Impact of an irreversible ß-galactosylceramidase inhibitor on the lipid profile of zebrafish embryos.

Krabbe disease is a sphingolipidosis characterized by the genetic deficiency of the acid hydrolase ß-galactosylceramidase (GALC). Most of the studies concerning the biological role of GALC performed on Krabbe patients and Galc-deficient twitcher mice (an authentic animal model of the disease) indicate that the pathogenesis of this disorder is the consequence of the accumulation of the neurotoxic GALC substrate ß-galactosylsphingosine (psychosine), ignoring the possibility that this enzyme may exert a wider biological impact. Indeed, limited information is available about the effect of GALC downregulation on the cell lipidome in adult and developing organisms. The teleost zebrafish (Danio rerio) has emerged as a useful platform to model human genetic diseases, including sphingolipidoses, and two GALC co-orthologs have been identified in zebrafish (galca and galcb). Here, we investigated the effect of the competitive and irreversible GALC inhibitor ß-galactose-cyclophellitol (GCP) on the lipid profile of zebrafish embryos. Molecular modelling indicates that GCP can be sequestered in the catalytic site of the enzyme and covalently binds human GALC, and the zebrafish Galca and Galcb proteins in a similar manner. Accordingly, GCP inhibits the ß-galactosylceramide hydrolase activity of zebrafish in vitro and in vivo, leading to significant alterations of the lipidome of zebrafish embryos. These results indicate that the lack of GALC activity deeply affects the lipidome during the early stages of embryonic development, and thereby provide insights into the pathogenesis of Krabbe disease.

The bacterial lysate OM-85 engages Toll-like receptors 2 and 4 triggering an immunomodulatory gene signature in human myeloid cells.

OM-85 is a bacterial lysate used in clinical practice to reduce duration and frequency of recurrent respiratory tract infections. Whereas knowledge of its regulatory effects in vivo has substantially advanced, the mechanisms of OM-85 sensing remain inadequately addressed. Here, we show that the immune response to OM-85 in the mouse is largely mediated by myeloid immune cells through Toll-like receptor (TLR) 4 in vitro and in vivo. Instead, in human immune cells, TLR2 and TLR4 orchestrate the response to OM-85, which binds to both receptors as shown by surface plasmon resonance assay. Ribonucleic acid-sequencing analyses of human monocyte-derived dendritic cells reveal that OM-85 triggers a pro-inflammatory signature and a unique gene set, which is not induced by canonical agonists of TLR2 or TLR4 and comprises tolerogenic genes. A largely overlapping TLR2/4-dependent gene signature was observed in individual subsets of primary human airway myeloid cells, highlighting the robust effects of OM-85. Collectively, our results suggest caution should be taken when relating murine studies on bacterial lysates to humans. Furthermore, our data shed light on how a standardized bacterial lysate shapes the response through TLR2 and TLR4, which are crucial for immune response, trained immunity, and tolerance.

Changes in Anti-OV-16 IgG4 Responses to Onchocerciasis after Elimination of Transmission in the Central Endemic Zone of Guatemala.

Current WHO guidelines for onchocerciasis elimination provide requirements for stopping mass drug administration of ivermectin and the verification of elimination of transmission. These guidelines also recommend post-elimination surveillance (PES) based on entomological surveys. Serological markers in humans could complement entomological PES once the longevity of anti-OV-16 antibody responses is better understood. In 2014-2015 we evaluated ELISA anti-OV-16 IgG4 antibody persistence among previously seropositive people from the central endemic zone of Guatemala. The country stopped all onchocerciasis program interventions in 2012 and was verified by WHO as having eliminated transmission of onchocerciasis in 2016. A total of 246 participants with prior OV-16 ELISA results from 2003, 2006, 2007, or 2009 were enrolled in a follow-up study. Of these, 77 people were previously OV-16 seropositive and 169 were previously seronegative. By 2014 and 2015, 56 (72.7%) previously seropositive individuals had sero-reverted, whereas all previous negatives remained seronegative. The progression of antibody responses over time was estimated using a mixed-effects linear regression model, using data from seropositive participants who had sero-reverted. The temporal variation showed a mean activity unit decay of 0.20 per year (95% credible interval [CrI]: 0.17, 0.23), corresponding to an estimated antibody response half-life of 3.3 years (95% CrI: 2.7, 4.1). These findings indicate that the majority of seropositive people will sero-revert over time.

