Two new earthworm species of the genus Amynthas Kinberg, 1867 (Annelida, Oligochaeta, Megascolecidae) from the Southeast region, Vietnam.

Two new earthworm species, namely Amynthas rarus sp. nov. and Amynthas tayninhensis sp. nov., are described from Tay Ninh Province, southeastern Vietnam. Amynthas rarus sp. nov. is recognized by having male pores in xix, two pairs of spermathecal pores in 6/7/8, and genital markings paired in vii, viii, 18/19, and 19/20. Amynthas tayninhensis sp. nov. is distinguished by having a pair of spermathecal pores in 5/6, numerous genital markings in transverse lines in 17/18 including three surrounding male pores, and spermatheca with fully developed diverticula. The COI fragments are also provided for those two new species.

A revision of the Metaphire peguana species-group (Oligochaeta: Megascolecidae) from Vietnam.

The Metaphire peguana species-group was revised from the earthworm fauna of Vietnam. As a result, a total of seven species/subspecies have been assigned to this species-gro+up, namely M. peguana peguana (Rosa, 1890), M. peguana laisonensis Nguyen & Nguyen, 2017, M. bahli (Gates, 1945), M. doiphamon Bantaowong & Panha, 2016, M. dorsomultitheca Nguyen & Nguyen, 2015, M. kiengiangensis Nguyen & Trinh, 2015, and M. nhuongi Nguyen, 2016. The species, M. pacseana (Thai & Samphon, 1988) was removed from the group. The species-group was diagnosed by having spermathecal pores in 6/7/8/9, sometimes 5/6/7/8/9 or 7/8/9; bithecate or polythecate; genital markings paired in 17/18 and 18/19 or in xvii and xix; accessory glands paired, coelomic, strongly covered by muscular-walled bursae; septum 10/11 only present ventrally; and intestinal caeca simple. The K2P genetic distances between the group members varies from 12.9% to 20.7%. The phylogenetic analysis clearly supports the members of this species-group.

Cataracts Across the Tree of Life: A Roadmap for Prevention and Biomedical Innovation.

PURPOSE: Spontaneous cataracts have been identified in the lenses of animals across a phylogenetically wide range of species. This can be a source of insights and innovation for human health professionals, but many persons may lack awareness of it. By providing a phylogenetic survey and analysis of species with cataract vulnerability, this paper demonstrates how a broad comparative perspective can provide critical information about environmental hazards to human visual health and can spark potential innovations in the prevention and treatment of cataracts in humans. DESIGN: Perspectives. METHODS: Review and synthesis of selected literature with interpretation and perspective. RESULTS: We found 273 recorded cases of spontaneously occurring cataracts in 113 species of birds, 83 species of mammals, 30 species of actinopterygii fish, 10 species of amphibians, 6 species of reptiles, and 1 species of cephalopod. CONCLUSION: A phylogenetically wide range of species, including many living in and around human environments, are vulnerable to cataracts. These animals may serve as sentinels for human visual health. Variation in cataract vulnerability across species may also facilitate the identification of resistance-conferring physiologies, leading to accelerated innovation in the prevention and treatment of cataracts in humans.

Two new earthworm species (Annelida, Oligochaeta, Megascolecidae) from the Mekong delta, Vietnam.

Two new earthworm species are described from the Mekong Delta, Southern Vietnam (An Giang and Ben Tre Provinces), namely Amynthas reductus sp. nov. and Metaphire giengensis sp. nov. The former is recognized by having a pair of spermathecal pores in 6/7, numerous genital markings in transverse lines in 17/18, and spermatheca with strongly reduced diverticula. The latter is distinguished by having two pairs of spermathecal pores in 5/6/7, two pairs of genital markings located closely to male pores in xviii, and the presence of septum 8/9. The COI fragments are also provided for Metaphire giengensis sp. nov.

Two new Drawida (Oligochaeta, Moniligastridae) earthworms from Vietnam.

Two new earthworm species are described, namely Drawidaangiang sp. nov. and Drawidacochinchina sp. nov. The former can be recognized by having male pores on spiniform penises in intersegment 10/11, an erect and sac-shaped spermathecal atrium, glandular prostate, the capsule coiled one round, the vas deferens strongly coiled but small, two large, round, genital markings on segments ix-x, and three gizzards in xiii-xv. The latter species is distinguished in having the male pores placed on highly elevated, backwardly directed, conical penises in 10/11, a slender spermathecal atrium, a glandular prostate, a somewhat folded capsule, the vas deferens strongly coiled as a bunch and equal size to the testis sacs, a pair of genital markings located closely anterior to the penises with 1-3 additional ones in xi-xii, and three or four gizzards in xiii-xvi. The DNA barcode fragment of the COI gene was extracted for each species, and the COI genetic distances and phylogenetic analysis also supported two new species..

