Integration of the Connectivity Map and Pathway Analysis to Predict Plant Extract's Medicinal Properties-The Study Case of Sarcopoterium spinosum L.

Medicinal properties of plants are usually identified based on knowledge of traditional medicine or using low-throughput screens for specific pharmacological activities. The former is very biased since it requires prior knowledge of plants' properties, while the latter depends on a specific screening system and will miss medicinal activities not covered by the screen. We sought to enrich our understanding of the biological activities of Sarcopoterium spinosum L. root extract based on transcriptome changes to uncover a plurality of possible pharmacological effects without the need for prior knowledge or functional screening. We integrated Gene Set Enrichment Analysis of the RNAseq data to identify pathways affected by the treatment of cells with the extract and perturbational signatures in the CMAP database to enhance the validity of the results. Activities of signaling pathways were measured using immunoblotting with phospho-specific antibodies. Mitochondrial membrane potential was assessed using JC-1 staining. SARS-CoV-2-induced cell killing was assessed in Vero E6 and A549 cells using an MTT assay. Here, we identified transcriptome changes following exposure of cultured cells to the medicinal plant Sarcopoterium spinosum L. root extract. By integrating algorithms of GSEA and CMAP, we confirmed known anti-cancer activities of the extract and predicted novel biological effects on oxidative phosphorylation and interferon pathways. Experimental validation of these pathways uncovered strong activation of autophagy, including mitophagy, and excellent protection from SARS-CoV-2 infection. Our study shows that gene expression analysis alone is insufficient for predicting biological effects since some of the changes reflect compensatory effects, and additional biochemical tests provide necessary corrections. This study defines the advantages and limitations of transcriptome analysis in predicting the biological and medicinal effects of the Sarcopoterium spinosum L. extract. Such analysis could be used as a general approach for predicting the medicinal properties of plants.

Novel Cyclic Peptides for Targeting EGFR and EGRvIII Mutation for Drug Delivery.

The epidermal growth factor-epidermal growth factor receptor (EGF-EGFR) pathway has become the main focus of selective chemotherapeutic intervention. As a result, two classes of EGFR inhibitors have been clinically approved, namely monoclonal antibodies and small molecule kinase inhibitors. Despite an initial good response rate to these drugs, most patients develop drug resistance. Therefore, new treatment approaches are needed. In this work, we aimed to find a new EGFR-specific, short cyclic peptide, which could be used for targeted drug delivery. Phage display peptide technology and biopanning were applied to three EGFR expressing cells, including cells expressing the EGFRvIII mutation. DNA from the internalized phage was extracted and the peptide inserts were sequenced using next-generation sequencing (NGS). Eleven peptides were selected for further investigation using binding, internalization, and competition assays, and the results were confirmed by confocal microscopy and peptide docking. Among these eleven peptides, seven showed specific and selective binding and internalization into EGFR positive (EGFR+ve) cells, with two of them-P6 and P9-also demonstrating high specificity for non-small cell lung cancer (NSCLC) and glioblastoma cells, respectively. These peptides were chemically conjugated to camptothecin (CPT). The conjugates were more cytotoxic to EGFR+ve cells than free CPT. Our results describe a novel cyclic peptide, which can be used for targeted drug delivery to cells overexpressing the EGFR and EGFRvIII mutation.

Ecogeographic Conditions Dramatically Affect Trans-Resveratrol and Other Major Phenolics' Levels in Wine at a Semi-Arid Area.

Grapevines are susceptible and responsive to their surrounding environment. Factors such as climate region and terroir are known to affect polyphenolic compounds in wine and therefore, its quality. The uniqueness of the terroir in Israel is the variety of soil types and the climatic conditions, ranging from Mediterranean to arid climates. Thus, understanding the effects of climate on grapevine performance in Israel may be a test case for the effect of climate change on grapevine at other areas in the future. First, we present a preliminary survey (2012-2014) in different climate zones and terroirs, which showed that trans-resveratrol concentrations in Merlot and Shiraz were high, while those of Cabernet Sauvignon were significantly lower. A further comprehensive countrywide survey (2016) of Merlot wines from 62 vineyards (53 wineries) compared several phenolic compounds' concentrations between five areas of different climate and terroir. Results show a connection between trans-resveratrol concentrations, variety, and terroir properties. Furthermore, we show that trans-resveratrol concentrations are strongly correlated to humidity levels at springtime, precipitation, and soil permeability. This work can be considered a glimpse into the possible alterations of wine composition in currently moderate-climate wine-growing areas.

