Zika Virus in the Americas: An Environmental Health Perspective.

Phylogenetic studies suggest that ZIKV may have been introduced to Brazil, and therefore to the Americas, in 2014 during the World Spring Canoe Championship held in the city of Rio de Janeiro. Since then the virus has spread across Latin America, Caribbean, and North America. It seems clear that Aedes aegypti and, to a lesser extent, Aedes albopictus are the main vectors of the pathogen. ZIKV infection symptoms are similar to other flaviviruses such as a dengue infection and therefore can be easily confounded. Currently, the ZIKV maintains two life cycles. The first, and the original one is the sylvatic/enzootic cycle that occurs in Africa. The second life cycle is the suburban-urban transmission cycle that emerged through natural evolution. ZIKV has gained the ability to maintain this human-endemic cycle in urban and suburban areas. ZIKV has never been isolated from non primates, so it is not clear whether other species can act as reservoir hosts. Several reports have been made of non-vector ZIKV transmission including breast-milk feeding, blood transfusion, sexual intercourse, saliva, urine, and physical contact (sweat, tears). A major global concern with ZIKV infection is the reported increase in cases of microcephaly and Guillain-Barre Syndrome (GBS) in the Americas after the recent ZIKV outbreak. Currently, there is no available vaccine for ZIKV. Therefore, prevention of ZIKV infection must be emphasized by local public health authorities promoting collective responsibility and engagement for integrated vector management through environmental management, biological control, and as a last resource chemical control.

The Role of Research in Policy and Practice: The Zika Phenomena in Puerto Rico.

On April 19, 2017, the 37th Medical Sciences Campus Annual Research and Education Forum opened with the discussion panel The role of research in policy and practice: The Zika phenomena in Puerto Rico, with Dr. Cruz M. Nazario Delgado, Dr. Carmen D. Zorrilla and Dr. Luis A. Bonilla Soto. In this article, we critically examine the public policy process of the Zika epidemic in Puerto Rico, as presented by authors Nazario and Bonilla. We argue that policymaking and the public health responses to confront the Zika epidemic in Puerto Rico took place in a political environment where different US and local actors operated to advance their goals and vision, undermining the role of knowledge, evidence and past experiences. We propose a bottom to top preventive and community empowerment approach to control the vector. This model must be built on successful policy implementation experiences with epidemics in the Island and strengthened by evidence, international guidelines and ethical principles.

Presence of Oil Mineral Aggregates (OMAs) in Surface Sediments from Mexico's Exclusive Economic Zone, NW Gulf of Mexico.

We assessed the presence and distribution of oil mineral aggregates (OMAs) in surficial sediments of Mexican waters in the NW Gulf of Mexico, their potential sources and their correlation with polycyclic aromatic hydrocarbons (PAH). In summer of 2010, OMAs were detected in three shallow sites. In winter of 2011, OMAs were observed in ten sites, two of them in the northernmost area at > 1500 m depth. These particles were possibly advected from the north Gulf and Mississippi area following the deep-water currents of the zone. The OMAs from shallower sites may reflect local pollution sources. PAHs displayed low concentrations in both surveys (from 0.01 to 0.7 µg g-1 in summer, and from 0.01 to 0.51 µg g-1 in winter), and showed rather a local origin. The expansion of the oil and port industry in the region is accountable for most of the OMAs detected.

Ammonia-oxidising archaea living at low pH: Insights from comparative genomics.

Obligate acidophilic members of the thaumarchaeotal genus Candidatus Nitrosotalea play an important role in nitrification in acidic soils, but their evolutionary and physiological adaptations to acidic environments are still poorly understood, with only a single member of this genus (Ca. N. devanaterra) having its genome sequenced. In this study, we sequenced the genomes of two additional cultured Ca. Nitrosotalea strains, extracted an almost complete Ca. Nitrosotalea metagenome-assembled genome from an acidic fen, and performed comparative genomics of the four Ca. Nitrosotalea genomes with 19 other archaeal ammonia oxidiser genomes. Average nucleotide and amino acid identities revealed that the four Ca. Nitrosotalea strains represent separate species within the genus. The four Ca. Nitrosotalea genomes contained a core set of 103 orthologous gene families absent from all other ammonia-oxidizing archaea and, for most of these gene families, expression could be demonstrated in laboratory culture or the environment via proteomic or metatranscriptomic analyses respectively. Phylogenetic analyses indicated that four of these core gene families were acquired by the Ca. Nitrosotalea common ancestor via horizontal gene transfer from acidophilic representatives of Euryarchaeota. We hypothesize that gene exchange with these acidophiles contributed to the competitive success of the Ca. Nitrosotalea lineage in acidic environments.

