Intratumoral heterogeneity of second-harmonic generation scattering from tumor collagen and its effects on metastatic risk prediction.

BACKGROUND: Metastases are the leading cause of breast cancer-related deaths. The tumor microenvironment impacts cancer progression and metastatic ability. Fibrillar collagen, a major extracellular matrix component, can be studied using the light scattering phenomenon known as second-harmonic generation (SHG). The ratio of forward- to backward-scattered SHG photons (F/B) is sensitive to collagen fiber internal structure and has been shown to be an independent prognostic indicator of metastasis-free survival time (MFS). Here we assess the effects of heterogeneity in the tumor matrix on the possible use of F/B as a prognostic tool. METHODS: SHG imaging was performed on sectioned primary tumor excisions from 95 untreated, estrogen receptor-positive, lymph node negative invasive ductal carcinoma patients. We identified two distinct regions whose collagen displayed different average F/B values, indicative of spatial heterogeneity: the cellular tumor bulk and surrounding tumor-stroma interface. To evaluate the impact of heterogeneity on F/B's prognostic ability, we performed SHG imaging in the tumor bulk and tumor-stroma interface, calculated a 21-gene recurrence score (surrogate for OncotypeDX®, or S-ODX) for each patient and evaluated their combined prognostic ability. RESULTS: We found that F/B measured in tumor-stroma interface, but not tumor bulk, is prognostic of MFS using three methods to select pixels for analysis: an intensity threshold selected by a blinded observer, a histogram-based thresholding method, and an adaptive thresholding method. Using both regression trees and Random Survival Forests for MFS outcome, we obtained data-driven prediction rules that show F/B from tumor-stroma interface, but not tumor bulk, and S-ODX both contribute to predicting MFS in this patient cohort. We also separated patients into low-intermediate (S-ODX < 26) and high risk (S-ODX ≥26) groups. In the low-intermediate risk group, comprised of patients not typically recommended for adjuvant chemotherapy, we find that F/B from the tumor-stroma interface is prognostic of MFS and can identify a patient cohort with poor outcomes. CONCLUSIONS: These data demonstrate that intratumoral heterogeneity in F/B values can play an important role in its possible use as a prognostic marker, and that F/B from tumor-stroma interface of primary tumor excisions may provide useful information to stratify patients by metastatic risk.

Evaluation of temocillin for phenotypic carbapenemase screening of Escherichia coli and Salmonella enterica isolates in relation to the presence of genes encoding ESBLs and carbapenemase production.

BACKGROUND: The expression of enzymes of the OXA-48 carbapenemase group is difficult to detect by phenotypic methods owing to frequent low levels of carbapenem resistance and negative results with some screening methods. Temocillin has been shown to be a good option for phenotypic screening as it is hydrolysed by the OXA-48-group enzymes, whereas ESBLs, AmpC and some other carbapenemases have a lower hydrolytic effect on this antimicrobial. However, no epidemiological cut-off for temocillin is available. OBJECTIVES: To evaluate temocillin MICs in relation to the presence or absence of genes encoding ESBLs and carbapenemases in Escherichia coli and Salmonella enterica. METHODS: In this study, 111 E. coli and 102 S. enterica isolates, including WT and well-characterized ESBL-, AmpC- or carbapenemase-producing isolates, were tested by three independent laboratories. MICs were determined according to the CLSI guidelines by agar dilution with the test range from 0.5 to 512 mg/L temocillin and WGS was performed and analysed with ResFinder. RESULTS: Some overlap was detected between temocillin MICs for WT and ESBL- or AmpC-producing isolates. However, isolates carrying genes encoding carbapenemases showed a broader range of MICs for both E. coli and S. enterica. Higher MICs were observed for the OXA-48 group, VIM and some NDM-producing isolates, whereas isolates harbouring KPC enzymes showed low MICs. CONCLUSIONS: The results indicate that temocillin MICs enable phenotypic distinction between strains producing OXA-48-group enzymes and both WT susceptible and ESBL/AmpC-carrying isolates, whereas the distinction from other carbapenemases likely requires genotypic testing.

