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Recombinant human erythropoietin (rHuEPO) is a new drug for treating anemia associated with end stage renal disease (ESRD). In a study of rHuEPO diffusion, costs, and effectiveness, we analyze ESRD program data and all claims submitted to Medicare for reimbursement of rHuEPO administered to ESRD dialysis patients. Access to rHuEPO was rapid and extensive during the first year of Medicare coverage. Dosing of rHuEPO and achieved hematocrit were lower than expected based on the results of clinical trials. rHuEPO cost Medicare $144 million in its first year. The analysis of insurance claims data allowed effective monitoring of access, costs, and effectiveness of this new biotechnology.

Objective: Glycemic control remains suboptimal despite the wide range of available medications. More effective medication prescription might result in better control. However, the process by which physicians choose glucose-lowering medicines is poorly understood. We sought to study the means by which physicians choose medications for type 2 diabetic patients.Research Design and Methods: We surveyed 886 physician members of either the Society of General Internal Medicine (academic generalists, response rate 30%) or the American Diabetes Association (specialists, response rate 23%) currently managing patients with type 2 diabetes. Respondents weighed the importance of 15 patient, physician, and nonclinical factors when deciding which medications to prescribe for type 2 diabetic subjects at each of three management stages (initiation, use of second-line oral agents, and insulin).Results: Respondents reported using a median of five major considerations (interquartile range 4-6) at each stage. Frequently cited major considerations included overall assessment of the patient's health/comorbidity, A1C level, and patient's adherence behavior but not expert guidelines/hospital algorithms or patient age. For insulin initiation, academic generalists placed greater emphasis on patient adherence (76 vs. 60% of specialists, P < 0.001). These generalists also identified patient fear of injections (68%) and patient desire to prolong noninsulin therapy (68%) as major insulin barriers. Overall, qualitative factors (e.g., adherence, motivation, overall health assessment) were somewhat more highly considered than quantitative factors (e.g., A1C, age, weight) with mean aggregate scores of 7.3 vs. 6.9 on a scale of 0-10, P < 0.001.Conclusions: The physicians in our survey considered a wide range of qualitative and quantitative factors when making medication choices for hyperglycemia management. The apparent complexity of the medication choice process contrasts with current evidence-based treatment guidelines. [ABSTRACT FROM AUTHOR]

Background: Colorectal cancer (CRC) is globally the third most prevalent cancer and a leading cause of cancer-related deaths. In Alberta, Canada, a significant portion of CRC diagnoses occur following emergency department (ED) presentations. Gaps remain in understanding patient's perspectives on CRC diagnosis after an ED visit. The aim of this study was to examine the experiences and perspectives of a group of patients diagnosed with CRC subsequent to an ED visit in Alberta and their close contacts., Methods: We conducted a qualitative study using in-depth, semi-structured interviews with patients diagnosed with CRC after an ED visit at the Rockyview General Hospital, Calgary, and their close contacts, from November 2022 to June 2023. Interviews focused on symptom recognition, healthcare interactions, and the decision-making process leading to an ED visit. They were conducted in-person or over the phone, and analysed using thematic analysis., Results: Eighteen participants (12 patients and 6 close contacts) were interviewed, revealing four main themes: (1) variability in symptom recognition and interpretation; (2) inconsistencies in primary care consultations; (3) factors influencing decision-making leading to an ED visit; and (4) recommendations for expedited diagnosis outside of EDs., Conclusion: The findings highlight the complexity of the diagnostic journey for CRC patients in Alberta, pointing to significant gaps in symptom recognition and response by patients and healthcare providers. Improved diagnostic protocols and targeted support for healthcare providers, as well as approaches to address systemic delays may help streamline the diagnostic journey. Future research should focus on exploring innovative interventions to address the identified barriers to timely CRC diagnosis., (© 2024. The Author(s).)

The Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus infects host cells by engaging its spike (S) protein with human ACE2 receptor. Recent studies suggest the involvement of integrins in SARS-CoV-2 infection through interaction with the S protein, but the underlying mechanism is not well understood. This study investigated the role of integrin α 5 β 1 , which recognizes the Arg-Gly-Asp (RGD) motif in its physiological ligands, in S-mediated virus entry and cell-cell fusion. Our results showed that α 5 β 1 does not directly contribute to S-mediated cell entry, but it enhances S-mediated cell-cell fusion in collaboration with ACE2. This effect cannot be inhibited by the putative α 5 β 1 inhibitor ATN-161 or the high-affinity RGD-mimetic inhibitor MK-0429 but requires the participation of α 5 cytoplasmic tail (CT). We detected a direct interaction between α 5 β 1 and the S protein, but this interaction does not rely on the RGD-containing receptor binding domain of the S1 subunit of the S protein. Instead, it involves the S2 subunit of the S protein and α 5 β 1 homo-oligomerization. Furthermore, we found that the S protein induces inflammatory responses in human endothelial cells, characterized by NF-κB activation, gasdermin D cleavage, and increased secretion of proinflammatory cytokines IL-6 and IL-1β. These effects can be attenuated by the loss of α 5 expression or inhibition of the α 5 CT binding protein phosphodiesterase-4D (PDE4D), suggesting the involvement of α 5 CT and PDE4D pathway. These findings provide molecular insights into the pathogenesis of SARS-CoV-2 mediated by a nonclassical RGD-independent ligand-binding and signaling function of integrin α 5 β 1 and suggest potential targets for antiviral treatment., Competing Interests: Competing interests statement:The authors declare no competing interest.