Effect of coadministration of neuronal growth factors on neuroglial differentiation of bone marrow-derived stem cells in the ischemic retina.

PURPOSE: Brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) have limited and transient supportive effects on retinal recovery from ischemia. The aim of this study was to investigate their effect on engrafted adult bone marrow-derived stem cells in a rodent model of anterior ischemic optic neuropathy (rAION). METHODS: Small cells were isolated from the bone marrow of green fluorescent protein expressing mice by counterflow centrifugal elutriation, depleted of cells expressing lineage markers, and grafted in conjunction with growth factors into the vitreous body of mice with unilateral rAION. Progenitors were mobilized with granulocyte macrophage colony-stimulating factor (GM-CSF) or stem cell factor (SCF). The contralateral eye served as a control. RESULTS: At 4 weeks, the quantitative incorporation of donor cells in the injured retina was increased by BDNF (P < 0.01 versus control) and decreased by CNTF (P < 0.01 versus control), with no notable difference at 24 weeks. Both growth factors improved the short-term and long-term qualitative engraftment of cells adopting neural phenotypes in the retinal ganglion cell (RGC) layer and astrocyte phenotypes in the anterior vasculature. The RGC-engrafted cells formed extensions toward the inner nuclear layer. In the presence of growth factors, donor cells migrated to the optic nerve and contributed to repair by gliosis. Mobilization with GM-CSF restricted cell fate to microglia, whereas SCF was associated with limited neuroglial differentiation. CONCLUSIONS: Both BDNF and CNTF enhance engraftment and neuroglial differentiation of adult bone marrow stem cells in injured retina, with BDNF having an early quantitative and qualitative advantage over CNTF. Mobilization with differentiation factors restricts cell fate in the injured retina.

VEGF induces neuroglial differentiation in bone marrow-derived stem cells and promotes microglia conversion following mobilization with GM-CSF.

PURPOSE: Evaluation of potential tropic effects of vascular endothelial growth factor (VEGF) on the incorporation and differentiation of bone-marrow-derived stem cells (BMSCs) in a murine model of anterior ischemic optic neuropathy (AION). METHODS: In the first approach, small-sized subset of BMCs were isolated from GFP donors mice by counterflow centrifugal elutriation and depleted of hematopoietic lineages (Fr25lin(-)). These cells were injected into a peripheral vein (1 × 10(6) in 0.2 ml) or inoculated intravitreally (2 × 10(5)) to syngeneic mice, with or without intravitreal injection of 5 µg/2µL VEGF, simultaneously with AION induction. In a second approach, hematopoietic cells were substituted by myelablative transplant of syngeseic GFP + bone marrow cells. After 3 months, progenitors were mobilized with granulocyte-macrophage colony-stimulating factor (GM-CSF) followed by VEGF inoculation into the vitreous body and AION induction . Engraftment and phenotype were examined by immunohistochemistry and FISH at 4 and 24 weeks post-transplantation, and VEGF receptors were determined by real time PCR. RESULTS: VEGF had no quantitative effect on incorporation of elutriated cells in the injured retina, yet it induced early expression of neuroal markers in cells incorporated in the RGC layer and promoted durable gliosis, most prominent perivascular astrocytes. These effects were mediated by VEGF-R1/Flt-1, which is constitutively expresses in the elutriated fraction of stem cells. Mobilization with GM-CSF limited the differentiation of bone marrow progenitors to microglia, which was also fostered by VEGF. CONCLUSIONS: VEGF signaling mediated by Flt-1 induces early neural and sustained astrocytic differentiation of stem cells elutriated from adult bone-marrow, with significant contribution to stabilization retinal architecture following ischemic injury.

Primitive stem cells derived from bone marrow express glial and neuronal markers and support revascularization in injured retina exposed to ischemic and mechanical damage.

