Multiview motion tracking based on a cartesian robot to monitor Caenorhabditis elegans in standard Petri dishes.

Data from manual healthspan assays of the nematode Caenorhabditis elegans (C. elegans) can be complex to quantify. The first attempts to quantify motor performance were done manually, using the so-called thrashing or body bends assay. Some laboratories have automated these approaches using methods that help substantially to quantify these characteristic movements in small well plates. Even so, it is sometimes difficult to find differences in motor behaviour between strains, and/or between treated vs untreated worms. For this reason, we present here a new automated method that increases the resolution flexibility, in order to capture more movement details in large standard Petri dishes, in such way that those movements are less restricted. This method is based on a Cartesian robot, which enables high-resolution images capture in standard Petri dishes. Several cameras mounted strategically on the robot and working with different fields of view, capture the required C. elegans visual information. We have performed a locomotion-based healthspan experiment with several mutant strains, and we have been able to detect statistically significant differences between two strains that show very similar movement patterns.

An Immunocompetent Microphysiological System to Simultaneously Investigate Effects of Anti-Tumor Natural Killer Cells on Tumor and Cardiac Microtissues.

Existing first-line cancer therapies often fail to cope with the heterogeneity and complexity of cancers, so that new therapeutic approaches are urgently needed. Among novel alternative therapies, adoptive cell therapy (ACT) has emerged as a promising cancer treatment in recent years. The limited clinical applications of ACT, despite its advantages over standard-of-care therapies, can be attributed to (i) time-consuming and cost-intensive procedures to screen for potent anti-tumor immune cells and the corresponding targets, (ii) difficulties to translate in-vitro and animal-derived in-vivo efficacies to clinical efficacy in humans, and (iii) the lack of systemic methods for the safety assessment of ACT. Suitable experimental models and testing platforms have the potential to accelerate the development of ACT. Immunocompetent microphysiological systems (iMPS) are microfluidic platforms that enable complex interactions of advanced tissue models with different immune cell types, bridging the gap between in-vitro and in-vivo studies. Here, we present a proof-of-concept iMPS that supports a triple culture of three-dimensional (3D) colorectal tumor microtissues, 3D cardiac microtissues, and human-derived natural killer (NK) cells in the same microfluidic network. Different aspects of tumor-NK cell interactions were characterized using this iMPS including: (i) direct interaction and NK cell-mediated tumor killing, (ii) the development of an inflammatory milieu through enrichment of soluble pro-inflammatory chemokines and cytokines, and (iii) secondary effects on healthy cardiac microtissues. We found a specific NK cell-mediated tumor-killing activity and elevated levels of tumor- and NK cell-derived chemokines and cytokines, indicating crosstalk and development of an inflammatory milieu. While viability and morphological integrity of cardiac microtissues remained mostly unaffected, we were able to detect alterations in their beating behavior, which shows the potential of iMPS for both, efficacy and early safety testing of new candidate ACTs.

Viability of a Serum Infliximab Concentration-Detecting Reagent as a Qualitative Assay for an Infliximab Biosimilar.

The efficacy of infliximab in treating rheumatoid arthritis depends on its serum trough concentration, which must be maintained at a minimum of 1 µg/mL to achieve the desired effects. However, Japan's National Health Insurance system does not cover tests for rheumatoid arthritis patients undergoing treatment with biosimilar infliximab because its performance as a biosimilar remains unclear. This study aimed to investigate whether the Remi-check Q qualitative assay yields comparable results for biosimilar infliximab and the originator product. Infliximab BS 100 "NK" and Remicade 100® were separately diluted in pooled human serum to yield test samples at the following concentrations: 0.30, 0.70, 1.20, and 3.00 µg/mL. Prepared samples were quantitatively assessed using an enzyme-linked immunosorbent assay (ELISA) and qualitatively using Remi-check Q, and the results obtained for the originator and biosimilar product were compared. For both originator and biosimilar infliximab, Remi-check Q yielded a negative result for all 0.30 and 0.70 µg/mL samples and a positive result for all 3.00 µg/mL samples. However, negative results were obtained with a fraction of the 1.20 µg/mL samples (biosimilar, 4/15; originator, 3/15). Concurrence rates between the results of quantitative ELISA and qualitative Remi-check Q analyses were comparable between originator and biosimilar infliximab at all tested concentrations. These results indicate that Remi-check Q yields comparable results for biosimilar infliximab and the originator product on being used as a qualitative assay for trough serum levels.

