Multiomics analysis revealed that the metabolite profile of raw milk is associated with lactation stage of dairy cows and could be affected by variations in the ruminal microbiota.

The nutritional components and quality of milk are influenced by the rumen microbiota and its metabolites at different lactation stages. Hence, rumen fluid and milk samples from 6 dairy cows fed the same diet were collected during peak, early mid- and later mid-lactation. Untargeted metabolomics and 16S rRNA sequencing were applied for analyzing milk and rumen metabolites, as well as rumen microbial composition, respectively. The levels of lipid-related metabolites, L-glutamate, glucose-1-phosphate and acetylphosphate in milk exhibited lactation-dependent attenuation. Maltol, N-acetyl-D-glucosamine, and choline, which are associated with milk flavor or coagulation properties, as well as L-valine, lansioside-A, clitocine and ginsenoside-La increased significantly in early mid- and later mid-lactation, especially in later mid-lactation. The obvious increase in rumen microbial diversities (Ace and Shannon indices) were observed in early mid-lactation compared with peak lactation. Twenty-one differential bacterial genera of the rumen were identified, with Succinivibrionaceae_UCG-001, Candidatus Saccharimonas, Fibrobacter, and SP3-e08 being significantly enriched in peak lactation. Rikenellaceae_RC9_gut_group, Eubacterium_ruminantium_group, Lachnospira, Butyrivibrio, Eubacterium_hallii_group, and Schwartzia were most significantly enriched in early mid-lactation. In comparison, only 2 bacteria (unclassified_f__Prevotellaceae and Prevotellaceae_UCG-001) were enriched in later mid-lactation. For rumen metabolites, LPE(16:0), L-glutamate and L-tyrosine had higher levels in peak lactation, whereas PE(17:0/0:0), PE(16:0/0:0), PS(18:1(9Z)/0:0), L-phenylalanine, dulcitol, 2-(methoxymethyl)furan and 3-phenylpropyl acetate showed higher levels in early mid- and later mid-lactation. Multiomics integrated analysis revealed that a greater abundance of Fibrobacter contributed to phospholipid content in milk by increasing ruminal acetate, L-glutamate and LysoPE(16:0). Prevotellaceae_UCG-001 and unclassified_f_Prevotellaceae provide substrates for milk metabolites of the same category by increasing ruminal L-phenylalanine and dulcitol contents. These results demonstrated that milk metabolomic fingerprints and critical functional metabolites during lactation, and the key bacteria in rumen related to them. These findings provide new insights into the development of functional dairy products.

Dietary fat and carbohydrate-balancing the lactation performance and methane emissions in the dairy cow industry: A meta-analysis.

For the agroecosystems of the dairy cow industry, dietary carbohydrate (starch, neutral detergent fiber [NDF]) and fat could directly affect rumen methane emissions and host energy utilization. However, the relationships among diet, lactation performance, and methane emissions need to be further determined to assist dairy farms to adjust diet formulations and feeding strategies for environmental and production management. A meta-analysis was conducted in the current study to explore quantitative patterns of dietary fat and carbohydrate at different levels in balancing lactation performance and environment sustainability of dairy cows, and to establish a methane emission prediction model using the artificial neural network (ANN) model. The results showed that the regression relationship between dietary fat, carbohydrate and methane emissions could be shown by the following models: methane = 106.78 + (14.86 × DMI), R2 = 0.80; methane = 443.17 - (46.41 × starch/NDF), R2 = 0.76; and methane = 388.91 + (31.40 × fat) - (5.42 × fat2), R2 = 0.80. The regression relationships between dietary fat, carbohydrate and lactation performance could be shown by the following models: milk fat yield = 1.08 + (0.43 × starch/NDF) - [0.34 × (starch/NDF)2], R2 = 0.79; milk protein yield = 0.68 + (0.15 × fat) - (0.016 × fat2), R2 = 0.82. In the structural equation model, we found that when formulating dietary carbohydrates and fats, it was necessary to balance the relationship between methane emissions and lactation performance. Specifically, dietary starch/NDF was lower than 0.63 (extremum point) and dietary fat was between 2.89% and 4.69% (extremum point), it could ensure that the aim of methane emission reduction (methane emissions decrease with increasing dietary starch/NDF and fat) was achieved without losing lactation performance of dairy cows (lactation performance increase with increasing dietary starch/NDF and fat). Finally, we established the ANN model to predict methane emissions (training set: R2 = 0.62; validation set: R2 = 0.61).

