Netrin signaling mediates survival of dormant epithelial ovarian cancer cells.

Dormancy in cancer is a clinical state in which residual disease remains undetectable for a prolonged duration. At a cellular level, rare cancer cells cease proliferation and survive chemotherapy and disseminate disease. We created a suspension culture model of high-grade serous ovarian cancer (HGSOC) dormancy and devised a novel CRISPR screening approach to identify survival genes in this context. In combination with RNA-seq, we discovered the Netrin signaling pathway as critical to dormant HGSOC cell survival. We demonstrate that Netrin-1, -3, and its receptors are essential for low level ERK activation to promote survival, and that Netrin activation of ERK is unable to induce proliferation. Deletion of all UNC5 family receptors blocks Netrin signaling in HGSOC cells and compromises viability during the dormancy step of dissemination in xenograft assays. Furthermore, we demonstrate that Netrin-1 and -3 overexpression in HGSOC correlates with poor outcome. Specifically, our experiments reveal that Netrin overexpression elevates cell survival in dormant culture conditions and contributes to greater spread of disease in a xenograft model of abdominal dissemination. This study highlights Netrin signaling as a key mediator HGSOC cancer cell dormancy and metastasis.

High-grade serous ovarian cancer (or HGSOC for short) is the fifth leading cause of cancer-related deaths in women. It is generally diagnosed at an advanced stage of disease when the cancer has already spread to other parts of the body. Surgical removal of tumors and subsequent treatment with chemotherapy often reduces the signs and symptoms of the disease for a time but some cancer cells tend to survive so that patients eventually relapse. The HGSOC cells typically spread from the ovaries by moving through the liquid surrounding organs in the abdomen. The cells clump together and enter an inactive state known as dormancy that allows them to survive chemotherapy and low-nutrient conditions. Understanding how to develop new drug therapies that target dormant cancer cells is thought to be an important step in prolonging the life of HGSOC patients. Cancer cells are hardwired to multiply and grow, so Perampalam et al. reasoned that becoming dormant poses challenges for HGSOC cells, which may create unique vulnerabilities not shared by proliferating cancer cells. To find out more, the researchers used HGSOC cells that had been isolated from patients and grown in the laboratory. The team used a gene editing technique to screen HGSOC cells for genes required by the cells to survive when they are dormant. The experiments found that genes involved in a cell signaling pathway, known as Netrin signaling, were critical for the cells to survive. Previous studies have shown that Netrin signaling helps the nervous system form in embryos and inhibits a program of controlled cell death in some cancers. Perampalam et al. discovered that Netrins were present in the environment immediately surrounding dormant HGSOC cells. Human HGSOC patients with higher levels of Netrin gene expression had poorer prognoses than patients with lower levels of Netrin gene expression. Further experiments demonstrated that Netrins help dormant HGSOC cells to spread around the body. These findings suggest that Netrin signalling may provide useful targets for future drug therapies against dormant cells in some ovarian cancers. This could include repurposing drugs already in development or creating new inhibitors of this pathway.

Spatiotemporal changes in Netrin/Dscam1 signaling dictate axonal projection direction in Drosophila small ventral lateral clock neurons.

Axon projection is a spatial- and temporal-specific process in which the growth cone receives environmental signals guiding axons to their final destination. However, the mechanisms underlying changes in axonal projection direction without well-defined landmarks remain elusive. Here, we present evidence showcasing the dynamic nature of axonal projections in Drosophila's small ventral lateral clock neurons (s-LNvs). Our findings reveal that these axons undergo an initial vertical projection in the early larval stage, followed by a subsequent transition to a horizontal projection in the early-to-mid third instar larvae. The vertical projection of s-LNv axons correlates with mushroom body calyx expansion, while the s-LNv-expressed Down syndrome cell adhesion molecule (Dscam1) interacts with Netrins to regulate the horizontal projection. During a specific temporal window, locally newborn dorsal clock neurons secrete Netrins, facilitating the transition of axonal projection direction in s-LNvs. Our study establishes a compelling in vivo model to probe the mechanisms of axonal projection direction switching in the absence of clear landmarks. These findings underscore the significance of dynamic local microenvironments in the complementary regulation of axonal projection direction transitions.

IGF2BP2 inhibits invasion and migration of clear cell renal cell carcinoma via targeting Netrin-4 in an m6A-dependent manner.

