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.

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.

Evaluating the Reproducibility of Mouse Anatomy under Rotation in a Custom Immobilization Device for Conformal FLASH Radiotherapy.

The observation of an enhanced therapeutic index for FLASH radiotherapy in mice has created interest in practical laboratory-based FLASH irradiators. To date, systems capable of 3D conformal FLASH irradiation in mice have been lacking. We are developing such a system, incorporating a high-current linear accelerator to produce a collimated X-ray beam in a stationary beamline design, rotating the mouse about a longitudinal axis to achieve conformal irradiation from multiple beam directions. The purpose of this work was to evaluate the reproducibility of mouse anatomy under rotation at speeds compatible with conformal FLASH delivery. Three short-hair mice and two hairless mice were immobilized under anesthesia in body weight-specific contoured plastic molds, and subjected to three rotational (up to 3 revolutions/s) and two non-rotational movement interventions. MicroCT images were acquired before and after each intervention. The displacements of 11 anatomic landmarks were measured on the image pairs. The displacement of the anatomical landmarks with any of the interventions was 0.5 mm or less for 92.4% of measurements, with a single measurement out of 275 (11 landmarks × 5 interventions × 5 mice) reaching 1 mm. There was no significant difference in the displacements associated with rotation compared to those associated with moving the immobilized mouse in and out of a scanner or with leaving the mouse in place for 5 min with no motion. There were no significant differences in displacements between mice with or without hair, although the analysis is limited by small numbers, or between different anatomic landmarks. These results show that anatomic reproducibility under rotation speed corresponding to FLASH irradiation times appears to be compatible with conformal/stereotactic irradiation in mice.

FLASH Irradiation Results in Reduced Severe Skin Toxicity Compared to Conventional-Dose-Rate Irradiation.

Radiation therapy, along with surgery and chemotherapy, is one of the main treatments for cancer. While radiotherapy is highly effective in the treatment of localized tumors, its main limitation is its toxicity to normal tissue. Previous preclinical studies have reported that ultra-high dose-rate (FLASH) irradiation results in reduced toxicity to normal tissues while controlling tumor growth to a similar extent relative to conventional-dose-rate (CONV) irradiation. To our knowledge this is the first report of a dose-response study in mice comparing the effect of FLASH irradiation vs. CONV irradiation on skin toxicity. We found that FLASH irradiation results in both a lower incidence and lower severity of skin ulceration than CONV irradiation 8 weeks after single-fraction hemithoracic irradiation at high doses (30 and 40 Gy). Survival was also higher after FLASH hemithoracic irradiation (median survival >180 days at doses of 30 and 40 Gy) compared to CONV irradiation (median survival 100 and 52 days at 30 and 40 Gy, respectively). No ulceration was observed at doses 20 Gy or below in either FLASH or CONV. These results suggest a shifting of the dose-response curve for radiation-induced skin ulceration to the right for FLASH, compared to CONV irradiation, suggesting the potential for an enhanced therapeutic index for radiation therapy of cancer.

A Near-Infrared Phosphorescent Nanoprobe Enables Quantitative, Longitudinal Imaging of Tumor Hypoxia Dynamics during Radiotherapy.

Hypoxia plays a key role in tumor resistance to radiotherapy. It is important to study hypoxia dynamics during radiotherapy to improve treatment planning and prognosis. Here, we describe a luminescent nanoprobe, composed of a fluorescent semiconducting polymer and palladium complex, for quantitative longitudinal imaging of tumor hypoxia dynamics during radiotherapy. The nanoprobe was designed to provide high sensitivity and reversible response for the subtle change in hypoxia over a narrow range (0-30 mmHg O2), which spans the oxygen range where tumors have limited radiosensitivity. Following intravenous administration, the nanoprobe efficiently accumulated in and distributed across the tumor, including the hypoxic region. The ratio between emissions at 700 and 800 nm provided quantitative mapping of hypoxia across the entire tumor. The nanoprobe was used to image tumor hypoxia dynamics over 7 days during fractionated radiotherapy and revealed that high fractional dose (10 Gy) was more effective in improving tumor reoxygenation than low dose (2 Gy), and the effect tended to persist longer in smaller or more radiosensitive tumors. Our results also indicated the importance of the reoxygenation efficiency of the first fraction in the prediction of the radiation treatment outcome. In summary, this work has established a new nanoprobe for highly sensitive, quantitative, and longitudinal imaging of tumor hypoxia dynamics following radiotherapy, and demonstrated its value for assessing the efficacy of radiotherapy and radiation treatment planning. SIGNIFICANCE: This study presents a novel nanoagent for the visualization and quantification of tumor hypoxia.

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.

Macrophages Promote Circulating Tumor Cell-Mediated Local Recurrence following Radiotherapy in Immunosuppressed Patients.

