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Chemical manipulations performed on the histone H3 lysine 9 methyltransferases (G9a/GLP) inhibitor BIX-01294 afforded novel desmethoxyquinazolines able to inhibit the DNA methyltransferase DNMT3A at low micromolar levels without any significant inhibition of DNMT1 and G9a. In KG-1 cells such compounds, when tested at sub-toxic doses, induced the luciferase re-expression in a stable construct controlled by a cytomegalovirus (CMV) promoter silenced by methylation (CMV-luc assay). Finally, in human lymphoma U-937 and RAJI cells, the N-(1-benzylpiperidin-4-yl)-2-(4-phenylpiperazin-1-yl)quinazolin-4-amine induced the highest proliferation arrest and cell death induction starting from 10 µM, in agreement with its DNMT3A inhibitory potency.

Abstract MicroRNAs are an emerging class of synaptic regulators. These small noncoding RNAs post-transcriptionally regulate gene expression, thereby altering neuronal pathways and shaping cell-to-cell communication. Their ability to rapidly alter gene expression and target multiple pathways makes them interesting candidates in the study of synaptic plasticity. Here, we demonstrate that the proconvulsive microRNA miR-324-5p regulates excitatory synapse structure and function in the hippocampus of mice. Both Mir324 knockout (KO) and miR-324-5p antagomir treatment significantly reduce dendritic spine density in the hippocampal CA1 subregion, and Mir324 KO, but not miR-324-5p antagomir treatment, shift dendritic spine morphology, reducing the proportion of thin, “unstable” spines. Western blot and quantitative Real-Time PCR revealed changes in protein and mRNA levels for potassium channels, cytoskeletal components, and synaptic markers, including MAP2 and Kv4.2, which are important for long-term potentiation (LTP). In line with these findings, slice electrophysiology revealed that LTP is severely impaired in Mir324 KO mice, while neurotransmitter release probability remains unchanged. Overall, this study demonstrates that miR-324-5p regulates dendritic spine density, morphology, and plasticity in the hippocampus, potentially via multiple cytoskeletal and synaptic modulators.

IntroductionPatients with head and neck cancer (PwHNC) benefit from targeted exercise interventions: symptom relief, compensation for dysfunction, improvement in quality of life (QoL). Data on acceptance physical interventions in PwHNC are rare. The 'OSHO #94' trial investigates the short- and medium-term effects of individualized home exercise in PwHNC on QoL, physical activity and functionality. The study includes a feasibility phase (proof of concept) in order to evaluate the acceptance. Here we present the study protocol as well as the feasibility results.Methods and analysisThis prospective, multicentre, single-arm intervention study includes PwHNC ≥18 years of age in aftercare or palliative care with stable remission under immunotherapy. The study opened in January 01, 2021, with estimated completion by December 31, 2024. The PwHNC receive an individualized home exercise program consisting of mobilization, coordination, strengthening and stretching exercises. This should be carried out at least three times a week over 12 weeks for 15 to 30 minutes, supplemented by aerobic training two to three times a week for 30 minutes (intervention). Once weekly telephone calls with a physiotherapist are performed. Subsequently, there is a 12-week follow-up (FU) without exercise specifications/contact. Outcomes are measured before and after the intervention and following the FU. Primary outcome of the feasibility phase (n = 25) was the determination of the dropout rate during the intervention with a termination cut off if more than 30% PwHNC withdrew premature. The primary outcome of the OSHO #94' trial (N = 53) is the change in global QoL score from pre- to post-intervention (EORTC QLQ-C30). Secondary outcomes include clinical and patient-reported measures, training details as well as functional diagnostic data (e.g. level of physical activity, training frequency, flexibility, fall risk and aerobic performance).Results25 PwHNC were enrolled onto the feasibility cohort. Only16% (4/25 patients) did not complete the study. Therefore, recruitment of PwHNC was continued. The dropout rate was adjusted from 30% (N = 60) to 20% (N = 53, calculated sample size n = 42 PwHNC and 20% (n = 11) to dropout).ConclusionsIndividualized home exercise programs in PwHNC in aftercare seem feasible. Consequently, the aim is now to evaluate the short and medium-term effects of individualized home exercise.

BackgroundAbdominal venous thromboses are rare thrombotic events with heterogeneous etiologies. They are related to myeloproliferative neoplasms (MPNs) in some patients and can occur as first signs of the disease. MPNs are characterized by mutations in the genes of Janus kinase 2 (JAK2), myeloproliferative leukemia virus oncogene (MPL), and calreticulin (CALR).MethodsWithin the prospective trial “Prevalence of JAK2 mutations in patients with abdominal venous thromboses” (JAK2 MV study; German Clinical Trials Register: DRKS00026943), the peripheral blood of patients with abdominal venous thromboses in Mecklenburg-West Pomerania, a federal state located in north-east Germany, was analyzed by next-generation ultradeep sequencing for MPN-associated mutations. Clinical characteristics and blood cell counts were also of interest. The primary endpoint was the detection of the mutation JAK2 p.V617F. Secondary endpoints were the detection of other acquired variants of JAK2, as well as MPL and CALR.ResultsA total of 68 patients with abdominal venous thromboses were included from February 2017 to January 2021, with splanchnic veins affected in 65 patients. The mutation JAK2 p.V617F was present in 13 patients (19%), with four patients showing low variant allele frequencies (VAF 0.1% to 1.9%). The time interval from the thrombotic event to analysis was longer for patients with the mutation. The mutation MPL p.W515R was detected in three cases, all of them with low VAF. One patient among them had a concurrent mutation of JAK2 p.V617F. The mutations CALR type I or type II were not found.DiscussionBy analyzing peripheral blood for the mutation JAK2 p.V617F, an important cause of these rare thrombotic events can be identified. The development of a diagnostic workup with next-generation ultradeep sequencing for the analysis of the JAK2 p.V617F mutation and further mutations has the potential to better understand the etiology of abdominal venous thromboses in individual patients in regional clinical care, as abdominal venous thromboses are diagnosed by various medical disciplines.

