Automated SEM analysis of particles in Hanford tank waste.

Multiple bench-scale filtration campaigns of Hanford tank waste supernatant on a backpulseable dead-end filtration skid have provided greater insight into the solids that cause fouling and reduce filter performance. The solids collected during each campaign were concentrated from the backpulse solutions and examined using automated particle analysis (APA) methods with scanning electron microscopy and X-ray energy dispersive spectroscopy to categorize particle types and their morphological characteristics. We show that with APA, thousands of particles can be analyzed to provide accurate insight into the phases that may be impacting filter performance.

Control of three-carbon amino acid homeostasis by promiscuous importers and exporters in Bacillus subtilis: role of the "sleeping beauty" amino acid exporters.

The Gram-positive model bacterium Bacillus subtilis can acquire amino acids by import, de novo biosynthesis, or degradation of proteins and peptides. The accumulation of several amino acids inhibits the growth of B. subtilis, probably due to misincorporation into cellular macromolecules such as proteins or peptidoglycan or due to interference with other amino acid biosynthetic pathways. Here, we studied the adaptation of B. subtilis to toxic concentrations of the three-carbon amino acids L-alanine, ß-alanine, and 2,3-diaminopropionic acid, as well as the two-carbon amino acid glycine. Resistance to the non-proteinogenic amino acid ß-alanine, which is a precursor for coenzyme A biosynthesis, is achieved by mutations that either activate a cryptic amino acid exporter, AexA (previously YdeD), or inactivate the amino acid importers AimA, AimB (previously YbxG), and BcaP. The aexA gene is very poorly expressed under most conditions studied. However, mutations affecting the transcription factor AerA (previously YdeC) can result in strong constitutive aexA expression. AexA is the first characterized member of a group of amino acid exporters in B. subtilis, which are all very poorly expressed. Therefore, we suggest to call this group "sleeping beauty amino acid exporters." 2,3-Diaminopropionic acid can also be exported by AexA, and this amino acid also seems to be a natural substrate of AerA/AexA, as it can cause a slight but significant induction of aexA expression, and AexA also provides some natural resistance toward 2,3-diaminopropionic acid. Moreover, our work shows how low-specificity amino acid transporters contribute to amino acid homeostasis in B. subtilis.IMPORTANCEEven though Bacillus subtilis is one of the most-studied bacteria, amino acid homeostasis in this organism is not fully understood. We have identified import and export systems for the C2 and C3 amino acids. Our work demonstrates that the responsible amino acid permeases contribute in a rather promiscuitive way to amino acid uptake. In addition, we have discovered AexA, the first member of a group of very poorly expressed amino acid exporters in B. subtilis that we call "sleeping beauty amino acid exporters." The expression of these transporters is typically triggered by mutations in corresponding regulator genes that are acquired upon exposure to toxic amino acids. These exporters are ubiquitous in all domains of life. It is tempting to speculate that many of them are not expressed until the cells experience selective pressure by toxic compounds, and they protect the cells from rare but potentially dangerous encounters with such compounds.

Clinicopathological Predictors of Positive Resection Margins in Breast-Conserving Surgery.

BACKGROUND: Ductal carcinoma in situ (DCIS) is associated with risk of positive resection margins following breast-conserving surgery (BCS) and subsequent reoperation. Prior reports grossly underestimate the risk of margin positivity with IBC containing a DCIS component (IBC + DCIS) due to patient-level rather than margin-level analysis. OBJECTIVE: The aim of this study was to delineate the relative risk of IBC + DCIS compared with pure IBC (without a DCIS component) on margin positivity through detailed margin-level interrogation. METHODS: A single institution, retrospective, observational cohort study was conducted in which pathology databases were evaluated to identify patients who underwent BCS over 5 years (2014-2019). Margin-level interrogation included granular detail into the extent, pathological subtype and grade of disease at each resection margin. Predictors of a positive margin were computed using multivariate regression analysis. RESULTS: Clinicopathological details were examined from 5454 margins from 909 women. The relative risk of a positive margin with IBC + DCIS versus pure IBC was 8.76 (95% confidence interval [CI] 6.64-11.56) applying UK Association of Breast Surgery guidelines, and 8.44 (95% CI 6.57-10.84) applying the Society of Surgical Oncology/American Society for Radiation Oncology guidelines. Independent predictors of margin positivity included younger patient age (0.033, 95% CI 0.006-0.060), lower specimen weight (0.045, 95% CI 0.020-0.069), multifocality (0.256, 95% CI 0.137-0.376), lymphovascular invasion (0.138, 95% CI 0.068-0.208) and comedonecrosis (0.113, 95% CI 0.040-0.185). CONCLUSIONS: Compared with pure IBC, the relative risk of a positive margin with IBC + DCIS is approximately ninefold, significantly higher than prior estimates. This margin-level methodology is believed to represent the impact of DCIS more accurately on margin positivity in IBC.

