GOVPH

Sex: Male

Education:

• Doctor of Philosophy in Organic Chemistry, University of California, Davis, 2009
• Master of Science in Natural Products Chemistry, University of Santo Tomas, 1999
• Bachelor of Science in Chemistry, University of San Agustin, 1991

Field of Specialization
Synthetic Organic Chemistry
Mass Spectrometry
Chromatography
High-Performance Liquid Chromatography
Nuclear Magnetic Resonance
Organic Chemistry Synthesis
Natural Product Chemistry
Liquid Chromatography

Researches:

Article title: The Isorhamnetin-Containing Fraction of Philippine Honey Produced by the Stingless Bee Tetragonula biroi Is an Antibiotic against Multidrug-Resistant Staphylococcus aureus
Authors: Angelica Faith L. Suarez, April Dawn G. Tirador, Zenith M. Villorente, Cathrina F. Bagarinao, et al.
Publication title: Molecules 26(6):1688, March 2021

Abstract:
Honey exhibits antibacterial and antioxidant activities that are ascribed to its diverse secondary metabolites. In the Philippines, the antibacterial and antioxidant activities, as well as the bioactive metabolite contents of the honey, have not been thoroughly described. In this report, we investigated the in vitro antibacterial and antioxidant activities of honey from Apis mellifera and Tetragonula biroi, identified the compound responsible for the antibacterial activity, and compared the observed bioactivities and metabolite profiles to that of Manuka honey, which is recognized for its antibacterial and antioxidant properties. The secondary metabolite contents of honey were extracted using a nonionic polymeric resin followed by antibacterial and antioxidant assays, and then spectroscopic analyses of the phenolic and flavonoid contents. Results showed that honey extracts produced by T. biroi exhibits antibiotic activity against Staphylococcal pathogens as well as high antioxidant activity, which are correlated to its high flavonoid and phenolic content as compared to honey produced by A. mellifera. The bioassay-guided fractionation paired with Liquid Chromatography Mass Spectrometry (LCMS) and tandem MS analyses found the presence of the flavonoid isorhamnetin (3-methylquercetin) in T. biroi honey extract, which was demonstrated as one of the compounds with inhibitory activity against multidrug-resistant Staphylococcus aureus ATCC BAA-44. Our findings suggest that Philippine honey produced by T. biroi is a potential nutraceutical that possesses antibiotic and antioxidant activities.
Full text link: https://tinyurl.com/4kvezk8a

Article title: SialoPen peptides are new cationic foldamers with remarkable cell permeability
Authors: I. Abrrey Monreala, Erik M. Contrerasa, Gary A. Waymanc, Hector C. Aguilar, et al.
Publication title: Heliyon 6(12), December 2020

Abstract:
The ability to access intracellular targets is of vital importance as the number of identified druggable intracellular targets increases every year. However, intracellular delivery poses a formidable barrier, as many potential therapeutics are impermeable to cell membranes, which hinders their practical application in drug development. Herein we present de novo-designed unnatural cell penetrating peptide foldamers utilizing a 2,3-Didehydro-2-deoxyneuraminic acid (Neu2en) scaffold. Conveniently, this scaffold is amenable to standard Fmoc-based solid-phase peptide synthesis, with the advantages of tunable secondary structures and enhanced biostability. Flow cytometry and live-cell confocal microscopy studies showed that these Neu2en-based peptides, hereinafter termed SialoPen peptides, have significantly superior uptake in HeLa and primary neuronal hippocampal cells, outperforming the classical cell permeable peptides penetratin and HIV-TAT.
Full text link: https://tinyurl.com/ym6s6hra

Article title: Bioconversion of agro-industry sourced biowaste into biomaterials via microbial factories – A viable domain of circular economy
Authors: Kee Seng Hon, Justin Brian V. Chiongson, Jonel P Saludes, Vigneswari Sevakumaran, et al.
Publication title: Environmental Pollution 271(1):116311, December 2020

Abstract:
Global increase in demand for food supply has resulted in surplus generation of wastes. What was once considered wastes, has now become a resource. Studies were carried out on the conversion of biowastes into wealth using methods such as extraction, incineration and microbial intervention. Agro-industry biowastes are promising sources of carbon for microbial fermentation to be transformed into value-added products. In the era of circular economy, the goal is to establish an economic system which aims to eliminate waste and ensure continual use of resources in a close-loop cycle. Biowaste collection is technically and economically practicable, hence it serves as a renewable carbon feedstock. Biowastes are commonly is biotransformed into value-added materials such as bioethanol, bioplastics, biofuels, biohydrogen, biobutanol and biogas. This review reveals the recent developments on microbial transformation of biowastes into biotechnologically important products. This approach addresses measures taken globally to valorize waste to achieve low carbon economy. The sustainable use of these renewable resources is a positive approach towards waste management and promoting circular economy.
Full text available upon request to the author

Article title: Marine Sediment-Derived Streptomyces Strain Produces Angucycline Antibiotics against Multidrug-Resistant Staphylococcus aureus Harboring SCCmec Type 1 Gene
Authors: Edna M. Sabido, Chuckcris P. Tenebro, Angelica Faith L. Suarez, Sarah Diane C. Ong, et al.
Publication title: Journal of Marine Science and Engineering 8(10):734, September 2020

Abstract:
The Philippine archipelago is geographically positioned in the tropics with rich areas of marine biodiversity. Its marine sediments harbor actinomycetes that exhibit antibacterial activity. Screening of actinomycetes isolated from marine sediments collected near the coast of Islas de Gigantes, Iloilo showed one isolate that exhibited high activity against the multidrug-resistant Staphylococcus aureus (MRSA) strain carrying the Staphylococcal Cassette Chromosome mec (SCCmec) type 1 gene, a biomarker for drug resistance. The isolate was identified as Streptomyces sp. strain DSD011 based on its 16s rRNA and protein-coding genes (atpD, recA, rpoB, and trpB) sequences, and was found to be a new species of salt-tolerant marine Streptomyces. Further, the strain harbors both non-ribosomal peptide synthetase (NRPS) and type II polyketide synthase (PKS) in its genome. The targeted chromatographic isolation and chemical investigations by Liquid Chromatography Mass Spectrometry-Time of Flight (LCMS-TOF), tandem mass spectrometry (MS/MS), and Global Natural Product Social molecular networking (GNPS) of the antibiotics produced by the strain afforded the two polycyclic aromatic polyketide angucycline glycosides, fridamycin A (1) and fridamycin D (2), which are products of type II PKS biosynthesis. Compounds 1 and 2 displayed antibacterial activity against MRSA with minimum inhibitory concentration (MIC) of 500 μg/mL and 62.5 μg/mL, respectively. These results suggest that the underexplored marine sediments near the coast of Islas de Gigantes, Iloilo offer access to undiscovered Streptomyces species that are invaluable sources of antibiotic leads.
Full text link: https://tinyurl.com/h42txyb2

