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The subtropical to subpolar planktic foraminifera Globigerina bulloides is a calcifying marine protist, and one of the dominant foraminiferal species of the Nordic Seas. Previously, the relative abundance and shell geochemistry of fossil G. bulloides have been studied for palaeoceanographic reconstructions. There is however a lack of biological observations on the species and a poor understanding of its ecological tolerances, especially for high latitude genotypes. Here, we present observations from the first extensive culturing of G. bulloides under subpolar conditions, including the first low temperature (6-13°C) and variable salinity (30-38) experiments. Carbonate chemistry (pH and [CO3 2-]) was also manipulated. Experimental conditions were chosen to reflect a range of plausible past and future scenarios for the Nordic Seas. We found G. bulloides to be tolerant of environmental conditions well outside their optimal range (<10°C, salinity <33, pH <8). Observed life span was up to three months, which was attributed to a microalgal diet. Two alternative life strategies were employed, whereby individuals either experienced rapid growth and death, or a prolonged lifespan with minimal growth and death via slow decay. We posit this could help explain differences in geochemical signals recorded from different size fractions of fossil specimens used for palaeoceanographic reconstructions.

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Anti-contactin associated protein receptor 2 (CASPR2) encephalitis is a severe autoimmune encephalitis with a variable clinical phenotype including behavioral abnormalities, cognitive decline, epileptic seizures, peripheral nerve hyperexcitability and neuropathic pain. The detailed mechanisms of how CASPR2 autoantibodies lead to synaptic dysfunction and clinical symptoms are largely unknown. Aiming for analyses from the molecular to the clinical level, we isolated antibody-secreting cells from the cerebrospinal fluid of two patients with CASPR2 encephalitis. From these we cloned four anti-CASPR2 human monoclonal autoantibodies (mAbs) with strong binding to brain and peripheral nerves. All were highly hypermutated and mainly of the IgG4 subclass. Mutagenesis studies determined selective binding to the discoidin domain of CASPR2. Surface plasmon resonance revealed affinities with dissociation constants KD in the pico- to nanomolar range. CASPR2 mAbs interrupted the interaction of CASPR2 with its binding partner contactin 2 in vitro and were internalized after binding to CASPR2-expressing cells. Electrophysiological recordings of rat hippocampal slices after stereotactic injection of CASPR2 mAbs showed characteristic afterpotentials following electrical stimulation. In vivo experiments with intracerebroventricular administration of human CASPR2 mAbs into mice and rats showed EEG-recorded brain hyperexcitability but no spontaneous recurrent seizures. Behavioral assessment of infused mice showed a subtle clinical phenotype, mainly affecting sociability. Mouse brain MRI exhibited markedly reduced resting-state functional connectivity without short-term structural changes. Together, the experimental data support the direct pathogenicity of CASPR2 autoantibodies. The minimally invasive EEG and MRI techniques applied here may serve as novel objective, quantifiable tools for improved animal models, in particular for subtle neuropsychiatric phenotypes or repeated measurements.

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Geochemical proxies of sea surface temperature (SST) and seawater pH (pHsw) in scleractinian coral skeletons are valuable tools for reconstructing tropical climate variability. However, most coral skeletal SST and pHsw proxies are univariate methods that are limited in their capacity to circumvent non-climate-related variability. Here we present a novel multivariate method for reconstructing SST and pHsw from the geochemistry of coral skeletons. Our Scleractinian Multivariate Isotope and Trace Element (SMITE) method optimizes reconstruction skill by leveraging the covariance across an array of coral elemental and isotopic data with SST and pHsw. First, using a synthetic proxy experiment, we find that SMITE SST reconstruction statistics (correlation, accuracy, and precision) are insensitive to noise and variable calibration period lengths relative to Sr/Ca. While SMITE pHsw reconstruction statistics remain relative to δ11B throughout the same synthetic experiment, the magnitude of the long-term trend in pHsw is progressively lost under conditions of moderate-to-high analytical uncertainty. Next, we apply the SMITE method to an array of seven coral-based geochemical variables (B/Ca, δ11B, Li/Ca, Mg/Ca, Sr/Ca, U/Ca & Li/Mg) measured from two Bermudan Porites astreoides corals. Despite a <3.5 year calibration period, SMITE SST and pHsw estimates exhibit significantly better accuracy, precision, and correlation with their respective climate targets than the best single- and dual-proxy estimators. Furthermore, SMITE model parameters are highly reproducible between the two coral cores, indicating great potential for fossil applications (when preservation is high). The results shown here indicate that the SMITE method can outperform the most common coral-based SST and pHsw reconstructions methods to date, particularly in datasets with a large variety of geochemical variables. We therefore provide a list of recommendations and procedures for users to begin implementing the SMITE method as well as an open-source software package to facilitate dissemination of the SMITE method.

