Multiple Stimulation Research Articles

Impact of number of stimulation sites on long-lasting desynchronization effects of coordinated reset stimulation.

Excessive neuronal synchrony is a hallmark of several neurological disorders, e.g., Parkinson's disease. An established treatment for medically refractory Parkinson's disease is high-frequency deep brain stimulation. However, it provides only acute relief, and symptoms return shortly after cessation of stimulation. A theory-based approach called coordinated reset (CR) has shown great promise in achieving long-lasting effects. During CR stimulation, phase-shifted stimuli are delivered to multiple stimulation sites to counteract neuronal synchrony. Computational studies in plastic neuronal networks reported that synaptic weights reduce during stimulation, which may cause sustained structural changes leading to stabilized desynchronized activity even after stimulation ceases. Corresponding long-lasting effects were found in recent preclinical and clinical studies. We study long-lasting desynchronization by CR stimulation in excitatory recurrent neuronal networks of integrate-and-fire neurons with spike-timing-dependent plasticity (STDP). We focus on the impact of the stimulation frequency and the number of stimulation sites on long-lasting effects. We compare theoretical predictions to simulations of plastic neuronal networks. Our results are important regarding CR calibration for two reasons. We reveal that long-lasting effects become most pronounced when stimulation parameters are adjusted to the characteristics of STDP-rather than to neuronal frequency characteristics. This is in contrast to previous studies where the CR frequency was adjusted to the dominant neuronal rhythm. In addition, we reveal a nonlinear dependence of long-lasting effects on the number of stimulation sites and the CR frequency. Intriguingly, optimal long-lasting desynchronization does not require larger numbers of stimulation sites.

Adaptive anti-oxidative responses to chronic exposure to stress-signaling molecule, oxidized cell-free DNA, in rat neural cells

Stress is an adaptation reaction to harmful environment changes. Both oxidized and non-oxidized cell-free DNA (oxo-cfDNA and no-cfDNA) liberating from damaged cells possesses a characteristic of stress-molecules. CfDNA molecules modified by oxidation exhibit more oxidative (increasing 8-oxodG in DNA) and antioxidative (enhanced transcription of NRF2 and NRF2-dependent genes) activities than no-cfDNA in non-neural cell cultures. Whether the oxo-cfDNA molecules affect brain cells under different regimens of treatment that may mimic multiple, chronically applied stressful insults remained unclear. We studied if multiple stimulations of nervous system cells with oxidized cfDNA may further augment the DNA oxidative damage by inducing 8-oxodG modifications, and increase antioxidant Hmox1 gene expression and the NRF2 protein level in a primary cell culture of rat cerebellum (RCC). We found that daily treatment of RCC with oxo-cfDNA for 3 days increased accumulation of 8-oxodG in intracellular DNA, activated transcription of Hmox1 gene and enhanced the NRF2 protein content. However, after eleven-day daily treatment, the opposite effect was observed, in which 8-oxodG level in intracellular DNA and Hmox1 gene expression and NRF2 protein level in cerebellar cells were significantly decreased. Therefore, under subchronic, 3-day regimen of treatment, oxidized cfDNA further augmented accumulation of 8-oxodG in cellular DNA and promoted activation of the antioxidant system whereas its 11-day chronic action decreased both activities presumably due to inducing tolerance to stress-signaling via overactivation and/or exhaustion of molecular targets of oxo-cfDNA in cerebellar cells.

Therapeutic Effects of Diagonal-Transcranial Direct Current Stimulation on Functional Recovery in Acute Stroke: A Pilot Study

