Serum levels of hydroxylated metabolites of arachidonic acid cross-sectionally and longitudinally predict knee pain progression: an observational cohort study.

OBJECTIVE: To examine associations between serum oxylipins, which regulate tissue repair and pain signalling, and knee pain/radiographic osteoarthritis (OA) at baseline and knee pain at 3 year follow-up. METHOD: Baseline, and 3 year follow-up, knee pain phenotypes were assessed from 154 participants in the Knee Pain in the Community (KPIC) cohort study. Serum and radiographic Kellgren and Lawrence (KL) and Nottingham line drawing atlas OA scores were collected at baseline. Oxylipin levels were quantified using liquid chromatography coupled with mass spectrometry. Associations were measured by linear regression and receiver operating characteristics (ROC). RESULTS: Serum levels of 8,9-epoxyeicosatrienoic acid (EET) (ß(95% confidence intervals (CI)) = 1.809 (-0.71 to 2.91)), 14,15-dihydroxyeicosatrienoic acid (DHET) (ß(95%CI) = 0.827 (0.34-1.31)), and 12-hydroxyeicosatetraenoic acid (HETE) (ß(95%CI) = 4.090 (1.92-6.26)) and anandamide (ß(95%CI) = 3.060 (1.35-4.77)) were cross-sectionally associated with current self-reported knee pain scores (numerical rating scale (NRS) item 3, average pain). Serum levels of 9- (ß(95%CI) = 0.467 (0.18-0.75)) and 15-HETE (ß(95%CI) = 0.759 (0.29-1.22)), 14-hydroxydocosahexaenoic acid (ß(95%CI) = 0.483(0.24-0.73)), and the ratio of 8,9-EET:DHET (ß(95%CI) = 0.510(0.19-0.82)) were cross-sectionally associated with KL scores. Baseline serum concentrations of 8,9-EET (ß(95%CI) = 2.166 (0.89-3.44)), 5,6-DHET (ß(95%CI) = 152.179 (69.39-234.97)), and 5-HETE (ß(95%CI) = 1.724 (0.677-2.77) showed positive longitudinal associations with follow-up knee pain scores (NRS item 3, average pain). Combined serum 8,9-EET and 5-HETE concentration showed the strongest longitudinal association (ß(95%CI) = 1.156 (0.54-1.77) with pain scores at 3 years, and ROC curves distinguished between participants with no pain and high pain scores at follow-up (area under curve (95%CI) = 0.71 (0.61-0.82)). CONCLUSIONS: Serum levels of a combination of hydroxylated metabolites of arachidonic acid may have prognostic utility for knee pain, providing a potential novel approach to identify people who are more likely to have debilitating pain in the future.

Impact of high-risk cytogenetics on outcomes for children and young adults receiving CD19-directed CAR T-cell therapy.

Chimeric antigen receptor (CAR) T-cell therapy can induce durable remissions of relapsed/refractory B-acute lymphoblastic leukemia (ALL). However, case reports suggested differential outcomes mediated by leukemia cytogenetics. We identified children and young adults with relapsed/refractory CD19+ ALL/lymphoblastic lymphoma treated on 5 CD19-directed CAR T-cell (CTL019 or humanized CART19) clinical trials or with commercial tisagenlecleucel from April 2012 to April 2019. Patients were hierarchically categorized according to leukemia cytogenetics: High-risk lesions were defined as KMT2A (MLL) rearrangements, Philadelphia chromosome (Ph+), Ph-like, hypodiploidy, or TCF3/HLF; favorable as hyperdiploidy or ETV6/RUNX1; and intermediate as iAMP21, IKZF1 deletion, or TCF3/PBX1. Of 231 patients aged 1 to 29, 74 (32%) were categorized as high risk, 28 (12%) as intermediate, 43 (19%) as favorable, and 86 (37%) as uninformative. Overall complete remission rate was 94%, with no difference between strata. There was no difference in relapse-free survival (RFS; P = .8112), with 2-year RFS for the high-risk group of 63% (95% confidence interval [CI], 52-77). There was similarly no difference seen in overall survival (OS) (P = .5488), with 2-year OS for the high-risk group of 70% (95% CI, 60-82). For patients with KMT2A-rearranged infant ALL (n = 13), 2-year RFS was 67% (95% CI, 45-99), and OS was 62% (95% CI, 40-95), with multivariable analysis demonstrating no increased risk of relapse (hazard ratio, 0.70; 95% CI, 0.21-2.90; P = .7040) but a higher proportion of relapses associated with myeloid lineage switch and a 3.6-fold increased risk of all-cause death (95% CI, 1.04-12.75; P = .0434). CTL019/huCART19/tisagenlecleucel are effective at achieving durable remissions across cytogenetic categories. Relapsed/refractory patients with high-risk cytogenetics, including KMT2A-rearranged infant ALL, demonstrated high RFS and OS probabilities at 2 years.