Growth rates of juvenile Boa constrictor under two feeding regimes.

Many husbandry routines in zoo herpetology are based on tradition, authoritarianism, anecdote, or speculation. However, relatively few empirical studies underlie many very common practices. We compared growth rates among littermates of Boa constrictor raised under two feeding regimes that were identical in terms of the mass of food ingested, but differed in weekly versus bi-weekly schedules. The growth rate of the group fed weekly was greater than the rate for the biweekly group. Snakes fed 10% of their body mass on a weekly regimen grew to a larger size, and at a faster rate, than did snakes fed 20% of their body mass on a biweekly regimen.

Cysteamine, an Endogenous Aminothiol, and Cystamine, the Disulfide Product of Oxidation, Increase Pseudomonas aeruginosa Sensitivity to Reactive Oxygen and Nitrogen Species and Potentiate Therapeutic Antibiotics against Bacterial Infection.

Cysteamine is an endogenous aminothiol produced in mammalian cells as a consequence of coenzyme A metabolism through the activity of the vanin family of pantetheinase ectoenzymes. It is known to have a biological role in oxidative stress, inflammation, and cell migration. There have been several reports demonstrating anti-infective properties targeting viruses, bacteria, and even the malarial parasite. We and others have previously described broad-spectrum antimicrobial and antibiofilm activities of cysteamine. Here, we go further to demonstrate redox-dependent mechanisms of action for the compound and how its antimicrobial effects are, at least in part, due to undermining bacterial defenses against oxidative and nitrosative challenges. We demonstrate the therapeutic potentiation of antibiotic therapy against Pseudomonas aeruginosa in mouse models of infection. We also demonstrate potentiation of many different classes of antibiotics against a selection of priority antibiotic-resistant pathogens, including colistin (often considered an antibiotic of last resort), and we discuss how this endogenous antimicrobial component of innate immunity has a role in infectious disease that is beginning to be explored and is not yet fully understood.

Validity of objective methods for measuring sedentary behaviour in older adults: a systematic review.

BACKGROUND: The evidence showing the ill health effects of prolonged sedentary behaviour (SB) is growing. Most studies of SB in older adults have relied on self-report measures of SB. However, SB is difficult for older adults to recall and objective measures that combine accelerometry with inclinometry are now available for more accurately assessing SB. The aim of this systematic review was to assess the validity and reliability of these accelerometers for the assessment of SB in older adults. METHODS: EMBASE, PubMed and EBSCOhost databases were searched for articles published up to December 13, 2017. Articles were eligible if they: a) described reliability, calibration or validation studies of SB measurement in healthy, community-dwelling individuals, b) were published in English, Portuguese or Spanish, and c) were published or in press as journal articles in peer-reviewed journals. RESULTS: The review identified 15 studies in 17 papers. Of the included studies, 11 assessed the ActiGraph accelerometer. Of these, three examined reliability only, seven (in eight papers) examined validity only and one (in two papers) examined both. The strongest evidence from the studies reviewed is from studies that assessed the validity of the ActiGraph. These studies indicate that analysis of the data using 60-s epochs and a vertical magnitude cut-point < 200 cpm or using 30- or 60-s epochs with a machine learning algorithm provides the most valid estimates of SB. Non-wear algorithms of 90+ consecutive zeros is also suggested for the ActiGraph. CONCLUSIONS: Few studies have examined the reliability and validity of accelerometers for measuring SB in older adults. Studies to date suggest that the criteria researchers use for classifying an epoch as sedentary instead of as non-wear time (e.g., the non-wear algorithm used) may need to be different for older adults than for younger adults. The required number of hours and days of wear for valid estimates of SB in older adults was not clear from studies to date. More older-adult-specific validation studies of accelerometers are needed, to inform future guidelines on the appropriate criteria to use for analysis of data from different accelerometer brands. TRIAL REGISTRATION: PROSPERO ID# CRD42017080754 registered December 12, 2017.

