The mTORC2/SGK1/NDRG1 Signaling Axis Is Critical for the Mesomesenchymal Transition of Pleural Mesothelial Cells and the Progression of Pleural Fibrosis.

Progressive lung scarring because of persistent pleural organization often results in pleural fibrosis (PF). This process affects patients with complicated parapneumonic pleural effusions, empyema, and other pleural diseases prone to loculation. In PF, pleural mesothelial cells undergo mesomesenchymal transition (MesoMT) to become profibrotic, characterized by increased expression of α-smooth muscle actin and matrix proteins, including collagen-1. In our previous study, we showed that blocking PI3K/Akt signaling inhibits MesoMT induction in human pleural mesothelial cells (HPMCs) (1). However, the downstream signaling pathways leading to MesoMT induction remain obscure. Here, we investigated the role of mTOR complexes (mTORC1/2) in MesoMT induction. Our studies show that activation of the downstream mediator mTORC1/2 complex is, likewise, a critical component of MesoMT. Specific targeting of mTORC1/2 complex using pharmacological inhibitors such as INK128 and AZD8055 significantly inhibited transforming growth factor ß (TGF-ß)-induced MesoMT markers in HPMCs. We further identified the mTORC2/Rictor complex as the principal contributor to MesoMT progression induced by TGF-ß. Knockdown of Rictor, but not Raptor, attenuated TGF-ß-induced MesoMT in these cells. In these studies, we further show that concomitant activation of the SGK1/NDRG1 signaling cascade is essential for inducing MesoMT. Targeting SGK1 and NDRG1 with siRNA and small molecular inhibitors attenuated TGF-ß-induced MesoMT in HPMCs. Additionally, preclinical studies in our Streptococcus pneumoniae-mediated mouse model of PF showed that inhibition of mTORC1/2 with INK128 significantly attenuated the progression of PF in subacute and chronic injury. In conclusion, our studies demonstrate that mTORC2/Rictor-mediated activation of SGK1/NDRG1 is critical for MesoMT induction and that targeting this pathway could inhibit or even reverse the progression of MesoMT and PF.

NOX1 Promotes Mesothelial-Mesenchymal Transition through Modulation of Reactive Oxygen Species-mediated Signaling.

Pleural organization may occur after empyema or complicated parapneumonic effusion and can result in restrictive lung disease with pleural fibrosis (PF). Pleural mesothelial cells (PMCs) may contribute to PF through acquisition of a profibrotic phenotype, mesothelial-mesenchymal transition (MesoMT), which is characterized by increased expression of α-SMA (α-smooth muscle actin) and other myofibroblast markers. Although MesoMT has been implicated in the pathogenesis of PF, the role of the reactive oxygen species and the NOX (nicotinamide adenine dinucleotide phosphate oxidase) family in pleural remodeling remains unclear. Here, we show that NOX1 expression is enhanced in nonspecific human pleuritis and is induced in PMCs by THB (thrombin). 4-Hydroxy-2-nonenal, an indicator of reactive oxygen species damage, was likewise increased in our mouse model of pleural injury. NOX1 downregulation blocked THB- and Xa (factor Xa)-mediated MesoMT, as did pharmacologic inhibition of NOX1 with ML-171. NOX1 inhibition also reduced phosphorylation of Akt, p65, and tyrosine 216-GSK-3ß, signaling molecules previously shown to be implicated in MesoMT. Conversely, ML-171 did not reverse established MesoMT. NOX4 downregulation attenuated TGF-ß- and THB-mediated MesoMT. However, NOX1 downregulation did not affect NOX4 expression. NOX1- and NOX4-deficient mice were also protected in our mouse model of Streptococcus pneumoniae-mediated PF. These data show that NOX1 and NOX4 are critical determinants of MesoMT.

Upregulated PTPN2 induced by inflammatory response or oxidative stress stimulates the progression of thyroid cancer.

