Selective whole-genome amplification reveals population genetics of Leishmania braziliensis directly from patient skin biopsies.

In Brazil, Leishmania braziliensis is the main causative agent of the neglected tropical disease, cutaneous leishmaniasis (CL). CL presents on a spectrum of disease severity with a high rate of treatment failure. Yet the parasite factors that contribute to disease presentation and treatment outcome are not well understood, in part because successfully isolating and culturing parasites from patient lesions remains a major technical challenge. Here we describe the development of selective whole genome amplification (SWGA) for Leishmania and show that this method enables culture-independent analysis of parasite genomes obtained directly from primary patient skin samples, allowing us to circumvent artifacts associated with adaptation to culture. We show that SWGA can be applied to multiple Leishmania species residing in different host species, suggesting that this method is broadly useful in both experimental infection models and clinical studies. SWGA carried out directly on skin biopsies collected from patients in Corte de Pedra, Bahia, Brazil, showed extensive genomic diversity. Finally, as a proof-of-concept, we demonstrated that SWGA data can be integrated with published whole genome data from cultured parasite isolates to identify variants unique to specific geographic regions in Brazil where treatment failure rates are known to be high. SWGA provides a relatively simple method to generate Leishmania genomes directly from patient samples, unlocking the potential to link parasite genetics with host clinical phenotypes.

Circadian control of heparan sulfate levels times phagocytosis of amyloid beta aggregates.

Alzheimer's Disease (AD) is a neuroinflammatory disease characterized partly by the inability to clear, and subsequent build-up, of amyloid-beta (Aß). AD has a bi-directional relationship with circadian disruption (CD) with sleep disturbances starting years before disease onset. However, the molecular mechanism underlying the relationship of CD and AD has not been elucidated. Myeloid-based phagocytosis, a key component in the metabolism of Aß, is circadianly-regulated, presenting a potential link between CD and AD. In this work, we revealed that the phagocytosis of Aß42 undergoes a daily circadian oscillation. We found the circadian timing of global heparan sulfate proteoglycan (HSPG) biosynthesis was the molecular timer for the clock-controlled phagocytosis of Aß and that both HSPG binding and aggregation may play a role in this oscillation. These data highlight that circadian regulation in immune cells may play a role in the intricate relationship between the circadian clock and AD.

CAnceR IN PreGnancy (CARING) - a retrospective study of cancer diagnosed during pregnancy in the United Kingdom.

BACKGROUND: The incidence of cancer diagnosed during pregnancy is increasing. Data relating to investigation and management, as well as maternal and foetal outcomes is lacking in a United Kingdom (UK) population. METHODS: In this retrospective study we report data from 119 patients diagnosed with cancer during pregnancy from 14 cancer centres in the UK across a five-year period (2016-2020). RESULTS: Median age at diagnosis was 33 years, with breast, skin and haematological the most common primary sites. The majority of cases were new diagnoses (109 patients, 91.6%). Most patients were treated with radical intent (96 patients, 80.7%), however, gastrointestinal cancers were associated with a high rate of palliative intent treatment (63.6%). Intervention was commenced during pregnancy in 68 (57.1%) patients; 44 (37%) had surgery and 31 (26.1%) received chemotherapy. Live births occurred in 98 (81.7%) of the cases, with 54 (55.1%) of these delivered by caesarean section. Maternal mortality during the study period was 20.2%. CONCLUSIONS: This is the first pan-tumour report of diagnosis, management and outcomes of cancer diagnosed during pregnancy in the UK. Our findings demonstrate proof of concept that data collection is feasible and highlight the need for further research in this cohort of patients.

A combined analysis of two prospective randomised studies exploring the impact of extended post-radiation temozolomide on survival outcomes in newly diagnosed glioblastoma.

