Evaluation of different isotope dilution mass spectrometry strategies for the characterization of naturally abundant and isotopically labelled peptide standards.

Natural abundance and isotopically labelled tryptic peptides are routinely employed as standards in quantitative proteomics. The certification of the peptide content is usually carried out by amino acid analysis using isotope dilution mass spectrometry (IDMS) after the acid hydrolysis of the peptide. For the validation and traceability of the amino acid analysis procedure, expensive certified peptides must be employed. In this work we evaluate different IDMS alternatives which will reduce the amount of certified peptide required for validation of the amino acid analysis procedure. In this context, the characterization of both natural and isotopically labelled synthetic angiotensin I peptides was carried out. First, we applied a fast procedure for peptide hydrolysis based on microwave-assisted digestion and employed two certified peptide reference materials SRM 998 angiotensin I and CRM 6901-b C-peptide for validation of the hydrolysis procedure. The amino acids proline, leucine, isoleucine, valine, tyrosine, arginine and phenylalanine were evaluated for their suitability for peptide certification by IDMS by both liquid chromatography with tandem mass spectrometry (LC-MS/MS) and gas chromatography with mass spectrometry (GC)-MS/MS. Then, natural angiotensin I and 13C1-labelled angiotensin I were synthesized in-house and purified by preparative liquid chromatography. The concentration of the 13C1-labelled angiotensin I peptide was established by reverse IDMS in its native form using SRM 998 angiotensin I as reference. The concentration of the natural synthesized peptide was determined by IDMS both using the 13C1-labelled peptide in its native form and by amino acid analysis showing comparable results. Finally, the synthetic naturally abundant angiotensin I peptide was employed as "in-house" standard for the validation of subsequent peptide characterization procedures. Therefore, the novelty of this work relies on, first, the development of a faster hydrolysis procedure assisted by focused microwaves, providing complete hydrolysis in 150 min, and secondly, a validation strategy combining GC-MS and LC-MS/MS that allowed us to certify the purity of an in-house-synthesized peptide standard that can be employed as quality control in further experiments.

Differential impacts of health systems and sociocultural environment on vulnerable populations during the COVID-19 pandemic: lessons from four Asia-Pacific countries.

BACKGROUND: This study aims to evaluate healthcare systems and pandemic responses in relation to marginalized and vulnerable groups, identify populations requiring urgent care, and assess the differential impacts on their health during the pandemic. METHODS: Data were collected by the Asia-Pacific Observatory on Health Systems and Policies (APO)-National University of Singapore and APO-International Health Policy Program consortium members: Korea, Indonesia, Philippines, and Singapore. Data were collected through a combination of semi-structured interviews, policy document reviews, and analysis of secondary data. RESULTS: Our findings reveal that the pandemic exacerbated existing health disparities, particularly affecting older adults, women, and children. Additionally, the study identified LGBTI individuals, healthcare workers, slum dwellers, and migrant workers as groups that faced particularly severe challenges during the pandemic. LGBTI individuals encountered heightened discrimination and limited access to health services tailored to their needs. Healthcare workers suffered from immense stress and risk due to prolonged exposure to the virus and critical working conditions. Slum dwellers struggled with healthcare access and social distancing due to high population density and inadequate sanitation. Migrant workers were particularly hard hit by high risks of virus transmission and stringent, often discriminatory, isolation measures that compounded their vulnerability. The study highlights the variation in the extent and nature of vulnerabilities, which were influenced by each country's specific social environment and healthcare infrastructure. It was observed that public health interventions often lacked the specificity required to effectively address the needs of all vulnerable groups, suggesting a gap in policy and implementation. CONCLUSIONS: The study underscores that vulnerabilities vary greatly depending on the social environment and context of each country, affecting the degree and types of vulnerable groups. It is critical that measures to ensure universal health coverage and equal accessibility to healthcare are specifically designed to address the needs of the most vulnerable. Despite commonalities among groups across different societies, these interventions must be adapted to reflect the unique characteristics of each group within their specific social contexts to effectively mitigate the impact of health disparities.

Determination of 3-monoiodotyrosine and 3,5-diiodotyrosine in newborn urine and dried urine spots by isotope dilution tandem mass spectrometry.

