Identifying risk zones and landscape features that affect common leopard depredation.

Human-wildlife conflict (HWC) is a pressing issue worldwide but varies by species over time and place. One of the most prevalent forms of HWC in the mid-hills of Nepal is human-common-leopard conflict (HLC). Leopard attacks, especially in forested areas, can severely impact villagers and their livestock. Information on HLC in the Gorkha district was scarce, thus making it an ideal location to identify high-risk zones and landscape variables associated with such events. Registered cases were collected and reviewed from the Division Forest Office (DFO) during 2019-2021. Claims from DFO records were confirmed with herders and villagers via eight focus group discussions. To enhance modeling success, researchers identified a total of 163 leopard attack locations on livestock, ensuring a minimum distance of at least 100 meters between locations. Using maximum entropy (MaxEnt) and considering 13 environmental variables, we mapped common leopard attack risk zones. True Skill Statistics (TSS) and area under receiver-operator curve (AUC) were used to evaluate and validate the Output. Furthermore, 10 replications, 1,000 maximum iterations, and 1000 background points were employed during modeling. The average AUC value for the model, which was 0.726 ± 0.021, revealed good accuracy. The model performed well, as indicated by a TSS value of 0.61 ± 0.03. Of the total research area (27.92 km2), about 74% was designated as a low-risk area, 19% as a medium-risk area, and 7% as a high-risk area. Of the 13 environmental variables, distance to water (25.2%) was the most significant predictor of risk, followed by distance to road (16.2%) and elevation (10.7%). According to response curves, the risk of common leopard is highest in the areas between 1.5 to 2 km distances from the water sources, followed by the closest distance from a road and an elevation of 700 to 800 m. Results suggest that managers and local governments should employ intervention strategies immediately to safeguard rural livelihoods in high-risk areas. Improvements include better design of livestock corrals, insurance, and total compensation of livestock losses. Settlements near roads and water sources should improve the design and construction of pens and cages to prevent livestock loss. More studies on the characteristics of victims are suggested to enhance understanding of common leopard attacks, in addition to landscape variables. Such information can be helpful in formulating the best management practices.

Unconventional Reactivity of a Grubbs Catalyst: Hydroalkylation Overriding Metathesis.

Unprecedented reactivity of a Grubbs catalyst has been disclosed in a reaction between vinyl azaarenes and alkenylnitriles under standard metathesis conditions. No metathesis was observed; only hydroalkylation products were obtained in high yields. The practical utility of this method has been demonstrated by the application of the products in useful transformations, e.g., the formation of cyclic iminoesters and highly challenging medium-sized carbocycles.

Palladium-Catalyzed Dearomative [4 + 2]-Cycloaddition toward Hydrocarbazoles.

An efficient method for the construction of hydrocarbazoles bearing three continuous sterically hindered stereocenters, two quaternary and one tertiary, via a highly diastereoselective palladium-catalyzed [4 + 2]-cycloaddition/dearomatization of 3-nitroindoles has been developed. The cycloaddition of 3-nitroindoles occurs at ambient conditions with a 1,4-zwitterionic intermediate, in situ generated from γ-methylidene-δ-valerolactones. The further synthetic utility of this method is demonstrated by the multifaceted transformations possible from the products. The catalytic asymmetric aspect of this transformation has also been explored.

Iridium-Catalyzed Enantioselective Ring Opening of Alkenyl Oxiranes by Unactivated Carboxylic Acids.

An iridium-catalyzed enantioselective ring-opening of alkenyl oxiranes by unactivated carboxylic acids has been developed. The reaction undergoes at ambient conditions between an in-situ-generated chiral iridium-π-allyl complex and carboxylic acids to provide rapid access to valuable alkenyl diols in high yields. The synthetic utility of this method is demonstrated by the elaboration of the products into various medium and large ring-sized compounds that are part of biologically relevant molecules.

Abrogation of Rb Tumor Suppression Initiates GBM in Differentiated Astrocytes by Driving a Progenitor Cell Program.

Glioblastoma (GBM) remains lethal with no effective treatments. Despite the comprehensive identification of commonly perturbed molecular pathways, little is known about the disease's etiology, particularly in early stages. Several studies indicate that GBM is initiated in neural progenitor and/or stem cells. Here, we report that differentiated astrocytes are susceptible to GBM development when initiated by perturbation of the RB pathway, which induces a progenitor phenotype. In vitro and in vivo inactivation of Rb tumor suppression (TS) induces cortical astrocytes to proliferate rapidly, express progenitor markers, repress differentiation markers, and form self-renewing neurospheres that are susceptible to multi-lineage differentiation. This phenotype is sufficient to cause grade II astrocytomas which stochastically progress to GBM. Together with previous findings, these results demonstrate that cell susceptibility to GBM depends on the initiating driver.

