Deep-time phylogenetic inference by paleoproteomic analysis of dental enamel.

In temperate and subtropical regions, ancient proteins are reported to survive up to about 2 million years, far beyond the known limits of ancient DNA preservation in the same areas. Accordingly, their amino acid sequences currently represent the only source of genetic information available to pursue phylogenetic inference involving species that went extinct too long ago to be amenable for ancient DNA analysis. Here we present a complete workflow, including sample preparation, mass spectrometric data acquisition and computational analysis, to recover and interpret million-year-old dental enamel protein sequences. During sample preparation, the proteolytic digestion step, usually an integral part of conventional bottom-up proteomics, is omitted to increase the recovery of the randomly degraded peptides spontaneously generated by extensive diagenetic hydrolysis of ancient proteins over geological time. Similarly, we describe other solutions we have adopted to (1) authenticate the endogenous origin of the protein traces we identify, (2) detect and validate amino acid variation in the ancient protein sequences and (3) attempt phylogenetic inference. Sample preparation and data acquisition can be completed in 3-4 working days, while subsequent data analysis usually takes 2-5 days. The workflow described requires basic expertise in ancient biomolecules analysis, mass spectrometry-based proteomics and molecular phylogeny. Finally, we describe the limits of this approach and its potential for the reconstruction of evolutionary relationships in paleontology and paleoanthropology.

Systematic Optimization of Automated Phosphopeptide Enrichment for High-Sensitivity Phosphoproteomics.

Improving coverage, robustness, and sensitivity is crucial for routine phosphoproteomics analysis by single-shot liquid chromatography-tandem mass spectrometry (LC-MS/MS) from minimal peptide inputs. Here, we systematically optimized key experimental parameters for automated on-bead phosphoproteomics sample preparation with a focus on low-input samples. Assessing the number of identified phosphopeptides, enrichment efficiency, site localization scores, and relative enrichment of multiply-phosphorylated peptides pinpointed critical variables influencing the resulting phosphoproteome. Optimizing glycolic acid concentration in the loading buffer, percentage of ammonium hydroxide in the elution buffer, peptide-to-beads ratio, binding time, sample, and loading buffer volumes allowed us to confidently identify >16,000 phosphopeptides in half-an-hour LC-MS/MS on an Orbitrap Exploris 480 using 30 µg of peptides as starting material. Furthermore, we evaluated how sequential enrichment can boost phosphoproteome coverage and showed that pooling fractions into a single LC-MS/MS analysis increased the depth. We also present an alternative phosphopeptide enrichment strategy based on stepwise addition of beads thereby boosting phosphoproteome coverage by 20%. Finally, we applied our optimized strategy to evaluate phosphoproteome depth with the Orbitrap Astral MS using a cell dilution series and were able to identify >32,000 phosphopeptides from 0.5 million HeLa cells in half-an-hour LC-MS/MS using narrow-window data-independent acquisition (nDIA).

Atypical cofilin signaling drives dendritic cell migration through the extracellular matrix via nuclear deformation.

To mount an adaptive immune response, dendritic cells must migrate to lymph nodes to present antigens to T cells. Critical to 3D migration is the nucleus, which is the size-limiting barrier for migration through the extracellular matrix. Here, we show that inflammatory activation of dendritic cells leads to the nucleus becoming spherically deformed and enables dendritic cells to overcome the typical 2- to 3-µm diameter limit for 3D migration through gaps in the extracellular matrix. We show that the nuclear shape change is partially attained through reduced cell adhesion, whereas improved 3D migration is achieved through reprogramming of the actin cytoskeleton. Specifically, our data point to a model whereby the phosphorylation of cofilin-1 at serine 41 drives the assembly of a cofilin-actomyosin ring proximal to the nucleus and enhances migration through 3D collagen gels. In summary, these data describe signaling events through which dendritic cells deform their nucleus and enhance their migratory capacity.

Protocol for high-throughput semi-automated label-free- or TMT-based phosphoproteome profiling.

Tandem mass tags data-dependent acquisition (TMT-DDA) as well as data-independent acquisition-based label-free quantification (LFQ-DIA) have become the leading workflows to achieve deep proteome and phosphoproteome profiles. We present a modular pipeline for TMT-DDA and LFQ-DIA that integrates steps to perform scalable phosphoproteome profiling, including protein lysate extraction, clean-up, digestion, phosphopeptide enrichment, and TMT-labeling. We also detail peptide and/or phosphopeptide fractionation and pre-mass spectrometry desalting and provide researchers guidance on choosing the best workflow based on sample number and input. For complete details on the use and execution of this protocol, please refer to Koenig et al.1 and Martínez-Val et al.2.