The neurofilament (NF) cytoskeleton is critical for neuronal morphology and function. In particular, the neurofilament-light (NF-L) subunit is required for NF assembly in vivo and is mutated in subtypes of Charcot-Marie-Tooth (CMT) disease. NFs are highly dynamic, and the regulation of NF assembly state is incompletely understood. Here, we demonstrate that human NF-L is modified in a nutrient-sensitive manner by O-linked-β-N-acetylglucosamine (O-GlcNAc), a ubiquitous form of intracellular glycosylation. We identify five NF-L O-GlcNAc sites and show that they regulate NF assembly state. NF-L engages in O-GlcNAc-mediated protein-protein interactions with itself and with the NF component α-internexin, implying that O-GlcNAc may be a general regulator of NF architecture. We further show that NF-L O-GlcNAcylation is required for normal organelle trafficking in primary neurons. Finally, several CMT-causative NF-L mutants exhibit perturbed O-GlcNAc levels and resist the effects of O-GlcNAcylation on NF assembly state, suggesting a potential link between dysregulated O-GlcNAcylation and pathological NF aggregation. Our results demonstrate that site-specific glycosylation regulates NF-L assembly and function, and aberrant NF O-GlcNAcylation may contribute to CMT and other neurodegenerative disorders., (© 2023. Springer Nature Limited.)

Homologous recombination (HR) is the major mechanism of rescue of stalled replication forks or repair of DNA double-strand breaks (DSBs) during S phase or mitosis. In human cells, HR is facilitated by the BRCA2-BRCA1-PALB2 module, which loads the RAD51 recombinase onto a resected single-stranded DNA end to initiate repair. Although the process is essential for error-free repair, unrestrained HR can cause chromosomal rearrangements and genome instability. F-box DNA Helicase 1 (FBH1) antagonizes the role of BRCA2-BRCA1-PALB2 to restrict hyper-recombination and prevent genome instability. Here, we analyzed reported FBH1 mutations in cancer cells using the Catalogue of Somatic Mutations in Cancers (COSMIC) to understand how they interact with the BRCA2-BRCA1-PALB2. Consistent with previous results from yeast, we find that FBH1 mutations co-occur with BRCA2 mutations and to some degree BRCA1 and PALB2. We also describe some co-occurring mutations with RAD52, the accessory RAD51 loader and facilitator of single-strand annealing, which is independent of RAD51. In silico modeling was used to investigate the role of key FBH1 mutations on protein function, and a Q650K mutation was found to destabilize the protein structure. Taken together, this work highlights how mutations in several DNA damage repair genes contribute to cellular transformation and immortalization.

Normalizing RT-qPCR miRNA datasets that encompass numerous preimplantation embryo stages requires the identification of miRNAs that may be used as stable reference genes. A need has also arisen for the normalization of the accompanying conditioned culture media as extracellular miRNAs may serve as biomarkers of embryo developmental competence. Here, we evaluate the stability of six commonly used miRNA normalization candidates, as well as small nuclear U6, using five different means of evaluation (BestKeeper, NormFinder, geNorm, the comparative Delta Ct method and RefFinder comprehensive analysis) to assess their stability throughout murine preimplantation embryo development from the oocyte to the late blastocyst stages, both in whole embryos and the associated conditioned culture media. In descending order of effectiveness, miR-16, miR-191 and miR-106 were identified as the most stable individual reference miRNAs for developing whole CD1 murine preimplantation embryos, while miR-16, miR-106 and miR-103 were ideal for the conditioned culture media. Notably, the widely used U6 reference was among the least appropriate for normalizing both whole embryo and conditioned media miRNA datasets. Incorporating multiple reference miRNAs into the normalization basis via a geometric mean was deemed beneficial, and combinations of each set of stable miRNAs are further recommended, pending validation on a per experiment basis.

