Journal Articles - UP - MSI
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- A conserved biosynthetic gene cluster is regulated by quorum sensing in a shipworm symbiontRobes, Jose Miguel D.; Altamia, Marvin A.; Murdock, Ethan G.; Concepcion, Gisela; Haygood, Margo G.; Puri, Aaron W. (American Society for Microbiology, 2022-06-14)Bacterial symbionts often provide critical functions for their hosts. For example, wood-boring bivalves called shipworms rely on cellulolytic endosymbionts for wood digestion. However, how the relationship between shipworms and their bacterial symbionts is formed and maintained remains unknown. Quorum sensing (QS) often plays an important role in regulating symbiotic relationships. We identified and characterized a QS system found in Teredinibacter sp. strain 2052S, a gill isolate of the wood-boring shipworm Bactronophorus cf. thoracites. We determined that 2052S produces the signal N-decanoyl-l-homoserine lactone (C10-HSL) and that this signal controls the activation of a biosynthetic gene cluster colocated in the symbiont genome that is conserved among all symbiotic Teredinibacter isolates. We subsequently identified extracellular metabolites associated with the QS regulon, including ones linked to the conserved biosynthetic gene cluster, using mass spectrometry-based molecular networking. Our results demonstrate that QS plays an important role in regulating secondary metabolism in this shipworm symbiont. This information provides a step toward deciphering the molecular details of the relationship between these symbionts and their hosts. Furthermore, because shipworm symbionts harbor vast yet underexplored biosynthetic potential, understanding how their secondary metabolism is regulated may aid future drug discovery efforts using these organisms.
- Somatostatin venom analogs evolved by fish-hunting cone snails: From prey capture behavior to identifying drug leadsRamiro, Iris Bea L.; Bjørn-Yoshimoto, Walden E.; Imperial, Julita S.; Gajewiak, Joanna; Salcedo, Paula Flórez; Watkins, Maren; Taylor, Dylan; Resager, William; Ueberheide, Beatrix; Bräuner-Osborne, Hans; Whitby, Frank G.; Hill, Christopher P.; Martin, Laurent F.; Patwardhan, Amol; Concepcion, Gisela; Olivera, Baldomero M.; Safavi-Hemami, Helena (American Association for the Advancement of Science, 2022-03-25)Somatostatin (SS) is a peptide hormone with diverse physiological roles. By investigating a deep-water clade of fish-hunting cone snails, we show that predator-prey evolution has generated a diverse set of SS analogs, each optimized to elicit specific systemic physiological effects in prey. The increased metabolic stability, distinct SS receptor activation profiles, and chemical diversity of the venom analogs make them suitable leads for therapeutic application, including pain, cancer, and endocrine disorders. Our findings not only establish the existence of SS-like peptides in animal venoms but also serve as a model for the synergy gained from combining molecular phylogenetics and behavioral observations to optimize the discovery of natural products with biomedical potential.
- In Silico supported nontarget analysis of contaminants of emerging concern: Increasing confidence in unknown identification in wastewater and surface watersAngeles, Luisa F.; Halwatura, Lahiruni M.; Antle, Jonathan P.; Simpson, Scott; Jaraula, Caroline M.B.; Aga, Diana S. (American Chemical Society, 2021-08-01)Nontarget analysis using liquid chromatography–high resolution mass spectrometry (LC–HRMS) is a valuable approach in characterizing for contaminants of emerging concern (CECs) in the environment. In this study, wastewater and surface water samples from three locations in Manila, Philippines were analyzed for CECs using a nontarget analysis approach with an LC-Orbitrap. A previously optimized semiautomated workflow was used for data processing with Compound Discoverer. A total of 157 compounds were identified, with 21 confirmed with reference standards, 83 confirmed with evidence from a mass spectral library (mzCloud), and 53 tentatively identified using in silico fragmentation (MetFrag). These compounds include pharmaceuticals such as antibiotics, antifungal, and antihypertensive compounds, human metabolites, natural products, pesticides, and industrial chemicals. Compounds confirmed with reference standards include antibiotics ciprofloxacin, clarithromycin, acetyl-sulfamethoxazole, and trimethoprim (2 to 19 ng/L), and antifungal compounds carbendazim and climbazole (3–47 ng/L). The pesticides diethyltoluamide (DEET) and diuron were also detected (37 ng/L). The utility of a preliminary multivariable linear regression quality structure-retention relationship (QSRR) model based on quantum chemical molecular descriptors is demonstrated. This study demonstrates the importance of using tools and software that are helpful for annotating HRMS data and reporting detections according to a standardized classification system. The detection of several CECs in wastewater and surface water samples show the importance of performing nontarget analysis in determining occurrence of CECs in the environment.We acknowledge support from the National Science Foundation PIRE-HEARD award number 1545756 and USAID PEER subaward number 2000009924. S.S. thanks the National Science Foundation (Award #1904825) for support of this research, and the Donors of the American Chemical Society Petroleum Research Fund (PRF-58954-UNI5). We also thank Shyrill Mae Mariano from the Marine Science Institute in the University of the Philippines − Diliman who helped with the sample collection and Rebecca Dickman from University at Buffalo for her help on technical work.
