Role of Csdc2 in Regulating Secondary Hair Follicle Growth in Cashmere Goats.

Cashmere goats possess two types of hair follicles, with the secondary hair follicles producing valuable cashmere fiber used for textiles. The growth of cashmere exhibits a seasonal pattern arising from photoperiod change. Transcription factors play crucial roles during this process. The transcription factor, cold-shock domain, containing C2 (Csdc2) plays a crucial role in modulating cell proliferation and differentiation. Our preceding research indicated that the expression of Csdc2 changes periodically during anagen to telogen. However, the mechanisms of Csdc2 in regulating SHF growth remain unclear. Here, we found that the knockdown of Csdc2 inhibits the proliferation of dermal papilla cells. ChIP-Seq analysis showed that Csdc2 had a unique DNA binding motif in SHFs. Through conjoint analysis of ChIP-Seq and RNA-Seq, we revealed a total of 25 candidate target genes of Csdc2. Notably, we discovered a putative Csdc2 binding site within roundabout guidance receptor 2 (Robo2) on chromosome 1 of the goat genome. Furthermore, qRT-PCR and dual-luciferase reporter assay confirmed Csdc2's positive regulatory influence on Robo2. These findings expand the research field of hair follicle transcriptional regulatory networks, offering insights into molecular breeding strategies to enhance cashmere production in goats.

Overexpression of miR-25 Downregulates the Expression of ROBO2 in Idiopathic Intellectual Disability.

Idiopathic intellectual disability (IID) encompasses the cases of intellectual disability (ID) without a known cause and represents approximately 50% of all cases. Neural progenitor cells (NPCs) from the olfactory neuroepithelium (NEO) contain the same information as the cells found in the brain, but they are more accessible. Some miRNAs have been identified and associated with ID of known etiology. However, in idiopathic ID, the effect of miRNAs is poorly understood. The aim of this study was to determine the miRNAs regulating the expression of mRNAs that may be involved in development of IID. Expression profiles were obtained using NPC-NEO cells from IID patients and healthy controls by microarray. A total of 796 miRNAs and 28,869 mRNAs were analyzed. Several miRNAs were overexpressed in the IID patients compared to controls. miR-25 had the greatest expression. In silico analysis showed that ROBO2 was the target for miR-25, with the highest specificity and being the most down-regulated. In vitro assay showed an increase of miR-25 expression induced a decrease in ROBO2 expression. In neurodevelopment, ROBO2 plays a crucial role in episodic learning and memory, so its down-regulation, caused by miR-25, could have a fundamental role in the intellectual disability that, until now, has been considered idiopathic.

SALL4 advances the proliferation and tumor cell stemness of colon cancer cells through the transcription and regulation of ROBO2.

SALL4 is a transcription factor highly expressed in diverse cancers and is implicated in the development of cancer. SALL4 has been implied to play a cancer-promoting role in colon cancer (CC), but the molecular mechanism remains unclear. Chromatin immunoprecipitation assay and dual-luciferase assay were conducted to verify the binding relationship of SALL4 and ROBO2. qRT-PCR detected the mRNA expression levels of SALL4 and ROBO2, and the flow cytometry analyzed the cell cycle distribution. Western blot examined SALL4 expression, and cell cycle/cell stemness-related proteins. The impact of SALL4 and ROBO2 on the proliferation capacity of cells and tumor cell stemness was elucidated by MTT, colony formation, and sphere-forming assays. SALL4 and ROBO2 were up-regulated in CC, and SALL4 could activate the transcription of ROBO2. Down-regulated SALL4 was able to significantly restrain the proliferation capacity of CC cells and arrest the cell cycle in G0/G1 phase by repressing the expression of cyclin B, cyclin E, and cyclin D1. Besides, the rescue assay results indicated that up-regulated ROBO2 could reverse the repressive impact of down-regulated SALL4 on the proliferation of CC cells and accelerate the progression of the cell cycle, thus promoting the sphere-forming of tumor stem cells. SALL4 advanced the proliferation of CC and cell stemness through direct activation of ROBO2 expression, implied the novel mechanism of SALL4 in CC, and pointed out that SALL4/ROBO2 axis was likely to be a potential target for clinical treatment of CC.

INF2 and ROBO2 gene mutation in an Indian family with end stage renal failure and follow-up of renal transplantation.

