Loss of TDP-43 oligomerization or RNA binding elicits distinct aggregation patterns.

Aggregation of the RNA-binding protein TAR DNA-binding protein 43 (TDP-43) is the key neuropathological feature of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). In physiological conditions, TDP-43 is predominantly nuclear, forms oligomers, and is contained in biomolecular condensates assembled by liquid-liquid phase separation (LLPS). In disease, TDP-43 forms cytoplasmic or intranuclear inclusions. How TDP-43 transitions from physiological to pathological states remains poorly understood. Using a variety of cellular systems to express structure-based TDP-43 variants, including human neurons and cell lines with near-physiological expression levels, we show that oligomerization and RNA binding govern TDP-43 stability, splicing functionality, LLPS, and subcellular localization. Importantly, our data reveal that TDP-43 oligomerization is modulated by RNA binding. By mimicking the impaired proteasomal activity observed in ALS/FTLD patients, we found that monomeric TDP-43 forms inclusions in the cytoplasm, whereas its RNA binding-deficient counterpart aggregated in the nucleus. These differentially localized aggregates emerged via distinct pathways: LLPS-driven aggregation in the nucleus and aggresome-dependent inclusion formation in the cytoplasm. Therefore, our work unravels the origins of heterogeneous pathological species reminiscent of those occurring in TDP-43 proteinopathy patients.

FTLD-TDP assemblies seed neoaggregates with subtype-specific features via a prion-like cascade.

Morphologically distinct TDP-43 aggregates occur in clinically different FTLD-TDP subtypes, yet the mechanism of their emergence and contribution to clinical heterogeneity are poorly understood. Several lines of evidence suggest that pathological TDP-43 follows a prion-like cascade, but the molecular determinants of this process remain unknown. We use advanced microscopy techniques to compare the seeding properties of pathological FTLD-TDP-A and FTLD-TDP-C aggregates. Upon inoculation of patient-derived aggregates in cells, FTLD-TDP-A seeds amplify in a template-dependent fashion, triggering neoaggregation more efficiently than those extracted from FTLD-TDP-C patients, correlating with the respective disease progression rates. Neoaggregates are sequentially phosphorylated with N-to-C directionality and with subtype-specific timelines. The resulting FTLD-TDP-A neoaggregates are large and contain densely packed fibrils, reminiscent of the pure compacted fibrils present within cytoplasmic inclusions in postmortem brains. In contrast, FTLD-TDP-C dystrophic neurites show less dense fibrils mixed with cellular components, and their respective neoaggregates are small, amorphous protein accumulations. These cellular seeding models replicate aspects of the patient pathological diversity and will be a useful tool in the quest for subtype-specific therapeutics.

Microgroove formation in thin copper by laser-induced cavitation bubble shock: numerical and experimental investigation.

A laser-induced cavitation bubble shock forming technology is proposed for microgroove formation in thin copper. It is stamped by using the impact pressure generated by the laser breakdown of liquid. The impact-induced micro-formation of thin copper is studied by numerical simulation and experimentation. A finite-element model is developed, and the impact pressure created by laser-induced cavitation is measured to study the spatial distribution of impact pressure. The laser-induced cavitation process of the high-speed impact on thin copper is numerically simulated. The results of simulations are consistent with those of experiments, confirming the model's accuracy. The simulation is then used to study the dynamic formation and deformation behavior of the laser-induced cavitation impact of thin copper. The stress and thickness distributions during the formation of microgrooves in thin copper are also investigated. Furthermore, the influence of laser fluence and copper thickness on formation is studied. The results reveal that the high-speed impact forming of thin copper by laser-induced cavitation is due to three impact forces: a plasma shock wave, a cavitation shock wave, and a microjet, and this technology can be used to form thin metal walls.

Experiment and Simulation Study of the Laser-Induced Cavitation Bubble Technique for Forming a Microgroove in Aluminum Foil.