Transcriptome in Liver of Periparturient Dairy Cows Differs between Supplementation of Rumen-Protected Niacin and Rumen-Protected Nicotinamide.

To investigate the difference between rumen-protected niacin (RPN) and rumen-protected nicotinamide (RPM) in the transcriptome of genes relating to the lipid metabolism of the liver of periparturient dairy cows, 10 healthy Chinese Holstein cows were randomly divided into two groups and fed diets supplemented with 18.4 g/d RPN or 18.7 g/d RPM, respectively. The experiment lasted from 14 days before to 21 days after parturition. Liver biopsies were taken 21 days postpartum for transcriptomic sequencing. In addition, human LO2 cells were cultured in a medium containing 1.6 mmol/L of non-esterified fatty acids and 1 mmol/L niacin (NA) or 2 mmol/L nicotinamide (NAM) to verify the expression of the 10 genes selected from the transcriptomic analysis of the liver biopsies. The expression of a total of 9837 genes was detected in the liver biopsies, among which 1210 differentially expressed genes (DEGs) were identified, with 579 upregulated and 631 downregulated genes. These DEGs were associated mainly with lipid metabolism, oxidative stress, and some inflammatory pathways. Gene ontology (GO) enrichment analysis showed that 355 DEGs were enriched in 38 GO terms. The differences in the expression of these DEGs between RPN and RPM were predominantly related to the processes of steroid catabolism, steroid hydroxylase, monooxygenase activity, oxidoreductase activity, hemoglobin binding, and ferric iron binding, which are involved mainly in lipid anabolism and redox processes. The expressions of FADS2, SLC27A6, ARHGAP24, and THRSP in LO2 cells were significantly higher (p < 0.05) while the expressions of BCO2, MARS1, GARS1, S100A12, AGMO, and OSBPL11 were significantly lower (p < 0.05) on the NA treatment compared to the NAM treatment, indicating that NA played a role in liver metabolism by directly regulating fatty acid anabolism and transport, inflammatory factor expression, and oxidative stress; and NAM functioned more as a precursor of nicotinamide adenine dinucleotide (NAD, coenzyme I) and nicotinamide adenine dinucleotide phosphate (NADP, coenzyme II) to participate indirectly in biological processes such as ether lipid metabolism, cholesterol metabolism, energy metabolism, and other processes.

Immune inhibitory receptor-mediated immune response, metabolic adaptation, and clinical characterization in patients with COVID-19.

Immune inhibitory receptors (IRs) play a critical role in the regulation of immune responses to various respiratory viral infections. However, in coronavirus disease 2019 (COVID-19), the roles of these IRs in immune modulation, metabolic reprogramming, and clinical characterization remain to be determined. Through consensus clustering analysis of IR transcription in the peripheral blood of patients with COVID-19, we identified two distinct IR patterns in patients with COVID-19, which were named IR_cluster1 and IR_cluster2. Compared to IR_cluster1 patients, IR_cluster2 patients with lower expressions of immune inhibitory receptors presented with a suppressed immune response, lower nutrient metabolism, and worse clinical manifestations or prognosis. Considering the critical influence of the integrated regulation of multiple IRs on disease severity, we established a scoring system named IRscore, which was based on principal component analysis, to evaluate the combined effect of multiple IRs on the disease status of individual patients with COVID-19. Similar to IR_cluster2 patients, patients with high IRscores had longer hospital-free days at day 45, required ICU admission and mechanical ventilatory support, and presented higher Charlson comorbidity index and SOFA scores. A high IRscore was also linked to acute infection phase and absence of drug intervention. Our investigation comprehensively elucidates the potential role of IR patterns in regulating the immune response, modulating metabolic processes, and shaping clinical manifestations of COVID-19. All of this evidence suggests the essential role of prognostic stratification and biomarker screening based on IR patterns in the clinical management and drug development of future emerging infectious diseases such as COVID-19.

Multi-omics reveals that the host-microbiome metabolism crosstalk of differential rumen bacterial enterotypes can regulate the milk protein synthesis of dairy cows.