Escherichia coli Export from Manured Fields Depends on the Time between the Start of Rainfall and Runoff Initiation.

After rainfall or irrigation begins, surface-applied chemicals and manure-borne microorganisms typically enter the soil with infiltration until the soil saturates, after which time the chemicals and microbes are exported from the field in the overland flow. This process is viewed as a reason for the dependence of chemical export on the time between rainfall start and runoff initiation that has been documented for agricultural chemicals. The objective of this work was to observe and quantify such dependence for released from solid farmyard dairy manure in field conditions. Experiments were performed for 6 yr and consisted of manure application followed by an immediate simulated rainfall event and a second event 1 wk later. The nonlinearity of the release seen in laboratory and plot studies did not manifest itself in the field. The number of exported cells in runoff was proportional to rainfall depth after runoff initiation in each trial. The proportionality coefficient, termed export rate, demonstrated a strong dependence on the runoff delay time that could be approximated with the exponential decrease. The export rate decreased by one order of magnitude when the rainfall depth at runoff initiation increased from 18 to 42 mm. The same dependence could approximate data from the simulated rainfall event 1 wk after the manure application, assuming that the initial content in manure after 1 wk of weathering was 10% of the initial content. Overall, accounting for the dependence of manure-borne export on the runoff delay time should improve the accuracy of export predictions related to the assessment of agricultural practices on microbial water quality.

Covert dissemination of carbapenemase-producing Klebsiella pneumoniae (KPC) in a successfully controlled outbreak: long- and short-read whole-genome sequencing demonstrate multiple genetic modes of transmission.

BACKGROUND: Carbapenemase-producing Enterobacteriaceae (CPE), including KPC-producing Klebsiella pneumoniae (KPC-Kpn), are an increasing threat to patient safety. OBJECTIVES: To use WGS to investigate the extent and complexity of carbapenemase gene dissemination in a controlled KPC outbreak. MATERIALS AND METHODS: Enterobacteriaceae with reduced ertapenem susceptibility recovered from rectal screening swabs/clinical samples, during a 3 month KPC outbreak (2013-14), were investigated for carbapenemase production, antimicrobial susceptibility, variable-number-tandem-repeat profile and WGS [short-read (Illumina), long-read (MinION)]. Short-read sequences were used for MLST and plasmid/Tn4401 fingerprinting, and long-read sequence assemblies for plasmid identification. Phylogenetic analysis used IQTree followed by ClonalFrameML, and outbreak transmission dynamics were inferred using SCOTTI. RESULTS: Twenty patients harboured KPC-positive isolates (6 infected, 14 colonized), and 23 distinct KPC-producing Enterobacteriaceae were identified. Four distinct KPC plasmids were characterized but of 20 KPC-Kpn (from six STs), 17 isolates shared a single pKpQIL-D2 KPC plasmid. All isolates had an identical transposon (Tn4401a), except one KPC-Kpn (ST661) with a single nucleotide variant. A sporadic case of KPC-Kpn (ST491) with Tn4401a-carrying pKpQIL-D2 plasmid was identified 10 months before the outbreak. This plasmid was later seen in two other species and other KPC-Kpn (ST14,ST661) including clonal spread of KPC-Kpn (ST661) from a symptomatic case to nine ward contacts. CONCLUSIONS: WGS of outbreak KPC isolates demonstrated blaKPC dissemination via horizontal transposition (Tn4401a), plasmid spread (pKpQIL-D2) and clonal spread (K. pneumoniae ST661). Despite rapid outbreak control, considerable dissemination of blaKPC still occurred among K. pneumoniae and other Enterobacteriaceae, emphasizing its high transmission potential and the need for enhanced control efforts.

A genomic portrait of the emergence, evolution, and global spread of a methicillin-resistant Staphylococcus aureus pandemic.

