Cystic echinococcosis (CE) is a prevalent zoonotic disease caused by Echinococcus granulosus, with cosmopolitan distribution. The parasite is transmitted cyclically between canines and numerous intermediate herbivorous livestock animals. Also other taeniid tapeworm could infect domestic dogs and they pose significant veterinary and public health concerns worldwide. This study aimed to develop a sensitive molecular method for detecting Echinococcus spp. DNA in dog fecal samples using next-generation sequencing (NGS). A set of PCR primers targeting conserved regions of Taeniid tapeworms’ 18s rRNA genes was designed and tested for amplifying genomic DNA from various tapeworm species. The PCR system demonstrated high sensitivity, amplifying DNA from all tested tapeworm species, with differences observed in amplified band sizes. The primers were adapted for NGS analysis by adding forward and reverse adapters, enabling sequencing of amplified DNA fragments. Application of the developed PCR system to dog fecal samples collected from Yatta town, Palestine, revealed the presence of E. granulosus DNA in five out of 50 samples. NGS analysis confirmed the specificity of the amplified DNA fragments, showing 98-99% similarity with the 18s rDNA gene of E. granulosus . This study demonstrates the utility of NGS-based molecular methods for accurate and sensitive detection of Echinococcus spp. in dog fecal samples, providing valuable insights for epidemiological surveillance and control programs of echinococcosis in endemic regions.Author Summary Cystic echinococcosis, or hydatidosis, is a serious and chronic zoonotic disease in humans caused by the dog tapeworm Echinococcus granulosus . The disease is transmitted cyclically between canines and numerous herbivorous livestock animals. Determining E. granulosus infection in dogs is crucial for assessing infection risk and identifying new foci of active infections. The infection rate in dogs is also necessary for evaluating transmission dynamics and assessing the efficacy of control programs. In this study, we present a PCR system based on amplification of the 18S rDNA. New primers were designed following an alignment of various taeniid tapeworms’ 18S rDNA sequences. The current PCR system was adapted to be used in amplicon sequencing utilizing next-generation sequencing technology. This strategy enables accurate detection of tapeworm DNA extracted from dogs’ fecal samples and provides quantitative measurement of taeniid infection in dogs.### Competing Interest StatementThe authors have declared no competing interest.

Increasing evidence has indicated that the non-coding RNA molecules play central roles in almost all biological processes and many pathological conditions including carcinogenesis. This review focuses on the pathological tumorigenic role of the first discovered long non-coding RNA gene called H19 and its pivotal contribution to the cancer axis of evil. H19 RNA utilizes a variety of mechanisms to perform its pathological function. Some key unanswered questions are presented by the end. Understanding the H19 RNA mechanisms of action will shed light into the class of long non-coding RNA which contains thousands of members mostly with unknown function and will help in delineating the pathological role played by at least some of them.

To date, classical deterministic Newtonian physics has been used by biologists to describe living processes. However, it is increasingly appreciated that the probabilistic view offered by quantum mechanics more accurately describes the behavior of atoms and materials in all systems. Here, we discuss how the concepts of quantum mechanics can be applied to biological processes involved in cancer. We present a concise summary inspired by Heisenberg's Uncertainty Principle to describe our «Genetic Environmental Field Hypothesis». Combining the uncertainties of genetic changes as expressed by epigenetic changes and/or somatic mutations with the uncertainties of environmental changes, cells may become cancerous as a way to increase entropy. Throughout the paper we will utilize the H19 gene system as an example. Using the concepts of quantum mechanics to describe oncological processes may provide novel directions in our understanding of cancer.

Context: Urothelial carcinoma (UC) is common and highly recurrent. Diagnosis and follow-up involve invasive cystoscopies. Objective: To evaluate H19 RNA in urine cells as diagnostic tool for UC. Materials and methods: RT-PCR analysis of urine samples from healthy volunteers and UC patients. Results: H19 RNA was unequivocally detected in the urine of 90.5% of patients and 25.9% of controls. H19 copies were three orders of magnitude higher in patients. Receiver operating characteristic analysis showed an area under the curve of 0.933. Conclusions: This pilot study shows that urinary cell H19 is a highly sensitive test for UC and pending verification could transform patient management.

