Integrated Microfluidics for Protein Modification Discovery.

Protein post-translational modifications mediate dynamic cellular processes with broad implications in human disease pathogenesis. There is a large demand for high-throughput technologies supporting post-translational modifications research, and both mass spectrometry and protein arrays have been successfully utilized for this purpose. Protein arrays override the major limitation of target protein abundance inherently associated with MS analysis. This technology, however, is typically restricted to pre-purified proteins spotted in a fixed composition on chips with limited life-time and functionality. In addition, the chips are expensive and designed for a single use, making complex experiments cost-prohibitive. Combining microfluidics with in situ protein expression from a cDNA microarray addressed these limitations. Based on this approach, we introduce a modular integrated microfluidic platform for multiple post-translational modifications analysis of freshly synthesized protein arrays (IMPA). The system's potency, specificity and flexibility are demonstrated for tyrosine phosphorylation and ubiquitination in quasicellular environments. Unlimited by design and protein composition, and relying on minute amounts of biological material and cost-effective technology, this unique approach is applicable for a broad range of basic, biomedical and biomarker research.

Shedding light on mitochondrial function by real time monitoring of NADH fluorescence: I. Basic methodology and animal studies.

Normal mitochondrial function in the process of metabolic energy production is a key factor in maintaining cellular activities. Many pathological conditions in animals, as well as in patients, are directly or indirectly related to dysfunction of the mitochondria. Monitoring the mitochondrial activity by measuring the autofluorescence of NADH has been the most practical approach since the 1950s. This review presents the principles and technological aspects, as well as typical results, accumulated in our laboratory since the early 1970s. We were able to apply the fiber-optic-based NADH fluorometry to many organs monitored in vivo under various pathophysiological conditions in animals. These studies were the basis for the development of clinical monitoring devices as presented in accompanying article. The encouraging experimental results in animals stimulated us to apply the same technology in patients after technological adaptations as described in the accompanying article. Our medical device was approved for clinical use by the FDA.

Shedding light on mitochondrial function by real time monitoring of NADH fluorescence: II: human studies.

Monitoring the mitochondrial function, alone or together with microcirculatory blood flow, volume and hemoglobin oxygenation in patients, is very rare. The integrity of microcirculation and mitochondrial activity is a key factor in keeping normal cellular activities. Many pathological conditions in patients are directly or indirectly related to dysfunction of the mitochondria. Evaluation of mitochondrial activity by measuring the autofluorescence of NADH has been the most practical approach since the 1950s. This review, which accompanies part I, presents the principles and technological aspects of various devices used in order to monitor mitochondrial NADH redox state and tissue viability in patients. In part I, the detailed technological aspects of NADH monitoring were described. Typical results accumulated in our studies since the mid-1990s are presented as well. We were able to apply the fiber optic based NADH fluorometry to several organs monitored in vivo in patients under various pathophysiological conditions.

PTOLEMI: Personalized Cancer Treatment through Machine Learning-Enabled Image Analysis of Microfluidic Assays.

Contemporary personalized cancer diagnostic approaches encounter multiple challenges. The presence of cellular and molecular heterogeneity in patient samples introduces complexities to analysis protocols. Conventional analyses are manual, reliant on expert personnel, time-intensive, and financially burdensome. The copious data amassed for subsequent analysis strains the system, obstructing real-time diagnostics at the "point of care" and impeding prompt intervention. This study introduces PTOLEMI: Python-based Tensor Oncological Locator Examining Microfluidic Instruments. PTOLEMI stands out as a specialized system designed for high-throughput image analysis, particularly in the realm of microfluidic assays. Utilizing a blend of machine learning algorithms, PTOLEMI can process large datasets rapidly and with high accuracy, making it feasible for point-of-care diagnostics. Furthermore, its advanced analytics capabilities facilitate a more granular understanding of cellular dynamics, thereby allowing for more targeted and effective treatment options. Leveraging cutting-edge AI algorithms, PTOLEMI rapidly and accurately discriminates between cell viability and distinct cell types within biopsy samples. The diagnostic process becomes automated, swift, precise, and resource-efficient, rendering it well-suited for point-of-care requisites. By employing PTOLEMI alongside a microfluidic cell culture chip, physicians can attain personalized diagnostic and therapeutic insights. This paper elucidates the evolution of PTOLEMI and showcases its prowess in analyzing cancer patient samples within a microfluidic apparatus. While the integration of machine learning tools into biomedical domains is undoubtedly in progress, this study's innovation lies in the fusion of PTOLEMI with a microfluidic platform-an integrated, rapid, and independent framework for personalized drug screening-based clinical decision-making.

