Magnetic Beads inside Droplets for Agitation and Splitting Manipulation by Utilizing a Magnetically Actuated Platform.

We successfully developed a platform for the magnetic manipulation of droplets containing magnetic beads and examined the washing behaviors of the droplets, including droplet transportation, magnetic bead agitation inside droplets, and separation from parent droplets. Magnetic field gradients were produced with two layers of 6 × 1 planar coils fabricated by using printed circuit board technology. We performed theoretical analyses to understand the characteristics of the coils and successfully predicted the magnetic field and thermal temperature of a single coil. We then investigated experimentally the agitation and splitting kinetics of the magnetic beads inside droplets and experimentally observed the washing performance in different neck-shaped gaps. The performance of the washing process was evaluated by measuring both the particle loss ratio and the optical density. The findings of this work will be used to design a magnetic-actuated droplet platform, which will separate magnetic beads from their parent droplets and enhance washing performance. We hope that this study will provide digital microfluidics for application in point-of-care testing. The developed microchip will be of great benefit for genetic analysis and infectious disease detection in the future.

R-spondin 3/LGR4 (Leucine-Rich Repeat-Containing G Protein-Coupled Receptor 4) Axis Is a Novel Inflammatory and Neurite Outgrowth Signaling System in the Ischemic Brain in Mice.

BACKGROUND: Although stimulation of Wnt/ß-catenin signaling is an important strategy to treat ischemic stroke, its signaling pathway has not been fully clarified yet. Recently, RSPO3 (R-spondin 3)/LGR4 (leucine-rich repeat-containing G protein-coupled receptor 4) signaling has resolved TLR4 (toll-like receptor 4)-induced inflammation in lung injury; however, whether this signal is critical in the ischemic brain remains unknown. Therefore, we investigated the role of RSPO3/LGR4 signaling in the ischemic brain. METHODS: BALB/c mice were exposed to permanent distal middle cerebral artery and common carotid artery occlusion. Temporal RSPO3 and LGR4 expressions were examined, and the mice were randomly assigned to receive vehicle or recombinant RSPO3. The underlying mechanisms were investigated using microglial cell lines and primary mixed glia-endothelia-neuron and primary neuronal cultures. RESULTS: In the ischemic brain, RSPO3 and LGR4 were expressed in endothelial cells and microglia/macrophages and neurons, respectively. Stimulation of RSPO3/LGR4 signaling by recombinant RSPO3 recovered neurological deficits with decreased Il1ß and iNOS mRNA on day 3 and increased Gap43 on day 9. In cultured cells, LGR4 was expressed in neuron and microglia, whereas RSPO3 promoted nuclear translocation of ß-catenin. Neuroprotective effects with reduced expression of inflammatory cytokines were observed in lipopolysaccharide-stimulated glia-endothelium-neuron cultures but not in glutamate-, CoCl2-, H2O2-, or oxygen glucose deprivation-stimulated neuronal cultures, indicating that RSPO3/LGR4 can protect neurons by regulating inflammatory cytokines. LGR4-Fc chimera, which was used to block endogenous RSPO3/LGR4 signaling, increased LPS-induced production of inflammatory cytokines, suggesting that endogenous RSPO3 suppresses inflammation. RSPO3 decreased TLR4-related inflammatory cytokine expression by decreasing TLR4 expression without affecting the M1/M2 phenotype. RSPO3 also inhibited TLR2- and TLR9-induced inflammation but not TLR7-induced inflammation, and promoted neurite outgrowth. CONCLUSIONS: RSPO3/LGR4 signaling plays a critical role in regulating TLR-induced inflammation and neurite outgrowth in the ischemic brain. Enhancing this signal will be a promising approach for treating ischemic stroke.

Force and Velocity Analysis of Particles Manipulated by Toroidal Vortex on Optoelectrokinetic Microfluidic Platform.

The rapid electrokinetic patterning (REP) technique has been demonstrated to enable dynamic particle manipulation in biomedical applications. Previous studies on REP have generally considered particles with a size less than 5 µm. In this study, a REP platform was used to manipulate polystyrene particles with a size of 3~11 µm in a microfluidic channel sandwiched between two ITO conductive glass plates. The effects of the synergy force produced by the REP electrothermal vortex on the particle motion were investigated numerically for fixed values of the laser power, AC driving voltage, and AC driving frequency, respectively. The simulation results showed that the particles were subject to a competition effect between the drag force produced by the toroidal vortex, which prompted the particles to recirculate in the bulk flow adjacent to the laser illumination spot on the lower electrode, and the trapping force produced by the particle and electrode interactions, which prompted the particles to aggregate in clusters on the surface of the illuminated spot. The experimental results showed that as the laser power increased, the toroidal flow range over which the particles circulated in the bulk flow increased, while the cluster range over which the particles were trapped on the electrode surface reduced. The results additionally showed that the particle velocity increased with an increasing laser power, particularly for particles with a smaller size. The excitation frequency at which the particles were trapped on the illuminated hot-spot reduced as the particle size increased. The force and velocity of polystyrene particles by the REP toroidal vortex has implications for further investigating the motion behavior at the biological cell level.

