Host and pathogen response to bacteriophage engineered against Mycobacterium abscessus lung infection.

Two mycobacteriophages were administered intravenously to a male with treatment-refractory Mycobacterium abscessus pulmonary infection and severe cystic fibrosis lung disease. The phages were engineered to enhance their capacity to lyse M. abscessus and were selected specifically as the most effective against the subject's bacterial isolate. In the setting of compassionate use, the evidence of phage-induced lysis was observed using molecular and metabolic assays combined with clinical assessments. M. abscessus isolates pre and post-phage treatment demonstrated genetic stability, with a general decline in diversity and no increased resistance to phage or antibiotics. The anti-phage neutralizing antibody titers to one phage increased with time but did not prevent clinical improvement throughout the course of treatment. The subject received lung transplantation on day 379, and systematic culturing of the explanted lung did not detect M. abscessus. This study describes the course and associated markers of a successful phage treatment of M. abscessus in advanced lung disease.

The nutritional quality of cereals varies geospatially in Ethiopia and Malawi.

Micronutrient deficiencies (MNDs) remain widespread among people in sub-Saharan Africa1-5, where access to sufficient food from plant and animal sources that is rich in micronutrients (vitamins and minerals) is limited due to socioeconomic and geographical reasons4-6. Here we report the micronutrient composition (calcium, iron, selenium and zinc) of staple cereal grains for most of the cereal production areas in Ethiopia and Malawi. We show that there is geospatial variation in the composition of micronutrients that is nutritionally important at subnational scales. Soil and environmental covariates of grain micronutrient concentrations included soil pH, soil organic matter, temperature, rainfall and topography, which were specific to micronutrient and crop type. For rural households consuming locally sourced food-including many smallholder farming communities-the location of residence can be the largest influencing factor in determining the dietary intake of micronutrients from cereals. Positive relationships between the concentration of selenium in grain and biomarkers of selenium dietary status occur in both countries. Surveillance of MNDs on the basis of biomarkers of status and dietary intakes from national- and regional-scale food-composition data1-7 could be improved using subnational data on the composition of grain micronutrients. Beyond dietary diversification, interventions to alleviate MNDs, such as food fortification8,9 and biofortification to increase the micronutrient concentrations in crops10,11, should account for geographical effects that can be larger in magnitude than intervention outcomes.

Group B Streptococcus transcriptome when interacting with brain endothelial cells.

Bacterial meningitis is a life-threatening infection of the central nervous system (CNS) that occurs when bacteria are able to cross the blood-brain barrier (BBB) or the meningeal-cerebrospinal fluid barrier (mBCSFB). The BBB and mBCSFB comprise highly specialized brain endothelial cells (BECs) that typically restrict pathogen entry. Group B Streptococcus (GBS or Streptococcus agalactiae) is the leading cause of neonatal meningitis. Until recently, identification of GBS virulence factors has relied on genetic screening approaches. Instead, we here conducted RNA-seq analysis on GBS when interacting with induced pluripotent stem cell-derived BECs (iBECs) to pinpoint virulence-associated genes. Of the 2,068 annotated protein-coding genes of GBS, 430 transcripts displayed significant changes in expression after interacting with BECs. Notably, we found that the majority of differentially expressed GBS transcripts were downregulated (360 genes) during infection of iBECs. Interestingly, codY, encoding a pleiotropic transcriptional repressor in low-G + C Gram-positive bacteria, was identified as being highly downregulated. We conducted qPCR to confirm the codY downregulation observed via RNA-seq during the GBS-iBEC interaction and obtained codY mutants in three different GBS background parental strains. As anticipated from the RNA-seq results, the [Formula: see text]codY strains were more adherent and invasive in two in vitro BEC models. Together, this demonstrates the utility of RNA-seq during the BEC interaction to identify GBS virulence modulators. IMPORTANCE: Group B Streptococcus (GBS) meningitis remains the leading cause of neonatal meningitis. Research work has identified surface factors and two-component systems that contribute to GBS disruption of the blood-brain barrier (BBB). These discoveries often relied on genetic screening approaches. Here, we provide transcriptomic data describing how GBS changes its transcriptome when interacting with brain endothelial cells. Additionally, we have phenotypically validated these data by obtaining mutants of a select regulator that is highly down-regulated during infection and testing on our BBB model. This work provides the research field with a validated data set that can provide an insight into potential pathways that GBS requires to interact with the BBB and open the door to new discoveries.

