Primary ciliary dyskinesia (PCD) is a genetic disease characterized by abnormalities in ciliary structure/function. We hypothesized that the major clinical and biologic phenotypic markers of the disease could be evaluated by studying a cohort of subjects suspected of having PCD. Of 110 subjects evaluated, PCD was diagnosed in 78 subjects using a combination of compatible clinical features coupled with tests of ciliary ultrastructure and function. Chronic rhinitis/sinusitis (n = 78; 100%), recurrent otitis media (n = 74; 95%), neonatal respiratory symptoms (n = 57; 73%), and situs inversus (n = 43; 55%) are strong phenotypic markers of the disease. Mucoid Pseudomonas aeruginosa (n = 12; 15%) and nontuberculous mycobacteria (n = 8; 10%) were present in older (> 30 years) patients with PCD. All subjects had defects in ciliary structure, 66% in the outer dynein arm. Nasal nitric oxide production was very low in PCD (nl/minute; 19 +/- 17 vs. 376 +/- 124 in normal control subjects). Rigorous clinical and ciliary phenotyping and measures of nasal nitric oxide are useful for the diagnosis of PCD. An increased awareness of the clinical presentation and diagnostic criteria for PCD will help lead to better diagnosis and care for this orphan disease.

Primary ciliary dyskinesia: diagnostic and phenotypic features.

Centrosomes, which were first described in the late 19th century, are found in most animal cells and undergo duplication once every cell cycle so that their number remains stable, like the genetic material of a cell. However, their function and regulation have remained elusive and controversial. Only recently has some understanding of these fundamental aspects of centrosome function and biogenesis been gained through the concerted application of genomics and proteomics, which we term 'centrosomics'. The identification of new molecules has highlighted the evolutionary conservation of centrosome function and provided a conceptual framework for understanding centrosome behaviour and how it can go awry in human disease.

Centrosome biogenesis and function: centrosomics brings new understanding

Hydrodynamic Flow-Mediated Protein Sorting on the Cell Surface of Trypanosomes

Joubert syndrome (JS) and related disorders are a group of autosomal-recessive conditions sharing the “molar tooth sign” on axial brain MRI, together with cerebellar vermis hypoplasia, ataxia, and psychomotor delay. JS is suggested to be a disorder of cilia function and is part of a spectrum of disorders involving retinal, renal, digital, oral, hepatic, and cerebral organs. We identified mutations in ARL13B in two families with the classical form of JS. ARL13B belongs to the Ras GTPase family, and in other species is required for ciliogenesis, body axis formation, and renal function. The encoded Arl13b protein was expressed in developing murine cerebellum and localized to the cilia in primary neurons. Overexpression of human wild-type but not patient mutant ARL13B rescued the Arl13b scorpion zebrafish mutant. Thus, ARL13B has an evolutionarily conserved role mediating cilia function in multiple organs.

https://depts.washington.edu/joubert/docs/publications/JS-ARL13B-Cantagrel-AJHG2008.pdf

Mutations in the Cilia Gene ARL13B Lead to the Classical Form of Joubert Syndrome

