Although tissue clearing and subsequent whole-brain imaging is now possible, standard protocols need to be adjusted to the innate properties of each specific tissue for optimal results. This work modifies exiting protocols to clear fragile brain samples and documents a downstream pipeline for image processing and data analysis.
 
  We developed a clearing protocol, CUBIC-f, which we optimized for fragile samples, such as the salamander brain. We modified hydrophilic and aqueous' tissue-clearing methods based on Advanced CUBIC by incorporating Omnipaque 350 for refractive index matching.
 
  By combining CUBIC-f, light sheet microscopy and bioinformatic pipelines, we quantified neuronal cell density, traced genetically marked fluorescent cells over long distance, and performed high resolution characterization of neural progenitor cells in the salamander brain. We also found that CUBIC-f is suitable for conserving tissue integrity in embryonic mouse brains.
 
  CUBIC-f shortens clearing and staining times, and requires less reagent use than Advanced CUBIC and Advanced CLARITY.
 
  CUBIC-f is suitable for conserving tissue integrity in embryonic mouse brains, larval and adult salamander brains which display considerable deformation using traditional CUBIC and CLARITY protocols.
 CUBIC-f is suitable for conserving tissue integrity in embryonic mouse brains, larval and adult salamander brains which display considerable deformation using traditional CUBIC and CLARITY protocols.Dysfunction of the endolysosomal system is often associated with neurodegenerative disease because postmitotic neurons are particularly reliant on the elimination of intracellular aggregates. Adequate function of endosomes and lysosomes requires finely tuned luminal ion homeostasis and transmembrane ion fluxes. Endolysosomal CLC Cl-/H+ exchangers function as electric shunts for proton pumping and in luminal Cl- accumulation. We now report three unrelated children with severe neurodegenerative disease, who carry the same de novo c.1658A>G (p.Tyr553Cys) mutation in CLCN6, encoding the late endosomal Cl-/H+-exchanger ClC-6. Whereas Clcn6-/- mice have only mild neuronal lysosomal storage abnormalities, the affected individuals displayed severe developmental delay with pronounced generalized hypotonia, respiratory insufficiency, and variable neurodegeneration and diffusion restriction in cerebral peduncles, midbrain, and/or brainstem in MRI scans. The p.Tyr553Cys amino acid substitution strongly slowed ClC-6 gating and increased current amplitudes, particularly at the acidic pH of late endosomes. Transfection of ClC-6Tyr553Cys, but not ClC-6WT, generated giant LAMP1-positive vacuoles that were poorly acidified. Their generation strictly required ClC-6 ion transport, as shown by transport-deficient double mutants, and depended on Cl-/H+ exchange, as revealed by combination with the uncoupling p.Glu200Ala substitution. Transfection of either ClC-6Tyr553Cys/Glu200Ala or ClC-6Glu200Ala generated slightly enlarged vesicles, suggesting that p.Glu200Ala, previously associated with infantile spasms and microcephaly, is also pathogenic. Bafilomycin treatment abrogated vacuole generation, indicating that H+-driven Cl- accumulation osmotically drives vesicle enlargement. Our work establishes mutations in CLCN6 associated with neurological diseases, whose spectrum of clinical features depends on the differential impact of the allele on ClC-6 function.A form-preparation task in the language production field was adopted to examine output phonological representations in Chinese dyslexia and their susceptibility to training. Forty-one Chinese children with dyslexia (7-11 years old) and 36 chronological age controls completed this task. The controls demonstrated a marginally significant syllable facilitation effect (d = -0.13), indicating their use of syllable-sized phonological representations during speech production, while the group with dyslexia showed a significantly different pattern (d = 0.04), opposite to the direction of a facilitation effect. The children with dyslexia were then randomly assigned to either metalinguistic training (N = 22) or working memory training (N = 19). Only the metalinguistic training subgroup demonstrated a significant syllable facilitation effect afterward (metalinguistic d = -0.13; working memory d = -0.01). The results suggest the presence of a phonological representation deficit at the syllable level in Chinese dyslexia and its possible remediation by metalinguistic training. Such a phonological deficit in readers of a logographic script strongly supports the impaired phonological representation view of developmental dyslexia. A video abstract of this article can be viewed at https//youtu.be/zT2Be0xMkh0.This study aimed to develop a highly selective, sensitive and fast liquid chromatography tandem mass spectrometric (LC-MS/MS) method for the determination of obacunone in rat plasma. Sample preparation was accomplished by a simple solid-phase extraction procedure. Chromatographic separation was carried out on an ACQUITY BEH C18 column using acetonitrile/methanol (11, v/v) and 0.1% formic acid in water as mobile phase at a flow rate of 0.4 mL/min. Quantification was performed with multiple reactions monitoring in positive ion mode with the precursor-to-product ion transitions at m/z 455.2 > 161.1 for obacunone and m/z 515.2 > 161.1 for nomilin (internal standard). The assay was demonstrated to be linear over the concentration range of 0.1-1,000 ng/mL with correlation coefficient >0.999 (r > 0.999). The intra- and inter-day accuracy ranged from -8.33 to 10.40%, while the precision was 75.32%, and the assay was free of matrix effect. The validated LC-MS/MS method was successfully applied to the pharmacokinetic study of obacunone in rats after oral and intravenous administrations.  https://www.selleckchem.com/products/cpi-0610.html  The oral bioavailability of obacunone was 13.59%.Progesterone has been recognized as essential for the establishment and maintenance of pregnancy, and is typically known as an immunosuppressive agent. However, its effects on mediating Brucella infection-induced inflammation have not been evaluated. Here we demonstrated that B. abortus infection inhibits progesterone levels in the pregnant mouse via suppressing the production of progesterone by placenta. Progesterone treatment significantly reduced the secretion of inflammatory cytokines in serum, macrophages and trophoblasts of B. abortus-infected mice leading to decreased placentitis and enhanced the pup viability. Mechanistically, this decreased inflammatory response results from inhibition of NF-kB activation by progesterone. Moreover, progesterone treatment suppresses B. abortus growth within trophoblasts associated with an inability of bacteria to escape the late endosome compartment in vitro. Collectively, our data illustrate that progesterone treatment might be useful therapeutically in protection against placentitis or abortion caused by B.