A Comprehensive Atlas of AAV Tropism in the Mouse.

Gene therapy with Adeno-Associated Viral (AAV) vectors requires knowledge of their tropism within the body. Here we analyze the tropism of ten naturally occurring AAV serotypes (AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAVrh10 and AAVrh74) following systemic delivery into male and female mice. A transgene expressing ZsGreen and Cre recombinase was used to identify transduction in a cell-dependent manner based on fluorescence. Cre-driven activation of tdTomato fluorescence offered superior sensitivity for transduced cells. All serotypes except AAV3B and AAV4 had high liver tropism. Fluorescence activation revealed transduction of unexpected tissues, including adrenals, testes and ovaries. Rare transduced cells within tissues were also readily visualized. Biodistribution of AAV genomes correlated with fluorescence, except in immune tissues. AAV4 was found to have a pan-endothelial tropism while also targeting pancreatic beta cells. This public resource enables selection of the best AAV serotypes for basic science and preclinical applications in mice.

In Vitro Bond Strength of Dentin Treated with Sodium Hypochlorite: Effects of Antioxidant Solutions.

This systematic review aims to evaluate whether the application of antioxidant solutions can enhance the bond strength of resin-based materials to sodium hypochlorite (NaOCl)-treated dentin. This study follows the PICOT strategy: population (sodium hypochlorite-treated dentin), intervention (application of antioxidants), control (distilled water), outcome (bond strength), and type of studies (in vitro studies). The systematic review and meta-analysis were conducted following PRISMA guidelines. Electronic databases were searched for in vitro studies evaluating the effects of antioxidants on bond strength to sodium hypochlorite-treated dentin. Two independent reviewers screened articles, extracted data, and assessed risk of bias. Meta-analyses were performed using a random-effects model to compare standardized mean differences in bond strength between antioxidant pretreatment and control groups. Inclusion criteria consisted of in vitro studies that examined the bond strength of resin-based materials to NaOCl-treated dentin with antioxidant application, while exclusion criteria included studies with incomplete data, those not using a control group, or those that did not directly measure bond strength. From 3041 initial records, 29 studies were included in the qualitative analysis and 25 in the meta-analysis. Ascorbic acid, sodium ascorbate, grape seed extract, green tea, and rosmarinic acid significantly improved bond strength to sodium hypochlorite-treated dentin (p < 0.05). The effectiveness of grape seed extract varied with adhesive system type. Hesperidin, p-toluene sulfonic acid, and sodium thiosulfate did not significantly improve bond strength. Most studies had a high risk of bias. This suggests that the conclusions drawn from these studies should be interpreted with caution, and further research with more robust methodologies may be needed to confirm the findings. In conclusion, this systematic review implies that certain antioxidants can improve bond strength to sodium hypochlorite-treated dentin, with efficacy depending on the specific agent and adhesive system used. Further standardized studies are needed to optimize protocols and confirm these findings.

Bioethanol production by immobilized co-culture of Saccharomyces cerevisiae and Scheffersomyces stipitis in a novel continuous 3D printing microbioreactor.

Biorefineries require low-cost production processes, low waste generation and equipment that can be used not only for a single process, but for the manufacture of several products. In this context, in this research a continuous 3D printing microbioreactor coupled to an Arduino-controlled automatic feeding system was developed for the intensification of the ethanol production process from xylose/xylulose (3:1), using a new biocatalyst containing the co-culture of Scheffersomyces stipitis and Saccharomyces cerevisiae (50/50). Initially, batch fermentations of monocultures of S. cerevisiae and S. stipitis and co-culture were carried out. Subsequently, the immobilized co-culture was used as a biocatalyst in continuous fermentations using the developed microreactor. Fermentations carried out in the microbioreactor presented a 2-fold increase in the ethanol concentration and a 3-fold increase in productivity when compared to monocultures. The microbioreactor developed proved to be efficient and can be extended for other bioproducts production. This approach proved to be a promising alternative for the use of the hemicellulose fraction of biomasses without the need to use modified strains.

Intravenous lipomas of head and neck: an exceptional entity and its clinical implications.

