Single-cell RNA sequencing reveals cellular and molecular immune profile in a Pembrolizumab-responsive PD-L1-negative lung cancer patient.

High expression of PD-L1 predicts PD-1/PD-L1 inhibitor benefit, meanwhile a few PD-L1-negative patients still benefit from these drugs. In this study, we aimed to explore the underlying cellular and molecular characteristics via single-cell sequencing. Before and after treatment with Pembrolizumab, peripheral blood mononuclear cells (PBMCs) were isolated via Ficoll gradient. Thereafter, single-cell RNA sequencing was performed, and clinical significance was validated with The Cancer Genome Atlas (TCGA) cohort. All 3423 cells of 16 clusters were classified into eight cell types, including NKG7+ T, NKG7+ NK, Naïve T, CDC1C+ dendritic cells, CD8+ T cells, B cells, macrophages and erythrocytes. Cell proportion, the clinical significance of differentially expressed genes and significant pathways of NKG7+ T, NKG7+ NK, Naïve T and CD8+ T cells were analyzed. Ubiquitin-mediated proteolysis/cell cycle/natural killer cell-mediated cytotoxicity were identified as PD-1 blockage-responsive pathways in NKG7+ NK cells. Apoptosis/Th1 and Th2 cell differentiation were proposed as Pembrolizumab-affected pathways in NKT cells. In gene level, ID2, PIK3CD, UQCR10, MATK, MZB1, IL7R and TRGC2 showed a significant correlation with PD-1 expression after TCGA dataset validation, which could possess potential as predictive markers for patients with PD-L1-negative lung squamous cell carcinoma who can benefit from Pembrolizumab.

Analysis of pathogens and risk factors of secondary pulmonary infection in patients with COVID-19.

To investigate the distribution and risk factors of pathogens in secondary pulmonary infection in patients with COVID-19.142 patients with confirmed COVID-19 from Shanghai Public Health Clinical Center were collected, and 32 patients with pulmonary infection were taken as the infection group. The distribution of pathogens in the sputum specimens was applied for retrospective analysis. Meanwhile, 110 patients diagnosed with COVID-19, but without pulmonary infection were regarded as the asymptomatic group. The risk factors of pulmonary infection were analyzed with generalized linear models and logistic regression. The pathogens in the lung infection group were mainly gram-negative bacteria (22, 68.8%), especially Klebsiella pneumoniae. Gram-positive bacteria and fungi accounted for 13 (40.6%), mainly Staphylococcus aureus, and 11 (34.4%), mainly Candida albicans. There were 14 cases (43.8%) infected with two or more pathogens. The comparison between the two groups found that, patients with elder age, underlying diseases, more lung lesions and low protein contents, were more likely to develop lung infections. At last, univariate analysis showed that 6 factors, including indwelling gastric catheter, the number of deep vein catheters, tracheal intubation tracheotomy, invasive mechanical ventilation, hormonal application, and the use of more than three antibacterial drugs, are risk factors for COVID-19 secondary pulmonary infection. Generalized linear models and logistic regression analysis showed antimicrobial use as an independent risk factor for COVID-19 secondary lung infection. There are many risk factors for secondary lung infection in severe COVID-19 patients, and it is recommended to use antibiotics reasonably.

Increased Th17 activation and gut microbiota diversity are associated with pembrolizumab-triggered tuberculosis.