Bilateral Total Hip Arthroplasty: Outcomes of Staged Versus Simultaneous Procedures Performed Using an Anterior-Based Muscle-Sparing Approach.

BACKGROUND: The incidence of total hip arthroplasty (THA) in the United States continues to increase due to its ability to markedly improve patients' quality of life. This study investigated and compared the perioperative and postoperative outcomes of simultaneous (SI-THA) and staged (ST-THA) bilateral THA procedures using an anterior-based muscle-sparing (ABMS) approach. METHODS: This retrospective case control study evaluated perioperative and postoperative outcomes from primary bilateral SI-THA or ST-THA (within 365 days) performed with the ABMS approach by 3 surgeons at a single institution between January 2013 and August 2020. A total of 226 patients (113 in each cohort) were matched based on age, sex, body mass index, and comorbidity score. RESULTS: Compared to the ST-THA group, the SI-THA had shorter anesthesia duration (P < .001) and shorter length of stay (P < .001), but longer length of surgery (P = .002). There was no statistical significance between groups in blood transfusion rates, discharge dispositions, emergency department visits, hospital readmissions, or postoperative complications within one year. CONCLUSIONS: The results of this study demonstrate that SI-THA and ST-THA yield comparable results using the ABMS approach. Our perioperative and postoperative results suggest low rates of complications, emergency department visits, readmissions, and high rates of patient satisfaction scores. Therefore, both SI-THA and ST-THA can be considered by experienced surgeons as treatment for advanced bilateral hip arthritis.

Phage Therapy of Mycobacterium Infections: Compassionate Use of Phages in 20 Patients With Drug-Resistant Mycobacterial Disease.

BACKGROUND: Nontuberculous Mycobacterium infections, particularly Mycobacterium abscessus, are increasingly common among patients with cystic fibrosis and chronic bronchiectatic lung diseases. Treatment is challenging due to intrinsic antibiotic resistance. Bacteriophage therapy represents a potentially novel approach. Relatively few active lytic phages are available and there is great variation in phage susceptibilities among M. abscessus isolates, requiring personalized phage identification. METHODS: Mycobacterium isolates from 200 culture-positive patients with symptomatic disease were screened for phage susceptibilities. One or more lytic phages were identified for 55 isolates. Phages were administered intravenously, by aerosolization, or both to 20 patients on a compassionate use basis and patients were monitored for adverse reactions, clinical and microbiologic responses, the emergence of phage resistance, and phage neutralization in serum, sputum, or bronchoalveolar lavage fluid. RESULTS: No adverse reactions attributed to therapy were seen in any patient regardless of the pathogen, phages administered, or the route of delivery. Favorable clinical or microbiological responses were observed in 11 patients. Neutralizing antibodies were identified in serum after initiation of phage delivery intravenously in 8 patients, potentially contributing to lack of treatment response in 4 cases, but were not consistently associated with unfavorable responses in others. Eleven patients were treated with only a single phage, and no phage resistance was observed in any of these. CONCLUSIONS: Phage treatment of Mycobacterium infections is challenging due to the limited repertoire of therapeutically useful phages, but favorable clinical outcomes in patients lacking any other treatment options support continued development of adjunctive phage therapy for some mycobacterial infections.

Whole-genome sequencing analysis of two heat-evolved Escherichia coli strains.