Clear cell renal cell carcinoma (ccRCC), the most common subtype of renal cell carcinoma, often leads to a poor prognosis due to metastasis. The investigation of N6-methyladenosine (m6A) methylation, a crucial RNA modification, and its role in ccRCC, particularly through the m6A reader insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2), revealed significant insights. We found that IGF2BP2 was notably downregulated in ccRCC, which correlated with tumor aggressiveness and poor prognosis. Thus, IGFBP2 has emerged as an independent prognostic factor of ccRCC. Moreover, a strong positive correlation was observed between the expression of IGF2BP2 and Netrin-4. Netrin-4 was also downregulated in ccRCC, and its lower levels were associated with increased malignancy and poor prognosis. Overexpression of IGF2BP2 and Netrin-4 suppressed the invasion and migration of ccRCC cells, while Netrin-4 knockdown reversed these effects in ccRCC cell lines. RNA immunoprecipitation (RIP)-quantitative polymerase chain reaction validated the robust enrichment of Netrin-4 mRNA in anti-IGF2BP2 antibody immunoprecipitates. MeRlP showed significantly increased Netrin4 m6A levels after lGF2BP2 overexpression. Moreover, we found that IGF2BP2 recognized and bound to the m6A site within the coding sequence of Netrin-4, enhancing its mRNA stability. Collectively, these results showed that IGF2BP2 plays a suppressive role in the invasion and migration of ccRCC cells by targeting Netrin-4 in an m6A-dependent manner. These findings underscore the potential of IGF2BP2/Netrin-4 as a promising prognostic biomarker and therapeutic target in patients with ccRCC metastasis.

Structural insights into the formation of repulsive netrin guidance complexes.

Netrins dictate attractive and repulsive responses during axon growth and cell migration, where the presence of the receptor Uncoordinated-5 (UNC-5) on target cells results in repulsion. Here, we showed that UNC-5 is a heparin-binding protein, determined its structure bound to a heparin fragment, and could modulate UNC-5-heparin affinity using a directed evolution platform or structure-based rational design. We demonstrated that UNC-5 and UNC-6/netrin form a large, stable, and rigid complex in the presence of heparin, and heparin and UNC-5 exclude the attractive UNC-40/DCC receptor from binding to UNC-6/netrin to a large extent. Caenorhabditis elegans with a heparin-binding-deficient UNC-5 fail to establish proper gonad morphology due to abrogated cell migration, which relies on repulsive UNC-5 signaling in response to UNC-6. Combining UNC-5 mutations targeting heparin and UNC-6/netrin contacts results in complete cell migration and axon guidance defects. Our findings establish repulsive netrin responses to be mediated through a glycosaminoglycan-regulated macromolecular complex.

EXOSC5 maintains cancer stem cell activity in endometrial cancer by regulating the NTN4/integrin ß1 signalling axis.

Endometrial carcinoma (EC) is a common type of uterine cancer in developed countries, originating from the uterine epithelium. The incidence rate of EC in Taiwan has doubled from 2005. Cancer stem cells (CSCs) are a subpopulation of cancer cells that have high tumorigenicity and play a crucial role in the malignant processes of cancer. Targeting molecules associated with CSCs is essential for effective cancer treatments. This study delves into the role of Exosome component 5 (EXOSC5) in EC. Data from The Cancer Genome Atlas suggests a correlation between high EXOSC5 mRNA expression and unfavorable EC prognosis. EXOSC5 knockdown diminished EC-CSC self-renewal and reduced expression of key cancer stemness proteins, including c-MYC and SOX2. Intriguingly, this knockdown significantly curtailed tumorigenicity and CSC frequency in EC tumor spheres. A mechanistic examination revealed a reduction in netrin4 (NTN4) levels in EXOSC5-depleted EC cells. Moreover, NTN4 treatment amplified EC cell CSC activity and, when secreted, NTN4 partnered with integrin ß1, subsequently triggering the FAK/SRC axis to elevate c-MYC activity. A clear positive relation between EXOSC5 and NTN4 was evident in 93 EC tissues. In conclusion, EXOSC5 augments NTN4 expression, activating c-MYC via the integrin ß1/FAK/SRC pathway, offering potential avenues for EC diagnosis and treatment.

Effects of cylindrospermopsin, chlorpyrifos and their combination in a SH-SY5Y cell model concerning developmental neurotoxicity.

