Programmable RNA sensing for cell monitoring and manipulation.

RNA is a central and universal mediator of genetic information underlying the diversity of cell types and cell states, which together shape tissue organization and organismal function across species and lifespans. Despite numerous advances in RNA sequencing technologies and the massive accumulation of transcriptome datasets across the life sciences1,2, the dearth of technologies that use RNAs to observe and manipulate cell types remains a bottleneck in biology and medicine. Here we describe CellREADR (Cell access through RNA sensing by Endogenous ADAR), a programmable RNA-sensing technology that leverages RNA editing mediated by ADAR to couple the detection of cell-defining RNAs with the translation of effector proteins. Viral delivery of CellREADR conferred specific cell-type access in mouse and rat brains and in ex vivo human brain tissues. Furthermore, CellREADR enabled the recording and control of specific types of neurons in behaving mice. CellREADR thus highlights the potential for RNA-based monitoring and editing of animal cells in ways that are specific, versatile, simple and generalizable across organ systems and species, with wide applications in biology, biotechnology and programmable RNA medicine.

Intratumoral NKT cell accumulation promotes antitumor immunity in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDA) is a potentially lethal disease lacking effective treatments. Its immunosuppressive tumor microenvironment (TME) allows it to evade host immunosurveillance and limits response to immunotherapy. Here, using the mouse KRT19-deficient (sgKRT19-edited) PDA model, we find that intratumoral accumulation of natural killer T (NKT) cells is required to establish an immunologically active TME. Mechanistically, intratumoral NKT cells facilitate type I interferon (IFN) production to initiate an antitumor adaptive immune response, and orchestrate the intratumoral infiltration of T cells, dendritic cells, natural killer cells, and myeloid-derived suppressor cells. At the molecular level, NKT cells promote the production of type I IFN through the interaction of their CD40L with CD40 on myeloid cells. To evaluate the therapeutic potential of these observations, we find that administration of folinic acid to mice bearing PDA increases NKT cells in the TME and improves their response to anti-PD-1 antibody treatment. In conclusion, NKT cells have an essential role in the immune response to mouse PDA and are potential targets for immunotherapy.

Carcinomas assemble a filamentous CXCL12-keratin-19 coating that suppresses T cell-mediated immune attack.

Cancer immunotherapy frequently fails because most carcinomas have few T cells, suggesting that cancers can suppress T cell infiltration. Here, we show that cancer cells of human pancreatic ductal adenocarcinoma (PDA), colorectal cancer, and breast cancer are coated with transglutaminase-2 (TGM2)-dependent covalent CXCL12-keratin-19 (KRT19) heterodimers that are organized as filamentous networks. Since a dimeric form of CXCL12 suppresses the motility of human T cells, we determined whether this polymeric CXCL12-KRT19 coating mediated T cell exclusion. Mouse tumors containing control PDA cells exhibited the CXCL12-KRT19 coating, excluded T cells, and did not respond to treatment with anti-PD-1 antibody. Tumors containing PDA cells not expressing either KRT19 or TGM2 lacked the CXCL12-KRT19 coating, were infiltrated with activated CD8+ T cells, and growth was suppressed with anti-PD-1 antibody treatment. Thus, carcinomas assemble a CXCL12-KRT19 coating to evade cancer immune attack.

Infection with 'Candidatus Liberibacter asiaticus' improves the fecundity of Diaphorina citri aiding its proliferation: A win-win strategy.

The evolution of insect vector-pathogen relationships has long been of interest in the field of molecular ecology. One system of special relevance, due to its economic impacts, is that between Diaphorina citri and 'Candidatus Liberibacter asiaticus' (CLas), the cause of the severe Asian form of huanglongbing. CLas-positive D. citri are more fecund than their CLas-negative counterparts, boosting opportunities for pathogens to acquire new vector hosts. The molecular mechanism behind this life-history shift remains unclear. Here, we found that CLas promoted ovarian development and increased the expression of the vitellogenin receptor (DcVgR) in ovaries. DcVgR RNAi significantly decreased fecundity and CLas titer in ovaries, extended the preoviposition period, shortened the oviposition period and blocked ovarian development. Given their importance in gene regulation, we explored the role of miRNAs in shaping these phenotypes and their molecular triggers. Our results showed that one miRNA, miR-275, suppressed DcVgR expression by binding to its 3' UTR. Overexpression of miR-275 knocked down DcVgR expression and CLas titer in ovaries, causing reproductive defects that mimicked DcVgR knockdown phenotypes. We focused, further, on roles of the Juvenile Hormone (JH) pathway in shaping the observed fecundity phenotype, given its known impacts on ovarian development. After CLas infection, this pathway was upregulated, thereby increasing DcVgR expression. From these combined results, we conclude that CLas hijacks the JH signalling pathway and miR-275, thereby targeting DcVgR to increase D. citri fecundity. These changes simultaneously increase CLas replication, suggesting a pathogen-vector host mutualism, or a seemingly helpful, but cryptically costly life-history manipulation.

