Early detection and surveillance of SARS-CoV-2 genomic variants in wastewater using COJAC

The continuing emergence of SARS-CoV-2 variants of concern and variants of interest emphasizes the need for early detection and epidemiological surveillance of novel variants. We used genomic sequencing of 122 wastewater samples from three locations in Switzerland to monitor the local spread of B.1.1.7 (Alpha), B.1.351 (Beta) and P.1 (Gamma) variants of SARS-CoV-2 at a population level. We devised a bioinformatics method named COJAC (Co-Occurrence adJusted Analysis and Calling) that uses read pairs carrying multiple variant-specific signature mutations as a robust indicator of low-frequency variants. Application of COJAC revealed that a local outbreak of the Alpha variant in two Swiss cities was observable in wastewater up to 13 d before being first reported in clinical samples. We further confirmed the ability of COJAC to detect emerging variants early for the Delta variant by analysing an additional 1,339 wastewater samples. While sequencing data of single wastewater samples provide limited precision for the quantification of relative prevalence of a variant, we show that replicate and close-meshed longitudinal sequencing allow for robust estimation not only of the local prevalence but also of the transmission fitness advantage of any variant. We conclude that genomic sequencing and our computational analysis can provide population-level estimates of prevalence and fitness of emerging variants from wastewater samples earlier and on the basis of substantially fewer samples than from clinical samples. Our framework is being routinely used in large national projects in Switzerland and the UK

Design Principles for Ligand-Sensing, Conformation-Switching Ribozymes

Nucleic acid sensor elements are proving increasingly useful in biotechnology and biomedical applications. A number of ligand-sensing, conformational-switching ribozymes (also known as allosteric ribozymes or aptazymes) have been generated by some combination of directed evolution or rational design. Such sensor elements typically fuse a molecular recognition domain (aptamer) with a catalytic signal generator (ribozyme). Although the rational design of aptazymes has begun to be explored, the relationships between the thermodynamics of aptazyme conformational changes and aptazyme performance in vitro and in vivo have not been examined in a quantitative framework. We have therefore developed a quantitative and predictive model for aptazymes as biosensors in vitro and as riboswitches in vivo. In the process, we have identified key relationships (or dimensionless parameters) that dictate aptazyme performance, and in consequence, established equations for precisely engineering aptazyme function. In particular, our analysis quantifies the intrinsic trade-off between ligand sensitivity and the dynamic range of activity. We were also able to determine how in vivo parameters, such as mRNA degradation rates, impact the design and function of aptazymes when used as riboswitches. Using this theoretical framework we were able to achieve quantitative agreement between our models and published data. In consequence, we are able to suggest experimental guidelines for quantitatively predicting the performance of aptazyme-based riboswitches. By identifying factors that limit the performance of previously published systems we were able to generate immediately testable hypotheses for their improvement. The robust theoretical framework and identified optimization parameters should now enable the precision design of aptazymes for biotechnological and clinical applications

Local adaptation in populations of Mycobacterium tuberculosis endemic to the Indian Ocean Rim

Background: Lineage 1 (L1) and 3 (L3) are two lineages of the Mycobacterium tuberculosis complex (MTBC) causing tuberculosis (TB) in humans. L1 and L3 are prevalent around the rim of the Indian Ocean, the region that accounts for most of the world's new TB cases. Despite their relevance for this region, L1 and L3 remain understudied. Methods: We analyzed 2,938 L1 and 2,030 L3 whole genome sequences originating from 69 countries. We reconstructed the evolutionary history of these two lineages and identified genes under positive selection. Results: We found a strongly asymmetric pattern of migration from South Asia toward neighboring regions, highlighting the historical role of South Asia in the dispersion of L1 and L3. Moreover, we found that several genes were under positive selection, including genes involved in virulence and resistance to antibiotics . For L1 we identified signatures of local adaptation at the esxH locus, a gene coding for a secreted effector that targets the human endosomal sorting complex, and is included in several vaccine candidates. Conclusions: Our study highlights the importance of genetic diversity in the MTBC, and sheds new light on two of the most important MTBC lineages affecting humans

NGS-pipe: a flexible, easily extendable, and highly configurable framework for NGS analysis

Next-generation sequencing is now an established method in genomics, and massive amounts of sequencing data are being generated on a regular basis. Analysis of the sequencing data is typically performed by lab-specific in-house solutions, but the agreement of results from different facilities is often small. General standards for quality control, reproducibility, and documentation are missing.; We developed NGS-pipe, a flexible, transparent, and easy-to-use framework for the design of pipelines to analyze whole-exome, whole-genome, and transcriptome sequencing data. NGS-pipe facilitates the harmonization of genomic data analysis by supporting quality control, documentation, reproducibility, parallelization, and easy adaptation to other NGS experiments. https://github.com/cbg-ethz/NGS-pipe [email protected]

Multiple introductions of Mycobacterium tuberculosis lineage 2-Beijing into Africa over centuries

The Lineage 2–Beijing (L2–Beijing) sub-lineage of Mycobacterium tuberculosis has received much attention due to its high virulence, fast disease progression, and association with antibiotic resistance. Despite several reports of the recent emergence of L2–Beijing in Africa, no study has investigated the evolutionary history of this sub-lineage on the continent. In this study, we used whole genome sequences of 781 L2 clinical strains from 14 geographical regions globally distributed to investigate the origins and onward spread of this lineage in Africa. Our results reveal multiple introductions of L2–Beijing into Africa linked to independent bacterial populations from East- and Southeast Asia. Bayesian analyses further indicate that these introductions occurred during the past 300 years, with most of these events pre-dating the antibiotic era. Hence, the success of L2–Beijing in Africa is most likely due to its hypervirulence and high transmissibility rather than drug resistance