BACKGROUND: Dairy cows' lactation performance is the outcome of the crosstalk between ruminal microbial metabolism and host metabolism. However, it is still unclear to what extent the rumen microbiome and its metabolites, as well as the host metabolism, contribute to regulating the milk protein yield (MPY). METHODS: The rumen fluid, serum and milk of 12 Holstein cows with the same diet (45% coarseness ratio), parity (2-3 fetuses) and lactation days (120-150 d) were used for the microbiome and metabolome analysis. Rumen metabolism (rumen metabolome) and host metabolism (blood and milk metabolome) were connected using a weighted gene co-expression network (WGCNA) and the structural equation model (SEM) analyses. RESULTS: Two different ruminal enterotypes, with abundant Prevotella and Ruminococcus, were identified as type1 and type2. Of these, a higher MPY was found in cows with ruminal type2. Interestingly, [Ruminococcus] gauvreauii group and norank_f_Ruminococcaceae (the differential bacteria) were the hub genera of the network. In addition, differential ruminal, serum and milk metabolome between enterotypes were identified, where the cows with type2 had higher L-tyrosine of rumen, ornithine and L-tryptophan of serum, and tetrahydroneopterin, palmitoyl-L-carnitine, S-lactoylglutathione of milk, which could provide more energy and substrate for MPY. Further, based on the identified modules of ruminal microbiome, as well as ruminal serum and milk metabolome using WGCNA, the SEM analysis indicated that the key ruminal microbial module1, which contains the hub genera of the network ([Ruminococcus] gauvreauii group and norank_f_Ruminococcaceae) and high abundance of bacteria (Prevotella and Ruminococcus), could regulate the MPY by module7 of rumen, module2 of blood, and module7 of milk, which contained L-tyrosine and L-tryptophan. Therefore, in order to more clearly reveal the process of rumen bacterial regulation of MPY, we established the path of SEM based on the L-tyrosine, L-tryptophan and related components. The SEM based on the metabolites suggested that [Ruminococcus] gauvreauii group could inhibit the energy supply of serum tryptophan to MPY by milk S-lactoylglutathione, which could enhance pyruvate metabolism. Norank_f_Ruminococcaceae could increase the ruminal L-tyrosine, which could provide the substrate for MPY. CONCLUSION: Our results indicated that the represented enterotype genera of Prevotella and Ruminococcus, and the hub genera of [Ruminococcus] gauvreauii group and norank_f_Ruminococcaceae could regulate milk protein synthesis by affecting the ruminal L-tyrosine and L-tryptophan. Moreover, the combined analysis of enterotype, WGCNA and SEM could be used to connect rumen microbial metabolism with host metabolism, which provides a fundamental understanding of the crosstalk between host and microorganisms in regulating the synthesis of milk composition.

Metabolomic profiling identifies hair as a robust biological sample for identifying women with cervical cancer.

Metabolomics serves as a useful tool for identifying biomarkers of disease and uncovering pathogenic mechanisms. However, most metabolomic studies use biological fluids such as blood and urine as biospecimens, which could be dramatically influenced by daily activities and dietary variation, resulting in measurement fluctuations. In contrast, hair may serve as a robust source of stable longitudinal metabolite information. Here, we conducted a pilot study to investigate the possibility of using hair as a biospecimen for the metabolomic analysis of cervical cancer. Hair, plasma, urine, and cervical tissue samples from cervical cancer and benign tumor patients were collected. Biospecimens were then tested using a gas chromatography-mass spectrometry-based metabolomic platform. The expressions of enzymatic genes related to metabolic changes were validated using qPCR. Statistical analyses were calculated via the R-console platform. Metabolite profiles in both hair and cervical tissue samples were significantly different between cancer and control groups, while no difference was observed in plasma and urine samples. Further analysis showed that most of the altered metabolites in hair were upregulated, and they had a negative correlation with those in the cervical tissue. Eight common metabolites showed an area under the Receiver Operating Characteristic curve greater than 0.95. These metabolites primarily participated in amino acid metabolism, cofactor synthesis, ferroptosis, and glycolysis. The gene expressions (IDH1, OGDH, GLUD1, ENO1, GSS, and GPX4) associated with the shortlisted metabolic pathways were also upregulated. Our study is the first to reveal metabolomic changes of hair in cervical cancer patients and demonstrates the potential for the hair metabolome to be used for biomarker identification in cervical cancer.