Mastitis and Mammary Abscess Management Audit (MAMMA) in the UK and Ireland.

BACKGROUND: The aim of this multicentre prospective audit was to describe the current practice in the management of mastitis and breast abscesses in the UK and Ireland, with a specific focus on rates of surgical intervention. METHODS: This audit was conducted in two phases from August 2020 to August 2021; a phase 1 practice survey and a phase 2 prospective audit. Primary outcome measurements for phase 2 included patient management pathway characteristics and treatment type (medical/radiological/surgical). RESULTS: A total of 69 hospitals participated in phase 2 (1312 patients). The key findings were a high overall rate of incision and drainage (21.0 per cent) and a lower than anticipated proportion of ultrasound-guided aspiration of breast abscesses (61.0 per cent). Significant variations were observed regarding the rate of incision and drainage (range 0-100 per cent; P < 0.001) and the rate of needle aspiration (range 12.5-100 per cent; P < 0.001) between individual units. Overall, 22.5 per cent of patients were admitted for inpatient treatment, out of whom which 72.9 per cent were commenced on intravenous antibiotics. The odds of undergoing incision and drainage for a breast abscess or being admitted for inpatient treatment were significantly higher if patients presented at the weekend compared with a weekday (P ≤ 0.023). Breast specialists reviewed 40.9 per cent of all patients directly, despite the majority of patients (74.2 per cent) presenting within working hours on weekdays. CONCLUSIONS: Variation in practice exists in the management of mastitis and breast abscesses, with high rates of incision and drainage in certain regions of the UK. There is an urgent need for a national best-practice toolbox to minimize practice variation and standardize patient care.

Mastitis and breast abscess is a painful infection of the breast. It is an extremely common breast problem. One in three women can get this condition at some stage in their life. To treat a breast abscess, the pus inside should be drained out of the body. This can be done either by cutting into the breast using surgery or by inserting a fine needle using an ultrasonography scan (which uses ultrasound). Fine-needle drainage has the benefit that it does not require admission to hospital. Surgery can cause the breast to look misshapen. It is unknown which method is used more often in the UK and Ireland. The aim of this study was to describe how mastitis and breast abscesses are treated in the UK and Ireland. This study involved a survey of practice (phase 1) and collection of data, which are routinely recorded for these patients (phase 2). This study involved 69 hospitals and 1312 patient records. One in five women had an operation for a breast abscess. This was higher than expected. Six in 10 women had a pus drainage using a fine needle. The chance of having an operation depended on the hospital. Women that came to hospital at the weekend were almost twice as likely to have an operation. One in five women were admitted to hospital. The chances of that more than doubled if a woman came to hospital at the weekend. There are differences in treatment of mastitis and breast abscesses across the UK and Ireland. Changes need to be put in place to make access to treatment more equal.

Insights into the Roles of Lipoteichoic Acids and MprF in Bacillus subtilis.

