Mycobacterium tuberculosis remains a leading cause of death worldwide, especially among individuals infected with HIV1. While phylogenetic analysis has revealed M. tuberculosis spread throughout history2–5 and in local outbreaks6–8, much less is understood about its dissemination within the body. Here, we report genomic analysis of 2693 samples collected postmortem from lung and extrapulmonary biopsies of 44 subjects in KwaZulu-Natal, South Africa who received minimal antitubercular treatment and most of whom were HIV seropositive. We found that purifying selection acted within individual patients, without the need for patient-to-patient transmission. Despite negative selection, mycobacteria diversified within individuals to form sub-lineages that co-existed for years. These sub-lineages, as well as distinct strains from mixed infections, were differentially distributed throughout the lung, suggesting temporary barriers to pathogen migration. As a consequence, samples taken from the upper airway often captured only a fraction of the population diversity, challenging current methods of outbreak tracing and resistance diagnostics. Phylogenetic analysis indicated that dissemination from the lungs to extrapulmonary sites was as frequent as between lung sites—supporting similar migration routes within and between organs, at least in subjects with HIV. Genomic diversity therefore provides a record of pathogen diversification and repeated dissemination across the body.
Background Low-frequency (delta/theta) oscillations in the thalamocortical system are elevated in schizophrenia during wakefulness and are also induced in the NMDAR hypofunction rat model. To determine whether abnormal delta oscillations might produce functional deficits, we used optogenetic methods in awake rats. We illuminated channelrhodopsin-2 in the thalamic nucleus reuniens (RE) at delta frequency and measured the effect on working memory performance (the RE is involved in working memory (WM), a process affected in schizophrenia (SZ)). Methods We injected RE with a virus (AAV) to transduce cells with channelrhodopsin-2. An optical fiber was implanted just dorsal to the hippocampus in order to illuminate RE axon terminals. Results During optogenetic delta frequency stimulation, rats displayed a strong WM deficit. On the following day, performance was normal if illumination was omitted. Conclusions The optogenetic experiments showed that delta frequency stimulation of a thalamic nucleus is sufficient to produce deficits in WM. This result supports the hypothesis that delta frequency bursting in particular thalamic nuclei has a causal role producing WM deficits in this SZ. The action potentials in these bursts may jam communication through the thalamus, thereby interfering with behaviors dependent on WM. Studies in thalamic slices using the NMDAR hypofunction model show that delta frequency bursting is dependent on T-type Ca2+ channels, a result that we confirmed here in vivo. These channels, which are strongly implicated in SZ by GWAS studies, may thus be a therapeutic target for treatment of SZ.
Temperature is a versatile input signal for the control of engineered cellular functions. Sharp induction of gene expression with heat has been established using bacteria- and phage-derived temperature-sensitive transcriptional repressors with tunable switching temperatures. However, few temperature-sensitive transcriptional activators have been reported that enable direct gene induction with cooling. Such activators would expand the application space for temperature control. In this technical note, we show that temperature-dependent versions of the Lambda phage repressor CI can serve as tunable cold-actuated transactivators. Natively, CI serves as both a repressor and activator of transcription. Previously, thermolabile mutants of CI, known as the TcI family, were used to repress the cognate promoters PR and PL. We hypothesized that TcI mutants can also serve as temperature-sensitive activators of transcription at CI’s natural PRM promoter, creating cold-inducible operons with a tunable response to temperature. Indeed, we demonstrate temperature-responsive activation by two variants of TcI with set points at 35.5 and 38.5 °C in E. coli . In addition, we show that TcI can serve as both an activator and a repressor of different genes in the same genetic circuit, leading to opposite thermal responses. Transcriptional activation by TcI expands the toolbox for control of cellular function using globally or locally applied thermal inputs.
Engineered living materials (ELMs) exhibit desirable characteristics of the living component, including growth and repair, and responsiveness to external stimuli.Escherichia coliare a promising constituent of ELMs because they are very tractable to genetic engineering, produce heterologous proteins readily, and grow exponentially. However, seasonal variation in ambient temperature presents a challenge in deploying ELMs outside of a laboratory environment, becauseE. coligrowth rate is impaired both below and above 37°C. Here, we develop a genetically-encoded mechanism for autonomous temperature homeostasis in ELMs containingE. coliby engineering circuits that control the expression of a light-absorptive chromophore in response to changes in temperature. We demonstrate that below 36°C, our engineeredE. coliincrease in pigmentation, causing an increase in sample temperature and growth rate above non-pigmented counterparts in a model planar ELM. On the other hand, above 36°C, they decrease in pigmentation, protecting their growth compared to bacteria with temperature-independent high pigmentation. Integrating our temperature homeostasis circuit into an ELM has the potential to improve living material performance by optimizing growth and protein production in the face of seasonal temperature changes.
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