A genetic algorithm-based approach for the prediction of metabolic drug-drug interactions involving CYP2C8 or CYP2B6.

BACKGROUND AND OBJECTIVES: A genetic algorithm (GA) approach was developed to predict drug-drug interactions (DDIs) caused by cytochrome P450 2C8 (CYP2C8) inhibition or cytochrome P450 2B6 (CYP2B6) inhibition or induction. Nighty-eight DDIs, obtained from published in vivo studies in healthy volunteers, have been considered using the area under the plasma drug concentration-time curve (AUC) ratios (i.e., ratios of AUC of the drug substrate administered in combination with a DDI perpetrator to AUC of the drug substrate administered alone) to describe the extent of DDI. METHODS: The following parameters were estimated in this approach: the contribution ratios (CRCYP2B6 and CRCYP2C8, i.e., the fraction of the dose metabolized via CYP2B6 or CYP2C8, respectively) and the inhibitory or inducing potency of the perpetrator drug (IRCYP2B6, IRCYP2C8 and ICCYP2B6, for inhibition of CYP2B6 and CYP2C8, and induction of CYP2B6, respectively). The workflow consisted of three main phases. First, the initial estimates of the parameters were estimated through GA. Then, the model was validated using an external validation. Finally, the parameter values were refined via a Bayesian orthogonal regression using all data. RESULTS: The AUC ratios of 5 substrates, 11 inhibitors and 19 inducers of CYP2B6, and the AUC ratios of 19 substrates and 23 inhibitors of CYP2C8 were successfully predicted by the developed methodology within 50-200% of observed values. CONCLUSIONS: The approach proposed in this work may represent a useful tool for evaluating the suitable doses of a CYP2C8 or CYP2B6 substrates co-administered with perpetrators.

Hybrid AI-enhanced lightning flash prediction in the medium-range forecast horizon.

Traditional fully-deterministic algorithms, which rely on physical equations and mathematical models, are the backbone of many scientific disciplines for decades. These algorithms are based on well-established principles and laws of physics, enabling a systematic and predictable approach to problem-solving. On the other hand, AI-based strategies emerge as a powerful tool for handling vast amounts of data and extracting patterns and relationships that might be challenging to identify through traditional algorithms. Here, we bridge these two realms by using AI to find an optimal mapping of meteorological features predicted two days ahead by the state-of-the-art numerical weather prediction model by the European Centre for Medium-range Weather Forecasts (ECMWF) into lightning flash occurrence. The prediction capability of the resulting AI-enhanced algorithm turns out to be significantly higher than that of the fully-deterministic algorithm employed in the ECMWF model. A remarkable Recall peak of about 95% within the 0-24 h forecast interval is obtained. This performance surpasses the 85% achieved by the ECMWF model at the same Precision of the AI algorithm.

Potential Factors Influencing Complete Functional Recovery in Traumatized Unowned Cats with Orthopedic Lesions-A Cohort Study.

The management of unowned cats is an emerging problem, with public institutions and citizens' concerns regarding their care and arrangement. Little is known regarding the outcome of traumatic orthopedic injuries in these patients. Indeed, complete functional recovery (CFR) should be the goal of treatment for return to their original location or adoption. The aim was to identify clinical factors influencing CFR in traumatized unowned cats with orthopedic lesions. This category of cats referred by the veterinary public service over three years was enrolled. Various clinical variables were retrospectively collected from the medical records and evaluated by nominal logistic analysis. Forty-eight unowned cats were enrolled, with a median estimated age of 24 (1-180) months and a body weight of 3 (0.7-5) kg. Thirty-four (71%) patients reached CFR. Estimated age, body weight, time from trauma to therapeutic intervention, spine involvement, presence of comorbidities, hospitalization time, and the radiographic score results were significantly associated with CFR. A longer time to therapeutic intervention seemed to be associated with a better outcome. Probably, cats severely traumatized did not live long enough to be evaluated and treated. Lighter cats experienced more severe consequences following blunt trauma. Younger and lighter cats bore a higher risk of panleukopenia-related death.

Leveraging Tissue-Specific Enhancer-Target Gene Regulatory Networks Identifies Enhancer Somatic Mutations That Functionally Impact Lung Cancer.