Introduction: Intrinsically photosensitive retinal ganglion cells (ipRGCs) integrate melanopsin and rod/cone-mediated inputs to signal to the brain. Whilst originally identified as a cell type specialised for encoding ambient illumination, several lines of evidence indicate a strong association between colour discrimination and ipRGC-driven responses. Thus, cone-mediated colour opponent responses have been widely found across ipRGC target regions in the mouse brain and influence a key ipRGC-dependent function, circadian photoentrainment. Although ipRGCs exhibiting spectrally opponent responses have also been identified, the prevalence of such properties have not been systematically evaluated across the mouse retina or yet been found in ipRGC subtypes known to influence the circadian system. Indeed, there is still uncertainty around the overall prevalence of cone-dependent colour opponency across the mouse retina, given the strong retinal gradient in S and M-cone opsin (co)-expression and overlapping spectral sensitivities of most mouse opsins. Methods: To address this, we use photoreceptor isolating stimuli in multielectrode recordings from human red cone opsin knock-in mouse (Opn1mwR) retinas to systematically survey cone mediated responses and the occurrence of colour opponency across ganglion cell layer (GCL) neurons and identify ipRGCs based on spectral comparisons and/or the persistence of light responses under synaptic blockade. Results: Despite detecting robust cone-mediated responses across the retina, we find cone opponency is rare, especially outside of the central retina (overall ~3% of GCL neurons). In keeping with previous suggestions we also see some evidence of rod-cone opponency (albeit even more rare under our experimental conditions), but find no evidence for any enrichment of cone (or rod) opponent responses among functionally identified ipRGCs. Conclusion: In summary, these data suggest the widespread appearance of cone-opponency across the mouse early visual system and ipRGC-related responses may be an emergent feature of central visual processing mechanisms., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Mouland, Watson, Martial, Lucas and Brown.)

Background: Cognitive impairment is a core feature of schizophrenia, associated with poor functional outcomes. The course of cognitive function in the years following illness onset has remained a subject of debate, with a previous analysis finding no worsening, providing support for the neurodevelopmental model of schizophrenia. Since then, many more studies have reported on longitudinal cognitive performance in early psychosis, with some indicating deterioration, which does not align with this view., Aims: This study aims to quantitatively review the literature on the longitudinal trajectory of cognitive deficits in the years following psychosis onset, in comparison with healthy controls. It is the first to also synthesise longitudinal data on social cognition., Method: Electronic databases ('PubMed', 'PsycInfo' and 'Scopus') were searched (to end September 2021). Meta-analyses of 25 longitudinal studies of cognition in early psychosis were conducted (1480 patients, 789 health controls). Unlike previous analyses, randomised controlled trials and those with multiple cognitive testing periods within the first year were excluded to minimise bias (PROSPERO, ID: CRD42021241525)., Results: Small improvements were observed for global cognition ( g = 0.25, 95% CI 0.17-0.33) and individual cognitive domains, but these were comparable with healthy controls and likely an artefact of practice effects., Conclusions: There is no evidence of continued cognitive decline or improvement in the early years following psychosis onset, with a need for more studies over longer follow-up periods. Practice effects highlight the importance of including control samples in longitudinal and intervention studies. Further data are needed to evaluate the course of social cognition subdomains.

Obese women experience greater incidence of infertility, with reproductive tracts exposing preimplantation embryos to elevated free fatty acids (FFA) such as palmitic acid (PA) and oleic acid (OA). PA treatment impairs mouse preimplantation development in vitro, while OA co-treatment rescues blastocyst development of PA treated embryos. In the present study, we investigated the effects of PA and OA treatment on NRF2/Keap1 localization, and relative antioxidant enzyme (Glutathione peroxidase; Gpx1, Catalase; Cat, Superoxide dismutase; Sod1 and γ-Glutamylcysteine ligase catalytic unit; Gclc) mRNA levels, during in vitro mouse preimplantation embryo development. Female mice were superovulated, mated, and embryos cultured in the presence of bovine Serum albumin (BSA) control or PA, or OA, alone (each at 100 μM) or PA + OA combined (each at 100 μM) treatment. NRF2 displayed nuclear localization at all developmental stages, whereas Keap1 primarily displayed cytoplasmic localization throughout control mouse preimplantation development in vitro. Relative transcript levels of Nrf2, Keap1, and downstream antioxidants significantly increased throughout control mouse preimplantation development in vitro. PA treatment significantly decreased blastocyst development and the levels of nuclear NRF2, while OA and PA + OA treatments did not. PA and OA treatments did not impact relative mRNA levels of Nrf2, Keap1, Gpx1, Cat, Sod1 or Gclc. Our outcomes demonstrate that cultured mouse embryos display nuclear NRF2, but that PA treatment reduces nuclear NRF2 and thus likely impacts NRF2/KEAP1 stress response mechanisms. Further studies should investigate whether free fatty acid effects on NRF2/KEAP1 contribute to the reduced fertility displayed by obese patients., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Throughout Earth's history, the abundance of oxygen in our atmosphere has varied, but by how much remains debated. Previously, an upper limit for atmospheric oxygen has been bounded by assumptions made regarding the fire window: atmospheric oxygen concentrations higher than 30-40% would threaten the regeneration of forests in the present world. Here we have tested these assumptions by adapting a Dynamic Global Vegetation Model to run over high atmospheric oxygen concentrations. Our results show that whilst global tree cover is significantly reduced under high O 2 concentrations, forests persist in the wettest parts of the low and high latitudes and fire is more dependent on fuel moisture than O 2 levels. This implies that the effect of fire on suppressing global vegetation under high O 2 may be lower than previously assumed and questions our understanding of the mechanisms involved in regulating the abundance of oxygen in our atmosphere, with moisture as a potentially important factor., (© 2022. The Author(s).)

