DNAJA1 positively regulates amino acid-stimulated milk protein and fat synthesis in bovine mammary epithelial cells.

Several cellular processes, including the recovery of misfolded proteins, the folding of polypeptide chains, transit of polypeptides across the membrane, construction and disassembly of protein complexes, and modulation of protein control, are carried out by DnaJ homolog subfamily A member 1 (DNAJA1), which belongs to the DnaJ heat-shock protein family. It is unknown if DNAJA1 regulates the production of milk in bovine mammary epithelium cells (BMECs). Methionine and leucine increased DNAJA1 expression and nuclear location, as seen by us. In contrast to DNAJA1 knockdown, overexpression of DNAJA1 boosted the production of milk proteins and fats as well as mammalian target of rapamycin (mTOR) and sterol regulatory element binding protein-1c (SREBP-1c). As a result of amino acids, mTOR and SREBP-1c gene expression are stimulated, and DNAJA1 is a positive regulator of BMECs' amino acid-induced controlled milk protein and fat production.

Genetic Variants of the COL4A3 , COL4A4 , and COL4A5 Genes Contribute to Thinned Glomerular Basement Membrane Lesions in Sporadic IgA Nephropathy Patients.

BACKGROUND: Thinned glomerular basement membrane (tGBM) lesions are not uncommon in IgA nephropathy (IgAN). Type IV collagen-built of α 3, α 4, and α 5 chains, encoded by COL4A3 / COL4A4 / COL4A5 genes-is the major component of glomerular basement membrane (GBM). In recent years, mutations in type IV collagen-encoding genes were also reported in patients with a histologic diagnosis of FSGS. Pathogenic COL4A3 / COL4A4 / COL4A5 variants were recently identified in familial cases of IgAN, but the contribution of these variants to sporadic IgAN is still unclear. METHODS: We compared 161 patients with sporadic IgAN with tGBM lesions (IgAN-tGBM) to matched patients with IgAN without tGBM lesions and matched patients with thin basement membrane nephropathy (TBMN). Variants of COL4A3 / COL4A4 / COL4A5 genes were screened and evaluated after whole-exome sequencing. GBM thickness was measured, and levels of circulating galactose-deficient IgA1 (Gd-IgA1) were assessed by ELISA. RESULTS: The patients with IgAN-tGBM manifested milder disease than did patients with IgAN without tGBM but had more severe features than the patients with TBMN. Exome sequence analysis of the 122 patients with IgAN-tGBM identified 37 diagnostic variants of the COL4A3 / COL4A4 / COL4A5 genes among 38 patients (31.1%). Furthermore, patients with IgAN-tGBM who had diagnostic variants had higher proportions of GBM thickness <250 nm and milder glomerular injury, whereas patients with IgAN-tGBM who did not have diagnostic variants showed more characteristic features of IgAN, including higher intensity of glomerular IgA deposits and elevated Gd-IgA1 levels. These findings suggest different mechanisms in patients with versus without diagnostic variants of these collagen genes. CONCLUSIONS: COL4A3 / COL4A4 / COL4A5 variant detection is essential in evaluating patients with sporadic IgAN with tGBM lesions.

Novel Near-Infrared Iridium(III) Complex for Chemiluminescence Imaging of Hypochlorous Acid.

Chemiluminescence (CL) probes that possess near-infrared (NIR) emission are highly desirable for in vivo imaging due to their deeper tissue penetration ability and intrinsically high sensitivity. Herein, a novel iridium-based CL probe (NIRIr-CL-1) with direct NIR emission was reported as the result of hypochlorous acid (HClO)-initiated oxidative deoximation. To improve its biocompatibility and extend the CL time for in vivo imaging applications, this NIRIr-CL-1 was prepared as a CL nanoparticle probe (NIRIr-CL-1 dots) through encapsulation by an amphiphilic polymer Pluronic F127 (F127). All results demonstrate that the NIRIr-CL-1 dots have good selectivity and sensitivity for visualization of HClO even at the depth of 1.2 cm. Owing to these advantages, the CL imaging of exogenous and endogenous HClO in mice was achieved. This study could provide new insights into the construction of new NIR emission CL probes and expand their applications in biomedical imaging.