Bioassay of Some Indigenous Entomopathogens for Controlling Rhynchophorus ferrugineus, Olivier in Saudi Arabia.

<b>Background and Objective:</b> The red palm weevil is a dangerous date palm pests that cannot be controlled with chemical pesticides only. As a result of the justified concerns of the negative use of synthetic insecticides on human health and the environment. So on, candidate eco-friendly micro-organisms isolated from KSA agri-ecosystems were evaluated in controlling RPW. <b>Materials and Methods:</b> Some indigenous entomopathogenic fungi and bacteria were isolated from naturally infected RPW larvae and adults and evaluated as alternative control methods. <b>Results:</b> The infection of RPW larvae with entomopathogenic fungi and bacteria under natural conditions was higher than in adults. <i>Beauveria bassiana </i>was the most prevalent followed by <i>Aspergillus </i>sp., <i>Metarhizium anisopliae</i>, <i>Mucor</i> sp., <i>Cladosporium chlorocephalum</i>. In contrast, both <i>Bacillus</i> <i>thuringiensis</i> and <i>Bacillus popilliae</i> formed 73.9 and 26.1%, respectively. From the 7th day, mortalities (%) increased gradually and recorded the highest mortalities with 21st days after treatment and recorded 93.33, 66.70, 53.36, 46.69 and 60.00% when treated with <i>B. bassiana</i>, <i>M. anisopliae</i>, <i>C. chlorosphalum</i>, <i>Mucor</i> sp. and <i>Aspergillus</i> sp., respectively. <b>Conclusion:</b> Although there was evidence indicating midgut damage and feeding inhibition among larvae that survived the treatments, instead of lower activity of <i>B. thuringiensis</i> against <i>R. ferrugineus</i> immature stages may refer to that, Both species of <i>Bacillus</i> were more virulent as the days 15-21 post-treatment.

Predictive biomarkers and clinical evidence.

Predictive biomarkers play an important role in our efforts to individualize pharmacotherapy, and within recent years, a number of different types of assays have been introduced. These biomarkers may potentially support the selection and dosage of specific drugs in order to maximize efficacy and minimize adverse reactions in the individual patient. However, in many instances, the scientific and clinical evidence is insufficient to support the prescribing decision. When predictive biomarkers are used to guide pharmacotherapy, it is important to secure that decisions are based on solid clinical evidence. Here, the regulatory authorities, especially the FDA, have been at the forefront in relation to regulate this type of biomarker assay in order to secure patient safety. The approval process for companion diagnostics is an example of this effort, where the scientific validity of the biomarker and assay is in focus. With the approaching implementation of the new IVD Regulation, greater attention will also be paid to analytical and clinical validity of biomarker assays in the EU. For any type of predictive biomarker assay, including pharmacogenetic and tumour profiling tests, the clinical evidence needs to be in place before they are used routinely in the clinic.

New mosquito repellency bioassay for evaluation of repellents and pyrethroids using an attractive blood-feeding device.