PTPN2 is one of the members of the protein Tyrosine Phosphatases (PTPs) family. To explore the promotive effect of upregulated PTPN2 induced by inflammatory response or oxidative stress on the progression of thyroid cancer. PTPN2 level in thyroid cancer tissues and cell lines was detected. Kaplan-Meier method was applied for evaluating the prognostic value of PTPN2 in thyroid cancer patients. After stimulation of inflammatory response (treatment of IFN-γ and TNF-α), or oxidative stress (treatment of H2O2), protein level of PTPN2 in K1 cells was measured by Western blot. Regulatory effects of PTPN2 on EdU-positive staining and Ki-67 positive cell ratio in K1 cells either with H2O2 stimulation or not were determined. PTPN2 was upregulated in thyroid cancer tissues and cell lines. Its level was higher in metastatic thyroid cancer patients than those of non-metastatic ones. High level of PTPN2 predicted worse prognosis of thyroid cancer. Treatment of either IFN-γ or TNF-α upregulated protein level of PTPN2 in K1 cells. Meanwhile, H2O2 stimulation upregulated PTPN2, which was reversed by NAC administration. With the stimulation of increased doses of H2O2, EdU-positive staining and Ki-67 positive cell ratio were dose-dependently elevated. Silence of PTPN2 attenuated proliferative ability and Ki-67 expression in K1 cells either with H2O2 stimulation or not. Inflammatory response or oxidative stress induces upregulation of PTPN2, thus promoting the progression of thyroid cancer.

Microglia TREM2 is required for electroacupuncture to attenuate neuroinflammation in focal cerebral ischemia/reperfusion rats.

Neuroinflammation plays a critical role in ischemic stroke pathology and could be a promising target in ischemic stroke. Triggering receptor expressed on myeloid cells 2 (TREM2) is a microglia-specific receptor in the CNS that is involved in regulating neuroinflammation in cerebral ischemia. However, the role of TREM2 in ischemic stroke is controversial. Electroacupuncture (EA) is an effective therapy for alleviating stroke-induced neuroinflammation. Here, we found that ischemic stroke induced an increased microglial TREM2 expression, and EA treatment can further promote microglial TREM2 expression following cerebral ischemia. TREM2 overexpression was observed to play a neuroprotective role by improving the neurobehavioral deficit and reducing the cerebral infarct volume 72 h after reperfusion, whereas TREM2 silencing had the opposite effects. Moreover, the effects of EA on improving stroke outcome and suppressing neuroinflammation in the brain were reversed by TREM2 silencing. Finally, TREM2 silencing also suppressed the ability of EA to regulate the PI3K/Akt and NF-κB signaling pathways. Altogether, the results show that TREM2 could be a potential target in EA treatment for attenuating inflammatory injury following cerebral ischemia/reperfusion.

Lentivirus-mediated overexpression of OTULIN ameliorates microglia activation and neuroinflammation by depressing the activation of the NF-κB signaling pathway in cerebral ischemia/reperfusion rats.

BACKGROUND: Ischemic stroke-induced neuroinflammation is mainly mediated by microglial cells. The nuclear factor kappa B (NF-κB) pathway is the key transcriptional pathway that initiates inflammatory responses following cerebral ischemia. OTULIN, a critical negative regulator of the NF-κΒ signaling pathway, exerts robust effects on peripheral immune cell-mediated inflammation and is regarded as an essential mediator for repressing inflammation in vivo. The effect of OTULIN on inflammatory responses in the central nervous system (CNS) was previously unstudied. This current study investigated the anti-inflammatory effect of OTULIN both in vitro and in vivo in ischemic stroke models. METHODS: Sprague-Dawley (SD) rats were subjected to transient middle cerebral artery occlusion (tMCAO) or an intraperitoneal injection of lipopolysaccharide (LPS). Overexpression of the OTULIN gene was utilized to observe the effect of OTULIN on ischemic stroke outcomes. The effect of OTULIN overexpression on microglia-mediated neuroinflammation was examined in rat primary microglia (PM) and in the microglial cell line N9 after induction by oxygen-glucose deprivation (OGD)-treated neuronal medium. The activation and inflammatory responses of microglia were detected using immunofluorescence, ELISA, and qRT-PCR. The details of molecular mechanism were assessed using Western blotting. RESULTS: In the tMCAO rats, the focal cerebral ischemia/reperfusion injury induced a continuous increase in OTULIN expression within 72 h, and OTULIN expression was increased in activated microglial cells. OTULIN overexpression obviously decreased the cerebral infarct volume, improved the neurological function deficits, and reduced neuronal loss at 72 h after reperfusion, and it also inhibited the activation of microglia and attenuated the release of TNF-α, IL-1ß, and IL-6 by suppressing the NF-κB pathway at 24 h after tMCAO. In vitro, OTULIN overexpression inhibited the microglia-mediated neuroinflammation by reducing the production of TNF-α, IL-1ß, and IL-6 via depressing the NF-κB pathway in both PM and N9 cells. CONCLUSIONS: OTULIN provides a potential therapeutic target for ischemic brain injury by ameliorating the excessive activation of microglial cells and neuroinflammation through repressing the NF-κB signaling pathway.