Article title: Multimeric TAT peptides are effective in vitro inhibitors of Staphylococcus saprophyticus
Authors: Justin Brian V. Chiongson, Edna M. Sabido, Kuo‐Ging Lin, Glenn V. Alea, et al.
Publication title: Chemical Biology & Drug Design 96(5), June 2020

Abstract:
TAT (48–60) is a tridecapeptide from the envelope protein of HIV that was previously shown to possess cell‐penetrating properties and antibacterial activity, making it a potential drug delivery agent for anticancer drugs and as antibacterial compound. Previous reports indicated that dimerization enhances the desired bioactivity of TAT; hence, we sought to synthesize multimeric TAT peptides. Herein, we describe the effects of multimerization on the antibacterial activity and secondary structure of the peptide. Terminal modifications such as N‐acetylation and C‐amidation were employed in the design. TATp monomer, dimer, and tetramer were synthesized using solid‐phase peptide synthesis, purified by reversed‐phase HPLC, and then characterized by mass spectrometry. Multimerization of the peptide did not change the secondary structure conformation. The CD analysis revealed a polyproline‐II conformation for all peptide designs. Thus, this study provides a method of increasing the biological activity of the peptide by multimerization while retaining the secondary conformation of its monomeric unit. Furthermore, the bacteria Staphylococcus saprophyticus was found to be susceptible to the dimer and tetramer, with MIC50 of 12.50 μm and <1.56 μm, respectively. This suggests a structure–activity relationship whereby the antibacterial activity increases with increase in valency. We designed and synthesized Tat (48–60) peptides using Fmoc solid phase peptide synthesis to produce the monomeric TATp, dimeric TATp‐D and tetrameric TATp‐T. These peptides were tested for their antibacterial properties against Gram positive and Gram negative bacteria and were found to be effective against Staphyloccocus saprophyticus, with the MIC50 of 12.50 μm and <1.56 μm for TATp‐D and TATp‐T, respectively. Circular dichroism also revealed that there is no change in the secondary structure of the Tat peptides upon multimerization.
Full text available upon request to the author

Article title: Barriers and Recommendations for Developing a Data Commons for the Implementation and Application of Cardiovascular Disease and Diabetes Risk Scoring in the Philippines
Authors: Gerard Dumancas, Romulo de Castro, Jonel P. Saludes, Bridith Penaranda, et al.
Publication title: Current Epidemiology Reports 7(2), April 2020

Abstract:
Purpose of Review Cardiovascular diseases (CVDs) and diabetes are the primary causes of death in the Philippines. This manuscript reviewed previous studies on the use of predictive analytics for CVD and diabetes risk scoring. This paper also discussed barriers and strategies on how to access/generate available data sets for CVDs and diabetes in the country. Recent Findings CVD and diabetes risk scoring requires the availability of data sets related to such diseases. Although the Philippines has taken strides to implement the Philippine National eHealth solution, such a program does not include strategies toward the use of predictive analytics for CVD and diabetes risk scoring. Summary CVD and diabetes risk scoring research is particularly limited in the Philippines due to challenges related to costs, gaps in policies, and stakeholder involvement. A possible theoretical framework for the analysis and utilization of data sets as well as recommendations and research directions were discussed in this manuscript.
Full text available upon request to the author

Article title: Anthracycline Shunt Metabolites From Philippine Marine Sediment-Derived Streptomyces Destroy Cell Membrane Integrity of Multidrug-Resistant Staphylococcus aureus
Authors: Melissa June V. Paderog, Angelica Faith L. Suarez, Edna M. Sabido, Low Zhen Jie, et al.
Publication title: Frontiers in Microbiology 11:743, April 2020

Abstract:
The rise of antibiotic resistance (ABR) and the drying up of the pipeline for the development of new antibiotics demands an urgent search for new antibiotic leads. While the majority of clinically available antibiotics were discovered from terrestrial Streptomyces, related species from marine sediments as a source of antibiotics remain underexplored. Here, we utilized culture-dependent isolation of thirty-five marine sediment-derived actinobacterial isolates followed by a screening of their antibacterial activity against multidrug-resistant S. aureus ATCC BAA-44. Our results revealed that the crude extract of Streptomyces griseorubens strain DSD069 isolated from marine sediments collected in Romblon, Philippines displays the highest antibacterial activity, with 96.4% growth inhibition. The S. aureus ATCC BAA-44 cells treated with crude extract of Streptomyces griseorubens strain DSD069 showed cell membrane damage as demonstrated by (a) leakage and loss of vital cell constituents, including DNA and proteins, (b) irregular shrinkage of cells, and (c) increase membrane permeability. The antibiotic compounds were identified as Bisanhydroaklavinone and 1-Hydroxybisanhydroaklavinone with MIC value of 6.25 μg/mL and 50.00 μg/mL, respectively. Bisanhydroaklavinone and 1-Hydroxybisanhydroaklavinone are shunt metabolites in the biosynthesis of anticancer anthracycline derivatives namely doxorubicin, daunorubicin, and cinerubins. It is rare, however, that shunt metabolites are accumulated during fermentation of marine sediment-derived Streptomyces strain without genetic modification. Thus, our study provides evidence that natural bacterial strain can produce Bisanhydroaklavinone and 1-Hydroxybisanhydroaklavinone as antibiotic leads to combat ABR.
Full text link: https://tinyurl.com/f2echs

Article title: The transmembrane domain of prostate specific membrane antigen: Deciphering the role of the Small-XXX-Small motif in oligomerization
Authors: Brianna S. Berg, Brandan M. Cook, Jack R. Hyder, James I. Godfroy, et al.
Publication title: Cancer Research 75(15 Supplement):2456-2456, August 2015