Ischemic or mechanical injury to the optic nerve is an irreversible cause of vision loss, associated with limited regeneration and poor response to neuroprotective agents. The aim of this study was to assess the capacity of adult bone marrow cells to participate in retinal regeneration following the induction of anterior ischemic optic neuropathy (AION) and optic nerve crush (ONC) in a rodent model. The small-sized subset of cells isolated by elutriation and lineage depletion (Fr25lin(-)) was found to be negative for the neuroglial markers nestin and glial fibrillary acidic protein (GFAP). Syngeneic donor cells, identified by genomic marker in sex-mismatched transplants and green fluorescent protein, incorporated into the injured retina (AION and ONC) at a frequency of 0.35%-0.45% after intravenous infusion and 1.8%-2% after intravitreous implantation. Perivascular cells with astrocytic morphology expressing GFAP and vimentin were of the predominant lineage that engrafted after AION injury; 10%-18% of the donor cells incorporated in the retinal ganglion cell layer and expressed NeuN, Thy-1, neurofilament, and beta-tubulin III. The Fr25lin(-) cells displayed an excellent capacity to migrate to sites of tissue disruption and developed coordinated site-specific morphological and phenotypic neural and glial markers. In addition to cellular reconstitution of the injured retinal layers, these cells contributed to endothelial revascularization and apparently supported remodeling by secretion of insulin-like growth factor-1. These results suggest that elutriated autologous adult bone marrow-derived stem cells may serve as an accessible source for cellular reconstitution of the retina following injury.

C1824T mutation in the LMNA gene has no association with senile cataract.

Mutations in the LMNA gene encoding lamins A/C are responsible for Hutchinson-Gilford syndrome (HGS), a disorder of premature aging. Cataract is 1 of the main manifestations. The most prevalent mutation in Hutchinson-Gilford syndrome is C1824T, which activates a cryptic splice donor site to produce an abnormal lamin A protein. The purpose of this study was to investigate a possible association of the C1824T mutation with age-related cataract. Anterior lens capsule material was collected during cataract extraction surgery from 178 patients with senile cataract during 2007-2008. DNA and mRNA were extracted and sequenced for the LMNA gene. DNA and cDNA were screened for the C1824T mutation, which was not detected. Messenger RNA (mRNA) expression was normal, with no truncation. We found that human age-related nuclear cataract is not associated with LMNA gene mutations or truncation of lamin A.

Reduction of apoptosis in ischemic retinas of two mouse models using hyperbaric oxygen treatment.

PURPOSE: To investigate the effect of hyperbaric oxygen (HBO) chamber treatment in mouse models of retinal ischemia. METHODS: Unilateral central retinal artery occlusion (CRAO) or optic nerve crush (ONC) was induced in 50 mice each, of which 30 were treated with 100% oxygen at 2 atm for 90 minutes immediately after injury and then daily for up to 14 days. Mice were euthanatized on days 1, 3, and 21 for histologic analysis, apoptosis assay, and quantitative real-time polymerase chain reaction test. Findings were analyzed by injury and by treatment. RESULTS: HBO treatment reduced cell loss from 58% to 30% in the CRAO model and from 52% to 32% in the ONC model. In both models, it was associated with significantly increased cell survival in the retinal ganglion cell layer. Expression levels of the proapoptosis genes (bax, caspase-3) decreased minimally in the HBO-treated CRAO mice on day 1, but this trend was reversed on day 3. In the ONC group, levels of caspase-3, bax, and bcl-x increased on day 1 and dropped below baseline on day 3. The pattern of changes in the expression levels of the ischemia- and oxidative-stress-related genes (HO-1, SOD-1, GPX-1, NOX-2) and the effectiveness of HBO treatment varied by model. Overall, however, gene expression levels that increased in the untreated mice increased further with HBO treatment and levels that decreased, decreased further with treatment. CONCLUSIONS: HBO treatment protects injured neuronal cells from apoptosis. Response to treatment differs molecularly after ONC or CRAO. These results should prompt clinical trials of acute ischemic retinal damage.

Neuroprotective effect of hyperbaric oxygen therapy on anterior ischemic optic neuropathy.

