Barakat syndrome diagnosed decades after initial presentation.

Summary: Barakat syndrome, also called HDR syndrome, is a rare genetic disorder encompassing hypoparathyroidism (H), sensorineural deafness (D) and renal disease (R). A 64-year-old woman was referred to our endocrinology clinic for a switch in treatment (from dihydrotachysterol to calcitriol). She had progressive sensorineural deafness since the age of 18 and idiopathic hypoparathyroidism diagnosed at age of 36. Her medical history included osteoporosis with hip/spine fractures, nephrolithiasis and a family history of hearing loss, osteoporosis and kidney disease. The patient's clinical presentation indicated Barakat syndrome. Genetic analysis found a GATA3:c.916C>T nonsense variant. Further tests such as audiometry, labs and renal imaging supported the diagnosis. Due to rarity and manifold symptoms, diagnosis can be challenging. Optional GATA3 testing was suggested in 2018, except in cases of isolated sensorineural deafness or renal disease with pertinent family history. In isolated 'H' cases without 'D' and 'R', GATA3 studies are not required, as no haploinsufficiency cases were reported. Given the rise in genetic disorders, physicians should consistently consider rare genetic disorders in patients with suggestive symptoms, even decades after onset. Although diagnosis might not always impact management directly, it aids patients in accepting their condition and has broader family implications. Learning points: There is currently an important increase in genetic and clinical characterization of new orphan diseases and their causative agents. Unbiased re-evaluation for possible genetic disorders is necessary at every consultation. It is essential to recognize the differential diagnosis of idiopathic hypoparathyroidism. The patient's clinical presentation and family history can be important to establish the correct diagnosis. Physicians should not hesitate to search a patient's signs and symptoms online.

Flipping the classroom in neurological bedside teaching: a prospective controlled study.

BACKGROUND: Bedside teaching is essential to foster core clinical competences in medical education, especially in Neurology. However, bedside skills are declining and new concepts to enhance the effectiveness of bedside teaching are needed, also in view of limited in-person teaching possibilities in the ongoing pandemic situation. If theoretical knowledge is taught prior to in-person sessions this might allow to better focus on practical application aspects during bedside teaching. We thus aimed to answer the question to what extent such an approach can enhance the effectiveness of neurological bedside teaching. METHODS: In this prospective controlled study, neurological bedside courses following a traditional and a flipped classroom (FC) approach were compared with regards to their effects on theoretical knowledge and practical skills of medical students. Evaluations were obtained from 161 students and their lecturers participating in a neurological bedside teaching course at a German university hospital between October 2020 and July 2021. Students were randomly assigned to course dates. However, the 74 students assigned to course dates from May to July 2021 completed a mandatory online preparation course prior to the bedside teaching. These students served as the interventional group (IG) and the remaining 87 students formed the control group (CG). Ratings of knowledge and skills provided by the students and their lecturers on numerical rating scales served as primary outcome measures. Moreover, the time needed to recapitulate theoretical contents during the in-person teaching session was assessed as a secondary outcome measure. Group comparisons were performed using t-statistics. RESULTS: Theoretical knowledge upon entering the course was rated significantly higher in the IG by the students (p < 0.001) and lecturers (p = 0.003). Lecturers also rated the practical skills of students in the IG significantly higher (p < 0.001). Furthermore, significantly less time was needed to recapitulate theoretical contents during the in-person session in the IG (p = 0.03). CONCLUSIONS: Using a FC approach enhances the effectiveness of in-person neurological bedside teaching. Thus, these concepts are particularly valuable in the ongoing pandemic situation. Moreover, they might allow to reuse e-learning contents developed during the pandemic and to develop future bedside teaching concepts.

Effectiveness of non-bedside teaching during the COVID-19 pandemic: a quasi-experimental study.

