Genome integrity sensing by the broad-spectrum Hachiman antiphage defense complex.

Hachiman is a broad-spectrum antiphage defense system of unknown function. We show here that Hachiman is a heterodimeric nuclease-helicase complex, HamAB. HamA, previously a protein of unknown function, is the effector nuclease. HamB is the sensor helicase. HamB constrains HamA activity during surveillance of intact double-stranded DNA (dsDNA). When the HamAB complex detects DNA damage, HamB helicase activity activates HamA, unleashing nuclease activity. Hachiman activation degrades all DNA in the cell, creating "phantom" cells devoid of both phage and host DNA. We demonstrate Hachiman activation in the absence of phage by treatment with DNA-damaging agents, suggesting that Hachiman responds to aberrant DNA states. Phylogenetic similarities between the Hachiman helicase and enzymes from eukaryotes and archaea suggest deep functional symmetries with other important helicases across domains of life.

A DNA barcoding framework for taxonomic verification in the Darwin Tree of Life Project.

Biodiversity genomics research requires reliable organismal identification, which can be difficult based on morphology alone. DNA-based identification using DNA barcoding can provide confirmation of species identity and resolve taxonomic issues but is rarely used in studies generating reference genomes. Here, we describe the development and implementation of DNA barcoding for the Darwin Tree of Life Project (DToL), which aims to sequence and assemble high quality reference genomes for all eukaryotic species in Britain and Ireland. We present a standardised framework for DNA barcode sequencing and data interpretation that is then adapted for diverse organismal groups. DNA barcoding data from over 12,000 DToL specimens has identified up to 20% of samples requiring additional verification, with 2% of seed plants and 3.5% of animal specimens subsequently having their names changed. We also make recommendations for future developments using new sequencing approaches and streamlined bioinformatic approaches.

Identifying species based solely on their morphology can be difficult. DNA-based identification using DNA barcoding can aid species identification, but can be challenging to implement in biodiversity projects sampling diverse organismal groups. Here, we describe the development and implementation of DNA barcoding for the Darwin Tree of Life Project (DToL), which aims to sequence and assemble high quality reference genomes for all eukaryotic species in Britain and Ireland. We discuss how a standardised approach has been adapted by each partner to suit different organismal groups, show the efficacy of this approach for confirming species identities and resolving taxonomic issues, and make recommendations for future developments.

Genomic analysis of intracranial and subcortical brain volumes yields polygenic scores accounting for variation across ancestries.

Subcortical brain structures are involved in developmental, psychiatric and neurological disorders. We performed GWAS meta-analyses of intracranial and nine subcortical brain volumes (brainstem, caudate nucleus, putamen, hippocampus, globus pallidus, thalamus, nucleus accumbens, amygdala and, for the first time, the ventral diencephalon) in 74,898 participants of European ancestry. We identified 254 independent loci associated with these brain volumes, explaining up to 35% of phenotypic variance. We observed gene expression in specific neural cell types across differentiation time points, including genes involved in intracellular signalling and brain ageing-related processes. Polygenic scores for brain volumes showed predictive ability when applied to individuals of diverse ancestries. We observed causal genetic effects of brain volumes with Parkinson's disease and ADHD. Findings implicate specific gene expression patterns in brain development and genetic variants in comorbid neuropsychiatric disorders, which could point to a brain substrate and region of action for risk genes implicated in brain diseases.

Surgery and postoperative definitive radiotherapy for management of canine soft tissue sarcoma: a multi-institutional retrospective study of 272 dogs (2010-2020).