Enhancers are noncoding regulatory DNA regions that modulate the transcription of target genes, often over large distances along with the genomic sequence. Enhancer alterations have been associated with various pathological conditions, including cancer. However, the identification and characterization of somatic mutations in noncoding regulatory regions with a functional effect on tumorigenesis and prognosis remain a major challenge. Here, we present a strategy for detecting and characterizing enhancer mutations in a genome-wide analysis of patient cohorts, across three lung cancer subtypes. Lung tissue-specific enhancers were defined by integrating experimental data and public epigenomic profiles, and the genome-wide enhancer-target gene regulatory network of lung cells was constructed by integrating chromatin three-dimensional architecture data. Lung cancers possessed a similar mutation burden at tissue-specific enhancers and exons but with differences in their mutation signatures. Functionally relevant alterations were prioritized on the basis of the pathway-level integration of the effect of a mutation and the frequency of mutations on individual enhancers. The genes enriched for mutated enhancers converged on the regulation of key biological processes and pathways relevant to tumor biology. Recurrent mutations in individual enhancers also affected the expression of target genes, with potential relevance for patient prognosis. Together, these findings show that noncoding regulatory mutations have a potential relevance for cancer pathogenesis and can be exploited for patient classification. SIGNIFICANCE: Mapping enhancer-target gene regulatory interactions and analyzing enhancer mutations at the level of their target genes and pathways reveal convergence of recurrent enhancer mutations on biological processes involved in tumorigenesis and prognosis.

Detection and genetic characterization of domestic cat hepadnavirus in cats with cavitary effusions.

After the identification of the novel domestic cat hepadnavirus (DCH) in 2018, its potential pathogenetic role in feline hepatic diseases has been suggested. Following the detection of DCH in a cat's serum and peritoneal effusion, the aim of this study was to retrospectively investigate the presence of DCH in cats with and without cavitary effusions along with DCH presence in effusions. Stored serum and effusion samples from cats with and without effusions admitted to the Veterinary Teaching Hospital of Lodi (Italy) in 2020-2022 were included based on results of hematobiochemical parameters. Effusions were classified based on cytological and physicochemical findings. The likelihood of liver damage was estimated based on clinical and laboratory findings. Samples were tested for DCH presence by quantitative PCR (qPCR). Positive samples were subjected to whole genome sequencing and phylogenetic analysis. DCH was detected in both serum and peritoneal effusion samples of 2/72 (2.8%) enrolled cats, included in the group with effusions (2/33; 6.1%), with one cat showing inflammatory and the other non-inflammatory effusion. Both DCH-positive cats belonged to the group with a likelihood of liver damage (2/22, 9.1%). Phylogeny showed that the DCH sequences from this study clustered with the prototypic Australian strain but were not included in the clade with other Italian DCH sequences. Results suggest the circulation of different DCH variants in Italy and show the presence of DCH in effusion samples from DCH-positive cats, mirroring the presence of HBV in body fluids from HBV-infected humans. Further studies are still recommended to define the pathogenic role of DCH in cats.

Comparison of intraperitoneal and incisional lidocaine or ropivacaine irrigation for postoperative analgesia in dogs undergoing major abdominal surgeries.

This study compared the postoperative analgesic efficacy of intraperitoneal and incisional lidocaine versus ropivacaine in dogs undergoing major abdominal surgeries. Dogs randomly received intraperitoneal lidocaine irrigation (4 mg kg-1, diluted to 5 ml kg-1, L group), ropivacaine (4 mg kg-1, diluted to 5 ml kg-1, R group) or 0.9% saline (5 ml kg-1, C group). Prior to skin closure, dogs received incisional lidocaine 2 mg kg-1 (group L), incisional ropivacaine 2 mg kg-1 (group R) or incisional saline 0.2 ml kg-1 (group C). Pain was assessed at different time points up to 24 hours after extubation, using the Short Form-Glasgow Composite Measure Pain Scale and VAS Scale. In group C, postoperative pain scores were significantly higher than in groups L and R from T0.5 to T6 (p < 0.05). In R group, postoperative pain scores were significantly lower than in groups L and C from T12 to T24 (p < 0.05). Rescue analgesia was administered to 5/11 dogs in L group, 1/10 dogs in R group and 8/10 dogs in C group. Groups L and R experienced a significantly lower postoperative pain during the first 6 hours after extubation, compared with group C. Ropivacaine provided lower postoperative pain scores than lidocaine and saline up to 24 hours after extubation. According to the obtained results, ropivacaine seemed to provide better and longer lasting postoperative analgesia compared with lidocaine. Therefore, intraperitoneal and incisional administration of ropivacaine in dogs undergoing major abdominal surgeries is recommended.

