Remifentanil-based sedation to treat noninvasive ventilation failure: a preliminary study

Objective To assess the feasibility and safety of remifentanil-based sedation during noninvasive ventilation (NIV) in patients with NIV failure. Design and setting Prospective clinical investigation in a 16-bed intensive care unit of a university hospital in France. Patients Thirteen patients in NIV failure due to discomfort and/or refusal to continue this ventilatory support: 10 with acute respiratory failure and 3 with acute hypercapnic respiratory failure. Intervention Patients were administered methylene blue and were sedated (Ramsay scale 2–3) by a continuous perfusion of remifentanil during NIV. Cardiorespiratory and ventilatory parameters, blood gas analysis, and adverse events were prospectively recorded. Measurements and results The 13 patients received a total of 125 NIV sessions, totaling 1200 h, of NIV under remifentanil-based sedation (mean remifentanil dose 0.1 ± 0.03 μg/kg per minute). Three patients also required propofol. PaO₂/FIO₂ ratio increased from 134 ± 69 to 187 ± 43 mmHg after 1 h. In patients with acute respiratory failure respiratory rate decreased from 34 ± 12 per minute before remifentanil to 25 ± 4 per minute after 1 h. In the three patients with acute hypercapnic respiratory failure PaCO₂ decreased from 69 ± 7 to 42 ± 5 mmHg. Four patients required endotracheal intubation without aspiration pneumonia. Twelve of the 13 patients left the ICU. Conclusion This pilot study shows that remifentanil-based sedation is safe and effective in the treatment of NIV failure due to low tolerance.     

Role for DNA repair factor XRCC4 in immunoglobulin class switch recombination

V(D)J recombination and immunoglobulin class switch recombination (CSR) are two somatic rearrangement mechanisms that proceed through the introduction of double-strand breaks (DSBs) in DNA. Although the DNA repair factor XRCC4 is essential for the resolution of DNA DSB during V(D)J recombination, its role in CSR has not been established. To bypass the embryonic lethality of XRCC4 deletion in mice, we developed a conditional XRCC4 knockout (KO) using LoxP-flanked XRCC4 cDNA lentiviral transgenesis. B lymphocyte restricted deletion of XRCC4 in these mice lead to an average two-fold reduction in CSR in vivo and in vitro. Our results connect XRCC4 and the nonhomologous end joining DNA repair pathway to CSR while reflecting the possible use of an alternative pathway in the repair of CSR DSB in the absence of XRCC4. In addition, this new conditional KO approach should be useful in studying other lethal mutations in mice.     

B cell antigen receptor cross-linking induces tyrosine phosphorylation and membrane translocation of a multimeric Shc complex that is augmented by CD19 co-ligation

The SH2 domain-containing transforming Shc protein has been implicated in mitogenic signaling via several surface receptors through p21^ras. Following tyrosine phosphorylation by either receptor or non-receptor tyrosine kinases, Shc may interact with the adaptor protein Grb2, which is linked to Sos1, a guanine nucleotide exchange factor for human ras. Ligation of the antigen receptor complex on B cells (BCR) is known to activate various intracellular signaling pathways, which may accumulate in mitogenic responses. With respect to the initial steps, the activation of BCR-associated non-receptor tyrosine kinases appears to be indispensible. In this report we show that Shc proteins become tyrosine phosphorylated after BCR ligation on both transformed and normal human B cells. This is accompanied by the association of Shc with Grb2 proteins and a yet unidentified 145-kDa tyrosine phosphorylated protein. Subcellular fractionation revealed that this activation-induced multimeric Shc complex rapidly translocates towards the plasma membrane. Co-ligation of the BCR with the CD19 molecule results in a marked increase of these events, whereas CD19 cross-linking alone does not induce Shc tyrosine phosphorylation or translocation. Thus, in B cells the Shc complex may represent a molecular junction between the BCR and the mitogenic p21^ras cascade.     