Propofol Inhibits Glioma Stem Cell Growth and Migration and Their Interaction with Microglia via BDNF-AS and Extracellular Vesicles.

Glioblastoma (GBM) is the most common and aggressive primary brain tumor. GBM contains a small subpopulation of glioma stem cells (GSCs) that are implicated in treatment resistance, tumor infiltration, and recurrence, and are thereby considered important therapeutic targets. Recent clinical studies have suggested that the choice of general anesthetic (GA), particularly propofol, during tumor resection, affects subsequent tumor response to treatments and patient prognosis. In this study, we investigated the molecular mechanisms underlying propofol's anti-tumor effects on GSCs and their interaction with microglia cells. Propofol exerted a dose-dependent inhibitory effect on the self-renewal, expression of mesenchymal markers, and migration of GSCs and sensitized them to both temozolomide (TMZ) and radiation. At higher concentrations, propofol induced a large degree of cell death, as demonstrated using microfluid chip technology. Propofol increased the expression of the lncRNA BDNF-AS, which acts as a tumor suppressor in GBM, and silencing of this lncRNA partially abrogated propofol's effects. Propofol also inhibited the pro-tumorigenic GSC-microglia crosstalk via extracellular vesicles (EVs) and delivery of BDNF-AS. In conclusion, propofol exerted anti-tumor effects on GSCs, sensitized these cells to radiation and TMZ, and inhibited their pro-tumorigenic interactions with microglia via transfer of BDNF-AS by EVs.

Microfluidic tool for rapid functional characterization of CRISPR complexes.

RNA guided nucleases are regarded as the future genome editing technologies. As such, they need to meet strong safety margins. Two major challenges in incorporating CRISPR technologies into the clinical world are off-target activity and editing efficiency. The common way to tackle such issues is to measure the binding and cleavage kinetics of the CRISPR enzyme. This can be challenging since, for example, DNA is not released from the CAS9 protein post cleavage. Here a promising new microfluidic approach to characterizing Enzymatic Interaction and Function of CRISPR complexes on a microfluidic platform (EnzyMIF) is presented. The method can rapidly detect the kd, koff, km and kcat for various RNA guided nucleases. In this work, two single guide RNAs with significantly different in-cell cleavage efficiency, RAG2 and RAG1, are used as proof-of-concept. The EnzyMIF assay results provide biochemical characterization of these guide RNAs that can explain the difference in cleavage using both wild type (WT) CAS9 and HiFi CAS9. Notably, it is shown that EnzyMIF characterization correlates with cell culture genomic editing efficiency results. It is suggested that EnzyMIF can predict the quality of cleavage rapidly and quantitatively.

Renal viability evaluated by the multiprobe assembly: a unique tool for the assessment of renal ischemic injury.

BACKGROUND: One of the major causes of transplanted organs' dysfunction is ischemia-reperfusion injury, where mitochondrial dysfunction is the primary contributor to cell damage. Mitochondrial NADH fluorescence reliably describes intracellular oxygen deficiency and mitochondrial function. Therefore, its monitoring at the tissue level, together with other physiological parameters, can serve to evaluate tissue vitality. METHODS: The multiprobe assembly (MPA) enabled the assessment of renal blood flow (RBF) using laser Doppler flowmetry, mitochondrial NADH redox state using the fluorometric technique, and ionic homeostasis using specific mini-electrodes (K(+) and H(+)). The MPA was utilized in two rat groups in which ischemia was induced for a period of 25-30 min (group 1) or for 60 min (group 2), and RBF and NADH were also monitored in a group of rats that underwent a complete kidney ischemia 24 h before the monitoring - a well-known model of acute renal failure. RESULTS: During ischemia, the RBF was completely abolished, NADH and extracellular potassium levels increased, and extracellular pH decreased. Immediately after the reperfusion, full recovery was observed; however, in the rats undergoing 60-min ischemia followed by 24-hour reperfusion, the tissue hemodynamic and mitochondrial functions were significantly impaired. CONCLUSION: This study demonstrates the advantage of using the MPA for real-time evaluation of kidney physiological state, which may serve as a practical instrument for the evaluation of graft viability during transplantation procedures.

Nitrite-induced improved blood circulation associated with an increase in a pool of RBC-NO with no bioactivity.

