Single-cell RNA-seq reveals the metabolic status of immune cells response to immunotherapy in triple-negative breast cancer.

Immune checkpoint blockade (ICB) therapy offers promise in the treatment of triple-negative breast cancer (TNBC); however, its limited efficacy in certain TNBC patients poses a challenge. In this study, we elucidated the metabolic mechanism at 'sub-subtype' resolution underlying the non-response to ICB therapy in TNBC. Here, an analytic pipeline was developed to reveal the metabolic heterogeneity, which is correlated with the ICB outcomes, within each immune cell subtype. First, we identified metabolic 'sub-subtypes' within certain cell subtypes, predominantly T cell subsets, which are enriched in ICB non-responders and named as non-responder-enriched (NR-E) clusters. Notably, most of NR-E T metabolic cells exhibit globally higher metabolic activities compared to other cells within the same individual subtype. Further, we investigated the extra-cellular signals that trigger the metabolic status of NR-E T cells. In detail, the prediction of cell-to-cell communication indicated that NR-E T cells are regulated by plasmatic dendritic cells (pDCs) through TNFSF9, as well as by macrophages expressing SIGLEC9. In addition, we also validate the communication between TNFSF9+ pDCs and NR-E T cells utilizing deconvolution of spatial transcriptomics analysis. In summary, our research identified specific metabolic 'sub-subtypes' associated with ICB non-response and uncovered the mechanisms of their regulation in TNBC. And the proposed analytical pipeline can be used to examine metabolic heterogeneity within cell types that correlate with diverse phenotypes.

[The neural basis underlying primary dysmenorrhea: evidence from neuroimaging and animal model studies].

Primary dysmenorrhea (PDM), cyclic menstrual pain in the absence of pelvic anomalies, is characterized by acute and chronic gynecological pain disorders in childbearing age women. PDM strongly affects the quality of life of patients and leads to economic losses. PDM generally do not receive radical treatment and often develop into other chronic pain disorders later in life. The clinical treatment status of PDM, the epidemiology of PDM and chronic pain comorbidities, and the abnormal physiological and psychological characteristics of patients with PDM suggest that PDM not only is related to the inflammation around the uterus, but also may be related to the abnormal pain processing and regulation function of patients' central system. Therefore, exploring the brain neural mechanism of PDM is indispensable and important to understand the pathological mechanism of PDM, and is also a hotspot of brain science research in recent years, which will bring new inspiration to explore the target of PDM intervention. Based on the progress of the neural mechanism of PDM, this paper systematically summarizes the evidence from neuroimaging and animal model studies.

Recombinant Human Clusterin Seals Damage to the Ocular Surface Barrier in a Mouse Model of Ophthalmic Preservative-Induced Epitheliopathy.

There is a significant unmet need for therapeutics to treat ocular surface barrier damage, also called epitheliopathy, due to dry eye and related diseases. We recently reported that the natural tear glycoprotein CLU (clusterin), a molecular chaperone and matrix metalloproteinase inhibitor, seals and heals epitheliopathy in mice subjected to desiccating stress in a model of aqueous-deficient/evaporative dry eye. Here we investigated CLU sealing using a second model with features of ophthalmic preservative-induced dry eye. The ocular surface was stressed by topical application of the ophthalmic preservative benzalkonium chloride (BAC). Then eyes were treated with CLU and sealing was evaluated immediately by quantification of clinical dye uptake. A commercial recombinant form of human CLU (rhCLU), as well as an rhCLU form produced in our laboratory, designed to be compatible with U.S. Food and Drug Administration guidelines on current Good Manufacturing Practices (cGMP), were as effective as natural plasma-derived human CLU (pCLU) in sealing the damaged ocular surface barrier. In contrast, two other proteins found in tears: TIMP1 and LCN1 (tear lipocalin), exhibited no sealing activity. The efficacy and selectivity of rhCLU for sealing of the damaged ocular surface epithelial barrier suggests that it could be of therapeutic value in treating BAC-induced epitheliopathy and related diseases.

Mixed lineage kinase 3 requires a functional CRIB domain for regulation of blood pressure, cardiac hypertrophy, and left ventricular function.