Gram-positive bacterial cells are protected from the environment by a cell envelope that is comprised of a thick layer of peptidoglycan that maintains cell shape and teichoic acid polymers whose biological function remains unclear. In Bacillus subtilis, the loss of all class A penicillin-binding proteins (aPBPs), which function in peptidoglycan synthesis, is conditionally lethal. Here, we show that this lethality is associated with an alteration of lipoteichoic acids (LTAs) and the accumulation of the major autolysin LytE in the cell wall. Our analysis provides further evidence that the length and abundance of LTAs act to regulate the cellular level and activity of autolytic enzymes, specifically LytE. Importantly, we identify a novel function for the aminoacyl-phosphatidylglycerol synthase MprF in the modulation of LTA biosynthesis in both B. subtilis and Staphylococcus aureus. This finding has implications for our understanding of antimicrobial resistance (particularly to daptomycin) in clinically relevant bacteria and the involvement of MprF in the virulence of pathogens such as methicillin-resistant S. aureus (MRSA). IMPORTANCE In Gram-positive bacteria such as Bacillus subtilis and Staphylococcus aureus, the cell envelope is a structure that protects the cells from the environment but is also dynamic in that it must be modified in a controlled way to allow cell growth. In this study, we show that lipoteichoic acids (LTAs), which are anionic polymers attached to the membrane, have a direct role in modulating the cellular abundance of cell wall-degrading enzymes. We also find that the apparent length of the LTA is modulated by the activity of the enzyme MprF, previously implicated in modifications of the cell membrane leading to resistance to antimicrobial peptides. These findings are important contributions to our understanding of how bacteria balance cell wall synthesis and degradation to permit controlled growth and division. These results also have implications for the interpretation of antibiotic resistance, particularly for the clinical treatment of MRSA infections.

Nipple aspirate fluid and its use for the early detection of breast cancer.

Nipple aspirate fluid is the physiological biofluid lining ductal epithelial cells. Historically, cytology of nipple fluid has been the gold standard diagnostic method for assessment of ductal fluid in patients with symptomatic nipple discharge. The role of biomarker discovery in nipple aspirate fluid for assessment of asymptomatic and high-risk patients is highly attractive but evaluation to date is limited by poor diagnostic accuracy. However, the emergence of new technologies capable of identifying metabolites that have been previously thought unidentifiable within such small volumes of fluid, has enabled testing of nipple biofluid to be re-examined. This review evaluates the use of new technologies to evaluate the components of nipple fluid and their potential to serve as biomarkers in screening.

The Effects of Commonly Consumed Dietary Fibres on the Gut Microbiome and Its Fibre Fermentative Capacity in Adults with Inflammatory Bowel Disease in Remission.

Introduction: It has been suggested that the gut microbiome of patients with inflammatory bowel disease (IBD) is unable to ferment dietary fibre. This project explored the in vitro effect of fibre fermentation on production of short-chain fatty acids (SCFA) and on microbiome composition. Methods: Faecal samples were collected from 40 adults (>16 y) with IBD (n = 20 with Crohn's disease and n = 20 with ulcerative colitis) in clinical remission and 20 healthy controls (HC). In vitro batch culture fermentations were carried out using as substrates maize starch, apple pectin, raftilose, wheat bran, α cellulose and a mixture of these five fibres. SCFA concentration (umol/g) was quantified with gas chromatography and microbiome was profiled with 16S rRNA sequencing. Results: Fibre fermentation did not correct the baseline microbial dysbiosis or lower diversity seen in either patients with CD or UC. For all fibres, up to 51% of baseline ASVs or genera changed in abundance in HC. In patients with IBD, fermentation of fibre substrates had no effect on species or genera abundance. Production of SCFA varied among the different fibre substrates but this was not different between the two IBD groups and compared to HC after either 5 or 24 h fermentation. Conclusions: Despite extensive microbial dysbiosis, patients with IBD have a similar capacity to ferment fibre and release SCFA as HC. Fibre supplementation alone may be unlikely to restore to a healthy status the compositional shifts characteristic of the IBD microbiome.

Experimental evaluation of respiratory droplet spread to rooms connected by a central ventilation system.

This article presents results from an experimental study to ascertain the transmissibility of the SARS-CoV-2 virus between rooms in a building that are connected by a central ventilation system. Respiratory droplet surrogates made of mucus and virus mimics were released in one room in a test building, and measurements of concentration levels were made in other rooms connected via the ventilation system. The paper presents experimental results for different ventilation system configurations, including ventilation rate, filtration level (up to MERV-13), and fractional outdoor air intake. The most important finding is that respiratory droplets can and do transit through central ventilation systems, suggesting a mechanism for viral transmission (and COVID-19 specifically) within the built environment in reasonable agreement with well-mixed models. We also find the deposition of small droplets (0.5-4 µm) on room walls to be negligibly small.