BACKGROUND: The COVID-19 pandemic poses a huge challenge for clinical teaching due to contact restrictions and social distancing. Medical teachers have to balance potential risks and benefits of bedside teaching, especially in course formats intended to foster practical clinical skills. In this context, we aimed to address the question, whether presence-based teaching formats without patient involvement are suitable to teach practical skills. METHODS: In this quasi-experimental study, presence-based teaching formats with and without patient contact were retrospectively compared regarding their effects on medical students' theoretical knowledge and practical skills, i.e. the performance and clinical interpretation of the neurological exam. To this end, evaluations from 102 students and their lecturers participating in a neurological bedside teaching course at a German university hospital between October 2020 and April 2021 were obtained. Students were initially randomly assigned to course dates. However, 53 students assigned to courses in November and December 2020, were not able to go bedside due to contact restrictions. These students formed the interventional group and the remaining 49 students the control group. The primary outcome measures were students' overall grading of the course (school grades, 1-6) as well as ratings of knowledge and skills provided by the students themselves and their lecturers on a numerical rating scale (0-10). Comparison between groups was performed using frequentist and Bayesian t-statistics. RESULTS: The teaching format without patient contact received a significantly poorer overall grade by the students (p = 0.018). However, improvements in the students' self-ratings of knowledge and skills did not differ between the two formats (all p > 0.05, BF10max = 0.42). Moreover, especially practical skills were even rated significantly better in the group without patient contact by the lecturers (p < 0.001). CONCLUSIONS: Teaching formats without patient contact are less well-received by the students. However, they are able to teach practical skills regarding the performance and clinical interpretation of examination techniques. Still, the evaluations obtained might not adequately capture the importance of bedside teaching in preparing future physicians for their practice. Perspectively, hybrid teaching approaches including flipped-classroom concepts hold considerable potential to enhance effectiveness of bedside teaching in the present pandemic situation and in the future.

Combined Application of Pan-AKT Inhibitor MK-2206 and BCL-2 Antagonist Venetoclax in B-Cell Precursor Acute Lymphoblastic Leukemia.

Aberrant PI3K/AKT signaling is a hallmark of acute B-lymphoblastic leukemia (B-ALL) resulting in increased tumor cell proliferation and apoptosis deficiency. While previous AKT inhibitors struggled with selectivity, MK-2206 promises meticulous pan-AKT targeting with proven anti-tumor activity. We herein, characterize the effect of MK-2206 on B-ALL cell lines and primary samples and investigate potential synergistic effects with BCL-2 inhibitor venetoclax to overcome limitations in apoptosis induction. MK-2206 incubation reduced AKT phosphorylation and influenced downstream signaling activity. Interestingly, after MK-2206 mono application tumor cell proliferation and metabolic activity were diminished significantly independently of basal AKT phosphorylation. Morphological changes but no induction of apoptosis was detected in the observed cell lines. In contrast, primary samples cultivated in a protective microenvironment showed a decrease in vital cells. Combined MK-2206 and venetoclax incubation resulted in partially synergistic anti-proliferative effects independently of application sequence in SEM and RS4;11 cell lines. Venetoclax-mediated apoptosis was not intensified by addition of MK-2206. Functional assessment of BCL-2 inhibition via Bax translocation assay revealed slightly increased pro-apoptotic signaling after combined MK-2206 and venetoclax incubation. In summary, we demonstrate that the pan-AKT inhibitor MK-2206 potently blocks B-ALL cell proliferation and for the first time characterize the synergistic effect of combined MK-2206 and venetoclax treatment in B-ALL.

Rhodopsin optogenetic toolbox v2.0 for light-sensitive excitation and inhibition in Caenorhabditis elegans.

In optogenetics, rhodopsins were established as light-driven tools to manipulate neuronal activity. However, during long-term photostimulation using channelrhodopsin (ChR), desensitization can reduce effects. Furthermore, requirement for continuous presence of the chromophore all-trans retinal (ATR) in model systems lacking sufficient endogenous concentrations limits its applicability. We tested known, and engineered and characterized new variants of de- and hyperpolarizing rhodopsins in Caenorhabditis elegans. ChR2 variants combined previously described point mutations that may synergize to enable prolonged stimulation. Following brief light pulses ChR2(C128S;H134R) induced muscle activation for minutes or even for hours ('Quint': ChR2(C128S;L132C;H134R;D156A;T159C)), thus featuring longer open state lifetime than previously described variants. Furthermore, stability after ATR removal was increased compared to the step-function opsin ChR2(C128S). The double mutants C128S;H134R and H134R;D156C enabled increased effects during repetitive stimulation. We also tested new hyperpolarizers (ACR1, ACR2, ACR1(C102A), ZipACR). Particularly ACR1 and ACR2 showed strong effects in behavioral assays and very large currents with fast kinetics. In sum, we introduce highly light-sensitive optogenetic tools, bypassing previous shortcomings, and thus constituting new tools that feature high effectiveness and fast kinetics, allowing better repetitive stimulation or investigating prolonged neuronal activity states in C. elegans and, possibly, other systems.

