Arsinothricin Inhibits Plasmodium falciparum Proliferation in Blood and Blocks Parasite Transmission to Mosquitoes.

Malaria, caused by Plasmodium protozoal parasites, remains a leading cause of morbidity and mortality. The Plasmodium parasite has a complex life cycle, with asexual and sexual forms in humans and Anopheles mosquitoes. Most antimalarials target only the symptomatic asexual blood stage. However, to ensure malaria eradication, new drugs with efficacy at multiple stages of the life cycle are necessary. We previously demonstrated that arsinothricin (AST), a newly discovered organoarsenical natural product, is a potent broad-spectrum antibiotic that inhibits the growth of various prokaryotic pathogens. Here, we report that AST is an effective multi-stage antimalarial. AST is a nonproteinogenic amino acid analog of glutamate that inhibits prokaryotic glutamine synthetase (GS). Phylogenetic analysis shows that Plasmodium GS, which is expressed throughout all stages of the parasite life cycle, is more closely related to prokaryotic GS than eukaryotic GS. AST potently inhibits Plasmodium GS, while it is less effective on human GS. Notably, AST effectively inhibits both Plasmodium erythrocytic proliferation and parasite transmission to mosquitoes. In contrast, AST is relatively nontoxic to a number of human cell lines, suggesting that AST is selective against malaria pathogens, with little negative effect on the human host. We propose that AST is a promising lead compound for developing a new class of multi-stage antimalarials.

Arsenic in medicine: past, present and future.

Arsenicals are one of the oldest treatments for a variety of human disorders. Although infamous for its toxicity, arsenic is paradoxically a therapeutic agent that has been used since ancient times for the treatment of multiple diseases. The use of most arsenic-based drugs was abandoned with the discovery of antibiotics in the 1940s, but a few remained in use such as those for the treatment of trypanosomiasis. In the 1970s, arsenic trioxide, the active ingredient in a traditional Chinese medicine, was shown to produce dramatic remission of acute promyelocytic leukemia similar to the effect of all-trans retinoic acid. Since then, there has been a renewed interest in the clinical use of arsenicals. Here the ancient and modern medicinal uses of inorganic and organic arsenicals are reviewed. Included are antimicrobial, antiviral, antiparasitic and anticancer applications. In the face of increasing antibiotic resistance and the emergence of deadly pathogens such as the severe acute respiratory syndrome coronavirus 2, we propose revisiting arsenicals with proven efficacy to combat emerging pathogens. Current advances in science and technology can be employed to design newer arsenical drugs with high therapeutic index. These novel arsenicals can be used in combination with existing drugs or serve as valuable alternatives in the fight against cancer and emerging pathogens. The discovery of the pentavalent arsenic-containing antibiotic arsinothricin, which is effective against multidrug-resistant pathogens, illustrates the future potential of this new class of organoarsenical antibiotics.

Regulation of arsenic methylation: identification of the transcriptional region of the human AS3MT gene.

The human enzyme As(III) S-adenosylmethionine methyltransferase (AS3MT) catalyzes arsenic biotransformations and is considered to contribute to arsenic-related diseases. AS3MT is expressed in various tissues and cell types including liver, brain, adrenal gland, and peripheral blood mononuclear cells but not in human keratinocytes, urothelial, or brain microvascular endothelial cells. This indicates that AS3MT expression is regulated in a tissue/cell type-specific manner, but the mechanism of transcriptional regulation of expression of the AS3MT gene is not known. In this study, we define the DNA sequence of the core promoter region of the human AS3MT gene. We identify a GC box in the promoter to which the stress-related transcription factor Sp1 binds, indicating involvement of regulatory elements in AS3MT gene expression.

Identification of the Biosynthetic Gene Cluster for the Organoarsenical Antibiotic Arsinothricin.

