Tricuspid transcatheter edge-to-edge repair in a 72-year-old patient with a left ventricular assist device and prior mitral edge-to-edge repair: a case report.

Background: We report a case of a 72-year-old patient developing a significant tricuspid regurgitation (TR) 6 years after a left ventricular assist device (LVAD) implantation. The aim of this case is to demonstrate the feasibility of transcatheter edge-to-edge repair (TEER) of the tricuspid valve and the excellent clinical benefit in long-term follow-up in an LVAD patient. Case summary: Our patient presented with recurrent acute heart failure syndrome. After a fulminant myocarditis in 2005, his previous treatment consisted of cardiac resynchronization therapy, TEER of the mitral valve, and LVAD (HeartMate III) implantation. At that point in time, his TR was only mild to moderate. Due to recurrent hospitalization despite optimized heart failure medication, we decided to treat the patient with a tricuspid TEER. His immediate post-interventional result and 1-year follow-up showed an excellent outcome with only minimal TR on transoesophageal echocardiogram. Discussion: In general, TR improves after LVAD implantation. However, there are two possible pathophysiological mechanisms, which result in an increasing TR: firstly, supporting LV dysfunction may lead to a leftward shift of the interventricular septum with restriction of the tricuspid leaflets. Secondly, the increase of venous preload with LVAD support may result in an annular dilatation with secondary TR, particularly in patients with pre-existing right ventricular dysfunction. According to the data currently available, the unpredictable course of developing TR necessitates regular clinical examination and echocardiographic investigation. Treatment with TEER appears to be feasible and safe, with excellent 1-year results in patients with previously implanted LVADs.

Digenic Leigh syndrome on the background of the m.11778G>A Leber hereditary optic neuropathy variant.

Leigh syndrome spectrum (LSS) is a primary mitochondrial disorder defined neuropathologically by a subacute necrotizing encephalomyelopathy and characterised by bilateral basal ganglia and/or brainstem lesions. LSS is associated with variants in several mitochondrial DNA (mtDNA) genes and more than 100 nuclear genes, most often related to mitochondrial complex I (CI) dysfunction. Rarely, LSS has been reported in association with primary Leber hereditary optic neuropathy (LHON) variants of the mtDNA, coding for CI subunits (m.3460G>A in MT-ND1, m.11778G>A in MT-ND4, and m.14484T>C in MT-ND6). The underlying mechanism by which these variants manifest as LSS, a severe neurodegenerative disease, as opposed to the LHON phenotype of isolated optic neuropathy, remains an open question. Here, we analyse the exome sequencing of six probands with LSS carrying primary LHON variants, and report digenic co-occurrence of the m.11778G>A variant with damaging heterozygous variants in nuclear disease genes encoding CI subunits as a plausible explanation. Our findings suggest a digenic mechanism of disease for m.11778G>A-associated LSS, consistent with recent reports of digenic disease in individuals manifesting with LSS due to biallelic variants in the recessive LHON-associated disease gene DNAJC30 in combination with heterozygous variants in CI subunits.

Developmental epileptic encephalopathy in DLG4-related synaptopathy.

OBJECTIVE: The postsynaptic density protein of excitatory neurons PSD-95 is encoded by discs large MAGUK scaffold protein 4 (DLG4), de novo pathogenic variants of which lead to DLG4-related synaptopathy. The major clinical features are developmental delay, intellectual disability (ID), hypotonia, sleep disturbances, movement disorders, and epilepsy. Even though epilepsy is present in 50% of the individuals, it has not been investigated in detail. We describe here the phenotypic spectrum of epilepsy and associated comorbidities in patients with DLG4-related synaptopathy. METHODS: We included 35 individuals with a DLG4 variant and epilepsy as part of a multicenter study. The DLG4 variants were detected by the referring laboratories. The degree of ID, hypotonia, developmental delay, and motor disturbances were evaluated by the referring clinician. Data on awake and sleep electroencephalography (EEG) and/or video-polygraphy and brain magnetic resonance imaging were collected. Antiseizure medication response was retrospectively assessed by the referring clinician. RESULTS: A large variety of seizure types was reported, although focal seizures were the most common. Encephalopathy related to status epilepticus during slow-wave sleep (ESES)/developmental epileptic encephalopathy with spike-wave activation during sleep (DEE-SWAS) was diagnosed in >25% of the individuals. All but one individual presented with neurodevelopmental delay. Regression in verbal and/or motor domains was observed in all individuals who suffered from ESES/DEE-SWAS, as well as some who did not. We could not identify a clear genotype-phenotype relationship even between individuals with the same DLG4 variants. SIGNIFICANCE: Our study shows that a subgroup of individuals with DLG4-related synaptopathy have DEE, and approximately one fourth of them have ESES/DEE-SWAS. Our study confirms DEE as part of the DLG4-related phenotypic spectrum. Occurrence of ESES/DEE-SWAS in DLG4-related synaptopathy requires proper investigation with sleep EEG.