OBJECTIVE: To report local progression and survival in dogs following surgery and postoperative definitive radiotherapy (dRT) for management of soft tissue sarcoma (STS) and to evaluate risk factors for local progression and survival. METHODS: Records were retrospectively reviewed at 9 referral hospitals for dogs managed with postoperative dRT between January 1, 2010, and January 1, 2020, following surgery for STS. Data related to presentation, surgery, dRT, systemic therapy, and outcome were abstracted. Selected variables were assessed for association with local progression and overall survival. RESULTS: 272 dogs were included. Histologic grade was reported in 249 dogs: 102 were grade 1 (40.9%), 120 were grade 2 (48.2%), and 27 were grade 3 (10.8%). Local progression was suspected or confirmed in 56 dogs. Local progression rates were similar for grade 1 (24 of 89 [26.7%]), grade 2 (23 of 111 [20.7%]), and grade 3 tumors (6 of 22 [27.3%]). Previous recurrence (P = .010) and subsequent distant metastasis (P = .014) were associated with more frequent local progression; intensity-modulated radiotherapy was associated with decreased local progression (P = .025) compared to other forms of delivery. Age (P = .049), grade (P = .009), previous recurrence (P = .009), and institution type for surgery (P = .043) were associated with overall survival. CONCLUSIONS: Outcomes for most dogs were good; however, the frequency of local progression indicates an ongoing need to critically appraise local management strategies, particularly for low-grade STS. Intensity-modulated radiotherapy was associated with lower rates of local progression and may be preferred to less precise forms of delivery. CLINICAL RELEVANCE: These data may guide clinicians when making decisions regarding dRT for management of STS.

NO EFFECT OF AEROBIC FITNESS ON EXERCISE-INDUCED DIAPHRAGM FATIGUE IN FEMALES.

We tested the hypothesis that the incidence and magnitude of diaphragm fatigue following high-intensity exercise would be lower in females with a high aerobic capacity (Hi-Fit) compared to healthy females with an average aerobic fitness (Avg-Fit). Participants were assigned to groups based on their peak O2 uptake (V̇O2peak) obtained during cycle exercise: Hi-Fit n=9, V̇O2peak > 56.1±3.4 mL·kg-1·min-1 vs. Avg-Fit n=9, V̇O2peak < 35.7±4.9 mL·kg-1·min-1. On a second day, diaphragm fatigue was assessed before and after constant load exercise test to exhaustion. Magnetic stimulation of the phrenic nerve roots was used to non-volitionally assess diaphragm fatigue by measurement of transdiaphragmatic twitch pressure (Pdi,tw). Both groups exercised at > 90% of V̇O2peak for a similar duration (Hi-Fit: 546.1 ± 177.8 vs Avg-Fit: 559.3 ± 175.0 seconds, p = 0.9). Diaphragm fatigue was defined as a > 15% reduction in Pdi,tw, approximately 2 times greater than the coefficient of variation. The mean group average reduction in Pdi,tw following exercise in the Hi-Fit (17.5%) and Avg-Fit groups (12.2%) was not different between groups (p = 0.2). The Hi-Fit group performed exercise at a higher absolute work rate that elicited significantly greater ventilatory work and inspiratory muscle force output. The Hi-Fit group did not experience greater fatigue compared to the Avg-Fit group, which we attribute to a greater reliance on accessory respiratory muscle recruitment, to training-induced increases in the aerobic capacity of the diaphragm, or a combination of the two. In summary, aerobic fitness is not predictive of exercise-induced diaphragm fatigue in healthy females.

Temporal variability in host availability alters the outcome of competition between two parasitoid species.

Variability in the availability of resources through time is a common attribute in trophic interactions, but its effects on the fitness of different consumer species and on interspecific competition between them are not clearly understood. To investigate this, we allowed two parasitoid species, Trichopria drosophilae and Pachycrepoideus vindemiae, to exploit Drosophila host pupae under different temporal variability treatments, either on their own or simultaneously. When tested individually (in the absence of interspecific competition), both parasitoid species had lower fitness when hosts were exposed for a short duration at high density than when exposed for a long duration at low density. When both parasitoid species exploited hosts simultaneously, interspecific competition significantly decreased the number of offspring for both parasitoid species. The outcome of this interspecific competition depended on host temporal variability, with T. drosophilae or P. vindemiae dominating in short and long host exposure treatments, respectively. These results can be explained by the combination of host availability and egg load of female adult parasitoids. When abundant hosts are provided for a short period, the ample mature eggs of the proovigenic T. drosophilae enable them to exploit hosts more efficiently than P. vindemiae, which is synovigenic. However, P. vindemiae is an intrinsically superior competitor and dominates when multiparasitism occurs. Multiparasitism is more frequent when hosts are at low levels relative to the egg load of the parasitoids. Our results clearly demonstrate that resource temporal availability can alter the outcome of competition between consumers with different reproductive traits.

