Precision medicine for KRAS wild-type pancreatic adenocarcinomas.

BACKGROUND: KRAS mutation is the most common molecular alteration in pancreatic adenocarcinoma (PDAC), and around 10% of patients harbor KRAS wild-type tumors (KRASWT). METHODS: A retrospective chart review of clinical/molecular data was performed including all PDAC patients with a determined KRAS status (tumor molecular profiling on tissue or liquid biopsy). RESULTS: 342 patients were included with 54 KRASWT PDAC (16%) compared to 288 patients with KRASm PDAC. Median age was 61 years [IQR:54.0;67.0] and 164 pts (48%) were female. At diagnosis, KRASWT patients (63%) were more frequently diagnosed at a non-metastatic stage compared to KRASm patients (41%) (p = 0.003). Regarding metastatic sites, liver was less frequent in KRASWT (39%, p < 0.0001). Median overall survival (mOS) from initial diagnosis was significantly higher in the KRASWT group compared to KRASm (50.8 months, CI95% [32.0-NR] vs 21.1 months, CI95% [18.9-23.4] (p < 0.004 after adjustment on age, ECOG and stage at diagnosis). In first-line systemic treatment, (mostly FOLFIRINOX) progression-free survival (PFS) was also higher in KRASWT. Based on ESCAT classification, a putative actionable alteration (ESCAT I-III) was identified in 19 (36%) KRASWT pts and 46 (16%) KRASm patients (p < 0.0001) with more alterations in FGFR2, BRAF(V600E), NRTK and more MSI tumors. KRASWT harbored also fewer alterations in TP53, CDKN2A, and SMAD4. 12 KRASWT patients received a molecularly-matched treatment with clinical benefit and improved outcomes compared to KRASm patients. CONCLUSIONS: KRASWT patients display distinct disease characteristics and outcomes with prolonged overall survival. KRASWT patients also harbor more actionable molecular alterations, leading to higher survival rates after receiving molecularly matched treatments.

Implementing patient derived organoids in functional precision medicine for patients with advanced colorectal cancer.

BACKGROUND: Patient Derived Organoids (PDOs) emerged as the best technology to develop ex vivo tumor avatars. Whether drug testing on PDOs to identify efficient therapies will bring clinical utility by improving patient survival remains unclear. To test this hypothesis in the frame of clinical trials, PDO technology faces three main challenges to be implemented in routine clinical practices: i) generating PDOs with a limited amount of tumor material; ii) testing a wide panel of anti-cancer drugs; and iii) obtaining results within a time frame compatible with patient disease management. We aimed to address these challenges in a prospective study in patients with colorectal cancer (CRC). METHODS: Fresh surgical or core needle biopsies were obtained from patients with CRC. PDOs were established and challenged with a panel of 25 FDA-approved anti-cancer drugs (chemotherapies and targeted therapies) to establish a scoring method ('chemogram') identifying in vitro responders. The results were analyzed at the scale of the cohort and individual patients when the follow-up data were available. RESULTS: A total of 25 PDOs were successfully established, harboring 94% concordance with the genomic profile of the tumor they were derived from. The take-on rate for PDOs derived from core needle biopsies was 61.5%. A chemogram was obtained with a 6-week median turnaround time (range, 4-10 weeks). At least one hit (mean 6.16) was identified for 92% of the PDOs. The number of hits was inversely correlated to disease metastatic dissemination and the number of lines of treatment the patient received. The chemograms were compared to clinical data obtained from 8 patients and proved to be predictive of their response with 75% sensitivity and specificity. CONCLUSIONS: We show that PDO-based drug tests can be achieved in the frame of routine clinical practice. The chemogram could provide clinicians with a decision-making tool to tailor patient treatment. Thus, PDO-based functional precision oncology should now be tested in interventional trials assessing its clinical utility for patients who do not harbor activable genomic alterations or have developed resistance to standard of care treatments.

Primary Colorectal Tumor Displays Differential Genomic Expression Profiles Associated with Hepatic and Peritoneal Metastases.