The widespread use of antibiotics in association with high-density clinical care has driven the emergence of drug-resistant bacteria that are adapted to thrive in hospitalized patients. Of particular concern are globally disseminated methicillin-resistant Staphylococcus aureus (MRSA) clones that cause outbreaks and epidemics associated with health care. The most rapidly spreading and tenacious health-care-associated clone in Europe currently is EMRSA-15, which was first detected in the UK in the early 1990s and subsequently spread throughout Europe and beyond. Using phylogenomic methods to analyze the genome sequences for 193 S. aureus isolates, we were able to show that the current pandemic population of EMRSA-15 descends from a health-care-associated MRSA epidemic that spread throughout England in the 1980s, which had itself previously emerged from a primarily community-associated methicillin-sensitive population. The emergence of fluoroquinolone resistance in this EMRSA-15 subclone in the English Midlands during the mid-1980s appears to have played a key role in triggering pandemic spread, and occurred shortly after the first clinical trials of this drug. Genome-based coalescence analysis estimated that the population of this subclone over the last 20 yr has grown four times faster than its progenitor. Using comparative genomic analysis we identified the molecular genetic basis of 99.8% of the antimicrobial resistance phenotypes of the isolates, highlighting the potential of pathogen genome sequencing as a diagnostic tool. We document the genetic changes associated with adaptation to the hospital environment and with increasing drug resistance over time, and how MRSA evolution likely has been influenced by country-specific drug use regimens.

Solid Manure As a Source of Fecal Indicator Microorganisms: Release under Simulated Rainfall.

Understanding and quantifying microbial release from manure is a precondition to estimation and management of microbial water quality. The objectives of this work were to determine the effects of rainfall intensity and surface slope on the release of Escherichia coli, enterococci, total coliforms, and dissolved chloride from solid dairy manure and to assess the performance of the one-parametric exponential model and the two-parametric Bradford-Schijven model when simulating the observed release. A controlled-intensity rainfall simulator induced 1 h of release in runoff/leachate partitioning boxes at three rainfall intensities (30, 60, and 90 mm h(-1)) and two surface slopes (5% and 20%). Bacterial concentrations in initial release were more than 1 order of magnitude lower than their starting concentrations in manure. As bacteria were released, they were partitioned into runoff and leachate at similar concentrations, but in different volumes, depending on slope. Bacterial release occurred in two stages that corresponded to mechanisms associated with release of manure liquid- and solid-phases. Parameters of the two models fitted to the bacterial release dependencies on rainfall depth were not significantly affected by rainfall intensity or slope. Based on model performance tests, the Bradford-Schijven model is recommended for simulating bacterial release from solid manure.

Release and Removal of Microorganisms from Land-Deposited Animal Waste and Animal Manures: A Review of Data and Models.

Microbial pathogens present a leading cause of impairment to rivers, bays, and estuaries in the United States, and agriculture is often viewed as the major contributor to such contamination. Microbial indicators and pathogens are released from land-applied animal manure during precipitation and irrigation events and are carried in overland and subsurface flow that can reach and contaminate surface waters and ground water used for human recreation and food production. Simulating the release and removal of manure-borne pathogens and indicator microorganisms is an essential component of microbial fate and transport modeling regarding food safety and water quality. Although microbial release controls the quantities of available pathogens and indicators that move toward human exposure, a literature review on this topic is lacking. This critical review on microbial release and subsequent removal from manure and animal waste application areas includes sections on microbial release processes and release-affecting factors, such as differences in the release of microbial species or groups; bacterial attachment in turbid suspensions; animal source; animal waste composition; waste aging; manure application method; manure treatment effect; rainfall intensity, duration, and energy; rainfall recurrence; dissolved salts and temperature; vegetation and soil; and spatial and temporal scale. Differences in microbial release from liquid and solid manures are illustrated, and the influential processes are discussed. Models used for simulating release and removal and current knowledge gaps are presented, and avenues for future research are suggested.

Colonisation with toxigenic Corynebacterium diphtheriae in a Scottish burns patient, June 2015.