Prevention of bleomycin-induced pulmonary fibrosis by a RANKL peptide in mice.

Despite the recent therapeutic developments for the treatment of pulmonary fibrosis, its prognosis is still not well controlled, and a novel therapeutic agent is needed. Recently, the critical role of Toll-like receptors (TLRs) in the pathophysiology of pulmonary fibrosis has been reported; however, the effects of multiple TLR signaling inhibition are still unknown. Here, we examined how the inhibition of multiple TLRs affects pulmonary fibrosis using a novel synthetic receptor activator of nuclear factor κB ligand (RANKL) partial peptide, MHP1-AcN, which could suppress TLR2, 3, 4, 7, and 9 signaling through CD14 and RANK. When MHP1-AcN was administered in the bleomycin-induced lung fibrosis model, reduced collagen deposition was observed, with suppressed fibrosis-related gene expression including Col1a1, Col1a2, Acta2, Tgfb1 and Tgfbr2. MHP1-AcN also decreased proinflammatory M1 and profibrotic M2 macrophage marker expression. Furthermore, MHP1-AcN treatment inhibited transforming growth factor (TGF-ß)-induced Smad2/3 phosphorylation and myofibroblast differentiation in human fetal lung fibroblast (MRC-5) cells. This effect was associated with decreased TGF-ß receptor levels and the upregulated Bmp7 and Smad7 expression. These findings suggest that MHP1-AcN protects mice against bleomycin-induced pulmonary fibrosis. MHP1-AcN might provide a novel therapeutic strategy for the pulmonary fibrosis.

Preclinical study of a DNA vaccine targeting SARS-CoV-2.

To fight against the worldwide COVID-19 pandemic, the development of an effective and safe vaccine against SARS-CoV-2 is required. As potential pandemic vaccines, DNA/RNA vaccines, viral vector vaccines and protein-based vaccines have been rapidly developed to prevent pandemic spread worldwide. In this study, we designed plasmid DNA vaccine targeting the SARS-CoV-2 Spike glycoprotein (S protein) as pandemic vaccine, and the humoral, cellular, and functional immune responses were characterized to support proceeding to initial human clinical trials. After intramuscular injection of DNA vaccine encoding S protein with alum adjuvant (three times at 2-week intervals), the humoral immunoreaction, as assessed by anti-S protein or anti-receptor-binding domain (RBD) antibody titers, and the cellular immunoreaction, as assessed by antigen-induced IFNγ expression, were up-regulated. In IgG subclass analysis, IgG2b was induced as the main subclass. Based on these analyses, DNA vaccine with alum adjuvant preferentially induced Th1-type T cell polarization. We confirmed the neutralizing action of DNA vaccine-induced antibodies by a binding assay of RBD recombinant protein with angiotensin-converting enzyme 2 (ACE2), a receptor of SARS-CoV-2, and neutralization assays using pseudo-virus, and live SARS-CoV-2. Further B cell epitope mapping analysis using a peptide array showed that most vaccine-induced antibodies recognized the S2 and RBD subunits. Finally, DNA vaccine protected hamsters from SARS-CoV-2 infection. In conclusion, DNA vaccine targeting the spike glycoprotein of SARS-CoV-2 might be an effective and safe approach to combat the COVID-19 pandemic.

Prevention of vascular dementia via immunotherapeutic blockade of renin-angiotensin system in a rat model.