Inhibition of Glycogen Synthase Kinase 3ß Blocks Mesomesenchymal Transition and Attenuates Streptococcus pneumonia-Mediated Pleural Injury in Mice.

Pleural loculation affects about 30,000 patients annually in the United States and in severe cases can resolve with restrictive lung disease and pleural fibrosis. Pleural mesothelial cells contribute to pleural rind formation by undergoing mesothelial mesenchymal transition (MesoMT), whereby they acquire a profibrotic phenotype characterized by increased expression of α-smooth muscle actin and collagen 1. Components of the fibrinolytic pathway (urokinase plasminogen activator and plasmin) are elaborated in pleural injury and strongly induce MesoMT in vitro. These same stimuli enhance glycogen synthase kinase (GSK)-3ß activity through increased phosphorylation of Tyr-216 in pleural mesothelial cells and GSK-3ß mobilization from the cytoplasm to the nucleus. GSK-3ß down-regulation blocked induction of MesoMT. Likewise, GSK-3ß inhibitor 9ING41 blocked induction of MesoMT and reversed established MesoMT. Similar results were demonstrated in a mouse model of Streptococcus pneumoniae-induced empyema. Intraperitoneal administration of 9ING41, after the induction of pleural injury, attenuated injury progression and improved lung function (lung volume and compliance; P < 0.05 compared with untreated and vehicle controls). MesoMT marker α-smooth muscle actin was reduced in 9ING41-treated mice. Pleural thickening was also notably reduced in 9ING41-treated mice (P < 0.05). Collectively, these studies identify GSK-3ß as a newly identified target for amelioration of empyema-related pleural fibrosis and provide a strong rationale for further investigation of GSK-3ß signaling in the control of MesoMT and pleural injury.

Complex-I Alteration and Enhanced Mitochondrial Fusion Are Associated With Prostate Cancer Progression.

Mitochondria (mt) encoded respiratory complex-I (RCI) mutations and their pathogenicity remain largely unknown in prostate cancer (PCa). Little is known about the role of mtDNA loss on mt integrity in PCa. We determined mtDNA mutation in human and mice PCa and assessed the impact of mtDNA depletion on mt integrity. We also examined whether the circulating exosomes from PCa patients are transported to mt and carry mtDNA or mt proteins. We have employed next generation sequencing of the whole mt genome in human and Hi-myc PCa. The impact of mtDNA depletion on mt integrity, presence of mtDNA, and protein in sera exosomes was determined. A co-culture of human PCa cells and the circulating exosomes followed by confocal imaging determined co-localization of exosomes and mt. We observed frequent RCI mutations in human and Hi-myc PCa which disrupted corresponding complex protein expression. Depletion of mtDNA in PCa cells influenced mt integrity, increased expression of MFN1, MFN2, PINK1, and decreased expression of MT-TFA. Increased mt fusion and expression of PINK1 and DNM1L were also evident in the Hi-myc tumors. RCI-mtDNA, MFN2, and IMMT proteins were detected in the circulating exosomes of men with benign prostate hyperplasia (BPH) and progressive PCa. Circulating exosomes and mt co-localized in PCa cells. Our study identified new pathogenic RCI mutations in PCa and defined the impact of mtDNA loss on mt integrity. Presence of mtDNA and mt proteins in the circulating exosomes implicated their usefulness for biomarker development.

Upregulation of neuronal zinc finger protein A20 expression is required for electroacupuncture to attenuate the cerebral inflammatory injury mediated by the nuclear factor-kB signaling pathway in cerebral ischemia/reperfusion rats.