Single-cell multiomics reveals increased plasticity, resistant populations, and stem-cell-like blasts in KMT2A-rearranged leukemia.

KMT2A-rearranged (KMT2A-r) infant acute lymphoblastic leukemia (ALL) is a devastating malignancy with a dismal outcome, and younger age at diagnosis is associated with increased risk of relapse. To discover age-specific differences and critical drivers that mediate poor outcome in KMT2A-r ALL, we subjected KMT2A-r leukemias and normal hematopoietic cells from patients of different ages to single-cell multiomics analyses. We uncovered the following critical new insights: leukemia cells from patients <6 months have significantly increased lineage plasticity. Steroid response pathways are downregulated in the most immature blasts from younger patients. We identify a hematopoietic stem and progenitor-like (HSPC-like) population in the blood of younger patients that contains leukemic blasts and form an immunosuppressive signaling circuit with cytotoxic lymphocytes. These observations offer a compelling explanation for the ability of leukemias in young patients to evade chemotherapy and immune-mediated control. Our analysis also revealed preexisting lymphomyeloid primed progenitors and myeloid blasts at initial diagnosis of B-ALL. Tracking of leukemic clones in 2 patients whose leukemia underwent a lineage switch documented the evolution of such clones into frank acute myeloid leukemia (AML). These findings provide critical insights into KMT2A-r ALL and have clinical implications for molecularly targeted and immunotherapy approaches. Beyond infant ALL, our study demonstrates the power of single-cell multiomics to detect tumor intrinsic and extrinsic factors affecting rare but critical subpopulations within a malignant population that ultimately determines patient outcome.

The state of cell and gene therapy in 2023.

Progress in the understanding of human diseases has coincided with the advent of precision medicine, whereby the underlying genetic and molecular contributors can be used as diagnostic and therapeutic biomarkers. To address these, drug developers have designed a range of different treatment strategies, including gene therapy, which the American Society of Gene and Cell Therapy defines as the use of genetic material to treat or prevent disease. A number of approaches exist, including the delivery of genetic material in vivo or ex vivo, as well as the use of RNA species to alter gene expression in particular disease states. Through the end of the first quarter of 2023, there were more than 100 different approved gene, cell, and RNA therapies throughout the world, with over 3,700 more in clinical and preclinical development. This review comprehensively captures the landscape for such advanced therapies, including the different genetic technologies used and diseases targeted in clinical trials.

Improving duplex ultrasound methods for diagnosing functional popliteal artery entrapment syndrome.

OBJECTIVES: Popliteal artery entrapment syndrome (PAES) is a rare condition where musculoskeletal structures compress the popliteal artery (POPA) leading to vascular compromise. This study investigates the effect of dynamic plantar- and dorsi-flexion loading on POPA hemodynamic parameters to develop a robust diagnostic ultrasound-based protocol for diagnosing functional PAES. METHODS: Healthy individuals (n = 20), recreational athletes (n = 20), and symptomatic (n = 20) PAES patients were consented. Triplex ultrasound imaging of lower limb arteries was performed (n = 120 limbs). Proximal and distal POPA's in dorsi-/plantar-flexion, in prone and erect positions, were imaged at rest and flexion. Peak systolic velocities (cm/s) and vessel diameter (antero-posterior, cm) was measured. RESULTS: Distal vessel occlusion was noted across all three groups whilst prone during plantar-flexion (62.7%). POPA occlusion was only noted in the proximal vessel within the patient group (15.8%). When prone, 50% of control (n = 40 limbs), 70% of athletes (n = 40 limbs), and 65% of patients (n = 40 limbs) had distal POPA occlusion in plantar-flexion. When prone, recreational athletes (5%), and patients (12.5%) had distal POPA compression under dorsi-flexion. POPA occlusions with the patient in erect position were only noted in the symptomatic patient group under both dorsi-flexion (15.8%) and plantar-flexion (23.7%). CONCLUSION: Compression of the POPA on ultrasound should not be the sole diagnostic criteria for PAES. POPA compression exists in asymptomatic individuals, primarily under prone plantar-flexion. To reduce false positives, ultrasound-based protocols should focus on scanning patients in the erect position only to diagnose PAES, rather than asymptomatic POPA compression. A distinction should be made between the two.