The soil bacterium Burkholderia gladioli GSRB05 produces the natural compound arsinothricin [2-amino-4-(hydroxymethylarsinoyl) butanoate] (AST), which has been demonstrated to be a broad-spectrum antibiotic. To identify the genes responsible for AST biosynthesis, a draft genome sequence of B. gladioli GSRB05 was constructed. Three genes, arsQML, in an arsenic resistance operon were found to be a biosynthetic gene cluster responsible for synthesis of AST and its precursor, hydroxyarsinothricin [2-amino-4-(dihydroxyarsinoyl) butanoate] (AST-OH). The arsL gene product is a noncanonical radical S-adenosylmethionine (SAM) enzyme that is predicted to transfer the 3-amino-3-carboxypropyl (ACP) group from SAM to the arsenic atom in inorganic arsenite, forming AST-OH, which is methylated by the arsM gene product, a SAM methyltransferase, to produce AST. Finally, the arsQ gene product is an efflux permease that extrudes AST from the cells, a common final step in antibiotic-producing bacteria. Elucidation of the biosynthetic gene cluster for this novel arsenic-containing antibiotic adds an important new tool for continuation of the antibiotic era. IMPORTANCE Antimicrobial resistance is an emerging global public health crisis, calling for urgent development of novel potent antibiotics. We propose that arsinothricin and related arsenic-containing compounds may be the progenitors of a new class of antibiotics to extend our antibiotic era. Here, we report identification of the biosynthetic gene cluster for arsinothricin and demonstrate that only three genes, two of which are novel, are required for the biosynthesis and transport of arsinothricin, in contrast to the phosphonate counterpart, phosphinothricin, which requires over 20 genes. Our discoveries will provide insight for the development of more effective organoarsenical antibiotics and illustrate the previously unknown complexity of the arsenic biogeochemical cycle, as well as bring new perspective to environmental arsenic biochemistry.

Integrated Collection of Stem Cell Bank Data, a Data Portal for Standardized Stem Cell Information.

The past decade has witnessed an extremely rapid increase in the number of newly established stem cell lines. However, due to the lack of a standardized format, data exchange among stem cell line resources has been challenging, and no system can search all stem cell lines across resources worldwide. To solve this problem, we have developed the Integrated Collection of Stem Cell Bank data (ICSCB) (http://icscb.stemcellinformatics.org/), the largest database search portal for stem cell line information, based on the standardized data items and terms of the MIACARM framework. Currently, ICSCB can retrieve >16,000 cell lines from four major data resources in Europe, Japan, and the United States. ICSCB is automatically updated to provide the latest cell line information, and its integrative search helps users collect cell line information for over 1,000 diseases, including many rare diseases worldwide, which has been a formidable task, thereby distinguishing itself from other database search portals.

Comparative Cytotoxicity of Inorganic Arsenite and Methylarsenite in Human Brain Cells.

The overall goal of this study is to elucidate the potential effect(s) of arsenic on a variety of human brain cells. Arsenic is the most pervasive Group A human environmental carcinogen. Long-term exposure to arsenic is associated with human diseases including cancer, cardiovascular disease, and diabetes. More immediate are the health effects on neurological development and associated disorders in infants and children exposed to arsenic in utero. Arsenic is metabolized in various organs and tissues into more toxic methylated species, including methylarsenite (MAs(III)), so the question arises whether the methylate species are responsible for the neurological effects of arsenic. Arsenic enters the brain through the blood-brain barrier and produces toxicity in the brain microvascular endothelial cells, glia (astrocytes and microglia), and neurons. In this study, we first assessed the toxicity in different types of brain cells exposed to either inorganic trivalent As(III) or MAs(III) using both morphological and cytotoxicity cell-based analysis. Second, we determined the methylation of arsenicals and the expression levels of the methylation enzyme, As(III) S-adenosylmethionine (SAM) methyltransferase (AS3MT), in several types of brain cells. We showed that the toxicity to neurons of MAs(III) was significantly higher than that of As(III). Interestingly, the differences in cytotoxicity between cell types was not due to expression of AS3MT, as this was expressed in neurons and glia but not in endothelial cells. These results support our hypothesis that MAs(III) is the likely physiological neurotoxin rather than inorganic arsenic species.

Arsinothricin, an arsenic-containing non-proteinogenic amino acid analog of glutamate, is a broad-spectrum antibiotic.

The emergence and spread of antimicrobial resistance highlights the urgent need for new antibiotics. Organoarsenicals have been used as antimicrobials since Paul Ehrlich's salvarsan. Recently a soil bacterium was shown to produce the organoarsenical arsinothricin. We demonstrate that arsinothricin, a non-proteinogenic analog of glutamate that inhibits glutamine synthetase, is an effective broad-spectrum antibiotic against both Gram-positive and Gram-negative bacteria, suggesting that bacteria have evolved the ability to utilize the pervasive environmental toxic metalloid arsenic to produce a potent antimicrobial. With every new antibiotic, resistance inevitably arises. The arsN1 gene, widely distributed in bacterial arsenic resistance (ars) operons, selectively confers resistance to arsinothricin by acetylation of the α-amino group. Crystal structures of ArsN1 N-acetyltransferase, with or without arsinothricin, shed light on the mechanism of its substrate selectivity. These findings have the potential for development of a new class of organoarsenical antimicrobials and ArsN1 inhibitors.