BACKGROUND: High temperatures cause a suite of problems for cells, including protein unfolding and aggregation; increased membrane fluidity; and changes in DNA supercoiling, RNA stability, transcription and translation. Consequently, enhanced thermotolerance can evolve through an unknown number of genetic mechanisms even in the simple model bacterium Escherichia coli. To date, each E. coli study exploring this question resulted in a different set of mutations. To understand the changes that can arise when an organism evolves to grow at higher temperatures, we sequenced and analyzed two previously described E. coli strains, BM28 and BM28 ΔlysU, that have been laboratory adapted to the highest E. coli growth temperature reported to date. RESULTS: We found three large deletions in the BM28 and BM28 ΔlysU strains of 123, 15 and 8.5 kb in length and an expansion of IS10 elements. We found that BM28 and BM28 ΔlysU have considerably different genomes, suggesting that the BM28 culture that gave rise to BM28 and BM28 ΔlysU was a mixed population of genetically different cells. Consistent with published findings of high GroESL expression in BM28, we found that BM28 inexplicitly carries the groESL bearing plasmid pOF39 that was maintained simply by high-temperature selection pressure. We identified over 200 smaller insertions, deletions, single nucleotide polymorphisms and other mutations, including changes in master regulators such as the RNA polymerase and the transcriptional termination factor Rho. Importantly, this genome analysis demonstrates that the commonly cited findings that LysU plays a crucial role in thermotolerance and that GroESL hyper-expression is brought about by chromosomal mutations are based on a previous misinterpretation of the genotype of BM28. CONCLUSIONS: This whole-genome sequencing study describes genetically distinct mechanisms of thermotolerance evolution from those found in other heat-evolved E. coli strains. Studying adaptive laboratory evolution to heat in simple model organisms is important in the context of climate change. It is important to better understand genetic mechanisms of enhancing thermotolerance in bacteria and other organisms, both in terms of optimizing laboratory evolution methods for various organisms and in terms of potential genetic engineering of organisms most at risk or most important to our societies and ecosystems.

Time-efficient, high-resistance inspiratory muscle strength training increases cerebrovascular reactivity in midlife and older adults.

Aging is associated with increased risk for cognitive decline and dementia due in part to increases in systolic blood pressure (SBP) and cerebrovascular dysfunction. High-resistance inspiratory muscle strength training (IMST) is a time-efficient, intensive respiratory training protocol (30 resisted inspirations/day) that lowers SBP and improves peripheral vascular function in midlife/older adults with above-normal SBP. However, whether, and by what mechanisms, IMST can improve cerebrovascular function is unknown. We hypothesized that IMST would increase cerebrovascular reactivity to hypercapnia (CVR to CO2), which would coincide with changes to the plasma milieu that improve brain endothelial cell function and enhance cognitive performance (NIH Toolbox). We conducted a 6-wk double-blind, randomized, controlled clinical trial investigating high-resistance IMST [75% maximal inspiratory pressure (PImax); 6×/wk; 4 females, 5 males] vs. low-resistance sham training (15% PImax; 6×/wk; 2 females, 5 males) in midlife/older adults (age 50-79 yr) with initial above-normal SBP. Human brain endothelial cells (HBECs) were exposed to participant plasma and assessed for acetylcholine-stimulated nitric oxide (NO) production. CVR to CO2 increased after high-resistance IMST (pre: 1.38 ± 0.66 cm/s/mmHg; post: 2.31 ± 1.02 cm/s/mmHg, P = 0.020). Acetylcholine-stimulated NO production increased in HBECs exposed to plasma from after vs. before the IMST intervention [pre: 1.49 ± 0.33; post: 1.73 ± 0.35 arbitrary units (AU); P < 0.001]. Episodic memory increased modestly after the IMST intervention (pre: 95 ± 13; post: 103 ± 17 AU; P = 0.045). Cerebrovascular and cognitive function were unchanged in the sham control group. High-resistance IMST may be a promising strategy to improve cerebrovascular and cognitive function in midlife/older adults with above-normal SBP, a population at risk for future cognitive decline and dementia.NEW & NOTEWORTHY Midlife/older adults with above-normal blood pressure are at increased risk of developing cognitive decline and dementia. Our findings suggest that high-resistance inspiratory muscle strength training (IMST), a novel, time-efficient (5-10 min/day) form of physical training, may increase cerebrovascular reactivity to CO2 and episodic memory in midlife/older adults with initial above-normal blood pressure.

The Importance of General Surgery Residency Program's Online Presence Varies Among Applicant Groups.