BACKGROUND: Accurate genetic diagnosis of end-stage renal disease patients with a family history of renal dysfunction is very essential. It not only helps in proper prognosis, but becomes crucial in designating donor for live related renal transplant. We here present a case of family with deleterious mutations in INF2 and ROBO2 and its importance of genetic testing before preparing for kidney transplantation. CASE PRESENTATION: We report the case of a 29-year-female with end-stage renal disease and rapidly progressive renal failure. Mutational analysis revealed an Autosomal Dominant inheritance pattern and mutation in exon 4 of the INF2 gene (p. Thr215Ser) and exon 26 of the ROBO2 gene (p. Arg1371Cys). Her mother was diagnosed for CKD stage 4 with creatinine level of 4.3 mg/dL. Genetic variants (INF2 and ROBO2) identified in proband were tested in her sisters and mother. Her elder sister was positive for both heterozygous variants (INF2 and ROBO2). Her mother was positive for mutation in INF2 gene, and her donor elder sister did not showed mutation in INF2 gene and had mutation in ROBO2 gene without any clinical symptoms. CONCLUSION: This case report emphasize that familial genetic screening has allowed us in allocating the donor selection in family where family member had history of genetic defect of Chronic Kidney Disease. Information of the causative renal disorder is extremely valuable for risk-assessment and planning of kidney transplantation.

Case Report: Decrypting an interchromosomal insertion associated with Marfan's syndrome: how optical genome mapping emphasizes the morbid burden of copy-neutral variants.

Optical genome mapping (OGM), which allows analysis of ultra-high molecular weight (UHMW) DNA molecules, represents a response to the restriction created by short-read next-generation-sequencing, even in cases where the causative variant is a neutral copy-number-variant insensitive to quantitative investigations. This study aimed to provide a molecular diagnosis to a boy with Marfan syndrome (MFS) and intellectual disability (ID) carrying a de novo translocation involving chromosomes 3, 4, and 13 and a 1.7 Mb deletion at the breakpoint of chromosome 3. No FBN1 alteration explaining his Marfan phenotype was highlighted. UHMW gDNA was isolated from both the patient and his parents and processed using OGM. Genome assembly was followed by variant calling and annotation. Multiple strategies confirmed the results. The 3p deletion, which disrupted ROBO2, (MIM*602431) included three copy-neutral insertions. Two came from chromosome 13; the third contained 15q21.1, including the FBN1 from intron-45 onwards, thus explaining the MFS phenotype. We could not attribute the ID to a specific gene variant nor to the reshuffling of topologically associating domains (TADs). Our patient did not have vesicular reflux-2, as reported by missense alterations of ROBO2 (VUR2, MIM#610878), implying that reduced expression of all or some isoforms has a different effect than some of the point mutations. Indeed, the ROBO2 expression pattern and its role as an axon-guide suggests that its partial deletion is responsible for the patient's neurological phenotype. Conclusion: OGM testing 1) highlights copy-neutral variants that could remain invisible if no loss of heterozygosity is observed and 2) is mandatory before other molecular studies in the presence of any chromosomal rearrangement for an accurate genotype-phenotype relationship.

Robo2 promotes osteoblast differentiation and mineralization through autophagy and is activated by parathyroid hormone induction.

