VEGFR2 Research Articles

A novel stop-gain pathogenic variant in FLT4 and a nonsynonymous pathogenic variant in PTPN11 associated with congenital heart defects.

Congenital heart defects (CHDs) are the most common congenital malformations, including structural malformations in the heart and great vessels. CHD complications such as low birth weight, prematurity, pregnancy termination, mortality, and morbidity depend on the type of defect. In the present research, genetic analyses via whole-exome sequencing (WES) was performed on 3 unrelated pedigrees with CHDs. The candidate variants were confirmed, segregated by PCR-based Sanger sequencing, and evaluated by bioinformatics analysis. A novel stop-gain c.C244T:p.R82X variant in the FLT4 gene, as well as a nonsynonymous c.C1403T:p.T468M variant in the PTPN11 gene, was reported by WES. FLT4 encodes a receptor tyrosine kinase involved in lymphatic development and is known as vascular endothelial growth factor 3. We are the first to report a novel c.C244T variant in the FLT4 gene associated with CHDs. Using WES, we also identified a nonsynonymous variant affecting protein-tyrosine phosphatase, the non-receptor type 11 (PTPN11) gene. The clinical implementation of WES can determine gene variants in diseases with high genetic and phenotypic heterogeneity like CHDs.

Pan-cancer analysis of DEPDC1 as a candidate prognostic biomarker and associated with immune infiltration.

DEP domain containing 1 (DEPDC1) gene is upregulated in several malignancies and contributes to tumorigenesis. Although the role of DEPDC1 in tumor is becoming increasingly popular, the function of DEPDC1 in pan-cancer still needs to be systematically elucidated. Data were downloaded from Genotype-Tissue Expression Data (GTEx), The Cancer Genome Atlas (TCGA) TIMER2.0, TISIDB, STRING, and CancerSEA databases and analyzed to determine the functionality of the DEPDC1. The results were visualized using tools provided by the databases and the R language. The results showed that DEPDC1 was significantly upregulated in 29 of the 33 human cancers analyzed. In addition, there were significant differences in DEPDC1 expression among cancer immune and molecular subtypes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that DEPDC1 was mainly involved in the cell cycle, and CancerSEA analysis showed that DEPDC1 promoted cell cycle, DNA repair, DNA damage, and proliferation in pan-cancer. Receiver operating characteristic (ROC) curve analysis showed high predictive accuracy for pan-cancer. DEPDC1 expression was positively correlated with activated CD4+ T helper 2 cells and common lymphoid progenitor cells, and negatively correlated with natural killer (NK) T cells, CD4+ central memory T cells, and CD4+ effector memory T cells. Furthermore, DEPDC1 was significantly positively correlated with T cell exhaustion marker genes, such as CD274, transforming growth factor beta receptor 1 (TGFBR1), kinase insert domain receptor (KDR), programmed cell death 1 ligand 2 (PDCD1LG2), granzyme B (GZMB), and granulysin (LAG2). Additionally, DEPDC1 was associated with overall survival (OS), disease-specific survival (DSS), and progress-free interval (PFI) prognosis in multiple tumor types. The ROC analysis showed high predictive accuracy for pan-cancer. Collectively, DEPDC1 is aberrantly expressed and plays animmune-oncogenic role in pan-cancer, and DEPDC1 may serve as a biomarker for cancer diagnosis and therapy.

Global DNA and protein interactomes of FLT1P1 (Fms-related tyrosine kinase 1 pseudogene 1) revealed its molecular regulatory functions associated with preeclampsia.