Long non-coding RNA (lncRNA) genes are emerging as key players in the metastatic cascade. Current evidence indicate that H19 lncRNA and the microRNA(miRNA) miR-675, which is processed from it, play crucial roles in metastasis, through the regulation of critical events specifically the epithelial to mesenchymal (EMT) and the mesenchymal to epithelial transitions (MET). This review summarizes recent mechanistic pathways and tries to put together seemingly conflicting data from different reports under one proposed general scheme underlying the various roles of H19/miR-675 in the metastatic cascade. We propose several approaches to harnessing this knowledge for translational medicine.

The imprinted oncofetal long non-coding RNA (lncRNA) H19 is expressed in the embryo, down-regulated at birth and then reappears in tumors. Its role in tumor initiation and progression has long been a subject of controversy, although accumulating data suggest that H19 is one of the major genes in cancer. It is actively involved in all stages of tumorigenesis and is expressed in almost every human cancer. In this review we delineate the various functions of H19 during the different stages in the complex process of tumor progression. H19 up-regulation allows cells to enter a "selfish" survival mode in response to stress conditions, such as destabilization of the genome and hypoxia, by accelerating their proliferation rate and increasing overall cellular resistance to stress. This response is tightly correlated with nullification, dysfunction or significant down-regulation of the master tumor suppressor gene P53. The growing evidence of H19's involvement in both proliferation and differentiation processes, together with its involvement in epithelial to mesenchymal transition (EMT) and also mesenchymal to epithelial transition (MET), has led us to conclude that some of the recent disputes and discrepancies arising from current research findings can be resolved from a viewpoint supporting the oncogenic properties of H19. According to a holistic approach, the versatile, seemingly contradictory functions of H19 are essential to, and differentially harnessed by, the tumor cell depending on its context within the process of tumor progression.

Since it was first described, the imprinted cluster 11p15.5 has been reported to be deregulated in a variety of pediatric and adult cancers including that of the lung. Both protein coding and non-coding genes functioning as oncogenes or as tumor suppressor genes reside within this cluster. Oncomirs that can function as oncogenes or as tumor suppressors have also been reported. While a complete account of the role played by the 11p15.5 imprinted cluster in lung cancer is beyond the scope of this review, we will focus on the role of the non-coding RNAs processed from the H19-IGF2 loci. A special emphasis will be given to the H19/miR-675 gene locus. Their potential diagnostic and therapeutic use in lung cancer will be described.

Concern over food authenticity has increased as a result of an increase in the consumption of processed foods containing meat or animal products. This raises a number of issues where the presence of pork in such foods is considered unacceptable in most Muslim and Jewish communities around the world. It also applied to the prohibition of beef consumption among Hindus. In order to ensure the absence of unwished meat products or mixing of meats from different sources in processed foods, a specific and sensitive test is essential. For this purpose we developed a molecular test based on DNA amplification by polymerase chain reaction (PCR) of the cytochrome b gene followed by reverse line blot analysis (RLB). Using this method many samples may be treated simultaneously and meat origins can easily be detected from processed foods or foods containing mixed meat sources; also, added pork components such as fat may be identified by this methodology. The PCR/RLB method is considered to be a sensitive and specific technique; it can detect one nucleotide change within the PCR-amplified DNA segment.

Specific targeting of cancer cells while sparing normal cells with therapeutic mediators is a critical advantage over the current conventional drugs. Although various strategies can potentially be followed to achieve this vital goal, this review focuses on the strategy of transcriptional targeting of tumor cells by employing a toxin gene. Tumor-specific regulatory sequences can be used for this purpose to drive the expression of a toxin specifically in tumor cells. It is of immense importance to choose regulatory sequences of a gene that is playing critical roles in various aspects of tumorigenesis to increase the therapeutic window. It would be beneficial to identify a strong cancer-specific promoter for treating a wide range of cancers while having minimal toxicity. In light of these prerequisites, we present our experience with the H19 gene and the successful use of its regulatory sequence to drive the expression of diphtheria toxin A in both preclinical and clinical studies.

