Enabling large-scale genome editing at repetitive elements by reducing DNA nicking.

To extend the frontier of genome editing and enable editing of repetitive elements of mammalian genomes, we made use of a set of dead-Cas9 base editor (dBE) variants that allow editing at tens of thousands of loci per cell by overcoming the cell death associated with DNA double-strand breaks and single-strand breaks. We used a set of gRNAs targeting repetitive elements-ranging in target copy number from about 32 to 161 000 per cell. dBEs enabled survival after large-scale base editing, allowing targeted mutations at up to ∼13 200 and ∼12 200 loci in 293T and human induced pluripotent stem cells (hiPSCs), respectively, three orders of magnitude greater than previously recorded. These dBEs can overcome current on-target mutation and toxicity barriers that prevent cell survival after large-scale genome engineering.

Knockdown of ß-Catenin through shRNA cause a reversal of EMT and metastatic phenotypes induced by HIF-1α.

OBJECTIVE: Wnt/ß-catenin signaling pathway regulates pattern formation during embryogenesis as well as tumor progression. Numbers of studies suggest that this signaling pathway may play an important role in Epithelial-Mesenchymal transition (EMT), however, there was no evidence that Wnt/ß-catenin signaling pathway directly controlled the EMT occurrence. Our previous research has successfully proved that overexpression of hypoxia inducible factor-1α (HIF-1α) could induce EMT in LNCaP cells, but not in PC-3. Consistently, the expression of ß-catenin protein increased in LNCaP/HIF-1α cells, but not in PC-3/HIF-1α. This study mainly aimed at exploring the essentiality and importance of Wnt/ß-catenin signaling pathway in HIF-1α-induced EMT. METHODS: Human prostate cancer cells (LNCaP) were stably transfected by recombinant plasmid pcDNA3.1(-)/HIF-1α. The positive clones were selected by G418 and confirmed through western blot analysis, reverse transcription-polymerase chain reaction (RT-PCR), and indirect immunofluoesence. Then LNCaP/HIF-1α was transiently transfected with ß-catenin shRNA (shRNA1 and shRNA2) and negative shRNA (shRNA-scr). The epithelial markers, mesenchymal markers, and critical proteins in Wnt/ß-catenin signaling pathway were separately detected by western blot analysis. Finally, the invasive potency of cells in different transfection group was examined by Matrgel transwell assay. RESULT: We successfully established prostate cancer cell line LNCaP/HIF-1α and LNCaP/HIF-1α/ß-catenin(-). LNCaP/HIF-1α displayed high expression of mesenchymal markers and low expression of epithelial markers. However, compared with LNCaP/HIF-1α, the epithelial marker E-cadherin was increased in LNCaP/HIF-1α/ß-catenin(-), whereas the expression of mesenchymal marker N-cadherin, vimentin, MMP-2 were significantly decreased. Inhibition of Wnt signal activity through ß-catenin shRNA cause a reversal of EMT induced by HIF-1α in human prostate cancer. CONCLUSION: Overexpression of HIF-1α stimulates the invasion potency of human prostate carcinoma cells through EMT pathway and Wnt/ß-catenin signaling pathway played a vital role in this process. Wnt/ß-catenin signaling pathway might be a necessary endogenous signal that directly controlled the EMT occurrence induced by HIF-1α.

Electrokinetic behavior of bullet-shaped nanopores modified by functional groups: Influence of finite thickness of modified layer.

We examined theoretically the electrokinetic behavior of a bullet-shaped nanopore modified by a functional layer, focusing on the influence of its thickness. The nanopore contains both fixed surface charge coming from the original bare surface, and space fixed charge from the modified layer. The results of numerical simulation reveal that the presence of this layer is crucial to the electrokinetic behavior of the nanopore. In particular, its softness is capable of influencing ionic profiles through electroosmotic flow (EOF). Unlike a conical nanopore where its surface normal vector is constant, that of the present bullet-shaped nanopore varies along the pore axis, thereby affecting the degree of EOF, which in turn, can make the ionic profile inside the modified layer more uniform. This is crucial to the applications of the nanopore, for example, in mimicking biological membranes and sensing metal ions.