Treatment of mouse preimplantation embryos with elevated palmitic acid (PA) reduces blastocyst development, whereas cotreatment with PA and oleic acid (OA) together rescues blastocyst development to control frequencies. To understand the mechanistic effects of PA and OA treatment on early mouse embryos, we investigated the effects of PA and OA, alone and in combination, on autophagy during preimplantation development in vitro. We hypothesized that PA would alter autophagic processes and that OA cotreatment would restore control levels of autophagy. Two-cell stage mouse embryos were placed into culture medium supplemented with 100 μM PA, 250 μM OA, 100 μM PA and 250 μM OA, or potassium simplex optimization media with amino acid (KSOMaa) medium alone (control) for 18-48 h. The results demonstrated that OA cotreatment slowed developmental progression after 30 h of cotreatment but restored control blastocyst frequencies by 48 h. PA treatment elevated light chain 3 (LC3)-II puncta and p62 levels per cell whereas OA cotreatment returned to control levels of autophagy by 48 h. Autophagic mechanisms are altered by nonesterified fatty acid (NEFA) treatments during mouse preimplantation development in vitro, where PA elevates autophagosome formation and reduces autophagosome degradation levels, whereas cotreatment with OA reversed these PA effects. Autophagosome-lysosome colocalization only differed between PA and OA alone treatment groups. These findings advance our understanding of the effects of free fatty acid exposure on preimplantation development, and they uncover principles that may underlie the associations between elevated fatty acid levels and overall declines in reproductive fertility.

As obese and overweight patients commonly display hyperlipidemia and are increasingly accessing fertility clinics for their conception needs, our studies are directed at understanding the effects of hyperlipidemia on early pregnancy. We have focused on investigating palmitic acid (PA) and oleic acid (OA) treatment alone and in combination from the mouse two-cell stage embryos as a model for understanding their effects on the mammalian preimplantation embryo. We recently reported that PA exerts a negative effect on mouse two-cell progression to the blastocyst stage, whereas OA co-treatment reverses that negative effect. In the present study, we hypothesized that PA treatment of mouse embryos would disrupt proper localization of cell fate determining and blastocyst formation gene products and that co-treatment with OA would reverse these effects. Our results demonstrate that PA treatment significantly (P < 0.05) reduces blastocyst development and cell number but did not prevent nuclear localization of YAP in outer cells. PA treatment significantly reduced the number of OCT4+ and CDX2+ nuclei. PA-treated embryos had lower expression of blastocyst formation proteins (E-cadherin, ZO-1 and Na/K-ATPase alpha1 subunit). Importantly, co-treatment of embryos with OA reversed PA-induced effects on blastocyst development and increased inner cell mass (ICM) and trophectoderm (TE) cell numbers and expression of blastocyst formation proteins. Our findings demonstrate that PA treatment does not impede cell fate gene localization but does disrupt proper blastocyst formation gene localization during mouse preimplantation development. OA treatment is protective and reverses PA's detrimental effects. The results advance our understanding of the impact of FFA exposure on mammalian preimplantation development.