BACKGROUND: With the increasing threat of the worldwide spread of mosquito-borne infectious diseases, consumer interest in anti-mosquito textiles that protect against mosquito bites is also increasing. Accordingly, repellent- or insecticide-treated textiles are gaining popularity. The standardization of commercial textile products is, therefore, indispensable for an authentic and objective evaluation of these products. Here we report a textile testing method using an artificial blood-feeding system that does not involve human volunteers or live animals, which aligns with the policy of protecting human and animal welfare. METHODS: The attractive blood-feeding device (ABFD) was designed using the Hemotek® membrane feeding system. The repellency of DEET, icaridin and permethrin was assayed using unfed female adults of Aedes albopictus (Skuse) under two different test conditions, namely choice and no-choice tests. The choice test consisted of two feeding units, one chemically treated and untreated, that were installed on the ABFD; mosquitoes attracted to and resting on the feeding units were counted and the overall blood-feeding rates recorded. The no-choice test consisted of two feeding units treated with the same chemical that were installed on the ABFD; mosquitoes attracted to and resting on the feeding units were counted and the blood-feeding rates were recorded. A control test was conducted using two feeding units, both sides of which were untreated. RESULTS: In the choice test, high repellency (> 95% inhibition of resting on the treated surface) of 1% DEET and 2% icaridin was observed, whereas 2% permethrin was not an effective repellent. Also, high blood-feeding inhibition (> 95%) was observed for 2% DEET and 2% icaridin. In the no-choice test, high repellency was observed for 1% DEET and 2% icaridin, whereas the repellency of 2% permethrin was low. Also, high blood-feeding inhibition was observed for 2% DEET, 4% icaridin and 2% permethrin. CONCLUSIONS: The accuracy and reproducibility of the developed method demonstrate that the ABFD may be widely used for fundamental experiments in the field of mosquito physiology, for the development of new repellent chemicals and in evaluation studies of mosquito repellent products, such as anti-mosquito textiles. The further development of the membrane and feeding unit systems will enable a more practical evaluation of mosquito repellents and blood-feeding inhibitors, such as pyrethroids.

A Portable Magnetic Particle Spectrometer for Future Rapid and Wash-Free Bioassays.

Nowadays, there is an increasing demand for more accessible routine diagnostics for patients with respect to high accuracy, ease of use, and low cost. However, the quantitative and high accuracy bioassays in large hospitals and laboratories usually require trained technicians and equipment that is both bulky and expensive. In addition, the multistep bioassays and long turnaround time could severely affect the disease surveillance and control especially in pandemics such as influenza and COVID-19. In view of this, a portable, quantitative bioassay device will be valuable in regions with scarce medical resources and help relieve burden on local healthcare systems. Herein, we introduce the MagiCoil diagnostic device, an inexpensive, portable, quantitative, and rapid bioassay platform based on the magnetic particle spectrometer (MPS) technique. MPS detects the dynamic magnetic responses of magnetic nanoparticles (MNPs) and uses the harmonics from oscillating MNPs as metrics for sensitive and quantitative bioassays. This device does not require trained technicians to operate and employs a fully automatic, one-step, and wash-free assay with a user friendly smartphone interface. Using a streptavidin-biotin binding system as a model, we show that the detection limit of the current portable device for streptavidin is 64 nM (equal to 5.12 pmole). In addition, this MPS technique is very versatile and allows for the detection of different diseases just by changing the surface modifications on MNPs. Although MPS-based bioassays show high sensitivities as reported in many literatures, at the current stage, this portable device faces insufficient sensitivity and needs further improvements. It is foreseen that this kind of portable device can transform the multistep, laboratory-based bioassays to one-step field testing in nonclinical settings such as schools, homes, offices, etc.

A Facile Strategy for Immobilizing GOD and HRP onto Pollen Grain and Its Application to Visual Detection of Glucose.

Pollen grain was explored as a new carrier for enzyme immobilization. After being modified with boric acid-functionalized titania, the pollen grain was able to covalently immobilize glycosylated enzymes by boronate affinity interaction under very mild experimental conditions (e.g., pH 7.0, ambient temperature and free of organic solvent). The glucose oxidase and horse radish peroxidase-immobilized pollen grain became a bienzyme system. The pollen grain also worked as an indicator of the cascade reaction by changing its color. A rapid, simple and cost-effective approach for the visual detection of glucose was then developed. When the glucose concentration exceeded 0.5 mM, the color change was observable by the naked eye. The assay of glucose in body fluid samples exhibited its great potential for practical application.

Lifetime encoding in flow cytometry for bead-based sensing of biomolecular interaction.