INTRODUCTION: A hypersensitivity response akin to immune reconstitution inflammatory syndrome (IRIS) has been proposed as a mechanism responsible for anti-PD-1 therapy-induced tuberculosis. IRIS is associated with enhanced activation of IL-17A-expressing CD4 + T cells (Th17). Gut microbiota is thought to be linked to pulmonary inflammation through the gut-lung axis. MATERIALS AND METHODS: We used ImmuCellAI to investigate the T cell population in lung cancer and tuberculosis samples. Then, we applied flow cytometry to monitor the expression levels of the Th17 cell activation marker CD38 in the peripheral blood of a patient experiencing adverse events, including tuberculosis, in response to pembrolizumab. The gut microbiome was examined by 16S rRNA sequencing to examine the alterations caused by pembrolizumab. RESULTS: The percentage of Th17 cells was increased in both lung cancer and tuberculosis. FACS analysis showed that pembrolizumab induced substantial CD38 expression in Th17 cells. The patient's fecal samples showed that the diversity of the gut microbiota was significantly increased in response to the pembrolizumab cycle. One enriched genus was Prevotella, which has previously been linked to lung inflammation and Th17 immune activation. DISCUSSION: The observed Th17 activation in our patient was consistent with a role of Th17-mediated IRIS in pembrolizumab-triggered tuberculosis. Pembrolizumab might trigger airway inflammation with a Th17 phenotype through microbiota interactions in the gut-lung axis.

Integrated transcriptomic and metabolomic responses in the hepatopancreas of kuruma shrimp (Marsupenaeus japonicus) under cold stress.

In aquatic ecosystems, the temperature of the water is an important ecological factor that modulates aquatic organisms' metabolism, growth, development, and reproduction. In this study, the morphological, transcriptomic, and metabolomic analyses of response of Marsupeneus japonicus to acute cold stress was investigated. The results revealed that low temperature caused profound morphological damage to the hepatopancreas. Transcriptomic responses suggested that energy and primary metabolism, cytoskeleton structure, and apoptosis signaling were altered. The metabolic responses to cold stress included changes of multiple amino acids and unsaturated fatty acids. Combined transcriptomic and metabolomic data indicated that energy metabolism pathways were downregulated in the hepatopancreas under cold stress. However, M. japonicus increased ATP and unsaturated fatty acids production to ameliorate. Moreover, cold stress caused significant attenuation of macrophage apoptosis. This study provides key information to increase our understanding of low-temperature tolerance in shrimp.

EBprotV2: A Perseus Plugin for Differential Protein Abundance Analysis of Labeling-Based Quantitative Proteomics Data.

We present EBprotV2, a Perseus plugin for peptide-ratio-based differential protein abundance analysis in labeling-based proteomics experiments. The original version of EBprot models the distribution of log-transformed peptide-level ratios as a Gaussian mixture of differentially abundant proteins and nondifferentially abundant proteins and computes the probability score of differential abundance for each protein based on the reproducible magnitude of peptide ratios. However, the fully parametric model can be inflexible, and its R implementation is time-consuming for data sets containing a large number of peptides (e.g., >100 000). The new tool built in the C++ language is not only faster in computation time but also equipped with a flexible semiparametric model that handles skewed ratio distributions better. We have also developed a Perseus plugin for EBprotV2 for easy access to the tool. In addition, the tool now offers a new submodule (MakeGrpData) to transform label-free peptide intensity data into peptide ratio data for group comparisons and performs differential abundance analysis using mixture modeling. This approach is especially useful when the label-free data have many missing peptide intensity data points.

Integrated bioinformatics analysis and validation revealed potential immune-regulatory miR-892b, miR-199b-5p and miR-582-5p as diagnostic biomarkers in active tuberculosis.

Tuberculosis (TB) is one of the most prevalent pulmonary diseases caused by Mycobacterium tuberculosis (Mtb). MiRNAs (miRNAs) participate in TB progression by modulating the host-pathogen interaction. Bioinformatics advancements provide basis for exploring novel immunoregulatory miRNAs and their performance as diagnostic biomarkers. Gene and miRNA expression datasets, GSE29190 and GSE54992, were downloaded from Gene Expression Omnibus (GEO) database. Based on fold changes and statistical significance, a total of 7463 differentially expressed mRNAs (DE-mRNAs) and 38 differentially expressed miRNAs (DE-miRNAs) were screened. Function annotation and protein-protein interaction (PPI) network were constructed to reveal underlying mechanisms of TB pathogenesis. Functional annotation identified the MAPK signalling pathway and leukocyte migration as the top enriched processes. The PPI and MGIP networks indicated that chemokine ligands like CXCL1/CXCL2 and receptors, like CCR7 were important down-regulated genes, implying that a protective mechanism against overdue inflammation induced cell death. MiRNA-gene-immune processes (MGIP) network enriched 7 deregulated miRNAs, and their expression was further examined with quantitative real-time PCR (qRT-PCR), in PBMC samples of 20 active TB patients and 20 healthy donors. The diagnostic performance was evaluated with ROC curves. MiR-892b; miR-199b-5p and miR-582-5p were significantly deregulated in TB patients, compared with healthy participants. The best overall performance was from miR-892b, with an area under curve (AUC) of 0.77, 55% sensitivity and 90% specificity. AUC of miR-199b-5p and miR-582-5p were 0.71 and 0.70, respectively. MiR-892b, miR-199b-5p and miR-582-5p could be considered promising novel diagnostic biomarkers for active tuberculosis.