Impact of catastrophic brain injury guidelines on organ donation rates: Results of an EAST multicenter trial.

BACKGROUND: One third of organ donors suffer catastrophic brain injury (CBI). There are no standard guidelines for the management of traumatic CBI prior to brain death, and not all trauma centers have institutional CBI guidelines. In addition, there is high variability in management between institutions with guidelines. Catastrophic brain injury guidelines vary and may include various combinations of hormone therapy, vasopressors, fluid resuscitation, and other practices. We hypothesized that centers with CBI guidelines have higher organ donation rates than those without. METHODS: This prospective, observational EAST-sponsored multicenter trial included adult (18+ years old) traumatic-mechanism CBI patients at 33 level I and II trauma centers from January 2022 to May 2023. Catastrophic brain injury was defined as a brain injury causing loss of function above the brain stem and subsequent death. Cluster analysis with linear mixed-effects model including UNOS regions and hospital size by bed count was used to determine whether CBI guidelines are associated with organ donation. RESULTS: A total of 790 CBI patients were included in this analysis. In unadjusted comparison, CBI guideline centers had higher rates of organ donation and use of steroids, whole blood, and hormone therapy. In a linear mixed-effects model, CBI guidelines were not associated with organ donation. Registered organ donor status, steroid hormones, and vasopressin were associated with increased relative risk of donation. CONCLUSION: There is high variability in management of CBI, even at centers with CBI guidelines in place. While the use of institutional CBI guidelines was not associated with increased organ donation, guidelines in this study were not identical. Hormone replacement with steroids and vasopressin was associated with increased donation. Hormone resuscitation is a common feature of CBI guidelines. Further analysis of individual practices that increase organ donation after CBI may allow for more effective guidelines and an overall increase in donation to decrease the long waiting periods for organ transplant recipients. LEVEL OF EVIDENCE: Prognostic; Level III.

Platelet-Rich Plasma as a Novel, Non-invasive Method to Treat Breast Capsular Contractures: a Case Report.

We describe the first examples of breast capsular contracture amelioration using a non-surgical, transdermal treatment with platelet-rich plasma. The treated patients did not experience any complications or significant pain. This report illustrates the potential of a non-invasive treatment option for a common complication of breast augmentation.

A Negatively Curved Nanographene with Four Embedded Heptagons.

Negatively curved nanographenes are considered as cutouts of three-dimensional fully sp2-hybridized carbon allotropes such as Schwarzites. Here we present the synthesis of a C76 cut-out of the Schwarzite 8-4-1-p proposed by Lenosky et al. and investigate its optical as well as electrochemical properties. Furthermore, supramolecular interactions with fullerenes C60 and C70 were studied.

Emerging SARS-CoV-2 Resistance After Antiviral Treatment.