Although radiotherapy (RT) decreases the incidence of locoregional recurrence in breast cancer, patients with triple-negative breast cancer (TNBC) have increased risk of local recurrence following breast-conserving therapy. The relationship between RT and local recurrence is unknown. Here, we tested the hypothesis that recurrence in some instances is due to the attraction of circulating tumor cells to irradiated tissues. To evaluate the effect of absolute lymphocyte count on local recurrence after RT in patients with TNBC, we analyzed radiation effects on tumor and immune cell recruitment to tissues in an orthotopic breast cancer model. Recurrent patients exhibited a prolonged low absolute lymphocyte count when compared with nonrecurrent patients following RT. Recruitment of tumor cells to irradiated normal tissues was enhanced in the absence of CD8+ T cells. Macrophages (CD11b+F480+) preceded tumor cell infiltration and were recruited to tissues following RT. Tumor cell recruitment was mitigated by inhibiting macrophage infiltration using maraviroc, an FDA-approved CCR5 receptor antagonist. Our work poses the intriguing possibility that excessive macrophage infiltration in the absence of lymphocytes promotes local recurrence after RT. This combination thus defines a high-risk group of patients with TNBC.Significance: This study establishes the importance of macrophages in driving tumor cell recruitment to sites of local radiation therapy and suggests that this mechanism contributes to local recurrence in women with TNBC that are also immunosuppressed.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/15/4241/F1.large.jpg Cancer Res; 78(15); 4241-52. ©2018 AACR.

Interannual variability of soft-bottom macrobenthic communities of the NW Gulf of Mexico in relationship to the Deepwater Horizon oil spill.

A 3-year research program was undertaken to assess potential environmental disturbance caused by the Deepwater Horizon oil spill to the soft-bottom macrobenthic communities within Mexican waters of the northwestern Gulf of Mexico. Community properties and temporal/spatial variability were analyzed besides toxicant parameters such as hydrocarbons and trace-metals. Overall infaunal density increased, taxa proportion changed, and small-size opportunistic organisms prevailed throughout the study. Annual abundance-biomass comparison (ABC) curves revealed progressive stress scenarios from moderate to severe. Concentrations of vanadium, nickel, cobalt, PAHs and AHs increased gradually over time. However, low correlations between benthic density and biogeochemical variables were determined. Initially, sedimentary properties were the main drivers of benthic community structure; subsequently, nickel, vanadium and PAHs, indicative of anthropogenic effect, were highlighted. Interannual variability in the macroinfauna was attributed to the synergy of several environmental factors. Undoubtedly, compounds derived from fossil fuels had a significant disturbance role, but their source remains uncertain.

Cycloheximide prevents the de novo polypeptide synthesis required to recover from acetylene inhibition in Nitrosopumilus maritimus.

Developing methods to differentiate the relative contributions of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) to ammonia (NH3) oxidation has been challenging due to the lack of compounds that selectively inhibit AOA. In this study, we investigated the effects of specific bacteria- and eukaryote-selective protein synthesis inhibitors on the recovery of acetylene (C2H2)-inactivated NH3 oxidation in the marine AOA Nitrosopumilus maritimus and compared the results with recovery of the AOB Nitrosomonas europaea. C2 H2 irreversibly inhibited N. maritimus NH3 oxidation in a similar manner to what was observed previously with N. europaea. However, cycloheximide (CHX), a widely used eukaryotic protein synthesis inhibitor, but not bacteria-specific protein synthesis inhibitors (kanamycin and gentamycin), inhibited the recovery of NH3-oxidizing activity in N. maritimus. CHX prevented the incorporation of (14)CO2 -labeling into cellular proteins, providing further evidence that CHX acts as a protein synthesis inhibitor in N. maritimus. If the effect of CHX on protein synthesis can be confirmed among other isolates of AOA, the combination of C2H2 inactivation followed by recovery of NH3 oxidation either in the presence of bacteria-selective protein synthesis inhibitors or CHX might be used to estimate the relative contributions of AOB and AOA to NH3 oxidation in natural environments.

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.

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.

Dissecting iron uptake and homeostasis in Nitrosomonas europaea.

The chemolithoautotroph Nitrosomonas europaea oxidizes about 25 mol of NH(3) for each mole of CO(2) that is converted to biomass using an array of heme and nonheme Fe-containing proteins. Hence mechanisms of efficient iron (Fe) uptake and homeostasis are particularly important for this Betaproteobacterium. Among nitrifiers, N.europaea has been the most studied to date. Characteristics that make N.europaea a suitable model to study Fe uptake and homeostasis are as follows: (a) its sequenced genome, (b) its capability to grow relatively well in 0.2 µM Fe in the absence of heterologous siderophores, and (c) its amenability to mutagenesis. In this chapter, we describe the methodology we use in our laboratory to dissect Fe uptake and homeostasis in the ammonia oxidizer N. europaea.

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.

Iron nutrition and physiological responses to iron stress in Nitrosomonas europaea.

Nitrosomonas europaea, as an ammonia-oxidizing bacterium, has a high Fe requirement and has 90 genes dedicated to Fe acquisition. Under Fe-limiting conditions (0.2 microM Fe), N. europaea was able to assimilate up to 70% of the available Fe in the medium even though it is unable to produce siderophores. Addition of exogenous siderophores to Fe-limited medium increased growth (final cell mass). Fe-limited cells had lower heme and cellular Fe contents, reduced membrane layers, and lower NH3- and NH2OH-dependent O2 consumption activities than Fe-replete cells. Fe acquisition-related proteins, such as a number of TonB-dependent Fe-siderophore receptors for ferrichrome and enterobactin and diffusion protein OmpC, were expressed to higher levels under Fe limitation, providing biochemical evidence for adaptation of N. europaea to Fe-limited conditions.