The Pro-Oncogenic Sphingolipid-Metabolizing Enzyme ß-Galactosylceramidase Modulates the Proteomic Landscape in BRAF(V600E)-Mutated Human Melanoma Cells.

ß-Galactosylceramidase (GALC) is a lysosomal enzyme involved in sphingolipid metabolism by removing ß-galactosyl moieties from ß-galactosylceramide and ß-galactosylsphingosine. Previous observations have shown that GALC may exert pro-oncogenic functions in melanoma and Galc silencing, leading to decreased oncogenic activity in murine B16 melanoma cells. The tumor-driving BRAF(V600E) mutation is present in approximately 50% of human melanomas and represents a major therapeutic target. However, such mutation is missing in melanoma B16 cells. Thus, to assess the impact of GALC in human melanoma in a more relevant BRAF-mutated background, we investigated the effect of GALC overexpression on the proteomic landscape of A2058 and A375 human melanoma cells harboring the BRAF(V600E) mutation. The results obtained by liquid chromatography-tandem mass spectrometry (LC-MS/MS) demonstrate that significant differences exist in the protein landscape expressed under identical cell culture conditions by A2058 and A375 human melanoma cells, both harboring the same BRAF(V600E)-activating mutation. GALC overexpression resulted in a stronger impact on the proteomic profile of A375 cells when compared to A2058 cells (261 upregulated and 184 downregulated proteins versus 36 and 14 proteins for the two cell types, respectively). Among them, 25 proteins appeared to be upregulated in both A2058-upGALC and A375-upGALC cells, whereas two proteins were significantly downregulated in both GALC-overexpressing cell types. These proteins appear to be involved in melanoma biology, tumor invasion and metastatic dissemination, tumor immune escape, mitochondrial antioxidant activity, endoplasmic reticulum stress responses, autophagy, and/or apoptosis. Notably, analysis of the expression of the corresponding genes in human skin cutaneous melanoma samples (TCGA, Firehose Legacy) using the cBioPortal for Cancer Genomics platform demonstrated a positive correlation between GALC expression and the expression levels of 14 out of the 27 genes investigated, thus supporting the proteomic findings. Overall, these data indicate for the first time that the expression of the lysosomal sphingolipid-metabolizing enzyme GALC may exert a pro-oncogenic impact on the proteomic landscape in BRAF-mutated human melanoma.

Zebra-Sphinx: Modeling Sphingolipidoses in Zebrafish.

Sphingolipidoses are inborn errors of metabolism due to the pathogenic mutation of genes that encode for lysosomal enzymes, transporters, or enzyme cofactors that participate in the sphingolipid catabolism. They represent a subgroup of lysosomal storage diseases characterized by the gradual lysosomal accumulation of the substrate(s) of the defective proteins. The clinical presentation of patients affected by sphingolipid storage disorders ranges from a mild progression for some juvenile- or adult-onset forms to severe/fatal infantile forms. Despite significant therapeutic achievements, novel strategies are required at basic, clinical, and translational levels to improve patient outcomes. On these bases, the development of in vivo models is crucial for a better understanding of the pathogenesis of sphingolipidoses and for the development of efficacious therapeutic strategies. The teleost zebrafish (Danio rerio) has emerged as a useful platform to model several human genetic diseases owing to the high grade of genome conservation between human and zebrafish, combined with precise genome editing and the ease of manipulation. In addition, lipidomic studies have allowed the identification in zebrafish of all of the main classes of lipids present in mammals, supporting the possibility to model diseases of the lipidic metabolism in this animal species with the advantage of using mammalian lipid databases for data processing. This review highlights the use of zebrafish as an innovative model system to gain novel insights into the pathogenesis of sphingolipidoses, with possible implications for the identification of more efficacious therapeutic approaches.

Performance of Existing and Novel Symptom- and Antigen Testing-Based COVID-19 Case Definitions in a Community Setting.

