Identification of drought responsive genes in aleppo pine (Pinus halepensis) and loblolly pine (Pinus taeda.L)

Drought is a major constraint for attaining economic yield in tree crops. As an initial step to understand molecular response to water-deficit-stress in trees, gene expression in response to water stress was quantified using real-time RT-PCR. The specific objectives established for this to were I. to identify and characterize the genes induced by drought stress in Aleppo pine (Pinus halepensis) and II to identify and quantify the differentially expressed genes in different populations of Loblolly pine (Pinus taeda.L) due to water deficit (chapter III). Results of these studies may be used to identify candidate genes for future breeding programs against water-deficit-stress

Landslide Susceptibility Assessment of a Part of the Western Ghats (India) Employing the AHP and F-AHP Models and Comparison with Existing Susceptibility Maps

Landslides are prevalent in the Western Ghats, and the incidences that happened in 2021 in the Koottickal area of the Kottayam district (Western Ghats) resulted in the loss of 10 lives. The objectives of this study are to assess the landslide susceptibility of the high-range local self-governments (LSGs) in the Kottayam district using the analytical hierarchy process (AHP) and fuzzy-AHP (F-AHP) models and to compare the performance of existing landslide susceptible maps. This area never witnessed any massive landslides of this dimension, which warrants the necessity of relooking into the existing landslide-susceptible models. For AHP and F-AHP modeling, ten conditioning factors were selected: slope, soil texture, land use/land cover (LULC), geomorphology, road buffer, lithology, and satellite image-derived indices such as the normalized difference road landslide index (NDRLI), the normalized difference water index (NDWI), the normalized burn ratio (NBR), and the soil-adjusted vegetation index (SAVI). The landslide-susceptible zones were categorized into three: low, moderate, and high. The validation of the maps created using the receiver operating characteristic (ROC) technique ascertained the performances of the AHP, F-AHP, and TISSA maps as excellent, with an area under the ROC curve (AUC) value above 0.80, and the NCESS map as acceptable, with an AUC value above 0.70. Though the difference is negligible, the map prepared using the TISSA model has better performance (AUC = 0.889) than the F-AHP (AUC = 0.872), AHP (AUC = 0.867), and NCESS (AUC = 0.789) models. The validation of maps employing other matrices such as accuracy, mean absolute error (MAE), and root mean square error (RMSE) also confirmed that the TISSA model (0.869, 0.226, and 0.122, respectively) has better performance, followed by the F-AHP (0.856, 0.243, and 0.147, respectively), AHP (0.855, 0.249, and 0.159, respectively), and NCESS (0.770, 0.309, and 0.177, respectively) models. The most landslide-inducing factors in this area that were identified through this study are slope, soil texture, LULC, geomorphology, and NDRLI. Koottickal, Poonjar-Thekkekara, Moonnilavu, Thalanad, and Koruthodu are the LSGs that are highly susceptible to landslides. The identification of landslide-susceptible areas using diversified techniques will aid decision-makers in identifying critical infrastructure at risk and alternate routes for emergency evacuation of people to safer terrain during an exigency

Radiogenomic Mapping of Edema/Cellular Invasion MRI-Phenotypes in Glioblastoma Multiforme

Despite recent discoveries of new molecular targets and pathways, the search for an effective therapy for Glioblastoma Multiforme (GBM) continues. A newly emerged field, radiogenomics, links gene expression profiles with MRI phenotypes. MRI-FLAIR is a noninvasive diagnostic modality and was previously found to correlate with cellular invasion in GBM. Thus, our radiogenomic screen has the potential to reveal novel molecular determinants of invasion. Here, we present the first comprehensive radiogenomic analysis using quantitative MRI volumetrics and large-scale gene- and microRNA expression profiling in GBM.Based on The Cancer Genome Atlas (TCGA), discovery and validation sets with gene, microRNA, and quantitative MR-imaging data were created. Top concordant genes and microRNAs correlated with high FLAIR volumes from both sets were further characterized by Kaplan Meier survival statistics, microRNA-gene correlation analyses, and GBM molecular subtype-specific distribution.The top upregulated gene in both the discovery (4 fold) and validation (11 fold) sets was PERIOSTIN (POSTN). The top downregulated microRNA in both sets was miR-219, which is predicted to bind to POSTN. Kaplan Meier analysis demonstrated that above median expression of POSTN resulted in significantly decreased survival and shorter time to disease progression (P<0.001). High POSTN and low miR-219 expression were significantly associated with the mesenchymal GBM subtype (P<0.0001).Here, we propose a novel diagnostic method to screen for molecular cancer subtypes and genomic correlates of cellular invasion. Our findings also have potential therapeutic significance since successful molecular inhibition of invasion will improve therapy and patient survival in GBM

