[Show abstract][Hide abstract] ABSTRACT: Fecal microbiota transplants (FMT) are an effective treatment for patients with gut microbe dysbiosis suffering from recurrent C. difficile infections. To further understand how FMT reconstitutes the patient’s gut commensal microbiota, we have analyzed the colonization potential of the donor, recipient and recipient post transplant fecal samples using transplantation in gnotobiotic mice.
A total of nine samples from three human donors, recipient’s pre and post FMT were transplanted into gnotobiotic mice. Microbiome analysis of three donor fecal samples revealed the presence of a high relative abundance of commensal microbes from the family Bacteriodaceae and Lachnospiraceae that were almost absent in the three recipient pre FMT fecal samples (<0.01 %). The microbe composition in gnotobiotic mice transplanted with the donor fecal samples was similar to the human samples. The recipient samples contained Enterobacteriaceae, Lactobacillaceae, Enterococcaceae in relative abundance of 43, 11, 8 %, respectively. However, gnotobiotic mice transplanted with the recipient fecal samples had an average relative abundance of unclassified Clostridiales of 55 %, approximately 7000 times the abundance in the recipient fecal samples prior to transplant. Microbiome analysis of fecal samples from the three patients early (2–4 weeks) after FMT revealed a microbe composition with the relative abundance of both Bacteriodaceae and Lachnospiraceae that was approximately 7 % of that of the donor. In contrast, gnotobioitc mice transplanted with the fecal samples obtained from the three at early times post FMT revealed increases in the relative abundance of Bacteriodaceae and Lachnospiraceae microbe compositions to levels similar to the donor fecal samples. Furthermore, the unclassified Clostridiales in the recipient samples post FMT was reduced to an average of 10 %.
We have used transplantation into gnotobiotic mice to evaluate the colonization potential of microbiota in FMT patients early after transplant. The commensal microbes present at early times post FMT out competed non-commensal microbes (e.g. such as unclassified Clostridiales) for niche space. The selective advantage of these commensal microbes to occupy niches in the gastrointestinal tract helps to explain the success of FMT to reconstitute the gut microbe community of patients with recurrent C. difficile infections.
[Show abstract][Hide abstract] ABSTRACT: Janthinobacterium
sp. Ant5-2-1, isolated from the Schirmacher Oasis of East Antarctica, produces a purple-violet pigment, manifests diverse energy metabolism abilities, and tolerates cold, ultraviolet radiation, and other environmental stressors. We report here the 6.19-Mb draft genome of strain Ant5-2-1, which will help understand its survival mechanisms in extreme Antarctic ecosystems.
Preview · Article · Feb 2016 · Genome Announcements
[Show abstract][Hide abstract] ABSTRACT: Background:
CD14, a co-receptor for several pattern recognition receptors and a widely used monocyte/macrophage marker, plays a key role in host responses to Gram-negative bacteria. Despite its central role in the inflammatory response to lipopolysaccharide and other microbial products, and in dissemination of bacteria in some infections, the signaling networks controlled by CD14 during urinary tract infection (UTI) are unknown.
We used uropathogenic Escherichia coli (UPEC) infection of wild-type (WT) C57BL/6 and Cd14-deficient mice and RNA-sequencing (RNA-seq) to define the CD14-dependent transcriptional signature, and role of CD14, in host defense against UTI in the bladder.
UPEC-induced the up-regulation of Cd14 and monocyte/macrophage related genes Emr1/F4/80 and Csf1r/c-fms, which was associated with lower UPEC burdens in WT compared to Cd14-deficient mice. Exacerbation of infection in Cd14-deficient mice was associated with the absence of a 491-gene transcriptional signature in the bladder that encompassed multiple host networks not previously associated with this receptor. CD14-dependent pathways included immune cell trafficking, differential cytokine production in macrophages, and IL-17 signaling. Depletion of monocytes/macrophages in the bladder by administration of liposomal clodronate led to higher UPEC burdens.
This study identifies new host protective and signaling roles for Cd14 in the bladder during UPEC UTI.
Full-text · Article · Aug 2015 · The Journal of Infectious Diseases
[Show abstract][Hide abstract] ABSTRACT: Although most hypocalcemia with hypomagenesemia in the neonatal period is due to transient neonatal hypoparathyroidism, magnesium channel defects should also be considered.
We report a case of persistent hypomagnesemia in an 8-day-old Hispanic male who presented with generalized seizures. He was initially found to have hypomagnesemia, hypocalcemia, hyperphosphatemia and normal parathyroid hormone. Serum calcium normalized with administration of calcitriol and calcium carbonate. Serum magnesium improved with oral magnesium sulfate. However, 1 week after magnesium was discontinued, serum magnesium declined to 0.5 mg/dL. Magnesium supplementation was immediately restarted, and periodic seizure activity resolved after serum magnesium concentration was maintained above 0.9 mg/dL. The child was eventually weaned off oral calcium and calcitriol with persistent normocalemia. However, supraphysiologic oral magnesium doses were necessary to prevent seizures and maintain serum magnesium at the low limit of normal.
