Analysis of Worldwide Greenhouse and Carbon Monoxide Gas Emissions: Which Countries Exhibit a Special Pattern? A Closer Look via Twitter.

There is significant global concern about the harmful effects of greenhouse gas and carbon monoxide emissions (deforestation, air pollution, global warming, etc.). The 2015 Paris Agreement on climate change aspires to reduce global warming by achieving a climate-neutral world. Research has been carried out to calculate and diminish the aforementioned emissions in waste, power industry, transport, building, in addition to other areas. The aim of this paper is to analyse the carbon and greenhouse gas emissions across countries around the globe in order to find patterns and correlate them to socio-economic indicators [gross national income (GNI), industrial production (IPI) and human development indexes (HDI)] as well as Twitter interactions regarding climate change. For this purpose, time series and socio-economic data have been downloaded from different repositories including EDGAR (Emissions Database for Global Atmospheric Research), World Bank and UNDP (United Nations Development Programme). Although classical clustering algorithms have already been used in the examination of some environmental issues, we use a non-parametric time series clustering method, which has been suggested in certain scientific literature as a more flexible approach, since any ad hoc parametric assumptions are required. The chosen socio-economic indicators have also demonstrated their relevance in pieces of research related to various fields. With respect to Twitter, which is one of the most popular social networks nowadays, significant analysis has also been performed on the basis of capturing citizens' perceptions on a multitude of matters. We found that several countries such as Brazil, India, China, Nigeria, Russia, United States, Spain, Andorra, Greece, and Qatar show differences in carbon and greenhouse gas emissions patterns. Besides, there does not seem to be a correlation between GNI, IPI and HDI as well as the above mentioned emissions ( correlation < 0.16 ) . Regarding Twitter interactions, a dissimilarity in the distribution of hashtags was detected between the aforementioned countries and the rest of the world. This research can help to identify countries in which more governmental measures are needed to reduce the type of emissions analysed in certain industrial sectors. In addition, it points out the topics related to climate change that seem to generate the most debate on Twitter for countries with an unusual pattern. Supplementary Information: The online version contains supplementary material available at 10.1007/s41742-023-00510-4.

Caracterización de la población que consulta por epistaxis al servicio de otorrinolaringología del Hospital Universitario Mayor Méderi (HUM) / Characterization of the population that consults by epistaxis to the Otolaryngology Service of the Hospital Universitario Mayor Méderi (HUM)

Introducción: la epistaxis es uno de los principales motivos de consulta por urgencias en el servicio de otorrinolaringología (ORL). La mayoría de los episodios se originan en el septo nasal anterior y su tratamiento tiende a ser conservador. Objetivo: caracterizar la población que consulta por epistaxis al servicio de otorrinolaringología del Hospital Universitario Mayor Méderi (HUM) entre marzo de 2017 y febrero de 2018. Metodología: estudio observacional, descriptivo y de corte transversal, con una recolección de datos de forma retrospectiva de pacientes que ingresan a urgencias del HUM por epistaxis, registrados en la base de datos del servicio de otorrinolaringología. Se analizaron datos demográficos, localización del sangrado, tratamiento, comorbilidades, fármacos y cifras tensionales. Resultados: un total de 587 pacientes; el 57 % son mujeres, con un promedio de 66 años +/- 16,15 años. La comorbilidad más frecuente fue la hipertensión arterial (60 %) y el 26 % de los pacientes presentaron cifras tensionales >140/90 mm Hg. En antecedentes farmacológicos, el 64,9 % pertenecían al grupo de antihipertensivos y el 37,6 % a los antiagregantes. La localización de los sangrados fue anterior (> área II de Cottle). El principal tratamiento fue el taponamiento nasal anterior con gasa (36,4 %), seguido por la cauterización con nitrato de plata (36,1 %). Conclusiones: en el HUM, los pacientes que consultan por epistaxis son adultos de edad media y avanzada, cuyo sangrado nasal tiene una localización anterior, que solamente requirieron tratamiento médico. Se requieren otros estudios para determinar la modalidad de tratamiento más efectiva según la gravedad de la epistaxis.