Chemiluminescence-assisted study on a peracetic acid-based advanced oxidation process activated with cobalt phosphide.

In recent years, the elimination of organic pollutants using advanced oxidation processes (AOPs) based on peracetic acid (PAA) has drawn increasing attention due to the high oxidative potential and low byproducts. However, to explore more efficient and stable PAA-based AOPs, there is still great room for study on the activation of PAA and degradation mechanism in the reaction process. In this study, a new PAA-based AOP activated by metal-organic framework-derived cobalt phosphide (CoP) and accompanied by chemiluminescence (CL) behaviour was explored. The CoP/PAA system could efficiently degrade 99.98% of RhB (20 mg L-1 ) within 5 min at pH 7 compared with the conventional Co3 O4 /PAA system (merely 17.29%), and the degradation process was matched well with the pseudo-first-order kinetic, and the kinetic constants was ~23.7 times higher than that of Co3 O4 (0.546 min-1 for CoP vs. 0.023 min-1 for Co3 O4 ). In the CoP/PAA/RhB process, the CL intensity was related to the concentration of 1 O2 , O2 •- and acetyl peroxyl radicals [CH3 C(O)OO• and CH3 C(O)O•]. Therefore, CL analysis, combined with quenching tests and electron paramagnetic resonance analysis, was used to study the degradation mechanism in detail, and 1 O2 was confirmed as the dominant contributor for the dye degradation.

Cardiac energetics alteration in a chronic hypoxia rat model: A non-invasive in vivo31P magnetic resonance spectroscopy study.

BACKGROUND: Energetics alteration plays a crucial role in the myocardial injury process in chronic hypoxia diseases (CHD). 31P magnetic resonance spectroscopy (MRS) can investigate alterations in cardiac energetics in vivo. OBJECTIVE: To characterize the potential value of 31P MRS in evaluating cardiac energetics alteration of chronic hypoxic rats (CHRs). METHODS: Twenty-four CHRs were induced by SU5416 combined with hypoxia and divided into four groups according to the modeling time of one, two, three and five weeks, respectively. Control group also contains six rats. 31P MRS was performed weekly and the ratio of concentrations of phosphocreatine (PCr) to adenosine triphosphate (ATP) (PCr/ATP) was obtained. In addition, the cardiac structure index and systolic function parameters, including the right ventricular ejection fraction (RVEF), right ventricular end-diastolic volume index (RVEDVi), right ventricular end-systolic volume index (RVESVi), and the left ventricular function parameters, were measured. RESULTS: Decreased resting cardiac PCr/ATP ratio in CHRs was observed at the first week, compared to the control group (2.90±0.35 vs. 3.31±0.45, p = 0.045), while the RVEF, RVEDVi, and RVESVi decreased at the second week (p < 0.05). The PCr/ATP ratio displayed a significant correlation with RVEF (r = 0.605, p = 0.001), RVEDVi, and RVESVi (r = -0.661, r = -0.703; p < 0.001). CONCLUSIONS: 31P MRS can easily detect the cardiac energetics alteration in a CHR model before the onset of ventricular dysfunction. The decreased PCr/ATP ratio likely reveales myocardial injury and cardiac dysfunction.

Fusarium solani G6, a novel vitexin-producing endophytic fungus: characterization, yield improvement and osteoblastic proliferation activity.

The study aimed to characterize a novel vitexin-producing endophytic fungus Fusarium solani G6 from Cajanus cajan, improve its capability for producing vitexin and evaluate its osteoblastic proliferation activity. A total of 153 endophytic fungi, classified into 6 genera, were isolated from C. cajan. Among them, only one strain, endophyte G6 identified as Fusarium solani, was found to produce vitexin. After the optimization of fermentation conditions, the highest vitexin yield (18.72 mg/L) for the strain was observed in PDB liquid medium containing 20.54 g/L of glucose and 8.90 g/L of ammonium sulfate, at an initial medium pH of 5.1 and at 28 °C for 6 days of cultivation. Moreover, the fungal vitexin exhibited notable osteoblastic proliferation stimulating activity. A novel vitexin-producing endophytic fungus F. solani G6 was characterized from C. cajan for the first time. The findings highlighted its potential use for large-scale production of vitexin and might have a promising use as therapeutic agent for osteoporosis.