Hybrid-DIA: intelligent data acquisition integrates targeted and discovery proteomics to analyze phospho-signaling in single spheroids.

Achieving sufficient coverage of regulatory phosphorylation sites by mass spectrometry (MS)-based phosphoproteomics for signaling pathway reconstitution is challenging, especially when analyzing tiny sample amounts. To address this, we present a hybrid data-independent acquisition (DIA) strategy (hybrid-DIA) that combines targeted and discovery proteomics through an Application Programming Interface (API) to dynamically intercalate DIA scans with accurate triggering of multiplexed tandem mass spectrometry (MSx) scans of predefined (phospho)peptide targets. By spiking-in heavy stable isotope labeled phosphopeptide standards covering seven major signaling pathways, we benchmark hybrid-DIA against state-of-the-art targeted MS methods (i.e., SureQuant) using EGF-stimulated HeLa cells and find the quantitative accuracy and sensitivity to be comparable while hybrid-DIA also profiles the global phosphoproteome. To demonstrate the robustness, sensitivity, and biomedical potential of hybrid-DIA, we profile chemotherapeutic agents in single colon carcinoma multicellular spheroids and evaluate the phospho-signaling difference of cancer cells in 2D vs 3D culture.

Optimal analytical strategies for sensitive and quantitative phosphoproteomics using TMT-based multiplexing.

In large-scale quantitative mass spectrometry (MS)-based phosphoproteomics, isobaric labeling with tandem mass tags (TMTs) coupled with offline high-pH reversed-phase peptide chromatographic fractionation maximizes depth of coverage. To investigate to what extent limited sample amounts affect sensitivity and dynamic range of the analysis due to sample losses, we benchmarked TMT-based fractionation strategies against single-shot label-free quantification with spectral library-free data independent acquisition (LFQ-DIA), for different peptide input per sample. To systematically examine how peptide input amounts influence TMT-fractionation approaches in a phosphoproteomics workflow, we compared two different high-pH reversed-phase fractionation strategies, microflow (MF) and stage-tip fractionation (STF), while scaling the peptide input amount down from 12.5 to 1 µg per sample. Our results indicate that, for input amounts higher than 5 µg per sample, TMT labeling, followed by microflow fractionation (MF) and phospho-enrichment, achieves the deepest phosphoproteome coverage, even compared to single shot direct-DIA analysis. Conversely, STF of enriched phosphopeptides (STF) is optimal for lower amounts, below 5 µg/peptide per sample. As a result, we provide a decision tree to help phosphoproteomics users to choose the best workflow as a function of sample amount.

Spatial-proteomics reveals phospho-signaling dynamics at subcellular resolution.

Dynamic change in subcellular localization of signaling proteins is a general concept that eukaryotic cells evolved for eliciting a coordinated response to stimuli. Mass spectrometry-based proteomics in combination with subcellular fractionation can provide comprehensive maps of spatio-temporal regulation of protein networks in cells, but involves laborious workflows that does not cover the phospho-proteome level. Here we present a high-throughput workflow based on sequential cell fractionation to profile the global proteome and phospho-proteome dynamics across six distinct subcellular fractions. We benchmark the workflow by studying spatio-temporal EGFR phospho-signaling dynamics in vitro in HeLa cells and in vivo in mouse tissues. Finally, we investigate the spatio-temporal stress signaling, revealing cellular relocation of ribosomal proteins in response to hypertonicity and muscle contraction. Proteomics data generated in this study can be explored through https://SpatialProteoDynamics.github.io .

Changes of Infant- and Family-Centered Care Practices Administered to Extremely Preterm Infants During Implementation of the NIDCAP Program.