Long non-coding RNA HOTAIR promotes cell migration by upregulating insulin growth factor-binding protein 2 in renal cell carcinoma.

Renal cell carcinoma (RCC) is one of the most lethal urologic cancers. About one-third of RCC patients already have distal metastasis at the time of diagnosis. There is growing evidence that Hox antisense intergenic RNA (HOTAIR) plays essential roles in metastasis in several types of cancers. However, the precise mechanism by which HOTAIR enhances malignancy remains unclear, especially in RCC. Here, we demonstrated that HOTAIR enhances RCC-cell migration by regulating the insulin growth factor-binding protein 2 (IGFBP2) expression. HOTAIR expression in tumors was significantly correlated with nuclear grade, lymph-node metastasis, and lung metastasis. High HOTAIR expression was associated with a poor prognosis in both our dataset and The Cancer Genome Atlas dataset. Migratory capacity was enhanced in RCC cell lines in a HOTAIR-dependent manner. HOTAIR overexpression accelerated tumorigenicity and lung metastasis in immunodeficient mice. Microarray analysis revealed that IGFBP2 expression was upregulated in HOTAIR-overexpressing cells compared with control cells. The enhanced migration activity of HOTAIR-overexpressing cells was attenuated by IGFBP2 knockdown. IGFBP2 and HOTAIR were co-expressed in clinical RCC samples. Our findings suggest that the HOTAIR-IGFBP2 axis plays critical roles in RCC metastasis and may serve as a novel therapeutic target for advanced RCC.

First Proposal of Minimum Information About a Cellular Assay for Regenerative Medicine.

: Advances in stem cell research have triggered scores of studies in regenerative medicine in a large number of institutions and companies around the world. However, reproducibility and data exchange among laboratories or cell banks are constrained by the lack of a standardized format for experiments. To enhance information flow in stem cell and derivative cell research, here we propose a minimum information standard to describe cellular assay data to facilitate practical regenerative medicine. Based on the existing Minimum Information About a Cellular Assay, we developed Minimum Information About a Cellular Assay for Regenerative Medicine (MIACARM), which allows for the description of advanced cellular experiments with defined taxonomy of human cell types. By using controlled terms, such as ontologies, MIACARM will provide a platform for cellular assay data exchange among cell banks or registries that have been established at more than 20 sites in the world. SIGNIFICANCE: Currently, there are more than 20 human cell information storage sites around the world. However, reproducibility and data exchange among different laboratories or cell information providers are usually inadequate or nonexistent because of the lack of a standardized format for experiments. This study, which is the fruit of collaborative work by scientists at stem cell banks and cellular information registries worldwide, including those in the U.S., the U.K., Europe, and Japan, proposes new minimum information guidelines, Minimum Information About a Cellular Assay for Regenerative Medicine (MIACARM), for cellular assay data deposition. MIACARM is intended to promote data exchange and facilitation of practical regenerative medicine.

RegenBase: a knowledge base of spinal cord injury biology for translational research.

Spinal cord injury (SCI) research is a data-rich field that aims to identify the biological mechanisms resulting in loss of function and mobility after SCI, as well as develop therapies that promote recovery after injury. SCI experimental methods, data and domain knowledge are locked in the largely unstructured text of scientific publications, making large scale integration with existing bioinformatics resources and subsequent analysis infeasible. The lack of standard reporting for experiment variables and results also makes experiment replicability a significant challenge. To address these challenges, we have developed RegenBase, a knowledge base of SCI biology. RegenBase integrates curated literature-sourced facts and experimental details, raw assay data profiling the effect of compounds on enzyme activity and cell growth, and structured SCI domain knowledge in the form of the first ontology for SCI, using Semantic Web representation languages and frameworks. RegenBase uses consistent identifier schemes and data representations that enable automated linking among RegenBase statements and also to other biological databases and electronic resources. By querying RegenBase, we have identified novel biological hypotheses linking the effects of perturbagens to observed behavioral outcomes after SCI. RegenBase is publicly available for browsing, querying and download.Database URL:http://regenbase.org.

Evolving BioAssay Ontology (BAO): modularization, integration and applications.