Siloxane-containing phosphine (oxide) ligands for enhanced catalytic activity of cobalt complexes for hydrosilylation reactions.

A series of siloxane-containing phosphine (oxide) ligands have been designed and synthesized. These phosphine (oxide) ligands contain silicon atoms, which can impart better solubility in the relevant media, thereby improving certain catalytic performances. The hydrosilylation of olefins catalyzed by these metal phosphine (oxide) complexes has been conducted under mild reaction conditions.

Pinctada martensii Hydrolysate Modulates the Brain Neuropeptidome and Proteome in Diabetic (db/db) Mice via the Gut-Brain Axis.

Pinctada martensii hydrolysate (PMH) has been proved to have the effect of ameliorating disorders of glucose and lipid metabolism in db/db mice, but the mechanism of its hyperglycemia effect is still unclear. Bacterial communities in fecal samples from a normal control group, a diabetic control group, and a PMH-treated diabetes mellitus type 2 (T2DM) group were analyzed by 16S gene sequencing. Nano LC-MS/MS was used to analyze mice neuropeptides and proteomes. The 16S rDNA sequencing results showed that PMH modulated the structure and composition of the gut microbiota and improved the structure and composition of Firmicutes and Bacteroidetes at the phylum level and Desulfovibrionaceae and Erysipelatoclostridiaceae at the family level. Furthermore, the expressions of functional proteins of the central nervous system, immune response-related protein, and proteins related to fatty acid oxidation in the brain disrupted by an abnormal diet were recovered by PMH. PMH regulates the brain neuropeptidome and proteome and further regulates blood glucose in diabetic mice through the gut-brain axis. PMH may be used as a prebiotic agent to attenuate T2DM, and target-specific microbial species may have unique therapeutic promise for metabolic diseases.

A Microvascular Segmentation Network Based on Pyramidal Attention Mechanism.

The precise segmentation of retinal vasculature is crucial for the early screening of various eye diseases, such as diabetic retinopathy and hypertensive retinopathy. Given the complex and variable overall structure of retinal vessels and their delicate, minute local features, the accurate extraction of fine vessels and edge pixels remains a technical challenge in the current research. To enhance the ability to extract thin vessels, this paper incorporates a pyramid channel attention module into a U-shaped network. This allows for more effective capture of information at different levels and increased attention to vessel-related channels, thereby improving model performance. Simultaneously, to prevent overfitting, this paper optimizes the standard convolutional block in the U-Net with the pre-activated residual discard convolution block, thus improving the model's generalization ability. The model is evaluated on three benchmark retinal datasets: DRIVE, CHASE_DB1, and STARE. Experimental results demonstrate that, compared to the baseline model, the proposed model achieves improvements in sensitivity (Sen) scores of 7.12%, 9.65%, and 5.36% on these three datasets, respectively, proving its strong ability to extract fine vessels.

Light Chain Q166C Mutation Permits One-step Site Specific Conjugation on Monoclonal Antibodies.