Utilization of Response Surface Methodology in Optimization of Polysaccharides Extraction from Vietnamese Red Ganoderma lucidum by Ultrasound-Assisted Enzymatic Method and Examination of Bioactivities of the Extract.

Red Ganoderma lucidum (G. lucidum) is a popular medicinal herb commonly used in Vietnamese traditional remedies due to its potential value for health. In this study, polysaccharides were extracted from G. lucidum using ultrasound-assisted enzymatic extraction method. The response surface methodology and Box-Behnken design were employed to investigate the effects of pH, extraction temperature, extraction time, and ultrasonic power on the content of polysaccharides. Based on ultraviolet-visible spectroscopy analysis, the highest content of polysaccharides in the extract was 32.08 mg/g under optimum experimental parameters including enzyme concentration of 3%, pH of 5.5, extraction temperature of 45°C, extraction time of 30 min, and ultrasonic power of 480 W. The Fourier-transform infrared spectroscopy was also used to identify the functional groups in the extracts. The molecular weights of polysaccharides were determined by gel permeation chromatography. The obtained extract was then evaluated for anticancer activities by using (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, showing the anticancer activities with the half-maximal inhibitory concentration value of more than 512 µg/mL. This result suggested that UAEE could be considered as an appropriate and effective extraction method for bioactive crude polysaccharides from G. lucidum.

Four new species of Amynthas earthworms in southeastern Vietnam (Annelida, Oligochaeta, Megascolecidae).

Four new earthworm species of the genus Amynthas Kinberg, 1867 are described from southeastern Vietnam, named A. longiprostaticus sp. nov., A. dorsomorrioides sp. nov., A. minhdam sp. nov., and A. ocularius sp. nov. A. longiprostaticus sp. nov. belongs to the A. minimus group of species, characterized by spermathecal pores in 5/6. It is distinguished by small body size with length 5-7 cm, and by elongate prostate glands, which extend over more than 10 segments. A. dorsomorrioides sp. nov. belongs to the A. morrisi group, characterized by holandry and spermathecal pores in 5/6/7. It is distinguished by a dorsal location of these pores, by one pair of large genital markings presetal in xvii, and by the absence of genital markings in the spermathecal region. A. minhdam sp. nov. also belongs to the A. morrisi group, and is distinguished by a pair of genital markings located medially in xviii and by the spermathecal diverticula, which have a small seminal chamber sub-divided by constrictions into 4 or 5 parts, in chain-form. A. ocularius sp. nov. belongs to the A. gracilis group by virtue of holandry and spermathecal pores in 5/6/7/8. It is distinguished by numerous small genital markings in the male region, postsetal in xviii and presetal in xix, and arranged in a peculiar fashion reminiscent of eyeglasses connected by a bridge. With these descriptions, there are now 119 species of Amynthas known in Vietnam.

The megascolecid earthworms (Annelida, Oligochaeta, Megascolecidae) in the Phu Quoc island, Vietnam, with descriptions of three new species.

The megascolecid earthworms of the Phu Quoc island are intensively investigated. Twelve species in three genera (Lampito Kinberg, 1867, Amynthas Kinberg, 1867, and Metaphire Sims & Easton, 1972) are recorded. Of these, Metaphire doiphamon Bantaowong & Panha, 2016 is recorded for the first time in Vietnam, and three species are newly described, namely Amynthas catenatus sp. nov., A. phuquocensis sp. nov., and A. poropapillatus sp. nov. An identification key to 12 megascolecid species is provided as well.

On the Giant Pheretimoid Earthworms From Vietnam (Clitellata: Megascolecidae), with Descriptions of Three New Species.

Tung T. Nguyen, Dang H. Lam, and Anh D. Nguyen (2016) The giant earthworms from Vietnam are being reviewed to consist of six species, Amynthas dangi (Thai, 1984), A. munglonganus (Thai & Tran, 1986) comb. nov., A. ghilarovi (Thai, 1982), and three new, A. munglongoides sp. nov., A. antoanensis sp. nov. and A. konkakinh sp. nov. All six species have lengths ranging 300-680 mm, diameter ca. 16-20 mm, and without copulatory pouches. The new species, A. munglongoides sp. nov. is characterised by three pairs of spermathecal pores in intersegment 5/6/7/8, genital markings paired in vii, viii and xviii. A. antoanensis sp. nov. is distinguished by four pairs of spermathecal pores in 5/6/7/8/9, genital marking paired in viii, ix, and xviii. A. konkakinh sp. nov. is recognised by four pairs of spermathecal pores in 5/6/7/8/9, genital marking single in viii, paired in xviii. In addition, a key to all six gigantic species in Vietnam is also provided.

