Cystatin C takes part in melanoma-microglia cross-talk: possible implications for brain metastasis

The development of melanoma brain metastasis is largely dependent on mutual interactions between the melanoma cells and cells in the brain microenvironment. Here, we report that the extracellular cysteine protease inhibitor cystatin C (CysC) is involved in these interactions. Microglia-derived factors upregulated CysC secretion by melanoma. Similarly, melanoma-derived factors upregulated CysC secretion by microglia. Whereas CysC enhanced melanoma cell migration through a layer of brain endothelial cells, it inhibited the migration of microglia cells toward melanoma cells. CysC was also found to promote the formation of melanoma three-dimensional structures in matrigel. IHC analysis revealed increased expression levels of CysC in the brain of immune-deficient mice bearing xenografted human melanoma brain metastasis compared to the brain of control mice. Based on these in vitro and in vivo experiments we hypothesize that CysC promotes melanoma brain metastasis. Increased expression levels of CysC were detected in the regenerating brain of mice after stroke. Post-stroke brain with melanoma brain metastasis showed an even stronger expression of CysC. The in vitro induction of stroke-like conditions in brain microenvironmental cells increased the levels of CysC in the secretome of microglia cells, but not in the secretome of brain endothelial cells. The similarities between melanoma brain metastasis and stroke with respect to CysC expression by and secretion from microglia cells suggest that CysC may be involved in shared pathways between brain metastasis and post-stroke regeneration. This manifests the tendency of tumor cells to highjack physiological molecular pathways in their progression.     