The oncofetal H19 gene transcribes a long non-coding RNA(lncRNA) that is essential for tumor growth. Here we found that numerous established inducers of epithelial to mesenchymal transition(EMT) also induced H19/miR-675 expression. Both TGF-β and hypoxia concomitantly induced H19 and miR-675 with the induction of EMT markers. We identified the PI3K/AKT pathway mediating the inductions of Slug, H19 RNA and miR-675 in response to TGF-β treatment, while Slug induction depended on H19 RNA. In the EMT induced multidrug resistance model, H19 level was also induced. In a mouse breast cancer model, H19 expression was tightly correlated with metastatic potential. In patients, we detected high H19 expression in all common metastatic sites tested, regardless of tumor primary origin. H19 RNA suppressed the expression of E-cadherin protein. H19 up-regulated Slug expression concomitant with the suppression of E-cadherin protein through a mechanism that involved miR-675. Slug also up-regulated H19 expression and activated its promoter. Altogether, these results may support the existence of a positive feedback loop between Slug and H19/miR-675, that regulates E-cadherin expression. H19 RNA enhanced the invasive potential of cancer cells in vitro and enhanced tumor metastasis in vivo. Additionally, H19 knockdown attenuated the scattering and tumorigenic effects of HGF/SF. Our results present novel mechanistic insights into a critical role for H19 RNA in tumor progression and indicate a previously unknown link between H19/miR-675, Slug and E-cadherin in the regulation of cancer cell EMT programs.

Specific targeting of cancer cells while sparing normal cells with therapeutic mediators is a critical advantage over the current conventional drugs. Although various strategies can potentially be followed to achieve this vital goal, this review focuses on the strategy of transcriptional targeting of tumor cells by employing a toxin gene. Tumor-specific regulatory sequences can be used for this purpose to drive the expression of a toxin specifically in tumor cells. It is of immense importance to choose regulatory sequences of a gene that is playing critical roles in various aspects of tumorigenesis to increase the therapeutic window. It would be beneficial to identify a strong cancer-specific promoter for treating a wide range of cancers while having minimal toxicity. In light of these prerequisites, we present our experience with the H19 gene and the successful use of its regulatory sequence to drive the expression of diphtheria toxin A in both preclinical and clinical studies.

The field of the long non-coding RNA (lncRNA) is advancing rapidly. Currently, it is one of the most popular fields in the biological and medical sciences. It is becoming increasingly obvious that the majority of the human transcriptome has little or no-protein coding capacity. Historically, H19 was the first imprinted non-coding RNA (ncRNA) transcript identified, and the H19/IGF2 locus has served as a paradigm for the study of genomic imprinting since its discovery. In recent years, we have extensively investigated the expression of the H19 gene in a number of human cancers and explored the role of H19 RNA in tumor development. Here, we discuss recently published data from our group and others that provide further support for a central role of H19 RNA in the process of tumorigenesis. Furthermore, we focus on major transcriptional modulators of the H19 gene and discuss them in the context of the tumor-promoting activity of the H19 RNA. Based on the pivotal role of the H19 gene in human cancers, we have developed a DNA-based therapeutic approach for the treatment of cancers that have upregulated levels of H19 expression. This approach uses a diphtheria toxin A (DTA) protein expressed under the regulation of the H19 promoter to treat tumors with significant expression of H19 RNA. In this review, we discuss the treatment of four cancer indications in human subjects using this approach, which is currently under development. This represents perhaps one of the very few examples of an existing DNA-based therapy centered on an lncRNA system. Apart from cancer, H19 expression has been reported also in other conditions, syndromes and diseases, where deregulated imprinting at the H19 locus was obvious in some cases and will be summarized below. Moreover, the H19 locus proved to be much more complicated than initially thought. It houses a genomic sequence that can transcribe, yielding various transcriptional outputs, both in sense and antisense directions. The major transcriptional outputs of the H19 locus are presented here.