- Characterization of Alexandrium tamutum (Dinophyceae) isolated from Philippine waters, with the rare detection of paralytic shellfish toxinBenico, Garry; Azanza, Rhodora (Association of Systematic Biologists of the Philippines, 2022-04-01)Alexandrium tamutum M.Montressor, A.Beran & U.John is a non-toxic, bloom-forming dinoflagellate species commonly reported in temperate waters. In this study, 8 cultures of A. tamutum established from Bolinao Channel and Manila Bay, Philippines were characterized in terms of their morphology, phylogeny and toxicity. Cells were roundish, measuring 25.5 –29.84 µm long and 26.2–28.45 µm wide. The nucleus is equatorially elongated and located at the center of the cell. The chloroplasts are numerous, golden brown in color and radially arranged. Thecal tabulation is typical of Alexandrium: APC, 4', 6'', 6c, 6s, 5''', 2''''. Shape of the taxonomically informative thecal plates such as sixth precingular plate (6'') and posterior sulcal plate (sp) was similar to A. tamutum, which confirms the species identity. However, the presence of anterior and posterior attachment pores observed in our cultured isolates is the first case in this species. Molecular phylogeny inferred from LSU rDNA and ITS supports our identification by forming a well-supported clade composed of A. tamutum strains from other geographic regions. HPLC analysis showed that A. tamutum is generally non-toxic except for strain ATC9 which has low amount of decarbamoylsaxitoxin (dcSTX), resulting to a toxicity of 0.07 fmole STX eq per cell. The present study reports the first verified occurrence of Philippine A. tamutum with reliable morphological and molecular information, including the first record in Manila Bay and first detection of PST in one strain at a certain culture period.We acknowledge the Department of Science and Technology Philippine Council for Agriculture, Aquatic and Natural Resources Research and Development (DOST PCAARRD) and the University of the Philippines-The Marine Science Institute (UP-MSI) for the funding support. We are grateful for the assistance of Joshua Vacarizas, Keith Pinto and Jenelyn Mendoza for the molecular and toxicity analyses of the cultures. We also acknowledge Estrelita Flores, Emelita Eugenio and Jayson Orpeza for their assistance during the fieldwork and other logistical support.