The lack of established standards to describe and annotate biological assays and screening outcomes in the domain of drug and chemical probe discovery is a severe limitation to utilize public and proprietary drug screening data to their maximum potential. We have created the BioAssay Ontology (BAO) project (http://bioassayontology.org) to develop common reference metadata terms and definitions required for describing relevant information of low-and high-throughput drug and probe screening assays and results. The main objectives of BAO are to enable effective integration, aggregation, retrieval, and analyses of drug screening data. Since we first released BAO on the BioPortal in 2010 we have considerably expanded and enhanced BAO and we have applied the ontology in several internal and external collaborative projects, for example the BioAssay Research Database (BARD). We describe the evolution of BAO with a design that enables modeling complex assays including profile and panel assays such as those in the Library of Integrated Network-based Cellular Signatures (LINCS). One of the critical questions in evolving BAO is the following: how can we provide a way to efficiently reuse and share among various research projects specific parts of our ontologies without violating the integrity of the ontology and without creating redundancies. This paper provides a comprehensive answer to this question with a description of a methodology for ontology modularization using a layered architecture. Our modularization approach defines several distinct BAO components and separates internal from external modules and domain-level from structural components. This approach facilitates the generation/extraction of derived ontologies (or perspectives) that can suit particular use cases or software applications. We describe the evolution of BAO related to its formal structures, engineering approaches, and content to enable modeling of complex assays and integration with other ontologies and datasets.

Formalization, annotation and analysis of diverse drug and probe screening assay datasets using the BioAssay Ontology (BAO).

Huge amounts of high-throughput screening (HTS) data for probe and drug development projects are being generated in the pharmaceutical industry and more recently in the public sector. The resulting experimental datasets are increasingly being disseminated via publically accessible repositories. However, existing repositories lack sufficient metadata to describe the experiments and are often difficult to navigate by non-experts. The lack of standardized descriptions and semantics of biological assays and screening results hinder targeted data retrieval, integration, aggregation, and analyses across different HTS datasets, for example to infer mechanisms of action of small molecule perturbagens. To address these limitations, we created the BioAssay Ontology (BAO). BAO has been developed with a focus on data integration and analysis enabling the classification of assays and screening results by concepts that relate to format, assay design, technology, target, and endpoint. Previously, we reported on the higher-level design of BAO and on the semantic querying capabilities offered by the ontology-indexed triple store of HTS data. Here, we report on our detailed design, annotation pipeline, substantially enlarged annotation knowledgebase, and analysis results. We used BAO to annotate assays from the largest public HTS data repository, PubChem, and demonstrate its utility to categorize and analyze diverse HTS results from numerous experiments. BAO is publically available from the NCBO BioPortal at http://bioportal.bioontology.org/ontologies/1533. BAO provides controlled terminology and uniform scope to report probe and drug discovery screening assays and results. BAO leverages description logic to formalize the domain knowledge and facilitate the semantic integration with diverse other resources. As a consequence, BAO offers the potential to infer new knowledge from a corpus of assay results, for example molecular mechanisms of action of perturbagens.

Synaptic formation in subsets of glutamatergic terminals in the mouse hippocampal formation is affected by a deficiency in the neural cell recognition molecule NB-3.

The neural cell recognition molecule NB-3, which is also referred to as contactin-6, is a member of the contactin subgroup molecules that are expressed prominently in the developing nervous system after birth. In mice, an NB-3 deficiency impairs motor coordination and reduces the synaptic density between parallel fibers and Purkinje cells in the cerebellum. Here, we studied the role of NB-3 in the formation of glutamatergic synapses in the hippocampal formation. At postnatal day 5, NB-3 immunoreactivity was detected in the subiculum, the stratum lacunosum-moleculare of the CA1 region and the hilus of the dentate gyrus. NB-3 expression in the strata radiatum and oriens was weak, and it was very weak in the granule cell layer of the dentate gyrus, the pyramidal cell layer of regions CA3 to CA1 and the stratum lucidum. NB-3-positive puncta partially overlapped with vesicular glutamate transporter 1 (VGLUT1) and 2 (VGLUT2), excitatory presynaptic markers, but not with vesicular GABA transporter (VGAT), an inhibitory presynaptic marker. The density of VGLUT1 and VGLUT2 puncta in the regions where NB-3 was strongly expressed in wild-type mice was reduced by approximately 20-30% in NB-3 knockout mice relative to wild-type mice, whereas that of VGAT puncta was not affected by NB-3 deficiency. Thus, NB-3 has key roles in the formation of glutamatergic, but not GABAergic, synapses during postnatal development of the hippocampal formation as well as the cerebellum.

