Shh-Gli2-Runx2 inhibits vascular calcification.

BACKGROUND: In patients with chronic kidney disease (CKD), vascular calcification (VC) is common and is associated with a higher risk of all-cause mortality. Shh, one ligand for Hedgehog (Hh) signaling, participates in osteogenesis and several cardiovascular diseases. However, it remains unclear whether Shh is implicated in the development of VC. METHODS: Inorganic phosphorus 2.6 mM was used to induce vascular smooth muscle cells (VSMCs) calcification. Mice were fed with adenine diet supplement with 1.2% phosphorus to induce VC. RESULTS: Shh was decreased in VSMCs exposed to inorganic phosphorus, calcified arteries in mice fed with an adenine diet, as well as radial arteries from patients with CKD presenting VC. Overexpression of Shh inhibited VSMCs ostosteoblastic differentiation and calcification, whereas its silencing accelerated these processes. Likewise, mice treated with smoothened agonist (SAG; Hh signaling agonist) showed alleviated VC, and mice treated with cyclopamine (CPN; Hh signaling antagonist) exhibited severe VC. Additionally, overexpression of Gli2 significantly reversed the pro-calcification effect of Shh silencing on VSMCs, suggesting that Shh inhibited VC via Gli2. Mechanistically, Gli2 interacted with Runx2 and promoted its ubiquitin proteasomal degradation, therefore protecting against VC. Of interest, the pro-degradation effect of Gli2 on Runx2 was independent of Smurf1 and Cullin4B. CONCLUSIONS: Our study provided deeper insight to the pathogenesis of VC, and Shh might be a novel potential target for VC treatment.

Cloning and Prokaryotic Expression of Carotenoid Cleavage Dioxygenases from Mulberry (Morus notabilis).

Carotenoid cleavage dioxygenase (CCD) is the key enzyme for carotenoid cleavage, and the products of carotenoid cleavage regulate the ability of plants to stress. In this paper, six CCD genes were obtained from Morus notabilis (Mn) by reverse transcription-polymerase chain reaction (RT-PCR) and we classified them into three subgroups based on gene structures and phylogenetic analysis. The CDS (coding sequence) regions of the six MnCCD genes were 1617, 1620, 1635, 1713, 1746, and 1791 bp in full length, encoding 538, 539, 544, 570, 581, and 596 amino acids, respectively. Then, Pcold-TF-MnCCD plasmids were constructed and independently transferred into E. coli BL21 (DE3), and the MnCCD proteins were successfully expressed by prokaryotic expression with an expected molecular weight of recombinant proteins (∼120 kDa) and high solubility. These results will lay a foundation for the identification of mulberry carotenoid products.

BVVLS2 overlooked for 3 years in a pediatric patient caused by novel compound heterozygous mutations in SLC52A2 gene.

BACKGROUND: Brown-Vialetto-Van Laere syndrome-2 (BVVLS2) is a rare autosomal recessive neurological disorder caused by mutations in the SLC52A2 gene, which is characterized by early childhood onset of sensorineural hearing loss, bulbar palsy, peripheral neuropathy, and respiratory insufficiency. We aimed to investigate the genetic cause of a 4-year-old boy who suffered from BVVLS2 whose initial presentation was severe normocytic anemia and had been overlooked for three years in a local hospital. He was misdiagnosed with pure red cell aplasia (PRCA) and treated with hormones and chemotherapy drugs, but there was no obvious effect. METHODS: The targeted capture of 927 genes associated with neuromuscular disorders and next-generation sequencing were performed. Sanger sequencing was employed to verify the variant mutations. RESULTS: The proband was found to be heterozygous for c.350T > C (p.L117P) in exon 3 and c.1135_1137delTGG (p.W379del) in exon 5 of SLC52A2 gene. His anemia and neurological symptoms improved significantly after treatment with low dose oral riboflavin. CONCLUSIONS: This study expands the mutational spectrum of SLC52A2 and phenotypic spectrum of BVVLS2, which provides a foundation for further investigations elucidating the SLC52A2 related mechanisms of BVVLS2. A low-dosage of riboflavin supplementation was used to obtain good curative effect, which provides further future references for the clinical treatments of BVVLS.