An Antagomir to MicroRNA Let7f Promotes Neuroprotection in an Ischemic Stroke Model

We previously showed that middle-aged female rats sustain a larger infarct following experimental stroke as compared to younger female rats, and paradoxically, estrogen treatment to the older group is neurotoxic. Plasma and brain insulin-like growth factor-1 (IGF-1) levels decrease with age. However, IGF-1 infusion following stroke, prevents estrogen neurotoxicity in middle-aged female rats. IGF1 is neuroprotective and well tolerated, but also has potentially undesirable side effects. We hypothesized that microRNAs (miRNAs) that target the IGF-1 signaling family for translation repression could be alternatively suppressed to promote IGF-1-like neuroprotection. Here, we report that two conserved IGF pathway regulatory microRNAs, Let7f and miR1, can be inhibited to mimic and even extend the neuroprotection afforded by IGF-1. Anti-mir1 treatment, as late as 4 hours following ischemia, significantly reduced cortical infarct volume in adult female rats, while anti-Let7 robustly reduced both cortical and striatal infarcts, and preserved sensorimotor function and interhemispheric neural integration. No neuroprotection was observed in animals treated with a brain specific miRNA unrelated to IGF-1 (anti-miR124). Remarkably, anti-Let7f was only effective in intact females but not males or ovariectomized females indicating that the gonadal steroid environment critically modifies miRNA action. Let7f is preferentially expressed in microglia in the ischemic hemisphere and confirmed in ex vivo cultures of microglia obtained from the cortex. While IGF-1 was undetectable in microglia harvested from the non-ischemic hemisphere, IGF-1 was expressed by microglia obtained from the ischemic cortex and was further elevated by anti-Let7f treatment. Collectively these data support a novel miRNA-based therapeutic strategy for neuroprotection following stroke

A Novel Volume-Age-KPS (VAK) Glioblastoma Classification Identifies a Prognostic Cognate microRNA-Gene Signature.

BACKGROUND: Several studies have established Glioblastoma Multiforme (GBM) prognostic and predictive models based on age and Karnofsky Performance Status (KPS), while very few studies evaluated the prognostic and predictive significance of preoperative MR-imaging. However, to date, there is no simple preoperative GBM classification that also correlates with a highly prognostic genomic signature. Thus, we present for the first time a biologically relevant, and clinically applicable tumor Volume, patient Age, and KPS (VAK) GBM classification that can easily and non-invasively be determined upon patient admission. METHODS: We quantitatively analyzed the volumes of 78 GBM patient MRIs present in The Cancer Imaging Archive (TCIA) corresponding to patients in The Cancer Genome Atlas (TCGA) with VAK annotation. The variables were then combined using a simple 3-point scoring system to form the VAK classification. A validation set (N = 64) from both the TCGA and Rembrandt databases was used to confirm the classification. Transcription factor and genomic correlations were performed using the gene pattern suite and Ingenuity Pathway Analysis. RESULTS: VAK-A and VAK-B classes showed significant median survival differences in discovery (P = 0.007) and validation sets (P = 0.008). VAK-A is significantly associated with P53 activation, while VAK-B shows significant P53 inhibition. Furthermore, a molecular gene signature comprised of a total of 25 genes and microRNAs was significantly associated with the classes and predicted survival in an independent validation set (P = 0.001). A favorable MGMT promoter methylation status resulted in a 10.5 months additional survival benefit for VAK-A compared to VAK-B patients. CONCLUSIONS: The non-invasively determined VAK classification with its implication of VAK-specific molecular regulatory networks, can serve as a very robust initial prognostic tool, clinical trial selection criteria, and important step toward the refinement of genomics-based personalized therapy for GBM patients