Deficiency of neural recognition molecule NB-2 affects the development of glutamatergic auditory pathways from the ventral cochlear nucleus to the superior olivary complex in mouse.

Neural recognition molecule NB-2/contactin 5 is expressed transiently during the first postnatal week in glutamatergic neurons of the central auditory system. Here, we investigated the effect of NB-2 deficiency on the auditory brainstem in mouse. While almost all principal neurons are wrapped with the calyces of Held in the medial nucleus of the trapezoid body (MNTB) in wild type, 8% of principal neurons in NB-2 knockout (KO) mice lack the calyces of Held at postnatal day (P) 6. At P10 and P15, apoptotic principal neurons were detected in NB-2 KO mice, but not in wild type. Apoptotic cells were also increased in the ventral cochlear nucleus (VCN) of NB-2 KO mice, which contains bushy neurons projecting to the MNTB and the lateral superior olive (LSO). At the age of 1 month, the number of principal neurons in the MNTB and of glutamatergic synapses in the LSO was reduced in NB-2 KO mice. Finally, interpeak latencies for auditory brainstem response waves II-III and III-IV were significantly increased in NB-2 KO mice. Together, these findings suggest that NB-2 deficiency causes a deficit in synapse formation and then induces apoptosis in MNTB and VCN neurons, affecting auditory brainstem function.

Contribution of the neural cell recognition molecule NB-3 to synapse formation between parallel fibers and Purkinje cells in mouse.

The neural cell recognition molecule NB-3, also referred to as contactin-6, is expressed prominently in the developing nervous system after birth and its deficiency has been shown to cause impairment in motor coordination. Here, we investigated the contribution of NB-3 to cerebellar development, focusing on lobule 3 where NB-3 was expressed in granule cells but not in Purkinje cells. In the developing molecular layer, the neural cell recognition molecules TAG-1, L1, and NB-3 formed distinct expression zones from the external granule cell layer to the internal granule cell layer (IGL), respectively. The NB-3-immunoreactive zone did not overlap with TAG-1-immunoreactive zone. By contrast, the L1-immunoreactive zone overlapped with both the TAG-1- and NB-3-immunoreactive zones. NB-3-positive puncta overlapped with vesicular glutamate transporter 1, a presynaptic marker and were apposed close to metabotropic glutamate receptor 1A, a postsynaptic marker, indicating that NB-3 is localized presynaptically at glutamatergic synapses between parallel fibers and Purkinje cells. In NB-3 knockout mice, L1 immunoreactive signals were increased in the IGL at postnatal day (P) 5, suggesting the increase in the number of immature granule cells of the IGL. In addition, the density of parallel fiber synaptic terminals was reduced in NB-3 knockout mice relative to wild-type mice at P5 to P10. In parallel with these findings, caspase-dependent cell death was significantly increased in the NB- 3-deficient cerebellum at P15. Collectively, our results indicate that NB-3 deficiency affects synapse formation during postnatal cerebellar development.

Preferential localization of neural cell recognition molecule NB-2 in developing glutamatergic neurons in the rat auditory brainstem.

NB-2 is a neuronal cell recognition molecule that is preferentially expressed in auditory pathways. Mice deficient in the NB-2 gene exhibit aberrant responses to acoustic stimuli. Here we examined the expression and localization of NB-2 in the auditory brainstem during development in the rat. NB-2 was strongly expressed in the ventral cochlear nucleus (VCN), ventral acoustic stria, lateral and medial superior olivary complex (SOC), superior paraolivary nucleus, medial nucleus of the trapezoid body (MNTB), ventrolateral lemniscus, and central nucleus of the inferior colliculus (CIC). In the VCN and CIC, NB-2 was expressed in the regions that are known to respond to high frequencies. In situ hybridization combined with immunohistochemistry suggested that NB-2 is expressed only in neurons. NB-2 was colocalized with glutamatergic elements in the neuropil and the calyces of Held but not with glycinergic or GABAergic elements. NB-2 expression in the SOC was first detected at embryonic day (E)19, reached a maximum level at postnatal day (P)7, and declined thereafter. Immunolabeling with VGLUT1 and VGLUT2, markers for mature and premature glutamatergic synapses, respectively, in combination with NB-2 immunolabeling revealed that NB-2 is expressed at glutamatergic synapses. Collectively, our findings suggest that NB-2 plays a key role in maturation of glutamatergic synapses in the brainstem during the final stages of auditory development.