A late-Ediacaran crown-group sponge animal.

Sponges are the most basal metazoan phylum1 and may have played important roles in modulating the redox architecture of Neoproterozoic oceans2. Although molecular clocks predict that sponges diverged in the Neoproterozoic era3,4, their fossils have not been unequivocally demonstrated before the Cambrian period5-8, possibly because Precambrian sponges were aspiculate and non-biomineralized9. Here we describe a late-Ediacaran fossil, Helicolocellus cantori gen. et sp. nov., from the Dengying Formation (around 551-539 million years ago) of South China. This fossil is reconstructed as a large, stemmed benthic organism with a goblet-shaped body more than 0.4 m in height, with a body wall consisting of at least three orders of nested grids defined by quadrate fields, resembling a Cantor dust fractal pattern. The resulting lattice is interpreted as an organic skeleton comprising orthogonally arranged cruciform elements, architecturally similar to some hexactinellid sponges, although the latter are built with biomineralized spicules. A Bayesian phylogenetic analysis resolves H. cantori as a crown-group sponge related to the Hexactinellida. H. cantori confirms that sponges diverged and existed in the Precambrian as non-biomineralizing animals with an organic skeleton. Considering that siliceous biomineralization may have evolved independently among sponge classes10-13, we question the validity of biomineralized spicules as a necessary criterion for the identification of Precambrian sponge fossils.

Vascular persistence following precision micropuncture.

OBJECTIVE: The success of engineered tissues continues to be limited by time to vascularization and perfusion. Recently, we described a simple microsurgical approach, termed micropuncture (MP), which could be used to rapidly vascularize an adjacently placed scaffold from the recipient macrovasculature. Here we studied the long-term persistence of the MP-induced microvasculature. METHODS: Segmental 60 µm diameter MPs were created in the recipient rat femoral artery and vein followed by coverage with a simple Type 1 collagen scaffold. The recipient vasculature and scaffold were then wrapped en bloc with a silicone sheet to isolate intrinsic vascularization. Scaffolds were harvested at 28 days post-implantation for detailed analysis, including using a novel artificial intelligence (AI) approach. RESULTS: MP scaffolds demonstrated a sustained increase of vascular density compared to internal non-MP control scaffolds (p < 0.05) secondary to increases in both vessel diameters (p < 0.05) and branch counts (p < 0.05). MP scaffolds also demonstrated statistically significant increases in red blood cell (RBC) perfused lumens. CONCLUSIONS: This study further highlights that the intrinsic MP-induced vasculature continues to persist long-term. Its combination of rapid and stable angiogenesis represents a novel surgical platform for engineered scaffold and graft perfusion.

Quick and affordable DNA cloning by reconstitution of Seamless Ligation Cloning Extract using defined factors.

The cloning of DNA fragments to plasmid vectors is at the heart of molecular biology. Recent developments have led to various methods utilizing homologous recombination of homology arms. Among them, Seamless Ligation Cloning Extract (SLiCE) is an affordable alternative solution that uses simple Escherichia coli lysates. However, the underlying molecular mechanisms remain unclear and the reconstitution of the extract by defined factors has not yet been reported. We herein show that the key factor in SLiCE is Exonuclease III (ExoIII), a double-strand (ds) DNA-dependent 3'-5' exonuclease, encoded by XthA. SLiCE prepared from the xthAΔ strain is devoid of recombination activity, whereas purified ExoIII alone is sufficient to assemble two blunt-ended dsDNA fragments with homology arms. In contrast to SLiCE, ExoIII is unable to digest (or assemble) fragments with 3' protruding ends; however, the addition of single-strand DNA-targeting Exonuclease T overcomes this issue. Through the combination of commercially available enzymes under optimized conditions, we achieved the efficient, reproducible, and affordable cocktail, "XE cocktail," for seamless DNA cloning. By reducing the cost and time required for DNA cloning, researchers will devote more resources to advanced studies and the careful validation of their own findings.

Characterisation and management of expected and unexpected urgent findings from positron emission tomography with 18F-fluorodeoxyglucose integrated with computed tomography in cardiovascular disease.