To demonstrate the potential of time-resolved flow cytometry (FCM) for bioanalysis, clinical diagnostics, and optically encoded bead-based assays, we performed a proof-of-principle study to detect biomolecular interactions utilizing fluorescence lifetime (LT)-encoded micron-sized polymer beads bearing target-specific bioligands and a recently developed prototype lifetime flow cytometer (LT-FCM setup). This instrument is equipped with a single excitation light source and different fluorescence detectors, one operated in the photon-counting mode for time-resolved measurements of fluorescence decays and three detectors for conventional intensity measurements in different spectral windows. First, discrimination of bead-bound biomolecules was demonstrated in the time domain exemplarily for two targets, Streptavidin (SAv) and the tumor marker human chorionic gonadotropin (HCG). In a second step, the determination of biomolecule concentration levels was addressed representatively for the inflammation-related biomarker tumor necrosis factor (TNF-α) utilizing fluorescence intensity measurements in a second channel of the LT-FCM instrument. Our results underline the applicability of LT-FCM in the time domain for measurements of biomolecular interactions in suspension assays. In the future, the combination of spectral and LT encoding and multiplexing and the expansion of the time scale from the lower nanosecond range to the longer nanosecond and the microsecond region is expected to provide many distinguishable codes. This enables an increasing degree of multiplexing which could be attractive for high throughput screening applications.

Assessing an alpha-defensin lateral flow device for diagnosing septic arthritis: reporting on a false-negative case and a false-positive case.

Alpha-defensin (αD), an antimicrobial peptide released by neutrophils in response to bacterial pathogens, was proposed as a novel diagnostic biomarker in synovial fluid. Several reports have shown that αD can serve as a reliable biomarker in the diagnosis of periprosthetic joint infection (PJI). We assessed whether αD could also serve to diagnosis of septic arthritis, a similarly difficult to diagnose PJI. To our knowledge, besides PJI, few reports exist assessing the utility of αD for septic arthritis. We have attempted to diagnose several cases of suspected septic arthritis using the Synovasure® αD detection lateral flow device. We report a false-positive case and a false-negative case. The false-negative case we experienced was caused by Staphylococcus capitis, which is coagulase-negative, and possibly represents a low virulence micro-organism infection. The false-positive case was ultimately diagnosed as seronegative rheumatoid arthritis and possessed calcium pyrophosphate depositions. False positives have been suggested to occur in conditions where neutrophils are mobilised. As for PJI, in cases where diagnosis is difficult, αD can be an additional diagnostic indicator. However, making a definitive diagnosis using the αD lateral flow device alone was found to be difficult. The utility of αD in assessing septic arthritis is inconclusive; therefore, larger prospective clinical studies should be considered for a better assessment.

A ferrobotic system for automated microfluidic logistics.

Automated technologies that can perform massively parallelized and sequential fluidic operations at small length scales can resolve major bottlenecks encountered in various fields, including medical diagnostics, -omics, drug development, and chemical/material synthesis. Inspired by the transformational impact of automated guided vehicle systems on manufacturing, warehousing, and distribution industries, we devised a ferrobotic system that uses a network of individually addressable robots, each performing designated micro-/nanofluid manipulation-based tasks in cooperation with other robots toward a shared objective. The underlying robotic mechanism facilitating fluidic operations was realized by addressable electromagnetic actuation of miniature mobile magnets that exert localized magnetic body forces on aqueous droplets filled with biocompatible magnetic nanoparticles. The contactless and high-strength nature of the actuation mechanism inherently renders it rapid (~10 centimeters/second), repeatable (>10,000 cycles), and robust (>24 hours). The robustness and individual addressability of ferrobots provide a foundation for the deployment of a network of ferrobots to carry out cross-collaborative logistics efficiently. These traits, together with the reconfigurability of the system, were exploited to devise and integrate passive/active advanced functional components (e.g., droplet dispensing, generation, filtering, and merging), enabling versatile system-level functionalities. By applying this ferrobotic system within the framework of a microfluidic architecture, the ferrobots were tasked to work cross-collaboratively toward the quantification of active matrix metallopeptidases (a biomarker for cancer malignancy and inflammation) in human plasma, where various functionalities converged to achieve a fully automated assay.

Comparison of bacterial endotoxin testing methods in purified pharmaceutical water matrices.