Background: Video consultations are increasingly used to communicate with patients, particularly during the current COVID-19 pandemic. However, training in video consultation skills receives scant attention in the literature. We sought to introduce this important topic to our undergraduate medical school curriculum., Objective: To increase final year medical students' video consultation skills and knowledge., Methods: We used Plan, Do, Study, Act (PDSA) quality improvement methodology with a pre-post study design to develop a teaching session for 5th year medical students, informed by a literature review and online clinician survey. The 2 hour session comprised an introduction and three practical stations: patient selection and ethics, technology and example videos, and simulation. Subjective pre- and post-session confidence was reported by students across seven domains using 5-point scales (1: not at all confident; 5: extremely confident). Students and facilitators completed post-session feedback forms., Results: The 40 students and 3 facilitators who attended, over two separate teaching sessions, provided unanimously positive feedback. All students considered the session relevant. Subjective confidence ratings (n = 34) significantly increased from pre- to post-session (mean increase 1.78, p < 0.001)., Conclusions: The inaugural teaching session was well-received and subjective assessment measures showed improvement in taught skills. This pilot has informed a UK-wide multi-centre study with subjective and objective data collection.

Introduction: Social cognition is an important area of mental functioning relevant to psychiatric disorders and social functioning, that may be affected by psychiatric drug treatments. The aim of this review was to investigate the effects of medications with sedative properties, on social cognition., Method: This systematic review included experimental and neuroimaging studies investigating drug effects on social cognition. Data quality was assessed using a modified Downs and Black checklist (Trac et al. CMAJ 188: E120-E129, 2016). The review used narrative synthesis to analyse the data., Results: 40 papers were identified for inclusion, 11 papers investigating benzodiazepine effects, and 29 investigating antipsychotic effects, on social cognition. Narrative synthesis showed that diazepam impairs healthy volunteer's emotion recognition, with supporting neuroimaging studies showing benzodiazepines attenuate amygdala activity. Studies of antipsychotic effects on social cognition gave variable results. However, many of these studies were in patients already taking medication, and potential practice effects were identified due to short-term follow-ups., Conclusion: Healthy volunteer studies suggest that diazepam reduces emotional processing ability. The effects of benzodiazepines on other aspects of social cognition, as well as the effects of antipsychotics, remain unclear. Interpretations of the papers in this review were limited by variability in measures, small sample sizes, and lack of randomisation. More robust studies are necessary to evaluate the impact of these medications on social cognition., (© 2021. The Author(s).)

Color vision, originating with opponent processing of spectrally distinct photoreceptor signals, plays important roles in animal behavior. 1-4 Surprisingly, however, comparatively little is understood about color processing in the brain, including in widely used laboratory mammals such as mice. The retinal gradient in S- and M-cone opsin (co-)expression has traditionally been considered an impediment to mouse color vision. 5-8 However, recent data indicate that mice exhibit robust chromatic discrimination within the central-upper visual field. 9 Retinal color opponency has been reported to emerge from superimposing inhibitory surround receptive fields on the cone opsin expression gradient, and by introducing opponent rod signals in retinal regions with sparse M-cone opsin expression. 10-13 The relative importance of these proposed mechanisms in determining the properties of neurons at higher visual processing stages remains unknown. We address these questions using multielectrode recordings from the lateral geniculate nucleus (LGN) in mice with altered M-cone spectral sensitivity (Opn1mw R ) and multispectral stimuli that allow selective modulation of signaling by individual opsin classes. Remarkably, we find many (∼25%) LGN cells are color opponent, that such cells are localized to a distinct medial LGN zone and that their properties cannot simply be explained by the proposed retinal opponent mechanisms. Opponent responses in LGN can be driven solely by cones, independent of cone-opsin expression gradients and rod input, with many cells exhibiting spatially congruent antagonistic receptive fields. Our data therefore suggest previously unidentified mechanisms may support extensive and sophisticated color processing in the mouse LGN., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Extracellular microRNA (miRNA) sequences derived from the pre-implantation embryo have attracted interest for their possible contributions to the ongoing embryonic-uterine milieu, as well as their potential for use as accessible biomarkers indicative of embryonic health. Spent culture media microdroplets used to culture late-stage E4.0 murine blastocysts were screened for 641 mature miRNA sequences using a reverse transcription-quantitative polymerase chain reaction-based array. We report here 39 miRNAs exclusively detected in the conditioned media, including the implantation-relevant miR-126a-3p, miR-101a, miR-143, and miR-320, in addition to members of the highly expressed embryonic miR-125 and miR-290 families. Based on these results, an miRNA panel was assembled comprising five members of the miR-290 family (miR-291-295) and five conserved sequences with significance to the embryonic secretome (miR-20a, miR-30c, miR-142-3p, miR-191, and miR-320). Panel profiling of developing embryo cohort lysates and accompanying conditioned media microdroplets revealed extensive similarities in relative quantities of miRNAs and, as a biomarker proof of concept, enabled distinction between media conditioned with differently staged embryos (zygote, 4-cell, and blastocyst). When used to assess media conditioned with embryos of varying degrees of degeneration, the panel revealed increases in all extracellular panel sequences, suggesting cell death is an influential and identifiable factor detectable by this assessment. In situ hybridization of three panel sequences (miR-30c, miR-294, and miR-295) in late-stage blastocysts revealed primarily inner cell mass expression with a significant presence of miR-294 throughout the blastocyst cavity. Furthermore, extracellular miR-290 sequences responded significantly to high centrifugal force, suggesting a substantial fraction of these sequences may exist within a vesicle such as an exosome, microvesicle, or apoptotic bleb. Together, these results support the use of extracellular miRNA to assess embryonic health and enable development of a non-invasive viability diagnostic tool for clinical use., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Hawke, Ahmed, Watson and Betts.)