On 12 June 2015, Corynebacterium diphtheriae was identified in a skin swab from a burns patient in Scotland. The isolate was confirmed to be genotypically and phenotypically toxigenic. Multilocus sequence typing of three patient isolates yielded sequence type ST 125. The patient was clinically well. We summarise findings of this case, and results of close contact identification and screening: 12 family and close contacts and 32 hospital staff have been found negative for C. diphtheriae.

Blood clotting and coagulation factors: the work of Yale Nemerson. 1980, 1982.

After developing a blood disorder, Yale Nemerson became interested in hematology. This led to his lifelong study of thrombogenic tissue factor and to his contributions to developing the modern theory of blood coagulation. The two Classic papers reprinted here detail some of Nemerson's studies on coagulation factors IX and VII.

Do differences in Panton-Valentine leukocidin production among international methicillin-resistant Staphylococcus aureus clones affect disease presentation and severity?

Panton-Valentine leukocidin (PVL) production by methicillin-resistant Staphylococcus aureus (MRSA) was determined in vitro using the enzyme-linked immunosorbent assay (ELISA), and associations with clinical presentation and bacterial genetic characteristics were examined. PVL production ranged from 0.02 to 4.865 µg/ml and correlated with a multilocus sequence type (MLST) clonal complex associated with specific PVL phage types. A relationship between PVL production and clinical presentation or patient demographics could not be demonstrated.

Intra-daily variation of Escherichia coli concentrations in agricultural irrigation ponds.

Microbial water quality is determined by comparing observed Escherichia coli concentrations with regulatory thresholds. Measured concentrations can be expected to change throughout the course of a day in response to diurnal variation in environmental conditions, such as solar radiation and temperature. Therefore, the time of day at which samples are taken is an important factor within microbial water quality measurements. However, little is known about the diurnal variations of E. coli concentrations in surface sources of irrigation water. The objectives of this work were to evaluate the intra-daily dynamics of E. coli in three irrigation ponds in Maryland over several years and to determine the water quality parameters to which E. coli populations are most sensitive. Water sampling was conducted across the ponds at 0900, 1200, and 1500 h on a total of 17 dates in the summers of 2019-2021. One-way ANOVA revealed significant diurnal variability in E. coli concentrations in Pond (P)1 and P2, whereas no significant effects were observed in P3. Escherichia coli die-off rates calculated between sampling time points in the same day were significantly higher in P2 than in P1 and P3, and these rates ranged from 0.005 to 0.799 h-1 across ponds. Concentrations of dissolved oxygen, pH, conductivity, and turbidity exerted the most control over E. coli populations. Results of this work demonstrate that sampling in the early-morning hours provides the most conservative assessment of the microbial quality of irrigation waters.

Second-Harmonic Generation Imaging Reveals Changes in Breast Tumor Collagen Induced by Neoadjuvant Chemotherapy.

Breast cancer is the most common invasive cancer in women, with most deaths attributed to metastases. Neoadjuvant chemotherapy (NACT) may be prescribed prior to surgical removal of the tumor for subsets of breast cancer patients but can have diverse undesired and off-target effects, including the increased appearance of the 'tumor microenvironment of metastasis', image-based multicellular signatures that are prognostic of breast tumor metastasis. To assess whether NACT can induce changes in two other image-based prognostic/predictive signatures derived from tumor collagen, we quantified second-harmonic generation (SHG) directionality and fiber alignment in formalin-fixed, paraffin-embedded sections of core needle biopsies and primary tumor excisions from 22 human epidermal growth factor receptor 2-overexpressing (HER2+) and 22 triple-negative breast cancers. In both subtypes, we found that SHG directionality (i.e., the forward-to-backward scattering ratio, or F/B) is increased by NACT in the bulk of the tumor, but not the adjacent tumor-stroma interface. Overall collagen fiber alignment is increased by NACT in triple-negative but not HER2+ breast tumors. These results suggest that NACT impacts the collagenous extracellular matrix in a complex and subtype-specific manner, with some prognostic features being unchanged while others are altered in a manner suggestive of a more metastatic phenotype.