INTRODUCTION: As several clinical trials have revealed that angiotensin-converting enzyme inhibitors and angiotensin II (Ang II) receptor blockers may be efficient in treating vascular dementia (VaD), the long-acting blockade of the renin-angiotensin system (RAS) would be useful considering the poor adherence of antihypertensive drugs. Accordingly, we continuously blocked RAS via vaccination and examined the effectiveness of the VaD model in rats. METHODS: Male Wistar rats were exposed to two-vessel occlusions (2VO) after three injections of Ang II peptide vaccine. The effects of the vaccine were evaluated in the novel object recognition test, brain RAS components, and markers for oligodendrocytes. RESULTS: In the vaccinated rats, anti-Ang II antibody titer level was increased in serum until Day 168, but not in cerebral parenchyma. Vaccinated rats showed better object recognition memory with inhibited demyelination in the corpus callosum and activation of astrocytes and microglia. Also, levels of BrdU/GSTπ-positive cells and the phosphorylation of cAMP response element binding protein was increased in vaccinated rats, indicating that the differentiation of oligodendrocyte progenitor cells to mature oligodendrocytes was accelerated. Vaccinated rats showed increased expression of fibroblast growth factor-2 (FGF2), which was observed in endothelial cells. Angiotensinogen mRNA was decreased at 7 days after 2VO but increased at 14 and 28 days. CONCLUSION: Ang II vaccine might have promoted oligodendrocyte differentiation and inhibited astrocytic and microglial activation by stimulating FGF2 signaling in the endothelial cells-oligodendrocyte/astrocyte/microglia coupling. These data indicate the feasibility of Ang II vaccine for preventing progression of vascular dementia.

Development of anti-thrombotic vaccine against human S100A9 in rhesus monkey.

In post-stroke patients, a decreased adherence to antiplatelet drugs is a major challenge in the prevention of recurrent stroke. Previously, we reported an antiplatelet vaccine against S100A9 in mice, but the use of Freund's adjuvant and the difference in amino acid sequences in epitopes between mice and humans were problematic for clinical use. Here, we redesigned the S100A9 vaccine for the common sequence in both humans and monkeys and examined its effects in cynomolgus monkeys with Alum adjuvant. First, we assessed several candidate epitopes and selected 102 to 112 amino acids as the suitable epitope, which could produce antibodies. When this peptide vaccine was intradermally injected into 4 cynomolgus monkeys with Alum, the antibody against human S100A9 was successfully produced. Anti-thrombotic effects were shown in two monkeys in a mixture of vaccinated serum and fresh whole blood from another cynomolgus monkey. Additionally, the anti-thrombotic effects were partially inhibited by the epitope peptide, indicating the feasibility of neutralizing anti-thrombotic effects of produced antibodies. Prolongation of bleeding time was not observed in vaccinated monkeys. Although further studies on increasing the effect of vaccine and safety are necessary, this vaccine will be a promising approach to improve adherence to antiplatelet drugs in clinical settings.

Prevention of Acute Lung Injury by a Novel CD14-Inhibitory Receptor Activator of the NF-κB Ligand Peptide in Mice.

Although CD14 has been implicated in the initiation of multiple TLR-mediated inflammatory responses to sepsis and sepsis-related acute lung injury (ALI), an inhibitor of CD14, except for neutralizing Abs, has not been developed. A partial peptide, microglial healing peptide 1 with N-terminal acetylation and C-terminal amidation (MHP1-AcN), derived from the receptor activator of the NF-кB ligand, was recently found to inhibit multiple TLR signaling in the macrophages. Therefore, we hypothesized that the inhibitory effect of MHP1-AcN might be through the inhibition of CD14, a common coreceptor for multiple TLRs. In cultured mouse macrophages, MHP1-AcN was shown to bind to CD14 and compete with LPS for competitive inhibition of CD14, resulting in inhibition of TLR4 signaling, including NF-кB and IFN regulatory factor 3 activation and nuclear translocation. In addition to TLR2, TLR4, and TLR7, MHP1-AcN also inhibited TLR3 signaling and Escherichia coli DNA-induced, CD14-dependent TLR9 signals; however, CpG oligodeoxynucleotide-induced, CD14-independent TLR9 signals were not inhibited in the mouse macrophages. In sepsis-induced ALI mouse model, MHP1-AcN treatment showed the reduction in the expression of IL-6 and CCL2 in both the serum and lung tissues. IL-6 levels in the bronchoalveolar lavage fluid and pathological score were also decreased by MHP1-AcN. Thus, MHP1-AcN, a novel CD14 inhibitor, could be a promising agent for treating sepsis-induced ALI.

Preventative effects of the partial RANKL peptide MHP1-AcN in a mouse model of imiquimod-induced psoriasis.