Rho GTPase activity crosstalk mediated by Arhgef11 and Arhgef12 coordinates cell protrusion-retraction cycles.

Rho GTPases play a key role in the spatio-temporal coordination of cytoskeletal dynamics during cell migration. Here, we directly investigate crosstalk between the major Rho GTPases Rho, Rac and Cdc42 by combining rapid activity perturbation with activity measurements in mammalian cells. These studies reveal that Rac stimulates Rho activity. Direct measurement of spatio-temporal activity patterns show that Rac activity is tightly and precisely coupled to local cell protrusions, followed by Rho activation during retraction. Furthermore, we find that the Rho-activating Lbc-type GEFs Arhgef11 and Arhgef12 are enriched at transient cell protrusions and retractions and recruited to the plasma membrane by active Rac. In addition, their depletion reduces activity crosstalk, cell protrusion-retraction dynamics and migration distance and increases migration directionality. Thus, our study shows that Arhgef11 and Arhgef12 facilitate exploratory cell migration by coordinating cell protrusion and retraction by coupling the activity of the associated regulators Rac and Rho.

Deoxyestrone-based lipofection agents with solution- and solid-state emission properties.

In this contribution, three deoxyestrone-based emissive lipofection agents are reported. Because of a centrally incorporated terephthalonitrile motif, these ligands can be classified as solution and solid-state emitters (SSSEs). With the attachment of tobramycin, these amphiphilic structures are able to form lipoplexes, mediating gene transfection of HeLa and HEK 293T cells.

KCNT2-Related Disorders: Phenotypes, Functional, and Pharmacological Properties.

OBJECTIVE: Pathogenic variants in KCNT2 are rare causes of developmental epileptic encephalopathy (DEE). We herein describe the phenotypic and genetic features of patients with KCNT2-related DEE, and the in vitro functional and pharmacological properties of KCNT2 channels carrying 14 novel or previously untested variants. METHODS: Twenty-five patients harboring KCNT2 variants were investigated: 12 were identified through an international collaborative network, 13 were retrieved from the literature. Clinical data were collected and included in a standardized phenotyping sheet. Novel variants were detected using exome sequencing and classified using ACMG criteria. Functional and pharmacological studies were performed by whole-cell electrophysiology in HEK-293 and SH-SY5Y cells. RESULTS: The phenotypic spectrum encompassed: (a) intellectual disability/developmental delay (21/22 individuals with available information), ranging from mild to severe/profound; (b) epilepsy (15/25); (c) neurological impairment, with altered muscle tone (14/22); (d) dysmorphisms (13/20). Nineteen pathogenic KCNT2 variants were found (9 new, 10 reported previously): 16 missense, 1 in-frame deletion of a single amino acid, 1 nonsense, and 1 frameshift. Among tested variants, 8 showed gain-of-function (GoF), and 6 loss-of-function (LoF) features when expressed heterologously in vitro. Quinidine and fluoxetine blocked all GoF variants, whereas loxapine and riluzole activated some LoF variants while blocking others. INTERPRETATION: We expanded the phenotypic and genotypic spectrum of KCNT2-related disorders, highlighting novel genotype-phenotype associations. Pathogenic KCNT2 variants cause GoF or LoF in vitro phenotypes, and each shows a unique pharmacological profile, suggesting the need for in vitro functional and pharmacological investigation to enable targeted therapies based on the molecular phenotype. ANN NEUROL 2023;94:332-349.

Quantum Rabi dynamics of trapped atoms far in the deep strong coupling regime.