IL-6 Blockade in Cytokine Storm Syndromes.

Interleukin-6 (IL-6) is a pro-inflammatory cytokine elevated in cytokine storm syndromes, including hemophagocytic lymphohistiocytosis (HLH) and macrophage activation syndrome (MAS). It is also elevated in cytokine release syndrome (CRS) after immune activating cancer therapies such as chimeric antigen receptor (CAR) T-cells or bispecific T-cell engagers (BITEs) and in some patients after infection with SARS-CoV-2. The interaction of IL-6 with its receptor complex can happen in several forms, making effectively blocking this cytokine's effects clinically challenging. Fortunately, effective clinical agents targeting the IL-6 receptor (tocilizumab) and IL-6 directly (siltuximab) have been developed and are approved for use in humans. IL-6 blockade has now been used to safely and effectively treat several cytokine storm syndromes (CSS). Other methods of investigation in effective IL-6 blockade are underway.

Growth rate and nutrient limitation as key drivers of extracellular quorum sensing signal molecule accumulation in Pseudomonas aeruginosa.

In Pseudomonas aeruginosa, quorum sensing (QS) depends on an interconnected regulatory hierarchy involving the Las, Rhl and Pqs systems, which are collectively responsible for the co-ordinated synthesis of a diverse repertoire of N-acylhomoserine lactones (AHLs) and 2-alkyl-4-quinolones (AQs). Apparent population density-dependent phenomena such as QS may, however, be due to growth rate and/or nutrient exhaustion in batch culture. Using continuous culture, we show that growth rate and population density independently modulate the accumulation of AHLs and AQs such that the highest concentrations are observed at a slow growth rate and high population density. Carbon source (notably succinate), nutrient limitation (C, N, Fe, Mg) or growth at 25 °C generally reduces AHL and AQ levels, except for P and S limitation, which result in substantially higher concentrations of AQs, particularly AQ N-oxides, despite the lower population densities achieved. Principal component analysis indicates that ~26 % variation is due to nutrient limitation and a further 30 % is due to growth rate. The formation of N-(3-oxododecanoyl)-l-homoserine lactone (3OC12-HSL) turnover products such as the ring opened form and tetramic acid varies with the limiting nutrient limitation and anaerobiosis. Differential ratios of N-butanoyl-homoserine lactone (C4-HSL), 3OC12-HSL and the AQs as a function of growth environment are clearly apparent. Inactivation of QS by mutation of three key genes required for QS signal synthesis (lasI, rhlI and pqsA) substantially increases the concentrations of key substrates from the activated methyl cycle and aromatic amino acid biosynthesis, as well as ATP levels, highlighting the energetic drain that AHL and AQ synthesis and hence QS impose on P. aeruginosa.

Nanoscale, antigen encounter-dependent, IL-12 delivery by CAR T cells plus PD-L1 blockade for cancer treatment.

