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Drugs active at G protein–coupled receptors (GPCRs) can differentially modulate either canonical or noncanonical signaling pathways via a phenomenon known as functional selectivity or biased signaling. We report biochemical studies showing that the hallucinogen lysergic acid diethylamide, its precursor ergotamine (ERG), and related ergolines display strong functional selectivity for β-arrestin signaling at the 5-HT 2B 5-hydroxytryptamine (5-HT) receptor, whereas they are relatively unbiased at the 5-HT 1B receptor. To investigate the structural basis for biased signaling, we determined the crystal structure of the human 5-HT 2B receptor bound to ERG and compared it with the 5-HT 1B /ERG structure. Given the relatively poor understanding of GPCR structure and function to date, insight into different GPCR signaling pathways is important to better understand both adverse and favorable therapeutic activities.

Using mRNA sequencing and de novo transcriptome assembly, we identified, cloned, and characterized 9 previously undiscovered fluorescent protein (FP) homologs from Aequorea victoria and a related Aequorea species, with most sequences highly divergent from A . victoria green fluorescent protein (avGFP). Among these FPs are the brightest green fluorescent protein (GFP) homolog yet characterized and a reversibly photochromic FP that responds to UV and blue light. Beyond green emitters, Aequorea species express purple- and blue-pigmented chromoproteins (CPs) with absorbances ranging from green to far-red, including 2 that are photoconvertible. X-ray crystallography revealed that Aequorea CPs contain a chemically novel chromophore with an unexpected crosslink to the main polypeptide chain. Because of the unique attributes of several of these newly discovered FPs, we expect that Aequorea will, once again, give rise to an entirely new generation of useful probes for bioimaging and biosensing.

G protein–coupled receptors (GPCRs) are important pharmaceutical targets. Knowledge of their 3D structures is critical to understanding mechanisms of drug action. Low cellular expression, purification yield, and in vitro instability are substantial hurdles to the successful determination of GPCR structure. Intense effort is required to optimize a receptor’s protein sequence and purification procedure, increasing the complexity of the precrystallization process. Here, we present a procedure for a small-scale precrystallization screen that involves detecting GPCR expression levels in Spodoptera frugiperda ( Sf 9) culture by flow cytometry and evaluating GPCR stability by size-exclusion chromatography and UV absorbance measurements. The example procedure uses the smallest volumes of Sf 9 cell culture that will yield sufficient quantities of purified protein for intrinsic UV absorbance analysis and is amenable to medium throughput with the same constructs and conditions that would be scaled up for crystallization trials. The protocol takes 8 d to complete and requires expertise in cell culture, protein purification, and chromatography. G protein–coupled receptors (GPCRs) play a central role in physiological processes and are common drug targets. This GPCR precrystallization screen uses small culture volumes to determine which conditions result in maximal protein expression and stability.

Purpose. To investigate the efficacy of XingZhi-YiNao (XZYN) granules and hyperbaric oxygenation (HBO) for cognition and motor dysfunction in patients with delayed encephalopathy after acute carbon monoxide poisoning (DEACMP). Methods. Eighty-nine patients with DEACMP were randomly divided into control group (n=19), HBO group (n=32), and XZYN group (n=38). All patients received conventional treatment. HBO group received HBO therapy once daily. XZYN group received extra XZYN granules plus HBO treatment. The related indexes including activity of daily living (ADL) scale, Montreal cognitive assessment (MoCA) scale, and mini mental state examination (MMSE) scale were measured. Cerebral white matter injury, age related white matter changes (ARWMC) scale, and the amplitude and latency of P300 were assessed. Results. Compared with control group, the neurological function scores of ADL, MoCA, and MMSE in HBO and XZYN groups were significantly improved, the impairment degree of brain white matter and cognition function were obviously alleviated, the latencies of P300 were significantly shortened, and the amplitudes of P300 were evidently increased (P<0.05). Treatment efficacy of XZYN group was superior to that of HBO group (P<0.05). Conclusion. Combined XZYN granules and HBO can significantly improve cognition and motor functions in patients with DEACMP.