Rare Pathogenic Variants in Mitochondrial and Inflammation-Associated Genes May Lead to Inflammatory Cardiomyopathy in Chagas Disease

Abstract

Cardiomyopathies are an important cause of heart failure and sudden cardiac death. Little is known about the role of rare genetic variants in inflammatory cardiomyopathy. Chronic Chagas disease cardiomyopathy (CCC) is an inflammatory cardiomyopathy prevalent in Latin America, developing in 30% of the 6 million patients chronically infected by the protozoan Trypanosoma cruzi, while 60% remain free of heart disease (asymptomatic (ASY)). The cytokine interferon-γ and mitochondrial dysfunction are known to play a major pathogenetic role. Chagas disease provides a unique model to probe for genetic variants involved in inflammatory cardiomyopathy.

Methods

We used whole exome sequencing to study nuclear families containing multiple cases of Chagas disease. We searched for rare pathogenic variants shared by all family members with CCC but absent in infected ASY siblings and in unrelated ASY.

Results

We identified heterozygous, pathogenic variants linked to CCC in all tested families on 22 distinct genes, from which 20 were mitochondrial or inflammation-related – most of the latter involved in proinflammatory cytokine production. Significantly, incubation with IFN-γ on a human cardiomyocyte line treated with an inhibitor of dihydroorotate dehydrogenase brequinar (enzyme showing a loss-of-function variant in one family) markedly reduced mitochondrial membrane potential (ΔψM), indicating mitochondrial dysfunction.

Conclusion

Mitochondrial dysfunction and inflammation may be genetically determined in CCC, driven by rare genetic variants. We hypothesize that CCC-linked genetic variants increase mitochondrial susceptibility to IFN-γ-induced damage in the myocardium, leading to the cardiomyopathy phenotype in Chagas disease. This mechanism may also be operative in other inflammatory cardiomyopathies.

     

Mitochondrial UCP4 and bcl-2 expression in imprints of breast carcinomas: relationship with DNA ploidy and classical prognostic factors

Mitochondria are the bioenergetic and metabolic centers of cells and play an important role in the regulation of cell death. The mitochondrial apoptosis pathway is controlled by the bcl-2 protein family. Overexpression of mitochondrial uncoupling protein 4 (UCP4) can promote proliferation and inhibit apoptosis and differentiation. Imprint smears obtained from 124 tumors were studied immunocytochemically, and results were correlated with prognostic markers. There were 112 ductal and 12 lobular carcinomas. The positivity of UCP4 was correlated with lymph node metastases (p = 0.005), positive ER and PR expression (p < 0.0001 for both), as well as positivity for p53 (p < 0.0001) and Ki-67 (p < 0.0001). Decreased expression of bcl-2 correlated with increased expression of UCP4 (p = 0.001). Regarding DNA ploidy, UCP4 positivity was correlated with aneuploid tumors (p = 0.002). Negative expression of bcl-2 was correlated with poorly differentiated carcinomas (p < 0.0001), as well as with positive expression of p53 (p < 0.0001) and Ki-67 (p < 0.0001). Logistic regression revealed that ploidy and p53 expression had an impact on UCP4. These findings encourage future investigations regarding the potential role of UCPs not only into mechanisms underlying breast cancer, but also as a novel candidate to the design and development of more effective therapeutic strategies.     

High levels of functional endopeptidase 24.11 (CD10) activity on human thymocytes: preferential expression on immature subsets.

Although it is now well established that cells of the immune system express most of the exopeptidases described so far, little information is available concerning the identification and the characterization of the peptidases associated with the surface of human thymocytes. In the present study we have focused on CD10 expression on thymocytes using both FACS and enzymatic analysis. Unfractionated intact human thymocytes were shown to express significant levels of CD10-specific enzymatic activity, as assessed by the hydrolysis of the neutral endopeptidase (NEP) substrate Suc-Ala-Ala-Phe-pNA and of D-Ala2-Leu-enkephalin, a typical NEP substrate. CD10 activity was abolished by specific NEP inhibitors, including thiorphan, retrothiorphan and phosphoramidon. Moreover, high performance liquid chromatography (HPLC) analysis showed that intact thymocytes and purified NEP hydrolysed thymopentin, a thymic factor known to induce the maturation of prothymocytes into thymocytes. Finally, CD 10/NEP was preferentially associated with CD3- CD3low and immature CD4- CD8- thymocytes. The data demonstrate for the first time that human thymocytes express functional NEP and suggest a role for this enzyme in the maturation of human thymocytes.     