Hydrothermal vent fields discovered in the southern Gulf of California clarify role of habitat in augmenting regional diversity.

Hydrothermal vent communities are distributed along mid-ocean spreading ridges as isolated patches. While distance is a key factor influencing connectivity among sites, habitat characteristics are also critical. The Pescadero Basin (PB) and Alarcón Rise (AR) vent fields, recently discovered in the southern Gulf of California, are bounded by previously known vent localities (e.g. Guaymas Basin and 21° N East Pacific Rise); yet, the newly discovered vents differ markedly in substrata and vent fluid attributes. Out of 116 macrofaunal species observed or collected, only three species are shared among all four vent fields, while 73 occur at only one locality. Foundation species at basalt-hosted sulfide chimneys on the AR differ from the functional equivalents inhabiting sediment-hosted carbonate chimneys in the PB, only 75 km away. The dominant species of symbiont-hosting tubeworms and clams, and peripheral suspension-feeding taxa, differ between the sites. Notably, the PB vents host a limited and specialized fauna in which 17 of 26 species are unknown at other regional vents and many are new species. Rare sightings and captured larvae of the 'missing' species revealed that dispersal limitation is not responsible for differences in community composition at the neighbouring vent localities. Instead, larval recruitment-limiting habitat suitability probably favours species differentially. As scenarios develop to design conservation strategies around mining of seafloor sulfide deposits, these results illustrate that models encompassing habitat characteristics are needed to predict metacommunity structure.

Acyl-Homoserine Lactone Production in Nitrifying Bacteria of the Genera Nitrosospira, Nitrobacter, and Nitrospira Identified via a Survey of Putative Quorum-Sensing Genes.

The genomes of many bacteria that participate in nitrogen cycling through the process of nitrification contain putative genes associated with acyl-homoserine lactone (AHL) quorum sensing (QS). AHL QS or bacterial cell-cell signaling is a method of bacterial communication and gene regulation and may be involved in nitrogen oxide fluxes or other important phenotypes in nitrifying bacteria. Here, we carried out a broad survey of AHL production in nitrifying bacteria in three steps. First, we analyzed the evolutionary history of AHL synthase and AHL receptor homologs in sequenced genomes and metagenomes of nitrifying bacteria to identify AHL synthase homologs in ammonia-oxidizing bacteria (AOB) of the genus Nitrosospira and nitrite-oxidizing bacteria (NOB) of the genera Nitrococcus, Nitrobacter, and Nitrospira Next, we screened cultures of both AOB and NOB with uncharacterized AHL synthase genes and AHL synthase-negative nitrifiers by a bioassay. Our results suggest that an AHL synthase gene is required for, but does not guarantee, cell density-dependent AHL production under the conditions tested. Finally, we utilized mass spectrometry to identify the AHLs produced by the AOB Nitrosospira multiformis and Nitrosospira briensis and the NOB Nitrobacter vulgaris and Nitrospira moscoviensis as N-decanoyl-l-homoserine lactone (C10-HSL), N-3-hydroxy-tetradecanoyl-l-homoserine lactone (3-OH-C14-HSL), a monounsaturated AHL (C10:1-HSL), and N-octanoyl-l-homoserine lactone (C8-HSL), respectively. Our survey expands the list of AHL-producing nitrifiers to include a representative of Nitrospira lineage II and suggests that AHL production is widespread in nitrifying bacteria.IMPORTANCE Nitrification, the aerobic oxidation of ammonia to nitrate via nitrite by nitrifying microorganisms, plays an important role in environmental nitrogen cycling from agricultural fertilization to wastewater treatment. The genomes of many nitrifying bacteria contain genes associated with bacterial cell-cell signaling or quorum sensing (QS). QS is a method of bacterial communication and gene regulation that is well studied in bacterial pathogens, but less is known about QS in environmental systems. Our previous work suggested that QS might be involved in the regulation of nitrogen oxide gas production during nitrite metabolism. This study characterized putative QS signals produced by different genera and species of nitrifiers. Our work lays the foundation for future experiments investigating communication between nitrifying bacteria, the purpose of QS in these microorganisms, and the manipulation of QS during nitrification.

