The Conserved Candida albicans CA3427 Gene Product Defines a New Family of Proteins Exhibiting the Generic Periplasmic Binding Protein Structural Fold

Nosocomial diseases due to Candida albicans infections are in constant rise in hospitals, where they cause serious complications to already fragile intensive care patients. Antifungal drug resistance is fast becoming a serious issue due to the emergence of strains resistant to currently available antifungal agents. Thus the urgency to identify new potential protein targets, the function and structure of which may guide the development of new antifungal drugs. In this context, we initiated a comparative genomics study in search of promising protein coding genes among the most conserved ones in reference fungal genomes. The CA3427 gene was selected on the basis of its presence among pathogenic fungi contrasting with its absence in the non pathogenic Saccharomyces cerevisiae. We report the crystal 3D-structure of the Candida albicans CA3427 protein at 2.1 Å resolution. The combined analysis of its sequence and structure reveals a structural fold originally associated with periplasmic binding proteins. The CA3427 structure highlights a binding site located between the two protein domains, corresponding to a sequence segment conserved among fungi. Two crystal forms of CA3427 were found, suggesting that the presence or absence of a ligand at the proposed binding site might trigger a “Venus flytrap” motion, coupled to the previously described activity of bacterial periplasmic binding proteins. The conserved binding site defines a new subfamily of periplasmic binding proteins also found in many bacteria of the bacteroidetes division, in a choanoflagellate (a free-living unicellular and colonial flagellate eukaryote) and in a placozoan (the closest multicellular relative of animals). A phylogenetic analysis suggests that this gene family originated in bacteria before its horizontal transfer to an ancestral eukaryote prior to the radiation of fungi. It was then lost by the Saccharomycetales which include Saccharomyces cerevisiae

Fully human IgG and IgM antibodies directed against the carcinoembryonic antigen (CEA) Gold 4 epitope and designed for radioimmunotherapy (RIT) of colorectal cancers

BACKGROUND: Human monoclonal antibodies (MAbs) are needed for colon cancer radioimmunotherapy (RIT) to allow for repeated injections. Carcinoembryonic antigen (CEA) being the reference antigen for immunotargeting of these tumors, we developed human anti-CEA MAbs. METHODS: XenoMouse(®)-G2 animals were immunized with CEA. Among all the antibodies produced, two of them, VG-IgG2κ and VG-IgM, were selected for characterization in vitro in comparison with the human-mouse chimeric anti-CEA MAb X4 using flow cytometry, surface plasmon resonance, and binding to radiolabeled soluble CEA and in vivo in human colon carcinoma LS174T bearing nude mice. RESULTS: Flow cytometry analysis demonstrated binding of MAbs on CEA-expressing cells without any binding on NCA-expressing human granulocytes. In a competitive binding assay using five reference MAbs, directed against the five Gold CEA epitopes, VG-IgG2κ and VG-IgM were shown to be directed against the Gold 4 epitope. The affinities of purified VG-IgG2κ and VG-IgM were determined to be 0.19 ± 0.06 × 10(8 )M(-1 )and 1.30 ± 0.06 × 10(8 )M(-1), respectively, as compared with 0.61 ± 0.05 × 10(8 )M(-1 )for the reference MAb X4. In a soluble phase assay, the binding capacities of VG-IgG2κ and VG-IgM to soluble CEA were clearly lower than that of the control chimeric MAb X4. A human MAb concentration of about 10(-7 )M was needed to precipitate approximatively 1 ng (125)I-rhCEA as compared with 10(-9 )M for MAb X4, suggesting a preferential binding of the human MAbs to solid phase CEA. In vivo, 24 h post-injection, (125)I-VG-IgG2κ demonstrated a high tumor uptake (25.4 ± 7.3%ID/g), close to that of (131)I-X4 (21.7 ± 7.2%ID/g). At 72 h post-injection, (125)I-VG-IgG2κ was still concentrated in the tumor (28.4 ± 11.0%ID/g) whereas the tumor concentration of (131)I-X4 was significantly reduced (12.5 ± 4.8%ID/g). At no time after injection was there any accumulation of the radiolabeled MAbs in normal tissues. A pertinent analysis of VG-IgM biodistribution was not possible in this mouse model in which IgM displays a very short half-life due to poly-Ig receptor expression in the liver. CONCLUSION: Our human anti-CEA IgG2κ is a promising candidate for radioimmunotherapy in intact form, as F(ab')(2 )fragments, or as a bispecific antibody

The exchange activities of [Fe] hydrogenase (iron–sulfur-cluster-free hydrogenase) from methanogenic archaea in comparison with the exchange activities of [FeFe] and [NiFe] hydrogenases

