Strong methane point sources contribute a disproportionate fraction of total emissions across multiple basins in the United States.

Understanding, prioritizing, and mitigating methane (CH4) emissions requires quantifying CH4 budgets from facility scales to regional scales with the ability to differentiate between source sectors. We deployed a tiered observing system for multiple basins in the United States (San Joaquin Valley, Uinta, Denver-Julesburg, Permian, Marcellus). We quantify strong point source emissions (>10 kg CH4 h-1) using airborne imaging spectrometers, attribute them to sectors, and assess their intermittency with multiple revisits. We compare these point source emissions to total basin CH4 fluxes derived from inversion of Sentinel-5p satellite CH4 observations. Across basins, point sources make up on average 40% of the regional flux. We sampled some basins several times across multiple months and years and find a distinct bimodal structure to emission timescales: the total point source budget is split nearly in half by short-lasting and long-lasting emission events. With the increasing airborne and satellite observing capabilities planned for the near future, tiered observing systems will more fully quantify and attribute CH4 emissions from facility to regional scales, which is needed to effectively and efficiently reduce methane emissions.

Comparison of two formulations of intravenous lipid emulsions in pediatric intestinal failure.

PURPOSE: The effect of different types of lipid emulsion may guide therapy of patients with intestinal failure (IF) to limit morbidity such as intestinal failure-associated liver disease (IFALD). METHODS: A retrospective chart review of pediatric patients with IF who received soybean oil lipid emulsion (SL) or mixed oil lipid emulsion (ML) was performed. Data over 1 year were collected. RESULTS: Forty-five patients received SL and 34 received ML. There were no differences in the incidence (82 versus 74%, P = 0.35) or resolution (86 versus 92%, P = 0.5) of IFALD between the cohorts. The median dose of ML was higher compared to SL (2 versus 1 g/kg/day, P < 0.001). If resolved, IFALD resolved rapidly in the ML cohort compared to the SL cohort (67 versus 37 days, P = 0.01). Weight gain was higher in the ML compared to the SL cohort at resolution of IFALD or 1 year from diagnosis of IF (P = 0.009). CONCLUSION: The administration of ML did not alter the incidence or resolution of IFALD compared to SL in pediatric IF. There was rapid resolution of IFALD and enhanced weight gain in the ML cohort compared to SL in pediatric IF.

Surgical Episodes of Care for Price Transparency Using the Episode Grouper for Medicare.

OBJECTIVE: To explore the use of an episode grouper to more accurately identify the complete set of surgical services typically provided in a surgical episode of care and the corresponding range of prices, using colectomy for cancer as the example. BACKGROUND: Price transparency is an important policy issue that will require surgeons to better understand the components and cost of care. METHODS: This study uses the Episode Grouper for Medicare business logic to construct colectomy surgical episodes of care for cancer using Medicare claims data for the Boston Hospital Referral Region from 2012 to 2015. Descriptive statistics show the mean reimbursement based on patient severity and stage of surgery, along with the number of unique clinicians billing for care and the mix of services provided. RESULTS: The Episode Grouper for Medicare episode grouper identified 3182 colectomies in Boston between 2012 and 2015, with 1607 done for cancer. The mean Medicare allowed amount per case is $29,954 and varies from $26,605 to $36,850 as you move from low to high-severity cases. The intrafacility stage is the most expensive ($23,175 on average) compared with the pre ($780) and post ($6,479) facility stages. There is tremendous heterogeneity in the service mix. CONCLUSIONS: Episode groupers are a potentially valuable tool for identifying variations in service mix and teaming patterns that correlate with a total price. By looking at patient care holistically, stakeholders can identify opportunities for price transparency and care redesign that have heretofore been hidden.

Estimating the Primary Care Workforce for Medicare Beneficiaries Using an Activity-Based Approach.