The cyanotoxin cylindrospermopsin (CYN) has been postulated to cause neurotoxicity, although the studies in this concern are very few. In addition, some studies in vitro indicate its possible effects on development. Furthermore, pesticides can be present in the same environmental samples as cyanotoxins. Therefore, chlorpyrifos (CPF) has been one of the most common pesticides used worldwide. The aim of this report was to study the effects of CYN, isolated and in combination with CPF, in a developmental neurotoxicity in vitro model. The human neuroblastoma SH-SY5Y cell line was exposed during 6 days of differentiation to both toxics to study their effects on cell viability and neurite outgrowth. To further evaluate effects of both toxicants on cholinergic signaling, their agonistic and antagonistic activities on the α7 homomeric nicotinic acetylcholine receptor (nAChR) were studied upon acute exposure. Moreover, a transcriptomic analysis by qPCR was performed after 6 days of CYN-exposure during differentiation. The results showed a concentration-dependent decrease on both cell viability and neurite outgrowth for both toxics isolated, leading to effective concentration 20 (EC20) values of 0.35 µM and 0.097 µM for CYN on cell viability and neurite outgrowth, respectively, and 100 µM and 58 µM for CPF, while the combination demonstrated no significant variations. In addition, 95 µM and 285 µM CPF demonstrated to act as an antagonist to nicotine on the nAChR, although CYN up to 2.4 µM had no effect on the efficacy of these receptors. Additionally, the EC20 for CYN (0.097 µM) on neurite outgrowth downregulated expression of the 5 genes NTNG2 (netrin G2), KCNJ11 (potassium channel), SLC18A3 (vesicular acetylcholine transporter), APOE (apolipoprotein E), and SEMA6B (semaphorin 6B), that are all important for neuronal development. Thus, this study points out the importance of studying the effects of CYN in terms of neurotoxicity and developmental neurotoxicity.

Comprehensive analysis of the role of Netrin G1 (NTNG1) in hepatocellular carcinoma cells.

Netrin G1 (NTNG1) is a member of the Netrin family and plays a crucial role in various human cancers. However, the molecular functions of NTNG1 in HCC and the underlying mechanisms remain unclear. HCC expression data was obtained from the GEO database and analyzed using various bioinformatics tools. The expression of NTNG1 in HCC tissues and liver cancer cells was evaluated through RT-qPCR and western blotting. Cells with stable NTNG1 overexpression and knockdown were established, and CCK-8, colony formation, and flow cytometry assays were conducted in vitro. The xenograft model was utilized to verify the tumorigenesis capacity of NTNG1 in vivo. IHC was employed to analyze the expression of NTNG1 and CD163 proteins. HCC-specific genes were screened, followed by functional enrichment and immune cell infiltration analysis. Finally, the Co-IP was used to detect the interaction between NTNG1 and N-cadherin. NTNG1 was highly expressed in HCC tissues and liver cancer cells, and associated with significantly poorer OS rates. In addition, NTNG1 overexpression in liver cancer cells significantly increased their proliferation, colony growth, invasion, migration, and EMT, while inhibiting apoptosis. Bioinformatics analyses indicated that NTNG1 was closely related to EMT and tumor infiltration. IHC staining revealed a positive correlation between NTNG1 expression and CD163 in HCC tissues. Additionally, an EMT inhibitor attenuated the expression levels of EMT-related markers and counteracted the effects of NTNG1 overexpression in liver cancer cells. This study is the first to identify NTNG1 as a potential therapeutic target in HCC, promoting tumor development and progression by regulating EMT.

RNA­binding protein quaking 5 inhibits the progression of non­small cell lung cancer by upregulating netrin­4 expression.

