Customization of an Ultrafast Thiol-Norbornene Photo-Cross-Linkable Hyaluronic Acid-Gelatin Bioink for Extrusion-Based 3D Bioprinting.

Light-based three-dimensional (3D) bioprinting has been widely studied in tissue engineering. Despite the fact that free-radical chain polymerization-based bioinks like hyaluronic acid methacrylate (HAMA) and gelatin methacryloyl (GelMA) have been extensively explored in 3D bioprinting, the thiol-ene hydrogel system has attracted increasing attention for its ability in building hydrogel scaffolds in an oxygen-tolerant and cell-friendly way. Herein, we report a superfast curing thiol-ene bioink composed of norbornene-modified hyaluronic acid (NorHA) and thiolated gelatin (GelSH) for 3D bioprinting. A new facile approach was first introduced in the synthesis of NorHA, which circumvented the cumbersome steps involved in previous works. Additionally, after mixing NorHA with macro-cross-linker GelSH, the customized NorHA/GelSH bioinks exhibited fascinating superiorities over the gold standard GelMA bioinks, such as an ultrafast curing rate (1-5 s), much lowered photoinitiator concentration (0.03% w/v), and flexible physical performances. Moreover, the NorHA/GelSH hydrogel greatly avoided excess ROS generation, which is important for the survival of the encapsulated cells. Last, compared with the GelMA scaffold, the 3D-printed NorHA/GelSH scaffold not only exhibited excellent cell viability but also guaranteed cell proliferation, revealing its superior bioactivity. In conclusion, the NorHA/GelSH system is a promising candidate for 3D bioprinting and tissue engineering applications.

The -250G/A and -514C/T Polymorphisms in Hepatic Lipase Gene Promoter Confers an Increased Risk of Hepatocellular Carcinoma in a Chinese Population.

INTRODUCTION AND AIM: Hepatocellular carcinoma (HCC) is a lethal malignancy, but the molecular mechanisms of hepatocarcinogenesis remain undefined. The present study aims to investigate the relationship between polymorphisms of the hepatic lipase (HL) gene promoters and risk of HCC. MATERIAL AND METHODS: Totally, 279 HCC patients and 200 healthy individuals were enrolled. Polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) was used to analyze the genotypes of HL gene. Logistic regression analysis was conducted to identify risk factors of HCC. RESULTS: There was significant difference in the distribution of smoking history, drinking history, and family history of subjects between the case and control groups (all p < 0.05). Difference in the -250G/A (p = 0.011; OR = 1.61; 95%CI: 1.11-2.34) and -514C/T (p = 0.007; OR = 1.65; 95%CI: 1.14-2.38) genotypes and allele frequencies between two groups was significant. A higher risk of HCC was identified in those with polymorphisms in the - 250G/A (p = 0.007; OR = 1.45; 95%CI: 1.11-1.89) and -514C/T (p = 0.003; OR = 1.51; 95%CI: 1.15-2.00). Polymorphisms at - 250G/A (GA + AA) (p = 0.025; OR = 1.55; 95%CI: 1.06-2.28), -514C/T (CT + TT) (p = 0.021; OR = 1.57; 95%CI: 1.07-2.29), smoking history (p = 0.017; OR = 1.70; 95%CI: 1.10-2.63) and drinking history (p = 0.003; OR = 2.04; 95%CI: 1.27-3.27) were significantly related to the risk of HCC (all p < 0.05). CONCLUSION: The results obtained from this study indicated that polymorphisms of -250G/A and -514C/T in HL gene promoters were associated with the risk of HCC.

Inhibition of Parkinsonian tremor with cutaneous afferent evoked by transcutaneous electrical nerve stimulation.