Engineered cysteines are frequently used for site-specific conjugation in antibody-drug conjugate (ADC) development. When cysteine-engineered mAbs are produced in the cell culture process, the sulfhydryl groups on the engineered cysteines are mostly in an oxidized form. The oxidized cysteines require multiple steps (such as reduction, reoxidation, and buffer exchanges) to reactivate for bioconjugation, which complicates the ADC production process and reduces yields. In this study, we identified a Q166C mutation in the light chain that allows the presence of free sulfhydryl groups during cell culture and purification process. This mutation is in the constant region and away from sites involved in antigen binding or Fc-mediated functions. The free sulfhydryl reacts readily with maleimide in a mild solution at a high conjugation rate. This is only the second such site reported (the first one is Q124C in the light chain). Using the Q166C mutation, we conjugated an anti-angiopoietin-2 (Ang-2) peptide on bevacizumab, an anti-vascular endothelial growth factor (VEGF) antibody, to construct a peptide antibody conjugate, Ava-Plus, which could block two pro-angiogenic factors simultaneously. Ava-Plus showed high affinity for both VEGF and Ang-2 and demonstrated higher activity than bevacizumab in in vitro cell migration and in vivo mouse xenograft models.

Dynamic alterations in metabolomics and transcriptomics associated with intestinal fibrosis in a 2,4,6-trinitrobenzene sulfonic acid-induced murine model.

BACKGROUND & AIMS: Intestinal fibrosis is a common and severe complication of inflammatory bowel disease without clear pathogenesis. Abnormal expression of host genes and metabolic perturbations might associate with the onset of intestinal fibrosis. In this study, we aimed to investigate the relationship between the development of intestinal fibrosis and the dynamic alterations in both fecal metabolites and host gene expression. METHODS: We induced intestinal fibrosis in a murine model using 2,4,6-trinitrobenzene sulfonic acid (TNBS). TNBS-treated or control mice were sacrificed after 4 and 6 weeks of intervention; alterations in colonic genes and fecal metabolites were determined by transcriptomics and metabolomics, respectively. Differential, tendency, enrichment, and correlation analyses were performed to assess the relationship between host genes and fecal metabolites. RESULTS: RNA-sequencing analysis revealed that 679 differential genes with enduring changes were mainly enriched in immune response-related signaling pathways and metabolism-related biological processes. Among them, 15 lipid metabolism-related genes were closely related to the development of intestinal fibrosis. Moreover, the fecal metabolic profile was significantly altered during intestinal fibrosis development, especially the lipid metabolites. Particularly, dynamic perturbations in lipids were strongly associated with alterations in lipid metabolism-related genes expression. Additionally, six dynamically altered metabolites might serve as biomarkers to identify colitis-related intestinal fibrosis in the murine model. CONCLUSIONS: Intestinal fibrosis in colitis mice might be related to dynamic changes in gene expression and metabolites. These findings could provide new insights into the pathogenesis of intestinal fibrosis.

Mitochondrial elongation factor 4 modulates energy metabolism and promotes breast cancer metastasis by orchestration of mitochondrial translation.

To cope with the requirements of energy and building blocks for rapid proliferation, cancer cells reprogram their metabolic pathways profoundly, especially in oxygen- and nutrients-deficient tumor microenvironments. However, functional mitochondria and mitochondria-dependent oxidative phosphorylation are still necessary for the tumorigenesis and metastasis of cancer cells. We show here that mitochondrial elongation factor 4 (mtEF4) is commonly upregulated in breast tumors compared to adjacent non-cancerous tissues, and is relevant to tumor progression and poor prognosis. Down regulation of mtEF4 in breast cancer cells impairs the assembly of mitochondrial respiration complexes, decreases mitochondrial respiration, reduces ATP production, attenuates the formation of lamellipodia, and suppresses cell motility in vitro and cancer metastasis in vivo. On the contrary, upregulation of mtEF4 elevates the mitochondrial oxidative phosphorylation, which contributes to the migratory capacities of breast cancer cells. mtEF4 also increases the potential of glycolysis, probably via an AMPK-related mechanism. In summary, we provide direct evidences that the aberrantly upregulated mtEF4 contributes to the metastasis of breast cancer by coordinating metabolic pathways.

Effects of acute normovolemic hemodilution on allogeneic blood transfusion & coagulation in orthognathic surgery: A randomized study.