It was recently reported that netrin­4 (Ntn­4), a component of the extracellular matrix, when downregulated, is involved in the progression of several types of cancer, including breast cancer, colorectal tumours, neuroblastoma and gastric cancer. In the present study, the level of Ntn­4 was examined in a public non­small cell lung cancer (NSCLC) dataset from the Netherlands Cancer Institute. This analysis revealed that the mRNA expression level of Ntn­4 was lower in the samples of patients with NSCLC compared with that in the control samples. Consistent with the mRNA level, the protein level of Ntn­4 was also found to be decreased in NSCLC cells. However, both the function of Ntn­4 and the underlying mechanisms of Ntn­4 downregulation in NSCLC have yet to be fully elucidated. As was anticipated, the overexpression of Ntn­4 led to a marked decrease in the proliferation, migration and invasion of NSCLC cells. Notably, RNA­binding protein quaking 5 (Qki­5) was found to exhibit antitumor activity in lung cancer, not only by enhancing the level of Ntn­4 by binding to Ntn­4 mRNA, but also by suppressing the proliferation, invasion and migration of NSCLC cells. However, Qki­5 is known to be frequently downregulated in NSCLC. Moreover, the knockdown of Ntn­4 was found to reverse the suppressive effects of Qki­5 on NSCLC progression both in vitro and in vivo. Taken together, the findings of the present study demonstrate that Ntn­4 is able to suppress the progression of NSCLC, and that the level of Ntn­4 can be regulated by Qki­5. Therefore, Ntn­4 may be a novel diagnostic and therapeutic target for the treatment of NSCLC.

Role of Axon Guidance Molecules in Ascending and Descending Paths in Spinal Cord Regeneration.

Axon guidance molecules (AGM) are critical regulators of neural development and play a vital role in guiding axons to their target regions during spinal cord development. The correct wiring of neural circuits depends on these molecules' precise expression and function. Defects in axonal pathfinding, growth cone navigation, axonal branching, and synapse formation have far-reaching implications for neuronal circuit construction and function after CNS traumas, such as spinal cord injury (SCI), which affect the expression or activity of AGM. Ascending and descending paths in the spinal cord have been found to include many AGM, including Netrins, Slits, Semaphorins (Sema), Ephrins, and their receptors. In contrast to the repulsive signals like Slits and Semaphorins, which restrict axonal growth and guide axons away from unsuitable locations, Netrins are appealing guidance cues that encourage axonal growth and guidance. Defects in motor function and sensory processing can result from changes in the expression or activity of Ephrins or their receptors, which play an essential role in axonal guidance and synaptic plasticity in the spinal cord. Herein, we highlighted the expressions, functions, and mechanisms of AGM in ascending and descending spinal cord tracts, which can help us identify novel therapeutic targets to improve axonal regeneration and functional recovery after SCI.

Systematic analysis of the Frazzled receptor interactome establishes previously unreported regulators of axon guidance.

The Netrin receptor Dcc and its Drosophila homolog Frazzled play crucial roles in diverse developmental process, including axon guidance. In Drosophila, Fra regulates midline axon guidance through a Netrin-dependent and a Netrin-independent pathway. However, what molecules regulate these distinct signaling pathways remain unclear. To identify Fra-interacting proteins, we performed affinity purification mass spectrometry to establish a neuronal-specific Fra interactome. In addition to known interactors of Fra and Dcc, including Netrin and Robo1, our screen identified 85 candidate proteins, the majority of which are conserved in humans. Many of these proteins are expressed in the ventral nerve cord, and gene ontology, pathway analysis and biochemical validation identified several previously unreported pathways, including the receptor tyrosine phosphatase Lar, subunits of the COP9 signalosome and Rho-5, a regulator of the metalloprotease Tace. Finally, genetic analysis demonstrates that these genes regulate axon guidance and may define as yet unknown signaling mechanisms for Fra and its vertebrate homolog Dcc. Thus, the Fra interactome represents a resource to guide future functional studies.

Oncogenic stress-induced Netrin is a humoral signaling molecule that reprograms systemic metabolism in Drosophila.

Cancer exerts pleiotropic, systemic effects on organisms, leading to health deterioration and eventually to organismal death. How cancer induces systemic effects on remote organs and the organism itself still remains elusive. Here we describe a role for NetrinB (NetB), a protein with a particularly well-characterized role as a tissue-level axon guidance cue, in mediating oncogenic stress-induced organismal, metabolic reprogramming as a systemic humoral factor. In Drosophila, Ras-induced dysplastic cells upregulate and secrete NetB. Inhibition of either NetB from the transformed tissue or its receptor in the fat body suppresses oncogenic stress-induced organismal death. NetB from the dysplastic tissue remotely suppresses carnitine biosynthesis in the fat body, which is critical for acetyl-CoA generation and systemic metabolism. Supplementation of carnitine or acetyl-CoA ameliorates organismal health under oncogenic stress. This is the first identification, to our knowledge, of a role for the Netrin molecule, which has been studied extensively for its role within tissues, in humorally mediating systemic effects of local oncogenic stress on remote organs and organismal metabolism.