- Genomic and targeted approaches unveil the cell membrane as a major target of the antifungal cytotoxin amantelide AElsadek, Lobna A.; Matthews, James H.; Nishimura, Shinichi; Nakatani, Takahiro; Ito, Airi; Gu, Tongjun; Luo, Danmeng; Salvador-Reyes, Lilibeth A.; Paul, Valerie J.; Kakeya, Hideaki; Luesch, Hendrik (Wiley, 2021-03-23)Amantelide A, a polyhydroxylated macrolide isolated from a marine cyanobacterium, displays broad-spectrum activity against mammalian cells, bacterial pathogens, and marine fungi. We conducted comprehensive mechanistic studies to identify the molecular targets and pathways affected by amantelide A. Our investigations relied on chemical structure similarities with compounds of known mechanisms, yeast knockout mutants, yeast chemogenomic profiling, and direct biochemical and biophysical methods. We established that amantelide A exerts its antifungal action by binding to ergosterol-containing membranes followed by pore formation and cell death, a mechanism partially shared with polyene antifungals. Binding assays demonstrated that amantelide A also binds to membranes containing epicholesterol or mammalian cholesterol, thus suggesting that the cytotoxicity to mammalian cells might be due to its affinity to cholesterol-containing membranes. However, membrane interactions were not completely dependent on sterols. Yeast chemogenomic profiling suggested additional direct or indirect effects on actin. Accordingly, we performed actin polymerization assays, which suggested that amantelide A also promotes actin polymerization in cell-free systems. However, the C-33 acetoxy derivative amantelide B showed a similar effect on actin dynamics in vitro but no significant activity against yeast. Overall, these studies suggest that the membrane effects are the most functionally relevant for amantelide A mechanism of action.This research was supported by the National Institutes of Health (grant R01CA172310 to H.L.), the Debbie and Sylvia DeSantis Chair Professorship (H.L), and a Grant-in Aid for Scientific Research (no. 17H06401 to S.N. and H.K.) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. L.E. was supported by the NIH/NIGMS T32GM136583 “Chemistry-Biology Interface Training Program at the University of Florida”. We thank Dr. Yanping Zhang (University of Florida) from the UF ICBR NextGen DNA Sequencing core facility for carrying out the nextgeneration sequencing, Dr. Maya Schuldiner (Weizmann Institute of Science, Israel) for providing the triple yeast deletion library, Dr. Kaoru Takegawa (Kyushu University, Japan) for providing the ergosterol mutants, Dr. Charles Boone laboratory (University of Toronto, Canada) for providing the wild-type S. cerevisiae Y7092 and Dr. Kalina Atanasova (CNPD3, University of Florida) for assistance with the imaging.
- The harmful unarmored Dinoflagellate Karlodinium in Japan and Philippines, with reference to ultrastructure and micropredation of Karlodinium azanzae sp. nov. (Kareniaceae, Dinophyceae)1Benico, Garry; Takahashi, Kazuya; Lum, Wai Mun; Yñiguez, Aletta T.; Iwataki, Mitsunori (Phycological Society of America, 2020)Twenty-six cultures of the harmful marine dinoflagellate Karlodinium, isolated from Japanese and Philippine coastal waters, were examined using LM, SEM, and molecular phylogeny inferred from ITS and LSU rDNA. Seven Karlodinium species (six from Japan and four from Philippines), K. australe, K. ballantinum, K. decipiens, K. gentienii, K. veneficum, K. zhouanum, and a novel species Karlodinium azanzae sp. nov., were identified based on their morphology and phylogenetic positions.Karlodinium azanzae from Manila Bay, Philippines was further characterized by TEM, HPLC (chloroplast pigment), and bioassay on brine shrimp and other marine zooplankton. Cells of K. azanzae were the largest (mean 25.3 µm long) in Karlodinium, possessed numerous tiny reflective particles, starch grains, and lipid granules, and usually swam at the bottom of the culture vessel. The straight apical structure complex and a ventral pore were common to the genus. The longitudinally elongated nucleus was located at the center, and the yellowish chloroplasts contained an embedded pyrenoid and carotenoid pigments typical of the genus (i.e., fucoxanthin as major carotenoid with its derivatives). TEM revealed a part of the flagellar apparatus, of which the long striated ventral connective is the first report in the Kareniaceae. Phylogenetic trees showed closest affinity of K. azanzae with K. australe and K. armiger. The new species could be differentiated from related species by cell size, position of the nucleus, and characteristic swimming behavior. Lethality of K. azanzae to large zooplankton and micropredation using a developed peduncle was also observed.