BACKGROUND: As a systemic skeletal disorder, osteoporosis can increase fracture risk. This study wants to discuss the mechanism of osteoporosis and find possible molecular therapy. Bone morphogenetic protein 2 (BMP2) was utilized to stimulate MC3T3-E1 to establish a cellular osteoporosis model in vitro. METHODS: Initially, the viability of BMP2-induced MC3T3-E1 was assessed with a Cell counting kit-8 (CCK-8) assay. By real-time quantitative PCR (RT-qPCR) and western blot, Robo2 expression after roundabout (Robo) silencing or overexpression was estimated. Besides, alkaline phosphatase (ALP) expression, mineralization level and LC3II green fluorescent protein (GFP) expression were evaluated using ALP assay, Alizarin red staining and immunofluorescence staining, separately. Additionally, the expression of proteins related to osteoblast differentiation and autophagy was analyzed by RT-qPCR and western blot. Then, following autophagy inhibitor 3-methyladenine (3-MA) treatment, osteoblast differentiation and mineralization were measured again. RESULTS: MC3T3-E1 cells were differentiated into osteoblasts under BMP2 induction and Robo2 expression was greatly ascended. After Robo2 silencing, Robo2 expression was markedly diminished. ALP activity and mineralization level in BMP2-induced MC3T3-E1 cells were declined after depleting Robo2. Robo2 expression was conspicuously enhanced after overexpressing Robo2. Robo2 overexpression promoted the differentiation and mineralization of BMP2-induced MC3T3-E1 cells. Rescue experiments revealed that Robo2 silence and its overexpression could regulate the autophagy of BMP2-stimulated MC3T3-E1 cells. After 3-MA treatment, the increased ALP activity and mineralization level of BMP2-induced MC3T3-E1 cells with Robo2 upregulation were reduced. Furthermore, parathyroid hormone 1-34 (PTH1-34) treatment enhanced the expression of ALP, Robo2, LC3II and Beclin-1 and reduced the levels of LC3I and p62 of MC3T3-E1 cells concentration-dependently. CONCLUSION: Collectively, Robo2, which was activated by PTH1-34, promoted osteoblast differentiation and mineralization through autophagy.

Characterization of enhancer fragments in Drosophila robo2.

Receptor proteins of the Roundabout (Robo) family regulate axon guidance decisions during nervous system development. Among the three Drosophila robo family genes (robo1, robo2 and robo3), robo2 displays a dynamic expression pattern and regulates multiple axon guidance outcomes, including preventing midline crossing in some axons, promoting midline crossing in others, forming lateral longitudinal axon pathways, and regulating motor axon guidance. The identity and location of enhancer elements regulating robo2's complex and dynamic expression pattern in different neural cell types are unknown. Here, we characterize a set of 17 transgenic lines expressing GAL4 under the control of DNA sequences derived from noncoding regions in and around robo2, to identify enhancers controlling specific aspects of robo2 expression in the embryonic ventral nerve cord. We identify individual fragments that confer expression in specific cell types where robo2 is known to function, including early pioneer neurons, midline glia and lateral longitudinal neurons. Our results indicate that robo2's dynamic expression pattern is specified by a combination of enhancer elements that are active in different subsets of cells. We show that robo2's expression in lateral longitudinal axons represents two genetically separable subsets of neurons, and compare their axon projections with each other and with Fasciclin II (FasII), a commonly used marker of longitudinal axon pathways. In addition, we provide a general description of each fragment's expression in embryonic tissues outside of the nervous system, to serve as a resource for other researchers interested in robo2 expression and its functional roles outside the central nervous system.

Circ_ROBO2/miR-186-5p/TRIM14 axis regulates oxidized low-density lipoprotein-induced cardiac microvascular endothelial cell injury.

Background: Coronary artery disease (CAD) is one of the main risks of death, which is mainly caused by coronary arteries arteriosclerosis. Circular RNAs (circRNAs) have shown important regulatory roles in cardiovascular diseases. We amid to explore the role of circ_ROBO2 in CAD. Methods: Cardiac microvascular endothelial cells (CMECs) stimulated by oxidized low-density lipoprotein (ox-LDL) were served as the cellular model of CAD. Real-time quantitative polymerase chain reaction (RT-qPCR) and western blot assay were performed to detect RNA levels and protein levels, respectively. Cell proliferation was assessed by 5-ethynyl-2'-deoxyuridine (EdU) assay and Cell Counting Kit-8 (CCK-8) assay. Flow cytometry was employed for measuring cell apoptosis. Matrigel tube formation assay was used to evaluate angiogenesis ability. The intermolecular interaction was predicted by bioinformatics analysis and verified by dual-luciferase reporter and RNA-pull down assays. Results: The expression of circ_ROBO2 was upregulated in CAD patients and ox-LDL-induced CMECs. Treatment of ox-LDL suppressed cell proliferation and angiogenic ability as well as promoted the apoptosis of CMECs partly by upregulating circ_ROBO2. MicroRNA-186-5p (miR-186-5p) was identified as a target of circ_ROBO2, and circ_ROBO2 knockdown attenuated ox-LDL-induced damage in CMECs by sponging miR-186-5p. Tripartite motif containing 14 (TRIM14) acted as a target of miR-186-5p, and TRIM14 overexpression alleviated miR-186-5p-mediated inhibitory effect on ox-LDL-induced injury in CMECs. Circ_ROBO2 positively regulated TRIM14 expression by sponging miR-186-5p. Conclusion: Circ_ROBO2 played a promoting role in ox-LDL-induced CMECs injury by sponging miR-186-5p and regulating TRIM14, providing a promising treatment strategy for CAD.