INTRODUCTION: The COVID-19 pandemic necessitated an exclusively virtual 2021 residency application cycle. We hypothesized that residency programs' online presence would have increased utility and influence for applicants. METHODS: Substantial surgery residency website modifications were undertaken in the summer of 2020. Page views were gathered by our institution's information technology office for comparison across years and programs. An anonymous, voluntary, online survey was sent to all interviewed applicants for our 2021 general surgery program match. Five-point Likert-scale questions evaluated applicants' perspective on the online experience. RESULTS: Our residency website received 10,650 page views in 2019 and 12,688 in 2020 (P = 0.14). Page views increased with a greater margin compared to a different specialty residency program's (P < 0.01). From 108 interviewees, 75 completed the survey (69.4%). Respondents indicated our website was satisfactory or very satisfactory compared to other programs (83.9%), and none found it unsatisfactory. Applicants overall stated our institution's online presence impacted their decision to interview (51.6%). Programs' online presence impacted the decision to interview for nonWhite applicants (68%) but significantly less for white applicants (31%, P < 0.03). We observed a trend that those with fewer than this cohort's median interviews (17 or less) put more weight on online presence (65%), compared to those with 18 or greater interviews (35%). CONCLUSIONS: Applicants utilized program websites more during the 2021 virtual application cycle; our data show most applicants depend on institutions' websites to supplement their decision-making; however, there are subgroup differences in the influence online presence has on applicant decisions. Efforts to enhance residency webpages and online resources for candidates may positively influence prospective surgical trainees, and especially those underrepresented in medicine, to decide to interview.

Ignorance is not bliss: evolutionary naiveté in an endangered desert fish and implications for conservation.

Predator naiveté has been invoked to explain the impacts of non-native predators on isolated populations that evolved with limited predation. Such impacts have been repeatedly observed for the endangered Pahrump poolfish, Empetrichthys latos, a desert fish species that evolved in isolation since the end of the Pleistocene. We tested Pahrump poolfish anti-predator responses to conspecific chemical alarm cues released from damaged epidermal tissue in terms of fish activity and water column position. Pahrump poolfish behavioural responses to conspecific alarm cues did not differ from responses to a dechlorinated tap water control. As a positive control, the well-studied fathead minnow, Pimephales promelas, showed significant alarm cue responses in terms of reduced activity and lowered water column position. The density of epidermal club cells, the presumptive source of alarm cues, was significantly lower in Pahrump poolfish relative to fathead minnows. Therefore, anti-predator competence mediated by conspecific alarm cues does not seem to be a component of the ecology of Pahrump poolfish. These findings provide a proximate mechanism for the vulnerability of Pahrump poolfish to non-native predators, with implications for the conservation and management of insular species.

Cetylpyridinium chloride (CPC) reduces zebrafish mortality from influenza infection: Super-resolution microscopy reveals CPC interference with multiple protein interactions with phosphatidylinositol 4,5-bisphosphate in immune function.

The COVID-19 pandemic raises significance for a potential influenza therapeutic compound, cetylpyridinium chloride (CPC), which has been extensively used in personal care products as a positively-charged quaternary ammonium antibacterial agent. CPC is currently in clinical trials to assess its effects on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) morbidity. Two published studies have provided mouse and human data indicating that CPC may alleviate influenza infection, and here we show that CPC (0.1 µM, 1 h) reduces zebrafish mortality and viral load following influenza infection. However, CPC mechanisms of action upon viral-host cell interaction are currently unknown. We have utilized super-resolution fluorescence photoactivation localization microscopy to probe the mode of CPC action. Reduction in density of influenza viral protein hemagglutinin (HA) clusters is known to reduce influenza infectivity: here, we show that CPC (at non-cytotoxic doses, 5-10 µM) reduces HA density and number of HA molecules per cluster within the plasma membrane of NIH-3T3 mouse fibroblasts. HA is known to colocalize with the negatively-charged mammalian lipid phosphatidylinositol 4,5-bisphosphate (PIP2); here, we show that nanoscale co-localization of HA with the PIP2-binding Pleckstrin homology (PH) reporter in the plasma membrane is diminished by CPC. CPC also dramatically displaces the PIP2-binding protein myristoylated alanine-rich C-kinase substrate (MARCKS) from the plasma membrane of rat RBL-2H3 mast cells; this disruption of PIP2 is correlated with inhibition of mast cell degranulation. Together, these findings offer a PIP2-focused mechanism underlying CPC disruption of influenza and suggest potential pharmacological use of this drug as an influenza therapeutic to reduce global deaths from viral disease.