BACKGROUND: Chimeric antigen receptor (CAR)-T cell therapies for the treatment of hematological malignancies experienced tremendous progress in the last decade. However, essential limitations need to be addressed to further improve efficacy and reduce toxicity to assure CAR-T cell persistence, trafficking to the tumor site, resistance to an hostile tumor microenvironment (TME), and containment of toxicity restricting production of powerful but potentially toxic bioproducts to the TME; the last could be achieved through contextual release upon tumor antigen encounter of factors capable of converting an immune suppressive TME into one conducive to immune rejection. METHODS: We created an HER2-targeting CAR-T (RB-312) using a clustered regularly interspaced short palindromic repeats (CRISPR) activation (CRISPRa) system, which induces the expression of the IL-12 heterodimer via conditional transcription of its two endogenous subunits p35 and p40. This circuit includes two lentiviral constructs. The first one (HER2-TEV) expresses an anti-human epidermal growth factor receptor 2 (HER2) CAR single chain variable fragment (scFv), with CD28 and CD3z co-stimulatory domains linked to the tobacco etch virus (TEV) protease and two single guide RNAs (sgRNA) targeting the interleukin (IL)-12A and IL12B transcription start site (TSS), respectively. The second construct (LdCV) encodes linker for activation of T cells (LAT) fused to nuclease-deactivated Streptococcus Pyogenes Cas9 (dCas9)-VP64-p65-Rta (VPR) via a TEV-cleavable sequence (TCS). Activation of the CAR brings HER2-TEV in close proximity to LdCV releasing dCas9 for nuclear localization. This conditional circuit leads to conditional and reversible induction of the IL-12/p70 heterodimer. RB-312 was compared in vitro to controls (cRB-312), lacking the IL-12 sgRNAs and conventional HER2 CAR (convCAR). RESULTS: The inducible CRISPRa system activated endogenous IL-12 expression resulting in enhanced secondary interferon (FN)-γ production, cytotoxicity, and CAR-T proliferation in vitro, prolonged in vivo persistence and greater suppression of HER2+ FaDu oropharyngeal cancer cell growth compared to the conventional CAR-T cell product. No systemic IL-12 was detected in the peripheral circulation. Moreover, the combination with programmed death ligand (PD-L1) blockade demonstrated robust synergistic effects. CONCLUSIONS: RB-312, the first clinically relevant product incorporating a CRISPRa system with non-gene editing and reversible upregulation of endogenous gene expression that promotes CAR-T cells persistence and effectiveness against HER2-expressing tumors. The autocrine effects of reversible, nanoscale IL-12 production limits the risk of off-tumor leakage and systemic toxicity.

Cellular therapy: Immune-related complications.

The advent of chimeric antigen receptor T (CAR-T) and the burgeoning field of cellular therapy has revolutionized the treatment of relapsed/refractory leukemia and lymphoma. This personalized "living therapy" is highly effective against a number of malignancies, but this efficacy is tempered by side effects relatively unique to immunotherapies, including CAR-T. The overwhelming release of cytokines and chemokines by activated CAR-T and other secondarily activated immune effector cells can lead to cytokine release syndrome (CRS), which can have clinical and pathophysiology similarities to systemic inflammatory response syndrome and macrophage activating syndrome/hemophagocytic lymphohistiocytosis. Tocilizumab, an anti-IL6 receptor antibody, was recently FDA approved for treatment of CRS after CAR-T based on its ability to mitigate CRS in many patients. Unfortunately, some patients are refractory and additional therapies are needed. Patients treated with CAR-T can also develop neurotoxicity and, as the biology is poorly understood, current therapeutic interventions are limited to supportive care. Nevertheless, a number of recent studies have shed new light on the pathophysiology of CAR-T-related neurotoxicity, which will hopefully lead to effective treatments. In this review we discuss some of the mechanistic contributions intrinsic to the CAR-T construct, the tumor being treated, and the individual patient that impact the development and severity of CRS and neurotoxicity. As CAR-T and cellular therapy have redefined the concept of personalized medicine, so too will personalization be necessary in managing the unique side effects of these therapies.

Fallen angels and forgotten heroes: A descriptive qualitative study exploring the impact of the angel and hero narrative on critical care nurses.

