The Impact of Left Ventricular Assist Device Outflow Graft Positioning on Aortic Hemodynamics: Improving Flow Dynamics to Mitigate Aortic Insufficiency.

Heart failure is a global health concern with significant implications for healthcare systems. Left ventricular assist devices (LVADs) provide mechanical support for patients with severe heart failure. However, the placement of the LVAD outflow graft within the aorta has substantial implications for hemodynamics and can lead to aortic insufficiency during long-term support. This study employs computational fluid dynamics (CFD) simulations to investigate the impact of different LVAD outflow graft locations on aortic hemodynamics. The introduction of valve morphology within the aorta geometry allows for a more detailed analysis of hemodynamics at the aortic root. The results demonstrate that the formation of vortex rings and subsequent vortices during the high-velocity jet flow from the graft interacted with the aortic wall. Time-averaged wall shear stress (TAWSS) and oscillatory shear index (OSI) indicate that modification of the outflow graft location changes mechanical states within the aortic wall and aortic valve. Among the studied geometric factors, both the height and inclination angle of the LVAD outflow graft are important in controlling retrograde flow to the aortic root, while the azimuthal angle primarily determines the rotational direction of blood flow in the aortic arch. Thus, precise positioning of the LVAD outflow graft emerges as a critical factor in optimizing patient outcomes by improving the hemodynamic environment.

Yeast Two-Hybrid Analysis for Ubiquitin Variant Inhibitors of Human Deubiquitinases.

We applied a yeast-two-hybrid (Y2H) analysis to screen for ubiquitin variant (UbV) inhibitors of a human deubiquitinase (DUB), ubiquitin-specific protease 2 (USP2). The Y2H screen used USP2 as the bait and a prey library consisting of UbVs randomized at four specific positions, which were known to interact with USP2 from phage display analysis. The screen yielded numerous UbVs that bound to USP2 both as a Y2H interaction in vivo and as purified proteins in vitro. The Y2H-derived UbVs inhibited the catalytic activity of USP2 in vitro with nanomolar-range potencies, and they bound and inhibited USP2 in human cells. Mutational and structural analysis showed that potent and selective inhibition could be achieved by just two substitutions in a UbV, which exhibited improved hydrophobic and hydrophilic contacts compared to the wild-type ubiquitin interaction with USP2. Our results establish Y2H as an effective platform for the development of UbV inhibitors of DUBs in vivo, providing an alternative strategy for the analysis of DUBs that are recalcitrant to phage display and other in vitro methods.

Traction cytometry: regularization in the Fourier approach and comparisons with finite element method.

Traction forces exerted by adherent cells are quantified using displacements of embedded markers on polyacrylamide substrates due to cell contractility. Fourier Transform Traction Cytometry (FTTC) is widely used to calculate tractions but has inherent limitations due to errors in the displacement fields; these are mitigated through a regularization parameter (γ) in the Reg-FTTC method. An alternate finite element (FE) approach computes tractions on a domain using known boundary conditions. Robust verification and recovery studies are lacking but essential in assessing the accuracy and noise sensitivity of the traction solutions from the different methods. We implemented the L2 regularization method and defined a maximum curvature point in the traction with γ plot as the optimal regularization parameter (γ*) in the Reg-FTTC approach. Traction reconstructions using γ* yield accurate values of low and maximum tractions (Tmax) in the presence of up to 5% noise. Reg-FTTC is hence a clear improvement over the FTTC method but is inadequate to reconstruct low stresses such as those at nascent focal adhesions. FE, implemented using a node-by-node comparison, showed an intermediate reconstruction compared to Reg-FTTC. We performed experiments using mouse embryonic fibroblast (MEF) and compared results between these approaches. Tractions from FTTC and FE showed differences of ∼92% and 22% as compared to Reg-FTTC. Selection of an optimum value of γ for each cell reduced variability in the computed tractions as compared to using a single value of γ for all the MEF cells in this study.

Evaluation and Design of Genome-Wide CRISPR/SpCas9 Knockout Screens.

The adaptation of CRISPR/SpCas9 technology to mammalian cell lines is transforming the study of human functional genomics. Pooled libraries of CRISPR guide RNAs (gRNAs) targeting human protein-coding genes and encoded in viral vectors have been used to systematically create gene knockouts in a variety of human cancer and immortalized cell lines, in an effort to identify whether these knockouts cause cellular fitness defects. Previous work has shown that CRISPR screens are more sensitive and specific than pooled-library shRNA screens in similar assays, but currently there exists significant variability across CRISPR library designs and experimental protocols. In this study, we reanalyze 17 genome-scale knockout screens in human cell lines from three research groups, using three different genome-scale gRNA libraries. Using the Bayesian Analysis of Gene Essentiality algorithm to identify essential genes, we refine and expand our previously defined set of human core essential genes from 360 to 684 genes. We use this expanded set of reference core essential genes, CEG2, plus empirical data from six CRISPR knockout screens to guide the design of a sequence-optimized gRNA library, the Toronto KnockOut version 3.0 (TKOv3) library. We then demonstrate the high effectiveness of the library relative to reference sets of essential and nonessential genes, as well as other screens using similar approaches. The optimized TKOv3 library, combined with the CEG2 reference set, provide an efficient, highly optimized platform for performing and assessing gene knockout screens in human cell lines.