Thiadiazoloquinoxaline-Based Semiconducting Polymer Nanoparticles for NIR-II Fluorescence Imaging-Guided Photothermal Therapy.

Phototheranostics have gained more and more attention in the field of cancer diagnosis and therapy. Among a variety of fluorophores for phototheranostics, semiconducting polymer nanoparticles (SPNs), which are usually constructed by encapsulating hydrophobic semiconducting polymers (SPs) with amphiphilic copolymers, have shown great promise. As second near-infrared (NIR-II) fluorescence imaging has both higher imaging resolution and deeper tissue penetration compared with first near-infrared (NIR-I) fluorescence imaging, NIR-II fluorescent SPNs have been widely designed and prepared. Among numerous structural units for semiconducting polymers (SPs) synthesis, thiadiazoloquinoxaline (TQ) has been proved as an efficient electron acceptor unit for constructing NIR-II fluorescent SPs by reacting with proper electron donor units. Herein, we summarize recent advances in TQ-based SPNs for NIR-II fluorescence imaging-guided cancer photothermal therapy. The preparation of TQ-based SPNs is first described. NIR-II fluorescence imaging-based and multimodal imaging-based phototheranostics are sequentially discussed. At last, the conclusion and future perspectives of this field are presented.

A molecular-beacon-based asymmetric PCR assay for detecting polymorphisms related to folate metabolism.

BACKGROUND: Polymorphisms (rs1801133 or C677T; rs1801131 or A1298C) of the MTHFR gene and rs1801394 (A66G) of the MTRR gene are important genetic determinants of folate metabolism. A convenient, sensitive, and reliable method is required to detect polymorphisms for the precise supplementation of folate. METHODS: A rapid detection method based on molecular beacon probes that can detect rs1801133, rs1801131, and rs1801394 simultaneously was developed in this study. Specific primers and probes were designed, and the amplification system and conditions were optimized. We applied our method to a group of 500 unrelated women of gestational age in the Dongguan region of Guangdong Province in China. The clinical performance of this assay was evaluated by testing 94 samples in comparison with Sanger sequencing. RESULTS: The molecular-beacon-based PCR assay we established is extremely sensitive, with a detection limit of 2 ng/µL of genomic DNA, and validated by direct sequencing in a blind study with 100% concordance. CONCLUSION: The results demonstrate that our molecular-beacon-based asymmetric PCR assay is an easy, reliable, high-yield, and cost-effective method for the simultaneous detection of three polymorphisms related to folate metabolism. It could help evaluate the risk of perinatal-neonatal neural tube malformation, pregnancy hypertension, and other diseases and guide the individualized supplementation of folic acid. Data on the spectrum of mutations in the Dongguan District in this study are beneficial for guiding the supplementation of folic acid.

Metabolomics Reveals that Cysteine Metabolism Plays a Role in Celastrol-Induced Mitochondrial Apoptosis in HL-60 and NB-4 Cells.

Recently, celastrol has shown great potential for inducing apoptosis in acute myeloid leukemia cells, especially acute promyelocytic leukaemia cells. However, the mechanism is poorly understood. Metabolomics provides an overall understanding of metabolic mechanisms to illustrate celastrol's mechanism of action. We treated both nude mice bearing HL-60 cell xenografts in vivo and HL-60 cells as well as NB-4 cells in vitro with celastrol. Ultra-performance liquid chromatography coupled with mass spectrometry was used for metabolomics analysis of HL-60 cells in vivo and for targeted L-cysteine analysis in HL-60 and NB-4 cells in vitro. Flow cytometric analysis was performed to assess mitochondrial membrane potential, reactive oxygen species and apoptosis. Western blotting was conducted to detect the p53, Bax, cleaved caspase 9 and cleaved caspase 3 proteins. Celastrol inhibited tumour growth, induced apoptosis, and upregulated pro-apoptotic proteins in the xenograft tumour mouse model. Metabolomics showed that cysteine metabolism was the key metabolic alteration after celastrol treatment in HL-60 cells in vivo. Celastrol decreased L-cysteine in HL-60 cells. Acetylcysteine supplementation reversed reactive oxygen species accumulation and apoptosis induced by celastrol and reversed the dramatic decrease in the mitochondrial membrane potential and upregulation of pro-apoptotic proteins in HL-60 cells. In NB-4 cells, celastrol decreased L-cysteine, and acetylcysteine reversed celastrol-induced reactive oxygen species accumulation and apoptosis. We are the first to identify the involvement of a cysteine metabolism/reactive oxygen species/p53/Bax/caspase 9/caspase 3 pathway in celastrol-triggered mitochondrial apoptosis in HL-60 and NB-4 cells, providing a novel underlying mechanism through which celastrol could be used to treat acute myeloid leukaemia, especially acute promyelocytic leukaemia.