BACKGROUND: Despite improvements in characterization of CRC heterogeneity, appropriate risk stratification tools are still lacking in clinical practice. This study aimed to elucidate the primary tumor transcriptomic signatures associated with distinct metastatic routes. METHODS: Primary tumor specimens obtained from CRC patients with either isolated LM (CRC-Liver) or PM (CRC-Peritoneum) were analyzed by transcriptomic mRNA sequencing, gene set enrichment analyses (GSEA) and immunohistochemistry. We further assessed the clinico-pathological associations and prognostic value of our signature in the COAD-TCGA independent cohort. RESULTS: We identified a significantly different distribution of Consensus Molecular Subtypes between CRC-Liver and CRC-peritoneum groups. A transcriptomic signature based on 61 genes discriminated between liver and peritoneal metastatic routes. GSEA showed a higher expression of immune response and epithelial invasion pathways in CRC-Peritoneum samples and activation of proliferation and metabolic pathways in CRC-Liver samples. The biological relevance of RNA-Seq results was validated by the immunohistochemical expression of three significantly differentially expressed genes (ACE2, CLDN18 and DUSP4) in our signature. In silico analysis of the COAD-TCGA showed that the CRC-Peritoneum signature was associated with negative prognostic factors and poor overall and disease-free survivals. CONCLUSIONS: CRC primary tumors spreading to the liver and peritoneum display significantly different transcriptomic profiles. The implementation of this signature in clinical practice could contribute to identify new therapeutic targets for stage IV CRC and to define individualized follow-up programs in stage II-III CRC.

Unexpected finding of Fusobacterium varium as the dominant Fusobacterium species in cattle rumen: potential implications for liver abscess etiology and interventions.

Fusobacterium varium has been generally overlooked in cattle rumen microbiome studies relative to the presumably more abundant liver abscess-causing Fusobacterium necrophorum. However, F. varium was found to be more abundant in the rumen fluid of cattle and under culture conditions tailored to enrich F. necrophorum. Using near-full length 16S ribosomal ribonucleic acid sequencing, we demonstrate that F. varium grows under restrictive conditions commonly used to enumerate F. necrophorum, suggesting that previous F. necrophorum abundance assessment may have been inaccurate and that F. varium may be an underestimated member of the ruminal bacterial community. Fusobacterium varium were not as susceptible as F. necrophorum to in-feed antibiotics conventionally used in feedlots. Exposure to tylosin, the current gold standard for liver abscess reduction strategies in cattle, consistently hindered growth of the F. necrophorum strains tested by over 67% (P < 0.05) relative to the unexposed control. In contrast, F. varium strains were totally or highly resistant (0%-13% reduction in maximum yield, P < 0.05). Monensin, an ionophore antibiotic, had greater inhibitory activity against F. necrophorum than F. varium. Finally, preliminary genomic analysis of two F. varium isolates from the rumen revealed the presence of virulence genes related to those of pathogenic human F. varium isolates associated with active invasion of mammalian cells. The data presented here encourage further investigation into the ecological role of F. varium within the bovine rumen and potential role in liver abscess development, and proactive interventions.

The conventional method of liver abscess prevention in feedlot cattle is in-feed use of tylosin to target Fusobacterium necrophorum, which has been presumed to be the most common Fusobacterium species within the ruminal compartment. Our investigation into ruminal Fusobacterium, however, revealed a different species, Fusobacterium varium, to be abundant and ubiquitous in ruminal content samples. Furthermore, growth conditions tailored to enrich F. necrophorum consistently promoted growth of F. varium, and the bovine isolates tested had much lower susceptibilities to the commonly fed antibiotics tylosin and monensin compared to F. necrophorum. Fusobacterium varium is an emerging pathogen in humans and preliminary genome sequencing of two ruminal F. varium isolates revealed genes linked to pathogenicity. While the ecological role of F. varium in the rumen is still not fully understood, our findings draw attention to this pathogen and its potential implication in liver abscesses.

Biofilm Control in Flow-Through Systems Using Polyvalent Phages Delivered by Peptide-Modified M13 Coliphages with Enhanced Polysaccharide Affinity.

