The Ubiquitous Dermokine Delta Activates Rab5 Function in the Early Endocytic Pathway

The expression of the recently identified dermokine (Dmkn) gene leads to four families of proteins with as yet unknown functions. The secreted α, β and γ isoforms share an epidermis-restricted expression pattern, whereas the δ isoform is intracellular and ubiquitous. To get an insight into Dmknδ function, we performed yeast two-hybrid screening and identified the small GTPases Rab5 as partners for Dmknδ. The Rab5 proteins are known to regulate membrane docking and fusion in the early endocytic pathway. GST pull-down assays confirmed the direct interaction between Rab5 and Dmknδ. Transient expression of Dmknδ in HeLa cells led to the formation of punctate structures colocalized with endogenous Rab5 and clathrin, indicating Dmknδ involvement in the early steps of endocytosis. Dmknδ indeed colocalized with transferrin at early stages of endocytosis, but did not modulate its endocytosis or recycling kinetics. We also showed that Dmknδ was able to bind both inactive (GDP-bound) and active (GTP-bound) forms of Rab5 in vitro but preferentially targeted GDP-bound form in HeLa cells. Interestingly, Dmknδ expression rescued the Rab5S34N-mediated inhibition of endosome fusion. Moreover, Dmknδ caused the enlargement of vesicles positive for Rab5 by promoting GTP loading onto the small GTPase. Together our data reveal that Dmknδ activates Rab5 function and thus is involved in the early endosomal trafficking

Precision medicine in cats:novel niemann-pick type C1 diagnosed by whole-genome sequencing

State-of-the-art health care includes genome sequencing of the patient to identify genetic variants that contribute to either the cause of their malady or variants that can be targeted to improve treatment. The goal was to introduce state-of-the-art health care to cats using genomics and a precision medicine approach. To test the feasibility of a precision medicine approach in domestic cats, a single cat that presented to the University of Missouri, Veterinary Health Center with an undiagnosed neurologic disease was whole-genome sequenced. The DNA variants from the cat were compared to the DNA variant database produced by the 99 Lives Cat Genome Sequencing Consortium. Approximately 25× genomic coverage was produced for the cat. A predicted p.H441P missense mutation was identified in NPC1, the gene causing Niemann-Pick type C1 on cat chromosome D3.47456793 caused by an adenine-to-cytosine transversion, c.1322A>C. The cat was homozygous for the variant. The variant was not identified in any other 73 domestic and 9 wild felids in the sequence database or 190 additionally genotyped cats of various breeds. The successful effort suggested precision medicine is feasible for cats and other undiagnosed cats may benefit from a genomic analysis approach. The 99 Lives DNA variant database was sufficient but would benefit from additional cat sequences. Other cats with the mutation may be identified and could be introduced as a new biomedical model for NPC1. A genetic test could eliminate the disease variant from the population

A deletion in GDF7 is associated with a heritable forebrain commissural malformation concurrent with ventriculomegaly and interhemispheric cysts in cats

Publisher Copyright: © 2020 by the authors.An inherited neurologic syndrome in a family of mixed-breed Oriental cats has been characterized as forebrain commissural malformation, concurrent with ventriculomegaly and interhemispheric cysts. However, the genetic basis for this autosomal recessive syndrome in cats is unknown. Forty-three cats were genotyped on the Illumina Infinium Feline 63K iSelect DNA Array and used for analyses. Genome-wide association studies, including a sib-transmission disequilibrium test and a case-control association analysis, and homozygosity mapping, identified a critical region on cat chromosome A3. Short-read whole genome sequencing was completed for a cat trio segregating with the syndrome. A homozygous 7 bp deletion in growth differentiation factor 7 (GDF7) (c.221_227delGCCGCGC [p.Arg74Profs]) was identified in affected cats, by comparison to the 99 Lives Cat variant dataset, validated using Sanger sequencing and genotyped by fragment analyses. This variant was not identified in 192 unaffected cats in the 99 Lives dataset. The variant segregated concordantly in an extended pedigree. In mice, GDF7 mRNA is expressed within the roof plate when commissural axons initiate ventrally-directed growth. This finding emphasized the importance of GDF7 in the neurodevelopmental process in the mammalian brain. A genetic test can be developed for use by cat breeders to eradicate this variant.Peer reviewe

Werewolf, there wolf : Variants in hairless associated with hypotrichia and roaning in the lykoi cat breed

Publisher Copyright: © 2020 by the authors. Licensee MDPI, Basel, Switzerland.A variety of cat breeds have been developed via novelty selection on aesthetic, dermatological traits, such as coat colors and fur types. A recently developed breed, the lykoi (a.k.a. werewolf cat), was bred from cats with a sparse hair coat with roaning, implying full color and all white hairs. The lykoi phenotype is a form of hypotrichia, presenting as a significant reduction in the average numbers of follicles per hair follicle group as compared to domestic shorthair cats, a mild to severe perifollicular to mural lymphocytic infiltration in 77% of observed hair follicle groups, and the follicles are often miniaturized, dilated, and dysplastic. Whole genome sequencing was conducted on a single lykoi cat that was a cross between two independently ascertained lineages. Comparison to the 99 Lives dataset of 194 non‐lykoi cats suggested two variants in the cat homolog for Hairless (HR) (HR lysine demethylase and nuclear receptor corepressor) as candidate causal gene variants. The lykoi cat was a compound heterozygote for two loss of function variants in HR, an exon 3 c.1255_1256dupGT (chrB1:36040783), which should produce a stop codon at amino acid 420 (p.Gln420Serfs*100) and, an exon 18 c.3389insGACA (chrB1:36051555), which should produce a stop codon at amino acid position 1130 (p.Ser1130Argfs*29). Ascertainment of 14 additional cats from founder lineages from Canada, France and different areas of the USA identified four additional loss of function HR variants likely causing the highly similar phenotypic hair coat across the diverse cats. The novel variants in HR for cat hypotrichia can now be established between minor differences in the phenotypic presentations.Peer reviewe