Background: epistaxis is one of the main reasons for emergency consultations in the Otolaryngology department. Most episodes originate in the anterior nasal septum and their management tends to be conservative. Aim: characterize the population that consults by epistaxis to the Otolaryngology department of the Hospital Universitario Mayor Méderi (HUM) between March 2017 and February 2018. Methods: observational, descriptive, cross-sectional study, with retrospective data collection of patients admitted to the emergency department of the HUM by epistaxis, registered in the database of the Otolaryngology Department. Demographic data, location of bleeding, treatment, comorbidities, drugs, and blood pressure were analyzed. Results: a total of 587 patients; 57 % women with an average of 66 years +/- 16.15 years. The most frequent comorbidity was arterial hypertension (60 %) and 26 % of the patients presented blood pressure levels >140/90 mm Hg. In pharmacological history, 64.9 % belonged to the group of antihypertensives and 37.6 % to antiplatelet agents. The location of the bleeds was anterior (> Cottle area II). The main treatment was the anterior nasal gauze packing (36.4 %), followed by silver nitrate cauterization (36.1 %). Conclusions: in the HUM, patients who consult for epistaxis are middle-aged and elderly adults, with anterior localization of nasal bleeding, which only required medical management. Other studies are required to determine the most effective treatment modality according to the severity of epistaxis.

Induction of DNA Hydroxymethylation Protects the Brain After Stroke.

Background and Purpose- Epigenetics play a significant role in brain pathologies. We currently evaluated the role of a recently discovered brain-enriched epigenetic modification known as 5-hydroxymethylcytosine (5hmC) in regulating transcriptomic and pathogenic mechanisms after focal ischemic injury. Methods- Young and aged male and female mice were subjected to transient middle cerebral artery occlusion, and the peri-infarct region was analyzed at various times of reperfusion. Two days before middle cerebral artery occlusion, short-interfering RNA against an isoform of the 5hmC producing enzyme TET (ten-eleven translocase) was injected intracerebrally. Ascorbate was injected intraperitoneally at 5 minutes, 30 minutes, or 2 hours of reperfusion. Motor function was tested with rotarod and beam-walk test. Results- Focal ischemia rapidly induced the activity of TET, the enzyme that catalyzes the formation of 5hmC and preferentially increased expression of the TET3 isoform in the peri-infarct region of the ischemic cortex. Levels of 5hmC were increased in a TET3-dependent manner, and inhibition of TET3 led to wide-scale reductions in the postischemic expression of neuroprotective genes involved in antioxidant defense and DNA repair. TET3 knockdown in adult male and female mice further increased brain degeneration after focal ischemia, demonstrating a role for TET3 and 5hmC in endogenous protection against stroke. Ascorbate treatment after focal ischemia enhanced TET3 activity and 5hmC enrichment in the peri-infarct region. TET3 activation by ascorbate provided robust protection against ischemic injury in young and aged mice of both sexes. Moreover, ascorbate treatment improved motor function recovery in both male and female mice. Conclusions- Collectively, these results indicate the potential of TET3 and 5hmC as novel stroke therapeutic targets. Visual Overview- An online visual overview is available for this article.

Opposing activities of oncogenic MIR17HG and tumor suppressive MIR100HG clusters and their gene targets regulate replicative senescence in human adult stem cells.

Growing evidence suggests that many diseases of aging, including diseases associated with robust changes and adipose deports, may be caused by resident adult stem cell exhaustion due to the process called cellular senescence. Understanding how microRNA pathways can regulate cellular senescence is crucial for the development of novel diagnostic and therapeutic strategies to combat these pathologies. Herein, using integrated transcriptomic and semi-quantitative proteomic analysis, we provide a system level view of the regulation of human adipose-derived stem cell senescence by a subset of mature microRNAs (termed senescence-associated-microRNAs) produced by biogenesis of oncogenic MIR17HG and tumor-suppressive MIR100HG clusters. We demonstrate functional significance of these mature senescence-associated-microRNAs in the process of replicative senescence of human adipose-derived stem cells ex-vivo and define a set of senescence-associated-microRNA gene targets that are able to elicit, modulate and, most importantly, balance intimate connections between oncogenic and senescent events.

Resveratrol preconditioning induces cerebral ischemic tolerance but has minimal effect on cerebral microRNA profiles.