BACKGROUND: Recent study suggests that tremor signals are transmitted by way of multi-synaptic corticospinal pathway. Neurophysiological studies have also demonstrated that cutaneous afferents exert potent inhibition to descending motor commands by way of spinal interneurons. We hypothesize in this study that cutaneous afferents could also affect the transmission of tremor signals, thus, inhibit tremor in patients with PD. METHODS: We tested this hypothesis by activating cutaneous afferents in the dorsal hand skin innervated by superficial radial nerve using transcutaneous electrical nerve stimulation (TENS). Eight patients with PD having tremor dominant symptom were recruited to participate in this study using a consistent experimental protocol for tremor inhibition. Resting tremor and electromyogram (EMG) of muscles in the upper extremity of these subjects with PD were recorded, while surface stimulation was applied to the dorsal skin of the hand. Fifteen seconds of data were recorded for 5 s prior to, during and post stimulation. Power spectrum densities (PSDs) of tremor and EMG signals were computed for each data segment. The peak values of PSDs in three data segments were compared to detect evidence of tremor inhibition. RESULTS: At stimulation intensity from 1.5 to 1.75 times of radiating sensation threshold, apparent suppressions of tremor at wrist, forearm and upper arm and in the EMGs were observed immediately at the onset of stimulation. After termination of stimulation, tremor and rhythmic EMG bursts reemerged gradually. Statistical analysis of peak spectral amplitudes showed a significant difference in joint tremors and EMGs during and prior to stimulation in all 8 subjects with PD. The average percentage of suppression was 61.56% in tremor across all joints of all subjects, and 47.97% in EMG of all muscles. The suppression appeared to occur mainly in distal joints and muscles. There was a slight, but inconsistent effect on tremor frequency in the 8 patients with PD tested. CONCLUSIONS: Our results provide direct evidence that tremor in the upper extremity of patients with PD can be inhibited to a large extent with evoked cutaneous reflexes via surface stimulation of the dorsal hand skin area innervated by the superficial radial nerve.

Carboxy-terminal telopeptide levels of type I collagen hydrogels modulated the encapsulated cell fate for regenerative medicine.

The cellular microenvironment has a profound impact on cell proliferation, interaction, and differentiation. In cell encapsulation for disease therapy, type I collagen is an important biomaterial due to its ability to mimic the extracellular matrix. Telopeptides (carboxy-terminal, CTX, and amino-terminal, NTX) protruding from the triple helix structure of type I collagen are cross-link sites, but also mediate the signal transmission in tissue homeostasis. It is worth investigating the features of the hydrogel microenvironment shaped by the tissue-derived type I collagen with various telopeptide levels, which is paramount for encapsulated cell development. Here, we found the fate of encapsulated human adipose-derived stem cells (hADSCs) and human umbilical vein endothelial cells (HUVECs) behaved differently towards decreasing CTX levels in the collagen hydrogels. Even among collagen hydrogels with a small magnitude of CTX variation, similar stiffness and microstructure, the apparent CTX modulation on the proliferation, cell-interaction, and genes expression of encapsulated hADSCs, as well as morphology and tubule structure formation of endothelial cells were observed, suggesting the biological roles of CTX and its modulation on microenvironment for cell development.

Human Adipose-Derived Mesenchymal Stem Cell-Based Microspheres Ameliorate Atherosclerosis Progression In Vitro.

Atherosclerosis (AS) is a chronic inflammatory disease associated with lipid deposition, which could be converted into acute clinical events by thrombosis or plaque rupture. Adipose-derived mesenchymal stem cell (ADSC)-encapsulated repair units could be an effective cure for the treatment of AS patients. In this study, we encapsulate human adipose-derived mesenchymal stem cells (hADSCs) in collagen microspheres to fabricate stem cell repair units. Besides, we show that encapsulation in collagen microspheres and cultured in vitro for 14 days maintain the viability and stemness of hADSCs. Moreover, we generate AS progression model and niche in vitro by combining hyperlipemia serum of AS patients with AS cell models. We further systematically demonstrate that hADSC-based microspheres could ameliorate AS progression by inhibiting oxidative stress injury, cell apoptosis, endothelial dysfunction, inflammation, and lipid accumulation. In addition, we perform transcriptomic analysis and functional studies to demonstrate how hADSCs (three dimensional cultured in microspheres) respond to AS niche compared with healthy microenvironment. These findings reveal a role for ADSC-based microspheres in the treatment of AS and provide new ideas for stem cell therapy in cardiovascular disease. The results may have implications for improving the efficiency of hADSC therapies by illuminating the mechanisms of hADSCs exposed in special pathological niche.