The study investigated the therapeutic effect of hyperbaric oxygen (HBO) on anterior ischemic optic neuropathy in a rodent model (rAION). rAION was laser-induced in one eye of 63 mice. The fellow (uninjured) eye served as an internal control. Thirty-three mice underwent two 90-min sessions of 100% oxygen (2 atm) treatment immediately following injury and one session daily thereafter for up to 14 days. The remaining mice were untreated. Retinas were harvested at different time points, and mRNA levels of various genes were analyzed by real-time polymerase chain reaction and histologic study. Untreated mice: day 1 post-rAION - SOD-1 (oxidative-stress-related) decreased to 82% of control (uninjured eye) levels (P < 0.05), Caspase-3 (proapoptotic) decreased to 89%, Bcl-xL mildly increased (117%; all NS); day 3 - HO-1 and endothelial nitric oxide synthase (eNOS; ischaemia-related) decreased to 74%, and Bcl-2-associated X protein, Caspase-3, and B-cell lymphoma 2 (Bcl-2; apoptotic) increased by 170, 120, and 111%, respectively (all NS); day21 - HO-1 increased to 222% (NS) and eNOS decreased to 48% (P < 0.05). Treated mice: day 1 - SOD-1 and Caspase-3 remained unchanged, Bcl-2 and Bcl-xL mildly increased (112 and 126% respectively); day 3 - HO-1 and eNOS increased, apoptosis-related gene decreased; day 21 - SOD-1 decreased whereas eNOS increased (P < 0.05), and HO-1 increased to a lesser degree than without treatment. None of the oxygen-treated animals had retinal ganglion cell loss or a decrease in Thy-1 expression. In conclusion, HBO treatment after rAION induction influences the expression of apoptosis-related genes as well as oxidative-stress-induced and ischaemia-related genes and may exert a neuroprotective effect.

Effect of subconjuctival and intraocular bevacizumab injection on angiogenic gene expression levels in a mouse model of corneal neovascularization.

PURPOSE: This study sought to characterize the expression of angiogenesis-related genes in a mouse model of corneal neovascularization, either untreated or after treatment with a single injection of bevacizumab by three different routes. In addition, the effectiveness of the treatment was compared to a rabbit model. METHODS: A chemical burn was induced in the mid-cornea of the right eye in 119 mice; 56 of them were untreated and 63 were bevacizumab-treated. Neovascularization was evaluated 2, 4, 8, 10, and 14 days later using digital photos, angiography and India ink perfusion. The relative area of new blood vessels was analyzed using slit-lamp examination in vivo and on histological and flat-mount sections. The levels of gene expression involved in the angiogenic process vascular endothelial growth factor [VEGF], insulin-like growth factor-1 [IGF-1], pigment epithelium derived factor [PEDF], and macrophage-inflammatory protein-2 [MIP-2]) were measured by a real-time polymerase chain reaction. Six rabbits underwent the same injury and treatment, and the response was compared to the mouse model. RESULTS: Neovascularization was first observed two days after injury. The affected section increased from 11.24% (+/-7.0) of the corneal area to 47.42% (+/-25.4) on day 8 and 50.62% (+/-24.7) on day 10. In the mice treated with bevacizumab, the relative area of neovascularization was significantly lower at the peak time points (p<0.005): 24.90% (+/-21.8) on day 8 and 28.29% (+/-20.9) on day 10. Spontaneous regression was observed on day 14 in both groups, to 26.98% (+/-19.9) in the untreated mice and 10.97% (+/-10.8) in the bevacizumab-treated mice (p<0.005). Rabbits also showed peak corneal neovascularization on days 8-10, with significant regression of the vessels following intracameral bevacizumab injection. In the mice, intraocular (intravitreal, intracameral) injection was more effective than subconjuctival injection. VEGF gene expression was upregulated in both the untreated and treated mice, but was slightly less in the treated mice. PEDF gene expression decreased in both the treated and untreated mice. In the untreated group, gene expression peaked (above baseline) at 14 days, and in the untreated mice, it had already peaked by day 8. IGF-1 was upregulated early in the model; at 8 days, there was only a slight change in the untreated group compared to a significant increase in the treated group. MIP-2 was upregulated in both groups in the early stage and returned to baseline on day 14. CONCLUSIONS: Bevacizumab treatment partially inhibits the progressive corneal neovascularization induced by chemical damage in a mouse model. Treatment is more effective when administered via the intraocular than the subconjunctival route. The clinical findings are compatible with the angiographic and histologic data and are supported by molecular analysis showing a partial change in expression of proangiogenic genes. The molecular mechanisms involved in corneal neovascularization and inflammation warrant further exploration. These findings may have important therapeutic implications in the clinical setting.

