Indirect CRISPR screening with photoconversion revealed key factors of drug resistance with cell-cell interactions.

Comprehensive screenings to clarify indirect cell-cell interactions, such as those in the tumor microenvironment, especially comprehensive assessments of supporting cells' effects, are challenging. Therefore, in this study, indirect CRISPR screening for drug resistance with cell-cell interactions was invented. The photoconvertible fluorescent protein Dendra2 was inducted to supporting cells and explored the drug resistance responsible factors of supporting cells with CRISPR screenings. Random mutated supporting cells co-cultured with leukemic cells induced drug resistance with cell-cell interactions. Supporting cells responsible for drug resistance were isolated with green-to-red photoconversion, and 39 candidate genes were identified. Knocking out C9orf89, MAGI2, MLPH, or RHBDD2 in supporting cells reduced the ratio of apoptosis of cancer cells. In addition, the low expression of RHBDD2 in supporting cells, specifically fibroblasts, of clinical pancreatic cancer showed a shortened prognosis, and a negative correlation with CXCL12 was observed. Indirect CRISPR screening was established to isolate the responsible elements of cell-cell interactions. This screening method could reveal unknown mechanisms in all kinds of cell-cell interactions by revealing live phenotype-inducible cells, and it could be a platform for discovering new targets of drugs for conventional chemotherapies.

Recurrent reactive hypoglycemia due to clozapine-induced glucose intolerance: A case report.

Background: Although diabetes is one of the most common side effects of clozapine, a medication for the treatment of schizophrenia, to the best of our knowledge no study exists on clozapine-induced glucose intolerance or hypoglycemia in patients with schizophrenia. Case Presentation: We report a case of schizophrenia with repeated reactive hypoglycemia due to abnormal glucose tolerance during clozapine treatment. During clozapine administration in patients with schizophrenia, it is necessary to monitor physical and psychiatric symptoms due to reactive hypoglycemia and hyperglycemia. If abnormal glucose tolerance is a concern, it should be promptly detected using blood or oral glucose tolerance tests. Conclusion: Early intervention for impaired glucose tolerance may prevent clozapine discontinuation due to diabetes or hyperglycemia.

Quantitative analysis of organophosphate pesticides and dialkylphosphates in duplicate diet samples to identify potential sources of measured urinary dialkylphosphates in Japanese women.

Increased levels of dialkylphosphates (DAP) in maternal urine are associated with a variety of adverse developmental outcomes in children. Although urinary DAP levels are usually considered to be a marker of exposure to organophosphate (OP) pesticides, excretion of DAP may also increase by ingesting preformed DAP. To date, no study has quantitatively assessed the possible contribution of the dietary intake of preformed DAP and OP pesticides to urinary levels of DAP. Therefore, we aimed to estimate the levels of 6 DAPs and 84 OP pesticides in duplicate diet samples and urine samples collected from 73 women living in urban areas of Japan in 2018. DAP and OP pesticides were detected in 94% and 45% of diet samples, while DAP was detected in 100% of urinary samples, respectively. The average daily intake of preformed DAP was significantly higher than that of parent OP pesticides in our participants. Dimethylphosphate and diethylphosphate were predominant in the preformed DAP, and the estimated average daily intake of total amount of DAP was 78.3 nmol. Fruits and vegetables were the major dietary sources of DAP. Dietary intake of DAP was positively associated with urinary DAP levels, suggesting that a considerable amount of urinary DAP was derived from ingesting preformed DAP. Our results show that attributing urinary DAP levels exclusively to OP pesticide exposure would result in a substantial overestimation of the exposure level. Therefore, the urinary levels of DAP may not be suitable for evaluating OP pesticide exposure in the general urban population.

Development and Validation of a Deep Neural Network for Accurate Evaluation of Endoscopic Images From Patients With Ulcerative Colitis.