NCOA5 is a master regulator of amino acid-induced mTOR activation and ß-casein synthesis in bovine mammary epithelial cells.

The nuclear receptor co-activator 5 (NCOA5) is known as a co-activator or co-repressor that influences gene expression and cellular physiology, but its roles and detailed molecular mechanism is still largely unknown. In this study, we explored the role and molecular mechanism of NCOA5 in amino acid-induced activation of the mechanistic target of rapamycin (mTOR) and milk protein synthesis in bovine mammary epithelial cells (BMECs). Methionine (Met) and leucine (Leu) significantly up-regulated the expression of NCOA5. NCOA5 overexpression increased mTOR phosphorylation and ß-casein synthesis, whereas its knockdown exhibited the opposite effects. Furthermore, inhibition of phosphatidylinositol 3-kinase (PI3K) completely abolished the stimulatory effects of Met and Leu on NCOA5 expression. ChIP-qPCR analysis detected that NCOA5 bound to the mTOR promoter, and this interaction was enhanced by the stimulation of Met and Leu. These above data reveal that NCOA5 is a key regulator of amino acid-induced PI3K-mediated mTOR activation and ß-casein synthesis in BMECs.

Transcription factor E2F4 is a positive regulator of milk biosynthesis and proliferation of bovine mammary epithelial cells.

The transcription factor E2F4 is a key determinant of cell differentiation and cell-cycle progression, but its function and regulatory mechanism are not completely understood. Here, we report that E2F4 acts as a positive regulator of the biosynthesis of milk components and proliferation of bovine mammary epithelial cells (BMECs). Overexpression of E2F4 in BMECs resulted in the upregulation of ß-casein, triglyceride, and lactose levels and increased cell proliferation, whereas E2F4 knockdown by small interfering RNA had the opposite effects. We further detected that overexpression of E2F4 significantly increased the messenger RNA expression of mTOR, SREBP-1c, and Cyclin D1, and increased protein levels of SREBP-1c, and Cyclin D1, and the ratio of p-mTOR/mTOR, whereas E2F4 knockdown had the opposite effects. E2F4 was almost entirely located in the nucleus, and we further identified, via ChIP-qPCR analysis, that mTOR, SREBP-1c, and Cyclin D1 were E2F4 target genes, and exogenous administration of methionine, leucine, ß-estradiol, and prolactin markedly increased the protein levels of E2F4 and its binding to the promoters of these three genes. In summary, our data reveal that E2F4 responds to extracellular stimuli and regulates the expression of mTOR, SREBP-1c, and Cyclin D1 for milk biosynthesis and proliferation of BMECs.

NUCKS1 is a novel regulator of milk synthesis in and proliferation of mammary epithelial cells via the mTOR signaling pathway.

Nuclear ubiquitous casein and cyclin-dependent kinase substrate 1 (NUCKS1) is a highly phosphorylated nuclear protein ubiquitously expressed in vertebrates. NUCKS1 has been reported to be a key chromatin modifier and transcriptional regulator of a number of signaling pathways, but the physiological role and detailed mechanism are still limited. In this study, we assessed the role of NUCKS1 on milk synthesis in and proliferation of mammary epithelial cells from a dairy cow. NUCKS1 was located in the nucleus of mammary epithelial cells, and the expression of NUCKS1 was stimulated by amino acids (Met and Leu) and hormones (estrogen and prolactin). Gene function study approaches detected that NUCKS1 positively regulated milk protein, milk fat, and lactose synthesis, and also increased the cell number, cell viability, and cell cycle progression. NUCKS1 mediated the stimulation of amino acids and hormones on the messenger RNA expression of the mechanistic target of rapamycin (mTOR), SREBP-1c, and Cyclin D1. The expression of NUCKS1 is dramatically higher in mouse mammary tissue of lactating period, compared with that in puberty and dry period. Taken together, these results reveal that NUCKS1 is a new mediator of milk synthesis in and proliferation of mammary epithelial cells via regulating the mTOR signaling pathway.