Reactivation of the multi-subunit ribonucleoprotein telomerase is the primary telomere maintenance mechanism in cancer, but it is rate-limited by the enzymatic component, telomerase reverse transcriptase (TERT). While regulatory in nature, TERT alternative splice variant/isoform regulation and functions are not fully elucidated and are further complicated by their highly diverse expression and nature. Our primary objective was to characterize TERT isoform expression across 7887 neoplastic and 2099 normal tissue samples using The Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression Project (GTEx), respectively. We confirmed the global overexpression and splicing shift towards full-length TERT in neoplastic tissue. Stratifying by tissue type we found uncharacteristic TERT expression in normal brain tissue subtypes. Stratifying by tumor-specific subtypes, we detailed TERT expression differences potentially regulated by subtype-specific molecular characteristics. Focusing on β-deletion splicing regulation, we found the NOVA1 trans -acting factor to mediate alternative splicing in a cancer-dependent manner. Of relevance to future tissue-specific studies, we clustered cancer cell lines with tumors from related origin based on TERT isoform expression patterns. Taken together, our work has reinforced the need for tissue and tumour-specific TERT investigations, provided avenues to do so, and brought to light the current technical limitations of bioinformatic analyses of TERT isoform expression.

The source of oxygen to Earth's atmosphere is organic carbon burial, whilst the main sink is oxidative weathering of fossil carbon. However, this sink is to insensitive to counteract oxygen rising above its current level of about 21%. Biogeochemical models suggest that wildfires provide an additional regulatory feedback mechanism. However, none have considered how the evolution of different plant groups through time have interacted with this feedback. The Cretaceous Period saw not only super-ambient levels of atmospheric oxygen but also the evolution of the angiosperms, that then rose to dominate Earth's ecosystems. Here we show, using the COPSE biogeochemical model, that angiosperm-driven alteration of fire feedbacks likely lowered atmospheric oxygen levels from ~30% to 25% by the end of the Cretaceous. This likely set the stage for the emergence of closed-canopy angiosperm tropical rainforests that we suggest would not have been possible without angiosperm enhancement of fire feedbacks.

Objective: To study the impact of follitropin delta for ovarian stimulation on embryo development and quality compared with that of follitropin alfa or beta in in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) cycles., Design: Retrospective cohort study., Setting: University-affiliated, hospital-based fertility clinic., Patients: A total of 403 IVF/ICSI cycles were conducted from September 1, 2018 to December 31, 2019. Cycles were grouped on the basis of stimulation with follitropin delta vs. follitropin alfa or beta., Interventions: None., Main Outcome Measures: Embryo parameters and clinical pregnancy and implantation rates., Results: Ovarian stimulation using follitropin delta resulted in no statistically significant difference in day 3 embryo quality between the control group and follitropin delta group (median 0.50 vs. 0.54 for good quality embryos and median 0.25 vs. 0.20 for intermediate quality embryos). Although on initial analysis there was a lower proportion of good quality blastocysts in the follitropin delta group than in the control group (0.11 vs. 0.22), this difference was no longer present when day 3 after fertilization vitrification and transfer cycles were excluded (0.26 vs. 0.33 follitropin delta vs. control). The clinical pregnancy rates and clinical implantation rates were similar in both groups in fresh transfer cycles., Conclusions: Stimulation with follitropin delta in IVF/ICSI cycles resulted in similar embryo development and pregnancy rates compared with those of stimulation with follitropin alfa or beta., (© 2020 The Authors.)

The ocean is a sink for ~25% of the atmospheric CO 2 emitted by human activities, an amount in excess of 2 petagrams of carbon per year (PgC yr -1 ). Time-resolved estimates of global ocean-atmosphere CO 2 flux provide an important constraint on the global carbon budget. However, previous estimates of this flux, derived from surface ocean CO 2 concentrations, have not corrected the data for temperature gradients between the surface and sampling at a few meters depth, or for the effect of the cool ocean surface skin. Here we calculate a time history of ocean-atmosphere CO 2 fluxes from 1992 to 2018, corrected for these effects. These increase the calculated net flux into the oceans by 0.8-0.9  PgC yr -1 , at times doubling uncorrected values. We estimate uncertainties using multiple interpolation methods, finding convergent results for fluxes globally after 2000, or over the Northern Hemisphere throughout the period. Our corrections reconcile surface uptake with independent estimates of the increase in ocean CO 2 inventory, and suggest most ocean models underestimate uptake.