Escherichia coli recombinant expression of SARS-CoV-2 protein fragments.

We have developed a method for the inexpensive, high-level expression of antigenic protein fragments of SARS-CoV-2 proteins in Escherichia coli. Our approach uses the thermophilic family 9 carbohydrate-binding module (CBM9) as an N-terminal carrier protein and affinity tag. The CBM9 module was joined to SARS-CoV-2 protein fragments via a flexible proline-threonine linker, which proved to be resistant to E. coli proteases. Two CBM9-spike protein fragment fusion proteins and one CBM9-nucleocapsid fragment fusion protein largely resisted protease degradation, while most of the CBM9 fusion proteins were degraded at some site in the SARS-CoV-2 protein fragment. All of the fusion proteins were highly expressed in E. coli and the CBM9-ID-H1 fusion protein was shown to yield 122 mg/L of purified product. Three purified CBM9-SARS-CoV-2 fusion proteins were tested and found to bind antibodies directed to the appropriate SARS-CoV-2 antigenic regions. The largest intact CBM9 fusion protein, CBM9-ID-H1, incorporates spike protein amino acids 540-588, which is a conserved region overlapping and C-terminal to the receptor binding domain that is widely recognized by human convalescent sera and contains a putative protective epitope.

Ambient air pollution and non-communicable respiratory illness in sub-Saharan Africa: a systematic review of the literature.

INTRODUCTION: Aerosol pollutants are known to raise the risk of development of non-communicable respiratory diseases (NCRDs) such as asthma, chronic bronchitis, chronic obstructive pulmonary disease, and allergic rhinitis. Sub-Saharan Africa's rapid pace of urbanization, economic expansion, and population growth raise concerns of increasing incidence of NCRDs. This research characterizes the state of research on pollution and NCRDs in the 46 countries of Sub-Saharan Africa (SSA). This research systematically reviewed the literature on studies of asthma; chronic bronchitis; allergic rhinitis; and air pollutants such as particulate matter, ozone, NOx, and sulfuric oxide. METHODS: We searched three major databases (PubMed, Web of Science, and Scopus) using the key words "asthma", "chronic bronchitis", "allergic rhinitis", and "COPD" with "carbon monoxide (CO)", "sulfuric oxide (SO)", "ozone (O3)", "nitrogen dioxide (NO2)", and "particulate matter (PM)", restricting the search to the 46 countries that comprise SSA. Only papers published in scholarly journals with a defined health outcome in individuals and which tested associations with explicitly measured or modelled air exposures were considered for inclusion. All candidate papers were entered into a database for review. RESULTS: We found a total of 362 unique research papers in the initial search of the three databases. Among these, 14 met the inclusion criteria. These papers comprised studies from just five countries. Nine papers were from South Africa; two from Malawi; and one each from Ghana, Namibia, and Nigeria. Most studies were cross-sectional. Exposures to ambient air pollutants were measured using spectrometry and chromatography. Some studies created composite measures of air pollution using a range of data layers. NCRD outcomes were measured by self-reported health status and measures of lung function (spirometry). Populations of interest were primarily schoolchildren, though a few studies focused on secondary school students and adults. CONCLUSIONS: The paucity of research on NCRDs and ambient air pollutant exposures is pronounced within the African continent. While capacity to measure air quality in SSA is high, studies targeting NCRDs should work to draw attention to questions of outdoor air pollution and health. As the climate changes and SSA economies expand and countries urbanize, these questions will become increasingly important.

Fasting prevents hypoxia-induced defects of proteostasis in C. elegans.

