Digital diffraction analysis enables low-cost molecular diagnostics on a smartphone.

The widespread distribution of smartphones, with their integrated sensors and communication capabilities, makes them an ideal platform for point-of-care (POC) diagnosis, especially in resource-limited settings. Molecular diagnostics, however, have been difficult to implement in smartphones. We herein report a diffraction-based approach that enables molecular and cellular diagnostics. The D3 (digital diffraction diagnosis) system uses microbeads to generate unique diffraction patterns which can be acquired by smartphones and processed by a remote server. We applied the D3 platform to screen for precancerous or cancerous cells in cervical specimens and to detect human papillomavirus (HPV) DNA. The D3 assay generated readouts within 45 min and showed excellent agreement with gold-standard pathology or HPV testing, respectively. This approach could have favorable global health applications where medical access is limited or when pathology bottlenecks challenge prompt diagnostic readouts.

Feeding intolerance in critically ill patients with COVID-19.

BACKGROUND & AIMS: Early reports suggest significant difficulty with enteral feeding in critically ill COVID-19 patients. This study aimed to characterize the prevalence, clinical manifestations, and outcomes of feeding intolerance in critically ill patients with COVID-19. METHODS: We examined 323 adult patients with COVID-19 admitted to the intensive care units (ICUs) of Massachusetts General Hospital between March 11 and June 28, 2020 who received enteral nutrition. Systematic chart review determined prevalence, clinical characteristics, and hospital outcomes (ICU complications, length of stay, and mortality) of feeding intolerance. RESULTS: Feeding intolerance developed in 56% of the patients and most commonly manifested as large gastric residual volumes (83.9%), abdominal distension (67.2%), and vomiting (63.9%). Length of intubation (OR 1.05, 95% CI 1.03-1.08), ≥1 GI symptom on presentation (OR 0.76, 95% CI 0.59-0.97), and severe obesity (OR 0.29, 95% CI 0.13-0.66) were independently associated with development of feeding intolerance. Compared to feed-tolerant patients, patients with incident feeding intolerance were significantly more likely to suffer cardiac, renal, hepatic, and hematologic complications during their hospitalization. Feeding intolerance was similarly associated with poor outcomes including longer ICU stay (median [IQR] 21.5 [14-30] vs. 15 [9-22] days, P < 0.001), overall hospitalization time (median [IQR] 30.5 [19-42] vs. 24 [15-35], P < 0.001) and in-hospital mortality (33.9% vs. 16.1%, P < 0.001). Feeding intolerance was independently associated with an increased risk of death (HR 3.32; 95% CI 1.97-5.6). CONCLUSIONS: Feeding intolerance is a frequently encountered complication in critically ill COVID-19 patients in a large tertiary care experience and is associated with poor outcomes.

Paramagnetic Fluorinated Nanoemulsions for in vivo F-19 MRI.

PURPOSE: We aim to develop perfluorocarbon-based nanoemulsions with improved sensitivity for detection of inflammatory macrophages in situ using F-19 MRI. Towards this goal, we evaluate the feasibility of nanoemulsion formulation incorporating a metal chelate in the fluorous phase which shortens the F-19 longitudinal relaxation rate and image acquisition time. PROCEDURES: Perfluorinated linear polymers were conjugated to metal-binding tris-diketonate, blended with unconjugated polymers, and emulsified in water. Phospholipid-based surfactant was used to stabilize nanoemulsion and provide biocompatibility. Nanoemulsions were metalated with the addition of ferric salt to the buffer. Physical stability of surfactant and nanoemulsion was evaluated by mass spectrometry and dynamic light scattering measurements. Nanoemulsions were injected intravenously into a murine granuloma inflammation model, and in vivo19F/1H MRI at 11.7 T was performed. RESULTS: We demonstrated stability and biocompatibility of lipid-based paramagnetic nanoemulsions. We investigated potential oxidation of lipid in the presence of metal chelate. As a proof of concept, we performed non-invasive monitoring of macrophage burden in a murine inflammation model following intravenous injection of nanoemulsion using in vivo F-19 MRI. CONCLUSION: Lipid-based nanoemulsion probes of perfluorocarbon synthesized with iron-binding fluorinated ß-diketones can be formulated for intravenous delivery and inflammation detection in vivo.