High levels of 3-mono- and 3,5-diiodotyrosine (MIT and DIT, respectively) in urine have been related to iodotyrosine dehalogenase 1 deficiency, a type of congenital hypothyroidism. However, the determination of MIT and DIT in urine is not included in newborn screening programs performed in clinical laboratories to detect inborn errors of metabolism. We report here on the development of an analytical method for the determination of MIT and DIT in newborn urine and dried urine spots (DUS) by Liquid Chromatography Isotope Dilution tandem Mass Spectrometry (LC-IDMSMS). The development included the synthesis of 15N-monoiodotyrosine and 13C2-diiodotyrosine through the iodination of 15N-tyrosine and 13C2-tyrosine, respectively, using bis(pyridine)iodonium(I) tetrafluoroborate (IPy2BF4). Both labelled analogues were added at the beginning of the sample preparation procedure and used to develop both single- and double-spike LC-IDMS methods for the determination of MIT and DIT. The developed double spike methodology was able to quantify and correct possible MIT ↔ DIT interconversions throughout the sample preparation, which was observed for concentrated urine samples but not for DUS. Suppression matrix effects on the absolute signals of MIT and DIT were observed in urine samples but did not affect the IDMS results as recoveries on urine samples at different dilution factors could be considered quantitative. Method detection limits were 0.018 and 0.046 ng g-1 (limits of quantification 0.06 and 0.15 ng g-1) by single-spike IDMS, for MIT and DIT, respectively, in the analysis of urine samples and 0.07 and 0.05 ng g-1 (limits of quantification 0.23 and 0.17 ng g-1) for MIT and DIT, respectively, in the analysis of DUS. No significant differences were obtained for MIT concentrations in the analysis of the same newborn samples stored as liquid urine or DUS when the results were corrected for the creatinine content. Finally, 36 DUS samples from healthy newborns were analyzed and MIT was detected in all samples at low ng mg-1creatinine levels.

Gold(I)-Catalyzed Intermolecular Formal [4+2] Cycloaddition of O-Aryl Ynol Ethers and Enol Ethers: Synthesis of Chromene Derivatives.

Gold(I)-catalyzed formal [4+2] cycloaddition of O-aryl ynol ethers 1 and enol ethers 2 is described. This intermolecular reaction between two electron-rich unsaturated systems takes place, under mild conditions, in the presence of 5 mol% [IPrAu(CH3 CN)]SbF6 as catalyst giving chromene derivatives with good yields. The cycloaddition is completely regio- and stereoselective, as well as versatile for both reactives. Silyl enol ethers can also react in the same way and under the same reaction conditions with quantitative yields. A plausible mechanism through a selective addition of the enol ether to the alkyne gold activated complex followed by an intramolecular aromatic electrophilic substitution is proposed. Several experimental results support the presence of a cationic oxonium intermediate prior to the aromatic substitution. The reaction represents a new entry to the chromene core.

Gold(I)-Catalyzed [8+2]-Cycloaddition of 8-Aryl-8-azaheptafulvenes with Allenamides and Ynamides: Regioselective Synthesis of Dihydrocycloheptapyrrole Derivatives.

Gold(I)-catalyzed higher-order [8+2] cycloadditions of 8-aryl-8-azaheptafulvenes 1 with allenamides 2 and ynamides 3 were studied. 1,8-Dihydrocycloheptapyrroles 4 were achieved by a regioselective [8+2] cycloaddition of azaheptafulvenes 1 and allenamides 2 in the presence of (2,4-ditBuC6 H3 O)3 PAuNTf2 as catalyst. Besides, ynamides 3 and 8-aryl-8-azaheptafulvenes 1, undergo a regioselective [8+2] cycloaddition, to give 2-amido-1,4-dihydrocycloheptapyrroles 7 in the presence of JohnPhosAuNTf2 as catalyst. Both reactions take place with good yields and with a variety of substituents. A plausible mechanism hypothesis suggests a nucleophilic attack of the 8-azaheptafulvene to the gold activated electron rich allene or alkyne moieties of the allenamide and ynamide, respectively.