Steady-State Growth under Inorganic Carbon Limitation Conditions Increases Energy Consumption for Maintenance and Enhances Nitrous Oxide Production in Nitrosomonas europaea.

UNLABELLED: Nitrosomonas europaea is a chemolithoautotrophic bacterium that oxidizes ammonia (NH3) to obtain energy for growth on carbon dioxide (CO2) and can also produce nitrous oxide (N2O), a greenhouse gas. We interrogated the growth, physiological, and transcriptome responses of N. europaea to conditions of replete (>5.2 mM) and limited inorganic carbon (IC) provided by either 1.0 mM or 0.2 mM sodium carbonate (Na2CO3) supplemented with atmospheric CO2 IC-limited cultures oxidized 25 to 58% of available NH3 to nitrite, depending on the dilution rate and Na2CO3 concentration. IC limitation resulted in a 2.3-fold increase in cellular maintenance energy requirements compared to those for NH3-limited cultures. Rates of N2O production increased 2.5- and 6.3-fold under the two IC-limited conditions, increasing the percentage of oxidized NH3-N that was transformed to N2O-N from 0.5% (replete) up to 4.4% (0.2 mM Na2CO3). Transcriptome analysis showed differential expression (P ≤ 0.05) of 488 genes (20% of inventory) between replete and IC-limited conditions, but few differences were detected between the two IC-limiting treatments. IC-limited conditions resulted in a decreased expression of ammonium/ammonia transporter and ammonia monooxygenase subunits and increased the expression of genes involved in C1 metabolism, including the genes for RuBisCO (cbb gene cluster), carbonic anhydrase, folate-linked metabolism of C1 moieties, and putative C salvage due to oxygenase activity of RuBisCO. Increased expression of nitrite reductase (gene cluster NE0924 to NE0927) correlated with increased production of N2O. Together, these data suggest that N. europaea adapts physiologically during IC-limited steady-state growth, which leads to the uncoupling of NH3 oxidation from growth and increased N2O production. IMPORTANCE: Nitrification, the aerobic oxidation of ammonia to nitrate via nitrite, is an important process in the global nitrogen cycle. This process is generally dependent on ammonia-oxidizing microorganisms and nitrite-oxidizing bacteria. Most nitrifiers are chemolithoautotrophs that fix inorganic carbon (CO2) for growth. Here, we investigate how inorganic carbon limitation modifies the physiology and transcriptome of Nitrosomonas europaea, a model ammonia-oxidizing bacterium, and report on increased production of N2O, a potent greenhouse gas. This study, along with previous work, suggests that inorganic carbon limitation may be an important factor in controlling N2O emissions from nitrification in soils and wastewater treatment.

Identifying Potential Mechanisms Enabling Acidophily in the Ammonia-Oxidizing Archaeon "Candidatus Nitrosotalea devanaterra".

