Modelled-Microgravity Reduces Virulence Factor Production in Staphylococcus aureus through Downregulation of agr-Dependent Quorum Sensing.

Bacterial contamination during space missions is problematic for human health and damages filters and other vital support systems. Staphylococcus aureus is both a human commensal and an opportunistic pathogen that colonizes human tissues and causes acute and chronic infections. Virulence and colonization factors are positively and negatively regulated, respectively, by bacterial cell-to-cell communication (quorum sensing) via the agr (accessory gene regulator) system. When cultured under low-shear modelled microgravity conditions (LSMMG), S. aureus has been reported to maintain a colonization rather than a pathogenic phenotype. Here, we show that the modulation of agr expression via reduced production of autoinducing peptide (AIP) signal molecules was responsible for this behavior. In an LSMMG environment, the S. aureus strains JE2 (methicillin-resistant) and SH1000 (methicillin-sensitive) both exhibited reduced cytotoxicity towards the human leukemia monocytic cell line (THP-1) and increased fibronectin binding. Using S. aureus agrP3::lux reporter gene fusions and mass spectrometry to quantify the AIP concentrations, the activation of agr, which depends on the binding of AIP to the transcriptional regulator AgrC, was delayed in the strains with an intact autoinducible agr system. This was because AIP production was reduced under these growth conditions compared with the ground controls. Under LSMMG, S. aureus agrP3::lux reporter strains that cannot produce endogenous AIPs still responded to exogenous AIPs. Provision of exogenous AIPs to S. aureus USA300 during microgravity culture restored the cytotoxicity of culture supernatants for the THP-1 cells. These data suggest that microgravity does not affect AgrC-AIP interactions but more likely the generation of AIPs.

Erratum: "Human lung-on-chips: Advanced systems for respiratory virus models and assessment of immune response" [Biomicrofluidics 15, 021501 (2021)].

[This corrects the article DOI: 10.1063/5.0038924.].

Human lung-on-chips: Advanced systems for respiratory virus models and assessment of immune response.

Respiratory viral infections are leading causes of death worldwide. A number of human respiratory viruses circulate in all age groups and adapt to person-to-person transmission. It is vital to understand how these viruses infect the host and how the host responds to prevent infection and onset of disease. Although animal models have been widely used to study disease states, incisive arguments related to poor prediction of patient responses have led to the development of microfluidic organ-on-chip models, which aim to recapitulate organ-level physiology. Over the past decade, human lung chips have been shown to mimic many aspects of the lung function and its complex microenvironment. In this review, we address immunological responses to viral infections and elaborate on human lung airway and alveolus chips reported to model respiratory viral infections and therapeutic interventions. Advances in the field will expedite the development of therapeutics and vaccines for human welfare.

Immunity in Space: Prokaryote Adaptations and Immune Response in Microgravity.

Immune dysfunction has long been reported by medical professionals regarding astronauts suffering from opportunistic infections both during their time in space and a short period afterwards once back on Earth. Various species of prokaryotes onboard these space missions or cultured in a microgravity analogue exhibit increased virulence, enhanced formation of biofilms, and in some cases develop specific resistance for specific antibiotics. This poses a substantial health hazard to the astronauts confined in constant proximity to any present bacterial pathogens on long space missions with a finite number of resources including antibiotics. Furthermore, some bacteria cultured in microgravity develop phenotypes not seen in Earth gravity conditions, providing novel insights into bacterial evolution and avenues for research. Immune dysfunction caused by exposure to microgravity may increase the chance of bacterial infection. Immune cell stimulation, toll-like receptors and pathogen-associated molecular patterns can all be altered in microgravity and affect immunological crosstalk and response. Production of interleukins and other cytokines can also be altered leading to immune dysfunction when responding to bacterial infection. Stem cell differentiation and immune cell activation and proliferation can also be impaired and altered by the microgravity environment once more adding to immune dysfunction in microgravity. This review elaborates on and contextualises these findings relating to how bacteria can adapt to microgravity and how the immune system subsequently responds to infection.