The health benefits of the plant-derived polyphenol resveratrol were established in multiple disease systems. Notably, pre-treatment with resveratrol was shown to be neuroprotective in several models of cerebral ischemia. Mechanisms of resveratrol-mediated neuroprotection have been explored in the context of canonical resveratrol targets, but epigenetic and non-coding RNA processes have not yet been evaluated. Resveratrol was shown to alter microRNAs in cancer and cardiac ischemia. Previous studies also showed that ischemic preconditioning that induces ischemic tolerance significantly alters cerebral microRNA levels, particularly those that target neuroprotective pathways. Therefore, we tested if resveratrol-mediated ischemic tolerance also alters microRNA expression with a goal to identify microRNAs that are amenable to manipulation to induce neuroprotection after cerebral ischemia. Hence, we tested the microRNA profiles in mouse brain following intraperitoneal administration of resveratrol that induced significant tolerance against transient focal ischemia. We analyzed microRNA profiles using microarrays from both Affymetrix and LC Sciences that contain probes for all known mouse microRNAs. The results show that there is no consistent change in any of the microRNAs tested between resveratrol and vehicle groups indicating that microRNAs play a minimal role in resveratrol-mediated cerebral ischemic tolerance.

Resveratrol neuroprotection in stroke and traumatic CNS injury.

Resveratrol, a stilbene formed in many plants in response to various stressors, elicits multiple beneficial effects in vertebrates. Particularly, resveratrol was shown to have therapeutic properties in cancer, atherosclerosis and neurodegeneration. Resveratrol-induced benefits are modulated by multiple synergistic pathways that control oxidative stress, inflammation and cell death. Despite the lack of a definitive mechanism, both in vivo and in vitro studies suggest that resveratrol can induce a neuroprotective state when administered acutely or prior to experimental injury to the CNS. In this review, we discuss the neuroprotective potential of resveratrol in stroke, traumatic brain injury and spinal cord injury, with a focus on the molecular pathways responsible for this protection.

A semi-automated mass spectrometric immunoassay coupled to selected reaction monitoring (MSIA-SRM) reveals novel relationships between circulating PCSK9 and metabolic phenotypes in patient cohorts.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a key regulator of circulating low density lipoprotein cholesterol (LDL-C) levels. Besides its full-length mature form, multiple variants of PCSK9 have been reported such as forms that are truncated, mutated and/or with posttranslational modifications (PTMs). Previous studies have demonstrated that most of these variants affect PCSK9's function and thereby LDL-C levels. Commercial ELISA kits are available for quantification of PCSK9, but do not allow discrimination between the various forms and PTMs of the protein. To address this issue and given the complexity and wide dynamic range of the plasma proteome, we have developed a mass spectrometric immunoassay coupled to selected reaction monitoring (MSIA-SRM) for the multiplexed quantification of several forms of circulating PCSK9 in human plasma. Our MSIA-SRM assay quantifies peptides spanning the various protein domains and the S688 phosphorylation site. The assay was applied in two distinct cohorts of obese patients and healthy pregnant women stratified by their circulating LDL-C levels. Seven PCSK9 peptides were monitored in plasma samples: one in the prodomain prior to the autocleavage site at Q152, one in the catalytic domain prior to the furin cleavage site at R218, two in the catalytic domain following R218, one in the cysteine and histidine rich domain (CHRD) and the C-terminal peptide phosphorylated at S688 and unmodified. The latter was not detectable in sufficient amounts to be quantified in human plasma. All peptides were measured with high reproducibility and with LLOQ and LOD below the clinical range. The abundance of 5 of the 6 detectable PCSK9 peptides was higher in obese patients stratified with high circulating LDL-C levels as compared to those with low LDL-C (p < 0.05). The same 5 peptides showed good and statistically significant correlations with LDL-C levels (0.55 < r < 0.65; 0.0002 ⩽ p ⩽ 0.002), but not the S688 phosphorylated peptide. However, this phosphopeptide was significantly correlated with insulin resistance (r = 0.48; p = 0.04). In the pregnant women cohort, none of the peptides were associated to LDL-C levels. However, the 6 detectable PCSK9 peptides, but not PCSK9 measured by ELISA, were significantly correlated with serum triglyceride levels in this cohort. Our results also suggest that PCSK9 circulates with S688 phosphorylated at high stoichiometry. In summary, we have developed and applied a robust and sensitive MSIA-SRM assay for the absolute quantification of all PCSK9 domains and a PTM in human plasma. This assay revealed novel relationships between PCSK9 and metabolic phenotypes, as compared to classical ELISA assays.