Ammonia oxidation is the first and rate-limiting step in nitrification and is dominated by two distinct groups of microorganisms in soil: ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB). AOA are often more abundant than AOB and dominate activity in acid soils. The mechanism of ammonia oxidation under acidic conditions has been a long-standing paradox. While high rates of ammonia oxidation are frequently measured in acid soils, cultivated ammonia oxidizers grew only at near-neutral pH when grown in standard laboratory culture. Although a number of mechanisms have been demonstrated to enable neutrophilic AOB growth at low pH in the laboratory, these have not been demonstrated in soil, and the recent cultivation of the obligately acidophilic ammonia oxidizer "Candidatus Nitrosotalea devanaterra" provides a more parsimonious explanation for the observed high rates of activity. Analysis of the sequenced genome, transcriptional activity, and lipid content of "Ca Nitrosotalea devanaterra" reveals that previously proposed mechanisms used by AOB for growth at low pH are not essential for archaeal ammonia oxidation in acidic environments. Instead, the genome indicates that "Ca Nitrosotalea devanaterra" contains genes encoding both a predicted high-affinity substrate acquisition system and potential pH homeostasis mechanisms absent in neutrophilic AOA. Analysis of mRNA revealed that candidate genes encoding the proposed homeostasis mechanisms were all expressed during acidophilic growth, and lipid profiling by high-performance liquid chromatography-mass spectrometry (HPLC-MS) demonstrated that the membrane lipids of "Ca Nitrosotalea devanaterra" were not dominated by crenarchaeol, as found in neutrophilic AOA. This study for the first time describes a genome of an obligately acidophilic ammonia oxidizer and identifies potential mechanisms enabling this unique phenotype for future biochemical characterization.

Hydroxylamine as an intermediate in ammonia oxidation by globally abundant marine archaea.

The ammonia-oxidizing archaea have recently been recognized as a significant component of many microbial communities in the biosphere. Although the overall stoichiometry of archaeal chemoautotrophic growth via ammonia (NH(3)) oxidation to nitrite (NO(2)(-)) is superficially similar to the ammonia-oxidizing bacteria, genome sequence analyses point to a completely unique biochemistry. The only genomic signature linking the bacterial and archaeal biochemistries of NH(3) oxidation is a highly divergent homolog of the ammonia monooxygenase (AMO). Although the presumptive product of the putative AMO is hydroxylamine (NH(2)OH), the absence of genes encoding a recognizable ammonia-oxidizing bacteria-like hydroxylamine oxidoreductase complex necessitates either a novel enzyme for the oxidation of NH(2)OH or an initial oxidation product other than NH(2)OH. We now show through combined physiological and stable isotope tracer analyses that NH(2)OH is both produced and consumed during the oxidation of NH(3) to NO(2)(-) by Nitrosopumilus maritimus, that consumption is coupled to energy conversion, and that NH(2)OH is the most probable product of the archaeal AMO homolog. Thus, despite their deep phylogenetic divergence, initial oxidation of NH(3) by bacteria and archaea appears mechanistically similar. They however diverge biochemically at the point of oxidation of NH(2)OH, the archaea possibly catalyzing NH(2)OH oxidation using a novel enzyme complex.

Nitrite-Oxidizing Bacterium Nitrobacter winogradskyi Produces N-Acyl-Homoserine Lactone Autoinducers.

Nitrobacter winogradskyi is a chemolithotrophic bacterium that plays a role in the nitrogen cycle by oxidizing nitrite to nitrate. Here, we demonstrate a functional N-acyl-homoserine lactone (acyl-HSL) synthase in this bacterium. The N. winogradskyi genome contains genes encoding a putative acyl-HSL autoinducer synthase (nwi0626, nwiI) and a putative acyl-HSL autoinducer receptor (nwi0627, nwiR) with amino acid sequences 38 to 78% identical to those in Rhodopseudomonas palustris and other Rhizobiales. Expression of nwiI and nwiR correlated with acyl-HSL production during culture. N. winogradskyi produces two distinct acyl-HSLs, N-decanoyl-l-homoserine lactone (C10-HSL) and a monounsaturated acyl-HSL (C10:1-HSL), in a cell-density- and growth phase-dependent manner, during batch and chemostat culture. The acyl-HSLs were detected by bioassay and identified by ultraperformance liquid chromatography with information-dependent acquisition mass spectrometry (UPLC-IDA-MS). The C=C bond in C10:1-HSL was confirmed by conversion into bromohydrin and detection by UPLC-IDA-MS.

Interactions of Nitrosomonas europaea and Nitrobacter winogradskyi grown in co-culture.