The present work introduces a laser-induced cavitation bubble technique for forming an axisymmetric structure (i.e., microgroove) and the dynamics of a cavitation bubble from initial expansion to the collapse stages that were also simulated. Furthermore, the shock wave signals and dynamic properties of the cavitation bubble were recorded using a hydrophone and a high-speed camera. The experiments on microgrooves formed by laser-induced cavitation bubble stamping were carried out, and the effects of laser energy, the initial position of the bubble, and the number of impacts on the microformability of aluminum sheets are discussed. The depth of the microgroove was investigated using experiments, and it was found that the process can serve as a rapid technique for impressing microfeatures on thin-sheet metals. The experimental results showed that as the initial position of the bubble increased, the deformation depth decreased. As the laser energy and number of impacts increased, the deformation depth increased. The results of the response surface experiments showed that a laser energy of 27 mJ, 3 impacts, and a bubble position of 3 mm were optimal for the process. By using the optimal parameters, flat and smooth microgrooves with a forming depth of 102.54 µm were successfully fabricated. Furthermore, the maximum thickness thinning of the microgroove section occurred at the entrance areas, and this area had the greatest hardness. This also indicated that the greatest amount of plastic deformation of the material and grain refinement occurred in this area. On the other hand, the aluminum foil did not undergo oxidation during the plastic deformation process. These results demonstrated that laser-induced bubble stamping is an advanced micromachining method with promising applications.

Herbal medicines for advanced colorectal cancer.

BACKGROUND: Herbal medicine has been widely used in patients with advanced colorectal cancer in China, but its efficacy has not been confirmed. OBJECTIVES: To evaluate the beneficial effect and safety on Chinese herbal medicine therapy for advanced stage colorectal cancer, and it's influence on the patients' quality of life. SEARCH METHODS: The following electronic databases were searched: BIOSIS Previews, Cochrane Controlled Trials Register, Medline EMBASE, Biological Abstracts, until Aug. 2011. Manual searching was performed on 16 types of Chinese journals which started from their respective first publication dates, as well as unpublished conference proceedings. No language restriction was applied. SELECTION CRITERIA: Randomized or quasi-randomised controlled trials on the treatment of advanced stage colorectal cancer by herbal medicines or herbal medicines combined with chemotherapy, regardless of blinding. DATA COLLECTION AND ANALYSIS: The data were extracted independently by two reviewers. Methodological quality of the included in trials was assessed according to the following parameters: randomisation, allocation concealment, double blinding, and drop-out rates. MAIN RESULTS: A total of 20 randomised controlled trials with 1304 participants were identified. All the 20 trials compared the use of herbal medicines with chemotherapy and chemotherapy alone in the treatment of advanced stage colorectal cancers.Compared with chemotherapy alone, the use of Quxie capsule combined with chemotherapy could decrease mortality rate (RR 0.17, 95% CI 0.03 to 0.97); the use of Jianpi Jiedu formula, Xiaozheng formula and Yiqi Huoxue herbal medicine combined with chemotherapy respectively could improve 1-year survival rate significantly; the use of Xiaozheng Formula in conjunction with chemotherapy could improve 3-year survival rate. There were 10 herbal medicines showing benefit in improving quality of life. Herbal medicines did not show additional benefit in response rate or stability rate. No trials reported serious adverse effect from herbal medicine. AUTHORS' CONCLUSIONS: Some herbal medicines combined chemotherapy compared with chemotherapy alone showed more beneficial effects in improving 1-year, 3-year survival and quality of life.

Multi-Ion-Based Modelling and Experimental Investigations on Consistent and High-Throughput Generation of a Micro Cavity Array by Mask Electrolyte Jet Machining.