Intravenous lipomas (IVLs) of the head and neck are uncommon benign tumors that develop within the venous walls, often detected incidentally during imaging for unrelated issues. While usually asymptomatic, these IVLs can cause congestive venous symptoms like swelling, paresthesia or pain in the head and neck and upper limbs, or even venous thromboembolism. The precise diagnosis of IVLs is predominantly achieved through computed tomography (CT) and magnetic resonance imaging (MRI), with CT being the most frequently used method. Symptomatic patients generally undergo open surgery with excision of the IVL followed by venous reconstruction, which has shown safe and effective outcomes. However, the management of asymptomatic IVLs remains controversial due to the limited number of reported cases. Despite this, there is a notable trend toward recommending surgical removal of IVLs to prevent complications and rule out malignancy, driven by the challenges of differentiating IVLs from malignant tumors using imaging alone. This review highlights the key differential imaging characteristics of IVLs and the main surgical techniques to remove the tumor and repair the vascular defect. Further research is necessary to establish a robust, evidence-based approach for treating asymptomatic IVLs, balancing the risks of surgery against the potential for future complications.

DNA-PKcs Inhibition Improves Sequential Gene Insertion of the Full-Length CFTR cDNA in Airway Stem Cells.

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Although many people with CF (pwCF) are treated using CFTR modulators, some are non-responsive due to their genotype or other uncharacterized reasons. Autologous airway stem cell therapies, in which the CFTR cDNA has been replaced, may enable a durable therapy for all pwCF. Previously, CRISPR-Cas9 with two AAVs was used to sequentially insert two halves of the CFTR cDNA and an enrichment cassette into the CFTR locus. However, the editing efficiency was <10% and required enrichment to restore CFTR function. Further improvement in gene insertion may enhance cell therapy production. To improve CFTR cDNA insertion in human airway basal stem cells (ABCs), we evaluated the use of the small molecules AZD7648 and ART558 which inhibit non-homologous end joining (NHEJ) and micro-homology mediated end joining (MMEJ). Adding AZD7648 alone improved gene insertion by 2-3-fold. Adding both ART558 and AZD7648 improved gene insertion but induced toxicity. ABCs edited in the presence of AZD7648 produced differentiated airway epithelial sheets with restored CFTR function after enrichment. Adding AZD7648 did not increase off-target editing. Further studies are necessary to validate if AZD7648 treatment enriches cells with oncogenic mutations.

New insights in the diagnosis and treatment of equine piroplasmosis: pitfalls, idiosyncrasies, and myths.

Equine piroplasmosis (EP) is a global tick-borne disease of equids caused by the intraerythrocytic apicomplexan parasites Theileria equi and Babesia caballi, and the more recently discovered Theileria haneyi. These parasites can be transmitted by several tick species, including Dermacentor, Hyalomma, and Rhipicephalus, but iatrogenic and vertical transmission are also common. Clinical signs of EP include poor performance, fever, icterus, abortions, among others, and peracute or acute forms of infection are associated with high mortality in non-endemic areas. EP is a reportable disease and represents an important barrier for the international trade of horses and other equids, causing disruption of international equine sports. Tick control measures, serological and molecular diagnostic methods, and parasiticidal drugs are currently used against EP, while vaccines remain unavailable. Since most acaricides used in equids are non-environmentally friendly and linked to drug resistances, this is considered as an unsustainable approach. Imidocarb dipropionate (ID) and buparvaquone (BPQ) are currently the main drugs used to control the disease. However, while ID has several side and toxic effects and recurrent failures of treatment have been reported, BPQ is less effective in the clearance of T. equi infection and not available in some countries. Thus, novel alternative and effective therapeutics are needed. While current trade regulations require testing equids for EP before exportation, the lack of standardized PCR tests and limitations of the currently recommended serological assays entail a risk of inaccurate diagnosis. Hereby, we propose a combination of standardized PCR-based techniques and improved serological tests to diminish the risks of exporting EP-infected animals making equid international trade safer. In addition, this review discusses, based on scientific evidence, several idiosyncrasies, pitfalls and myths associated with EP, and identifies weaknesses of current methods of control and gaps of research, as initial steps toward developing novel strategies leading to control this disease.

Role of Rhipicephalus bursa larvae in transstadial transmission and endemicity of Babesia ovis in chronically infected sheep.