Background: Peak neurologic recovery from acute stroke occurs within the first 3 months, and continues at a slower pace for 6 months. Objective/Hypothesis: The aim of this pilot study is to clarify the safety and feasibility of multiple diagonal-transcranial direct current stimulation (d-tDCS) sessions up to 3 months with electrodes placed diagonally over the lesional dorsolateral prefrontal cortex and contralesional primary motor cortex for upper limb hemiparesis in acute stroke. Methods: Five patients with acute stroke (2 with intracerebral hemorrhage and 3 with cerebral infarction) with upper limb paresis participated. d-tDCS (1 mA, 20 min per day) combined with conventional rehabilitation was given starting 7–21 days after stroke onset. Each session consisted of 10 d-tDCS over 2 weeks and patients received 2 sessions in the acute phase and 2 sessions in the subacute phase for a total 40 treatments. Motor function was assessed using Fugl-Meyer Assessment for upper extremity (FMA-UE) before and after each session, and the period to achieve 70% of maximal potential recovery in FMA-UE was evaluated. Results: All 5 patients completed the intervention and showed no adverse effects throughout the protocol. Of these, 3 (60%) achieved 70% predicted scores within 2 months. Regarding therapeutic gains of FMA-UE in each of the 10 sessions in the acute phase, 4 sessions showed great recovery, 3 sessions showed moderate recovery, and 3 sessions showed poor recovery. Conclusion: These findings suggest that d-tDCS over 3 months may be safe and feasible for acute stroke patients in the acute to subacute phases and have therapeutic potential to promote recovery of upper limb function, although further randomized, double-blind, sham-controlled trial is warranted with larger sample size.

Effects of fatigue on steady state motion visual evoked potentials: Optimised stimulus parameters for a zoom motion-based brain-computer interface

Background and ObjectiveIn flicker-based steady-state visual evoked potentials (SSVEP) brain-computer interface (BCI), the system performance decreases due to prolonged repeated visual stimulation. To reduce the performance decrease due to visual fatigue, the zoom motion based steady-state motion visual evoked potentials (SSMVEPs) paradigm had been proposed. In this study, the stimulation parameters of the paradigm are optimised to mitigate the decrease in detection accuracy for SSMVEP due to visual fatigue. MethodsEight zoom motion-based SSMVEP paradigms with different stimulation parameters were compared. The graph size, luminance, colour, and shape, as well as the frequency range and interval of the stimulation and refresh rate of the screen was changed to determine the optimal paradigm with high recognition accuracy and reduced fatigue effects. The signal-to-noise ratio (SNR) of SSMVEP was also calculated for four fatigue levels. Moreover, the power spectral density of electroencephalograph (EEG) alpha and theta bands during ongoing activity was calculated for the stimulation experiment to evaluate fatigue at the start and end of the stimulation task. ResultsAll stimulation SSMVEP paradigms exhibited high accuracies. Changes in luminance, colour, and shape did not impact the recognition accuracy, nor did a higher stimulation frequency or lower frequency interval of each stimulation block. However, the paradigm with smaller stimulus achieved the highest average target selection accuracy of 97.2%, compared to 94.9% for the standard paradigm. Furthermore, it exhibited almost zero reduction in recognition accuracy due to fatigue. From fatigue level 1 to level 4, the smaller zoom motion-based SSMVEP exhibited a lower decrease in the SNR of SSMVEP and a lower alpha/theta ratio decrease during ongoing stimulation activity compared to the standard paradigm. ConclusionsFor a zoom motion-based SSMVEP paradigm, changing multiple stimulation parameters can lead to the same high performance as the standard paradigm. Moreover, using a smaller stimulus can reduce the accuracy decrease caused by fatigue because the SNR decrease in the evoked SSMVEP state was negligible and the alpha/theta index decrease during ongoing activity was lower than that for the standard paradigm.

Multiple nanosecond pulsed electric fields stimulation with conductive poly(l-lactic acid)/carbon nanotubes films maintains the multipotency of mesenchymal stem cells during prolonged in vitro culture.

Mesenchymal stem cells (MSCs) gradually lose multipotency when cultured for prolonged durations in vitro, which significantly hinders subsequent clinical applications. Nanosecond pulsed electric fields (nsPEFs) have been recently investigated to overcome this problem in our lab; however, the differentiation potency of MSCs could only be partially and transiently recovered because the nsPEFs can only be delivered to suspended cells once. Here, we develop a new strategy to apply multiple nsPEFs to adherent MSCs with conductive films to mitigate the decreasing multipotency of prolonged cultured MSCs. The poly(l-lactic acid)/graphitized-carboxylated functionalized carbon nanotubes (PLLA/CNT) films were fabricated as conductive cell culture platforms. Both single and multiple nsPEFs stimulation could significantly increase the differentiation potential of MSCs, as evidenced by upregulated expression of chondrogenic, osteogenic, and adipogenic-related gene (SOX9, RUNX2, and PPAR-γ), as well as increased production of proteoglycans, mineralized calcium, and triglycerides. Multiple nsPEFs stimulation demonstrated significant efficacy in upregulating expression of pluripotency genes of OCT4A (3.5- to 4.5-folds), NANOG (3.5- to 4.0-folds), and SOX2 (1.5- to 2.0-folds) and stably maintaining high expression of these genes for nearly 23 days. Notably, nsPEFs stimulation did not significantly shorten telomere length. In conclusion, multiple nsPEFs stimulation could effectively mitigate decreasing multipotency of MSCs during prolonged in vitro culture.

