Targeting and monitoring ovarian cancer invasion with an RNAi and peptide delivery system.

RNA interference (RNAi) therapeutics are an emerging class of medicines that selectively target mRNA transcripts to silence protein production and combat disease. Despite the recent progress, a generalizable approach for monitoring the efficacy of RNAi therapeutics without invasive biopsy remains a challenge. Here, we describe the development of a self-reporting, theranostic nanoparticle that delivers siRNA to silence a protein that drives cancer progression while also monitoring the functional activity of its downstream targets. Our therapeutic target is the transcription factor SMARCE1, which was previously identified as a key driver of invasion in early-stage breast cancer. Using a doxycycline-inducible shRNA knockdown in OVCAR8 ovarian cancer cells both in vitro and in vivo, we demonstrate that SMARCE1 is a master regulator of genes encoding proinvasive proteases in a model of human ovarian cancer. We additionally map the peptide cleavage profiles of SMARCE1-regulated proteases so as to design a readout for downstream enzymatic activity. To demonstrate the therapeutic and diagnostic potential of our approach, we engineered self-assembled layer-by-layer nanoparticles that can encapsulate nucleic acid cargo and be decorated with peptide substrates that release a urinary reporter upon exposure to SMARCE1-related proteases. In an orthotopic ovarian cancer xenograft model, theranostic nanoparticles were able to knockdown SMARCE1 which was in turn reported through a reduction in protease-activated urinary reporters. These LBL nanoparticles both silence gene products by delivering siRNA and noninvasively report on downstream target activity by delivering synthetic biomarkers to sites of disease, enabling dose-finding studies as well as longitudinal assessments of efficacy.

Microenvironment-triggered multimodal precision diagnostics.

Therapeutic outcomes in oncology may be aided by precision diagnostics that offer early detection, localization and the opportunity to monitor response to therapy. Here, we report a multimodal nanosensor engineered to target tumours through acidosis, respond to proteases in the microenvironment to release urinary reporters and (optionally) carry positron emission tomography probes to enable localization of primary and metastatic cancers in mouse models of colorectal cancer. We present a paradigm wherein this multimodal sensor can be employed longitudinally to assess burden of disease non-invasively, including tumour progression and response to chemotherapy. Specifically, we showed that acidosis-mediated tumour insertion enhanced on-target release of matrix metalloproteinase-responsive reporters in urine. Subsequent on-demand loading of the radiotracer 64Cu allowed pH-dependent tumour visualization, enabling enriched microenvironmental characterization when compared with the conventional metabolic tracer 18F-fluorodeoxyglucose. Through tailored target specificities, this modular platform has the capacity to be engineered as a pan-cancer test that may guide treatment decisions for numerous tumour types.

Using tele-ultrasound to teach medical students: A randomised control equivalence study.

Objectives: Undergraduate ultrasound education is becoming increasingly important, but its expansion is limited by time, space and the availability of trained faculty. In order to validate an alternative and more accessible teaching model, our aim was to assess whether combining teleguidance and peer-assisted learning to teach ultrasound is as effective as traditional in-person methods. Methods: Peer instructors taught 47 second-year medical students ocular ultrasound via either teleguidance or traditional in-person methods. Proficiency was assessed using a multiple-choice knowledge test and objective structured clinical examination (OSCE). Confidence, overall experience, and experience with a peer instructor were measured using a 5-point Likert scale. Two one-sided t-tests were used to measure equivalency between the two groups. The null hypothesis that the two groups were not different was rejected when P < 0.05. Results: The teleguidance group performed as well as the traditional in-person group in terms of knowledge change, confidence change, OSCE time and OSCE score (p = 0.011, p = 0.006, p = 0.005 and  = 0.004, respectively, indicating the two groups are statistically equivalent). The teleguidance group rated the experience highly overall (4.06/5), but less than the traditional group (4.47/5; P = 0.448, indicating statistical difference). Peer instruction was rated 4.35/5 overall. Conclusion: Peer-instructed teleguidance was equivalent to in-person instruction with respect to knowledge change, confidence gain and OSCE performance in basic ocular ultrasound.

CRISPR-Cas-mediated Multianalyte Synthetic Urine Biomarker Test for Portable Diagnostics.

Creating synthetic biomarkers for the development of precision diagnostics has enabled detection of disease through pathways beyond those used for traditional biofluid measurements. Synthetic biomarkers generally make use of reporters that provide readable signals in the biofluid to reflect the biochemical alterations in the local disease microenvironment during disease incidence and progression. The pharmacokinetic concentration of the reporters and biochemical amplification of the disease signal are paramount to achieving high sensitivity and specificity in a diagnostic test. Here, a cancer diagnostic platform is built using one format of synthetic biomarkers: activity-based nanosensors carrying chemically stabilized DNA reporters that can be liberated by aberrant proteolytic signatures in the tumor microenvironment. Synthetic DNA as a disease reporter affords multiplexing capability through its use as a barcode, allowing for the readout of multiple proteolytic signatures at once. DNA reporters released into the urine are detected using CRISPR nucleases via hybridization with CRISPR RNAs, which in turn produce a fluorescent or colorimetric signal upon enzyme activation. In this protocol, DNA-barcoded, activity-based nanosensors are constructed and their application is exemplified in a preclinical mouse model of metastatic colorectal cancer. This system is highly modifiable according to disease biology and generates multiple disease signals simultaneously, affording a comprehensive understanding of the disease characteristics through a minimally invasive process requiring only nanosensor administration, urine collection, and a paper test which enables point-of-care diagnostics.