As his clinical presentation suggested primary renal magnesium wastage, TRPM6 gene mutations were suspected; subsequent genetic testing revealed the child to be compound heterozygous for TRPM6 mutations.
Two novel TRPM6 mutations are described with a new geographic and ethnic origin. This case highlights the importance of recognizing disorders of magnesium imbalance and describing new genetic mutations.
[Show abstract][Hide abstract] ABSTRACT: Somatic mosaicism for DNA copy number alterations (SMC-CNAs) is defined as gain or loss of chromosomal segments in somatic cells within a single organism. As cells harboring SMC-CNAs can undergo clonal expansion, it has been proposed that SMC-CNAs may contribute to the predisposition of these cells to genetic disease including cancer. Herein, the gross genomic alterations (>500 kbp) were characterized in uninvolved mammary glandular tissue from 59 breast cancer patients and matched samples of primary tumors and lymph node metastases. Array based comparative genomic hybridization showed 10% (6/59) of patients harbored 1 - 359 large SMC-CNAs (mean: 1328 kbp; median: 961 kbp) in a substantial portion of glandular tissue cells, distal from the primary tumor site. SMC-CNAs were partially recurrent in tumors, albeit with considerable contribution of stochastic SMC-CNAs indicating genomic destabilization. Targeted resequencing of 301 known predisposition and somatic driver loci revealed mutations and rare variants in genes related to maintenance of genomic integrity: BRCA1 (p.Gln1756Profs*74, p.Arg504Cys), BRCA2 (p.Asn3124Ile), NCOR1 (p.Pro1570Glnfs*45), PALB2 (p.Ser500Pro) and TP53 (p.Arg306*). Co-occurrence of gross SMC-CNAs along with point mutations or rare variants in genes responsible for safeguarding genomic integrity highlights the temporal and spatial neoplastic potential of uninvolved glandular tissue in breast cancer patients. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: Hymenobacter sp. IS2118, isolated from a freshwater lake in Schirmacher Oasis, Antarctica, produces extracellular polymeric substance
(EPS) and manifests tolerance to cold, UV radiation (UVR), and oxidative stress. We report the 5.26-Mb draft genome of strain
IS2118, which will help us to understand its adaptation and survival mechanisms in Antarctic extreme ecosystems.
[Show abstract][Hide abstract] ABSTRACT: Pseudomonas sp. strain Ant30-3, isolated from fuel-contaminated Antarctic soil, exhibited distinctive psychrotolerant attributes and
the potential for degrading aromatic hydrocarbon compounds at cold temperatures. We report here the 6.14-Mb draft genome of
Ant30-3, which will provide insights into the genomic basis for the psychrotolerant and biodegradative properties of this
Full-text · Article · May 2014 · Genome Announcements
[Show abstract][Hide abstract] ABSTRACT: Constitutional SMARCB1 mutations at 22q11.23 have been found in ∼50% of familial and <10% of sporadic schwannomatosis cases. We sequenced highly conserved regions along 22q from eight individuals with schwannomatosis whose schwannomas involved somatic loss of one copy of 22q, encompassing SMARCB1 and NF2, with a different somatic mutation of the other NF2 allele in every schwannoma but no mutation of the remaining SMARCB1 allele in blood and tumor samples. LZTR1 germline mutations were identified in seven of the eight cases. LZTR1 sequencing in 12 further cases with the same molecular signature identified 9 additional germline mutations. Loss of heterozygosity with retention of an LZTR1 mutation was present in all 25 schwannomas studied. Mutations segregated with disease in all available affected first-degree relatives, although four asymptomatic parents also carried an LZTR1 mutation. Our findings identify LZTR1 as a gene predisposing to an autosomal dominant inherited disorder of multiple schwannomas in ∼80% of 22q-related schwannomatosis cases lacking mutation in SMARCB1.
[Show abstract][Hide abstract] ABSTRACT: Wnt5a is a non-canonical signaling Wnt. Low expression of WNT5A is correlated with poor prognosis in breast cancer patients. The highly invasive breast cancer cell lines, MDA-MB-231 and 4T1, express very low levels of WNT5A. To determine if enhanced expression of WNT5A would affect metastatic behavior, we generated WNT5A expressing cells from the 4T1 and MDA-MB-231 parental cell lines. WNT5A expressing cells demonstrated cobblestone morphology and reduced in vitro migration relative to controls. Cell growth was not altered. Metastasis to the lung via tail vein injection was reduced in the 4T1-WNT5A expressing cells relative to 4T1-vector controls. To determine the mechanism of WNT5A action on metastasis, we performed microarray and whole-transcriptome sequence analysis (RNA-seq) to compare gene expression in 4T1-WNT5A and 4T1-vector cells. Analysis indicated highly significant alterations in expression of genes associated with cellular movement. Down-regulation of a subset of these genes, Mmp13, Nos2, Il1a, Cxcl2, and Lamb3, in WNT5A expressing cells was verified by semi-quantitative RT-PCR. Significant differences in transcript splicing were also detected in cell movement associated genes including Cd44. Cd44 is an adhesion molecule with a complex genome structure. Variable exon usage is associated with metastatic phenotype. Alternative spicing of Cd44 in WNT5A expressing cells was confirmed using RT-PCR. We conclude that WNT5A inhibits metastasis through down-regulation of multiple cell movement pathways by regulating transcript levels and splicing of key genes like Cd44.