Determinants of adherence to treatment in first-episode psychosis: a comprehensive review

Objective:To conduct a comprehensive review of current evidence on factors for nonadherence to treatment in individuals with first-episode psychosis (FEP).Methods:MEDLINE, LILACS, PsycINFO, and SciELO databases were searched with the keywords first episode psychosis, factor, adherence, nonadherence, engagement, disengagement, compliance, and intervention. References of selected studies were consulted for relevant articles.Results:A total of 157 articles were screened, of which 33 articles were retained for full review. The factors related to nonadherence were: a) patient-related (e.g., lower education level, persistent substance use, forensic history, unemployment, history of physical abuse); b) environment-related (e.g., no family involved in treatment, social adjustment difficulties); c) medication-related (e.g., rapid remission of negative symptoms when starting treatment, therapeutic alliance); and d) illness-related (e.g., more positive symptoms, more relapses). Treatment factors that improve adherence include a good therapeutic alliance and a voluntary first admission when hospitalization occurs.Conclusion:The results of this review suggest that nonadherence to treatment in FEP is multifactorial. Many of these factors are modifiable and can be specifically targeted in early intervention programs. Very few studies have assessed strategies to raise adherence in FEP

Rab5 is a partner of Dmknδ5.

<p><i>A.</i> By yeast two-hybrid screening, positive clones were identified as Rab5b and Rab5c. Rab5a, subsequently tested, was also able to grow on selective medium and was positive for the β–galactosidase filter assay. Three representative clones of each double transformant corresponding to Dmknδ5/Rab5b, -c or -a are shown. <i>B.</i> GST-Dmknδ5 fusion protein or GST alone were captured on glutathione-sepharose beads before loading HeLa protein extract (HeLa) or purified recombinant wild-type Rab5 (Rab5). Proteins initially loaded onto the column (input) or eluted from the column (output) were detected by immunoblotting with an antibody directed against Rab5. <i>C.</i> HeLa cells were transiently transfected with GFP-Dmknδ5 (green) and processed for immunofluorescence analysis using an anti-Rab5 antibody (red). Representative transfected cells are shown, where GFP-Dmknδ5 is found in punctate structures (arrowheads) partially colocalized with endogenous Rab5.</p

The N-terminal region of Dmknδ5 is responsible for the interaction with Rab5.

<p><i>A.</i> δ5-Nt and δ5-Ct were tested for interaction with Rab5 using the yeast two-hybrid system. Expression of the reporter genes assay is shown for three representative clones of each double transformant (δ5-Nt/Rab5 and δ5-Ct/Rab5). <i>B.</i> HeLa cells were transiently transfected with DsRed-Rab5wt (red) and GFP-δ5-Nt or GFP-δ5-Ct (green) and observed by confocal microscopy. Bar, 5 µm</p

Dmknδ5 colocalizes early with endocytosed transferrin and does not influence transferrin uptake or recycling kinetics.

<p><i>A</i>. HeLa cells transiently transfected with GFP-Dmknδ5 were incubated with AlexaFluor-555 conjugated-transferrin at 4°C (<i>0 min</i>). Transferrin uptake was then carried out for 4, 10, 15 or 30 min at 37°C, as indicated. Localization of GFP-Dmknδ5 (green) and Alexa Fluor-555 conjugated-transferrin (red) was then observed by confocal microscopy. Arrowheads show colocalization between GFP-Dmknδ5 and transferrin. Bars, 5 µm. <i>B, C.</i> Kinetics of endocytosis (<i>B</i>) and recycling (<i>C</i>) of transferrin in HeLa cells transiently expressing GFP-Dmknδ5 (black square) or GFP alone (open circle). For transferrin endocytosis, results are expressed as the percentage of internalized transferrin with respect to the prebound transferrin at +4°C (<i>B</i>). For recycling of intracellular transferrin, results are expressed as the percentage of initial (time 0, 100%) intracellular tranferrin (<i>C</i>). In <i>B</i> and <i>C</i>, the graphs are mean ± SD of three independent experiments.</p

Dmknδ interacts with active and inactive Rab5.

<p><i>A.</i> After capture of GST-Dmknδ5 fusion protein or GST alone on glutathione-sepharose beads, purified recombinant wild-type Rab5 (Rab5), incubated beforehand with GppNHp (active conformation) or GDP (inactive conformation), was loaded. Proteins initially loaded onto the column (input) or eluted from the column (output) were detected by immunoblotting with an antibody directed against Rab5. <i>B.</i> HeLa cells were transiently transfected with GFP-Dmknδ5 (green) and DsRed-Rab5Q79L or DsRed-Rab5S34N (red), respectively. Cells were then visualized by confocal microscopy. Bars, 5 µm</p

Characterization of Dmknδ5 positive vesicles.

<p>HeLa cells transiently transfected with GFP-Dmknδ5 (green) were processed for immunofluorescence analysis using antibodies directed against the endogenous organelle markers (red) clathrin (<i>A</i>), EEA1 (<i>B</i>), Rab11 (<i>C</i>), Rab7 (<i>D</i>) or LAMP1 (<i>E</i>). Cells were then visualized by confocal microscopy. Colocalization with GFP-Dmknδ5 was obvious only with clathrin as seen in the merged images (<i>A, arrowheads</i>). Bars, 5 µm</p