In addition to transcripts encoding an FP clearly homologous to A. victoria green fluorescent protein (avGFP), as we expected, the A. cf. australis transcriptome also contains several transcripts encoding other, much more divergent avGFP homologs. Photophysical properties of fluorescent proteins described in this study derived from A. victoria and A. cf. australis. https://doi.org/10.1371/journal.pbio.3000936.t001 Intrigued by the diversity of optical properties in the A. cf. australis FP homologs, we next investigated a sample of A. victoria from the Crystal Jelly exhibit at the Birch Aquarium at Scripps to determine whether this species also contained multiple diverse FPs. The data underlying this figure (nucleotide sequences of the FPs from this study) may be found in GenBank, accession numbers MN114103 through MN114112. avGFP, Aequorea victoria green fluorescent protein; FP, fluorescent protein. https://doi.org/10.1371/journal.pbio.3000936.g003 Multiple, diverse Aequorea GFPs As expected, both Aequorea species abundantly express close homologs of avGFP. Though brightly fluorescent, AausFP1 is largely insoluble in this context, and when purified, the soluble fraction of the protein runs as a high-molecular-weight aggregate on size exclusion chromatography (Fig BB in S1 Text).

The nucleocapsid protein (NCP) from Mason-Pfizer monkey virus (MPMV) contains two evolutionary invariant Cys-X2–Cys-X4–His-X4–Cys retroviral-type zinc finger structures, where the Cys and His residues provide ligands to a tetrahedrally coordinated Zn(II) ion. The N-terminal zinc finger (F1) of NCP from MPMV contains an immediately contiguous Cys in the –1 position relative to the start of this conserved motif: Cys-Cys-X2–Cys-X4–His-X4–Cys. Metal complexes of 18-amino acid peptides which model the native zinc finger sequence, SER-Cys-X2–Cys-X4–His-X4–Cys (F1_SC), and non-native Cys-SER-X2–Cys-X4–His-X4–Cys (F1_CS) and SER-SER-X2–Cys-X4–His-X4–Cys (F1_SS) sequences have been spectroscopically characterized and compared to the native two-zinc-finger protein fragment, MPMV NCP 21–80. All Co(II)-substituted peptide complexes adopt tetrahedral ligand geometries and have S–→Co(II) ligand-to-metal charge-transfer (LMCT) transition intensities consistent with three Co(II)-S bonds for F1_SC and F1_CS. The non-native F1_CS peptide binds Co(II) with KCo=1.5×106 M–1, comparable to that of the native complex, and ≈100-fold tighter than F1_SS. Like the Co(II) derivative, the absorption spectrum of Ni(II)-substituted NCP 21–80 is most consistent with tetrahedral Ni(II) complexes with multiple thiolate donors. In contrast, Ni(II) complexes of F1_SC and F1_CS exhibit a single absorption band in the 400–550 nm region (ε≈200–300 M–1 cm–1), distinct in the two complexes, assignable to a degenerate d-d transition envelope characteristic of non-native square-planar coordination geometry, and an intense LMCT transition in the UV (ε255≈14,000 M–1 cm–1). Cd(II) complexes have intense absorption in the UV (λmax=233 nm), with absolute intensities consistent with ≈5000 M–1 cm–1 per Cd(II)-S bond. 113Cd NMR spectroscopy of 113Cd MPMV NCP gives δ=649 ppm, consistent with S3N coordination. Co(II) and Cd(II) complexes of non-native F1_CS peptides are more sensitive to oxidation by O2, relative to F1_SC, suggestive of a higher lability in the non-native chelate. The implications of these findings for the evolutionary conservation of this motif are discussed.

Background Von Hippel–Lindau (VHL) syndrome is a dominantly inherited multisystem cancer syndrome caused by a heterozygous mutation in the VHL tumor suppressor gene. Previous studies suggested that similar populations of Caucasian and Japanese patients have similar genotype or phenotype characteristics. In this comprehensive study of East Asian patients, we investigated the genetic and clinical characteristics of patients with VHL syndrome. Methods To create a registry of clinical characteristics and mutations reported in East Asian patients with VHL syndrome, we conducted a comprehensive review of English language and non‐English language articles identified through a literature search. Publications in Japanese or Chinese language were read by native speakers of the language, who then performed the data extraction. Results Of 237 East Asian patients with VHL syndrome, 154 unique kindreds were identified for analysis. Analyzed by kindred, missense mutations were the most common (40.9%, 63/154), followed by large/complete deletions (32.5%, 50/154) and nonsense mutations (11.7%, 18/154). Compared with a previously reported study of both East Asian and non‐East Asian patients, we found several key differences. First, missense and frameshift mutations in the VHL gene occurred less commonly in our population of East Asian patients (40.9% vs. 52.0%; P = 0.012 and 8.4% vs. 13.0%; P < 0.001, respectively). Second, large/complete deletions were more common in our population of East Asian patients (32.5% vs. 10.5%; P < 0.001). Third, phenotypically, we observed that, in our population of East Asian patients with VHL syndrome, the incidence of retinal capillary hemangioblastoma was lower, whereas the incidence of renal cell carcinoma was higher. Conclusions Evidence suggests that the genotypic and phenotypic characteristics of East Asian patients with VHL syndrome differ from other populations. This should be considered when making screening recommendations for VHL syndrome in Asia.