BACKGROUND & AIMS: There are intra- and interobserver variations in endoscopic assessment of ulcerative colitis (UC) and biopsies are often collected for histologic evaluation. We sought to develop a deep neural network system for consistent, objective, and real-time analysis of endoscopic images from patients with UC. METHODS: We constructed the deep neural network for evaluation of UC (DNUC) algorithm using 40,758 images of colonoscopies and 6885 biopsy results from 2012 patients with UC who underwent colonoscopy from January 2014 through March 2018 at a single center in Japan (the training set). We validated the accuracy of the DNUC algorithm in a prospective study of 875 patients with UC who underwent colonoscopy from April 2018 through April 2019, with 4187 endoscopic images and 4104 biopsy specimens. Endoscopic remission was defined as a UC endoscopic index of severity score of 0; histologic remission was defined as a Geboes score of 3 points or less. RESULTS: In the prospective study, the DNUC identified patients with endoscopic remission with 90.1% accuracy (95% confidence interval [CI] 89.2%-90.9%) and a kappa coefficient of 0.798 (95% CI 0.780-0.814), using findings reported by endoscopists as the reference standard. The intraclass correlation coefficient between the DNUC and the endoscopists for UC endoscopic index of severity scoring was 0.917 (95% CI 0.911-0.921). The DNUC identified patients in histologic remission with 92.9% accuracy (95% CI 92.1%-93.7%); the kappa coefficient between the DNUC and the biopsy result was 0.859 (95% CI 0.841-0.875). CONCLUSIONS: We developed a deep neural network for evaluation of endoscopic images from patients with UC that identified those in endoscopic remission with 90.1% accuracy and histologic remission with 92.9% accuracy. The DNUC can therefore identify patients in remission without the need for mucosal biopsy collection and analysis. Trial number: UMIN000031430.

iPSC-derived functional human neuromuscular junctions model the pathophysiology of neuromuscular diseases.

The control of voluntary skeletal muscle contraction relies on action potentials, which send signals from the motor neuron through the neuromuscular junction (NMJ). Although dysfunction of the NMJ causes various neuromuscular diseases, a reliable in vitro system for disease modeling is currently unavailable. Here, we present a potentially novel 2-step, self-organizing approach for generating in vitro human NMJs from human induced pluripotent stem cells. Our simple and robust approach results in a complex NMJ structure that includes functional connectivity, recapitulating in vivo synapse formation. We used these in vitro NMJs to model the pathological features of spinal muscular atrophy, revealing the developmental and functional defects of NMJ formation and NMJ-dependent muscular contraction. Our differentiation system is therefore useful for investigating and understanding the physiology and pathology of human NMJs.

Development of red blood cell-photon simulator for optical propagation analysis in blood using Monte Carlo method.

We have developed a "red blood cell (RBC)-photon simulator" to reveal optical propagation in prethrombus blood for various levels of RBC density and aggregation. The simulator investigates optical propagation in the prethrombus blood and will be applied to detect it noninvasively for thrombosis prevention in an earlier stage. In our simulator, Lambert-Beer's law is employed to simulate the absorption of RBCs with hemoglobin, while the Monte Carlo method is applied to simulate scattering through iterative calculations. One advantage of our simulator is that concentrations and distributions of RBCs can be arbitrarily chosen to exhibit the prethrombus, while conventional models cannot. Using the simulator, we found that various levels of RBC density and aggregation have different effects on the optical propagation of near-infrared response light in blood. The same different effects were acquired in in vitro experiments with 12 bovine blood samples, which were performed to evaluate the simulator. We measured RBC density using the clinical hematocrit index and RBC aggregation using activated whole blood clotting time. The experimental results correspond to the simulator results well. Therefore, we could show that our simulator exhibits the correct optical propagation for prethrombus blood and is applicable for the prethrombus detection using multiple detectors.

Development of optical sensing system for noninvasive and dynamic monitoring of thrombogenic process.

We developed a compact optical sensing system to monitor thrombogenic process noninvasively and dynamically by attaching a sensor on a blood circulating tubing. The system monitors the process for normal, erythrocyte aggregation, emboli, and thrombus formation stages with a continuously measured backscattered light. The system consists of an optical sensing unit, which has an 810-nm-wavelength chip light-emitting diode (LED) and a photodiode, and real-time monitoring software. The erythrocyte aggregation is verified by using the baseline transition of optical intensity, which is influenced by the apparent low number of red blood cells in erythrocyte-aggregated situation. Meanwhile, the emboli are evaluated by counting light-detected microembolic signals (LMES) induced by the flowing emboli. The performance of the system was evaluated using in vitro experiments with eight bovine blood samples. We performed optical sensing during thrombogenic process, which was intentionally generated by controlling the ratio of sodium citrate and calcium chloride solution. The activated whole blood clotting time (ACT) has gradually decreased in the process. The optical baseline transition has continuously increased at the beginning. After the increasing stopped, the LMES was observed. Finally, we found thrombus inside the mock-up circuit. As a result, we confirmed that our system is capable of monitoring the thrombogenic process noninvasively and dynamically.

Optical measurement of blood hematocrit on medical tubing with dual wavelength and detector model.

