Signal Transduction in Human Cell Lysate via Dynamic RNA Nanotechnology.

Dynamic RNA nanotechnology with small conditional RNAs (scRNAs) offers a promising conceptual approach to introducing synthetic regulatory links into endogenous biological circuits. Here, we use human cell lysate containing functional Dicer and RNases as a testbed for engineering scRNAs for conditional RNA interference (RNAi). scRNAs perform signal transduction via conditional shape change: detection of a subsequence of mRNA input X triggers formation of a Dicer substrate that is processed to yield small interfering RNA (siRNA) output anti-Y targeting independent mRNA Y for destruction. Automated sequence design is performed using the reaction pathway designer within NUPACK to encode this conditional hybridization cascade into the scRNA sequence subject to the sequence constraints imposed by X and Y. Because it is difficult for secondary structure models to predict which subsequences of mRNA input X will be accessible for detection, here we develop the RNAhyb method to experimentally determine accessible windows within the mRNA that are provided to the designer as sequence constraints. We demonstrate the programmability of scRNA regulators by engineering scRNAs for transducing in both directions between two full-length mRNAs X and Y, corresponding to either the forward molecular logic "if X then not Y" (X [Formula: see text] Y) or the reverse molecular logic "if Y then not X" (Y [Formula: see text] X). In human cell lysate, we observe a strong OFF/ON conditional response with low crosstalk, corresponding to a ≈20-fold increase in production of the siRNA output in response to the cognate versus noncognate full-length mRNA input. 2'OMe-RNA chemical modifications protect signal transduction reactants and intermediates against RNase degradation while enabling Dicer processing of signal transduction products. Because diverse biological pathways interact with RNA, scRNAs that transduce between detection of endogenous RNA inputs and production of biologically active RNA outputs hold great promise as a synthetic regulatory paradigm.

Comparison of precision and speed in laparoscopic and robot-assisted surgical task performance.

BACKGROUND: Robotic platforms have the potential advantage of providing additional dexterity and precision to surgeons while performing complex laparoscopic tasks, especially for those in training. Few quantitative evaluations of surgical task performance comparing laparoscopic and robotic platforms among surgeons of varying experience levels have been done. We compared measures of quality and efficiency of Fundamentals of Laparoscopic Surgery task performance on these platforms in novices and experienced laparoscopic and robotic surgeons. METHODS: Fourteen novices, 12 expert laparoscopic surgeons (>100 laparoscopic procedures performed, no robotics experience), and five expert robotic surgeons (>25 robotic procedures performed) performed three Fundamentals of Laparoscopic Surgery tasks on both laparoscopic and robotic platforms: peg transfer (PT), pattern cutting (PC), and intracorporeal suturing. All tasks were repeated three times by each subject on each platform in a randomized order. Mean completion times and mean errors per trial (EPT) were calculated for each task on both platforms. Results were compared using Student's t-test (P < 0.05 considered statistically significant). RESULTS: Among novices, greater errors were noted during laparoscopic PC (Lap 2.21 versus Robot 0.88 EPT, P < 0.001). Among expert laparoscopists, greater errors were noted during laparoscopic PT compared with robotic (PT: Lap 0.14 versus Robot 0.00 EPT, P = 0.04). Among expert robotic surgeons, greater errors were noted during laparoscopic PC compared with robotic (Lap 0.80 versus Robot 0.13 EPT, P = 0.02). Among expert laparoscopists, task performance was slower on the robotic platform compared with laparoscopy. In comparisons of expert laparoscopists performing tasks on the laparoscopic platform and expert robotic surgeons performing tasks on the robotic platform, expert robotic surgeons demonstrated fewer errors during the PC task (P = 0.009). CONCLUSIONS: Robotic assistance provided a reduction in errors at all experience levels for some laparoscopic tasks, but no benefit in the speed of task performance. Robotic assistance may provide some benefit in precision of surgical task performance.

The Exosome Total Isolation Chip.

Circulating tumor-derived extracellular vesicles (EVs) have emerged as a promising source for identifying cancer biomarkers for early cancer detection. However, the clinical utility of EVs has thus far been limited by the fact that most EV isolation methods are tedious, nonstandardized, and require bulky instrumentation such as ultracentrifugation (UC). Here, we report a size-based EV isolation tool called ExoTIC (exosome total isolation chip), which is simple, easy-to-use, modular, and facilitates high-yield and high-purity EV isolation from biofluids. ExoTIC achieves an EV yield ∼4-1000-fold higher than that with UC, and EV-derived protein and microRNA levels are well-correlated between the two methods. Moreover, we demonstrate that ExoTIC is a modular platform that can sort a heterogeneous population of cancer cell line EVs based on size. Further, we utilize ExoTIC to isolate EVs from cancer patient clinical samples, including plasma, urine, and lavage, demonstrating the device's broad applicability to cancers and other diseases. Finally, the ability of ExoTIC to efficiently isolate EVs from small sample volumes opens up avenues for preclinical studies in small animal tumor models and for point-of-care EV-based clinical testing from fingerprick quantities (10-100 µL) of blood.

Transfer and priming of surgical skills across minimally invasive surgical platforms.

BACKGROUND: Robot-assisted laparoscopic surgery (RALS) uses 3-dimensional visualization and wristed instruments that provide more degrees of freedom than rigid traditional laparoscopic (TLS) instrumentation. These features have been touted to improve accuracy and efficiency during surgical task performance. Little is known, however, about the transferability of skills between the two platforms or whether task performance on one platform primes surgeons for task performance on the other. METHODS: Twenty-six subjects naïve to RALS were recruited to perform three Fundamentals of Laparoscopic Surgery tasks on both TLS and RALS platforms: peg transfer, pattern cutting (PC), and intracorporeal suturing. All tasks were performed within Fundamentals of Laparoscopic Surgery testing parameters and repeated three times by each subject on each platform. Platform and task order were randomized. Errors in task performance were defined as drops in the peg transfer task, faults 5 mm or more from the defined pattern during PC, and faults greater than 1 mm in suture placement from the defined points in intracorporeal suturing. Mean completion times and mean errors per trial (EPT) were calculated for each task on both platforms. Results were compared between those who performed TLS first (LF) and those who performed RALS first (RF) using unpaired Student's t-test (P < 0.05 considered statistically significant). RESULTS: No statistically significant differences in task completion time were noted between the LF and RF groups. RF subjects had fewer errors during robotic PC than LF subjects (1.02 EPT versus 1.86 EPT, respectively; P = 0.02). No other differences in task quality were noted. CONCLUSIONS: In surgeon's naïve to RALS, there is no evidence that skills acquired on RALS or TLS platforms are transferable to the other platform or that performing tasks on one platform primes a subject for task performance on the other. Performing TLS PC may have had a negative impact on subsequent RALS PC performance. These findings suggest that distinct programs for skills acquisition are necessary for both the TLS and RALS platforms.