Cyclase-associated protein 1 is a key negative regulator of milk synthesis and proliferation of bovine mammary epithelial cells.

Adenylyl cyclase-associated protein (CAP) is a highly conserved protein. Previous reports have suggested that CAP1 may be a negative regulator of cellular proliferation, migration, and adhesion and the development of cell carcinomas. The molecular mechanism of CAP1 regulation of downstream pathways, as well as how CAP1 is regulated by environmental stimuli and upstream signalling, is not well understood. In this present study, we assessed the role of CAP1 in milk synthesis and proliferation of bovine mammary epithelial cells. Using gene overexpression and silencing methods, CAP1 was found to negatively regulate milk synthesis and proliferation of cells via the PI3K-mTOR/SREBP-1c/Cyclin D1 signalling pathway. Hormones, such as prolactin and oestrogen, and amino acids, such as methionine and leucine, stimulate MMP9 expression and trigger CAP1 degradation, and thus, abrogate its inhibition of synthesis of milk protein, fat, and lactose by and proliferation of bovine mammary epithelial cells. The results of our study help deepen our understanding of the regulatory mechanisms underlying milk synthesis and aid in characterizing the molecular mechanisms of CAP1. Previous reports have suggested that CAP1 is a negative regulator of cellular proliferation and anabolism, but the molecular mechanisms are largely unknown. In this present study, we identified CAP1 as a negative regulator of milk synthesis and proliferation of bovine mammary epithelial cells. Our results will deepen our understanding of the regulatory mechanisms underlying milk synthesis and aid in exploring the molecular mechanisms of CAP1.

U2AF65 enhances milk synthesis and growth of bovine mammary epithelial cells by positively regulating the mTOR-SREBP-1c signalling pathway.

U2 snRNP auxiliary factor 65 kDa (U2AF65) is a splicing factor that promotes prespliceosome assembly. The function of U2AF65 in alternative splicing has been identified; however, the essential physiological role of U2AF65 remains poorly understood. In this study, we investigated the regulatory role of U2AF65 in milk synthesis and growth of bovine mammary epithelial cells (BMECs). Our results showed that U2AF65 localizes in the nucleus. Treatment with amino acids (Met and Leu) and hormones (prolactin and ß-estradiol) upregulated the expression of U2AF65 in these cells. U2AF65 overexpression increased the synthesis of ß-casein, triglycerides, and lactose; increased cell viability; and promoted proliferation of BMECs. Furthermore, our results showed that U2AF65 positively regulated mTOR phosphorylation and expression of mature mRNA of mTOR and SREBP-1c. Collectively, our findings demonstrate that U2AF65 regulates the mRNA expression of signalling molecules (mTOR and SREBP-1c) involved in milk synthesis and growth of BMECs, possibly via controlling the splicing and maturation of these mRNAs. U2 snRNP auxiliary factor 65 kDa (U2AF65) is a splicing factor that promotes prespliceosome assembly. The essential physiological role of U2AF65 remains poorly understood. In the present study, we confirmed that U2AF65 functions as a positive regulator of milk synthesis in and proliferation of bovine mammary epithelial cells via the mTOR-SREBP-1c signalling pathway. Therefore, our study uncovers the regulatory role of U2AF65 in milk synthesis and cell proliferation.

Annexin A2 positively regulates milk synthesis and proliferation of bovine mammary epithelial cells through the mTOR signaling pathway.

Annexin A2 (AnxA2) has been shown to play multiple roles in growth, development, and metabolism, but the functions of AnxA2 and the signaling pathways associated with AnxA2 are still not fully understood. In this study, we aim to reveal whether and how AnxA2 could be involved in milk synthesis and proliferation of bovine mammary epithelial cells (BMECs). Using gene function study approaches, we found that AnxA2 positively regulates PIP3 level, phosphorylation of mTOR, and protein levels of SREBP-1c and Cyclin D1 leading to milk synthesis and cell proliferation. We further observed that both AnxA2-36 kD phosphorylated form and AnxA2-33 kD protein could be induced from AnxA2-36 kD protein in BMECs under methionine, leucine, estrogen or prolactin stimulation. These above results strongly demonstrate that AnxA2 functions as a critical regulator for amino acid or hormone-induced milk synthesis and cell proliferation via the PI3K-mTOR-SREBP-1c/Cyclin D1 signaling pathway.