Research priority-setting for human, plant, and animal virology: an online experience for the Virology Institute of the Philippines.

BACKGROUND: Viral pandemics have had catastrophic consequences on population health and economies. The Philippine government intends to establish the Virology Institute of the Philippines, one of the key areas of which will be virology research. This project aimed to develop the institute's research agenda across the fields of human, plant, and animal virology. METHODOLOGY: Key considerations for the prioritization methodology were (1) the imminent establishment of the Virology Institute of the Philippines, (2) mobility restrictions caused by the coronavirus disease 2019 (COVID-19) pandemic, (3) the timeline to develop the research agenda, and (4) the need to separate the research agenda for the three fields of human, plant, and animal virology. The process was fully conducted online in four steps: stakeholder identification, soliciting research priorities, generating initial research priorities, and final prioritization consultations conducted on Zoom Pro. RESULTS: Twenty-eight participants attended three online consultations between 21 and 27 July 2020 through Zoom Pro. Participants selected the research prioritization criteria and its weights, and used these to evaluate the research priorities. The final research agenda covers topics in epidemiology, diagnostics, surveillance, biosafety, and genomics. CONCLUSION: This initiative resulted in the first research agenda for the Virology Institute of the Philippines across the three fields of human, plant, and animal virology. An expert-driven process which places a premium on consensus-building facilitated through online platforms was the most feasible approach to develop the research agenda. This process resulted in an agenda aligned with the mandates of national research councils but leaves gaps on areas such as emerging infectious diseases. Pre-COVID-19 literature expressed apprehensions on the online medium that weakens social ties necessary for consensus. Our experience with changing the mode of consensus-building shows that users will continually adapt to technology. Online tools are currently able to address the limitations of the virtual space.

Propargylsilanes as Reagents for Synergistic Gold(I)-Catalyzed Propargylation of Carbonyl Compounds: Isolation and Characterization of σ-Gold(I) Allenyl Intermediates.

Reported herein is the isolation and characterization, for the first time, of a σ-gold allenyl complex as an intermediate in gold catalysis. This intermediate was captured during the study of a novel gold(I)-catalyzed propargylation of carbonyl compounds with propargylsilanes. Notably, the gold-catalyzed propargylation reaction, which proceeds with aldehydes and ketones, can be driven to the formation of either homopropargyl silyl ethers or the in situ synthesis of corresponding 2-silyl-4,5-dihydrofurans.

Direct Synthesis of α-Iodoenones by IPy2BF4-Promoted Rearrangement of Propargylic Esters.

A direct access to α-iodoeones from iodonium ion and propargylic tosylates or acetates is described. Bis(pyridine) iodonium tetrafluoroborate (IPy2BF4, Barluenga's reagent) promotes the rearrangement of these propargylic alcohol derivatives in mild conditions. The transformation gives ß-unsubstituted, ß-monosubstituted, and ß,ß-disubstituted α-iodoenones in high yields. ß-Substituted α-iodoenones are obtained with excellent ( Z)-selectivity.

Gold(I)-Catalyzed Addition of Silylacetylenes to Acylsilanes: Synthesis of Indanones by C-H Functionalization through a Gold(I) Carbenoid.

A gold(I)-catalyzed synthesis of indanones from trimethylsilylacetylenes and acylsilanes is presented. The reaction is initiated through a synergistic acylsilane activation-gold acetylide formation and involves consecutive alkyne σ-gold(I) addition, π-activation, and 1,2-migration of a silyl group. Studies performed on the reaction mechanism allowed to establish the nature of the silyl migrating group and invoke the participation of a gold(I) carbenoid intermediate. The reaction is completed by a gold(I) C-H functionalization step.

Mechanistic studies on the rearrangement of 1-alkenyl-2-alkynylcyclopropanes: from allylic gold(I) cations to stable carbocations.

An allylic gold(I) cation, proposed as key intermediate in the gold-promoted rearrangement of 1,5-enynes bearing a fixed conformation, has been detected and characterized by NMR spectroscopy. Moreover, its participation in the overall transformation was confirmed. Computational studies indicate that the gold-catalyzed transformation occurs through an uncommon rearrangement. Additionally, this study led us to isolate and characterize a stable homoantiaromatic carbocation.