An optogenetic arrhythmia model to study catecholaminergic polymorphic ventricular tachycardia mutations.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a condition of abnormal heart rhythm (arrhythmia), induced by physical activity or stress. Mutations in ryanodine receptor 2 (RyR2), a Ca2+ release channel located in the sarcoplasmic reticulum (SR), or calsequestrin 2 (CASQ2), a SR Ca2+ binding protein, are linked to CPVT. For specific drug development and to study distinct arrhythmias, simple models are required to implement and analyze such mutations. Here, we introduced CPVT inducing mutations into the pharynx of Caenorhabditis elegans, which we previously established as an optogenetically paced heart model. By electrophysiology and video-microscopy, we characterized mutations in csq-1 (CASQ2 homologue) and unc-68 (RyR2 homologue). csq-1 deletion impaired pharynx function and caused missed pumps during 3.7 Hz pacing. Deletion mutants of unc-68, and in particular the point mutant UNC-68(R4743C), analogous to the established human CPVT mutant RyR2(R4497C), were unable to follow 3.7 Hz pacing, with progressive defects during long stimulus trains. The pharynx either locked in pumping at half the pacing frequency or stopped pumping altogether, possibly due to UNC-68 leakiness and/or malfunctional SR Ca2+ homeostasis. Last, we could reverse this 'worm arrhythmia' by the benzothiazepine S107, establishing the nematode pharynx for studying specific CPVT mutations and for drug screening.

A severe pediatric infection with a novel enterovirus A71 strain, Thuringia, Germany.

Infection by Enterovirus A71 (EV-A71) is an important cause of hand, foot, and mouth disease (HFMD). Outbreaks including severe cases with neurological and cardiopulmonary complications have been reported particularly from Southeast Asia. In Europe, the epidemiology of EV-A71 is not well understood. In summer 2015, a two-year-old girl from Thuringia, Germany, presented with rhombencephalitis/brainstem encephalitis associated with severe neurological and cardiopulmonary complications. EV-A71 was detected in stool and almost the entire viral genome was amplified and sequenced. While the capsid protein VP1-encoding region belongs to the EV-A71 subgenogroup C1, the 3D polymerase encoding region represents a unique lineage. Thus, the data suggest that the Thuringian EV-A71 sequence likely represents a recombinant. The case underlines the importance of intensified EV-A71 surveillance in Germany and Europe including analysis of full-genome data.

Staccato/Unc-13-4 controls secretory lysosome-mediated lumen fusion during epithelial tube anastomosis.

A crucial yet ill-defined step during the development of tubular networks, such as the vasculature, is the formation of connections (anastomoses) between pre-existing lumenized tubes. By studying tracheal tube anastomosis in Drosophila melanogaster, we uncovered a key role of secretory lysosome-related organelle (LRO) trafficking in lumen fusion. We identified the conserved calcium-binding protein Unc-13-4/Staccato (Stac) and the GTPase Rab39 as critical regulators of this process. Stac and Rab39 accumulate on dynamic vesicles, which form exclusively in fusion tip cells, move in a dynein-dependent manner, and contain late-endosomal, lysosomal, and SNARE components characteristic of LROs. The GTPase Arl3 is necessary and sufficient for Stac LRO formation and promotes Stac-dependent intracellular fusion of juxtaposed apical plasma membranes, thereby forming a transcellular lumen. Concomitantly, calcium is released locally from ER exit sites and apical membrane-associated calcium increases. We propose that calcium-dependent focused activation of LRO exocytosis restricts lumen fusion to appropriate domains within tip cells.

Arrhythmogenic effects of mutated L-type Ca 2+-channels on an optogenetically paced muscular pump in Caenorhabditis elegans.

Cardiac arrhythmias are often associated with mutations in ion channels or other proteins. To enable drug development for distinct arrhythmias, model systems are required that allow implementing patient-specific mutations. We assessed a muscular pump in Caenorhabditis elegans. The pharynx utilizes homologues of most of the ion channels, pumps and transporters defining human cardiac physiology. To yield precise rhythmicity, we optically paced the pharynx using channelrhodopsin-2. We assessed pharynx pumping by extracellular recordings (electropharyngeograms--EPGs), and by a novel video-microscopy based method we developed, which allows analyzing multiple animals simultaneously. Mutations in the L-type VGCC (voltage-gated Ca(2+)-channel) EGL-19 caused prolonged pump duration, as found for analogous mutations in the Cav1.2 channel, associated with long QT syndrome. egl-19 mutations affected ability to pump at high frequency and induced arrhythmicity. The pharyngeal neurons did not influence these effects. We tested whether drugs could ameliorate arrhythmia in the optogenetically paced pharynx. The dihydropyridine analog Nemadipine A prolonged pump duration in wild type, and reduced or prolonged pump duration of distinct egl-19 alleles, thus indicating allele-specific effects. In sum, our model may allow screening of drug candidates affecting specific VGCCs mutations, and permit to better understand the effects of distinct mutations on a macroscopic level.