[Economic Benefit of Air Quality Improvement During Implementation of the Air Pollution Prevention and Control Action Plan in Beijing].

Air quality in Beijing has been improved significantly since the implementation of the Air Pollution Prevention and Control Action Plan ('Action Plan'). To evaluate the effect of the Action Plan, the cost-of-illness, human capital, and market value approaches were used to estimate air quality improvement benefits including human health, agriculture, building materials, and cleanliness. The policy benefit through monetization was also evaluated, which can affirm the positive effect of air pollution prevention and control by local government. The results illustrated that:① Since the policy implementation, air quality in Beijing has improved significantly and the improvement efficiency had been growing steadily. From 2013 to 2017, air quality had reached -1.982, -1.893, 15.707, 15.264, and 22.330 billion yuan respectively, accounting for -0.85‰, -0.81‰, 6.68‰, 6.16‰, and 8.77‰ of GDP in the fiscal year. The total profit during the five years was 49.426 billion yuan, accounting for 4.11‰ of the 5-year total GDP; ② The total improvement benefit was mainly linked to reductions in health and cleaning costs, primarily associated with a reduction in particulate matter concentrations; and ③ The majority of 16 administrative regions in Beijing reached an ideal level of benefit during the late stages of policy implementation, including Yanqing, Miyun, Pinggu, Changping, Tongzhou, Shijingshan, and Chaoyang Districts, which have retained a positive improvement index for five years. In contrast, Chaoyang and Haidian Districts have benefited relatively little as a result of the exposure of high-density populations to atmospheric pollution. These research results demonstrate the effectiveness and necessity of the Air Pollution Prevention and Control Action Plan in Beijing.

Controlling the Poly(ε-caprolactone) Degradation to Maintain the Stemness and Function of Adipose-Derived Mesenchymal Stem Cells in Vascular Regeneration Application.

Biodegradable poly(ε-caprolactone) (PCL) scaffolds with adipose-derived mesenchymal stem cells (ADSCs) have been used in vascular regeneration studies. An evaluation method of the effect of PCL degradation products (DP) on the viability, stemness, and differentiation capacities of ADSCs is established. ADSCs are cultured in medium containing different concentrations of PCL DP before evaluating the effect of PCL DP on the cell apoptosis and proliferation, cell surface antigens, adipogenic and osteogenic differentiation capacities, and capacities to differentiate into endothelial cells and smooth muscle cells. The results demonstrate that PCL DP exceed 0.05 mg mL-1 may change the stemness and differentiation capacities of ADSCs. Therefore, to control the proper concentration of PCL DP is essential for ADSCs in vascular regeneration application.

Optimization of 5-phenyl-3-pyridinecarbonitriles as PKCtheta inhibitors.

The key intermediate, 4-chloro-5-iodo-3-pyridinecarbonitrile, allowed for ready optimization of the PKCtheta inhibitory activity of a series of 3-pyridinecarbonitriles. Analog 13b with a 4-methylindol-5-ylamino group at C-4 and a 4-(2-(4-methylpiperazin-1-yl)ethoxy)phenyl group at C-5 had an IC(50) value of 7.4nM for the inhibition of PKCtheta.

Clinical efficacy of absolute ethanol combined with n-butyl cyanoacrylate sclerotherapy in the treatment of Puig's classified advanced venous malformation in children.