Robo2 Drives Target-Selective Peripheral Nerve Regeneration in Response to Glia-Derived Signals.

Peripheral nerves are divided into multiple branches leading to divergent synaptic targets. This poses a remarkable challenge for regenerating axons as they select their original trajectory at nerve branch-points. Despite implications for functional regeneration, the molecular mechanisms underlying target selectivity are not well characterized. Danio Rerio (zebrafish) motor nerves are composed of a ventral and a dorsal branch that diverge at a choice-point, and we have previously shown that regenerating axons faithfully select their original branch and targets. Here we identify robo2 as a key regulator of target-selective regeneration (sex of experimental subjects unknown). We demonstrate that robo2 function in regenerating axons is required and sufficient to drive target-selective regeneration, and that robo2 acts in response to glia located precisely where regenerating axons select the branch-specific trajectory to prevent and correct axonal errors. Combined, our results reveal a glia-derived mechanism that acts locally via axonal robo2 to promote target-selective regeneration.SIGNIFICANCE STATEMENT Despite its relevance for functional recovery, the molecular mechanisms that direct regenerating peripheral nerve axons toward their original targets are not well defined. Zebrafish spinal motor nerves are composed of a dorsal and a ventral branch that diverge at a stereotyped nerve branch-point, providing a unique opportunity to decipher the molecular mechanisms critical for target-selective regeneration. Using a combination of live cell imaging and molecular-genetic manipulations, we demonstrate that the robo2 guidance receptor is necessary and sufficient to promote target-selective regeneration. Moreover, we demonstrate that robo2 is part of a genetic pathway that generates transient, spatially restricted, and tightly coordinated signaling events that direct axons of the dorsal nerve branch toward their original, pre-injury targets.

Epigenetic adaptations of the masticatory mucosa to periodontal inflammation.

BACKGROUND: In mucosal barrier interfaces, flexible responses of gene expression to long-term environmental changes allow adaptation and fine-tuning for the balance of host defense and uncontrolled not-resolving inflammation. Epigenetic modifications of the chromatin confer plasticity to the genetic information and give insight into how tissues use the genetic information to adapt to environmental factors. The oral mucosa is particularly exposed to environmental stressors such as a variable microbiota. Likewise, persistent oral inflammation is the most important intrinsic risk factor for the oral inflammatory disease periodontitis and has strong potential to alter DNA-methylation patterns. The aim of the current study was to identify epigenetic changes of the oral masticatory mucosa in response to long-term inflammation that resulted in periodontitis. METHODS AND RESULTS: Genome-wide CpG methylation of both inflamed and clinically uninflamed solid gingival tissue biopsies of 60 periodontitis cases was analyzed using the Infinium MethylationEPIC BeadChip. We validated and performed cell-type deconvolution for infiltrated immune cells using the EpiDish algorithm. Effect sizes of DMPs in gingival epithelial and fibroblast cells were estimated and adjusted for confounding factors using our recently developed "intercept-method". In the current EWAS, we identified various genes that showed significantly different methylation between periodontitis-inflamed and uninflamed oral mucosa in periodontitis patients. The strongest differences were observed for genes with roles in wound healing (ROBO2, PTP4A3), cell adhesion (LPXN) and innate immune response (CCL26, DNAJC1, BPI). Enrichment analyses implied a role of epigenetic changes for vesicle trafficking gene sets. CONCLUSIONS: Our results imply specific adaptations of the oral mucosa to a persistent inflammatory environment that involve wound repair, barrier integrity, and innate immune defense.

Soluble TREM-1, as a new ligand for the membrane receptor Robo2, promotes hepatic stellate cells activation and liver fibrosis.