Importance: Previous studies have identified mutations in SARS-CoV-2 strains that confer resistance to nirmatrelvir, yet how often this resistance arises and its association with posttreatment virologic rebound is not well understood. Objective: To examine the prevalence of emergent antiviral resistance after nirmatrelvir treatment and its association with virologic rebound. Design, Setting, and Participants: This cohort study enrolled outpatient adults with acute COVID-19 infection from May 2021 to October 2023. Participants were divided into those who received antiviral therapy and those who did not. The study was conducted at a multicenter health care system in Boston, Massachusetts. Exposure: Treatment regimen, including none, nirmatrelvir, and remdesivir. Main Outcomes and Measures: The primary outcome was emergent SARS-CoV-2 antiviral resistance, defined as the detection of antiviral resistance mutations, which were not present at baseline, were previously associated with decreased antiviral efficacy, and emerged during or after completion of a participant's treatment. Next-generation sequencing was used to detect low frequency mutations down to 1% of the total viral population. Results: Overall, 156 participants (114 female [73.1%]; median [IQR] age, 56 [38-69] years) were included. Compared with 63 untreated individuals, the 79 who received nirmatrelvir were older and more commonly immunosuppressed. After sequencing viral RNA from participants' anterior nasal swabs, nirmatrelvir resistance mutations were detected in 9 individuals who received nirmatrelvir (11.4%) compared with 2 of those who did not (3.2%) (P = .09). Among the individuals treated with nirmatrelvir, those who were immunosuppressed had the highest frequency of resistance emergence (5 of 22 [22.7%]), significantly greater than untreated individuals (2 of 63 [3.1%]) (P = .01). Similar rates of nirmatrelvir resistance were found in those who had virologic rebound (3 of 23 [13.0%]) vs those who did not (6 of 56 [10.7%]) (P = .86). Most of these mutations (10 of 11 [90.9%]) were detected at low frequencies (<20% of viral population) and reverted to the wild type at subsequent time points. Emerging remdesivir resistance mutations were only detected in immunosuppressed individuals (2 of 14 [14.3%]) but were similarly low frequency and transient. Global Initiative on Sharing All Influenza Data analysis showed no evidence of increased nirmatrelvir resistance in the United States after the authorization of nirmatrelvir. Conclusions and Relevance: In this cohort study of 156 participants, treatment-emergent nirmatrelvir resistance mutations were commonly detected, especially in individuals who were immunosuppressed. However, these mutations were generally present at low frequencies and were transient in nature, suggesting a low risk for the spread of nirmatrelvir resistance in the community with the current variants and drug usage patterns.

Local delivery of cell surface-targeted immunocytokines programs systemic antitumor immunity.

Systemically administered cytokines are potent immunotherapeutics but can cause severe dose-limiting toxicities. To overcome this challenge, cytokines have been engineered for intratumoral retention after local delivery. However, despite inducing regression of treated lesions, tumor-localized cytokines often elicit only modest responses at distal untreated tumors. In the present study, we report a localized cytokine therapy that safely elicits systemic antitumor immunity by targeting the ubiquitous leukocyte receptor CD45. CD45-targeted immunocytokines have lower internalization rates relative to wild-type counterparts, leading to sustained downstream cis and trans signaling between lymphocytes. A single intratumoral dose of αCD45-interleukin (IL)-12 followed by a single dose of αCD45-IL-15 eradicated treated tumors and untreated distal lesions in multiple syngeneic mouse tumor models without toxicity. Mechanistically, CD45-targeted cytokines reprogrammed tumor-specific CD8+ T cells in the tumor-draining lymph nodes to have an antiviral transcriptional signature. CD45 anchoring represents a broad platform for protein retention by host immune cells for use in immunotherapy.

CRISPR-Cas12a bends DNA to destabilize base pairs during target interrogation.

RNA-guided endonucleases are involved in processes ranging from adaptive immunity to site-specific transposition and have revolutionized genome editing. CRISPR-Cas9, -Cas12 and related proteins use guide RNAs to recognize ~20-nucleotide target sites within genomic DNA by mechanisms that are not yet fully understood. We used structural and biochemical methods to assess early steps in DNA recognition by Cas12a protein-guide RNA complexes. We show here that Cas12a initiates DNA target recognition by bending DNA to induce transient nucleotide flipping that exposes nucleobases for DNA-RNA hybridization. Cryo-EM structural analysis of a trapped Cas12a-RNA-DNA surveillance complex and fluorescence-based conformational probing show that Cas12a-induced DNA helix destabilization enables target discovery and engagement. This mechanism of initial DNA interrogation resembles that of CRISPR-Cas9 despite distinct evolutionary origins and different RNA-DNA hybridization directionality of these enzyme families. Our findings support a model in which RNA-mediated DNA engineering begins with local helix distortion by transient CRISPR-Cas protein binding.