The aim of the present retrospective study was to investigate the clinical safety and efficacy of absolute ethanol combined with n-butyl cyanoacrylate sclerotherapy in the treatment of Puig's classified advanced venous malformation. Sclerotherapy was performed in 121 children (52 males and 69 females; age range, 5 months to 16 years) with venous malformations under general anesthesia between April 2009 and October 2014 at the Department of Interventional Radiology and Vascular Anomalies, Guangzhou Women and Children's Medical Center, Guangzhou, China. The patients with venous malformations were diagnosed and classified according to the diagnostic criteria of the International Society for the Study of Vascular Anomalies. According to the characteristics of intraoperative percutaneous angiography, 21 patient cases (9 males and 12 females; age range, 6 months to 14 years) were classified as advanced Puig's venous malformation. These 21 patients were treated with absolute ethanol combined with n-butyl cyanoacrylate. The patients were followed-up for 6-24 months (average, 15 months) after treatment. Following treatment with absolute ethanol combined with n-butyl cyanoacrylate, 15 cases were controlled and the total effective rate was 71% (15/21). However, 1 patient developed skin ulcerations, which was classed as a minor complication, 1 patient developed ectopic embolism caused by n-butyl cyanoacrylate reflux, and 1 patient developed transient pulmonary hypertension, the latter two complications were classified as major. Notably, the incidence rate of minor and major complications were 14.3%. To conclude, the present findings indicated that absolute ethanol combined with n-butyl cyanoacrylate sclerotherapy was a safe and effective method with a low complication rate in the treatment of Puig's classified advanced venous malformation in patients.

Neural Correlation between Evoked Tactile Sensation and Central Activities in the Somatosensory Cortex.

Evoked tactile sensation (ETS) is induced with stimulation of areas in the projected finger map (PFM) in the stump skin of forearm amputees. The aim of the present study is to further explore the neural correlation of the ETS with central activities in the somatosensory cortex (SI). Two forearm amputees were recruited to participate in this study. The neuroimaging technique of Magnetoencephalography (MEG) was employed to reveal the activities in the somatosensory cortex (SI), while the thumb and little finger areas in the PFM and the thumb and little finger of the contralateral hand were stimulated with a bi-phasic current pulse train. In one subject, local anesthesia was applied to the skin areas of the thumb and little finger of the PFM to examine the effects of blocking peripheral nerve transmission on the central activities in SI. Results obtained in the two subjects indicated that stimulation of the thumb and little finger areas in the PFM of amputated side produced the similar neural activities in the somatosensory cortex as those of stimulating the thumb and little finger of the contralateral hand, both of which were consistent with the homunculus organization of the SI cortex. The intensity of SI cortical activities was proportional to the intensity of the amplitude of peripheral stimulation. In particular, local anesthesia reduced the intensity of central activities in SI as revealed by the MEG response, as well as the sensitivity of ETS as reported by the subject. This neural correlation appears to suggest that the finger areas in the PFM in the stump skin are neuroanatomically connected to the finger areas of the somatosensory cortex. Thus, electrical stimulation of the PFM can induce natural sensation as that of normal fingers. This establishes the neural basis of natural sensory feedback from the prosthetic hand to the forearm amputee with finger-to-finger specificity.

Comparative Analysis of the Complete Chloroplast Genome of Four Endangered Herbals of Notopterygium.

Notopterygium H. de Boissieu (Apiaceae) is an endangered perennial herb endemic to China. A good knowledge of phylogenetic evolution and population genomics is conducive to the establishment of effective management and conservation strategies of the genus Notopterygium. In this study, the complete chloroplast (cp) genomes of four Notopterygium species (N. incisum C. C. Ting ex H. T. Chang, N. oviforme R. H. Shan, N. franchetii H. de Boissieu and N. forrestii H. Wolff) were assembled and characterized using next-generation sequencing. We investigated the gene organization, order, size and repeat sequences of the cp genome and constructed the phylogenetic relationships of Notopterygium species based on the chloroplast DNA and nuclear internal transcribed spacer (ITS) sequences. Comparative analysis of plastid genome showed that the cp DNA are the standard double-stranded molecule, ranging from 157,462 bp (N. oviforme) to 159,607 bp (N. forrestii) in length. The circular DNA each contained a large single-copy (LSC) region, a small single-copy (SSC) region, and a pair of inverted repeats (IRs). The cp DNA of four species contained 85 protein-coding genes, 37 transfer RNA (tRNA) genes and 8 ribosomal RNA (rRNA) genes, respectively. We determined the marked conservation of gene content and sequence evolutionary rate in the cp genome of four Notopterygium species. Three genes (psaI, psbI and rpoA) were possibly under positive selection among the four sampled species. Phylogenetic analysis showed that four Notopterygium species formed a monophyletic clade with high bootstrap support. However, the inconsistent interspecific relationships with the genus Notopterygium were identified between the cp DNA and ITS markers. The incomplete lineage sorting, convergence evolution or hybridization, gene infiltration and different sampling strategies among species may have caused the incongruence between the nuclear and cp DNA relationships. The present results suggested that Notopterygium species may have experienced a complex evolutionary history and speciation process.