BACKGROUND: The enumeration of the primary care workforce relies on potentially inaccurate specialty designations sourced from licensure registries and clinician surveys. OBJECTIVE: To use an activity-based measure of primary care to estimate the number of physicians, nurse practitioners (NPs), and physician assistants (PAs) providing primary care to Medicare beneficiaries. DESIGN: Observational study using Medicare fee-for-service (FFS) claims data. SUBJECTS: All clinicians in the US billing Medicare in 2019 and their fee-for-service Medicare patients. MAIN MEASURES: We construct three measures that together distinguish primary care from specialty clinicians: (1) presence of evaluation and management (E&M) services in a setting consistent with primary care, (2) the dispersion of clinical care across International Classification of Diseases-10 (ICD-10) chapters, and (3) the extent of provided services that are atypical of primary care (e.g., surgical procedure). We apply parameters to the measures to identify the clinicians likely providing primary care and compare the resulting classifications across provider type. KEY RESULTS: Of physicians with at least 50 Medicare beneficiaries, 19-22% provide primary care. Of medical generalists (i.e., family medicine, internal medicine) with at least 50 beneficiaries, 61-68% provide primary care. We estimate that 40-45% of NPs and 27-30% of PAs meeting the panel size threshold are primary care providers in FFS Medicare. CONCLUSIONS: Our findings suggest that based on a primary care practice style, the number of primary care physicians in FFS Medicare is likely smaller than conventional estimates. However, compared to prior estimates, the number of primary care NPs is larger and the number of PAs is similar.

Comparable outcomes with intramedullary nail and plate constructs for Schatzker VI tibial plateau fractures.

BACKGROUND: Outcomes data of intramedullary nail fixation (IMN) constructs for complex Schatzker VI tibial plateau fractures are scant in the literature. This study compares the clinical and radiographic outcomes of IMN, dual plate, and single plate constructs for Schatzker IV tibial plateau fractures. METHODS: Retrospective cohort study of sixty-two patients at a University-based Level 1 trauma center who underwent open reduction internal fixation for Schatzker VI tibial plateau fracture. Constructs evaluated were IMN (with or without raft screws), dual plating, and single plating. Demographic, clinical, and radiographic outcomes were recorded. All fractures were additionally classified based on the OTA classification for sub analyses. Mean follow-up was 13.2 (SD 13.3) months. Predictors of construct selection and outcomes were evaluated with bivariate logistic regression. Outcomes were compared between groups with independent samples t-tests and Chi Square tests. RESULTS: No significant demographic differences were found between IMN, dual plate or single plate construct cohorts. There was a higher proportion of open fractures within the IMN construct group versus the dual plate cohort (21.1% vs 3.6%). No statistically significant differences in radiographic outcomes were observed between cohort groups except for small but statistically significant differences in condylar width (CW) ratio change and tibial slope; when fracture cohorts were sub analyzed by specific OTA classification, there were no significant differences in any radiographic outcomes. There was a significant difference between the ratio of OTA 41C1, C2 and C3 fractures regarding treatment allocation (p = 0.004), favoring dual plate fixation for OTA 41C3 fractures. There were no significant differences found between treatment cohorts in terms of all cause complications (p > 0.05). IMN and single plate constructs were utilized when posteromedial condyle fractures were nondisplaced or minimally displaced. CONCLUSION: Intramedullary nail fixation with or without supplemental raft screws produced similar short-term clinical and radiographic results compared to dual and single plate constructs among patients with Schatzker VI fracture types, regardless of OTA classification. Level of Evidence Level III retrospective cohort.

Macromolecular Crowding as an Intracellular Stimulus for Responsive Nanomaterials.

Stimuli-responsive materials are exploited in biological, materials, and sensing applications. We introduce a new endogenous stimulus, biomacromolecule crowding, which we achieve by leveraging changes in thermoresponsive properties of polymers upon high concentrations of crowding agents. We prepare poly(2-oxazoline) amphiphiles that exhibit lower critical solution temperatures (LCST) in serum above physiological temperature. These amphiphiles stabilize oil-in-water nanoemulsions at temperatures below the LCST but are ineffective surfactants above the LCST, resulting in emulsion fusion. We find that the transformations observed upon heating nanoemulsions above their surfactant's LCST can instead be induced at physiological temperatures through the addition of polymers and protein, rendering thermoresponsive materials "crowding responsive." We demonstrate that the cytosol is a stimulus for nanoemulsions, with droplet fusion occurring upon injection into cells of living zebrafish embryos. This report sets the stage for classes of thermoresponsive materials to respond to macromolecule concentration rather than temperature changes.

