Minimally instrumented SHERLOCK (miSHERLOCK) for CRISPR-based point-of-care diagnosis of SARS-CoV-2 and emerging variants.

The COVID-19 pandemic highlights the need for diagnostics that can be rapidly adapted and deployed in a variety of settings. Several SARS-CoV-2 variants have shown worrisome effects on vaccine and treatment efficacy, but no current point-of-care (POC) testing modality allows their specific identification. We have developed miSHERLOCK, a low-cost, CRISPR-based POC diagnostic platform that takes unprocessed patient saliva; extracts, purifies, and concentrates viral RNA; performs amplification and detection reactions; and provides fluorescent visual output with only three user actions and 1 hour from sample input to answer out. miSHERLOCK achieves highly sensitive multiplexed detection of SARS-CoV-2 and mutations associated with variants B.1.1.7, B.1.351, and P.1. Our modular system enables easy exchange of assays to address diverse user needs and can be rapidly reconfigured to detect different viruses and variants of concern. An adjunctive smartphone application enables output quantification, automated interpretation, and the possibility of remote, distributed result reporting.

Phosphorylation of ACTN4 Leads to Podocyte Vulnerability and Proteinuric Glomerulosclerosis.

BACKGROUND: Genetic mutations in α-actinin-4 (ACTN4)-an important actin crosslinking cytoskeletal protein that provides structural support for kidney podocytes-have been linked to proteinuric glomerulosclerosis in humans. However, the effect of post-translational modifications of ACTN4 on podocyte integrity and kidney function is not known. METHODS: Using mass spectrometry, we found that ACTN4 is phosphorylated at serine (S) 159 in human podocytes. We used phosphomimetic and nonphosphorylatable ACTN4 to comprehensively study the effects of this phosphorylation in vitro and in vivo. We conducted x-ray crystallography, F-actin binding and bundling assays, and immunofluorescence staining to evaluate F-actin alignment. Microfluidic organ-on-a-chip technology was used to assess for detachment of podocytes simultaneously exposed to fluid flow and cyclic strain. We then used CRISPR/Cas9 to generate mouse models and assessed for renal injury by measuring albuminuria and examining kidney histology. We also performed targeted mass spectrometry to determine whether high extracellular glucose or TGF-ß levels increase phosphorylation of ACTN4. RESULTS: Compared with the wild type ACTN4, phosphomimetic ACTN4 demonstrated increased binding and bundling activity with F-actin in vitro. Phosphomimetic Actn4 mouse podocytes exhibited more spatially correlated F-actin alignment and a higher rate of detachment under mechanical stress. Phosphomimetic Actn4 mice developed proteinuria and glomerulosclerosis after subtotal nephrectomy. Moreover, we found that exposure to high extracellular glucose or TGF-ß stimulates phosphorylation of ACTN4 at S159 in podocytes. CONCLUSIONS: These findings suggest that increased phosphorylation of ACTN4 at S159 leads to biochemical, cellular, and renal pathology that is similar to pathology resulting from human disease-causing mutations in ACTN4. ACTN4 may mediate podocyte injury as a consequence of both genetic mutations and signaling events that modulate phosphorylation.

Author Correction: On-chip recapitulation of clinical bone marrow toxicities and patient-specific pathophysiology.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

On-chip recapitulation of clinical bone marrow toxicities and patient-specific pathophysiology.

The inaccessibility of living bone marrow (BM) hampers the study of its pathophysiology under myelotoxic stress induced by drugs, radiation or genetic mutations. Here, we show that a vascularized human BM-on-a-chip (BM chip) supports the differentiation and maturation of multiple blood cell lineages over 4 weeks while improving CD34+ cell maintenance, and that it recapitulates aspects of BM injury, including myeloerythroid toxicity after clinically relevant exposures to chemotherapeutic drugs and ionizing radiation, as well as BM recovery after drug-induced myelosuppression. The chip comprises a fluidic channel filled with a fibrin gel in which CD34+ cells and BM-derived stromal cells are co-cultured, a parallel channel lined by human vascular endothelium and perfused with culture medium, and a porous membrane separating the two channels. We also show that BM chips containing cells from patients with the rare genetic disorder Shwachman-Diamond syndrome reproduced key haematopoietic defects and led to the discovery of a neutrophil maturation abnormality. As an in vitro model of haematopoietic dysfunction, the BM chip may serve as a human-specific alternative to animal testing for the study of BM pathophysiology.

Robotic fluidic coupling and interrogation of multiple vascularized organ chips.

Organ chips can recapitulate organ-level (patho)physiology, yet pharmacokinetic and pharmacodynamic analyses require multi-organ systems linked by vascular perfusion. Here, we describe an 'interrogator' that employs liquid-handling robotics, custom software and an integrated mobile microscope for the automated culture, perfusion, medium addition, fluidic linking, sample collection and in situ microscopy imaging of up to ten organ chips inside a standard tissue-culture incubator. The robotic interrogator maintained the viability and organ-specific functions of eight vascularized, two-channel organ chips (intestine, liver, kidney, heart, lung, skin, blood-brain barrier and brain) for 3 weeks in culture when intermittently fluidically coupled via a common blood substitute through their reservoirs of medium and endothelium-lined vascular channels. We used the robotic interrogator and a physiological multicompartmental reduced-order model of the experimental system to quantitatively predict the distribution of an inulin tracer perfused through the multi-organ human-body-on-chips. The automated culture system enables the imaging of cells in the organ chips and the repeated sampling of both the vascular and interstitial compartments without compromising fluidic coupling.