PURPOSE: The optimal duration of post-radiation temozolomide in newly diagnosed glioblastoma remains unclear, with no published phase III randomised trials. Standard-of-care stipulates 6 months. However, in routine care, it is often extended to 12 months, despite lacking robust supporting data. METHODS: GEINO14-01 (Spain) and EX-TEM (Australia) studies enrolled glioblastoma patients without progression at the end of 6 months post-radiation temozolomide. Participants were randomised 1:1 to six additional months of temozolomide or observation. Primary endpoint was 6-month progression free survival from date of randomisation (6mPFS). Secondary endpoints included overall survival (OS) and toxicity. 204 patients were required to detect an improvement in 6mPFS from 50 to 60% (80% power). Neither study recruited sufficient patients. We performed a combined analysis of individual patient data. RESULTS: 205 patients were recruited: 159 in GEINO14-01 (2014-2018) and 46 in EX-TEM (2019-2022). Median follow-up was 20.0 and 14.5 months. Baseline characteristics were balanced. There was no significant improvement in 6mPFS (57.2% vs 64.0%, OR0.75, p = 0.4), nor across any subgroups, including MGMT methylated; PFS (HR0.92, p = 0.59, median 7.8 vs 9.7 months); or OS (HR1.03, p = 0.87, median 20.1 vs 19.4 months). During treatment extension, 64% experienced any grade adverse event, mainly fatigue and gastrointestinal (both 54%). Only a minority required treatment changes: 4.5% dose delay, 7.5% dose reduction, 1.5% temozolomide discontinuation. CONCLUSION: For glioblastoma patients, extending post-radiation temozolomide from 6 to 12 months is well tolerated but does not improve 6mPFS. We could not identify any subset that benefitted from extended treatment. Six months should remain standard-of-care.

Exploring management and outcomes of elderly patients with glioblastoma using data from two randomised trials (GEINO1401/EX-TEM).

PURPOSE: The impact of age on optimal management of glioblastoma remains unclear. A recent combined analysis of two randomised trials, GEINO14-01 and EX-TEM, found no benefit from extending post-radiation temozolomide in newly diagnosed glioblastoma. Here, we explore the impact of age. METHODS: Relevant intergroup statistics were used to identify differences in tumour, treatment and outcome characteristics based on age with elderly patients (EP) defined as age 65 years and over. Survival was estimated using the Kaplan Meier method. RESULTS: Of the combined 205 patients, 57 (28%) were EP. Of these, 95% were ECOG 0-1 and 65% underwent macroscopic resection compared with 97% and 61% of younger patients (YP) respectively. There were numerically less MGMT-methylated (56% vs. 63%, p = 0.4) and IDH-mutated (4% vs. 13%, p = 0.1) tumours in EP vs. YP. Following surgery, EP were more likely to receive short course chemoradiation (17.5% vs. 6%, p = 0.017). At recurrence, EP tended to receive or best supportive care (28.3% vs. 15.4%, p = 0.09) or non-surgical options (96.2% vs. 84.6%, p = 0.06), but were less likely to receive bevacizumab (23.1% vs. 49.5%, p < 0.01). Median PFS was similar at 9.3months in EP and 8.5months in YP, with similar median OS at 20months. CONCLUSION: In this trial population of predominantly fit EP, survival was similar to YP despite a proportion receiving less aggressive therapy at diagnosis and recurrence. Advancing age does not appear to be an adverse prognostic factor for glioblastoma when patients are fit for treatment, and a less aggressive approach in selected patients may not compromise outcomes.

Volatile capture technology in sustainable anaesthetic practice: a narrative review.

Anaesthetic practice contributes to climate change. Volatile capture technology, typically based on adsorption to a carbon- or silica-based substrate, has the potential to mitigate some of the harmful effects of using halogenated hydrocarbons. Anaesthetists have a professional responsibility to use anaesthetic agents which offer the greatest safety and clinical benefit with the lowest financial cost and environmental impacts. Inhalational anaesthetics should be used at an appropriate concentration with a minimal fresh gas flow via a circle system to minimise unnecessary waste. Once practice efficiencies have been maximised, only then should technical solutions such as volatile capture be employed. In this narrative review, we focus on the available literature relating to volatile capture technology, obtained via a targeted literature search and through contacting manufacturers and researchers. We found six studies focusing on the Blue-Zone Technologies Deltasorb®, SageTech Medical SID and Baxter/ZeoSys CONTRAfluran™ volatile capture systems. Though laboratory analyses of available systems suggest that > 95% in vitro mass transfer is possible for all three systems, the in vivo results for capture efficiency vary from 25% to 73%. Currently, there is no financial incentive for healthcare organisations to capture waste anaesthetic gases, and so the value of volatile capture technology requires quantification. System-level organisations, such as Greener NHS, are best positioned to commission such evaluations and make policy decisions to guide investment. Further research using volatile capture technology in real-world settings is necessary and we highlight some priority research questions to improve our understanding of the utility of this group of technologies.