BACKGROUND: Cardiac 18F-fluorodeoxyglucose (FDG)-PET-CT plays an important role in the assessment of cardiovascular diseases. Effective management of urgent scan findings facilitates optimal patient care. METHODS: We characterised the management of urgent, expected and unexpected findings in patients referred for cardiac [18F]fluorodeoxyglucose integrated with computed tomography (FDG-PET-CT) at the Royal Brompton Hospital (United Kingdom). Urgent findings are escalated by the reporting physicians/radiologists raising RadAlert notifications to the referring clinician. We characterised the indications and time to management (TTM) between the RadAlert and the resulting management. As controls, we characterised the TTM of 33 urgent findings identified before the RadAlert system was implemented. RESULTS: Of the 1497 consecutive FDG-PET-CT scans screened (April 2021 to February 2023), 93 RadAlerts were suitable for analysis (TTM 7 days [interquartile range: 2-14]). Expected urgent findings included active cardiac sarcoidosis (56%; TTM 8 days [5-18]), heart transplant rejection (12%; 6 ± 4 days), infective endocarditis (9%; 2 days [1-12]), cardiac device infections (5%; 1 day [0-2]), acute myocarditis (2%; 5 and 14 days) and epicardial mass (1%; 1 day). TTM did not differ significantly between indications (P = 0.06). RadAlert cases had significantly shorter TTM than controls without RadAlert, P = 0.001. After the RadAlerts, 81% of patients had clinical reviews, and 55% had escalation of medical/surgical therapies. Unexpected findings (total N = 45; median TTM 6 days [1-10]) included malignancies (N = 3), infections (N = 2), pneumothorax (N = 1), benign diagnosis (N = 30), unclear diagnosis (N = 5) and 4 findings disappeared on repeat imaging. CONCLUSIONS: Cardiac FDG-PET-CT identifies expected and unexpected findings in a range of cardiovascular diseases. Serious, unexpected findings are rare and can be effectively escalated by the RadAlert system.

Retromuscular drain output at removal does not influence adverse outcome rate in open ventral hernia repairs.

BACKGROUND: Retromuscular drains are commonly placed during retromuscular hernia repair (RHR) to decrease postoperative wound complications and help mesh in-growth. Drains are traditionally removed when output is low but the relationship between drain output at the time of removal and postoperative complications has yet to be delineated. This study aimed to investigate outcomes of RHR patients with drain removal at either high or low output volume. METHODS: An institutional review board-approved retrospective chart review evaluated adult patients undergoing open RHR with retromuscular drain placement between 2013 and 2022 at a single academic medical center. Patients were stratified into low output drainage (LOD, < 50 mL/day) or high output drainage (HOD, ≥ 50 mL/day) groups based on volume on the day of drain removal. RESULTS: We identified 336 patients meeting inclusion criteria: 58% LOD (n = 195) and 42% HOD (n = 141). Demographics and risk factors pertaining to hernia complexity were similar between cohorts. Low-drain output at the time of removal was associated with a significantly longer drain duration (6.3 ± 4.5 vs. 4.4 ± 1.6 days, p < 0.001) and postoperative hospital stay (5.9 ± 3.6 vs. 4.8 ± 2.8 days, p < 0.001). With a 97% 30-day follow-up, incidence of surgical site occurrence (SSO) was not statistically different between groups (29.2% LOD, 26.2% HOD, p = 0.63). Surgical site infection and SSO requiring procedural intervention was also not statistically significant between cohort. At 1-year follow-up, hernia recurrence rates were the same between groups (4.2% LOD, 1.4% HOD, p = 0.25). CONCLUSION: Following open ventral hernia repair with retromuscular mesh placement, the rate of postoperative wound complications was not statistically different based on volume of drain output day of removal. These results suggest that removing drains earlier despite higher output is safe and has no effect on short- or long-term hernia outcomes.

Viewing images of foods evokes taste quality-specific activity in gustatory insular cortex.