Association between caloric adequacy and short-term clinical outcomes in critically ill patients using a weight-based equation: Secondary analysis of a cluster-randomized controlled trial.

Background: There is controversy over the optimal energy delivery in intensive care units (ICUs). In this study, we aimed to evaluate the association between different caloric adequacy assessed by a weight-based equation and short-term clinical outcomes in a cohort of critically ill patients. Methods: This is a secondary analysis of a cluster-randomized controlled trial (N = 2,772). The energy requirement was estimated as 25 kcal/kg of body weight. The study subjects were divided into three groups according to their caloric adequacy as calculated by the mean energy delivered from days 3 to 7 of enrollment divided by the estimated energy requirements: (1) received < 70% of energy requirement (hypocaloric), (2) received 70-100% of energy requirement (normocaloric), and (3) received > 100% of energy requirement (hypercaloric). Cox proportional hazards models were used to analyze the association between caloric adequacy and 28-day mortality and time to discharge alive from the ICU. Results: A total of 1,694 patients were included. Compared with normocaloric feeding, hypocaloric feeding significantly increased the risk of 28-day mortality (hazard ratio [HR] = 1.590, 95% confidence interval [CI]: 1.162-2.176, p = 0.004), while hypercaloric feeding did not. After controlling for potential confounders, the association remained valid (adjusted HR = 1.596, 95% CI: 1.150-2.215, p = 0.005). The caloric adequacy was not associated with time to discharge alive from the ICU in the unadjusted and the adjusted models. Conclusion: Energy delivery below 70% of the estimated energy requirement during days 3-7 of critical illness is associated with 28-day mortality. Clinical trial registration: [https://www.isrctn.com/ISRCTN12233792], identifier [ISRCTN12233792].

Trajectories of protein intake and 28-day mortality in critically ill patients: A secondary analysis of a cluster-randomized controlled trial.

BACKGROUND & AIMS: The optimal protein intake approach during the early phase of critical illness remains controversial. This study aimed to evaluate the association between different trajectories of protein intake and 28-day mortality in a cohort of critically ill patients. METHODS: The NEED trial is a multicenter, cluster-randomized controlled trial assessing the impact of an actively implemented evidence-based nutrition guideline on mortality in critically ill patients. This secondary analysis included the patients who stayed in ICU for at least seven days. Group-based trajectory modeling was applied to identify subgroups with similar protein intake trajectories in this cohort. Cox proportional hazards models were used to analyze the impact of different trajectories on 28-day mortality. RESULTS: Overall, 2191 patients were included for analysis. A distinct triple-group trajectory of protein intake was identified, with 919 patients categorized into the low-level protein intake group, 1146 the medium-level group, and 126 the high-level group. The mean daily protein intake from the low-to high-level protein intake group during the first week of enrollment were 0.38 ± 0.14, 0.8 ± 0.18, and 1.68 ± 0.39 g/kg/d, respectively. Compared with the medium-level protein intake group, the low-level or high-level protein intake group was associated with significantly increased 28-day mortality (hazard ratio [HR] = 1.348, 95% confidence interval [CI]: 1.067-1.704; HR = 2.291, 95% CI: 1.533-3.423, respectively). After controlling for potential confounders, the adjusted HRs were 1.365 (95% CI: 1.032-1.807) for the low-level group and 1.921 (95% CI: 1.274-2.896) for the high-level group. However, when taking energy intake into account, low-level protein intake was no longer related to mortality. In contrast, the detrimental effects of high-level protein intake remain tenable (Adjusted HR = 2.324, 95% CI: 1.524-3.543, P < 0.001). CONCLUSIONS: Low-level or high-level protein intake in the early phase of critical illness was associated with increased 28-day mortality than medium-level protein intake. However, when adjusted for energy intake, low-level protein intake in the early phase was no longer associated with increased 28-day mortality.

Comparative Transcriptomic and Proteomic Analyses Prove that IFN-λ1 is a More Potent Inducer of ISGs than IFN-α against Porcine Epidemic Diarrhea Virus in Porcine Intestinal Epithelial Cells.