Abstract:
Prostate cancer (PCa) is the second leading cause of cancer-related deaths among males in the United States. The prevalence of this disease and the benefits associated with early detection and treatment call for closer investigation into potential biomarkers and therapeutics targets. The high expression of the integral membrane protein called prostate specific membrane antigen (PSMA, EC 3.4.17.21) is implicated in PCa invasiveness as well as neovasculature metastasis of nonprostatic solid tumors. Little is known about the exact role of PSMA in PCa progression albeit it is one of the most validated biomarker for the diagnosis and detection of PCa. We have demonstrated that the isolated PSMA transmembrane domain (TMD) is capable of oligomerization under reducing gel electrophoresis conditions, interacting independently of extracellular domain dimerization. Following this discovery, we proceeded to determine the factor(s) responsible for its oligomerization. Since it is known that certain motifs are critical for TMD oligomerization of many transmembrane proteins, we hypothesized that the Small-XXX-Small motif in PSMA TMD may play a role in the oligomerization and subsequent activation of PSMA. Thus, to gain insight into the assembly of PSMA and the factors responsible for this assembly, we investigated if mutations at this TM motif will disrupt TM oligomerization. Our initial studies revealed that PSMA TMD has two repeat units of the Small-XXX-Small motif. This motif is known to be crucial in inducing a strong peptide self-assembly, analogous to the bitopic protein Glycophorin A. We synthesized the PSMA TMD mutant peptides using Biotage Alstra microwave peptide synthesizer, purified by high performance liquid chromatography, and characterized by MALDI-TOF mass spectrometry to establish homogeneity. SDS-PAGE gel shift assays revealed a direct correlation of oligomerization propensity and the presence of the motif, while motif knockdown led to observed loss of this property. ToxR reporter assay that qualitatively assesses relative TMD interactions revealed that oligomerization of PSMA TMD is ∼150% stronger than Glycophorin A. These findings will serve as a road map in the design of tight-binding exogenous anti-TMD peptides that target this oligomerization hot spot for possible diagnostic and therapeutic applications. Citation Format: Brianna S. Berg, Brandan M. Cook, Jack R. Hyder, James I. Godfroy, Hubert Yin, Jonel P. Saludes. The transmembrane domain of prostate specific membrane antigen: Deciphering the role of the Small-XXX-Small motif in oligomerization. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2456. doi:10.1158/1538-7445.AM2015-2456
Full text available upon request to the author

Article title: Branched dimerization of Tat peptide improves permeability to HeLa and hippocampal neuronal cells
Authors: Abrrey Monreal, Qian Liu, Katherine Tyson, Tyler Bland, et al.
Publication title: Chemical Communications 51(25), February 2015

Abstract:
A dimeric branched peptide TATp-D designed as an analogue of the HIV-Tat protein transduction domain (TATp), a prototypical cell penetrating peptide (CPP), demonstrates significantly enhanced cell uptake at 0.25 to 2.5 μM. Live cell confocal laser scanning microscopy revealed that multivalency dramatically improved the permeation potency of TATp-D to HeLa and primary hippocampal neuronal cells. The observed enhanced ability of TATp-D to translocate through the membrane is highlighted by a non-linear dependence on concentration, exhibiting the greatest uptake at sub-micromolar concentrations as compared to TATp. Multimerization via bis-Fmoc Lysine offered a synthetically straightforward method to investigate the effects of multivalent CPPs while offering orthogonal handles for cargo attachment, increasing the utility of CPPs at significantly lower concentrations.
Full text link: https://tinyurl.com/ym7hm5s7

Article title: Nipah Virus Attachment Glycoprotein Stalk C-Terminal Region Links Receptor Binding to Fusion Triggering
Authors: Qian Liu, Birgit Bradel-Tretheway, Abrrey I. Monreal, Jonel P. Saludes, et al.
Publication title: Journal of Virology, November 2014

Abstract:
Unlabelled: Membrane fusion is essential for paramyxovirus entry into target cells and for the cell-cell fusion (syncytia) that results from many paramyxoviral infections. The concerted efforts of two membrane-integral viral proteins, the attachment (HN, H, or G) and fusion (F) glycoproteins, mediate membrane fusion. The emergent Nipah virus (NiV) is a highly pathogenic and deadly zoonotic paramyxovirus. We recently reported that upon cell receptor ephrinB2 or ephrinB3 binding, at least two conformational changes occur in the NiV-G head, followed by one in the NiV-G stalk, that subsequently result in F triggering and F execution of membrane fusion. However, the domains and residues in NiV-G that trigger F and the specific events that link receptor binding to F triggering are unknown. In the present study, we identified a NiV-G stalk C-terminal region (amino acids 159 to 163) that is important for multiple G functions, including G tetramerization, conformational integrity, G-F interactions, receptor-induced conformational changes in G, and F triggering. On the basis of these results, we propose that this NiV-G region serves as an important structural and functional linker between the NiV-G head and the rest of the stalk and is critical in propagating the F-triggering signal via specific conformational changes that open a concealed F-triggering domain(s) in the G stalk. These findings broaden our understanding of the mechanism(s) of receptor-induced paramyxovirus F triggering during viral entry and cell-cell fusion. Importance: The emergent deadly viruses Nipah virus (NiV) and Hendra virus belong to the Henipavirus genus in the Paramyxoviridae family. NiV infections target endothelial cells and neurons and, in humans, result in 40 to 75% mortality rates. The broad tropism of the henipaviruses and the unavailability of therapeutics threaten the health of humans and livestock. Viral entry into host cells is the first step of henipavirus infections, which ultimately cause syncytium formation. After attaching to the host cell receptor, henipaviruses enter the target cell via direct viral-cell membrane fusion mediated by two membrane glycoproteins: the attachment protein (G) and the fusion protein (F). In this study, we identified and characterized a region in the NiV-G stalk C-terminal domain that links receptor binding to fusion triggering via several important glycoprotein functions. These findings advance our understanding of the membrane fusion-triggering mechanism(s) of the henipaviruses and the paramyxoviruses.
Full text link: https://tinyurl.com/3247judk