Previous studies have shown that the conceptual representation of food involves brain regions associated with taste perception. The specificity of this response, however, is unknown. Does viewing pictures of food produce a general, nonspecific response in taste-sensitive regions of the brain? Or is the response specific for how a particular food tastes? Building on recent findings that specific tastes can be decoded from taste-sensitive regions of insular cortex, we asked whether viewing pictures of foods associated with a specific taste (e.g., sweet, salty, and sour) can also be decoded from these same regions, and if so, are the patterns of neural activity elicited by the pictures and their associated tastes similar? Using ultrahigh-resolution functional magnetic resonance imaging at high magnetic field strength (7-Tesla), we were able to decode specific tastes delivered during scanning, as well as the specific taste category associated with food pictures within the dorsal mid-insula, a primary taste responsive region of brain. Thus, merely viewing food pictures triggers an automatic retrieval of specific taste quality information associated with the depicted foods, within gustatory cortex. However, the patterns of activity elicited by pictures and their associated tastes were unrelated, thus suggesting a clear neural distinction between inferred and directly experienced sensory events. These data show how higher-order inferences derived from stimuli in one modality (i.e., vision) can be represented in brain regions typically thought to represent only low-level information about a different modality (i.e., taste).

Naturalistic food categories are driven by subjective estimates rather than objective measures of food qualities.

Food-related studies often categorize foods using criteria such as fat and sugar content (e.g., high-fat, high-sugar foods; low-fat, low-sugar foods), and use these categorizations for further analyses. While these criteria are relevant to nutritional health, it is unclear whether they agree with the ways in which we typically group foods. Do these objective categories correspond to our subjective sense? To address this question, we recruited a group of 487 online participants to perform a triplet comparison task involving implicit object similarity judgements on images of 36 foods, which varied in their levels of fat and sugar. We also acquired subjective ratings of other food properties from another set of 369 online participants. Data from the online triplet task was used to generate a similarity matrix of these 36 foods. Principal Components Analysis (PCA) of this matrix identified that the strongest determinant of food similarity (the first PC) was most highly related to participants' judgements of how processed the foods were, while the second component was most related to estimates of sugar and fat content. K-means clustering analysis revealed five emergent food groupings along these PC axes: sweets, fats, starches, fruits, and vegetables. Our results suggest that naturalistic categorizations of food are driven primarily by knowledge of the origin of foods (i.e., grown or manufactured), rather than by their sensory or macronutrient properties. These differences should be considered and explored when developing methods for scientific food studies.

Effective prognostic and clinical risk stratification in COVID-19 using multimodality biomarkers.

In acute coronavirus disease 19 (COVID-19) patients, effective clinical risk stratification has important implications on treatment and therapeutic resource distribution. This article reviews the evidence behind a wide range of biomarkers with prognostic value in COVID-19. Patient characteristics and co-morbidities, such as cardiovascular and respiratory diseases, are associated with increased mortality risk. Peripheral oxygen saturation and arterial oxygenation are predictive of severe respiratory compromise, whereas risk scores such as the 4C-score enable multi-factorial prognostic risk estimation. Blood tests such as markers of inflammation, cardiac injury and d-dimer and abnormalities on electrocardiogram are linked to inpatient prognosis. Of the imaging modalities, lung ultrasound and echocardiography enable the bedside assessment of prognostic abnormalities in COVID-19. Chest radiograph (CXR) and computed tomography (CT) can inform about prognostic pulmonary pathologies, whereas cardiovascular CT detects high-risk features such as coronary artery and aortic calcification. Dynamic changes in biomarkers, such as blood tests, CXR, CT and electrocardiogram findings, can further inform about disease severity and prognosis. Despite the vast volumes of existing evidence, several gaps exist in our understanding of COVID-19 biomarkers. First, the pathophysiological basis on which these markers can foretell prognosis in COVID-19 remains poorly understood. Second, certain under-explored tests such as thoracic impedance assessment and cardiovascular magnetic resonance imaging deserve further investigation. Lastly, the prognostic values of most biomarkers in COVID-19 are derived from retrospective analyses. Prospective studies are required to validate these markers for guiding clinical decision-making and to facilitate their translation into clinical management pathways.

Long-read single-molecule maps of the functional methylome.

We report on the development of a methylation analysis workflow for optical detection of fluorescent methylation profiles along chromosomal DNA molecules. In combination with Bionano Genomics genome mapping technology, these profiles provide a hybrid genetic/epigenetic genome-wide map composed of DNA molecules spanning hundreds of kilobase pairs. The method provides kilobase pair-scale genomic methylation patterns comparable to whole-genome bisulfite sequencing (WGBS) along genes and regulatory elements. These long single-molecule reads allow for methylation variation calling and analysis of large structural aberrations such as pathogenic macrosatellite arrays not accessible to single-cell second-generation sequencing. The method is applied here to study facioscapulohumeral muscular dystrophy (FSHD), simultaneously recording the haplotype, copy number, and methylation status of the disease-associated, highly repetitive locus on Chromosome 4q.