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The skeletons of long-lived bamboo coral (Family Keratoisididae) are promising archives for deep-water palaeoceanographic reconstructions as they can record environmental variation at sub-decadal resolution in locations where in-situ measurements lack temporal coverage. Yet, detailed three dimensional (3D) characterisations of bamboo coral skeletal architecture are not routinely available and non-destructive investigations into microscale variations in calcification are rare. Here, we provide high-resolution micro-focus computed tomography (µCT) data of skeletal density for two species of bamboo coral (Acanella arbuscula: 5 specimens, voxel size, 15 µm (central branch scans) and 50 µm (complete structure scan); Keratoisis sp.: 4 specimens, voxel size, 15 µm) collected from the Labrador Sea and Baffin Bay deep-water basins, Eastern Canadian Arctic. These data provide reference models useful for developing methods to assess structural integrity and other fine-scale complexities in many biological, geological, and industrial systems. This will be of wider value to those investigating structural composition, arrangement and/or composition of complex architecture within the fields and subdisciplines of biology, ecology, medicine, environmental geology, and structural engineering.

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Glaucoma is a progressive optic neuropathy resulting from pathological changes at the optic disc due to elevated intraocular pressure. Its diagnosis, treatment and follow-up are almost entirely conducted in ophthalmology clinics, with screening conducted by community optometrists. Despite this, neurologists may encounter glaucoma in both its acute presentation (as angle closure, presenting as acute headache) and its chronic forms (often as optic neuropathy of unknown cause). An awareness of the underlying pathological process, and the key distinguishing signs and symptoms, will allow neurologists to identify the glaucomatous process rapidly. Timely referral is essential as glaucoma invariably results in progressive visual loss without treatment. This review therefore condenses the wide field of glaucoma into a practical summary, aimed at practitioners with limited clinical experience of this ophthalmic condition.

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The chemical and isotopic composition of stony coral skeletons form an important archive of past climate. However, these reconstructions are largely based on empirical relationships often complicated by "vital effects" arising from uncertain physiological processes of the coral holobiont. The skeletons of deep-sea corals, such as Desmophyllum dianthus, are characterised by micron-scale or larger geochemical heterogeneity associated with: (1) centres of calcification (COCs) where nucleation of new skeleton begins, and (2) fibres that thicken the skeleton. These features are difficult to sample cleanly using traditional techniques, resulting in uncertainty surrounding both the causes of geochemical differences and their influence on environmental signals. Here we combine optical, and in-situ chemical and isotopic, imaging tools across a range of spatial resolutions (~ 100 nm to 10 s of µm) in a correlative multimodal imaging (CMI) approach to isolate the microstructural geochemistry of each component. This reveals COCs are characterised by higher organic content, Mg, Li and Sr and lower U, B and δ11B compared to fibres, reflecting the contrasting biomineralisation mechanisms employed to construct each feature. CMI is rarely applied in Environmental/Earth Sciences, but here we illustrate the power of this approach to unpick the "vital effects" in D. dianthus, and by extension, other scleractinian corals.