Blood hematocrit (Hct) is a clinically important index to detect death-dealing symptoms such as anemia and thrombosis, especially for patients with artificial heart, in dialysis and during open-heart surgery. Optical technology has been applied to monitor hematocrit noninvasively, continuously and conveniently, however, it was not well established for actual use. The purpose of this study is to develop an accurate and stable optical hematocrit measurement without any calibrations for device-mounting errors. To this end, we propose a theoretical method. In this method, disturbances are cancelled by using dual detector and optical path changes are calibrated by dual wavelength so that we can measure hematocrit without calibrations of these. Based on the method, a measurement unit that has two LEDs (805/1300[nm]) and four photo detectors was developed. Then, we performed experiments with 38 blood samples from five bovines' blood (Hct: 19-55%). These blood samples were circulated in a mock-up circuit by a blood pump. During the experiment, we measured hematocrit on medical tubing with the developed measurement system. As a result, we could measure hematocrit within 1.7 Hct% mean errors for 38 blood samples without any calibrations. The result indicates that the proposed method is applicable for hematocrit measurement on medical tube in enough small error. We found this proposed method is effective for developments of clinically workable hematocrit measurement/monitoring system.

Evaluation of optical propagation in blood for noninvasive detection of prethrombus blood condition.

This article evaluates the optical propagation to detect a "prethrombus" blood noninvasively. Thrombosis is still an inevitable issue in use of blood pumps, and it is required to predict thrombus formation as early as possible. We focused on the red blood cell (RBC) aggregation that is one of the features of thrombogenic process. First, by using a computer simulation, we calculated the optical propagations in blood for the RBC aggregation and nonaggregation blood. This simulation is based on the Monte-Carlo method and attempts to calculate the optical characteristics of the blood stochastically. In our simulation, the optical propagation with the RBC aggregation showed a different characteristic from that of the nonaggregation. Next, we examined the optical propagation in bovine blood with various activated whole blood clotting time (ACT). The blood mixed with sodium citrate was circulated by a blood pump. The ACT was adjusted between 1,000 and 50 seconds by controlling the ratio of calcium chloride solution to sodium citrate. We confirmed the RBC aggregation by using microscopic images and microthromboses in the pump directly. As a result, we evaluated that the change of the optical propagation has a correlation with thrombogenic process just as it was observed in our computer simulation. Our data indicate that the measurement of optical propagation can detect a prethrombous blood condition with RBC aggregation. Our study will help to establish optical technologies to detect prethrombous continuously and noninvasively.

Improvement of the accuracy in the optical hematocrit measurement by optimizing mean optical path length.

Optical techniques have been developed to acquire blood information (e.g., hematocrit [Hct], saturation of oxygen, thrombus) noninvasively and continuously in an artificial heart. For the practical use of an optical Hct measurement, Twersky's theory has been shown to be useful and have a good agreement in forward-scattered measurements. However, it was not applied to backward-scattered measurements, which can provide the measurement with a less demanding spatial requirement. Additionally, optimal measurement for accuracy is not well examined. Therefore, we developed an accurate Hct measurement in an artificial heart using current optical devices. To this end, we focused on optimizing an emitter-detector distance to provide a maximum optical path length. We attached optical emitter and detector fibers on Tygon tubing at various distances to measure forward- and backward-scattered light. Fresh bovine blood (Hct: 30-50%) was circulated in the tubing by a nonpulsatile artificial heart. We calculated the optical path length at various emitter-detector distances by fitting the measured optical outputs and the reference Hcts to Twersky's theory. Then, we performed Hct measurements. As a result, Twersky's theory is applicable not only to forward- but also to backward-scattered measurements in the physiogical Hct range. In both forward- and backward-scattered measurements, calculated optical path lengths become maximum at the same emitter-detector distance. The accuracy of Hct measurement is improved two to three times with the emitter-detector distance compared with other distances. The mean error is less than 1 Hct%. This result shows that an accurate Hct measurement is realized by selecting the optimal emitter-detector distance, which provides maximum optical path length defined by Twersky's theory. Our study provides a framework for the practical and less restrictive application of the optical Hct measurement to patients with an artificial heart.

Simulator with photon and arbitrarily arranged RBC for hematocrit estimation.

This paper introduces a simulator to estimate density of red blood cells (RBCs) from optical intensity based on blood properties of optical absorption and scattering model. The simulator will be directly used to estimate density of RBC, which is an important index for preventing thrombosis. In this simulator, Lambert-beer's law is used to acquire transmittance of RBCs with hemoglobin absorptance, while Monte Carlo method is used to obtain scattering property through iterative calculations. One of the advantages of this simulator is that position and number of RBCs are arbitrarily arranged for hematocrit estimation, while the conventional model can not deal with. Using the simulator, transitions of optical intensity were simulated with two variables: emitter-detector distance and hematocrit. These transitions were also measured in experiments with bovine blood to evaluate the simulator model. As a result, these experiment data corresponded with the simulated data in 2.5% error of converting hematocrit. We confirmed the developed simulator model was precise enough in optical propagation, and it indicated that this simulator could be applicable to hematocrit estimation.