A qualitative study of older adults' responses to sitting-time questions: do we get the information we want?

BACKGROUND: In the last decade, there has been increasing interest in the health effects of sedentary behavior, which is often assessed using self-report sitting-time questions. The aim of this qualitative study was to document older adults' understanding of sitting-time questions from the International Physical Activity (PA) Questionnaire (IPAQ) and the PA Scale for the Elderly (PASE). METHODS: Australian community-dwelling adults aged 65+ years answered the IPAQ and PASE sitting questions in face-to-face semi-structured interviews. IPAQ uses one open-ended question to assess sitting on a weekday in the last 7 days 'at work, at home, while doing coursework and during leisure time'; PASE uses a three-part closed question about daily leisure-time sitting in the last 7 days. Participants expressed their thoughts out loud while answering each question. They were then probed about their responses. Interviews were recorded, transcribed and coded into themes. RESULTS: Mean age of the 28 male and 27 female participants was 73 years (range 65-89). The most frequently reported activity was watching TV. For both questionnaires, many participants had difficulties understanding what activities to report. Some had difficulty understanding what activities should be classified as 'leisure-time sitting'. Some assumed they were being asked to only report activities provided as examples. Most reported activities they normally do, rather than those performed on a day in the previous week. Participants used a variety of strategies to select 'a day' for which they reported their sitting activities and to calculate sitting time on that day. Therefore, many different ways of estimating sitting time were used. Participants had particular difficulty reporting their daily sitting-time when their schedules were not consistent across days. Some participants declared the IPAQ sitting question too difficult to answer. CONCLUSION: The accuracy of older adults' self-reported sitting time is questionable given the challenges they have in answering sitting-time questions. Their responses to sitting-time questions may be more accurate if our recommendations for clarifying the sitting domains, providing examples relevant to older adults and suggesting strategies for formulating responses are incorporated. Future quantitative studies should include objective criterion measures to assess validity and reliability of these questions.

Prediction of E. coli Concentrations in Agricultural Pond Waters: Application and Comparison of Machine Learning Algorithms.

The microbial quality of irrigation water is an important issue as the use of contaminated waters has been linked to several foodborne outbreaks. To expedite microbial water quality determinations, many researchers estimate concentrations of the microbial contamination indicator Escherichia coli (E. coli) from the concentrations of physiochemical water quality parameters. However, these relationships are often non-linear and exhibit changes above or below certain threshold values. Machine learning (ML) algorithms have been shown to make accurate predictions in datasets with complex relationships. The purpose of this work was to evaluate several ML models for the prediction of E. coli in agricultural pond waters. Two ponds in Maryland were monitored from 2016 to 2018 during the irrigation season. E. coli concentrations along with 12 other water quality parameters were measured in water samples. The resulting datasets were used to predict E. coli using stochastic gradient boosting (SGB) machines, random forest (RF), support vector machines (SVM), and k-nearest neighbor (kNN) algorithms. The RF model provided the lowest RMSE value for predicted E. coli concentrations in both ponds in individual years and over consecutive years in almost all cases. For individual years, the RMSE of the predicted E. coli concentrations (log10 CFU 100 ml-1) ranged from 0.244 to 0.346 and 0.304 to 0.418 for Pond 1 and 2, respectively. For the 3-year datasets, these values were 0.334 and 0.381 for Pond 1 and 2, respectively. In most cases there was no significant difference (P > 0.05) between the RMSE of RF and other ML models when these RMSE were treated as statistics derived from 10-fold cross-validation performed with five repeats. Important E. coli predictors were turbidity, dissolved organic matter content, specific conductance, chlorophyll concentration, and temperature. Model predictive performance did not significantly differ when 5 predictors were used vs. 8 or 12, indicating that more tedious and costly measurements provide no substantial improvement in the predictive accuracy of the evaluated algorithms.