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Anthropogenic threats such as water infrastructure, land-use changes, overexploitation of fishes and other biological resources, invasive species and climate change present formidable challenges to freshwater biodiversity. Historically, management of fish and fishery species has largely been based on studies of population- and community-level dynamics; however, the emerging field of conservation physiology promotes the assessment of individual fish health as a key management tool. Fish health is highly sensitive to environmental disturbances and is also a fundamental driver of fitness, with implications for population dynamics such as recruitment and resilience. However, the mechanistic links between particular anthropogenic disturbances and changes in fish health, or impact pathways, are diverse and complex. The diversity of ways in which fish health can be measured also presents a challenge for researchers deciding on methods to employ in studies seeking to understand the impact of these threats. In this review, we aim to provide an understanding of the pathway through which anthropogenic threats in freshwater ecosystems impact fish health and the ways in which fish health components impacted by anthropogenic threats can be assessed. We employ a quantitative systematic approach to a corpus of papers related to fish health in freshwater and utilize a framework that summarizes the impact pathway of anthropogenic threats through environmental alterations and impact mechanisms that cause a response in fish health. We found that land-use changes were the most prolific anthropogenic threat, with a range of different health metrics being suitable for assessing the impact of this threat. Almost all anthropogenic threats impacted fish health through two or more impact pathways. A robust understanding of the impact pathways of anthropogenic threats and the fish health metrics that are sensitive to these threats is crucial for fisheries managers seeking to undertake targeted management of freshwater ecosystems.

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Coral reefs provide ecosystem benefits to millions of people but are threatened by rapid environmental change and ever-increasing human pressures. Restoration is becoming a priority strategy for coral reef conservation, yet implementation remains challenging and it is becoming increasingly apparent that indirect conservation and restoration approaches will not ensure the long-term sustainability of coral reefs. The important role of environmental conditions in restoration practice are currently undervalued, carrying substantial implications for restoration success. Giving paramount importance to environmental conditions, particularly during the pre-restoration planning phase, has the potential to bring about considerable improvements in coral reef restoration and innovation. This Essay argues that restoration risk may be reduced by adopting an environmentally aware perspective that gives historical, contemporary, and future context to restoration decisions. Such an approach will open up new restoration opportunities with improved sustainability that have the capacity to dynamically respond to environmental trajectories.

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INTRODUCTION: The humerus is a common site of metastases and primary tumors. For some patients with a segmental defect and/or diaphyseal cortical destruction a cemented intercalary device may provide a more reliable construct, however data on their use is limited. METHODS: We reviewed 43 (28 male and 15 female) patients treated with an intercalary humeral spacer at a single tertiary referral center between 1989 and 2022. Humeral lesions were most commonly secondary to metastatic disease (n = 29, 68%), with 25 (58%) patients presenting with a pathologic fracture. Mean age and body mass index were 66 years and 27.9 kg/m2 . First generation taper joint device were used in 22 patients and second-generation lap device in 21 patients. RESULTS: Following reconstruction the 2-year overall survival was 30%. Mechanical complications occurred in 11 patients, most commonly aseptic loosening (n = 6, 14%). With death as a competing risk, the cumulative incidence of mechanical failure was 28% at 2-years postoperative. Following the procedure, mean Musculoskeletal Tumor Society scores was 70% and mean shoulder elevation was 87°. CONCLUSION: Reconstruction of the humeral diaphysis with an intercalary endoprosthesis provides restoration of function of the upper extremity, however, is associated with one in four patients having mechanical failure.

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BACKGROUND: Multiple hereditary exostosis (MHE) is a rare autosomal dominant disorder characterized by multiple osteochondromas. There is a paucity of literature concerning total hip arthroplasty (THA) in patients with MHE. The aim of this study is to report long-term outcomes of THA in patients with MHE. METHODS: Fourteen patients undergoing 15 THA's for the treatment of osteoarthritis in the presence of osteochondromas and proximal femoral deformity secondary to MHE were reviewed. Mean age at the time of surgery and follow-up was 56 and 12 years. Seven (47%) had uncemented femoral components. Eleven hips had coxa valga on preoperative imaging. Clinical outcomes were assessed with both Harris hip scores (HHS) and Musculoskeletal Tumor Society Scores (MSTS). RESULTS: Following surgery, there was an improvement in the HHS (48-82, p < 0.01) and MSTS scores (41-70%, p < 0.01). Complications occurred in 5 patients leading to reoperation in 3 patients, of which 2 patients underwent a revision procedure at 19 and 20-years postoperative. The 10-year revision free survival was 100%. CONCLUSIONS: THA in the setting of MHE reliably improves patient function. One in three patients will have a postoperative complication; however, the long-term incidence of revision is low.