Comparative proteomic investigation of Marsupenaeus japonicus hepatopancreas challenged with Vibrio parahaemolyticus and white spot syndrome virus.

This study aimed to use isobaric tags (IBTs) to investigate the immune response of the hepatopancreas of Marsupenaeus japonicas infected with Vibrio parahaemolyticus or white spot syndrome virus (WSSV). Liquid chromatography-tandem mass spectrometry and protein sequencing identified 1005 proteins. Among them, 109 proteins were upregulated and 94 were downregulated after V. parahaemolyticus infection. After WSSV infection, 130 proteins were identified as differentially abundant, including 88 that were upregulated and 42 were downregulated. Fifty-four proteins were identified as differentially abundant after both V. parahaemolyticus and WSSV infection. A number of proteins related to cytoskeletal processes, including actin and myosin, and apoptosis-related proteins were upregulated in shrimp after V. parahaemolyticus and WSSV infection, indicating that phagocytosis and apoptosis may be involved in the response to in V. parahaemolyticus or WSSV infection. Quantitative real-time PCR was carried out to verify the reliability of the proteomic data. These data provide a basis to characterize the immunity-related processes of shrimp in response to infection with WSSV or V. parahaemolyticus.

Prmt7 regulates epiboly and gastrulation cell movements by facilitating syntenin.

Epiboly spreads and thins the blastoderm over the yolk cell during zebrafish gastrulation. Despite of its fundamental function, little is known about the molecular mechanisms that control this coordinated cell movement. In this study, we investigated protein arginine methyltransferase 7 (Prmt7) morphants with an epibolic delay defect in zebrafish. The ratio of morphants with epiboly delay phenotypes increased as the dose of the injected morpholino (MO) increased. Here, syntenin transcripts are maternally deposited and ubiquitously expressed from the oocyte period to the early larva stage. Furthermore, we demonstrated that Prmt7 modulates epibolic movements of the enveloping layer by regulating F-actin organization. These defects can be partially rescued by re-expression of Prmt7 or syntenin protein. Analysis of the earliest cellular defects suggested a role of Prmt7 in the autonomous vegetal expansion of the yolk syncytial layer and the rearrangement of the actin cytoskeleton in extra-embryonic tissues. By a combination of knockdown studies and rescue experiments in zebrafish, we showed that epiboly relies on the molecular networking of Prmt7 by facilitating syntenin, which acts as a regulator for cytoskeleton. This study identifies the important function of the Prmt7 for the progression of zebrafish epiboly and establishes its key role in directional cell movements during early development.

Protein Arginine Methyltransferase 6 (Prmt6) Is Essential for Early Zebrafish Development through the Direct Suppression of gadd45αa Stress Sensor Gene.

