Effects of Age on the Biological Properties of Cryopreserved Adipose-Derived Stem Cells and ASC-Enriched Fat Grafts.

BACKGROUND: Donor age is an important factor affecting the biological characteristics of human adipose-derived stem cells. The aim of this study was to compare the effects of age on the biological properties of cryopreserved adipose-derived stem cells and fat survival of cell-assisted lipotransfer. METHODS: Human lipoaspirates were obtained from 60 healthy female patients (aged 18-65 years) who underwent abdominal liposuction. Samples were divided into three groups according to donor age: group A, 18-29 years; group B, 30-49 years; and group C, 50-65 years. Adipose-derived stem cells were obtained by in vitro culture at the second passage and cryopreserved for 4 weeks. The cryopreserved ASCs were examined for biological characteristics, including cell proliferation, wound healing and adipogenic differentiation. Then, the fat survival of cryopreserved ASC-assisted fat transplantation was compared at different ages. RESULTS: SVF viability decreased with increasing age. Moreover, there was a decline in cell proliferation and migration of ASCs with increasing age. A significant difference was found in the adipogenic differentiation of ASCs in the three groups. There were significant differences in graft retention in different age groups. ASC-assisted fat grafting was more effective in young people than in elderly people. CONCLUSIONS: Honor age affects the proliferation and migration of adipose-derived stem cells but not the adipogenic differentiation potential of ASCs. Cryopreserved ASCs from younger people more effectively improved the fat survival of grafts. LEVEL OF EVIDENCE V: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Investigating the Adipogenic Effects of Different Tissue-Derived Decellularized Matrices.

Objective: Decellularized adipose-derived matrix (DAM) can promote adipogenic differentiation and adipose tissue remodeling, but the biological impact of tissue origin on DAM remains unknown. The present study aimed to investigate the effects of tissue origins on the adipogenic capacity of the decellularized matrix by comparing the cellular and tissue responses of DAM versus acellular dermal matrix (ADM). Methods: The in vitro response of adipose-derived stem/stromal cells (ADSCs) to DAM and ADM was characterized by proliferation and differentiation. The in vivo remodeling response was evaluated in the subcutaneous injection model of immunocompromised mice, using histology, protein expression, and transcriptome analysis. Results: Both DAM and ADM exhibited excellent decellularization effects and cytocompatibility. In the absence of exogenous stimuli, DAM could induce adipogenic differentiation of ADSCs compared with ADM. In the animal model, the levels of PDGF, VEGF, and ACRP30 were higher in the DAM groups than in the ADM group, and more neovascularization and extensive adipose tissue remodeling were observed. The mRNA-seq analysis indicated that the DAM implant regulated tissue remodeling by modulating Lat1/2 expression along with Hippo Signaling pathway in the early stage. Conclusion: Tissue origin can influence the biological response of the decellularized matrix. DAM can retain favorable tissue-specific characteristics after the decellularization process and have unique adipogenic effects in vitro and vivo, which can be fully utilized for soft tissue repair and regeneration.

Geranylgeranylation signaling promotes breast cancer cell mitosis via the YAP-activated transcription of kinetochore/centromere genes.

Geranylgeranylation signaling plays an important role in cancer cell proliferation. Our previous studies have shown that the YAP is one of the geranylgeranylation signal transducers in breast cancer cells (Mi W, et al., Oncogene. 2015; 34(24): 3095-3106). However, the downstream effectors that mediate the promoting effect of the geranylgeranylation/YAP signal axis on breast cancer cell proliferation remain elusive. In this report, we investigated the pathway that mediates the effect of the geranylgeranylation on breast cancer cell proliferation. The results have shown that inhibition of geranylgeranyl biosynthesis inactivates transcription of a set of kinetochore/centromere genes. Further biochemical and cell biological studies demonstrated that inhibition of geranylgeranyl biosynthesis significantly reduced the level of key kinetochore/centromere proteins, thus caused a defect in mitosis. Knockdown of YAP caused similar inhibitory effects on the kinetochore/centromere gene expression and mitosis to that of inhibition of geranylgeranyl biosynthesis. Furthermore, we found that E2F1, the gene coding for E2F1 that is known to activate expression of cell cycle genes, is a target gene of YAP. Knockdown of E2F1 also reduced expression of the kinetochore/centromere genes, suggesting that the activation effect of YAP on expression of the kinetochore/centromere genes may be mediated by E2F1. Our studies have proposed a novel geranylgeranylation-dependent cancer cell proliferation signaling pathway in which geranylgeranylation signaling promotes cancer cell mitosis via the YAP-activated transcription of kinetochore/centromere genes.