BACKGROUND: Zinc finger protein A20 (tumor necrosis factor alpha-induced protein 3) functions as a potent negative feedback inhibitor of the nuclear factor-kB (NF-kB) signaling. It exerts these effects by interrupting the activation of IkB kinase beta (IKKß), the most critical kinase in upstream of NF-kB, and thereby controlling inflammatory homeostasis. We reported previously that electroacupuncture (EA) could effectively suppress IKKß activation. However, the mechanism underlying these effects was unclear. Therefore, the current study further explored the effects of EA on A20 expression in rat brain and investigated the possible mechanism of A20 in anti-neuroinflammation mediated by EA using transient middle cerebral artery occlusion (MCAO) rats. METHODS: Rats were treated with EA at the "Baihui (GV20)," "Hegu (L14)," and "Taichong (Liv3)" acupoints once a day starting 2 h after focal cerebral ischemia. The spatiotemporal expression of A20, neurobehavioral scores, infarction volumes, cytokine levels, glial cell activation, and the NF-kB signaling were assessed at the indicated time points. A20 gene interference (overexpression and silencing) was used to investigate the role of A20 in mediating the neuroprotective effects of EA and in regulating the interaction between neuronal and glial cells by suppressing neuronal NF-kB signaling during cerebral ischemia/reperfusion-induced neuroinflammation. RESULTS: EA treatment increased A20 expression with an earlier peak and longer lasting upregulation. The upregulated A20 protein was predominantly located in neurons in the cortical zone of the ischemia/reperfusion. Furthermore, neuronal A20 cell counts were positively correlated with neurobehavioral scores but negatively correlated with infarct volume, the accumulation of pro-inflammatory cytokines, and glial cell activation. Moreover, the effects of EA on improving the neurological outcome and suppressing neuroinflammation in the brain were reversed by A20 silencing. Finally, A20 silencing also suppressed the ability of EA to inhibit neuronal NF-kB signaling pathway. CONCLUSIONS: Ischemia/reperfusion cortical neurons in MCAO rats are the main cell types that express A20, and there is a correlation between A20 expression and the suppression of neuroinflammation and the resulting neuroprotective effects. EA upregulated neuronal A20 expression, which played an essential role in the anti-inflammatory effects of EA by suppressing the neuronal NF-kB signaling pathway in the brains of MCAO rats.

[Effects of Minocycline on the Neuronal Plasticity in Rats after Focal Cerebral Ischemia Reperfusion Injury].

OBJECTIVE: To observe the effects of minocycline on morphology and the expression of synaptophysin in cortical tissues of rats after cerebral ischemia reperfusion injury. METHODS: 36 male SD rats were randomly divided into 3 groups: sham group, model group [ischemia reperfusion (I/R)] and minocycline (Min) group (treated with minocycline for 14 d, 3 mg/kg, 2 times/d ). Middle cerebral artery occlusion (MCAO) was used to established as focal cerebral I/R model. At 14 d after I/R. Neurological functional recovery was evaluated using the staircase test, the cell morphology in cortex was evaluated by HE staining, the neurite growth was observed by immunostaining with anti-microtubule-associated protein-2 (MAP-2) antibody, the expression of synaptophysin in pei-infarct region was tested by Western blot. RESULTS: In the sham group, the rats did not show any neurological deficits. The neurons in the cortex were arranged in neat rows and the morphology were normal, the MAP-2 positive neurons showed longer neuronal processes than the model group. Compared to the model group, minocycline significantly improved forelimb motor function, increased the expression of synaptophysin and the number of MAP-2-positive cells in peri-infarct region (P < 0.05). CONCLUSION: Minocycline could improve the neurite regrowth and the expression of synaptophysin of neuron in ischemic cortex, promote neurological functional recovery of rats after MCAO, which is related to regulate the neuronal plasticity.

Methylation and miRNA effects of resveratrol on mammary tumors vs. normal tissue.

We reported that resveratrol decreased DNA methyltransferase (DNMT) 1 and 3b expression in vitro and demethylates tumor suppressor RASSF-1a in women at increased breast cancer risk. We investigated the effects of resveratrol on DNMT and miRNA expression in normal and tumor mammary tissue in a rodent model of estrogen dependent mammary carcinoma. Eighty-nine female ACI rats received estradiol plus: low dose (lo) resveratrol, high dose (hi) resveratrol, 5-aza-2-deoxycytidine (Aza), a known inhibitor of DNMTs, or control (no additional treatment). After 21 wk of treatment, animals were sacrificed and mammary glands harvested. Matched tumor/normal tissues were available from 36 rats. DMNT3b (but not DNMT1) differed in tumor vs. normal tissue after lo (P = .04) and hi (P = .007) resveratrol and Aza treatment. With hi resveratrol, DNMT3b decreased in tumor but increased normal tissue. Hi resveratrol increased miR21, -129, -204, and -489 >twofold in tumor and decreased the same miRs in normal tissue 10-50% compared to control. There was an inverse association between DNMT3b and miR129, -204, and -489 in normal and/or tumor tissue. Treatment with resveratrol differentially influences tumor vs. normal tissue DNMT3b and miRNA expression. This mechanism of action of resveratrol to influence mammary carcinogenesis warrants further investigation.