Neuroenhancement of surgeons during robotic suturing.

BACKGROUND: The initial phases of robotic surgical skills acquisition are associated with poor technical performance, such as low knot-tensile strength (KTS). Transcranial direct-current stimulation (tDCS) can improve force and accuracy in motor tasks but research in surgery is limited to open and laparoscopic tasks in students. More recently, robotic surgery has gained traction and is now the most common approach for certain procedures (e.g. prostatectomy). Early-phase robotic suturing performance is dependent on prefrontal cortex (PFC) activation, and this study aimed to determine whether performance can be improved with prefrontal tDCS. METHODS: Fifteen surgical residents were randomized to either active then sham tDCS or sham then active tDCS, in two counterbalanced sessions in a double-blind crossover study. Within each session, participants performed a robotic suturing task repeated in three blocks: pre-, intra- and post-tDCS. During the intra-tDCS block, participants were randomized to either active tDCS (2 mA for 15 min) to the PFC or sham tDCS. Primary outcome measures of technical quality included KTS and error scores. RESULTS: Significantly faster completion times were observed longitudinally, regardless of active (p < 0.001) or sham stimulation (p < 0.001). KTS was greater following active compared to sham stimulation (median: active = 44.35 N vs. sham = 27.12 N, p < 0.001). A significant reduction in error scores from "pre-" to "post-" (p = 0.029) were only observed in the active group. CONCLUSION: tDCS could reduce error and enhance KTS during robotic suturing and warrants further exploration as an adjunct to robotic surgical training.

Corrigendum to "A novel mechanism of inhibiting in-stent restenosis with arsenic trioxide drug-eluting stent: Enhancing contractile phenotype of vascular smooth muscle cells via YAP pathway" [Bioact. Mater. 6 2 (February 2021) 375-385].

[This corrects the article DOI: 10.1016/j.bioactmat.2020.08.018.].

Diagnostic Accuracy of Nipple Discharge Fluid Cytology: A Meta-Analysis and Systematic Review of the Literature.

BACKGROUND: Nipple discharge is the third most frequent complaint of women attending rapid diagnostic breast clinics. Nipple smear cytology remains the single most used diagnostic method for investigating fluid content. This study aimed to conduct a systematic review and meta-analysis of the diagnostic accuracy of nipple discharge fluid assessment. METHODS: The study incorporated searches for studies interrogating the diagnostic data of nipple discharge fluid cytology compared with the histopathology gold standard. Data from studies published from 1956 to 2019 were analyzed. The analysis included 8648 cytology samples of women with a presenting complaint of nipple discharge. Both hierarchical and bivariate models for diagnostic meta-analysis were used to attain overall pooled sensitivity and specificity. RESULTS: Of 837 studies retrieved, 45 fulfilled the criteria for inclusion. The diagnostic accuracy of the meta-analysis examining nipple discharge fluid had a sensitivity of 75 % (95 % confidence interval [CI], 0.74-0.77) and a specificity of 87 % (95 % CI, 0.86-0.87) for benign breast disease. For breast cancer, it had a sensitivity of 62 % (95 % CI, 0.53-0.71) and a specificity 71 % (95 % CI, 0.57-0.81). Furthermore, patients presenting with blood-stained discharge yielded an overall malignancy rate of 58 % (95 % CI, 0.54-0.60) with a positive predictive value (PPV) of 27 % (95 % CI, 0.17-0.36). CONCLUSIONS: Pooled data from studies encompassing nipple discharge fluid assessment suggest that nipple smear cytology is of limited diagnostic accuracy. The authors recommend that a tailored approach to diagnosis be required given the variable sensitivities of currently available tests.

Gossypol acetate: A natural polyphenol derivative with antimicrobial activities against the essential cell division protein FtsZ.