Babesia ovis, transmitted by Rhipicephalus bursa ticks, is the causative agent of ovine babesiosis, a disease characterized by fever, anemia, hemoglobinuria, and high mortality in sheep. This study investigates whether sheep that survived babesiosis without treatment can serve as a source of infection for B. ovis-free host-seeking R. bursa larvae in a later season. Three donor sheep were experimentally infected with B. ovis, and after six months, persistence of B. ovis was assessed through blood and tick transmission experiments. Blood from donor sheep was intravenously injected into three recipient sheep, while donor sheep were also infested with B. ovis-free R. bursa larvae. Engorged nymphs molted to adults, and new recipient sheep were infested with these ticks. All recipient sheep were monitored for B. ovis for 100 days using microscopic, serological, and molecular approaches. The presence of B. ovis was confirmed in the recipient sheep that received blood, leading to clinical infection in two. However, no B. ovis was detected in recipient sheep infested with ticks. These results suggest that sheep recovering from B. ovis infection do not serve as a source of infection for R. bursa larvae in subsequent seasons.

Adhesive Performance of Pit and Fissure Sealants on Deproteinized Enamel with Different Proteolytic Agents: In Vitro Study.

The objective of this work was to assess the efficacy of different proteolytic agents on the bond strength of pit and fissure sealants to bovine enamel. Eighty-four bovine enamel specimens were randomly assigned in groups according to the pit and fissure sealant applied (HelioSeal F or Dyad Flow). Then, the specimens were subdivided according to the proteolytic agent used (n = 7): Group 1, distilled water (control); Group 2, 10 wt.% Tergazyme®; Group 3, 10 wt.% ZYME®; Group 4, 10% papain gel; Group 5, 10% bromelain gel; and Group 6, 5.25 wt.% sodium hypochlorite. The cell viability of the proteolytic solutions was assessed through the MTT assay. The proteolytic agents were applied on the enamel surface prior to the acid-etching procedure; then, the pit and fissure sealants were placed. The micro-shear bond strength was evaluated after 24 h or 6 months of water storing at 37 °C. Representative SEM images were taken for each experimental group. The bond strength data were statistically analyzed by a three-way ANOVA test using a significance level of α = 0.05. Bromelain and papain proteolytic solutions did not exert any cytotoxic effect on the human dental pulp cells. After 24 h and 6 months of aging, for both pit and fissure sealants, sodium hypochlorite, papain, bromelain, and Tergazyme® achieved statistically significant higher bond strength values (p < 0.05). Irrespective of the deproteinizing agent used, Dyad Flow resulted in a better bond strength after 6 months of aging. The type 1 etching pattern was identified for sodium hypochlorite, papain, and bromelain. Tergazyme®, papain, and bromelain demonstrated efficacy in deproteinizing enamel surfaces prior to acid etching, leading to the improved bond strength of pit and fissure sealants. Clinically, this suggests that these proteolytic agents can be considered viable alternatives to traditional methods for enhancing sealant retention and longevity. Utilizing these agents in dental practice could potentially reduce sealant failures.

Bioaccumulation of trace elements in marine mammals: New data and transplacental transfer on threatened species.

Coastal areas are affected by urban, industrial and agriculture pollutants runoff, wastewater and stormwater discharges, making this environment the final repository of chemical contaminants. These contaminants have the potential to spread out to the entire food chain, impacting marine life and the quality of their habitat. In this aspect, the concept of marine mammals as bioindicators provides an approach to the degree of contamination in the environment and to the identification and management of multiple sources of contaminants. The present study analyzed several elements like As, Ba, Cd, Cr, Cu, Hg, Mn, Mo, Ni, Pb, V and Zn in liver tissue from two dolphin species: Sotalia guianensis, a near-threatened species, and the vulnerable Pontoporia blainvillei. In the study, we also investigated if dolphins (population) recorded using the heaviest urban areas have higher concentrations of contaminants in their tissues. Dolphin samples (n = 40 S. guianensis; n = 97 P. blainvillei) were collected by daily monitoring carried out by Santos Basin Beach Monitoring Project (PMP-BS), from stranded individuals found in São Paulo state. The Spearman's rank correlation showed distinct correlations in the accumulation of trace elements by both species, indicating different sources of exposure to the elements studied or distinct biochemical processes between species. Interspecific and intraspecific variations were observed, possibly related to the individual distribution and feeding habits. Correlations were observed between age and concentrations of trace elements, positive for Cd, Hg and Mo. Finally, our findings indicate high levels of Cu, Zn, and concentrations of As, V and Hg in fetuses, in particular, an analysis was performed on a fetus found inside a stranded individual, indicating placental transfer as the first route of exposure for some elements.