Is preparation of a sterile operative field before percutaneous endoscopic gastrostomy necessary? A cost-effectiveness analysis.

BACKGROUND: Despite the non-sterile nature of the alimentary tract, percutaneous endoscopic gastrostomy (PEG) procedures are often performed after creating a sterile surgical field to reduce infection risk. Our group has previously demonstrated no statistically significant difference in the rate of surgical site infection (SSI) following PEG performed with or without sterile operative fields. The purpose of this study is to evaluate the cost-effectiveness of working with or without sterile operative fields during PEG. METHODS: A decision tree model of PEG with and without sterile operative fields was created to evaluate the cost-effectiveness of these two methods. The primary outcome was incremental cost-effectiveness ratio with a ceiling willingness to pay threshold assumed of $100,000 per quality-adjusted life year (QALY). Costs were from the perspective of the healthcare system with a time horizon for the model of 90 days. Sensitivity analyses were performed on all model parameters. RESULTS: In the base case analysis, sterile operative field PEG was cost-effective resulting in 0.2225 QALYs at an expected cost of $2,099. PEG procedures without a sterile operative field yielded less QALYs (0.2224) at a higher cost ($2,199) and were dominated. These procedures became cost-effective when the expected cost was < $1618 (e.g., $140 less than sterile operative field PEG) or when the SSI rate was < 1.6% (e.g., 0.2% higher than sterile operative field PEG) while holding all other variables constant. CONCLUSION: PEG procedures performed without a sterile operative field can be cost-effective compared to procedures performed with a sterile operative field if the rates of SSI remain similar. Cost savings from foregoing a sterile operative field appear to be offset if the SSI rate increases > 0.2% above that of sterile operative field PEG procedures.

Development of the Bereavement and Interpersonal Domains codebook.

We describe the development of an empirically-derived codebook for qualitative data concerning the impact of grief on the interpersonal relationships of bereaved individuals. Relatives (N = 39) of deceased military service members participated in focus groups concerning how grief influenced their relationships across multiple interpersonal domains, including family, friends, community, and with the deceased. Focus group transcripts were coded using a stepwise process consistent with grounded theory to identify and categorize recurrent themes. The process yielded a comprehensive codebook containing 44 nodes with definitions and examples. The codebook provides researchers with an empirically-grounded analytic tool for future studies on bereavement.

Taste Quality Representation in the Human Brain.

In the mammalian brain, the insula is the primary cortical substrate involved in the perception of taste. Recent imaging studies in rodents have identified a "gustotopic" organization in the insula, whereby distinct insula regions are selectively responsive to one of the five basic tastes. However, numerous studies in monkeys have reported that gustatory cortical neurons are broadly-tuned to multiple tastes, and tastes are not represented in discrete spatial locations. Neuroimaging studies in humans have thus far been unable to discern between these two models, though this may be because of the relatively low spatial resolution used in taste studies to date. In the present study, we examined the spatial representation of taste within the human brain using ultra-high resolution functional magnetic resonance imaging (MRI) at high magnetic field strength (7-tesla). During scanning, male and female participants tasted sweet, salty, sour, and tasteless liquids, delivered via a custom-built MRI-compatible tastant-delivery system. Our univariate analyses revealed that all tastes (vs tasteless) activated primary taste cortex within the bilateral dorsal mid-insula, but no brain region exhibited a consistent preference for any individual taste. However, our multivariate searchlight analyses were able to reliably decode the identity of distinct tastes within those mid-insula regions, as well as brain regions involved in affect and reward, such as the striatum, orbitofrontal cortex, and amygdala. These results suggest that taste quality is not represented topographically, but by a distributed population code, both within primary taste cortex as well as regions involved in processing the hedonic and aversive properties of taste.SIGNIFICANCE STATEMENT The insula is the primary cortical substrate involved in taste perception, yet some question remains as to whether this region represents distinct tastes topographically or via a population code. Using high field (7-tesla), high-resolution functional magnetic resonance imaging in humans, we examined the representation of different tastes delivered during scanning. All tastes activated primary taste cortex within the bilateral mid-insula, but no brain region exhibited any consistent taste preference. However, multivariate analyses reliably decoded taste quality within the bilateral mid-insula as well as the striatum, orbitofrontal cortex, and bilateral amygdala. This suggests that taste quality is represented by a spatial population code within regions involved in sensory and appetitive properties of taste.