Clinical outcomes for sexual and gender minority adolescents in a dialectical behaviour therapy programme.

BACKGROUND: Sexuality and gender minoritised (SGM) adolescents are at increased risk of self-injury and suicide, and experience barriers to accessing mental health support. Dialectical behaviour therapy (DBT) is an effective treatment for self-injury and emotion dysregulation in adolescent populations, but few studies have published outcomes of DBT for SGM young people. AIMS: This study aimed to investigate treatment outcomes and completion for SGM adolescents and their cisgender and heterosexual peers, in the National & Specialist CAMHS, DBT service (UK). METHOD: Treatment completion, and opting out before and during treatment were examined for sexual and gender identity groups, as well as changes by the end of treatment in emotion dysregulation, self-injury, in-patient bed-days, emergency department attendances, and borderline personality disorder, depression and anxiety symptoms. RESULTS: SGM adolescents were over-represented in this service, even after considering their increased risk for self-injury. No statistically significant differences were found for treatment completion between the sexual orientation and gender identity groups, although there were patterns indicating possible lower treatment uptake and completion that warrant further investigation. Clinical outcomes for treatment-completers showed improvement by the end of DBT for each group, with few exceptions. DISCUSSION: These results are from relatively small subsamples, and it was not possible to separate by sex assigned at birth. Findings should be treated tentatively and as early indications of effect sizes to inform future studies. This study suggests that DBT could be a useful treatment for SGM adolescents in a highly specialist treatment setting.

Integrin ß3 regulates apical dendritic morphology of pyramidal neurons throughout hippocampal CA3.

In excitatory hippocampal pyramidal neurons, integrin ß3 is critical for synaptic maturation and plasticity in vitro. Itgb3 is a potential autism susceptibility gene that regulates dendritic morphology in the cerebral cortex in a cell-specific manner. However, it is unknown what role Itgb3 could have in regulating hippocampal pyramidal dendritic morphology in vivo, a key feature that is aberrant in many forms of autism and intellectual disability. We found that Itgb3 mRNA is expressed in the stratum pyramidale of CA3. We examined the apical dendritic morphology of CA3 hippocampal pyramidal neurons in conditional Itgb3 knockouts and controls, utilizing the Thy1-GFP-M line. We fully reconstructed the apical dendrite of each neuron and determined each neuron's precise location along the dorsoventral, proximodistal, and radial axes of the stratum pyramidale. We found a very strong effect for Itgb3 expression on CA3 apical dendritic morphology: neurons from conditional Itgb3 knockouts had longer and thinner apical dendrites than controls, particularly in higher branch orders. We also assessed potential relationships between pairs of topographic or morphological variables, finding that most variable pairs were free from any linear relationships to each other. We also found that some neurons from controls, but not conditional Itgb3 knockouts, had a graded pattern of overall diameter along the dorsoventral and proximodistal axes of the stratum pyramidale of CA3. Taken together, Itgb3 is essential for constructing normal dendritic morphology in pyramidal neurons throughout CA3.

Photocatalytic conversion of aryl diazonium salts to sulfonyl fluorides.

Sulfonyl fluorides have emerged as powerful tools in chemical biology for the selective labelling of proteins. A photocatalytic method is described for the conversion of aryl diazonium salts to aryl sulfonyl fluorides. The diazonium substrates are easily obtained in one step from functionalized anilines. We present the optimization of this mild method for the synthesis of sulfonyl fluorides, the scope of the transformation with a series of functionalized diazonium salts, and we discuss photophysical measurements that provide detailed information about the mechanism of the photochemical process.

Designing heterotropically activated allosteric conformational switches using supercharging.

