopathy. Last, silencing DCLK1 in human neurons derived from individuals with SNCA triplications reduces phosphorylated and total α-Syn, thereby highlighting DCLK1 as a potential therapeutic target to reduce pathological α-Syn in disease.SIGNIFICANCE STATEMENT DCLK1 regulates α-Syn protein levels, and Dclk1 knockdown rescues α-Syn toxicity in mice. This study provides evidence for a novel function for DCLK1 in the mature brain, and for its potential as a new therapeutic target for synucleinopathies.

Mechanisms Underlying Cardiomyocyte Development: Can We Exploit Them to Regenerate the Heart?

PURPOSE OF REVIEW: It is well established that the adult mammalian cardiomyocytes retain a low capacity for cell cycle activity; however, it is insufficient to effectively respond to myocardial injury and facilitate cardiac regenerative repair. Lessons learned from species in which cardiomyocytes do allow for proliferative regeneration/repair have shed light into the mechanisms underlying cardiac regeneration post-injury. Importantly, many of these mechanisms are conserved across species, including mammals, and efforts to tap into these mechanisms effectively within the adult heart are currently of great interest. RECENT FINDINGS: Targeting the endogenous gene regulatory networks (GRNs) shown to play roles in the cardiac regeneration of conducive species is seen as a strong approach, as delivery of a single or combination of genes has promise to effectively enhance cell cycle activity and CM proliferation in adult hearts post-myocardial infarction (MI). In situ re-induction of proliferative gene regulatory programs within existing, local, non-damaged cardiomyocytes helps overcome significant technical hurdles, such as successful engraftment of implanted cells or achieving complete cardiomyocyte differentiation from cell-based approaches. Although many obstacles currently exist and need to be overcome to successfully translate these approaches to clinical settings, the current efforts presented here show great promise.

SenSARS: A Low-Cost Portable Electrochemical System for Ultra-Sensitive, Near Real-Time, Diagnostics of SARS-CoV-2 Infections.

A critical path to solving the SARS-CoV-2 pandemic, without further socioeconomic impact, is to stop its spread. For this to happen, pre- or asymptomatic individuals infected with the virus need to be detected and isolated opportunely. Unfortunately, there are no current ubiquitous (i.e., ultra-sensitive, cheap, and widely available) rapid testing tools capable of early detection of SARS-CoV-2 infections. In this article, we introduce an accurate, portable, and low-cost medical device and bio-nanosensing electrode dubbed SenSARS and its experimental validation. SenSARS' device measures the electrochemical impedance spectra of a disposable bio-modified screen-printed carbon-based working electrode (SPCE) to the changes in the concentration of SARS-CoV-2 antigen molecules ("S" spike proteins) contained within a sub-microliter fluid sample deposited on its surface. SenSARS offers real-time diagnostics and viral load tracking capabilities. Positive and negative control tests were performed in phosphate-buffered saline (PBS) at different concentrations (between 1 and 50 fg/mL) of SARS-CoV-2(S), Epstein-Barr virus (EBV) glycoprotein gp350, and Influenza H1N1 M1 recombinant viral proteins. We demonstrate that SenSARS is easy to use, with a portable and lightweight (< 200 g) instrument and disposable test electrodes (

Novel REEP6 gene mutation associated with autosomal recessive retinitis pigmentosa.

PURPOSE: This study reports the ophthalmic and genetic findings of a Cameroonian patient with autosomal recessive retinitis pigmentosa (arRP) caused by a novel Receptor Expression Enhancing Protein 6 (REEP6) homozygous mutation. PATIENT AND METHODS: A 33-year-old man underwent comprehensive ophthalmic examinations, including visual acuity measurements, dilated fundus imaging, electroretinography (ERG), and spectral-domain optical coherence tomography (SD-OCT). Short-wavelength fundus autofluorescence (SW-AF) and near-infrared fundus autofluorescence (NIR-AF) were also evaluated. Whole exome sequencing (WES) was used to identify potential pathogenic variants. RESULTS: Fundus examination revealed typical RP findings with additional temporal ten micron yellow dots. SD-OCT imaging revealed cystoid macular edema and perifoveal outer retinal atrophy with centrally preserved inner segment ellipsoid zone (EZ) bands. Hyperreflective spots were seen in the inner retinal layers. On SW-AF images, a hypoautofluorescent area in the perifoveal area was observed. NIR-AF imaging revealed an irregularly shaped hyperautofluorescent ring. His visual acuity was mildly affected. ERG showed undetectable rod responses and intact cone responses. Genetic testing via WES revealed a novel homozygous mutation (c.295G>A, p.Glu99Lys) in the gene encoding REEP6, which is predicted to alter the charge in the transmembrane helix. CONCLUSIONS: This report is not only the first description of a Cameroonian patient with arRP associated with a REEP6 mutation, but also this particular genetic alteration. Substitution of p.Glu99Lys in REEP6 likely disrupts the interactions between REEP6 and the ER membrane. NIR-AF imaging may be particularly useful for assessing functional photoreceptor cells and show an "avocado" pattern of hyperautofluorescence in patients with the REEP6 mutation.