Mitochondrial ATAD3A regulates milk biosynthesis and proliferation of mammary epithelial cells from dairy cow via the mTOR pathway.

ATPase family AAA-domain containing protein 3A (ATAD3A) is a nuclear-encoded mitochondrial membrane protein, which is essential for cell growth and metabolism. The mechanism by which ATAD3A acts is still not fully understood. In this study, we explored the regulatory role of ATAD3A on milk biosynthesis and proliferation of bovine mammary epithelial cell. We showed that ATAD3A is localized in mitochondria and the expression of ATAD3A was up-regulated in response to extracellular stimuli such as amino acids and hormones. We observed that ATAD3A positively regulated milk protein, fat, and lactose biosynthesis, and cell proliferation. We further revealed that ATAD3A promoted the expressions of mTOR, SREBP-1c, and Cyclin D1, and triggers mTOR phosphorylation. In summary, our data reveal that ATAD3A regulates the mTOR, SREBP-1c, and Cyclin D1 signaling pathways for milk biosynthesis and cell proliferation.

Promoting Effect of Pinostrobin on the Proliferation, Differentiation, and Mineralization of Murine Pre-osteoblastic MC3T3-E1 Cells.

Pinostrobin (PI), a natural flavonoid found in a variety of plants, is well known for its rich pharmacological activities. However, its osteogenic function remains unclear. The aim of this study is to evaluate the effect of PI on the proliferation, differentiation, and mineralization of murine pre-osteoblastic MC3T3-E1 cells in vitro using MTT, alkaline phosphatase (ALP) activity, the synthesis of collagen I (Col I) assay, and Von-Kossa staining, respectively. The expression of osteocalcin (OCN) mRNA in cells was detected by real-time PCR. The effect of PI on the differentiation of dexamethasone (DEX)-suppressed cells was also investigated. The results showed that PI greatly promoted the proliferation of MC3T3-E1 cells at 5-80 µg/mL (p < 0.05 or p < 0.01), and caused a significant elevation of ALP activity, Col I content, and mineralization of osteoblasts at 10-40 µg/mL (p < 0.05 or p < 0.01), and the expression levels of OCN gene were greatly upregulated after PI treatment (p < 0.01). Furthermore, PI could rescue the inhibition effect of cell differentiation induced by DEX. Taken together, these results indicated that PI could directly promote proliferation, differentiation, and mineralization of MC3T3-E1 cells and has potential for use as a natural treatment for osteoporosis.

Preparation of Vitexin Nanoparticles by Combining the Antisolvent Precipitation and High Pressure Homogenization Approaches Followed by Lyophilization for Dissolution Rate Enhancement.

Vitexin, a natural flavonoid found in many medicinal plants, is well known for its rich pharmacological activities. However, the poor water solubility of vitexin has limited its therapeutic application. The aim of this study was to prepare the nanoparticles of vitexin by combining the antisolvent precipitation (ASP) and high pressure homogenization (HPH) approaches followed by lyophilization for improving the dissolution rate of this poorly water-soluble drug. The effects of main factors influencing the mean particle size (MPS) of vitexin were investigated and optimized. Under optimum conditions, vitexin nanosuspensions with an MPS of 80.5 nm were obtained and then lyophilized to form nanoparticles. The obtained vitexin nanoparticles were further characterized by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), mass spectrometry (MS), X-ray powder diffraction (XRPD), gas chromatography (GC) and dissolution testing. The results showed that the nanoparticles of vitexin were converted into an amorphous form, with its chemical structure unchanged. Additionally, the residual dimethyl sulfoxide (DMSO) is lower than the International Conference on Harmonization (ICH) limit for class 3 solvents. The dissolution rate of processed vitexin was significantly higher (5.58-fold) than that of raw drug. Overall, the combinative process we developed is an effective way to produce vitexin nanoparticles with markedly enhanced dissolution rate.