The reduction of nitrite by RBCs producing NO can play a role in regulating vascular tone. This hypothesis was investigated in rats by measuring the effect of nitrite infusion on mean arterial blood pressure (MAP), cerebral blood flow (CBF) and cerebrovascular resistance (CVR) in conjunction with the accumulation of RBC-NO. The nitrite infusion reversed the increase in MAP and decrease in CBF produced by L-NAME inhibition of e-NOS. At the same time there was a dramatic increase in RBC-NO. Correlations of RBC-NO for individual rats support a role for the regulation of vascular tone by this pool of NO. Furthermore, data obtained prior to treatment with L-NAME or nitrite are consistent with a contribution of RBC reduced nitrite in regulating vascular tone even under normal conditions. The role of the RBC in delivering NO to the vasculature was explained by the accumulation of a pool of bioactive NO in the RBC when nitrite is reduced by deoxygenated hemoglobin chains. A comparison of R and T state hemoglobin demonstrated a potential mechanism for the release of this NO in the T-state present at reduced oxygen pressures when blood enters the microcirculation. Coupled with enhanced hemoglobin binding to the membrane under these conditions the NO can be released to the vasculature.

Effects of anesthesia on brain mitochondrial function, blood flow, ionic and electrical activity monitored in vivo.

Thiopental, a well-known barbiturate, is often used in patients who are at high risk of developing cerebral ischemia, especially during brain surgery. Although barbiturates are known to affect a variety of processes in the cerebral cortex, including oxygen consumption by the mitochondria, the interrelation between mitochondrial function and anesthetics has not been investigated in detail under in vivo conditions. The aim of this study was to examine the effects of thiopental on brain functions in normoxia and under partial or complete ischemia. The use of the multiparametric monitoring system permitted simultaneous measurements of microcirculatory blood flow, NADH fluorescence, tissue reflectance, and ionic and electrical activities of the cerebral cortex. Thiopental caused a significant, dose-dependent decrease in blood flow and a significant decrease in extracellular levels of potassium, with no significant changes in NADH levels in normoxic and ischemic rats. Following complete ischemia (death), the increase in the reflectance was significantly smaller in the anesthetized normoxic group versus the awake normoxic group. The time until the secondary increase in reflectance, seen in death, was significantly shorter in the anesthetized ischemic group. In conclusion, it seems that the protective effect of thiopental occurs only under partial ischemia and not under complete ischemia.

An Integrated Microfluidics Approach for Personalized Cancer Drug Sensitivity and Resistance Assay.

Cancer is the second leading cause of death globally. Matching proper treatment and dosage is crucial for a positive outcome. Any given drug may affect patients with similar tumors differently. Personalized medicine aims to address this issue. Unfortunately, most cancer samples cannot be expanded in culture, limiting conventional cell-based testing. Herein, presented is a microfluidic device that combines a drug microarray with cell microscopy. The device can perform 512 experiments to test chemosensitivity and resistance to a drug array. MCF7 and 293T cells are cultured inside the device and their chemosensitivity and resistance to docetaxel, applied at various concentrations, are determined. Cell mortality is determined as a function of drug concentration and exposure time. It is found that both cell types form cluster morphology within the device, not evident in conventional tissue culture under similar conditions. Cells inside the clusters are less sensitive to drugs than dispersed cells. These findings support a heterogenous response of cancer cells to drugs. Then demonstrated is the principle of drug microarrays by testing cell response to four different drugs at four different concentrations. This approach may enable the personalization of treatment to the particular tumor and patient and may eventually improve final patient outcome.

A high-throughput integrated microfluidics method enables tyrosine autophosphorylation discovery.

Autophosphorylation of receptor and non-receptor tyrosine kinases is a common molecular switch with broad implications for pathogeneses and therapy of cancer and other human diseases. Technologies for large-scale discovery and analysis of autophosphorylation are limited by the inherent difficulty to distinguish between phosphorylation and autophosphorylation in vivo and by the complexity associated with functional assays of receptors kinases in vitro. Here, we report a method for the direct detection and analysis of tyrosine autophosphorylation using integrated microfluidics and freshly synthesized protein arrays. We demonstrate the efficacy of our platform in detecting autophosphorylation activity of soluble and transmembrane tyrosine kinases, and the dependency of in vitro autophosphorylation assays on membranes. Our method, Integrated Microfluidics for Autophosphorylation Discovery (IMAD), is high-throughput, requires low reaction volumes and can be applied in basic and translational research settings. To our knowledge, it is the first demonstration of posttranslational modification analysis of membrane protein arrays.

Real-time multi-site multi-parametric monitoring of rat brain subjected to traumatic brain injury.