Molecular Characterization of Differences between the Tomato Immune Receptors Flagellin Sensing 3 and Flagellin Sensing 2.

Plants mount defense responses by recognizing indicators of pathogen invasion, including microbe-associated molecular patterns (MAMPs). Flagellin, from the bacterial pathogen Pseudomonas syringae pv. tomato (Pst), contains two MAMPs, flg22 and flgII-28, that are recognized by tomato (Solanum lycopersicum) receptors Flagellin sensing2 (Fls2) and Fls3, respectively, but to what degree each receptor contributes to immunity and whether they promote immune responses using the same molecular mechanisms are unknown. Here, we characterized CRISPR/Cas9-generated Fls2 and Fls3 tomato mutants and found that the two receptors contribute equally to disease resistance both on the leaf surface and in the apoplast. However, we observed striking differences in certain host responses mediated by the two receptors. Compared to Fls2, Fls3 mediated a more sustained production of reactive oxygen species and an increase in transcript abundance of 44 tomato genes, with two genes serving as specific reporters for the Fls3 pathway. Fls3 had greater in vitro kinase activity than Fls2 and could transphosphorylate a substrate. Using chimeric Fls2/Fls3 proteins, we found no evidence that a single receptor domain is responsible for the Fls3-sustained reactive oxygen species, suggesting involvement of multiple structural features or a nullified function of the chimeric construct. This work reveals differences in certain immunity outputs between Fls2 and Fls3, suggesting that they might use distinct molecular mechanisms to activate pattern-triggered immunity in response to flagellin-derived MAMPs.

Reconstructing the reproductive mode of an Ediacaran macro-organism.

Enigmatic macrofossils of late Ediacaran age (580-541 million years ago) provide the oldest known record of diverse complex organisms on Earth, lying between the microbially dominated ecosystems of the Proterozoic and the Cambrian emergence of the modern biosphere. Among the oldest and most enigmatic of these macrofossils are the Rangeomorpha, a group characterized by modular, self-similar branching and a sessile benthic habit. Localized occurrences of large in situ fossilized rangeomorph populations allow fundamental aspects of their biology to be resolved using spatial point process techniques. Here we use such techniques to identify recurrent clustering patterns in the rangeomorph Fractofusus, revealing a complex life history of multigenerational, stolon-like asexual reproduction, interspersed with dispersal by waterborne propagules. Ecologically, such a habit would have allowed both for the rapid colonization of a localized area and for transport to new, previously uncolonized areas. The capacity of Fractofusus to derive adult morphology by two distinct reproductive modes documents the sophistication of its underlying developmental biology.

Cotton Leaf Curl Multan virus C4 protein suppresses both transcriptional and post-transcriptional gene silencing by interacting with SAM synthetase.

Gene silencing is a natural antiviral defense mechanism in plants. For effective infection, plant viruses encode viral silencing suppressors to counter this plant antiviral response. The geminivirus-encoded C4 protein has been identified as a gene silencing suppressor, but the underlying mechanism of action has not been characterized. Here, we report that Cotton Leaf Curl Multan virus (CLCuMuV) C4 protein interacts with S-adenosyl methionine synthetase (SAMS), a core enzyme in the methyl cycle, and inhibits SAMS enzymatic activity. By contrast, an R13A mutation in C4 abolished its capacity to interact with SAMS and to suppress SAMS enzymatic activity. Overexpression of wild-type C4, but not mutant C4R13A, suppresses both transcriptional gene silencing (TGS) and post-transcriptional gene silencing (PTGS). Plants infected with CLCuMuV carrying C4R13A show decreased levels of symptoms and viral DNA accumulation associated with enhanced viral DNA methylation. Furthermore, silencing of NbSAMS2 reduces both TGS and PTGS, but enhanced plant susceptibility to two geminiviruses CLCuMuV and Tomato yellow leaf curl China virus. These data suggest that CLCuMuV C4 suppresses both TGS and PTGS by inhibiting SAMS activity to enhance CLCuMuV infection in plants.

Effect of comorbid anxiety and depression in complicated grief on perceived cognitive failures.