- Transcriptomic profiling reveals extraordinary diversity of venom peptides in unexplored predatory gastropods of the genus ClavusLu, Aiping; Watkins, Maren; Li, Qing; Robinson, Samuel D.; Concepcion, Gisela; Yandell, Mark; Weng, Zhiping; Olivera, Baldomero M.; Safavi-Hemami, Helena; Fedosov, Alexander E. (Oxford University Press, 2020)Predatory gastropods of the superfamily Conoidea number over 12,000 living species. The evolutionary success of this lineage can be explained by the ability of conoideans to produce complex venoms for hunting, defense, and competitive interactions. Whereas venoms of cone snails (family Conidae) have become increasingly well studied, the venoms of most other conoidean lineages remain largely uncharacterized. In the present study, we present the venom gland transcriptomes of two species of the genus Clavus that belong to the family Drilliidae. Venom gland transcriptomes of two specimens of Clavus canalicularis and two specimens of Clavus davidgilmouri were analyzed, leading to the identification of a total of 1,176 putative venom peptide toxins (drillipeptides). Based on the combined evidence of secretion signal sequence identity, entire precursor similarity search (BLAST), and the orthology inference, putative Clavus toxins were assigned to 158 different gene families. The majority of identified transcripts comprise signal, pro-, mature peptide, and post-regions, with a typically short (<50 amino acids) and cysteine-rich mature peptide region. Thus, drillipeptides are structurally similar to conotoxins. However, convincing homology with known groups of Conus toxins was only detected for very few toxin families. Among these are Clavus counterparts of Conus venom insulins (drillinsulins), porins (drilliporins), and highly diversified lectins (drillilectins). The short size of most drillipeptides and structural similarity to conotoxins were unexpected, given that most related conoidean gastropod families (Terebridae and Turridae) possess longer mature peptide regions. Our findings indicate that, similar to conotoxins, drillipeptides may represent a valuable resource for future pharmacological exploration.
- Multifaceted assessment of wastewater-based epidemiology for SARS-CoV-2 in selected urban communities in Davao City, Philippines: A pilot studyOtero, Maria Catherine B.; Murao, Lyre Anni E.; Limen, Mary Antoinette G.; Caalim, Daniel Rev A.; Gaite, Paul Lorenzo A.; Bacus, Michael G.; Acaso, Joan T.; Miguel, Refeim M.; Corazo, Kahlil; Knot, Ineke E.; Sajonia, Homer; de los Reyes, Francis L.; Jaraula, Caroline Marie B.; Baja, Emmanuel S.; Del Mundo, Dann Marie N. (MDPI, 2022-07-19)Over 60 countries have integrated wastewater-based epidemiology (WBE) in their COVID-19 surveillance programs, focusing on wastewater treatment plants (WWTP). In this paper, we piloted the assessment of SARS-CoV-2 WBE as a complementary public health surveillance method in susceptible communities in a highly urbanized city without WWTP in the Philippines by exploring the extraction and detection methods, evaluating the contribution of physico-chemical–anthropogenic factors, and attempting whole-genome sequencing (WGS). Weekly wastewater samples were collected from sewer pipes or creeks in six communities with moderate-to-high risk of COVID-19 transmission, as categorized by the City Government of Davao from November to December 2020. Physico-chemical properties of the wastewater and anthropogenic conditions of the sites were noted. Samples were concentrated using a PEG-NaCl precipitation method and analyzed by RT-PCR to detect the SARS-CoV-2 N, RdRP, and E genes. A subset of nine samples were subjected to WGS using the Minion sequencing platform. SARS-CoV-2 RNA was detected in twenty-two samples (91.7%) regardless of the presence of new cases. Cycle threshold values correlated with RNA concentration and attack rate. The lack of a sewershed map in the sampled areas highlights the need to integrate this in the WBE planning. A combined analysis of wastewater physico-chemical parameters such as flow rate, surface water temperature, salinity, dissolved oxygen, and total dissolved solids provided insights on the ideal sampling location, time, and method for WBE, and their impact on RNA recovery. The contribution of fecal matter in the wastewater may also be assessed through the coliform count and in the context of anthropogenic conditions in the area. Finally, our attempt on WGS detected single-nucleotide polymorphisms (SNPs) in wastewater which included clinically reported and newly identified mutations in the Philippines. This exploratory report provides a contextualized framework for applying WBE surveillance in low-sanitation areas.The authors thank the Davao City Health Office, the local government units under the City Government of Davao, the partner hospitals for their support and assistance, and Diana Aga for discussions on sample collection and analyses. The authors would also like to thank the members of the Bortz Virology Laboratory at the University of Alaska Anchorage- Ralf Dagdag and Matthew Redlinger, Amanda Warr from the Roslin Institute, Nicole Wheeler from the University of Birmingham, Lara Urban, co-founder of PuntSeq, and Joe Russell from MRI Global for their expert advice in nanopore and wastewater sequencing.