Distribution of catecholaminergic and peptidergic cells in the gerbil medial amygdala, caudal preoptic area and caudal bed nuclei of the stria terminalis with a focus on areas activated at ejaculation

The posterodorsal preoptic nucleus (PdPN), lateral part of the posterodorsal medial amygdala (MeApd) and medial part of the medial preoptic nucleus (MPNm) are activated at ejaculation in male gerbils as assessed by Fos expression. We sought to immunocytochemically visualize substance P (SP), cholecystokinin (CCK), oxytocin, vasopressin and tyrosine hydroxylase (TH), a catecholaminergic marker, in the mating-activated cells, but the need for colchicine precluded behavioral testing. Instead, we detailed distributions of cells containing these molecules in the medial amygdala, caudal preoptic area and caudal bed nuclei of the stria terminalis (BST) and quantified their densities in the PdPN, MPNm and lateral MeApd for comparison to densities previously assessed for mating-activated efferents from these sites. TH cells were as dense in the PdPN and lateral MeApd as activated efferents to the anteroventral periventricular nucleus. In the lateral MeApd, TH cells were grouped where cells activated at ejaculation are clustered and where CCK cells form a ball. Lateral MeApd CCK cells and PdPN SP cells were as dense as activated efferents to the principal BST. Oxytocinergic PdPN cells and SP cells in the MPNm were as dense as mating-activated efferents to the lateral MeApd. If some oxytocin cells in the PdPN project to the neurohypophysis, as in rats, they could be a source of the oxytocin secreted at ejaculation. Since gerbils are monogamous and biparental, it was also interesting that, unlike monogamous prairie voles, they had few TH cells in the MeApd or dorsal BST, resembling promiscuous rats, hamsters and meadow voles.     

Recent Advances in Archaeological Science Techniques Special Feature: A Neandertal dietary conundrum: Insights provided by tooth enamel Zn isotopes from Gabasa,Spain

The characterization of Neandertals’ diets has mostly relied on nitrogen isotope analyses of bone and tooth collagen. However, few nitrogen isotope data have been recovered from bones or teeth from Iberia due to poor collagen preservation at Paleolithic sites in the region. Zinc isotopes have been shown to be a reliable method for reconstructing trophic levels in the absence of organic matter preservation. Here, we present the results of zinc (Zn), strontium (Sr), carbon (C), and oxygen (O) isotope and trace element ratio analysis measured in dental enamel on a Pleistocene food web in Gabasa, Spain, to characterize the diet and ecology of a Middle Paleolithic Neandertal individual. Based on the extremely low δ⁶⁶Zn value observed in the Neandertal’s tooth enamel, our results support the interpretation of Neandertals as carnivores as already suggested by δ¹⁵N isotope values of specimens from other regions. Further work could help identify if such isotopic peculiarities (lowest δ⁶⁶Zn and highest δ¹⁵N of the food web) are due to a metabolic and/or dietary specificity of the Neandertals.