Triggering receptor expressed on myeloid cells-1 (TREM-1) exists in two forms: a transmembrane form and a soluble form (sTREM-1). The levels of sTREM-1 are elevated in supernatants of activated HSCs. However, the role of sTREM-1 in HSC activation and liver fibrosis remains undefined. Previous studies have primarily focused on the transmembrane form of TREM-1; we innovatively observed the function of sTREM-1 as a ligand in liver fibrosis and screened its receptor. Here, recombinant sTREM-1 was used as a stimulator which induced HSC activation and further aggravated liver fibrosis. Then, screening for sTREM-1 interacting membrane receptors was performed using pull-down assay followed by mass spectrometry, and the membrane receptor roundabout guidance receptor 2 (Robo2) was identified as a candidate receptor for sTREM-1. The interaction between sTREM-1 and Robo2 was verified by pull-down and immunofluorescence. The role of Robo2 on sTREM-1-induced HSC activation and its downstream signal pathways was assessed by knockdown of Robo2 in LX-2 cells. Furthermore, HSC-specific knockdown of Robo2 was achieved in a mouse model of liver fibrosis by using a recombinant adeno-associated virus (AAV) vector to confirm the role of the receptor, and we proved that Robo2 knockdown inhibited the activation of HSC and liver fibrosis, which also led to the inactivation of Smad2/3 and PI3K/Akt pathways in sTREM-1-induced HSC activation and liver fibrosis. In conclusion, sTREM-1 acts as a new ligand of Robo2; the binding of sTREM-1 to Robo2 initiates the activation of the downstream Smad2/3 and PI3K/Akt signalling pathways, thereby promoting HSC activation and liver fibrosis.

Circ_ROBO2/miR-149 Axis Promotes the Proliferation and Migration of Human Aortic Smooth Muscle Cells by Activating NF-κB Signaling.

Atherosclerosis is the leading global cause of mortality. The occurrence of coronary artery disease (CAD) is regulated by a diversity of pathways, including circRNAs. However, the potential mechanisms of circRNAs in CAD remain unclear. Here, qRT-PCR was used to examine the expressions of miR-149 and circ_ROBO2. Their influences on cell proliferation, migration, and apoptosis were measured by CCK-8, trans-well, and flow cytometry assays, respectively. The protein levels of p-IκBα and NF-κB p65 were examined using western blot. The molecular interactions were validated using dual luciferase reporter and RNA pull-down assays. The expression patterns of circ_ROBO2 and miR-149 in CAD patients and PDGF-BB-treated human aortic smooth muscle cells (HASMCs) were upregulated and downregulated, respectively. Knockdown of circ_ROBO2 could markedly inhibit the capabilities of proliferation and migration, enhance the apoptotic rate, and suppress NF-κB signaling in PDGF-BB-treated HASMCs. Mechanistically, circ_ROBO2 acted as a sponge of miR-149 to activate TRAF6/NF-κB signaling. Rescue studies demonstrated that neither silencing miR-149 nor activation of NF-κB signaling obviously abolished the biological roles of circ_ROBO2 knockdown in PDGF-BB treated-HASMCs. This discovery elucidated a functional mechanism of circ_ROBO2 in CAD, suggesting that circRNAs serve a vital role in the progression of CAD.

SLIT3 promotes myogenic differentiation as a novel therapeutic factor against muscle loss.