Possible neuroprotective effect of brimonidine in a mouse model of ischaemic optic neuropathy.

PURPOSE: To investigate the neuroprotective effect of brimonidine following induction of ischaemic optic neuropathy in rodents (rAION). METHODS: Mice were treated with an intraperitoneal injection of brimonidine 48, 24 or 0 h before rAION induction or eye drops for 5 days after rAION induction. Retinal ganglion cell (RGC) loss and expression of genes involved in the angiogenesis (vascular endothelial growth factor [VEGF], pigment epithelium-derived factor [PEDF], The epidermal growth factor homology domains-2 [Tie-2]), ischaemia (haem oxygense-1 [HO-1], hypoxia-inducible factor 1alpha[HIF-1alpha], endothelial nitric oxide synthase [eNOS]) and oxidative stress (superoxide dismutase-1 [SOD-1], glutathione peroxidase-1 [GPX-1]) response to ischaemic damage were compared with sham or rAION-untreated mice. RESULTS: No RGC loss was detected in the brimonidine-treated mice. Effect of post-rAION eye drops: day 1--no decrease in retinal mRNA levels of angiogenesis-related genes, increase in ischaemia- and oxidative stress-related genes except HIF-1alpha; day 3--baseline or higher levels of oxidative and ischaemia-related genes except HIF-1alpha, increase in VEGF, decrease in PEDF; day 21--no change in angiogenesis-related genes. Effect of pre-rAION injection: baseline levels of angiogenesis-related genes with all injection schedules; increase in ischaemia-related genes with 48-h and 0-h pretreatment; decrease in HO-1 and eNOS with 24-h pretreatment; increase in oxidative-related genes except GPX-1. In optic nerve tissue, HO-1, HIF-1alpha and SOD-1 decreased on day 1 after topical administration and were still below baseline on day 3. CONCLUSIONS: The increase in HO-1 associated with rAION is mitigated with brimonidine treatment, especially when administered intraperitoneally. Topical brimonidine apparently reduces VEGF, Tie-2, HIF-1alpha and GPX-1 expression on day 21. These results agree with published data and may have therapeutic implications for patients diagnosed with AION in the acute phase.

PI3K/Akt pathway mutations in retinoblastoma.

PURPOSE: Many malignancies are known to be associated with abnormal activation of the PI3K-AKT pathway. Recently, a somatic mutation in the AKT1 gene (E17K) was identified in a small proportion of human tumors. This mutation activated AKT1 by means of abnormal membrane recruitment and stimulated downstream signaling. This study was designed to analyze AKT1 mutations in retinoblastoma and gain insights into the role PI3K-AKT pathway plays in the development of this tumor. METHODS: Twenty-four samples of retinoblastoma from children were analyzed for mutations in the AKT1, PTEN and K-RAS genes, using a chip-based matrix-assisted laser desorption-time-of-flight (MALDI-TOF) mass spectrometer. Mutations in the PIK3CA gene were analyzed in 16 retinoblastoma samples using direct sequencing. RESULTS: These results show that the mutation E17K/AKT1 was not detected in the 24 samples of retinoblastoma analyzed. K-RAS mutations were identified in two samples. There were no mutations in any of the other genes analyzed by a mass array system. On direct sequencing of 16 samples for the PIK3CA gene, one sample showed gain of function mutation in exon 9. In another sample, a genetic polymorphism of unknown significance (rs17849079) was detected in exon 20. CONCLUSIONS: Although the PI3K-AKT pathway is known to be dysregulated in retinoblastoma, the low frequency of oncogenic mutations in the AKT1, PIK3CA, and PTEN genes, suggests a different activating mechanism.