No abstract

Discovery of the genetic components underpinning fundamental and disease-related processes is being rapidly accelerated by combining efficient, programmable genetic engineering with phenotypic readouts of high spatial, temporal, and/or molecular resolution. Microscopy is a fundamental tool for studying cell biology, but its lack of high-throughput sequence readouts hinders integration in large-scale genetic screens. Optical pooled screens using in situ sequencing provide massively scalable integration of barcoded lentiviral libraries (e.g., CRISPR perturbation libraries) with high-content imaging assays, including dynamic processes in live cells. The protocol uses standard lentiviral vectors and molecular biology, providing single-cell resolution of phenotype and engineered genotype, scalability to millions of cells, and accurate sequence reads sufficient to distinguish >10 6 perturbations. In situ amplification takes ~2 days, while sequencing can be performed in ~1.5 hours per cycle. The image analysis pipeline provided enables fully parallel automated sequencing analysis using a cloud or cluster computing environment.

Understanding the basis for cellular growth, proliferation, and function requires determining the contributions of essential genes to diverse cellular processes. Here, we combined pooled CRISPR/Cas9-based functional screening of 5,072 fitness-conferring genes in human cells with microscopy-based visualization of DNA, DNA damage, actin, and microtubules. Analysis of >31 million individual cells revealed measurable phenotypes for >90% of genes. Using multi-dimensional clustering based on hundreds of quantitative phenotypic parameters, we identified co-functional genes across diverse cellular activities, revealing novel gene functions and associations. Pooled live-cell screening of ∼450,000 cell division events for 239 genes further identified functional contributions to chromosome segregation. Our work creates a resource for the phenotypic analysis of core cellular processes and defines the functional landscape of essential human genes.

Many instances of cellular signaling and transcriptional regulation involve switch-like molecular responses to the presence or absence of input ligands. To understand how these responses come about and how they can be harnessed, we develop a statistical mechanical model to characterize the types of Boolean logic that can arise from allosteric molecules following the Monod-Wyman-Changeux (MWC) model. Building upon previous work, we show how an allosteric molecule regulated by two inputs can elicit AND, OR, NAND and NOR responses, but is unable to realize XOR or XNOR gates. Next, we demonstrate the ability of an MWC molecule to perform ratiometric sensing - a response behavior where activity depends monotonically on the ratio of ligand concentrations. We then extend our analysis to more general schemes of combinatorial control involving either additional binding sites for the two ligands or an additional third ligand and show how these additions can cause a switch in the logic behavior of the molecule. Overall, our results demonstrate the wide variety of control schemes that biological systems can implement using simple mechanisms.

Purpose The Lower Limb Function Questionnaire (LLFQ) was developed as a self-report assessment of lower-limb functional ability for orthotic and prosthetic (O&P) device users to be suitable for a wide range of conditions, cultures, and ages. The measure aims to address an existing gap in tools for the assessment of functional ability in this population. The purpose of this study is to evaluate LLFQ reliability and validity in a sample of young adult O&P users. Methods Adolescents from a secondary school in Kenya completed the LLFQ twice, 6 d apart, and test-retest reliability was assessed using intra-class correlation coefficients. Validity evaluations involved Timed Up-and-Go, 6-min walk, 6-min obstacle course, and/or spatiotemporal gait assessments. Oxygen consumption was measured during walk tests. Associations between the LLFQ and each measure were evaluated using Pearson correlation coefficients for construct validity. Results LLFQ reliability was acceptable (ICC = 0.79, 95% CIs 0.64-0.89). Construct validity was demonstrated via moderate correlation (r > 0.60) with obstacle course distance, gait velocity, stride length, and stance/single support/double support percent of gait cycle. Conclusions Both LLFQ reliability and validity were acceptable in the sample of youth in Kenya. Further testing is required to determine applicability in other cultural contexts. Implications for Rehabilitation The LLFQ may be clinically useful across a variety of cultures and conditions to provide feedback on the effectiveness of rehabilitative treatment or assistive devices for youth with lower limb impairments. The LLFQ may enable specific strengths and challenges to lower limb function to be identified to enable planning of well-targeted rehabilitation.

Many instances of cellular signaling and transcriptional regulation involve switchlike molecular responses to the presence or absence of input ligands. To understand how these responses come about and how they can be harnessed, we develop a statistical mechanical model to characterize the types of Boolean logic that can arise from allosteric molecules following the Monod-Wyman-Changeux (MWC) model. Building upon previous work, we show how an allosteric molecule regulated by two inputs can elicit AND, OR, NAND and NOR responses, but is unable to realize XOR or XNOR gates. Next, we demonstrate the ability of an MWC molecule to perform ratiometric sensing -a response behavior where activity depends monotonically on the ratio of ligand concentrations. We then extend our analysis to more general schemes of combinatorial control involving either additional binding sites for the two ligands or an additional third ligand and show how these additions can cause a switch in the logic behavior of the molecule. Overall, our results demonstrate the wide variety of control schemes that biological systems can implement using simple mechanisms.