Does Water-Jet Force Affect Cryopreserved Adipose-Derived Stem Cells? Evidence of Improved Cell Viability and Fat Graft Survival.

BACKGROUND: Adipose tissue harvested by liposuctions is an available source of adipose-derived stem cells (ASCs). Water-jet-assisted liposuction is a favorable method for fat collection with little mechanical damage. This study aimed to investigate whether or not the water-jet-assisted liposuction made a difference in the biological characteristics of cryopreserved ASCs and fat graft survival in cell-assisted lipotransfer. METHODS: Human lipoaspirates were obtained from the abdomen or thighs of 20 female participants for body contouring. A single surgeon randomly harvested 50 mL of adipose tissue by the water-jet-assisted liposuction and the conventional liposuction, respectively. Adipose-derived stem cells were isolated from lipoaspirates and then cryopreserved for 4 weeks. Cryopreserved ASCs were used to examine the surface markers, cell proliferation, migration, and adipogenic differentiation in vitro. The fat survival of ASCs-enriched grafts from different liposuctions was measured in animal models. RESULTS: The cryopreserved ASCs with the water-jet assistance had better capacities of cell proliferation, migration, and adipogenic differentiation and achieved a better survival result of ASCs-enriched fat grafting. CONCLUSIONS: Cryopreservation of ASCs with the water-jet force showed more excellent biological characteristics. The water-jet-assisted liposuction was superior to the conventional liposuction in obtaining ASCs and fat survival of coimplantation with grafts.

ASK1 inhibits proliferation and migration of lung cancer cells via inactivating TAZ.

ASK1 (Apoptosis Signal-regulating Kinase 1, also MEKK5) is known to mediate cellular stress signaling pathways through activating p38 kinase. We here observed that ectopically expression of ASK1, but not its kinase-dead mutant, impaired cell proliferation and migration in lung cancer A549 and NCI-H1975 cells. To our surprise, this inhibitory effect of ASK1 is independent on activation of p38 kinase. We further discovered that ASK1 interacts with the WW domain of YAP and TAZ (also WWTR1) that are transcriptional co-activators and the Hippo signaling effectors. Overexpression of wild type ASK1, but not the kinase-dead mutant, in the lung cancer cells down-regulated the expression of the YAP/TAZ target genes CYR61 and CTGF. It seems that ASK1 specifically inactivates TAZ, not YAP, as ASK1 blocked nuclear translocation of TAZ only, while had no effect on YAP. Furthermore, knockdown of TAZ in the lung cancer cells caused the same inhibitory effect on cell proliferation and migration as that of overexpression of ASK1. Thus, our studies have defined a new signaling pathway of ASK1 for regulation of lung cancer cell proliferation and migration via interacting with and inactivating TAZ.

Effects of Harvest Sites on Cryopreserved Adipose-Derived Stem Cells and ASC-Enriched Fat Grafts.