Characterization of polymorphic microsatellite markers in Pinus armandii (Pinaceae), an endemic conifer species to China.

PREMISE OF THE STUDY: Pinus armandii (Pinaceae) is an important conifer tree species in central and southwestern China, and it plays a key role in the local forest ecosystems. To investigate its population genetics and design effective conservation strategies, we characterized 18 polymorphic microsatellite markers for this species. METHODS AND RESULTS: Eighteen novel polymorphic and 16 monomorphic microsatellite loci of P. armandii were isolated using Illumina MiSeq technology. The number of alleles per locus ranged from two to five. The expected heterozygosity ranged from 0.061 to 0.609 with an average of 0.384, and the observed heterozygosity ranged from 0.063 to 0.947 with an average of 0.436. Seventeen loci could be successfully transferred to five related Pinus species (P. koraiensis, P. griffithii, P. sibirica, P. pumila, and P. bungeana). CONCLUSIONS: These novel microsatellites could potentially be used to investigate the population genetics of P. armandii and related species.

Synthesis and biological activity of analogues of the antimicrotubule agent N,beta,beta-trimethyl-L-phenylalanyl-N(1)-[(1S,2E)-3-carboxy-1-isopropylbut-2-enyl]- N(1),3-dimethyl-L-valinamide (HTI-286).

Hemiasterlin, a tripeptide isolated from marine sponges, induces microtubule depolymerization and mitotic arrest in cells. HTI-286, an analogue from an initial study of the hemiasterlins, is presently in clinical trials. In addition to its potent antitumor effects, 2 has the advantage of circumventing the P-glycoprotein-mediated resistance that hampers the efficacy of other antimicrotubule agents such as paclitaxel and vincristine in animal models. This paper describes an in-depth study of the structure--activity relationships of analogues of 2, their effects on microtubule polymerization, and their in vitro and in vivo anticancer activity. Regions of the molecule necessary for potent activity are identified. Groups tolerant of modification, leading to novel analogues, are reported. Potent analogues identified through in vivo studies in tumor xenograft models include one superior analogue, HTI-042.

[Construction and growth properties of recombinant pseudorabies virus expressing modified enhanced green fluorescent protein].

The 1.0 kb DNA fragment containing the modified enhanced green fluorescent protein CDNA M1 (EGFP S147/P) and SV40 poly(A) signal sequence w as amplified and cloned in frame behind the first eight codons of the nonessential glycoprotein G (gG) of pseudorabies virus (PRV) to yield a transfer plasmid. The transfer plasmid was linearized and cotransfected with the genomic DNA of P RV Ea mutant gG(-)/LacZ(+). The resulting recombinant virus expressing M1, designated as gG(-)/LacZ(+), was isolated and confirmed by plaque purification, PCR, Southern blot and Western blot. PK-15 cells were infected with the purified recombinant virus at 0.1 pfu/cell and fluorescence emission was monitored at different times post-infection (p.i.) using fluorescence microscopy. Fluorescence emission could be detected as early as 6 h p.i. when there was no apparent cytopathic effects (CPE) yet. Fluorescence intensity increased drastically later. Maximum intensity was achieved at 24-36 h p.i. and fluorescence was stable. With the progress of the CPE, fluorescence vanished. The growth properties of gG(-)/M1(+) in tissue cultures were further examined and the titer of gG(-)/M1(+) was similar to that of PRV Ea wild strain and the parental strain gG(-)/LacZ(+). The above results indicated that the recombinant virus expressing the modified EGFP can be used as an in vivo marker to monitor replication and spread, as well as t o study the molecular pathogenesis of PRV.

Enhancement of activated sludge activity by 10-50 mT static magnetic field intensity at low temperature.