BACKGROUND: Sarcopenia and osteoporosis frequently co-occur in the elderly and have common pathophysiological determinants. Slit guidance ligand 3 (SLIT3) has been recently discovered as a novel therapeutic factor against osteoporosis, and a SLIT3 fragment containing the second leucine-rich repeat domain (LRRD2) had a therapeutic efficacy against osteoporosis. However, a role of SLIT3 in the skeletal muscle is unknown. METHODS: Skeletal muscle mass, strength, and/or physical activity were evaluated in Slit3-/- , ovariectomized, and aged mice, based on the measurements of muscle weight and grip strength, Kondziella's inverted hanging test, and/or wheel-running test. Skeletal muscles were also histologically evaluated by haematoxylin and eosin staining and/or immunofluorescence. The ovariectomized and aged mice were intravenously injected with recombinant SLIT3 LRRD2 for 4 weeks. C2C12 cells were used to know cellular effects of SLIT3, such as in vitro myogenesis, fusion, cell viability, and proliferation, and also used to evaluate its molecular mechanisms by immunocytochemistry, immunoprecipitation, western blotting, real-time PCR, siRNA transfection, and receptor-ligand binding ELISA. RESULTS: Slit3-deficient mice exhibited decreased skeletal muscle mass, muscle strength, and physical activity. The relative masses of gastrocnemius and soleus were lower in the Slit3-/- mice (0.580 ± 0.039% and 0.033 ± 0.003%, respectively) than those in the WT littermates (0.622 ± 0.043% and 0.038 ± 0.003%, respectively) (all, P < 0.05). Gastrocnemius of Slit3-/- mice showed the reduced number of Type I and Type IIa fibres (all, P < 0.05), but not of Type IIb and Type IIx fibres. SLIT3 activated ß-catenin signalling by promoting its release from M-cadherin, thereby increasing myogenin expression to stimulate myoblast differentiation. In vitro experiments involving ROBO2 expression, knockdown, and interaction with SLIT3 indicated that ROBO2 functions as a SLIT3 receptor to aid myoblast differentiation. SLIT3 LRRD2 dissociated M-cadherin-bound ß-catenin and up-regulated myogenin expression to increase myoblast differentiation, in a manner similar to full-length SLIT3. Systemic treatment with SLIT3 LRRD2 increased skeletal muscle mass in both ovariectomized and aged mice (all, P < 0.05). The relative masses of gastrocnemius and soleus were higher in the treated aged mice (0.548 ± 0.045% and 0.033 ± 0.005%, respectively) than in the untreated aged mice (0.508 ± 0.016% and 0.028 ± 0.003%, respectively) (all, P < 0.05). SLIT3 LRRD2 treatment increased the hanging duration of the aged mice by approximately 1.7-fold (P < 0.05). CONCLUSIONS: SLIT3 plays a sarcoprotective role by activating ß-catenin signalling. SLIT3 LRRD2 can potentially be used as a therapeutic agent against muscle loss.

A Phase 1 first-in-human study of the safety, tolerability, and pharmacokinetics of the ROBO2 fusion protein PF-06730512 in healthy participants.

Proteinuria associated with podocyte effacement is a hallmark of focal segmental glomerulosclerosis (FSGS). Preclinical studies implicated ROBO2/SLIT2 signaling in the regulation of podocyte adhesion, and inhibition of this pathway is a novel target to slow FSGS disease progression. This first-in-human dose-escalation study evaluated the safety, tolerability, pharmacokinetics, and immunogenicity of PF-06730512, an Fc fusion protein that targets the ROBO2/SLIT2 pathway, in healthy adults. In this Phase 1, double-blind, sponsor-open study, single ascending dose (SAD) cohorts were randomized to receive up to 1000 mg or placebo intravenously (IV); multiple ascending dose (MAD) cohorts were randomized to receive up to 400 mg subcutaneous (SC) doses, 1000 mg IV dose, or matching placebo. Safety evaluations were performed up to 71 (SAD) and 113 (MAD) days after dosing; blood samples were collected to measure serum PF-06730512 concentrations and antidrug antibodies (ADA) to PF-06730512. Seventy-nine participants (SAD, 47; MAD, 32) were enrolled. There were 108 mild (SAD, 46; MAD, 62) and 21 moderate (SAD, 13; MAD, 8) treatment-emergent adverse events (TEAEs); no deaths, treatment-related serious AEs, severe TEAEs, or infusion reactions were reported. PF-06730512 exposure generally increased in an approximately dose-proportional manner; mean t1/2 ranged from 12-15 days across 50-1000 mg doses. Immunogenicity incidence was low (SAD, 0 ADA+; MAD, 2 ADA+). In conclusion, single IV doses of PF-06730512 up to 1000 mg and multiple IV and SC dosing up to 1000 and 400 mg, respectively, were safe and well tolerated in healthy participants. Further trials in patients with FSGS are warranted. Clinical trial registration: Clinicaltrials.gov: NCT03146065.

PODO: Trial Design: Phase 2 Study of PF-06730512 in Focal Segmental Glomerulosclerosis.