A microfluidic platform enabling real-time control of dynamic biochemical stimuli to biological cells

In the present study, we report a microfluidic platform that enables real-time control of biochemical stimuli to biological cells. The microfluidic platform is designed by integrating a ‘Christmas tree’ inlet for pre-generating spatially linear concentration gradient, with a Y-shaped channel to modulate dynamic signals with an external programmable air pump. The proposed design is simple and straightforward, has negligible response time compared to the traditional Y-shaped channel, and for the first time, provides the capability of generating versatile waveforms of dynamic stimuli and implementing multiple dynamic stimulating signals with different amplitudes synchronously. The feasibility of the microfluidic platform is proved by both computational fluid dynamics simulation and fluorescein experiment. The applicability of the proposed platform is demonstrated by characterizing the intracellular calcium ion dynamics in human umbilical vein endothelial cells in response to multiple dynamic stimuli within a single run. The proposed platform provides a simple approach to rapidly control dynamic stimuli with versatile waveforms to biological cells and shows the potential for the quantitative study of biological cellular dynamic responses.

Three-dimensional Map of Lumbar Spinal Cord Motor Function for Intraspinal Microstimulation in Rats.

Intraspinal microstimulation is an effective method to rebuild motor function after spinal cord injury. However, in the implementation, available map of stimulation sites is lacking for reference. The location of electrode implantation can only be determined through multiple stimulation, causing secondary damage to the spinal cord. Therefore, in this paper, SD rats were chosen as the research subject, and the intraspinal microstimulation was used to perform three-dimensional scanning electrical stimulation on the lumbar spinal cord that controls the hindlimb motion. The site coordinates and corresponding threshold current that can induce motion of hip, knee and ankle joints were recorded. In order to reduce the individual variances and improve the universality and applicability of the map, the results of 6 groups were normalized, and three-dimensional map of spinal motor function were drawn in the same coordinate system. The overlap of the distribution area of the same motion in each group was defined as the core region. The threshold current of all sites were analyzed statistically to obtain the most appropriate range of current intensity required to induce hindlimb motion. Using appropriate current for intraspinal microstimulation in the core region can selectively induce desired hindlimb motion, greatly improving the accuracy and reliability of electrode implantation.

Study and Pilot Test of Multiple Thermal-Fluid Stimulation in Offshore Nanpu Oilfield

Summary Heavy-oil resources whose underground oil viscosity is greater than 350 mPa·s is abundant in the Bohai oilfield. Because of the lack of effective exploitation technology, production performance with cold-production methods was not satisfactory. Seeking an effective method of heavy-oil exploitation, the multiple thermal-fluid stimulation was proposed and studied, in which hot water or steam mixed with carbon dioxide (CO2) and nitrogen (N2) would be injected into the formation for heating the oil and improving heavy-oil production. Considering the limitation of the offshore platform, a miniaturized multiple thermal-fluid generator was designed and developed. Integrated technologies such as seawater desalinization, heat insulation, and anticorrosion methods were also studied and developed. A pilot test of multiple thermal-fluid stimulation was conducted in Nanpu oilfield, starting in 2008. Until now, the pilot test has lasted for more than 10 years, and a total of 27 cycles of multiple thermal-fluid stimulation have been carried out. The oil-production rate and periodic-oil production amount of the thermal wells were both greatly improved. The cumulative oil produced by multiple thermal-fluid stimulation reached approximately 5.7×105 m3, and the incremental oil production is approximately 2.6×105 m3. The performance of oil production was satisfactory. As the first offshore thermal pilot area, the application experience showed that multiple thermal-fluid stimulation was effective for exploitation of heavy oilfields. With the increase of the stimulation cycles, a gas-channeling problem emerged and resulted in a decrease in the oil-production rate. New methods need to be studied and used for further enhancing oil recovery in Nanpu oilfield at the late stage of multiple thermal-fluid stimulation. Correction Notice: This paper has been updated from its originally published version to change the spelling of "Nanbao" to "Nanpu" throughout. No other changes to the paper were made.

