Unexpected role of anticoagulant protein C in controlling epithelial barrier integrity and intestinal inflammation

The protein C (PC) pathway is a well-characterized coagulation system. Endothelial PC receptors and thrombomodulin mediate the conversion of PC to its activated form, a potent anticoagulant and anti-inflammatory molecule. Here we show that the PC pathway is expressed on intestinal epithelial cells. The epithelial expression of PC and endothelial PC receptor is down-regulated In patients with inflammatory bowel disease. PC(-/-)/PC(Tg) mice, expressing only 3% of WT PC, developed spontaneous intestinal inflammation and were prone to severe experimental colitis. These mice also demonstrated spontaneous elevated production of inflammatory cytokines and increased intestinal permeability. Structural analysis of epithelial tight junction molecules revealed that lack of PC leads to decreased JAM-A and claudin-3 expression and an altered pattern of ZO-1 expression. In vitro, treatment of epithelial cells with activated PC led to protection of tight junction disruption induced by TNF-α, and in vivo, topical treatment with activated PC led to mucosal healing and amelioration of colitis. Taken together, these findings demonstrate that the PC pathway is a unique system involved in controlling intestinal homeostasis and inflammation by regulating epithelial barrier function.     

Adjunctive tests for diagnosis of tuberculosis: serology, ELISPOT for site-specific lymphocytes, urinary lipoarabinomannan, string test, and fine needle aspiration

The diagnostic gold standard for active tuberculosis (TB) is the detection of Mycobacterium tuberculosis (MTB) by culture or molecular methods. However, despite its limited sensitivity, sputum smear microscopy is still the mainstay of TB diagnosis in resource-limited settings. Consequently, diagnosis of smear-negative pulmonary and extrapulmonary TB remains challenging in such settings. A number of novel or alternative techniques could provide adjunctive diagnostic use in the context of difficult-to-diagnose TB. These may be especially useful in certain patient groups such as persons infected with human immunodeficiency virus (HIV) and children, who are disproportionably affected by smear-negative and extrapulmonary disease and who are also most adversely affected by delays in TB diagnosis and treatment. We review a selection of these methods that are independent of nucleic acid amplification techniques and could largely be implemented in resource-limited settings in current or adapted versions. Specifically, we discuss the diagnostic use and potential of serologic tests based on detection of antibodies to MTB antigens; interferon gamma release assays using site-specific lymphocytes; detection of lipoarabinomannan, a glycolipid of MTB, in urine; the string test, a novel technique to retrieve lower respiratory tract samples; and fine needle aspiration biopsy of lymph nodes.     

Geographic variation of Trypanosoma cruzi discrete typing units from Triatoma infestans at different spatial scales

We assessed the diversity and distribution of Trypanosoma cruzi discrete typing units (DTU) in Triatoma infestans populations and its association with local vector-borne transmission levels at various geographic scales. At a local scale, we found high predominance (92.4%) of TcVI over TcV in 68 microscope-positive T. infestans collected in rural communities in Santiago del Estero province in northern Argentina. TcV was more often found in communities with higher house infestation prevalence compatible with active vector-borne transmission. Humans and dogs were the main bloodmeal sources of the TcV- and TcVI-infected bugs. At a broader scale, the greatest variation in DTU diversity was found within the Argentine Chaco (227 microscope-positive bugs), mainly related to differences in equitability between TcVI and TcV among study areas. At a country-wide level, a meta-analysis of published data revealed clear geographic variations in the distribution of DTUs across countries. A correspondence analysis showed that DTU distributions in domestic T. infestans were more similar within Argentina (dominated by TcVI) and within Bolivia (where TcI and TcV had similar relative frequencies), whereas large heterogeneity was found within Chile. DTU diversity was lower in the western Argentine Chaco region and Paraguay (D = 0.14–0.22) than in the eastern Argentine Chaco, Bolivia and Chile (D = 0.20–0.68). Simultaneous DTU identifications of T. cruzi-infected hosts and triatomines across areas differing in epidemiological status are needed to shed new light on the structure and dynamics of parasite transmission cycles.     

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.     