Enhanced Antibacterial Properties of Titanium Surfaces through Diversified Ion Plating with Silver Atom Deposition.

In this study, we investigate the antibacterial effect of silver atoms implanted into a thin surface layer of titanium at low energies using an alternative ion plating technology called Diversified Ion Plating. Silver atoms were incorporated into titanium samples using reactive low-voltage ion plating at 2 keV and 4 keV. Surface modifications and morphology were evaluated using wettability, profilometry measurements, and energy-dispersive spectroscopy. For a precise determination of the quantity and depth of implanted silver atoms on titanium surfaces, a combination of experimental techniques such as Rutherford Backscattering Spectrometry along with Monte Carlo simulations were utilized. To assess the antibacterial effects of the silver atoms incorporated into pure titanium surfaces, bacterial suspension immersion tests were performed with a standard strain of Staphylococcus aureus (ATCC 12600). The outcomes indicate that titanium surfaces implanted with silver atoms were more effective in inhibiting the growth of Staphylococcus aureus than pure titanium surfaces. Better results were found when the deposition was performed at 4 keV, indicating that a deeper implantation of silver, spanning a few nanometers, can result in a longer and more effective release of silver atoms. These findings suggest the potential for the development of new, cost-effective biomaterials, paving the way for improved implant materials in various health-related applications.

Molecular classification of metastatic and recurrent endometrial endometrioid carcinoma: prognostic relevance among low- and high-stage tumours.

AIMS: Molecular classification according to The Cancer Genome Atlas (TCGA) improves endometrial endometrioid carcinoma (EEC) prognostication and has specific treatment implications; however, original data were skewed towards low-grade and low-stage tumours. Herein, we molecularly classify EECs metastatic at the time of diagnosis or with subsequently documented recurrent/metastatic disease to examine correlation with clinical outcomes. METHODS: TCGA categories include POLE-mutated, microsatellite instability (MSI), p53 abnormal (p53 abnl) and no specific molecular profile (NSMP). POLE targeted sequencing at exons 9, 11, 13 and 14 and immunohistochemistry (IHC) for PMS2, MSH6 and p53 were performed to establish molecular classification. RESULTS: The distribution in our cohort of 141 EECs was similar to that generally reported in EEC, with nine POLE-mutated (6%), 45 MSI (32%), 16 p53 abnl (11%) and 71 NSMP (50%), with similar distributions between low- and high-stage cohorts. We demonstrate that when stratified by molecular subtype, disease-specific survival from the time of high-stage (stages III-IV) presentation or time of recurrence in low-stage (stages I-II) disease among metastatic and/or recurrent EEC is strongly associated with TCGA classification (high-stage P = 0.02, low-stage P = 0.017). Discordant molecular classification between primary and metastatic/recurrent tumours occurred in four of 105 (3.8%) patients, two related to PMS2/MSH6 IHC and two related to p53 IHC. CONCLUSIONS: We demonstrate that molecular classification is prognostically relevant not only at the time of diagnosis, but also at the time of recurrence and in the metastatic setting. Rare subclonal alterations occur and suggest a role for confirming TCGA classification in recurrent/metastatic tumours.

General Applicability of Existing College of American Pathologists Accreditation Requirements to Clinical Implementation of Machine Learning-Based Methods in Molecular Oncology Testing.

CONTEXT.­: The College of American Pathologists (CAP) accreditation requirements for clinical laboratory testing help ensure laboratories implement and maintain systems and processes that are associated with quality. Machine learning (ML)-based models share some features of conventional laboratory testing methods. Accreditation requirements that specifically address clinical laboratories' use of ML remain in the early stages of development. OBJECTIVE.­: To identify relevant CAP accreditation requirements that may be applied to the clinical adoption of ML-based molecular oncology assays, and to provide examples of current and emerging ML applications in molecular oncology testing. DESIGN.­: CAP accreditation checklists related to molecular pathology and general laboratory practices (Molecular Pathology, All Common and Laboratory General) were reviewed. Examples of checklist requirements that are generally applicable to validation, revalidation, quality management, infrastructure, and analytical procedures of ML-based molecular oncology assays were summarized. Instances of ML use in molecular oncology testing were assessed from literature review. RESULTS.­: Components of the general CAP accreditation framework that exist for traditional molecular oncology assay validation and maintenance are also relevant for implementing ML-based tests in a clinical laboratory. Current and emerging applications of ML in molecular oncology testing include DNA methylation profiling for central nervous system tumor classification, variant calling, microsatellite instability testing, mutational signature analysis, and variant prediction from histopathology images. CONCLUSIONS.­: Currently, much of the ML activity in molecular oncology is within early clinical implementation. Despite specific considerations that apply to the adoption of ML-based methods, existing CAP requirements can serve as general guidelines for the clinical implementation of ML-based assays in molecular oncology testing.