A Double-Switch Cell Fusion-Inducible Transgene Expression System for Neural Stem Cell-Based Antiglioma Gene Therapy.

Recent progress in neural stem cell- (NSC-) based tumor-targeted gene therapy showed that NSC vectors expressing an artificially engineered viral fusogenic protein, VSV-G H162R, could cause tumor cell death specifically under acidic tumor microenvironment by syncytia formation; however, the killing efficiency still had much room to improve. In the view that coexpression of another antitumoral gene with VSV-G can augment the bystander effect, a synthetic regulatory system that triggers transgene expression in a cell fusion-inducible manner has been proposed. Here we have developed a double-switch cell fusion-inducible transgene expression system (DoFIT) to drive transgene expression upon VSV-G-mediated NSC-glioma cell fusion. In this binary system, transgene expression is coregulated by a glioma-specific promoter and targeting sequences of a microRNA (miR) that is highly expressed in NSCs but lowly expressed in glioma cells. Thus, transgene expression is "switched off" by the miR in NSC vectors, but after cell fusion with glioma cells, the miR is diluted and loses its suppressive effect. Meanwhile, in the syncytia, transgene expression is "switched on" by the glioma-specific promoter. Our in vitro and in vivo experimental data show that DoFIT successfully abolishes luciferase reporter gene expression in NSC vectors but activates it specifically after VSV-G-mediated NSC-glioma cell fusion.

Antitumor effects of CD40 ligand-expressing endothelial progenitor cells derived from human induced pluripotent stem cells in a metastatic breast cancer model.

Given their intrinsic ability to home to tumor sites, endothelial progenitor cells (EPCs) are attractive as cellular vehicles for targeted cancer gene therapy. However, collecting sufficient EPCs is one of the challenging issues critical for effective clinical translation of this new approach. In this study, we sought to explore whether human induced pluripotent stem (iPS) cells could be used as a reliable and accessible cell source to generate human EPCs suitable for cancer treatment. We used an embryoid body formation method to derive CD133(+)CD34(+) EPCs from human iPS cells. The generated EPCs expressed endothelial markers such as CD31, Flk1, and vascular endothelial-cadherin without expression of the CD45 hematopoietic marker. After intravenous injection, the iPS cell-derived EPCs migrated toward orthotopic and lung metastatic tumors in the mouse 4T1 breast cancer model but did not promote tumor growth and metastasis. To investigate their therapeutic potential, the EPCs were transduced with baculovirus encoding the potent T cell costimulatory molecule CD40 ligand. The systemic injection of the CD40 ligand-expressing EPCs stimulated the secretion of both tumor necrosis factor-α and interferon-γ and increased the caspase 3/7 activity in the lungs with metastatic tumors, leading to prolonged survival of the tumor bearing mice. Therefore, our findings suggest that human iPS cell-derived EPCs have the potential to serve as tumor-targeted cellular vehicles for anticancer gene therapy.

Induced pluripotent stem cell-derived neural stem cells transduced with baculovirus encoding CD40 ligand for immunogene therapy in mouse models of breast cancer.

The interaction between CD40 ligand (CD40L) and CD40 can directly inhibit growth of CD40-positive carcinoma cells and may indirectly inhibit tumor growth through coordination of immune responses. Many efforts in CD40L cancer gene therapy have been focused on direct CD40L gene transfer into malignant target cells. This in vivo gene therapy approach relies on high-efficiency gene transfer and could be technically challenging for the treatment of certain cancers, especially multisite metastases. We report herein an alternative means of using the tumor-homing property of neural stem cells (NSCs) to deliver CD40L molecules into tumor tissues. NSCs were derived from human induced pluripotent stem cells, transduced in vitro with a baculoviral vector encoding CD40L, and intravenously injected into immunocompetent mice with orthotopic and metastatic breast cancers. Through a bystander mechanism of intercellular transfer of CD40L from the donor NSCs to tumor target cells, the treatment impeded tumor growth, leading to prolonged survival of the tumor-bearing mice. We further showed that compared with the stem cell-based gene therapy that employed a suicide gene, the CD40L immunogene therapy did not cause liver and kidney injury in the treated mice. This new approach may be particularly valuable for metastatic cancer treatments after systemic stem cell administration.