The coupling of a two-level system with an electromagnetic field, whose fully quantized version is the quantum Rabi model, is among the central topics of quantum physics. When the coupling strength becomes large enough that the field mode frequency is reached, the deep strong coupling regime is approached, and excitations can be created from the vacuum. Here we demonstrate a periodic variant of the quantum Rabi model in which the two-level system is encoded in the Bloch band structure of cold rubidium atoms in optical potentials. With this method we achieve a Rabi coupling strength of 6.5 times the field mode frequency, which is far in the deep strong coupling regime, and observe a subcycle timescale raise in bosonic field mode excitations. In a measurement recorded in the basis of the coupling term of the quantum Rabi Hamiltonian, a freezing of dynamics is revealed for small frequency splittings of the two-level system, as expected when the coupling term dominates over all other energy scales, and a revival for larger splittings. Our work demonstrates a route to realize quantum-engineering applications in yet unexplored parameter regimes.

Temporal Dynamics of MOG Antibodies in Children With Acquired Demyelinating Syndrome.

BACKGROUND AND OBJECTIVE: The spectrum of myelin oligodendrocyte glycoprotein (MOG) antibody-associated disorder (MOGAD) comprises monophasic diseases such as acute disseminated encephalomyelitis (ADEM), optic neuritis (ON), and transverse myelitis and relapsing courses of these presentations. Persistently high MOG antibodies (MOG immunoglobulin G [IgG]) are found in patients with a relapsing disease course. Prognostic factors to determine the clinical course of children with a first MOGAD are still lacking. The objective of the study is to assess the clinical and laboratory prognostic parameters for a risk of relapse and the temporal dynamics of MOG-IgG titers in children with MOGAD in correlation with clinical presentation and disease course. METHODS: In this prospective multicenter hospital-based study, children with a first demyelinating attack and complete data set comprising clinical and radiologic findings, MOG-IgG titer at onset, and clinical and serologic follow-up data were included. Serum samples were analyzed by live cell-based assay, and a titer level of ≥1:160 was classified as MOG-IgG-positive. RESULTS: One hundred sixteen children (f:m = 57:59) with MOGAD were included and initially diagnosed with ADEM (n = 59), unilateral ON (n = 12), bilateral ON (n = 16), myelitis (n = 6), neuromyelitis optica spectrum disorder (n = 8) or encephalitis (n = 6). The median follow-up time was 3 years in monophasic and 5 years in relapsing patients. There was no significant association between disease course and MOG-IgG titers at onset, sex, age at presentation, or clinical phenotype. Seroconversion to MOG-IgG-negative within 2 years of the initial event showed a significant risk reduction for a relapsing disease course. Forty-two/one hundred sixteen patients (monophasic n = 26, relapsing n = 16) had serial MOG-IgG testing in years 1 and 2 after the initial event. In contrast to relapsing patients, monophasic patients showed a significant decrease of MOG-IgG titers during the first and second years, often with seroconversion to negative titers. During the follow-up, MOG-IgG titers were persistently higher in relapsing than in monophasic patients. Decrease in MOG-IgG of ≥3 dilution steps after the first and second years was shown to be associated with a decreased risk of relapses. In our cohort, no patient experienced a relapse after seroconversion to MOG-IgG-negative. DISCUSSION: In this study, patients with declining MOG-IgG titers, particularly those with seroconversion to MOG-IgG-negative, are shown to have a significantly reduced relapse risk.

Innate immune receptor signaling induces transient melanoma dedifferentiation while preserving immunogenicity.

BACKGROUND: Immune-stimulatory agents, like agonists of the innate immune receptor RIG-I, are currently tested in clinical trials as an intratumoral treatment option for patients with unresectable melanoma, aiming to enhance anti-tumor T cell responses. Switching of melanoma toward a dedifferentiated cell state has recently been linked to T cell and therapy resistance. It remains to be determined whether RIG-I agonists affect melanoma differentiation, potentially leading to T cell resistance. METHODS: Patient metastases-derived melanoma cell lines were treated with the synthetic RIG-I agonist 3pRNA, and effects on tumor cell survival, phenotype and differentiation were determined. Transcriptomic data sets from cell lines and metastases were analyzed for associations between RIG-I (DDX58) and melanoma differentiation markers and used to define signaling pathways involved in RIG-I-driven dedifferentiation. The impact of 3pRNA-induced melanoma dedifferentiation on CD8 T cell activation was studied in autologous tumor T cell models. RESULTS: RIG-I activation by 3pRNA induced apoptosis in a subpopulation of melanoma cells, while the majority of tumor cells switched into a non-proliferative cell state. Those persisters displayed a dedifferentiated cell phenotype, marked by downregulation of the melanocytic lineage transcription factor MITF and its target genes, including melanoma differentiation antigens (MDA). Transition into the MITFlow/MDAlow cell state was JAK-dependent, with some cells acquiring nerve growth factor receptor expression. MITFlow/MDAlow persisters switched back to the proliferative differentiated cell state when RIG-I signaling declined. Consistent with our in vitro findings, an association between melanoma dedifferentiation and high RIG-I (DDX58) levels was detected in transcriptomic data from patient metastases. Notably, despite their dedifferentiated cell phenotype, 3pRNA-induced MITFlow/MDAlow persisters were still efficiently targeted by autologous CD8 tumor-infiltrating T lymphocytes (TILs). CONCLUSIONS: Our results demonstrate that RIG-I signaling in melanoma cells drives a transient phenotypic switch toward a non-proliferative dedifferentiated persister cell state. Despite their dedifferentiation, those persisters are highly immunogenic and sensitive toward autologous TILs, challenging the concept of melanoma dedifferentiation as a general indicator of T cell resistance. In sum, our findings support the application of RIG-I agonists as a therapeutic tool for the generation of long-term clinical benefit in non-resectable melanoma.