Type III interferon (IFN-λ) is currently considered to be largely nonredundant to type I interferon (IFN-α) in antivirus infection, especially in epithelial cells. Previous studies reported that, compared with IFN-α, IFN-λ exhibited stronger induction of interferon-stimulated genes (ISGs) at the transcriptional level in intestinal epithelial cells and stronger inhibition of porcine epidemic diarrhea virus (PEDV). In this study, the different mechanisms of ISG upregulation induced by IFN-α and IFN-λ1 were compared at the mRNA and protein levels in the porcine intestinal epithelial cell model (IPEC-J2). It was proved that IFN-λ1 consistently exhibited stronger stimulation effects at both levels. At the mRNA level, 132 genes were significantly upregulated upon IFN-λ1 stimulation, while 42 genes upon IFN-α stimulation. At the protein level, 47 proteins were significantly upregulated upon IFN-λ1 stimulation, but only 8 proteins were upregulated upon IFN-α stimulation. The shared upregulated genes/proteins by IFN-λ1 in both transcriptional and translational omics, especially the regulation factors of ISG15, were involved in the JAK-STAT signaling pathway. Compared to IFN-α, IFN-λ1 could induce more consistent upregulation of the key ISGs (ISG15, USP18, OASL, and RSAD2) at 3-24 h postinduction as measured by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) validation. It was further confirmed through functional analysis that ISG15 and RSAD2 could inhibit PEDV infection in dose-dependent manners. This study provided solid evidence that IFN-λ1 could induce a more unique and higher ISG expression level, which exhibited anti-PEDV effects on porcine intestinal epithelial cells.

CircMUC16 promotes autophagy of epithelial ovarian cancer via interaction with ATG13 and miR-199a.

BACKGROUND: Circular RNA (circRNA) has been proven to play a significant role in multiple types of cancer. However, the expression and role of circRNAs in epithelial ovarian cancer (EOC) remains elusive. METHODS: CircRNA and mRNA expression profiles of EOC were screened with sequencing analysis. Gene silencing and over-expression were used to study circRNA function. Cell proliferation and Matrigel invasion assays were used to detect cell proliferation and invasion, respectively. The expression of circRNAs, mRNAs and miRNAs was detected using qPCR. The location of circRNAs was detected using FISH. The expression of proteins was detected using western blot and immunohistochemistry. RESULTS: CircMUC16 had increased expression in EOC tissues as compared to healthy ovarian tissues. The expression of circMUC16 was linked to the progression in stage and grade of EOC. Hence, silencing circMUC16 suppressed autophagy flux of SKOV3 cells. In contrast, ectopic expression of circMUC16 promoted autophagy flux of A2780 cells. CircMUC16-mediated autophagy exacerbated EOC invasion and metastasis. Mechanistically, circMUC16 could directly bind to miR-199a-5p and relieve suppression of target Beclin1 and RUNX1. In turn, RUNX1 elevated the expression of circMUC16 via promotion of its transcription. CircMUC16 could directly bind to ATG13 and promote its expression. CONCLUSION: This study demonstrated that circMUC16 regulated Beclin1 and RUNX1 by sponging miR-199a-5p. The data suggested that circMUC16 could be a potential target for EOC diagnosis and therapy.

Overexpression of TIM-3 in Macrophages Aggravates Pathogenesis of Pulmonary Fibrosis in Mice.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive lung disorder and lacks effective treatments because of unclear mechanisms. Aberrant function of alveolar macrophages is directly linked to pulmonary fibrosis. Here, we show TIM-3 (T-cell immunoglobulin domain and mucin domain-3), a key regulator of macrophage function, aggravates pulmonary fibrosis. TIM-3 mRNA of patients with IPF was analyzed based on the Gene Expression Omnibus and Array Express databases. Lung pathology and profibrotic molecules were assessed in a bleomycin (BLM)-induced pulmonary fibrosis model using wild-type (WT) and TIM-3 transgenic (TIM-3-TG) mice. Macrophage cells, RAW264.7, were then applied to investigate the effect of macrophage TIM-3 under BLM exposure in vitro. Macrophage depletion and adoptive-transfer experiments were finally performed to examine lung morphology and profibrotic molecules. TIM-3 expression was increased both in patients with IPF and in our BLM-induced mouse model. TIM-3-TG mice developed more serious pathological changes in lung tissue and higher expressions of TGF-ß1 (transforming growth factor-ß1) and IL-10 than WT mice. After BLM treatment, TGF-ß1 and IL-10 expression was significantly decreased in RAW264.7 cells after TIM-3 knock-out, whereas it was increased in TIM-3-TG peritoneal macrophages. The scores of pulmonary fibrosis in WT and TIM-3-TG mice were significantly reduced, and there was no difference between them after macrophage depletion. Furthermore, WT mice receiving adoptive macrophages from TIM-3-TG mice also had more serious lung fibrosis and increased expression of TGF-ß1 and IL-10 than those receiving macrophages from WT mice. Our findings revealed that overexpressed TIM-3 in alveolar macrophages aggravated pulmonary fibrosis.