Abstract Fragile X syndrome (FXS) is the most common inherited intellectual disability. FXS is caused by a trinucleotide repeat expansion in the 5′ untranslated region of the FMR1 gene, which leads to gene methylation, transcriptional silencing, and lack of expression of Fragile X Messenger Riboprotein (FMRP). Currently available FXS therapies are inefficient, and the disease severity is highly variable, making it difficult to predict disease trajectory and treatment response. We and others have recently shown that a subset of full-mutation, fully-methylated (FM–FM) males with FXS express low amounts of FMRP which could contribute to phenotypic variability. To better understand the underlying mechanisms, we developed a sensitive qRT-PCR assay to detect FMR1 mRNA in blood. This assay reproducibly detects trace amounts of FMR1 mRNA in a subset of FM–FM males, suggesting that current Southern Blot and PCR determination of FM–FM status is not always associated with complete transcriptional silencing. The functional relevance of trace-level FMR1 mRNA is confirmed by showing a positive correlation with cognitive function; however, phenotypic variability is not fully explained by FMR1 expression. These results corroborate the need for better molecular assays for FXS diagnosis and encourage studies to elucidate the factors contributing to the phenotypic variability of FXS.

BackgroundAcquired epilepsies are caused by an initial brain insult that is followed by epileptogenesis and finally the development of spontaneous recurrent seizures. The mechanisms underlying epileptogenesis are not fully understood. MicroRNAs regulate mRNA translation and stability and are frequently implicated in epilepsy. For example, antagonism of a specific microRNA, miR-324-5p, before brain insult and in a model of chronic epilepsy decreases seizure susceptibility and frequency, respectively. Here, we tested whether antagonism of miR-324-5p during epileptogenesis inhibits the development of epilepsy.MethodsWe used the intrahippocampal kainic acid (IHpKa) model to initiate epileptogenesis in male wild type C57BL/6 J mice aged 6–8 weeks. Twenty-four hours after IHpKa, we administered a miR-324-5p or scrambled control antagomir intracerebroventricularly and implanted cortical surface electrodes for EEG monitoring. EEG data was collected for 28 days and analyzed for seizure frequency and duration, interictal spike activity, and EEG power. Brains were collected for histological analysis.ResultsHistological analysis of brain tissue showed that IHpKa caused characteristic hippocampal damage in most mice regardless of treatment. Antagomir treatment did not affect latency to, frequency, or duration of spontaneous recurrent seizures or interictal spike activity but did alter the temporal development of frequency band-specific EEG power.ConclusionThese results suggest that miR-324-5p inhibition during epileptogenesis induced by status epilepticus does not convey anti-epileptogenic effects despite having subtle effects on EEG frequency bands. Our results highlight the importance of timing of intervention across epilepsy development and suggest that miR-324-5p may act primarily as a proconvulsant rather than a pro-epileptogenic regulator.

The assembly of the mammalian brain is orchestrated by temporally coordinated waves of gene expression. Post-transcriptional regulation by microRNAs (miRNAs) is a key aspect of this program. Indeed, deletion of neuron-enriched miRNAs induces strong developmental phenotypes, and miRNA levels are altered in patients with neurodevelopmental disorders. However, the mechanisms used by miRNAs to instruct brain development remain largely unexplored. Here, we identified miR-218 as a critical regulator of hippocampal assembly. MiR-218 is highly expressed in the hippocampus and enriched in both excitatory principal neurons (PNs) and GABAergic inhibitory interneurons (INs). Early life inhibition of miR-218 results in an adult brain with a predisposition to seizures. Changes in gene expression in the absence of miR-218 suggest that network assembly is impaired. Indeed, we find that miR-218 inhibition results in the disruption of early depolarizing GABAergic signaling, structural defects in dendritic spines, and altered intrinsic membrane excitability. Conditional knockout of Mir218-2 in INs, but not PNs, is sufficient to recapitulate long-term instability. Finally, de-repressing Kif21b and Syt13, two miR-218 targets, phenocopies the effects on early synchronous network activity induced by miR-218 inhibition. Taken together, the data suggest that miR-218 orchestrates formative events in PNs and INs to produce stable networks.

Abstract Fragile X Syndrome (FXS) is caused by a trinucleotide expansion leading to silencing of the FMR1 gene and lack of expression of Fragile X Protein (FXP, formerly known as Fragile X Mental Retardation Protein, FMRP). Phenotypic presentation of FXS is highly variable, and the lack of reproducible, sensitive assays to detect FXP makes evaluation of peripheral FXP as a source of clinical variability challenging. We optimized a Luminex-based assay to detect FXP in dried blot spots for increased reproducibility and sensitivity by improving reagent concentrations and buffer conditions. The optimized assay was used to quantify FXP in 187 individuals. We show that the optimized assay is highly reproducible and detects a wide range of FXP levels. Mosaic individuals had, on average, higher FXP levels than fully methylated individuals, and trace amounts of FXP were consistently detectable in a subset of individuals with full mutation FXS. IQ scores were positively correlated with FXP levels in males and females with full mutation FXS demonstrating the clinical utility of this method. Our data suggest trace amounts of FXP detectable in dried blood spots of individuals with FXS could be clinically relevant and may be used to stratify individuals with FXS for optimized treatment.