[Association Between Grounds for Legal Commitment to a Psychiatric Facility and Assessment of Fitness to Drive: An Orientating Analysis of the Relevance of Driving-Related Medicine to the National Health Service and Other Implicated Actors]. / Öffentlich-rechtliche Unterbringung und Fahreignung ­ Eine orientierende Untersuchung von verkehrsmedizinischer Relevanz für den Öffentlichen Gesundheitsdienst und andere Akteure.

AIM OF THE STUDY: The objective of this analysis was to record the social and epidemiological characteristics of a specific sample population, as well as to identify any associations between a previous commitment to a public facility on legal grounds and subsequent assessments of an individual's fitness to drive as per the National Health Service (or "ÖGD"). METHODS: For the retrospective data analysis, the documents of 87 subjects were evaluated who had been committed to public psychiatric institutions on legal grounds between 2015 and 2019. Using the SAS software package, frequency distributions and statistical relationships were identified between specific features of the commitment to accommodation and the assessment of fitness to drive by means of Chi-squared testing. RESULTS: The average age of the study cohort was 43.5 years (range: 16-82 years; male: 59%). The most frequent grounds for commitment to a facility were suicidal intentions expressed by the person in question. In one third of the cases, these individuals were under the influence of alcohol at the time of commitment to the facility, and drug use was documented in 3 of the 87 cases. In 74% of cases, confinement was solely due to an individual's risk to themselves; in 26% a risk to others was (additionally) identified; and in 20% of those affected, there was verbal and/or physical resistance to commitment to the accommodation facility. In 57% of cases, the medical evaluation raised doubts about the individual's fitness to drive, resulting in the matter being referred on to the driving license authority. Statistically significant associations were demonstrated between: a) the grounds for commitment to a facility; the type of risk; and resistance to commitment being enforced, and b) the results of a fitness-to-drive assessment carried out by the ÖGD. CONCLUSION: The data available on individuals committed to public facilities on legal grounds in connection with driving-related medical issues should be optimised to improve quality, whereby the anonymous registration system, introduced on the basis of the Bavarian Mental Health Act ("BayPsychoKHG"), can make a contribution in this regard. In addition, further qualification measures for effective quality management are necessary for all actors involved.

Variants in Mitochondrial ATP Synthase Cause Variable Neurologic Phenotypes.