Preeclampsia (PE) is one of the most serious pregnancy complications with unknown pathogenesis. Emerging evidence has demonstrated that Fms-related tyrosine kinase 1 (FLT1) is highly involved in PE development. As a pseudogene of FLT1, FLT1P1 increased in PE samples. However, its functions remain largely unknown. In this study, co-expression analysis was performed to identify the potential target genes of FTL1P1. Then chromatin isolation using RNA purification (ChIRP) method was employed to explore the interactomes of FLT1P1, including interacting with DNA fragments and proteins. We found that in PE samples, both FLT1P1 and FLT1 were highly expressed and closely correlated. ChIRP-protein data revealed that FLT1P1 interacts with translation- and transcription-related proteins, including 4 transcription factors (TFs). ChIRP-DNA analysis revealed that FLT1P1 preferentially interacted with DNA fragments downstream of transcription start sites (TSSs). Functional analysis of its interacting genes revealed that they were enriched in transcriptional regulation and apoptosis-related pathways. Twenty-six TFs, including CREB1 and SRF, were extracted from the potential FLT1P1-interacting gene sets and were potential targets of FLT1P1. CREB1 could bind to FLT1 promoter, and was negatively correlated with FLT1 at the expression level, making it a potential regulator of FLT1. Our study extensively investigated the interactome profiles of FLT1P1, especially the prompter region of TF gene CREB1, and revealed the potential molecular regulatory mechanisms of FLT1 expression in PE samples. Our results provide a novel view of PE pathogenesis, and suggest that FLT1P1 could serve as a potential therapeutic target in PE diagnosis and treatment.

Attenuated inflammatory profile following single and repeated handgrip exercise and remote ischemic preconditioning in patients with cerebral small vessel disease.

Background: Similar to remote ischemic preconditioning bouts of exercise may possess immediate protective effects against ischemia-reperfusion injury. However, underlying mechanisms are largely unknown. This study compared the impact of single and repeated handgrip exercise versus remote ischemic preconditioning on inflammatory biomarkers in patients with cerebral small vessel disease (cSVD). Methods: In this crossover study, 14 patients with cSVD were included. All participants performed 4-day of handgrip exercise (4x5-minutes at 30% of maximal handgrip strength) and remote ischemic preconditioning (rIPC; 4x5-minutes cuff occlusion around the upper arm) twice daily. Patients were randomized to start with either handgrip exercise or rIPC and the two interventions were separated by > 9days. Venous blood was drawn before and after one intervention, and after 4-day of repeated exposure. We performed a targeted proteomics on inflammation markers in all blood samples. Results: Targeted proteomics revealed significant changes in 9 out of 92 inflammatory proteins, with four proteins demonstrating comparable time-dependent effects between handgrip and rIPC. After adjustment for multiple testing we found significant decreases in FMS-related tyrosine kinase-3 ligand (Flt3L; 16.2% reduction; adjusted p-value: 0.029) and fibroblast growth factor-21 (FGF-21; 32.8% reduction adjusted p-value: 0.029) after single exposure. This effect did not differ between handgrip and rIPC. The decline in Flt3L after repeated handgrip and rIPC remained significant (adjusted p-value = 0.029), with no difference between rIPC and handgrip (adjusted p-value = 0.98). Conclusion: Single handgrip exercise and rIPC immediately attenuated plasma Flt3L and FGF-21, with the reduction of Flt3L remaining present after 4-day of repeated intervention, in people with cSVD. This suggests that single and repeated handgrip exercise and rIPC decrease comparable inflammatory biomarkers, which suggests activation of shared (anti-)inflammatory pathways following both stimuli. Additional studies will be needed to exclude the possibility that this activation is merely a time effect.

CDX-301 prevents radiation-induced dysregulation of miRNA expression and biogenesis

Risks of radiation exposure necessitate the development ofradioprophylactic drugs. We have reported the efficacy of CDX-301, a recombinantly developed human protein form of Fms-related tyrosine kinase 3 ligand (Flt3L), as a radioprophylactic and radiomitigatory agent. Here, we performed global microRNA profiling to further understand the mechanism of action of CDX-301. We find that CDX-301 administration 24h prior to total body irradiation prevents radiation-induced dysregulation of microRNA biogenesis and expression in murine serum and spleen samples in a time- and tissue-dependent manner. Further analysis shows that activation of the HOTAIR regulatory pathway has a prominent function in radiation-induced injury responses, which is inhibited by pre-treatment with CDX-301. Moreover, CDX-301 attenuates radiation-induced dysregulation of several cellular functions such as inflammatory and immune responses. In corroboration, we also find that pre-treatment with CDX-301 restores the expression of bone marrow aplasia markers and inflammatory cytokines and growth factors, as well as the expression of genes associated with MAP kinase and TGF-β pathways that are altered by radiation. Our findings provide new insights into CDX-301-mediated molecular and cellular mechanisms and point to a possible novel radioprotective drug for the prevention of irradiation-induced injury and hematopoietic acute radiation syndrome.