BACKGROUND: Bereavement is associated with cognitive difficulties, but it is unclear whether these difficulties are associated with normative and/or complicated grief (CG) and how comorbid depression and anxiety contribute to them. Self-reported "minor errors in thinking" (i.e., cognitive failures) may manifest following bereavement and be differentially affected by CG, anxiety, and depression. METHODS: Associations between perceived cognitive failures and CG, anxiety, and depression were investigated in 581 bereaved participants. To examine both single and comorbid conditions across the spectrum of bereaved participants, these relationships were examined using both linear regressions and group comparisons. RESULTS: Continuous measures of depression, anxiety, and grief each independently predicted perceived cognitive failures. Group comparisons indicated that the group with three comorbid conditions had the highest frequency of perceived cognitive failures and the group with no conditions had the lowest. In addition, groups with threshold depression levels (both alone and comorbid with another condition) had higher frequencies of perceived cognitive failures than other groups, suggesting that depression was more strongly associated with perceived cognitive failures than CG or anxiety. CONCLUSIONS: Future research about cognition following bereavement should address how multiple mental health symptoms or conditions combine to affect perceived and actual cognitive capacity.

The importance of neutral over niche processes in structuring Ediacaran early animal communities.

The relative influence of niche vs. neutral processes in ecosystem dynamics is an on-going debate, but the extent to which they structured the earliest animal communities is unknown. Some of the oldest known metazoan-dominated paleocommunities occur in Ediacaran age (~ 565 million years old) strata in Newfoundland, Canada and Charnwood Forest, UK. These comprise large and diverse populations of sessile organisms that are amenable to spatial point process analyses, enabling inference of the most likely underlying niche or neutral processes governing community structure. We mapped seven Ediacaran paleocommunities using LiDAR, photogrammetry and a laser line probe. We found that neutral processes dominate these paleocommunities, with niche processes exerting limited influence, in contrast with the niche-dominated dynamics of modern marine ecosystems. The dominance of neutral processes suggests that early metazoan diversification may not have been driven by systematic adaptations to the local environment, but instead may have resulted from stochastic demographic differences.

Natural variation for unusual host responses and flagellin-mediated immunity against Pseudomonas syringae in genetically diverse tomato accessions.

The interaction between tomato and Pseudomonas syringae pv tomato (Pst) is a well-developed model for investigating the molecular basis of the plant immune system. There is extensive natural variation in Solanum lycopersicum (tomato) but it has not been fully leveraged to enhance our understanding of the tomato-Pst pathosystem. We screened 216 genetically diverse accessions of cultivated tomato and a wild tomato species for natural variation in their response to three strains of Pst. The host response to Pst was investigated using multiple Pst strains, tomato accessions with available genome sequences, reactive oxygen species (ROS) assays, reporter genes and bacterial population measurements. The screen uncovered a broad range of previously unseen host symptoms in response to Pst, and one of these, stem galls, was found to be simply inherited. The screen also identified tomato accessions that showed enhanced responses to flagellin in bacterial population assays and in ROS assays upon exposure to flagellin-derived peptides, flg22 and flgII-28. Reporter genes confirmed that the host responses were due primarily to pattern recognition receptor-triggered immunity. This study revealed extensive natural variation in tomato for susceptibility and resistance to Pst and will enable elucidation of the molecular mechanisms underlying these host responses.

The origin of animals: Can molecular clocks and the fossil record be reconciled?

The evolutionary emergence of animals is one of the most significant episodes in the history of life, but its timing remains poorly constrained. Molecular clocks estimate that animals originated and began diversifying over 100 million years before the first definitive metazoan fossil evidence in the Cambrian. However, closer inspection reveals that clock estimates and the fossil record are less divergent than is often claimed. Modern clock analyses do not predict the presence of the crown-representatives of most animal phyla in the Neoproterozoic. Furthermore, despite challenges provided by incomplete preservation, a paucity of phylogenetically informative characters, and uncertain expectations of the anatomy of early animals, a number of Neoproterozoic fossils can reasonably be interpreted as metazoans. A considerable discrepancy remains, but much of this can be explained by the limited preservation potential of early metazoans and the difficulties associated with their identification in the fossil record. Critical assessment of both records may permit better resolution of the tempo and mode of early animal evolution.