A novel canine kidney cell line model for the evaluation of neoplastic development: karyotype evolution associated with spontaneous immortalization and tumorigenicity.

The molecular mechanisms underlying spontaneous neoplastic transformation in cultured mammalian cells remain poorly understood, confounding recognition of parallels with the biology of naturally occurring cancer. The broad use of tumorigenic canine cell lines as research tools, coupled with the accumulation of cytogenomic data from naturally occurring canine cancers, makes the domestic dog an ideal system in which to investigate these relationships. We developed a canine kidney cell line, CKB1-3T7, which allows prospective examination of the onset of spontaneous immortalization and tumorigenicity. We documented the accumulation of cytogenomic aberrations in CKB1-3T7 over 24 months in continuous culture. The majority of aberrations emerged in parallel with key phenotypic changes in cell morphology, growth kinetics, and tumor incidence and latency. Focal deletion of CDKN2A/B emerged first, preceding the onset and progression of tumorigenic potential, and progressed to a homozygous deletion across the cell population during extended culture. Interestingly, CKB1-3T7 demonstrated a tumorigenic phenotype in vivo prior to exhibiting loss of contact inhibition in vitro. We also performed the first genome-wide characterization of the canine tumorigenic cell line MDCK, which also exhibited CDKN2A/B deletion. MDCK and CKB1-3T7 cells shared several additional aberrations that we have reported previously as being highly recurrent in spontaneous canine cancers, many of which, as with CDKN2A/B deletion, are evolutionarily conserved in their human counterparts. The conservation of these molecular events across multiple species, in vitro and in vivo, despite their contrasting karyotypic architecture, is a powerful indicator of a common mechanism underlying emerging neoplastic activity. Through integrated cytogenomic and phenotypic characterization of serial passages of CKB1-3T7 from initiation to development of a tumorigenic phenotype, we present a robust and readily accessible model (to be made available through the American Type Culture Collection) of spontaneous neoplastic transformation that overcomes many of the limitations of earlier studies.

Training pathologists in mouse pathology.

Expertise in the pathology of mice has expanded from traditional regulatory and drug safety screening (toxicologic pathology) primarily performed by veterinary pathologists to the highly specialized area of mouse research pathobiology performed by veterinary and medical pathologists encompassing phenotyping of mutant mice and analysis of research experiments exploiting inbred mouse strains and genetically engineered lines. With increasing use of genetically modified mice in research, mouse pathobiology and, by extension, expert mouse research-oriented pathologists have become integral to the success of basic and translational biomedical research. Training for today's research-oriented mouse pathologist must go beyond knowledge of anatomic features of mice and strain-specific background diseases to the specialized genetic nomenclature, husbandry, and genetics, including the methodology of genetic engineering and complex trait analysis. While training can be accomplished through apprenticeships in formal programs, these are often heavily service related and do not provide the necessary comprehensive training. Specialty courses and short-term mentoring with expert specialists are opportunities that, when combined with active practice and publication, will lead to acquisition of the skills required for cutting-edge mouse-based experimental science.

Quality of teaching in chemical pathology: ability of interns to order and interpret laboratory tests.

BACKGROUND AND OBJECTIVE: There has been a steady decline in the overt teaching of many basic and pathology sciences in the medical curriculum worldwide. As interns are the doctors most likely to request and act on tests, an assessment of their confidence in dealing with laboratory investigations was undertaken. METHODS: Interns at two hospitals in Cape Town, South Africa, were asked to complete a structured questionnaire designed to assess their confidence in ordering and interpreting a number of tests. The questionnaire also probed their desire for further teaching and the preferred delivery vehicle for such teaching. RESULTS AND CONCLUSIONS: 61 out of 117 questionnaires were returned. Interns were confident in the use of common tests, but 23% were not confident in interpreting a test that they were confident in ordering. All respondents felt they would benefit from teaching in at least one area and lectures were the preferred method, although the majority felt it very likely that they would complete an online tutorial if available. The results suggest that institutions need to devise strategies to fulfil the learning needs of new graduates in the area of chemical pathology and clinical biochemistry.