Infection of Tabasco pepper (Capsicum frutescens L. cv. Tabasco) by tobacco etch virus (TEV) typically causes wilting due to root necrosis. A recently isolated strain of TEV, designated TEV non-wilting (TEV NW), causes mosaic symptoms but not wilting in Tabasco pepper. It also induces larger local lesions on Chenopodium amaranticolor than the type strain of TEV, TEV HAT. In order to find the mutations responsible for the inability of TEV NW to cause wilting, the complete nucleotide sequence of TEV NW was determined. TEV NW has 9496 nucleotides and a polyadenylated tail; translation of a single putative polyprotein can be deduced. Compared to the published sequence of TEV HAT, there are 767 nucleotide and 126 amino acid changes scattered throughout the TEV NW genome. By using the available TEV NW sequence and its derived restriction map, a TEV NW infectious clone, pTEVNW, was constructed based on the TEV HAT infectious clone, pTEV7DA. The progeny virus generated from pTEVNW caused mosaic but not wilting symptoms on Tabasco pepper, and produced the same size local lesions on C. amaranticolor as TEV NW. The location of the wilting determinants was pursued by exchanging DNA fragments between pTEVNW and pTEV7DA. Substitutions of the individual P1, HC, P3, NIb and CP coding regions of TEV NW into TEV HAT did not alter the wilting response. When the pTEV7DA EcoR I (4264)-BamH I (5916) or Eco47 III (4967)-BamH I (5916) fragments, containing the C-terminal portion of CI and the entire 6-kDa coding sequence, were inserted into pTEVNW, the resulting chimeric viruses elicited wilting in Tabasco pepper. Therefore, the EcoR I (4264)-BamH I (5916) region contains sufficient sequence for eliciting the wilting response. With further exchange experiments, another wilting determinant was located within the EcoR I fragment (2383-4264) of TEV HAT. This fragment encompasses the entire P3 and the N-terminal CI coding sequence.

The liver’s exceptional capacity sets it apart from other organs in its response to various acute and chronic injuries, known as “liver regeneration”. Liver regeneration is not driven by a single pathway, but is achieved through a multi-level network including hepatocyte dedifferentiation, liver progenitor cell (LPCs)activation, non-coding RNA regulation, and metabolic reprogramming. Moreover, liver regeneration research still faces challenges: precise regulation of regeneration termination signals, the tumorigenic risk of stem cell therapy, and immune rejection in personalized treatment, among other issues, need to be addressed urgently. In this review, we delineate the cellular dynamics of liver regeneration and synthesize numerous signaling pathways and factors that prominently contribute to liver regeneration alongside recent research advancements. As well as its current clinical application including molecular therapy, stem cell therapy, and the development of artificial livers. We also discuss some of the current problems and look forward to new treatments. By integrating findings from numerous studies, it provides a comprehensive understanding of liver regeneration, highlighting its significance in treating liver diseases and guiding future research. Graphical abstract

Facial expression recognition under classroom scenes can help the teacher to understand students’ classroom learning status and improve teaching effectiveness. Aiming at the problem of low expression recognition accuracy in classroom scenarios, a novel multi-scale facial expression recognition algorithm based on improved Res2Net is proposed. Firstly, a bi-directional residual BiRes2Net module is proposed to achieve bi-directional multi-scale expression feature extraction at the fine-grained level, while a short-directed connection path is introduced to make the network have the self-closing capability and avoid extracting redundant information of expressions; Then the Fine-Grained Coordinate Attention (FGCA) mechanism is embedded to extract expression spatial location features and channel features at a fine-grained level by making full use of the prior knowledge of facial expressions; Finally, a multi-classification Focalloss loss function is used to alleviate the imbalance of expression data, and different weights are assigned to expression samples with different recognition difficulty so that the network is biased towards difficult sample feature extraction. The experimental results show that the recognition accuracy of the  proposed method is 79.47%, 94.06%, and 96.67% in RAF-DB, JAFFE, and CK+ datasets respectively, and up to 72.71% in real classroom scenes, which are better than other comparative algorithms significantly.