Abstract Background Head and neck squamous cell carcinoma (HNSCC) is heterogeneous in etiology, phenotype and biology. Patient-derived xenografts (PDX) maintain morphology and molecular profiling of the original tumors and have become a standard “Avatar” model for human cancer research. However, restricted availability of tumor samples hindered the widespread use of PDX. Most PDX-projects include only surgical specimens because reliable engraftment from biopsies is missing. Therefore, sample collection is limited and excludes recurrent and metastatic, non-resectable cancer from preclinical models as well as future personalized medicine. Methods This study compares the PDX-take rate, -growth, histopathology, and molecular characteristics of endoscopic specimens with surgical specimens. HNSCC samples (n = 55) were collected ad hoc, fresh frozen and implanted into NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ mice. Results Engraftment was successful in both sample types. However, engraftment rate was lower (21 vs. 52%) and growth delayed (11.2 vs. 6.7 weeks) for endoscopic biopsies. Following engraftment, growth kinetic was similar. Comparisons of primary tumors and corresponding PDX models confirmed preservation of histomorphology (HE histology) and molecular profile (Illumina Cancer Hotspot Panel) of the patients’ tumors. Accompanying flow cytometry on primary tumor specimens revealed a heterogeneous tumor microenvironment among individual cases and identified M2-like macrophages as positive predictors for engraftment. Vice versa, a high PD-L1 expression (combined positive score on tumor/immune cells) predicted PDX rejection. Conclusion Including biopsy samples from locally advanced or metastatic lesions from patients with non-surgical treatment strategies, increases the availability of PDX for basic and translational research. This facilitates (pre-) clinical studies for individual response prediction based on immunological biomarkers.

Background: Sensory impairments commonly occur in patients with autism or intellectual disability. Fragile X syndrome (FXS) is one form of intellectual disability that is often comorbid with autism. In electroencephalographic (EEG) recordings obtained from humans with FXS, the ability of cortical regions to consistently synchronize, or “phase-lock”, to modulated auditory stimuli is reduced compared to that of typically developing individuals. At the same time, less time-locked, “non-phase-locked” power induced by sounds is higher. The same changes occur in the Fmr1 knockout (KO) mouse – an animal model of FXS. We determined if Fmr1 deletion in a subset of brainstem auditory neurons plays any role in these EEG changes in the mouse. Methods: We reinstated FMRP expression in a subpopulation of brainstem auditory neurons in an otherwise Fmr1 KO control (conditional on; cON Fmr1) mouse and used EEG recordings to determine if reinstatement normalized, or “rescued”, the phase-locking phenotype observed in the cON Fmr1 mouse. In determining rescue, this also meant that Fmr1 deletion in the same neuron population was necessary for the phenotype to occur. Results: We find that Fmr1 reinstatement in a subset of brainstem neurons rescues certain aspects of the phase-locking phenotype but does not rescue the increase in non-phase-locked power. Unexpectedly, not all electrophysiological phenotypes observed in the Fmr1 KO were observed in the cON Fmr1 mouse used for the reinstatement experiments, and this was likely due to residual expression of FMRP in these Fmr1 KO controls. Conclusions: Fmr1 deletion in brainstem neurons is necessary for certain aspects of the decreased phase-locking phenotype in the Fmr1 KO, but not necessary for the increase in non-phase-locked power induced by a sound. The most likely brainstem structure underlying these results is the inferior colliculus. We also demonstrate that low levels of FMRP can rescue some EEG phenotypes but not others. This latter finding provides a foundation for how symptoms in FXS individuals may vary due to FMRP levels and that reinstatement of low FMRP levels may be sufficient to alleviate particular symptoms.

Abstract Myeloproliferative neoplasms are characterized by mutations in JAK2, MPL and CALR genes. Commonly in diagnostics and previous studies mainly sequencing and common PCR techniques under conventional detection limits are used. Splanchnic vein thromboses are rare, but often appear associated with myeloproliferative neoplasms and represent serious complications. Herein, blood from patients with abdominal vein thromboses in Mecklenburg-West Pomerania (federal district of northern Germany), included in an ongoing prospective prevalence study, was analyzed by next generation sequencing representing the complete protein coding regions of JAK2, MPL and CALR genes with a coverage of > 2000 reads, therefore an ultradeep targeting approach. JAK2 V617F mutations were detected in 11/44 patients. In four of these cases allele frequencies ranged below the conventional cut off of 2%. MPL W515R was detected in 3/44 cases in low frequencies. Very low allele frequencies of JAK2 and MPL variants in patients with abdominal vein thromboses may indicate early manifestations of myeloproliferative neoplasms.