Mechanisms of neurodegeneration in a preclinical autosomal dominant retinitis pigmentosa knock-in model with a RhoD190N mutation.

D190N, a missense mutation in rhodopsin, causes photoreceptor degeneration in patients with autosomal dominant retinitis pigmentosa (adRP). Two competing hypotheses have been developed to explain why D190N rod photoreceptors degenerate: (a) defective rhodopsin trafficking prevents proteins from correctly exiting the endoplasmic reticulum, leading to their accumulation, with deleterious effects or (b) elevated mutant rhodopsin expression and unabated signaling causes excitotoxicity. A knock-in D190N mouse model was engineered to delineate the mechanism of pathogenesis. Wild type (wt) and mutant rhodopsin appeared correctly localized in rod outer segments of D190N heterozygotes. Moreover, the rhodopsin glycosylation state in the mutants appeared similar to that in wt mice. Thus, it seems plausible that the injurious effect of the heterozygous mutation is not related to mistrafficking of the protein, but rather from constitutive rhodopsin activity and a greater propensity for chromophore isomerization even in the absence of light.

A Druggable Genome Screen Identifies Modifiers of α-Synuclein Levels via a Tiered Cross-Species Validation Approach.

Accumulation of α-Synuclein (α-Syn) causes Parkinson's disease (PD) as well as other synucleopathies. α-Syn is the major component of Lewy bodies and Lewy neurites, the proteinaceous aggregates that are a hallmark of sporadic PD. In familial forms of PD, mutations or copy number variations in SNCA (the α-Syn gene) result in a net increase of its protein levels. Furthermore, common risk variants tied to PD are associated with small increases of wild-type α-Syn levels. These findings are further bolstered by animal studies which show that overexpression of α-Syn is sufficient to cause PD-like features. Thus, increased α-Syn levels are intrinsically tied to PD pathogenesis and underscore the importance of identifying the factors that regulate its levels. In this study, we establish a pooled RNAi screening approach and validation pipeline to probe the druggable genome for modifiers of α-Syn levels and identify 60 promising targets. Using a cross-species, tiered validation approach, we validate six strong candidates that modulate α-Syn levels and toxicity in cell lines, Drosophila, human neurons, and mouse brain of both sexes. More broadly, this genetic strategy and validation pipeline can be applied for the identification of therapeutic targets for disorders driven by dosage-sensitive proteins.SIGNIFICANCE STATEMENT We present a research strategy for the systematic identification and validation of genes modulating the levels of α-Synuclein, a protein involved in Parkinson's disease. A cell-based screen of the druggable genome (>7,500 genes that are potential therapeutic targets) yielded many modulators of α-Synuclein that were subsequently confirmed and validated in Drosophila, human neurons, and mouse brain. This approach has broad applicability to the multitude of neurological diseases that are caused by mutations in genes whose dosage is critical for brain function.

CAPN5 genetic inactivation phenotype supports therapeutic inhibition trials.

Small molecule pharmacological inhibition of dominant human genetic disease is a feasible treatment that does not rely on the development of individual, patient-specific gene therapy vectors. However, the consequences of protein inhibition as a clinical therapeutic are not well-studied. In advance of human therapeutic trials for CAPN5 vitreoretinopathy, genetic inactivation can be used to infer the effect of protein inhibition in vivo. We created a photoreceptor-specific knockout (KO) mouse for Capn5 and compared the retinal phenotype to both wild-type and an existing Capn5 KO mouse model. In humans, CAPN5 loss-of-function (LOF) gene variants were ascertained in large exome databases from 60,706 unrelated subjects without severe disease phenotypes. Ocular examination of the retina of Capn5 KO mice by histology and electroretinography showed no significant abnormalities. In humans, there were 22 LOF CAPN5 variants located throughout the gene and in all major protein domains. Structural modeling of coding variants showed these LOF variants were nearby known disease-causing variants within the proteolytic core and in regions of high homology between human CAPN5 and 150 homologs, yet the LOF of CAPN5 was tolerated as opposed to gain-of-function disease-causing variants. These results indicate that localized inhibition of CAPN5 is a viable strategy for hyperactivating disease alleles.

In trans variant calling reveals enrichment for compound heterozygous variants in genes involved in neuronal development and growth.