INTRODUCTION: Traumatic brain injury (TBI) is one of the major causes of death in the world, with at least ten million serious traumatic brain injuries occurring annually; nevertheless, the pathophysiologic events taking place immediately after the injury are not yet fully known. OBJECTIVE: To study the effects of TBI on brain hemodynamic, metabolic and ionic homeostasis using the multi-parametric monitoring system. This system enables real-time monitoring of cerebral blood flow (CBF), mitochondrial NADH redox state, extracellular levels of K+, H+, DC potential, ECoG and ICP. METHODS: In order to find the best brain location for the monitoring device in relation to the fluid percussion injury site, we used the multi-site multi-parametric monitoring system. Two groups of rats were connected to four monitoring probes at four different locations near the injury site, two in each hemisphere. We monitored CBF, NADH redox state, tissue reflectance and DC steady potential in each of the four sites. RESULTS: Under anoxia, the initial CBF decrease was followed by an increase, NADH level increased, the reflectance decreased and dc potential showed a biphasic response, in all 4 locations. However, following fluid percussion injury, there was a significant variability in the responses in each of the 4 monitored locations. CONCLUSION: The advantage of the multi-parametric-monitoring approach for enhanced understanding of the injured brain was indicated. Moreover, we showed that contralateral monitoring of the injured brain gives good indication for the events taking place following fluid percussion brain injury.

Neuregulin 1 discovered as a cleavage target for the HCV NS3/4A protease by a microfluidic membrane protein array.

The hepatitis C virus (HCV) non-structural protein 3 (NS3) is essential for HCV maturation. The NS3/4A protease is a target for several HCV treatments and is a well-known target for HCV drug discovery. The protein is membrane associated and thus probably interacts with other membrane proteins. However, the vast majority of known NS3 host partners are soluble proteins rather than membrane proteins, most likely due to lack of appropriate platforms for their discovery. Utilization of an integrated microfluidics platform enables analysis of membrane proteins in their native form. We screened over 2800 membrane proteins for interaction with NS3 and 90 previously unknown interactions were identified. Of these, several proteins were selected for validation by co-immunoprecipitation and for NS3 proteolytic activity. Bearing in mind the considerable number of interactions formed, together with the popularity of NS3/4A protease as a drug target, it was striking to note its lack of proteolytic activity. Only a single protein, Neuregulin1, was observed to be cleaved, adding to the 3 known NS3/4A cleavage targets. Neuregulin1 participates in neural proliferation. Recent studies have shown its involvement in HCV infection and hepatocellular carcinoma. We showed that NS3/4A triggers an increase in neuregulin1 mRNA levels in HCV infected cells. Despite this increase, its protein concentration is decreased due to proteolytic cleavage. Additionally, its EGF-like domain levels were increased, possibly explaining the ErbB2 and EGFR upregulation in HCV infected cells. The newly discovered protein interactions may provide insights into HCV infection mechanisms and potentially provide new therapeutic targets against HCV.

Draft Genome Sequence of a New Bacterium Named Rappaport israeli, gen. nov., sp. nov., Isolated from a Blood Culture.

Here, we report the draft genome sequence of a Gram-negative microbe found in a blood culture (B08008) from a patient. The organism was proposed to be from a new unknown genus and species. This publication will increase worldwide microbial knowledge and may improve microbial identification and antibiotic treatment for patients.

Draft Genome Sequence of the Suttonellaornithocola Bacterium.

We report here the draft genome sequence of the Suttonella ornithocola bacterium. To date, this bacterium, found in birds, passed only phylogenetic and phenotypic analyses. To our knowledge, this is the first publication of the Suttonella ornithocola genome sequence. The genetic profile provides a basis for further analysis of its infection pathways.

Control and automation of multilayered integrated microfluidic device fabrication.

Integrated microfluidics is a sophisticated three-dimensional (multi layer) solution for high complexity serial or parallel processes. Fabrication of integrated microfluidic devices requires soft lithography and the stacking of thin-patterned PDMS layers. Precise layer alignment and bonding is crucial. There are no previously reported standards for alignment of the layers, which is mostly performed using uncontrolled processes with very low alignment success. As a result, integrated microfluidics is mostly used in academia rather than in the many potential industrial applications. We have designed and manufactured a semiautomatic Microfluidic Device Assembly System (µDAS) for full device production. µDAS comprises an electrooptic mechanical system consisting of four main parts: optical system, smart media holder (for PDMS), a micropositioning xyzθ system and a macropositioning XY mechanism. The use of the µDAS yielded valuable information regarding PDMS as the material for device fabrication, revealed previously unidentified errors, and enabled optimization of a robust fabrication process. In addition, we have demonstrated the utilization of the µDAS technology for fabrication of a complex 3 layered device with over 12 000 micromechanical valves and an array of 64 × 64 DNA spots on a glass substrate with high yield and high accuracy. We increased fabrication yield from 25% to about 85% with an average layer alignment error of just ∼4 µm. It also increased our protein expression yields from 80% to over 90%, allowing us to investigate more proteins per experiment. The µDAS has great potential to become a valuable tool for both advancing integrated microfluidics in academia and producing and applying microfluidic devices in the industry.

