Amber suppression

Calmodulin (CaM) binds to a linker between the oxygenase and reductase domains of nitric oxide synthase (NOS) to regulate the functional conformational dynamics. Specific residues on the interdomain interface guide the domain-domain docking to facilitate the electron transfer in NOS. Notably, the docking interface between CaM and the heme-containing oxygenase domain of NOS is isoform specific, which is only beginning to be investigated. Toward advancing understanding of the distinct CaM-NOS docking interactions by infrared spectroscopy, we introduced a cyano-group as frequency-resolved vibrational probe into CaM individually and when associated with full-length and a bi-domain oxygenase/FMN construct of the inducible NOS isoform (iNOS)...

BACKGROUND: The metabolically demanding nature of immune response requires nutrients to be preferentially directed towards the immune system at the expense of peripheral tissues. We study the mechanisms by which this metabolic reprograming occurs using the parasitoid infection of Drosophila larvae. To overcome such an immune challenge hemocytes differentiate into lamellocytes, which encapsulate and melanize the parasitoid egg. Hemocytes acquire the energy for this process by expressing JAK/STAT ligands upd2 and upd3, which activates JAK/STAT signaling in muscles and redirects carbohydrates away from muscles in favor of immune cells...

CONTEXT: The body has evolved homeostatic mechanisms to maintain free levels of Ca+2 and 1,25-dihydroxyvitamin D [1,25(OH)2D] within narrow physiological ranges. Clinical guidelines emphasize important contributions of PTH in maintaining this homeostasis. OBJECTIVE: To investigate mechanisms of homeostatic regulation of vitamin D (VitD) metabolism and to apply mechanistic insights to improve clinical assessment of VitD status. DESIGN: Crossover clinical trial studying participants before and after VitD3-supplementation...

Emerging microorganism Pseudomonas putida KT2440 is utilized for the synthesis of biobased chemicals from renewable feedstocks and for bioremediation. However, the methods for analyzing, engineering, and regulating the biosynthetic enzymes and protein complexes in this organism remain underdeveloped.Such attempts can be advanced by the genetic code expansion-enabled incorporation of noncanonical amino acids (ncAAs) into proteins, which also enables further controls over the strain's biological processes. Here, we give a step-by-step account of the incorporation of two ncAAs into any protein of interest (POI) in response to a UAG stop codon by two commonly used orthogonal archaeal tRNA synthetase and tRNA pairs...

INTRODUCTION: The BIN1 coding variant rs138047593 (K358R) is linked to Late-Onset Alzheimer's Disease (LOAD) via targeted exome sequencing. METHODS: To elucidate the functional consequences of this rare coding variant on brain amyloidosis and neuroinflammation, we generated BIN1K358R knock-in mice using CRISPR/Cas9 technology. These mice were subsequently bred with 5xFAD transgenic mice, which serve as a model for Alzheimer's pathology. RESULTS: The presence of the BIN1K358R variant leads to increased cerebral amyloid deposition, with a dampened response of astrocytes and oligodendrocytes, but not microglia, at both the cellular and transcriptional levels...

The Sambia Peninsula (Kaliningrad region) is historically well known for its amber mining. The 2019 year was the last year of direct overburden disposal into the Baltic Sea as a part of technological amber mining process. The extremely high-suspended particulate matter concentrations during that disposal were recorded immediately after the discharge of significant volumes of pulp and reached 200 mg/L. The impact of pulp discharge had sequentially suppressed plankton communities development due to the high content of suspended solids and afterwards stimulated plankton development due to the glauconite infusion...

Ferroptosis has been recognized as a unique cell death modality driven by excessive lipid peroxidation and unbalanced cellular metabolism. In this study, we established a protein interaction landscape for ferroptosis pathways through proteomic analyses, and identified choline/ethanolamine phosphotransferase 1 (CEPT1) as a lysophosphatidylcholine acyltransferase 3 (LPCAT3)-interacting protein that regulates LPCAT3 protein stability. In contrast to its known role in promoting phospholipid synthesis, we showed that CEPT1 suppresses ferroptosis potentially by interacting with phospholipases and breaking down certain pro-ferroptotic polyunsaturated fatty acid (PUFA)-containing phospholipids...

Neighborhood is a key context where children learn to process social information; however, the field has largely overlooked the ways children's individual characteristics might be moderated by neighborhood effects. We examined 1,030 six- to 11-year-olds (48.7% female; 82% White) twin pairs oversampled for neighborhood disadvantage from the Twin Study of Behavioral and Emotional Development in Children. We evaluated neighbor reports ( N = 1,880) of neighborhood structural and social characteristics as moderators of genetic and environmental influences on children's social processing...

Intracellular aggregation of transactive response DNA binding protein of 43 kDa (TDP-43) is a hallmark of neurodegenerative diseases such as amyotrophic lateral sclerosis. While primarily a nuclear protein, TDP-43 translocates to the cytosol during cellular stress. Consequences of cytosolic accumulation of TDP-43 is difficult to evaluate in the absence of exogenous toxins. Here, we demonstrate spatiotemporal control over the nuclear import of TDP-43 by installing a photocage (ortho-nitrobenzyl ester) on a single lysine residue (K84) through amber codon suppression in HEK293T cells...

