General anesthetics selectively modulate glutamatergic and dopaminergic signaling via site-specific phosphorylation in vivo

Isoflurane, propofol and ketamine are representative general anesthetics with distinct molecular mechanisms of action that have neuroprotective properties in models of excitotoxic ischemic damage. We characterized the effects of these agents on neuronal glutamate and dopamine signaling by profiling drug-induced changes in brain intracellular protein phosphorylation in vivo to test the hypothesis that they affect common downstream effectors. Anesthetic-treated and control mice were killed instantly by focused microwave irradiation, frontal cortex and striatum were removed, and the phosphorylation profile of specific neuronal signaling proteins was analyzed by immunoblotting with a panel of phospho-specific antibodies. At anesthetic doses that produced loss of righting reflex, isoflurane, propofol, and ketamine all reduced phosphorylation of the activating residue T183 of ERK2 (but not of ERK1); S897 of the NR1 NMDA receptor subunit; and S831 (but not S845) of the GluR1 AMPA receptor subunit in cerebral cortex. At sub-anesthetic doses, these drugs only reduced phosphorylation of ERK2. Isoflurane and ketamine also reduced phosphorylation of spinophilin at S94, but oppositely regulated phosphorylation of presynaptic (tyrosine hydroxylase) and postsynaptic (DARPP-32) markers of dopaminergic neurotransmission in striatum. These data reveal both shared and agent-specific actions of CNS depressant drugs on critical intracellular protein phosphorylation signaling pathways that integrate multiple second messenger systems. Reduced phosphorylation of ionotropic glutamate receptors by all three anesthetics indicates depression of normal glutamatergic synaptic transmission and reduced potential excitotoxicity. This novel approach indicates a role for phosphorylation-mediated down-regulation of glutamatergic synaptic transmission by general anesthetics and identifies specific in vivo targets for focused evaluation of anesthetic mechanisms.     

Accuracy and reliability of forensic handwriting comparisons

Forensic handwriting examination involves the comparison of writing samples by forensic document examiners (FDEs) to determine whether or not they were written by the same person. Here we report the results of a large-scale study conducted to assess the accuracy and reliability of handwriting comparison conclusions. Eighty-six practicing FDEs each conducted up to 100 handwriting comparisons, resulting in 7,196 conclusions on 180 distinct comparison sets, using a five-level conclusion scale. Erroneous “written by” conclusions (false positives) were reached in 3.1% of the nonmated comparisons, while 1.1% of the mated comparisons yielded erroneous “not written by” conclusions (false negatives). False positive rates were markedly higher for nonmated samples written by twins (8.7%) compared to nontwins (2.5%). Notable associations between training and performance were observed: FDEs with less than 2 y of formal training generally had higher error rates, but they also had higher true positive and true negative rates because they tended to provide more definitive conclusions; FDEs with at least 2 y of formal training were less likely to make definitive conclusions, but those definitive conclusions they made were more likely to be correct (higher positive predictive and negative predictive values). We did not observe any association between writing style (cursive vs. printing) and rates of errors or incorrect conclusions. This report also provides details on the repeatability and reproducibility of conclusions, and reports how conclusions are affected by the quantity of writing and the similarity of content.

Forensic science is under scrutiny, particularly for pattern-based disciplines in which source conclusions are reported. The National Research Council report Strengthening Forensic Science in the United States: A Path Forward (1) stated that “The scientific basis for handwriting comparisons needs to be strengthened” and noted that “there has been only limited research to quantify the reliability and replicability of the practices used by trained document examiners.” The President’s Council of Advisors on Science and Technology (PCAST) report Forensic Science in Criminal Courts: Ensuring Scientific Validity of Feature-Comparison Methods (2) expressed concerns regarding the validity and reliability of conclusions made by forensic examiners, and called for empirical testing: “The only way to establish the scientific validity and degree of reliability of a subjective forensic feature-comparison method—that is, one involving significant human judgment—is to test it empirically by seeing how often examiners actually get the right answer. Such an empirical test of a subjective forensic feature-comparison method is referred to as a ‘black-box test.’” The National Commission on Forensic Science also called for such testing (3). Although the accuracy and reliability of conclusions made by forensic document examiners (FDEs) have been the focus of multiple studies over the years (4–10), the designs of those studies are notably different from this study (and from PCAST’s recommendations), and therefore the resulting rates are not directly comparable (in particular, when comparing open-set to closed-set studies, comparing studies based on one-to-one vs. one-to-many examinations, and comparing studies that use notably different conclusion scales; see SI Appendix, Appendix B for a summary).This study was conducted to provide data that can be used to assess the scientific validity of handwriting comparisons, for use by policy makers, laboratory managers, the legal community, and FDEs. This study follows the approach used in the previous FBI Laboratory–Noblis latent print black box study (11) and later recommended by the PCAST report. The design utilizes open-set, one-to-one document comparisons to evaluate the conclusions reached by practicing FDEs when comparing writing samples selected to be broadly comparable to casework. The primary purposes of the study are to measure the accuracy of conclusions by FDEs when comparing handwriting samples and to assess reliability by measuring the reproducibility (interexaminer variability) and repeatability (intraexaminer variability) of those conclusions. Secondary purposes include reporting any associations between the accuracy of the decisions in this study, factors related to the participants (such as training or experience), and factors related to the samples (such as quantity of writing, comparability of content, limitations, or style of writing).     