MiR-301a Suppression within Fibroblasts Limits the Progression of Fibrosis through the TSC1/mTOR Pathway

Pulmonary fibrosis has been characterized by abnormal proliferation of fibroblasts and massive deposition of the extracellular matrix, which results from a complex interplay of chronic injury and inflammatory responses. MicroRNA-301a (miR-301a) is activated by multiple inflammatory stimulators, contributing to multiple tumorigenesis and autoimmune diseases. This study showed that miR-301a was overexpressed in a bleomycin-induced murine model of pulmonary fibrosis and patients with idiopathic pulmonary fibrosis (IPF). In addition, miR-301a was activated by transforming growth factor β (TGF-β) and interleukin 6 (IL-6) in normal and IPF fibroblasts, which was markedly reversed by the signal transducer and activator of transcription 3 (STAT3) inhibitor. The genetic ablation of miR-301a in mice reduced bleomycin-induced lung fibrosis, and the downregulation of miR-301a restrained proliferation and activation of fibroblasts. Furthermore, this study demonstrated that TSC1 was a functional target of miR-301a in fibroblasts, and the negative regulation of TSC1 by miR-301a promoted the severity of pulmonary fibrosis through the mammalian target of rapamycin (mTOR) signaling pathway. The blocking of miR-301a by the intravenous injection of antagomiR-301a inhibited the proliferation of fibroblasts and the structural destruction of lung tissues in the bleomycin-induced lung fibrosis mouse model. The findings revealed the crucial role of the miR-301a/TSC1/mTOR axis in the pathogenesis of pulmonary fibrosis, suggesting that miR-301a might serve as a potential therapeutic target.

Assisted reproductive technology outcomes in childhood cancer survivors: A report from the Childhood Cancer Survivor Study.

10528 Background: Some treatment exposures for childhood cancer reduce ovarian reserve. Registry-based evaluation has not been conducted for assisted reproductive technology (ART) outcomes of female survivors. Methods: The Childhood Cancer Survivor Study, a retrospective cohort of five-year survivors and siblings, was linked to the Society for Assisted Reproductive Technology Clinic Outcome Reporting System (SART CORS), which captures nationwide, CDC-required reporting of ART outcomes. We assessed live birth rate and relative risk (RR, 95% CI) as a function of treatment exposure, using generalized estimating equation to account for multiple ovarian stimulations per subject. Results: Among 9885 female survivors, 137 (1.4%; median age at diagnosis 10 years, range 0-20; 11 years of follow-up, 2-11) underwent 243 ART cycles (mean 1.8 cycles) and among 2419 siblings, 33 (1.4%) underwent 60 ART cycles (mean 1.8). Median age at autologous egg retrieval was 30 years (19-44) for survivors and 34 (24-43) for siblings. In the subset using autologous eggs (Table), 99 survivors underwent 155 ovarian stimulation cycles that resulted in 113 embryo transfers and 49 live births for a live birth rate of 32% per ovarian stimulation and 43% per transfer. Sibling live birth rate was 38% (p = 0.39 compared to survivors) per autologous ovarian stimulation and 53% (p = 0.33) per transfer. 38 survivors and 1 sibling underwent egg donor ovarian stimulation cycles. Two survivors used autologous eggs with gestational carriers and one cycle resulted in live birth. Cranial radiation therapy (RT) [RR 0.48 (0.27-0.87) p = 0.02] and pelvic RT [0.30 (0.14-0.66) p = 0.002], compared with no RT, resulted in lower RR of live birth in survivors. The likelihood of live birth after ART in survivors was not impacted by alkylator exposure [CED < 8000 mg/m2 vs. none: 1.14 (0.65-2.02); CED >8000 mg/m2 vs none: 1.07 (0.06-1.91)]. Conclusions: While live birth rates among survivors were lower compared with siblings, differences were not statistically significant. Pelvic and cranial RT were associated with a decreased likelihood of live birth, with no association with alkylator exposure identified. [Table: see text]

An MRI and MEG Compatible Device for the Study of Somatosensory Information Processing.

