Explore the vast range of titles available.

The unprecedented demand for rapid diagnostics in response to the COVID‐19 pandemic has brought the spotlight onto clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated systems (Cas)‐assisted nucleic acid detection assays. Already benefitting from an elegant detection mechanism, fast assay time, and low reaction temperature, these assays can be further advanced via integration with powerful, digital‐based detection. Thus motivated, the first digital CRISPR/Cas‐assisted assay—coined digitization‐enhanced CRISPR/Cas‐assisted one‐pot virus detection (deCOViD)—is developed and applied toward SARS‐CoV‐2 detection. deCOViD is realized through tuning and discretizing a one‐step, fluorescence‐based, CRISPR/Cas12a‐assisted reverse transcription recombinase polymerase amplification assay into sub‐nanoliter reaction wells within commercially available microfluidic digital chips. The uniformly elevated digital concentrations enable deCOViD to achieve qualitative detection in <15 min and quantitative detection in 30 min with high signal‐to‐background ratio, broad dynamic range, and high sensitivity—down to 1 genome equivalent (GE) µL−1 of SARS‐CoV‐2 RNA and 20 GE µL−1 of heat‐inactivated SARS‐CoV‐2, which outstrips its benchtop‐based counterpart and represents one of the fastest and most sensitive CRISPR/Cas‐assisted SARS‐CoV‐2 detection to date. Moreover, deCOViD can detect RNA extracts from clinical samples. Taken together, deCOViD opens a new avenue for advancing CRISPR/Cas‐assisted assays and combating the COVID‐19 pandemic and beyond. A digital clustered regularly interspaced short palindromic repeats (CRISPR)/Cas‐assisted nucleic acid detection assay is created by discretizing a one‐step, fluorescence‐based, CRISPR/Cas12a‐assisted reverse transcription recombinase polymerase amplification assay within sub‐nanoliter reaction wells of a commercial microfluidic digital chip, which enables quantitative detection of 1 genome equivalent µL−1 of SARS‐CoV‐2 RNA in <30 min—one of the fastest and most sensitive CRISPR/Cas‐assisted detection to date.

Injury and loss of oligodendrocytes can cause demyelinating diseases such as multiple sclerosis. To improve our understanding of human oligodendrocyte development, which could facilitate development of remyelination-based treatment strategies, here we describe time-course single-cell-transcriptomic analysis of developing human stem cell-derived oligodendrocyte-lineage-cells (hOLLCs). The study includes hOLLCs derived from both genome engineered embryonic stem cell (ESC) reporter cells containing an Identification-and-Purification tag driven by the endogenous PDGFRα promoter and from unmodified induced pluripotent (iPS) cells. Our analysis uncovers substantial transcriptional heterogeneity of PDGFRα-lineage hOLLCs. We discover sub-populations of human oligodendrocyte progenitor cells (hOPCs) including a potential cytokine-responsive hOPC subset, and identify candidate regulatory genes/networks that define the identity of these sub-populations. Pseudotime trajectory analysis defines developmental pathways of oligodendrocytes vs astrocytes from PDGFRα-expressing hOPCs and predicts differentially expressed genes between the two lineages. In addition, pathway enrichment analysis followed by pharmacological intervention of these pathways confirm that mTOR and cholesterol biosynthesis signaling pathways are involved in maturation of oligodendrocytes from hOPCs. Brain myelinating oligodendrocytes are rare and difficult to isolate, which has limited data on their development. Here the authors develop a reporter for scalable purification of human pluripotent stem cell derived oligodendrocyte lineage cells, and use this to map differentiation using single cell RNA-sequencing,