Mitochondria are emerging as important players in the transformation process of cells, maintaining the biosynthetic and energetic capacities of cancer cells and serving as one of the primary sites of apoptosis and autophagy regulation. Although several avenues of cancer therapy have focused on mitochondria, progress in developing mitochondria-targeting anticancer drugs nonetheless has been slow, owing to the limited number of known mitochondrial target proteins that link metabolism with autophagy or cell death. Recent studies have demonstrated that two members of the newly discovered family of NEET proteins, NAF-1 (CISD2) and mitoNEET (mNT; CISD1), could play such a role in cancer cells. NAF-1 was shown to be a key player in regulating autophagy, and mNT was proposed to mediate iron and reactive oxygen homeostasis in mitochondria. Here we show that the protein levels of NAF-1 and mNT are elevated in human epithelial breast cancer cells, and that suppressing the level of these proteins using shRNA results in significantly reduced cell proliferation and tumor growth, decreased mitochondrial performance, uncontrolled accumulation of iron and reactive oxygen in mitochondria, and activation of autophagy. Our findings highlight NEET proteins as promising mitochondrial targets for cancer therapy.

Pancreatic cancer is the eighth cancer leading cause of cancer-related death in the world and has a 5-year survival rate of 1–4% only. Gemcitabine is a first line agent for advanced pancreatic therapy; however, its efficacy is limited by its poor intracellular metabolism and chemoresistance. Studies have been conducted in an effort to improve gemcitabine treatment results by adding other chemotherapeutic agents, but none of them showed any significant advantage over gemcitabine monotherapy. We found that 85% of human pancreatic tumors analyzed by in situ hybridization analyses showed moderated to strong expression of the H19 gene. We designed a preclinical study combining gemcitabine treatment and a DNA-based therapy for pancreatic cancer using a non viral vector BC-819 (also known as DTA-H19), expressing the diphtheria toxin A chain under the control of the H19 gene regulatory sequences. The experiments conducted either in an orthotopic and heterotopic pancreatic carcinoma animal model showed better antitumor activity following the sequential administration of the vector BC-819 and gemcitabine as compared to the effect of each of them alone. The results presented in the current study indicate that treatment with BC-819 in combination with gemcitabine might be a viable new therapeutic option for patients with advanced pancreatic cancer.

The purpose of this review is to demonstrate a successful use of preclinical models of bladder cancer to confirm the therapeutic potential of new promising drug candidates. The bladder has long been thought to be an ideal target for investigating therapies. When developing a new antineoplastic pharmaceutical agent, the bladder should be considered for use as an experimental model demonstrating initial proof of concept that if successful can be later assessed in further cancer indications. Non-muscle-invasive bladder carcinoma can be removed by transurethral resection but these cancers tend to recur in most patients. Conventional treatments decrease the recurrence rate but are associated with side effects and frequent failures. Thus, there is an obvious need for the development of highly effective targeted therapies with limited side effects. Accordingly, a double-promoter vector was developed, expressing diphtheria toxin A (DTA) under control of two different regulatory promoter sequences, H19 and IGF2. This vector was then used to transfect and to eradicate tumor cells in bladder cancer models, effectively destroying tumor cells without affecting normal cells. Our studies demonstrate the potential efficacy of the therapeutic vector and should be a solid base for future clinical studies. These models illuminate the path for future investigations of new drug candidates for bladder cancer.