Introduction: Many studies have evaluated the Neonatal Individualized Developmental Care and Assessment Program (NIDCAP), but few studies have assessed changes in infant- and family-centered developmental care (IFCDC) practices during its implementation. Objectives: The primary objective of this single center study was to investigate the impact of the implementation of the NIDCAP program on IFCDC practices used for management of extremely preterm infants (EPIs). The secondary objective was to determine during implementation the impact of this program on the short-term medical outcomes of all EPIs hospitalized at our center. Methods: All EPIs (<28 weeks gestational age) who were hospitalized at Strasbourg University Hospital from 2007 to 2014 were initially included. Outborn infants were excluded. The data of EPIs were compared for three time periods: 2007 to 2008 (pre-NIDCAP), 2010 to 2011, and 2013 to 2014 (during-NIDCAP implementation) using appropriate statistical tests. The clinical and caring procedures used during the first 14 days of life were analyzed, with a focus on components of individualized developmental care (NIDCAP observations), infant pain management (number of painful procedures, clinical pain assessment), skin-to-skin contact (SSC; frequency, day of initiation, and duration), and family access and involvement in the care of their children (duration of parental presence, parental participation in care). The short-term mortality and morbidity at discharge were evaluated. Results: We examined 228 EPIs who received care during the three time periods. Over time, painful procedures decreased, but pain evaluations, parental involvement in care, individualized observations, and SSC increased (all p < 0.01). In addition, the first SSC was performed earlier (p = 0.03) and lasted longer (p < 0.01). There were no differences in mortality and morbidity, but there were reductions in the duration of mechanical ventilation (p = 0.02) and the time from birth to first extubation (p = 0.02), and an increase of weight gain at discharge (p = 0.02). Conclusion: NIDCAP implementation was accompanied by progressive, measurable, and significant changes in IFCDC strategies. There were, concomitantly, moderate but statistically significant improvements in multiple important outcome measures of all hospitalized EPI.

Recommendations on neonatal light environment from the French Neonatal Society.

AIM: Hospital light may affect neonatal neurosensory development and the well-being of parents and caregivers. We aimed to issue practical recommendations regarding the optimal light environment for neonatal units. METHODS: A systematic evaluation was performed using PubMed to identify relevant papers published in English or French up to July 2018, and the different grades of evidence were evaluated. RESULTS: We identified 89 studies and one meta-analysis and examined 31 eligible studies. The major results were that natural or artificial light should not exceed 1000 lux and that all changes in light level should be gradual. Light protection should be used for infants of <32 weeks of postmenstrual age and but must be individualised to each infant. Infants should not be exposed to continuous high light levels regardless of their term and postnatal age. Cycled light before discharge seemed to be safe and beneficial. For medical caregivers' well-being, higher light levels and access to natural light are recommended. Special attention should be given to protecting neonatal patients from high light levels that may be necessary when performing specific care procedures. CONCLUSION: Consideration of general principles and practical applications can improve the neonatal light environment for newborn infants, parents and caregivers.

Palaeoproteomic identification of breast milk protein residues from the archaeological skeletal remains of a neonatal dog.

Accurate postmortem estimation of breastfeeding status for archaeological or forensic neonatal remains is difficult. Confident identification of milk-specific proteins associated with these remains would provide direct evidence of breast milk consumption. We used liquid chromatography coupled to tandem mass spectrometry (MS) to confidently identify beta-lactoglobulin-1 (LGB1) and whey acidic protein (WAP), major whey proteins associated with a neonatal dog (Canis lupus familiaris) skeleton (430-960 cal AD), from an archaeological site in Hokkaido, Japan. The age at death of the individual was estimated to be approximately two weeks after birth. Protein residues extracted from rib and vertebra fragments were analyzed and identified by matching tandem MS spectra against the dog reference proteome. A total of 200 dog protein groups were detected and at least one peptide from canine LGB1 and two peptides from canine WAP were confidently identified. These milk proteins most probably originated from the mother's breast milk, ingested by the neonate just before it died. We suggest the milk diffused outside the digestive apparatus during decomposition, and, by being absorbed into the bones, it partially preserved. The result of this study suggests that proteomic analysis can be used for postmortem reconstruction of the breastfeeding status at the time of death of neonatal mammalian, by analyzing their skeletal archaeological remains. This method is also applicable to forensic and wildlife studies.

Assessment of the mutagenic potential of para-chloroaniline and aniline in the liver, spleen, and bone marrow of Big Blue® rats with micronuclei analysis in peripheral blood.