Functional characterization and development of novel human kinase insert domain receptor chimeric antigen receptor T-cells for immunotherapy of non-small cell lung cancer

CAR-T cell therapy, in which T cells are transfected or transduced with a chimeric antigen receptor (CAR), is a transformative type of cancer immunotherapy. Despite outstanding success in hematological malignancies, their efficacy against solid tumors has been limited. Here, we aimed to explore whether T cells modified by a CAR targeting the vascular endothelial growth factor 2 receptor/ kinase insert domain receptor (KDR) could destroy tumors and their vasculature. A second-generation KDR-CAR was constructed and transfected into T cells using lentivirus. The 3D structure of the CAR construct and target antigen was predicted. Moreover, in silico analysis, including molecular docking and molecular dynamics (MD) simulation, were used to evaluate the minimum energy of interaction and stability of the complex. The anti-cancer effect of KDR-specific CAR-T cells was tested with KDR-expressing and KDR overexpressing A549 cell line. The in-silico study suggested that this CAR construct could be effective for lung cancer therapy. We evaluated this using both in vitro and in vivo experiments. The KDR-CAR-T cells targeted and killed KDR-A549 with high efficiency by expressing IFN-γ and releasing granzyme B. The in vivo study showed that KDR-CAR-T cells dramatically inhibited the growth of lung cancer KDR-A549 xenografts in BALB/c-nu mice at day 10. The characterization of T cells modified by KDR-CAR by computational biology and wet-lab experiments suggested its applicability as a new treatment strategy for lung cancer and, potentially, for other vascularized solid tumors.

Leukoreductive response to the combination of sorafenib and chemotherapy in hyperleukocytosis of FLT3-ITD mutated pediatric AML.

Twelve to 22% of pediatric acute myeloid leukemia (AML) patients present with hyperleukocytosis, which is one of the main risk factors of early death due to its clinical complications: leukostasis, causing pulmonary or central nervous system injuries, tumor lysis syndrome, and disseminated intravascular coagulation. Sorafenib is a multi-kinase inhibitor that blocks the Fms-Related Tyrosine Kinase 3 receptor (FLT3) in AML patients with a FLT3-internal tandem duplication (FLT3-ITD), leading to a reduction of proliferation. Here we report four de novo diagnosed or relapsed pediatric FLT3-ITD-positive AML patients with hyperleukocytosis, which were treated with sorafenib in combination with cytoreductive chemotherapy prior to the start of the induction phase. We observed a fast reduction of white blood cells in peripheral blood and bone marrow. This resulted in a rapid clinical stabilization of the patients. Adverse side effects-such as dermatologic toxicity, elevation of transaminases and hypertension-occurred but were mild and inductive chemotherapy could be started in parallel or subsequently. This implies sorafenib as a safe and effective treatment option in combination with chemotherapy during cytoreductive prephase for children with this life-threatening condition.

WT1 and DNMT3A play essential roles in the growth of certain patient AML cells in mice

WT1 and DNMT3A play essential roles in the growth of certain patient AML cells in mice

Real world molecular characterisation and clonal evolution of acute myeloid leukaemia reveals therapeutic opportunities and challenges