The Proteome of the Mouse Photoreceptor Sensory Cilium Complex

There is little hope for a vaccine for African sleeping sickness, and most of the drugs currently used to treat it are old, not particularly effective and difficult to use in the conditions that prevail in sub-Saharan Africa, where the disease is endemic. So the discovery of a new class of molecule that might be targeted by drug intervention could be an important boost to the field. The sleeping sickness parasite Trypanosoma brucei is a protozoon equipped with a whip-like flagellum. RNA interference (RNAi) knock-down experiments show that a functioning flagellum is essential for Trypanosoma's survival in the bloodstream. That makes the flagellum a possible point of therapeutic attack, and proteomic analysis points to a number of trypanosome-specific flagellar proteins that could be targeted. On the cover, monstrous cells of bloodstream-form trypanosomes formed by a failure of cell division in cells with defective flagella. The 9 + 2 microtubule axoneme of flagella and cilia represents one of the most iconic structures built by eukaryotic cells and organisms. Both unity and diversity are present among cilia and flagella on the evolutionary as well as the developmental scale. Some cilia are motile, whereas others function as sensory organelles and can variously possess 9 + 2 and 9 + 0 axonemes and other associated structures1. How such unity and diversity are reflected in molecular repertoires is unclear. The flagellated protozoan parasite Trypanosoma brucei is endemic in sub-Saharan Africa, causing devastating disease in humans and other animals2. There is little hope of a vaccine for African sleeping sickness and a desperate need for modern drug therapies3. Here we present a detailed proteomic analysis of the trypanosome flagellum. RNA interference (RNAi)-based interrogation of this proteome provides functional insights into human ciliary diseases and establishes that flagellar function is essential to the bloodstream-form trypanosome. We show that RNAi-mediated ablation of various proteins identified in the trypanosome flagellar proteome leads to a rapid and marked failure of cytokinesis in bloodstream-form (but not procyclic insect-form) trypanosomes, suggesting that impairment of flagellar function may provide a method of disease control. A postgenomic meta-analysis, comparing the evolutionarily ancient trypanosome with other eukaryotes including humans, identifies numerous trypanosome-specific flagellar proteins, suggesting new avenues for selective intervention.

/pdf/flagellar-motility-is-required-for-the-viability-of-the-56dj552b7n.pdf

Flagellar motility is required for the viability of the bloodstream trypanosome

https://link.springer.com/content/pdf/10.1016%2Fj.jasms.2008.08.014.pdf

Analysis of the trypanosome flagellar proteome using a combined electron transfer collisionally activated dissociation strategy.

The coronavirus disease of 2019 (COVID-19) pandemic launched an unprecedented global effort to rapidly develop vaccines to stem the spread of the novel severe acute respiratory syndrome coronavirus (SARS-CoV-2). Messenger ribonucleic acid (mRNA) vaccines were developed quickly by companies that were actively developing mRNA therapeutics and vaccines for other indications, leading to two mRNA vaccines being not only the first SARS-CoV-2 vaccines to be approved for emergency use but also the first mRNA drugs to gain emergency use authorization and to eventually gain full approval. This was possible partly because mRNA sequences can be altered to encode nearly any protein without significantly altering its chemical properties, allowing the drug substance to be a modular component of the drug product. Lipid nanoparticle (LNP) technology required to protect the ribonucleic acid (RNA) and mediate delivery into the cytoplasm of cells is likewise modular, as are technologies and infrastructure required to encapsulate the RNA into the LNP. This enabled the rapid adaptation of the technology to a new target. Upon the coattails of the clinical success of mRNA vaccines, this modularity will pave the way for future RNA medicines for cancer, gene therapy, and RNA engineered cell therapies. In this review, trends in the publication records and clinical trial registrations are tallied to show the sharp intensification in preclinical and clinical research for RNA medicines. Demand for the manufacturing of both the RNA drug substance (DS) and the LNP drug product (DP) has already been strained, causing shortages of the vaccine, and the rise in development and translation of other mRNA drugs in the coming years will exacerbate this strain. To estimate demand for DP manufacturing, the dosing requirements for the preclinical and clinical studies of the two approved mRNA vaccines were examined. To understand the current state of mRNA-LNP production, current methods and technologies are reviewed, as are current and announced global capacities for commercial manufacturing. Finally, a vision is rationalized for how emerging technologies such as self-amplifying mRNA, microfluidic production, and trends toward integrated and distributed manufacturing will shape the future of RNA manufacturing and unlock the potential for an RNA medicine revolution.

Richard Broadhead

Papers

Flagellar motility is required for the viability of the bloodstream trypanosome

Analysis of the trypanosome flagellar proteome using a combined electron transfer collisionally activated dissociation strategy.

Current Status and Future Perspectives on MRNA Drug Manufacturing