Point-of-care antigen tests are an important tool for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection, yet are less clinically sensitive than real-time reverse-transcription polymerase chain reaction (RT-PCR), affecting their efficacy as screening procedures. Our goal in this analysis was to see whether we could improve this sensitivity by considering antigen test results in combination with other relevant information, namely exposure status and reported symptoms. In November 2020, we collected 3,419 paired upper respiratory specimens tested by RT-PCR and the Abbott BinaxNOW (Abbott Laboratories, Abbott Park, Illinois) antigen test at 2 community testing sites in Pima County, Arizona. We used symptom, exposure, and antigen-testing data to evaluate the sensitivity and specificity of various symptom definitions in predicting RT-PCR positivity. Our analysis yielded 6 novel multisymptom case definitions with and without antigen test results, the best of which overall achieved a Youden's J index of 0.66, as compared with 0.53 for antigen testing alone. Using a random forest as a guide, we show that this definition, along with our others, does not lose the ability to generalize well to new data despite achieving optimal performance in our sample. Our methodology is broadly applicable, and our code is publicly available to aid public health practitioners in developing or fine-tuning their own case definitions.

Evaluation of self-administered antigen testing in a college setting.

BACKGROUND: The objective of our investigation was to better understand barriers to implementation of self-administered antigen screening testing for SARS-CoV-2 at institutions of higher education (IHE). METHODS: Using the Quidel QuickVue At-Home COVID-19 Test, 1347 IHE students and staff were asked to test twice weekly for seven weeks. We assessed seroconversion using baseline and endline serum specimens. Online surveys assessed acceptability. RESULTS: Participants reported 9971 self-administered antigen test results. Among participants who were not antibody positive at baseline, the median number of tests reported was eight. Among 324 participants seronegative at baseline, with endline antibody results and ≥ 1 self-administered antigen test results, there were five COVID-19 infections; only one was detected by self-administered antigen test (sensitivity = 20%). Acceptability of self-administered antigen tests was high. CONCLUSIONS: Twice-weekly serial self-administered antigen testing in a low prevalence period had low utility in this investigation. Issues of testing fatigue will be important to address in future testing strategies.

Performance Characteristics of the Abbott BinaxNOW SARS-CoV-2 Antigen Test in Comparison to Real-Time Reverse Transcriptase PCR and Viral Culture in Community Testing Sites during November 2020.

Point-of-care antigen tests are an important tool for SARS-CoV-2 detection. Antigen tests are less sensitive than real-time reverse transcriptase PCR (rRT-PCR). Data on the performance of the BinaxNOW antigen test compared to rRT-PCR and viral culture by symptom and known exposure status, timing during disease, or exposure period and demographic variables are limited. During 3 to 17 November 2020, we collected paired upper respiratory swab specimens to test for SARS-CoV-2 by rRT-PCR and Abbott BinaxNOW antigen test at two community testing sites in Pima County, Arizona. We administered a questionnaire to capture symptoms, known exposure status, and previous SARS-CoV-2 test results. Specimens positive by either test were analyzed by viral culture. Previously we showed overall BinaxNOW sensitivity was 52.5%. Here, we showed BinaxNOW sensitivity increased to 65.7% among currently symptomatic individuals reporting a known exposure. BinaxNOW sensitivity was lower among participants with a known exposure and previously symptomatic (32.4%) or never symptomatic (47.1%) within 14 days of testing. Sensitivity was 71.1% in participants within a week of symptom onset. In participants with a known exposure, sensitivity was highest 8 to 10 days postexposure (75%). The positive predictive value for recovery of virus in cell culture was 56.7% for BinaxNOW-positive and 35.4% for rRT-PCR-positive specimens. Result reporting time was 2.5 h for BinaxNOW and 26 h for rRT-PCR. Point-of-care antigen tests have a shorter turnaround time than laboratory-based nucleic acid amplification tests, which allows for more rapid identification of infected individuals. Antigen test sensitivity limitations are important to consider when developing a testing program.

Heparin Insensitivity and Thrombotic Risk Associated With Sequential Uses of Prothrombin Complex Concentrate and Andexanet Alfa for Apixaban Reversal During Acute Type A Aortic Dissection Repair: A Case Report.

The management of patients on direct oral anticoagulants (DOACs) who require emergent cardiac surgery is slowly evolving. The introduction of andexanet alfa, a novel antidote for apixaban and rivaroxaban, added a specific reversal agent to our armamentarium, but its safety and efficacy are still being investigated. We report 2 patients on DOAC treatment who required emergency cardiac surgery. Both received perioperative andexanet alfa together with prothrombin complex concentrate (PCC) at some time during 6 hours before operative management. Heparin resistance was noted in each instance, and pump thrombosis developed in 1 case.