Nitrosomonas europaea and Nitrobacter winogradskyi were grown singly and in co-culture in chemostats to probe for physiological differences between the two growth conditions. Co-culture growth medium containing 60 mM NH4 (+) resulted in a cell density (0.20-0.29 OD600) greater than the sum of the densities in single chemostat cultures, i.e., 0.09-0.14 OD600 for N. europaea with 60 mM NH4 (+)and 0.04-0.06 OD600 for N. winogradskyi with 60 mM NO2 (-). The NO2 (-)- and NH4 (+)-dependent O2 uptake rates, qRT-PCR, and microscopic observations indicated that in co-culture, N. europaea contributed ~0.20 OD600 (~80 %) and N. winogradskyi ~0.05 OD600 (~20 %). In co-culture, the transcriptomes showed that the mRNA levels of 773 genes in N. europaea (30.2 % of the genes) and of 372 genes in N. winogradskyi (11.8 % of the genes) changed significantly. Total cell growth and the analysis of the transcriptome revealed that in co-culture, N. europaea benefits more than N. winogradskyi.

Characterization of a nitrite reductase involved in nitrifier denitrification.

Nitrifier denitrification is the conversion of nitrite to nitrous oxide by ammonia-oxidizing organisms. This process, which is distinct from denitrification, is active under aerobic conditions in the model nitrifier Nitrosomonas europaea. The central enzyme of the nitrifier dentrification pathway is a copper nitrite reductase (CuNIR). To understand how a CuNIR, typically inactivated by oxygen, functions in this pathway, the enzyme isolated directly from N. europaea (NeNIR) was biochemically and structurally characterized. NeNIR reduces nitrite at a similar rate to other CuNIRs but appears to be oxygen tolerant. Crystal structures of oxidized and reduced NeNIR reveal a substrate channel to the active site that is much more restricted than channels in typical CuNIRs. In addition, there is a second fully hydrated channel leading to the active site that likely acts a water exit pathway. The structure is minimally affected by changes in pH. Taken together, these findings provide insight into the molecular basis for NeNIR oxygen tolerance.

A severe acute respiratory coronavirus virus 2 (SARS-CoV-2) nosocomial cluster with inter-facility spread: Lessons learned.

BACKGROUND: Despite infection control guidance, sporadic nosocomial coronavirus disease 2019 (COVID-19) outbreaks occur. We describe a complex severe acute respiratory coronavirus virus 2 (SARS-CoV-2) cluster with interfacility spread during the SARS-CoV-2 δ (delta) pandemic surge in the Midwest. SETTING: This study was conducted in (1) a hematology-oncology ward in a regional academic medical center and (2) a geographically distant acute rehabilitation hospital. METHODS: We conducted contact tracing for each COVID-19 case to identify healthcare exposures within 14 days prior to diagnosis. Liberal testing was performed for asymptomatic carriage for patients and staff. Whole-genome sequencing was conducted for all available clinical isolates from patients and healthcare workers (HCWs) to identify transmission clusters. RESULTS: In the immunosuppressed ward, 19 cases (4 patients, 15 HCWs) shared a genetically related SARS-CoV-2 isolate. Of these 4 patients, 3 died in the hospital or within 1 week of discharge. The suspected index case was a patient with new dyspnea, diagnosed during preprocedure screening. In the rehabilitation hospital, 20 cases (5 patients and 15 HCWs) positive for COVID-19, of whom 2 patients and 3 HCWs had an isolate genetically related to the above cluster. The suspected index case was a patient from the immune suppressed ward whose positive status was not detected at admission to the rehabilitation facility. Our response to this cluster included the following interventions in both settings: restricting visitors, restricting learners, restricting overflow admissions, enforcing strict compliance with escalated PPE, access to on-site free and frequent testing for staff, and testing all patients prior to hospital discharge and transfer to other facilities. CONCLUSIONS: Stringent infection control measures can prevent nosocomial COVID-19 transmission in healthcare facilities with high-risk patients during pandemic surges. These interventions were successful in ending these outbreaks.

Exploring Deep Learning for Estimating the Isoeffective Dose of FLASH Irradiation From Mouse Intestinal Histological Images.