Histone lysine methylation is important in early zebrafish development; however, the role of histone arginine methylation in this process remains unclear. H3R2me2a, generated by protein arginine methyltransferase 6 (Prmt6), is a repressive mark. To explore the role of Prmt6 and H3R2me2a during zebrafish embryogenesis, we identified the maternal characteristic of prmt6 and designed two prmt6-specific morpholino-oligos (MOs) to study its importance in early development, application of which led to early epiboly defects and significantly reduced the level of H3R2me2a marks. prmt6 mRNA could rescue the epiboly defects and the H3R2me2a reduction in the prmt6 morphants. Functionally, microarray data demonstrated that growth arrest and DNA damage-inducible, α, a (gadd45αa) was a significantly up-regulated gene in MO-treated embryos, the activity of which was linked to the activation of the p38/JNK pathway and apoptosis. Importantly, gadd45αa MO and p38/JNK inhibitors could partially rescue the defect of prmt6 morphants, the downstream targets of Prmt6, and the apoptosis ratios of the prmt6 morphants. Moreover, the results of ChIP quantitative real time PCR and luciferase reporter assay indicated that gadd45αa is a repressive target of Prmt6. Taken together, these results suggest that maternal Prmt6 is essential to early zebrafish development by directly repressing gadd45αa.

Conservation of the STING-Mediated Cytosolic DNA Sensing Pathway in Zebrafish.

UNLABELLED: Zebrafish (Danio rerio) is a unique potential model animal for dissecting innate immune signaling. Here we demonstrate that herpes simplex virus 1 (HSV-1) could infect zebrafish at its different developmental stages and trigger the expression of type I interferons (IFNs) as well as interferon-stimulated genes (ISGs) in zebrafish larvae. Silencing of zSTING, but not zMAVS, markedly attenuates the DNA virus-induced antiviral responses. Notably, a conserved serine residue (S373) is essential for the action of zSTING. Unexpectedly, zebrafish cyclic GMP-AMP synthase (cGAS) is dispensable for the STING signaling, whereas zDHX9 and zDDX41 are potential sensors for HSV-1 infection in vivo. Taken together, this proof-of-concept study establishes the zebrafish larva as a feasible model for investigating the cytosolic DNA sensing mechanism, shedding light on the conservation of the STING antiviral signaling pathway. IMPORTANCE: The zebrafish larva provides technical advantages for understanding host-pathogen interactions. In this study, we established the zebrafish larva as a useful model for studying HSV-1 infection. HSV-1 infection triggers strong type I interferon production, which depends on STING expression. In addition, STING-mediated antiviral signaling is conserved in zebrafish. Interestingly, zDHX9 and zDDX41 are indispensable for detecting HSV-1, while cGAS is dispensable. This proof-of-concept study indicates that the zebrafish represents an amenable model for the investigation of cytosolic DNA sensing mechanisms.

Evaluation of cinnamon essential oil microemulsion and its vapor phase for controlling postharvest gray mold of pears (Pyrus pyrifolia).

BACKGROUND: Essential oil of cinnamon (CM) is a potential alternative to chemical fungicides. Thus this work aimed to investigate the possible effects of CM microemulsions on decay developments and qualitative properties of pears. RESULTS: The decay incidence of samples treated with 500 µg L⁻¹ microemulsion was significantly reduced by 18.7% in comparison to that of 500 µg L⁻¹ non-microemulsion after 4 days' storage at 20 °C. In the vapor phase, the CM microemulsion with the lowest concentration had the best control for decay incidence and lesion diameter. The interval between inoculations also influenced decay development. Pears treated with Botrytis cinerea and immediately followed by CM microemulsion showed the lowest decay incidence. Moreover, in the natural decay experiment, the percentage of rotted pears was 3.8% in the CM microemulsion treatment and 5.8% in the control. CM microemulsion delayed the loss of ascorbic acid, yet it had no significant influence on pear qualities such as firmness and color. CONCLUSION: CM microemulsion may be an alternative way to control the gray mold of pears without a negative influence on its qualities.

Establishment and transcriptome analysis of single blastomere-derived cell lines from zebrafish.

Maintaining chromosome euploidy in zebrafish embryonic cells is challenging because of the degradation of genomic integrity during cell passaging. In this study, we report the derivation of zebrafish cell lines from single blastomeres. These cell lines have a stable chromosome status attributed to BMP4 and exhibit continuous proliferation in vitro. Twenty zebrafish cell lines are successfully established from single blastomeres. Single-cell transcriptome sequencing analysis confirms the fidelity of gene expression profiles throughout long-term culturing of at least 45 passages. The long-term cultured cells are specialized into epithelial cells, exhibiting similar expression patterns validated by integrative transcriptomic analysis. Overall, this work provides a protocol for establishing zebrafish cell lines from single blastomeres, which can serve as valuable tools for in vitro investigations of epithelial cell dynamics in terms of life-death balance and cell fate determination during normal homeostasis.