Kikuchi-Fujimoto disease evolves into lupus encephalopathy characterized by venous sinus thrombosis: a case report.

Kikuchi-Fujimoto disease (KFD) is a benign, self-limiting illness that can progress to systemic lupus erythematosus (SLE) in approximately 30% of cases. Neurological injuries can occur in both diseases, albeit with distinct presentations. Venous sinus thrombosis is a serious cerebrovascular complication in patients with neuropsychiatric SLE but is rarely observed in patients with KFD. The involvement of various antibodies, particularly antiphospholipid antibodies, can cause vascular endothelial cell injury, resulting in focal cerebral ischemia and intracranial vascular embolism in SLE. However, there are cases in which thrombotic pathology occurs without antiphospholipid antibody positivity, attributed to vascular lesions. In this report, we present a case of KFD and lupus encephalopathy featuring cerebral venous sinus thrombosis, despite the patient being negative for antiphospholipid antibody. We also conducted a comparative analysis of C3 and C4 levels in cerebrospinal fluid (CSF) and peripheral blood, along with the protein ratio in CSF and serum, to elucidate the pathological changes and characteristics of lupus encephalopathy.

Bioorthogonal click labeling of an amber-free HIV-1 provirus for in-virus single molecule imaging.

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. Here, we develope an intact amber-free HIV-1 system that overcomes hurdles of preexisting viral amber codons. We achieved dual-ncAA incorporation into Env on amber-free virions, enabling single-molecule Förster resonance energy transfer (smFRET) studies of click-labeled Env that validated the previous peptide-based labeling approaches by confirming the intrinsic propensity of Env to dynamically sample multiple conformational states. Amber-free click-labeled Env also enabled real-time tracking of single virion internalization and trafficking in cells. Our system thus permits in-virus bioorthogonal labeling of proteins, compatible with studies of virus entry, trafficking, and egress from cells.

Electroacupuncture improves behavioral recovery and increases SCF/c-kit expression in a rat model of focal cerebral ischemia/reperfusion.

This study aims to investigate the mechanism of electroacupuncture (EA) in promoting behavioral recovery after focal cerebral ischemia/reperfusion. The SD rats received filament occlusion of the right middle cerebral artery for 2 h followed by reperfusion for 1, 3, 7, 14, and 21 days, respectively. Rats were randomly divided into sham group, model group and EA group. After 2 h of the reperfusion, EA was given at bilateral "Hegu" point (LI 4) in the EA group. Neurobehavioral evaluation, the expression of stem cell factor (SCF), its receptor c-kit and matrix metalloproteinase-9 (MMP-9) protein and mRNA in the cortical ischemic region were measured. EA treatment can improve behavioral recovery after ischemia/reperfusion. Compared with the sham group, the positive cells and mRNA expression of SCF, c-kit, MMP-9, the protein expression of SCF were increased significantly in the model and EA groups (P < 0.001). Compared with the model group, the positive cells, protein and mRNA expression of SCF were increased significantly in EA groups (P < 0.01). The positive cells and mRNA expression of c-kit were increased in EA groups beginning at 3 day and remained significantly high thereafter. The expression of MMP-9 positive cells and mRNA were deceased significantly in the 1 day subgroup in EA (P < 0.01), but increased significantly in the 3, 7 days subgroups (P < 0.01). We conclude that EA treatment up-regulates the positive cells and mRNA expression of SCF, c-kit and MMP-9 after cerebral ischemia/reperfusion. EA may promote neurobehavioral recovery by increasing the protein and mRNA expression of SCF, c-kit and MMP-9 after cerebral ischemia/reperfusion.

An intact amber-free HIV-1 system for in-virus protein bioorthogonal click labeling that delineates envelope conformational dynamics.