Droplet microfluidics has in recent years found a wide range of analytical and bioanalytical applications. In droplet microfluidics, the samples that are discretized into droplets within the devices are predominantly loaded through tubings, but such tubing-based sample loading has drawbacks such as limited scalability for processing many samples, difficulty for automation, and sample wastage. While advances in autosamplers have alleviated some of these drawbacks, sample loading that can instead obviate tubings offers a potentially promising alternative but has been underexplored. To fill the gap, we introduce herein a droplet device that features a new Tubing Eliminated Sample Loading Interface (TESLI). TESLI integrates a network of programmable pneumatic microvalves that regulate vacuum and pressure sources so that successive sub-microliter samples can be directly spotted onto the open-to-atmosphere TESLI inlet, vacuumed into the device, and pressurized into nanoliter droplets within the device with minimal wastage. The same vacuum and pressure regulation also endows TESLI with cleaning and sample switching capabilities, thus enabling scalable processing of many samples in succession. Moreover, we implement a pair of TESLIs in our device to parallelize and alternate their operation as means to minimizing idle time. For demonstration, we use our device to successively process 44 samples into droplets—a number that can further scale. Our results demonstrate the feasibility of tubing-free sample loading and a promising approach for advancing droplet microfluidics.

Empiric broad‐spectrum antimicrobial treatments of urinary tract infections (UTIs) have contributed to widespread antimicrobial resistance. Clinical adoption of evidence‐based treatments necessitates rapid diagnostic methods for pathogen identification (ID) and antimicrobial susceptibility testing (AST) with minimal sample preparation. In response, a microfluidic droplet‐based platform is developed for achieving both ID and AST from urine samples within 30 min. In this platform, fluorogenic hybridization probes are utilized to detect 16S rRNA from single bacterial cells encapsulated in picoliter droplets, enabling molecular identification of uropathogenic bacteria directly from urine in as little as 16 min. Moreover, in‐droplet single‐bacterial measurements of 16S rRNA provide a surrogate for AST, shortening the exposure time to 10 min for gentamicin and ciprofloxacin. A fully integrated device and screening workflow were developed to test urine specimens for one of seven unique diagnostic outcomes including the presence/absence of Gram‐negative bacteria, molecular ID of the bacteriaas Escherichia coli, an Enterobacterales, or other organism, and assessment of bacterial susceptibility to ciprofloxacin. In a 50‐specimen clinical comparison study, the platform demonstrates excellent performance compared to clinical standard methods (areas‐under‐curves, AUCs >0.95), within a small fraction of the turnaround time, highlighting its clinical utility. Single‐cell measurements of bacterial 16S rRNA are leveraged in picoliter droplets to achieve simultaneous molecular detection of bacteria and phenotypic assessment of antimicrobial susceptibility, unlocking a 30 min sample‐to‐answer diagnostic platform for urinary tract infections.

Photoreceptor loss is a leading cause of blindness, but mechanisms underlying photoreceptor degeneration are not well understood. Treatment strategies would benefit from improved understanding of gene-expression patterns directing photoreceptor development, as many genes are implicated in both development and degeneration. Neural retina leucine zipper (NRL) is critical for rod photoreceptor genesis and degeneration, with NRL mutations known to cause enhanced S-cone syndrome and retinitis pigmentosa. While murine Nrl loss has been characterized, studies of human NRL can identify important insights for human retinal development and disease. We utilized iPSC organoid models of retinal development to molecularly define developmental alterations in a human model of NRL loss. Consistent with the function of NRL in rod fate specification, human retinal organoids lacking NRL develop S-opsin dominant photoreceptor populations. We report generation of two distinct S-opsin expressing populations in NRL null retinal organoids and identify MEF2C as a candidate regulator of cone development. Kallman et al. showed the effect of Nrl in human PSC-derived retinal organoids. Using histological and single cell transcriptomics, they identified an intermediate “cod” subpopulation in the predominant S-opsin population. Their findings provide important insights for human retinal development and degeneration.