Acute myeloid leukaemia (AML) is an aggressive haematological malignancy with poor prognosis. Increasing understanding of the molecular mechanisms driving clonal proliferation has resulted in advancements in classification and available therapeutic targets. Fms-related tyrosine kinase 3 (FLT3) mutations are prognostically important and offer options for targeted inhibition, however they are not stable and can emerge or disappear at relapse. Our aim was to review diagnostic testing of consecutive cases of newly diagnosed and relapsed AML reported across Queensland in comparison to available literature. We conducted a retrospective review of 1531 samples from 1231 patients to identify patterns of molecular testing and AML subtypes in our cohort. Outcomes included World Health Organization (WHO) classification, European LeukaemiaNet (ELN) risk category and rates of missed FLT3 mutation testing. Patients aged <60 years had significantly more favourable risk AML (48% vs 25%, p<0.01), with favourable risk chromosomal translocations [t(8;21) and inv(16)] being more common. Thirteen patients (1%) did not have FLT3 mutation testing at diagnosis, with 103 relapse samples (39%) not being tested. Eighteen patients (10%) had FLT3 mutations lost at relapse, with five patients (3%) developing new FLT3 mutations at relapse. This study identifies the subtypes and risk stratification of a large cohort of AML patients over an extended period. The relatively high rate of absent FLT3 mutation testing at relapse as well as FLT3 loss or gain highlights the potential missed opportunities for salvage treatment strategies.

Targeting FLT3-specific chimeric antigen receptor T cells for acute lymphoblastic leukemia with KMT2A rearrangement.

CD19-specific chimeric antigen receptor T (CAR T) immunotherapy is used to treat B-cell malignancies. However, antigen-escape mediated relapse following CAR T therapy has emerged as a major concern. In some relapsed cases, especially KMT2A rearrangement-positive B-acute lymphoblastic leukemia (KMT2A-r B-ALL), most of the B-cell antigens are lost via lineage conversion to the myeloid phenotype, rendering multi-B-cell-antigen-targeted CAR T cell therapy ineffective. Fms-related tyrosine kinase-3 (FLT3) is highly expressed in KMT2A-r B-ALL; therefore, in this study, we aimed to evaluate the antitumor efficacy of CAR T cells targeting both CD19 and FLT3 in KMT2A-r B-ALL cells. We developed piggyBac transposon-mediated CAR T cells targeting CD19, FLT3, or both (dual) and generated CD19-negative KMT2A-r B-ALL models through CRISPR-induced CD19 gene-knockout (KO). FLT3 CAR T cells showed antitumor efficacy against CD19-KO KMT2A-r B-ALL cells both in vitro and in vivo; dual-targeted CAR T cells showed cytotoxicity against wild-type (WT) and CD19-KO KMT2A-r B-ALL cells, whereas CD19 CAR T cells demonstrated cytotoxicity only against WT KMT2A-r B-ALL cells in vitro. Therefore, targeting FLT3-specific CAR T cells would be a promising strategy for KMT2A-r B-ALL cells even with CD19-negative relapsed cases.

Low Level FLT3LG is a Novel Poor Prognostic Biomarker for Cervical Cancer with Immune Infiltration.

IntroductionThe FMS-related tyrosine kinase 3 (FLT3) ligand (FLT3LG), a growth factor, binds to FLT3 on dendritic cell (DCs) to enhance their differentiation and expansion. It has shown great potential as an immunotherapy target for cancers. However, the expression and function of FLT3LG in cervical cancer remain largely unknown.Materials and MethodsIn this study, we obtained the expression of FLT3LG, the clinical prognosis in cervical cancer, via multiple databases, including The Cancer Genome Atlas (TCGA), the TISIDB database, and Tumor Immune Estimate Resource (TIMER). The results were further investigated using real-time quantitative PCR (qPCR) cytology specimens in 489 patients. Furthermore, Kaplan-Meier Cox regression and prognostic nomogram analyses were used to assess FLT3LG’s clinical significance in cervical cancer patients. All calculations used the R package.ResultsAs a result, FLT3LG expression decreased in cervical cancer compared with standard samples. And the low expression of FLT3LG was associated with a poor prognosis. Furthermore, Receiver Operating Characteristics (ROC) analysis indicated that FLT3LG might serve as a valuable diagnostic biomarker for cervical cancer. Additionally, it indicated that the FLT3LG had the highest odds ratio (OR=10.519; (7.371–27.071)) for detecting CIN 2+. In addition, our result also demonstrated that expression of FLT3LG was closely related to immune cells, immune inhibitors, immunostimulators, receptors, and chemokines in CESC.ConclusionResearch on FLT3LG provided insight into its critical function. Hence, the low expression of FLT3LG may be a valuable biomarker in CESC patients linked with immune infiltration.