ALKBH5 inhibited autophagy of epithelial ovarian cancer through miR-7 and BCL-2.

BACKGROUND: ALKBH5 regulated the malignant behavior of breast cancer and glioblastoma. However, the expression and function of ALKBH5 in epithelial ovarian cancer have not yet been determined. In the present study, we investigated the expression and function of ALKBH5 in epithelial ovarian cancer with respect to its potential role in the tumorigenesis of the disease as well as an early diagnostic marker. METHODS: Immunohistochemistry and western blot were used to detect protein expression. Gene silencing and over-expression experiment were used to study gene function. Cell proliferation assay and Matrigel invasion assays were used to detect cell proliferation and invasion, respectively. The nude mouse tumor formation experiment was used to evaluate the growth of cells in vivo. RESULTS: The expression of ALKBH5 was found to be increased in epithelial ovarian cancer tissue as compared to the normal ovarian tissues. The silencing of ALKBH5 in SKOV3 cells enhanced the autophagy and inhibited the proliferation and invasion in vitro and in vivo, whereas the ectopic expression of ALKBH5 in A2780 cells exerted an opposite effect. Mechanical study revealed that ALKBH5 physically interacted with HuR. ALKBH5 activated EGFR-PIK3CA-AKT-mTOR signaling pathway. Also, ALKBH5 enhanced the stability of BCL-2 mRNA and promoted the interaction between Bcl-2 and Beclin1. CONCLUSION: Overall, the present study identified ALKBH5 as a candidate oncogene in epithelial ovarian cancer and a potential target for ovarian cancer therapy.

Cyanidin-3-O-glucoside protects against cadmium-induced dysfunction of sex hormone secretion via the regulation of hypothalamus-pituitary-gonadal axis in male pubertal mice.

Cadmium (Cd) has been generally recognized as an endocrine-disrupting chemical for its toxic effects on the hypothalamus-pituitary-gonadal (HPG) axis accompanied by dysfunction in sex hormone secretion. Particularly, exposure to Cd during puberty versus post-puberty exhibits differing age-dependent effects that require further examination. This study sought to determine if cyanidin-3-O-glucoside (C3G), a typical anthocyanin with neuroprotective bioactivity, could protect against Cd-induced sex hormone-disorder in Pubertal male mice. C3G treatment reversed the disruption of hormone levels and increased Gnrh1 gene expression in the hypothalamus. In addition, the levels of gonadotropins, including luteinizing hormone (LH) and follicle stimulating hormone (FSH), were reversed by C3G. Interestingly, C3G improved the expression of LH and FSH receptor in the testis in mice exposed to Cd. Furthermore, C3G activated the signaling pathway related to the synthesis of testosterone processing. In conclusion, C3G protected against Cd-induced dysfunction of sex hormone secretion through the regulation of the HPG axis in male mice during puberty. The results of this study suggest that consumption of anthocyanins can be protective against metal-induced male reproductive dysfunction.

Co-Delivery of Gemcitabine and Mcl-1 SiRNA via Cationic Liposome-Based System Enhances the Efficacy of Chemotherapy in Pancreatic Cancer.