Predictive imaging of chemotherapeutic response in a transgenic mouse model of pancreatic cancer

The underglycosylated mucin 1 tumor antigen (uMUC1) is a biomarker that forecasts the progression of adenocarcinomas. In this study, we evaluated the utility of a dual‐modality molecular imaging approach based on targeting uMUC1 for monitoring chemotherapeutic response in a transgenic murine model of pancreatic cancer (KCM triple transgenic mice). An uMUC1‐specific contrast agent (MN‐EPPT) was synthesized for use with magnetic resonance imaging (MRI) and fluorescence optical imaging. It consisted of dextran‐coated iron oxide nanoparticles conjugated to the near infrared fluorescent dye Cy5.5 and to a uMUC1‐specific peptide (EPPT). KCM triple transgenic mice were given gemcitabine as chemotherapy while control animals received saline injections following the same schedule. Changes in uMUC1 levels following chemotherapy were monitored using T2‐weighted MRI and optical imaging before and 24 hr after injection of the MN‐EPPT. uMUC1 expression in tumors from both groups was evaluated by histology and qRT‐PCR. We observed that the average delta‐T2 in the gemcitabine‐treated group was significantly reduced compared to the control group indicating lower accumulation of MN‐EPPT, and correspondingly, a lower level of uMUC1 expression. In vivo optical imaging confirmed the MRI findings. Fluorescence microscopy of pancreatic tumor sections showed a lower level of uMUC1 expression in the gemcitabine‐treated group compared to the control, which was confirmed by qRT‐PCR. Our data proved that changes in uMUC1 expression after gemcitabine chemotherapy could be evaluated using MN‐EPPT‐enhanced in vivo MR and optical imaging. These results suggest that the uMUC1‐targeted imaging approach could provide a useful tool for the predictive assessment of therapeutic response.     

High-Throughput Generation of P. falciparum Functional Molecules by Recombinational Cloning

Large-scale functional genomics studies for malaria vaccine and drug development will depend on the generation of molecular tools to study protein expression. We examined the feasibility of a high-throughput cloning approach using the Gateway system to create a large set of expression clones encoding Plasmodium falciparum single-exon genes. Master clones and their ORFs were transferred en masse to multiple expression vectors. Target genes (n = 303) were selected using specific sets of criteria, including stage expression and secondary structure. Upon screening four colonies per capture reaction, we achieved 84% cloning efficiency. The genes were subcloned in parallel into three expression vectors: a DNA vaccine vector and two protein expression vectors. These transfers yielded a 100% success rate without any observed recombination based on single colony screening. The functional expression of 95 genes was evaluated in mice with DNA vaccine constructs to generate antibody against various stages of the parasite. From these, 19 induced antibody titers against the erythrocytic stages and three against sporozoite stages. We have overcome the potential limitation of producing large P. falciparum clone sets in multiple expression vectors. This approach represents a powerful technique for the production of molecular reagents for genome-wide functional analysis of the P. falciparum genome and will provide for a resource for the malaria resource community distributed through public repositories.     

BRCA1 and BRCA2 mutations in Russian familial breast cancer

We have screened index cases from 25 Russian breast/ovarian cancer families for germ‐line mutations in all coding exons of the BRCA1 and BRCA2 genes, using multiplex heteroduplex analysis. In addition we tested 22 patients with breast cancer diagnosed before age 40 without family history and 6 patients with bilateral breast cancer. The frequency of families with germline mutations in BRCA was 16% (4/25). One BRCA1 mutation, 5382insC, was found in three families. The results of present study, and those of a separate study of 19 breast‐ovarian cancer families, suggest that BRCA1 5382insC is a founder mutation in the Russian population. Three BRCA2 mutations were found in patients with breast cancer without family history: two in young patients and one in patients with bilateral breast cancer. Four novel BRCA2 mutations were identified: three frameshift (695insT, 1528del4, 9318del4) and one nonsense (S1099X). © 2002 Wiley‐Liss, Inc.     

Bilateral hydrosalpinx in patients with Hirschsprung’s disease

Purpose

Hirschsprung's disease (HD) is uncommon in females. There are very few reports on the patients' obstetric and gynecological outcome. Hydrosalpinx causes pain and infertility. It is rare in nonsexually active teenagers. It may be because of an intrinsic disease of the fallopian tubes or secondary to surgery. Aim: to describe the relationship between hydrosalpinx and HD or its surgical approach; to report the impact of bilateral hydrosalpinx on fertility in HD.