BACKGROUND: Enrichment of adipose-derived stem cells (ASCs) with fat grafts has demonstrated benefit for graft retention and histologic appearance. There is no consensus on the optimal harvest site for adipose-derived stem cells. This study aimed to investigate the effects of harvest sites on the characteristics of cryopreserved adipose-derived stem cells and the graft retention of cell-assisted lipotransfer. METHODS: Lipoaspirates were harvested from 18 healthy volunteers who underwent liposuctions for body contouring. Twenty milliliters of lipoaspirates was, respectively, obtained from four sites, including the upper limb, abdomen, waist, and thighs, by the Coleman technique. Adipose-derived stem cells were ex vivo cultured and cryopreserved for four weeks. The biological characteristics of ASCs from four harvest sites were analyzed: MSC surface markers, cell proliferation, migration ability, and multipotential differentiation. The fat grafts were co-implanted with ASCs from four harvest sites and injected subcutaneously in mice. The ASC-enriched fat grafts were analyzed three months after transplantation. RESULTS: Cryopreserved ASCs from the abdomen and thighs maintained more significant cell proliferation, migration ability, and differentiation potential, compared with cells from the upper limb and waist. Moreover, we achieved better graft retention of cell-assisted fat grafts with cryopreserved ASC from the abdomen and thighs. CONCLUSIONS: The harvest site of adipose tissue affects the cellular activity and differentiation potential of cryopreserved ASCs. Improved understanding of harvest sites for ASCs can optimize the outcomes of cell-assisted fat grafts. Fat grafts enriched with cryopreserved ASCs from the abdomen or thighs are the optimal choices. LEVEL OF EVIDENCE V: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Amelioration of paraquat-induced pulmonary fibrosis in mice by regulating miR-140-5p expression with the fibrogenic inhibitor Xuebijing.

Intravenous Xuebijing (XBJ) therapy suppresses paraquat (PQ)-induced pulmonary fibrosis. However, the mechanism underlying this suppression remains unknown. This work aimed to analyze the miR-140-5p-induced effects of XBJ injection on PQ-induced pulmonary fibrosis in mice. The mice were arbitrarily assigned to four groups. The model group was administered with PQ only. The PQ treatment group was administered with PQ and XBJ. The control group was administered with saline only. The control treatment group was administered with XBJ only. The miR-140-5p and miR-140-5p knockout animal models were overexpressed. The gene expression levels of miR-140-5p, transglutaminase-2 (TG2), ß-catenin, Wnt-1, connective tissue growth factor (CTGF), mothers against decapentaplegic homolog (Smad), and transforming growth factor-ß1 (TGF-ß1) in the lungs were assayed with quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot analysis. The levels of TGF-ß1, CTGF, and matrix metalloproteinase-9 (MMP-9) in the bronchoalveolar lavage fluid were assessed by enzyme-linked immunosorbent assay (ELISA). Hydroxyproline (Hyp) levels and pulmonary fibrosis were also scored. After 14 days of PQ induction of pulmonary fibrosis, AdCMV-miR-140-5p, and XBJ upregulated miR-140-5p expression; blocked the expressions of TG2, Wnt-1, and ß-catenin; and decreased p-Smad2, p-Smad3, CTGF, MMP-9, and TGF-ß1 expressions. In addition, Hyp and pulmonary fibrosis scores in XBJ-treated mice decreased. Histological results confirmed that PQ-induced pulmonary fibrosis in XBJ-treated lungs was attenuated. TG2 expression and the Wnt-1/ß-catenin signaling pathway were suppressed by the elevated levels of miR-140-5p expression. This inhibition was pivotal in the protective effect of XBJ against PQ-induced pulmonary fibrosis. Thus, XBJ efficiently alleviated PQ-induced pulmonary fibrosis in mice.

The Effect of Thymosin beta4 on the Survival of Autologous Fat Grafting: A Preliminary Study.

BACKGROUND: Autologous fat grafting is a common procedure to improve tissue deficiencies. However, the survival rate of fat grafting is unpredictable. Thymosin beta 4 (Tß4), a multifunctional peptide containing 43 amino acids, is effective in angiogenesis, inhibiting apoptosis and inflammation. OBJECTIVES: The authors initially investigated the potential effect of Tß4 in fat grafting. METHODS: Adipose tissue premixed exogenous Tß4 were transplanted into rabbit ears. Rabbits were randomly assigned to 3 groups: group A, 5 µg/mL Tß4; group B, 10 µg/mL Tß4; and group C, phosphate-buffered saline buffer as a blank control. The fat grafts were subjected to magnetic resonance imaging at 2, 4, and 12 weeks in vivo. Each harvested graft was analyzed at 3 time points after transplantation. RESULTS: The fat grafts in the Tß4-treated groups showed better volume and weight retention, greater adipose tissue integrity, adipocyte viability, and angiogenesis. The results of dynamic contrast-enhanced magnetic resonance imaging also showed that the experimental groups increased microcirculation perfusion of the grafts. CONCLUSIONS: The study proved that Tß4 could improve adipose tissue survival and neovascularization. It may be useful for fat grafting as a potential protective reagent.

