FACS purification and transcriptome analysis of drosophila neural stem cells reveals a role for Klumpfuss in self-renewal

Drosophila neuroblasts (NBs) have emerged as a model for stem cell biology that is ideal for genetic analysis but is limited by the lack of cell-type-specific gene expression data. Here, we describe a method for isolating large numbers of pure NBs and differentiating neurons that retain both cell-cycle and lineage characteristics. We determine transcriptional profiles by mRNA sequencing and identify 28 predicted NB-specific transcription factors that can be arranged in a network containing hubs for Notch signaling, growth control, and chromatin regulation. Overexpression and RNA interference for these factors identify Klumpfuss as a regulator of self-renewal. We show that loss of Klumpfuss function causes premature differentiation and that overexpression results in the formation of transplantable brain tumors. Our data represent a valuable resource for investigating Drosophila developmental neurobiology, and the described method can be applied to other invertebrate stem cell lineages as well

Ketamine Alters Functional Gamma and Theta Resting-State Connectivity in Healthy Humans: Implications for Schizophrenia Treatment Targeting the Glutamate System

Disturbed functional connectivity is assumed to cause neurocognitive deficits in patients suffering from schizophrenia. A Glutamate N-methyl-D-aspartate receptor (NMDAR) dysfunction has been suggested as a possible mechanism underlying altered connectivity in schizophrenia, especially in the gamma- and theta-frequency range. The present study aimed to investigate the effects of the NMDAR-antagonist ketamine on resting-state power, functional connectivity, and schizophrenia-like psychopathological changes in healthy volunteers. In a placebo-controlled crossover design, 25 healthy subjects were recorded using resting-state 64-channel-electroencephalography (EEG) (eyes closed). The imaginary coherence-based Multivariate Interaction Measure (MIM) was used to measure gamma and theta connectivity across 80 cortical regions. The network-based statistic was applied to identify involved networks under ketamine. Psychopathology was assessed with the Positive and Negative Syndrome Scale (PANSS) and the 5-Dimensional Altered States of Consciousness Rating Scale (5D-ASC). Ketamine caused an increase in all PANSS (p < 0.001) as well as 5D-ASC scores (p < 0.01). Significant increases in resting-state gamma and theta power were observed under ketamine compared to placebo (p < 0.05). The source-space analysis revealed two distinct networks with an increased mean functional gamma- or theta-band connectivity during the ketamine session. The gamma-network consisted of midline regions, the cuneus, the precuneus, and the bilateral posterior cingulate cortices, while the theta-band network involved the Heschl gyrus, midline regions, the insula, and the middle cingulate cortex. The current source density (CSD) within the gamma-band correlated negatively with the PANSS negative symptom score, and the activity within the gamma-band network correlated negatively with the subjective changed meaning of percepts subscale of the 5D-ASC. These results are in line with resting-state patterns seen in people who have schizophrenia and argue for a crucial role of the glutamate system in mediating dysfunctional gamma- and theta-band-connectivity in schizophrenia. Resting-state networks could serve as biomarkers for the response to glutamatergic drugs or drug development efforts within the glutamate system

A community effort towards a knowledge-base and mathematical model of the human pathogen Salmonella Typhimurium LT2

<p>Abstract</p> <p>Background</p> <p>Metabolic reconstructions (MRs) are common denominators in systems biology and represent biochemical, genetic, and genomic (BiGG) knowledge-bases for target organisms by capturing currently available information in a consistent, structured manner. <it>Salmonella enterica </it>subspecies I serovar Typhimurium is a human pathogen, causes various diseases and its increasing antibiotic resistance poses a public health problem.</p> <p>Results</p> <p>Here, we describe a community-driven effort, in which more than 20 experts in <it>S</it>. Typhimurium biology and systems biology collaborated to reconcile and expand the <it>S</it>. Typhimurium BiGG knowledge-base. The consensus MR was obtained starting from two independently developed MRs for <it>S</it>. Typhimurium. Key results of this reconstruction jamboree include i) development and implementation of a community-based workflow for MR annotation and reconciliation; ii) incorporation of thermodynamic information; and iii) use of the consensus MR to identify potential multi-target drug therapy approaches.</p> <p>Conclusion</p> <p>Taken together, with the growing number of parallel MRs a structured, community-driven approach will be necessary to maximize quality while increasing adoption of MRs in experimental design and interpretation.</p