INTRODUCTION: Focal segmental glomerulosclerosis (FSGS) is characterized by proteinuria and a histologic pattern of glomerular lesions of diverse etiology that share features including glomerular scarring and podocyte foot process effacement. Roundabout guidance receptor 2 (ROBO2)/slit guidance ligand 2 (SLIT2) signaling destabilizes the slit diaphragm and reduces podocyte adhesion to the glomerular basement membrane (GBM). Preclinical studies suggest that inhibition of glomerular ROBO2/SLIT2 signaling can stabilize podocyte adhesion and reduce proteinuria. This clinical trial evaluates the preliminary efficacy and safety of ROBO2/SLIT2 inhibition with the ROBO2 fusion protein PF-06730512 in patients with FSGS. METHODS: The Study to Evaluate PF-06730512 in Adults With FSGS (PODO; ClinicalTrials.gov identifier NCT03448692), an open-label, phase 2a, multicenter trial in adults with FSGS, will enroll patients into 2 cohorts (n = 22 per cohort) to receive either high- or low-dose PF-06730512 (intravenous) every 2 weeks for 12 weeks. Key inclusion criteria include a confirmed biopsy diagnosis of FSGS, an estimated glomerular filtration rate (eGFR) ≥45 ml/min/1.73 m2 based on the Chronic Kidney Disease Epidemiology Collaboration formula (30-45 with a recent biopsy), and urinary protein-to-creatinine ratio (UPCR) >1.5 g/g. Key exclusion criteria include collapsing FSGS, serious/active infection, ≥50% tubulointerstitial fibrosis on biopsy, and organ transplantation. The primary endpoint is change from baseline to week 13 in UPCR; secondary endpoints include safety, changes in eGFR, and PF-06730512 serum concentration. RESULTS: This ongoing trial will report the efficacy, safety, pharmacokinetics, and biomarker results of PF-06730512 for patients with FSGS. CONCLUSION: Findings from this proof-of-concept study may support further development and evaluation of PF-06730512 to treat FSGS and warrant assessment in phase 3 clinical trials.

Genomic Regions and Candidate Genes Linked to Capped Hock in Pig.

Capped hock affects the exterior of pedigree pigs, making them unsalable and resulting in a negative impact on the efficiency of pig-breeding centers. The purpose of this paper was to carry out pilot studies aimed at finding genomic regions and genes linked to the capped hock in pigs. The studies were carried out on Landrace pigs (n = 75) and Duroc pigs (n = 70). To identify genomic regions linked to capped hock in pigs, we used smoothing FST statistics. Genotyping was performed with GeneSeek® GGP Porcine HD Genomic Profiler v1 (Illumina Inc, USA). The research results showed 70 SNPs linked to capped hock in Landrace (38 SNPs) and Duroc (32 SNPs). The identified regions overlapped with QTLs related with health traits (blood parameters) and meat and carcass traits (fatness). In total, 31 genes were identified (i.e., 17 genes in Landrace, 14 genes in Durocs). Three genes appeared in both the Landrace and Duroc groups, including A2ML1 (SSC5), ROBO2 (SSC13), and MSI1 (SSC14). We identified genomic regions directly or indirectly linked to capped hock, which thus might contribute to identifying genetic variants and using them as genetic markers in pig breeding.

ROBO2 hampers malignant biological behavior and predicts a better prognosis in pancreatic adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a fatalmalignant cancer with extremely poor prognosis and high mortality. Genome wide studies show that Slit/Robo signaling pathway takes a major effect in the oncogenesis and progression of pancreatic cancer. However, the function and mechanism of ROBO2 in the development of PDAC remains unclear. METHODS: In present study, we use Western blot and real-time polymerase chain reaction (RT-PCR) to detect the expression of ROBO2 in pancreatic cell lines. Cell proliferation,Transwellmigration and invasion were conducted inAsPC-1, MIA PaCa-2 and PANC-1cell lines. RNA sequencing, bioinformatics analysisand Western blot were used to explore its mechanism and potential target molecules. The expression of ROBO2 in 95 tumor tissues was detected by immunohistochemistry. RESULTS: ROBO2 expression was downregulated in PDAC cell lines and tissue samples. A high expression of ROBO2 was associated with better prognosis. Upregulation of ROBO2 inhibited PDAC cell proliferation, migration, and invasion. However, we found theoppositeresults in the ROBO2 downregulation group. In addition, the function of ROBO2 on cell proliferation was further affirmed by the animal model. Finally, the results of RNA sequencing indicated that ROBO2 partly promoted the antitumor activity by inhibiting ECM1 in PDAC. CONCLUSIONS: Our work suggests that ROBO2 inhibits tumor progression in PDAC and may serve as a predictive biomarker and therapeutic target in PDAC.