Iron oxides stimulate sulfate-driven anaerobic methane oxidation in seeps

Seep sediments are dominated by intensive microbial sulfate reduction coupled to the anaerobic oxidation of methane (AOM). Through geochemical measurements of incubation experiments with methane seep sediments collected from Hydrate Ridge, we provide insight into the role of iron oxides in sulfate-driven AOM. Seep sediments incubated with ¹³C-labeled methane showed co-occurring sulfate reduction, AOM, and methanogenesis. The isotope fractionation factors for sulfur and oxygen isotopes in sulfate were about 40‰ and 22‰, respectively, reinforcing the difference between microbial sulfate reduction in methane seeps versus other sedimentary environments (for example, sulfur isotope fractionation above 60‰ in sulfate reduction coupled to organic carbon oxidation or in diffusive sedimentary sulfate–methane transition zone). The addition of hematite to these microcosm experiments resulted in significant microbial iron reduction as well as enhancing sulfate-driven AOM. The magnitude of the isotope fractionation of sulfur and oxygen isotopes in sulfate from these incubations was lowered by about 50%, indicating the involvement of iron oxides during sulfate reduction in methane seeps. The similar relative change between the oxygen versus sulfur isotopes of sulfate in all experiments (with and without hematite addition) suggests that oxidized forms of iron, naturally present in the sediment incubations, were involved in sulfate reduction, with hematite addition increasing the sulfate recycling or the activity of sulfur-cycling microorganisms by about 40%. These results highlight a role for natural iron oxides during bacterial sulfate reduction in methane seeps not only as nutrient but also as stimulator of sulfur recycling.Microbial dissimilatory processes generate energy through the decomposition of substrates, whereas assimilatory processes use substrates for intracellular biosynthesis of macromolecules. The most known and energetically favorable dissimilatory process is the oxidation of organic carbon coupled to oxygen as terminal electron acceptor (Eq. 1). In sediments with a high supply of organic carbon, oxygen can be depleted within the upper few millimeters, leading to anoxic conditions deeper in the sediment column. Under these conditions, microbial dissimilatory processes are coupled to the reduction of a series of other terminal electron acceptors besides oxygen (1). The largest free-energy yields are associated with nitrate reduction (denitrification), followed by manganese and iron oxide reduction, and then sulfate reduction. Due to the high concentration of sulfate in the ocean, dissimilatory bacterial sulfate reduction (Eq. 2) is responsible for the majority of organic matter oxidation in marine sediments (2). Below the depth of sulfate depletion, traditionally the only presumed process is methanogenesis (methane production), where its main pathways are fermentation of organic matter, mainly acetate (Eq. 3), or the reduction of carbon dioxide with hydrogen as substrate (Eq. 4) (3):O₂ + CH₂O → H₂O + CO₂[1]SO42−+2CH2O→H2S+2HCO3−[2]CH₃COOH→CH₄ + CO₂[3]CO₂ + 4H₂→CH₄ + 2H₂O[4]When methane that has been produced deep in sediments diffuses into contact with an available electron acceptor, it can be oxidized (methanotrophy). Methanotrophy is the main process that prevents the escape of methane produced within marine and fresh water sediments into the atmosphere. In fresh water systems, methanotrophic bacteria are responsible for oxidizing methane to dissolved inorganic carbon (DIC) typically using oxygen as an electron acceptor (4, 5). In marine sediments, however, where oxygen diffusion is limited, anaerobic oxidation of methane (AOM) coupled to sulfate reduction [e.g., refs. 6 and 7 (Eq. 5)] has been shown to consume up to 90% of the methane produced within the subseafloor environment (8). Often, when methane is present, the majority of sulfate available in marine pore fluids is reduced through sulfate-driven AOM (9–13):CH4+SO42−→HS−+HCO3−+H2O.[5]Other electron acceptors such as nitrate and oxides of iron and manganese, could also oxidize methane anaerobically and provide a greater free-energy yield than sulfate-coupled methane oxidation (14). Indeed, Beal et al. (15) showed the potential for iron- and manganese-driven AOM in microcosm experiments with methane seep sediments from Eel River Basin and Hydrate Ridge, and iron-driven AOM has been interpreted from modeling geochemical profiles in deep-sea sediments (13, 16). AOM has been shown to occur in nonmarine sediments via denitrification (17–21) and iron reduction (22, 23). However, all geochemical and microbiological studies point to sulfate-driven AOM as the dominant sink for methane in marine sediments.Sulfate-driven AOM is understood to involve microbial consortia of archaea and bacteria affiliated with archaeal methanotrophs (“methane oxidizers”) and sulfate-reducing bacteria (11, 24). A common view is that anaerobic methanotrophic archaea (ANME) oxidize methane, while the sulfate-reducing syntrophic partner scavenges the resulting reducing equivalents to reduce sulfate to sulfide (7, 25, 26). Recently, however, cultured AOM enrichments from seeps were reported to be capable of direct coupling of methane oxidation and sulfate reduction by the ANME-2 archaea, with the passage of zero valent sulfur to a disproportionating bacterial partner, capable of simultaneously oxidizing and reducing this substrate to sulfate and sulfide in a ratio of 1:7, respectively (27). Whether this “single organism mechanism” for sulfate-driven AOM is widespread in the natural environment, or whether there is a diversity of mechanisms for sulfate-driven AOM, remains enigmatic.Carbon isotopes provide a good constraint on the depth distribution and location of methanogenesis and methanotrophy because of the carbon isotope fractionation associated with these processes (e.g., refs. 28 and 29). During methanogenesis, ¹²C is strongly partitioned into methane; the δ¹³C of the methane produced can be between −50‰ to −110‰. In parallel, the residual DIC pool in methanogenic zones becomes highly enriched in ¹³C, occasionally by as much as 50‰ to 70‰ (e.g., ref. 28). Oxidizing this methane on the other hand, results in ¹³C-depleted DIC and slightly heavier δ¹³C values of the residual methane, caused by a fractionation of 0‰ to 10‰ during methane oxidation and the initial negative δ¹³C value of the methane itself (30, 31).The sulfur and oxygen isotopes in dissolved sulfate (δ³⁴S_SO4 and δ¹⁸O_SO4) may also be a diagnostic tool for tracking the pathways of sulfate reduction by methane or other organic compounds. Sulfur isotope fractionation during dissimilatory bacterial sulfate reduction, which partitions ³²S into the sulfide, leaving ³⁴S behind in the residual sulfate, can be as high as 72‰ (32–35). As sulfate is reduced to sulfide via intracellular intermediates (34, 36–40), the magnitude of this sulfur isotope fractionation depends upon the isotope partitioning at each of the intercellular steps and on the ratio between the backward and forward sulfur fluxes within the bacterial cells (34, 36).Oxygen isotopes in sulfate, however, have been shown to be strongly influenced by the oxygen isotope composition of water in which the bacteria are grown (41–45). The consensus is that, within the cell, sulfur compounds, such as sulfite, and water exchange oxygen atoms; some of these isotopically equilibrated molecules return to the extracellular sulfate pool. As all of the intercellular steps are considered to be reversible (e.g., refs. 34, 36, 46, and 47), water–oxygen is also incorporated during the oxidation of these sulfur intermediates back to sulfate (41–43, 48–51).Therefore, both oxygen and sulfur isotopes in the residual sulfate during dissimilatory sulfate reduction are affected by the changes in the intracellular fluxes of sulfur species. However, these isotopes in the residual sulfate are affected in different ways, and thus the change of one isotope vs. the other helps uniquely solve for the relative change in the flux of each intracellular step as sulfate is being reduced (42, 43, 50). The sulfur and oxygen isotope composition of residual sulfate has been used to explore the mechanism of traditional (organoclastic) sulfate reduction both in pure culture (e.g., refs. 44, 45, and 52) and in the natural environment (e.g., refs. 12, 49, 50, and 53–55). The coupled isotope approach has been used specifically to study sulfate-driven AOM recently in estuaries (56). In the work of Antler et al. (56), it was shown that the oxygen and sulfur isotopes in the residual sulfate in the pore fluids are linearly correlated during sulfate-driven AOM, whereas during organoclastic bacterial sulfate reduction, the isotopes exhibit a concaved curve relationship.Although iron and manganese oxides should be reduced before the onset of dissimilatory bacterial sulfate reduction in the natural environment from thermodynamic considerations, due to their low solubility, they may not be completely reduced through dissimilatory respiration when sulfate reduction starts (e.g., ref. 22). These lower reactivity manganese and iron oxides therefore may still be present during the lower-energy yielding anaerobic processes such as sulfate reduction, methanotrophy, and methanogenesis. Indeed, iron oxides have been shown to serve as electron acceptors for methane oxidation even in the sulfate “zone” (15, 16, 22, 57), although the mechanism of this coupling remains enigmatic. In the context of deep-sea methane seep ecosystems, earlier work by Beal et al. (15) demonstrated stimulation of AOM by the addition of iron and manganese oxides in sediment incubation experiments. In that work, however, the nature of the coupling between methane oxidation and metal oxides was not ascertained, and the multiple links between the sediment sulfur, iron, and methane cycles are equivocal.Here, we conducted microcosm experiments with sediments collected from Hydrate Ridge South (Fig. S1) and used synergistic combinations of isotope analyses (δ³⁴S_SO4, δ¹⁸O_SO4, and δ¹³C_DIC) to aid in assessing whether methane oxidation is directly coupled to the respiration of iron oxides or whether stimulation in methanotrophy is a result of the coupling between iron and sulfate. We provide compelling evidence for the stimulation of AOM in seep sediments through the coupling between iron and sulfate, and propose a mechanism for iron involvement in sulfate-driven AOM. Using microcosm experiments with seep sediments dominated by sulfate-driven AOM and amended with hematite and ¹³C-labeled methane and glucose, we are able to demonstrate the role of iron in sulfate-driven AOM. Hematite is a less reactive form of iron oxide than, for example, amorphous iron (58), and it was used to prevent the microbial populations from “switching” completely to the more energetically favorable process of iron reduction.     