Continuous high-frequency repetitive transcranial magnetic stimulation at extremely low intensity affects exploratory behavior and spatial cognition in mice.

High-frequency repetitive transcranial magnetic stimulation (HF-rTMS) has been shown to be effective for cognitive intervention. However, whether HF-rTMS with extremely low intensity could influence cognitive functions is still under investigation. The present study systematically investigated the effects of continuous 40 Hz and 10 Hz rTMS on cognition in young adult mice at extremely low intensity (10 mT and 1 mT) for 11 days (30 min/day). Cognitive functions were assessed using diverse behavioral tasks, including the open field, Y-maze, and Barnes maze paradigms. We found that 40 Hz rTMS significantly impaired exploratory behavior and spatial memory in both 10 mT and 1 mT conditions. In addition, 40 Hz rTMS induced remarkably different effects on exploratory behavior between 10 mT and 1mT, compared to 10 Hz stimulation. Our results indicate that extremely low intensity rTMS can significantly alter cognitive performance depending on intensity and frequency, shedding light on the understanding of the mechanism of rTMS effects.

Historical and contemporary applications of moxibustion at Gaohuang (BL 43). / 膏肓灸古今应用探析.

The relevant passages on moxibustion at Gaohuang (BL 43) in the Chinese Medical Code (fifth edition), and relevant literature on moxibustion at Gaohuang (BL 43) published up to January 17th, 2023 in the CNKI, Wanfang, VIP, SinoMed, PubMed, Web of Science, EMbase and Scopus were searched. The localization and selection methods of Gaohuang (BL 43), types of moxibustion at Gaohuang (BL 43), moxibustion quantity, and the main clinical indications were analyzed. As a result, a total of 227 ancient passages were included, with 51 related to moxibustion quantity and 171 related to clinical indications, encompassing 33 different diseases. A total of 50 modern articles were reviewed, covering 26 different diseases. The key of selection methods of Gaohuang (BL 43) is exploring sensitivity around the scapula, with direct moxibustion as a preferred technique; the optimal moxibustion dose is detenuined by arrival and withdrawal of deqi, and primary indications were related to syndrome of heart and lung deficiencies.

Role of GABAB receptors in cognition and EEG activity in aged APP and PS1 transgenic mice.

Alzheimer's disease (AD) is the most common cause of dementia in the elderly. Recent evidence suggests that gamma-aminobutyric acid B (GABAB) receptor-mediated inhibition is a major contributor to AD pathobiology, and GABAB receptors have been hypothesized to be a potential target for AD treatment. The aim of this study is to determine how GABAB regulation alters cognitive function and brain activity in an AD mouse model. Early, middle and late stage (8-23 months) amyloid precursor protein (APP) and presenilin 1 (PS1) transgenic mice were used for the study. The GABAB agonist baclofen (1 and 2.5 mg/kg, i. p.) and the antagonist phaclofen (0.5 mg/kg, i. p.) were used. Primarily, we found that GABAB activation was able to improve spatial and/or working memory performance in early and late stage AD animals. In addition, GABAB activation and inhibition could regulate global and local EEG oscillations in AD animals, with activation mainly regulating low-frequency activity (delta-theta bands) and inhibition mainly regulating mid- and high-frequency activity (alpha-gamma bands), although the regulated magnitude at some frequencies was reduced in AD. The cognitive improvements in AD animals may be explained by the reduced EEG activity in the theta frequency band (2-4 Hz). This study provides evidence for a potential therapeutic effect of baclofen in the elderly AD brain and for GABAB receptor-mediated inhibition as a potential therapeutic target for AD.

Spatiotemporal control of RNA metabolism and CRISPR-Cas functions using engineered photoswitchable RNA-binding proteins.