Over the last 30 years, analyses of nitrogen isotopes in collagen (δ¹⁵N_collagen) have provided direct evidence for Neandertal diets across Europe and Asia. These studies all indicate a carnivorous (1–12), or at least a meat-heavy, diet for European Neandertals. However, one peculiarity of Neandertal δ¹⁵N_collagen remains the subject of an ongoing debate. From the one Siberian and eight western European sites, where both Neandertal and associated fauna have been analyzed, nitrogen isotope ratios in Neandertal collagen are systematically higher than that of other carnivores (3, 6–8, 10, 11, 13, 14). An explanation for such elevated values could be the consumption of herbivores, such as mammoths, which themselves exhibit elevated δ¹⁵N values due to the consumption of plants growing on arid soils (1, 2, 7). While mammoth remains are usually scarce at Neandertal fossil localities, they were nonetheless occasionally consumed, as suggested by remains with cut marks and other human butchery signatures (15). The absence of mammoth remains at Middle Paleolithic sites could be a result of 1) Neandertals chose to leave large bone elements at the kill site and transport other edible carcass products, mainly meat, back to the habitation site (15), or 2) mammoths were not frequently consumed, and the δ¹⁵N peculiarity consequently reflects the consumption of other resources enriched in ¹⁵N.Alongside this δ¹⁵N peculiarity, one major obstacle to our knowledge of Neandertals’ subsistence patterns is that the preservation of organic matter limits the application of collagen-bound nitrogen isotope analysis to fossil specimens. Collagen degrades over time at a varying speed depending on climatic and environmental conditions (16). The oldest hominin specimen in which bone protein is preserved is that of Scladina (Belgium), which dates to 90,000 cal BP (calibrated years before the present) (17), but most studied specimens are younger than 50,000 cal BP (1–3, 6–8, 10–13, 18). Furthermore, these specimens are only from sites in northwestern and central Europe and Siberia, where climatic conditions favored collagen preservation. As a result, the variability of Neandertals’ diet over time and between regions may not accurately be reflected by the currently available isotope data. In Iberia, where the latest surviving Neandertals have been discovered (19, 20), collagen was successfully extracted for only one site (21). Therefore, our knowledge of Iberian Neandertal diets mostly relies on zooarcheological and dental calculus data, which show some inconsistencies (21–25). For instance, similar to other western European sites, zooarcheological studies emphasize the consumption of terrestrial mammals and birds (21). In contrast, analysis of dental calculus for microremains and ancient DNA metagenomic analysis (26–28) highlight the frequent consumption of plants and mushrooms. Indeed, Weyrich et al. (26) even suggest that Neandertals at El Sidrón (Fig. 1) rarely consumed meat but often ate mushrooms, which would also result in elevated δ¹⁵N values (29). The consumption of marine foods is also attested for coastal Neandertals, but its frequency cannot be truly assessed in the absence of isotope studies (21, 23–25, 30). Finally, cannibalism has been documented at two Iberian sites (El Sidrón and Zafarraya) (22, 31) (Fig. 1), though such practices appear limited and most likely occurred only during periods of nutritional stress (32). Therefore, it is challenging to confirm the homogeneity of Neandertals’ diets across time and space, calling into question a direct link between their subsistence strategy and disappearance.Open in a separate windowFig. 1.(A) Location of the Gabasa site as well as other Neandertal sites mentioned in the text. (B) Detailed map of the Gabasa region. San Estaban de Litera and Benabarre are nearby modern cities.This study aims to investigate if the Zn isotope proxy could help elucidate the dietary behaviors of Neandertals and the source of their δ¹⁵N peculiarity, specifically by studying a Late Pleistocene Iberian food web where the presence of mammoth has not been documented (33). The development of Zn isotope analysis (⁶⁶Zn/⁶⁴Zn, expressed as δ⁶⁶Zn) has proven that trophic level information can be retrieved from mammalian tooth enamel (δ⁶⁶Zn_enamel) (34, 35), including fossil samples from Pleistocene food webs where organic matter is typically not preserved (36, 37). Previous studies have demonstrated that δ⁶⁶Zn_enamel decreases by ca. 0.30 to 0.60 ‰ with each step in archeological and modern food webs (34–38) and that δ⁶⁶Zn values associated with breastfeeding are higher than postweaning-associated values (39). While the main source of variation of δ⁶⁶Zn_enamel values is diet, local geology can also likely influence the isotope ratio of a given animal (36, 39). To date, three modern assemblages from Koobi Fora (Kenya), Kruger Park, and the western Cape (South Africa) (40), a few animals from a historical site (Rennes, France) (41), and three Late Pleistocene sites (Tam Hay Marklot, Nam Lot, and Tam Pa Ling, Laos) (36, 37) are the only terrestrial food webs for which Zn isotope data in teeth and/or bones have been published (SI Appendix, Fig. S14). In the modern Koobi Fora savannah food web, δ⁶⁶Zn_enamel differences have been observed between browsers and grazers (35), but this pattern was not seen in any of the three Pleistocene Asian forest food webs (36, 37). Among modern and historical human populations, historically documented diets relying on plants are associated with higher δ⁶⁶Zn values than those that include a substantial quantity of animal products (41–44). Zinc isotopes of ancient hominins have been analyzed only in one Pleistocene Homo sapiens individual (37) from Southeast Asia, but not yet in any Neandertal specimen.This current study contributes significantly to our understanding of Iberian Neandertal diets by providing information on their trophic ecology for a region where traditional nitrogen isotope analyses are unfeasible due to the poor preservation of organic matter. We use the Zn isotopic tool as well as other mobility, ecological, and dietary proxies applied on tooth enamel from hominin and animal remains from the cave site Cueva de los Moros 1 (Gabasa, Pyrenees, Spain; Fig. 1). The site, excavated in the 1980s, is very well documented [for stratigraphic context, see Montes and Utrilla (45) and SI Appendix, Section S1]. All remains come from layers e, f, and g of a single stratigraphic layer directly above layer h dated to 143 ± 43 ka. Numerous carnivore remains were recovered along with Neandertal remains (layers e and f), allowing for comparison of the different meat-eating taxa. Coexisting herbivores from three different types of environmental contexts are homogeneously represented in layers e, f, and g: mountains (Iberian ibex [Capra pyrenaica], chamois [Rupicapra rupicapra]), forest (cervids including red deer [Cervus elaphus]), and open environments (horses [Equus ferus], European wild asses [Equus hydruntinus]).     