Abstract Background Head and neck squamous cell cancer (HNSCC) frequently causes severe symptoms that may be reduced, when the tumor is successfully treated. The SOCCER trial studied the association of treatment response with patient reported tumor symptom burden in first line treatment of recurrent and/or metastatic HNSCC. Methods In this prospective, multi-center, non-interventional trial patients were treated either with platinum-based chemotherapy and cetuximab or radiotherapy and cetuximab. Tumor symptom burden was assessed every four weeks with a questionnaire containing ten visual analogue scales (VAS, range 0–100), which were summarized to the overall VAS score. Results Fourhundred seventy patients were registered in 97 German centers. A total of 315 patients with at least the baseline and one subsequent questionnaire were available for analysis. Changes in the VAS score were rated as absolute differences from baseline. Negative values indicate improvement of symptoms. The overall VAS score improved significantly at the first post-baseline assessment in responders (− 2.13 vs. non-responders + 1.15, p = 0.048), and even more for the best post-baseline assessment (− 7.82 vs. non-responders − 1.97, p = 0.0005). The VAS for pain (− 16.37 vs. non-responders − 8.89, p = 0.001) and swallowing of solid food (− 16.67 vs. non-responders − 5.06, p = 0.002) improved significantly more in responders (best post-baseline assessment). In the multivariable Cox regression analysis, worse overall VAS scores were associated with worse overall survival (hazard ratio for death 1.12 per 10 points increment on the overall VAS scale, 95% CI 1.05–1.20, p = 0.0009). Conclusion In unselected patients beyond randomized controlled trials, treatment response lowers tumor symptom burden in recurrent and/or metastatic HNSCC. Trial registration ClinicalTrials.gov, NCT00122460 . Registered 22 Juli 2005,

Abstract Objective The pathogenesis of attention‐deficit/hyperactivity disorder (ADHD) is currently unclear. We hypothesized that chronic immune activation, as indexed by T and B cells, plays a role in the pathophysiology of attention problems. Therefore, we examined T and B cell subsets in a general pediatric population with information on attention problems. Methods We included 756 10‐year‐old children from the Generation R population‐based cohort. Eleven‐color flow cytometry was performed on peripheral blood samples to determine T and B cell subsets. The Child Behavior Checklist rated by parents was used to measure attention problems. Data were analyzed using linear regression analyses, adjusting for maternal and child covariates and co‐occurring childhood psychopathology. Results For T helper 1 (Th1) cells, one standard deviation (SD) increase was associated with 5.3% (95%CI 0.3; 10.5) higher attention problem scores. Furthermore, 1SD increase in CD8+ T cells was associated with 7.5% (95%CI 2.4; 12.7) higher attention problem scores. Within total CD8+ T cells, 1SD increase in naive or central memory cells was associated with 6.9% (95%CI 2.0; 12.1) and 6.4% (95%CI 1.5; 11.6) higher attention problem scores, respectively. No associations between Th2, Treg or B memory cells and attention problem scores were observed. Conclusion Higher Th1 and cytotoxic T cell numbers are associated with higher attention problem scores independent of co‐occurring psychopathology. This might indicate a possible role of a pro‐inflammatory immune profile in childhood attention problems.

Fragile X syndrome (FXS) is the most common cause of inherited intellectual disability. FXS is caused by functional loss of the Fragile X Protein (FXP), also known as Fragile X Mental Retardation Protein (FMRP). In humans and animal models, loss of FXP leads to sensory hypersensitivity, increased susceptibility to seizures and cortical hyperactivity. Several components of the GABAergic system, the major inhibitory system in the brain, are dysregulated in FXS, and thus modulation of GABAergic transmission was suggested and tested as a treatment strategy. However, so far, clinical trials using broad spectrum GABAA or GABAB receptor-specific agonists have not yielded broad improvement of FXS phenotypes in humans. Here, we tested a more selective strategy in Fmr1 knockout (KO) mice using the experimental drug BAER-101, which is a selective GABAA α2/α3 agonist. Our results suggest that BAER-101 reduces hyperexcitability of cortical circuits, partially corrects increased frequency-specific baseline cortical EEG power, reduces susceptibility to audiogenic seizures and improves novel object memory. Other Fmr1 KO-specific phenotypes were not improved by the drug, such as increased hippocampal dendritic spine density, open field activity and marble burying. Overall, this work shows that BAER-101 improves select phenotypes in Fmr1 KO mice and encourages further studies into the efficacy of GABAA-receptor subunit-selective agonists for the treatment of FXS.

Epilepsy affects all ages, races, genders, and socioeconomic groups. In about one third of patients, epilepsy is uncontrolled with current medications, leaving a vast need for improved therapies. The causes of epilepsy are diverse and not always known but one gene mutated in a small subpopulation of patients is phosphatase and tensin homolog (PTEN). Moreover, focal cortical dysplasia, which constitutes a large fraction of refractory epilepsies, has been associated with signaling defects downstream of PTEN. So far, most preclinical attempts to reverse PTEN deficiency-associated neurological deficits have focused on mTOR, a signaling hub several steps downstream of PTEN. Phosphoinositide 3-kinases (PI3Ks), by contrast, are the direct enzymatic counteractors of PTEN, and thus may be alternative treatment targets. PI3K activity is mediated by four different PI3K catalytic isoforms. Studies in cancer, where PTEN is commonly mutated, have demonstrated that inhibition of only one isoform, p110β, reduces progression of PTEN-deficient tumors. Importantly, inhibition of a single PI3K isoform leaves critical functions of general PI3K signaling throughout the body intact. Here, we show that this disease mechanism-targeted strategy borrowed from cancer research rescues or ameliorates neuronal phenotypes in male and female mice with neuron-specific PTEN deficiency. These phenotypes include cell signaling defects, protein synthesis aberrations, seizures, and cortical dysplasia. Of note, p110β is also dysregulated and a promising treatment target in the intellectual disability Fragile X syndrome, pointing towards a shared biological mechanism that is therapeutically targetable in neurodevelopmental disorders of different etiologies. Overall, this work advocates for further assessment of p110β inhibition not only in PTEN deficiency-associated neurodevelopmental diseases but also other brain disorders characterized by defects in the PI3K/mTOR pathway.