BACKGROUND: Acute normovolemic hemodilution (ANH) is one of the important techniques predominantly used in cardiac, hepatic, and vascular surgery for decreasing allogeneic blood transfusion. However, the effect of ANH in orthognathic surgery has been rarely studied. Therefore, this study aims to assess the ANH-mediated reduction in the allogeneic red blood cell transfusion for orthognathic surgery patients. STUDY DESIGN AND METHODS: In this single-center study, 18-80 years old patients were recruited. Patients with hemoglobin ≥11 g/dL and normal coagulation function were randomly divided into ANH or standard treatment group. RESULTS: Ninety six patients underwent ANH, and 101 patients received standard treatment. No differences in demographic or major pre-operative characteristics were observed between the two groups. One patient in the ANH and three patients in the standard treatment group received allogeneic blood [3(2.97%) vs. 1(1.16%), control vs. ANH, p = .395]. Multivariate logistic regression analysis revealed that ANH treatment was not associated with transfusion of allogeneic blood (p = .763). After retransfusing autologous blood, PT and APTT in the ANH group significantly increased compared to standard treatment group (PT: -1.73 ± 1.09 vs. -2.15 ± 1.06, p = .035; APTT: -6.39 ± 5.76 vs. -8.16 ± 5.70, p = .031; control vs. ANH). No significant differences between the two groups were observed for changes in coagulation parameters at first postoperative day. However, platelet counts in the ANH group decreased compared to the standard group. No significant difference in major adverse outcomes was observed between the two groups. CONCLUSION: ANH did not reduce the incidence of allogeneic transfusion in patients undergoing orthognathic surgery.

PNP-type ligands enabled copper-catalyzed N-formylation of amines with CO2 in the presence of silanes.

The sustainable catalytic transformation of carbon dioxide into valuable fine chemicals with high efficiency is a global challenge as although CO2 is an abundant, nontoxic, and sustainable carbon feedstock it is also the most important factor behind the Greenhouse Effect. We describe herein a PNP-type ligand-enabled copper-catalyzed N-formylation of amines utilizing CO2 as the building block in the presence of hydrosilane as the reductant. Our current protocol featured newly synthesized PNP-type ligands with broad substrate scope under mild reaction conditions.

Potential influence of fine aerosol chemistry on the optical properties in a semi-arid region.

The current understanding regarding the potential influence of aerosol chemistry on the optical properties does not satisfy accurate evaluation of aerosol radiative effects and precise determination of aerosol sources. We conducted a comprehensive study of the potential influence of aerosol chemistry on the optical properties in a semi-arid region based on various observations. Organic matter was the main contributor to the scattering coefficients followed by secondary inorganic aerosols in all seasons. We further related aerosol absorption to elemental carbon, organic matter, and mineral dust. Results showed that organic matter and mineral dust contributed to >40% of the aerosol absorption in the ultraviolet wavelengths. Therefore, it is necessary to consider the absorption of organic matter and mineral dust in addition to that of elemental carbon. We further investigated the potential influence of chemical composition, especially of organic matter and mineral dust on the optical parameters. Mineral dust contributed to higher absorption efficiency and lower scattering efficiency in winter. The absorption Ångström exponent (AAE) was mostly sensitive to organic matter and mineral dust in winter and spring, respectively; it was relatively high (i.e., 1.68) in winter and moderate (i.e., 1.42) in spring. Unlike in the other seasons, mineral dust contributed to higher mass absorption efficiency in winter. This work reveals the complexity of the relationship between aerosol chemistry and optical properties, and especially the influence of organic matter and mineral dust on aerosol absorption. The results are highly important regarding both regional air pollution and climate.

Spatial Distribution and Temporal Dynamics of Candidatus Liberibacter Asiaticus in Different Stages of Embryos, Nymphs and Adults of Diaphorina citri.

Huanglongbing, a globally devastating citrus disease, is associated with Candidatus Liberibacter asiaticus (CLas) and is mainly transmitted by Diaphorina citri. Verification of the distribution and dynamics of CLas in D. citri is critical to understanding CLas transmitted by vectors in nature. Here, the distribution and titers of CLas in different sexes and tissues of D. citri adults were investigated by fluorescence in-situ hybridization (FISH) and quantitative real-time PCR (qRT-PCR). Results showed that CLas had widespread distribution in the brain, salivary glands, digestive system, and reproductive system of both females and males, indicating a systemic infection of CLas in D. citri. Moreover, CLas fluorescence intensity and titers were significantly increased in both the digestive system and the female reproductive system with development and there was a marked decreased in both the salivary glands and the male brain, but there was no significant change in the female brain or the male reproductive system. Furthermore, the distribution and dynamics of CLas in embryos and nymphs were investigated. CLas was observed in all laid eggs and subsequent first-second-instar nymphs, indicating that a high percentage of embryos and nymphs resulting from infected D. citri mothers were infected with CLas.

