Axoneme-specific β-tubulin specialization a conserved C-terminal motif specifies the central pair

AbstractAxonemes are ancient organelles that mediate motility of cilia and flagella in animals, plants, and protists. The long evolutionary conservation of axoneme architecture, a cylinder of nine doublet microtubules surrounding a central pair of singlet microtubules, suggests all motile axonemes may share common assembly mechanisms. Consistent with this, α- and β-tubulins utilized in motile axonemes fall among the most conserved tubulin sequences [1, 2], and the β-tubulins contain a sequence motif at the same position in the carboxyl terminus [3]. Axoneme doublet microtubules are initiated from the corresponding triplet microtubules of the basal body [4], but the large macromolecular “central apparatus” that includes the central pair microtubules and associated structures [5] is a specialization unique to motile axonemes. In Drosophila spermatogenesis, basal bodies and axonemes utilize the same α-tubulin but different β-tubulins [6–13]. β1 is utilized for the centriole/basal body, and β2 is utilized for the motile sperm tail axoneme. β2 contains the motile axoneme-specific sequence motif, but β1 does not [3]. Here, we show that the “axoneme motif” specifies the central pair. β1 can provide partial function for axoneme assembly but cannot make the central microtubules [14]. Introducing the axoneme motif into the β1 carboxyl terminus, a two amino acid change, conferred upon β1 the ability to assemble 9 + 2 axonemes. This finding explains the conservation of the axoneme-specific sequence motif through 1.5 billion years of evolution

Protective effect of Toll-like receptor 4 in pulmonary vaccinia infection.

Innate immune responses are essential for controlling poxvirus infection. The threat of a bioterrorist attack using Variola major, the smallpox virus, or zoonotic transmission of other poxviruses has renewed interest in understanding interactions between these viruses and their hosts. We recently determined that TLR3 regulates a detrimental innate immune response that enhances replication, morbidity, and mortality in mice in response to vaccinia virus, a model pathogen for studies of poxviruses. To further investigate Toll-like receptor signaling in vaccinia infection, we first focused on TRIF, the only known adapter protein for TLR3. Unexpectedly, bioluminescence imaging showed that mice lacking TRIF are more susceptible to vaccinia infection than wild-type mice. We then focused on TLR4, the other Toll-like receptor that signals through TRIF. Following respiratory infection with vaccinia, mice lacking TLR4 signaling had greater viral replication, hypothermia, and mortality than control animals. The mechanism of TLR4-mediated protection was not due to increased release of proinflammatory cytokines or changes in total numbers of immune cells recruited to the lung. Challenge of primary bone marrow macrophages isolated from TLR4 mutant and control mice suggested that TLR4 recognizes a viral ligand rather than an endogenous ligand. These data establish that TLR4 mediates a protective innate immune response against vaccinia virus, which informs development of new vaccines and therapeutic agents targeted against poxviruses

DNA methylation differentiates smoking from vaping and non-combustible tobacco use

Increasing use of non-combusted forms of nicotine such as e-cigarettes poses important public health questions regarding their specific risks relative to combusted tobacco products such as cigarettes. To fully delineate these risks, improved biomarkers that can distinguish between these forms of nicotine use are needed. Prior work has suggested that methylation status at cg05575921 may serve as a specific biomarker of combusted tobacco smoke exposure. We hypothesized combining this epigenetic biomarker with conventional metabolite assays could classify the type of nicotine product consumption. Therefore, we determined DNA methylation and serum cotinine values in samples from 112 smokers, 35 e-cigarette users, 19 smokeless tobacco users, and 269 controls, and performed mass spectroscopy analyses of urine samples from all nicotine users and 22 verified controls to determine urinary levels of putatively nicotine product-specific substances; propylene glycol, 2-cyanoethylmercapturic acid (CEMA), and anabasine. 1) Cigarette smoking was associated with a dose dependent demethylation of cg05575921 and increased urinary CEMA and anabasine levels, 2) e-cigarette use did not demethylate cg05575921, 3) smokeless tobacco use also did not demethylate cg05575921 but was positively associated with anabasine levels 4) CEMA and cg05575921 levels were highly correlated and 5) propylene glycol levels did not reliably distinguish use groups. Cg05575921 assessments distinguish exposure to tobacco smoke from smokeless sources of nicotine including e-cigarettes and smokeless tobacco, neither of which are associated with cg05575921 demethylation. A combination of methylomic and metabolite profiling may allow for accurate classification use status of a variety of nicotine containing products

Increased viral replication in TLR4 mutant mice is more pronounced at higher viral dose.

<p>C3HeB/FeJ and C3H/HeJ mice were infected with 5×10⁵ pfu Vac-GFL intranasally. (A) Head luminescence. (B) Chest luminescence. (C) Representative chest images. C3HeB/FeJ (left) and C3H/HeJ (right), 30 s exposure, f-stop 1. Purple denotes lower luminescence intensity; red, higher luminescence intensity.*<i>p</i><0.05. Error bars denote SEM.</p

Lack of TLR4 does not impair IFN-β production.

<p>IFN-β concentrations in lung homogenate supernatants were measured by ELISA. Points represent individual mice. Dashed line represents lower limit of reliable detection on standard curve. Solid lines represent mean IFN-β concentration.</p

Lack of TLR4 does not impair proinflammatory cytokine production.

<p>IL-6 levels in the lung homogenate supernatants measured by ELISA. Error bars denote SEM. *<i>p</i><0.05.</p