Eradication of biofilms that may harbor pathogens in water distribution systems is an elusive goal due to limited penetration of residual disinfectants. Here, we explore the use of engineered filamentous coliphage M13 for enhanced biofilm affinity and precise delivery of lytic polyvalent phages (i.e., broad-host-range phages lysing multiple host strains after infection). To promote biofilm attachment, we modified the M13 major coat protein (pVIII) by inserting a peptide sequence with high affinity for Pseudomonas aeruginosa (P. aeruginosa) extracellular polysaccharides (commonly present on the surface of biofilms in natural and engineered systems). Additionally, we engineered the M13 tail fiber protein (pIII) to contain a peptide sequence capable of binding a specific polyvalent lytic phage. The modified M13 had 102- and 5-fold higher affinity for P. aeruginosa-dominated mixed-species biofilms than wildtype M13 and unconjugated polyvalent phage, respectively. When applied to a simulated water distribution system, the resulting phage conjugates achieved targeted phage delivery to the biofilm and were more effective than polyvalent phages alone in reducing live bacterial biomass (84 vs 34%) and biofilm surface coverage (81 vs 22%). Biofilm regrowth was also mitigated as high phage concentrations induced residual bacteria to downregulate genes associated with quorum sensing and extracellular polymeric substance secretion. Overall, we demonstrate that engineered M13 can enable more accurate delivery of polyvalent phages to biofilms in flow-through systems for enhanced biofilm control.

A Carotenoid- and Nuclease-Producing Bacterium Can Mitigate Enterococcus faecalis Transformation by Antibiotic Resistance Genes.

Dissemination of antibiotic resistance genes (ARGs) through natural transformation is facilitated by factors that stabilize extracellular DNA (eDNA) and that induce reactive oxygen species (ROS) that permeabilize receptor cells and upregulate transformation competence genes. In this study, we demonstrate that Deinococcus radiodurans can mitigate this ARG dissemination pathway by removing both eDNA and ROS that make recipient cells more vulnerable to transformation. We used plasmid RP4 as source of extracellular ARGs (tetA, aphA, and blaTEM-2) and the opportunistic pathogen Enterococcus faecalis as receptor. The presence of D. radiodurans significantly reduced the transformation frequency from 2.5 ± 0.7 × 10-6 to 7.4 ± 1.4 × 10-7 (p < 0.05). Based on quantification of intracellular ROS accumulation and superoxide dismutase (SOD) activity, and quantitative polymerase chain reaction (qPCR) and transcriptomic analyses, we propose two mechanisms by which D. radiodurans mitigates E. faecalis transformation by ARGs: (a) residual antibiotics induce D. radiodurans to synthesize liposoluble carotenoids that scavenge ROS and thus mitigate the susceptibility of E. faecalis for eDNA uptake, and (b) eDNA induces D. radiodurans to synthesize extracellular nucleases that degrade eARGs. This mechanistic insight informs biological strategies (including bioaugmentation) to curtail the spread of ARGs through transformation.

Patient-derived organoids identify an apico-basolateral polarity switch associated with survival in colorectal cancer.

The metastatic progression of cancer remains a major issue in patient treatment. However, the molecular and cellular mechanisms underlying this process remain unclear. Here, we use primary explants and organoids from patients harboring mucinous colorectal carcinoma (MUC CRC), a poor-prognosis histological form of digestive cancer, to study the architecture, invasive behavior and chemoresistance of tumor cell intermediates. We report that these tumors maintain a robust apico-basolateral polarity as they spread in the peritumoral stroma or organotypic collagen-I gels. We identified two distinct topologies - MUC CRCs either display a conventional 'apical-in' polarity or, more frequently, harbor an inverted 'apical-out' topology. Transcriptomic analyses combined with interference experiments on organoids showed that TGFß and focal adhesion signaling pathways are the main drivers of polarity orientation. Finally, we show that the apical-out topology is associated with increased resistance to chemotherapeutic treatments in organoids and decreased patient survival in the clinic. Thus, studies on patient-derived organoids have the potential to bridge histological, cellular and molecular analyses to decrypt onco-morphogenic programs and stratify cancer patients. This article has an associated First Person interview with the first author of the paper.

Renaissance for Phage-Based Bacterial Control.