The HIV-1 envelope (Env) glycoprotein is conformationally dynamic and mediates membrane fusion required for cell entry. Single-molecule fluorescence resonance energy transfer (smFRET) of Env using peptide tags has provided mechanistic insights into the dynamics of Env conformations. Nevertheless, using peptide tags risks potential effects on structural integrity. Here, we aim to establish minimally invasive smFRET systems of Env on the virus by combining genetic code expansion and bioorthogonal click chemistry. Amber stop-codon suppression allows site-specifically incorporating noncanonical/unnatural amino acids (ncAAs) at introduced amber sites into proteins. However, ncAA incorporation into Env (or other HIV-1 proteins) in the virus context has been challenging due to low copies of Env on virions and incomplete amber suppression in mammalian cells. Here, we developed an intact amber-free virus system that overcomes impediments from preexisting ambers in HIV-1. Using this system, we successfully incorporated dual ncAAs at amber-introduced sites into Env on intact virions. Dual-ncAA incorporated Env retained similar neutralization sensitivities to neutralizing antibodies as wildtype. smFRET of click-labeled Env on intact amber-free virions recapitulated conformational profiles of Env. The amber-free HIV-1 infectious system also permits in-virus protein bioorthogonal labeling, compatible with various advanced microscopic studies of virus entry, trafficking, and egress in living cells. Amber-free HIV-1 infectious systems actualized minimal invasive Env tagging for smFRET, versatile for in-virus bioorthogonal click labeling in advanced microscopic studies of virus-host interactions.

Proteins and carbohydrates in nipple aspirate fluid predict the presence of atypia and cancer in women requiring diagnostic breast biopsy.

BACKGROUND: Herein we present the results of two related investigations. The first study determined if concentrations in breast nipple discharge (ND) of two proteins (urinary plasminogen activator, uPA and its inhibitor, PAI-1) predicted the presence of breast atypia and cancer in pre- and/or postmenopausal women requiring surgery because of a suspicious breast lesion. The second study assessed if these proteins increased the predictive ability of a carbohydrate (Thomsen Friedenreich, TF) which we previously demonstrated predicted the presence of disease in postmenopausal women requiring surgery. METHODS: In the first study we prospectively enrolled 79 participants from whom we collected ND, measured uPA and PAI-1 and correlated expression with pathologic findings. In the second study we analyzed 35 (uPA and PAI-1 in 24, uPA in an additional 11) ND samples collected from different participants requiring breast surgery, all of whom also had TF results. RESULTS: uPA expression was higher in pre- and PAI-1 in postmenopausal women with 1) cancer (DCIS or invasive) vs. either no cancer (atypia or benign pathology, p = .018 and .025, respectively), or benign pathology (p = .017 and .033, respectively); and 2) abnormal (atypia or cancer) versus benign pathology (p = .018 and .052, respectively). High uPA and PAI-1 concentrations and age were independent predictors of disease in premenopausal women, with an area under the curve (AUC) of 83-87% when comparing diseased vs. benign pathology. uPA, TF, and age correctly classified 35 pre- and postmenopausal women as having disease or not 84-91% of the time, whereas combining uPA+PAI-1+TF correctly classified 24 women 97-100% of the time. CONCLUSIONS: uPA and PAI-1 concentrations in ND were higher in women with atypia and cancer compared to women with benign disease. Combining uPA, PAI-1 and TF in the assessment of women requiring diagnostic breast surgery maximized disease prediction. The assessment of these markers may prove useful in early breast cancer detection.

Differential expression of cancer associated proteins in breast milk based on age at first full term pregnancy.

BACKGROUND: First full term pregnancy (FFTP) completed at a young age has been linked to low long term breast cancer risk, whereas late FFTP pregnancy age confers high long term risk, compared to nulliparity. Our hypothesis was that proteins linked to breast cancer would be differentially expressed in human milk collected at three time points during lactation based on age at FFTP. METHODS: We analyzed breast milk from 72 lactating women. Samples were collected within 10 days of the onset of lactation (baseline-BL), two months after lactation started and during breast weaning (W). We measured 16 proteins (11 kallikreins (KLKs), basic fibroblast growth factor, YKL-40, neutrophil gelatinase-associated lipocalin and transforming growth factor (TGF) ß-1 and -2) associated with breast cancer, most known to be secreted into milk. RESULTS: During lactation there was a significant change in the expression of 14 proteins in women < 26 years old and 9 proteins in women > = 26 at FFTP. The most significant (p < .001) changes from BL to W in women divided by FFTP age (< 26 vs. > = 26) were in KLK3,6, 8, and TGFß2 in women < 26; and KLK6, 8, and TGFß2 in women > = 26. There was a significant increase (p = .022) in KLK8 expression from BL to W depending on FFTP age. Examination of DNA methylation in the promoter region of KLK6 revealed high levels of methylation that did not explain the observed changes in protein levels. On the other hand, KLK6 and TGFß1 expression were significantly associated (r2 = .43, p = .0050). CONCLUSIONS: The expression profile of milk proteins linked to breast cancer is influenced by age at FFTP. These proteins may play a role in future cancer risk.