Ancient pigs reveal a near-complete genomic turnover following their introduction to Europe.

Archaeological evidence indicates that pig domestication had begun by ∼10,500 y before the present (BP) in the Near East, and mitochondrial DNA (mtDNA) suggests that pigs arrived in Europe alongside farmers ∼8,500 y BP. A few thousand years after the introduction of Near Eastern pigs into Europe, however, their characteristic mtDNA signature disappeared and was replaced by haplotypes associated with European wild boars. This turnover could be accounted for by substantial gene flow from local European wild boars, although it is also possible that European wild boars were domesticated independently without any genetic contribution from the Near East. To test these hypotheses, we obtained mtDNA sequences from 2,099 modern and ancient pig samples and 63 nuclear ancient genomes from Near Eastern and European pigs. Our analyses revealed that European domestic pigs dating from 7,100 to 6,000 y BP possessed both Near Eastern and European nuclear ancestry, while later pigs possessed no more than 4% Near Eastern ancestry, indicating that gene flow from European wild boars resulted in a near-complete disappearance of Near East ancestry. In addition, we demonstrate that a variant at a locus encoding black coat color likely originated in the Near East and persisted in European pigs. Altogether, our results indicate that while pigs were not independently domesticated in Europe, the vast majority of human-mediated selection over the past 5,000 y focused on the genomic fraction derived from the European wild boars, and not on the fraction that was selected by early Neolithic farmers over the first 2,500 y of the domestication process.

Robustness and reproducibility of an artificial intelligence-assisted online segmentation and adaptive planning process for online adaptive radiation therapy.

Clinical implementation of online adaptive radiation therapy requires initial and ongoing performance assessment of the underlying auto-segmentation and adaptive planning algorithms, although a straightforward and efficient process for this in phantom is lacking. The purpose of this work was to investigate robustness and repeatability of the artificial intelligence-assisted online segmentation and adaptive planning process on the Varian Ethos adaptive platform, and to develop an end-to-end test strategy for online adaptive radiation therapy. Five synthetic deformations were generated and applied to a computed tomography image of an anthropomorphic pelvis phantom, and reference treatment plans were generated from each of the resulting deformed images. The undeformed phantom was repeatedly imaged, and the online adaptive process was performed including auto-segmentation, review and manual correction of contours, and adaptive plan creation. One adaptive fractions in five different deformation scenarios were performed. The manually corrected contours had a high degree of consistency (> 93% Dice similarity coefficient and < 1.0 mm mean surface distance) across repeated fractions, with no significant variation across the synthetic deformation instance except for bowel (p = 0.026, one-way ANOVA). Adaptive treatment plans also resulted in highly consistent dose-volume values for targets and organs at risk. A straightforward and efficient process was developed and used to quantify a set of organ specific contouring and dosimetric action levels to help establish uncertainty bounds for an end-to-end test on the Varian Ethos system.

Autologous thrombin preparations: Biocompatibility and growth factor release.

Platelet-rich plasma (PRP) has been investigated to promote wound healing in a variety of tissues. Thrombin, another essential component of wound healing, is sometimes combined with PRP to generate a fibrin clot in order to retain the sample at the delivery site and to stimulate growth factor release. Using a fully autologous approach, autologous serum (AS) with thrombin activity can be prepared using a one-step procedure by supplementing with ethanol (E+ AS) to prolong room temperature stability or prepared ethanol free (E- AS) by utilizing a two-step procedure to prolong stability. The objective of this study was to evaluate potential wound healing mechanisms of these two preparations using commercially available devices. A variety of tests were conducted to assess biocompatibility and growth factor release from PRP at various ratios. It was found that E- AS contained greater leukocyte viability in the product (97.1 ± 2.0% compared to 41.8 ± 11.5%), supported greater bone marrow mesenchymal stem cell proliferation (3.7× vs 0.8× at a 1:4 ratio and 3.6× vs 1.6× at a 1:10 ratio), and stimulated release of growth factors and cytokines from PRP to a greater extent than E+ AS. Of the 36 growth factors and cytokines evaluated, release of 27 of them were significantly reduced by the presence of ethanol in at least one of the tested configurations. It is concluded that the high concentrations of ethanol needed to stabilize point of care autologous thrombin preparations could be detrimental to normal wound healing processes.