Systemic delivery of fusogenic membrane glycoprotein-expressing neural stem cells to selectively kill tumor cells.

Intravenously injected neural stem cells (NSCs) can infiltrate both primary and metastatic tumor sites; thus, they are attractive tumor-targeting vehicles for delivering anticancer agents. However, because the systemic distribution of the injected NSCs involves normal organs and might induce off-target actions leading to unintended side effects, clinical applications of this approach is impeded. Given that the vesicular stomatitis virus glycoprotein (VSV-G) can promote the formation of multinucleated syncytia to kill cells in a pH-dependent manner, we engineered a pH sensor of VSV-G and generated a novel VSV-G mutant that efficiently promotes syncytium formation at the tumor extracellular pH (pHe) but not at pH 7.4. Using transduced NSCs derived from induced pluripotent stem cells (iPSCs), the VSV-G mutant was delivered into mice with metastatic breast cancers in the lung through tail vein injection. Compared with the conventional stem cell-based gene therapy that uses the herpes simplex virus thymidine kinase (HSVtk) suicide gene, this treatment did not display toxicity to normal non-targeted organs while retaining therapeutic effects in tumor-bearing organs. Our findings demonstrate the effectiveness of a new approach for achieving tumor-selective killing effects following systemic stem cell administration. Its potential in stem cell-based gene therapy for metastatic cancer is worthy of further exploration.

Quantum dot coating of baculoviral vectors enables visualization of transduced cells and tissues.

Imaging of transduced cells and tissues is valuable in developing gene transfer vectors and evaluating gene therapy efficacy. We report here a simple method to use bright and photostable quantum dots to label baculovirus, an emerging gene therapy vector. The labeling was achieved through the non-covalent interaction of glutathione-capped CdTe quantum dots with the virus envelope, without the use of chemical conjugation. The quantum dot labeling was nondestructive to viral transduction function and enabled the identification of baculoviral vector-transduced, living cells based on red fluorescence. When the labeled baculoviral vectors were injected intravenously or intraventricularly for in vivo delivery of a transgene into mice, quantum dot fluorescence signals allow us monitor whether or not the injected tissues were transduced. More importantly, using a dual-color whole-body imaging technology, we demonstrated that in vivo viral transduction could be evaluated in a real-time manner in living mice. Thus, our method of labeling a read-to-use gene delivery vector with quantum dots could be useful towards the improvement of vector design and will have the potential to assess baculovirus-based gene therapy protocols in future.

Inhibition of neuronal nitric oxide synthase activity promotes migration of human-induced pluripotent stem cell-derived neural stem cells toward cancer cells.

The breakthrough in derivation of human-induced pluripotent stem cells (hiPSCs) provides an approach that may help overcome ethical and allergenic challenges posed in numerous medical applications involving human cells, including neural stem/progenitor cells (NSCs). Considering the great potential of NSCs in targeted cancer gene therapy, we investigated in this study the tumor tropism of hiPSC-derived NSCs and attempted to enhance the tropism by manipulation of biological activities of proteins that are involved in regulating the migration of NSCs toward cancer cells. We first demonstrated that hiPSC-NSCs displayed tropism for both glioblastoma cells and breast cancer cells in vitro and in vivo. We then compared gene expression profiles between migratory and non-migratory hiPSC-NSCs toward these cancer cells and observed that the gene encoding neuronal nitric oxide synthase (nNOS) was down-regulated in migratory hiPSC-NSCs. Using nNOS inhibitors and nNOS siRNAs, we demonstrated that this protein is a relevant regulator in controlling migration of hiPSC-NSCs toward cancer cells, and that inhibition of its activity or down-regulation of its expression can sensitize poorly migratory NSCs and be used to improve their tumor tropism. These findings suggest a novel application of nNOS inhibitors in neural stem cell-mediated cancer therapy.

Attenuated adenosine-to-inosine editing of microRNA-376a* promotes invasiveness of glioblastoma cells.