Analysis of two measures of child behavior problems by African American, Latino, and non-Hispanic Caucasian parents of young children: a focus group study

This study examined how parents from different racial/ethnic, income, and language groups viewed two widely used parent-report measures of child behavior problems, the Child Behavior Checklist and the Eyberg Child Behavior Inventory. Seventy African American, Latino, and non-Latino Caucasian parents of preschoolers stratified by income met in 1 of 15 focus groups to discuss their perceptions of the instruments. Participants agreed that items on these instruments were relevant indicators of child behavior problems. Overall, parents found the items on both instruments useful, comprehensible, and acceptable. The findings support the use of these instruments in pediatric practice with ethnically diverse parents of young children.     

Astrocytes facilitate melanoma brain metastasis via secretion of IL‐23

Melanoma is the leading cause of skin cancer mortality. The major cause of melanoma mortality is metastasis to distant organs, frequently to the brain. The microenvironment plays a critical role in tumourigenesis and metastasis. In order to treat or prevent metastasis, the interactions of disseminated tumour cells with the microenvironment at the metastatic organ have to be elucidated. However, the role of brain stromal cells in facilitating metastatic growth is poorly understood. Astrocytes are glial cells that function in repair and scarring of the brain following injury, in part via mediating neuroinflammation, but the role of astrocytes in melanoma brain metastasis is largely unresolved. Here we show that astrocytes can be reprogrammed by human brain‐metastasizing melanoma cells to express pro‐inflammatory factors, including the cytokine IL‐23, which was highly expressed by metastases‐associated astrocytes in vivo. Moreover, we show that the interactions between astrocytes and melanoma cells are reciprocal: paracrine signalling from astrocytes up‐regulates the secretion of the matrix metalloproteinase MMP2 and enhances the invasiveness of brain‐metastasizing melanoma cells. IL‐23 was sufficient to increase melanoma cell invasion, and neutralizing antibodies to IL‐23 could block this enhanced migration, implying a functional role for astrocyte‐derived IL‐23 in facilitating the progression of melanoma brain metastasis. Knocking down the expression of MMP2 in melanoma cells resulted in inhibition of IL‐23‐induced invasiveness. Thus, our study demonstrates that bidirectional signalling between melanoma cells and astrocytes results in the formation of a pro‐inflammatory milieu in the brain, and in functional enhancement of the metastatic potential of disseminated melanoma cells. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Maternal medical compromise during pregnancy and pregnancy outcomes