BACKGROUND: During the COVID-19 pandemic, the use of the labels 'heroes' and 'angels' to describe nurses (and especially critical care nurses) became prevalent. While often well intentioned, the use of these labels may not be the most positive image of nurses and the nursing profession. Critical care nurses have not previously been given the opportunity to provide their perceptions of the angel/hero narrative and the impact this may have on their practice and working environments. OBJECTIVES: The objectives of this study were to explore the perspectives of critical care nurses and discover their perceptions about the angel/hero narrative and its impact on their clinical practice, safe working environments, and professional development during the COVID-19 pandemic. METHODS: A semistructured qualitative virtual interview study was conducted with critical care nurses from the United Kingdom, Australia, and North America. Digital audio data were transcribed verbatim. Thematic analysis of the transcribed data was performed. The COREQ guidelines were used to report the study. FINDINGS: Twenty-three critical care nurses located in the United Kingdom, Australia, and North America participated. Four themes were synthesised: history repeating, gender stereotypes, political pawns, and forgotten heroes. CONCLUSIONS: Critical care nurses did not perceive the hero and angel labels positively. Participants were concerned about unrealistic expectations, potential safety workplace risks, and poor remuneration related to these narratives. Participants perceived that context and intention were important in the interpretation of these narratives; they spoke with pride about their work and called for improved representations of their role, recognition, and work conditions.

Serum Levels of Proinflammatory Lipid Mediators and Specialized Proresolving Molecules Are Increased in Patients With Severe Acute Respiratory Syndrome Coronavirus 2 and Correlate With Markers of the Adaptive Immune Response.

BACKGROUND: Specialized proresolution molecules (SPMs) halt the transition to chronic pathogenic inflammation. We aimed to quantify serum levels of pro- and anti-inflammatory bioactive lipids in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) patients, and to identify potential relationships with innate responses and clinical outcome. METHODS: Serum from 50 hospital admitted inpatients (22 female, 28 male) with confirmed symptomatic SARS-CoV-2 infection and 94 age- and sex-matched controls collected prior to the pandemic (SARS-CoV-2 negative), were processed for quantification of bioactive lipids and anti-nucleocapsid and anti-spike quantitative binding assays. RESULTS: SARS-CoV-2 serum had significantly higher concentrations of omega-6-derived proinflammatory lipids and omega-6- and omega-3-derived SPMs, compared to the age- and sex-matched SARS-CoV-2-negative group, which were not markedly altered by age or sex. There were significant positive correlations between SPMs, proinflammatory bioactive lipids, and anti-spike antibody binding. Levels of some SPMs were significantly higher in patients with an anti-spike antibody value >0.5. Levels of linoleic acid and 5,6-dihydroxy-8Z,11Z,14Z-eicosatrienoic acid were significantly lower in SARS-CoV-2 patients who died. CONCLUSIONS: SARS-CoV-2 infection was associated with increased levels of SPMs and other pro- and anti-inflammatory bioactive lipids, supporting the future investigation of the underlying enzymatic pathways, which may inform the development of novel treatments.

A cellular antidote to specifically deplete anti-CD19 chimeric antigen receptor-positive cells.

Unintentional transduction of B-cell acute lymphoblastic leukemia blasts during CART19 manufacturing can lead to CAR19+ leukemic cells (CARB19) that are resistant to CART19 killing. We developed an anti-CAR19 idiotype chimeric antigen receptor (αCAR19) to specifically recognize CAR19+ cells. αCAR19 CAR T cells efficiently lysed CARB19 cells in vitro and in a primary leukemia-derived xenograft model. We further showed that αCAR19-CART cells could be used as an "antidote" to deplete CART19 cells to reduce long-term side effects, such as B-cell aplasia.

Physicochemical and metabolic constraints for thermodynamics-based stoichiometric modelling under mesophilic growth conditions.

Metabolic engineering in the post-genomic era is characterised by the development of new methods for metabolomics and fluxomics, supported by the integration of genetic engineering tools and mathematical modelling. Particularly, constraint-based stoichiometric models have been widely studied: (i) flux balance analysis (FBA) (in silico), and (ii) metabolic flux analysis (MFA) (in vivo). Recent studies have enabled the incorporation of thermodynamics and metabolomics data to improve the predictive capabilities of these approaches. However, an in-depth comparison and evaluation of these methods is lacking. This study presents a thorough analysis of two different in silico methods tested against experimental data (metabolomics and 13C-MFA) for the mesophile Escherichia coli. In particular, a modified version of the recently published matTFA toolbox was created, providing a broader range of physicochemical parameters. Validating against experimental data allowed the determination of the best physicochemical parameters to perform the TFA (Thermodynamics-based Flux Analysis). An analysis of flux pattern changes in the central carbon metabolism between 13C-MFA and TFA highlighted the limited capabilities of both approaches for elucidating the anaplerotic fluxes. In addition, a method based on centrality measures was suggested to identify important metabolites that (if quantified) would allow to further constrain the TFA. Finally, this study emphasised the need for standardisation in the fluxomics community: novel approaches are frequently released but a thorough comparison with currently accepted methods is not always performed.