α-Arbutin Protects Against Parkinson's Disease-Associated Mitochondrial Dysfunction In Vitro and In Vivo.

Parkinson's disease (PD), the most common neurodegenerative movement disorder, is characterized by the progressive loss of dopaminergic neurons in substantia nigra. The underlying mechanisms of PD pathogenesis have not been fully illustrated and currently PD remains incurable. Accumulating evidences suggest that mitochondrial dysfunction plays pivotal role in the dopaminergic neuronal death. Therefore, discovery of novel and safe agent for rescuing mitochondrial dysfunction would benefit PD treatment. Here we demonstrated for the first time that α-Arbutin (Arb), a natural polyphenol extracted from Ericaceae species, displayed significant protective effect on the rotenone (Rot)-induced mitochondrial dysfunction and apoptosis of human neuroblastoma cell (SH-SY5Y). We further found that the neuroprotective effect of Arb was associated with ameliorating oxidative stress, stabilizing of mitochondrial membrane potential, and enhancing adenosine triphosphate production. To investigate the underlying mechanism, we checked the AMP-activated protein kinase and autophagy pathway and we found that both were involved in the neuroprotection of Arb. Moreover, we explored the protective effect of Arb in drosophila PD model and found that Arb rescued parkin deficiency-induced motor function disability and mitochondrial abnormality of drosophila. Taken together, our study demonstrated that Arb got excellent neuroprotective effect on PD models both in vitro and in vivo and Arb might serve as a potent therapeutic agent for the treatment of PD.

Gadolinium-Chelated Conjugated Polymer-Based Nanotheranostics for Photoacoustic/Magnetic Resonance/NIR-II Fluorescence Imaging-Guided Cancer Photothermal Therapy.

Our exploiting versatile multimodal theranostic agent aims to integrate the complementary superiorities of photoacoustic imaging (PAI), second near-infrared (NIR-II, 1000-1700) fluorescence and T1-weighted magnetic resonance imaging (MRI) with an ultimate objective of perfecting cancer diagnosis, thus improving cancer therapy efficacy. Herein, we engineered and prepared a water-soluble gadolinium-chelated conjugated polymer-based theranostic nanomedicine (PFTQ-PEG-Gd NPs) for in vivo tri-mode PA/MR/NIR-II imaging-guided tumor photothermal therapy (PTT). Methods: We firstly constructed a semiconducting polymer composed of low-bandgap donor-acceptor (D-A) which afforded the strong NIR absorption for PAI/PTT and long fluorescence emission to NIR-II region for in vivo imaging. Then, the remaining carboxyl groups of the polymeric NPs could effectively chelate with Gd3+ ions for MRI. The in vitro characteristics of the PFTQ-PEG-Gd NPs were studied and the in vivo multimode imaging as well as anti-tumor efficacy of the NPs was evaluated using 4T1 tumor-bearing mice. Results: The obtained theranostic agent showed excellent chemical and optical stability as well as low biotoxicity. After 24 h of systemic administration using PQTF-PEG-Gd NPs, the tumor sites of living mice exhibited obvious enhancement in PA, NIR-II fluorescence and positive MR signal intensities. Better still, a conspicuous tumor growth restraint was detected under NIR light irradiation after administration of PQTF-PEG-Gd NPs, indicating the efficient photothermal potency of the nano-agent. Conclusion: we triumphantly designed and synthesized a novel and omnipotent semiconducting polymer nanoparticles-based theranostic platform for PAI, NIR-II fluorescence imaging as well as positive MRI-guided tumor PTT in living mice. We expect that such a novel organic nano-platform manifests a great promise for high spatial resolution and deep penetration cancer theranostics.