We recently developed a partial peptide of receptor activator of nuclear factor-кB ligand (RANKL) known as microglial healing peptide 1 (MHP1-AcN), that inhibits Toll-like receptor (TLR)-related inflammation through RANKL/RANK signaling in microglia and macrophages without promoting osteoclast activation. The abnormal activation of TLRs contributes to the initiation and maintenance of psoriasis, which is a chronic inflammatory skin disease that involves the aberrant expression of proinflammatory cytokines and the subsequent dermal γδ T cell and T helper 17 (Th17) cell responses. The inhibition of TLR-mediated inflammation provides an important strategy to treat psoriasis. Here, we examined the preventative effects of MHP1-AcN in a mouse model of imiquimod (a TLR 7/8 agonist)-induced psoriasis. Topical imiquimod application induced psoriasis-like skin lesions on the ear and dorsal skin. Systemic administration of MHP1-AcN by daily subcutaneous injection significantly prevented the development of skin lesions, including erythema, scaling and thickening. Mice treated with MHP1-AcN showed reduced levels of skin Il6 mRNA at 32 h and reduced levels of Il23 and Il17a mRNA at d9. Serum levels of IL-6 and IL-23 were reduced at 32 h, and IL-17A was reduced at d9. These results indicated that MHP1-AcN could decrease imiquimod-induced IL-6, IL-23 and IL-17A production. MHP1-AcN is potentially an alternative treatment for psoriasis.

Design and fabrication of a personalized anthropomorphic phantom using 3D printing and tissue equivalent materials.

To fabricate an individualized anthropomorphic lung phantom with tissue-equivalent radiation attenuation properties using a cost-effective three-dimensional (3D) printing technique. Based on anonymized human chest CT images, the phantom contained a 3D-printed skin shell, filled with tissue equivalent materials with similar radiation attenuation characteristics. The filling materials were a mixture of CaCO3, MgO, agarose, NaCl, pearl powder and silica gel. The dose calculation accuracy of different treatment planning system (TPS) algorithms was validated and compared with the ion chamber measurements in the phantom, including tumor and surrounding normal tissues. The chest phantom was shown to represent a human's chest in terms of radiation attenuation property and human anatomy. The Hounsfield unit ranges were -60 to -100, 20 to 60, and 120 to 300 for fat, muscle, and bone, respectively. The actual measured values of the ionization chamber were 213.7 cGy for the tumor, 53.85 cGy for normal lung tissue, and 4.1 cGy for the spinal cord, compared to 214.1, 55.2, and 4.5 cGy, respectively, with use of the Monte Carlo algorithm in TPS. The application of 3D printing in anthropomorphic phantoms can improve personalized medical need and efficiency with reduce costs thus, can be used for radiation dose verification.

Development of a novel RANKL-based peptide, microglial healing peptide1-AcN (MHP1-AcN), for treatment of ischemic stroke.

Although the regulation of post-ischemic inflammation is an important strategy to treat ischemic stroke, all clinical trials have failed to show its efficacy. To solve the problem, we previously developed a novel partial peptide of RANKL, microglial healing peptide 1 (MHP1), which could reduce ischemic injury by inhibiting Toll-like receptor (TLR) induced inflammation. However, optimization of the peptide was necessary to increase the stability and efficacies for clinical use. According to information gathered through HPLC/MS in serum, we have newly designed a series of modified MHP1 peptides and have found that N-terminal acetylation and C-terminal amidation in MHP1 (MHP1-AcN), can strengthen its anti-inflammatory effects and increase its stability with anti-osteoclastogenic effects. Anti-TLR activity was reported to be reduced in MHP1 when incubated at 37 °C for 24 hrs, but MHP1-AcN could keep the activity under the same condition. The therapeutic effect of MHP1-AcN was observed in transient ischemic stroke model at lower dose than MHP1. Importantly, MHP1-AcN did not affect thrombolytic effects of tissue plasminogen activator (tPA) and inhibited tPA-induced hemorrhagic transformation. These findings indicated that MHP1-AcN was stable and effective anti-TLR signal peptide and could be a promising agent for treating stroke patients receiving tPA and endovascular therapy.

Lab-on-CD microfluidic platform for rapid separation and mixing of plasma from whole blood.

Traditional clinical methods for separating whole blood into blood cells and cell-free plasma are labor intensive and time consuming. Accordingly, the present study proposes a simple compact disk (CD) microfluidic platform for the rapid separation of plasma from whole human blood and the subsequent mixing of the plasma with a suitable reagent. The performance of three CD microfluidic platforms incorporating square-wave mixing channels with different widths is evaluated both numerically and experimentally. The results show that given an appropriate specification of the microchannel geometry and a CD rotation speed of 2000 rpm, a separation efficiency of 95 % can be achieved within 5 ~ 6 s given a diluted blood sample with a hematocrit concentration of 6 %. Moreover, a mixing efficiency of more than 96 % can be obtained within 5 s given a CD rotation speed of 2200 rpm. The practical feasibility of the proposed device is demonstrated by performing a prothrombin time (PT) test. It is shown that while the time required to perform the PT test using a conventional bench top system is around 15 min, the proposed CD microfluidic platform allows the test to be completed within 1 min.