An Orthotopic Model of Uveal Melanoma in Zebrafish Embryo: A Novel Platform for Drug Evaluation.

Uveal melanoma is a highly metastatic tumor, representing the most common primary intraocular malignancy in adults. Tumor cell xenografts in zebrafish embryos may provide the opportunity to study in vivo different aspects of the neoplastic disease and its response to therapy. Here, we established an orthotopic model of uveal melanoma in zebrafish by injecting highly metastatic murine B16-BL6 and B16-LS9 melanoma cells, human A375M melanoma cells, and human 92.1 uveal melanoma cells into the eye of zebrafish embryos in the proximity of the developing choroidal vasculature. Immunohistochemical and immunofluorescence analyses showed that melanoma cells proliferate during the first four days after injection and move towards the eye surface. Moreover, bioluminescence analysis of luciferase-expressing human 92.1 uveal melanoma cells allowed the quantitative assessment of the antitumor activity exerted by the canonical chemotherapeutic drugs paclitaxel, panobinostat, and everolimus after their injection into the grafted eye. Altogether, our data demonstrate that the zebrafish embryo eye is a permissive environment for the growth of invasive cutaneous and uveal melanoma cells. In addition, we have established a new luciferase-based in vivo orthotopic model that allows the quantification of human uveal melanoma cells engrafted in the zebrafish embryo eye, and which may represent a suitable tool for the screening of novel drug candidates for uveal melanoma therapy.

Point-of-Care Antigen Test for SARS-CoV-2 in Asymptomatic College Students.

We used the BinaxNOW COVID-19 Ag Card to screen 1,540 asymptomatic college students for severe acute respiratory syndrome coronavirus 2 in a low-prevalence setting. Compared with reverse transcription PCR, BinaxNOW showed 20% overall sensitivity; among participants with culturable virus, sensitivity was 60%. BinaxNOW provides point-of-care screening but misses many infections.

Caffeine Inhibits Direct and Indirect Angiogenesis in Zebrafish Embryos.

In this study, we report the effects of caffeine on angiogenesis in zebrafish embryos both during normal development and after exposure to Fibroblast Growth Factor 2 (FGF2). As markers of angiogenesis, we measured the length and width of intersegmental vessels (ISVs), performed whole-mount in situ hybridization with fli1 and cadh5 vascular markers, and counted the number of interconnecting vessels (ICVs) in sub-intestinal venous plexus (SIVP). In addition, we measured angiogenesis after performing zebrafish yolk membrane (ZFYM) assay with microinjection of fibroblast growth factor 2 (FGF2) and perivitelline tumor xenograft assay with microinjection of tumorigenic FGF2-overexpressing endothelial (FGF2-T-MAE) cells. The results showed that caffeine treatment causes a shortening and thinning of ISVs along with a decreased expression of the vascular marker genes and a decrease in the number of ICVs in the SIVP. Caffeine was also able to block angiogenesis induced by exogenous FGF2 or FGF2-producing cells. Overall, our results are suggestive of the inhibitory effect of caffeine in both direct and indirect angiogenesis.

VEGF-Independent Activation of Müller Cells by the Vitreous from Proliferative Diabetic Retinopathy Patients.

Proliferative diabetic retinopathy (PDR), a major complication of diabetes mellitus, results from an inflammation-sustained interplay among endothelial cells, neurons, and glia. Even though anti-vascular endothelial growth factor (VEGF) interventions represent the therapeutic option for PDR, they are only partially efficacious. In PDR, Müller cells undergo reactive gliosis, produce inflammatory cytokines/chemokines, and contribute to scar formation and retinal neovascularization. However, the impact of anti-VEGF interventions on Müller cell activation has not been fully elucidated. Here, we show that treatment of MIO-M1 Müller cells with vitreous obtained from PDR patients stimulates cell proliferation and motility, and activates various intracellular signaling pathways. This leads to cytokine/chemokine upregulation, a response that was not mimicked by treatment with recombinant VEGF nor inhibited by the anti-VEGF drug ranibizumab. In contrast, fibroblast growth factor-2 (FGF2) induced a significant overexpression of various cytokines/chemokines in MIO-M1 cells. In addition, the FGF receptor tyrosine kinase inhibitor BGJ398, the pan-FGF trap NSC12, the heparin-binding protein antagonist N-tert-butyloxycarbonyl-Phe-Leu-Phe-Leu-Phe Boc2, and the anti-inflammatory hydrocortisone all inhibited Müller cell activation mediated by PDR vitreous. These findings point to a role for various modulators beside VEGF in Müller cell activation and pave the way to the search for novel therapeutic strategies in PDR.