Accurate brain tumour segmentation is critical for tasks such as surgical planning, diagnosis, and analysis, with magnetic resonance imaging (MRI) being the preferred modality due to its excellent visualisation of brain tissues. However, the wide intensity range of voxel values in MR scans often results in significant overlap between the density distributions of different tumour tissues, leading to reduced contrast and segmentation accuracy. This paper introduces a novel framework based on conditional generative adversarial networks (cGANs) aimed at enhancing the contrast of tumour subregions for both voxel-wise and region-wise segmentation approaches...

The HIV-1 core consists of a cone-shaped capsid shell made of capsid protein (CA) hexamers and pentamers encapsulating the viral genome. HIV-1 capsid disassembly, referred to as uncoating, is important for productive infection; however, the location, timing, and regulation of uncoating remain controversial. Here, we employ amber codon suppression to directly label CA. In addition, a fluid phase fluorescent probe is incorporated into the viral core to detect small defects in the capsid lattice. This double-labeling strategy enables the visualization of uncoating of single cores in vitro and in living cells, which we found to always proceed through at least two distinct steps─the formation of a defect in the capsid lattice that initiates gradual loss of CA below a detectable level...

BACKGROUND: T cell responses are impaired in Staphylococcus aureus-infected children, highlighting a potential mechanism of immune evasion. This study tested the hypotheses that toxin-specific antibodies protect immune cells from bacterial killing and are associated with improved T cell function following infection. METHODS: S. aureus-infected and healthy children (n = 33 each) were prospectively enrolled. During acute infection and convalescence, we quantified toxin-specific IgG levels by ELISA, antibody function using a cell-killing assay, and functional T cell responses by ELISpot...

Structural dynamics of human immunodeficiency virus 1 (HIV-1) envelope (Env) glycoprotein mediate cell entry and facilitate immune evasion. Single-molecule FRET using peptides for Env labeling revealed structural dynamics of Env, but peptide use risks potential effects on structural integrity/dynamics. While incorporating noncanonical amino acids (ncAAs) into Env by amber stop-codon suppression, followed by click chemistry, offers a minimally invasive approach, this has proved to be technically challenging for HIV-1...

How cells coordinate cell cycling with cell survival and death remains incompletely understood. Here, we show that cell cycle arrest has a potent suppressive effect on ferroptosis, a form of regulated cell death induced by overwhelming lipid peroxidation at cellular membranes. Mechanistically, cell cycle arrest induces diacylglycerol acyltransferase (DGAT)-dependent lipid droplet formation to sequester excessive polyunsaturated fatty acids (PUFAs) that accumulate in arrested cells in triacylglycerols (TAGs), resulting in ferroptosis suppression...

Photochromic fluorescent proteins (FPs) have proved to be indispensable luminous probes for sophisticated and advanced bioimaging techniques. Among them, an interplay between photoswitching and photoconversion has only been observed in a limited subset of Kaede-like FPs that show potential for discovering the key mechanistic steps during green-to-red photoconversion. Various spectroscopic techniques including femtosecond stimulated Raman spectroscopy (FSRS), X-ray crystallography, and femtosecond transient absorption were employed on a set of five related FPs with varying photoconversion and photoswitching efficiencies...

(1) Introduction: Claudin-9 (CLDN9) is a member of the claudin protein family, a critical transmembrane protein family for tight junctions that are implemented in the progression of numerous cancer types. The present study investigated the role that CLDN9, along with the subcoat proteins, Zonula Occludens (ZOs), plays in clinical breast cancer and subsequent impact on drug response of patients. (2) Methods: CLDN9 protein and CLDN9 transcript were determined and correlated with clinical and pathological indicators, together with the status of hormonal receptors...

Deep eutectic solvents (DESs) emerged as potential alternative solvent media in multiple areas, including biomolecular (cryo)preservation. Herein, we studied the stability of a small protein (ubiquitin) in water and a betaine-glycerol-water (B:G:W) (1:2:ζ; ζ = 0, 1, 2, 5, 10) DES, through molecular dynamics. An AMBER-based model that accurately describes the density and shear viscosity of the DES is proposed. We find that water molecules are largely trapped in the solvent, precluding the formation of a full hydration layer, seemingly opposite to osmolytes' preferential exclusion/preferential hydration mechanism...

Lipid droplets (LDs) are dynamic organelles with a neutral lipid core surrounded by a phospholipid monolayer. Solid tumors exhibit LD accumulation, and it is believed that LDs promote cell survival by providing an energy source during energy deprivation. However, the precise mechanisms controlling LD accumulation and utilization in prostate cancer are not well known. Here, we show peroxisome proliferator-activated receptor α (PPARα) acts downstream of PIM1 kinase to accelerate LD accumulation and promote cell proliferation in prostate cancer...

Functionalization of proteins by incorporating reactive non-canonical amino acids (ncAAs) has been widely applied for numerous biological and therapeutic applications. The requirement not to lose the intrinsic properties of these proteins is often underestimated and not considered. Main purpose of this study was to answer the question whether functionalization via residue-specific incorporation of the ncAA N6 -[(2-Azidoethoxy) carbonyl]-l-lysine (Azk) influences the properties of the anti-tumor-necrosis-factor-α-Fab (FTN2)...

BACKGROUND: Multisystem inflammatory syndrome in children (MIS-C) represents a hyperinflammatory state that can result in multi-organ dysfunction and death. Myeloid-derived suppressor cells (MDSC) are an immunosuppressive cell population that expands under inflammatory conditions and suppresses T cell function. We hypothesized that MDSC would be increased in children with MIS-C and that MDSC expansion would be associated with T cell lymphopenia. METHODS: We conducted a prospective, observational study...