Current bacterial endotoxin testing systems can be labor-intensive and time-consuming, involving several manual pipetting steps. In our quality control laboratory, annually, we test about 15,000 samples of different grades of purified water, WFI and water samples taken to validate cleaning procedures for endotoxins. We are currently using the Kinetic-QCL™ assay which is a pharmacopeia method that provides reliable results. We compared this assay with another Limulus amebocyte lysate (LAL)-based assay (Endosafe®-MCS) and an alternative endpoint fluorescent recombinant Factor C (rFC) assay (ENDOZYME II GO®). Both these assays have been developed to reduce analyst preparation time. Our objective was to assess if they could increase the throughput of our testing while maintaining low rates of invalid results. The results demonstrated that the two most appropriate methods for rapid endotoxin detection in water are our current assay, K-QCL, and the rFC-based assay, ENDOZYME II GO. This latter assay was found to be less sensitive to interference than our current assay, particularly in cleaning validation water samples. It also showed better performance, accuracy, repeatability and had a shorter time-to-results. ENDOZYME II GO assay allows quick testing of large numbers of samples with reliable results and is a good alternative for conventional LAL assays.

Evaluation and modeling of direct membrane-feeding assay with Plasmodium vivax to support development of transmission blocking vaccines.

Standard and direct membrane-feeding assays (SMFA and DMFA) are fundamental assays to evaluate efficacy of transmission-blocking intervention (TBI) candidates against Plasmodium falciparum and vivax. To compare different candidates precisely, it is crucial to understand the error range of measured activity, usually expressed as percent inhibition in either oocyst intensity (% transmission reducing activity, %TRA), or in prevalence of infected mosquitoes (% transmission blocking activity, %TBA). To this end, mathematical models have been proposed for P. falciparum SMFA (PfSMFA), but such study for DMFA is limited. In this study, we analyzed P. vivax DMFA (PvDMFA) data from 22,236 mosquitoes tested from 96 independent assays. While the two assays are quite different, a zero-inflated negative binomial (ZINB) model could reasonably explain the PvDMFA results, as it has for PfSMFA. Our simulation studies based on the ZINB model revealed it is better to report %TRA values with a proper error range, rather than observed %TBA both in SMFA and DMFA. Furthermore, the simulations help in designing a better assay and aid in estimating an error range of a %TRA value when the uncertainty is not reported. This study strongly supports future TBI development by providing a rational method to compare different candidates.

Chromatographic methods development for clinical practice: requirements and limitations.

Development of a chromatographic method in bioanalysis is a challenging and complex procedure with many pitfalls and often unexpected reversals that can require several months to accomplish. Even an experienced analytical team must contend many limitations mainly in connection with the strict requirements imposed on current clinical research. These restrictions typically persist throughout the whole development process, from clinical trial assignment, across optimization of extraction of biological materials and chromatographic separation, to validation and data interpretation. This paper describes questions and their possible answers raised during the pre-analytical phase such as use of modern sample preparation techniques in clinical methods, application of internal standards, as well as selection of stationary phases and detection techniques in the analytical phase. Validation problems and interpretation of results are demonstrated with three typical examples of characteristics to be considered, i.e. recovery, matrix effect, and limit of detection vs. lower limit of quantification.

Assessing motor-related phenotypes of Caenorhabditis elegans with the wide field-of-view nematode tracking platform.

Caenorhabditis elegans is a valuable model organism in biomedical research that has led to major discoveries in the fields of neurodegeneration, cancer and aging. Because movement phenotypes are commonly used and represent strong indicators of C. elegans fitness, there is an increasing need to replace manual assessments of worm motility with automated measurements to increase throughput and minimize observer biases. Here, we provide a protocol for the implementation of the improved wide field-of-view nematode tracking platform (WF-NTP), which enables the simultaneous analysis of hundreds of worms with respect to multiple behavioral parameters. The protocol takes only a few hours to complete, excluding the time spent culturing C. elegans, and includes (i) experimental design and preparation of samples, (ii) data recording, (iii) software management with appropriate parameter choices and (iv) post-experimental data analysis. We compare the WF-NTP with other existing worm trackers, including those having high spatial resolution. The main benefits of WF-NTP relate to the high number of worms that can be assessed at the same time on a whole-plate basis and the number of phenotypes that can be screened for simultaneously.