Introduction: Head and neck cancer patients often suffer from physical and cognitive impairments after cancer treatment. During rehabilitation, exercise therapy can improve physical function and quality of life (QoL). Surveys demonstrated patients’ preference for home training with low- to moderate-intensity. This study was conducted in order to develope a suitable home-based training program. Therefore, the feasibility and effects of a low- to moderate-intensity exercise intervention on physical functions and QoL were evaluated. Methods: Training was conducted as supervised group training and consisted of mobilization, coordination, resistance, stretching, and relaxation exercises. The intervention lasted 12 weeks with 2 training sessions per week. Feasibility, attendance rate, physical function (eg, range of motion, 6-minute walk test [6MWT]), and QoL (eg, EORTC QLQ-30) were analyzed. Results: Ten out of 12 participants completed the intervention (83%) with an average attendance rate of 83%. Participants showed significant improvements in selected physical functions. For example, head rotation increased by 11.2° ( P = .042), walking distance in the 6MWT increased by an average of 43.3 m ( P = .010), and the global QoL scale improved by 8.2 points ( P = .059). Additionally, there were positive changes in the physical function scale ( P = .008), cognitive function scale ( P = .015), and social function scale ( P = .031) of the EORTC QLQ-30. Conclusion : Data indicate that the exercise program was feasible and had positive effects on physical function and QoL. Future research will analyze the effects of a home-based exercise program on physical function and QoL in a large-scale study.

Antibody formation to human(ized) therapeutic antibodies in humans is highly skewed toward anti-idiotype responses, probably because the idiotype is the only ‘foreign’ part of the antibody molecule. Here, we analyzed antibody responses to F(ab’)2 fragments of a panel of 17 human(ized) therapeutic antibodies in rabbits. Homology between the rabbit germline and the human(ized) antibodies is moderate not only for the variable domains (both the complementarity-determining regions and the framework regions), but also for the constant domains (66% or less). Nevertheless, we observed a highly skewed anti-idiotype response in all cases, with up to >90% of the antibodies directed toward the idiotype. These results indicate that the idiotype may be inherently immunodominant. We used these biased responses to raise monoclonal rabbit anti-idiotype antibodies against secukinumab, ustekinumab, reslizumab, mepolizumab, palivizumab, and dupilumab and demonstrate the potential to develop sensitive pharmacokinetic assays with these antibodies.

Abstract Objective Selective IgA deficiency (sIgAD) is the most common primary immunodeficiency in Western countries. Patients can suffer from recurrent infections and autoimmune diseases because of a largely unknown aetiology. To increase insights into the pathophysiology of the disease, we studied memory B and T cells and cytokine concentrations in peripheral blood. Methods We analysed 30 sIgAD patients (12 children, 18 adults) through detailed phenotyping of peripheral B‐cell, CD8+ T‐cell and CD4+ T‐cell subsets, sequence analysis of IGA and IGG transcripts, in vitro B‐cell activation and blood cytokine measurements. Results All patients had significantly decreased numbers of T‐cell‐dependent (TD; CD27+) and T‐cell‐independent (TI; CD27−) IgA memory B cells and increased CD21low B‐cell numbers. IgM+IgD− memory B cells were decreased in children and normal in adult patients. IGA and IGG transcripts contained normal SHM levels. In sIgAD children, IGA transcripts more frequently used IGA2 than controls (58.5% vs. 25.1%), but not in adult patients. B‐cell activation after in vitro stimulation was normal. However, adult sIgAD patients exhibited increased blood levels of TGF‐β1, BAFF and APRIL, whereas they had decreased Th1 and Th17 cell numbers. Conclusion Impaired IgA memory formation in sIgAD patients is not due to a B‐cell activation defect. Instead, decreased Th1 and Th17 cell numbers and high blood levels of BAFF, APRIL and TGF‐β1 might reflect disturbed regulation of IgA responses in vivo. These insights into B‐cell extrinsic immune defects suggest the need for a broader immunological focus on genomics and functional analyses to unravel the pathogenesis of sIgAD.

Due to their large inner surface, nanofiber mats are often used in tissue engineering and examined with respect to cell adhesion, e.g., for cultivation of fibroblasts. The combination of different polymers with a large contact area, however, could also be used for growth of different plants including green microalgae. Here, the cultivation of the microalga Chlamydomonas reinhardtii on different polymer substrates was examined. We investigated growth on two nanofiber mats consisting of polyamide (PA6) and polyacrylonitrile (PAN) and a polypropylene (PP) microfiber mat as substrates, compared with a pure multi-well plate. It was found that the algae were able to grow on all textile mats without change in morphology, indicating that all polymers were non-toxic to the cells. Thus, these nonwovens might be suitable filters for the separation of microalgae in biotechnological processes.