The use of next generation sequencing for improving food safety: Translation into practice.

Next Generation Sequencing (NGS) combined with powerful bioinformatic approaches are revolutionising food microbiology. Whole genome sequencing (WGS) of single isolates allows the most detailed comparison possible hitherto of individual strains. The two principle approaches for strain discrimination, single nucleotide polymorphism (SNP) analysis and genomic multi-locus sequence typing (MLST) are showing concordant results for phylogenetic clustering and are complementary to each other. Metabarcoding and metagenomics, applied to total DNA isolated from either food materials or the production environment, allows the identification of complete microbial populations. Metagenomics identifies the entire gene content and when coupled to transcriptomics or proteomics, allows the identification of functional capacity and biochemical activity of microbial populations. The focus of this review is on the recent use and future potential of NGS in food microbiology and on current challenges. Guidance is provided for new users, such as public health departments and the food industry, on the implementation of NGS and how to critically interpret results and place them in a broader context. The review aims to promote the broader application of NGS technologies within the food industry as well as highlight knowledge gaps and novel applications of NGS with the aim of driving future research and increasing food safety outputs from its wider use.

Acquired Versus Congenital Neonatal Purpura Fulminans: A Case Report and Literature Review.

Neonatal purpura fulminans (PF) is a life-threatening disorder caused by congenital or acquired deficiencies of protein C (PC) or S. PF presents as a cutaneous manifestation of disseminated intravascular coagulation. We describe a case of PF in a newborn with left leg ischemia and undetectable PC levels soon after birth. Despite anticoagulation therapy and PC concentrate, left foot amputation was required. Genetic testing of PROC for congenital PC deficiency was normal. This case highlights the course of PF due to acquired PC deficiency in a newborn treated with PC concentrate which is rarely described in the literature.

An Ethanol-Free Autologous Thrombin System.

Thrombin is a coagulation protein of central importance to hemostasis and wound healing that can be sourced from human blood, bovine blood, and engineered cell lines. Only autologous thrombin lacks the risks of transmitting emergent pathogens or eliciting an immunogenic response. Previous commercial autologous thrombin devices require the use of high concentrations of ethanol to achieve thrombin stability, introducing cytotoxicity risks. A new point of care device for preparing an ethanol-free autologous thrombin serum was investigated. The ethanol-free autologous serum (AS) was prepared using the Thrombinator™ System (Arthrex, Inc., Naples, FL). A total of 120 devices were tested with the blood of 30 healthy donors to determine the reliability and flexibility of the procedure. AS was prepared from both whole blood (WB) and platelet-poor plasma (PPP). Study endpoints were thrombin activity determined using a coagulation analyzer and formation of cohesive bone graft composites objectively measured using a durometer. The average thrombin activity produced by this system from 24 donors was 20.6 ± 2.7 IU/mL for WB and 13.4 ± 3.8 IU/mL for PPP which correlated to clot times of 3.9 and 5.9 seconds, respectively. The device tolerated use of varying volumes of blood to prepare AS. In addition, the system was able to generate four successive and comparable AS productions. When combined with platelet-rich plasma and bone graft material, cohesive scaffolds were always formed. A new device and method for preparing single donor, ethanol-free, AS with thrombin activity was demonstrated.

Extended-Release Niacin/Laropiprant Improves Overall Efficacy of Postprandial Reverse Cholesterol Transport.