Background: Primary cilia have been shown to play a central role in regulating epithelial cell differentiation during injury and repair. Growing evidence implicates structural and functional abnormalities of primary cilia in kidney epithelial cells in the onset and development of various kidney diseases including polycystic kidney disease (PKD). Neutrophil-gelatinase associated lipocalin (NGAL) has been identified as a reliable urinary biomarker of kidney injury. However, the mechanism by which this protein accumulates in patient urine samples has not been fully elucidated., Methods: Human renal tubular epithelial cells (RPTECs) were exposed to previously characterized deciliating agents to assess mechanisms of primary cilium loss. Confocal immunofluorescent imaging was employed to visualise the effects on cilia. Western blot analysis was utilised to quantify the ciliary protein Arl13b in both RPTEC whole cell lysates and supernatants. Co-immunoprecipitation was used to demonstrate co-localisation of Arl13b and NGAL in urinary samples from a clinical Chronic Allograft Nephropathy (CAN) cohort., Results: Immunofluorescent analysis revealed that NGAL was localised to the primary cilium in RPTECs, co-localizing with a ciliary specific protein, Arl13b. Deciliation experiments showed that loss of the cilia coincided with loss of NGAL from the cells., Conclusion: The accumulation of NGAL in supernatants in vitro and in the urine of CAN patients was concurrent with loss of Arl13b, a specific ciliary protein. The findings of this study propose that increased NGAL urinary concentrations are directly linked to deciliation of the renal epithelial cells as a result of injury., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Haemorrhoids are a benign anorectal condition and are highly prevalent in the UK population. Treatments involve clinic-based procedures and surgery. The surgical procedures available include stapled haemorrhoidopexy (SH) and traditional haemorrhoidectomy (TH), and over 25,000 operations are performed for haemorrhoids annually in the UK. The disease is therefore important both to patients and to health service commissioners. Debate remains as to which of these surgical procedures is the most clinically effective and cost-effective., Objective: The aim of this study was to compare the clinical effectiveness and cost-effectiveness of SH with that of TH., Design: A large, open two-arm parallel-group pragmatic multicentre randomised controlled trial involving 32 UK hospitals and a within-trial cost-benefit analysis. A discrete choice experiment was conducted to estimate benefits (willingness to pay)., Participants: Patients with grades II-IV haemorrhoids who had not previously undergone SH or TH were included in the study., Interventions: Participants were randomised to receive either SH or TH. Randomisation was minimised at 1 : 1, in accordance with baseline EuroQol-5 Dimensions, three-level version (EQ-5D-3L) score, haemorrhoid grade, sex and centre, via an automated system., Main Outcome Measures: The primary outcome was area under the quality-of-life curve measured using the EQ-5D-3L descriptive system over 24 months, and the primary economic outcome was the incremental cost-effectiveness ratio. Secondary outcomes included disease-specific quality of life, recurrence, complications, further interventions and costs., Results: Between January 2011 and August 2014, 777 patients were randomised (389 to receive SH and 388 to receive TH). There were 774 participants included in the analysis as a result of one post-randomisation exclusion in the SH arm and two in the TH arm. SH was less painful than TH in the short term. Surgical complications were similar in both arms. EQ-5D-3L score was higher for the SH arm in the first 6 weeks after surgery, but over 24 months the TH group had significantly better EQ-5D-3L scores (-0.073, 95% confidence interval -0.140 to -0.006; p  = 0.0342). Symptoms and further interventions were significantly fewer in the TH arm at 24 months. Continence was better in the TH arm and tenesmus occurred less frequently. The number of serious adverse events reported was 24 out of 337 (7.1%) for participants who received SH and 33 out of 352 (9.4%) for those who received TH. There were two deaths in the SH arm, both unrelated to the eTHoS (either Traditional Haemorrhoidectomy or Stapled haemorrhoidopexy for haemorrhoidal disease) study. Patient preference did not seem to influence the treatment difference. SH was dominated by TH as it cost more and was less effective. The net benefit for the TH arm was higher than that for the SH arm., Limitations: Neither the participants nor the assessors were masked to treatment assignment and final recruitment was slightly short of the total target of 800. There were also substantial missing follow-up data., Conclusions: While patients who received SH had less short-term pain, after 6 weeks, recurrence rates, symptoms, re-interventions and quality-of-life measures all favoured TH. In addition, TH is cheaper. As part of a tailored management plan for haemorrhoids, TH should be considered over SH as the surgical treatment of choice for haemorrhoids refractory to clinic-based interventions., Future Work: Perform an updated meta-analysis incorporating recently conducted European trials [eTHoS, HubBLe (haemorrhoidal artery ligation versus rubber band ligation for the management of symptomatic second-degree and third-degree haemorrhoids) and LingaLongo (Cost-effectiveness of New Surgical Treatments for Haemorrhoidal Disease)]., Trial Registration: Current Controlled Trials ISRCTN80061723., Funding: This project was funded by the National Institute for Health Research (NIHR) Health Technology Assessment programme and will be published in full in Health Technology Assessment ; Vol. 21, No. 70. See the NIHR Journals Library website for further project information.