Antimicrobial resistance has attracted worldwide attention and remains an urgent issue to resolve. Discovery of novel compounds is regarded as one way to circumvent the development of resistance and increase the available treatment options. Gossypol is a natural polyphenolic aldehyde, and it has attracted increasing attention as a possible antibacterial drug. In this paper, we studied the antimicrobial properties (minimum inhibitory concentrations) of gossypol acetate against both Gram-positive and Gram-negative bacteria strains and dig up targets of gossypol acetate using in vitro assays, including studying its effects on functions (GTPase activity and polymerization) of Filamenting temperature sensitive mutant Z (FtsZ) and its interactions with FtsZ using isothermal titration calorimetry (ITC), and in vivo assays, including visualization of cell morphologies and proteins localizations using a microscope. Lastly, Bacterial membrane permeability changes were studied, and the cytotoxicity of gossypol acetate was determined. We also estimated the interactions of gossypol acetate with the promising target. We found that gossypol acetate can inhibit the growth of Gram-positive bacteria such as the model organism Bacillus subtilis and the pathogen Staphylococcus aureus [both methicillin-sensitive (MSSA) and methicillin-resistant (MRSA)]. In addition, gossypol acetate can also inhibit the growth of Gram-negative bacteria when the outer membrane is permeabilized by Polymyxin B nonapeptide (PMBN). Using a cell biological approach, we show that gossypol acetate affects cell division in bacteria by interfering with the assembly of the cell division FtsZ ring. Biochemical analysis shows that the GTPase activity of FtsZ was inhibited and polymerization of FtsZ was enhanced in vitro, consistent with the block to cell division in the bacteria tested. The binding mode of gossypol acetate in FtsZ was modeled using molecular docking and provides an understanding of the compound mode of action. The results point to gossypol (S2303) as a promising antimicrobial compound that inhibits cell division by affecting FtsZ polymerization and has potential to be developed into an effective antimicrobial drug by chemical modification to minimize its cytotoxic effects in eukaryotic cells that were identified in this work.

Regulatory dissection of the severe COVID-19 risk locus introgressed by Neanderthals

Individuals infected with the SARS-CoV-2 virus present with a wide variety of phenotypes ranging from asymptomatic to severe and even lethal outcomes. Past research has revealed a genetic haplotype on chromosome 3 that entered the human population via introgression from Neanderthals as the strongest genetic risk factor for the severe COVID-19 phenotype. However, the specific variants along this introgressed haplotype that contribute to this risk and the biological mechanisms that are involved remain unclear. Here, we assess the variants present on the risk haplotype for their likelihood of driving the severe COVID-19 phenotype. We do this by first exploring their impact on the regulation of genes involved in COVID-19 infection using a variety of population genetics and functional genomics tools. We then perform an locus-specific massively parallel reporter assay to individually assess the regulatory potential of each allele on the haplotype in a multipotent immune-related cell line. We ultimately reduce the set of over 600 linked genetic variants to identify 4 introgressed alleles that are strong functional candidates for driving the association between this locus and severe COVID-19. These variants likely drive the locus impact on severity by putatively modulating the regulation of two critical chemokine receptor genes: CCR1 and CCR5. These alleles are ideal targets for future functional investigations into the interaction between host genomics and COVID-19 outcomes.

Intronic regulation of SARS-CoV-2 receptor (ACE2) expression mediated by immune signaling and oxidative stress pathways