Article title: The transmembrane domain of prostate-specific membrane antigen: Its role in oligomerization and activation in prostate cancer and nonprostatic neovasculature
Authors: Brianna S. Berg, Brandan M. Cook, Nathan Beattie, Clifford E. Berkman, et al.
Publication title: Cancer Research 74(19 Supplement):3238-3238, October 2014

Abstract:
Prostate cancer (PCa) is the second leading cause of cancer-related deaths among American men. The high expression of the membrane protein called prostate specific membrane antigen (PSMA) is implicated in PCa invasiveness as well as neovasculature of nonprostatic solid tumors. Little is known about the exact role of PSMA in PCa progression albeit it is one of the most validated biomarker for the diagnosis and detection of PCa. The functional form of PSMA occurs as a non-covalent homodimer, which is a common motif among bitopic membrane proteins. Further, it is widely recognized that many membrane proteins participate in intracellular signal transduction through the lateral association of their transmembrane domains (TMDs), but this has not been directly demonstrated in PSMA. We hypothesize that PSMA TMD participates in oligomerization and subsequent activation of PSMA. Thus, to gain insight into the non-covalent assembly of PSMA, we aim to determine if the isolated PSMA TMD peptide forms a dimer and if the treatment of full-length PSMA with exogenous PSMA TMD peptide will perturb its properties. Studies using isolated TMDs have been widely used to probe protein-protein interactions and provide useful information on protein assembly under membrane mimetic systems. We identified the amino acid sequence and designed the isolated PSMA TMD peptide based on a known human PSMA sequence (UniProt accession code: Q04609). Two Lysine residues ware appended at both the N- and C-termini to increase the solubility of this highly hydrophobic peptide in polar solvents. Our initial studies revealed that the TMD has twenty-four residues and contains two repeat units of the Small-XXX-Small motif. This sequence is known to be crucial in inducing a strong self-assembly as observed in the bitopic protein Glycophorin A. We prepared the PSMA TMD peptide by microwave-assisted solid phase synthesis and purified by high performance liquid chromatography. The peptide was analyzed by MALDI-TOF mass spectrometry to establish homogeneity and by circular dichroism to examine its secondary structure in membrane mimetic environment. We investigated the oligomerization of the peptide using SDS-PAGE gel shift and fluorescence self-quenching assays. We tested the effect of the peptide against PSMA using an established assay. Herein we report on the oligomerization and PSMA binding properties of PSMA TMD. Our findings may shed light on the role of PSMA TMD in PSMA oligomerization and its potential as a model peptide that target transmembrane helices. It may also aid in the rational design and development of peptide probes for protein-protein interactions as well as therapeutics that target cancer- and clinically-relevant membrane proteins. Citation Format: Brianna S. Berg, Brandan M. Cook, Nathan Beattie, Cliff E. Berkman, Jonel P. Saludes. The transmembrane domain of prostate-specific membrane antigen: Its role in oligomerization and activation in prostate cancer and nonprostatic neovasculature.
Full text available upon request to the author

Article title: Acute Stressor Exposure Modifies Plasma Exosome-Associated Heat Shock Protein 72 (Hsp72) and microRNA (miR-142-5p and miR-203)
Authors: Lida A. Beninson, Peter N. Brown, Alice B. Loughridge, Jonel P. Saludes, et al.
Publication title: PLoS ONE 9(9):e108748, September 2014

Abstract:
Exosomes, biologically active nanoparticles (40–100 nm) released by hematopoietic and non-hematopoietic cells, contain a variety of proteins and small, non-coding RNA known as microRNA (miRNA). Exposure to various pathogens and disease states modifies the composition and function of exosomes, but there are no studies examining in vivo exosomal changes evoked by the acute stress response. The present study reveals that exposing male Fisher 344 rats to an acute stressor modulates the protein and miRNA profile of circulating plasma exosomes, specifically increasing surface heat shock protein 72 (Hsp72) and decreasing miR-142-5p and -203. The selected miRNAs and Hsp72 are associated with immunomodulatory functions and are likely a critical component of stress-evoked modulation of immunity. Further, we demonstrate that some of these stress-induced modifications in plasma exosomes are mediated by sympathetic nervous system (SNS) activation of alpha-1 adrenergic receptors (ADRs), since drug-mediated blockade of the receptors significantly attenuates the stress-induced modifications of exosomal Hsp72 and miR-142-5p. Together, these findings demonstrate that activation of the acute stress response modifies the proteomic and miRNA profile of exosomes released into the circulation.
Full text link: https://tinyurl.com/49dmuh75

Article title: One-pot SSA-catalyzed β-elimination: An efficient and inexpensive protocol for easy access to the glycal of sialic acid
Authors: Erickson M. Paragas, Abrrey Monreal, Chris M. Vasil, Jonel P Saludes, et al.
Publication title: Carbohydrate Research 402, September 2014

Abstract:
Neu5Ac2en1Me per-OAc, the fully protected glycal of sialic acid, is a key intermediate in the discovery of therapeutics and diagnostics, including anti-influenza drugs and proteolysis resistant peptidomimetic foldamers. The synthesis of this sialic acid derivative, however, still relies on standard sugar chemistry that utilizes multi-step methodologies. Herein we report a facile and highly efficient microwave-assisted preparation of Neu5Ac1Me using silica sulfuric acid (SSA) as solid-supported acid catalyst that is one- to two-orders of magnitude faster than standard procedures. We also describe the microwave-assisted and SSA-catalyzed one-pot, rapid, solvent free reaction that combines both peracetylation and β-elimination reactions in one step to generate the glycal from Neu5Ac1Me. We coined the term One-pot SSA-catalyzed Technology for β-Elimination Protocol (OneSTEP) to describe this least laborious, most efficient, and practical preparation to date of Neu5Ac2en1Me per-OAc in terms of yield, time, reagent cost, and waste generation. Copyright © 2014 Elsevier Ltd. All rights reserved.
Full text link: https://tinyurl.com/vzhu7rxd

Article title: Exposure to acute stress modifies plasma exosomes capable of improving host defense to bacterial challenge
Authors: L.A. Beninson, Jay Campisi, Peter N. Brown, Alice B Loughridge, et al.
Publication title: Brain Behavior and Immunity 40:e4, September 2014