Evaluation of Abbott BinaxNOW Rapid Antigen Test for SARS-CoV-2 Infection at Two Community-Based Testing Sites - Pima County, Arizona, November 3-17, 2020.

Rapid antigen tests, such as the Abbott BinaxNOW COVID-19 Ag Card (BinaxNOW), offer results more rapidly (approximately 15-30 minutes) and at a lower cost than do highly sensitive nucleic acid amplification tests (NAATs) (1). Rapid antigen tests have received Food and Drug Administration (FDA) Emergency Use Authorization (EUA) for use in symptomatic persons (2), but data are lacking on test performance in asymptomatic persons to inform expanded screening testing to rapidly identify and isolate infected persons (3). To evaluate the performance of the BinaxNOW rapid antigen test, it was used along with real-time reverse transcription-polymerase chain reaction (RT-PCR) testing to analyze 3,419 paired specimens collected from persons aged ≥10 years at two community testing sites in Pima County, Arizona, during November 3-17, 2020. Viral culture was performed on 274 of 303 residual real-time RT-PCR specimens with positive results by either test (29 were not available for culture). Compared with real-time RT-PCR testing, the BinaxNOW antigen test had a sensitivity of 64.2% for specimens from symptomatic persons and 35.8% for specimens from asymptomatic persons, with near 100% specificity in specimens from both groups. Virus was cultured from 96 of 274 (35.0%) specimens, including 85 (57.8%) of 147 with concordant antigen and real-time RT-PCR positive results, 11 (8.9%) of 124 with false-negative antigen test results, and none of three with false-positive antigen test results. Among specimens positive for viral culture, sensitivity was 92.6% for symptomatic and 78.6% for asymptomatic individuals. When the pretest probability for receiving positive test results for SARS-CoV-2 is elevated (e.g., in symptomatic persons or in persons with a known COVID-19 exposure), a negative antigen test result should be confirmed by NAAT (1). Despite a lower sensitivity to detect infection, rapid antigen tests can be an important tool for screening because of their quick turnaround time, lower costs and resource needs, high specificity, and high positive predictive value (PPV) in settings of high pretest probability. The faster turnaround time of the antigen test can help limit transmission by more rapidly identifying infectious persons for isolation, particularly when used as a component of serial testing strategies.

Inhibition of the FGF/FGFR System Induces Apoptosis in Lung Cancer Cells via c-Myc Downregulation and Oxidative Stress.

Lung cancer represents an extremely diffused neoplastic disorder with different histological/molecular features. Among the different lung tumors, non-small-cell lung cancer (NSCLC) is the most represented histotype, characterized by various molecular markers, including the expression/overexpression of the fibroblast growth factor receptor-1 (FGFR1). Thus, FGF/FGFR blockade by tyrosine kinase inhibitors (TKi) or FGF-ligand inhibitors may represent a promising therapeutic approach in lung cancers. In this study we demonstrate the potential therapeutic benefit of targeting the FGF/FGFR system in FGF-dependent lung tumor cells using FGF trapping (NSC12) or TKi (erdafitinib) approaches. The results show that inhibition of FGF/FGFR by NSC12 or erdafitinib induces apoptosis in FGF-dependent human squamous cell carcinoma NCI-H1581 and NCI-H520 cells. Induction of oxidative stress is the main mechanism responsible for the therapeutic/pro-apoptotic effect exerted by both NSC12 and erdafitinib, with apoptosis being abolished by antioxidant treatments. Finally, reduction of c-Myc protein levels appears to strictly determine the onset of oxidative stress and the therapeutic response to FGF/FGFR inhibition, indicating c-Myc as a key downstream effector of FGF/FGFR signaling in FGF-dependent lung cancers.