Lipoprotein concentration, particle number, size and cholesterol efflux capacity are associated with mitochondrial oxidative stress and function in an HIV positive cohort.

BACKGROUND: Association of lipoprotein particle size/number and HDL function with mitochondrial oxidative stress and function may underlie the excess cardiovascular (CVD) risk in HIV. METHODS AND RESULTS: Among HIV infected individuals on stable highly active antiretroviral therapy, we related standard and novel lipid measures [plasma total cholesterol, triglycerides, HDL-C, LDL-C, lipoprotein particle (-P) subclass size and number and HDL function (via cholesterol-efflux capacity)] with oxidative stress [peripheral blood mononuclear cell's mitochondrial-specific 8-oxo-deoxyguanine (8-oxo-dG)] and function markers [oxidative phosphorylation (OXPHOS) NADH dehydrogenase (Complex I) and cytochrome c oxidase (Complex IV) enzyme activities]. Multivariable-adjusted logistic and linear regression analyses were employed adjusting for age, gender, CD4 nadir, viral load, smoking, diabetes, HOMA-IR, hypertension and lipid medications. Among 150 HIV-infected persons (mean age 52 years, 12% women, median CD4 count 524 cell/mm3), low HDL-C and high total cholesterol/HDL-C ratio were related to PBMC 8-oxo-deoxyguanine (p = 0.01 and 0.02 respectively). Large HDL-P and HDL-P size were inversely related to PBMC 8-oxo-deoxyguanine (p = 0.04). Small LDL-P (p = 0.01) and total LDL-P (p = 0.01) were related to decreased OXPHOS Complex I activity. LDL-P was related to decreased OXPHOS Complex IV activity (p = 0.02). Cholesterol efflux capacity was associated with increased OXPHOS Complex IV activity. CONCLUSIONS: HDL concentration and particle size and number are related to decreased PBMC mitochondrial oxidative stress whereas HDL function is positively related to mitochondrial oxidative function. The association we find between atherogenic lipoprotein profile and increased oxidative stress and function suggests these pathways may be important in the pathogenesis of cardiometabolic disease in HIV disease.

Delineating monoclonal antibody specificity by mass spectrometry.

Generation of monoclonal antibody (mAb) libraries against antigens in complex matrices can prove a valuable analytical tool. However, delineating the specificity of newly generated antibodies is the limiting step of the procedure. Here, we propose a strategy for mAb production by injecting mice with complex biological fluid and mAb characterization by coupling immunoaffinity techniques with Mass spectrometry (immuno-MS). Mice were immunized against fractionated seminal plasma and mAbs were produced. Different immuno-MS protocols based on four types of solid support (i.e. polystyrene microtiter plates, NHS-activated agarose beads, tosyl-activated magnetic beads and MSIA™ pipette tips) were established. A well-characterized mouse monoclonal anti-KLK3 (PSA) Ab was used as a model to evaluate each protocol's robustness and reproducibility and to establish a set of criteria which would allow antigen characterization of newly developed Abs. Three of the newly generated Abs were analyzed using our optimized protocols. Analysis revealed that all assay configurations used were capable of antibody characterization. Furthermore, low-abundance antigens (e.g. ribonuclease T2) could be identified as efficiently as the high-abundance ones. Our data suggest that complex biological samples can be used for the production of mAbs, which will facilitate the analysis of their proteome, while the established immuno-MS protocols can offer efficient mAb characterization. BIOLOGICAL SIGNIFICANCE: The inoculation of animals with complex biological samples is aiming at the discovery of novel disease biomarkers, present in the biological specimens, as well as the production of rare reagents that will facilitate the ultra-sensitive analysis of the biomolecules' native form. In the present study, we initially propose a general workflow concerning the handling of biological samples, as well as the monoclonal antibody production. Furthermore, we established protocols for the reliable and reproducible identification of antibody specificity using various immuno-affinity purification techniques coupled to mass spectrometry. Our data suggest that processed biological fluids can be used for the production of mAbs targeting proteins of varying abundance, and that various immuno-MS protocols can offer great capabilities for the mAb characterization procedure.