Norepinephrine: material-independent, multifunctional surface modification reagent.

A facile approach for material-independent surface modification using norepinephrine was investigated. pH-induced oxidative polymerization of norepinephrine forms adherent films on vastly different types of material surfaces of noble metals, metal oxides, semiconductors, ceramics, shape-memory alloys, and synthetic polymers. Secondary biochemical functionalizations such as immobilization of proteins and growth of biodegradable polyester on the poly(norepinephrine) films were demonstrated.

Magnetic nanosensor for detection and profiling of erythrocyte-derived microvesicles.

During the course of their lifespan, erythrocytes actively shed phospholipid-bound microvesicles (MVs). In stored blood, the number of these erythrocyte-derived MVs has been observed to increase over time, suggesting their potential value as a quality metric for blood products. The lack of sensitive, standardized MV assays, however, poses a significant barrier to implementing MV analyses into clinical settings. Here, we report on a new nanotechnology platform capable of rapid and sensitive MV detection in packed red blood cell (pRBC) units. A filter-assisted microfluidic device was designed to enrich MVs directly from pRBC units, and label them with target-specific magnetic nanoparticles. Subsequent detection using a miniaturized nuclear magnetic resonance system enabled accurate MV quantification as well as the detection of key molecular markers (CD44, CD47, CD55). When the developed platform was applied, MVs in stored blood units could also be monitored longitudinally. Our results showed that MV counts increase over time and, thus, could serve as an effective metric of blood aging. Furthermore, our studies found that MVs have the capacity to generate oxidative stress and consume nitric oxide. By advancing our understanding of MV biology, we expect that the developed platform will lead to improved blood product quality and transfusion safety.

Live cell imaging distinguishes bona fide human iPS cells from partially reprogrammed cells.

Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells by enforced expression of transcription factors. Using serial live imaging of human fibroblasts undergoing reprogramming, we identified distinct colony types that morphologically resemble embryonic stem (ES) cells yet differ in molecular phenotype and differentiation potential. By analyzing expression of pluripotency markers, methylation at the OCT4 and NANOG promoters and differentiation into teratomas, we determined that only one colony type represents true iPS cells, whereas the others represent reprogramming intermediates. Proviral silencing and expression of TRA-1-60, DNMT3B and REX1 can be used to distinguish the fully reprogrammed state, whereas alkaline phosphatase, SSEA-4, GDF3, hTERT and NANOG are insufficient as markers. We also show that reprogramming using chemically defined medium favors formation of fully reprogrammed over partially reprogrammed colonies. Our data define molecular markers of the fully reprogrammed state and highlight the need for rigorous characterization and standardization of putative iPS cells.

Differential methylation of tissue- and cancer-specific CpG island shores distinguishes human induced pluripotent stem cells, embryonic stem cells and fibroblasts.

Induced pluripotent stem (iPS) cells are derived by epigenetic reprogramming, but their DNA methylation patterns have not yet been analyzed on a genome-wide scale. Here, we find substantial hypermethylation and hypomethylation of cytosine-phosphate-guanine (CpG) island shores in nine human iPS cell lines as compared to their parental fibroblasts. The differentially methylated regions (DMRs) in the reprogrammed cells (denoted R-DMRs) were significantly enriched in tissue-specific (T-DMRs; 2.6-fold, P < 10(-4)) and cancer-specific DMRs (C-DMRs; 3.6-fold, P < 10(-4)). Notably, even though the iPS cells are derived from fibroblasts, their R-DMRs can distinguish between normal brain, liver and spleen cells and between colon cancer and normal colon cells. Thus, many DMRs are broadly involved in tissue differentiation, epigenetic reprogramming and cancer. We observed colocalization of hypomethylated R-DMRs with hypermethylated C-DMRs and bivalent chromatin marks, and colocalization of hypermethylated R-DMRs with hypomethylated C-DMRs and the absence of bivalent marks, suggesting two mechanisms for epigenetic reprogramming in iPS cells and cancer.