Glycogen storage disease type 1b (GSD-1b) is proposed to be caused by a deficiency in microsomal glucose 6-phosphate (G6P) transport, causing a loss of glucose-6-phosphatase activity and glucose homeostasis. However, for decades, this disorder has defied molecular characterization. In this study, we characterize the structural organization of the G6P transporter gene and identify mutations in the gene that segregate with the GSD-1b disorder. We report the functional characterization of the recombinant G6P transporter and demonstrate that mutations uncovered in GSD-1b patients disrupt G6P transport. Our results, for the first time, define a molecular basis for functional deficiency in GSD-1b and raise the possibility that the defective G6P transporter contributes to neutropenia and neutrophil/monocyte dysfunctions characteristic of GSD-1b patients.

Biocathode extracellular electron transfer (EET) may be exploited for biotechnology applications, including microbially mediated O 2 reduction in microbial fuel cells and microbial electrosynthesis. However, biocathode mechanistic studies needed to improve or engineer functionality have been limited to a few select species that form sparse, homogeneous biofilms characterized by little or no growth. Attempts to cultivate isolates from biocathode environmental enrichments often fail due to a lack of some advantage provided by life in a consortium, highlighting the need to study and understand biocathode consortia in situ. Here, we present metagenomic and metaproteomic characterization of a previously described biocathode biofilm (؉310 mV versus a standard hydrogen electrode [SHE]) enriched from seawater, reducing O 2 , and presumably fixing CO 2 for biomass generation. Metagenomics identified 16 distinct cluster genomes, 15 of which could be assigned at the family or genus level and whose abundance was roughly divided between Alpha-and Gammaproteobacteria. A total of 644 proteins were identified from shotgun metaproteomics and have been deposited in the the ProteomeXchange with identifier PXD001045. Cluster genomes were used to assign the taxonomic identities of 599 proteins, with Marinobacter, Chromatiaceae, and Labrenzia the most represented. RubisCO and phosphoribulokinase, along with 9 other Calvin-Benson-Bassham cycle proteins, were identified from Chromatiaceae. In addition, proteins similar to those predicted for iron oxidation pathways of known iron-oxidizing bacteria were observed for Chromatiaceae. These findings represent the first description of putative EET and CO 2 fixation mechanisms for a selfregenerating, self-sustaining multispecies biocathode, providing potential targets for functional engineering, as well as new insights into biocathode EET pathways using proteomics. Bioelectrochemical systems (BES) use microorganisms as catalysts to drive complex electrochemical reactions, such as electricity generation by microbial fuel cells (MFCs) (1), wastewater treatment (2), and microbial electrosynthesis (3-6), that would not be possible without living cells. The term "biocathode" refers to a biofilm, constituted of a single organism or microbial consortium, that has formed on the cathode of a BES and consumes electrons (e Ϫ ). Cathodes hold great potential as a stable electron source to drive microbial metabolism (7); however, little is known about the underlying extracellular electron transfer (EET) pathways that could be exploited for biocathode functional engineering. Although biocathode EET has been demonstrated for a variety of microorganisms, including acetogens (5) and a methanogenic archaeon (6), studies aimed at identifying EET conduits from the electrode to cells have mostly been confined to the model organisms Geobacter (8) and Shewanella (9), due to the massive effort put forth to understand how these iron-reducing bacteria are able to catalyze EET at bioanodes (10-12). The ability of iron-...

Three notable members of the Harveyi clade, Vibrio harveyi, Vibrio alginolyticus and Vibrio parahaemolyticus, are best known as marine pathogens of commercial and medical import. In spite of this fact, the discrimination of Harveyi clade members remains difficult due to genetic and phenotypic similarities, and this has led to misidentifications and inaccurate estimations of a species' involvement in certain environments. To begin to understand the underlying genetics that complicate species level discrimination, we compared the genomes of Harveyi clade members isolated from different environments (seawater, shrimp, corals, oysters, finfish, humans) using microarray-based comparative genomic hybridization (CGH) and multilocus sequence analyses (MLSA). Surprisingly, we found that the only two V. harveyi strains that have had their genomes sequenced (strains BAA-1116 and HY01) have themselves been misidentified. Instead of belonging to the species harveyi, they are actually members of the species campbellii. In total, 28% of the strains tested were found to be misidentified and 42% of these appear to comprise a novel species. Taken together, our findings correct a number of species misidentifications while validating the ability of both CGH and MLSA to distinguish closely related members of the Harveyi clade.