Large-scale differential proteome analysis in Plasmodium falciparum under drug treatment.

Here, we establish a methodology for large-scale quantitative proteomics using SIL (stable isotope labeling) to examine protein expression changes in trophozoite stages of the malaria parasite Plasmodium falciparum following drug treatment. For this purpose, exposure to (13)C6 (15)N1-isoleucine was optimized in order to obtain 99% atomic enrichment. Proteome fractionation with anion exchange chromatography was used to reduce sample complexity and increase quantitative coverage of protein expression. Tryptic peptides of subfractions were subjected to SCX/RP separation, measured by LC-MS/MS, and quantified using the software tool Census. In drug-treated parasites, we identified a total number of 1,253 proteins, thus increasing the overall number of proteins so far identified in the trophozoite stage by 30% in the previous literature. A relative quantification was obtained for more than 800 proteins. About 5% of proteins showed a clear up- or downregulation upon drug treatment.

Mobile money, smallholder farmers, and household welfare in Kenya.

The use of mobile phones has increased rapidly in many developing countries, including in rural areas. Besides reducing the costs of communication and improving access to information, mobile phones are an enabling technology for other innovations. One important example are mobile phone based money transfers, which could be very relevant for the rural poor, who are often underserved by the formal banking system. We analyze impacts of mobile money technology on the welfare of smallholder farm households in Kenya. Using panel survey data and regression models we show that mobile money use has a positive impact on household income. One important pathway is through remittances received from relatives and friends. Such remittances contribute to income directly, but they also help to reduce risk and liquidity constraints, thus promoting agricultural commercialization. Mobile money users apply more purchased farm inputs, market a larger proportion of their output, and have higher profits than non-users of this technology. These results suggest that mobile money can help to overcome some of the important smallholder market access constraints that obstruct rural development and poverty reduction.

Thrombolysis in patients with lacunar stroke is safe: an observational study.

Research suggests that the etiology of lacunar stroke is different from that of other stroke subtypes. This could imply an altered response to thrombolysis, but data concerning the efficacy of rt-PA in lacunar stroke is limited and inconsistent. From our prospectively collected stroke database, we identified patients with an MRI-confirmed purely lacunar stroke that were treated in our Stroke Unit between 2004 and 2011. We compared both the clinical course (NIHSS, deterioration, mRS at 3 months) and the MRI findings between patients who either received or did not receive rt-PA. In comparison to patients who obtained standard medical care (n = 468), acute lacunar stroke patients treated with rt-PA (n = 69) were more severely affected on admission (median NIHSS of 5 vs. 3; p < 0.001) and presented less frequently with a lacunar syndrome (74 vs. 88 %; p = 0.003). The clinical course was more favorable in patients treated with rt-PA (median NIHSS improvement of 3 vs. 1; p < 0.001), while functional deficit after 3 months was similar in both groups (median mRS of 2; p = 0.211). Overall complication rates did not differ significantly between the two groups, but while we did not detect symptomatic intracranial hemorrhage, hemorrhagic transformation was more frequent in thrombolyzed patients (11.6 vs. 1.9 %; p = 0.001). Patients with acute lacunar stroke benefited from thrombolysis without additional complications. Thus, patients with suspected acute lacunar stroke or lacunar syndrome should not be treated differently than other stroke populations.

Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics.

Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Here we describe red, single-wavelength GECIs, "RCaMPs," engineered from circular permutation of the thermostable red fluorescent protein mRuby. High-resolution crystal structures of mRuby, the red sensor RCaMP, and the recently published red GECI R-GECO1 give insight into the chromophore environments of the Ca(2+)-bound state of the sensors and the engineered protein domain interfaces of the different indicators. We characterized the biophysical properties and performance of RCaMP sensors in vitro and in vivo in Caenorhabditis elegans, Drosophila larvae, and larval zebrafish. Further, we demonstrate 2-color calcium imaging both within the same cell (registering mitochondrial and somatic [Ca(2+)]) and between two populations of cells: neurons and astrocytes. Finally, we perform integrated optogenetics experiments, wherein neural activation via channelrhodopsin-2 (ChR2) or a red-shifted variant, and activity imaging via RCaMP or GCaMP, are conducted simultaneously, with the ChR2/RCaMP pair providing independently addressable spectral channels. Using this paradigm, we measure calcium responses of naturalistic and ChR2-evoked muscle contractions in vivo in crawling C. elegans. We systematically compare the RCaMP sensors to R-GECO1, in terms of action potential-evoked fluorescence increases in neurons, photobleaching, and photoswitching. R-GECO1 displays higher Ca(2+) affinity and larger dynamic range than RCaMP, but exhibits significant photoactivation with blue and green light, suggesting that integrated channelrhodopsin-based optogenetics using R-GECO1 may be subject to artifact. Finally, we create and test blue, cyan, and yellow variants engineered from GCaMP by rational design. This engineered set of chromatic variants facilitates new experiments in functional imaging and optogenetics.

[Fixation of cells for analysis by laser microdissection--comparative studies in forensic trace material]. / Fixierung von Zellen zur Analyse mittels Laser-Mikrodissektion: Vergleichende Untersuchungen an forensischem Spurenmaterial.

This paper is focused on the preparation of samples for laser microdissection (LM) in forensic casework. In forensic genetics, it is essential to preserve and separate cellular traces during sample preparation, as they are usually gathered in very small amounts and are often contaminated with undesired cells. This is made possible by laser microdissection, a technique developed to cut cells or tissue of a certain type from a microscopical specimen by UV laser and catapult them directly into a PCR reactor. This method minimizes the risk of getting inconclusive, mixed DNA profiles due to contamination by foreign DNA and also supplies information about the cellular origin of a DNA profile. A method for optimized fixation and staining of spermatozoa for laser microdissection was established. Four different fixation methods combined with two staining methods were tested on two different microscope slides. Moreover, the effect of a blocker pen to contain the specimen on the slide was investigated.

High-throughput screening for small-molecule inhibitors of plasmodium falciparum glucose-6-phosphate dehydrogenase 6-phosphogluconolactonase.

Plasmodium falciparum causes severe malaria infections in millions of people every year. The parasite is developing resistance to the most common antimalarial drugs, which creates an urgent need for new therapeutics. A promising and attractive target for antimalarial drug design is the bifunctional enzyme glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase (PfGluPho) of P. falciparum, which catalyzes the key step in the parasites' pentose phosphate pathway. In this study, we describe the development of a high-throughput screening assay to identify small-molecule inhibitors of recombinant PfGluPho. The optimized assay was used to screen three small-molecule compound libraries-namely, LOPAC (Sigma-Aldrich, 1280 compounds), Spectrum (MicroSource Discovery Systems, 1969 compounds), and DIVERSet (ChemBridge, 49 971 compounds). These pilot screens identified 899 compounds that inhibited PfGluPho activity by at least 50%. Selected compounds were further studied to determine IC(50) values in an orthogonal assay, the type of inhibition and reversibility, and effects on P. falciparum growth. Screening results and follow-up studies for selected PfGluPho inhibitors are presented. Our high-throughput screening assay may provide the basis to identify novel and urgently needed antimalarial drugs.

In vitro assessment of methylene blue on chloroquine-sensitive and -resistant Plasmodium falciparum strains reveals synergistic action with artemisinins.

Methylene blue (MB) represents a promising antimalarial drug candidate for combination therapies against drug-resistant parasite strains. To support and facilitate the application of MB in future field trials, we studied its antiparasitic effects in vitro. MB is active against all blood stages of both chloroquine (CQ)-sensitive and CQ-resistant P. falciparum strains with 50% inhibitory concentration (IC50) values in the lower nanomolar range. Ring stages showed the highest susceptibility. As demonstrated by high-performance liquid chromatography-tandem mass spectrometry on different cell culture compartments, MB is accumulated in malarial parasites. In drug combination assays, MB was found to be antagonistic with CQ and other quinoline antimalarials like piperaquine and amodiaquine; with mefloquine and quinine, MB showed additive effects. In contrast, we observed synergistic effects of MB with artemisinin, artesunate, and artemether for all tested parasite strains. Artemisinin/MB combination concentration ratios of 3:1 were found to be advantageous, demonstrating that the combination of artemisinin with a smaller amount of MB can be recommended for reaching maximal therapeutic effects. Our in vitro data indicate that combinations of MB with artemisinin and related endoperoxides might be a promising option for treating drug-resistant malaria and should be studied in future field trials. Resistance development under this drug combination is unlikely to occur.