Robo2 and Gen1 Coregulate Ureteric Budding by Activating the MAPK/ERK Signaling Pathway in Mice.

Congenital anomalies of the kidney and urinary tract (CAKUT) are some of the most common developmental defects and have a complicated etiology, indicating an interaction of (epi-) genetic and environmental factors. Single gene mutations and copy number variations (CNVs) do not explain most cases of CAKUT, and simultaneous contributions of more than one gene (di-, oligo-, or polygenic effects; i.e., complex genetics) may lead to the pathogenesis of CAKUT. Robo2 plays a key role in regulating ureteric bud (UB) formation in the embryo, with mutations leading to supernumerary kidneys. Gen1 is a candidate gene associated with CAKUT because of its important role in early metanephric development in mice. We established a mouse model with double disruption of Robo2 and Gen1 using a piggyBac transposon and found that double gene mutation led to significantly increased CAKUT phenotypes in Robo2 PB/+ Gen1 PB/+ mouse offspring, especially a duplicated collecting system. Increased ectopic UB formation was observed in the Robo2 PB/+ Gen1 PB/+ mice during the embryonic period. Robo2 and Gen1 exert synergistic effects on mouse kidney development, promoting cell proliferation by activating the GDNF/RET pathway and downstream MAPK/ERK signaling. Our findings provide a disease model for CAKUT as an oligogenic disorder.

Neuronal NADPH oxidase 2 regulates growth cone guidance downstream of slit2/robo2.

NADPH oxidases (Nox) are membrane-bound multi-subunit protein complexes producing reactive oxygen species (ROS) that regulate many cellular processes. Emerging evidence suggests that Nox-derived ROS also control neuronal development and axonal outgrowth. However, whether Nox act downstream of receptors for axonal growth and guidance cues is presently unknown. To answer this question, we cultured retinal ganglion cells (RGCs) derived from zebrafish embryos and exposed these neurons to netrin-1, slit2, and brain-derived neurotrophic factor (BDNF). To test the role of Nox in cue-mediated growth and guidance, we either pharmacologically inhibited Nox or investigated neurons from mutant fish that are deficient in Nox2. We found that slit2-mediated growth cone collapse, and axonal retraction were eliminated by Nox inhibition. Though we did not see an effect of either BDNF or netrin-1 on growth rates, growth in the presence of netrin-1 was reduced by Nox inhibition. Furthermore, attractive and repulsive growth cone turning in response to gradients of BDNF, netrin-1, and slit2, respectively, were eliminated when Nox was inhibited in vitro. ROS biosensor imaging showed that slit2 treatment increased growth cone hydrogen peroxide levels via mechanisms involving Nox2 activation. We also investigated the possible relationship between Nox2 and slit2/Robo2 signaling in vivo. astray/nox2 double heterozygote larvae exhibited decreased area of tectal innervation as compared to individual heterozygotes, suggesting both Nox2 and Robo2 are required for establishment of retinotectal connections. Our results provide evidence that Nox2 acts downstream of slit2/Robo2 by mediating growth and guidance of developing zebrafish RGC neurons.

ROBO2-mediated RALDH2 signaling is required for common nephric duct fusion with primitive bladder.

Congenital anomalies of the urinary tract are a significant cause of morbidity in infancy, and many congenital anomalies are linked to ureter development; however, the mechanism by which congenital anomalies control ureter development remains unknown. The loss of Robo2 can cause ureter defects and vesicoureteral reflux. However, how Robo2 impacts ureter development is unclear. We found that ROBO2 is expressed in the common nephric duct (CND) and primitive bladder, and impacts CND migration and fusion with the primitive bladder via its novel binding partner retinaldehyde dehydrogenase-2 (RALDH2). Delayed apoptosis that is due to the failure of CND fusion with the primitive bladder in the Robo2-/-embryo results in an abnormal ureter connection to the CND, which is required for ureter development. We define a novel pathway in which the CND is remodeled by ROBO2 and retinoic acid rescued the ureter anomalies in the Robo2-/-embryo. These findings may be relevant to diverse disease conditions that are associated with altered signaling in the primitive bladder.