Trans-resveratrol alters mammary promoter hypermethylation in women at increased risk for breast cancer.

Trans-resveratrol, present in high concentration in the skin of red grapes and red wine, has a dose-dependent antiproliferative effect in vitro, prevents the formation of mammary tumors, and has been touted as a chemopreventive agent. Based upon in vitro studies demonstrating that trans-resveratrol downregulates the expression of 1) DNA methyltransferases and 2) the cancer promoting prostaglandin (PG)E(2), we determined if trans-resveratrol had a dose-related effect on DNA methylation and prostaglandin expression in humans. Thirty-nine adult women at increased breast cancer risk were randomized in double-blind fashion to placebo, 5 or 50 mg trans-resveratrol twice daily for 12 wk. Methylation assessment of 4 cancer-related genes (p16, RASSF-1α, APC, CCND2) was performed on mammary ductoscopy specimens. The predominant resveratrol species in serum was the glucuronide metabolite. Total trans-resveratrol and glucuronide metabolite serum levels increased after consuming both trans-resveratrol doses (P < .001 for both). RASSF-1α methylation decreased with increasing levels of serum trans-resveratrol (P = .047). The change in RASSF-1α methylation was directly related to the change in PGE(2) (P = .045). This work provides novel insights into the effects of trans-resveratrol on the breast of women at increased breast cancer risk, including a decrease in methylation of the tumor suppressor gene RASSF-1α. Because of the limited sample size, our findings should be validated in a larger study.

Knockout of SLAMF8 attenuates collagen-induced rheumatoid arthritis in mice through inhibiting TLR4/NF-κB signaling pathway.

Rheumatoid arthritis (RA) is a chronic autoimmune disorder characterized by synovial hyperplasia, cartilage damage, and ultimate bone destruction. The signaling lymphocytic activation molecule family member 8 (SLAMF8) is a cell surface receptor expressed on various immune cells. This study aimed to investigate the role of SLAMF8 in the pathogenesis of RA. The SLAMF8 gene was identified as a differentially expressed gene in RA by analyzing the Gene Expression Omnibus database and synovial tissue samples collected from RA patients. Upregulation of SLAMF8 was associated with increased disease activity and inflammation in RA. Mice with collagen type II-induced arthritis (CIA) showed highly expressed SLAMF8, severe paw swelling, elevated inflammatory cytokine production, and excessive accumulation of immune cells. However, knockout of SLAMF8 alleviated collagen type II immunization-induced synovial hyperplasia and joint arthritis in mice. The in-vitro and in-vivo study showed that genetic deletion of SLAMF8 significantly inhibited upregulation of Toll-like receptor 4 (TLR4) and activation of the nuclear factor kappa B (NF-κB) pathway in fibroblast-like synoviocytes and bone marrow-derived macrophages derived from WT and SLAMF8 knockout mice under lipopolysaccharide stimulation. In conclusion, SLAMF8 was aberrantly expressed in RA patient and played an indispensable role in initiating inflammation and maintaining the pro-inflammatory environment in the inflamed joint. Targeted inhibition of SLAMF8 attenuated the severity of RA via blocking the TLR4/NF-κB signaling pathway. These data suggested that SLAMF8 may be a potential target for the treatment of RA.

Guizhi-Shaoyao-Zhimu decoction attenuates monosodium urate crystal-induced inflammation through inactivation of NF-κB and NLRP3 inflammasome.

