In situ hybridization chain reaction (HCR) is a highly sensitive method for visualizing single-molecule messenger RNA (mRNA) using self-assembling hairpin DNA pairs. However, conventional in situ HCR is primarily optimized for fluorescence detection, limiting its applicability in routine pathology and in tissues with strong autofluorescence. In this study, we established a chromogenic in situ HCR protocol using short hairpin DNA that achieves sensitivity comparable to fluorescent in situ HCR. To achieve efficient amplification and avoid steric hindrance, we adopted an indirect labeling strategy in which hairpin DNA conjugated with haptens (biotin, digoxigenin, or fluorescein) is detected using enzyme-conjugated streptavidin or antibodies. We validated this approach across various mouse tissues, including the brain, kidney, and liver, and on both slide-mounted and free-floating sections. The protocol enabled the clear visualization of low-abundance transcripts such as Oxtr and Esr1. Furthermore, we demonstrated the versatility of this approach by performing duplex chromogenic staining for two mRNA targets and by combining in situ HCR with immunohistochemistry to visualize mRNA and protein simultaneously. This chromogenic in situ HCR retains the high sensitivity of HCR and adds the practical advantages of bright-field detection, thereby expanding the utility of in situ HCR in histological research.
The new genus Pseudoangiostoma gen. n. (Nematoda: Angiostomatidae) was established on the basis of the type species Pseudoangiostoma onychodactyla comb. n. (syn. Angiostoma onychodactyla), which parasitizes the stomach and intestine of some species of the genus Onychodactylus (Amphibia: Hynobiidae) in Japan. Pseudoangiostoma gen. n. differs from the genus Angiostoma by having a lower inverted truncated cone-shaped buccal cavity, a thicker and strongly curved buccal wall, and for parasitizing hosts of the order Caudata. The phylogenetic pattern provided substantial suppot for the new genus, exhibiting two major divergent lineages (diverged in terrestrial gastropods vs. salamanders). This study indicated that P. onychodactyla comb. n. did not originate from host-switching event from terrestrial gastropods to salamanders. However, other species of Angiostoma parasitic in amphibians and reptiles are still considered insertae sedis.
Thioether analogs of vitamin K3 (VK3) promote the generation of reactive oxygen species (ROS) and enhance the expression of the apoptosis-inducing receptor Fas and its ligand, thereby inducing apoptosis through activation of mitogen-activated protein kinase (MAPK) and caspase-3. They can also induce apoptosis without generating ROS by maintaining phosphorylation via inhibition of mitochondrial or tyrosine and serine/threonine phosphatases involved in intracellular signal transduction. Moreover, VK3 analogs have been shown to upregulate death receptor 5 (DR5) and induce apoptosis in human leukemia-derived cells through the intracellular DR signaling pathway. In this study, we aimed to investigate the effect of the VK3 analog 2-(2-mer-captoethanol)-3-methyl-1,4-naphthoquinone (CPD5) on the expression of factors acting further upstream in this signaling pathway. Materials and Methods: Apoptosis induction in human leukemia-derived cells following CPD5 treatment was examined by assessing intracellular protein expression levels using Western blotting. The analyzed proteins were factors involved in apoptosis signaling upstream of DR5. Results: Treatment with CPD5 (10 & micro;M) activated caspase-3 and caspase-8 and increased the expression of DR5. Further investigation of upstream signaling revealed elevated levels of tumor necrosis factor alpha-related apoptosis-inducing ligand (TRAIL), which acts on death receptors. Expression of the TRAIL-cleaving enzyme was also increased. Conclusions: These findings indicate that CPD5 acts on the TRAIL-cleaving enzyme in leukemia-derived cells, leading to the excessive release of soluble TRAIL (sTRAIL) and subsequent apoptosis via DR5. By elucidating its upstream effects and site of action, CPD5 can be proposed as a potential anti-tumor agent.
To investigate the early macular choroidal hemodynamic changes induced by faricimab loading in treatment-naïve diabetic macular edema (DME) and to determine potential systemic crossover effects by comparing faricimab-injected eyes with the contralateral non-injected eyes. This retrospective, single-center cohort study analyzed data of a total of 92 eyes from 46 treatment-naïve patients with DME. All patients received three consecutive monthly intravitreal faricimab injections in the study eye. The primary outcome was the longitudinal change in macular choroidal blood flow (CBF) in injected and contralateral non-injected eyes, together with changes in central retinal thickness (CRT) and subfoveal choroidal thickness (SFCT). Macular CBF was quantified as mean blur rate (MBR) using laser speckle flowgraphy. Data were analyzed using a linear mixed-effects model. In the 46 injected eyes, faricimab treatment resulted in significant monthly decreases in CRT and SFCT. This anatomical improvement was accompanied by a modest but statistically significant reduction in macular CBF, whereas ocular perfusion pressure remained stable. Visual acuity significantly improved. In contrast, no significant changes occurred in macular CBF, CRT, or SFCT in the 46 contralateral non-injected eyes. Initial faricimab loading therapy yielded marked anatomical improvements with only modest changes in macular choroidal perfusion in treatment-naïve DME. No measurable changes were detected in the contralateral non-injected eyes, suggesting that the early effects of faricimab are predominantly localized to the treated eye. Faricimab substantially reduces central retinal thickness and improves visual acuity in treatment-naïve diabetic macular edema (DME) through dual inhibition of anti-vascular endothelial growth factor-A and angiopoietin-2. Conventional monotherapies with anti-vascular endothelial growth factor-A reduce choroidal blood flow; however, the specific hemodynamic profile of faricimab during the loading phase has not been fully characterized. In this study of 46 injected eyes with treatment-naïve DME, the initial faricimab loading phase resulted in marked anatomical improvements with unchanged vascular resistance and only modest changes in macular choroidal blood flow. These results suggest that faricimab functions via a vascular stabilization mechanism rather than excessive vasoconstriction. Quantitative assessment using laser speckle flowgraphy revealed no hemodynamic or structural changes in the contralateral non-injected eyes, providing novel evidence that faricimab exerts a potent localized effect with negligible systemic influence during the induction phase.