Compound heterozygotes occur when different variants at the same locus on both maternal and paternal chromosomes produce a recessive trait. Here we present the tool VarCount for the quantification of variants at the individual level. We used VarCount to characterize compound heterozygous coding variants in patients with epileptic encephalopathy and in the 1000 Genomes Project participants. The Epi4k data contains variants identified by whole exome sequencing in patients with either Lennox-Gastaut Syndrome (LGS) or infantile spasms (IS), as well as their parents. We queried the Epi4k dataset (264 trios) and the phased 1000 Genomes Project data (2504 participants) for recessive variants. To assess enrichment, transcript counts were compared between the Epi4k and 1000 Genomes Project participants using minor allele frequency (MAF) cutoffs of 0.5 and 1.0%, and including all ancestries or only probands of European ancestry. In the Epi4k participants, we found enrichment for rare, compound heterozygous variants in six genes, including three involved in neuronal growth and development - PRTG (p = 0.00086, 1% MAF, combined ancestries), TNC (p = 0.022, 1% MAF, combined ancestries) and MACF1 (p = 0.0245, 0.5% MAF, EU ancestry). Due to the total number of transcripts considered in these analyses, the enrichment detected was not significant after correction for multiple testing and higher powered or prospective studies are necessary to validate the candidacy of these genes. However, PRTG, TNC and MACF1 are potential novel recessive epilepsy genes and our results highlight that compound heterozygous variants should be considered in sporadic epilepsy.

SCAPER-associated nonsyndromic autosomal recessive retinitis pigmentosa.

Mutations in the gene SCAPER (S-phase CyclinA Associated Protein residing in the Endoplasmic Reticulum) have recently been identified as causing syndromic autosomal recessive retinitis pigmentosa with the extraocular manifestations of intellectual disability and attention-deficit/hyperactivity disorder. We present the case of an 11-year-old boy that presented to our clinic with the complaint of decreased night vision. Clinical presentation, family history, and diagnostic imaging were congruent with the diagnosis of autosomal recessive retinitis pigmentosa. Genetic testing of the patient and both parents via whole-exome sequencing revealed the homozygous mutation c.2023-2A>G in SCAPER. Unique to our patient's presentation is the absence of intellectual disability and attention-deficit/hyperactivity disorder, suggesting that SCAPER-associated retinitis pigmentosa can also present without systemic manifestations.

BESTROPHIN1 mutations cause defective chloride conductance in patient stem cell-derived RPE.

Bestrophin1 (BEST1) is expressed in human retinal pigment epithelium (RPE) and mutations in the BEST1 gene commonly cause retinal dysfunction and macular degeneration. BEST1 is presumed to assemble into a calcium-activated chloride channel and be involved in chloride transport but there is no direct evidence in live human RPE cells to support this idea. To test whether BEST1 functions as a chloride channel in living tissue, BEST1-mutant RPE (R218H, L234P, A243T) were generated from patient-derived induced pluripotent stem cells and compared with wild-type RPE in a retinal environment, using a biosensor that visualizes calcium-induced chloride ion flux in the cell. Calcium stimulation elicited chloride ion export in normal RPE but not in RPE derived from three patients with BEST1 mutations. These data, along with three-dimensional modeling, provide evidence that BEST1 assembles into a key calcium-sensing chloride channel in human RPE.

Diagnostic accuracy of loop-mediated isothermal amplification (LAMP) for screening malaria in peripheral and placental blood samples from pregnant women in Colombia.

BACKGROUND: Pregnant women frequently show low-density Plasmodium infections that require more sensitive methods for accurate diagnosis and early treatment of malaria. This is particularly relevant in low-malaria transmission areas, where intermittent preventive treatment is not recommended. Molecular methods, such as polymerase chain reaction (PCR) are highly sensitive, but require sophisticated equipment and advanced training. Instead, loop mediated isothermal amplification (LAMP) provides an opportunity for molecular detection of malaria infections in remote endemic areas, outside a reference laboratory. The aim of the study is to evaluate the performance of LAMP for the screening of malaria in pregnant women in Colombia. METHODS: This is a nested prospective study that uses data and samples from a larger cross-sectional project conducted from May 2016 to January 2017 in three Colombian endemic areas (El Bagre, Quibdó, and Tumaco). A total of 531 peripheral and placental samples from pregnant women self-presenting at local hospitals for antenatal care visits, at delivery or seeking medical care for suspected malaria were collected. Samples were analysed for Plasmodium parasites by light microscopy (LM), rapid diagnostic test (RDT) and LAMP. Diagnostic accuracy endpoints (sensitivity, specificity, predictive values, and kappa scores) of LM, RDT and LAMP were compared with nested PCR (nPCR) as the reference standard. RESULTS: In peripheral samples, LAMP showed an improved sensitivity (100.0%) when compared with LM 79.5% and RDT 76.9% (p < 0.01), particularly in afebrile women, for which LAMP sensitivity was two-times higher than LM and RDT. Overall agreement among LAMP and nPCR was high (kappa value = 1.0). Specificity was similar in all tests (100%). In placental blood, LAMP evidenced a four-fold improvement in sensitivity (88.9%) when compared with LM and RDT (22.2%), being the only method, together with nPCR, able to detect placental infections in peripheral blood. CONCLUSIONS: LAMP is a simple, rapid and accurate molecular tool for detecting gestational and placental malaria, being able to overcome the limited sensitivity of LM and RDT. These findings could guide maternal health programs in low-transmission settings to integrate LAMP in their surveillance systems for the active detection of low-density infections and asymptomatic malaria cases.