Low oxygen conditions (hypoxia) can impair essential physiological processes and cause cellular damage and death. We have shown that specific hypoxic conditions disrupt protein homeostasis in C. elegans, leading to protein aggregation and proteotoxicity. Here, we show that nutritional cues regulate this effect of hypoxia on proteostasis. Animals fasted prior to hypoxic exposure develop dramatically fewer polyglutamine protein aggregates compared to their fed counterparts, indicating that the effect of hypoxia is abrogated. Fasting also reduced the hypoxia-induced exaggeration of proteostasis defects in animals that express Aß1-42 and in animals with a temperature-sensitive mutation in dyn-1, suggesting that this effect was not specific to polyglutamine proteins. Our data also demonstrate that the nutritional environment experienced at the onset of hypoxia dictates at least some aspects of the physiological response to hypoxia. We further demonstrate that the insulin/IGF-like signaling pathway plays a role in mediating the protective effects of fasting in hypoxia. Animals with mutations in daf-2, the C. elegans insulin-like receptor, display wild-type levels of hypoxia-induced protein aggregation upon exposure to hypoxia when fed, but are not protected by fasting. DAF-2 acts independently of the FOXO transcription factor, DAF-16, to mediate the protective effects of fasting. These results suggest a non-canonical role for the insulin/IGF-like signaling pathway in coordinating the effects of hypoxia and nutritional state on proteostasis.

The structure of a family 110 glycoside hydrolase provides insight into the hydrolysis of α-1,3-galactosidic linkages in λ-carrageenan and blood group antigens.

α-Linked galactose is a common carbohydrate motif in nature that is processed by a variety of glycoside hydrolases from different families. Terminal Galα1-3Gal motifs are found as a defining feature of different blood group and tissue antigens, as well as the building block of the marine algal galactan λ-carrageenan. The blood group B antigen and linear α-Gal epitope can be processed by glycoside hydrolases in family GH110, whereas the presence of genes encoding GH110 enzymes in polysaccharide utilization loci from marine bacteria suggests a role in processing λ-carrageenan. However, the structure-function relationships underpinning the α-1,3-galactosidase activity within family GH110 remain unknown. Here we focus on a GH110 enzyme (PdGH110B) from the carrageenolytic marine bacterium Pseudoalteromonas distincta U2A. We showed that the enzyme was active on Galα1-3Gal but not the blood group B antigen. X-ray crystal structures in complex with galactose and unhydrolyzed Galα1-3Gal revealed the parallel ß-helix fold of the enzyme and the structural basis of its inverting catalytic mechanism. Moreover, an examination of the active site reveals likely adaptations that allow accommodation of fucose in blood group B active GH110 enzymes or, in the case of PdGH110, accommodation of the sulfate groups found on λ-carrageenan. Overall, this work provides insight into the first member of a predominantly marine clade of GH110 enzymes while also illuminating the structural basis of α-1,3-galactoside processing by the family as a whole.

Mendelian Randomization Study of Obesity and Cerebrovascular Disease.

OBJECTIVE: To systematically investigate causal relationships between obesity and cerebrovascular disease and the extent to which hypertension and hyperglycemia mediate the effect of obesity on cerebrovascular disease. METHODS: We used summary statistics from genome-wide association studies for body mass index (BMI), waist-to-hip ratio (WHR), and multiple cerebrovascular disease phenotypes. We explored causal associations with 2-sample Mendelian randomization (MR) accounting for genetic covariation between BMI and WHR, and we assessed what proportion of the association between obesity and cerebrovascular disease was mediated by systolic blood pressure (SBP) and blood glucose levels, respectively. RESULTS: Genetic predisposition to higher BMI did not increase the risk of cerebrovascular disease. In contrast, for each 10% increase in WHR there was a 75% increase (95% confidence interval [CI] = 44-113%) in risk for large artery ischemic stroke, a 57% (95% CI = 29-91%) increase in risk for small vessel ischemic stroke, a 197% increase (95% CI = 59-457%) in risk of intracerebral hemorrhage, and an increase in white matter hyperintensity volume (ß = 0.11, 95% CI = 0.01-0.21). These WHR associations persisted after adjusting for genetic determinants of BMI. Approximately one-tenth of the observed effect of WHR was mediated by SBP for ischemic stroke (proportion mediated: 12%, 95% CI = 4-20%), but no evidence of mediation was found for average blood glucose. INTERPRETATION: Abdominal adiposity may trigger causal pathological processes, partially independent from blood pressure and totally independent from glucose levels, that lead to cerebrovascular disease. Potential targets of these pathological processes could represent novel therapeutic opportunities for stroke. ANN NEUROL 2020;87:516-524.