Profiling oxylipins released from human platelets activated through the GPVI collagen receptor.

In addition to haemostasis, platelets are involved in pathological processes, often driven by material released upon activation. Interaction between collagen and glycoprotein VI (GPVI) is a primary platelet stimulus that liberates arachidonic acid and linoleic acid from membrane phospholipids. These are oxidised by cyclooxygenase-1 (COX-1) and 12-lipoxygenase (12-LOX) to eicosanoids and other oxylipins with various biological properties. Using liquid chromatography-tandem mass spectrometry we found that GPVI-stimulated platelets released significant levels of ten oxylipins; the well documented TxA2 and 12-HETE, PGD2 and PGE2, as well as 8-, 9-, 11-, and 15-HETE, 9- and 13-HODE.1 Levels of oxylipins released from washed platelets mirrored those from platelets stimulated in the presence of plasma, indicating generation from intracellular, rather than exogenous AA/LA. Inhibition of COX-1 with aspirin, as expected, completely abolished production of TxA2 and PGD/E2, but also significantly inhibited the release of 11-HETE (89 ± 3%) and 9-HODE (74 ± 6%), and reduced 15-HETE and 13-HODE by ∼33 %. Inhibition of 12-LOX by either esculetin or ML355 inhibited the release of all oxylipins apart from 15-HETE. These findings suggest routes to modify the production of bioactive molecules released by activated platelets.

Human Adenovirus 7-Associated Hemophagocytic Lymphohistiocytosis-like Illness: Clinical and Virological Characteristics in a Cluster of Five Pediatric Cases.

BACKGROUND: Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening condition of immune dysregulation. Children often suffer from primary genetic forms of HLH, which can be triggered by infection. Others suffer from secondary HLH as a complication of infection, malignancy, or rheumatologic disease. Identifying the exact cause of HLH is crucial, as definitive treatment for primary disease is hematopoietic stem cell transplant. Adenoviruses have been associated with HLH but molecular epidemiology data are lacking. METHODS: We describe the clinical and virologic characteristics of 5 children admitted with adenovirus infection during 2018-2019 who developed HLH or HLH-like illness. Detailed virologic studies, including virus isolation and comprehensive molecular typing were performed. RESULTS: All patients recovered; clinical management varied but included immunomodulating and antiviral therapies. A genetic predisposition for HLH was not identified in any patient. Adenovirus isolates were recovered from 4/5 cases; all were identified as genomic variant 7d. Adenovirus type 7 DNA was detected in the fifth case. Phylogenetic analysis of genome sequences identified 2 clusters-1 related to strains implicated in 2016-2017 outbreaks in Pennsylvania and New Jersey, the other related to a 2009 Chinese strain. CONCLUSIONS: It can be challenging to determine whether HLH is the result of an infectious pathogen alone or genetic predisposition triggered by an infection. We describe 5 children from the same center presenting with an HLH-like illness after onset of adenovirus type 7 infection. None of the patients were found to have a genetic predisposition to HLH. These findings suggest that adenovirus 7 infection alone can result in HLH.

Limited phylogenetic overlap between fluoroquinolone-resistant Escherichia coli isolated on dairy farms and those causing bacteriuria in humans living in the same geographical region.