Development and Characterization of an HCMV Multi-Antigen Therapeutic Vaccine for Glioblastoma Using the UNITE Platform.

Glioblastoma multiforme (GBM) is an aggressive form of brain cancer with a median survival of 15 months that has remained unchanged despite advances in the standard of care. GBM cells express human cytomegalovirus (HCMV) proteins, providing a unique opportunity for targeted therapy. We utilized our UNITE (UNiversal Intracellular Targeted Expression) platform to develop a multi-antigen DNA vaccine (ITI-1001) that codes for the HCMV proteins pp65, gB, and IE-1. The UNITE platform involves lysosomal targeting technology, fusing lysosome-associated membrane protein 1 (LAMP1) with target ntigens. We demonstrate evidence of increased antigen presentation by both MHC-I and -II, delivering a robust antigen-specific CD4 and CD8 T-cell response in addition to a strong humoral response. Using a syngeneic orthotopic GBM mouse model, therapeutic treatment with the ITI-1001 vaccine resulted in ~56% survival of tumor-bearing mice. Investigation of the tumor microenvironment showed significant CD4 infiltration as well as enhanced Th1 and cytotoxic CD8 T activation. Regulatory T cells were also upregulated after ITI-1001 vaccination. In addition, tumor burden negatively correlated with activated interferon (IFN)γ+ CD4 T cells, reiterating the importance of CD4 activation in ITI-1001 efficacy and in identifying treatment responders and non-responders. Further characterization of these two groups showed high infiltration of CD3+, CD4+, and CD8+ T cells in responders compared to non-responders. Thus, we show that vaccination with HCMV antigens using the ITI-1001-UNITE platform generates strong cellular and humoral immune responses, triggering significant antitumor activity, leading to enhanced survival in a mouse model of GBM.

Novel siRNA formulation to effectively knockdown mutant p53 in osteosarcoma.

OBJECTIVES: The tumor suppressor p53 plays a crucial role in the development of osteosarcoma. The primary objective of this study is to develop and optimize lipid based nanoparticle formulations that can carry siRNA and effectively silence mutant p53 in 318-1, a murine osteosarcoma cell line. METHODS: The nanoparticles were composed of a mixture of two lipids (cholesterol and DOTAP) and either PLGA or PLGA-PEG and prepared by using an EmulsiFlex-B3 high pressure homogenizer. A series of studies that include using different nanoparticles, different amount of siRNAs, cell numbers, incubation time, transfection media volume, and storage temperature was performed to optimize the gene silencing efficiency. KEY FINDINGS: Replacement of lipids by PLGA or PLGA-PEG decreased the particle size and overall cytotoxicity. Among all lipid-polymer nanoformulations, nanoparticles with 10% PLGA showed highest mutant p53 knockdown efficiency while maintaining higher cell viability when a nanoparticle to siRNA ratio equal to 6.8:0.66 and 75 nM siRNA was used. With long term storage the mutant p53 knockdown efficiency decreased to a greater extent. CONCLUSIONS: This study warrants a future evaluation of this formulation for gene silencing efficiency of mutant p53 in tissue culture and animal models for the treatment of osteosarcoma.

Synergistic targeted inhibition of MEK and dual PI3K/mTOR diminishes viability and inhibits tumor growth of canine melanoma underscoring its utility as a preclinical model for human mucosal melanoma.

Human mucosal melanoma (MM), an uncommon, aggressive and diverse subtype, shares characteristics with spontaneous MM in dogs. Although BRAF and N-RAS mutations are uncommon in MM in both species, the majority of human and canine MM evaluated exhibited RAS/ERK and/or PI3K/mTOR signaling pathway activation. Canine MM cell lines, with varying ERK and AKT/mTOR activation levels reflective of naturally occurring differences in dogs, were sensitive to the MEK inhibitor GSK1120212 and dual PI3K/mTOR inhibitor NVP-BEZ235. The two-drug combination synergistically decreased cell survival in association with caspase 3/7 activation, as well as altered expression of cell cycle regulatory proteins and Bcl-2 family proteins. In combination, the two drugs targeted their respective signaling pathways, potentiating reduction of pathway mediators p-ERK, p-AKT, p-S6, and 4E-BP1 in vitro, and in association with significantly inhibited solid tumor growth in MM xenografts in mice. These findings provide evidence of synergistic therapeutic efficacy when simultaneously targeting multiple mediators in melanoma with Ras/ERK and PI3K/mTOR pathway activation.