Mixed lineage kinase 3 (MLK3) modulates blood pressure and left ventricular function, but the mechanisms governing these effects remain unclear. In the current study, we therefore investigated the role of the MLK3 Cdc42/Rac interactive binding (CRIB) domain in cardiovascular physiology. We examined baseline and left ventricular pressure overload responses in a MLK3 CRIB mutant (MLK3C/C) mouse, which harbors point mutations in the CRIB domain to disrupt MLK3 activation by Cdc42. Male and female MLK3C/C mice displayed increased invasively measured blood pressure compared with wild-type (MLK3+/+) littermate controls. MLK3C/C mice of both sexes also developed left and right ventricular hypertrophy but normal baseline LV function by echocardiography and invasive hemodynamics. In LV tissue from MLK3C/C mice, map3k11 mRNA, which encodes MLK3, and MLK3 protein were reduced by 74 ± 6% and 73 ± 7%, respectively. After 1-wk LV pressure overload with 25-gauge transaortic constriction (TAC), male MLK3C/C mice developed no differences in LV hypertrophy but displayed reduction in the LV systolic indices ejection fraction and dP/dt normalized to instantaneous pressure. JNK activation was also reduced in LV tissue of MLK3C/C TAC mice. TAC induced MLK3 translocation from cytosolic fraction to membrane fraction in LV tissue from MLK3+/+ but not MLK3C/C mice. These findings identify a role of the MLK3 CRIB domain in MLK3 regulation of basal blood pressure and cardiac morphology, and in promoting the compensatory LV response to pressure overload.NEW & NOTEWORTHY Here, we identified that the presence of two discrete point mutations within the Cdc42/Rac interaction and binding domain of the protein MLK3 recapitulates the effects of whole body MLK3 deletion on blood pressure, cardiac hypertrophy, and left ventricular compensation after pressure overload. These findings implicate the CRIB domain, and thus MLK3 activation by this domain, as critical for maintenance of cardiovascular homeostasis.

3-MCPD and glycidol coexposure induces systemic toxicity and synergistic nephrotoxicity via NLRP3 inflammasome activation, necroptosis, and autophagic cell death.

3-Monochloropropane-1,2-diol (3-MCPD), 2,3-epoxy-1-propanol (glycidol), and their esters are well-known food contaminants mainly formed by the heat processing of certain refined oils and coexist in various kinds of foodstuffs. However, the combined health effect and the underlying mechanism of 3-MCPD and glycidol coexposure are not well-understood. In this study, we investigated the systemic toxicity effects and the nephrotoxicity mechanisms of 3-MCPD and glycidol coexposure with in vitro and in vivo models, and next-generation sequencing (NGS) analysis. It was found that 3-MCPD and glycidol coexposure for 28 days synergistically induced toxicity in the kidney, lung, testis, and heart in C57BL/6 mice. Kidney was the most sensitive organ to coexposure, and the coexposure had a synergistic effect on inflammation and cytotoxicity through activation of the NLRP3 inflammasome, and the induction of necroptosis, and autophagic cell death in NRK-52E cells. Moreover, the NGS results revealed the genes changes associated with nephrotoxicity, inflammation and with the broad toxicity effects induced by 3-MCPD or glycidol alone or in combination, which were consistent with the results of in vitro and in vivo models. In summary, we report for the first time of the comprehensive toxicity effects and the mechanisms caused by 3-MCPD and glycidol coexposure.

Construction of an efficient genomic editing system with CRISPR/Cas9 in the vector mosquito Aedes albopictus.

Aedes (Stegomyia) albopictus, also known as the Asian tiger mosquito, is a mosquito which originated in Asia. In recent years, it has become increasingly rampant throughout the world. This mosquito can transmit several arboviruses, including dengue, Zika and chikungunya viruses, and is considered a public health threat. Despite the urgent need of genome engineering to analyze specific gene functions, progress in genetical manipulation of Ae. albopictus has been slow due to a lack of efficient methods and genetic markers. In the present study, we established targeted disruptions in two genes, kynurenine hydroxylase (kh) and dopachrome conversion enzyme (yellow), to analyze the feasibility of generating visible phenotypes with genome editing by the clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated protein 9 (Cas9) system in Ae. albopictus. Following Cas9 single guide RNA ribonucleoprotein injection into the posterior end of pre-blastoderm embryos, 30%-50% of fertile survivors produced alleles that failed to complement existing kh and yellow mutations. Complete eye and body pigmentation defects were readily observed in G1 pupae and adults, indicating successful generation of highly heritable mutations. We conclude that the CRISPR/Cas9-mediated gene editing system can be used in Ae. albopictus and that it can be adopted as an efficient tool for genome-scale analysis and biological study.

Use of a Recombinant Mosquito Densovirus As a Gene Delivery Vector for the Functional Analysis of Genes in Mosquito Larvae.