- Anti-inflammatory activity of monosubstituted xestoquinone analogues from the marine sponge Neopetrosia compactaSusana, Shalice R.; Salvador-Reyes, Lilibeth A. (MDPI, 2022-03-22)Chronic inflammation is recognized as a contributor to multiple chronic diseases, such as cancer, cardiovascular, and autoimmune disorders. Here, a natural products-initiated discovery of anti-inflammatory agents from marine sponges was undertaken. From the screening of 231 crude extracts, a total of 30 extracts showed anti-inflammatory activity with no direct cytotoxic effects at 50 μg/mL on RAW 264.7 (ATCC®TIB-71™) murine macrophage cells stimulated with 1 μg/mL lipopolysaccharide (LPS). Bioactivity-guided purification of the anti-inflammatory extract from the sponge Neopetrosia compacta led to the isolation of xestoquinone (1), adociaquinone B (2), adociaquinone A (3), 14-hydroxymethylxestoquinone (4), 15-hydroxymethylxestoquinone (5), and an inseparable 2:1 mixture of 14-methoxyxestoquinone and 15-methoxyxestoquinone (6). Compounds 1–6 caused a concentration-dependent reduction of nitric oxide (NO) production in LPS-stimulated RAW 264.7 cells, with 4–6 having low micromolar IC50 and acceptable selectivity index. Gene expression analysis using qRT-PCR showed that 1, 5, and 6 downregulated Il1b and Nos2 expression by 2.1- to 14.8-fold relative to the solvent control at 10 μM. Xestoquinone (1) and monosubstituted analogues (4–6), but not the disubstituted adociaquinones (2 and 3), caused Nrf2 activation in a luciferase reporter MCF7 stable cells. Compounds 5 and 6 caused a modest increase in Nqo1 gene expression at 10 μM. The anti-inflammatory activity of xestoquinone (1) and monosubstituted analogues (4–6) may, in part, be mediated by Nrf2 activation, leading to attenuation of inflammatory mediators such as IL-1β and NOS2.Samples were collected under gratuitous permit numbers GP-0084-15 and GP-0123-17, issued by the Department of Agriculture of the Philippines. We thank the municipalities of Bolinao, Pangasinan, and Puerto Galera, Oriental Mindoro for permission for sample collection. We acknowledge assistance from Z. L. Malto and DDHP chemical ecology group in obtaining the mass spectrometric data and sample collection, respectively.
- Global mass spectrometric analysis reveals chemical diversity of secondary metabolites and 44-Methylgambierone production in Philippine Gambierdiscus strainsMalto, Zabrina Bernice L.; Benico, Garry A.; Batucan, Jeremiah D.; Dela Cruz, James; Romero, Marc Lawrence J.; Azanza, Rhodora V.; Salvador-Reyes, Lilibeth A. (Frontiers Media SA, 2022-02-04)Surveillance and characterization of emerging marine toxins and toxigenic dinoflagellates are warranted to evaluate their associated health risks. Here, we report the occurrence of the ciguatera poisoning-causative dinoflagellate Gambierdiscus balechii in the Philippines. Toxin production and chemical diversity of secondary metabolites in G. balechii GtoxSAM092414, G. balechii Gtox112513, and the recently reported Gambierdiscus carpenteri Gam1BOL080513 were assessed using targeted and untargeted UPLC-MS/MS analysis and radioligand receptor-binding assay (RBA). 44-methylgambierone was produced by all three strains, albeitwith different levels based on RBA and UPLC-HRMS/MS analysis. The fatty acid composition was similar in all strains, while subtle differences in monosaccharide content were observed, related to the collection site rather than the species. Molecular networking using the GNPS database identified 45 clusters belonging to at least ten compound classes, with terpene glycosides, carbohydrate conjugates, polyketides, and macrolides as major convergence points. Species-specific peptides and polyhydroxylated compounds were identified in G. balechii GtoxSAM092414 and G. carpenteri Gam1BOL080513, respectively. These provide a glimpse of the uncharacterized biosynthetic potential of benthic dinoflagellates and highlight the intricate and prolific machinery for secondary metabolites production in these organisms.We would like to thank H. Junio and the Secondary Metabolites Profiling Laboratory of the Institute of Chemistry, University of the Philippines Diliman and K. B. Davis for assistance in the conduct of this study.