PURPOSE: Ultrahigh-dose-rate (FLASH) irradiation has been reported to reduce normal tissue damage compared with conventional dose rate (CONV) irradiation without compromising tumor control. This proof-of-concept study aims to develop a deep learning (DL) approach to quantify the FLASH isoeffective dose (dose of CONV that would be required to produce the same effect as the given physical FLASH dose) with postirradiation mouse intestinal histology images. METHODS AND MATERIALS: Eighty-four healthy C57BL/6J female mice underwent 16 MeV electron CONV (0.12 Gy/s; n = 41) or FLASH (200 Gy/s; n = 43) single fraction whole abdominal irradiation. Physical dose ranged from 12 to 16 Gy for FLASH and 11 to 15 Gy for CONV in 1 Gy increments. Four days after irradiation, 9 jejunum cross-sections from each mouse were hematoxylin and eosin stained and digitized for histological analysis. CONV data set was randomly split into training (n = 33) and testing (n = 8) data sets. ResNet101-based DL models were retrained using the CONV training data set to estimate the dose based on histological features. The classical manual crypt counting (CC) approach was implemented for model comparison. Cross-section-wise mean squared error was computed to evaluate the dose estimation accuracy of both approaches. The validated DL model was applied to the FLASH data set to map the physical FLASH dose into the isoeffective dose. RESULTS: The DL model achieved a cross-section-wise mean squared error of 0.20 Gy2 on the CONV testing data set compared with 0.40 Gy2 of the CC approach. Isoeffective doses estimated by the DL model for FLASH doses of 12, 13, 14, 15, and 16 Gy were 12.19 ± 0.46, 12.54 ± 0.37, 12.69 ± 0.26, 12.84 ± 0.26, and 13.03 ± 0.28 Gy, respectively. CONCLUSIONS: Our proposed DL model achieved accurate CONV dose estimation. The DL model results indicate that in the physical dose range of 13 to 16 Gy, the biologic dose response of small intestinal tissue to FLASH irradiation is represented by a lower isoeffective dose compared with the physical dose. Our DL approach can be a tool for studying isoeffective doses of other radiation dose modifying interventions.

Tracing the origin of SARS-CoV-2 omicron-like spike sequences detected in an urban sewershed: a targeted, longitudinal surveillance study of a cryptic wastewater lineage.

BACKGROUND: The origin of novel SARS-CoV-2 spike sequences found in wastewater, without corresponding detection in clinical specimens, remains unclear. We sought to determine the origin of one such cryptic wastewater lineage by tracking and characterising its persistence and genomic evolution over time. METHODS: We first detected a cryptic lineage, WI-CL-001, in municipal wastewater in Wisconsin, USA, in January, 2022. To determine the source of WI-CL-001, we systematically sampled wastewater from targeted sub-sewershed lines and maintenance holes using compositing autosamplers. Viral concentrations in wastewater samples over time were measured by RT digital PCR. In addition to using metagenomic 12s rRNA sequencing to determine the virus's host species, we also sequenced SARS-CoV-2 spike receptor binding domains, and, where possible, whole viral genomes to identify and characterise the evolution of this lineage. FINDINGS: We traced WI-CL-001 to its source at a single commercial building. There we detected the cryptic lineage at concentrations as high as 2·7 × 109 genome copies per L. The majority of 12s rRNA sequences detected in wastewater leaving the identified source building were human. Additionally, we generated over 100 viral receptor binding domain and whole-genome sequences from wastewater samples containing the cryptic lineage collected over the 13 consecutive months this virus was detectable (January, 2022, to January, 2023). These sequences contained a combination of fixed nucleotide substitutions characteristic of Pango lineage B.1.234, which circulated in humans in Wisconsin at low levels from October, 2020, to February, 2021. Despite this, mutations in the spike gene and elsewhere resembled those subsequently found in omicron variants. INTERPRETATION: We propose that prolonged detection of WI-CL-001 in wastewater indicates persistent shedding of SARS-CoV-2 from a single human initially infected by an ancestral B.1.234 virus. The accumulation of convergent omicron-like mutations in WI-CL-001's ancestral B.1.234 genome probably reflects persistent infection and extensive within-host evolution. People who shed cryptic lineages could be an important source of highly divergent viruses that sporadically emerge and spread. FUNDING: The Rockefeller Foundation, Wisconsin Department of Health Services, Centers for Disease Control and Prevention, National Institute on Drug Abuse, and the Center for Research on Influenza Pathogenesis and Transmission.