Detection of FMS-Like Tyrosine Kinase 3 (FLT3) and Nucleophosmin 1 (NPM1) Mutations from Marrow Tissues in Patients with Acute Myeloid Leukaemia

INTRODUCTION: Acute myeloid leukaemia is a haematological malignancy with diverse cytogenetic abnormalities and molecular mutations. Amongst the important mutations are FMS-related tyrosine kinase 3 (FLT3) and nucleophosmin 1 (NPM1) gene mutations. These mutations have been shown to be of prognostic significance. A cross-sectional study to examine the frequency of these mutations and their association with the haematological and cytogenetic characteristics of the cases was carried out in Kuantan, Pahang, Malaysia. MATERIALS AND METHODS: A total of 43 cases were included in the study. Polymerase chain reaction-based assays were employed for mutation detection from the retrieved trephine biopsy tissue blocks. Mutation positivity was subsequently validated by Sanger DNA sequencing. RESULTS: Six of the 43 cases (14.0%) of the acute myeloid leukaemia were positive for FLT3-type internal tandem duplications (FLT3-ITD) and a similar proportion (6/43, 14.0%) were positive for NPM1 mutations. FLT3 mutations at codon D835 (FLT3-D835) mutation was identified in three of the cases (7.0%) while concurrent mutations of NPM1 and FLT3-ITD were seen in two of the mutation-positive cases (4.7%). The total white cell count was found to be significantly higher in patients with FLT3 mutations (p=0.001). Other haematological parameters and the cytogenetic results did not reveal any significant association with the mutational status. CONCLUSION: The frequency of FLT3-ITD, FLT3-D835, and NPM1 mutations among AML patients were 14%, 7%, and 14% respectively. Follow-up studies to include the clinical parameters and the treatment outcomes are advocated.

IgA-Based Secretory Response in Tears of COVID-19 Patients: A Potential Biomarker of Pro-Inflammatory State in Course of SARS-CoV-2 Infection.

Mucosal immunity, including secretory IgA (sIgA), plays an important role in the early defence against SARS-CoV-2 infection. However, a comprehensive evaluation of the local immune response in tears in relation to blood antibody reservoirs has not yet been conducted. A total of 179 symptomatic laboratory-confirmed COVID-19 patients were included in this single-centre study. Conjunctival swabs were analysed by a reverse transcription polymerase chain reaction for the detection of SARS-CoV-2 RNA. In parallel, tear samples collected by Schirmer test strips and plasma samples were analysed by ELISA to detect anti-S1 IgA levels. The concentrations of selected inflammatory cytokines in tears were determined by a magnetic bead assay. Anti-SARS-CoV-2 sIgA was present in the tears of 81 (45.25%) confirmed COVID-19 patients, and the tear IgA levels were correlated with the plasma IgA levels (Rs = +0.29, p = 0.0003). SARS-CoV-2 RNA in the conjunctival sac was identified in 18 COVID-19 patients (10%). Positive correlations between the tear IgA level and the concentrations of several cytokines TNF-α (Rs = +0.23, p = 0.002), IL-1β (Rs = +0.25, p < 0.001), IL-2 (Rs = +0.20, p = 0.007), IL-4 (Rs = +0.16, p = 0.04), IL-5 (Rs = +0.36, p < 0.001), IL-6 (Rs = +0.32, p < 0.001), IL-8 (Rs = +0.31, p < 0.001), VEGF (Rs = +0.25, p < 0.001) and GM-CSF (Rs = +0.27, p < 0.001) were also found. Quantitative tear film-based sIgA could potentially serve as a rapid and easily accessible biomarker of external mucosal immunity to SARS-CoV-2. The concentration of sIgA is directly related to individual host immune responses to SARS-CoV-2 infection.