In vivo microinjection is the most commonly used gene transfer technique for analyzing the gene functions in individual mosquitoes. However, this method requires a more technically demanding operation and involves complicated procedures, especially when used in larvae due to their small size, relatively thin and fragile cuticle, and high mortality, which limit its application. In contrast, viral vectors for gene delivery have been developed to surmount extracellular and intracellular barriers. These systems have the advantages of easy manipulation, high gene transduction efficiency, long-term maintenance of gene expression, and the ability to produce persistent effects in vivo. Mosquito densoviruses (MDVs) are mosquito-specific, small single-stranded DNA viruses that can effectively deliver foreign nucleic acids into mosquito cells; however, the replacement or insertion of foreign genes to create recombinant viruses typically causes a loss of packaging and/or replication abilities, which is a barrier to the development of these viruses as delivery vectors. Herein, we report using an artificial intronic small-RNA expression strategy to develop a non-defective recombinant Aedes aegypti densovirus (AaeDV) in vivo delivery system. Detailed procedures for the construction, packaging and quantitative analysis of the rAaeDV vectors, and for larval infection are described. This study demonstrates, for the first time, the feasibility of developing a non-defective recombinant MDV micro RNA (miRNA) expression system, and thus providing a powerful tool for the functional analysis of genes in mosquito and establishing a basis for the application of viral paratransgenesis for controlling mosquito-borne diseases. We demonstrated that Aedes albopictus 1st instar larvae could be easily and effectively infected by introducing the virus into the water body of the larvae breeding site and that the developed rAaeDVs could be used to overexpress or knock down the expression of a specific target gene in larvae, providing a tool for the functional analysis of mosquito genes.

P2Y12 and P2Y13 receptors involved in ADPßs induced the release of IL-1ß, IL-6 and TNF-α from cultured dorsal horn microglia.

OBJECTIVE: P2 receptors have been implicated in the release of neurotransmitter and pro-inflammatory cytokines due to their response to neuroexcitatory substances in the microglia. Dorsal horn P2Y12 and P2Y13 receptors are involved in the development of pain behavior induced by peripheral nerve injury. However, it is not known whether P2Y12 and P2Y13 receptors activation is associated with the expression and the release of interleukin-1B (IL-1ß), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) in cultured dorsal spinal cord microglia. For this reason, we examined the effects of ADPßs (ADP analog) on the expression and the release of IL-1ß, IL-6, and TNF-α. METHODS AND RESULTS: In this study, we observed the effect of P2Y receptor agonist ADPßs on the expression and release of IL-1ß, IL-6 and TNF-α by using real-time fluorescence quantitative polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). ADPßs induced the increased expression of Iba-1, IL-1ß, IL-6 and TNF-α at the level of messenger RNA (mRNA). ADPßs-evoked increase in Iba-1, IL-1ß, IL-6 and TNF-α mRNA expression was inhibited only partially by P2Y12 receptor antagonist MRS2395 or P2Y13 receptor antagonist MRS2211, respectively. Similarly, ADPßs-evoked release of IL-1ß, IL-6 and TNF-α was inhibited only partially by MRS2395 or MRS2211. Furthermore, ADPßs-evoked increased expression of Iba-1, IL-1ß, IL-6 and TNF-α mRNA, and release of IL-1ß, IL-6 and TNF-α were nearly all blocked after co-administration of MRS2395 plus MRS2179. Further evidence indicated that P2Y12 and P2Y13 receptor-evoked increased gene expression of IL-1ß, IL-6 and TNF-α were inhibited by Y-27632 (ROCK inhibitor), SB203580 (P38MAPK inhibitor) and PDTC (NF-κb inhibitor), respectively. Subsequently, P2Y12 and P2Y13 receptor-evoked release of IL-1ß, IL-6 and TNF-α, were also inhibited by Y-27632, SB203580 and PDTC, respectively. CONCLUSION: These observations suggest that P2Y12 and P2Y13 receptor-evoked gene expression and release of IL-1ß, IL-6 and TNF-α are associated with ROCK/P38MAPK/NF-κb signaling pathway.

Serum uric acid and metabolic syndrome in Taiwanese adults.

A positive association between serum uric acid and metabolic syndrome has been reported, but little information is available about the association between serum uric acid and metabolic syndrome in Taiwanese adults. The purpose of this study was to investigate the association between serum uric acid levels and metabolic syndrome in Taiwanese adults. We performed a cross-sectional study of 2085 men and 1557 women. All of the participants underwent a health screening during the period from January 2005 to December 2005 at a health center of the Shin Kong Wu Ho-Su Memorial Hospital. Metabolic syndrome was defined according to the National Cholesterol Education Program Adult Treatment Panel III criteria. The results showed that hyperuricemia was significantly associated with increased risk for hypertriglyceridemia, low high-density lipoprotein cholesterol level, and high blood pressure in men and women. The risk of metabolic syndrome was significantly higher in the fourth quartile than in the first quartile of uric acid level in men (odds ratio [OR], 1.50; 95% confidence interval [CI], 1.06-2.14) and women (OR, 2.33; 95% CI, 1.39-3.93). In addition, uric acid level was inversely associated with hyperglycemia in men. The ORs of hyperglycemia for the second, third, and fourth quartile of uric acid were 0.69 (95% CI, 0.46-1.03), 0.55 (95% CI, 0.37-0.83), and 0.45 (95% CI, 0.29-0.69), respectively, compared with the lowest quartile of uric acid. The results demonstrate that there is a positive association between serum uric acid levels and metabolic syndrome and an inverse association between uric acid and fasting plasma glucose in Taiwanese adults.