RNA molecules perform various crucial roles in diverse cellular processes, from translating genetic information to decoding the genome, regulating gene expression and catalyzing chemical reactions. RNA-binding proteins (RBPs) play an essential role in regulating the diverse behaviors and functions of RNA in live cells, but techniques for the spatiotemporal control of RBP activities and RNA functions are rarely reported yet highly desirable. We recently reported the development of LicV, a synthetic photoswitchable RBP that can bind to a specific RNA sequence in response to blue light irradiation. LicV has been used successfully for the optogenetic control of RNA localization, splicing, translation and stability, as well as for the photoswitchable regulation of transcription and genomic locus labeling. Compared to classical genetic or pharmacologic perturbations, LicV-based light-switchable effectors have the advantages of large dynamic range between dark and light conditions and submicron and millisecond spatiotemporal resolutions. In this protocol, we provide an easy, efficient and generalizable strategy for engineering photoswitchable RBPs for the spatiotemporal control of RNA metabolism. We also provide a detailed protocol for the conversion of a CRISPR-Cas system to optogenetic control. The protocols typically take 2-3 d, including transfection and results analysis. Most of this protocol is applicable to the development of novel LicV-based photoswitchable effectors for the optogenetic control of other RNA metabolisms and CRISPR-Cas functions.

Comprehensive strength evaluation system of commercial centres based on multi-source data: a case of Hefei central city.

Urban commercial centres are the most concentrated areas of economic activity. Understanding the spatial distribution pattern and comprehensive strength of urban commercial centres is important to guide the reasonable graded allocation of urban commercial space, spatial structure optimization and sustainable development of the commercial economy. Herein, mobile phone signalling data are used to identify the functional connection between recreational and residential places and local spatial autocorrelation analysis is used to identify the 24 commercial centres in the central city of Hefei. The comprehensive strength evaluation system of commercial centres is constructed from their basic conditions and customer consumption behaviours, and their comprehensive strength indices are accordingly measured and graded. The spatial distribution characteristics of commercial centres at all levels are analysed, and optimisation suggestions are made for the whole area and region. The following conclusions are drawn from the results of this study. (1) Compared to traditional single-perspective evaluations, the developed comprehensive strength evaluation system considers supply and demand perspectives for commercial centres, providing a more holistic and accurate portrayal of the strengths of various centres within a region. (2) The current commercial centres are characterised by 'large dispersion and small concentration' in spatial and hierarchical distribution. (3) The commercial centres in Hefei have formed a relatively complete 'first, second, and third level' commercial centre system, with the first level as the core and relying on the urban road system to form a network spatial connection. (4) Most of the commercial centres are concentrated in the First Ring Road, Swan Lake in the Government Affairs District and Binhu Century Town Estate, while most areas north of the Second Ring Road and Binhu New District still lack large-scale commercial centres. This study provides a technical reference for analysing urban commercial spatial structure patterns and provides decision support for optimising the spatial layout of urban commercial service functions.

Infrared protein crystallography.

We consider the application of infrared spectroscopy to protein crystals, with particular emphasis on exploiting molecular orientation through polarization measurements on oriented single crystals. Infrared microscopes enable transmission measurements on individual crystals using either thermal or nonthermal sources, and can accommodate flow cells, used to measure spectral changes induced by exposure to soluble ligands, and cryostreams, used for measurements of flash-cooled crystals. Comparison of unpolarized infrared measurements on crystals and solutions probes the effects of crystallization and can enhance the value of the structural models refined from X-ray diffraction data by establishing solution conditions under which they are most relevant. Results on several proteins are consistent with similar equilibrium conformational distributions in crystal and solutions. However, the rates of conformational change are often perturbed. Infrared measurements also detect products generated by X-ray exposure, including CO(2). Crystals with favorable symmetry exhibit infrared dichroism that enhances the synergy with X-ray crystallography. Polarized infrared measurements on crystals can distinguish spectral contributions from chemically similar sites, identify hydrogen bonding partners, and, in opportune situations, determine three-dimensional orientations of molecular groups. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.