Heterotropic allosteric activation of protein function, in which binding of one ligand thermodynamically activates the binding of another, different ligand or substrate, is a fundamental control mechanism in metabolism and as such has been a long-aspired capability in protein design. Here we show that greatly increasing the magnitude of a protein's net charge using surface supercharging transforms that protein into an allosteric ligand- and counterion-gated conformational molecular switch. To demonstrate this we first modified the designed helical bundle hemoprotein H4, creating a highly charged protein which both unfolds reversibly at low ionic strength and undergoes the ligand-induced folding transition commonly observed in signal transduction by intrinsically disordered proteins in biology. As a result of the high surface-charge density, ligand binding to this protein is allosterically activated up to 1,300-fold by low concentrations of divalent cations and the polyamine spermine. To extend this process further using a natural protein, we similarly modified Escherichia coli cytochrome b562 and the resulting protein behaves in a like manner. These simple model systems not only establish a set of general engineering principles which can be used to convert natural and designed soluble proteins into allosteric molecular switches useful in biodesign, sensing, and synthetic biology, the behavior we have demonstrated--functional activation of supercharged intrinsically disordered proteins by low concentrations of multivalent ions--may be a control mechanism utilized by Nature which has yet to be appreciated.

Compromised fractalkine signaling delays microglial occupancy of emerging modules in the multisensory midbrain.

Microglial cells (MGCs) are highly dynamic and have been implicated in shaping discrete neural maps in several unimodal systems. MGCs respond to numerous cues in their microenvironment, including the neuronally expressed chemokine, fractalkine (CX3CL1), via interactions with its corresponding fractalkine receptor (CX3CR1). The present study examines microglial and CX3CL1 patterns with regard to the emerging modular-extramodular matrix organization within the lateral cortex of the inferior colliculus (LCIC). The LCIC is a multisensory shell region of the midbrain inferior colliculus where discrete compartments receive modality-specific connections. Somatosensory inputs terminate within modular confines, while auditory inputs target the surrounding matrix. Glutamic acid decarboxylase (GAD) is an established marker of LCIC modules in developing mouse. During early postnatal development, multimodal LCIC afferents segregate into discrete, neurochemically defined compartments. Here, we analyzed neonatal GAD67-GFP (GFP is defined as green fluorescent protein) and CX3CR1-GFP mice to assess: (1) whether MGCs are recruited to distinct LCIC compartments known to be undergoing active circuit assembly, and (2) if such behaviors are fractalkine signaling-dependent. MGCs colonize the nascent LCIC by birth and increase in density until postnatal day 12 (P12). At the peak critical period (P4-P8), MGCs conspicuously border emerging LCIC modules, prior to their subsequent invasion by P12. CX3CL1 expression becomes distinctly modular at P12, in keeping with the notion of fractalkine-mediated recruitment of microglia to modular centers. In CX3CR1GFP/GFP mice with compromised fractalkine signaling, microglial recruitment into modules is delayed. Taken together, these results suggest a potential role for microglia and fractalkine signaling in sculpting multisensory LCIC maps during an early critical period.

Warming signals in temperate reef communities following more than a decade of ecological stability.

Ecosystem structure and function are increasingly threatened by changing climate, with profound effects observed globally in recent decades. Based on standardized visual censuses of reef biodiversity, we describe 27 years of community-level change for fishes, mobile macroinvertebrates and macroalgae in the Tasmanian ocean-warming hotspot. Significant ecological change was observed across 94 reef sites (5-10 m depth range) spanning four coastal regions between three periods (1992-95, 2006-07, 2017-19), which occurred against a background of pronounced sea temperature rise (+0.80°C on average). Overall, fish biomass increased, macroinvertebrate species richness and abundance decreased and macroalgal cover decreased, particularly during the most recent decade. While reef communities were relatively stable and warming was slight between the 1990s and mid-2000s (+0.12°C mean temperature rise), increased abundances of warm affinity fishes and invertebrates accompanied warming during the most recent decade (+0.68°C rise). However, significant rises in the community temperature index (CTI) were only found for fishes, invertebrates and macroalgae in some regions. Coastal warming was associated with increased fish biomass of non-targeted species in fished zones but had little effect on reef communities within marine reserves. Higher abundances of larger fishes and lobsters inside reserves appeared to negate impacts of 'thermophilization'.

Why don't we share data and code? Perceived barriers and benefits to public archiving practices.

The biological sciences community is increasingly recognizing the value of open, reproducible and transparent research practices for science and society at large. Despite this recognition, many researchers fail to share their data and code publicly. This pattern may arise from knowledge barriers about how to archive data and code, concerns about its reuse, and misaligned career incentives. Here, we define, categorize and discuss barriers to data and code sharing that are relevant to many research fields. We explore how real and perceived barriers might be overcome or reframed in the light of the benefits relative to costs. By elucidating these barriers and the contexts in which they arise, we can take steps to mitigate them and align our actions with the goals of open science, both as individual scientists and as a scientific community.