[Research progress on plant resources distribution of vitexin and its pharmacological effects].

Vitexin, a naturally occurring flavone glycoside in plants, has many pharmacological effects, which is widely distributed in nature. This paper reviewed the research progress of the distribution of vitexin in the plant resources and its pharmacological effects, and summarized its application prospects, aiming to provide a useful reference for the development of vitexin-enriched plant resources.

Zonulin, as a marker of intestinal permeability, is elevated in IgA nephropathy and IgA vasculitis with nephritis.

Background: Immunoglobulin A nephropathy (IgAN) and IgA vasculitis with nephritis (IgAV-N) are considered related diseases and share some similar clinicopathologic phenotypes. Elevated circulating galactose-deficient IgA1 (Gd-IgA1)-containing immune complexes and mucosal immunity were associated with the pathogenesis of IgAN and IgAV-N. Recently, studies have identified that the zonulin level, as a modulator of intestinal permeability, is significantly elevated in several inflammatory and autoimmune-related diseases. However, whether zonulin also plays a role in IgAN and IgAV-N is not clear. Methods: A total of 73 IgAV-N patients, 68 IgAN patients and 54 healthy controls were assessed for circulating zonulin and Gd-IgA1 levels by enzyme-linked immunosorbent assay. The diagnostic efficiency of the combination of zonulin with Gd-IgA1 was evaluated by the area under the receiver operating characteristic curve (AUC) and integrated discrimination improvement (IDI) analysis. Results: Compared with healthy controls, we found that both IgAV-N and IgAN patients had elevated zonulin and Gd-IgA1 levels (P < .001). Additionally, patients with IgAV-N presented with even higher circulating zonulin levels than patients with IgAN (P = .020). The addition of zonulin to Gd-IgA1 showed better predictive performance than Gd-IgA1 alone in the diagnosis of both IgAN and IgAV-N, as illustrated by a significantly increased AUC (IgAN: 0.805 versus 0.708, P = .0021; IgAV-N: 0.886 versus 0.673, P < .001) and significant IDI (IgAN: IDI 0.136, P < .001; IgAV-N: IDI 0.281, P < .001). Conclusion: Elevated circulating zonulin levels were detected in both patients with IgAV-N and those with IgAN. Combined detection of circulating zonulin and Gd-IgA1 is recommended as a noninvasive diagnostic biomarker for IgAV-N and IgAN.

Alternaria alternata F3, a Novel Taxol-Producing Endophytic Fungus Isolated from the Fruits of Taxus cuspidata: Isolation, Characterization, Taxol Yield Improvement, and Antitumor Activity.

In this study, a novel taxol-producing endophytic fungus, strain F3, was isolated from the fruits of Taxus cuspidata and identified as Alternaria alternata according to its macroscopic and microscopic traits and sequence analysis of internal transcribed spacer (ITS). The presence of taxol was detected by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) and confirmed by ultra-high-performance liquid chromatography-electrospray coupled to tandem mass spectrometry (UPLC-ESI-MS/MS) and nuclear magnetic resonance (NMR). The fermentation parameters of strain F3 were then optimized for high taxol production. The maximum taxol yield of 195.4 µg L-1 by A. alternata F3 was observed in 200-mL yeast peptone dextrose (YPD) broth, at an initial pH value of 6.0, supplemented with 0.1 g L-1 sodium acetate, 0.25 g L-1 salicylic acid, and 0.00125 g L-1 silver nitrate and inoculum size 2%, and incubated at 28 °C and 150 rpm for 8 days, which was 2.12-fold compared with the initial yield of taxol. Also, fungal taxol exhibited antitumor activity towards human lung carcinoma (A549) cell line and human cervical carcinoma (Hela) cell line with IC50 values of 3.98 µg mL-1 and 0.35 µg mL-1. Overall, this is the first report on taxol-producing endophytic fungus isolated from the fruits of Taxus. This study offers a novel source for the production of taxol for anticancer treatment.