BACKGROUND: Our primary aim was to test whether cattle-associated fluoroquinolone-resistant (FQ-R) Escherichia coli found on dairy farms are closely phylogenetically related to those causing bacteriuria in humans living in the same 50 × 50 km geographical region suggestive of farm-human sharing. Another aim was to identify risk factors for the presence of FQ-R E. coli on dairy farms. METHODS: FQ-R E. coli were isolated during 2017-18 from 42 dairy farms and from community urine samples. Forty-two cattle and 489 human urinary isolates were subjected to WGS, allowing phylogenetic comparisons. Risk factors were identified using a Bayesian regularization approach. RESULTS: Of 489 FQ-R human isolates, 255 were also third-generation-cephalosporin-resistant, with strong genetic linkage between aac(6')Ib-cr and blaCTX-M-15. We identified possible farm-human sharing for pairs of ST744 and ST162 isolates, but minimal core genome SNP distances were larger between farm-human pairs of ST744 and ST162 isolates (71 and 63 SNPs, respectively) than between pairs of isolates from different farms (7 and 3 SNPs, respectively). Total farm fluoroquinolone use showed a positive association with the odds of isolating FQ-R E. coli, while total dry cow therapy use showed a negative association. CONCLUSIONS: This work suggests that FQ-R E. coli found on dairy farms have a limited impact on community bacteriuria within the local human population. Reducing fluoroquinolone use may reduce the on-farm prevalence of FQ-R E. coli and this reduction may be greater when dry cow therapy is targeted to the ecology of resistant E. coli on the farm.

Reduced Antibacterial Drug Resistance and blaCTX-M ß-Lactamase Gene Carriage in Cattle-Associated Escherichia coli at Low Temperatures, at Sites Dominated by Older Animals, and on Pastureland: Implications for Surveillance.

Little is known about the drivers of critically important antibacterial resistance in species with zoonotic potential present on farms (e.g., CTX-M ß-lactamase-positive Escherichia coli). We collected samples monthly between January 2017 and December 2018 on 53 dairy farms in South West England, along with data for 610 variables concerning antibacterial usage, management practices, and meteorological factors. We detected E. coli resistant to amoxicillin, ciprofloxacin, streptomycin, and tetracycline in 2,754/4,145 (66%), 263/4,145 (6%), 1,475/4,145 (36%), and 2,874/4,145 (69%), respectively, of samples from fecally contaminated on-farm and near-farm sites. E. coli positive for blaCTX-M were detected in 224/4,145 (5.4%) of samples. Multilevel, multivariable logistic regression showed antibacterial dry cow therapeutic choice (including use of cefquinome or framycetin) to be associated with higher odds of blaCTX-M positivity. Low average monthly ambient temperature was associated with lower odds of blaCTX-ME. coli positivity in samples and with lower odds of finding E. coli resistant to each of the four test antibacterials. This was in addition to the effect of temperature on total E. coli density. Furthermore, samples collected close to calves had higher odds of having E. coli resistant to each antibacterial, as well as E. coli positive for blaCTX-M Samples collected on pastureland had lower odds of having E. coli resistant to amoxicillin or tetracycline, as well as lower odds of being positive for blaCTX-MIMPORTANCE Antibacterial resistance poses a significant threat to human and animal health and global food security. Surveillance for resistance on farms is important for many reasons, including tracking impacts of interventions aimed at reducing the prevalence of resistance. In this longitudinal survey of dairy farm antibacterial resistance, we showed that local temperature-as it changes over the course of a year-was associated with the prevalence of antibacterial-resistant E. coli We also showed that prevalence of resistant E. coli was lower on pastureland and higher in environments inhabited by young animals. These findings have profound implications for routine surveillance and for surveys carried out for research. They provide important evidence that sampling at a single time point and/or single location on a farm is unlikely to be adequate to accurately determine the status of the farm regarding the presence of samples containing resistant E. coli.

Metabolic signatures of osteoarthritis in urine using liquid chromatography-high resolution tandem mass spectrometry.