We present a method to induce electric fields and drive electrotaxis (galvanotaxis) without the need for electrodes to be in contact with the media containing the cell cultures. We report experimental results using a modification of the transmembrane assay, demonstrating the hindrance of migration of breast cancer cells (SCP2) when an induced a.c. electric field is present in the appropriate direction (i.e. in the direction of migration). Of significance is that migration of these cells is hindered at electric field strengths many orders of magnitude (5 to 6) below those previously reported for d.c. electrotaxis and even in the presence of a chemokine (SDF-1α) or a growth factor (EGF). Induced a.c. electric fields applied in the direction of migration are also shown to hinder motility of non-transformed human mammary epithelial cells (MCF10A) in the presence of the growth factor EGF. In addition, we also show how our method can be applied to other cell migration assays (scratch assay) and by changing the coil design and holder, that it is also compatible with commercially available multi-well culture plates.

Droplet microfluidic technology is becoming increasingly useful for high-throughput and high-sensitivity detection of biological and biochemical reactions. Most current droplet devices function by passively discretizing a single sample subject to a homogeneous or random reagent/reaction condition into tens of thousands of picoliter-volume droplets for analysis. Despite their apparent advantages in speed and throughput, these droplet devices inherently lack the capability to customize the contents of droplets in order to test a single sample against multiple reagent conditions or multiple samples against multiple reagents. In order to incorporate such combinatorial capability into droplet platforms, we have developed the fully Integrated Programmable Picodroplet Assembler. Our platform is capable of generating customized picoliter-volume droplet groups from nanoliter-volume plugs which are assembled in situ on demand. By employing a combination of microvalves and flow-focusing-based discretization, our platform can be used to precisely control the content and volume of generated nanoliter-volume plugs, and thereafter the content and the effective dynamic range of picoliter-volume droplets. Furthermore, we can use a single integrated device for continuously generating, incubating, and detecting multiple distinct droplet groups. The device successfully marries the precise control and on-demand capability of microvalve-based platforms with the sensitivity and throughput of picoliter droplet platforms in a fully automated monolithic device. The device ultimately will find important applications in single-cell and single-molecule analyses.

The emergence of multi-drug resistant bacteria due to indiscriminate use of broad-spectrum antibiotics has grown into a global healthcare crisis. This is due in part to clinical reliance on century-old culture-based methods for pathogen identification (ID) and antimicrobial susceptibility testing (AST) which necessitate several days for definitively diagnosing patients and guiding targeted antimicrobial treatments. To overcome this bottleneck, improve patient outcomes, and increase antimicrobial stewardship, there is an urgent need for rapid diagnostic methods for pathogen ID and AST that can be easily operated, with minimal sample preparation. This thesis investigates the use of droplet microfluidics for expediting definitive diagnoses of infectious diseases, via single-cell detection of bacterial pathogens. We demonstrate the utility of droplet-based measurements of single-bacterial cells towards achieving multiplexed and quantitative molecular detection (ID) of bacteria and towards accelerating AST to the timescale of bacterial replication. In expanding the clinical utility of these methods, we present facile filtration-based strategies for direct evaluation of clinical urine samples using our droplet platforms. Next, we integrate our findings to develop a rapid sample-to-answer platform to achieve both ID and AST directly from patient urine samples for urinary tract infections. Finally, we present a strategy for further enhancing the throughput, multiplexability, and hence the clinical translatability of our sample-to-answer platform. The strategies outlined herein represent an important step in bringing droplet-based technologies to the clinic, and we hope that this work can help pave the way for droplet-based solutions to increasingly life-threatening infectious diseases.

Atypical hemolytic uremic syndrome (aHUS) is a rare disorder resulting from a dysregulated activation of the alternative pathway of the complement system. It results in significant morbidity and mortality if not diagnosed and treated promptly. It lends itself to myriad renal and extrarenal manifestations, all potentially disabling. Eculizumab, a monoclonal antibody to complement C5 is now the widely accepted norm for treatment. However, in resource-limited settings, plasma exchange if instituted early may be as beneficial. We report a case of aHUS treated with extended plasma exchange with excellent results. Critical care monitoring is essential for the management of the disease in view of a tendency to develop multiple complications. Long-term immunosuppression may be successful in maintaining remission.