Renal failure in sick hypertensive premature infants receiving captopril therapy

A retrospective study of nine sick premature infants with chronic lung disease who received captopril for control of systemic hypertension (systolic blood pressure (BP) greater than 113 mm Hg) was carried out to determine efficacy of therapy and associated complications. All nine infants had markedly elevated peripheral renin values, 134.3 +/- 128.1 ng/mL/hr (mean +/- SD). Five infants had abnormal renal sonographic and perfusion scans with evidence of renal artery thrombosis, parenchymal disease, or both. Captopril therapy (0.3 mg/kg) was instituted at a postnatal age of 123 +/- 108 days. After the initial dose, the systolic BP decreased significantly in all infants, the decrease ranging from 21% to 58% of the pretreatment value. Dosage was subsequently halved in all infants. Seventeen episodes of unpredictable decreases in BP more than 40% from baseline occurred during the reduced maintenance therapy. Four infants had a total of seven episodes during which the BP decreased by 57 +/- 10% from baseline; this decrease persisted for 17 +/- 6 hours and was unresponsive to volume reexpansion and inotropic therapy. All seven episodes were accompanied by oliguria (urine output less than 1 mL/kg/hr) that persisted for 18 +/- 12 hours. These episodes were accompanied by neurologic signs (subtle seizures, lethargy, and/or apnea) within 18 +/- 6 hours after the onset of oliguria. The remaining five infants had a total of 13 episodes of decreased BP of 50 +/- 8% of baseline, which were of significantly shorter duration and responded to volume reexpanders, inotropic therapy, or both and were unaccompanied by oliguria. These data suggest the need for close observation of BP in infants receiving maintenance captopril therapy.     

Timely follow‐up of positive cancer screening results: A systematic review and recommendations from the PROSPR Consortium

Timely follow‐up for positive cancer screening results remains suboptimal, and the evidence base to inform decisions on optimizing the timeliness of diagnostic testing is unclear. This systematic review evaluated published studies regarding time to follow‐up after a positive screening for breast, cervical, colorectal, and lung cancers. The quality of available evidence was very low or low across cancers, with potential attenuated or reversed associations from confounding by indication in most studies. Overall, evidence suggested that the risk for poorer cancer outcomes rises with longer wait times that vary within and across cancer types, which supports performing diagnostic testing as soon as feasible after the positive result, but evidence for specific time targets is limited. Within these limitations, we provide our opinion on cancer‐specific recommendations for times to follow‐up and how existing guidelines relate to the current evidence. Thresholds set should consider patient worry, potential for loss to follow‐up with prolonged wait times, and available resources. Research is needed to better guide the timeliness of diagnostic follow‐up, including considerations for patient preferences and existing barriers, while addressing methodological weaknesses. Research is also needed to identify effective interventions for reducing wait times for diagnostic testing, particularly in underserved or low‐resource settings. CA Cancer J Clin 2018;68:199–216 . © 2018 American Cancer Society .     

Cytokine expression in response to bacterial antigens in preterm and term infant cord blood monocytes

BACKGROUND: Neonatal susceptibility to bacterial infection is associated with an immature immune system, but the role of different bacterial antigens in specific responses is largely unknown. OBJECTIVE: To evaluate differences in intracellular cytokine response to physiologically relevant bacterial antigens in term and preterm infants as compared with adults. METHODS: Cord blood samples from preterm and term neonates and adult peripheral blood samples were cultured ex vivo with and without whole heat-killed bacteria. Intracellular leukocyte production of interleukin (IL)-6, IL-10, IL-12, and IL-8 responses was assessed by flow cytometry. RESULTS: Monocytes were the primary producers of all mediators. Escherichia coli was the most potent stimulant. Lactobacillus plantarum 299v activated fewer monocytes as compared with E. coli for all responses (p < 0.05), except for IL-12 in term neonates. IL-6 response to Staphylococcus epidermidis was lower in both groups of neonates as compared with adults (p = 0.023 and p = 0.001). IL-8 response to S. epidermidis was lower in term as compared with preterm neonates and adults (p = 0.003). IL-10 response to group B streptococci was lower in term neonates as compared with adults and higher in preterm as compared with term neonates (p = 0.015). CONCLUSIONS: Monocytes from term neonates compared to preterm neonates show a downregulated anti-inflammatory response to specific bacteria. High neonatal response to pathogenic E. coli in the preterm infant could cause uncontrolled inflammatory response, while lower IL-6 response to S. epidermidis in neonates may indicate a basis for vulnerability to S. epidermidis infection.     

