Effects of α-crystallin gene knockout on zebrafish lens development

The α-crystallin small heat shock proteins contribute to the transparency and refractive properties of the vertebrate eye lens and prevent the protein aggregation that would otherwise produce lens cataract, the leading cause of human blindness. There are conflicting data in the literature as to what role the α-crystallins may play in early lens development. In this study we used CRISPR gene editing to produce zebrafish lines with null mutations for each of the three α-crystallin genes (cryaa, cryaba and cryabb). Absence of protein was confirmed by mass spectrometry and lens phenotypes were assessed with differential interference contrast microscopy and histology. Loss of αA-crystallin produced a variety of lens defects with varying severity in larval lenses at 3 and 4 dpf, but little significant change in normal fiber cell denucleation. Loss of either αBa- or αBb-crystallin produced no significant lens defects. Mutation of each α-crystallin gene did not alter the expression levels of the remaining two, suggesting a lack of genetic compensation. These data confirm a developmental role for αA-crystallin in lens development, but the range of phenotype severity suggests its loss simply increases the chance for defect, and that the protein is not essential. Our finding that cryaba and cryabb null mutants lack noticeable lens defects is congruent with insignificant transcript levels in lens epithelial and fiber cells. Future experiments can explore the molecular consequences of cryaa mutation and causes of lens defects in this null mutant, as well as the roles of other genes in lens development and function.

Oncholaimus tripapillatus sp. nov., a New Free-Living Marine Nematode of the Genus Oncholaimus Dujardin, 1845 (Nematoda: Enoplida: Oncholaimidae) from the Subtidal Sediment of Dokdo Island, East Sea, Korea, with a New Record of O. qingdaoensis Zhang & Platt, 1983

During a survey of the free-living marine nematodes of Korea, two species belonging to the genus Oncholaimus Dujardin, 1845 were discovered. A new species, Oncholaimus tripapillatus sp. nov. and a newly recorded species, Oncholaimus qingdaoensis Zhang & Platt, 1983, are reported. Oncholaimus tripapillatus sp. nov. was collected from the washing of subtidal coarse sediments around Dokdo Island of the East Sea, Korea. Oncholaimus tripapillatus sp. nov. is characterized by a relatively long (4071–4435 µm in males and 4514–4661 µm in females) and slender body, a slightly constricted head region, relatively long cephalic setae (10–12 µm), males having a precloacal sexual protuberance bearing two small cone-shaped supplementary spines, five pairs of long cloacal setae (three pairs of precloacal and two pairs of postcloacal setae in subventral position), and three remarkable papillae near the end of the tail, with two pairs of subventral setae. The Korean specimens of Oncholaimus qingdaoensis Zhang & Platt, 1983 are almost identical to the Chinese specimens of the original description from the intertidal sand of Qingdao, China. However, the Korean specimens differ from the Chinese specimens in the longer body length in males (3379–3715 µm vs. 2380–2640 µm), the larger spicule length (47–52 µm vs. 34–36 µm), and the presence of ventral tail papillae situated around 14–16 µm from the tail tip. Detailed morphological features and illustrations of two Oncholaimus species from Korea were obtained by differential interference contrast microscopy and scanning electron microscopy. A pictorial key to the species group with distinct tail papillae among the genus Oncholaimus is also provided.

Enhanced Loading of Paclitaxel in Cationic Liposomes by Replacement of Oleoyl with Linoleoyl Lipid Tails with Benefits in Cancer Therapeutics from In Vitro Studies

Lipid-based carriers of the hydrophobic drug paclitaxel (PTX) are used in clinical trials as next-generation agents for cancer chemotherapy. Improving the loading capacity of these carriers requires enhanced PTX solubilization. We compared the solubility of PTX in cationic liposomes (CLs) with lipid tails containing one (oleoyl; C18:1 Δ9; DOTAP/DOPC) or two (linoleoyl; C18:2 Δ9; DLinTAP/DLinPC) cis double bonds with newly synthesized cationic DLinTAP (2,3-dilinoleoyloxypropyltrimethylammonium methylsufate). We used differential-interference-contrast microscopy to directly observe PTX crystal formation and generate kinetic phase diagrams representing the time-dependence of PTX solubility as a function of PTX content in the membrane. Replacing tails bearing one cis double bond (DO lipids) with those bearing two (DLin lipids) significantly increased PTX membrane solubility in CLs. Remarkably, 8 mol% PTX in DLinTAP/DLinPC CLs remained soluble for approximately as long as 3 mol% PTX (the membrane solubility limit which has been the focus of most previous fundamental studies and clinical trials) in DOTAP/DOPC CLs. The large increase in solubility is likely caused by enhanced molecular affinity between lipid tails and PTX upon replacement of oleoyl by linoleoyl tails, rather than by the transition in membrane structure from lipid bilayers to inverse cylindrical micelles observed in small-angle X-ray scattering. Importantly, the efficacy of PTX-loaded CLs against human prostate cancer (PC3) cells from measurements of the IC50 of PTX cytotoxicity was unaffected by changing the lipid tails, and toxicity of the CL carrier alone was negligible. Moreover, efficacy was approximately doubled against human melanoma (M21) cells for PTX-loaded DLinTAP/DLinPC over DOTAP/DOPC CLs. The findings demonstrate the potential of chemical modifications of the lipid tails to increase the PTX membrane loading well over the typically used 3 mol% while maintaining (and in some cases even increasing) the efficacy of CLs. The increased PTX solubility will aid the development of liposomal PTX carriers that require significantly less lipid to deliver a given amount of PTX, reducing side effects and costs.