The CO oxidation mechanism on the W(111) surface and the W helical nanowire investigated by the density functional theory calculation.

Two CO oxidation reactions (CO + O2 → CO2 + O and CO + O → CO2) were considered in the Eley-Rideal (ER) reaction mechanism. These oxidation processes on the W(111) surface and the W helical nanowire were investigated by the density functional theory (DFT) calculation. The stable adsorption sites of O2 and O as well as their adsorption energies were obtained first. In order to understand the catalytic properties of the W helical nanowire, the Fukui function and local density of state (LDOS) profiles were determined. The nudged elastic band (NEB) method was applied to locate transition states and minimum energy pathways (MEPs) of CO oxidation processes on the W helical nanowire and on the W(111) surface. In this study, we have demonstrated that the catalytic ability of the W helical nanowire is superior to that of the W(111) surface for CO oxidation.

Recombinant MPT83 derived from Mycobacterium tuberculosis induces cytokine production and upregulates the function of mouse macrophages through TLR2.

TLR2 recognizes components of Mycobacterium tuberculosis and initiates APC activities that influence both innate and adaptive immunity. M. tuberculosis lipoproteins are an important class of TLR2 ligands. In this study, we focused on recombinant MPT83 (rMPT83) to determine its effects on mouse macrophages. We demonstrated that rMPT83 induced the production of TNF-α, IL-6, and IL-12 p40 and that cytokine induction depended on activated MAPKs, because we observed the rapid phosphorylation of ERK1/2, p38, and JNK in macrophages. Additionally, neutralizing Abs against TLR2 significantly inhibited cytokine secretion and reduced or attenuated the rMPT83-induced activation of p38 and JNK in RAW264.7 cells, a mouse macrophage cell line. Furthermore, rMPT83-induced cytokine production was significantly lower in macrophages from TLR2(-/-) mice than in macrophages from wild-type mice. We further found that prolonged exposure (>24 h) of RAW264.7 cells or macrophages from wild-type and TLR2(-/-) mice to rMPT83 resulted in a significant enhancement of IFN-γ-induced MHC class II expression and an enhanced ability of macrophages to present the rMPT83 peptide to CD4(+) T cells. These results indicated that rMPT83 is a TLR2 agonist that induces the production of cytokines by macrophages and upregulates macrophage function.

The effects of a web-based 24-hour movement behavior lifestyle education program on mental health and psychological well-being in parents of children with autism spectrum disorder: A randomized controlled trial.

BACKGROUND AND PURPOSE: Compared with parents of neurotypical children or children diagnosed with other disabilities, parents of children with autism spectrum disorder (ASD) often experience poorer mental health, greater stress, and more depression and anxiety symptoms. This study aimed to assess the effects of a web-based 24-h movement behavior lifestyle education program on mental health and psychological well-being in parents of children with ASD. METHODS: This study employed a randomized controlled trial utilizing the Health Action Process Approach (HAPA) as a theoretical framework. A total of 318 parents of children with ASD were enrolled and randomly assigned to the experimental or control group. The experimental group received an 8-week web-based 24-h movement behavior lifestyle education program, while the control group followed their usual routine. Two instruments, the Depression Anxiety and Stress Scale (DASS-21) and the Satisfaction With Life Scale (SWLS), were used to measure mental health and psychological well-being, respectively. The data were collected at two time points-at the beginning and the end of the intervention. RESULTS: Compared with the baseline and control groups, the experimental group demonstrated significant improvements in all outcome measures (p < 0.01). There were significant differences in the DASS-21 and SWLS scores between the two groups before and after the intervention (p ≤ 0.01). CONCLUSION: This study represents the first randomized controlled trial involving a web-based 24-h movement behavior lifestyle education program specifically designed to address the mental health and psychological well-being of parents of children with ASD. The findings confirm the potential impact of 24-h movement behavior lifestyle education as a functional and effective strategy for parents of children with ASD.

Integrating sodium cholate-modified MOF hybrid lipase and solubilization of hydrophobic candidates into a step for liganding fishing lipase inhibitors from Nelumbinis Folium.