Molecular Classification Outperforms Histologic Classification in Prognostication of High-grade Endometrial Carcinomas With Undifferentiated and Sarcomatous Components.

Since the establishment of 4 molecular subgroups of endometrial carcinoma (EC), there has been significant interest in understanding molecular classification in the context of histologic features and diagnoses. ECs with undifferentiated, spindle, and/or sarcomatous components represent a diagnostically challenging subset of tumors with overlapping clinical and histologic features. We examined the clinicopathologic, morphologic, immunohistochemical, and molecular features of these tumors identified in our institutions' pathology databases using immunohistochemistry and targeted sequencing. Disease-specific survival (DSS) and progression-free survival (PFS) were analyzed using Kaplan-Meier curves and log-rank tests. One hundred sixty-two ECs were included: carcinosarcomas (UCS; n=96), dedifferentiated/undifferentiated EC (DDEC/UDEC; n=49), and grade 3 endometrioid EC with spindled growth (GR3spEEC) (n=17). All molecular subgroups were represented in all histologic subtypes and included 12 (7%) POLE -mutated ( POLE mut), 43 (27%) mismatch repair-deficient (MMRd), 77 (48%) p53-abnormal (p53abn), and 30 (19%) no specific molecular profile (NSMP) tumors. However, the molecular classification (irrespective of histologic diagnosis) was a significant predictor for both DSS ( P =0.008) and P≤0.0001). POLE mut EC showed an excellent prognosis with no recurrences or deaths from the disease. MMRd tumors also showed better outcomes relative to NSMP and p53abn tumors. In conclusion, molecular classification provides better prognostic information than histologic diagnosis for high-grade EC with undifferentiated and sarcomatous components. Our study strongly supports routine molecular classification of these tumors, with emphasis on molecular group, rather than histologic subtyping, in providing prognostication.

Comparative efficacy of buparvaquone and imidocarb in inhibiting the in vitro growth of Babesia bovis.

Introduction: B. bovis is an apicomplexan parasite responsible for bovine babesiosis, a tick-borne disease with a worldwide impact. The disease remains inefficiently controlled, and few effective drugs, including imidocarb dipropionate (ID), are currently available in endemic areas. The objective of this study was to evaluate whether buparvaquone (BPQ), a drug currently used to treat cattle infected with the Babesia-related Theileria spp. parasites, could be active against Babesia parasites. Herein, we compared the effect of ID and BPQ on B. bovis growth in vitro erythrocyte culture. Methods: We compared the effect of ID and BPQ on the culture-adapted Texas T2Bo strain of B. bovis. In vitro cultured parasites were incubated with ID and BPQ at two starting parasitemia levels (PPE), 0.2% and 1%. In vitro cultured parasites were treated with ID or BPQ at concentrations ranging from 10 to 300 nM, during 4 consecutive days. Parasitemia levels were daily evaluated using microscopic examination. Data was compared using the independent Student's t-test. Results and discussion: Both ID and BPQ significantly inhibited (p < 0.05) the growth of B. bovis, regardless of the initial parasitemia used. At 1% parasitemia, BPQ had lower calculated inhibitory concentration 50 (IC50: 50.01) values than ID (IC50: 117.3). No parasites were found in wells with 0.2% starting parasitemia, treated previously with 50 nM of BPQ or ID, after 2 days of culture without drugs. At 1% parasitemia, no parasite survival was detected at 150 nM of BPQ or 300 nM of ID, suggesting that both drugs acted as babesiacidals. Conclusion: Overall, the data suggests that BPQ is effective against B. bovis and shows a residual effect that seems superior to ID, which is currently the first-line drug for treating bovine babesiosis globally.