Objective: To examine the outcomes of pregnancy and newborn following an event of maternal medical compromise during pregnancy.

Methods: A retrospective study was performed on all patients hospitalized following an event of medical compromise during pregnancy. Medical compromise was divided to acute or chronic bleeding, major or complicated operations, and admission to intensive care unit (ICU). Data collected included maternal, fetal, neonatal and child’s follow-up.

Results: The study included 51 pregnant patients and 58 fetuses. The study group had increased risk of preterm deliveries (35.0 versus 6.5%, p?p?p?=?0.002). Patients with acute bleeding had higher rates of cesarean sections, preterm deliveries, admissions to neonatal ICU and neonatal mortality. Two cases of fetal abnormalities included brain abnormalities and pericardial effusion. Three terminations of pregnancies were performed: two in patients in ICU due to severe maternal medical condition and one in the fetus with brain abnormalities.

Conclusions: Maternal medical compromise during pregnancy increases the risk for preterm deliveries, cesarean delivery and low Apgar scores. Acute bleeding was the main cause of medical compromised and with the higher rates of adverse outcomes.     

Melatonin receptors in Atlantic salmon stimulate cAMP levels in heterologous cell lines and show season‐dependent daily variations in pituitary expression levels

The hormone melatonin connects environmental cues, such as photoperiod and temperature, with a number of physiological and behavioural processes, including seasonal reproduction, through binding to their cognate receptors. This study reports the structural, functional and physiological characterization of five high‐affinity melatonin receptors (Mtnr1aaα, Mtnr1aaβ, Mtnr1ab, Mtnr1al, Mtnr1b) in Atlantic salmon. Phylogenetic analysis clustered salmon melatonin receptors into three monophyletic groups, Mtnr1A, Mtnr1Al and Mtnr1B, but no functional representative of the Mtnr1C group. Contrary to previous studies in vertebrates, pharmacological characterization of four receptors in COS‐7, CHO and SH‐SY5Y cell lines (Mtnr1Aaα, Mtnr1Aaβ, Mtnr1Ab, Mtnr1B) showed induction of intracellular cAMP levels following 2‐iodomelatonin or melatonin exposure. No consistent response was measured after N‐acetyl‐serotonin or serotonin exposure. Melatonin receptor genes were expressed at all levels of the hypothalamo‐pituitary‐gonad axis, with three genes (mtnr1aaβ, mtnr1ab and mtnr1b) detected in the pituitary. Pituitary receptors displayed daily fluctuations in mRNA levels during spring, prior to the onset of gonadal maturation, but not in autumn, strongly implying a direct involvement of melatonin in seasonal processes regulated by the pituitary. To the best of our knowledge, this is the first report of cAMP induction mediated via melatonin receptors in a teleost species.     

Consistency of strength curves for determining maximal effort production during isokinetic knee testing of anterior cruciate ligament-deficient patients

The purpose of this investigation was to attempt to establish decision rules for determining maximal effort production during isokinetic strength testing of unilateral anterior cruciate ligament-deficient patients based on the degree of strength curve consistency within a set. Thirty-three participants performed six bilateral knee extension and flexion exertions at maximal effort and at 80% of perceived maximum at testing velocities of 60 and 180°s^–1. Within-set consistency was quantified by computation of the variance ratio across strength curves. Tolerance interval-based cutoff scores covering 99% of the population were calculated for declaring efforts as being maximal or not at confidence levels of 90%, 95%, and 99%. The sensitivity percentages attained for the injured knee for both testing velocities ranged between 9.1% and 27.2%, while specificity percentages ranged between 84.8% and 100%. For the non-injured knee, sensitivity values for both testing velocities ranged between 21.2% and 45.0%, while specificity percentages ranged between 97.0% and 100%. The developed decision rules do not effectively discriminate on an individual patient basis between maximal and non-maximal isokinetic knee musculature efforts. Further research is needed for development of methods that would enable to ascertain maximal effort production in this patient population during knee muscle strength testing.     