Effects of polymerization of casein and sources of lysine on amino acid bioavailability among calves fed liquid-based diets.

Two experiments were conducted to evaluate the bioavailability of AA between polymerized and less polymerized or unpolymerized sources of AA. In the first experiment, 6 bull calves (53.8 ± 0.6 kg of body weight) were bottle-fed milk replacer that contained 0, 60, or 120 additional grams of AA from casein or acid hydrolyzed casein every 12 h. Plasma essential AA increased linearly with increasing intake of casein from either source. Branched-chain amino acids accounted for 74% of increases in essential AA, regardless of source of AA. Concentrations of nonessential AA increased linearly with increased intake of AA from acid hydrolyzed casein but only tended to increase in response to casein. Also, the rate of increase in total plasma AA concentration in response to acid hydrolyzed casein (4.3 µM increase per g of supplemental AA) tended to be 145% greater than casein (3.0 µM per g of supplemental AA). In a separate experiment, 6 additional bull calves (52.1 ± 0.9 kg of body weight) were bottle-fed milk replacer that contained 0, 4.8, or 9.6 additional grams of Lys from ε-polylysine or Lys-HCl each 12 h to measure Lys bioavailability between a polymerized and unpolymerized source of Lys. Plasma Lys concentrations increased linearly in response to greater Lys intake from Lys-HCl (slope = 13.51 µM/g Lys,), but plasma Lys concentrations did not change in response to increased intake of Lys from ε-polylysine. Plasma concentrations of Thr, Met, Glu, and Gln decreased linearly with increasing ε-polylysine intake, whereas concentrations of His, Val, Leu, and Ile increased linearly with increasing ε-polylysine intake. Data from these experiments suggest that the form of AA provided to calves should be considered when formulating diets to meet AA requirements.

Problematic substance use: an assessment of workplace implications in midwifery.

BACKGROUND: Problematic substance use (PSU) poses occupational, personal and professional risks. As an occupational group, midwives have been under-represented in research on PSU. AIMS: The aim of this study was to assess self-reported occurrences of PSU, help-seeking behaviours and barriers, and perceptions of impairment in UK-based midwives. METHODS: Self-selecting registered midwives were anonymously surveyed using the Tobacco, Alcohol, Prescription Medications, and Substance Use/Misuse (TAPS) tool, the Perceptions of Nursing Impairment Inventory (PNII) and open-ended/closed questions. Quantitative data were used to explore PSU, help-seeking and attitudes to impairment. Qualitative responses were used to provide richer understandings. RESULTS: From 623 completed surveys, 28% (n = 176) self-reported PSU in response to work-related stress and anxiety, bullying, traumatic clinical incidents and maintenance of overall functioning. PSU was related to alcohol and a range of restricted drugs. While 11% of those affected indicated they had sought help, 27% felt they should seek help but did not. Reported barriers to help-seeking included fear of repercussions, shame, stigma, practicalities and a perceived lack of support either available or required. Perceptions of impairment were predominantly compassionate with a minority of stigmatizing attitudes displayed. CONCLUSIONS: Overall, 10% of the sample reported they had attended work under the influence of alcohol, and 6% under the influence of drugs other than tobacco or those as prescribed to them. Furthermore, 37% indicated concern about a colleague's substance use. As stigmatizing attitudes and punitive actions can dissuade help-seeking, changed perceptions and policies which favour alternatives to discipline are suggested to reduce the risk overall.

Towards Intraoperative Quantification of Atrial Fibrosis Using Light-Scattering Spectroscopy and Convolutional Neural Networks.