The ecological condition of Veracruz, Mexico estuaries.

During June and July, 2002, forty-seven stations were sampled within estuaries along the gulf coast of the state of Veracruz, MX, using a probabilistic survey design and a common set of response indicators. The objective of the study was to collect information to assess the condition of estuarine waters within the state of Veracruz, and to provide data that would strengthen future assessments of Gulf of Mexico estuaries. Samples for water quality, sediment contaminants, sediment toxicity, and benthic populations were collected in a manner consistent with EPA's National Coastal Assessment (NCA). Data were evaluated by comparing indicator measurements to tropical waters threshold values cited in US EPA's National Coastal Condition Report II, 2004, for tropical waters. In Veracruz, 75% of the area sampled rated poor for water quality, attributed primarily to high concentrations reported for chlorophyll a, and dissolved nutrients. One percent of the area exhibited poor sediment quality, based on PAH and metals concentrations. Compared to US estuaries of the Gulf of Mexico, water quality observed in Veracruz estuaries was more affected by nutrient over-enrichment. The probabilitistic nature of the survey design allowed for the comparison of the condition of Veracruz and the US GOM estuaries.

Prediction of preterm delivery in twin pregnancy: a prospective, observational study of cervical length and fetal fibronectin testing.

OBJECTIVE: To evaluate prospectively cervical length measurements and fetal fibronectin detection as predictors of spontaneous preterm delivery in an unselected population of twin pregnancies. METHODS: Transvaginal ultrasound assessments of cervical length were performed serially at 18, 24, 28 and 32 weeks' gestation. Receiver-operating characteristic curves were generated at each time point to determine the optimal cut-off for cervical length and rate of change in cervical length in the prediction of preterm delivery (< 35 weeks). A bedside assay for the detection of fetal fibronectin in the maternal vaginal secretions was performed prior to cervical length measurements from 24 weeks. Likelihood ratios (LRs) were used to assess the performance of each test. RESULTS: The spontaneous preterm delivery rate was 16.5% in 91 studied twin pregnancies. A cervical length /= 2.5 mm per week between 18 and 28 weeks' gestation also predicted preterm delivery (LR+ 10.8, sensitivity 16.7%). There was no relationship between the detection of fetal fibronectin and preterm delivery. CONCLUSIONS: This study confirms the value of transvaginal ultrasound assessment of cervical length as a predictor of preterm delivery in twin pregnancies. However, the poor sensitivity of this test makes it unsuitable as a single predictor of preterm delivery. Fetal fibronectin testing does not identify twin pregnancies destined to deliver prematurely.

Toxicological studies in tropical ecosystems: an ecotoxicological risk assessment of pesticide runoff in South Florida estuarine ecosystems.

A multiyear study in the C-111 canal system and associated sites in Florida Bay was undertaken to determine the potential pesticide risk that exists in South Florida. After the examination of extensive pesticide concentration data in surface water, tissues, and semipermeable membrane devices (SPMDs), canal contamination seems to be derived from the extensive agricultural production that drains into the C-111 canal. The results of this study indicate that runoff from agricultural processes led to quantifiable pesticide residues in both canal and bay surface water, which occasionally exceeded current water quality criteria. The major pesticide of concern was endosulfan, which was detected at 100% of the sites sampled. Endosulfan exposure did not cause any acute effects in fish and crustaceans deployed in field bioassays. Chronic effects were observed in copepods, clams, and oysters but could not be attributed to endosulfan exposure. The decision to alter the C-111 canal flow and allow increased freshwater flow into the adjacent Everglades National Park may result in discharges of pesticides into the Everglades. Continued monitoring in this area is needed during this change in flow regime.

The ecological condition of south Florida estuaries.