NMR spectroscopic study of the self-aggregation of 3-hexen-1,5-diyne derivatives.

The self-assembly of polycatenar molecules derived from 1,6-diphenyl-3,4-dipropyl-3-hexen-1,5-diyne has been studied in detail by solution NMR spectroscopy. The analysis of the concentration- and temperature-dependent evolution of the chemical shifts and the diffusion coefficients in [D12]cyclohexane agrees well with an isodesmic model of association in this solvent. The association constants for the stacking and entropy and enthalpy of the process have been obtained. The driving force for the aggregation process is provided by a negative enthalpy (ΔH), which is partially compensated by a negative entropy (ΔS). A structural study of the self-assembly in solution has been carried out with the help of NOESY NMR spectroscopic experiments.

Silylium-Catalyzed Regio- and Stereoselective Carbosilylation of Ynamides with Allylic Trimethylsilanes.

The regio- and stereoselective carbosilylation of tosylynamides with allylic trimethylsilanes takes place under mild conditions in the presence of catalytic TMSNTf2 or HNTf2 to give (Z)-α-allyl-ß-trimethylsilylenamides with good yields. Theoretical calculations show the activation of the C-C triple bond of the ynamides by the trimethylsilylium ion and formation of a ß-trimethylsilylketenimonium cation. Further transformations of the products demonstrate the synthetic utility of this reaction.

Gold-catalysed rearrangement of unconventional cyclopropane-tethered 1,5-enynes.

The synthesis of particular cyclopropane-tethered 1,5-enynes, namely 6-alkynyl-4-alkylidenebicyclo[3.1.0]hex-2-enes, enabled the discovery of unprecedented gold-catalyzed rearrangment to indenes. A computational study of the mechanism of this profound skeleton rearrangement is also disclosed.

COVID-19 public health and social measures: a comprehensive picture of six Asian countries.

The COVID-19 pandemic will not be the last of its kind. As the world charts a way towards an equitable and resilient recovery, Public Health and Social Measures (PHSMs) that were implemented since the beginning of the pandemic need to be made a permanent feature of health systems that can be activated and readily deployed to tackle sudden surges in infections going forward. Although PHSMs aim to blunt the spread of the virus, and in turn protect lives and preserve health system capacity, there are also unintended consequences attributed to them. Importantly, the interactions between PHSMs and their accompanying key indicators that influence the strength and duration of PHSMs are elements that require in-depth exploration. This research employs case studies from six Asian countries, namely Indonesia, Singapore, South Korea, Thailand, the Philippines and Vietnam, to paint a comprehensive picture of PHSMs that protect the lives and livelihoods of populations. Nine typologies of PHSMs that emerged are as follows: (1) physical distancing, (2) border controls, (3) personal protective equipment requirements, (4) transmission monitoring, (5) surge health infrastructure capacity, (6) surge medical supplies, (7) surge human resources, (8) vaccine availability and roll-out and (9) social and economic support measures. The key indicators that influence the strength and duration of PHSMs are as follows: (1) size of community transmission, (2) number of severe cases and mortality, (3) health system capacity, (4) vaccine coverage, (5) fiscal space and (6) technology. Interactions between PHSMs can be synergistic or inhibiting, depending on various contextual factors. Fundamentally, PHSMs do not operate in silos, and a suite of PHSMs that are complementary is required to ensure that lives and livelihoods are safeguarded with an equity lens. For that to be achieved, strong governance structures and community engagement are also required at all levels of the health system.

Control of self-assembly of a 3-hexen-1,5-diyne derivative: toward soft materials with an aggregation-induced enhancement in emission.

The supramolecular architectures of a fluorophore are controlled through the design of a conjugated polycatenar molecule, the self-assembly of which can be addressed toward a columnar liquid-crystalline phase and organogels. Thus, depending on the environmental conditions for self-assembly, compound CA9 organizes into an unprecedented hexagonal columnar mesophase in the condensed state, in which half a molecule constitutes the slice of the column, or into a rectangular mesomorphic-like organization in the presence of apolar solvents such as cyclohexane and dodecane, at a concentration in which fibers form and gelling conditions are fulfilled. In this Col(r)-type arrangement, the organization within the columns depends on the solvent. All of the materials prepared show luminescence, and moreover, a remarkable 3-fold increase in fluorescence intensity was observed in going from the solution to the gel state.