Comparison of video-assisted thyroidectomy and traditional thyroidectomy for the treatment of papillary thyroid carcinoma.

BACKGROUND: There is concern regarding the oncological effectiveness of minimally invasive video-assisted thyroidectomy (VAT) for thyroid carcinoma. This study compared the surgical results of VAT and traditional thyroidectomy in patients with small papillary thyroid carcinomas (PTC). METHODS: Of 44 patients with PTC offered the choice between VAT and traditional thyroidectomy, 21 underwent VAT and 23 underwent traditional thyroidectomy. Residual thyroid tissue and function were assessed postoperatively by measuring thyroid-stimulating hormone (TSH), T4, thyroglobulin, and anti-thyroglobulin antibody levels and with sonographic examination. RESULTS: Operative time, maximum tumor size, number of positive lymph nodes, and TSH and T4 levels 4 weeks postoperatively were similar in the two groups (all p > 0.05). Patients in the traditional thyroidectomy group had significantly higher postoperative serum thyroglobulin levels 4 weeks after surgery than did patients in the VAT group (5.4 versus 0.5 ng/ml, respectively; p = 0.007). Postoperative ultrasonography showed no residual thyroid tissue or evidence of recurrence in any of the patients. The median follow-up period was 60 months (range 31-77 month) for the VAT group and 53 months (range 31-80 months) for the traditional thyroidectomy group. Thyroglobulin levels of all patients in both groups decreased to <0.2 ng/ml at last follow-up. CONCLUSIONS: VAT is safe and effective for the treatment of small papillary thyroid carcinomas, and has similar oncological effectiveness to traditional thyroidectomy.

[Effect of retroviral vector mediating RNA interference on the release of tumor necrosis factor-alpha in lipopolysaccharide-stimulating macrophage].

OBJECTIVE: To explore the effect of small interfering RNA (siRNA) targeting NF-kappaB signal pathway on the expression level of tumor necrosis factor alpha (TNF-alpha) released by lipopolysaccharides (LPS)-stimulating-macrophages. METHODS: Human monocytic THP-1 cell was induced by phorbol myristate acetate (PMA) and transformed into macrophage. Two groups of macrophage were infected by siRNA retroviral expression vector specific to NF-kappaB functional subunit P65 (siRNA group) and Scramble control vector (Scramble control group) constructed by molecular cloning technology. Lipopolysaccharide (50 microg/ml) was used to treat the macrophages continuously. RT-PCR was performed to detect the expression level of NF-kappaB P65 mRNA and TNF-alpha mRNA at different time-points of LPS stimulation. Western blotting was used to analyze the protein level of NF-kappaB P65. Enzyme-linked immunosorbent assay was applied to analyze the expression level of TNF-alpha released by LPS-stimulated macrophages. RESULTS: At Hours 12 and 24 after LPS stimulation, the expression level of NF-kappaB P65 mRNA in siRNA group (0.97 +/- 0.02, 0.89 +/- 0.01) was significantly less than that in Scramble control group (1.01 +/- 0.03, 0.97 +/- 0.01, both P < 0.05). At Hours 24 and 72 after LPS stimulation, the expression level of NF-kappaB P65 protein in siRNA group (0.95 +/- 0.04, 0.94 +/- 0.01) was obviously less than that in Scramble control group (1.07 +/- 0.06, 1.03 +/- 0.05, both P < 0.05). At Hours 4, 8, 12 and 24 after LPS stimulation, TNF-alpha mRNA released by siRNA group macrophages was far less than that by Scramble control group macrophages (0.92 +/- 0.02 vs 0.98 +/- 0.01, 0.86 +/- 0.02 vs 1.00 +/- 0.01, 0.79 +/- 0.03 vs 1.01 +/- 0.01, 0.78 +/- 0.03 vs 1.02 +/- 0.01, all P < 0.05). At Hours 2, 4, 8, 24, 36, 48, 54 and 72 after LPS stimulation, the TNF-alpha content in culture medium supernatant in siRNA group macrophage was less than that in scramble control group (P < 0.05). CONCLUSION: NF-kappaB P65 siRNA inhibits the functional activity of NF-kappaB signal pathway in PMA-induced macrophage. Then it blocks the activation of macrophage and the excessive release of TNF-alpha due to endotoxin stimulation. The RNA interference technology may be applied to prevent and treat excessive inflammatory reaction in acute lung injury.