The controllability and consistency in the fabrication of micro-textures on large-scale remains a challenge for existing production processes. Mask electrolyte jet machining (MEJM) is an alternative to Jet-ECM for controllable and high-throughput surface microfabrication with more consistency of dimensional tolerances. This hybrid configuration combines the high-throughput of masked-ECM and the adjustable flow-field of jet-ECM. In this work, a duckbill jet nozzle was introduced to make MEJM more capable of batch micro-structuring. A multiphysics model was built to simulate the distribution of electrochemical reaction ions, the current density distribution, and the evolution of the shape of the machined cavity. Experimental investigations are presented showing the influence of the machining voltage and nozzle moving speed on the micro cavity. Several 35×35 micro cavity arrays with a diameter of 11.73-24.92 µm and depth of 7.24-15.86 µm are generated on 304 stainless steel.

The clinical population pharmacokinetics, metabolomics and therapeutic analysis of alkaloids from Alstonia scholaris leaves in acute bronchitis patients.

BACKGROUND: Capsule of alkaloids from leaf of Alstonia scholaris (CALAS) is a new investigational botanical drug (No. 2011L01436) for respiratory disease. Clinical population pharmacokinetics (PK), metabolomics and therapeutic data are essential to guide dosing in patients. Previous research has demonstrated the potential therapeutic effect of CALAS on acute bronchitis. Further clinical trial data are needed to verify its clinical efficacy, pharmacokinetics behavior, and influence of dosage and other factors. PURPOSE: To verify the clinical efficacy and explore the potential biomarkers related to CALAS treatment for acute bronchitis. MATERIALS AND METHODS: Oral CALAS was assessed in a randomized, double-blind, placebo-controlled trial. Fifty-five eligible patients were randomly assigned to four cohorts to receive 20, 40 or 80 mg, of CALAS three times daily for seven days, or placebo. Each CALAS cohort included 15 subjects, and the placebo group included 10 subjects. A population PK model of CALAS was developed using plasma with four major alkaloid components. Metabolomics analysis was performed to identify biomarkers correlated with the therapeutic effect of CALAS, and efficacy and safety were assessed based on clinical symptoms and adverse events. RESULTS: The symptoms of acute bronchitis were alleviated by CALAS treatment without serious adverse events or clinically significant changes in vital signs, electrocardiography or upper abdominal Doppler ultrasonography. Moreover, one compartment model with first-order absorption showed that an increase in aspartate transaminase will reduce the clearance (CL) of scholaricine, and picrinine CL was inversely proportional to body mass index, while 19-epischolaricine and vallesamine CL increased with aging. The serum samples from acute bronchitis patients at different time points were analyzed using UPLC-QTOF in combination with the orthogonal projection to latent structures-discriminant analysis, which indicated higher levels of lysophosphatidylcholines, lysophosphatidylethanolamines and amino acids with CALAS treatment than with placebo. CONCLUSION: This is the first study to evaluate the clinical efficacy and explored the potential biomarkers related to CALAS therapeutic mechanism of acute bronchitis by means of clinical trial combined the metabolomics study. This exploratory study provides a basis for further research on clinical efficacy and optimal dosing regimens based on pharmacokinetics behavior. Additional acute bronchitis patients and CALAS PK samples collected in future studies may be used to improve model performance and maximize its clinical value.

Investigation on the Electrochemical Deposition of Nanocrystalline Zinc with Cationic Polyacrylamide (CPAM)-ZnSO4 Electrolyte.

The electrochemical deposition of nanocrystalline zinc has high potential to deposit zinc coatings, which have improved wear and corrosion properties compared to conventional coating methods. Conventionally, two or more additives are used in the electrolyte for the formation nanocrystalline zinc; these electrolyte components are complex, and their maintenance is inconvenient, making it unstable and not suitable for industrial scale production. This paper proposes an electrochemical deposition technique for nanocrystalline zinc using a ZnSO4 solution with cationic polyacrylamide (CPAM) as the unique additive. The results reveal that the cationic degree of CPAM has a significant influence on the deposition process and that the cationic degree of 20% enhances the electrolyte conductivity and improves the density of the deposited coating. The concentration of CPAM affects the electrolyte viscosity and conductivity. CPAM with a concentration of 20 g/L could simultaneously improve the electrolyte conductivity and maintain the viscosity at a low value, which promotes the formation of a bright deposited coating with a grain size of 87 nm. Additionally, the current density affects the grain structure of the deposited coating. With a current density of 0.5 A/dm2, a dense coating with lamellar grains and a grain size of 54.5 nm was obtained, which has, and the surface roughness was reduced to 0.162 µm. Moreover, the corrosion resistant property of the deposited coating was also improved.