Bacteriophages (phages) are an underutilized biological resource with vast potential for pathogen control and microbiome editing. Phage research and commercialization have increased rapidly in biomedical and agricultural industries, but adoption has been limited elsewhere. Nevertheless, converging advances in DNA sequencing, bioinformatics, microbial ecology, and synthetic biology are now poised to broaden phage applications beyond pathogen control toward the manipulation of microbial communities for defined functional improvements. Enhancements in sequencing combined with network analysis make it now feasible to identify and disrupt microbial associations to elicit desirable shifts in community structure or function, indirectly modulate species abundance, and target hub or keystone species to achieve broad functional shifts. Sequencing and bioinformatic advancements are also facilitating the use of temperate phages for safe gene delivery applications. Finally, integration of synthetic biology stands to create novel phage chassis and modular genetic components. While some fundamental, regulatory, and commercialization barriers to widespread phage use remain, many major challenges that have impeded the field now have workable solutions. Thus, a new dawn for phage-based (chemical-free) precise biocontrol and microbiome editing is on the horizon to enhance, suppress, or modulate microbial activities important for public health, food security, and more sustainable energy production and water reuse.

Bioaugmenting the poplar rhizosphere to enhance treatment of 1,4-dioxane.

1,4-Dioxane is a highly mobile and persistent groundwater pollutant that often forms large dilute plumes. Because of this, utilizing aggressive pump-and-treat and ex-situ technologies such as advanced oxidation can be prohibitively expensive. In this study, we bioaugmented the poplar rhizosphere with dioxane-degrading bacteria Mycobacterium dioxanotrophicus PH-06 or Pseudonocardia dioxanivorans CB1190 to enhance treatment of 1,4-dioxane in bench-scale experiments. All treatments tested removed 10 mg/L dioxane to near health advisory levels (<4 µg/L). However, PH-06-bioaugmented poplar significantly outperformed all other treatments, reaching <4 µg/L in only 13 days. Growth curve experiments confirmed that PH-06 could not utilize root extract as an auxiliary carbon source for growth. Despite this limitation, our findings suggest that PH-06 is a strong bioaugmentation candidate to enhance the treatment of dioxane by phytoremediation. In addition, we confirmed that CB1190 could utilize both 1,4-dioxane and root extract as substrates. Finally, we demonstrated the large-scale production of these two strains for use in the field. Overall, this study shows that combining phytoremediation and bioaugmentation is an attractive strategy to treat dioxane-contaminated groundwater to low risk-based concentrations (~1 µg/L).

Enhanced long-term attenuation of 1,4-dioxane in bioaugmented flow-through aquifer columns.

Long term natural attenuation of 1,4-dioxane (dioxane) and its enhanced biodegradation after bioaugmentation with Pseudonocardia dioxanivorans CB1190 were assessed using flow-through aquifer columns. Natural attenuation of dioxane was not observed even after 2 years of acclimation. However, dioxane removal was observed in the bioaugmented columns (34% when the influent was 200 µg/L and 92% for 5 mg/L). The thmA gene that encodes the tetrahydrofuran monooxygenase that initiates dioxane degradation by CB1190 was only detected at the inoculation port and persisted for months after inoculation, implying the resiliency of bioaugmentation and its potential to offer long-term enhanced biodegradation capabilities. However, due to extensive clumping and limited mobility of CB1190, the augmented catabolic potential may be restricted to the immediate vicinity of the inoculation port. Accordingly, bioaugmentation with CB1190 seems more appropriate for the establishment of biobarriers. Bioaugmentation efficiency was associated with the availability of oxygen. Aeration of the column influent to increase dissolved oxygen significantly improved dioxane removal (p < 0.05), suggesting that (for sites with oxygen-limiting conditions) bioaugmentation can benefit from engineered approaches for delivering additional oxygen.

Dendritic cell-derived hepcidin sequesters iron from the microbiota to promote mucosal healing.

Bleeding and altered iron distribution occur in multiple gastrointestinal diseases, but the importance and regulation of these changes remain unclear. We found that hepcidin, the master regulator of systemic iron homeostasis, is required for tissue repair in the mouse intestine after experimental damage. This effect was independent of hepatocyte-derived hepcidin or systemic iron levels. Rather, we identified conventional dendritic cells (cDCs) as a source of hepcidin that is induced by microbial stimulation in mice, prominent in the inflamed intestine of humans, and essential for tissue repair. cDC-derived hepcidin acted on ferroportin-expressing phagocytes to promote local iron sequestration, which regulated the microbiota and consequently facilitated intestinal repair. Collectively, these results identify a pathway whereby cDC-derived hepcidin promotes mucosal healing in the intestine through means of nutritional immunity.