[Expression of COX-2 in different human prostate cancer cell lines and its significance].

OBJECTIVE: To investigate the expression of cyclooxygenase-2 (COX-2) in different prostate cancer (PCa) cell lines and its role in the acquisition of invasive and metastatic potentials of PCa. METHODS: We detected the expressions of COX-2 in prostate cancer cell lines LNCaP, C4-2, IF11, IA8 and PC-3 with different metastatic potentials by Western blotting and RT-PCR, and analyzed their roles in the invasion and metastasis of different PCa cell lines. RESULTS: Western blotting and RT-PCR showed that the expression of the COX-2 protein was high in PC-3, but absent in IF11, IA8, LNCaP and C4-2 (P < 0.05), and it was consistent with the expression of COX-2 mRNA. CONCLUSION: COX-2 expresses differently in PCa cell lines with different metastatic potentials. The overexpression of COX-2 may be associated with the high invasion and metastasis of PC-3, but not with the metastasis of other cell lines.

Estimating the thermodynamic equilibrium constants of metal oxide particles through a general electrophoresis model.

We propose an efficient and convenient procedure for estimating the thermodynamic equilibrium constants of the surface dissociation/association reactions of metal oxide particles through electrophoresis measurements and a general electrophoresis model, which takes account of essentially all the relevant factors. These constants are usually estimated experimentally through a tedious potentiometric acid-base titration procedure. In addition, since several assumptions need be made in applying this procedure, the results obtained can be unreliable, especially when the difference between the equilibrium constant of the dissociation reaction and that of the association reaction is small. Another merit of the procedure proposed is that the site density of the surface dissociation/associations functional groups need not be known in advance so that it becomes much more convenient and efficient than previous procedures. The applicability of the present procedure is examined by applying it to the cases of SiO2 and TiO2 nanoparticles dispersed in an aqueous NaCl solution.

Usability of Wearable Devices With a Novel Cardiac Force Index for Estimating the Dynamic Cardiac Function: Observational Study.

BACKGROUND: Long-distance running can be a form of stress to the heart. Technological improvements combined with the public's gradual turn toward mobile health (mHealth), self-health, and exercise effectiveness have resulted in the widespread use of wearable exercise products. The monitoring of dynamic cardiac function changes during running and running performance should be further studied. OBJECTIVE: We investigated the relationship between dynamic cardiac function changes and finish time for 3000-meter runs. Using a wearable device based on a novel cardiac force index (CFI), we explored potential correlations among 3000-meter runners with stronger and weaker cardiac functions during running. METHODS: This study used the American product BioHarness 3.0 (Zephyr Technology Corporation), which can measure basic physiological parameters including heart rate, respiratory rate, temperature, maximum oxygen consumption, and activity. We investigated the correlations among new physiological parameters, including CFI = weight * activity / heart rate, cardiac force ratio (CFR) = CFI of running / CFI of walking, and finish times for 3000-meter runs. RESULTS: The results showed that waist circumference, smoking, and CFI were the significant factors for qualifying in the 3000-meter run. The prediction model was as follows: ln (3000 meters running performance pass probability / fail results probability) = -2.702 - 0.096 × [waist circumference] - 1.827 × [smoke] + 0.020 × [ACi7]. If smoking and the ACi7 were controlled, contestants with a larger waist circumference tended to fail the qualification based on the formula above. If waist circumference and ACi7 were controlled, smokers tended to fail more often than nonsmokers. Finally, we investigated a new calculation method for monitoring cardiac status during exercise that uses the CFI of walking for the runner as a reference to obtain the ratio between the cardiac force of exercise and that of walking (CFR) to provide a standard for determining if the heart is capable of exercise. A relationship is documented between the CFR and the performance of 3000-meter runs in a healthy 22-year-old person. During the running period, data are obtained while participant slowly runs 3000 meters, and the relationship between the CFR and time is plotted. The runner's CFR varies with changes in activity. Since the runner's acceleration increases, the CFR quickly increases to an explosive peak, indicating the runner's explosive power. At this period, the CFI revealed a 3-fold increase (CFR=3) in a strong heart. After a time lapse, the CFR is approximately 2.5 during an endurance period until finishing the 3000-meter run. Similar correlation is found in a runner with a weak heart, with the CFR at the beginning period being 4 and approximately 2.5 thereafter. CONCLUSIONS: In conclusion, the study results suggested that measuring the real-time CFR changes could be used in a prediction model for 3000-meter running performance.