Ultrasonic Particle Manipulation in Glass Capillaries: A Concise Review.

Ultrasonic particle manipulation (UPM), a non-contact and label-free method that uses ultrasonic waves to manipulate micro- or nano-scale particles, has recently gained significant attention in the microfluidics community. Moreover, glass is optically transparent and has dimensional stability, distinct acoustic impedance to water and a high acoustic quality factor, making it an excellent material for constructing chambers for ultrasonic resonators. Over the past several decades, glass capillaries are increasingly designed for a variety of UPMs, e.g., patterning, focusing, trapping and transporting of micron or submicron particles. Herein, we review established and emerging glass capillary-transducer devices, describing their underlying mechanisms of operation, with special emphasis on the application of glass capillaries with fluid channels of various cross-sections (i.e., rectangular, square and circular) on UPM. We believe that this review will provide a superior guidance for the design of glass capillary-based UPM devices for acoustic tweezers-based research.

Drug-drug interactions between salvianolate injection and aspirin based on their metabolic enzymes.

BACKGROUND: It is unclear whether the combination of traditional Chinese medicine and Western medicine leads to interactions in pharmacokinetics (PKs) and pharmacodynamics (PDs). In this study, the influence of salvianolate and aspirin on metabolic enzymes, and the relationship between the blood concentration and pharmacodynamic indexes, were determined. METHOD: In this, randomized, parallel-grouped, single-center clinical trial, 18 patients with coronary heart disease were randomly allocated into three groups: aspirin (AP) group, salvianolate (SV) group, and combination (A + S) group. All treatment courses lasted for 10 days, and blood samples were acquired before and after administration at different timepoints. The expression of catechol-O-methyltransferase (COMT), CD62p, procaspase-activating compound 1 (PAC-1), P2Y12, phosphodiesterase, and mitogen-activated protein kinase 8 (MAPK8) were compared with variance analysis The blood concentrations were analyzed by ultra-performance liquid chromatography-tandem mass spectrometry. RESULTS: Sixteen subjects completed the study. No significant difference in COMT was found among groups, although there was a decrease in the SV group. The PK results indicated that the absorption time of salicylic acid was shortened and the AUC0-∞ decreased and the elimination time of salvianolic acid B was prolonged and the AUC0-∞ decreased. The PD results declined after administration. A significant difference was found in MAPK8, CD62p, and P2Y12 expression. Compared with the SV group, a significant difference in P2Y12 in the A + S group was found. CONCLUSION: A pharmacokinetic drug-drug interaction was found in the aspirin and salvianolate combination. Pharmacodynamically, there was no difference between the A + S and AP groups. However, P2Y12 expression in the combination group was superior to that in the SV group. TRIAL REGISTRATION NUMBERS: The trial was registered on October 9, 2017, ClinicalTrials.gov, NCT03306550. https://register.clinicaltrials.gov/prs/app/action/SelectProtocol?sid=S0007D8H&selectaction=Edit&uid=U0003QY8&ts=2&cx=oiuc9g.

Precision EDM of Micron-Scale Diameter Hole Array Using in-Process Wire Electro-Discharge Grinding High-Aspect-Ratio Microelectrodes.