Gold(I)-Catalyzed One-Pot and Diastereoselective Synthesis of trans-2-Silyl-4,5-dihydrofurans from Propargylsilanes and Aldehydes.

A diastereoselective and high-yielding gold-catalyzed synthesis of trans-2-silyl-4,5-dihydrofurans is described. In addition to a sequential manner, this reaction could be performed in a one-pot procedure from propargylsilanes and aldehydes. A mechanistic proposal for the cis-trans isomerization step is formulated. To provide experimental support for this proposal, which involves ring opening/ring closing steps of the dihydrofuran, several isotopically labeled experiments, intramolecular capture of a proposed intermediate, and construction of a Hammett plot have been performed.

Asymmetric C2-C3 cyclopentannulation of the indole ring.

Chiral nonracemic alkynyl(alkoxy)carbene complexes of tungsten(0) undergo the [3 + 2] cyclization toward alpha-methylindoles and 1,6-dimethyl-1,2,3,4-tetrahydropyridine to provide 2,3-indoline-fused cyclopentenone and 2,3-piperidine-fused cyclopentenone skeletons, respectively, with very high enantiomeric purities (96-99% ee).

Capillary HPLC-ICPMS and tyrosine iodination for the absolute quantification of peptides using generic standards.

The validity of using tyrosine iodination chemistry for the absolute and generic quantification of peptides by capillary high-performance liquid chromatography (capHPLC) coupled to inductively coupled plasma mass spectrometry (ICPMS) is investigated in detail. In this approach, two iodine atoms are specifically bioconjugated to the meta positions of the aromatic ring of every tyrosine residue. Characterization studies by capHPLC with parallel ICPMS and electrospray ionization tandem mass spectrometry (ESIMS/MS) detection clearly showed that such labeling iodination reaction affords one to obtain most accurate peptide determinations (after translation of the picomoles of iodine, quantified by ICPMS in each chromatographic peak, into picomoles of the corresponding labeled peptide). It is demonstrated that only, but every, tyrosine residue present in the peptide is completely diiodinated. The excellent detection limits for iodine using ICPMS allowed robust and highly sensitive tyrosine-containing peptide quantification (480 pM, 480 amol absolute). Derivatization is easily accomplished in a water/acetonitrile solution in only 2 min. Moreover, since the signal in ICPMS is completely independent from the chemical species containing the detected element, any iodine-containing standard (e.g., iodobenzoic acid) could be used as internal standard for the absolute quantification of every iodine-labeled tyrosine-containing peptide separated and detected along the gradient. The approach was optimized for tyrosine labeling and then validated by application to the absolute quantification of the three standard peptides present in the only reference material for peptide quantity (NIST 8327) commercially available. Identification of the species quantified by ICPMS was carried out by parallel capHPLC-ESI quadrupole time-of-flight (Q/TOF) analysis and corresponded, as expected, to the diiodinated peptides. The collision-induced dissociation (CID) spectra obtained demonstrated unequivocally the specific and complete derivatization of the tyrosine residues. The obtained quantitative results closely matched the reference values reported by the National Institute of Standards and Technology (NIST). In terms of precision, the relative standard deviation was as low as 3% RSD. Finally the approach was tested for the absolute quantification of proteins using a model standard protein (beta-casein). Results agreed again with the value specified showing that this labeling reaction is compatible with tryptic digestion.

Arylation of Phe and Tyr side chains of unprotected peptides by a Suzuki-Miyaura reaction in water.

An efficient arylation in water of tyrosine and phenylalanine side chains from unprotected iodopeptides is accomplished by using Suzuki-Miyaura cross-coupling processes. The method is compatible with the hydrophilic and thermolabile nature of biologically active peptides. Also of interest, the arylated tyrosine peptides can be accessed in one-pot mode starting from native peptides.