Allele-specific silencing of mutant p53 attenuates dominant-negative and gain-of-function activities.

Many p53 hotspot mutants not only lose the transcriptional activity, but also show dominant-negative (DN) and oncogenic gain-of-function (GOF) activities. Increasing evidence indicates that knockdown of mutant p53 (mutp53) in cancer cells reduces their aggressive properties, suggesting that survival and proliferation of cancer cells are, at least partially, dependent on the presence of mutp53. However, these p53 siRNAs can downregulate both wild-type p53 (wtp53) and mutp53, which limits their therapeutic applications. In order to specifically deplete mutp53, we have developed allele-specific siRNAs against p53 hotspot mutants and validated their biological effects in the absence or presence of wtp53. First, the mutp53-specific siRNAs selectively reduced protein levels of matched p53 mutants with minimal reduction in wtp53 levels. Second, downregulation of mutp53 in cancer cells expressing a mutp53 alone (p53mut) resulted in significantly decreased cell proliferation and migration. Third, transfection of mutp53-specific siRNAs in cancer cells expressing both wtp53 and mutp53 also reduced cell proliferation and migration with increased transcripts of p53 downstream target genes, which became further profound when cells were treated with an MDM2 inhibitor Nutlin-3a or a chemotherapeutic agent doxorubicin. These results indicate that depletion of mutp53 by its specific siRNA restored endogenous wtp53 activity in cells expressing both wtp53 and mutp53. This is the first study demonstrating biological effects and therapeutic potential of allele-specific silencing of mutp53 by mutp53-specific siRNAs in cancer cells expressing both wtp53 and mutp53, thus providing a novel strategy towards targeted cancer therapies.

αB-crystallin, a small heat shock protein, modulates NF-κB activity in a phosphorylation-dependent manner and protects muscle myoblasts from TNF-α induced cytotoxicity.

αB-crystallin, a member of the small heat shock protein family, has been implicated in various biological functions including response to heat shock, differentiation and apoptosis, the mechanisms of which have not been well understood. Myoblasts, the precursor cells in muscle regeneration, when subjected to growth factor deprivation differentiate to form myotubes or undergo apoptosis. During differentiation, myoblasts express elevated levels of αB-crystallin as well as TNF-α but the connecting link between these proteins in cell signaling is not clearly understood. We have therefore investigated the role of αB-crystallin in TNF-α induced regulation of NF-κB. We demonstrate that in response to TNF-α treatment, αB-crystallin associates with IKKß and activate its kinase activity, facilitating the degradation of phosphorylated I-kBα, a prime step in NF-κB activation. Reducing the level of αB-crystallin using the RNAi approach reduces the translocation of p65, further confirming the role of αB-crystallin in NF-κB activation. Our study shows that the ability of αB-crystallin to activate NF-κB depends on its phosphorylation status. The present study shows that αB-crystallin-dependent NF-κB activation protects myoblasts from TNF-α induced cytoxicity by enhancing the expression of the anti-apoptotic protein, Bcl 2. Thus, our study identifies yet another mechanism by which αB-crystallin exerts its anti-apoptotic activity.

Metastatic potential of tumor-initiating cells in solid tumors.

The lethality of cancer is mainly caused by its properties of metastasis, drug resistance, and subsequent recurrence. Understanding the mechanisms governing these properties and developing novel strategies to overcome them will greatly improve the survival of cancer patients. Recent findings suggest that tumors are comprised of heterogeneous cell populations, and only a small fraction of these are tumorigenic with the ability to self-renew and produce phenotypically diverse tumor cell populations. Cells in this fraction are called tumor-initiating cells (TICs) or cancer stem cells (CSCs). TICs have been identified from many types of cancer. They share several similarities with normal adult stem cells including sphere-forming ability, self-renewability, and expression of stem cell surface markers and transcription factors. TICs have also been proposed to be responsible for cancer metastasis, however, scarce evidence for their metastatic potential has been provided. In this review article, we have attempted to summarize the studies which have examined the metastatic potential of TICs in solid tumors.