OBJECTIVES: Postprandial atherogenic lipoproteins, characterizing high-risk patients, correlate positively with cardiovascular events. Although the effect of niacin on fasting lipids is well established, its impact on atheroprotective reverse cholesterol transport (RCT) pathway and on functional features of circulating lipoproteins during the postprandial state remains indeterminate. APPROACH AND RESULTS: We evaluated RCT pathway during postprandial phase in dyslipidemic patients displaying a low high-density lipoprotein (HDL) cholesterol phenotype. Ten subjects on stable statin therapy received 1 g/20 mg extended-release niacin/laropiprant (ERN/LRPT) for 4 weeks followed by 2 g/40 mg ERN/LRPT for additional 8 weeks. At each experimental period, postprandial hypertriglyceridemia and major steps of RCT, including cholesterol efflux from human macrophages, cholesteryl ester transfer protein-mediated cholesteryl ester transfer, and hepatic HDL-cholesteryl ester selective uptake were evaluated. Equally, the capacity of postprandial HDL particles isolated from patients before and after ERN/LRPT treatment to mediate RCT to feces was evaluated in vivo in human apolipoprotein B/cholesteryl ester transfer protein double transgenic mouse model. Compared with baseline, ERN/LRPT significantly reduced postprandial hypertriglyceridemia (incremental area under the curve-triglyceride: -53%; P=0.02). Postprandial increase in endogenous plasma cholesteryl ester transfer protein activity was completely abolished after ERN/LRPT treatment. Despite a slight reduction in plasma cholesterol efflux capacity from human THP-1 macrophages, evaluation of global RCT efficacy by combining both ex vivo and in vivo approaches indicate that postprandial HDL particles formed under ERN/LRPT therapy displayed a greater capacity for HDL-mediated RCT to feces. CONCLUSIONS: ERN/LRPT treatment efficiently attenuates atherogenic postprandial lipemia and stimulates HDL-mediated cholesterol return to the liver and elimination into feces during postprandial phase, thus maintaining an efficient removal of cholesterol from the body.

Evaluating a social problem solving intervention for juvenile detainees: Depressive outcomes and moderators of effectiveness.

This study reports findings from the administration of a social problem-solving training (SPST) intervention to juvenile detainees in the Connecticut Youth Detainee Program. SPST is a cognitive behavioral intervention that teaches children and youth how to more effectively cope with interpersonal stress and conflict. In the current study, we tested whether SPST could decrease depressive symptoms in a sample of detained adolescent offenders. The study used a randomized-control design with detention staff administering the intervention. The results showed that SPST, as a main effect, was not more effective in reducing depressive symptoms than treatment as usual. However, the effectiveness of SPST was moderated by fluid intelligence. Juvenile detainees with high intelligence scores were most likely to benefit from SPST compared to treatment as usual. It was surprising that, for those with lower intelligence scores, SPST increased depressive symptoms relative to treatment as usual. These results help fill a critical need for intervention effectiveness data on juvenile detainees and indicate that SPST may not be useful for reducing outcomes such as depression.

Violent offending among juveniles: A 7-year longitudinal study of recidivism, desistance, and associations with mental health.

Serious and violent offending among juveniles is a consistent concern of researchers, practitioners, and policymakers, yet the development of violent offending remains poorly understood because of limited availability of relevant data, small sample sizes, and shortage of longitudinal data sets. This study analyzed developmental patterns of violent offending over 7 years in the complete population of court-referred youth in Connecticut between 2006 and 2012 (N = 58,678; mean age at first offense = 14.7 years). This unique dataset provided several key findings: First, results from a latent class growth analysis showed that violent crimes peaked at age 14-15, with high-rate adolescent offenders (3.7% of the sample) accounting for 31.9% of all violent offenses. Further, 74.2% of this group desisted from violent crimes in adulthood. Higher levels of self-reported anger/irritability slightly increased the odds of violent recidivism (odds ratio, OR = 1.09), where higher levels of depression/anxiety depressed the odds (OR = 0.89). The overrepresentation of males, non-Hispanic Black, and Hispanic youth among high-rate adolescence offenders were traceable through adolescence but not beyond the age of 18. Together, these finding may help to inform new delinquency interventions that target the needs of this proportionally small group of violent adolescent offenders accounting for a large amount of violent crimes. (PsycINFO Database Record

All-Trans-Retinoic Acid Enhances Mitochondrial Function in Models of Human Liver.