Abstract:
Exosomes, biologically active nanoparticles (40–100 nm) released by hematopoietic and non-hematopoietic cells, contain a variety of proteins and small, non-coding RNA known as microRNA (miRNA). Exposure to various pathogens and disease states modifies the composition and function of exosomes, but recent studies reveal that the acute stress response can also evoke in vivo exosomal changes in the absence of disease. The present study demonstrates that exposing male Fisher 344 rats to an acute stressor modulates proteins and miRNA in their circulating plasma exosomes, specifically up-regulating heat shock protein 72 (Hsp72) and decreasing miR-142-5p and −203 expression. The selected miRNAs and Hsp72 are associated with immunomodulatory functions and are likely a critical component of stress-enhanced immunity. Further, we demonstrate that some of these stress-induced modifications in plasma exosomes are mediated by sympathetic nervous system (SNS) activation of alpha-1 adrenergic receptors (ADRs), since drug-mediated blockade of the receptors significantly attenuates the stress-induced modifications of exosomal Hsp72 and miR-142-5p. Moreover, in vivo analysis reveals that administering rats stress modified plasma exosomes improves their inflammatory response to subcutaneous injections of Escherichia coli (E. coli) compared to rats administered non-stressed plasma exosomes or saline. Together, these findings suggest that activation of the acute stress response potentiates innate immunity by modifying the proteomic and miRNA profile of exosomes released into the circulation. Supported by NSF IOS 1022451.
Full text available upon request to the author

Article title: A facile microwave-assisted protocol for rapid synthesis of N-acetylneuraminic acid congeners
Authors: Jonel P. Saludes, Dhananjaya Sahoo, Abrrey Monreal, et al.
Publication title: New Journal of Chemistry 38(2):507-510, February 2014

Abstract:
We developed a simple, rapid and efficient microwave irradiation-assisted protocol that is 1- to 2-orders of magnitude faster than conventional techniques, providing an expedient access to the sialic acid congeners Neu5Ac1Me (1), Neu5Acβ1,2Me2 (2), Neu5Ac1Me O-peracetate (3) and 4,5-oxazoline of Neu5Ac2en1Me O-peracetate (4).
Full text link: https://tinyurl.com/y4tbpsuj

Article title: Solution Phase Conformation and Proteolytic Stability of Amide-Linked Neuraminic Acid Analogues
Authors: Jonel P. Saludes, Travis Q. Gregar, Abrrey Monreal, Brandan M Cook, et al.
Publication title: Biopolymers 99(10), October 2013

Abstract:
Amide-linked homopolymers of sialic acid offer the advantages of stable secondary structure and increased bioavailability making them useful constructs for pharmaceutical design and drug delivery. Defining the structural characteristics that give rise to secondary structure in aqueous solution is challenging in homopolymeric material due to spectral overlap in NMR spectra. Having previously developed computational tools for heteroologomers with resolved spectra, we now report that application of these methods in combination with circular dichroism, NH/ND NMR exchange rates and nOe data has enabled the structural determination of a neutral, δ-amide-linked homopolymer of a sialic acid analogue called Neu2en. The results show that the inherent planarity of the pyranose ring in Neu2en brought about by the α,β-conjugated amide bond serves as the primary driving force of the overall conformation of the homooligomer. This peptide surrogate has an excellent bioavailability profile, with half-life of ~12 hours in human blood serum, which offers a viable peptide scaffold that is resistant to proteolytic degradation. Furthermore, a proof-of-principle study illustrates that Neu2en oligomers are functionalizable with small molecule ligands using 1,3-dipolar cycloaddition chemistry.
Full text available upon request to the author

Article title: Multivalency Amplifies the Selection and Affinity of Bradykinin-Derived Peptide for Lipid Nanovesicles
Authors: Jonel P. Saludes, Leslie A. Morton, Sara K. Coulup, Zeno Fiorini, et al.
Publication title: Molecular BioSystems 9(8), May 2013

Abstract:
The trimer of a bradykinin derivative displayed a more than five-fold increase in binding affinity for phosphatidylserine-enriched nanovesicles as compared to its monomeric precursor. The nanovesicle selection is directly correlated with multivalency, which amplifies the electrostatic attraction. This strategy may lead to the development of novel molecular probes for detecting highly curved membrane bilayers.
Full text available upon request to the author

Article title: Computationally Designed Peptide Inhibitors of the Ubiquitin E3 Ligase SCF Fbx4
Authors: Junglim Lee, Deanne Sammond, Zeno Fiorini, Jonel P. Saludes
Publication title: ChemBioChem 14(4):445-51, March 2013

Abstract:
A structure-based computational approach was used to rationally design peptide inhibitors that can target an E3 ligase (SCF(Fbx4) )-substrate (TRF1) interface and subsequent ubiquitylation. Characterization of the inhibitors demonstrates that our sequence-optimization protocol results in an increase in peptide-TRF1 affinity without compromising peptide-protein specificity.
Full text available upon request to the author

Article title: Detection of Highly Curved Membrane Surfaces Using a Cyclic Peptide Derived from Synaptotagmin-I
Authors: Jonel P. Saludes, Leslie A Morton, Nilanjan Ghosh, Lida A Beninson
Publication title: ACS Chemical Biology 7(10):1629-35, July 2012

Abstract:
The generation of highly curved membranes is essential to cell growth, division, and movement. Recent research in the field is focused to answer questions related to the consequences of changes in the topology of the membrane once it is created, broadly termed as membrane curvature sensing. Most probes that are used to study curvature sensing are intact membrane active proteins such as DP1/Yop1p, ArfGAP1, BAR domains, and Synaptotagmin-I (Syt1). Taking a cue from nature, we created the cyclic peptide C2BL3C based on the membrane penetration C2B loop 3 of Syt1 via "Click" chemistry. Using a combination of spectroscopic techniques, we investigated the peptide-lipid interactions of this peptide with synthetic phospholipid vesicles and exosomes from rat blood plasma. We found that the macrocycle peptide probe was selective for lipid vesicles with highly curved surfaces (d < 100 nm). These results suggested that C2BL3C functions as a selective detector of highly curved phospholipid bilayers.
Full text available upon request to the author

Article title: Constant Pressure-controlled Extrusion Method for the Preparation of Nano-sized Lipid Vesicles
Authors: Leslie A. Morton, Jonel P. Saludes, Hang Hubert Yin
Publication title: Journal of Visualized Experiments 64(64), June 2012