Single nanoparticles as versatile phototheranostics for tri-modal imaging-guided photothermal therapy.

Development of versatile phototheranostics is highly desirable for cancer theranostics. Herein, a novel organic conjugated polymer (named DPP-TT) with excellent optical properties was designed and prepared. Based on single-component DPP-TT, single DPP-TT NPs as versatile phototheranostics were developed by a simple nanoprecipitation method. The obtained NPs exhibited good water solubility, excellent biocompatibility, outstanding photostability, strong NIR-I light absorption, and appropriate NIR-II fluorescence emission. Importantly, such NPs presented high photothermal conversion efficiency. From the investigations performed in vitro and in vivo, it was observed that DPP-TT NPs show remarkable performance for cancer theranostics, benefiting from single 808 nm laser-induced tri-modal (NIR-II fluorescence/photoacoustic/thermal) imaging-guided photothermal therapy.

Semiconducting polymer nanotheranostics for NIR-II/Photoacoustic imaging-guided photothermal initiated nitric oxide/photothermal therapy.

Gasotransmitters with their cytotoxicity in high concentration have become the focus of attention. For such concentration depended therapy, how to effectively deliver gases and precisely control gases release to the lesion as well as combine them with other therapy to achieve precise therapeutics is still a big challenge. Herein, we realize single near-infrared (NIR) laser-initiated nitric oxide (NO) therapy/photothermal therapy (PTT) using semiconducting polymer nanoparticles (SPNs, PFTDPP) combing s-nitrosothiol groups (the NO donor, SNAP). By the good photothermal conversion effect of SPNs, NIR laser energy can be spatio-temporally controlled to convert into heat to decompose s-nitrosothiol. Meanwhile, considering the accompanied PTT produced by photothermal, we can easily and precisely conduct a dual therapy (NO therapy/PTT) under single NIR laser irradiation. Additionally, semiconducting polymer with its structural modifiability and spectral adjustability can provide a second NIR window & photoacoustic (NIR II/PA) imaging for guiding photothermal initiated NO/photothermal therapy. PFTDPP showed a high photothermal conversion efficiency of 48% and good dual-mode imaging signals (NIR-II/photoacoustic). Cellular test illustrated that NO combined photothermal presented more prominent cytotoxicity than any one of them individually. As the tumor pinpointed in vivo by dual-mode imaging (NIR II/PA), this nanotheranostics provided a tumor inhibition of 77%. Consequently, such phototheranostics produced a new design thought for effectively deliver and precisely controlled release of drugs for oncology. And also, it expanded the application range of gasotransmitters combined therapy that shall have a promising application foreground.

Danshen (Radix Salviae Miltiorrhizae) reverses renal injury induced by myocardial infarction.

OBJECTIVE: To evaluate the protection of danshen (Radix Salviae Miltiorrhizae) (SM) injection in myocardial infarction (MI) induced renal damage. METHODS: Forty male C57 mice were divided into control group, MI group and SM group. In MI group, the left coronary artery was occluded for 8 weeks; the same procedure was used for the SM group, with the additional step of SM (0.2 mL) administered intraperitoneally for 56 days. Before surgery and 8 weeks later, transthoracic echocardiography was performed and urine protein and albumin was measured. At the end of the time, all mice were killed and kidneys removed for reactive oxygen species (ROS) and fibrosis analysis, plasma was collected for blood urea nitrogen and serum creatinine determination. RESULTS: MI slightly decreased renal function and increased production of ROS, accompanied with renal fibrosis. Administration of SM reduced production of ROS and increased renal function, it also reduced renal fibrosis. CONCLUSION: MI plays a causal role in renal injury and SM exerts renal-protective effects, probably by its antioxidant activities.