TOM-1/tomosyn acts with the UNC-6/netrin receptor UNC-5 to inhibit growth cone protrusion in Caenorhabditis elegans.

In the polarity/protrusion model of growth cone repulsion from UNC-6/netrin, UNC-6 first polarizes the growth cone of the VD motor neuron axon via the UNC-5 receptor, and then regulates protrusion asymmetrically across the growth cone based on this polarity. UNC-6 stimulates protrusion dorsally through the UNC-40/DCC receptor, and inhibits protrusion ventrally through UNC-5, resulting in net dorsal growth. Previous studies showed that UNC-5 inhibits growth cone protrusion via the flavin monooxygenases and potential destabilization of F-actin, and via UNC-33/CRMP and restriction of microtubule plus-end entry into the growth cone. We show that UNC-5 inhibits protrusion through a third mechanism involving TOM-1/tomosyn. A short isoform of TOM-1 inhibited protrusion downstream of UNC-5, and a long isoform had a pro-protrusive role. TOM-1/tomosyn inhibits formation of the SNARE complex. We show that UNC-64/syntaxin is required for growth cone protrusion, consistent with a role of TOM-1 in inhibiting vesicle fusion. Our results are consistent with a model whereby UNC-5 utilizes TOM-1 to inhibit vesicle fusion, resulting in inhibited growth cone protrusion, possibly by preventing the growth cone plasma membrane addition required for protrusion.

Axon targeting of Drosophila medulla projection neurons requires diffusible Netrin and is coordinated with neuroblast temporal patterning.

How axon guidance pathways are utilized in coordination with temporal and spatial patterning of neural progenitors to regulate neuropil assembly is not well understood. We study this question in the Drosophila medulla using the transmedullary (Tm) projection neurons that target lobula through the inner optic chiasm (IOC). We demonstrate that the Netrin pathway plays multiple roles in guidance of Tm axons and that temporal patterning of medulla neuroblasts determines pioneer versus follower Tm neurons during this process. Loss of Frazzled (Fra) in early-born pioneer Tm neurons leads to IOC defects, while loss of Fra from follower neurons does not affect the IOC. In the follower projection neurons, Fra is required in other targeting steps including lobula branch extension and layer-specific targeting. Furthermore, different from other identified scenarios of Netrin/Fra involved axon guidance in Drosophila, we demonstrate that diffusible Netrin is required for the correct axon targeting and optic lobe organization.

Notch-dependent binary fate choice regulates the Netrin pathway to control axon guidance of Drosophila visual projection neurons.

Notch-dependent binary fate choice between sister neurons is one of the mechanisms to generate neural diversity. How these upstream neural fate specification programs regulate downstream effector genes to control axon targeting and neuropil assembly remains less well understood. Here, we report that Notch-dependent binary fate choice in Drosophila medulla neurons is required to regulate the Netrin axon guidance pathway, which controls targeting of transmedullary (Tm) neurons to lobula. In medulla neurons of Notch-on hemilineage composed of mostly lobula-targeting neurons, Notch signaling is required to activate the expression of Netrin-B and repress the expression of its repulsive receptor Unc-5. Turning off Unc-5 is necessary for Tm neurons to target lobula. Furthermore, Netrin-B provided by Notch-on medulla neurons is required for correct targeting of Tm axons from later-generated medulla columns. Thus, the coordinate regulation of Netrin pathway components by Notch signaling ensures correct targeting of Tm axons and contributes to the neuropil assembly.

Netrins and Netrin Receptors are Essential for Normal Targeting of Sensory Axons in the Zebrafish Olfactory Bulb.

Olfactory sensory neurons that express related odorant receptors specifically target large identifiable neuropils called protoglomeruli when they first reach the olfactory bulb in the zebrafish. This crude odorant receptor-related mapping is further refined as odorant receptor-specific glomeruli segregate from protoglomeruli later in development. Netrins are a prominent class of axon guidance molecules whose contribution to olfactory circuit formation is poorly studied. Morpholino knock down experiments have suggested that Netrin/Dcc signaling is involved in normal protoglomerular targeting. Here we extend these findings with more detailed characterization and modeling of netrin expression, and by examining protoglomerular targeting in mutant lines fornetrin1a (ntn1a), netrin1b (ntn1b), and their receptorsunc5b,dcc, andneo1a. We confirm thatntn1a,ntn1b, anddccare required for normal protoglomerular guidance of a subset of olfactory sensory neurons that are labeled with the Tg(or111-7:IRES:Gal4) transgene. We also observe errors in the targeting of these axons inunc5bmutants, but not inneo1a mutants. Our findings are consistent with ntn1a andntn1bacting primarily as attractants for olfactory sensory neurons targeting the central zone protoglomerulus.