A conserved motif in the immune-subdominant RAP-1 related antigen of Babesia bovis contains a B-cell epitope recognized by antibodies from protected cattle.

Introduction: Babesia bovis, a tick-borne apicomplexan parasite causing bovine babesiosis, remains a significant threat worldwide, and improved and practical vaccines are needed. Previous studies defined the members of the rhoptry associated protein-1 (RAP-1), and the neutralization-sensitive rhoptry associated protein-1 related antigen (RRA) superfamily in B. bovis, as strong candidates for the development of subunit vaccines. Both RAP-1 and RRA share conservation of a group of 4 cysteines and amino acids motifs at the amino terminal end (NT) of these proteins. Methods and results: Sequence comparisons among the RRA sequences of several B. bovis strains and other Babesia spp parasites indicate a high level of conservation of a 15-amino acid (15-mer) motif located at the NT of the protein. BlastP searches indicate that the 15-mer motif is also present in adenylate cyclase, dynein, and other ATP binding proteins. AlphaFold2 structure predictions suggest partial exposure of the 15-mer on the surface of RRA of three distinct Babesia species. Antibodies in protected cattle recognize a synthetic peptide representing the 15-mer motif sequence in iELISA, and rabbit antibodies against the 15-mer react with the surface of free merozoites in immunofluorescence. Discussion and conclusion: The presence of the 15-mer-like regions in dynein and ATP-binding proteins provides a rationale for investigating possible functional roles for RRA. The demonstrated presence of a surface exposed B-cell epitope in the 15-mer motif of the B. bovis RRA, which is recognized by sera from protected bovines, supports its inclusion in future subunit epitope-based vaccines against B. bovis.

In situ study of the effect of endogenous and exogenous agents on color stability, hardness, and surface roughness of an elastomer for facial prostheses.

PURPOSE: To evaluate in situ the influence of sweat, oil, sunscreen, and disinfectant solution on the color stability, hardness, and roughness of elastomer for facial prostheses. MATERIALS AND METHODS: Standardized and intrinsically pigmented specimens remained in contact with human skin from the same person for 30 days, considering exposures (n = 36 per group), absent of exposition (Control, C); sweat and oiliness contact (SO); sweat and oiliness associated with sunscreen (SOS); 0.12% chlorhexidine digluconate immersion (CD0.12%); and all agents exposed (SOSCD). The main variables were color change (CIELab and National Standard Bureau system, NBS), Shore A hardness, and surface roughness, measured at baseline and 30 days. Qualitative analyses were performed by atomic force microscopy (AFM) and scanning electron microscopy (SEM). The data were analyzed by Kruskal-Wallis tests (color) and two-way ANOVA (hardness and roughness) with Sidak post-test (α = 0.05). RESULTS: CD0.12% (1.54 ± 0.49) and SOSCD (2.10 ± 1.03) had similar effects and caused the smallest color changes, considered mild and noticeable (NBS), respectively. SOS promoted the greatest color change (6.99 ± 1.43, NBS: large) and hardness (17.97 ± 0.56); SOS promoted intermediate roughness (3.48 ± 1.05) between SOSCD (2.25 ± 0.53), and two similar groups: C (4.46 ± 0.95), and CD0.12% (4.39 ± 1.26). The qualitative analysis showed an irregular, dense, dry, and whitish layer on the surface of the specimens exposed to sunscreen, which was reduced when in contact with 0.12% chlorhexidine digluconate. CONCLUSIONS: Endogenous and exogenous factors are capable of altering elastomer properties. The 0.12% chlorhexidine digluconate minimized the changes caused by sweat, oil, and sunscreen.

Expression of IL-10 and TGF-ß1 in horses experimentally infected with T. equi merozoites is associated with antibody production but not modulation of pro-inflammatory responses.

Theileria equi (T. equi) is an apicomplexan parasite that causes severe hemolytic anemia in equids. Presently, there is inadequate knowledge of the immune responses induced by T. equi in equid hosts impeding understanding of the host parasite relationship and development of potent vaccines for control of T. equi infections. The objective of this study was to evaluate the host-parasite dynamics between T. equi merozoites and infected horses by assessing cytokine expression during primary and secondary parasite exposure, and to determine whether the pattern of expression correlated with clinical indicators of disease. Our findings showed that the expression of pro-inflammatory cytokines was very low and inconsistent during both primary and secondary infection. There was also no correlation between the symptoms observed during primary infection and expression of the cytokines. This suggests that the symptoms might have occurred primarily due to hemolysis and likely not the undesirable effects of pro-inflammatory responses. However, IL-10 and TGF-ß1 were highly expressed in both phases of infection, and their expression was linked to antibody production but not moderation of pro-inflammatory cytokine responses.