Genetic changes in neoplasms arising in congenital melanocytic nevi: differences between nodular proliferations and melanomas

Large congenital melanocytic nevi (CMN) are at an increased risk of developing melanoma. Several forms of secondary proliferations can arise in congenital nevi on rare occasions. Although some of these closely resemble melanoma both clinically and histologically, metastasis is rare. We used comparative genomic hybridization to analyze chromosomal aberrations in different types of proliferations arising in CMN and compared them to typical congenital nevi, clear-cut melanomas arising in congenital nevi, as well as primary cutaneous melanomas that were not associated with a CMN. Cases of CMN and CMN with secondary proliferations were assigned to six groups according to the predominant histological pattern: group I, bland congenital nevi (n = 6); group II, congenital nevi with foci of increased cellularity (n = 4); group III, CMN with a proliferation simulating superficial spreading melanoma in situ (n = 3); group IV, CMN with a proliferation simulating nodular melanoma (n = 9); group V, proliferating neurocristic hamartoma (n = 1); and group VI, melanoma arising in congenital nevus (n = 6). No aberrations were found in groups I to III, whereas seven of nine cases of group IV, and one of one case of group V, showed aberrations. In group IV six of seven cases with aberrations (86%) showed numerical aberrations of whole chromosomes exclusively. This pattern differed significantly from the findings in melanoma that arose within CMN (n = 6), group VI, or independent of CMN (n = 122) in which only 5% showed numerical changes only. The single case in group V showed aberrations similar to melanoma. The finding of frequent numerical chromosomal aberrations in atypical nodular proliferations arising in CMN identifies these as clonal neoplasms with a genomic instability consistent with a mitotic spindle checkpoint defect. This difference compared to the aberration pattern found in melanoma might explain their more benign clinical behavior and may be of diagnostic value in ambiguous cases.     

Crystal structures of TAFI elucidate the inactivation mechanism of activated TAFI: a novel mechanism for enzyme autoregulation

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a pro-metallocarboxypeptidase that can be proteolytically activated (TAFIa). TAFIa is unique among carboxypeptidases in that it spontaneously inactivates with a short half-life, a property that is crucial for its role in controlling blood clot lysis. We studied the intrinsic instability of TAFIa by solving crystal structures of TAFI, a TAFI inhibitor (GEMSA) complex and a quadruple TAFI mutant (70-fold more stable active enzyme). The crystal structures show that TAFIa stability is directly related to the dynamics of a 55-residue segment (residues 296-350) that includes residues of the active site wall. Dynamics of this flap are markedly reduced by the inhibitor GEMSA, a known stabilizer of TAFIa, and stabilizing mutations. Our data provide the structural basis for a model of TAFI auto-regulation: in zymogen TAFI the dynamic flap is stabilized by interactions with the activation peptide. Release of the activation peptide increases dynamic flap mobility and in time this leads to conformational changes that disrupt the catalytic site and expose a cryptic thrombin-cleavage site present at Arg302. This represents a novel mechanism of enzyme control that enables TAFI to regulate its activity in plasma in the absence of specific inhibitors.