The H19-IGF2 locus within the imprinted cluster of the human chromosome 11, has been implicated in a variety of disorders and cancer pre-disposition including bladder cancer. BBN induced bladder cancer model in rats has identified both H19 and IGF2 among differentially expressed genes that are induced in response to carcinogen exposure. In this chapter, the role of both H19 and IGF2 genes in cancer will be handled in general with special focus on bladder cancer. Although IGF2 role in human cancers is relatively well established, recent data from our laboratory and others have just revealed a critical role for H19 RNA in the process of tumorigenicity including that of the bladder. H19 functions as a stress modulator, being induced by hypoxia, and a survival factor that is involved in several fundamental processes of tumorigenesis. Furthermore, we uncovered a molecular mechanism that integrates H19, p53 and HIF1-┙ to hypoxic stress response. Placing the H19 gene product in this deadly circuit undoubtedly will have major impacts in its utility as a target for cancer gene therapy. Regulatory sequences of both H19 and IGF2 have already been used to successfully target expression of a toxic protein, diphtheria toxin A (DT-A), in carcinoma cells in culture, in several xenograft, orthotopic animal models, and in chemically induced BBN model of bladder cancer. In case of H19, it is successfully used in patients with bladder carcinoma for a period of over 5 years and recently a clinical trial phase I/IIa using this therapeutic approach has been successfully completed. It is also successfully used in other types of human cancers but will not be handled in the current chapter. We will discuss also novel approaches, to create a new family of plasmids. In one approach a cytotoxic gene is driven by two different regulatory sequences, selected from the cancerspecific promoters H19, IGF2-P3 and IGF2-P4 carried on a single construct. In a second

BACKGROUND:The H19-IGF2 locus is either highly expressed and/or shows aberrant allelic pattern of expression in a large array of human cancers, while rarely expressed in the corresponding normal tissue. Preclinical, clinical studies and human compassionate using a DNA plasmid containing H19 and/or IGF2-P4 regulatory sequences that drive the expression of an intracellular toxin [diphtheria toxin A-fragment (DTA)] have demonstrated promising results in several types of carcinomas. Recently we reported that a single construct that expresses DTA under the control of both H19 and IGF2 P4 promoters showed superior efficacy in vitro as well as in vivo, in comparison to a single promoter construct in bladder carcinoma. Here we extended this approach to glioblastoma and tested the antitumor efficacy of the double promoter DTA-expressing vector (H19-DTA-P4-DTA) in vitro as well as in heterotopic animal model. H19 gene expression was tested by in-situ hybridization (ISH) and by quantitative Real-Time PCR (qRT-PCR) in samples of diffuse glioma.METHODS:IGF2-P4 gene expression was tested by qRT-PCR as well.RESULTS:Both H19 and IGF2-P4 transcripts were highly expressed in high grade gliomas. Furthermore, significant H19 expression in other types of primary brain tumors as well as in brain metastases was detected by ISH. Both A172 and U87 human glioblastoma cell lines showed high expression of IGF2-P4 while the A172 cell line showed high expression of H19 RNA as well. H19-DTA-P4-DTA exhibited superior cytotoxic activity compared to the single promoter expression vectors, in U87 and A172 glioblastoma cell lines in vitro and showed antitumoral efficacy in heterotopic glioblastoma animal model.CONCLUSIONS:Our findings indicate antitumoral efficacy against glioblastoma of the targeted double promoter vector H19-DTA-P4-DTA, both in-vitro and in-vivo. Thus, its test in orthotopic animal model of glioblastoma as well as in clinical trials is warranted.

Curative surgery is possible in barely 10% of patients with colorectal liver metastases and combined treatment modalities scarcely improve survival in this group of patients. Hence, investigations of new therapeutic modalities are crucial. Overexpression of the H19 gene in liver metastases points to H19 as a target for cancer gene therapy. Here we have evaluated the possibility of regional intra-arterial treatment of liver meta-stases with the DTA-H19 plasmid. Intra-arterial treatment of a total dose of 2.5 mg (repeated injections of 500 µg DTA-H19 plasmid each dose after the first injection of 1000 µg) caused a significant delay in the tumor growth compared to control group. All of the tumors treated with the control vector increased in size, whereas 35.7% of the tumors in the groups treated with a total amount of 2.5 mg DTA-H19 plasmid shrank in size. The present study showed that the DTA-H19 plasmid administered intra-arterially significantly delayed the tumor growth and even resulted in tumor regression in high percentage of the treated animals with liver metastases of colon cancer. Since human liver metastases demonstrated overexpression of the H19 gene, regional administration of the plasmid seems to be a promising therapeutic approach.