FLASH-RT does not affect chromosome translocations and junction structures beyond that of CONV-RT dose-rates.

BACKGROUND AND PURPOSE: The impact of radiotherapy (RT) at ultra high vs conventional dose rate (FLASH vs CONV) on the generation and repair of DNA double strand breaks (DSBs) is an important question that remains to be investigated. Here, we tested the hypothesis as to whether FLASH-RT generates decreased chromosomal translocations compared to CONV-RT. MATERIALS AND METHODS: We used two FLASH validated electron beams and high-throughput rejoin and genome-wide translocation sequencing (HTGTS-JoinT-seq), employing S. aureus and S. pyogenes Cas9 "bait" DNA double strand breaks (DSBs) in HEK239T cells, to measure differences in bait-proximal repair and their genome-wide translocations to "prey" DSBs generated after various irradiation doses, dose rates and oxygen tensions (normoxic, 21% O2; physiological, 4% O2; hypoxic, 2% and 0.5% O2). Electron irradiation was delivered using a FLASH capable Varian Trilogy and the eRT6/Oriatron at CONV (0.08-0.13 Gy/s) and FLASH (1x102-5x106 Gy/s) dose rates. Related experiments using clonogenic survival and γH2AX foci in the 293T and the U87 glioblastoma lines were also performed to discern FLASH-RT vs CONV-RT DSB effects. RESULTS: Normoxic and physioxic irradiation of HEK293T cells increased translocations at the cost of decreasing bait-proximal repair but were indistinguishable between CONV-RT and FLASH-RT. Although no apparent increase in chromosome translocations was observed with hypoxia-induced apoptosis, the combined decrease in oxygen tension with IR dose-rate modulation did not reveal significant differences in the level of translocations nor in their junction structures. Furthermore, RT dose rate modality on U87 cells did not change γH2AX foci numbers at 1- and 24-hours post-irradiation nor did this affect 293T clonogenic survival. CONCLUSION: Irrespective of oxygen tension, FLASH-RT produces translocations and junction structures at levels and proportions that are indistinguishable from CONV-RT.

FLASH-RT does not affect chromosome translocations and junction structures beyond that of CONV-RT dose-rates.

The molecular and cellular mechanisms driving the enhanced therapeutic ratio of ultra-high dose-rate radiotherapy (FLASH-RT) over slower conventional (CONV-RT) radiotherapy dose-rate remain to be elucidated. However, attenuated DNA damage and transient oxygen depletion are among several proposed models. Here, we tested whether FLASH-RT under physioxic (4% O 2 ) and hypoxic conditions (≤2% O 2 ) reduces genome-wide translocations relative to CONV-RT and whether any differences identified revert under normoxic (21% O 2 ) conditions. We employed high-throughput rejoin and genome-wide translocation sequencing ( HTGTS-JoinT-seq ), using S. aureus and S. pyogenes Cas9 "bait" DNA double strand breaks (DSBs), to measure differences in bait-proximal repair and their genome-wide translocations to "prey" DSBs generated by electron beam CONV-RT (0.08-0.13Gy/s) and FLASH-RT (1×10 2 -5×10 6 Gy/s), under varying ionizing radiation (IR) doses and oxygen tensions. Normoxic and physioxic irradiation of HEK293T cells increased translocations at the cost of decreasing bait-proximal repair but were indistinguishable between CONV-RT and FLASH-RT. Although no apparent increase in chromosome translocations was observed with hypoxia-induced apoptosis, the combined decrease in oxygen tension with IR dose-rate modulation did not reveal significant differences in the level of translocations nor in their junction structures. Thus, Irrespective of oxygen tension, FLASH-RT produces translocations and junction structures at levels and proportions that are indistinguishable from CONV-RT.