In previous publications we have reported methods for applying multi-site vibratory stimuli to the fingertips. Typically, this involves the use of multiple, individual vibrotactile stimulator and limitations of such an arrangement include difficulty with both positioning the stimuli as well as ensuring that stimuli are delivered in a synchronized and deliberate manner. The device that we reported is a significant improvement on multiple independent stimulators (Holden et al, 2011), and due to both the success of that stimulator and the consequent need to validate a number of findings that have been made with both that device and the precursor of that device (Tannan et al, 2007a), we designed and fabricated a four-site stimulator that could be used in MRI and MEG compatible environments. The device can stimulate four independent skin sites and is primarily designed for stimulating the digit tips. The device is similar to the previously reported device in that it is portable and is ergonomically suited for delivering stimuli to the finger tips, but it has the advantage of being MRI and MEG compatible. However, the fundamental mechanisms of the device are significantly different from the device that we recently reported since the device is piezo-based rather than VCA based. The device was tested in both MEG and MRI environments and demonstrated that no detectable signal (or noise) was introduced by the stimulator in those environments. To demonstrate the reliability of the device for delivering tactile stimulation in a magnetic field, tactile stimuli were presented in an MRI to a single individual. The study produced results that were consistent with prior studies that produced activation of cortical ensembles.

Genetic Influence on Efficacy of Pharmacotherapy for Pediatric Attention-Deficit/Hyperactivity Disorder: Overview and Current Status of Research.

Multiple stimulant and non-stimulant medications are approved for the treatment of attention-deficit/hyperactivity disorder (ADHD), one of the most prevalent childhood neurodevelopmental disorders. Choosing among the available agents and determining the most effective ADHD medication for a given child can be a time-consuming process due to the high inter-individual variability in treatment efficacy. As a result, there is growing interest in identifying predictors of ADHD medication response in children through the burgeoning field of pharmacogenomics. This article reviews childhood ADHD pharmacogenomics efficacy studies published during the last decade (2009-2019), which have largely focused on pharmacodynamic candidate gene investigations of methylphenidate and atomoxetine response, with a smaller number investigating pharmacokinetic candidate genes and genome-wide approaches. Findings from studies which have advanced the field of ADHD pharmacogenomics through investigation of meta-analytic approaches and gene-gene interactions are also overviewed. Despite recent progress, no one genetic variant or currently available pharmacogenomics test has demonstrated clinical utility in pinpointing the optimal ADHD medication for a given individual patient, highlighting the need for further investigation.

Normal faulting activated by hydraulic fracturing: A case study from the Barnett Shale, Fort Worth Basin

Surface microseismic arrays enable long-term field-scale monitoring over multiple stimulations during the life of an unconventional field. In this study, we show highly economic methods of monitoring with sparse surface arrays in the Barnett Shale and develop an alternatative method of processing to enable good vertical and horizontal resolution of located events. We show that sparse surface monitoring arrays enable not only the detection and location of high numbers of microseismic events but also source mechanism characterization. This case study illustrates how hydraulic fracturing activated normal faulting at a distance of approximately 1 mile from stimulated wells. We show that the source mechanism enables us to resolve between newly created hydraulic fractures and activated faults. The differences in source mechanisms and b-values of newly created fractures and activated faults are consistent with independently processed temporary star-like arrays, which are also deployed over the same stimulation.