Micro-electrical discharge machining (micro-EDM) is a good candidate for processing micro-hole arrays, which are critical features of micro-electro-mechanical systems (MEMS), diesel injector nozzles, inkjet printheads and turbine blades, etc. In this study, the wire vibration of the wire electro-discharge grinding (WEDG) system has been analyzed theoretically, and, accordingly, an improved WEDG method was developed to fabricate micron-scale diameter and high-aspect-ratio microelectrodes for the in-process micro-EDM of hole array with hole diameter smaller than 20 µm. The improved method has a new feature of a positioning device to address the wire vibration problem, and thus to enhance microelectrodes fabrication precision. Using this method, 14 µm diameter microelectrodes with less than 0.4 µm deviation and an aspect ratio of 142, which is the largest aspect ratio ever reported in the literature, were successfully fabricated. These microelectrodes were then used to in-process micro-EDM of hole array in stainless steel. The effects of applied voltage, current and pulse frequency on hole dimensional accuracy and microelectrode wear were investigated. The optimal processing parameters were selected using response-surface experiments. To improve machining accuracy, an in-process touch-measurement compensation strategy was applied to reduce the cumulative compensation error of the micro-EDM process. Using such a system, micro-hole array (2 × 80) with average entrance diameter 18.91 µm and average exit diameter 17.65 µm were produced in 50 µm thickness stainless steel sheets, and standard deviations of hole entrance and exit sides of 0.44 and 0.38 µm, respectively, were achieved.

Fast Fabrication of Complex Surficial Micro-Features Using Sequential Lithography and Jet Electrochemical Machining.

This paper presents fabrication of complex surficial micro-features employing a cross-innovative hybrid process inspired from lithography and Jet-ECM. The process is referred here as mask electrolyte jet machining (MEJM). MEJM is a non-contact machining process which combines high resolution of lithography and greater flexibility of Jet-ECM. It is a non-contact process which can fabricate variety of microstructures on difficult-to-machine materials without need of expensive tooling. The presented work demonstrates the process performance of this technology by statistical analysis and multivariate kernel density estimation (KDE) based on probabilistic density function. Micro-letters are fabricated as an example of complex surficial structure comprising of multiple intersecting, straight and curved grooves. The processing response is characterized in terms of geometrical size, similarity ratio, and cumulative shape deviation. Experimental results demonstrated that micro letters with good repeatability (minimum SD of shape error ratio 0.297%) and shape accuracy (minimum shape error of 0.039%) can be fabricated with this technology. The results suggest MEJM could be a promising technology for batch manufacturing of surface microstructures with high productivity.

Jet Electrochemical Micromachining of Micro-Grooves with Conductive-Masked Porous Cathode.

Surface structures with micro-grooves have been reported to be an effective way for improving the performance of metallic components. Through-mask electrochemical micromachining (TMEMM) is a promising process for fabricating micro-grooves. Due to the isotropic nature of metal dissolution, the dissolution of a workpiece occurs both along the width and depth. Overcut is generated inevitably with increasing depth, which makes it difficult to enhance machining localization. In this paper, a method of electrochemical machining using a conductive masked porous cathode and jet electrolyte supply is proposed to generate micro-grooves with high machining localization. In this configuration, the conductive mask is directly attached to the workpiece, thereby replacing the traditional insulated mask. This helps in achieving a reduction in overcut and an improvement in machining localization. Moreover, a metallic nozzle is introduced to supply a jetted electrolyte in the machining region with enhanced mass transfer via a porous cathode. The simulation and experimental results indicate that as compared with an insulated mask, the use of a conductive mask weakens the electric field intensity on both sides of machining region, which is helpful to reduce overcut and enhance machining localization. The effect of electrolyte pressure is investigated for this process configuration, and it has been observed that high electrolyte pressure enhances the mass transfer and improves the machining quality. In addition, as the pulse duty cycle is decreased, the dimensional standard deviation and roughness of the fabricated micro-groove are improved. The results suggest the feasibility and reliability of the proposed method.

Effect of heat-clearing and detoxifying Chinese medicines combined with conventional therapy on mild hand, foot, and mouth disease with fever: An individual patient data meta-analysis.