Induction and regulation of CAMP gene expression

Örverudrepandi peptíðið LL-37 er af flokki cathelicidina og er mikilvægt sem fyrsta vörn okkar gegn bakteríum. Örvun á tjáningu varnarþátta eins og LL-37 gæti verið ný leið til að ráða niðurlögum baktería þar sem vanaleg sýklalyf hafa brugðist. Áhrif 4-phenylbutyrate (PBA) á tjáningu bakteríudrepandi peptíða var athuguð. Fyrst voru áhrif PBA á tjáningu cathelicidin antimicrobial peptide (CAMP) mRNAsins greind í nokkrum frumulínum með PCR greiningu í rauntíma. Tjáning gensins reyndist örvuð í öllum frumulínunum. Annað gen DEFB1 sem skráir bakteríudrepandi peptíðið human beta defensin 1 sýndi líka aukna tjáningu í lungnaþekjufrumulínu (VA10) en minnkaða í monocytum (U937). Framhaldstilraunir í verkefninu lutu eingöngu að spurningum tengdum tjáningu CAMP genins í VA10 frumulínunni. Þessar tilraunir sýndu að örvun af völdum PBA magnast með 1,25-dihydroxyvítamín D3 viðbót hvort sem litið er á CAMP mRNA eða prótín. Þá kom í ljós að PBA örvunin er óbein og háð virkri þýðingu en cyclohexamide sem hindrar prótínþýðingu kom í veg fyrir örvunina. Einnig var unnt að draga úr örvuninni með hindrum fyrir prótín kínasana MEK1/2 og JNK. Í lokin sýndum við að -methylhydrocinnamate (ST7), sem líkist PBA, gat aukið genatjáninguna. Niðurstöður rannsóknanna auka skilning okkar á tjáningu bakteríudrepandi peptíða og gefa vísbendingu um að PBA og/eða ST7 gætu virkað sem lyf gegn bakteríusýkingum með því að efla náttúrulegt varnarkerfi okkar

Virtual Screening for Promising Kinetic Stabilizers of Light Chains in Immunoglobulin Light Chain Amyloidosis Through Drug Repurposing

In immunoglobulin light chain amyloidosis, unstable immunoglobulin light chains aggregate as amyloid fibrils in organs, leading to organ failure and death in more than 50% of untreated patients. Research by Morgan et al. (Morgan, G. J. et. al., Proceedings of the National Academy of Sciences 2019, 116, 8360–8369) and Yan et al. (Yan, N. L. et. al., Journal of Medicinal Chemistry 2021, 64, 6273–6299) has shown that several classes of small molecules can be used as kinetic stabilizers of native light chains and potentially slow or stop the disease. Based on their criteria of a clinically successful kinetic stabilizer, this study includes an in-silico virtual screening of drugs approved by the U.S. Food and Drug Administration to find promising candidates of kinetic stabilizers through drug repurposing. Of these candidates, molecular dynamics simulations and subsequent studies of molecular mechanics with generalized Born and surface area solvation suggest that delavirdine and pazopanib can potentially be used as kinetic stabilizers of native light chains

Phenylbutyrate Induces Antimicrobial Peptide Expression ▿

Antimicrobial peptides (AMPs) are important components of our first line of defense. Induction of AMPs such as LL-37 of the cathelicidin family might provide a novel approach in treating bacterial infections. In this study we identified 4-phenylbutyrate (PBA) as a novel inducer of AMP expression and investigated affected regulatory pathways. We treated various cell lines with PBA and assessed mRNA expression by real-time reverse transcriptase PCR (RT-PCR). Cathelicidin AMP (CAMP) gene expression was found to be upregulated in all four cell lines tested. Additionally, we found that the beta-defensin 1 gene was upregulated in the lung epithelial cell line VA10 while being downregulated in the monocytic cell line U937. Further we found that PBA induced CAMP gene expression synergistically with 1,25-dihydroxyvitamin D3 at both protein and mRNA levels. The general mechanism of induction of CAMP gene expression by PBA was found to be dependent on protein synthesis. Results from quantitative chromatin immunoprecipitation experiments challenge the common view that histone deacetylase inhibitors directly increase CAMP gene expression. Furthermore, we have demonstrated that inhibition of the mitogen-activated protein kinases MEK1/2 and c-Jun N-terminal kinase attenuate PBA-induced CAMP gene expression. Similarly, α-methylhydrocinnamate (ST7), an analogue of PBA, increases CAMP gene expression. Our findings contribute to understanding of the regulation of AMP expression and suggest that PBA and/or ST7 is a promising drug candidate for treatment of microbial infections by strengthening the epithelial antimicrobial barriers

Opposite Modulation of the NMDA Receptor by Glycine and S-Ketamine and the Effects on Resting State EEG Gamma Activity: New Insights into the Glutamate Hypothesis of Schizophrenia

NMDA-receptor hypofunction is increasingly considered to be an important pathomechanism in schizophrenia. However, to date, it has not been possible to identify patients with relevant NMDA-receptor hypofunction who would respond to glutamatergic treatments. Preclinical models, such as the ketamine model, could help identify biomarkers related to NMDA-receptor function that respond to glutamatergic modulation, for example, via activation of the glycine-binding site. We, therefore, aimed to investigate the effects of opposing modulation of the NMDA receptor on gamma activity (30–100 Hz) at rest, the genesis of which appears to be highly dependent on NMDA receptors. The effects of subanesthetic doses of S-ketamine and pretreatment with glycine on gamma activity at rest were examined in twenty-five healthy male participants using 64-channel electroencephalography. Psychometric scores were assessed using the PANSS and the 5D-ASC. While S-ketamine significantly increased psychometric scores and gamma activity at the scalp and in the source space, pretreatment with glycine did not significantly attenuate any of these effects when controlled for multiple comparisons. Our results question whether increased gamma activity at rest constitutes a suitable biomarker for the target engagement of glutamatergic drugs in the preclinical ketamine model. They might further point to a differential role of NMDA receptors in gamma activity generation