Isolating Synaptic Vesicles from Neurospheres for Proteomics.

This chapter presents an optimized method for isolating synaptic vesicles (SVs) from neurospheres derived from human induced pluripotent stem cells (hiPSCs). The protocol begins with neurosphere cultivation to achieve mature neurons, which is essential for the functional studies of neuronal activity. Following this, neurosphere-derived synaptosomes are isolated, and SVs are enriched through differential centrifugation.

A proteome-wide yeast degron collection for the dynamic study of protein function.

Genome-wide collections of yeast strains, known as libraries, revolutionized the way systematic studies are carried out. Specifically, libraries that involve a cellular perturbation, such as the deletion collection, have facilitated key biological discoveries. However, short-term rewiring and long-term accumulation of suppressor mutations often obscure the functional consequences of such perturbations.

Gα and Gα/Gα deletion differentially affect hippocampal mossy fiber tract anatomy and neuronal morphogenesis.

The heterotrimeric G-protein αo subunit is ubiquitously expressed in the CNS as two splice variants Gα and Gα, regulating various brain functions. Here, we investigated the effect of single Gα, Gα, and double Gα knockout on the postnatal development of the murine mossy fiber tract, a central pathway of the hippocampal connectivity circuit. The size of the hippocampal synaptic termination fields covered by mossy fiber boutons together with various fiber length parameters of the tract was analyzed by immunohistochemical staining of the vesicular Zinc transporter 3 (ZnT3) or Synaptoporin at postnatal days 2, 4, 8, 12, 16, and in the adult.

A type III-Dv CRISPR-Cas system is controlled by the transcription factor RpaB and interacts with the DEAD-box RNA helicase CrhR.

How CRISPR-Cas systems defend bacteria and archaea against invading genetic elements is well understood, but less is known about their regulation. In the cyanobacterium Synechocystis sp. PCC 6803, the expression of one of the three different CRISPR-Cas systems responds to changes in environmental conditions.

Proteome profiling of Campylobacter jejuni 81-176 at 37 °C and 42 °C by label-free mass spectrometry.

Background: The main natural reservoir for Campylobacter jejuni is the avian intestinal tract. There, C. jejuni multiplies optimally at 42 °C - the avian body temperature.

All SNAP25 molecules in the vesicle-plasma membrane contact zone change conformation during vesicle priming.

In neuronal cell types, vesicular exocytosis is governed by the SNARE (soluble NSF attachment receptor) complex consisting of synaptobrevin2, SNAP25, and syntaxin1. These proteins are required for vesicle priming and fusion. We generated an improved SNAP25-based SNARE COmplex Reporter (SCORE2) incorporating mCeruelan3 and Venus and overexpressed it in SNAP25 knockout embryonic mouse chromaffin cells.

Yeast gene (alias ) operates in the initiation step of diphthamide synthesis on elongation factor 2.

In yeast, Elongator-dependent tRNA modifications are regulated by the Kti11•Kti13 dimer and hijacked for cell killing by zymocin, a tRNase ribotoxin. Kti11 (alias Dph3) also controls modification of elongation factor 2 (EF2) with diphthamide, the target for lethal ADP-ribosylation by diphtheria toxin (DT). Diphthamide formation on EF2 involves four biosynthetic steps encoded by the network and an ill-defined function.

A Study of the Activity of Adamantyl Amines against Mutant Influenza A M2 Channels Identified a Polycyclic Cage Amine Triple Blocker, Explored by Molecular Dynamics Simulations and Solid-State NMR.

We compared the anti-influenza potencies of 57 adamantyl amines and analogs against influenza A virus with serine-31 M2 proton channel, usually termed as WT M2 channel, which is amantadine sensitive. We also tested a subset of these compounds against viruses with the amantadine-resistant L26F, V27A, A30T, G34E M2 mutant channels. Four compounds inhibited WT M2 virus in vitro with mid-nanomolar potency, with 27 compounds showing sub-micromolar to low micromolar potency.

Tfap2b acts in GABAergic neurons to control sleep in mice.

Sleep is a universal state of behavioral quiescence in both vertebrates and invertebrates that is controlled by conserved genes. We previously found that AP2 transcription factors control sleep in C. elegans, Drosophila, and mice.

Inositol pyrophosphates activate the vacuolar transport chaperone complex in yeast by disrupting a homotypic SPX domain interaction.

Many proteins involved in eukaryotic phosphate homeostasis are regulated by SPX domains. In yeast, the vacuolar transporter chaperone (VTC) complex contains two such domains, but mechanistic details of its regulation are not well understood. Here, we show at the atomic level how inositol pyrophosphates interact with SPX domains of subunits Vtc2 and Vtc3 to control the activity of the VTC complex.

Protein-metabolite interactomics of carbohydrate metabolism reveal regulation of lactate dehydrogenase.

Metabolic networks are interconnected and influence diverse cellular processes. The protein-metabolite interactions that mediate these networks are frequently low affinity and challenging to systematically discover. We developed mass spectrometry integrated with equilibrium dialysis for the discovery of allostery systematically (MIDAS) to identify such interactions.

Bacterial Cellulose-Based Materials as Dressings for Wound Healing.

Bacterial cellulose (BC) is produced by several microorganisms as extracellular structures and can be modified by various physicochemical and biological strategies to produce different cellulosic formats. The main advantages of BC for biomedical applications can be summarized thus: easy moldability, purification, and scalability; high biocompatibility; and straightforward tailoring. The presence of a high amount of free hydroxyl residues, linked with water and nanoporous morphology, makes BC polymer an ideal candidate for wound healing.

Conserved reduction of mA RNA modifications during aging and neurodegeneration is linked to changes in synaptic transcripts.

N-methyladenosine (mA) regulates mRNA metabolism. While it has been implicated in the development of the mammalian brain and in cognition, the role of mA in synaptic plasticity, especially during cognitive decline, is not fully understood. In this study, we employed methylated RNA immunoprecipitation sequencing to obtain the mA epitranscriptome of the hippocampal subregions CA1, CA3, and the dentate gyrus and the anterior cingulate cortex (ACC) in young and aged mice.

ORE-Seq: Genome-Wide Absolute Occupancy Measurement by Restriction Enzyme Accessibilities.

Digestion with restriction enzymes is a classical approach for probing DNA accessibility in chromatin. It allows to monitor both the cut and the uncut fraction and thereby the determination of accessibility or occupancy (= 1 - accessibility) in absolute terms as the percentage of cut or uncut molecules, respectively, out of all molecules. The protocol presented here takes this classical approach to the genome-wide level.

Pharmacological applications of nitric oxide-releasing biomaterials in human skin.

Nitric oxide (NO) is an important endogenous molecule that plays several roles in biological systems. NO is synthesized in human skin by three isoforms of nitric oxide synthase (NOS) and, depending on the produced NO concentration, it can actuate in wound healing, dermal vasodilation, or skin defense against different pathogens, for example. Besides being endogenously produced, NO-based pharmacological formulations have been developed for dermatological applications targeting diverse pathologies such as bacterial infection, wound healing, leishmaniasis, and even esthetic issues such as acne and skin aging.