ETHNOPHARMACOLOGICAL RELEVANCE: Guizhi-Shaoyao-Zhimu decoction (GSZD), a classical traditional Chinese medicine (TCM) prescription, is used empirically to treat various types of arthritis in TCM clinical practice. However, the underlying mechanisms of GSZD on gouty inflammation are not totally elucidated. AIM OF STUDY: The purpose of this study is to investigate the effects of GSZD on peritoneal recruitment of neutrophils, production of proinflammatory mediators, activations of nuclear factor (NF)-κB and nucleotide oligomerization domain-like receptor protein-3 (NLRP3) inflammasome in mice with monosodium urate crystal (MSU)-induced peritonitis (MIP). MATERIALS AND METHODS: Mice were intragastrically administered with GSZD for 7 days. After the last administration, mice were intraperitoneally injected with MSU. Peritoneal exudates of mice were harvested, and total peritoneal cells were calculated. Levels of interleukin (IL)-1ß, IL-6 and monocyte chemotactic protein (MCP)-1 in peritoneal exudates were tested by enzyme-linked immunosorbent assay. Expressions of IL-1ß, NLRP3, cysteinyl aspartate specific proteinase (caspase)-1, apoptosis-associated speck-like protein containing the caspase activation and recruitment domain (ASC), phosphorylated (p)-p65, inhibitor of NF-κB (IκB)α, p-IκB kinase (IKK)ß, nuclear p65, p-mitogen-activated protein kinases (MAPKs) in peritoneal cells were analyzed by Western blot. Binding activity of NF-κB to DNA was measured by a Trans AM™ kit for p65. Interaction between ASC and pro-caspase-1 was assessed by co-immunoprecipitation assay. RESULTS: Total peritoneal cells, levels of IL-1ß, IL-6 and MCP-1 were significantly reduced by GSZD treatment in peritoneal exudates of MIP mice. As for the activation of NF-κB, GSZD treatment significantly reduced the levels of p-p65, p-IKKß, nuclear p65 and p-MAPKs, enhanced the level of IκBα and abated the binding ability of NF-κB to DNA in peritoneal cells of MIP mice. As for the activation of NLRP3 inflammasome, GSZD treatment significantly reduced the levels of IL-1ß, NLRP3 and caspase-1, and alleviated the interaction between ASC and pro-caspase-1 in peritoneal cells of MIP mice. Nevertheless, GSZD didn't remarkably change the level of ASC. CONCLUSIONS: These results suggest that GSZD attenuates the MSU-induced inflammation through inhibiting the activations of NF-κB and NLRP3 inflammasome.

[Modeling spatial distributions of hydrogen and oxygen stable isotopes in surface soils of the upper reaches of Minjiang River based on machine languages]. / åŸºäºŽæœºå™¨è¯­è¨€çš„å²·æ±Ÿä¸Šæ¸¸æµåŸŸè¡¨å±‚åœŸå£¤æ°¢æ°§ç¨³å®šåŒä½ç´ ç©ºé—´åˆ†å¸ƒæ¨¡æ‹Ÿ.

To study the feasibility of simulating the spatial distribution of hydrogen and oxygen stable isotopes composition (δ2H and δ18O) in the surface soil based on the machine learning method and to investigate large-scale distribution of δ2H and δ18O in the upper reaches of Minjiang River, 183 soil samples were collected from the 0-10 cm soil layer. After variable selection, back propagation (BP) neural network, random forests (RF) and support vector machine (SVM) were used to model the δ2H and δ18O of the study area, with the accuracies being evaluated. The structural equation model (SEM) was used to reveal the mechanism between the auxiliary variables and the δ2H and δ18O of soil water. The results showed that the RF model had the highest prediction accuracy, and could explain 75.0% and 64.0% of the variations of δ2H and δ18O in the surface soil, respectively. In this model, soil water content was the most important auxiliary variable, contributing 48.9% and 37.4% to δ2H and δ18O. Vegetation factors had stronger influence on δ2H and δ18O in the surface soil than climate factors, and the influence of climate factors on δ2H and δ18O was media-ted by vegetation factors. Among all the auxiliary variables, hydrogen/oxygen isotope of precipitation had the lowest effect on δ2H and δ18O due to the fractionation. The δ2H and δ18O in the surface soil of the upper reaches of the Minjiang River changed significantly across different months during the growing season. The increases of δ2H and δ18O in the early growing season and the decreases in the late growing season were mainly affected by vegetation, while climate change led to a small fluctuation in the middle growing season.