Modular construction of mammalian gene circuits using TALE transcriptional repressors

An important goal of synthetic biology is the rational design and predictable implementation of synthetic gene circuits using standardized and interchangeable parts. However, engineering of complex circuits in mammalian cells is currently limited by the availability of well-characterized and orthogonal transcriptional repressors. Here, we introduce a library of 26 reversible transcription activator–like effector repressors (TALERs) that bind newly designed hybrid promoters and exert transcriptional repression through steric hindrance of key transcriptional initiation elements. We demonstrate that using the input-output transfer curves of our TALERs enables accurate prediction of the behavior of modularly assembled TALER cascade and switch circuits. We also show that TALER switches using feedback regulation exhibit improved accuracy for microRNA-based HeLa cancer cell classification versus HEK293 cells. Our TALER library is a valuable toolkit for modular engineering of synthetic circuits, enabling programmable manipulation of mammalian cells and helping elucidate design principles of coupled transcriptional and microRNA-mediated post-transcriptional regulation.National Institutes of Health (U.S.) (Grant 5R01CA155320-04)National Institutes of Health (U.S.) (Grant P50GM098792)National Institutes of Health (U.S.) (Grant 1R01CA173712-01

Long-Distance Three-Color Neuronal Tracing in Fixed Tissue Using NeuroVue Dyes

Dissecting development of neuronal connections is critical for understanding neuronal function in both normal and diseased states. Charting the development of the multitude of connections is a monumental task, since a given neuron typically receives hundreds of convergent inputs from other neurons and provides divergent outputs for hundreds of other neurons. Although progress is being made utilizing various mutants and/or genetic constructs expressing fluorescent proteins like GFP, substantial work remains before a database documenting the development and final location of the neuronal pathways in an adult animal is completed. The vast majority of developing neurons cannot be specifically labeled with antibodies and making specific GFP-expressing constructs to tag each of them is an overwhelming task. Fortunately, fluorescent lipophilic dyes have emerged as very useful tools to systematically compare changes in neuronal networks between wild-type and mutant mice. These dyes diffuse laterally along nerve cell membranes in fixed preparations, allowing tracing of the position of a given neuron within the neuronal network in murine mutants fixed at various stages of development. Until recently, however, most evaluations have been limited to one, or at most, two color analyses. We have previously reported three color neuronal profiling using the novel lipophilic dyes NeuroVue (NV) Green, Red and Maroon (Fritzsch et al., Brain. Res. Bull. 66:249–258, 2005). Unfortunately such three color experiments have been limited by the fact that NV Green and its brighter successor, NV Emerald, both exhibit substantially decreased signal intensities when times greater than 48 hours at 37°C are required to achieve neuronal profile filling (unpublished observations). Here we describe a standardized test system developed to allow comparison of candidate dyes and its use to evaluate a series of 488 nm-excited green-emitting lipophilic dyes. The best of these, NV Jade, has spectral properties well matched to NV Red and NV Maroon, better solubility in DMF than DiO or DiA, improved thermostability compared with NV Emerald, and the ability to fill neuronal profiles at rates of 1 mm per day for periods of at least 5 days. Use of NV Jade in combination with NV Red and NV Maroon substantially improves the efficiency of connectional analysis in complex mutants and transgenic models where limited numbers of specimens are available

Synthetic human cell fate regulation by protein-driven RNA switches

Understanding how to control cell fate is crucial in biology, medical science and engineering. In this study, we introduce a method that uses an intracellular protein as a trigger for regulating human cell fate. The ON/OFF translational switches, composed of an intracellular protein L7Ae and its binding RNA motif, regulate the expression of a desired target protein and control two distinct apoptosis pathways in target human cells. Combined use of the switches demonstrates that a specific protein can simultaneously repress and activate the translation of two different mRNAs: one protein achieves both up- and downregulation of two different proteins/pathways. A genome-encoded protein fused to L7Ae controlled apoptosis in both directions (death or survival) depending on its cellular expression. The method has potential for curing cellular defects or improving the intracellular production of useful molecules by bypassing or rewiring intrinsic signal networks

Mechanistic Insights into a Novel Exporter-Importer System of Mycobacterium tuberculosis Unravel Its Role in Trafficking of Iron

Elucidation of the basic mechanistic and biochemical principles underlying siderophore mediated iron uptake in mycobacteria is crucial for targeting this principal survival strategy vis-à-vis virulence determinants of the pathogen. Although, an understanding of siderophore biosynthesis is known, the mechanism of their secretion and uptake still remains elusive.Here, we demonstrate an interplay among three iron regulated Mycobacterium tuberculosis (M.tb) proteins, namely, Rv1348 (IrtA), Rv1349 (IrtB) and Rv2895c in export and import of M.tb siderophores across the membrane and the consequent iron uptake. IrtA, interestingly, has a fused N-terminal substrate binding domain (SBD), representing an atypical subset of ABC transporters, unlike IrtB that harbors only the permease and ATPase domain. SBD selectively binds to non-ferrated siderophores whereas Rv2895c exhibits relatively higher affinity towards ferrated siderophores. An interaction between the permease domain of IrtB and Rv2895c is evident from GST pull-down assay. In vitro liposome reconstitution experiments further demonstrate that IrtA is indeed a siderophore exporter and the two-component IrtB-Rv2895c system is an importer of ferrated siderophores. Knockout of msmeg_6554, the irtA homologue in Mycobacterium smegmatis, resulted in an impaired M.tb siderophore export that is restored upon complementation with M.tb irtA.Our data suggest the interplay of three proteins, namely IrtA, IrtB and Rv2895c in synergizing the balance of siderophores and thus iron inside the mycobacterial cell