An assessment of the ecological condition of south Florida estuaries based on regional probabilistic monitoring was conducted during the summer of 1995. Samples and data were collected on water and sediment quality, benthos, and fish tissue contaminants. Elevated concentrations of metals and pesticides were measured in both sediments and fish tissue with some exceedances of guidance values. Bottom dissolved oxygen levels over 23-37% of the area were below state criteria. Eighty-eight percent of surface waters had greater than 10% penetration of ambient light to a depth of 1.0 m. Nine percent of the area studied in South Florida exhibited degraded biology and impaired use based on a calculated index of ecological condition. Using the probability-based monitoring design, useful information of this type can be provided to resource managers regarding estuarine condition on a regional scale.

A model system for studying the integration of molecular biology databases.

MOTIVATION: Integration of molecular biology databases remains limited in practice despite its practical importance and considerable research effort. The complexity of the problem is such that an experimental approach is mandatory, yet this very complexity makes it hard to design definitive experiments. This dilemma is common in science, and one tried-and-true strategy is to work with model systems. We propose a model system for this problem, namely a database of genes integrating diverse data across organisms, and describe an experiment using this model. RESULTS: We attempted to construct a database of human and mouse genes integrating data from GenBank and the human and mouse genome-databases. We discovered numerous errors in these well-respected databases: approximately 15% of genes are apparently missing from the genome-databases; links between the sequence and genome-databases are missing for another 5-10% of the cases; about a third of likely homology links are missing between the genome-databases; 10-20% of entries classified as 'genes' are apparently misclassified. By using a model system, we were able to study the problems caused by anomalous data without having to face all the hard problems of database integration. CONTACT: nat@jax.org

Distribution of total mercury and methyl mercury in water, sediment, and fish from south Florida estuaries.

Concentrations of total mercury and methyl mercury were determined in sediment and fish collected from estuarine waters of Florida to understand their distribution and partitioning. Total mercury concentrations in sediments ranged from 1 to 219 ng/g dry wt. Methyl mercury accounted for, on average, 0.77% of total mercury in sediment. Methyl mercury concentrations were not correlated with total mercury or organic carbon content in sediments. The concentrations of total mercury in fish muscle were between 0.03 and 2.22 (mean: 0.31) micrograms/g, wet wt, with methyl mercury contributing 83% of total mercury. Methyl mercury concentrations in fish muscle were directly proportional to total mercury concentrations. The relationship of total and methyl mercury concentrations in fish to those of sediments from corresponding locations was fish-species dependent, in addition to several abiotic factors. Among fish species analyzed, hardhead catfish, gafftopsail catfish, and sand seatrout contained the highest concentrations of mercury. Filtered water samples from canals and creeks that discharge into the Florida Bay showed mercury concentrations of 3-7.4 ng/L, with methyl mercury accounting for < 0.03-52% of the total mercury. Consumption of fish containing 0.31 microgram mercury/g wet wt, the mean concentration found in this study, at rates greater than 70 g/day, was estimated to be hazardous to human health.

Adherence-dependent calcium signaling in monocytes: induction of a CD14-high phenotype, stimulus-responsive subpopulation.

Isolation of monocytes by plastic adherence alters cell morphology and function. In order to study the effects of cell isolation procedures and subsequent culture on monocyte function, we examined cytoplasmic calcium concentration changes (delta[Ca2+]in) in human monocytes isolated by either negative (magnetic bead) or positive (plastic adherence) selection then stimulated with formyl-Met-Leu-Phe (fMLP), either immediately after isolation, or after 48 h in culture. We have previously shown that fresh adherence-isolated monocytes respond to fMLP with small delta[Ca2+]in and oxidative burst responses, exhibiting larger responses following 48 h of incubation. We now demonstrate that fresh monocytes, prevented from adhering by negative selection, exhibit an even smaller fMLP-induced delta[Ca2+]in, which does not increase during 48 h in culture if cells are kept nonadherent, in Teflon. Calcium responses of adherent, fresh monocytes do not increase if cells are subsequently placed into suspension and maintained nonadherent, but increase if nonadherent cells are permitted to adhere to plastic. Furthermore, augmented fMLP-[Ca2+]in and oxidative burst responses in plastic-adherent cells are restricted to a CD14-high phenotype subpopulation. The CD14-high phenotype also describes a subpopulation of cells that responds to CD4 crosslinking with a rapid delta[Ca2+]in. Induction of a subpopulation of CD14-high expressing cells by adherence may explain in part maturation-induced response changes observed in macrophage but not in monocyte in vitro systems.

