The Response of Caenorhabditis elegans to Hydrogen Sulfide and Hydrogen Cyanide

Hydrogen sulfide (H2S), an endogenously produced small molecule, protects animals from various stresses. Recent studies demonstrate that animals exposed to H2S are long lived, resistant to hypoxia, and resistant to ischemia–reperfusion injury. We performed a forward genetic screen to gain insights into the molecular mechanisms Caenorhabditis elegans uses to appropriately respond to H2S. At least two distinct pathways appear to be important for this response, including the H2S-oxidation pathway and the hydrogen cyanide (HCN)-assimilation pathway. The H2S-oxidation pathway requires two distinct enzymes important for the oxidation of H2S: the sulfide:quinone reductase sqrd-1 and the dioxygenase ethe-1. The HCN-assimilation pathway requires the cysteine synthase homologs cysl-1 and cysl-2. A low dose of either H2S or HCN can activate hypoxia-inducible factor 1 (HIF-1), which is required for C. elegans to respond to either gas. sqrd-1 and cysl-2 represent the entry points in the H2S-oxidation and HCN-assimilation pathways, respectively, and expression of both of these enzymes is highly induced by HIF-1 in response to both H2S and HCN. In addition to their role in appropriately responding to H2S and HCN, we found that cysl-1 and cysl-2 are both essential mediators of innate immunity against fast paralytic killing by Pseudomonas. Furthermore, in agreement with these data, we showed that growing worms in the presence of H2S is sufficient to confer resistance to Pseudomonas fast paralytic killing. Our results suggest the hypoxia-independent hif-1 response in C. elegans evolved to respond to the naturally occurring small molecules H2S and HCN

Imaging cell lineage with a synthetic digital recording system

Cell lineage plays a pivotal role in cell fate determination. Chow et al. demonstrate the use of an integrase-based synthetic barcode system called intMEMOIR, which uses the serine integrase Bxb1 to perform irreversible nucleotide edits. Inducible editing either deletes or inverts its target region, thus encoding information in three-state memory elements, or trits, and avoiding undesired recombination events. Using intMEMOIR combined with single-molecule fluorescence in situ hybridization, the authors were able to identify clonal structures as well as gene expression patterns in the fly brain, enabling both clonal analysis and expression profiling with intact spatial information. The ability to visualize cell lineage relationships directly within their native tissue context provides insights into development and disease

Biomechanical Analysis of Stoop and Free-Style Squat Lifting and Lowering with a Generic Back-Support Exoskeleton Model

Musculoskeletal disorders (MSDs) induced by industrial manual handling tasks are a major issue for workers and companies. As flexible ergonomic solutions, occupational exoskeletons can decrease critically high body stress in situations of awkward postures and motions. Biomechanical models with detailed anthropometrics and motions help us to acquire a comprehension of person- and application-specifics by considering the intended and unintended effects, which is crucial for effective implementation. In the present model-based analysis, a generic back-support exoskeleton model was introduced and applied to the motion data of one male subject performing symmetric and asymmetric dynamic manual handling tasks. Different support modes were implemented with this model, including support profiles typical of passive and active systems and an unconstrained optimal support mode used for reference to compare and quantify their biomechanical effects. The conducted simulations indicate that there is a high potential to decrease the peak compression forces in L4/L5 during the investigated heavy loaded tasks for all motion sequences and exoskeleton support modes (mean reduction of 13.3% without the optimal support mode). In particular, asymmetric motions (mean reduction of 14.7%) can be relieved more than symmetric ones (mean reduction of 11.9%) by the exoskeleton support modes without the optimal assistance. The analysis of metabolic energy consumption indicates a high dependency on lifting techniques for the effectiveness of the exoskeleton support. While the exoskeleton support substantially reduces the metabolic cost for the free-squat motions, a slightly higher energy consumption was found for the symmetric stoop motion technique with the active and optimal support mode

Synthetic mammalian signaling circuits for robust cell population control

In multicellular organisms, cells actively sense, respond to, and control their own population density. Synthetic mammalian quorum sensing circuits could provide insight into principles of population control and improve cell therapies. However, a key challenge is avoiding their inherent sensitivity to “cheater” mutations that evade control. Here, we repurposed the plant hormone auxin to enable orthogonal mammalian cell-cell communication and quorum sensing. Further, we show that a “paradoxical” circuit design, in which auxin stimulates and inhibits net cell growth at different concentrations, achieves population control that is robust to cheater mutations, controlling growth for 43 days of continuous culture. By contrast, a non-paradoxical control circuit limited growth but was susceptible to mutations. These results establish a foundation for future cell therapies that can respond to and control their own population sizes

In situ readout of DNA barcodes and single base edits facilitated by in vitro transcription

