Biology subjects

Watson, P. R.

Publications and source records attributed to Watson, P. R..

3 recordsLinked to original sources

bioRxiv2025

Polar tube firing dynamics and ultrastructure from the shrimp microsporidian Ecytonucleospora hepatopenaei (EHP)

Ecytonucleospora (Enterocytozoon) hepatopenaei or EHP is an obligate intracellular parasite that belongs to phylum Microsporidia. EHP infection in shrimp results in growth retardation and size variation, leading to severe economic loss to shrimp aquaculture worldwide. Similar to other microsporidian species, EHP utilizes a harpoon-like invasion apparatus called the polar tube in order to infect host cells. The polar tube typically coils inside the spore and rapidly transits to a long, extended tube after being activated with proper stimuli. However, the mechanism and physical conditions affecting the polar tube firing in EHP are poorly understood. Here, we screened several germination buffers and found that a buffer containing potassium hydrogen phthalate (KHP) at pH 3.0 yielded the highest germination rate. The optimal temperatures for EHP germination range between 25-28{degrees}C, similar to the shrimps body temperature. The spores require at least 30 seconds to be activated, suggesting that the stimuli could rapidly move across the spore wall. We utilized high-speed live-cell imaging to study the dynamics of the EHP polar tube firing and compared the dynamics when the spores were treated with KHP and a previously reported stimulus, phloxine B. The polar tube firing dynamics between these two conditions are different. The total firing time was [~]100 milliseconds with the maximum velocity of [~]300 m*s-1 in KHP condition. Further investigation on the EHP polar tube ultrastructure using cryo-electron microscopy revealed that the EHP polar tube was composed of a membrane layer with an additional repetitive protein-array on the outermost surface. The distance between each repetitive unit ranges between 52-66 [A]. These repetitive units could possibly be polar tube proteins (PTPs). Altogether, this study provides insights into EHP polar tube firing dynamics and its architecture which are important foundations for understanding the biophysical factors governing EHP pathogenesis and in developing EHP control strategies. Author summaryThe microsporidian Ecytonucleospora hepatopenaei (EHP) poses a significant threat to global shrimp aquaculture, causing substantial economic losses. Many studies on EHP have been focused on development of detection methods, and farm management. Little is known about EHP pathogenesis and how it infects shrimp cells. Here, we identified laboratory conditions to activate the parasites infection process, which occurs on a millisecond timescale. We utilized a high-speed live-cell imaging to further characterize the rapid EHP infection process. The architecture and ultrastructure of EHP invasion organelle were also investigated using cryo-electron microscopy. Our findings provide a better understanding of the EHP infection mechanisms that are crucial for developing effective control strategies.

microbiology↗

bioRxiv2023

Crystal Structure of Histone Deacetylase 6 Complexed with (R)-Lipoic Acid, an Essential Cofactor in Central Carbon Metabolism

The enzyme cofactor (R)-lipoic acid plays a critical role in central carbon metabolism due to its catalytic function in the generation of acetyl-CoA, which links glycolysis with the tricarboxylic acid cycle. This cofactor is also essential for the generation of succinyl CoA within the tricarboxylic acid cycle. However, the biological functions of (R)-lipoic acid extend beyond metabolism owing to its facile redox chemistry. Most recently, the reduced form of (R)-lipoic acid, (R)-dihydrolipoic acid, has been shown to inhibit histone deacetylases (HDACs) with selectivity for the inhibition of HDAC6. Here, we report the 2.4 [A]-resolution X-ray crystal structure of the HDAC6-(R)-dihydrolipoic acid complex, and we report a dissociation constant (KD) of 350 nM for this complex as determined by isothermal titration calorimetry. The crystal structure illuminates key affinity determinants in the enzyme active site, including thiolate-Zn2+ coordination and S-{pi} interactions in the F583-F643 aromatic crevice. This study provides the first visualization of the connection between HDAC function and the biological response to oxidative stress: the dithiol moiety of (R)-dihydrolipoic acid can serve as a redox-regulated pharmacophore capable of simultaneously targeting the catalytic Zn2+ ion and the aromatic crevice in the active site of HDAC6.

biochemistry↗

bioRxiv2017

Adapterama III: Quadruple-indexed, triple-enzyme RADseq libraries for about $1USD per Sample (3RAD)

Molecular ecologists frequently use genome reduction strategies that rely upon restriction enzyme digestion of genomic DNA to sample consistent portions of the genome from many individuals (e.g., RADseq, GBS). However, researchers often find the existing methods expensive to initiate and/or difficult to implement consistently, especially due to the inability to highly-multiplex samples to fill entire sequencing lanes. Here, we introduce a low-cost and highly robust approach for the construction of dual-digest RADseq libraries that relies on adapters and primers designed in Adapterama I. Major features of our method include: 1) minimizing the number of processing steps; 2) focusing on a single strand of sample DNA for library construction, allowing the use of a non-phosphorylated adapter on one end; 3) ligating adapters in the presence of active restriction enzymes, thereby reducing chimeras; 4) including an optional third restriction enzyme to cut apart adapter-dimers formed by the phosphorylated adapter, thus increasing the efficiency of adapter ligation to sample DNA, which is particularly effective when only low quantity/quality DNA samples are available; 5) interchangeable adapter designs; 6) incorporating variable-length internal indexes within the adapters to increase the scope of sample indexing, facilitate pooling, and increase sequence diversity; 7) maintaining compatibility with universal dual-indexed primers and thus, Illumina sequencing reagents and libraries; and, 8) easy modification for the identification of PCR duplicates. We present eight adapter designs that work with 72 restriction enzyme combinations. We demonstrate the efficiency of our approach by comparing it with existing methods, and we validate its utility through the discovery of many variable loci in a variety of non-model organisms. Our 2RAD/3RAD method is easy to perform, has low startup costs, has increased utility with low-concentration input DNA, and produces libraries that can be highly-multiplexed and pooled with other Illumina libraries.

genomics↗