Mass spectrometry assays of plasma biomarkers to predict radiographic progression of knee osteoarthritis.

INTRODUCTION: Biomarkers to identify osteoarthritis (OA) patients at risk for disease progression are needed. As part of a proteomic analysis of knee synovial fluid from normal and OA patients, differentially expressed proteins were identified that could represent potential biomarkers for OA. This study aimed to use mass spectrometry assays to identify representative peptides from several proteins in synovial fluid and peripheral blood, and assess their levels as biomarkers of OA progression. METHODS: Multiplexed high throughput selected reaction monitoring (SRM) assays were developed to measure tryptic peptides representative of 23 proteins in matched serum and synovial fluid samples from late OA subjects at the time of joint replacement. Subsequently plasma samples from the baseline visit of 173 subjects in an observational OA cohort were tested by SRM for peptides from nine of these proteins: afamin, clusterin, cartilage oligomeric matrix protein, hepatocyte growth factor, kallistatin, insulin-like growth factor binding protein, acid labile subunit, lubricin, lumican, and pigment epithelium-derived factor. Linear regression was used to determine the association between the peptide biomarker level at baseline and change in joint space width (ΔJSW) from baseline to 30 months, adjusting for age and sex. RESULTS: In the matched cohort, 17 proteins could be identified in synovial fluid and 16 proteins were detected in serum. For the progression cohort, the average age was 62 and average ΔJSW over 30 months was 0.68 mm. A high correlation between different peptides from individual proteins was observed, indicating our assays correctly measured their target proteins. Peptides representative of clusterin, lumican and lubricin showed statistically significant associations with joint space narrowing after adjustment for age and sex. Partial R2 values showed clusterin FMETVAEK and lubricin LVEVNPK peptide biomarkers explains about 2 to 3% of the variability of ΔJSW, similar to that explained by age. A biomarker score combining normalized data for both lubricin and clusterin peptides increased the model R2 to 0.079. CONCLUSIONS: Our results suggest that when combined, levels of peptides representative of clusterin and lubricin in plasma are as predictive of OA progression as age. Replication of these findings in other prospective OA cohorts is planned.

Hybrid data acquisition and processing strategies with increased throughput and selectivity: pSMART analysis for global qualitative and quantitative analysis.

Data-dependent acquisition (DDA) and data-independent acquisition strategies (DIA) have both resulted in improved understanding of proteomics samples. Both strategies have advantages and disadvantages that are well-published, where DDA is typically applied for deep discovery and DIA may be used to create sample records. In this paper, we present a hybrid data acquisition and processing strategy (pSMART) that combines the strengths of both techniques and provides significant benefits for qualitative and quantitative peptide analysis. The performance of pSMART is compared to published DIA strategies in an experiment that allows the objective assessment of DIA performance with respect to interrogation of previously acquired MS data. The results of this experiment demonstrate that pSMART creates fewer decoy hits than a standard DIA strategy. Moreover, we show that pSMART is more selective, sensitive, and reproducible than either standard DIA or DDA strategies alone.

Assessment of peptide chemical modifications on the development of an accurate and precise multiplex selected reaction monitoring assay for apolipoprotein e isoforms.