[Show abstract][Hide abstract] ABSTRACT: Generation and characterization of Wnt5 expressing MDA-MB-231 cells. (A) Western blot was used to show expression of the WNT5A transgene in MDA-MB-231 cells (231) relative to vector transduced (V) and WNT5A transduced cells. (B) A transwell migration assay indicated that WNT5A expressing MDA-MD-231 cells had reduced migration toward serum free media (SFM), 10% FBS and 20% FBS relative to vector only control cells. (C) The transwell migration assay was used to determine the effects of WNT5A conditioned media (WNT5A CM) on cell migration towards 10% FBS. Treatment of cells with WNT5A conditioned medium inhibited cell migration. (D) MDA-MB-231 cells were placed in SFM in the top well of the transwell while either parental or WNT5A conditioned media was placed in the bottom well. Migration towards WNT5A CM was inhibited (D, left). Migration was also inhibited if conditioned media was placed in both top and bottom chambers (D, right). * = T-test p-value <0.05, **p<0.01, ***p<0.001.
[Show abstract][Hide abstract] ABSTRACT: The most common causes of urinary tract infections (UTIs) are Gram-negative pathogens such as Escherichia coli; however, Gram-positive organisms, including Streptococcus agalactiae, or group B streptococcus (GBS), also cause UTI. In GBS infection, UTI progresses to cystitis once the bacteria colonize the bladder, but the host responses triggered in the bladder immediately following infection are largely unknown. Here, we used genome-wide expression profiling to map the bladder transcriptome of GBS UTI in mice infected transurethrally with uropathogenic GBS that was cultured from a 35-year-old women with cystitis. RNA from bladders was applied to Affymetrix Gene-1.0ST microarrays; quantitative reverse transcriptase PCR (qRT-PCR) was used to analyze selected gene responses identified in array data sets. A surprisingly small significant-gene list of 172 genes was identified at 24 h; this compared to 2,507 genes identified in a side-by-side comparison with uropathogenic E. coli (UPEC). No genes exhibited significantly altered expression at 2 h in GBS-infected mice according to arrays despite high bladder bacterial loads at this early time point. The absence of a marked early host response to GBS juxtaposed with broad-based bladder responses activated by UPEC at 2 h. Bioinformatics analyses, including integrative system-level network mapping, revealed multiple activated biological pathways in the GBS bladder transcriptome that regulate leukocyte activation, inflammation, apoptosis, and cytokine-chemokine biosynthesis. These findings define a novel, minimalistic type of bladder host response triggered by GBS UTI, which comprises collective antimicrobial pathways that differ dramatically from those activated by UPEC. Overall, this study emphasizes the unique nature of bladder immune activation mechanisms triggered by distinct uropathogens.
Full-text · Article · Jun 2012 · Infection and immunity
[Show abstract][Hide abstract] ABSTRACT: Early transcriptional activation events that occur in bladder immediately following bacterial urinary tract infection (UTI) are not well defined. In this study, we describe the whole bladder transcriptome of uropathogenic Escherichia coli (UPEC) cystitis in mice using genome-wide expression profiling to define the transcriptome of innate immune activation stemming from UPEC colonization of the bladder. Bladder RNA from female C57BL/6 mice, analyzed using 1.0 ST-Affymetrix microarrays, revealed extensive activation of diverse sets of innate immune response genes, including those that encode multiple IL-family members, receptors, metabolic regulators, MAPK activators, and lymphocyte signaling molecules. These were among 1564 genes differentially regulated at 2 h postinfection, highlighting a rapid and broad innate immune response to bladder colonization. Integrative systems-level analyses using InnateDB (http://www.innatedb.com) bioinformatics and ingenuity pathway analysis identified multiple distinct biological pathways in the bladder transcriptome with extensive involvement of lymphocyte signaling, cell cycle alterations, cytoskeletal, and metabolic changes. A key regulator of IL activity identified in the transcriptome was IL-10, which was analyzed functionally to reveal marked exacerbation of cystitis in IL-10-deficient mice. Studies of clinical UTI revealed significantly elevated urinary IL-10 in patients with UPEC cystitis, indicating a role for IL-10 in the innate response to human UTI. The whole bladder transcriptome presented in this work provides new insight into the diversity of innate factors that determine UTI on a genome-wide scale and will be valuable for further data mining. Identification of protective roles for other elements in the transcriptome will provide critical new insight into the complex cascade of events that underpin UTI.
Full-text · Article · Dec 2011 · The Journal of Immunology