The genome sequence of the Mottled Pug, Eupithecia exiguata (Hübner, 1813).

We present a genome assembly from an individual male Eupithecia exiguata (the Mottled Pug; Arthropoda; Insecta; Lepidoptera; Geometridae). The genome sequence is 372.9 megabases in span. Most of the assembly is scaffolded into 31 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 16.39 kilobases in length. Gene annotation of this assembly on Ensembl identified 11,194 protein coding genes.

Functional and perceptive differences between conventional and advanced ankle foot orthoses in community ambulators post-limb trauma: the injuries managed with advanced bracing of the lower extremity (IM ABLE) study.

Introduction: Many military service members and civilians suffer from lower extremity trauma. Despite recent advancements in lower limb bracing technology, it remains unclear whether these newer advanced braces offer improved comfort and functionality compared to conventional options. The IDEO (Intrepid Dynamic Exoskeletal Orthosis), a type of "advanced" orthosis was developed to assist in maintaining high functional performance in patients who have experienced high-energy lower extremity trauma and underwent limb salvage surgeries. Methods: A cross-sector multi-site initiative was completed to study the efficacy of advanced ankle foot orthoses (AFO) for lower limb trauma and injury compared to a conventional AFO. Following fitting, training, and accommodation, the subjects were assessed in each AFO system for mobility, self-reported function, safety and pain, and preference. Results: They preferred the advanced over the conventional AFO and the mobility and exertion perception improved with the advanced AFO with no difference in pain or overall health status scores. Discussion: Thus, an advanced AFO is an option for trauma affecting the lower limb. Long-term studies are required to better understand the accommodation and learning process of using an advanced AFO.

Deformed wing virus genotypes A and B do not elicit immunologically different responses in naïve honey bee hosts.

Iflavirus aladeformis (Picornavirales: Iflaviridae), commonly known as deformed wing virus(DWV), in association with Varroa destructor Anderson and Trueman (Mesostigmata: Varroidae), is a leading factor associated with honey bee (Apis mellifera L. [Hymenoptera: Apidae]) deaths. The virus and mite have a near global distribution, making it difficult to separate the effect of one from the other. The prevalence of two main DWV genotypes (DWV-A and DWV-B) has changed over time, leading to the possibility that the two strains elicit a different immune response by the host. Here, we use a honey bee population naïve to both the mite and the virus to investigate if honey bees show a different immunological response to DWV genotypes. We examined the expression of 19 immune genes by reverse transcription quantitative PCR (RT-qPCR) and analysed small RNA after experimental injection with DWV-A and DWV-B. We found no evidence that DWV-A and DWV-B elicit different immune responses in honey bees. RNA interference genes were up-regulated during DWV infection, and small interfering RNA (siRNA) responses were proportional to viral loads yet did not inhibit DWV accumulation. The siRNA response towards DWV was weaker than the response to another honey bee pathogen, Triatovirus nigereginacellulae (Picornavirales: Dicistroviridae; black queen cell virus), suggesting that DWV is comparatively better at evading host antiviral defences. There was no evidence for the production of virus-derived Piwi-interacting RNAs (piRNAs) in response to DWV. In contrast to previous studies, and in the absence of V. destructor, we found no evidence that DWV has an immunosuppressive effect. Overall, our results advance our understanding of the immunological effect that DWV in isolation elicits in honey bees.

Associations of Epigenetic Age Acceleration With CVD Risks Across the Lifespan: The Bogalusa Heart Study.