Abstract:
Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform to study protein-protein and protein-lipid interactions, monitor drug delivery, and encapsulation. Phospholipids naturally create curved lipid bilayers, distinguishing itself from a micelle. Liposomes are traditionally classified by size and number of bilayers, i.e. large unilamellar vesicles (LUVs), small unilamellar vesicles (SUVs) and multilamellar vesicles (MLVs). In particular, the preparation of homogeneous liposomes of various sizes is important for studying membrane curvature that plays a vital role in cell signaling, endo- and exocytosis, membrane fusion, and protein trafficking. Several groups analyze how proteins are used to modulate processes that involve membrane curvature and thus prepare liposomes of diameters <100 - 400 nm to study their behavior on cell functions. Others focus on liposome-drug encapsulation, studying liposomes as vehicles to carry and deliver a drug of interest. Drug encapsulation can be achieved as reported during liposome formation. Our extrusion step should not affect the encapsulated drug for two reasons, i.e. (1) drug encapsulation should be achieved prior to this step and liposomes should retain their natural biophysical stability, securely carrying the drug in the aqueous core. These research goals further suggest the need for an optimized method to design stable sub-micron lipid vesicles. Nonetheless, the current liposome preparation technologies (sonication, freeze-and-thaw, sedimentation) do not allow preparation of liposomes with highly curved surface (i.e. diameter <100 nm) with high consistency and efficiency, which limits the biophysical studies of an emerging field of membrane curvature sensing. Herein, we present a robust preparation method for a variety of biologically relevant liposomes. Manual extrusion using gas-tight syringes and polycarbonate membranes, is a common practice but heterogeneity is often observed when using pore sizes <100 nm due to due to variability of manual pressure applied. We employed a constant pressure-controlled extrusion apparatus to prepare synthetic liposomes whose diameters range between 30 and 400 nm. Dynamic light scattering (DLS), electron microscopy and nanoparticle tracking analysis (NTA) were used to quantify the liposome sizes as described in our protocol, with commercial polystyrene (PS) beads used as a calibration standard. A near linear correlation was observed between the employed pore sizes and the experimentally determined liposomes, indicating high fidelity of our pressure-controlled liposome preparation method. Further, we have shown that this lipid vesicle preparation method is generally applicable, independent of various liposome sizes. Lastly, we have also demonstrated in a time course study that these prepared liposomes were stable for up to 16 hours. A representative nano-sized liposome preparation protocol is demonstrated below.
Full text link: https://tinyurl.com/sunhxj23

Article title: Exosomes in motion: Visualizing cancer metastasis via the Synaptotagmin C2B domain
Authors: Jonel P. Saludes, Leslie A. Morton, Nilanjan Ghosh, Lida Beninson
Publication title: Cancer Research 72(8 Supplement):4743-4743, June 2012

Abstract:
Exosomes play pivotal roles in intercellular communication and cancer progression. Recent findings have shown exosomes to incite proangiogenesis and prepare lymph nodes as remote niches for accumulation and migration of melanoma cells. Exosomes present a highly curved morphology with a size range of ∼30-100 nm that is distinct from other lipid vesicles. Furthermore, exosomes have the potential as a general metastasis biomarker since their increased secretion in the peripheral blood has been correlated with cancer metastasis. Since aqueous suspensions of exosomes cannot be seen by conventional optical methods, we hypothesized that a molecular probe that binds to exosomes could be a novel tool for detecting these vesicles. We aim to develop a minimally-invasive diagnostic tool to detect and measure secreted exosomes as biomarkers of cancer metastasis that may help clinicians and patients decide on an appropriate therapeutic action. Taking a cue from nature and using solid phase ‘Click’ chemistry as a technique for peptide modification, we created a fluorophore-tagged cyclic peptide based on the membrane penetrating C2B domain of Synaptotagmin I. We investigated the peptide-lipid interactions using synthetic liposomes as preliminary models of exosomes through a combination of spectroscopic techniques. We found that the peptide probe was selective for highly curved liposomes (d <100 nm) and does not bind to low curvature liposomes (d >100 nm). We tested blood plasma samples from rat models to find out if our preliminary findings would translate to the detection of exosomes. Our results showed that blood plasma treated with the peptide probe showed fluorescence intensity that was remarkably higher than the untreated peptide, which indicated binding to exosomes. Furthermore, we used a real time nanoparticle tracking analysis system to visualize the exosomes in a dynamic and physiologically-relevant medium. The peptide-treated exosomes were detected as fast-moving, fluorescent particles even in the complex matrix of blood plasma. This technique provided us with a platform to study the size distribution and concentration of exosomes in blood plasma. We have created a peptide probe that selectively binds to exosomes from an animal model. The investigation of the exosome binding property of this probe on clinically relevant samples is ongoing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4743. doi:1538-7445.AM2012-4743
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Article title: Multivalent dendrimeric peptides as new biomarker probes for the detection of cancer metastasis
Authors: Sara K. Coulup, Jonel P. Saludes, Hang Hubert Yin
Publication title: Cancer Research 72(8 Supplement):4745-4745, June 2012