REST–miR-21–SOX2 axis maintains pluripotency in E14Tg2a.4 embryonic stem cells

Our previous studies have shown that the regulatory network that maintains pluripotency in mouse embryonic stem cells (mESCs) is regulated in a context-dependent manner and can be modulated, at least in part, by re-calibration of an intracellular network of pluripotency factors as well as cues arising from the extracellular matrix. The transcriptional repressor REST represses miR-21 and, thus, regulates self-renewal in E14Tg2a.4 mESCs cultured in the absence of mouse embryonic fibroblast feeder cell effects. However, how miR-21 connects to the nuclear regulatory network has not been clear. Here, we show that miR-21, a direct target of REST-mediated repression, directly targets Sox2. Exogenously added miR-21 to mESCs decreases the expression of Sox2, decreasing mESC self-renewal, and this effect of miR-21 on mESC self-renewal can be blocked by expression of exogenous Sox2. Conversely, destabilization of Sox2 by miR-21 can be blocked by anti-miR-21. Thus, the REST–miR-21–Sox2 axis connects REST to the core nuclear pluripotency regulators in E14Tg2a.4 mESCs cultured in the absence of feeder cells

Anti-Let7f regulates IGF-1 in microglia.

<p>A. IGF-1 levels in vivo: Cortical lysates from the ischemic and non ischemic hemisphere of scrambled and anti-Let7f treated animals were analyzed for IGF-1 by ELISA. Ischemia induced a 4-fold elevation of IGF-1 in both scrambled and anti-Let7f treated animals. However, IGF-1 levels in the anti-Let7f treated group was no different from that of the scramble controls. Histogram depicts mean ± SEM of 4–5 animals per group. B. IGF-1 levels in microglia: Microglia were harvested from the ischemic cortex of adult rats and treated in culture with anti-Let7f or scrambled oligonucleotides for 48 h. Anti-Let7f treatment significantly increased IGF-1 levels in microglial media. Ex vivo experiments were performed 6 times using cells harvested from 1–2 animals for each run; histogram depicts mean ± SEM of all experiments. *: p<0.05.</p

Cellular localization of Let7f in vivo and ex vivo.

<p>A. Sections (40 um) from the ischemic cortex were probed for Let7f by in situ hybridization (red) and either GFAP (green, Ai) or CD11b (green, Aii) immunohistochemistry. While Let7f was occasionally colocalized to GFAP-positive cells, virtually all CD11b-positive cells were also Let7f labeled. In a parallel analysis, Let7f (green) was also localized to cells expressing moderate levels of Iba1 (red; red arrow), but not in cells expressing high levels of Iba1 (white arrow). Bar: 200 µm. B. Microglial cells were harvested from the ischemic cortex and cultured <i>ex vivo</i> for 2–4 days. (Bi) Combined <i>in situ</i> hybridization and immunohistochemistry indicated that all CD11b-positive cells were also positively labeled with Let7f probes. (Bii) Microglial cultures exposed to FAM-labeled anti-Let7f in media readily incorporated the antagomir. Shown here are confocal images of microglia (DIC illumination) with FAM-labeled antagomirs (green) Bar: 30 µm.</p

Behavioral assessment of post-stroke anti-Let7f treatment.

<p>Animals injected with anti-Let7f or scrambled oligonucleotides 4 h post MCAo were assessed on the vibrissae evoked forelimb placement task and the rotarod task. A. Control animals were impaired post stroke (blue bars) on the cross midline task when the left vibrissa was stimulated (and required right paw placement), as compared to their pre-stroke (green bars) performance. Anti-Let7f treated animals performed no differently from their pre-stroke performance indicating significant functional neuroprotection. Histogram depicts percent correct responses+SEM, N = 4–5/group, *: p<0.05. B. Animals were placed on a slowly-accelerating rod and the amount of time spent on the rod was recorded. Post-stroke, anti-Let7f treated animals were able to maintain their balance on the rotating rod for significantly longer than control treated animals. Histogram depicts time in seconds+SEM, N = 5/group, *: p<0.05.</p

Effect of anti-sense miRNA on infarct volume.

<p>Female rats were injected with either anti-sense miRNA or scrambled oligonucleotides <u>(control)</u> 4 h after ET-1 induced middle cerebral artery occlusion (MCAo). Infarct volume was assessed using TTC-stained brain sections and quantitative morphometry. A. Animals injected with anti-miR1 had significantly lower cortical infarct volume as compared to controls injected with a scrambled oligo. Striatal infarct was not significantly different between the two groups. B. Animals injected with anti-Let7f had significantly reduced cortical and striatal infarct volumes as compared to controls. C. Animals injected with anti-miR124 had infarct volumes that were similar to the control group. Histograms depict mean ± SEM. *: p<0.05, n = 5 per group.</p