Epidermal hepcidin is required for neutrophil response to bacterial infection.

Novel approaches for adjunctive therapy are urgently needed for complicated infections and patients with compromised immunity. Necrotizing fasciitis (NF) is a destructive skin and soft tissue infection. Despite treatment with systemic antibiotics and radical debridement of necrotic tissue, lethality remains high. The key iron regulatory hormone hepcidin was originally identified as a cationic antimicrobial peptide (AMP), but its putative expression and role in the skin, a major site of AMP production, have never been investigated. We report here that hepcidin production is induced in the skin of patients with group A Streptococcus (GAS) NF. In a GAS-induced NF model, mice lacking hepcidin in keratinocytes failed to restrict systemic spread of infection from an initial tissue focus. Unexpectedly, this effect was due to its ability to promote production of the CXCL1 chemokine by keratinocytes, resulting in neutrophil recruitment. Unlike CXCL1, hepcidin is resistant to degradation by major GAS proteases and could therefore serve as a reservoir to maintain steady-state levels of CXCL1 in infected tissue. Finally, injection of synthetic hepcidin at the site of infection can limit or completely prevent systemic spread of GAS infection, suggesting that hepcidin agonists could have a therapeutic role in NF.

Going Viral: Emerging Opportunities for Phage-Based Bacterial Control in Water Treatment and Reuse.

Water security to protect human lives and support sustainable development is one of the greatest global challenges of this century. While a myriad of water pollutants can impact public health, the greatest threat arises from pathogenic bacteria that can be harbored in different components of water treatment, distribution, and reuse systems. Bacterial biofilms can also promote water infrastructure corrosion and biofouling, which substantially increase the cost and complexity of many critical operations. Conventional disinfection and microbial control approaches are often insufficient to keep up with the increasing complexity and renewed relevance of this pressing challenge. For example, common disinfectants cannot easily penetrate and eradicate biofilms, and are also relatively ineffective against resistant microorganisms. The use of chemical disinfectants is also curtailed by regulations aimed at minimizing the formation of harmful disinfection byproducts. Furthermore, disinfectants cannot be used to kill problematic bacteria in biological treatment processes without upsetting system performance. This underscores the need for novel, more precise, and more sustainable microbial control technologies. Bacteriophages (phages), which are viruses that exclusively infect bacteria, are the most abundant (and perhaps the most underutilized) biological resource on Earth, and hold great promise for targeting problematic bacteria. Although phages should not replace broad-spectrum disinfectants in drinking water treatment, they offer great potential for applications where selective targeting of problematic bacteria is warranted and antimicrobial chemicals are either relatively ineffective or their use would result in unintended detrimental consequences. Promising applications for phage-based biocontrol include selectively suppressing bulking and foaming bacteria that hinder activated sludge clarification, mitigating proliferation of antibiotic resistant strains in biological wastewater treatment systems where broad-spectrum antimicrobials would impair pollutant biodegradation, and complementing biofilm eradication efforts to delay corrosion and biofouling. Phages could also mitigate harmful cyanobacteria blooms that produce toxins in source waters, and could also serve as substitutes for the prophylactic use of antibiotics and biocides in animal agriculture to reduce their discharge to source waters and the associated selective pressure for resistant bacteria. Here, we consider the phage life cycle and its implications for bacterial control, and elaborate on the biochemical basis of such potential application niches in the water supply and reuse cycle. We also discuss potential technological barriers for phage-based bacterial control and suggest strategies and research needs to overcome them.

Iron Regulator Hepcidin Impairs Macrophage-Dependent Cardiac Repair After Injury.