Intra‐follicular temperature acts to regulate mammalian ovulation

Writing as one with a long-standing interest in the process of mammalian ovulation,1, 2 any new evidence as to factors underlying the release of an oocyte from a mature Graafian follicle is welcome. We now wish to report that one such factor is temperature of the antral fluid, and that this temperature corresponds closely with, or mirrors, that of the ovarian follicular surface. There is a cumulative body of evidence that pre-ovulatory follicles may be more than 1.0°C cooler than ovarian stroma in eutherian mammals, and both compartments are cooler than jugular and deep rectal temperatures.3, 4 Greater confidence in the work and its potential significance has now been achieved in mature cows.5-7 Our recent findings can be summarized in a single sentence. Follicles having a reduced antral fluid temperature compared with that of neighboring tissues invariably ovulated whereas follicles not revealing such cooling did not. This evidence comes from cow ovaries which, it should be noted, have various features in common with human ovaries: these include the pre-ovulatory follicular diameter (commonly 14-22 mm) and the time interval from the gonadotropin peak to ovulation itself (approximately 26-36 hours). Previous studies in rabbits (n = 40),8 pigs (n = 89),1, 9 cows (n = 6)10 and women (n = 26)11 employed infra-red sensing of surface temperatures8-10, 12 or follicular puncture with thermoelectrodes11 in ovaries exposed during mid-ventral or flank laparotomy under systemic or local anesthesia. All follicles in the different studies except one (from an infertile woman)11 were cooler than the ovarian stroma. In a series of recent studies, by contrast, a fine thermistor probe was introduced into the follicular antrum of dairy cows (n = 80) without the use of sedatives, anesthetics or surgery.5-7 The only tissue invasion involved passing a 17G needle containing the probe across the anterior vaginal wall followed by puncture of the ovarian surface. The procedure was monitored under ultrasonic scanning. None of the animals revealed any form of discomfort or stress during or after this straightforward approach. Results are summarized in Table 1. Graafian follicles that ovulated within 24 hours of temperature measurement were a mean of 1.12°C cooler than deep rectal temperatures, whereas no significant differences were found for non-ovulating follicles. This lower temperature was a consistent observation throughout the studies,5-7 enabling follicular cooling to be correlated positively with the potential for pregnancy.7 Evidence in support of this point can be detected in infra-red images of pig ovaries.12 By contrast, the temperature of newly ovulated follicles—ie, without their content of cumulus-oocyte complex and follicular fluid—was similar to that of other ovarian tissues. Pregnancies n (%) The specific mechanisms involved in follicular cooling have not been fully revealed, although they currently focus on endothermic reactions within pre-ovulatory follicles and would require effective local heat exchange.13, 14 The present results should stimulate further examination of antral fluid contents, especially large glycoprotein molecules, during the remodeling that leads to increased viscosity. At a more practical level, these results provide much-needed inspiration for human in vitro fertilization clinics because they offer a meaningful route to oocyte selection in protocols of multiple follicle stimulation by systemic gonadotropin treatment.15 Of course, non-invasive infra-red scanning of ovarian surface temperature would be a more attractive approach than multiple follicle puncture, but this awaits an endoscope capable of transmitting infra-red radiation. Relationships between temperature, follicular growth in vitro and oocyte quality would also be considered in the procedures of fertility preservation.16-19

Optogenetic modulation of cortical neurons using organic light emitting diodes (OLEDs)

Objective: There is a need for low power, scalable photoelectronic devices and systems for emerging optogenetic needs in neuromodulation. Conventional light emitting diodes (LEDs) are constrained by power and lead-counts necessary for scalability. Organic LEDs (OLEDs) offer an exciting approach to decrease power and lead-counts while achieving high channel counts on thin, flexible substrates that conform to brain surfaces or peripheral neuronal fibers. In this study, we investigate the potential for using OLEDs to modulate neuronal networks cultured in vitro on a transparent microelectrode array (MEA) and subsequently validate neurostimulation in vivo in a transgenic mouse model. Approach: Cultured mouse cortical neurons were transfected with light-sensitive opsins such as blue-light sensitive channel-rhodopsin (ChR2) and green-light sensitive chimeric channel-rhodopsin (C1V1tt) and stimulated using blue and green OLEDs (with 455 and 520 nm peak emission spectra respectively) at a power of ~1 mW mm−2 under pulsed conditions. Main results: We demonstrate neuromodulation and optostimulus-locked, single unit-neuronal activity in neurons expressing stimulating opsins (34 units on n = 4 MEAs, each with 16 recordable channels). We also validated the optostimulus-locked response in preliminary experiments in a channel-rhodopsin expressing transgenic mouse model, where at least three isolatable single neuronal cortical units respond to OLED stimulation. Significance: The above results indicate the feasibility of generating sufficient luminance from OLEDs to perform neuromodulation both in vitro and in vivo. This opens up the possibility of developing thin, flexible OLED films with multiple stimulation sites that can conform to the shape of the neuronal targets in the brain or the peripheral nervous system. However, stability of these OLEDs under chronic conditions still needs to be carefully assessed with appropriate packaging approaches.