Integrin ß3 in forebrain Emx1-expressing cells regulates repetitive self-grooming and sociability in mice.

BACKGROUND: Autism spectrum disorder (ASD) is characterized by repetitive behaviors, deficits in communication, and overall impaired social interaction. Of all the integrin subunit mutations, mutations in integrin ß3 (Itgb3) may be the most closely associated with ASD. Integrin ß3 is required for normal structural plasticity of dendrites and synapses specifically in excitatory cortical and hippocampal circuitry. However, the behavioral consequences of Itgb3 function in the forebrain have not been assessed. We tested the hypothesis that behaviors that are typically abnormal in ASD-such as self-grooming and sociability behaviors-are disrupted with conditional Itgb3 loss of function in forebrain circuitry in male and female mice. METHODS: We generated male and female conditional knockouts (cKO) and conditional heterozygotes (cHET) of Itgb3 in excitatory neurons and glia that were derived from Emx1-expressing forebrain cells during development. We used several different assays to determine whether male and female cKO and cHET mice have repetitive self-grooming behaviors, anxiety-like behaviors, abnormal locomotion, compulsive-like behaviors, or abnormal social behaviors, when compared to male and female wildtype (WT) mice. RESULTS: Our findings indicate that only self-grooming and sociability are altered in cKO, but not cHET or WT mice, suggesting that Itgb3 is specifically required in forebrain Emx1-expressing cells for normal repetitive self-grooming and social behaviors. Furthermore, in cKO (but not cHET or WT), we observed an interaction effect for sex and self-grooming environment and an interaction effect for sex and sociability test chamber. LIMITATIONS: While this study demonstrated a role for forebrain Itgb3 in specific repetitive and social behaviors, it was unable to determine whether forebrain Itgb3 is required for a preference for social novelty, whether cHET are haploinsufficient with respect to repetitive self-grooming and social behaviors, or the nature of the interaction effect for sex and environment/chamber in affected behaviors of cKO. CONCLUSIONS: Together, these findings strengthen the idea that Itgb3 has a specific role in shaping forebrain circuitry that is relevant to endophenotypes of autism spectrum disorder.

Correction: Attention promotes the neural encoding of prediction errors.

[This corrects the article DOI: 10.1371/journal.pbio.2006812.].

Attention promotes the neural encoding of prediction errors.

The encoding of sensory information in the human brain is thought to be optimised by two principal processes: 'prediction' uses stored information to guide the interpretation of forthcoming sensory events, and 'attention' prioritizes these events according to their behavioural relevance. Despite the ubiquitous contributions of attention and prediction to various aspects of perception and cognition, it remains unknown how they interact to modulate information processing in the brain. A recent extension of predictive coding theory suggests that attention optimises the expected precision of predictions by modulating the synaptic gain of prediction error units. Because prediction errors code for the difference between predictions and sensory signals, this model would suggest that attention increases the selectivity for mismatch information in the neural response to a surprising stimulus. Alternative predictive coding models propose that attention increases the activity of prediction (or 'representation') neurons and would therefore suggest that attention and prediction synergistically modulate selectivity for 'feature information' in the brain. Here, we applied forward encoding models to neural activity recorded via electroencephalography (EEG) as human observers performed a simple visual task to test for the effect of attention on both mismatch and feature information in the neural response to surprising stimuli. Participants attended or ignored a periodic stream of gratings, the orientations of which could be either predictable, surprising, or unpredictable. We found that surprising stimuli evoked neural responses that were encoded according to the difference between predicted and observed stimulus features, and that attention facilitated the encoding of this type of information in the brain. These findings advance our understanding of how attention and prediction modulate information processing in the brain, as well as support the theory that attention optimises precision expectations during hierarchical inference by increasing the gain of prediction errors.

MRI safety, imaging artefacts, and grid distortion evaluated for FFP3 respiratory masks worn throughout the COVID-19 pandemic.