H19 is an imprinted non-coding RNA gene that encodes for perhaps one of the most abundant mRNA-like transcript in fetal life expressed predominantly in tissues of mesodermal and endodermal origin and down regulated at birth. It is located within the imprinted cluster on chromosome 11p15.5 in human and shares common regulatory sequences with other genes within the cluster, including insulin-like growth factor-2 (IGF2). H19 gene is either highly expressed and/or shows aberrant allelic pattern of expression in a large array of human cancers, while not expressed in the corresponding normal tissues. It possesses diagnostic, prognostic and therapeutic values in many cancers and its essential role in tumorigenesis has been demonstrated by using RNA interference and knockdown technologies. Modulation of the imprinting status of H 19 and IGF2 may play an important role in the development of brain tumors including meningiomas, medulloblastomas, and gliomas. High levels of H19 expression have been shown by utilizing gene array in glioblastoma-derived cell lines, which are related to CD133 positive cells. This might be related to the modulation of H19 by hypoxia through upregulation of its non-coding RNA. There appears to be a crucial link between the well-established tumor suppressor p53 gene and the oncogenic H19 RNA that could determine vital processes in the tumorigenic pathway including response to hypoxic stress. The expression of H 19 in a large array of human tumors and at different stages of tumor development, its hypoxic induction and its tight association with mutant form of p53, make it an ideal new target for cancer therapy, including low grade astrocytomas, which show frequent p53 mutations, and glioblastomas, which show unique hypoxia-driven angiogenesis (microvascular proliferation) and necrosis (pseudopalisading necrosis). Clinical studies and human compassionate use treatments using a DNA plasmid containing H19 gene regulatory sequences that drive the expression of an intracellular toxin [diphtheria toxin A (DTA) chain] and preclinical studies with siRNAs specifically targeting the H19 RNA have demonstrated promising results in several types of carcinomas. In preliminary studies, we have found expression of H 19 and IGF2 by RT-PCR in several glioma cell lines and also significant expression of H19 by in situ hybridization performed on paraffin sections from several brain tumors including high-grade astrocytic neoplasms. In some of the glioblastomas, expression of H19 was observed in neoplastic cells as well as in proliferating microvasculture. The A 172 human astrocytoma cell line has shown high expression of H19, IGF2-P3 and IGF2-P4 by using semiquantitative RT-PCR. Significant reduction in the number of cells was observed after transfection with a double promoter expressing vector, carrying two separate genes expressing DTA on a single construct, from two different regulatory sequences, namely H19-DTA-P4-DTA. In conclusion, H19-DTA-P4-DTA plasmid, which is based upon the toxic efficacy of diphtheria-toxin and H19-siRNA, which appears to be a promising therapeutic agent, are putative therapeutic candidates for treating brain tumors either with intra-tumoral convection-enhanced delivery or with better tolerated and more efficient modes of delivery yet to be developed.

Expression of the imprinted H19 gene is remarkably elevated in a large number of human cancers. Recently, we reported that H19 RNA is up-regulated in hypoxic stress and furthermore, it possesses oncogenic properties. However, the underlying mechanism(s) of these phenomena remain(s) unknown. Here we demonstrate a tight correlation between H19 RNA elevation by hypoxia and the status of the p53 tumor suppressor. Wild type p53 (p53wt) prevents the induction of H19 upon hypoxia, and upon its reconstitution in p53null cells. The last case is accompanied by a decrease in cell viability. The p53 effect is nuclear and seems independent of its tetramerization. Furthermore, using knockdown and over-expression approaches we identified HIF1-α as a critical factor that is responsible for H19 induction upon hypoxia. Knocking down HIF1-α abolishes H19 RNA induction, while its over-expression significantly enhances the H19 elevation in p53null hypoxic cells. In p53wt hypoxic cells simultaneous suppression of p53 and over-expression of HIF1-α are needed to induce H19 significantly, while each treatment separately resulting in a mild induction, indicating that the molecular mechanism of p53 suppression effect on H19 may at least in part involve interfering with HIF1-α activity. In vivo a significant increase in H19 expression occurred in tumors derived from p53null cells but not in p53wt cells. Taken together, our results indicate that a functional link exists between p53, HIF1-α and H19 that determines H19 elevation in hypoxic cancer cells. We suggest that this linkage plays a role in tumor development.