Myeloid cell leukemia 1 (Mcl-1) overexpression is found in various human tumors and has emerged as a promising new target for pancreatic cancer treatment. Recent research has found that most pancreatic cancers develop resistance to the current first-line chemotherapeutic drug, gemcitabine (Gem), and high expression of Mcl-1 can reduce the sensitivity of pancreatic cancer cells to Gem chemotherapy. Therefore, novel strategies, such as combination therapy, to overcome resistance of Gem chemotherapy are needed urgently. Here, we employed a lipid-based delivery system (LPs) to codeliver Mcl-1 siRNA and Gem for pancreatic cancer treatment, named LP-Gem-siMcl-1. LP-Gem-siMcl-1 exhibited an increased cellular uptake, enhanced Mcl-1 down-regulation efficacy, and significant cytotoxicity in the human pancreatic carcinoma cell lines PANC-1 and BxPC-3. Furthermore, tumor inhibition in vivo proved that LP-Gem-siMcl-1 has higher anti-tumor efficiency than LP-siMcl-1 plus LP-Gem, indicating the synergistic anti-tumor effects of Gem and siMcl-1. Meanwhile, histological analysis demonstrated that LPs could efficiently co-deliver Gem and Mcl-1 siRNA to cancerous cells and overcome the resistance of Gem. Taken together, our results offer proof that LP-Gem-siMcl-1 is an effective co-delivery system to treat pancreatic cancers and may serve as a valuable tool for developing new strategies for cancer therapy.

Peptide Self-Assembly Nanoparticles Loaded with Panobinostat to Activate Latent Human Immunodeficiency Virus.

Highly active antiretroviral therapy (HAART) can turn human immunodeficiency virus-1 (HIV-1) infection into a controllable chronic disease, but because of the presence of an HIV reservoir, it cannot completely eliminate the virus in HIV-infected patients. The activation of latent reservoirs is the key to the successful treatment of acquired immune deficiency syndrome (AIDS). As a class of latency-reversing agents (LRAs), histone deacetylase inhibitors (HDACis), such as panobinostat, have been the most widely investigated, but most of them have resulted in only a modest and transient activation of HIV latency. To improve the potency of latency activation, an injectable peptide self-assembly nanoparticle loaded with panobinostat (PNP-P) was designed with the ability to efficiently penetrate the cell to achieve better drug delivery and activation of latent HIV. The results confirmed that these nanoparticles could activate latently infected cells in vitro and in vivo and activate peripheral blood mononuclear cells (PBMCs) from latently infected patients ex vivo. Increased cellular drug uptake made the PNP-P more effective than panobinostat alone. Therefore, this strategy demonstrates that nanotechnology can help improve the activation of latent HIV, and this study lays a foundation for further development of LRA delivery systems for use against an HIV reservoir.

Ubiquitin E3 Ligase MARCH7 promotes proliferation and invasion of cervical cancer cells through VAV2-RAC1-CDC42 pathway.

Ubiquitin E3 Ligase MARCH7 is involved in T cell proliferation and neuronal development. In our previous study, we demonstrated MARCH7 promoted malignant behavior of ovarian cancer via the nuclear factor (NF)-κB and Wnt/ß-catenin signaling pathway. However, the expression and function of MARCH7 in cervical cancer remains unknown. The present study aimed to unravel the expression and function of MARCH7 in cervical cancer to elucidate its potential role in the diagnosis and pathogenesis of cervical cancer. Results indicated that the expression of MARCH7 was abnormally high in cervical cancer tissues than normal cervical tissues. However, silencing the expression of MARCH7 in HeLa cells resulted in decreased cell proliferation and invasion. Mechanistic investigations revealed that MARCH7 interacted with VAV2. Silencing the expression of MARCH7 in HeLa cells inhibited the VAV2-RAC1-CDC42 signaling pathway. Overall, the results of the present study identified MARCH7 as a candidate oncogene in cervical cancer, and a potential target for cervical cancer therapy.

Interaction of E3 Ubiquitin Ligase MARCH7 with Long Noncoding RNA MALAT1 and Autophagy-Related Protein ATG7 Promotes Autophagy and Invasion in Ovarian Cancer.