AIM: To determine which filtering face piece (FFP3) respirators worn throughout the COVID-19 pandemic are safe for magnetic resonance imaging (MRI). MATERIALS AND METHODS: Three clinical MRI sequences were performed to assess imaging artefacts, grid distortion, and local heating for eight commercially available FFP3 respirators. All examinations were performed at Cardiff University Brain Research Imaging Centre using a 3 T Siemens Magnetom Prisma with a 64-channel head and neck coil. Each FFP3 mask was positioned on a custom-developed three-dimensional (3D) head phantom for testing. RESULTS: Five of the eight FFP3 masks contained ferromagnetic components and were regarded as "MRI unsafe". One mask was considered "MRI conditional" and only two masks were deemed "MRI safe" for both MRI staff and patients. Temperature strips positioned at the nasal bridge of the phantom did not exhibit local heating. A maximum grid distortion of 5 mm was seen in the anterior portion of the head of the ferromagnetic FFP3 masks. CONCLUSION: This study has demonstrated the importance of assessing respiratory FFP3 masks for use in and around MRI machines. Future research involving FFP3 masks can be conducted safely by following the procedures laid out in this study.

Realist analysis of whether emergency departments with primary care services generate 'provider-induced demand'.

BACKGROUND: It is not known whether emergency departments (EDs) with primary care services influence demand for non-urgent care ('provider-induced demand'). We proposed that distinct primary care services in EDs encourages primary care demand, whereas primary care integrated within EDs may be less likely to cause additional demand. We aimed to explore this and explain contexts (C), mechanisms (M) and outcomes (O) influencing demand. METHODS: We used realist evaluation methodology and observed ED service delivery. Twenty-four patients and 106 staff members (including Clinical Directors and General Practitioners) were interviewed at 13 EDs in England and Wales (240 hours of observations across 30 days). Field notes from observations and interviews were analysed by creating 'CMO' configurations to develop and refine theories relating to drivers of demand. RESULTS: EDs with distinct primary care services were perceived to attract demand for primary care because services were visible, known or enabled direct access to health care services. Other influencing factors included patients' experiences of accessing primary care, community care capacity, service design and population characteristics. CONCLUSIONS: Patient, local-system and wider-system factors can contribute to additional demand at EDs that include primary care services. Our findings can inform service providers and policymakers in developing strategies to limit the effect of potential influences on additional demand when demand exceeds capacity.

Understanding the Structure and Apo Dynamics of the Functionally Active JIP1 Fragment.

Recent experiments indicate that the C-Jun amino-terminal kinase-interacting protein 1 (JIP1) binds to and activates the c-Jun N-terminal kinase (JNK) protein. JNK is an integral part of cell apoptosis, and misregulation of this process is a causative factor in diseases such as Alzheimer's disease (AD), obesity, and cancer. It has also been shown that JIP1 may increase the phosphorylation of tau by facilitating the interaction between the tau protein and JNK, which could also be a causative factor in AD. Very little is known about the structure and dynamics of JIP1; however, the amino acid composition of the first 350 residues suggests that it contains an intrinsically disordered region. Molecular dynamics (MD) simulations using AMBER 14 were used to study the structure and dynamics of a functionally active JIP1 10mer fragment to better understand the solution behavior of the fragment. Two microseconds of unbiased MD was performed on the JIP1 10mer fragment in 10 different seeds for a total of 20 µs of simulation time, and from this, seven structurally stable conformations of the 10mer fragment were identified via classical clustering. The 10mer ensemble was also used to build a Markov state model (MSM) that identified four metastable states that encompassed six of the seven conformational families identified by classical dimensional reduction. Based on this MSM, conformational interconversions between the four states occur via two dominant pathways with probability fluxes of 55 and 44% for each individual pathway. Transitions between the initial and final states occur with mean first passage times of 31 (forward) and 16 (reverse) µs.

Total Synthesis of the Meroterpenoid Manginoid†A as Fueled by a Challenging Pinacol Coupling and Bicycle-forming Etherification.

The manginoids are a unique collection of bioactive natural products whose structures fuse an oxa-bridged spirocyclohexanedione with a heavily substituted trans-hydrindane framework. Herein, we show that such architectures can be accessed through a strategy combining a challenging pinacol coupling and bicycle-forming etherification with several additional chemo- and regioselective reactions. The success of these key events proved to be highly substrate and condition specific, affording insights for their application to other targets. As a result, not only has a 19-step total synthesis of manginoid A been achieved, but a potential roadmap to access other members of the family and related natural products has also been identified.