Molecular barcoding technologies that uniquely identify single cells are hampered by limitations in barcode measurement. Readout by sequencing does not preserve the spatial organization of cells in tissues, whereas imaging methods preserve spatial structure but are less sensitive to barcode sequence. Here we introduce a system for image-based readout of short (20-base-pair) DNA barcodes. In this system, called Zombie, phage RNA polymerases transcribe engineered barcodes in fixed cells. The resulting RNA is subsequently detected by fluorescent in situ hybridization. Using competing match and mismatch probes, Zombie can accurately discriminate single-nucleotide differences in the barcodes. This method allows in situ readout of dense combinatorial barcode libraries and single-base mutations produced by CRISPR base editors without requiring barcode expression in live cells. Zombie functions across diverse contexts, including cell culture, chick embryos and adult mouse brain tissue. The ability to sensitively read out compact and diverse DNA barcodes by imaging will facilitate a broad range of barcoding and genomic recording strategies

Pydna: a simulation and documentation tool for DNA assembly strategies using python

Background: Recent advances in synthetic biology have provided tools to efficiently construct complex DNA molecules which are an important part of many molecular biology and biotechnology projects. The planning of such constructs has traditionally been done manually using a DNA sequence editor which becomes error-prone as scale and complexity of the construction increase. A human-readable formal description of cloning and assembly strategies, which also allows for automatic computer simulation and verification, would therefore be a valuable tool. Results: We have developed pydna, an extensible, free and open source Python library for simulating basic molecular biology DNA unit operations such as restriction digestion, ligation, PCR, primer design, Gibson assembly and homologous recombination. A cloning strategy expressed as a pydna script provides a description that is complete, unambiguous and stable. Execution of the script automatically yields the sequence of the final molecule(s) and that of any intermediate constructs. Pydna has been designed to be understandable for biologists with limited programming skills by providing interfaces that are semantically similar to the description of molecular biology unit operations found in literature. Conclusions: Pydna simplifies both the planning and sharing of cloning strategies and is especially useful for complex or combinatorial DNA molecule construction. An important difference compared to existing tools with similar goals is the use of Python instead of a specifically constructed language, providing a simulation environment that is more flexible and extensible by the user

Computed tomography to predict pacemaker need after transcatheter aortic valve replacement

Transcatheter aortic valve replacement (TAVR) is preferred therapy for elderly patients with severe aortic stenosis (AS) and increasingly used in younger patient populations with good safety and efficacy outcomes. However, cardiac conduction abnormalities remain a frequent complication after TAVR ranging from relative benign interventriculair conduction delays to prognostically relevant left bundle branch block and complete atrio-ventricular (AV) block requiring permanent pacemaker implantation (PPI). Although clinical, procedural and electrocardiographic factors have been identified as predictors of this complication, there is a need for advanced strategies to control the burden of conduction defects particularly as TAVR shifts towards younger populations. This state of the art review highlights the value of ECG-synchronized computed tomographic angiography (CTA) evaluation of the aortic root to better understand and manage conduction problems post-TAVR. An update on CTA derived anatomic features related to conduction issues is provided and complemented with computational framework modelling. This CTA-derived 3-dimensional anatomical reconstruction tool generates patient-specific TAVR simulations enabling operators to adapt procedural strategy and implantation technique to mitigate conduction abnormality risks.</p

Evaluating fibre orientation dispersion in white matter: comparison of diffusion MRI, histology and polarized light imaging

Diffusion MRI is an exquisitely sensitive probe of tissue microstructure, and is currently the only non-invasive measure of the brain’s fibre architecture. As this technique becomes more sophisticated and microstructurally informative, there is increasing value in comparing diffusion MRI with microscopic imaging in the same tissue samples. This study compared estimates of fibre orientation dispersion in white matter derived from diffusion MRI to reference measures of dispersion obtained from polarized light imaging and histology. Three post-mortem brain specimens were scanned with diffusion MRI and analyzed with a two-compartment dispersion model. The specimens were then sectioned for microscopy, including polarized light imaging estimates of fibre orientation and histological quantitative estimates of myelin and astrocytes. Dispersion estimates were correlated on region – and voxel-wise levels in the corpus callosum, the centrum semiovale and the corticospinal tract. The region-wise analysis yielded correlation coefficients of r=0.79 for the diffusion MRI and histology comparison, while r=0.60 was reported for the comparison with polarized light imaging. In the corpus callosum, we observed a pattern of higher dispersion at the midline compared to its lateral aspects. This pattern was present in all modalities and the dispersion profiles from microscopy and diffusion MRI were highly correlated. The astrocytes appeared to have minor contribution to dispersion observed with diffusion MRI. These results demonstrate that fibre orientation dispersion estimates from diffusion MRI represents the tissue architecture well. Dispersion models might be improved by more faithfully incorporating an informed mapping based on microscopy data