Apolipoprotein E (ApoE) is a polymorphic protein that plays a major role in lipid metabolism in the central nervous system and periphery. It has three common allelic isoforms, ApoE2, ApoE3, and ApoE4, that differ in only one or two amino acids. ApoE isoforms have been associated with the occurrence and progression of several pathological conditions, such as coronary atherosclerosis and Alzheimer's disease. The aim of this study was to develop a mass spectrometry (MS)-based assay for absolute quantification of ApoE isoforms in cerebrospinal fluid and plasma samples using isotope-labeled peptides. The assay included five tryptic peptides: CLAVYQAGAR (ApoE2), LGADMEDVCGR (ApoE2 and 3), LAVYQAGAR (ApoE3 and 4), LGADMEDVR (ApoE4), and LGPLVEQGR (total ApoE). Both cerebrospinal fluid and plasma samples were assayed to validate the method. The digestion yield and the extension of chemical modifications in selected amino acid residues (methionine oxidation, glutamine deamidation, and cyclization of N-terminus carbamidomethylcysteine) were also studied. The ApoE phenotype was successfully assigned to all samples analyzed in a blinded manner. The method showed good linearity (R(2) > 0.99) and reproducibility (within laboratory imprecision <13%). The comparison of the MS-based assay with an ELISA for total ApoE concentration showed a moderate correlation (R(2) = 0.59). This MS-based assay can serve as an important tool in clinical studies aiming to elucidate the association between ApoE genotype, total ApoE, and ApoE isoform concentrations in various disorders related to ApoE polymorphisms.

SIV antigen-specific effects on immune responses induced by vaccination with DNA electroporation and plasmid IL-12.

Molecular adjuvants are important for augmenting or modulating immune responses induced by DNA vaccination. Promising results have been obtained using IL-12 expression plasmids in a variety of disease models including the SIV model of HIV infection. We used a mouse model to evaluate plasmid IL-12 (pIL-12) in a DNA prime, recombinant adenovirus serotype 5 (rAd5) boost regimen specifically to evaluate the effect of IL-12 expression on cellular and humoral immunity induced against both SIVmac239 Gag and Env antigens. Priming with electroporated (EP) DNA+pIL-12 resulted in a 2-4-fold enhanced frequency of Gag-specific CD4 T cells which was maintained through the end of the study irrespective of the pIL-12 dose, while memory Env-specific CD4+T cells were maintained only at the low dose of pIL-12. There was little positive effect of pIL-12 on the humoral response to Env, and in fact, high dose pIL-12 dramatically reduced SIV Env-specific IgG. Additionally, both doses of pIL-12 diminished the frequency of CD8 T-cells after DNA prime, although a rAd5 boost recovered CD8 responses regardless of the pIL-12 dose. In this prime-boost regimen, we have shown that a high dose pIL-12 can systemically reduce Env-specific humoral responses and CD4T cell frequency, but not Gag-specific CD4+ T cells. These data indicate that it is important to independently characterize individual SIV or HIV antigen immunogenicity in multi-antigenic vaccines as a function of adjuvant dose.

Rapid development of sensitive, high-throughput, quantitative and highly selective mass spectrometric targeted immunoassays for clinically important proteins in human plasma and serum.

OBJECTIVES: The aim of this study was to develop high-throughput, quantitative and highly selective mass spectrometric, targeted immunoassays for clinically important proteins in human plasma or serum. DESIGN AND METHODS: The described method coupled mass spectrometric immunoassay (MSIA), a previously developed technique for immunoenrichment on a monolithic microcolumn activated with an anti-protein antibody and fixed in a pipette tip, to selected reaction monitoring (SRM) detection and accurate quantification of targeted peptides, including clinically relevant sequence or truncated variants. RESULTS: In this report, we demonstrate the rapid development of MSIA-SRM assays for sixteen different target proteins spanning seven different clinically important areas (including neurological, Alzheimer's, cardiovascular, endocrine function, cancer and other diseases) and ranging in concentration from pg/mL to mg/mL. The reported MSIA-SRM assays demonstrated high sensitivity (within published clinical ranges), precision, robustness and high-throughput as well as specific detection of clinically relevant isoforms for many of the target proteins. Most of the assays were tested with bona-fide clinical samples. In addition, positive correlations, (R2 0.67-0.87, depending on the target peptide), were demonstrated for MSIA-SRM assay data with clinical analyzer measurements of parathyroid hormone (PTH) and insulin growth factor 1 (IGF1) in clinical sample cohorts. CONCLUSIONS: We have presented a practical and scalable method for rapid development and deployment of MS-based SRM assays for clinically relevant proteins and measured levels of the target analytes in bona fide clinical samples. The method permits the specific quantification of individual protein isoforms and addresses the difficult problem of protein heterogeneity in clinical proteomics applications.