Study Question: What is the impact of adenosine monophosphate-activated protein kinase (AMPK) activation on blastocyst formation, gene expression, and tight junction formation and function?, Summary Answer: AMPK activity must be tightly controlled for normal preimplantation development and blastocyst formation to occur., What Is Known Already: AMPK isoforms are detectable in oocytes, cumulus cells and preimplantation embryos. Cultured embryos are subject to many stresses that can activate AMPK., Study Design, Size, Duration: Two primary experiments were carried out to determine the effect of AICAR treatment on embryo development and maintenance of the blastocoel cavity. Embryos were recovered from superovulated mice. First, 2-cell embryos were treated with a concentration series (0-2000 μM) of AICAR for 48 h until blastocyst formation would normally occur. In the second experiment, expanded mouse blastocysts were treated for 9 h with 1000 μM AICAR., Participants/materials, Setting, Methods: Outcomes measured included development to the blastocyst stage, cell number, blastocyst volume, AMPK phosphorylation, Cdx2 and blastocyst formation gene family expression (mRNAs and protein measured using quantitative RT-PCR, immunoblotting, immunofluorescence), tight junction function (FITC dextran dye uptake assay), and blastocyst ATP levels. The reversibility of AICAR treatment was assessed using Compound C (CC), a well-known inhibitor of AMPK, alone or in combination with AICAR., Main Results and the Role of Chance: Prolonged treatment with AICAR from the 2-cell stage onward decreases blastocyst formation, reduces total cell number, embryo diameter, leads to loss of trophectoderm cell contacts and membrane zona occludens-1 staining, and increased nuclear condensation. Treatment with CC alone inhibited blastocyst development only at concentrations that are higher than normally used. AICAR treated embryos displayed altered mRNA and protein levels of blastocyst formation genes. Treatment of blastocysts with AICAR for 9 h induced blastocyst collapse, altered blastocyst formation gene expression, increased tight junction permeability and decreased CDX2. Treated blastocysts displayed three phenotypes: those that were unaffected by treatment, those in which treatment was reversible, and those in which effects were irreversible., Large Scale Data: Not applicable., Limitations, Reasons for Caution: Our study investigates the effects of AICAR treatment on early development. While AICAR does increase AMPK activity and this is demonstrated in our study, AICAR is not a natural regulator of AMPK activity and some outcomes may result from off target non-AMPK AICAR regulated events. To support our results, blastocyst developmental outcomes were confirmed with two other well-known small molecule activators of AMPK, metformin and phenformin., Wider Implications of the Findings: Metformin, an AMPK activator, is widely used to treat type II diabetes and polycystic ovarian disorder (PCOS). Our results indicate that early embryonic AMPK levels must be tightly regulated to ensure normal preimplantation development. Thus, use of metformin should be carefully considered during preimplantation and early post-embryo transfer phases of fertility treatment cycles., Study Funding and Competing Interest(s): Canadian Institutes of Health Research (CIHR) operating funds. There are no competing interests., (© The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com)

The model organism Dictyostelium discoideum has greatly facilitated our understanding of the signal transduction and cytoskeletal pathways that govern cell motility. Cell-substrate adhesion is downstream of many migratory and chemotaxis signaling events. Dictyostelium cells lacking the tumor suppressor PTEN show strongly impaired migratory activity and adhere strongly to their substrates. We reasoned that other regulators of migration could be obtained through a screen for overly adhesive mutants. A screen of restriction enzyme-mediated integration mutagenized cells yielded numerous mutants with the desired phenotypes, and the insertion sites in 18 of the strains were mapped. These regulators of adhesion and motility mutants have increased adhesion and decreased motility. Characterization of seven strains demonstrated decreased directed migration, flatness, increased filamentous actin-based protrusions, and increased signal transduction network activity. Many of the genes share homology to human genes and demonstrate the diverse array of cellular networks that function in adhesion and migration., Competing Interests: The authors declare no conflict of interest.