Dissection of the ascending aorta: a late complication of coronary artery bypass grafting.

Acute dissection of the ascending aorta as a late complication of coronary artery bypass grafting has been rarely reported. We report a case of a 61 year-old man in whom acute dissection of the ascending aorta developed 2 years after coronary artery bypass grafting. The ascending aorta was replaced with a Dacron graft, and an island of the aortic wall, on which previous proximal anastomoses had been placed, was implanted into the Dacron graft successfully.

An assessment of water quality and primary productivity in Perdido Bay, a Northern Gulf of Mexico Estuary.

Perdido Bay is a shallow estuarine system of approximately 130 km(2) with three major freshwater inputs. On a seasonal basis the productivity and chlorophyll a concentration of phytoplankton in Perdido Bay are controlled by temperature. One input, Eleven Mile Creek, is influenced by a paper mill discharge. Eleven Mile Creek exhibits high levels of light attenuation, high concentrations of dissolved nutrients, and low rates of carbon fixation that are significantly different from the other inputs or areas of Perdido Bay and productivity in Eleven Mile Creek is light limited. Upper Perdido Bay had slightly elevated concentrations of dissolved nutrients which correlate with significantly higher rates of carbon fixation and phytoplankton biomass. Nutrients and color from Eleven Mile Creck are diluted by the Perdido River inflow, restricting nutrient and light limitations to the area at the mouth of Eleven Mile Creek.

Influence of dietary fat, vitamin E, ethoxyquin and indomethacin on the synthesis of prostaglandin E2 in brain regions of mice.

The regional brain synthesis of prostaglandin E2 (PGE2) of rats varies with age and dietary vitamin E. We investigated ex-vivo synthesis of PGE2 in brain regions of 24-mo-old male C57BL/6Nia mice. Mice were fed semipurified diets containing 1) 5% by weight of vitamin E-stripped corn oil, nonhydrogenated coconut oil or fish oil, or 2) 3.8% lard + 1.2% stripped corn oil (control), each supplemented with 30 or 500 mg of vitamin E per kg for 6 wk. In addition, two groups of mice were fed control diets containing 2500 mg of ethoxyquin/kg or 1 mg of indomethacin/(kg.day). Mice fed fish oil had lower (P less than 0.01) plasma alpha-tocopherol than those fed coconut oil. Vitamin E supplementation had no effect on the concentration of alpha-tocopherol in most brain regions. The rank order of PGE2 synthesis among all groups was cerebrum = cerebellum greater than midbrain greater than brainstem. Vitamin E supplementation tended to reduce brain PGE2 synthesis. Fat with 30 mg/kg of vitamin E in their diet. In the brainstem, fish oil significantly reduced PGE2 relative to mice fed corn oil. Ethoxyquin lowered plasma alpha-tocopherol by 42% and reduced the PGE2 synthesis in brain regions, and indomethacin reduced it by 50%. Thus, ethoxyquin and indomethacin significantly reduced the brain's regional capacity for PGE2 synthesis, whereas vitamin E was not effective for the period tested. The reduction of PGE2 synthesis in the brain with fish oil feeding in this study was not as notable as reported for other tissues unless combined with high-dose vitamin E supplementation.

Postprandial changes in the plasma concentration of alpha- and gamma-tocopherol in human subjects fed a fat-rich meal supplemented with fat-soluble vitamins.