The Drosophila dNab2 protein is an ortholog of human ZC3H14, a poly(A) RNA binding protein required for intellectual function. dNab2 supports memory and axon projection, but its molecular role in neurons is undefined. Here, we present a network of interactions that links dNab2 to cytoplasmic control of neuronal mRNAs in conjunction with the fragile X protein ortholog dFMRP. dNab2 and dfmr1 interact genetically in control of neurodevelopment and olfactory memory, and their encoded proteins co-localize in puncta within neuronal processes. dNab2 regulates CaMKII, but not futsch, implying a selective role in control of dFMRP-bound transcripts. Reciprocally, dFMRP and vertebrate FMRP restrict mRNA poly(A) tail length, similar to dNab2/ZC3H14. Parallel studies of murine hippocampal neurons indicate that ZC3H14 is also a cytoplasmic regulator of neuronal mRNAs. Altogether, these findings suggest that dNab2 represses expression of a subset of dFMRP-target mRNAs, which could underlie brain-specific defects in patients lacking ZC3H14.

Epilepsy is often associated with altered expression or function of ion channels. One example of such a channelopathy is the reduction of A-type potassium currents in the hippocampal CA1 region. The underlying mechanisms of reduced A-type channel function in epilepsy are unclear. Here, we show that inhibiting a single microRNA, miR-324-5p, which targets the pore-forming A-type potassium channel subunit Kv4.2, selectively increased A-type potassium currents in hippocampal CA1 pyramidal neurons in mice. Resting membrane potential, input resistance and other potassium currents were not altered. In a mouse model of acquired chronic epilepsy, inhibition of miR-324-5p reduced the frequency of spontaneous seizures and interictal epileptiform spikes supporting the physiological relevance of miR-324-5p-mediated control of A-type currents in regulating neuronal excitability. Mechanistic analyses demonstrated that microRNA-induced silencing of Kv4.2 mRNA is increased in epileptic mice leading to reduced Kv4.2 protein levels, which is mitigated by miR-324-5p inhibition. By contrast, other targets of miR-324-5p were unchanged. These results suggest a selective miR-324-5p-dependent mechanism in epilepsy regulating potassium channel function, hyperexcitability and seizures.

Fragile X syndrome (FXS) is the most common inherited form of intellectual disability and is associated with increased risk for autism spectrum disorder (ASD), anxiety, ADHD, and epilepsy. While our understanding of FXS pathophysiology has improved, a lack of validated blood-based biomarkers of disease continues to impede bench-to-bedside efforts. To meet this demand, there is a growing effort to discover a reliable biomarker to inform treatment discovery and evaluate treatment target engagement. Such a marker, amyloid-beta precursor protein (APP), has shown potential dysregulation in the absence of fragile X mental retardation protein (FMRP) and may therefore be associated with FXS pathophysiology. While APP is best understood in the context of Alzheimer disease, there is a growing body of evidence suggesting the molecule and its derivatives play a broader role in regulating neuronal hyperexcitability, a well-characterized phenotype in FXS. To evaluate the viability of APP as a peripheral biological marker in FXS, we conducted an exploratory ELISA-based evaluation of plasma APP-related species involving 27 persons with FXS (mean age: 22.0 ± 11.5) and 25 age- and sex-matched persons with neurotypical development (mean age: 21.1 ± 10.7). Peripheral levels of both Aβ(1–40) and Aβ(1–42) were increased, while sAPPα was significantly decreased in persons with FXS as compared to control participants. These results suggest that dysregulated APP processing, with potential preferential β-secretase processing, may be a readily accessible marker of FXS pathophysiology.

Changes in the intestinal microbiota have been associated with the development of immune-mediated diseases in humans. Additionally, the introduction of defined bacterial species into the mouse intestinal microbiota has been shown to impact on the adaptive immune response. However, how much impact the intestinal microbiota composition actually has on regulating adaptive immunity remains poorly understood. Therefore, we studied aspects of the adaptive immunity in healthy adults possessing distinct intestinal microbiota profiles. The intestinal microbiota composition was determined via Illumina sequencing of bacterial 16S rRNA genes extracted from the feces of 35 individuals. Blood B-cell and T-cell subsets from the same individuals were studied using flow cytometry. Finally, the binding of fecal and plasma Immunoglobulin A (IgA) to intestinal bacteria (associated with health and disease) Bacteroides fragilis, Prevotella copri, Bifidobacterium longum, Clostridium difficile, and Escherichia coli was analyzed using ELISA. Unsupervised clustering of microbiota composition revealed the presence of three clusters within the cohort. Cluster 1 and 2 were similar to previously-described enterotypes with a predominance of Bacteroides in Cluster 1 and Prevotella in Cluster 2. The bacterial diversity (Shannon index) and bacterial richness of Cluster 3 was significantly higher than observed in Clusters 1 and 2, with the Ruminococacceae tending to predominate. Within circulating B- and T-cell subsets, only Th subsets were significantly different between groups of distinct intestinal microbiota. Individuals of Cluster 3 have significantly fewer Th17 and Th22 circulating cells, while Th17.1 cell numbers were increased in individuals of Cluster 1. IgA reactivity to intestinal bacteria was higher in plasma than feces, and individuals of Cluster 1 had significant higher plasma IgA reactivity against B. longum than individuals of Cluster 2. In conclusion, we identified three distinct fecal microbiota clusters, of which two clusters resembled previously-described “enterotypes”. Global T-cell and B-cell immunity seemed unaffected, however, circulating Th subsets and plasma IgA reactivity were significantly different between Clusters. Hence, the impact of intestinal bacteria composition on human B cells, T cells and IgA reactivity appears limited in genetically-diverse and environmentally-exposed humans, but can skew antibody reactivity and Th cell subsets.