Glycogen storage disease type Ib (GSD-Ib) is caused by a deficiency in the glucose-6-phosphate transporter (G6PT). In addition to disrupted glucose homeostasis, GSD-Ib patients have unexplained and unexpected defects in neutrophil respiratory burst, chemotaxis and calcium flux, in response to the bacterial peptide f-Met-Leu-Phe, as well as intermittent neutropenia. We generated a G6PT knockout (G6PT-/-) mouse that mimics all known defects of the human disorder and used the model to further our understanding of the pathogenesis of GSD-Ib. We demonstrate that the neutropenia is caused directly by the loss of G6PT activity; that chemotaxis and calcium flux, induced by the chemokines KC and macrophage inflammatory protein-2, are defective in G6PT-/- neutrophils; and that local production of these chemokines and the resultant neutrophil trafficking in vivo are depressed in G6PT-/- ascites during an inflammatory response. The bone and spleen of G6PT-/- mice are developmentally delayed and accompanied by marked hypocellularity of the bone marrow, elevation of myeloid progenitor cell frequencies in both organs and a corresponding dramatic increase in granulocyte colony stimulating factor levels in both GSD-Ib mice and humans. So, in addition to transient neutropenia, a sustained defect in neutrophil trafficking due to both the resistance of neutrophils to chemotactic factors, and reduced local production of neutrophil-specific chemokines at sites of inflammation, may underlie the myeloid deficiency in GSD-Ib. These findings demonstrate that G6PT is not just a G6P transport protein but also an important immunomodulatory protein whose activities need to be addressed in treating the myeloid complications in GSD-Ib patients.

Simultaneous testing for detection of infectious pathogens that cause similar symptoms (e.g., acute respiratory infections) is invaluable for patient treatment, outbreak prevention, and efficient use of antibiotic and antiviral agents. In addition, such testing may provide information regarding possible coinfections or induced secondary infections, such as virally induced bacterial infections. Furthermore, in many cases, detection of a pathogen requires more than genus/species-level resolution, since harmful agents (e.g., avian influenza virus) are grouped with other, relatively benign common agents, and for every pathogen, finer resolution is useful to allow tracking of the location and nature of mutations leading to strain variations. In this study, a previously developed resequencing microarray that has been demonstrated to have these capabilities was further developed to provide individual detection sensitivity ranging from 10 1 to 10 3 genomic copies for more than 26 respiratory pathogens while still retaining the ability to detect and differentiate between close genetic neighbors. In addition, the study demonstrated that this system allows unambiguous and reproducible sequence-based strain identification of the mixed pathogens. Successful proof-of-concept experiments using clinical specimens show that this approach is potentially very useful for both diagnostics and epidemic surveillance.Accurate and rapid identification of infectious pathogens that cause similar symptoms, such as acute respiratory infections (ARIs), can be a critical factor in the successful treatment of the illness, outbreak control measures, and the efficient use of precious antibiotics and antiviral drugs (30, 40). Simultaneous testing for all possible pathogens is an efficient means to obtain a conclusive result. In addition, assaying for all potential pathogens may yield information regarding possible coinfections or induced secondary infections (e.g., virally induced bacterial infections). Currently, many promising approaches using reverse transcription-PCR (RT-PCR)/PCR amplification strategies as multiplexed approaches for testing several organisms are being developed (1, 2, 4-10, 14-16, 22, 23, 25, 28, 36, 37, 41, 42). While these approaches are versatile, additional strategies must be implemented to ensure good specificity for the assay. In cases where closely related organisms can have very different clinical consequences and epidemiological patterns (e.g., Bordetella pertussis versus Bordetella parapertussis), discrimination to the species, serotype/subtype, or even strain level is required to reduce the incidence of false positives (22). Influenza virus detection is a case where serotype/subtype discrimination, at a minimum, is required. In addition, strain-level information can be used to quickly discern the effectiveness of vaccines and make proper recommendations for appropriate outbreak control measures. Furthermore, the sequence information of new circulating human isolates and possible zoonotic strains [e.g., avian infl...