Global analysis of the Nitrosomonas europaea iron starvation stimulon.

The importance of iron to the metabolism of the ammonia-oxidizing bacterium Nitrosomonas europaea is well known. However, the mechanisms by which N. europaea acquires iron under iron limitation are less well known. To obtain insight into these mechanisms, transcriptional profiling of N. europaea was performed during growth under different iron availabilities. Of 2,355 N. europaea genes on DNA microarrays, transcripts for 247 genes were identified as differentially expressed when cells were grown under iron limitation compared to cells grown under iron-replete conditions. Genes with higher transcript levels in response to iron limitation included those with confirmed or assigned roles in iron acquisition. Genes with lower transcript levels included those encoding iron-containing proteins. Our analysis identified several potentially novel iron acquisition systems in N. europaea and provided support for the primary involvement of a TonB-dependent heme receptor gene in N. europaea iron homeostasis. We demonstrated that hemoglobin can act as an iron source under iron-depleted conditions for N. europaea. In addition, we identified a hypothetical protein carrying a lipocalin-like domain that may have the ability to chelate iron for growth in iron-limited media.

Role of a Fur homolog in iron metabolism in Nitrosomonas europaea.

BACKGROUND: In response to environmental iron concentrations, many bacteria coordinately regulate transcription of genes involved in iron acquisition via the ferric uptake regulation (Fur) system. The genome of Nitrosomonas europaea, an ammonia-oxidizing bacterium, carries three genes (NE0616, NE0730 and NE1722) encoding proteins belonging to Fur family. RESULTS: Of the three N. europaea fur homologs, only the Fur homolog encoded by gene NE0616 complemented the Escherichia coli H1780 fur mutant. A N. europaea fur:kanP mutant strain was created by insertion of kanamycin-resistance cassette in the promoter region of NE0616 fur homolog. The total cellular iron contents of the fur:kanP mutant strain increased by 1.5-fold compared to wild type when grown in Fe-replete media. Relative to the wild type, the fur:kanP mutant exhibited increased sensitivity to iron at or above 500 µM concentrations. Unlike the wild type, the fur:kanP mutant was capable of utilizing iron-bound ferrioxamine without any lag phase and showed over expression of several outer membrane TonB-dependent receptor proteins irrespective of Fe availability. CONCLUSIONS: Our studies have clearly indicated a role in Fe regulation by the Fur protein encoded by N. europaea NE0616 gene. Additional studies are required to fully delineate role of this fur homolog.

Role of Nitrosomonas europaea NitABC iron transporter in the uptake of Fe3+-siderophore complexes.

Nitrosomonas europaea has a single three-gene operon (nitABC) encoding an iron ABC transporter system (NitABC). Phylogenetic analysis clustered the subunit NitB with Fe(3+)-ABC transporter permease components from other organisms. The N. europaea strain deficient in nitB (nitB::kan) grew well in either Fe-replete or Fe-limited media and in Fe-limited medium containing the catecholate-type siderophore, enterobactin or the citrate-based dihydroxamate-type siderophore, aerobactin. However, the nitB::kan mutant strain was unable to grow in Fe-limited media containing either the hydroxamate-type siderophores, ferrioxamine and ferrichrome or the mixed-chelating type siderophore, pyoverdine. Exposure of N. europaea cells to a ferrichrome analog coupled to the fluorescent moiety naphthalic diimide (Fhu-NI) led to increase in fluorescence in the wild type but not in nitB::kan mutant cells. Spheroplasts prepared from N. europaea wild type exposed to Fhu-NI analog retained the fluorescence, while spheroplasts of the nitB::kan mutant were not fluorescent. NitABC transports intact Fe(3+)-ferrichrome complex into the cytoplasm and is an atypical ABC type iron transporter for Fe(3+) bound to ferrioxamine, ferrichrome or pyoverdine siderophores into the cytoplasm. The mechanisms to transport iron in either the Fe(3+) or Fe(2+) forms or Fe(3+) associated with enterobactin or aerobactin siderophores into the cell across the cytoplasmic membrane are as yet undetermined.