CD117 and Stro-1 identify osteosarcoma tumor-initiating cells associated with metastasis and drug resistance.

Emerging evidence indicates the presence of tumor-initiating cells (TIC) or cancer stem cells in osteosarcoma. However, no study has shown specific markers to identify osteosarcoma TICs with in vivo tumor formation ability. Additionally, there has been a lack of investigations gauging the contribution of osteosarcoma TICs to metastatic and drug-resistant properties. In this study, we have identified mouse and human osteosarcoma TICs using mesenchymal stem cell markers CD117 and Stro-1. These markers were preferentially expressed in spheres and doxorubicin-resistant cells. Both mouse and human cells expressing these markers were sorted and analyzed for their abilities of tumor formation with as few as 200 cells, self-renewability, multipotency, drug resistance, metastatic potential, and enrichment of a metastasis-associated marker (CXCR4) and a drug resistance marker (ABCG2). CD117(+)Stro-1(+) cells efficiently formed serially transplantable tumors, whereas CD117(-)Stro-1(-) cells rarely initiated tumors. On orthotopic injections, CD117(+)Stro-1(+ )cell-derived tumors metastasized at a high frequency. Further, CD117(+)Stro-1(+) cells showed high invasive and drug-resistant properties and were efficiently enriched for CXCR4 (20-90%) and ABCG2 (60-90%). These results suggest possible mechanisms for the high metastatic and drug-resistant properties of osteosarcoma TICs. In summary, CD117 and Stro-1 identify osteosarcoma TICs associated with the most lethal characteristics of the disease-metastasis and drug resistance-and these markers offer candidates for TIC-targeted drug delivery aimed at eradicating osteosarcoma.

Mutant p53 gain of oncogenic function: in vivo evidence, mechanism of action and its clinical implications.

p53 is an indispensible tumor suppressor and exerts this function by transactivating numerous downstream target genes that play vital roles in controlling cell proliferation, apoptosis, senescence, and DNA repair. Mutations in the p53 gene, which are frequently seen in human tumors, impair its tumor suppressor function. Several of these tumor-derived p53 mutants can confer further aggressive oncogenic properties such as exacerbated malignant transformation and metastatic phenotype when overexpressed in p53-null cells. This oncogene-like behavior of mutant p53 is referred to as gain of function. The exact mechanism underlying gain-of-function phenotypes, however, remains enigmatic. Recently, we have generated mice with a point mutation (p53(R172H)) in their endogenous p53 loci as a model for the human Li-Fraumeni syndrome. The mutant p53(R172H) knock-in mice spontaneously develop tumors with high frequency of metastasis, contrary to that observed in mice with p53 deletion, indicating gain of function by the mutant p53R172H. In addition, our results show that other p53 family members, p63 and p73, are involved in the gain-of-function phenotypes. We further demonstrate that mutant p53(R172H) is inherently unstable and its stabilization is required for its gain-of-function phenotypes. This review focuses on recent reports regarding the potential molecular pathways for mutant p53 gain of oncogenic function and discusses its clinical implications.

Heat stress-induced localization of small heat shock proteins in mouse myoblasts: intranuclear lamin A/C speckles as target for alphaB-crystallin and Hsp25.

We examined the effect of heat stress on localization of two sHsps, alphaB-crystallin and Hsp25, and of Hsc70, a member of a different class of heat shock proteins (Hsps), in both undifferentiated and differentiated mouse C2C12 cells. Under normal conditions, alphaB-crystallin and Hsp25 are found in the cytoplasm; only alphaB-crystallin is also found in the nucleus, distributed in a speckled pattern. Hsc70 is found to be homogeneously distributed throughout the cell. On heat stress, all these proteins translocate almost entirely into the nucleus and upon recovery relocate to the cytoplasm. Dual staining experiments using C2C12 myoblasts show that alphaB-crystallin and Hsp25, but not Hsc70, colocalize with the intranuclear lamin A/C and the splicing factor SC-35, suggesting interactions of sHsps and intranuclear lamin A/C. Interestingly, none of these proteins are found in the myotube nuclei. Upon heat stress, only Hsc70 translocates into the myotube nuclei. This differential entry of alphaB-crystallin and Hsp25 into the nuclei of myoblasts and myotubes upon heat stress may have functional role in the development and/or in the maintenance of muscle cells. Our study therefore suggests that these sHsps may be a part of the intranuclear lamin A/C network or stabilizing this specific network.