[Fe] hydrogenase (iron–sulfur-cluster-free hydrogenase) catalyzes the reversible reduction of methenyltetrahydromethanopterin (methenyl-H4MPT+) with H2 to methylene-H4MPT, a reaction involved in methanogenesis from H2 and CO2 in many methanogenic archaea. The enzyme harbors an iron-containing cofactor, in which a low-spin iron is complexed by a pyridone, two CO and a cysteine sulfur. [Fe] hydrogenase is thus similar to [NiFe] and [FeFe] hydrogenases, in which a low-spin iron carbonyl complex, albeit in a dinuclear metal center, is also involved in H2 activation. Like the [NiFe] and [FeFe] hydrogenases, [Fe] hydrogenase catalyzes an active exchange of H2 with protons of water; however, this activity is dependent on the presence of the hydride-accepting methenyl-H4MPT+. In its absence the exchange activity is only 0.01% of that in its presence. The residual activity has been attributed to the presence of traces of methenyl-H4MPT+ in the enzyme preparations, but it could also reflect a weak binding of H2 to the iron in the absence of methenyl-H4MPT+. To test this we reinvestigated the exchange activity with [Fe] hydrogenase reconstituted from apoprotein heterologously produced in Escherichia coli and highly purified iron-containing cofactor and found that in the absence of added methenyl-H4MPT+ the exchange activity was below the detection limit of the tritium method employed (0.1 nmol min−1 mg−1). The finding reiterates that for H2 activation by [Fe] hydrogenase the presence of the hydride-accepting methenyl-H4MPT+ is essentially required. This differentiates [Fe] hydrogenase from [FeFe] and [NiFe] hydrogenases, which actively catalyze H2/H2O exchange in the absence of exogenous electron acceptors

Vampires in the village Žrnovo on the island of Korčula: following an archival document from the 18th century

Središnja tema rada usmjerena je na raščlambu spisa pohranjenog u Državnom arhivu u Mlecima (fond: Capi del Consiglio de’ Dieci: Lettere di Rettori e di altre cariche) koji se odnosi na događaj iz 1748. godine u korčulanskom selu Žrnovo, kada su mještani – vjerujući da su se pojavili vampiri – oskvrnuli nekoliko mjesnih grobova. U radu se podrobno iznose osnovni podaci iz spisa te rečeni događaj analizira u širem društvenom kontekstu i prate se lokalna vjerovanja.The main interest of this essay is the analysis of the document from the State Archive in Venice (file: Capi del Consiglio de’ Dieci: Lettere di Rettori e di altre cariche) which is connected with the episode from 1748 when the inhabitants of the village Žrnove on the island of Korčula in Croatia opened tombs on the local cemetery in the fear of the vampires treating. This essay try to show some social circumstances connected with this event as well as a local vernacular tradition concerning superstitions

High-Fiber, Whole-Food Dietary Intervention Alters the Human Gut Microbiome but Not Fecal Short-Chain Fatty Acids.

Dietary shifts can have a direct impact on the gut microbiome by preferentially selecting for microbes capable of utilizing the various dietary nutrients. The intake of dietary fiber has decreased precipitously in the last century, while consumption of processed foods has increased. Fiber, or microbiota-accessible carbohydrates (MACs), persist in the digestive tract and can be metabolized by specific bacteria encoding fiber-degrading enzymes. The digestion of MACs results in the accumulation of short-chain fatty acids (SCFAs) and other metabolic by-products that are critical to human health. Here, we implemented a 2-week dietary fiber intervention aiming for 40 to 50 g of fiber per day within the context of a course-based undergraduate research experience (CURE) (n = 20). By coupling shotgun metagenomic sequencing and targeted gas chromatography-mass spectrometry (GC-MS), we found that the dietary intervention significantly altered the composition of individual gut microbiomes, accounting for 8.3% of the longitudinal variability within subjects. Notably, microbial taxa that increased in relative abundance as a result of the diet change included known MAC degraders (i.e., Bifidobacterium and Lactobacillus). We further assessed the genetic diversity within Bifidobacterium, assayed by amplification of the groEL gene. Concomitant with microbial composition changes, we show an increase in the abundance of genes involved in inositol degradation. Despite these changes in gut microbiome composition, we did not detect a consistent shift in SCFA abundance. Collectively, our results demonstrate that on a short-term timescale of 2 weeks, increased fiber intake can induce compositional changes of the gut microbiome, including an increase in MAC-degrading bacteria.IMPORTANCE A profound decrease in the consumption of dietary fiber in many parts of the world in the last century may be associated with the increasing prevalence of type II diabetes, colon cancer, and other health problems. A typical U.S. diet includes about ∼15 g of fiber per day, far less fiber than the daily recommended allowance. Changes in dietary fiber intake affect human health not only through the uptake of nutrients directly but also indirectly through changes in the microbial community and their associated metabolism. Here, we conducted a 2-week diet intervention in healthy young adults to investigate the impact of fiber consumption on the gut microbiome. Participants increased their average fiber consumption by 25 g/day on average for 2 weeks. The high-fiber diet intervention altered the gut microbiome of the study participants, including increases in known fiber-degrading microbes, such as Bifidobacterium and Lactobacillus