INTRODUCTION: Osteoarthritis (OA) is a common cause of disability in older people, but its aetiology is not yet fully understood. Biomarkers of OA from metabolomics studies have shown potential use in understanding the progression and pathophysiology of OA. OBJECTIVES: To investigate possible surrogate biomarkers of knee OA in urine using metabolomics to contribute towards a better understanding of OA progression and possible targeted treatment. METHOD: Liquid chromatography-high resolution mass spectrometry (LC-HRMS) was applied in a case-control approach to explore the possible metabolic differences between the urinary profiles of symptomatic knee OA patients (n = 74) (subclassified into inflammatory OA, n = 22 and non-inflammatory OA, n = 52) and non-OA controls (n = 68). Univariate, multivariate and pathway analyses were performed with a rigorous validation including cross-validation, permutation test, prediction and receiver operating characteristic curve to identify significantly altered metabolites and pathways in OA. RESULTS: OA datasets generated 7405 variables and multivariate analysis showed clear separation of inflammatory OA, but not non-inflammatory OA, from non-OA controls. Adequate cross-validation (R2Y = 0.874, Q2 = 0.465) was obtained. The prediction model and the ROC curve showed satisfactory results with a sensitivity of 88%, specificity of 71% and accuracy of 77%. 26 metabolites were identified as potential biomarkers of inflammatory OA using HMDB, authentic standards and/or MS/MS database. CONCLUSION: Urinary metabolic profiles were altered in inflammatory knee OA subjects compared to those with non-inflammatory OA and non-OA controls. These altered profiles associated with perturbed activity of the TCA cycle, pyruvate and amino acid metabolism linked to inflammation, oxidative stress and collagen destruction. Of note, 2-keto-glutaramic acid level was > eightfold higher in the inflammatory OA patients compared to non-OA control, signalling a possible perturbation in glutamine metabolism related to OA progression.

Potential sealing and repair of human FM defects after trauma with peptide amphiphiles and Cx43 antisense.

OBJECTIVE: We examined whether peptide amphiphiles functionalised with adhesive, migratory or regenerative sequences could be combined with amniotic fluid (AF) to form plugs that repair fetal membrane (FM) defects after trauma and co-culture with connexin 43 (Cx43) antisense. METHODS: We assessed interactions between peptide amphiphiles and AF and examined the plugs in FM defects after trauma and co-culture with the Cx43antisense. RESULTS: Confocal microscopy confirmed directed self-assembly of peptide amphiphiles with AF to form a plug within minutes, with good mechanical properties. SEM of the plug revealed a multi-layered, nanofibrous network that sealed the FM defect after trauma. Co-culture of the FM defect with Cx43 antisense and plug increased collagen levels but reduced GAG. Culture of the FM defect with peptide amphiphiles incorporating regenerative sequences for 5 days, increased F-actin and nuclear cell contraction, migration and polarization of collagen fibers across the FM defect when compared to control specimens with minimal repair. CONCLUSIONS: Whilst the nanoarchitecture revealed promising conditions to seal iatrogenic FM defects, the peptide amphiphiles need to be designed to maximize repair mechanisms and promote structural compliance with high mechanical tolerance that maintains tissue remodeling with Cx43 antisense for future treatment.

Interspecific Variation in One-Carbon Metabolism within the Ovarian Follicle, Oocyte, and Preimplantation Embryo: Consequences for Epigenetic Programming of DNA Methylation.

One-carbon (1C) metabolism provides methyl groups for the synthesis and/or methylation of purines and pyrimidines, biogenic amines, proteins, and phospholipids. Our understanding of how 1C pathways operate, however, pertains mostly to the (rat) liver. Here we report that transcripts for all bar two genes (i.e., BHMT, MAT1A) encoding enzymes in the linked methionine-folate cycles are expressed in all cell types within the ovarian follicle, oocyte, and blastocyst in the cow, sheep, and pig; as well as in rat granulosa cells (GCs) and human KGN cells (a granulosa-like tumor cell line). Betaine-homocysteine methyltransferase (BHMT) protein was absent in bovine theca and GCs, as was activity of this enzyme in GCs. Mathematical modeling predicted that absence of this enzyme would lead to more volatile S-adenosylmethionine-mediated transmethylation in response to 1C substrate (e.g., methionine) or cofactor provision. We tested the sensitivity of bovine GCs to reduced methionine (from 50 to 10 µM) and observed a diminished flux of 1C units through the methionine cycle. We then used reduced-representation bisulfite sequencing to demonstrate that this reduction in methionine during bovine embryo culture leads to genome-wide alterations to DNA methylation in >1600 genes, including a cohort of imprinted genes linked to an abnormal fetal-overgrowth phenotype. Bovine ovarian and embryonic cells are acutely sensitive to methionine, but further experimentation is required to determine the significance of interspecific variation in BHMT expression.