OBJECTIVE: ATP synthase (ATPase) is responsible for the majority of ATP production. Nevertheless, disease phenotypes associated with mutations in ATPase subunits are extremely rare. We aimed at expanding the spectrum of ATPase-related diseases. METHODS: Whole-exome sequencing in cohorts with 2,962 patients diagnosed with mitochondrial disease and/or dystonia and international collaboration were used to identify deleterious variants in ATPase-encoding genes. Findings were complemented by transcriptional and proteomic profiling of patient fibroblasts. ATPase integrity and activity were assayed using cells and tissues from 5 patients. RESULTS: We present 10 total individuals with biallelic or de novo monoallelic variants in nuclear ATPase subunit genes. Three unrelated patients showed the same homozygous missense ATP5F1E mutation (including one published case). An intronic splice-disrupting alteration in compound heterozygosity with a nonsense variant in ATP5PO was found in one patient. Three patients had de novo heterozygous missense variants in ATP5F1A, whereas another 3 were heterozygous for ATP5MC3 de novo missense changes. Bioinformatics methods and populational data supported the variants' pathogenicity. Immunohistochemistry, proteomics, and/or immunoblotting revealed significantly reduced ATPase amounts in association to ATP5F1E and ATP5PO mutations. Diminished activity and/or defective assembly of ATPase was demonstrated by enzymatic assays and/or immunoblotting in patient samples bearing ATP5F1A-p.Arg207His, ATP5MC3-p.Gly79Val, and ATP5MC3-p.Asn106Lys. The associated clinical profiles were heterogeneous, ranging from hypotonia with spontaneous resolution (1/10) to epilepsy with early death (1/10) or variable persistent abnormalities, including movement disorders, developmental delay, intellectual disability, hyperlactatemia, and other neurologic and systemic features. Although potentially reflecting an ascertainment bias, dystonia was common (7/10). INTERPRETATION: Our results establish evidence for a previously unrecognized role of ATPase nuclear-gene defects in phenotypes characterized by neurodevelopmental and neurodegenerative features. ANN NEUROL 2022;91:225-237.

Luminescent Amphiphilic Aminoglycoside Probes to Study Transfection.

We report the characterization of amphiphilic aminoglycoside conjugates containing luminophores with aggregation-induced emission properties as transfection reagents. These inherently luminescent transfection vectors are capable of binding plasmid DNA through electrostatic interactions; this binding results in an emission "on" signal due to restriction of intramolecular motion of the luminophore core. The luminescent cationic amphiphiles effectively transferred plasmid DNA into mammalian cells (HeLa, HEK 293T), as proven by expression of a red fluorescent protein marker. The morphologies of the aggregates were investigated by microscopy as well as ζ-potential and dynamic light-scattering measurements. The transfection efficiencies using luminescent cationic amphiphiles were similar to that of the gold-standard transfection reagent Lipofectamine® 2000.

Imaging Erythrocyte Sedimentation in Whole Blood.

The erythrocyte sedimentation rate (ESR) is one of the oldest medical diagnostic tools. However, currently there is some debate on the structure formed by the cells during the sedimentation process. While the conventional view is that erythrocytes sediment as separate aggregates, others have suggested that they form a percolating gel, similar to other colloidal suspensions. However, visualization of aggregated erythrocytes, which would settle the question, has always been challenging. Direct methods usually study erythrocytes in 2D situations or low hematocrit (∼1%). Indirect methods, such as scattering or electric measurements, provide insight on the suspension evolution, but cannot directly discriminate between open or percolating structures. Here, we achieved a direct probing of the structures formed by erythrocytes in blood at stasis. We focused on blood samples at rest with controlled hematocrit of 45%, from healthy donors, and report observations from three different optical imaging techniques: direct light transmission through thin samples, two-photon microscopy and light-sheet microscopy. The three techniques, used in geometries with thickness from 150 µm to 3 mm, highlight that erythrocytes form a continuous network with characteristic cracks, i.e., a colloidal gel. The characteristic distance between the main cracks is of the order of ∼100 µm. A complete description of the structure then requires a field of view of the order of ∼1 mm, in order to obtain a statistically relevant number of structural elements. A quantitative analysis of the erythrocyte related processes and interactions during the sedimentation need a further refinement of the experimental set-ups.

The role of clinical response to treatment in determining pathogenicity of genomic variants.

PURPOSE: The 2015 American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines for the interpretation of sequence variants provide a framework to standardize terminology in the classification of variants uncovered through genetic testing. We aimed to assess the validity of utilizing clinical response to therapies specifically targeted to a suspected disease in clarifying variant pathogenicity. METHODS: Five families with disparate clinical presentations and different genetic diseases evaluated and treated in multiple diagnostic settings are summarized. RESULTS: Extended evaluations indicated possible genetic diagnoses and assigned candidate causal variants, but the cumulative clinical, biochemical, and molecular information in each instance was not completely consistent with the identified disease. Initiation of treatment specific to the suspected diagnoses in the affected individuals led to clinical improvement in all five families. CONCLUSION: We propose that the effect of therapies that are specific and targeted to treatable genetic diseases embodies an in vivo physiological response and could be considered as additional criteria within the 2015 ACMG/AMP guidelines in determining genomic variant pathogenicity.

Optogenetic Tuning Reveals Rho Amplification-Dependent Dynamics of a Cell Contraction Signal Network.