The angiotensin-converting enzyme 2 (ACE2) protein has been highly studied as a key catalytic regulator of the renin-angiotensin system (RAS), involved in fluid homeostasis and blood pressure modulation. In addition to its important physiological role as a broadly-expressed membrane-bound protein, ACE2 serves as a cell-surface receptor for some viruses - most notably, coronaviruses such as SARS-CoV and SARS-CoV-2. Differing levels of ACE2 expression may impact viral susceptibility and subsequent changes to expression may be a pathogenic mechanism of disease risk and manifestation. Therefore, an improved understanding of how ACE2 expression is regulated at the genomic and transcriptional level may help us understand not only how the effects of pre-existing conditions (e.g., chronic obstructive pulmonary disease) may manifest with increased COVID-19 incidence, but also the mechanisms that regulate ACE2 levels following viral infection. Here, we initially perform bioinformatic analyses of several datasets to generate hypotheses about ACE2 gene-regulatory mechanisms in the context of immune signaling and chronic oxidative stress. We then identify putative non-coding regulatory elements within ACE2 intronic regions as potential determinants of ACE2 expression activity. We perform functional validation of our computational predictions in vitro via targeted CRISPR-Cas9 deletions of the identified ACE2 cis-regulatory elements in the context of both immunological stimulation and oxidative stress conditions. We demonstrate that intronic ACE2 regulatory elements are responsive to both immune signaling and oxidative-stress pathways, and this contributes to our understanding of how expression of this gene may be modulated at both baseline and during immune challenge. Our work supports the further pursuit of these putative mechanisms in our understanding, prevention, and treatment of infection and disease caused by ACE2-utilizing viruses such as SARS-CoV, SARS-CoV-2, and future emerging SARS-related viruses. Author SummaryThe recent emergence of the virus SARS-CoV-2 which has caused the COVID-19 pandemic has prompted scientists to intensively study how the virus enters human host cells. This work has revealed a key protein, ACE2, that acts as a receptor permitting the virus to infect cells. Much research has focused on how the virus physically interacts with ACE2, yet little is known on how ACE2 is turned on or off in human cells at the level of the DNA molecule. Understanding this level of regulation may offer additional ways to prevent or lower viral entry into human hosts. Here, we have examined the control of the ACE2 gene, the DNA sequence that instructs ACE2 protein receptor formation, and we have done so in the context of immune stimulation. We have indeed identified a number of DNA on/off switches for ACE2 that appear responsive to immuno-logical and oxidative stress. These switches may fine-tune how ACE2 is turned on or off before, during, and/or after infection by SARS-CoV-2 or other related coronaviruses. Our studies help pave the way for additional functional studies on these switches, and their potential therapeutic targeting in the future.

A novel mechanism of inhibiting in-stent restenosis with arsenic trioxide drug-eluting stent: Enhancing contractile phenotype of vascular smooth muscle cells via YAP pathway.

OBJECTIVE: Arsenic trioxide (ATO or As2O3) has beneficial effects on suppressing neointimal hyperplasia and restenosis, but the mechanism is still unclear. The goal of this study is to further understand the mechanism of ATO's inhibitory effect on vascular smooth muscle cells (VSMCs). METHODS AND RESULTS: Through in vitro cell culture and in vivo stent implanting into the carotid arteries of rabbit, a synthetic-to-contractile phenotypic transition was induced and the proliferation of VSMCs was inhibited by ATO. F-actin filaments were clustered and the elasticity modulus was increased within the phenotypic modulation of VSMCs induced by ATO in vitro. Meanwhile, Yes-associated protein (YAP) nuclear translocation was inhibited by ATO both in vivo and in vitro. It was found that ROCK inhibitor or YAP inactivator could partially mask the phenotype modulation of ATO on VSMCs. CONCLUSIONS: The interaction of YAP with the ROCK pathway through ATO seems to mediate the contractile phenotype of VSMCs. This provides an indication of the clinical therapeutic mechanism for the beneficial bioactive effect of ATO-drug eluting stent (AES) on in-stent restenosis (ISR).

Diagnostic Accuracy of Nipple Aspirate Fluid Cytology in Asymptomatic Patients: A Meta-analysis and Systematic Review of the Literature.

PURPOSE: To calculate the diagnostic accuracy of nipple aspirate fluid (NAF) cytology. BACKGROUND: Evaluation of NAF cytology in asymptomatic patients conceptually offers a non-invasive method for either screening for breast cancer or else predicting or stratifying future cancer risk. METHODS: Studies were identified by performing electronic searches up to August 2019. A meta-analysis was conducted to attain an overall pooled sensitivity and specificity of NAF for breast cancer detection. RESULTS: A search through 938 studies yielded a total of 19 studies. Overall, 9308 patients were examined, with cytology results from 10,147 breasts [age (years), mean ± SD = 49.73 ± 4.09 years]. Diagnostic accuracy meta-analysis of NAF revealed a pooled specificity of 0.97 (95% CI 0.97-0.98), and sensitivity of 0.64 (95% CI 0.62-0.66). CONCLUSIONS: The diagnostic accuracy of nipple smear cytology is limited by poor sensitivity. If nipple fluid assessment is to be used for diagnosis, then emerging technologies for fluid biomarker analysis must supersede the current diagnostic accuracy of NAF cytology.