Expression of netrin and its receptors uncoordinated-5 and frazzled in arthropods and onychophorans suggests conserved and diverged functions in neuronal pathfinding and synaptogenesis.

BACKGROUND: Development of the nervous system and the correct connection of nerve cells require coordinated axonal pathfinding through an extracellular matrix. Outgrowing axons exhibit directional growth toward or away from external guidance cues such as Netrin. Guidance cues can be detected by growth cones that are located at the end of growing axons through membrane-bound receptors such as Uncoordianted-5 and Frazzled. Binding of Netrin causes reformation of the cytoskeleton and growth of the axon toward (or away from) the source of Netrin production. RESULTS: Here, we investigate the embryonic mRNA expression patterns of netrin genes and their potential receptors, uncoordinated-5 and frazzled in arthropod species that cover all main branches of Arthropoda, that is, Pancrustacea, Myriapoda, and Chelicerata. We also studied the expression patterns in a closely related outgroup species, the onychophoran Euperipatoides kanangrensis, and provide data on expression profiles of these genes in larval tissues of the fly Drosophila melanogaster including the brain and the imaginal disks. CONCLUSION: Our data reveal conserved and diverged aspects of neuronal guidance in Drosophila with respect to the other investigated species and suggest a conserved function in nervous system patterning of the developing appendages.

A role for axon-glial interactions and Netrin-G1 signaling in the formation of low-threshold mechanoreceptor end organs.

Low-threshold mechanoreceptors (LTMRs) and their cutaneous end organs convert light mechanical forces acting on the skin into electrical signals that propagate to the central nervous system. In mouse hairy skin, hair follicle-associated longitudinal lanceolate complexes, which are end organs comprising LTMR axonal endings that intimately associate with terminal Schwann cell (TSC) processes, mediate LTMR responses to hair deflection and skin indentation. Here, we characterized developmental steps leading to the formation of Aß rapidly adapting (RA)-LTMR and Aδ-LTMR lanceolate complexes. During early postnatal development, Aß RA-LTMRs and Aδ-LTMRs extend and prune cutaneous axonal branches in close association with nascent TSC processes. Netrin-G1 is expressed in these developing Aß RA-LTMR and Aδ-LTMR lanceolate endings, and Ntng1 ablation experiments indicate that Netrin-G1 functions in sensory neurons to promote lanceolate ending elaboration around hair follicles. The Netrin-G ligand (NGL-1), encoded by Lrrc4c, is expressed in TSCs, and ablation of Lrrc4c partially phenocopied the lanceolate complex deficits observed in Ntng1 mutants. Moreover, NGL-1-Netrin-G1 signaling is a general mediator of LTMR end organ formation across diverse tissue types demonstrated by the fact that Aß RA-LTMR endings associated with Meissner corpuscles and Pacinian corpuscles are also compromised in the Ntng1 and Lrrc4c mutant mice. Thus, axon-glia interactions, mediated in part by NGL-1-Netrin-G1 signaling, promote LTMR end organ formation.

Identification, molecular characterization, and in silico structural analysis of larval salivary glands Netrin-A as a potent biomarker from Lucilia sericata (Diptera: Calliphoridae).