Local cell contraction pulses play important roles in tissue and cell morphogenesis. Here, we improve a chemo-optogenetic approach and apply it to investigate the signal network that generates these pulses. We use these measurements to derive and parameterize a system of ordinary differential equations describing temporal signal network dynamics. Bifurcation analysis and numerical simulations predict a strong dependence of oscillatory system dynamics on the concentration of GEF-H1, an Lbc-type RhoGEF, which mediates the positive feedback amplification of Rho activity. This prediction is confirmed experimentally via optogenetic tuning of the effective GEF-H1 concentration in individual living cells. Numerical simulations show that pulse amplitude is most sensitive to external inputs into the myosin component at low GEF-H1 concentrations and that the spatial pulse width is dependent on GEF-H1 diffusion. Our study offers a theoretical framework to explain the emergence of local cell contraction pulses and their modulation by biochemical and mechanical signals.

Expanding the clinical and genetic spectrum of CAD deficiency: an epileptic encephalopathy treatable with uridine supplementation.

PURPOSE: Biallelic CAD variants underlie CAD deficiency (or early infantile epileptic encephalopathy-50, [EIEE-50]), an error of pyrimidine de novo biosynthesis amenable to treatment via the uridine salvage pathway. We further define the genotype and phenotype with a focus on treatment. METHODS: Retrospective case series of 20 patients. RESULTS: Our study confirms CAD deficiency as a progressive EIEE with recurrent status epilepticus, loss of skills, and dyserythropoietic anemia. We further refine the phenotype by reporting a movement disorder as a frequent feature, and add that milder courses with isolated developmental delay/intellectual disability can occur as well as onset with neonatal seizures. With no biomarker available, the diagnosis relies on genetic testing and functional validation in patient-derived fibroblasts. Underlying pathogenic variants are often rated as variants of unknown significance, which could lead to underrecognition of this treatable disorder. Supplementation with uridine, uridine monophosphate, or uridine triacetate in ten patients was safe and led to significant clinical improvement in most patients. CONCLUSION: We advise a trial with uridine (monophosphate) in all patients with developmental delay/intellectual disability, epilepsy, and anemia; all patients with status epilepticus; and all patients with neonatal seizures until (genetically) proven otherwise or proven unsuccessful after 6 months. CAD deficiency might represent a condition for genetic newborn screening.

A pH-sensitive fluorescent protein sensor to follow the pathway of calcium phosphate nanoparticles into cells.

Calcium phosphate nanoparticles (100 nm) were fluorescently labelled with poly(ethyleneimine) (PEIATTO490LS; red fluorescence). They were loaded with a Tandem fusion protein consisting of mRFP1-eGFP (red and green fluorescence in the same molecule)that acts as smart biological pH sensor to trace nanoparticles inside cells. Its fluorescence is also coupled to the structural integrity of the protein, i.e. it is also a label for a successful delivery of a functional protein into the cell. At pH 7.4, the fluorescence of both proteins (red and green) is detectable. At a pH of 4.5-5 inside the lysosomes, the green fluorescence is quenched due to the protonation of the eGFP chromophore, but the pH-independent red fluorescence of mRFP1 remains. The nanoparticles were taken up by cells (cell lines: HeLa, Caco-2 and A549) via endocytic pathways and then directed to lysosomes. Time-resolved confocal laser scanning microscopy confirmed mRFP1 and nanoparticles co-localizing with lysosomes. The fluorescence of eGFP was only detectable outside lysosomes, i.e. most likely inside early endosomes or at the cell membrane during the uptake, indicating the neutral pH at these locations. The Tandem fusion protein provides a versatile platform to follow the intracellular pathway of bioactive nanocarriers, e.g. therapeutic proteins. The transfection with a Tandem-encoding plasmid by calcium phosphate nanoparticles led to an even intracellular protein distribution in cytosol and nucleoplasm, i.e. very different from direct protein uptake. Neither dissolved protein nor dissolved plasmid DNA were taken up by the cells, underscoring the necessity for a suitable carrier like a nanoparticle. STATEMENT OF SIGNIFICANCE: A pH-sensitive protein ("tandem") was used to follow the pathway of calcium phosphate nanoparticles. This protein consists of a pH-sensitive fluorophore (eGFP; green) and a pH-independent fluorophore (mRFP1; red). This permits to follow the pathway of a nanoparticle inside a cell. At a low pH inside an endolysosome, the green fluorescence vanishes but the red fluorescence persists. This is also a very useful model for the delivery of therapeutic proteins into cells. The delivery by nanoparticles was compared with the protein expression after cell transfection with plasmid DNA encoding for the tandem protein. High-resolution image analysis gave quantitative data on the intracellular protein distribution.