BACKGROUND/AIMS: Ubiquitin E3 ligase MARCH7 plays an important role in T cell proliferation and neuronal development. But its role in ovarian cancer remains unclear. This study aimed to investigate the role of Ubiquitin E3 ligase MARCH7 in ovarian cancer. METHODS: Real-time PCR, immunohistochemistry and western blotting analysis were performed to determine the expression of MARCH7, MALAT1 and ATG7 in ovarian cancer cell lines and clinical specimens. The role of MARCH7 in maintaining ovarian cancer malignant phenotype was examined by Wound healing assay, Matrigel invasion assays and Mouse orthotopic xenograft model. Luciferase reporter assay, western blot analysis and ChIP assay were used to determine whether MARCH7 activates TGF-ß-smad2/3 pathway by interacting with TGFßR2. RESULTS: MARCH7 interacted with MALAT1 by miR-200a (microRNA-200a). MARCH7 may function as a competing endogenous RNA (ceRNA) to regulate the expression of ATG7 by competing with miR-200a. MARCH7 regulated TGF-ß-smad2/3 pathway by interacting with TGFßR2. Inhibition of TGF-ß-smad2/3 pathway downregulated MARCH7, MALAT1 and ATG7. MiR-200a regulated TGF-ß induced autophagy, invasion and metastasis of SKOV3 cells by targeting MARCH7. MARCH7 silencing inhibited autophagy invasion and metastasis of SKOV3 cells both in vitro and in vivo. In contrast, MARCH7 overexpression promoted TGF-ß induced autophagy, invasion and metastasis of A2780 cells in vitro by depending on MALAT1 and ATG7. We also found that TGF-ß-smad2/3 pathway regulated MARCH7 and ATG7 through MALAT1. CONCLUSIONS: These findings suggested that TGFßR2-Smad2/3-MALAT1/MARCH7/ATG7 feedback loop mediated autophagy, migration and invasion in ovarian cancer.

Negative regulation of Nod-like receptor protein 3 inflammasome activation by T cell Ig mucin-3 protects against peritonitis.

The Nod-like receptor protein 3 (NLRP3) inflammasome plays roles in host defence against invading pathogens and in the development of autoimmune damage. Strict regulation of these responses is important to avoid detrimental effects. Here, we demonstrate that T cell Ig mucin-3 (Tim-3), an immune checkpoint inhibitor, inhibits NLRP3 inflammasome activation by damping basal and lipopolysaccharide-induced nuclear factor-κB-mediated up-regulation of NLRP3 and interleukin-1ß during the priming step and basal and ATP/lipopolysaccharide-induced ATP production, K+ efflux, and reactive oxygen species production during the activation step. Residues Y256/Y263 in the C-terminal region of Tim-3 are required for these inhibitory effects on the NLRP3 inflammasome. In mice with alum-induced peritonitis, blockade of Tim-3 exacerbates peritonitis by overcoming the inhibitory effect of Tim-3 on NLRP3 inflammasome activation, while transgenic expression of Tim-3 attenuates inflammation by inhibiting NLRP3 inflammasome activation. Our results show that Tim-3 is a critical negative regulator of NLRP3 inflammasome and provides a potential target for intervention of diseases with uncontrolled inflammasome activation.

MARCH5 RNA promotes autophagy, migration, and invasion of ovarian cancer cells.

MARCH5 is a crucial regulator of mitochondrial fission. However, the expression and function of MARCH5 in ovarian cancer have not been determined. This study investigated the expression and function of MARCH5 in ovarian cancer with respect to its potential role in the tumorigenesis of the disease as well as its usefulness as an early diagnostic marker. We found that the expression of MARCH5 was substantially upregulated in ovarian cancer tissue in comparison with the normal control. Silencing MARCH5 in SKOV3 cells decreased TGFB1-induced cell macroautophagy/autophagy, migration, and invasion in vitro and in vivo, whereas the ectopic expression of MARCH5 in A2780 cells had the opposite effect. Mechanistic investigations revealed that MARCH5 RNA may function as a competing endogenous RNA (ceRNA) to regulate the expression of SMAD2 and ATG5 by competing for MIR30A. Knocking down SMAD2 or ATG5 can block the effect of MARCH5 in A2780 cells. Also, silencing the expression of MARCH5 in SKOV3 cells can inhibit the TGFB1-SMAD2/3 pathway. In contrast, the ectopic expression of MARCH5 in A2780 cells can activate the TGFB1-SMAD2/3 pathway. In turn, the TGFB1-SMAD2/3 pathway can regulate MARCH5 and ATG5 through MIR30A. Overall, the results of this study identified MARCH5 as a candidate oncogene in ovarian cancer and a potential target for ovarian cancer therapy.