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ABSTRACT: Recombination-activating genes (RAG1 and RAG2) are critical for lymphoid cell development and function by initiating the variable (V), diversity (D), and joining (J) (V(D)J)-recombination process to generate polyclonal lymphocytes with broad antigen specificity. The clinical manifestations of defective RAG1/2 genes range from immune dysregulation to severe combined immunodeficiencies (SCIDs), causing life-threatening infections and death early in life without hematopoietic cell transplantation (HCT). Despite improvements, haploidentical HCT without myeloablative conditioning carries a high risk of graft failure and incomplete immune reconstitution. The RAG complex is only expressed during the G0-G1 phase of the cell cycle in the early stages of T- and B-cell development, underscoring that a direct gene correction might capture the precise temporal expression of the endogenous gene. Here, we report a feasibility study using the CRISPR/Cas9-based "universal gene-correction" approach for the RAG2 locus in human hematopoietic stem/progenitor cells (HSPCs) from healthy donors and RAG2-SCID patient. V(D)J-recombinase activity was restored after gene correction of RAG2-SCID-derived HSPCs, resulting in the development of T-cell receptor (TCR) αß and γδ CD3+ cells and single-positive CD4+ and CD8+ lymphocytes. TCR repertoire analysis indicated a normal distribution of CDR3 length and preserved usage of the distal TRAV genes. We confirmed the in vivo rescue of B-cell development with normal immunoglobulin M surface expression and a significant decrease in CD56bright natural killer cells. Together, we provide specificity, toxicity, and efficacy data supporting the development of a gene-correction therapy to benefit RAG2-deficient patients.

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The planktic foraminifera Neogloboquadrina pachyderma is a calcifying marine protist and the dominant planktic foraminifera species in the polar oceans, making it a key species in marine polar ecosystems. The calcium carbonate shells of foraminifera are widely used in palaeoclimate studies because their chemical composition reflects the seawater conditions in which they grow. This species provides unique proxy data for past surface ocean hydrography, which can provide valuable insight to future climate scenarios. However, little is known about the response of N. pachyderma to variable and changing environmental conditions. Here, we present observations from large-scale culturing experiments where temperature, salinity and carbonate chemistry were altered independently. We observed overall low mortality, calcification of new chambers and addition of secondary calcite crust in all our treatments. In-culture asexual reproduction events also allowed us to monitor the variable growth of N. pachyderma's offspring. Several specimens had extended periods of dormancy or inactivity after which they recovered. These observations suggest that N. pachyderma can tolerate, adapt to and calcify within a wide range of environmental conditions. This has implications for the species-level response to ocean warming and acidification, for future studies aiming to culture N. pachyderma and use in palaeoenvironmental reconstruction.

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Volatile phenols possess "smoky, spicy" aromas and are routinely measured in grapes, wines and other foodstuffs for quality control. Routine analyses of volatile phenols rely on gas chromatography - mass spectrometry (GC-MS), but slow throughput of GC-MS can cause challenges during times of surge demand, i.e. following 'smoke taint' events involving forest fires near vineyards. Parallel extraction of headspace volatiles onto sorbent sheets (HS-SPMESH) followed by direct analysis in real time mass spectrometry (DART-MS) is a rapid alternative to conventional GC-MS approaches. However, HS-SPMESH extraction is poorly suited for lower volatility odorants, including volatile phenols. This work reports development and validation of an HS-SPMESH-DART-MS approach for five volatile phenols (4-ethylphenol, 4-ethylguiacol, guaiacol, 4-methylguaiacol, and cresols). Prior to HS-SPMESH extraction, volatile phenols were acetylated to facilitate their extraction. A unique feature of this work was the use of d6-Ac2O as a derivatizing agent to overcome issues with isobaric interferences inherent to chromatography-free MS techniques. The use of alkaline conditions during derivatization resulted in cumulative measurement of both free and bound forms of volatile phenols. The validated HS-SPMESH-DART-MS method achieved a throughput of 24 samples in ∼60 min (including derivatization and extraction time) with low limits of detection (<1 µg L-1) and good repeatability (3-6% RSD) in grape and wine matrices. Validation experiments with smoke-tainted grape samples indicated good correlation between total (free + bound) volatile phenols measured by HS-SPMESH-DART-MS and a gold standard GC-MS method.