Lipofection with estrone-based luminophores featuring aggregation-induced emission properties.

In this communication we present the use of a novel class of luminophores with aggregation-induced emission (AIE) properties based on the steroid estrone. These molecules were equipped with cationic residues yielding amphiphiles suitable for lipofection. To this end, self-assembled luminescent structures were formed in aqueous media and mixed with a red-fluorescent protein expressing plasmid, yielding lipoplexes with increased emission intensity. These luminescent lipoplexes were able to efficiently transfect HeLa and HEK 293T cells and were able to be tracked due to the aggregation induced-emission properties.

Computer-Assisted Propofol Sedation for Esophagogastroduodenoscopy Is Effective, Efficient, and Safe.

BACKGROUND AND AIMS: Computer-assisted propofol sedation (CAPS) allows non-anesthesiologists to administer propofol for gastrointestinal procedures in relatively healthy patients. As the first US medical center to adopt CAPS technology for routine clinical use, we report our 1-year experience with CAPS for esophagogastroduodenoscopy (EGD). METHODS: Between September 2014 and August 2015, 926 outpatients underwent elective EGDs with CAPS at our center. All EGDs were performed by 1 of 17 gastroenterologists certified in the use of CAPS. Procedural success rates, procedure times, and recovery times were compared against corresponding historical controls done with midazolam and fentanyl sedation from September 2013 to August 2014. Adverse events in CAPS patients were recorded. RESULTS: The mean age of the CAPS cohort was 56.7 years (45% male); 16.2% of the EGDs were for variceal screening or Barrett's surveillance and 83.8% for symptoms. The procedural success rates were similar to that of historical controls (99.0% vs. 99.3%; p = 0.532); procedure times were also similar (6.6 vs. 7.4 min; p = 0.280), but recovery time was markedly shorter (31.7 vs. 52.4 min; p < 0.001). There were 11 (1.2%) cases of mild transient oxygen desaturation (< 90%), 15 (1.6%) cases of marked agitation due to undersedation, and 1 case of asymptomatic hypotension. In addition, there were six (0.6%) patients with more pronounced desaturation episodes that required brief (< 1 min) mask ventilation. There were no other serious adverse events. CONCLUSIONS: CAPS appears to be a safe, effective, and efficient means of providing sedation for EGD in healthy patients. Recovery times were much shorter than historical controls.

Phenotype, treatment practice and outcome in the cobalamin-dependent remethylation disorders and MTHFR deficiency: Data from the E-HOD registry.

AIM: To explore the clinical presentation, course, treatment and impact of early treatment in patients with remethylation disorders from the European Network and Registry for Homocystinurias and Methylation Defects (E-HOD) international web-based registry. RESULTS: This review comprises 238 patients (cobalamin C defect n = 161; methylenetetrahydrofolate reductase deficiency n = 50; cobalamin G defect n = 11; cobalamin E defect n = 10; cobalamin D defect n = 5; and cobalamin J defect n = 1) from 47 centres for whom the E-HOD registry includes, as a minimum, data on medical history and enrolment visit. The duration of observation was 127 patient years. In 181 clinically diagnosed patients, the median age at presentation was 30 days (range 1 day to 42 years) and the median age at diagnosis was 3.7 months (range 3 days to 56 years). Seventy-five percent of pre-clinically diagnosed patients with cobalamin C disease became symptomatic within the first 15 days of life. Total homocysteine (tHcy), amino acids and urinary methylmalonic acid (MMA) were the most frequently assessed disease markers; confirmatory diagnostics were mainly molecular genetic studies. Remethylation disorders are multisystem diseases dominated by neurological and eye disease and failure to thrive. In this cohort, mortality, thromboembolic, psychiatric and renal disease were rarer than reported elsewhere. Early treatment correlates with lower overall morbidity but is less effective in preventing eye disease and cognitive impairment. The wide variation in treatment hampers the evaluation of particular therapeutic modalities. CONCLUSION: Treatment improves the clinical course of remethylation disorders and reduces morbidity, especially if started early, but neurocognitive and eye symptoms are less responsive. Current treatment is highly variable. This study has the inevitable limitations of a retrospective, registry-based design.