Thallium exposure at low concentration leads to early damage on multiple organs in children: A case study followed-up for four years.

Thallium (TI) is one of the most toxic heavy metals and priority pollutant metals. The emerging TI environmental pollution worldwide has posed a great threat to human health. However, based on the World Health Organization (WHO), the risk and severity of adverse health effects of TI in the range of 5-500 µg/L are uncertain. Moreover, evidence regarding the adverse impacts of TI on children's health is still insufficient. Herein, we aim to investigate the early adverse effects of TI on children's health and provide references for the WHO to establish stricter safety limits of TI. From 2015 to 2019, urinary TI and many clinical laboratory parameters related to blood routine, hepatic, renal, myocardial, coagulation function and serum electrolyte were measured in six children aged 1-9 years. The urinary TI concentration ranged from 13.4 µg/L to 60.1 µg/L with a mean of 36.1 µg/L and a median of 34.8 µg/L in six children in 2015. Although only four children felt a little poor appetite, several laboratory abnormalities indicated early damage in liver, renal, and myocardial functions in all children in 2015. After treatment and following up for four years, although the children's TI concentration decreased below 5 µg/L, their liver and renal functions did not completely recover, and their myocardial function worsened. Results indicated that impaired liver, renal, and myocardial functions were closely associated with elevated urinary TI concentration in children. Considering the increasing use of TI in high-technology industries and emerging TI environmental-contamination zones worldwide, establishing stricter safety limits of TI and paying more attention to the adverse health effects of TI on children are urgently required. SUMMARY: We found that a relatively low concentration of thallium (13.4 µg/L to 60.1 µg/L) impaired liver, renal, and myocardial function in six children. After treatment and following up these children for four years, although their urinary TI concentration decreased below 5 µg/L, their liver and renal functions did not completely recover, and their myocardial function worsened.

Identification of the Optimal Recipient Layer for Transplanted Fat: A Prospective Study on Breast Lipoaugmentation.

BACKGROUND: Fat grafting has become a popular procedure in aesthetic and reconstructive surgeries due to its safety, minimal invasiveness, and favorable visual outcomes, although the volume retention rate is unpredictable. OBJECTIVES: A prospective clinical study on lipoaugmentation of the breast was conducted to compare fat retention rates in the pectoralis muscle and the periglandular area. METHODS: This prospective study included 20 breasts from 11 patients who underwent primary lipoaugmentation. Volume retention rate and percentage augmentation among different recipient layers, as well as complications and patient satisfaction, were evaluated. Magnetic resonance imaging was performed preoperatively and at 1 day and 3 months postoperatively. Complications were recorded, and patient satisfaction was appraised through the use of the Breast-Q questionnaire. RESULTS: Breasts were injected with 207 ± 29 mL of fat, achieving overall volume retention rates of 56.63% ± 16.40%. The overall augmentation was 21.53% ± 10.27%. Volume retention rate was significantly higher (59.00% ± 13.84%) in the periglandular area than in the pectoralis muscle (47.21% ± 22.41%) (P = 0.04). Augmentation was significantly higher (32.13% ± 12.96%) in the periglandular area than in the pectoralis muscle (4.95% ± 4.23%) (P = 0.00). Pain and numbness were the only reported complications. The Breast-Q score increased significantly for the measures "satisfaction with breasts," "psychosocial well-being," and "sexual well-being." CONCLUSIONS: Fat transfer is a safe and acceptable method for aesthetic and reconstructive breast surgery. The periglandular area was a better recipient site than muscle for transferred fat.