EVA-1 functions as an UNC-40 Co-receptor to enhance attraction to the MADD-4 guidance cue in Caenorhabditis elegans.

We recently discovered a secreted and diffusible midline cue called MADD-4 (an ADAMTSL) that guides migrations along the dorsoventral axis of the nematode Caenorhabditis elegans. We showed that the transmembrane receptor, UNC-40 (DCC), whose canonical ligand is the UNC-6 (netrin) guidance cue, is required for extension towards MADD-4. Here, we demonstrate that MADD-4 interacts with an EVA-1/UNC-40 co-receptor complex to attract cell extensions. EVA-1 is a conserved transmembrane protein with predicted galactose-binding lectin domains. EVA-1 functions in the same pathway as MADD-4, physically interacts with both MADD-4 and UNC-40, and enhances UNC-40's sensitivity to the MADD-4 cue. This enhancement is especially important in the presence of UNC-6. In EVA-1's absence, UNC-6 interferes with UNC-40's responsiveness to MADD-4; in UNC-6's absence, UNC-40's responsiveness to MADD-4 is less dependent on EVA-1. By enabling UNC-40 to respond to MADD-4 in the presence of UNC-6, EVA-1 may increase the precision by which UNC-40-directed processes can reach their MADD-4-expressing targets within a field of the UNC-6 guidance cue.

MADD-4 is a secreted cue required for midline-oriented guidance in Caenorhabditis elegans.

The netrins and slits are two families of widely conserved cues that guide axons and cells along the dorsal-ventral (D-V) axis of animals. These cues typically emanate from the dorsal or ventral midlines and provide spatial information to migrating cells by forming gradients along the D-V axis. Some cell types, however, extend processes to both the dorsal and ventral midlines, suggesting the existence of additional guidance cues that are secreted from both midlines. Here, we report that a previously uncharacterized protein called MADD-4 is secreted by the dorsal and ventral nerve cords of the nematode C. elegans to attract sensory axons and muscle membrane extensions called muscle arms. MADD-4's activity is dependent on UNC-40/DCC, a netrin receptor, which functions cell-autonomously to direct membrane extension. The biological role of MADD-4 orthologs, including ADAMTSL1 and 3 in mammals, is unknown. MADD-4 may therefore represent the founding member of a family of guidance proteins.

MADD-2, a homolog of the Opitz syndrome protein MID1, regulates guidance to the midline through UNC-40 in Caenorhabditis elegans.

The body muscles of Caenorhabditis elegans extend plasma membrane extensions called muscle arms to the midline motor axons to form the postsynaptic membrane of the neuromuscular junction. Through a screen for muscle arm development defective (Madd) mutants, we previously discovered that the UNC-40/DCC guidance receptor directs muscle arm extension through the Rho-GEF UNC-73. Here, we describe a gene identified through our mutant screen called madd-2, and show that it functions in an UNC-40 pathway. MADD-2 is a C1-TRIM protein and a homolog of human MID1, mutations in which cause Opitz Syndrome. We demonstrate that MADD-2 functions cell autonomously to direct muscle and axon extensions to the ventral midline of worms. Our results suggest that MADD-2 may enhance UNC-40 pathway activity by facilitating an interaction between UNC-40 and UNC-73. The analogous phenotypes that result from MADD-2 and MID1 mutations suggest that C1-TRIM proteins may have a conserved biological role in midline-oriented developmental events.

Conserved mechanism of Wnt signaling function in the specification of vulval precursor fates in C. elegans and C. briggsae.

The C. elegans hermaphrodite vulva serves as a paradigm for understanding how signaling pathways control organ formation. Previous studies have shown that Wnt signaling plays important roles in vulval development. To understand the function and evolution of Wnt signaling in Caenorhabditis nematodes we focused on C. briggsae, a species that is substantially divergent from C. elegans in terms of the evolutionary time scale yet shares almost identical morphology. We isolated mutants in C. briggsae that display multiple pseudo-vulvae resulting from ectopic VPC induction. We cloned one of these loci and found that it encodes an Axin homolog, Cbr-PRY-1. Our genetic studies revealed that Cbr-pry-1 functions upstream of the canonical Wnt pathway components Cbr-bar-1 (beta-catenin) and Cbr-pop-1(tcf/lef) as well as the Hox target Cbr-lin-39 (Dfd/Scr). We further characterized the pry-1 vulval phenotype in C. briggsae and C. elegans using 8 cell fate markers, cell ablation, and genetic interaction approaches. Our results show that ectopically induced VPCs in pry-1 mutants adopt 2° fates independently of the gonad-derived inductive and LIN-12/Notch-mediated lateral signaling pathways. We also found that Cbr-pry-1 mutants frequently show a failure of P7.p induction. A similar, albeit low penetrant, defect is also observed in C. elegans pry-1 mutants. The genetic analysis of the P7.p induction defect revealed that it was caused by altered regulation of lin-12 and its transcriptional target lip-1 (MAP kinase phosphatase). Thus, our results provide evidence for LIN-12/Notch-dependent and independent roles of Wnt signaling in promoting 2 degrees VPC fates in both nematode species.