An approach for evaluating the effects of dietary fiber polysaccharides on the human gut microbiome and plasma proteome

Increases in snack consumption associated with Westernized lifestyles provide an opportunity to introduce nutritious foods into poor diets. We describe two 10-wk-long open label, single group assignment human studies that measured the effects of two snack prototypes containing fiber preparations from two sustainable and scalable sources; the byproducts remaining after isolation of protein from the endosperm of peas and the vesicular pulp remaining after processing oranges for the manufacture of juices. The normal diets of study participants were supplemented with either a pea- or orange fiber-containing snack. We focused our analysis on quantifying the abundances of genes encoding carbohydrate-active enzymes (CAZymes) (glycoside hydrolases and polysaccharide lyases) in the fecal microbiome, mass spectrometric measurements of glycan structures (glycosidic linkages) in feces, plus aptamer-based assessment of levels of 1,300 plasma proteins reflecting a broad range of physiological functions. Computational methods for feature selection identified treatment-discriminatory changes in CAZyme genes that correlated with alterations in levels of fiber-associated glycosidic linkages; these changes in turn correlated with levels of plasma proteins representing diverse biological functions, including transforming growth factor type β/bone morphogenetic protein-mediated fibrosis, vascular endothelial growth factor-related angiogenesis, P38/MAPK-associated immune cell signaling, and obesity-associated hormonal regulators. The approach used represents a way to connect changes in consumer microbiomes produced by specific fiber types with host responses in the context of varying background diets.

Advances in our understanding of the role of the gut microbiome in regulating many aspects of human physiology hold the promise of evolving our view of human nutrition by establishing mechanistic connections between the foods we consume and how they affect health status. One manifestation of this effort is a series of studies, performed on well-phenotyped cohorts, that seek to relate features of gut microbial community composition (organisms, genes), dietary practices, and pre- and postprandial cardiometabolic responses to test meals (1–4). A key question raised by these initiatives relates to the nature of the “bioactive” components of foods. Specifically, what are the nutrients utilized by various gut community members or microbiome-encoded metabolic pathways? What products are produced by biotransformation of these nutrients? How are these products linked to specific host physiologic (or pathophysiologic) processes?Plant-derived dietary fibers represent a “poster child” for these efforts and illustrate the formidable challenges faced. The health benefits of dietary fibers are widely known, as is their inadequate representation in Western diets. However, natural fibers are structurally complex and highly diverse. They contain numerous, typically undefined polysaccharide structures and largely unspecified protein, lipid, and small molecule constituents. Their composition varies as a function of their origin (food staple and cultivar), the different methods employed to recover them from these sources, as well as the different techniques used to incorporate them into processed foods with acceptable organoleptic properties (5). Moreover, analyzing the host effects of metabolism of different fibers is confounded by the fact that there is substantial intra- and interpersonal variation in microbiome configuration (6, 7).Snacking is becoming an ever more dominant feature of daily life worldwide and thus provides an opportunity to introduce nutritious ingredients, such as fibers, into diets. However, obtaining structure-activity relationships for specific fiber types and their corresponding targets in the gut community is foundational for designing snack foods that evoke and/or reinforce microbiome responses that are beneficial to the host.Degradation of dietary polysaccharides is a function primarily performed by bacterial carbohydrate-active enzymes (CAZymes). The gut microbiome harbors tens of thousands of CAZyme genes belonging to at least 136 glycoside hydrolase (GH) and 29 polysaccharide lyase (PL) families [extrapolated and updated from El Kaoutari et al. (8)]. In contrast, the human genome only contains 98 GH and no PL genes (9), of which <20% contribute to the processing of dietary glycans.In the current study, we test the effects of dietary supplementation with two snack food prototypes, one containing pea fiber and the other orange fiber, in two pilot studies of overweight and obese individuals consuming their normal, unrestricted diets. Our strategy was to focus on fiber-associated changes in the abundances of microbial GH and PL genes to determine whether responses to the pea or orange fiber prototypes in the gut microbiome and host are decipherable against a background of varying dietary practices and starting microbiome configurations. Higher order singular value decomposition (10) was utilized as a feature selection tool to identify treatment-discriminating changes in GH and PL gene representation. Mass spectrometric assays of the levels of fecal glycan structures (glycosidic linkages) were subsequently performed and the results were correlated with changes in the abundances of treatment-discriminating GH and PL genes with known or predicted substrate specificities. Our analysis concluded by measuring changes in levels of 1,305 plasma proteins in each study participant as a function of fiber treatment and applying computational tools to identify links between these microbiome and plasma proteome changes in response to fiber consumption. Our results provide an approach, using pilot human studies, for selecting specific fiber preparations, plus informative microbiome and host biomarkers, that can be advanced to proof-of-concept clinical trials which assess their capacity for precise manipulation of microbiome and host features.