Abstract:
Malignant tumor cells overexpress and release lipid vesicles called exosomes into the body fluids to facilitate their movement and metastasis to other parts of the body. It was found that an increased secretion of exosomes in the peripheral blood is correlated with lung cancer and melanoma metastasis. The hallmarks of exosomes are their highly curved surface (d = ∼30-100 nm) that is distinct from other extracellular lipid vesicles and their enrichment with the anionic lipid phosphatidylserine (PS) in the outer leaflet of the membrane bilayer. These properties provide an opportunity in selectively targeting exosomes as potential cancer biomarkers. Current clinical methods that are being used to classify metastatic patients are not accurate or reproducible, are often problematic, and do not indicate whether or not treatment is reducing metastasis. Furthermore, these methods involve invasive procedures. We aim to develop a novel, minimally invasive, peptide-based diagnostic tool to detect and visualize exosomes and measure exosome oversecretion in cancer patients. The successful creation of this diagnostic tool for cancer metastasis will help in the proper diagnosis and therapy for cancer patients. We chose the small peptide Bradykinin (BK) as our core molecule because it was recently reported that this peptide differentially interacts with nanometer size synthetic lipid vesicles, with preferential binding for vesicles with higher anionic lipid composition. Our earlier studies on a BK analogue showed that it selectively binds to synthetic lipid vesicles with d = 55 +/− 5 nm, dimensions that mimic the size of exosomes. We hypothesized that by creating a library of multivalent, dendrimeric analogues of BK, we could increase its binding affinity to lipid vesicle models that would translate to the detection of exosomes in body fluids. The peptide was prepared by microwave-assisted solid phase synthesis, was modified with Lys-Gly spacers, the epsilon-amino of the Lys residue was deprotected and converted into an azide, and the peptide was labeled with a fluorophore, cleaved from the resin, and purified by high performance liquid chromatography. We also synthesized a series of dendrimer-type, alkyne functionalized, aromatic small molecules as scaffolds in the preparation of the multivalent dendrimeric peptides. The azide-functionalized BK analogue was conjugated to the scaffolds using solution phase Copper-catalyzed azide-alkyne cycloaddition reaction (‘Click’ chemistry). We investigated the curvature sensing ability of the peptides using synthetic liposomes and exosomes from the plasma of a rat model using a combination of spectroscopic techniques. The lipid vesicle detecting properties of these peptides and their potential as new diagnostic tool for cancer metastasis shall be reported herein
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Article title: Investigation of homo-oligomeric interface and binding hotspot of Latent membrane protein-1 (LMP-1) of Epstein-Barr virus (EBV)
Authors: Guiin Lee, Deanne Sammond, Catherine Joce, Ryan Takeshita, et al.
Publication title: Rapid Communication in Photoscience 1(2), June 2012

Abstract:
A human herpesvirus, Epstein Barr virus (EBV), establishes lifelong infection through memory B cells and often ‐ leads to lymphoid malignancies and lymphoproliferative syndromes. Although the detailed mechanism of LMP 1 ‐ activity is not clearly known, previous studies indicate that EBV uses the viral latent membrane protein 1 (LMP 1) for B lymphocyte immortalization. Our study demonstrates the fifth transmembrane helix (TM5) of ‐ LMP 1 form homotrimeric complexes. The polar aspartic acid residue (D150) of TM5 embedded in the membrane ‐ mediates the self association of TM5. ‐ In vivo and in vitro studies indicate that the trimerization of TM5 plays a key role in constitutive activation of signaling of LMP 1. In addition, we developed small molecule inhibitors ‐ specifically disrupting the TM5 trimerization, suggesting a new strategy for drug development targeting transmembrane protein protein interactions.
Full text link: https://tinyurl.com/495dup5v

Article title: Targeting the lateral interactions of transmembrane domain 5 of Epstein–Barr virus latent membrane protein 1
Authors: Xiaohui Wang, Jonel P. Saludes, Tina X. Zhao, Adam Csakai, et al.
Publication title: Biochimica et Biophysica Acta 1818(9):2282-9, May 2012

Abstract:
The lateral transmembrane protein-protein interaction has been regarded as "undruggable" despite its importance in many biological processes. The homo-trimerization of transmembrane domain 5 (TMD-5) of latent membrane protein 1 (LMP-1) is critical for the constitutive oncogenic activation of the Epstein-Barr virus (EBV). Herein, we report a small molecule agent, NSC 259242 (compound 1), to be a TMD-5 self-association disruptor. Both the positively charged acetimidamide functional groups and the stilbene backbone of compound 1 are essential for its inhibitory activity. Furthermore, cell-based assays revealed that compound 1 inhibits full-length LMP-1 signaling in EBV infected B cells. These studies demonstrated a new strategy for identifying small molecule disruptors for investigating transmembrane protein-protein interactions.
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Article title: Ptilomycalin A inhibits laccase and melanization in Cryptococcus neoformans
Authors: Doralyn Dalisay, Jonel P. Saludes, Tadeusz F. Molinski
Publication title: Bioorganic & Medicinal Chemistry 19(22):6654-7, May 2011

Abstract:
The antifungal spirocyclic guanidine alkaloid, ptilomycalin A, from marine sponge Monanchora arbuscula, inhibits melanogenesis of Cryptococcus neoformans in vitro through inhibition of biosynthesis of laccase in the melanin biosynthetic pathway with an IC(50) of 7.3 μM.
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Article title: The Remarkable Stability of Chimeric, Sialic Acid-derived α/δ-Peptides in Human Blood Plasma
Authors: Jonel P. Saludes, Arutselvan Natarajan, Sally J. DeNardo, Jacquelyn Gervay-Hague
Publication title: Chemical Biology & Drug Design 75(5):455-60, May 2010

Abstract:
Peptides are labile toward proteolytic enzymes, and structural modifications are often required to prolong their metabolic half-life and increase resistance. One modification is the incorporation of non-alpha-amino acids into the peptide to deter recognition by hydrolytic enzymes. We previously reported the synthesis of chimeric alpha/delta-peptides from glutamic acids (Glu) and the sialic acid derivative Neu2en. Conformational analyses revealed these constructs adopt secondary structures in water and may serve as conformational surrogates of polysialic acid. Polysialic acid is a tumor-associated polysaccharide and is correlated with cancer metastasis. Soluble polysialic acid is rapidly cleared from the blood limiting its potential for vaccine development. One motivation in developing structural surrogates of polysialic acid was to create constructs with increased bioavailability. Here, we report plasma stability profiles of Glu/Neu2en alpha/delta-peptides. DOTA was conjugated at the peptide N-termini by solid phase peptide synthesis, radiolabeled with (111)In, incubated in human blood plasma at 37 degrees C, and their degradation patterns monitored by cellulose acetate electrophoresis and radioactivity counting. Results indicate that these peptides exhibit a long half-life that is two- to three-orders of magnitude higher than natural alpha-peptides. These findings provide a viable platform for the synthesis of plasma stable, sialic acid-derived peptides that may find pharmaceutical application.
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Article title: P19-15. Synthesis of multivalent mimotopes as potential vaccine candidates
Authors: JG Go, Jonel P. Saludes, Jessica Hoch Brown, JW Oliver, et al.
Publication title: Retrovirology 6(Suppl 3), October 2009