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BACKGROUND/AIM: Pre-emptive targeted muscle reinnervation (TMR) at the time of amputation results in less phantom limb pain (PLP) compared with untreated amputee controls. There is limited literature describing the technique in patients undergoing hindquarter amputation despite up to 90% of these patients reporting PLP and 50% presenting with painful neuroma. The purpose of the current study was to describe the motor nerves accessible through a primary hind-quarter amputation to be used for TMR and review pain outcomes in clinical case correlates of patients with TMR. PATIENTS AND METHODS: Six limbs were obtained from three fresh adult cadavers and proximal sensory and motor nerves were dissected. A review of patients undergoing hindquarter amputation with TMR was conducted. RESULTS: Transfers for the sciatic, femoral, and obturator nerves were identified in cadavers. In reviews of patients, they were taking narcotic and neuro-leptic pain medication for a mean of 23 days and 168 days. At most recent follow-up, no patient reported debilitating phantom pain nor pain associated with neuromas. CONCLUSION: Given the positive preliminary results in our study group as well as the accessible neuroanatomy, pre-emptive TMR should be considered at the time of surgery to limit PLP and dependence on pain medications.

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[This corrects the article DOI: 10.3389/fmicb.2023.1018242.].

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Age structure information of animal populations is fundamental to their conservation and management. In fisheries, age is routinely obtained by counting daily or annual increments in calcified structures (e.g., otoliths) which requires lethal sampling. Recently, DNA methylation has been shown to estimate age using DNA extracted from fin tissue without the need to kill the fish. In this study we used conserved known age-associated sites from the zebrafish (Danio rerio) genome to predict the age of golden perch (Macquaria ambigua), a large-bodied native fish from eastern Australia. Individuals aged using validated otolith techniques from across the species' distribution were used to calibrate three epigenetic clocks. One clock was calibrated using daily (daily clock) and another with annual (annual clock) otolith increment counts, respectively. A third used both daily and annual increments (universal clock). We found a high correlation between the otolith and epigenetic age (Pearson correlation > 0.94) across all clocks. The median absolute error was 2.4 days in the daily clock, 184.6 days in the annual clock, and 74.5 days in the universal clock. Our study demonstrates the emerging utility of epigenetic clocks as non-lethal and high-throughput tools for obtaining age estimates to support the management of fish populations and fisheries.

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BACKGROUND: Chimeric Antigen Receptor (CAR) T cells are now standard of care (SOC) for some patients with B cell and plasma cell malignancies and could disrupt the therapeutic landscape of solid tumors. However, access to CAR-T cells is not adequate to meet clinical needs, in part due to high cost and long lead times for manufacturing clinical grade virus. Non-viral site directed CAR integration can be accomplished using CRISPR/Cas9 and double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA) via homology-directed repair (HDR), however yields with this approach have been limiting for clinical application (dsDNA) or access to large yields sufficient to meet the manufacturing demands outside early phase clinical trials is limited (ssDNA). METHODS: We applied homology-independent targeted insertion (HITI) or HDR using CRISPR/Cas9 and nanoplasmid DNA to insert an anti-GD2 CAR into the T cell receptor alpha constant (TRAC) locus and compared both targeted insertion strategies in our system. Next, we optimized post-HITI CRISPR EnrichMENT (CEMENT) to seamlessly integrate it into a 14-day process and compared our knock-in with viral transduced anti-GD2 CAR-T cells. Finally, we explored the off-target genomic toxicity of our genomic engineering approach. RESULTS: Here, we show that site directed CAR integration utilizing nanoplasmid DNA delivered via HITI provides high cell yields and highly functional cells. CEMENT enriched CAR T cells to approximately 80% purity, resulting in therapeutically relevant dose ranges of 5.5 × 108-3.6 × 109 CAR + T cells. CRISPR knock-in CAR-T cells were functionally comparable with viral transduced anti-GD2 CAR-T cells and did not show any evidence of off-target genomic toxicity. CONCLUSIONS: Our work provides a novel platform to perform guided CAR insertion into primary human T-cells using nanoplasmid DNA and holds the potential to increase access to CAR-T cell therapies.