Optimizing Insertion and Deletion Detection Using Next-Generation Sequencing in the Clinical Laboratory

Detection of insertions and deletions (InDels) by short-read next-generation sequencing (NGS) technology can be challenging because of frequent misaligned reads. A systematic analysis of short InDels (1 to 30 bases) and fms-related receptor tyrosine kinase 3 (FLT3) internal tandem duplications (ITDs; 6 to 183 bases) from 46 clinical cases of solid or hematologic malignancy processed with a clinical NGS assay identified misaligned reads in every case, ranging from 3% to 100% of reads with the InDel showing mismapped bases. Mismaps also increased with InDel size. As a consequence, the clinical NGS bioinformatics pipeline undercalled the variant allele frequency by 1% to 84%, incorrectly called simultaneous single-base substitutions along with InDels, or did not report an FLT3 ITD that had been detected by capillary electrophoresis. To improve the ability of the pipeline to better detect and quantify InDels, we utilized a software program called Assembly-Based ReAligner (ABRA2) to more accurately remap reads. ABRA2 was able to correct 41% to 100% of the reads with mismapped bases and led to absolute increases in the variant allele frequency from 1% to 61% along with correction of all of the single-base substitutions except for two cases. ABRA2 could also detect multiple FLT3 ITD clones except for one 183-base ITD. Our analysis has found that ABRA2 performs well on short InDels as well as FLT3 ITDs that are <100 bases.

Association between inflammatory cytokines in the aqueous humor and hyperreflective foci on optical coherence tomography in patients with neovascular age-related macular degeneration and polypoidal choroidal vasculopathy.

PurposeTo investigate the associations between cytokine levels in the aqueous humor (AH) and hyperreflective foci (HF) on spectral-domain optical coherence tomography (SD-OCT) in neovascular age-related macular degeneration (nAMD) and polypoidal choroidal vasculopathy (PCV).MethodsThe prospective study included 63 eyes with nAMD, 44 with PCV, and 43 with cataracts (Controls). AH samples were obtained before anti-vascular endothelial growth factor (VEGF) therapy or cataract surgery. Cytokines interleukin 6 (IL-6), IL-8, IL-10, interferon-inducible protein 10 (IP-10), monocyte chemotactic protein 1 (MCP-1), and VEGF were measured by multiplex bead assay. Best-corrected visual acuity (BCVA), central macular thickness (CMT), and the number of HF were evaluated at baseline and 1 month after anti-VEGF treatment.ResultsNo significances difference in IL-6 and IL-8 levels were noted among the three groups (P = 0.370 and P = 0.067). VEGF, IP-10, and IL-10 levels were significantly higher in nAMD and PCV groups than in Controls (all P < 0.05). In nAMD, HF was positively correlated with VEGF (rs = 0.300, P = 0.025) and in eyes with HF group, VEGF and IL-10 were significantly higher than those without HF (P = 0.008 and P = 0.022). In PCV, no correlation was observed between HF and cytokines (all P > 0.05). After anti-VEGF treatment, patients with HF in nAMD and PCV were predisposed to worse visual outcomes (P = 0.022 and P = 0.015) and a significantly greater reduction in CMT (P = 0.001 and P = 0.057). And nAMD patients with HF were more sensitive to anti-VEGF treatment than those without HF (P = 0.029).ConclusionsIn the nAMD group, HF was positively correlated with VEGF. Patients in nAMD with HF had elevated levels of VEGF and IL-10 and responded favorably to anti-VEGF. HF might serve as an inflammatory biomarker and a predictive factor for therapeutic efficacy in patients with nAMD.