Long-Pentraxin 3 Affects Primary Cilium in Zebrafish Embryo and Cancer Cells via the FGF System.

Primary cilium drives the left-right asymmetry process during embryonic development. Moreover, its dysregulation contributes to cancer progression by affecting various signaling pathways. The fibroblast growth factor (FGF)/FGF receptor (FGFR) system modulates primary cilium length and plays a pivotal role in embryogenesis and tumor growth. Here, we investigated the impact of the natural FGF trap long-pentraxin 3 (PTX3) on the determination of primary cilium extension in zebrafish embryo and cancer cells. The results demonstrate that down modulation of the PTX3 orthologue ptx3b causes the shortening of primary cilium in zebrafish embryo in a FGF-dependent manner, leading to defects in the left-right asymmetry determination. Conversely, PTX3 upregulation causes the elongation of primary cilium in FGF-dependent cancer cells. Previous observations have identified the PTX3-derived small molecule NSC12 as an orally available FGF trap with anticancer effects on FGF-dependent tumors. In keeping with the non-redundant role of the FGF/FGR system in primary cilium length determination, NSC12 induces the elongation of primary cilium in FGF-dependent tumor cells, thus acting as a ciliogenic anticancer molecule in vitro and in vivo. Together, these findings demonstrate the ability of the natural FGF trap PTX3 to exert a modulatory effect on primary cilium in embryonic development and cancer. Moreover, they set the basis for the design of novel ciliogenic drugs with potential implications for the therapy of FGF-dependent tumors.

Both Handwashing and an Alcohol-Based Hand Sanitizer Intervention Reduce Soil and Microbial Contamination on Farmworker Hands during Harvest, but Produce Type Matters.

Hand hygiene interventions are critical for reducing farmworker hand contamination and preventing the spread of produce-associated illness. Hand hygiene effectiveness may be produce-commodity specific, which could influence implementation strategies. This study's goal was to determine if produce commodity influences the ability of handwashing with soap and water or two-step alcohol-based hand sanitizer (ABHS) interventions to reduce soil and bacteria on farmworker hands. Farmworkers (n = 326) harvested produce (cantaloupe, jalapeño, and tomato) for 30 to 90 minutes before engaging in handwashing, two-step ABHS (jalapeño and cantaloupe), or no hand hygiene. Hands were rinsed to measure amounts of soil (absorbance at 600 nm) and indicator bacteria (coliforms, Enterococcus sp., generic Escherichia coli, and Bacteroidales universal [AllBac] and human-specific [BFD] 16S rRNA gene markers). Without hand hygiene, bacterial concentrations (0.88 to 5.1 log10 CFU/hand) on hands significantly differed by the produce commodity harvested. Moderate significant correlations (ρ = -0.41 to 0.56) between soil load and bacterial concentrations were observed. There were significant produce-commodity-specific differences in the ability of handwashing and two-step ABHS interventions to reduce soil (P < 0.0001), coliforms (P = 0.002), and Enterococcus sp. (P = 0.003), but not the Bacteroidales markers AllBac (P = 0.4) or BFD (P = 0.3). Contamination on hands of farmworkers who harvested cantaloupe was more difficult to remove. Overall, we found that a two-step ABHS intervention was similar to handwashing with soap and water at reducing bacteria on farmworker hands. In summary, produce commodity type should be considered when developing hand hygiene interventions on farms.IMPORTANCE This study demonstrated that the type of produce commodity handled influences the ability of handwashing with soap and water or a two-step alcohol-based hand sanitizer (ABHS) intervention to reduce soil and bacterial hand contamination. Handwashing with soap and water, as recommended by the FDA's Produce Safety Rule, when tested in three agricultural environments, does not always reduce bacterial loads. Consistent with past results, we found that the two-step ABHS method performed similarly to handwashing with soap and water but also does not always reduce bacterial loads in these contexts. Given the ease of use of the two-step ABHS method, which may increase compliance, the two-step ABHS method should be further evaluated and possibly considered for implementation in the agricultural environment. Taken together, these results provide important information on hand hygiene effectiveness in three agricultural contexts.

Atypical Chemokine Receptor 3 Generates Guidance Cues for CXCL12-Mediated Endothelial Cell Migration.