In vivo multiparametric monitoring of brain functions under intracranial hypertension following mannitol administration.

OBJECTIVE: Over the last 20 years, mannitol has replaced other osmotic diuretics. Its beneficial effects on intracranial pressure (ICP), cerebral perfusion pressure (CPP), cerebral blood flow (CBF) and brain metabolism are widely accepted. In the present study, we tested the effect of mannitol injection on brain hemodynamic, metabolic, ionic and electrical state in rats exposed to intracranial hypertension. METHODS: The parameters monitored simultaneously included ICP, CBF using the laser Doppler flowmetry, mitochondrial NADH redox state by the fluorometric technique, extracellular K(+) and H(+) levels, DC potential, ECoG, blood pressure and calculated CPP. ICP was elevated to 30 mmHg for 30 minutes and mannitol was injected 15 minutes post-ICP elevation. RESULTS: Our results showed that mannitol decreased ICP, and improved the levels of MAP, CPP and CBF. Moreover, mannitol completely prevented mortality following intracranial hypertension in rats. CONCLUSION: It seems that the multiparametric monitoring approach, used in intracranial hypertension models, is an important tool for brain functional state evaluation.

An off-the-shelf integrated microfluidic device comprising self-assembled monolayers for protein array experiments.

Microfluidic-based protein arrays are promising tools for life sciences, with increased sensitivity and specificity. One of the drawbacks of this technology is the need to create fresh surface chemistry for protein immobilization at the beginning of each experiment. In this work, we attempted to include the process of surface functionalization as part of the fabrication of the device, which would substitute the time consuming step of surface functionalization at the beginning of each protein array experiment. To this end, we employed a novel surface modification using self-assembled monolayers (SAMs) to immobilize biomolecules within the channels of a polydimethylsiloxane (PDMS) integrated microfluidic device. As a model, we present a general method for depositing siloxane-anchored SAMs, with 1-undecyl-thioacetate-trichlorosilane (C11TA) on the silica surfaces. The process involved developing PDMS-compatible conditions for both SAM deposition and functional group activation. We successfully demonstrated the ability to produce, within an integrated microfluidic channel, a C11TA monolayer with a covalently conjugated antibody. The antibody could then bind its antigen with a high signal to background ratio. We further demonstrated that the antibody was still active after storage of the device for a week. Integration of the surface chemistry into the device as part of its fabrication process has potential to significantly simplify and shorten many experimental procedures involving microfluidic-based protein arrays. In turn, this will allow for broader dissemination of this important technology.

Effects of elevated ICP on brain function: can the multiparametric monitoring system detect the 'Cushing Response'?

The 'Cushing Response' is a significant phenomenon associated with elevated ICP. The purpose of our study was to examine the effects of the intracranial hypertension level and duration on the cerebral tissue physiology, using a Multiprobe assembly (MPA). The parameters monitored simultaneously included ICP, CBF, mitochondrial NADH redox state, extracellular K+ and H+ levels, DC potential and ECoG, calculated CPP and blood pressure. Two groups of rats were used. In one group, ICP was elevated to 50-60 mmHg for 13-15 min and, in the second group, ICP was elevated to 20 mmHg for 30 min. The results show that ICP of 50-60 mmHg led to CPP reduction below the lower limits of autoregulation. However, ICP of 20 mmHg, even for a prolonged period of time is completely tolerated. Additionally, we found that the 'Cushing Response', developed in the moderate treatment (ICP = 20 mmHg) is beneficial, assuring high CBF levels under intracranial hypertension. Furthermore, CBF and CPP monitoring, apparently, are not sufficient for autoregulation assessment; more parameters are needed.

Effects of euthanasia on brain physiological activities monitored in real-time.

Animal experimentation is terminated by the euthanasia procedure in order to avoid pain and minimize suffering. Very little is known about the real time physiological changes taking place in the brain of animals during the euthanasia. Since there is no way to evaluate the suffering of animals under euthanasia, it is assumed that objective physiological changes taking place could serve as a good way to compare various types of euthanasia procedures. In the present study we compared the effect of euthanasia induced by i. v. injection of concentrated KCL to that of Taxan T-61 (a standard mixture used by veterinarians). The responses of the cat brain were evaluated by monitoring the hemodynamic (CBF), metabolic (NADH redox state), electrical (EcoG) and extracellular ion levels, as an indicator to the ionic homeostasis.