Scalable Fabrication of Stretchable, Dual Channel, Microfluidic Organ Chips.

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human patients. Human Organ-on-a-Chip (Organ Chip) microfluidic cell culture devices, which provide an experimental in vitro platform to assess efficacy, toxicity, and pharmacokinetic (PK) profiles in humans, may be better predictors of therapeutic efficacy and safety in the clinic compared to animal studies. These devices may be used to model the function of virtually any organ type and can be fluidically linked through common endothelium-lined microchannels to perform in vitro studies on human organ-level and whole body-level physiology without having to conduct experiments on people. These Organ Chips consist of two perfused microfluidic channels separated by a permeable elastomeric membrane with organ-specific parenchymal cells on one side and microvascular endothelium on the other, which can be cyclically stretched to provide organ-specific mechanical cues (e.g., breathing motions in lung). This protocol details the fabrication of flexible, dual channel, Organ Chips through casting of parts using 3D printed molds, enabling combination of multiple casting and post-processing steps. Porous poly (dimethyl siloxane) (PDMS) membranes are cast with micrometer sized through-holes using silicon pillar arrays under compression. Fabrication and assembly of Organ Chips involves equipment and steps that can be implemented outside of a traditional cleanroom. This protocol provides researchers with access to Organ Chip technology for in vitro organ- and body-level studies in drug discovery, safety and efficacy testing, as well as mechanistic studies of fundamental biological processes.

Rapid Prototyping of Thermoplastic Microfluidic Devices.

Microfluidic systems can be applied to develop unique tools for cell culture, low-cost diagnostics, and precision experimentation by leveraging microscale fluid flow. As the field has expanded and matured, there is a need for rapid prototyping that is both accessible to most research groups and can readily translate toward scalable commercial manufacturing. Here, we describe a protocol that incorporates rapid computer numerical control (CNC) milling of positive molds, casting of a negative high-durometer silicone mold, and hot embossing to produce microfluidic devices composed of virtually any thermoplastic material. The method bypasses the need for high-precision machining of the bonding surfaces by using a cast acrylic stock and only milling channels, thus expanding this protocol to any CNC platform This technique represents a versatile, high-fidelity prototyping method that enables fast turnaround of prototype devices in a standard laboratory setting, while offering scalability for commercial manufacturing.

Proatherogenic Flow Increases Endothelial Stiffness via Enhanced CD36-Mediated Uptake of Oxidized Low-Density Lipoproteins.

OBJECTIVE: Disturbed flow (DF) is well-known to induce endothelial dysfunction and synergistically with plasma dyslipidemia facilitate plaque formation. Little is known, however, about the synergistic impact of DF and dyslipidemia on endothelial biomechanics. Our goal was to determine the impact of DF on endothelial stiffness and evaluate the role of dyslipidemia/oxLDL (oxidized low-density lipoprotein) in this process. APPROACH AND RESULTS: Endothelial elastic modulus of intact mouse aortas ex vivo and of human aortic endothelial cells exposed to laminar flow or DF was measured using atomic force microscopy. Endothelial monolayer of the aortic arch is found to be significantly stiffer than the descending aorta (4.2+1.1 versus 2.5+0.2 kPa for aortic arch versus descending aorta) in mice maintained on low-fat diet. This effect is significantly exacerbated by short-term high-fat diet (8.7+2.5 versus 4.5+1.2 kPa for aortic arch versus descending aorta). Exposure of human aortic endothelial cells to DF in vitro resulted in 50% increase in oxLDL uptake and significant endothelial stiffening in the presence but not in the absence of oxLDL. DF also increased the expression of oxLDL receptor CD36 (cluster of differentiation 36), whereas downregulation of CD36 abrogated DF-induced endothelial oxLDL uptake and stiffening. Furthermore, genetic deficiency of CD36 abrogated endothelial stiffening in the aortic arch in vivo in mice fed either low-fat diet or high-fat diet. We also show that the loss of endothelial stiffening in CD36 knockout aortas is not mediated by the loss of CD36 in circulating cells. CONCLUSIONS: DF facilitates endothelial CD36-dependent uptake of oxidized lipids resulting in local increase of endothelial stiffness in proatherogenic areas of the aorta.

Bubble removal with the use of a vacuum pressure generated by a converging-diverging nozzle.

Bubbles are an intrinsic problem in microfluidic devices and they can appear during the initial filling of the device or during operation. This report presents a generalizable technique to extract bubbles from microfluidic networks using an adjacent microfluidic negative pressure network over the entire microfluidic channel network design. We implement this technique by superimposing a network of parallel microchannels with a vacuum microfluidic channel and characterize the bubble extraction rates as a function of negative pressure applied. In addition, we generate negative pressure via a converging-diverging (CD) nozzle, which only requires inlet gas pressure to operate. Air bubbles generated during the initial liquid filling of the microfluidic network are removed within seconds and their volume extraction rate is calculated. This miniaturized vacuum source can achieve a vacuum pressure of 7.23 psi which corresponds to a bubble extraction rate of 9.84 pL/s, in the microfluidic channels we characterized. Finally, as proof of concept it is shown that the bubble removal system enables bubble removal on difficult to fill microfluidic channels such as circular or triangular shaped channels. This method can be easily integrated into many microfluidic experimental protocols.