CRMP/UNC-33 maintains neuronal microtubule arrays by promoting individual microtubule rescue.

Microtubules (MTs) are intrinsically dynamic polymers. In neurons, staggered individual microtubules form stable, polarized acentrosomal MT arrays spanning the axon and dendrite to support long-distance intracellular transport. How the stability and polarity of these arrays are maintained when individual MTs remain highly dynamic is still an open question. Here we visualize MT arrays in vivo in C. elegans neurons with single microtubule resolution. We find that the CRMP family homolog, UNC-33, is essential for the stability and polarity of MT arrays in neurites. In unc-33 mutants, MTs exhibit dramatically reduced rescue after catastrophe, develop gaps in coverage, and lose their polarity, leading to trafficking defects. UNC-33 is stably anchored on the cortical cytoskeleton and forms patch-like structures along the dendritic shaft. These discrete and stable UNC-33 patches concentrate free tubulins and correlate with MT rescue sites. In vitro , purified UNC-33 preferentially associates with MT tips and increases MT rescue frequency. Together, we propose that UNC-33 functions as a microtubule-associated protein (MAP) to promote individual MT rescue locally. Through this activity, UNC-33 prevents the loss of individual MTs, thereby maintaining the coverage and polarity of MT arrays throughout the lifetime of neurons.

Randomized controlled field trial to assess the efficacy of an intranasal Moraxella bovis cytotoxin vaccine against naturally occurring infectious bovine keratoconjunctivitis.

Background: Infectious bovine keratoconjunctivitis (IBK; pinkeye) is generally considered to be caused by corneal infections with Moraxella bovis. Previous studies demonstrated that M. bovis cytotoxin-specific mucosal immune responses in the bovine eye can be stimulated by intranasal vaccination with a recombinant M. bovis cytotoxin subunit adjuvanted with polyacrylic acid. Methods: A randomized controlled field trial (two-arm parallel design with blinding) was conducted in beef steers in Northern California to determine if this vaccine could prevent naturally occurring IBK and/or reduce morbidity rates associated with this disease. Beef steers were vaccinated intranasally on days 0 and 21 with either a recombinant M. bovis cytotoxin subunit adjuvanted with polyacrylic acid (Vaccine group) or adjuvant alone (Control group). Eye examinations were performed on all steers every 7 days for 16 weeks to document the occurrence of IBK and to determine sizes of corneal ulcers. Serum and tear samples were collected on days 0, 42, and 112 from a subset of animals to measure changes in systemic and ocular immune responses to M. bovis cytotoxin. Results: The cumulative proportion of steers that developed IBK after 16 weeks did not differ between groups. Variables related to disease severity were numerically lower in steers that received the experimental vaccine. IBK-affected Vaccine group steers had a significantly lower number of observation weeks with severe ulcers versus Control group steers. Cytotoxin-specific tear IgA was significantly higher in Vaccine group compared to Control group steers on day 112. Conclusion: Although the proportion of animals that developed corneal ulcers associated with IBK did not differ between groups, the lowered metrics of disease severity in vaccinated steers suggests that intranasal vaccination with recombinant M. bovis cytotoxin can reduce the severity of IBK in cattle.

Relatedness of type IV pilin PilA amongst geographically diverse Moraxella bovoculi isolated from cattle with infectious bovine keratoconjunctivitis.

Introduction. Moraxella bovoculi is frequently isolated from the eyes of cattle with infectious bovine keratoconjunctivitis (IBK; pinkeye). As with M. bovis, which has been causally linked to IBK, M. bovoculi expresses an RTX (repeats in the structural toxin) cytotoxin that is related to M. bovis cytotoxin. Pilin, another pathogenic factor in M. bovis, is required for corneal attachment. Seven antigenically distinct pilin serogroups have been described in M. bovis.Hypothesis/Gap Statement. Multiple different serogroups exist amongst type IV pilin encoded by M. bovis, however, it is not known whether M. bovoculi exhibits a similar degree of diversity in type IV pilin that it encodes.Aim. This study was done to characterize a structural pilin (PilA) encoded by M. bovoculi isolated from cases of IBK to determine if diversity exists amongst PilA sequences.Methodology. Ninety-four isolates of M. bovoculi collected between 2002 and 2017 from 23 counties throughout California and from five counties in four other Western states were evaluated.Results. DNA sequencing and determination of deduced amino acid sequences revealed ten (designated groups A through J) unique PilA sequences that were ~96.1-99.3 % identical. Pilin groups A and C matched previously reported putative PilA sequences from M. bovoculi isolated from IBK-affected cattle in the USA (Virginia, Nebraska, and Kansas) and Asia (Kazakhstan). The ten pilin sequences identified were only ~74-76 % identical to deduced amino acid sequences of putative pilin proteins identified from the previously reported whole-genome sequences of M. bovoculi derived from deep nasopharyngeal swabs of IBK-asymptomatic cattle.Conclusions. Compared to the diversity reported between structural pilin proteins amongst different serogroups of M. bovis, M. bovoculi PilA from geographically diverse isolates derived from IBK-affected cattle are more conserved.

Autoregulatory control of microtubule binding in doublecortin-like kinase 1.

The microtubule-associated protein, doublecortin-like kinase 1 (DCLK1), is highly expressed in a range of cancers and is a prominent therapeutic target for kinase inhibitors. The physiological roles of DCLK1 kinase activity and how it is regulated remain elusive. Here, we analyze the role of mammalian DCLK1 kinase activity in regulating microtubule binding. We found that DCLK1 autophosphorylates a residue within its C-terminal tail to restrict its kinase activity and prevent aberrant hyperphosphorylation within its microtubule-binding domain. Removal of the C-terminal tail or mutation of this residue causes an increase in phosphorylation within the doublecortin domains, which abolishes microtubule binding. Therefore, autophosphorylation at specific sites within DCLK1 has diametric effects on the molecule's association with microtubules. Our results suggest a mechanism by which DCLK1 modulates its kinase activity to tune its microtubule-binding affinity. These results provide molecular insights for future therapeutic efforts related to DCLK1's role in cancer development and progression.

Automation of PacBio SMRTbell NGS library preparation for bacterial genome sequencing.

BACKGROUND: The PacBio RS II provides for single molecule, real-time DNA technology to sequence genomes and detect DNA modifications. The starting point for high-quality sequence production is high molecular weight genomic DNA. To automate the library preparation process, there must be high-throughput methods in place to assess the genomic DNA, to ensure the size and amounts of the sheared DNA fragments and final library. FINDINGS: The library construction automation was accomplished using the Agilent NGS workstation with Bravo accessories for heating, shaking, cooling, and magnetic bead manipulations for template purification. The quality control methods from gDNA input to final library using the Agilent Bioanalyzer System and Agilent TapeStation System were evaluated. CONCLUSIONS: Automated protocols of PacBio 10 kb library preparation produced libraries with similar technical performance to those generated manually. The TapeStation System proved to be a reliable method that could be used in a 96-well plate format to QC the DNA equivalent to the standard Bioanalyzer System results. The DNA Integrity Number that is calculated in the TapeStation System software upon analysis of genomic DNA is quite helpful to assure that the starting genomic DNA is not degraded. In this respect, the gDNA assay on the TapeStation System is preferable to the DNA 12000 assay on the Bioanalyzer System, which cannot run genomic DNA, nor can the Bioanalyzer work directly from the 96-well plates.