Depletion of nuclear histone H2A variants is associated with chronic DNA damage signaling upon drug-evoked senescence of human somatic cells.

Cellular senescence is associated with global chromatin changes, altered gene expression, and activation of chronic DNA damage signaling. These events ultimately lead to morphological and physiological transformations in primary cells. In this study, we show that chronic DNA damage signals caused by genotoxic stress impact the expression of histones H2A family members and lead to their depletion in the nuclei of senescent human fibroblasts. Our data reinforce the hypothesis that progressive chromatin destabilization may lead to the loss of epigenetic information and impaired cellular function associated with chronic DNA damage upon drug-evoked senescence. We propose that changes in the histone biosynthesis and chromatin assembly may directly contribute to cellular aging. In addition, we also outline the method that allows for quantitative and unbiased measurement of these changes.

Interlaboratory reproducibility of selective reaction monitoring assays using multiple upfront analyte enrichment strategies.

Over the past few years, mass spectrometry has emerged as a technology to complement and potentially replace standard immunoassays in routine clinical core laboratories. Application of mass spectrometry to protein and peptide measurement can provide advantages including high sensitivity, the ability to multiplex analytes, and high specificity at the amino acid sequence level. In our previous study, we demonstrated excellent reproducibility of mass spectrometry-selective reaction monitoring (MS-SRM) assays when applying standardized standard operating procedures (SOPs) to measure synthetic peptides in a complex sample, as lack of reproducibility has been a frequent criticism leveled at the use of mass spectrometers in the clinical laboratory compared to immunoassays. Furthermore, an important caveat of SRM-based assays for proteins is that many low-abundance analytes require some type of enrichment before detection with MS. This adds a level of complexity to the procedure and the potential for irreproducibility increases, especially across different laboratories with different operators. The purpose of this study was to test the interlaboratory reproducibility of SRM assays with various upfront enrichment strategies and different types of clinical samples (representing real-world body fluids commonly encountered in routine clinical laboratories). Three different, previously published enrichment strategies for low-abundance analytes and a no-enrichment strategy for high-abundance analytes were tested across four different laboratories using different liquid chromatography-SRM (LC-SRM) platforms and previously developed SOPs. The results demonstrated that these assays were indeed reproducible with coefficients of variation of less than 30% for the measurement of important clinical proteins across all four laboratories in real world samples.

Enhanced control of pathogenic Simian immunodeficiency virus SIVmac239 replication in macaques immunized with an interleukin-12 plasmid and a DNA prime-viral vector boost vaccine regimen.

DNA priming has previously been shown to elicit augmented immune responses when administered by electroporation (EP) or codelivered with a plasmid encoding interleukin-12 (pIL-12). We hypothesized that the efficacy of a DNA prime and recombinant adenovirus 5 boost vaccination regimen (DNA/rAd5) would be improved when incorporating these vaccination strategies into the DNA priming phase, as determined by pathogenic simian immunodeficiency virus SIVmac239 challenge outcome. The whole SIVmac239 proteome was delivered in 5 separate DNA plasmids (pDNA-SIV) by EP with or without pIL-12, followed by boosting 4 months later with corresponding rAd5-SIV vaccine vectors. Remarkably, after repeated low-dose SIVmac239 mucosal challenge, we demonstrate 2.6 and 4.4 log reductions of the median SIV peak and set point viral loads in rhesus macaques (RMs) that received pDNA-SIV by EP with pIL-12 compared to the median peak and set point viral loads in mock-immunized controls (P < 0.01). In 5 out of 6 infected RMs, strong suppression of viremia was observed, with intermittent "blips" in virus replication. In 2 RMs, we could not detect the presence of SIV RNA in tissue and lymph nodes, even after 13 viral challenges. RMs immunized without pIL-12 demonstrated a typical maximum of 1.5 log reduction in virus load. There was no significant difference in the overall magnitude of SIV-specific antibodies or CD8 T-cell responses between groups; however, pDNA delivery by EP with pIL-12 induced a greater magnitude of SIV-specific CD4 T cells that produced multiple cytokines. This vaccine strategy is relevant for existing vaccine candidates entering clinical evaluation, and this model may provide insights into control of retrovirus replication.