In the human brain, microRNAs (miRNAs) from the microRNA-376 (miR-376) cluster undergo programmed "seed" sequence modifications by adenosine-to-inosine (A-to-I) editing. Emerging evidence suggests a link between impaired A-to-I editing and cancer, particularly in high-grade gliomas. We hypothesized that disruption of A-to-I editing alters expression of genes regulating glioma tumor phenotypes. By sequencing the miR-376 cluster, we show that the overall miRNA editing frequencies were reduced in human gliomas. Specifically in high-grade gliomas, miR-376a* accumulated entirely in an unedited form. Clinically, a significant correlation was found between accumulation of unedited miR-376a* and the extent of invasive tumor spread as measured by magnetic resonance imaging of patient brains. Using both in vitro and orthotopic xenograft mouse models, we demonstrated that the unedited miR-376a* promoted glioma cell migration and invasion, while the edited miR-376a* suppressed these features. The effects of the unedited miR-376a* were mediated by its sequence-dependent ability to target RAP2A and concomitant inability to target AMFR. Thus, the tumor-dependent introduction of a single base difference in the miR-376a* sequence dramatically alters the selection of its target genes and redirects its function from inhibiting to promoting glioma cell invasion. These findings uncover a new mechanism of miRNA deregulation and identify unedited miR-376a* as a potential therapeutic target in glioblastoma cells.

Tumor tropism of intravenously injected human-induced pluripotent stem cell-derived neural stem cells and their gene therapy application in a metastatic breast cancer model.

Human pluripotent stem cells can serve as an accessible and reliable source for the generation of functional human cells for medical therapies. In this study, we used a conventional lentiviral transduction method to derive human-induced pluripotent stem (iPS) cells from primary human fibroblasts and then generated neural stem cells (NSCs) from the iPS cells. Using a dual-color whole-body imaging technology, we demonstrated that after tail vein injection, these human NSCs displayed a robust migratory capacity outside the central nervous system in both immunodeficient and immunocompetent mice and homed in on established orthotopic 4T1 mouse mammary tumors. To investigate whether the iPS cell-derived NSCs can be used as a cellular delivery vehicle for cancer gene therapy, the cells were transduced with a baculoviral vector containing the herpes simplex virus thymidine kinase suicide gene and injected through tail vein into 4T1 tumor-bearing mice. The transduced NSCs were effective in inhibiting the growth of the orthotopic 4T1 breast tumor and the metastatic spread of the cancer cells in the presence of ganciclovir, leading to prolonged survival of the tumor-bearing mice. The use of iPS cell-derived NSCs for cancer gene therapy bypasses the sensitive ethical issue surrounding the use of cells derived from human fetal tissues or human embryonic stem cells. This approach may also help to overcome problems associated with allogeneic transplantation of other types of human NSCs.

Isolation of three important types of stem cells from the same samples of banked umbilical cord blood.

It is known that umbilical cord blood (UCB) is a rich source of stem cells with practical and ethical advantages. Three important types of stem cells which can be harvested from umbilical cord blood and used in disease treatment are hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs). Since these stem cells have shown enormous potential in regenerative medicine, numerous umbilical cord blood banks have been established. In this study, we examined the ability of banked UCB collected to produce three types of stem cells from the same samples with characteristics of HSCs, MSCs and EPCs. We were able to obtain homogeneous plastic rapidly-adherent cells (with characteristics of MSCs), slowly-adherent (with characteristics of EPCs) and non-adherent cells (with characteristics of HSCs) from the mononuclear cell fractions of cryopreserved UCB. Using a protocol of 48 h supernatant transferring, we successfully isolated MSCs which expressed CD13, CD44 and CD90 while CD34, CD45 and CD133 negative, had typical fibroblast-like shape, and was able to differentiate into adipocytes; EPCs which were CD34, and CD90 positive, CD13, CD44, CD45 and CD133 negative, adherent with cobble-like shape; HSCs which formed colonies when cultured in MethoCult medium.

Glioma gene therapy using induced pluripotent stem cell derived neural stem cells.

Using neural stem cells (NSCs) with tumor tropic migratory capacity to deliver therapeutic genes is an attractive strategy in eliminating metastatic or disseminated tumors. While different methods have been developed to isolate or generate NSCs, it has not been assessed whether induced pluripotent stem (iPS) cells, a type of pluripotent stem cells that hold great potential for regenerative medicine, can be used as a source for derivation of NSCs with tumor tropism. In this study, we used a conventional lentivirus transduction method to derive iPS cells from primary mouse embryonic fibroblasts and then generated NSCs from the iPS cells. To investigate whether the iPS cell derived NSCs can be used in the treatment of disseminated brain tumors, the cells were transduced with a baculoviral vector containing the herpes simplex virus thymidine kinase suicide gene and injected into the cerebral hemisphere contralateral to a tumor inoculation site in a mouse intracranial human glioma xenograft model. We observed that NSCs expressing the suicide gene were, in the presence of ganciclovir, effective in inhibiting the growth of the glioma xenografts and prolonging survival of tumor-bearing mice. Our findings provide evidence for the feasibility of using iPS cell derived NSCs as cellular vehicles for targeted anticancer gene therapy.