Although epigenetic age acceleration (EAA) might serve as a molecular signature of childhood cardiovascular disease (CVD) risk factors and further promote midlife subclinical CVD, few studies have comprehensively examined these life course associations. This study sought to test whether childhood CVD risk factors predict EAA in adulthood and whether EAA mediates the association between childhood CVD risks and midlife subclinical disease. Among 1,580 Bogalusa Heart Study participants, we estimated extrinsic EAA, intrinsic EAA, PhenoAge acceleration (PhenoAgeAccel), and GrimAge acceleration (GrimAgeAccel) during adulthood. We tested prospective associations of longitudinal childhood body mass index (BMI), blood pressure, lipids, and glucose with EAAs using linear mixed effects models. After confirming EAAs with midlife carotid intima-media thickness and carotid plaque, structural equation models examined mediating effects of EAAs on associations of childhood CVD risk factors with subclinical CVD measures. After stringent multiple testing corrections, each SD increase in childhood BMI was significantly associated with 0.6-, 0.9-, and 0.5-year increases in extrinsic EAA, PhenoAgeAccel, and GrimAgeAccel, respectively (P < 0.001 for all 3 associations). Likewise, each SD increase in childhood log-triglycerides was associated with 0.5- and 0.4-year increases in PhenoAgeAccel and GrimAgeAccel (P < 0.001 for both), respectively, whereas each SD increase in childhood high-density lipoprotein cholesterol was associated with a 0.3-year decrease in GrimAgeAccel (P = 0.002). Our findings indicate that PhenoAgeAccel mediates an estimated 27.4% of the association between childhood log-triglycerides and midlife carotid intima-media thickness (P = 0.022). Our data demonstrate that early life CVD risk factors may accelerate biological aging and promote subclinical atherosclerosis.

Prospective assessment of canine thyroid cancer-part I: nodal metastatic rate and impact of nodal immunohistochemistry in 70 dogs.

OBJECTIVE: To determine the rate of nodal metastasis in dogs with thyroid cancer and evaluate whether immunohistochemistry (IHC) identifies additional metastases beyond evaluation with H&E. ANIMALS: 70 prospectively enrolled client-owned dogs with thyroid cancer managed with thyroidectomy. METHODS: Dogs underwent thyroidectomy with concurrent elective bilateral medial retropharyngeal (MRP) ± deep cervical lymphadenectomy. Thyroid tumors and associated lymph nodes were reviewed by a single board-certified pathologist. Immunohistochemistry was used for all primary tumors (thyroid transcription factor-1 and calcitonin) to support a diagnosis of follicular or medullary carcinoma. Lymph nodes without evidence of metastasis after H&E review were labeled with the antibody associated with the wider uptake in the primary tumor. RESULTS: 77 thyroid cancers were resected from the 70 dogs enrolled, including 61 (79.2%) follicular, 8 (10.7%) medullary, and 7 (9.3%) mixed follicular/medullary carcinomas, with 1 (1.3%) carcinosarcoma. Twelve dogs had evidence of nodal metastasis following H&E review. Occult micrometastasis was identified in 1 dog following nodal IHC, resulting in documented metastasis in 13 of 70 (18.6%) dogs. Metastasis was more common with medullary (5/8) and follicular/medullary carcinoma (3/7) than follicular carcinoma (5/61). All MRP metastases were ipsilateral (7/77 [9.1%]), without contralateral MRP metastases (0/62). Fourteen of 41 (34.1%) deep cervical lymph nodes were metastatic. CLINICAL RELEVANCE: Nodal metastasis was uncommon for follicular carcinoma but was seen in > 50% of dogs with thyroid cancer involving a medullary component. Routine nodal IHC appears to be low yield for thyroid carcinoma. Extirpation of ipsilateral MRP and identifiable deep cervical lymph nodes is recommended with thyroidectomy until detailed preoperative risk stratification becomes available.

The genome sequence of the Marbled White Spot, Protodeltote pygarga (Hufnagel, 1766).

We present a genome assembly from an individual male Protodeltote pygarga (the Marbled White Spot; Arthropoda; Insecta; Lepidoptera; Noctuidae). The genome sequence is 421.1 megabases in span. Most of the assembly is scaffolded into 31 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 15.48 kilobases in length. Gene annotation of this assembly on Ensembl identified 17,784 protein coding genes.