All-trans-retinoic acid (atRA) is the active metabolite of vitamin A. The liver is the main storage organ of vitamin A, but activation of the retinoic acid receptors (RARs) in mouse liver and in human liver cell lines has also been shown. AlthoughatRA treatment improves mitochondrial function in skeletal muscle in rodents, its role in modulating mitochondrial function in the liver is controversial, and little data are available regarding the human liver. The aim of this study was to determine whetheratRA regulates hepatic mitochondrial activity.atRA treatment increased the mRNA and protein expression of multiple components of mitochondrialß-oxidation, tricarboxylic acid (TCA) cycle, and respiratory chain. Additionally,atRA increased mitochondrial biogenesis in human hepatocytes and in HepG2 cells with and without lipid loading based on peroxisome proliferator activated receptor gamma coactivator 1αand 1ßand nuclear respiratory factor 1 mRNA and mitochondrial DNA quantification.atRA also increasedß-oxidation and ATP production in HepG2 cells and in human hepatocytes. Knockdown studies of RARα, RARß, and PPARδrevealed that the enhancement of mitochondrial biogenesis andß-oxidation byatRA requires peroxisome proliferator activated receptor delta. In vivo in mice,atRA treatment increased mitochondrial biogenesis markers after an overnight fast. Inhibition ofatRA metabolism by talarozole, a cytochrome P450 (CYP) 26 specific inhibitor, increased the effects ofatRA on mitochondrial biogenesis markers in HepG2 cells and in vivo in mice. These studies show thatatRA regulates mitochondrial function and lipid metabolism and that increasingatRA concentrations in human liver via CYP26 inhibition may increase mitochondrial biogenesis and fatty acidß-oxidation and provide therapeutic benefit in diseases associated with mitochondrial dysfunction.

Use of captive spray ionization to increase throughput of the data-independent acquisition technique PAcIFIC.

RATIONALE: The Precursor Acquisition Independent From Ion Count (PAcIFIC) method is a data-independent acquisition technique capable of identifying proteins over eight orders of magnitude in a single analysis in human plasma. Widespread application of this technique in proteomic studies is hindered by its time-intensive nature. There exists a need to explore strategies to increase the throughput of the PAcIFIC method. METHODS: The PAcIFIC acquisition technique was optimized for use with an Orbitrap mass spectrometer fitted with a captive spray ionization (CSI) source. Chromatographic methods, PAcIFIC acquisition parameters, for example, channels interrogated per chromatographic gradient, time span of chromatographic gradient, and sample loading amount, were investigated to achieve a maximum number of peptide and protein identifications on a yeast proteome where protein copy number had been previously determined. RESULTS: A 24-hour CSI PAcIFIC method was developed with minimal reduction of peptide and protein identifications from the 4.2-day nano-electrospray ionization (nESI) PAcIFIC method. Analysis of a yeast cell lysate with the 4.2-day nESI PAcIFIC method resulted in 13,468 peptide and 2120 protein identifications. A 24-hour CSI PAcIFIC method resulted in 11,277 peptide and 1753 protein identifications. Increased sample loading of the CSI system allowed for an 8% increase in peptide and protein identifications. CONCLUSIONS: A dramatic decrease in the overall analysis time of the PAcIFIC method (24 h with CSI versus 100.8 h with nESI) was achieved with minimal reduction of peptide and protein identifications. Furthermore, the CSI PAcIFIC method demonstrated a high degree of sensitivity and capability of identifying proteins across a large dynamic range observed with the nESI PAcIFIC method (CSI PAcIFIC identified proteins as low as 46 molecules per cell).

Defining recurrence of nonmelanoma skin cancer after Mohs micrographic surgery: Report of the American College of Mohs Surgery Registry and Outcomes Committee.