126 Antioxidant-Containing Kibble Supplemented to Adult Cats Elicited Improvements in Antioxidant Outcomes and C-Reactive Protein

Abstract The objective of this study was to evaluate oxidative health biomarkers in healthy male and female adult cats (n=30; 10 male and 20 female, avg age and BW + SD 3.34 ± 3.19 years; 3.85 ± 1.07 kg) fed an adult maintenance diet (Blue Buffalo Healthy Living Adult Chicken and Brown Rice) without (control diet) and supplemented with an antioxidant-containing kibble (test diet; proprietary blend containing blueberries, cranberries, and other antioxidant-containing ingredients). Both diets analyzed as 35.9% crude protein, 19.8% fat, 2.1% crude fiber, 6.4% moisture, 28.6% carbohydrate as-is. We hypothesized that test feeding would reduce serum pro-oxidative biomarkers, compared with control feeding. The study protocol was first approved by the Institutional Animal Care and Use Committee. All cats were fed a base kennel maintenance diet (no added antioxidants beyond maintenance requirements) for 14d before treatment phase. Animals were then randomized to control or test for a 28d treatment period in a randomized cross-over design. Base kennel maintenance diet was provided for a 14d washout, followed by another treatment period. Serum was collected on days 0, 14, and 28 of for biomarkers analysis. Data were analyzed as a mixed models 2-way ANCOVA (SAS v9.4). Significant differences between treatments were set at P&amp;lt;0.05. Average daily food intake and body weight were similar between treatments for the duration of the study. Serum advanced glycation end-products and malondialdehyde were significantly (P&amp;lt;0.05) less when animals consumed test diet compared with control diet. Interestingly, serum C-reactive protein (CRP) was decreased and Fms-related tyrosine kinase 3 ligand was greater (P&amp;lt;0.05) when cats were fed test diet compared with control diet. These data suggest that inclusion of an antioxidant-containing kibble extruded to preserve antioxidant activity to a base kibble exhibits some favorable modulation to serum antioxidant markers and CRP in cats. These observations warrant additional research to elucidate the effects.

COMPOUND HETEROZYGOSITY FOR A VARIABLY PENETRANT VARIANT AND A VARIANT OF UNKNOWN SIGNIFICANCE IN FLT4 CAUSES FULLY PENETRANT MILROY'S LYMPHEDEMA

Milroy disease, known as primary congenital lymphedema, is characterized by chronic tissue swelling due to impaired lymphatic drainage and is inherited in an autosomal dominant manner. This study reports a rare case of Milroy disease affecting siblings from unaffected parents. A one-month-old female infant presented with swelling of the bilateral calf and the dorsum of the feet which had been present since birth. Her 14-month-old brother had a similar presentation since birth with swelling of the bilateral calf and the dorsum of the feet. Milroy disease was diagnosed based on the clinical findings of bilateral lower limb swelling and confirmed by molecular genetic testing. The patient and her family, including her brother, parents, and maternal grandfather, were genetically tested, and two novel missense mutations (NM_182925.4: c.2534T>C; p.Leu845Pro, c.4006G>A; p.Glu1336Lys) were found in the Fms-related tyrosine kinase (FLT4) gene. Mutations segregated by the parents who carried each mutation in the heterozygous state were identified in the patient and her brother. The present case report in which Milroy disease developed in all offspring of parents with a normal phenotype suggests the possibility of a compound heterozygous mutation or non-penetrance during the process of inheritance of Milroy disease.

A novel, liposome-loaded, injectable hydrogel for enhanced treatment of choroidal neovascularization by sub-tenon's injection

As a common multifactor fundus lesion, choroidal neovascularization (CNV) has become the leading cause of severe vision loss in the elderly. The mainstay treatment is an invasive intravitreal injection of anti-VEGF drug, which leads to a short half-life, incomplete response, and severe ocular complications. In this work, we have developed a novel, liposome-loaded, injectable hydrogel (cSA@Lip-HAC) by a minimally invasive sub-tenon's injection for CNV treatment. First, the multitarget angiogenic inhibitor (sunitinib) and hypoxia-inducible factor inhibitor (acriflavine) were co-loaded in the liposome, which was then loaded in the injectable hydrogel. The in vitro results showed that the cSA@Lip-HAC group had a strong anti-angiogenesis effect. After sub-tenon's injection, the hydrogel endowed the drug-loaded liposome with a longer retention time in the target area. In the CNV model, cSA@Lip-HAC showed a significant anti-CNV effect, which was superior to that of intravitreal injection of a commercial product (Conbercept). Exploration of the molecular mechanism revealed that cSA@Lip-HAC did not only inhibit CNV through the AKT/mTOR/HIF-1α/VEGF signal pathways but also inhibit VEGF R1 and VEGF R2. In conclusion, this novel drug delivery system, combining the merits of the liposome and hydrogel, showed an enhanced anti-CNV effect, thus providing a new and minimally invasive alternative for the treatment of neovascular ocular diseases.