The plasma concentrations of alpha (alpha)- and gamma (gamma)-tocopherol in 10 male and 15 female subjects (n = 14) received 1 g fat/kg body wt as soybean oil, and the meal was supplemented with 100% of the RDA for fat-soluble vitamins. In expt. 2, the subjects (n = 11) received 1 g fat/kg body wt as 50% soybean oil + 50% cream, and the meal was supplemented with 200% of the RDA for fat-soluble vitamins. The ratio of gamma- :alpha-tocopherol given in the test meal of expt. 1 was 2.8:1 and in expt. 2 was 0.9:1. Blood samples were obtained 0, 3, 6, 9 and 12 h after the meal. Tocopherol concentration was measured in plasma and lipoprotein fractions. In both studies, plasma triglyceride concentration increased significantly after the meal (P less than 0.001). Mean plasma cholesterol and alpha-tocopherol concentrations were unchanged, but plasma gamma-tocopherol concentration was significantly increased at 6, 9 and 12 h after the meal (P less than 0.05). The increase in plasma gamma-tocopherol was due to increases within the triglyceride-rich lipoprotein (TRL) fraction (d less than 1.006 g/ml) at earlier timepoints, followed by a sustained increase within low density lipoprotein (LDL) and high density lipoprotein (HDL) fractions at later timepoints. In contrast, alpha-tocopherol in LDL and HDL decreased postprandially, concomitant with a rise in TRL alpha-tocopherol. Our results are consistent with the concept that there are differences in the distribution of alpha- and gamma-tocopherol in postprandial lipoproteins.

Effect of dietary vitamin E and selenium on susceptibility of brain regions to lipid peroxidation.

The effect of dietary vitamin E and/or selenium (Se) supplementation (200 IU and/or 0.2 ppm, respectively) or deficiency for two months on lipid peroxidation in cerebrum, cerebellum, mid-brain, and brain stem of one-month-old male F344 rats was investigated. Dietary treatment had a minimal effect on weight gain of rats for the period tested. Plasma alpha-tocopherol (alpha-T) concentration and glutathione peroxidase (GSH-Px) activity were reflective of dietary treatments. Supplementation of diets with vitamin E and/or Se increased plasma alpha-T and/or GSH-Px activity, while diets devoid of these nutrients reduced them significantly. Increased GSH-Px activity in Se-supplemented rats was further enhanced by vitamin E supplementation. Differential concentrations of alpha-T among brain regions were affected by dietary vitamin E but not by Se. In vitro lipid peroxidation of brain homogenates was inhibited by dietary vitamin E supplementation and increased by deficiency. Addition of 0.25 mM ascorbic acid or 0.1 mM of Fe2+ to brain homogenates markedly increased in vitro lipid peroxidation. Ascorbic acid-induced lipid peroxidation was inversely correlated with dietary vitamin E and Se in cerebrum. In vitro Fe2+-addition induced the greatest stimulation of lipid peroxidation, with cerebellum and brain stem of vitamin E-deficient rats showing the highest response to Fe2+ challenge. These findings indicate that concentrations of alpha-T among the brain regions are different and can be altered by dietary vitamin E treatments, cerebellum and brain stem are more susceptible to in vitro challenge by peroxidative agents than other regions, and the degree of lipid peroxidation of brain regions is partially affected by dietary vitamin E but not by Se in the levels tested.

Effect of age and dietary fat (fish, corn and coconut oils) on tocopherol status of C57BL/6Nia mice.

The effect of age and dietary fat type on tocopherol status was investigated using young and old C57BL/6Nia mice fed semipurified diets containing 5% (by weight) fish, corn or coconut oils and supplemented with 30, 100 or 500 ppm dl-alpha-tocopheryl acetate for 6 wk. Tocopherol levels in the diets, plasma, liver, kidney and lung were measured by high performance liquid chromatography following appropriate extractions. The results indicate that mice fed fish oil maintain lower plasma and tissue tocopherol concentrations than those fed corn and coconut oils (fish less than corn oil less than coconut oil). The difference was not due to a loss of tocopherol prior to consumption, but rather appeared to occur during the absorption process. Old mice had lower plasma and liver tocopherol concentrations than young mice. Old mice fed fish oil, however, maintained plasma tocopherol levels better than young mice fed fish oil, presumably due to their larger tocopherol pool. No age effect was detected on kidney and lung tocopherol levels. It is concluded that tocopherol status is affected by age and dietary fat type, especially fish oil.