Is There a Role for Oral Antihyperglycemics in Gestational Diabetes and Type 2 Diabetes during Pregnancy?

Diabetes mellitus is a heterogeneous disorder of glucose intolerance that is generally classified into the following categories: type 1 and type 2 diabetes and gestational diabetes (GDM). Currently, the number of pregnancies complicated by type 2 diabetes and GDM exceed those affected by type 1 diabetes. Numerous studies have established a direct relationship between maternal glycemic control and neonatal outcomes for all types of diabetes. Therefore, modern treatment protocols during pregnancy emphasize strict glycemic control by a combination of diet and medication. Traditionally, insulin therapy has been considered the gold standard for management because of its efficacy in achieving tight glucose control and the fact that it does not cross the placenta. Since GDM and type 2 diabetes are characterized by insulin resistance and relatively decreased insulin secretion, treatment with oral antihyperglycemic agents that target these defects is of potential interest. However, because of concerns regarding transplacental passage and, therefore, the possibility of fetal teratogenesis and prolonged neonatal hypoglycemia, these agents are not currently recommended in pregnancy. There are no randomized controlled trials on which to draw conclusions regarding the teratogenicity of these oral agents. However, most retrospective studies and the published clinical experience have not demonstrated an increased risk of malformed infants among women treated with oral antihyperglycemic agents. Rather, the data indicate that the increased risk for major congenital anomalies appears to be related to maternal glycemic control prior to and during conception. These studies and currently available data on the use of both metformin and sulfonylureas in pregnancy have also failed to demonstrate an increased risk of neonatal hypoglycemia and other neonatal morbidities. To date, there has only been one randomized controlled trial to test the effectiveness and safety of sulfonylurea therapy (glyburide [glibenclamide]) in the management of women with GDM. Both the insulin- and glyburide-treated women were able to achieve satisfactory glucose control and had similar perinatal outcomes. Glyburide was not detected in the cord serum of any infant in the glyburide group. In summary, based on the currently available data, it appears that glyburide could be safely and effectively utilized in the management of GDM. However, more intensive investigation regarding the safety and feasibility of oral agents in pregnancies complicated by type 2 diabetes is necessary. It is important to emphasize that it is the level of metabolic control achieved and not the mode of therapy that is crucial to improving outcomes in these pregnancies.     

Efficient maternal to neonatal transfer of antibodies against SARS-CoV-2 and BNT162b2 mRNA COVID-19 vaccine

BACKGROUNDThe significant risks posed to mothers and fetuses by COVID-19 in pregnancy have sparked a worldwide debate surrounding the pros and cons of antenatal SARS-CoV-2 inoculation, as we lack sufficient evidence regarding vaccine effectiveness in pregnant women and their offspring. We aimed to provide substantial evidence for the effect of the BNT162b2 mRNA vaccine versus native infection on maternal humoral, as well as transplacentally acquired fetal immune response, potentially providing newborn protection.METHODSA multicenter study where parturients presenting for delivery were recruited at 8 medical centers across Israel and assigned to 3 study groups: vaccinated (n = 86); PCR-confirmed SARS-CoV-2 infected during pregnancy (n = 65), and unvaccinated noninfected controls (n = 62). Maternal and fetal blood samples were collected from parturients prior to delivery and from the umbilical cord following delivery, respectively. Sera IgG and IgM titers were measured using the Milliplex MAP SARS-CoV-2 Antigen Panel (for S1, S2, RBD, and N).RESULTSThe BNT162b2 mRNA vaccine elicits strong maternal humoral IgG response (anti-S and RBD) that crosses the placenta barrier and approaches maternal titers in the fetus within 15 days following the first dose. Maternal to neonatal anti-COVID-19 antibodies ratio did not differ when comparing sensitization (vaccine vs. infection). IgG transfer ratio at birth was significantly lower for third-trimester as compared with second trimester infection. Lastly, fetal IgM response was detected in 5 neonates, all in the infected group.CONCLUSIONAntenatal BNT162b2 mRNA vaccination induces a robust maternal humoral response that effectively transfers to the fetus, supporting the role of vaccination during pregnancy.FUNDINGIsrael Science Foundation and the Weizmann Institute Fondazione Henry Krenter.