MicroRNA-326 aggravates acute lung injury in septic shock by mediating the NF-κB signaling pathway.

Our previous studies have demonstrated that the activation of the nuclear factor-kappa B (NF-κB) signaling pathway contributes to the development of lipopolysaccharide (LPS)-induced acute lung injury (ALI) as well as an inflammatory reaction, and its inhibition may provide future therapeutic values. Thereby, this study aims to explore the effects of miR-326 on inflammatory response and ALI in mice with septic shock via the NF-κB signaling pathway. The study included normal mice and LPS-induced mouse models of septic shock with ALI. Modeled mice were transfected with the blank plasmid, miR-326 mimic, miR-326 inhibitor, si-BCL2A1 and miR-326 inhibitor + si-BCL2A1. Mean arterial pressure (MAP), airway pressure (AP), heart rate (HR) and lung wet dry (W/D) ratio were determined. Serum levels of interleukin (IL)-6, IL-10, IL-1ß, and tumor necrosis factor-α (TNF-α) were detected using ELISA. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis were performed to detect the miR-326 expression and expression levels of BCL2A1, related genes of inflammatory response and the NF-κB signaling pathway in lung tissues. Cell viability and apoptosis were measured using the CCK-8 assay and flow cytometry, respectively. Compared to the ALI models and those transfected with blank plasmid, the up-regulated miR-326 expression and silenced BCL2A1 lead to decreased levels of MAP, increased AP, HR and lung W/D, increased serum levels of IL-6, IL-10, IL-1ß and TNF-α, increased expressions of IL-6, IL-1ß, TNF-α, NF-κB p65 (p-NF-κB p65), and iNOS with decreased expressions of BCL2A1s as well as inhibition of cell viability and enhanced cell apoptosis; the down-regulated miR-326 expression reversed the aforementioned situation. MiR-326 targeting the BCL2A1 gene activated the NF-κB signaling pathway, resulting in aggravated inflammatory response and lung injury of septic shock with ALI in mice.

Spin-orbit torque magnetometry by wide-field magneto-optical Kerr effect.

Magneto-optical Kerr effect (MOKE) is an efficient approach to probe surface magnetization in thin film samples. Here we present a wide-field MOKE technique that adopts a Köhler illumination scheme to characterize the current-induced damping-like spin-orbit torque (DL-SOT) in micron-sized and unpatterned magnetic heterostructures with perpendicular magnetic anisotropy. Through a current-induced hysteresis loop shift analysis, we quantify the DL-SOT efficiency of a Ta-based heterostructure with bar-shaped geometry, Hall-cross geometry, and unpatterned geometry to be |ξ DL | ≈ 0.08. The proposed wide-field MOKE approach therefore provides an instant and direct characterization of DL-SOT, without the need of any further interpretation on electrical signals.

Hippocampal awake replay in fear memory retrieval.

Hippocampal place cells are key to episodic memories. How these cells participate in memory retrieval remains unclear. After rats acquired a fear memory by receiving mild footshocks in a shock zone on a track, we analyzed place cells when the animals were placed on the track again and displayed an apparent memory retrieval behavior: avoidance of the shock zone. We found that place cells representing the shock zone were reactivated, despite the fact that the animals did not enter the shock zone. This reactivation occurred in ripple-associated awake replay of place cell sequences encoding the paths from the animal's current positions to the shock zone but not in place cell sequences within individual cycles of theta oscillation. The result reveals a specific place-cell pattern underlying inhibitory avoidance behavior and provides strong evidence for the involvement of awake replay in fear memory retrieval.

Eriodictyol, a plant flavonoid, attenuates LPS-induced acute lung injury through its antioxidative and anti-inflammatory activity.