Splenic tumors have been reported in rat cancer bioassays with para-chloroaniline (PCA) and aniline. Development of these tumors is hypothesized to be due to hematotoxicity via the formation of methemoglobin (MetHb) and not direct DNA reactivity. To evaluate the mode of action (MOA) for tumor formation a transgenic rodent (TGR) in vivo gene mutation assay in Big Blue® TgF344 rats was performed with parallel micronuclei analysis in peripheral blood. Male rats were gavaged daily for 28 d to 0.5, 15, and 60 mg/kg PCA and 100 mg/kg aniline, the base molecular structure of PCA. On test day 10, the 60 mg/kg PCA dose was reduced to 30 mg/kg due to toxicity. On test day 4 and 29 peripheral blood micronucleus analysis was performed and on test day 29 clinical chemistry, hematology, and MetHb measurements were taken. At study termination, on test day 31, spleen, bone marrow, and liver (control tissue) were analyzed for cII transgene mutant frequency (MF). Repeat gavage exposure to PCA and aniline for 28 d did not produce an increase in cII transgene MF in analyzed tissues. An increase in micronuclei was seen at both time points at ≥15 mg/kg PCA and 100 mg/kg aniline. At the same dose levels, significant reductions in red blood cells, increases in absolute reticulocytes (ABRET), and increased levels of MetHb were observed. Together these results support that generation of micronuclei and tumorigenicity following exposure to PCA and aniline is due to compensatory mechanisms (e.g. increased cellular turnover) and not direct DNA reactivity. Environ. Mol. Mutagen. 59:785-797, 2018. © 2018 Wiley Periodicals, Inc.

Neural-specific deletion of Htra2 causes cerebellar neurodegeneration and defective processing of mitochondrial OPA1.

HTRA2, a serine protease in the intermembrane space, has important functions in mitochondrial stress signaling while its abnormal activity may contribute to the development of Parkinson's disease. Mice with a missense or null mutation of Htra2 fail to thrive, suffer striatal neuronal loss, and a parkinsonian phenotype that leads to death at 30-40 days of age. While informative, these mouse models cannot separate neural contributions from systemic effects due to the complex phenotypes of HTRA2 deficiency. Hence, we developed mice carrying a Htra2-floxed allele to query the consequences of tissue-specific HTRA2 deficiency. We found that mice with neural-specific deletion of Htra2 exhibited atrophy of the thymus and spleen, cessation to gain weight past postnatal (P) day 18, neurological symptoms including ataxia and complete penetrance of premature death by P40. Histologically, increased apoptosis was detected in the cerebellum, and to a lesser degree in the striatum and the entorhinal cortex, from P25. Even earlier at P20, mitochondria in the cerebella already exhibited abnormal morphology, including swelling, vesiculation, and fragmentation of the cristae. Furthermore, the onset of these structural anomalies was accompanied by defective processing of OPA1, a key molecule for mitochondrial fusion and cristae remodeling, leading to depletion of the L-isoform. Together, these findings suggest that HTRA2 is essential for maintenance of the mitochondrial integrity in neurons. Without functional HTRA2, a lifespan as short as 40 days accumulates a large quantity of dysfunctional mitochondria that contributes to the demise of mutant mice.

Using mouse models of autism spectrum disorders to study the neurotoxicology of gene-environment interactions.

To better study the role of genetics in autism, mouse models have been developed which mimic the genetics of specific autism spectrum and related disorders. These models have facilitated research on the role genetic susceptibility factors in the pathogenesis of autism in the absence of environmental factors. Inbred mouse strains have been similarly studied to assess the role of environmental agents on neurodevelopment, typically without the complications of genetic heterogeneity of the human population. What has not been as actively pursued, however, is the methodical study of the interaction between these factors (e.g., gene and environmental interactions in neurodevelopment). This review suggests that a genetic predisposition paired with exposure to environmental toxicants plays an important role in the etiology of neurodevelopmental disorders including autism, and may contribute to the largely unexplained rise in the number of children diagnosed with autism worldwide. Specifically, descriptions of the major mouse models of autism and toxic mechanisms of prevalent environmental chemicals are provided followed by a discussion of current and future research strategies to evaluate the role of gene and environment interactions in neurodevelopmental disorders.

Maternal transfer of BDE-47 to offspring and neurobehavioral development in C57BL/6J mice.