Since the discovery of the first microRNA 25 years ago, microRNAs (miRNAs) have emerged as critical regulators of gene expression within the mammalian brain. miRNAs are small non-coding RNAs that direct the RNA induced silencing complex to complementary sites on mRNA targets, leading to translational repression and/or mRNA degradation. Within the brain, intra- and extracellular signaling events tune the levels and activities of miRNAs to suit the needs of individual neurons under changing cellular contexts. Conversely, miRNAs shape neuronal communication by regulating the synthesis of proteins that mediate synaptic transmission and other forms of neuronal signaling. Several miRNAs have been shown to be critical for brain function regulating, for example, enduring forms of synaptic plasticity and dendritic morphology. Deficits in miRNA biogenesis have been linked to neurological deficits in humans, and widespread changes in miRNA levels occur in epilepsy, traumatic brain injury, and in response to less dramatic brain insults in rodent models. Manipulation of certain miRNAs can also alter the representation and progression of some of these disorders in rodent models. Recently, microdeletions encompassing MIR137HG, the host gene which encodes the miRNA miR-137, have been linked to autism and intellectual disability, and genome wide association studies have linked this locus to schizophrenia. Recent studies have demonstrated that miR-137 regulates several forms of synaptic plasticity as well as signaling cascades thought to be aberrant in schizophrenia. Together, these studies suggest a mechanism by which miRNA dysregulation might contribute to psychiatric disease and highlight the power of miRNAs to influence the human brain by sculpting communication between neurons.

Seizures are bursts of excessive synchronized neuronal activity, suggesting that mechanisms controlling brain excitability are compromised. The voltage-gated potassium channel Kv4.2, a major mediator of hyperpolarizing A-type currents in the brain, is a crucial regulator of neuronal excitability. Kv4.2 expression levels are reduced following seizures and in epilepsy, but the underlying mechanisms remain unclear. Here, we report that Kv4.2 mRNA is recruited to the RNA-induced silencing complex shortly after status epilepticus in mice and after kainic acid treatment of hippocampal neurons, coincident with reduction of Kv4.2 protein. We show that the microRNA miR-324-5p inhibits Kv4.2 protein expression and that antagonizing miR-324-5p is neuroprotective and seizure suppressive. MiR-324-5p inhibition also blocks kainic-acid-induced reduction of Kv4.2 protein in vitro and in vivo and delays kainic-acid-induced seizure onset in wild-type but not in Kcnd2 knockout mice. These results reveal an important role for miR-324-5p-mediated silencing of Kv4.2 in seizure onset.

Distinct isoforms of the PI3K catalytic subunit have specialized functions in the brain, but their role in cognition is unknown. Here, we show that the catalytic subunit p110β plays an important role in prefrontal cortex (PFC)-dependent cognitive defects in mouse models of Fragile X syndrome (FXS), an inherited intellectual disability. FXS is caused by loss of function of the fragile X mental retardation protein (FMRP), which binds and translationally represses mRNAs. PFC-selective knockdown of p110β, an FMRP target that is translationally upregulated in FXS, reverses deficits in higher cognition in Fmr1 knockout mice. Genetic full-body reduction of p110β in Fmr1 knockout mice normalizes excessive PI3K activity, restores stimulus-induced protein synthesis, and corrects increased dendritic spine density and behavior. Notably, adult-onset PFC-selective Fmr1 knockdown mice show impaired cognition, which is rescued by simultaneous p110β knockdown. Our results suggest that FMRP-mediated control of p110β is crucial for neuronal protein synthesis and cognition.

The PI3K enhancer PIKE links PI3K catalytic subunits to group 1 metabotropic glutamate receptors (mGlu1/5) and activates PI3K signaling. The roles of PIKE in synaptic plasticity and the etiology of mental disorders are unknown. Here, we show that increased PIKE expression is a key mediator of impaired mGlu1/5-dependent neuronal plasticity in mouse and fly models of the inherited intellectual disability fragile X syndrome (FXS). Normalizing elevated PIKE protein levels in FXS mice reversed deficits in molecular and cellular plasticity and improved behavior. Notably, PIKE reduction rescued PI3K-dependent and -independent neuronal defects in FXS. We further show that PI3K signaling is increased in a fly model of FXS and that genetic reduction of the Drosophila ortholog of PIKE, CenG1A rescued excessive PI3K signaling, mushroom body defects, and impaired short-term memory in these flies. Our results demonstrate a crucial role of increased PIKE expression in exaggerated mGlu1/5 signaling causing neuronal defects in FXS.

Carbon fibers belong to the materials of high interest in medical application due to their good mechanical properties and because they are chemically inert at room temperature. Carbon nanofiber mats, which can be produced by electrospinning diverse precursor polymers, followed by thermal stabilization and carbonization, are under investigation as possible substrates for cell growth, especially for possible 3D cell growth applications in tissue engineering. However, such carbon nanofiber mats may be too brittle to serve as a reliable substrate. Here we report on a simple method of creating highly robust carbon nanofiber mats by using electrospun polyacrylonitrile/ZnO nanofiber mats as substrates. We show that the ZnO-blended polyacrylonitrile (PAN) nanofiber mats have significantly increased fiber diameters, resulting in enhanced mechanical properties and thus supporting tissue engineering applications.