Dual pH-responsive charge-reversal and photo-crosslinkable polymer nanoparticles for controlled drug release

In this article, we introduce a pH-responsive charge-reversible and photo-crosslinkable polymer nanoparticle. It is prepared via typical self-assembly from a block copolymer poly((7-(4-vinyl-benzyloxyl)-4-methylcoumarin)-co-acrylicacid)-b-poly((2-dimethylamino) ethyl methacrylate)-co-styrene) (P(VBMC-co-AA)-b-P(DMAEMA-co-St)), whose two blocks have different ionizable moieties. In an aqueous solution of pH ≤ 4, the cationic polymer nanoparticles are formed due to the fully protonated PDMAEMA. At a pH ranging from 5.0 to 7.8, partially ionized PAA and protonated PDMAEMA lead to the formation of polymer nanoparticles with a mixed shell. In a pH range of 8–10, a large amount of precipitation is produced within the isoelectric point (IEP) region because of the weak hydrophilic two blocks. In an aqueous solution of pH ≥ 10, polymer nanoparticles are reformed with PAA shell and P(DMAEMA-co-St) core. The coumarin groups of polymer can undergo photo-crosslinking and photo-cleavage reactions under UV light irradiation at λ = 365 nm and λ = 254 nm, respectively. The reversible nature of the photo-reaction can regulate the reversal of polymer nanoparticles. Furthermore, the aggregation-induced fluorescence emission (AIFE) property of polymer nanoparticles at different pH is tested by fluorescence emission spectra. The results indicate that the aggregation state of coumarin blocks in solution also changes with the pH value. The DOX release experiment in vitro shows that the release behavior of DOX-loaded nanoparticles can be adjusted by pH and light to achieve significant control. The inhibitory effect on the growth of tumor cells is displayed by cellular uptake and cytotoxicity test in vitro. The self-assembly system of polymer nanoparticles can be cooperatively controlled by multiple stimulations and displays potential applications in controlled drug delivery.

Sustained, cell-intrinsic versus intermittent, cell-extrinsic checkpoint blockade in solid tumor CAR T-cell therapy.

16 Background: We and others have published that antigen-stress induced functional exhaustion of chimeric antigen receptor (CAR) T cells can be rescued by addition of anti-PD1 agents. To avoid the need for multiple administrations of anti-PD1 agent, we developed CAR T-cell intrinsic PD1 dominant negative receptor (PD1 DNR). Herein, we investigated the anti-tumor efficacy of cell-extrinsic versus intrinsic anti-PD1 strategies. Methods: Human T cells transduced with CD28 costimulated mesothelin-targeted CAR T cells (M28z) with or without anti-PD1 agent, and M28zPD1DNR CAR T cells were investigated against mesothelin-expressing cancer cells with inducible or constitutive PDL1 over expression. In vitro, cytotoxicity upon single and multiple antigen stimulation and proliferation, inhibitory receptor expression (PD1, TIM3 LAG3) were tested. In vivo, tumor burden regression kinetics and median survival were determined in an orthotopic model of pleural mesothelioma. Results: Following single antigen stimulation, both CAR T cells exhibited equivalent cytotoxicity. Following multiple antigen stimulations (antigen stress test), M28z PD1DNR CARs showed sustained and relatively higher cytotoxicity and proliferation compared to M28z CAR T cells, even when combined with anti-PD1 agent. PD1, TIM3 and LAG3 expression upregulation was noticed in both CAR T cells. CAR T cell therapy combined with extrinsic PD1 blockade or intrinsic PD1 DNR demonstrated enhanced and sustained tumor regression and prolonged survival (median survival 66 and 60 days) compared to CAR T cell therapy alone (27.5 days). In mouse model with constitutive PDL1 overexpressing cancer, similar trend is observed (74, 69 versus 28 days). Conclusions: A single dose of M28z CAR T cells with cell-intrinsic PD1DNR demonstrated equal anti-tumor efficacy compared to multiple doses of extrinsic anti-PD1 agent administration in models with inducible and constitutive high-PDL1 expressing cancer with no on-target, off-tumor toxicity with either strategy. This data provides the rationale for our ongoing phase II trial of combination therapy with M28z CAR T cells and anti-PD1 agent, and upcoming M28zPD1DNR CAR T cell trial in 2020.