Polybrominated diphenyl ethers (PBDEs) are flame retardants used worldwide in a variety of commercial goods, and are now widely found in both environmental and biological samples. BDE-47 is one of the most pervasive of these PBDE congeners and therefore is of particular concern. In this study C57BL/6J mice were exposed perinatally to 0.03, 0.1 or 1mg/kg/day of BDE-47, a dose range chosen to encompass human exposure levels. Tissue levels of BDE-47 were measured in the blood, brain, fat and milk of dams and in whole fetal homogenate and blood and brain of pups on gestational day (GD) 15, and postnatal days (PNDs) 1, 10 and 21. From GD 15 to PND 1 levels of BDE-47 increased within dam tissues and then decreased from PNDs 1 to 21. Over the period of lactation levels in dam milk were comparatively high when compared to both brain and blood for all dose groups. Measurable levels of BDE-47 were found in the fetus on GD 15 confirming gestational exposure. From PNDs 1 to 21, levels of BDE-47 in pup tissue increased over the period of lactation due to the transfer of BDE-47 through milk. Behavioral tests of fine motor function and learning and memory were carried out between postnatal weeks 5-17 in order to evaluate the neurobehavioral toxicity of BDE-47. Behavioral deficits were only seen in the Barnes spatial maze where mice in the three exposure groups had longer latencies and traveled longer distances to find the escape hole when compared to vehicle control mice. These results support the conclusions that perinatal exposure to BDE-47 can have neurodevelopmental consequences, and that lactational exposure represents a significant exposure risk during development.

Maternal autism-associated IgG antibodies delay development and produce anxiety in a mouse gestational transfer model.

A murine passive transfer model system was employed to ascertain the effects of gestational exposure to a single, intravenous dose of purified, brain-reactive IgG antibodies from individual mothers of children with autism (MAU) or mothers with typically developing children (MTD). Growth and behavioral outcomes in offspring were measured from postnatal days 8 to 65 in each group. Comparisons revealed alterations in early growth trajectories, significantly impaired motor and sensory development, and increased anxiety. This report demonstrates for the first time the effects of a single, low dose gestational exposure of IgG derived from individual MAU on their offspring's physical and social development.

Lack of evidence for neonatal misoprostol neurodevelopmental toxicity in C57BL6/J mice.

Misoprostol is a synthetic analogue of prostaglandin E1 that is administered to women at high doses to induce uterine contractions for early pregnancy termination and at low doses to aid in cervical priming during labor. Because of the known teratogenic effects of misoprostol when given during gestation and its effects on axonal growth in vitro, we examined misoprostol for its potential as a neurodevelopmental toxicant when administered to neonatal C57BL6/J mice. Mice were injected subcutaneously (s.c.) with 0.4, 4 or 40 µg/kg misoprostol on postnatal day 7, the approximate developmental stage in mice of human birth, after which neonatal somatic growth, and sensory and motor system development were assessed. These doses were selected to span the range of human exposure used to induce labor. In addition, adult mice underwent a battery of behavioral tests relevant to neurodevelopmental disorders such as autism including tests for anxiety, stereotyped behaviors, social communication and interactions, and learning and memory. No significant effects of exposure were found for any measure of development or behavioral endpoints. In conclusion, the results of the present study in C57BL/6J mice do not provide support for neurodevelopmental toxicity after misoprostol administration approximating human doses and timed to coincide with the developmental stage of human birth.

Bioaccumulation and behavioral effects of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) in perinatally exposed mice.

Polybrominated diphenyl ethers (PBDEs) are widely used flame retardants that have become pervasive environmental contaminants and may contribute to adverse health outcomes. We evaluated in mice the developmental neurotoxicity of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), one of the most abundant PBDE congeners detected in animal and human tissues. Female C57BL/6J mice were exposed to daily doses of 0, 0.03, 0.1 or 1mg/kg beginning 4 weeks prior to conception, continuing through gestation and lactation, and ending at weaning on postnatal day (PND) 21. Levels of BDE-47 in blood, brain, liver and adipose tissues of dams were markedly increased after 4 weeks of exposure, around the time of mating, and continued to increase through the time of parturition. Blood levels of BDE-47 in the dosed dams were within the range reported in humans. BDE-47 tissue levels in the dams decreased between parturition and weaning, possibly reflecting mobilization during lactation. Brain BDE-47 levels in the offspring at PND 1 approached those of the dams at parturition. Perinatal exposure to BDE-47 resulted in significant dose dependent growth retardation, slower motor performance in several behavioral tests, and mice exposed to 1mg/kg/day BDE-47 showed altered performance in the Morris water maze. There were no differences between groups in the numbers of pyramidal neurons in hippocampus CA1. These results document accumulation of BDE-47 in several organ systems following exposure to low-levels of BDE-47, and provide evidence that such exposure is associated with early behavioral deficits in exposed neonates.