BACKGROUND: Defective systemic and local iron metabolism correlates with cardiac disorders. Hepcidin, a master iron sensor, actively tunes iron trafficking. We hypothesized that hepcidin could play a key role to locally regulate cardiac homeostasis after acute myocardial infarction. METHODS: Cardiac repair was analyzed in mice harboring specific cardiomyocyte or myeloid cell deficiency of hepcidin and challenged with acute myocardial infarction. RESULTS: We found that the expression of hepcidin was elevated after acute myocardial infarction and the specific deletion of hepcidin in cardiomyocytes failed to improve cardiac repair and function. However, transplantation of bone marrow-derived cells from hepcidin-deficient mice ( Hamp-/-) or from mice with specific deletion of hepcidin in myeloid cells (LysMCRE/+/ Hampf/f) improved cardiac function. This effect was associated with a robust reduction in the infarct size and tissue fibrosis in addition to favoring cardiomyocyte renewal. Macrophages lacking hepcidin promoted cardiomyocyte proliferation in a prototypic model of apical resection-induced cardiac regeneration in neonatal mice. Interleukin (IL)-6 increased hepcidin levels in inflammatory macrophages. Hepcidin deficiency enhanced the number of CD45+/CD11b+/F4/80+/CD64+/MHCIILow/chemokine (C-C motif) receptor 2 (CCR2)+ inflammatory macrophages and fostered signal transducer and activator of transcription factor-3 (STAT3) phosphorylation, an instrumental step in the release of IL-4 and IL-13. The combined genetic suppression of hepcidin and IL-4/IL-13 in macrophages failed to improve cardiac function in both adult and neonatal injured hearts. CONCLUSIONS: Hepcidin refrains macrophage-induced cardiac repair and regeneration through modulation of IL-4/IL-13 pathways.

Bacterial Endospores as Phage Genome Carriers and Protective Shells.

Bacterial endospores can serve as phage genome protection shells against various environmental stresses to enhance microbial control applications. The genomes of polyvalent lytic Bacillus phages PBSC1 and PBSC2, which infect both B. subtilis subsp. subtilis and B. cereus NRS 248, were incorporated into B. subtilis endospores (without integration into the host chromosome). When PBSC1 and PBSC2 were released from germinating endospores, they significantly inhibited the growth of the targeted opportunistic pathogen B. cereus Optimal endospore entrapment was achieved when phages were introduced to the fast-sporulating prespores at a multiplicity of infection of 1. Longer endospore maturation (48 h versus 24 h) increased both spore yield and efficiency of entrapment. Compared with free phages, spore-protected phage genomes showed significantly higher resistance toward high temperatures (60 to 80°C), extreme pH (pH 2 or pH 12), and copper ions (0.1 to 10 mg/liter). Endospore germination is inducible by low concentrations of l-alanine or by a germinant mixture (l-asparagine, d-glucose, d-fructose, and K+) to trigger the expression, assembly, and consequent release of phage particles within 60 to 90 min. Overall, the superior resiliency of polyvalent phages protected by endospores might enable nonrefrigerated phage storage and enhance phage applications after exposure to adverse environmental conditions.IMPORTANCE Bacteriophages are being considered for the control of multidrug-resistant and other problematic bacteria in environmental systems. However, the efficacy of phage-based microbial control is limited by infectivity loss during phage delivery and/or storage. Here, we exploit the pseudolysogenic state of phages, which involves incorporation of their genome into bacterial endospores (without integration into the host chromosome), to enhance survival in unfavorable environments. We isolated polyvalent (broad-host-range) phages that efficiently infect both benign and opportunistically pathogenic Bacillus strains and encapsulated the phage genomes in B. subtilis endospores to significantly improve resistance to various environmental stressors. Encapsulation by spores also significantly enhanced phage genome viability during storage. We also show that endospore germination can be induced on demand with nutrient germinants that trigger the release of active phages. Overall, we demonstrate that encapsulation of polyvalent phage genomes into benign endospores holds great promise for broadening the scope and efficacy of phage biocontrol.

Detection and cell sorting of Pseudonocardia species by fluorescence in situ hybridization and flow cytometry using 16S rRNA-targeted oligonucleotide probes.