BACKGROUND: In China, heat-clearing and detoxifying Chinese medicines combined with conventional therapy are commonly applied to treat the mild hand, foot, and mouth disease (HFMD). However, there is lack of solid evidence on the efficacy and safety of such therapies. METHODS: We conducted a pooled analysis with individual patient data from 5 strictly randomized controlled clinical trials to assess the efficacy and safety of this combination therapy for mild HFMD. An intention-to-treat analysis was performed. A 2-stage meta-analysis method was adopted to analyze the pooled effect size. RESULTS: In total, 947 patients were included. Compared with conventional therapy, the combination therapy significantly reduced the progression rate of HFMD from mild to severe (odds ratio [OR] 0.43, 95% confidence interval [CI]: 0.22 to 0.83, P = .01). Meanwhile, the healing time of skin rash and oral ulcer in the combination therapy group was significantly shorter than that of conventional therapy. The overall hazard ratio (HR) of healing time of the skin rash or oral ulcer was 1.22 (95%CI: 1.04 to 1.43; P = .02). However, except Jinlianqingre effervescent tablets, the combination therapy cannot shorten the time to fever resolution (HR 1.12, 95%CI: 0.97 to 1.29, P = .14). Because of the heterogeneity, Jinlianqingre effervescent tablets were analyzed separately and the HRs of the time to fever resolution and the healing time of skin rash or oral ulcer were 3.88 (95%CI: 3.19 to 4.72; P < .0001) and 3.79 (95%CI: 2.81 to 5.11; P < .0001), respectively. There were 30 adverse events reported in total; 2 cases were related to Chinese medicines. CONCLUSION: In conclusion, the heat-clearing and detoxifying Chinese medicines on top of conventional therapy can effectively reduce the progressive rate of mild HFMD and improve healing of skin and oral mucosal lesions. More studies are needed for the time to fever resolution.

Extracorporeal photopheresis in a rat model of pulmonary fibrosis.

Extracorporeal photopheresis has anti-inflammatory properties. The development of pulmonary fibrosis includes inflammatory episodes. This study evaluates effects of extracorporeal photopheresis in experimental pulmonary fibrosis. The bleomycin model of pulmonary fibrosis was used. Two groups of 4 rats received intratracheal bleomycin to induce fibrosis. The treatment group received infusions of photochemically treated leukocytes harvested from syngeneic animals. All animals were sacrificed at day 21 after fibrosis induction and analyzed with respect to lung histology and hydroxyproline content, cellular composition of bronchoalveolar lavages, serum and lavage concentrations of transforming growth factor-beta, interferon-gamma, and interleukin-10, and expression of selected genes in the lung. Interleukin-10 and transforming growth factor-beta protein concentrations increased in the plasma of treated animals, whereas the interferon-gamma protein concentration was higher in bronchoalveolar lavages. Interferon-gamma gene expression was up-regulated in the lung tissue of treated animals. No significant differences between treated and untreated animals were found with respect to hydroxyproline, histology, and lavage cell count. To conclude, extracorporeal photopheresis has positive molecular effects but does not attenuate experimental lung fibrosis with respect to histology, hydroxyproline, and lavage cell count in the applied treatment regimen. Further investigations of extracorporeal photopheresis in experimental pulmonary fibrosis are justified.

Experimental Mouse Model of Bleomycin-Induced Skin Fibrosis.

Systemic sclerosis (SSc) refers to an autoimmune disease, which is manifested by inflammation, vasculopathy, and fibrosis of the skin and internal organs. There are a number of different animal models recapitulating specific aspects of SSc. The experimental mouse model of bleomycin-induced skin fibrosis is commonly used to study the pathogenesis observed in SSc. In this model, repetitive intradermal injections of the cytotoxic agent bleomycin trigger progressive skin thickening, associated with excessive accumulation of collagen, infiltration of immune cells, and formation of α-smooth muscle actin (α-SMA)-positive myofibroblasts. In this article, we provide a detailed protocol for the induction of skin fibrosis in experimental mice by bleomycin. Moreover, we describe procedures for processing and analyzing affected skin tissue, provide troubleshooting, highlight advantages and limitations of the presented model, and critically discuss representative results. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Intradermal bleomycin injections to induce skin fibrosis in mice Support Protocol: Mouse tissue collection for fibrosis evaluation and for other molecular assays Basic Protocol 2: Evaluation of mouse skin thickness using Masson's trichrome staining Basic Protocol 3: Measurement of hydroxyproline content in skin tissue using a colorimetric assay Basic Protocol 4: Evaluation of myofibroblasts in mouse skin by immunohistochemistry.