Light-scattering spectroscopy (LSS) is an established optical approach for characterization of biological tissues. Here, we investigated the capabilities of LSS and convolutional neural networks (CNNs) to quantitatively characterize the composition and arrangement of cardiac tissues. We assembled tissue constructs from fixed myocardium and the aortic wall with a thickness similar to that of the atrial free wall. The aortic sections represented fibrotic tissue. Depth, volume fraction, and arrangement of these fibrotic insets were varied. We gathered spectra with wavelengths from 500-1100 nm from the constructs at multiple locations relative to a light source. We used single and combinations of two spectra for training of CNNs. With independently measured spectra, we assessed the accuracy of the CNNs for the classification of tissue constructs from single spectra and combined spectra. Combined spectra, including the spectra from fibers distal from the illumination fiber, typically yielded the highest accuracy. The maximal classification accuracy of the depth detection, volume fraction, and permutated arrangements was (mean ± standard deviation (stddev)) 88.97 ± 2.49%, 76.33 ± 1.51%, and 84.25 ± 1.88%, respectively. Our studies demonstrate the reliability of quantitative characterization of tissue composition and arrangements using a combination of LSS and CNNs. The potential clinical applications of the developed approach include intraoperative quantification and mapping of atrial fibrosis, as well as the assessment of ablation lesions.

Interobserver reliability of Feline Dermatitis Extent and Severity Index (FEDESI) and Scoring Feline Allergic Dermatitis (SCORFAD) and the relationship between lesion scores and pruritus.

BACKGROUND: Feline Dermatitis Extent and Severity Index (FEDESI) and Scoring Feline Allergic Dermatitis (SCORFAD) are scales used to assess lesion severity in cats with allergic dermatitis. Interobserver reliability has not been assessed for either. HYPOTHESIS AND OBJECTIVES: To determine interobserver reliability for FEDESI and SCORFAD, and the relationship between lesion scores and pruritus. ANIMALS: Thirty-eight cats presenting for pruritus. METHODS AND MATERIALS: Each cat's lesions were scored by two observers at each visit using both FEDESI and SCORFAD (n = 117 paired observations). Spearman's rho was calculated to assess correlation between scales and between each scale and the owner-reported pruritus Visual Analog Scale (pVAS). Concordance correlation coefficients were calculated between observers for each scale, and Bland-Altman plots were used to visually represent the relationship between paired scores. RESULTS: FEDESI and SCORFAD were strongly positively correlated with one another (rho = 0.84, P < 0.001). Each scale showed fair correlation with pVAS (rho = 0.42, P < 0.001; rho = 0.38, P < 0.001, respectively). There was good concordance between observers for both scales, with a correlation coefficient of 0.77 for FEDESI and 0.80 for SCORFAD [intraclass correlation coefficient (ICC) 95%, confidence interval (CI) 0.69-0.83; ICC 95%, CI 0.72-0.86, respectively]. Median lesion score was low (FEDESI 20; SCORFAD 4), which may improve interobserver reliability. CONCLUSIONS AND CLINICAL IMPORTANCE: There is good interobserver reliability for both FEDESI and SCORFAD. FEDESI and SCORFAD are positively correlated with one another and with pVAS. These findings support use of both scales in clinical research and assessment.

Estimating Arterial Cyclic Strain from the Spacing of Endothelial Nuclei.

BACKGROUND: The non-uniform distribution of atherosclerosis within the arterial system is widely attributed to variation in haemodynamic wall shear stress. It may also depend on variation in pressure-induced stresses and strains within the arterial wall; these have been less widely investigated, at least in part because of a lack of suitable techniques. OBJECTIVES: Here we show that local arterial strain can be determined from impressions left by endothelial cells on the surface of vascular corrosion casts made at different pressures, even though only one pressure can be examined in each vessel. The pattern of pits in the cast caused by protruding endothelial nuclei was subject to "retro-deformation" to identify the pattern that would have occurred in the absence of applied stresses. METHODS: Retaining the nearest-neighbour pairs found under this condition, changes in nearest-neighbour vectors were calculated for the pattern seen in the cast, and the ratio of mean changes at different pressures determined. This approach removes errors in simple nearest-neighbour analyses caused by the nearest neighbour changing as deformation occurs. RESULTS: The accuracy, precision and robustness of the approach were validated using simulations. The method was implemented using confocal microscopy of casts of the rabbit aorta made at systolic and diastolic pressures; results agreed well with the ratio of the macroscopic dimensions of the casts. CONCLUSIONS: Applying the new technique to areas around arterial branches could support or refute the hypothesis that the development of atherosclerosis is influenced by mural strain, and the method may be applicable to other tissues.