[Expression, purification and characterization of arabinose-5-phosphate isomerase from Arabidopsis thaliana].

Arabinose-5-phosphate isomerase (KdsD) is the first key limiting enzyme in the biosynthesis of 3-deoxy-D-manno-octulosonate (KDO). KdsD gene was cloned into prokaryotic expression vector pET-HTT by seamless DNA cloning method and the amount of soluble recombinant protein was expressed in a soluble form in E. coli BL21 (DE3) after induction of Isopropyl ß-D-1-thiogalactopyranoside (IPTG). The target protein was separated and purified by Ni-NTA affinity chromatography and size exclusion chromatography, and its purity was more than 85%. Size exclusion chromatography showed that KdsD protein existed in three forms: polymers, dimmers, and monomers in water solution, different from microbial KdsD enzyme with the four polymers in water solution. Further, the purified protein was identified through Western blotting and MALDI-TOF MASS technology. The results of activity assay showed that the optimum pH and temperature of AtKdsD isomerase activities were 8.0 and 37 ℃, respectively. The enzyme was activated by metal protease inhibitor EDTA (5 mmol/L) and inhibited by some metal ions at lower concentration, especially with Co²âº and Cd²âº metal ion. Furthermore, when D-arabinose-5-phosphate (A5P) was used as substrate, Km and Vmax of AtKdsD values were 0.16 mmol/L, 0.18 mmol/L·min. The affinity of AtKdsD was higher than KdsD in E. coli combined with substrate. Above results have laid a foundation for the KdsD protein structure and function for its potential industrial application.

Effects on decreasing upper-limb poststroke muscle tone using transcranial direct current stimulation: a randomized sham-controlled study.

OBJECTIVE: To assess the efficacy of transcranial direct current stimulation (tDCS) on decreasing upper-limb (UL) muscle tone after stroke. DESIGN: A prospective, sham-controlled, randomized controlled trial with 4-weeks follow-up. Randomization into the tDCS group or the control group. SETTING: Rehabilitation education and research hospital. PARTICIPANTS: Inpatients (N=90, 45 per group; age range, 15-70y; 69 men, 21 women; duration of stroke, 2-12mo) with poststroke UL spasticity. No participant withdrew because of adverse effects. INTERVENTION: The tDCS group received tDCS to the primary sensorimotor cortex of the affected side with cathodal stimulation, 20 minutes per day, 5 days per week, for 4 weeks and conventional physical therapy. The control group received sham stimulation (same area as the tDCS group) and conventional physical therapy. MAIN OUTCOME MEASURES: Modified Ashworth scale (MAS), Fugl-Meyer Assessment of motor recovery, and Barthel Index. All outcomes were measured at admission, after treatment, and after follow-up. A clinically important difference (CID) was defined as a reduction of ≥1 in the MAS score. RESULTS: Compared with the sham tDCS group, the active tDCS group had significantly more patients with a clinically important difference after treatment (80% and 78% vs 6% and 9%) and at 4-week follow-up (84% and 82% vs 7% and 4%), and UL motor function and activities of daily living (ADL) assessment improved more significantly in the active tDCS group (Fugl-Meyer Assessment of motor recovery from 12 [range, 4-26] to 22 [range, 7-50] to 32 [range, 28-41], Barthel Index from 55 [range, 0-85] to 85 [range, 5-100] to 90 [range, 10-100 vs Fugl-Meyer Assessment of motor recovery from 8 [range, 3-34] to 10 [range, 8-25] to 15 [range, 6-40], Barthel Index from 55 [range, 25-95] to 65 [range, 30-100] to 75 [range, 40-100], respectively, P<.01). CONCLUSIONS: UL muscle tone after stroke can be decreased using cathodal tDCS. Combined with conventional physical therapy, tDCS appears to improve motor function and ADL. Cathodal tDCS over ipsilesional primary sensorimotor cortex may inhibit primary sensorimotor cortex hyperactivation, resulting in significant reductions in muscle tone.