[Effects of electroacupuncture on urinary bladder function after radical hysterectomy].

OBJECTIVE: To observe the effect of electroacupuncture on recovery of urinary bladder function after radical hysterectomy. METHODS: One hundred and ten cases were randomly divided into an electroacupuncture (EA) group and a control group, 55 cases in each group. In the control group, the urinary tube was placed and kept with routine method and the urinary bladder was rinsed, and from the eighth day the abdomen was radiated with TDP, 30 min each day, for 5 days. In the EA group, on the basis of treatment in the control group EA was given at Sanyinjiao (SP 6), Zusanli (ST 36), Waiguan (TE 5), Shuidao (ST 28), Guilai (ST 29), etc. from the eighth day to twelfth day after operation. The recovery time of urinary bladder function after radical hysterectomy, urine dynamic indexes and hospitalization days were compared between the two groups. RESULTS: The cases of the bladder function recovery, retention of urine, urinary incontinence were 51(51/55), 4(4/55), 0 on the 14 th day after operation and 53(53/55), 2(2/55), 0 on the 28 th day in the EA group, and 27(27/55), 25(25/55), 3(3/55) on the 14 th day and 43(43/55), 11(11/55), 1(1/55) on the 28th day in the control group, respectively, with a very significant difference between the two groups (P < 0.01); the EA group in residual urine volume, bladder volume, mean urinary flowing rate was better than the control group on the 14 th day after operation (P < 0.01 or P < 0.05); the hospitalization days after operation was (21.1 +/- 3.3) days in the EA group and (25.5 +/- 3.5) days in the control group, the former being shorter than the later (P < 0.01). CONCLUSION: EA can promote recovery of bladder function, shorten the keeping time of urinary tube after radical hysterectomy, which is benefit to decreasing incidence rate of urinary system infection and shortening hospitalization days.

[Effect of glucosidorum tripterygii tororum on acute graft-versus-host disease in mice].

To prevent acute graft-versus-host disease (aGVHD), glucosidorum tripterygii tororum (GTT) was used in the murine model. The lethally irradiated C57BL/6 recipients were injected with bone marrow and lymphocyte grafts from BALB/c donors and were treated intraperitoneally with GTT, cyclosporine A (CsA), or methotrexate (MTX). T lymphocytes, adhesion molecules and cytokines were detected by immunohistochemical method, flow cytometry, ELISA and RT-PCR, respectively. The results showed that all the control recipient mice (21/21) died of aGVHD within 30 days, but many recipient mice treated with GTT (19/21), CsA + MTX (13/21) and GTT + CsA (17/21) survived beyond 30 days without obvious signs of aGVHD. The numbers of CD3(+), CD4(+), CD8(+), CD11a(+), CD18(+) lymphocytes in skin and lung decreased markedly by GTT, GTT + CsA and CsA + MTX treatments. The numbers of CD3(+), CD4(+), CD8(+), CD4(+)CD11a(+), CD4(+)CD18(+), CD8(+)CD11a(+), CD8(+)CD18(+) lymphocytes in spleen decreased markedly by GTT, GTT + CsA and CsA + MTX treatments. and the changes of CD3(+), CD4(+), CD8(+) cells in small intestine were not remarkable (P > 0.05) by above mentioned GTT, GTT + CsA and CsA + MTX treatments. The serum concentrations and mRNA expressions of IL-2 and TNFalpha in spleens decreased significantly (P < 0.05); the concentration of IL-10 increased significantly (P < 0.05), the change of IL-4 was not remarkable (P > 0.05) by GTT treatment. It is concluded that the GTT may retain the graft-versus-leukemia (GVL) effect of transplant without aGVHD. The role of GTT in prevention of murine aGVHD is mediated by reduction of T lymphocytes and their subgroups, expression of adhesion molecule, and regulation of cytokine secretion.