The greenbottle blowfly Lucilia sericata (L. sericata) is increasingly used in larval therapy of chronic wounds. Netrins as bifunctional proteins are in the superfamily of Laminins secreted from larval salivary glands. The Netrin protein has a significant instructive role in axon guidance, causing neuronal outgrowth, angiogenesis, and cell migration. It seems to be crucial in wound healing and acts as a potential biomarker in diagnosing some clinical diseases. This survey aimed to identify molecular features and analyze in silico structural configuration of Netrin-A in L. sericata larvae. The larvae were reared under standard maggotarium conditions. The nucleic acid sequence of L. sericata Netrin-A (LSN-A) was then identified using rapid amplification of circular DNA ends (RACE) and rapid amplification of genomic ends (RAGE). Parts of the Netrin-A gene, including the middle, 3'-, and 5'-ends, were identified, TA cloned in pTG19 plasmid, and transferred into DH5ɑ Escherichia coli. Each part was sequenced and assembled using SeqMan software. This gene structure was further subjected to in silico analysis. The DNA of LSN-A was identified to be 2407 bp, while its mRNA sequence was recognized as 2115 bp by Oligo0.7 software. It translated the Netrin-A protein with 704 amino acid residues. Its estimated molecular weight was 78.6 kDa. Sequencing of this fragment and its BLAST analysis revealed laminin-based high (95%) similarity with the mRNA sequence of Lucilia cuprina Netrin-A. The 3-D structure of Netrin-A drawn by SWISS-MODEL exhibited its partial resemblance to the reference molecule Netrin-1 of Homo sapiens. This study supports the molecular and structural analyses of LSN-A protein, which could lead to wound treatment. Ultimately, it can be an effective candidate to ameliorate injury. Our next attempt is to produce LSN-A recombinant protein for use in biomedical sciences.

Recreational physical activity before and during pregnancy and placental DNA methylation-an epigenome-wide association study.

BACKGROUND: Physical activity (PA) prior to and during pregnancy may have intergenerational effects on offspring health through placental epigenetic modifications. We are unaware of epidemiologic studies on longitudinal PA and placental DNA methylation. OBJECTIVES: We evaluated the association between PA before and during pregnancy and placental DNA methylation. METHODS: Placental tissues were obtained at delivery and methylation was measured using HumanMethylation450 Beadchips for participants in the Eunice Kennedy Shriver National Institute of Child Health and Human Development Fetal Growth Studies-Singletons among 298 participants. Using the Pregnancy Physical Activity Questionnaire, women recalled periconception PA (past 12 mo) at 8-13 wk of gestation and PA since last visit at 4 follow-up visits at 16-22, 24-29, 30-33, and 34-37 wk. We conducted linear regression for associations of PA at each visit with methylation controlling for false discovery rate (FDR). Top 100 CpGs were queried for enrichment of functional pathways using Ingenuity Pathway Analysis. RESULTS: Periconception PA was significantly associated with 1 CpG site. PA since last visit for visits 1-4 was associated with 2, 2, 8, and 0 CpGs (log fold changes ranging from -0.0319 to 0.0080, after controlling for FDR). The largest change in methylation occurred at a site in TIMP2 , which is known to encode a protein critical for vasodilation, placentation, and uterine expansion during pregnancy (log fold change: -0.05; 95% CI: -0.06, -0.03 per metabolic equivalent of task-h/wk at 30-33 wk). Most significantly enriched pathways include cardiac hypertrophy signaling, B-cell receptor signaling, and netrin signaling. Significant CpGs and enriched pathways varied by visit. CONCLUSIONS: Recreational PA in the year prior and during pregnancy was associated with placental DNA methylation. The associated CpG sites varied based on timing of PA. If replicated, the findings may inform the mechanisms underlying the impacts of PA on placenta health. This study was registered at clinicaltrials.gov as NCT00912132.

The risk variant rs11836367 contributes to breast cancer onset and metastasis by attenuating Wnt signaling via regulating NTN4 expression.

Most genome-wide association study (GWAS)-identified breast cancer-associated causal variants remain uncharacterized. To provide a framework of understanding GWAS-identified variants to function, we performed a comprehensive study of noncoding regulatory variants at the NTN4 locus (12q22) and NTN4 gene in breast cancer etiology. We find that rs11836367 is the more likely causal variant, disrupting enhancer activity in both enhancer reporter assays and endogenous genome editing experiments. The protective T allele of rs11837367 increases the binding of GATA3 to the distal enhancer and up-regulates NTN4 expression. In addition, we demonstrate that loss of NTN4 gene in mice leads to tumor earlier onset, progression, and metastasis. We discover that NTN4, as a tumor suppressor, can attenuate the Wnt signaling pathway by directly binding to Wnt ligands. Our findings bridge the gaps among breast cancer-associated single-nucleotide polymorphisms, transcriptional regulation of NTN4, and breast cancer biology, which provides previously unidentified insights into breast cancer prediction and prevention.