The Influence of Multiple Stimulations on the Unusual Delamination Phenomenon of a Li-Ion Battery Separator Prepared by a Wet Process

In this article, the unusual delamination phenomenon of LIB separators prepared by a wet process is reported. Meanwhile, a simple tearing test is proposed to quantify the delamination trend from the energy point of view and the effects of the tearing rate and temperature on the delamination trend of the separator were researched. Increasing the temperature makes separator delamination much more difficult, while the tearing rate has weaker effects on the separator delamination trend at lower temperatures but has a stronger influence at higher temperatures. In addition, the delamination trend of the separator in various electrolytes and under repeated compression cycles, which happened to the separator during battery cycles, is also investigated in detail. Also, the results show that the energy consumption per unit area during delamination decreases distinctly with increasing number of compression cycles, which was also influenced severely by different kinds of electrolytes with different Hildebrand interaction parameter.

Reply to Comment on "Kadomoto, S. et al. Tumor-Associated Macrophages Induce Migration of Renal Cell Carcinoma Cells via Activation of the CCL20-CCR6 Axis" Cancers 2020 12, 89.

We appreciate Zins and Abraham [1] commenting on our paper studying the role of the CCL20-CCR6 axis on renal cell carcinoma (RCC) cells [2]. As they pointed out, our study has certain limitations. Although M1- and M2-types cannot be separated clearly and a consecutive change of character might exist between them, it has been reported that plural specific markers express on M1- and M2-types. Unfortunately, a definite difference between M1 and M2 macrophages was not confirmed in our study. For more differentiation, multiple stimulations, such as suggested in the comments of Zins and Abraham, might be needed. Hence, we needed to expediently use "M1-like" and "M2-like" to mention specific status of these macrophage-like cells. Meanwhile, CCL20 expression levels of M2-like-THP-1 cells co-cultured with RCC cells were dramatically increased compared with parental THP-1 cells, indicating that certain stimulations within the tumor microenvironment rather than theoretical stimulations make macrophages differentiated; however, further studies are needed to clarify this mechanism using a more appropriate co-culture system mimicking the tumor microenvironment. Immunohistochemistry of CCL20 and M2 markers will help to better understand the role of tissue infiltrating macrophages, even tissue CD68 staining intensity itself was reported to correlate with prognosis of RCC patients [3]. [...].

An automatic detection method for 40-Hz auditory steady state response and its application in prognosis of comatose patients

ObjectiveWe proposed a 40-Hz auditory steady-state response (ASSR) automatic detection method, and studied the prognosis of comatose patients by combining the 40-Hz ASSR detection results of multiple paradigms of auditory stimulation. MethodsThe 40-Hz ASSR elicitation experiments were carried out on 32 comatose patients, with the detection results used as prognosis predictors. To achieve automatic detection, the detection was modeled as a binary hypothesis test for a sinusoidal waveform with unknown amplitude and phase, based on the generalized likelihood ratio test (GLRT). The patients were followed up for 6 months, and each patient’s outcome was classified as either favorable outcome (severe disability, moderate disability or good recovery) or unfavorable outcome (vegetative state/unresponsive wakefulness syndrome or death) according to the Glasgow outcome scale (GOS). The performance of the prognosis predictors was assessed using the area under the receiver operating characteristic curve (AUC-ROC). ResultsThe largest AUC in univariate analysis involving a single stimulation paradigm was 0.849, while the AUC obtained by combining multiple predictors was increased to 0.966. ConclusionsFor comatose patients, the absence of 40-Hz ASSR in multiple stimulation paradigms may indicate an unfavorable prognosis. Furthermore, the combination of multiple auditory stimulation paradigms may increase the outcome prediction accuracy. SignificanceThe combination of multi-paradigm 40-Hz ASSR automatic detection results may provide a feasible automatic outcome prediction method for comatose patients.