Pseudonocardia spp. are receiving increasing attention due to their ability to biodegrade recalcitrant cyclic ether pollutants (e.g., 1,4-dioxane and tetrahydrofuran), as well as for their distinctive ecological niches (e.g., symbiosis with ants/plants and production of antibiotics). Isolating and characterizing Pseudonocardia spp. is thus important to discern their metabolic and physiological idiosyncrasies and advance their potential applications. However, slow growth, low cell yield, and dissimilar colony morphology hinder efficient isolation of Pseudonocardia using conventional plating methods. Here, we develop the first fluorescent probe (Pse631) targeting the 16S rRNA of Pseudonocardia members. In combination with flow cytometry and cell sorting, in situ hybridization with this probe enables sensitive and specific detection of Pseudonocardia cells in mixed cultures and enriched environmental samples without significant false positives, using Escherichia coli, Bacillus subtilis, and Mycobacterium spp. as negative controls. Pseudonocardia dioxanivorans CB1190 cells labeled with Pse631 as a positive control were detected when their relative abundance in the total bacterial community was as low as 0.1%. Effective separation of Pseudonocardia cells from the mixed consortium was confirmed by quantitative PCR analysis of sorted cells. This study provides a culture-independent high-throughput molecular approach enabling effective separation of Pseudonocardia populations from complex microbial communities. This approach will not only facilitate subsequent molecular analyses including species identification and quantification, but also advance understanding of their catabolic capacities and functional molecular diversity.

1,4-Dioxane-degrading consortia can be enriched from uncontaminated soils: prevalence of Mycobacterium and soluble di-iron monooxygenase genes.

Two bacterial consortia were enriched from uncontaminated soil by virtue of their ability to grow on 1,4-dioxane (dioxane) as a sole carbon and energy source. Their specific dioxane degradation rates at 30°C, pH = 7 (i.e. 5.7 to 7.1 g-dioxane per g-protein per day) were comparable to those of two dioxane-metabolizing archetypes: Pseudonocardia dioxanivoransCB1190 and Mycobacterium dioxanotrophicusPH-06. Based on 16S rRNA sequencing, Mycobacterium was the dominant genus. Acetylene inhibition tests suggest that dioxane degradation was mediated by monooxygenases. However, qPCR analyses targeting the tetrahydrofuran/dioxane monooxygenase gene (thmA/dxmA) (which is, to date, the only sequenced dioxane monooxygenase gene) were negative, indicating that other (as yet unknown) catabolic gene(s) were responsible. DNA sequence analyses also showed threefold to sevenfold enrichment of group 5 and group 6 soluble di-iron monooxygenase (SDIMO) genes relative to the original soil samples. Whereas biodegradation of trace levels of dioxane is a common challenge at contaminated sites, both consortia degraded dioxane at low initial concentrations (300 µg l-1 ) below detectable levels (5 µg l-1 ) in bioaugmented microcosms prepared with impacted groundwater. Overall, this work shows that dioxane-degrading bacteria (and the associated natural attenuation potential) exist even in some uncontaminated soils, and may be enriched to broaden bioaugmentation options for sites experiencing insufficient dioxane catabolic capacity.

Pulmonary Iron Homeostasis in Hepcidin Knockout Mice.

Pulmonary iron excess is deleterious and contributes to a range of chronic and acute inflammatory diseases. Optimal lung iron concentration is maintained through dynamic regulation of iron transport and storage proteins. The iron-regulatory hormone hepcidin is also expressed in the lung. In order to better understand the interactions between iron-associated molecules and the hepcidin-ferroportin axis in lung iron balance, we examined lung physiology and inflammatory responses in two murine models of systemic iron-loading, either hepcidin knock-out (Hepc KO) or liver-specific hepcidin KO mice (Hepc KOliv), which do (Hepc KOliv) or do not (Hepc KO) express lung hepcidin. We have found that increased plasma iron in Hepc KO mice is associated with increased pulmonary iron levels, consistent with increased cellular iron uptake by pulmonary epithelial cells, together with an increase at the apical membrane of the cells of the iron exporter ferroportin, consistent with increased iron export in the alveoli. Subsequently, alveolar macrophages (AM) accumulate iron in a non-toxic form and this is associated with elevated production of ferritin. The accumulation of iron in the lung macrophages of hepcidin KO mice contrasts with splenic and hepatic macrophages which contain low iron levels as we have previously reported. Hepc KOliv mice with liver-specific hepcidin deficiency demonstrated same pulmonary iron overload profile as the Hepc KO mice, suggesting that pulmonary hepcidin is not critical in maintaining local iron homeostasis. In addition, the high iron load in the lung of Hepc KO mice does not appear to enhance acute lung inflammation or injury. Lastly, we have shown that intraperitoneal LPS injection is not associated with pulmonary hepcidin induction, despite high levels of inflammatory cytokines. However, intranasal LPS injection stimulates a hepcidin response, likely derived from AM, and alters pulmonary iron content in Hepc KO mice.