Ropivacaine plus dexamethasone infiltration reduces postoperative pain after tonsillectomy and adenoidectomy.

OBJECTIVE: To compare the effect of ropivacaine plus dexamethasone and ropivacaine alone as infiltration anesthesia on postoperative pain, nausea and vomiting, and oral intake in children after tonsillectomy and adenoidectomy. METHODS: Two hundred pediatric patients scheduled for tonsillectomy and adenoidectomy were prospectively enrolled and randomly placed in a ropivacaine with dexamethasone group (RD) or a ropivacaine alone group (R). Treatment for both groups was administered by local infiltration, and pain scores were recorded at various intervals. Primary outcomes were pain scores recorded 4-24h postoperation. Secondary outcomes included time to the first administration of analgesic and total consumption of analgesics for all children, time to first water request, first oral intake, incidence of nausea or vomiting, and time to discharge. RESULTS: From postoperative hours 4-24, children in the RD group had lower pain scores than children in the R group (P < 0.05). Total fentanyl consumption was significantly decreased in the RD group compared to the R group (50.9 ± 9.3 vs. 103.9 ± 11.5 µg, P < 0.001). The time to first water request and first oral intake were significantly shorter in the RD group [(40 min (27-64) vs. 64 min (43-89); P < 0.001) and (54 min (40-91) vs. 85 min (67-127); P < 0.001), respectively]. Oral intake was significantly improved, and the incidence of nausea and vomiting were reduced in the RD group (P < 0.05). The time to discharge was shorter in the RD group when compared with the R group (9.06 ± 0.89 d vs. 7.05 ± 0.71 d; P < 0.001). CONCLUSIONS: Ropivacaine plus dexamethasone infiltration effectively lowers pain, improves oral intake, lowers postoperative nausea and vomiting, and decreases the time to discharge.

Resection of a giant renal angiomyolipoma in a solitary kidney with preoperative arterial embolization.

Renal angiomyolipoma is a type of benign tumor that occurs sporadically in addition to being associated with tuberous sclerosis. Preoperative embolization of large tumors is important to avoid excessive blood loss during surgery. We reported a patient with a 5505-g giant renal angiomyolipoma in a solitary kidney. The patient was treated with preoperative embolization and radical nephrectomy without complications. This type of treatment for an enormous angiomyolipoma can reduce the risk of uncontrolled hemorrhage caused by rupture of the tumor during the operation and should be considered for the treatment of similar tumors.

Resection of a giant renal angiomyolipoma in a solitary kidney with preoperative arterial embolization / 中华医学杂志(英文版)

Renal angiomyolipoma is a type of benign tumor that occurs sporadically in addition to being associated with tuberous sclerosis. Preoperative embolization of large tumors is important to avoid excessive blood loss during surgery. We reported a patient with a 5505-g giant renal angiomyolipoma in a solitary kidney. The patient was treated with preoperative embolization and radical nephrectomy without complications. This type of treatment for an enormous angiomyolipoma can reduce the risk of uncontrolled hemorrhage caused by rupture of the tumor during the operation and should be considered for the treatment of similar tumors.

Optical projection display systems integrated with three-color-mixing waveguides and grating-light-valve devices.

An integrated optical projection display technique utilizing three-color-mixing waveguides and grating-light-valve devices is demonstrated. This new projection display system comprises an optical lens, a microscanner, a grating light valve, and a 3x1 planar waveguide device. The planar waveguide device is fabricated using a SU-8 negative photoresist process, which is suitable material for rapid prototyping of integrated optical circuits. It works as a three-color-mixer and is successfully used for color image generation. The intensity of color for each pixel in the display picture is tuned by groups of movable ribbons comprising a grating light valve and image generating diffraction gratings. This study also demonstrates a surface-micromachined optical scanner using four stress-actuated polysilicon plates to raise a horizontal mirror. The electrostatically driven mirror can be used for scanning projection display applications. Experimental data show that the optical scanner has a mirror scanning angle up to +/-15(o) using an operating voltage of 25 V. A sub-millisecond switching time (<900 mus) and an optical insertion loss of 0.85 dB is achieved for multi-mode waveguides. The development of the proposed integrated optical system could be promising for an image generation system.