Fabrication of a Metal Micro Mold by Using Pulse Micro Electroforming.

Microfluidic devices have been widely used for biomedical and biochemical applications. Due to its unique characteristics, polymethyl methacrylate (PMMA) show great potential in fabricating microfluidic devices. Hot embossing technology has established itself as a popular method of preparing polymer microfluidic devices in both academia and industry. However, the fabrication of the mold used in hot embossing is time-consuming in general and often impractical for economically efficient prototyping. This paper proposes a modified technology for preparing metal micro molds by using pulse micro electroforming directly on metallic substrate, which could save time and reduce costs. In this method, an additive was used to avoid surface defect on deposited nickel. A chemical etching process was performed on the metallic substrate before the electroforming process in order to improve the bonding strength between the deposited structure and substrate. Finally, with the aim of obtaining a metal micro mold with high surface quality (low surface roughness), an orthogonal experiment was designed and conducted to optimize the electroforming parameters. Additionally, metal micro molds with different structures were well prepared by using the optimized parameters.

Improving the Cell Viability and Isolating Precision of Laser-induced Forward Transfer Process by Maintaining a Proper Environment with a Microchip.

BACKGROUND: Aiming to improve the laser-induced forward transfer (LIFT) cell isolation process, a polydimethylsiloxane (PDMS) layer with micro-hole arrays was employed to improve the cell separation precision, and a microchip with heater was developed to maintain the working area at 100% humidity and 37°C with the purpose to preserve the viability of the isolated cells. METHODS: A series of experiments were conducted to verify the contributions of the optimization to LIFT cell isolation process as well as to study the effect of laser pulse energy, laser spot size and the titanium thickness on cell isolation. With 40µm laser spot size and 40nm thick of titanium, laser energy threshold for 100% single cell isolating succeed ratio is 7µJ. RESULTS: According to the staining images and proliferation ratios, the chip did help to improve the cell availability and the cells can recover from the juries at least a day earlier comparing to the samples processed without the chip. CONCLUSION: With a Lattice Boltzmann model, the cell isolation process is numerically studied and it turns out that the micro-hole makes the isolation process shift to a micro-syringe injection model leading to the lower laser energy threshold for cell separation and fewer injuries.

Single cell isolation process with laser induced forward transfer.

BACKGROUND: A viable single cell is crucial for studies of single cell biology. In this paper, laser-induced forward transfer (LIFT) was used to isolate individual cell with a closed chamber designed to avoid contamination and maintain humidity. Hela cells were used to study the impact of laser pulse energy, laser spot size, sacrificed layer thickness and working distance. The size distribution, number and proliferation ratio of separated cells were statistically evaluated. Glycerol was used to increase the viscosity of the medium and alginate were introduced to soften the landing process. RESULTS: The role of laser pulse energy, the spot size and the thickness of titanium in energy absorption in LIFT process was theoretically analyzed with Lambert-Beer and a thermal conductive model. After comprehensive analysis, mechanical damage was found to be the dominant factor affecting the size and proliferation ratio of the isolated cells. An orthogonal experiment was conducted, and the optimal conditions were determined as: laser pulse energy, 9 µJ; spot size, 60 µm; thickness of titanium, 12 nm; working distance, 700 µm;, glycerol, 2% and alginate depth, greater than 1 µm. With these conditions, along with continuous incubation, a single cell could be transferred by the LIFT with one shot, with limited effect on cell size and viability. CONCLUSION: LIFT conducted in a closed chamber under optimized condition is a promising method for reliably isolating single cells.