Optical Single Cell Resolution Cytotoxicity Biosensor Based on Single Cell Adhesion Dot Arrays.

Low cost, easy to use cell viability tests are needed in the pharmaceutical, biomaterial, and environmental industries to measure adverse cellular effects. We present a new methodology to track cell death with high resolution. Adherent cells commonly detach from the surface when they die, but some toxic compounds promote cell adhesion. A methodology that enables both dynamic detachment monitoring but also rapid detection of toxic effects of compounds that promote cell adhesion would constitute a step forward toward high-throughput cytotoxicity measurements. We achieved dynamic digital quantification of cell viability by simple optical imaging using "single cell adhesion dot arrays" (SCADA), fibronectin (FN) dot arrays designed to accommodate a single cell on each fibronectin dot. For cytotoxicity measurements, cell-filled SCADA substrates were exposed to K2CrO4, HgSO4 salts, and dimethyl sulfoxide (DMSO). The toxic effect of DMSO and K2CrO4 was dynamically monitored by measuring the cell detachment rate during more than 30 h by quantifying the number of occupied dots in the SCADA array. HgSO4 inhibited cellular detachment from the surface, and cytotoxicity was monitored using the trypan blue life/death assay directly on the surface. In all cases, the cytotoxicity effects were easily monitored with single cell resolution, and the results were comparable to previous reports. SCADA enabled dynamic measurements at the highest resolution due to the digital measuring in this method. The integration of SCADA substrates into microfluidic platforms will provide a practical tool that will extend to fundamental research and commercial applications.

Rapid lipid bilayer membrane formation on Parylene coated apertures to perform ion channel analyses.

We present a chip design allowing rapid and robust lipid bilayer (LBL) membrane formation using a Parylene coated thin silicon nitride aperture. After bilayer formation, single membrane channels can be reconstituted and characterized by electrophysiology. The ability for robust reconstitution will allow parallelization and enhanced screening of small molecule drugs acting on or permeating across the membrane channel. The aperture was realized on a microfabricated silicon nitride membrane by using standard clean-room fabrication processes. To ensure the lipid bilayer formation, the nitride membrane was coated with a hydrophobic and biocompatible Parylene layer. We tested both Parylene-C and Parylene-AF4. The contact angle measurements on both Parylene types showed very good hydrophobic properties and affinity to lipids. No precoating of the Parylene with an organic solvent is needed to make the aperture lipophilic, in contradiction to Teflon membranes. The chips can be easily placed in an array utilizing a 3D printed platform. Experiments show repetitive LBL formation and destruction (more than 6 times) within a very short time (few seconds). Through measurements we have established that the LBL layers are very thin. This allows the investigation of the fusion process of membrane proteins i.e. outer membrane protein (OmpF) in the LBL within a few minutes.

Resistance to deltamethrin in Triatoma infestans: microgeographical distribution, validation of a rapid detection bioassay and evaluation of a fumigant canister as control alternative strategy.

Triatoma infestans (Klug) (Hemiptera: Reduviidae) is the main vector of Chagas disease in the Southern Cone of America and resistance to pyrethroid insecticides has been detected in several areas from its geographical distribution. Pyrethroid resistance presents a complex geographical pattern at different spatial scales. However, it is still unknown if the toxicological variability is a common feature within villages of the Gran Chaco were high resistance was descripted. The objectives of this study were to determine: (a) the microgeographical distribution of the deltamethrin-resistance in insects from Pampa Argentina village, (b) the performance of the insecticide impregnated paper bioassay to evaluate deltamethrin-resistance in field collected insects and (c) the lethal activity of the fumigant canister containing DDVP against insects resistant to deltamethrin. High survival of T. infestans exposed to discriminant dose was observed in the samples of all the evaluated dwellings, suggesting that the resistance to deltamethrin is homogeneous at the microgeographical level. Resistance determination by impregnated paper bioassay was similar to traditional topical determination, highlighting the use of this rapid methodology in field large-scale monitoring. The fumigant canister was not effective against resistant insects, remarking the need to develop suitable formulations that ensure minimal toxicological risk and high effectivity.