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Introduction: Ulcerative colitis (UC) is characterized by chronic inflammation in the colonic epithelium and has a blurred etiology. A western diet and microbial dysbiosis in the colon were reported to play a role in UC development. In this study, we investigated the effect of a westernized diet, i.e., increasing fat and protein content by including ground beef, on the colonic bacterial composition in a dextran sulfate sodium (DexSS) challenged pig study. Methods: The experiment was carried out in three complete blocks following a 2×2 factorial design including 24 six-week old pigs, fed either a standard diet (CT) or the standard diet substituted with 15% ground beef to simulate a typical westernized diet (WD). Colitis was induced in half of the pigs on each dietary treatment by oral administration of DexSS (DSS and WD+DSS, respectively). Samples from proximal and distal colon and feces were collected. Results and discussion: Bacterial alpha diversity was unaffected by experimental block, and sample type. In proximal colon, WD group had similar alpha diversity to CT group and the WD+DSS group showed the lowest alpha diversity compared to the other treatment groups. There was a significant interaction between western diet and DexSS for beta diversity, based on Bray-Curtis dissimilarly. The westernized diet and DexSS resulted in three and seven differentially abundant phyla, 21 and 65 species, respectively, mainly associated with the Firmicutes and Bacteroidota phyla followed by Spirochaetota, Desulfobacterota, and Proteobacteria. The concentration of short-chain fatty acids (SCFA) was lowest in the distal colon. Treatment had a slight effect on the estimates for microbial metabolites that might have valuable biological relevance for future studies. The concentration of putrescine in the colon and feces and that of total biogenic amines was highest in the WD+DSS group. We conclude that a westernized diet could be a potential risk factor and an exacerbating agent for UC by reducing the abundance of SCFA-producing bacteria, increasing the abundance of pathogens such as Helicobacter trogontum, and by increasing the concentration of microbial proteolytic-derived metabolites in the colon.

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RATIONALE: Organisms that grow a hard carbonate shell or skeleton, such as foraminifera, corals or molluscs, incorporate trace elements into their shell during growth that reflect the environmental change and biological activity they experienced during life. These geochemical signals locked within the carbonate are archives used in proxy reconstructions to study past environments and climates, to decipher taxonomy of cryptic species and to resolve evolutionary responses to climatic changes. METHODS: Here, we use laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) as a time-resolved acquisition to quantify the elemental composition of carbonate shells and skeletons. We present the LABLASTER (Laser Ablation BLASt Through Endpoint in R) package, which imports a single time-resolved LA-ICP-MS analysis, then detects when the laser has ablated through the carbonate as a function of change in signal over time and outputs key summary statistics. We provide two examples within the package: a fossil planktic foraminifer and a tropical coral skeleton. RESULTS: We present the first R package that automates the selection of desired data during data reduction workflows. This is achieved by automating the detection of when the laser has ablated through a sample using a smoothed time series, followed by removal of off-target data points. The functions are flexible and adjust dynamically to maximise the duration of the desired geochemical target signal, making this package applicable to a wide range of heterogenous bioarchives. Visualisation tools for manual validation are also included. CONCLUSIONS: LABLASTER increases transparency and repeatability by algorithmically identifying when the laser has either ablated fully through a sample or across a mineral boundary and is thus no longer documenting a geochemical signal associated with the desired sample. LABLASTER's focus on better data targeting means more accurate extraction of biological and geochemical signals.

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Particulate Matter (PM) low-cost sensors (LCS) present a cost-effective opportunity to improve the spatiotemporal resolution of airborne PM data. Previous studies focused on PM-LCS-reported hourly data and identified, without fully addressing, their limitations. However, PM-LCS provide measurements at finer temporal resolutions. Furthermore, government bodies have developed certifications to accompany new uses of these sensors, but these certifications have shortcomings. To address these knowledge gaps, PM-LCS of two models, 8 Sensirion SPS30 and 8 Plantower PMS5003, were collocated for one year with a Fidas 200S, MCERTS-certified PM monitor and were characterised at 2 min resolution, enabling replication of certification processes, and highlighting their limitations and improvements. Robust linear models using sensor-reported particle number concentrations and relative humidity, coupled with 2-week biannual calibration campaigns, achieved reference-grade performance, at median PM2.5 background concentration of 5.5 µg/m3, demonstrating that, with careful calibration, PM-LCS may cost-effectively supplement reference equipment in multi-nodes networks with fine spatiotemporality.