Evolution and Potential Subfunctionalization of Duplicated fms-Related Class III Receptor Tyrosine Kinase flt3s and Their Ligands in the Allotetraploid Xenopus laevis.

The fms-related tyrosine kinase 3 (Flt3) and its ligand (Flt3lg) are important regulators of hematopoiesis and dendritic cell (DC) homeostasis with unsettled coevolution. Gene synteny and deduced amino acid sequence analyses identified conserved flt3 gene orthologs across all jawed vertebrates. In contrast, flt3lg orthologs were not retrieved in ray-finned fish, and the gene locus exhibited more variability among species. Interestingly, duplicated flt3/flt3lg genes were maintained in the allotetraploid Xenopus laevis Comparison of modeled structures of X. laevis Flt3 and Flt3lg homoeologs with the related diploid Xenopus tropicalis and with humans indicated a higher conformational divergence between the homoeologous pairs than their respective counterparts. The distinctive developmental and tissue expression patterns of Flt3 and Flt3lg homoeologs in tadpoles and adult frogs suggest a subfunctionalization of these homoeologs. To characterize Flt3 cell surface expression, X. laevis-tagged rFlt3lg.S and rFlt3lg.L were produced. Both rFlt3lg.S and rFlt3lg.L bind in vitro Flt3.S and Flt3.L and can trigger Erk1/2 signaling, which is consistent with a partial overlapping function between homoeologs. In spleen, Flt3.S/L cell surface expression was detected on a fraction of B cells and a population of MHC class IIhigh/CD8+ leukocytes phenotypically similar to the recently described dual follicular/conventional DC-like XL cells. Our result suggests that 1) Flt3lg.S and Flt3lg.L are both involved in XL cell homeostasis and that 2) XL cells have hematopoietic origin. Furthermore, we detected surface expression of the macrophage/monocyte marker Csf1r.S on XL cells as in mammalian and chicken DCs, which points to a common evolutionary origin in vertebrate DCs.

Differential dependency of human glioblastoma cells on vascular endothelial growth factor‑A signaling via neuropilin‑1.

Despite the high expression of neuropilin-1 (NRP-1) in human glioblastoma (GB), the understanding of its function as a co-receptor of vascular endothelial growth factor receptors (VEGFRs) in angiogenesis is currently limited. Therefore, the aim of the present study was to elucidate the non-classical function of NRP-1 expression in human GB. Expression patterns of NRP-1 and VEGF-A were determined by sandwich ELISA, western blot analysis, or immunohistochemistry. Differential dependency of GB cells following ablation of VEGF-A signaling was validated in vitro and in vivo. Cellular mechanism responsible for distinct response to VEGF-A signaling was evaluated by western blotting and immune-precipitation analysis. Prognostic implications were assessed using IHC analysis. GB cells exhibited differing sensitivity to silencing of vascular endothelial growth factor (VEGF)-A signaling, which resulted in a distinct expression pattern of wild-type or chondroitin-sulfated NRP-1. VEGF-A-sensitive GB exhibited the physical interaction between wild-type NRP-1 and FMS related receptor tyrosine kinase 1 (Flt-1) whereas VEGF-A-resistant GB exhibited chondroitin-sulfated NRP-1 without interaction with Flt-1. Eliminating the chondroitin sulfate modification in NRP-1 led to re-sensitization to VEGF-A signaling, and chondroitin sulfate modification was found to be associated with an adverse prognosis in patients with GB. The present study identified the distinct functions of NRP-1 in VEGF-A signaling in accordance with its unique expression type and interaction with Flt-1. The present research is expected to provide a strong basis for targeting VEGF-A signaling in patients with GB, with variable responses.