Effects of different static magnetic field (MF) intensities (10-50 mT) on the activity of activated sludge were compared at low temperature (5°C). The TTC dehydrogenase activity (TTC-DHA) achieved the maximum of 8.12 mg TF/gSS at the MF intensity of 40 mT. MF intensity enhanced from 20 mT to 40 mT could promote microorganisms to produce more unsaturated fatty acids (UFAs) to stimulate the TTC-DHA and adapt to low temperature. At 30 mT, the Gram-negative bacteria with better cold adaptability were greatly enriched, which ensured the cold resistance of low-temperature reactor. The PLFA diversity strengthened by MF was higher than that of control when MF intensities changed from 20 mT to 50 mT and reached the maximum of 1.31 at 20 mT. Optimal MF intensity is supposed to be a potential pathway to strengthen the activity and cold resistance of activated sludge microorganisms in biological wastewater treatment under low temperature.

The strengthening effect of a static magnetic field on activated sludge activity at low temperature.

The strengthening effects of static (homopolar and heteropolar) magnetic fields (MF) on microorganisms were compared in activated sludge degrading organic matter at low temperature. The TTC dehydrogenase activity improved substantially through external heteropolar MF intensification, and led to the highest COD removal rate of 94.9% at 5°C. Phospholipid fatty acid analysis showed that C16:1ω7c, C18:1ω7c and C18:1ω9c were the essential unsaturated fatty acids in cell membrane at low temperature (4-15°C), accounting for the majority of the whole unsaturated fatty acids. The MF effect increased the Gram-negative bacteria content to improve the cold adaptability. Shannon-Wiener diversity analysis demonstrated the samples with heteropolar MF had a higher PLFA diversity index (1.17-1.25) than that with homopolar MF (0.89-1.13). AFM observation showed MF smoothed part of the microbial cell surface, with some remaining distinct protuberances. Heteropolar MF enhancement performance is much more effective than that of the homopolar MF with identical plate distance.

Comparison between absolute ethanol and bleomycin for the treatment of venous malformation in children.

The aim of this study was to investigate the therapeutic efficacies and treatment effects of absolute ethanol and bleomycin for the treatment of venous malformation (VM) in children. A total of 138 children with VM were randomly divided into two groups; 75 patients were treated with absolute ethanol, while a further 63 were treated with bleomycin under general anesthesia between February 2009 and February 2012. The treatment outcome and complications were observed in the two groups and the treatment efficacy was classified as one of four categories: cured, markedly effective, effective and ineffective. The curative effect was analyzed 6-24 months after treatment, with a mean of 15 months. Absolute ethanol was effective (cured, markedly effective or effective) in 71 cases and bleomycin was effective in 41 cases, and the difference between the effective rates was considered to be statistically significant (χ2=19.6, P<0.05). In the absolute ethanol group there were 14 cases with skin necrosis, 17 patients had serious localized swelling which required additional treatment, three patients developed muscle fibrosis and one patient suffered a brain embolism. In the bleomycin group there were five cases with skin necrosis and the difference in the incidence of adverse reactions was considered to be statistically significant (χ2=18.8, P<0.05). The curative effect of sclerotherapy for VM is clear, and absolute ethanol is the most effective sclerosing agent, but has a greater incidence of adverse side-effects than bleomycin. The major side-effect is skin necrosis. The choice of sclerotherapy depends on the classification of VM in children.

The cold adaptability of microorganisms with different carbon source in activated sludge treating synthetical wastewater.

The cold adaptability of microorganisms with different carbon source under 5°C was studied in activated sludge for treating synthetical wastewater. Phospholipid fatty acid (PLFA) analysis indicated contents of unsaturated fatty acids in cell membrane at 5°C were 13.66% and 24.96% higher for glucose and sodium acetate source than that at 25°C. PLFA biomarkers showed more Gram-negative bacteria enriched than Gram-positive bacteria in low-temperature activated sludge. The Shannon-Wiener diversity analysis demonstrated glucose fed reactor in low temperature had lower PLFA diversity index (1.21-1.30) than that at 25°C and sodium acetate source was reverse (1.08-0.69). The 16S rRNA analysis manifested certain microbes were considerably suitable for existence under cold environment, most of which belong to Gram-negative bacteria.