Abstract:
No available
Full text link: https://tinyurl.com/ypt9kdve

Article title: Synthesis and Structural Characterization of Sialic Acid−Glutamic Acid Hybrid Foldamers as Conformational Surrogates of α-2,8-Linked Polysialic Acid
Authors: Jonel P. Saludes, J.B. Ames, Jacquelyn Gervay-Hague
Publication title: Journal of the American Chemical Society 131(15):5495-505, April 2009

Abstract:
Surface expression of alpha-(2,8)-linked polymers of sialic acid in adult tissues has been correlated with metastasis of several human cancers. One approach to chemotherapeutic intervention against the spread of these cancers involves the development of immunogenic molecules that elicit an antibody response against alpha-(2,8)-linked polysialic acids. Naturally occurring polysialic acids are not viable candidates because they are present during embryonic development and are recognized as self by the immune system. These natural polymers also have poor pharmacokinetic properties because they are readily degraded by neuraminidase enzymes. We have been interested in developing structural surrogates of polysialic acids in an effort to overcome these limitations. Reported herein are microwave-assisted solid-phase peptide syntheses and structural characterization studies of a series of alpha/delta hybrid peptides derived from Fmoc-Neu2en and Fmoc-Glu(OtBu)-OH. Conformational experiments including circular dichroism, NH/ND exchange, and ROESY in aqueous solution were performed to study the secondary structures of these hybrid foldamers. ROESY data were analyzed with the assistance of XPLOR-NIH that was modified to include parameter and topology files to accommodate unnatural amino acids and the delta amide linkages. The results indicate that stable secondary structure is dependent upon both the amino acid sequence and the configuration of Glu. The most stable foldamer was composed of a total of 6 residues beginning with L-Glu at the carboxy terminus and alternating Neu2en and L-Glu residues. In water, this foldamer adopts a right-handed helical conformation with 3.7 residues per turn, 7.4 A pitch, 5.8 A diameter, and a length of 18.5 A, which is stabilized by both classical C=O...H-N backbone interactions and by pyranose ring O and L-Glu HN H-bonding. These structural features orient the L-Glu carboxylates along the helical backbone with a periodicity that matches the carboxylate positions along the reported G2(+) left-handed helix of alpha-(2,8)-polysialic acid. However, the charge density of the foldamer is one-half that of the natural polymer. These findings provide a fundamental understanding of the factors that influence stable secondary structure in hybrid Neu2en/Glu systems, and the tools we have developed establish a viable platform for the rational design of alpha-(2,8)-polysialic acid surrogates.
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Article title: Drug development from marine natural products
Authors: Tadeusz F. Molinski, Doralyn Dalisay, Sarah C. Lievens, Jonel P. Saludes
Publication title: Nature Reviews Drug Discovery 8(1):69-85, January 2009

Abstract:
Drug discovery from marine natural products has enjoyed a renaissance in the past few years. Ziconotide (Prialt; Elan Pharmaceuticals), a peptide originally discovered in a tropical cone snail, was the first marine-derived compound to be approved in the United States in December 2004 for the treatment of pain. Then, in October 2007, trabectedin (Yondelis; PharmaMar) became the first marine anticancer drug to be approved in the European Union. Here, we review the history of drug discovery from marine natural products, and by describing selected examples, we examine the factors that contribute to new discoveries and the difficulties associated with translating marine-derived compounds into clinical trials. Providing an outlook into the future, we also examine the advances that may further expand the promise of drugs from the sea.
Full text link: https://tinyurl.com/c3wrr6s5

Article title: Investigation into an efficient synthesis of 2,3-dehydro-N-acetyl neuraminic acid leads to three decarboxylated sialic acid dimers
Authors: Evan J. Horn, Jonel P. Saludes, Jacquelyn Gervay-Hague
Publication title: Carbohydrate Research 343(5):936-40, May 2008

Abstract:
Sialic acid, an important carbohydrate found incorporated on the cell surface of many organisms, has been modified for use in a wide range of biological and pharmaceutical applications. We hypothesized that 4,7,8,9-tetra-O-acetyl-2-deoxy-2,3-dehydro-N-acetyl neuraminic acid methyl ester (4) could be efficiently synthesized in a one-pot reaction by heating peracetylated sialic acid (2) in pyridine and acetic anhydride to induce beta-elimination. When reduced to practice, this reaction produced only modest yields of 4. Six compounds, including three new decarboxylated sialic acid dimers, were also found to have been synthesized in the reaction. In an effort to better understand the chemistry and the mechanisms of this reaction, all of the side products were isolated and fully characterized.
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Article title: Occurrence of the alpha-Glucosidase Inhibitor 1,4-Dideoxy-1,4-imino- d -arabinitol and Related Iminopentitols in Marine Sponges
Authors: Jonel P. Saludes, Sarah C. Lievens, Tadeusz F. Molinski
Publication title: Journal of Natural Products 70(3):436-438, March 2007

Abstract:
The alpha-glucosidase inhibitor 1,4-dideoxy-1,4-imino-D-arabinitol (1) was isolated from two marine sponges collected in Western Australia and shown by LC-MS to be responsible for the alpha-glycosidase inhibitory activity in different sponge extracts collected over a wide geographic area. The configuration of 1 was determined by application of Marfey's method. The two most inhibitory extracts contained only 1, while the less inhibitory extracts contained 1,4-dideoxy-1,4-imino-D-xylitol (2) or the putative diastereomeric imino pentitols 3 and 4. The least active or inactive extracts showed no detectable imino pentitols. While both 1 and 2 are known from plants, this is the first report on the isolation and detection of 1 and 2 in marine invertebrates.
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Article title: Antitubercular constituents from the hexane fraction of Morinda citrifolia Linn. (Rubiaceae)
Authors: Jonel P. Saludes, Mary J. Garson, Scott G. Franzblau, Alicia Aguinaldo
Publication title: Phytotherapy Research 16(7):683-5, November 2002

Abstract:
A crude ethanol extract and hexane fraction from Morinda citrifolia Linn. (Rubiaceae) show antitubercular activity. The major constituents of the hexane fraction are E-phytol, cycloartenol, stigmasterol, beta-sitosterol, campesta-5,7,22-trien-3beta-ol and the ketosteroids stigmasta-4-en-3-one and stigmasta-4-22-dien-3-one. E-Phytol, a mixture of the two ketosteroids, and the epidioxysterol derived from campesta-5,7,22-trien-3beta-ol all show pronounced antitubercular activity.
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