BACKGROUND: Standardized definitions and methods of surveillance for local recurrence of nonmelanoma skin cancer are critical in determining cure rates attributed to treatment modalities. OBJECTIVE: We sought to offer a standard definition of local recurrence after surgical treatment of nonmelanoma skin cancer and to propose an acceptable surveillance period and tracking methods. METHODS: A literature search was performed for background definitions of local recurrence and tracking methods. The American College of Mohs Surgery (ACMS) Registry and Outcomes Committee then conducted a modified Delphi process to arrive at consensus definitions. RESULTS: We define local recurrence as a tumor with comparable histology, with contiguity to the surgical scar after treatment, and that arises within the area of the previously treated tumor. LIMITATIONS: This project reports the results of a modified Delphi method process involving members of the ACMS. The model described may not be useful for nonexcision type treatments such as topical chemotherapy, electrodessication and curettage, or radiation treatment. CONCLUSIONS: Previous definitions of recurrence and surveillance methods after surgical treatment of nonmelanoma skin cancer are variable and nonstandard. We describe consensus standards for defining and tracking recurrence that should allow for consistent scientific evaluation and development of performance data in skin cancer outcomes registries.

Multiplexed and data-independent tandem mass spectrometry for global proteome profiling.

One of the most important early developments in the field of proteomics was the advent of automated data acquisition routines that allowed high-throughput unattended data acquisition during HPLC introduction of peptide mixtures to a tandem mass spectrometer. Prior to this, data acquisition was orders of magnitude less efficient being based entirely on lists of predetermined ions generated in a prior HPLC-MS experiment. This process, known generically as data-dependent analysis, empowered the development of shotgun proteomics where hundreds to thousands of peptide sequences are matched per experiment. In their most popular implementation, the most abundant ionized species from every precursor ion scan at each moment in chromatographic time are successively selected for isolation, activation and tandem mass analysis. While extremely powerful, this strategy has one primary limitation in that detectable dynamic range is restricted (in a top-down manner) to the peptides that ionize the best. To circumvent the serial nature of the data-dependent process and increase detectable dynamic range, the concepts of multiplexed and data-independent acquisition (DIA) have emerged. Multiplexed-data acquisition is based on more efficient co-selection and co-dissociation of multiple precursor ions in parallel, the data from which is subsequently de-convoluted to provide polypeptide sequences for each individual precursor ion. DIA has similar goals, but there is no real-time ion selection based on prior precursor ion scans. Instead, predefined m/z ranges are interrogated either by fragmenting all ions entering the mass spectrometer at every single point in chromatographic time; or by dividing the m/z range into smaller m/z ranges for isolation and fragmentation. These approaches aim to fully utilize the capabilities of mass spectrometers to maximize tandem MS acquisition time and to address the need to expand the detectable dynamic range, lower the limit of detection, and improve the overall confidence of peptide identifications and relative protein quantification measurements. This review covers all aspects of multiplexed- and data-independent tandem mass spectrometry in proteomics, from experimental implementations to advances in software for data interpretation.

The production of alpha/beta and gamma/delta double negative (DN) T-cells and their role in the maintenance of pregnancy.

The ability of the thymus gland to convert bone marrow-derived progenitor cells into single positive (SP) T-cells is well known. In this review we present evidence that the thymus, in addition to producing SP T-cells, also has a pathway for the production of double negative (DN) T-cells. The existence of this pathway was noted during our examination of relevant literature to determine the cause of sex steroid-induced thymocyte loss. In conducting this search our objective was to answer the question of whether thymocyte loss is the end product of a typical interaction between the reproductive and immune systems, or evidence that the two systems are incompatible. We can now report that "thymocyte loss" is a normal process that occurs during the production of DN T-cells. The DN T-cell pathway is unique in that it is mediated by thymic mast cells, and becomes functional following puberty. Sex steroids initiate the development of the pathway by binding to an estrogen receptor alpha located in the outer membrane of the mast cells, causing their activation. This results in their uptake of extracellular calcium, and the production and subsequent release of histamine and serotonin. Lymphatic vessels, located in the subcapsular region of the thymus, respond to the two vasodilators by undergoing a substantial and preferential uptake of gamma/delta and alpha/beta DN T- cells. These T- cells exit the thymus via efferent lymphatic vessels and enter the lymphatic system.The DN pathway is responsible for the production of three subsets of gamma/delta DN T-cells and one subset of alpha/beta DN T-cells. In postpubertal animals approximately 35 % of total thymocytes exit the thymus as DN T-cells, regardless of sex. In pregnant females, their levels undergo a dramatic increase. Gamma/delta DN T-cells produce cytokines that are essential for the maintenance of pregnancy.