CRISPR-Cas-amplified urinary biomarkers for multiplexed and portable cancer diagnostics.

Synthetic biomarkers, bioengineered sensors that generate molecular reporters in diseased microenvironments, represent an emerging paradigm in precision diagnostics. Despite the utility of DNA barcodes as a multiplexing tool, their susceptibility to nucleases in vivo has limited their utility. Here we exploit chemically stabilized nucleic acids to multiplex synthetic biomarkers and produce diagnostic signals in biofluids that can be 'read out' via CRISPR nucleases. The strategy relies on microenvironmental endopeptidase to trigger the release of nucleic acid barcodes and polymerase-amplification-free, CRISPR-Cas-mediated barcode detection in unprocessed urine. Our data suggest that DNA-encoded nanosensors can non-invasively detect and differentiate disease states in transplanted and autochthonous murine cancer models. We also demonstrate that CRISPR-Cas amplification can be harnessed to convert the readout to a point-of-care paper diagnostic tool. Finally, we employ a microfluidic platform for densely multiplexed, CRISPR-mediated DNA barcode readout that can potentially evaluate complex human diseases rapidly and guide therapeutic decisions.

Acute injuries in American collegiate taekwondo competitions: a prospective study spanning four competitive seasons.

BACKGROUND: Injury patterns are well-documented for taekwondo competitions prior to the use of an electronic chest protector for scoring tabulation. To see if injury rates and types changed following this rule change that transformed the fighting style in taekwondo, we investigated injuries in collegiate taekwondo competitions in the USA. METHODS: Data were collected at eight collegiate taekwondo tournaments from April 2018 to December 2019. All injured athletes seen at the first-aid station were invited to complete a survey that included injury location, type, and mechanism of injury. Injury rates were calculated per 1000 athlete-exposures (A-Es) and minute-exposures (M-Es). Risk factors were modeled using logistic regression and χ2 analysis. RESULTS: Out of 1096 athletes, 194 athletes reported 275 acute injuries. We found an injury risk of 17.7/100 athletes (95% CI: 15.4, 20.0) and injury rate of 68.9/1000 A-E (95% CI: 60.7, 77.0) which was comparable to previous studies. The most common injuries were contusions to the lower limbs. In contrast to prior reports, men were injured more frequently from delivering a kick and women from receiving a kick. Populations at higher risk for injury included those with low belt rank and middle weight class for women. CONCLUSIONS: It appears that the new fighting style did not affect injury rates. Injury locations and types remain similar, but the mechanisms of injury have reversed as men are more injured from attacking and women from defending. There remains a strong need for research to improve protective equipment and safety rules in taekwondo.

Cancer Cell-Derived Matrisome Proteins Promote Metastasis in Pancreatic Ductal Adenocarcinoma.

The prognosis for pancreatic ductal adenocarcinoma (PDAC) remains poor despite decades of effort. The abundant extracellular matrix (ECM) in PDAC comprises a major fraction of the tumor mass and plays various roles in promoting resistance to therapies. However, nonselective depletion of ECM has led to poor patient outcomes. Consistent with that observation, we previously showed that individual matrisome proteins derived from stromal cells correlate with either long or short patient survival. In marked contrast, those derived from cancer cells correlate strongly with poor survival. Here, we studied three cancer cell-derived matrisome proteins that are significantly overrepresented during PDAC progression, AGRN (agrin), SERPINB5 (serine protease inhibitor B5), and CSTB (cystatin B). Using both overexpression and knockdown experiments, we demonstrate that all three are promoters of PDAC metastasis. Furthermore, these proteins operate at different metastatic steps. AGRN promoted epithelial-to-mesenchymal transition in primary tumors, whereas SERPINB5 and CSTB enhanced late steps in the metastatic cascade by elevating invadopodia formation and in vivo extravasation. All three genes were associated with a poor prognosis in human patients and high levels of SERPINB5, secreted by cancer cells and deposited in the ECM, correlated with poor patient prognosis. This study provides strong evidence that cancer cell-derived matrisome proteins can be causal in promoting tumorigenesis and metastasis and lead to poor patient survival. Therefore, compared with the bulk matrix, mostly made by stromal cells, precise interventions targeting cancer cell-derived matrisome proteins, such as AGRN, SERPINB5, and CSTB, may represent preferred potential therapeutic targets. SIGNIFICANCE: This study provides insights into the biological roles of cancer cell-derived matrisome proteins in PDAC and supports the notion that these proteins are protumorigenic and better therapeutic targets.