Acute lung injury (ALI) is characterized by excessive inflammatory responses and oxidative injury in the lung tissue. It has been suggested that anti-inflammatory or antioxidative agents could have therapeutic effects in ALI, and eriodictyol has been reported to exhibit antioxidative and anti-inflammatory activity in vitro. The aim of the present study was to investigate the effect of eriodictyol on lipopolysaccharide (LPS)-induced ALI in a mouse model. The mice were divided into four groups: Phosphate-buffered saline-treated healthy control, LPS-induced ALI, vehicle-treated ALI (LPS + vehicle) and eriodictyol-treated ALI (LPS + eriodictyol). Eriodictyol (30 mg/kg) was administered orally once, 2 days before the induction of ALI. The data showed that eriodictyol pretreatment attenuated LPS-induced ALI through its antioxidative and anti-inflammatory activity. Furthermore, the eriodictyol pretreatment activated the nuclear factor erythroid-2-related factor 2 (Nrf2) pathway in the ALI mouse model, which attenuated the oxidative injury and inhibited the inflammatory cytokine expression in macrophages. In combination, the results of the present study demonstrated that eriodictyol could alleviate the LPS-induced lung injury in mice by regulating the Nrf2 pathway and inhibiting the expression of inflammatory cytokines in macrophages, suggesting that eriodictyol could be used as a potential drug for the treatment of LPS-induced lung injury.

Effect of siRNA against NF-κB on sepsis­induced acute lung injury in a mouse model.

The aim of the present study was to explore the protective effect of small interfering RNA (siRNA) against nuclear factor κB (NF-κB) p65 on sepsis-induced acute lung injury (ALI) in mice. In total, 70 male Kunming mice were randomly divided into a healthy control group, a sepsis group, a specific interfering group and a scrambled control group (Sc), and the latter three groups were divided into post-operational 6 and 12 h subgroups, each of which consisted of 10 mice. The mice were administered with NF-κB siRNA, scrambled siRNA and normal saline via tail vein injection. Following 1 h, a mouse model of septic ALI was produced by cecal ligation and puncture (CLP) in the two siRNA groups and the sepsis control group. At 6 and 12 h post­operation, the experimental mice were sacrificed and the lung tissue samples were collected. Histopathological changes, wet/dry ratio of lung weight, NF-κB protein and NF-κB p65 mRNA levels, matrix metalloproteinase-9 (MMP-9) mRNA and protein activity were detected. Compared with the sepsis group and the Sc at the corresponding time, the expression levels of NF-κB p65 mRNA, the lung injury of experimental mice, the wet/dry ratio and the levels of MMP-9 mRNA and protein activity decreased, and significant differences were observed at 6 h post-operation (P<0.05). RNA interference against NF-κB p65 was able to decrease the expression of NF-κB and further inhibit the early phasic excessive inflammatory reaction in sepsis, which may alleviate ALI.

Occurrence of aryl hydrocarbon receptor agonists and genotoxic compounds in the river systems in Southern Taiwan.

Water and sediment samples from river systems located in Southern Taiwan were investigated for the presence of aryl hydrocarbon receptor (AhR) agonists and genotoxicants by a combination of recombinant cell assays and gas chromatography-mass spectrometry analysis. AhR agonist activity and genotoxic response were frequently detected in samples collected during different seasons. In particular, dry-season water and sediment samples from Erren River showed strong AhR agonist activity (201-1423 ng L(-1) and 1374-5631 ng g(-1) ß-naphthoflavone equivalents) and high genotoxic potential. Although no significant correlation was found between AhR agonist activity and genotoxicity, potential genotoxicants in sample extracts were suggested to be causative agents for yeast growth inhibition in the AhR-responsive reporter gene assay. After high performance liquid chromatography fractionation, AhR agonist candidates were detected in several fractions of Erren River water and sediment extracts, while possible genotoxicants were only found in water extracts. In addition, polycyclic aromatic hydrocarbons, the typical contaminants showing high AhR binding affinity, were only minor contributors to the AhR agonist activity detected in Erren River sediment extracts. Our findings displayed the usefulness of bioassays in evaluating the extent of environmental contamination, which may be helpful in reducing the chances of false-negative results obtained from chemical analysis of conventional contaminants. Further research will be undertaken to identify major candidates for xenobiotic AhR agonists and genotoxicants to better protect the aquatic environments in Taiwan.