Enzyme immobilization by metal organic frameworks (MOFs) is an efficient way for screening active constituents in natural products. However, the enzyme's biocatalysis activity is usually decreased due to unfavorable conformational changes during the immobilization process. In this study, sodium cholate was firstly used as the modifier for zeolitic imidazolate framework-8 (ZIF-8) immobilized lipase to increase both the stability and activity. More importantly, with the help of solubilization of sodium cholate, a total of 3 flavonoids and 6 alkaloids candidate compounds were fished out. Their structures were identified and the enzyme inhibitory activities were verified. In addition, the binding information between the candidate compound and the enzyme was displayed by molecular docking. This study provides valuable information for the improvement of immobilized enzyme activity and functional active ingredients in complicated medicinal plant extracts.

Comprehensive evaluation and screening of phytochemical compounds and their hypolipidemic activities of lotus leaf based on HPLC-Q-TOF-MS and spectral-effect analysis.

This study aimed to identify and quantify the primary components in lotus leaf and to explore the hypolipidemic components through spectral-effect relationships and chemometric methods. Utilizing a data-dependent acquisition-diagnostic fragment ion/characteristic neutral loss screening strategy (DFI-NLS), a reliable HPLC-Q-TOF-MS analysis was conducted, identifying 77 compounds, including 36 flavonoids, 21 alkaloids, 3 terpenoids, 11 organic acids, 4 phenols, 1 lignin and 1 unsaturated hydrocarbon. A straightforward HPLC-DAD method was developed for the simultaneous determination of seven major components in lotus leaf, and quercetin-3-O-glucuronide (Q3GA) was identified as the most abundant component. The HPLC fingerprints of 36 lotus leaf sample batches were assessed using chemometric approaches such as principal component analysis and hierarchical cluster analysis. The hypolipidemic effect of these samples was analyzed by measuring total cholesterol (TC) and total triglycerides (TG) levels in palmitic acid (PA) and oleic acid (OA)-induced lipid modeling in HepG-2 cells, employing partial least squares regression and grey relation analysis to investigate the spectral-effect relationship of the lotus leaf. The in vivo hypolipidemic effect of these compounds was assessed using an egg yolk powder-induced high-fat zebrafish model. The findings indicated that peak No.11 (Q3GA) in the chemical fingerprint was significantly associated with hypolipidemic activity, suggesting it as a potential hypolipidemic compound in lotus leaf. In summary, this study facilitates the exploration of the phytochemical compounds and their bioactive properties in the lotus leaf.

Adipokinetic hormone signaling mediates the enhanced fecundity of Diaphorina citri infected by 'Candidatus Liberibacter asiaticus'.

Diaphorina citri serves as the primary vector for 'Candidatus Liberibacter asiaticus (CLas),' the bacterium associated with the severe Asian form of huanglongbing. CLas-positive D. citri are more fecund than their CLas-negative counterparts and require extra energy expenditure. Therefore, understanding the molecular mechanisms linking metabolism and reproduction is of particular importance. In this study, we found adipokinetic hormone (DcAKH) and its receptor (DcAKHR) were essential for increasing lipid metabolism and fecundity in response to CLas infection in D. citri. Knockdown of DcAKH and DcAKHR not only resulted in the accumulation of triacylglycerol and a decline of glycogen, but also significantly decreased fecundity and CLas titer in ovaries. Combined in vivo and in vitro experiments showed that miR-34 suppresses DcAKHR expression by binding to its 3' untranslated region, whilst overexpression of miR-34 resulted in a decline of DcAKHR expression and CLas titer in ovaries and caused defects that mimicked DcAKHR knockdown phenotypes. Additionally, knockdown of DcAKH and DcAKHR significantly reduced juvenile hormone (JH) titer and JH signaling pathway genes in fat bodies and ovaries, including the JH receptor, methoprene-tolerant (DcMet), and the transcription factor, Krüppel homolog 1 (DcKr-h1), that acts downstream of it, as well as the egg development related genes vitellogenin 1-like (DcVg-1-like), vitellogenin A1-like (DcVg-A1-like) and the vitellogenin receptor (DcVgR). As a result, CLas hijacks AKH/AKHR-miR-34-JH signaling to improve D. citri lipid metabolism and fecundity, while simultaneously increasing the replication of CLas, suggesting a mutualistic interaction between CLas and D. citri ovaries.