MEK signaling represents a viable therapeutic vulnerability of KRAS-driven somatic brain arteriovenous malformations.

Brain arteriovenous malformations (bAVMs) are direct connections between arteries and veins that remodel into a complex nidus susceptible to rupture and hemorrhage. Most sporadic bAVMs feature somatic activating mutations within KRAS, and endothelial-specific expression of the constitutively active variant KRASG12D models sporadic bAVM in mice. By leveraging 3D-based micro-CT imaging, we demonstrate that KRASG12D-driven bAVMs arise in stereotypical anatomical locations within the murine brain, which coincide with high endogenous Kras expression. We extend these analyses to show that a distinct variant, KRASG12C, also generates bAVMs in predictable locations. Analysis of 15,000 human patients revealed that, similar to murine models, bAVMs preferentially occur in distinct regions of the adult brain. Furthermore, bAVM location correlates with hemorrhagic frequency. Quantification of 3D imaging revealed that G12D and G12C alter vessel density, tortuosity, and diameter within the mouse brain. Notably, aged G12D mice feature increased lethality, as well as impaired cognition and motor function. Critically, we show that pharmacological blockade of the downstream kinase, MEK, after lesion formation ameliorates KRASG12D-driven changes in the murine cerebrovasculature and may also impede bAVM progression in human pediatric patients. Collectively, these data show that distinct KRAS variants drive bAVMs in similar patterns and suggest MEK inhibition represents a non-surgical alternative therapy for sporadic bAVM.

Understanding the Regiodivergence between Hydroarylation and Trifluoromethylarylation of 1,3-Dienes Using Anilines in HFIP.

Conjugated dienes (1,3-dienes) are versatile and valuable chemical feedstocks that can be used as two-carbon or four-carbon synthons with vast applications across the chemical industry. However, the main challenge for their productive incorporation in synthetic routes is their chemo-, regio-, and stereoselective functionalization. Herein, we introduce a unified strategy for the 1,2-hydroarylation and 1,4-trifluoromethylarylation of 1,3-dienes using anilines in hexafluoroisopropanol. DFT calculations point toward a kinetically controlled process in both transformations, particularly in the trifluoromethylarylation, to explain the regiodivergent outcome. In addition, we perform an extensive program of functionalization and diversification of the products obtained, including hydrogenation, oxidation, cyclizations, or cross-coupling reactions, that allows access to a library of high-value species in a straightforward manner.

Targeted mutational profiling of Epstein Barr virus-positive mucocutaneous ulcer: Implications for differential diagnosis with EBV-positive diffuse large B-cell lymphoma.

Epstein Barr Virus-positive mucocutaneous ulcer (EBVMCU) can be difficult to distinguish from EBV-positive diffuse large B cell lymphoma (DLBCL). We used targeted next-generation sequencing (NGS) to explore genetic alterations in EBVMCU to aid in this diagnostic challenge. Ten cases of EBVMCU were evaluated by a targeted NGS panel of 164 genes. Targeted NGS identified 18 variants in 15 genes in eight cases of EBVMCU. Loss of function TET2 variants were most frequently identified (3 of 10 cases, 30 %). One TET2 variant occurred at low variant allele frequency (VAF) of 3 %, which may be suggestive of clonal hematopoiesis of indeterminate potential. One case harbored a loss of function DNMT3A variant at low VAF. Two cases demonstrated missense variants in the IRF8 gene. Both variants occurred at a VAF close to 50 % and with an estimated high burden of disease (75 %). Two cases of mucosal gastrointestinal involvement had no reportable variants. Mutational profiling of EBVMCU identified TET2 loss of function variants at an elevated frequency in our cohort; however, the findings are not specific and its clinical significance cannot be completely elucidated. Further studies are needed to confirm the findings in an independent and larger cohort of EBVMCU, to determine the cell of origin of the variants, and to further assess their significance in the pathogenesis of this disorder.