Chemokine receptor CXCR4, its ligand stromal cell-derived factor-1 (CXCL12) and the decoy receptor atypical chemokine receptor 3 (ACKR3, also named CXCR7), are involved in the guidance of migrating cells in different anatomical districts. Here, we investigated the role of the ACKR3 zebrafish ortholog ackr3b in the vascularization process during embryonic development. Bioinformatics and functional analyses confirmed that ackr3b is a CXCL12-binding ortholog of human ACKR3. ackr3b is transcribed in the endoderm of zebrafish embryos during epiboly and is expressed in a wide range of tissues during somitogenesis, including central nervous system and somites. Between 18 somite and 26 h-post fertilization stages, the broad somitic expression of ackr3b becomes restricted to the basal part of the somites. After ackr3b knockdown, intersomitic vessels (ISVs) lose the correct direction of migration and are characterized by the presence of aberrant sprouts and ectopic filopodia protrusions, showing downregulation of the tip/stalk cell marker hlx1. In addition, ackr3b morphants show significant alterations of lateral dorsal aortae formation. In keeping with a role for ackr3b in endothelial cell guidance, CXCL12 gradient generated by ACKR3 expression in CHO cell transfectants guides human endothelial cell migration in an in vitro cell co-culture chemotaxis assay. Our results demonstrate that ackr3b plays a non-redundant role in the guidance of sprouting endothelial cells during vascular development in zebrafish. Moreover, ACKR3 scavenging activity generates guidance cues for the directional migration of CXCR4-expressing human endothelial cells in response to CXCL12.

Laryngotracheal separation in pediatric patients: 13-year experience in a reference service.

OBJECTIVE: To evaluate clinical stability of neurologically impaired children and adolescents with recurrent pneumonia submitted to laryngotracheal separation. METHODS: Between October 2002 and June 2015, 92 neurologically impaired children from a reference service, with median age of 68.5 months were submitted to laryngotracheal separation. Data were evaluated and statistical analysis was made by Student's t test and Pearson's χ2 test (significance level adopted of 95%). RESULTS: Fifty-three children were male (57.6%). Forty-six children required admission to intensive care, and 42.4% needed mechanical ventilation. We observed that 90.2% of patients were exclusively fed by gastrostomy and 72.4% of the gastrostomies were performed before the tracheal surgery. Thirteen (14.1%) children had postoperative complications as follows: fistulae (5.4%), bleeding (4.3%), granuloma (2.2%) and stenosis (3.2%). A total of 24 patients had pneumonia in the postoperative period (26.1%), but there was a significant drop in occurrence of this condition after surgery (100% versus 26.1%; p<0.001). Twenty-three patients (25%) died. Postoperative complications were similar when comparing patients who died and those that presented good outcome (16.7% versus 13.2%; p=0.73). CONCLUSION: When well-indicated, the laryngotracheal separation reduces the incidence of postoperative pulmonary infections, thus improving quality of life and reducing admissions to hospital. Laryngotracheal separation should be indicated as a primary procedure in patients with cerebral palsy and recurrent aspiration pneumonia.

Exploring the Antiangiogenic Potential of Solomonamide A Bioactive Precursors: In Vitro and in Vivo Evidences of the Inhibitory Activity of Solo F-OH During Angiogenesis.

Marine sponges are a prolific source of bioactive compounds. In this work, the putative antiangiogenic potential of a series of synthetic precursors of Solomonamide A, a cyclic peptide isolated from a marine sponge, was evaluated. By means of an in vitro screening, based on the inhibitory activity of endothelial tube formation, the compound Solo F-OH was selected for a deeper characterization of its antiangiogenic potential. Our results indicate that Solo F-OH is able to inhibit some key steps of the angiogenic process, including the proliferation, migration, and invasion of endothelial cells, as well as diminish their capability to degrade the extracellular matrix proteins. The antiangiogenic potential of Solo F-OH was confirmed by means of two different in vivo models: the chorioallantoic membrane (CAM) and the zebrafish yolk membrane (ZFYM) assays. The reduction in ERK1/2 and Akt phosphorylation in endothelial cells treated with Solo F-OH denotes that this compound could target the upstream components that are common to both pathways. Taken together, our results show a new and interesting biological activity of Solo F-OH as an inhibitor of the persistent and deregulated angiogenesis that characterizes cancer and other pathologies.