Modulation of inositol polyphosphate levels regulates neuronal differentiation.

The binding of neurotrophins to tropomyosin receptor kinase receptors initiates several signaling pathways, including the activation of phospholipase C-γ, which promotes the release of diacylglycerol and inositol 1,4,5-trisphosphate (IP(3)). In addition to recycling back to inositol, IP(3) serves as a precursor for the synthesis of higher phosphorylated inositols, such as inositol 1,3,4,5,6-pentakisphosphate (IP(5)) and inositol hexakisphosphate (IP(6)). Previous studies on the effect of neurotrophins on inositol signaling were limited to the analysis of IP(3) and its dephosphorylation products. Here we demonstrate that nerve growth factor (NGF) regulates the levels of IP(5) and IP(6) during PC12 differentiation. Furthermore, both NGF and brain-derived neurotrophic factor alter IP(5) and IP(6) intracellular ratio in differentiated PC12 cells and primary neurons. Neurotrophins specifically regulate the expression of IP(5)-2 kinase (IP(5)-2K), which phosphorylates IP(5) into IP(6). IP(5)-2K is rapidly induced after NGF treatment, but its transcriptional levels sharply decrease in fully differentiated PC12 cells. Reduction of IP(5)-2K protein levels by small interfering RNA has an effect on the early stages of PC12 cell differentiation, whereas fully differentiated cells are not affected. Conversely, perturbation of IP(5)-2K levels by overexpression suggests that both differentiated PC12 cells and sympathetic neurons require low levels of the enzyme for survival. Therefore maintaining appropriate intracellular levels of inositol polyphosphates is necessary for neuronal survival and differentiation.

Patterns of target tissue reinnervation and trophic factor expression after nerve grafting.

BACKGROUND: Reinnervation of target tissues determines functional outcomes after nerve grafting, which is important in traumatic injury caused by accidents or consequences resulting from surgical removal of tumors. Previous studies documented the influences of nerve repair mainly based on nerve morphometry but rarely compared the final outcomes according to target reinnervation patterns by nerve fibers of different categories. METHODS: In a mouse model of nerve grafting, the authors analyzed the innervation indexes of different target tissues after transection-reimplantation on the sciatic nerve, which were defined as a parameter on the operated side normalized to that on the control side. RESULTS: Muscle reinnervation appeared to be the best compared with skin reinnervation (p < 0.0001) and sweat gland reinnervation (p < 0.0001) at postoperative month 3. The sudomotor reinnervation was relatively higher than the cutaneous reinnervation (p = 0.014). The abundance of trophin transcripts for brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), and neurotrophin 3 (NT3) was higher in plantar muscles on the operated side than those on the control side. In contrast, transcripts of BDNF, GDNF, nerve growth factor, and NT3 were all similar in the footpad skin between the operated and control sides. CONCLUSIONS: The results suggested that, compared with the skin, muscles achieved the best reinnervation after nerve grafting, which was related to higher expression of BDNF, GDNF, and NT3 in muscles than in the skin.

All pedicle screw fixation technique in correcting severe kyphoscoliosis in an osteogenesis imperfecta patient: a case report.

Spinal deformity in patients with osteogenesis imperfecta demonstrates a high prevalence. The surgical treatment of this problem had numerous difficulties, which included breakage of bone, dislodgement of implants, and late loss of correction. We reported the correction of severe kyphoscoliosis in a patient with osteogenesis imperfecta using 3-rod all pedicle screw fixation technique. In this case, the 2 main goals of spinal operation, stabilization and correction of spinal curvatures, were achieved. The Cobb angles of scoliosis and thoracic kyphosis were corrected from 110 degrees to 68 degrees and from 107 degrees to 39 degrees, respectively. One and a half years after the operation, spinal radiographs showed no loss of correction, either on coronal or sagittal planes. The predicted forced vital capacity, predicted forced expiratory volume in 1 second and vital capacity of the lung of the patient had improved 2-fold. The usage of pedicle screw, with its conical core and cylindrical thread design, and 3-rod technique in fixation, together with cyclic intravenous bisphosphonate administration and halo-gravity traction preoperatively, contributed to the success in this case.