TiO2 is a semiconductor that is commonly used in dye-sensitized solar cells (DSSCs). However, the necessity of sintering the TiO2 layer is usually problematic due to the desired temperatures of typically 500 °C in cells that are prepared on polymeric or textile electrodes. This is why textile-based DSSCs often use metal fibers or metallic woven fabrics as front electrodes on which the TiO2 is coated. Alternatively, several research groups investigate the possibilities to reduce the necessary sintering temperatures by chemical or other pre-treatments of the TiO2. Here, we report on a simple method to avoid the sintering step by using a nanofiber mat as a matrix embedding TiO2 nanoparticles. The TiO2 layer can be dyed with natural dyes, resulting in a similar bathochromic shift of the UV/Vis spectrum, as it is known from sintered TiO2 on glass substrates, which indicates an equivalent chemical bonding. Our results indicate a new possibility for producing textile-based DSSCs with TiO2, even on textile fabrics that are not high-temperature resistant.

LysM receptor kinases were identified as receptors of acylated chitin (Nod factors) or chitin produced by plant-interacting microbes. Here, we present the identification and characterization of the LysM receptor kinase gene (Lys) family (17 members) in Lotus japonicus. Comprehensive phylogenetic analysis revealed a correlation between Lys gene structure and phylogeny. Further mapping coupled with sequence-based anchoring on the genome showed that the family has probably expanded by a combination of tandem and segmental duplication events. Using a sliding-window approach, we identified distinct regions in the LysM and kinase domains of recently diverged Lys genes where positive selection may have shaped ligand interaction. Interestingly, in the case of NFR5 and its closest paralog, LYS11, one of these regions coincides with the predicted Nod-factor binding groove and the suggested specificity determining area of the second LysM domain. One hypothesis for the evolutionary diversification of this receptor family in legumes is their unique capacity to decipher various structures of chitin-derived molecules produced by an extended spectrum of interacting organisms: symbiotic, associative, endophytic, and parasitic. In a detailed expression analysis, we found several Lotus Lys genes regulated not only during the symbiotic association with Mesorhizobium loti but also in response to chitin treatment.

Clinical, epidemiological, and genetic evidence suggest overlapping pathogenic mechanisms between autism spectrum disorder (ASD) and schizophrenia. We tested this hypothesis by asking if mutations in the ASD gene MECP2 which cause Rett syndrome affect the expression of genes encoding the schizophrenia risk factor dysbindin, a subunit of the biogenesis of lysosome-related organelles complex-1 (BLOC-1), and associated interacting proteins. We measured mRNA and protein levels of key components of a dysbindin interaction network by, quantitative real time PCR and quantitative immunohistochemistry in hippocampal samples of wild-type and Mecp2 mutant mice. In addition, we confirmed results by performing immunohistochemistry of normal human hippocampus and quantitative qRT-PCR of human inducible pluripotent stem cells (iPSCs)-derived human neurons from Rett syndrome patients. We defined the distribution of the BLOC-1 subunit pallidin in human and mouse hippocampus and contrasted this distribution with that of symptomatic Mecp2 mutant mice. Neurons from mutant mice and Rett syndrome patients displayed selectively reduced levels of pallidin transcript. Pallidin immunoreactivity decreased in the hippocampus of symptomatic Mecp2 mutant mice, a feature most prominent at asymmetric synapses as determined by immunoelectron microcopy. Pallidin immunoreactivity decreased concomitantly with reduced BDNF content in the hippocampus of Mecp2 mice. Similarly, BDNF content was reduced in the hippocampus of BLOC-1 deficient mice suggesting that genetic defects in BLOC-1 are upstream of the BDNF phenotype in Mecp2 deficient mice. Our results demonstrate that the ASD-related gene Mecp2 regulates the expression of components belonging to the dysbindin interactome and these molecular differences may contribute to synaptic phenotypes that characterize Mecp2 deficiencies and ASD.

In legumes rhizobial infection during root nodule symbiosis (RNS) is controlled by a conserved set of receptor proteins and downstream components. MtSYMREM1, a protein of the Remorin family in Medicago truncatula, was shown to interact with at least three receptor-like kinases (RLKs) that are essential for RNS. Remorins are comprised of a conserved C-terminal domain and a variable N-terminal region that defines the six different Remorin groups. While both N- and C-terminal regions of Remorins belonging to the same phylogenetic group are similar to each other throughout the plant kingdom, the N-terminal domains of legume-specific group 2 Remorins show exceptional high degrees of sequence divergence suggesting evolutionary specialization of this protein within this clade. We therefore identified and characterized the MtSYMREM1 ortholog from Lotus japonicus (LjSYMREM1), a model legume that forms determinate root nodules. Here, we resolved its spatio-temporal regulation and showed that over-expression of LjSYMREM1 increases nodulation on transgenic roots. Using a structure-function approach we show that protein interactions including Remorin oligomerization are mainly mediated and stabilized by the Remorin C-terminal region with its coiled-coil domain while the RLK kinase domains transiently interact in vivo and phosphorylate a residue in the N-terminal region of the LjSYMREM1 protein in vitro. These data provide novel insights into the mechanism of this putative molecular scaffold protein and underline its importance during rhizobial infection.