Tissue fluidification promotes a cGAS-STING cytosolic DNA response in invasive breast cancer.

The process in which locally confined epithelial malignancies progressively evolve into invasive cancers is often promoted by unjamming, a phase transition from a solid-like to a liquid-like state, which occurs in various tissues. Whether this tissue-level mechanical transition impacts phenotypes during carcinoma progression remains unclear. Here we report that the large fluctuations in cell density that accompany unjamming result in repeated mechanical deformations of cells and nuclei. This triggers a cellular mechano-protective mechanism involving an increase in nuclear size and rigidity, heterochromatin redistribution and remodelling of the perinuclear actin architecture into actin rings. The chronic strains and stresses associated with unjamming together with the reduction of Lamin B1 levels eventually result in DNA damage and nuclear envelope ruptures, with the release of cytosolic DNA that activates a cGAS-STING (cyclic GMP-AMP synthase-signalling adaptor stimulator of interferon genes)-dependent cytosolic DNA response gene program. This mechanically driven transcriptional rewiring ultimately alters the cell state, with the emergence of malignant traits, including epithelial-to-mesenchymal plasticity phenotypes and chemoresistance in invasive breast carcinoma.

Surgical complications following sentinel lymph node biopsy guided by γ-probe and methylene blue in 113 tumour-bearing dogs.

Sentinel lymph node biopsy (SLNB) is an accepted veterinary surgical procedure given the impact of early detection of nodal metastases on staging of several canine malignancies. This study aims at reporting the incidence and risk factors for surgical complications of SLNB in tumour-bearing dogs. A total of 113 client-owned dogs that underwent tumour excision and SLNB guided by γ-probing and blue dye were retrospectively enrolled. Recorded variables included: signalment, location and number of extirpated lymphocenters and nodes, time for SLNB, histopathological status of excised nodes. Incidence of SLNB complications was calculated. They were classified as minor and major based on severity and required treatment, and as short-term (0-30 days) and long-term (31-90 days). Univariate analysis with generalized linear model with binomial error estimated the association between variables and incidence of SLNB complications. Significance was set at 5%. Median overall time for SLNB was 25 min. Surgeons excised one node in 38% of dogs and multiple nodes in 62% of cases, belonging to one (62%) or multiple (38%) lymphocenters. Metastases were detected in 45% of nodes. No intraoperative complications occurred. The overall incidence of postoperative complications of SLNB was 21,24%, the majority of which (91.67%) were minor. Only increasing dogs' weight was associated with an increased incidence of SLNB complications (p = .00976). Sentinel lymphadenectomy was associated with a relatively low incidence of complications, most of which were self-limiting. The low morbidity and previously reported impact on staging of SLNB justify its implementation to collect data for prognostic studies.

CHD8 suppression impacts on histone H3 lysine 36 trimethylation and alters RNA alternative splicing.

Disruptive mutations in the chromodomain helicase DNA-binding protein 8 gene (CHD8) have been recurrently associated with autism spectrum disorders (ASDs). Here we investigated how chromatin reacts to CHD8 suppression by analyzing a panel of histone modifications in induced pluripotent stem cell-derived neural progenitors. CHD8 suppression led to significant reduction (47.82%) in histone H3K36me3 peaks at gene bodies, particularly impacting on transcriptional elongation chromatin states. H3K36me3 reduction specifically affects highly expressed, CHD8-bound genes and correlates with altered alternative splicing patterns of 462 genes implicated in 'regulation of RNA splicing' and 'mRNA catabolic process'. Mass spectrometry analysis uncovered a novel interaction between CHD8 and the splicing regulator heterogeneous nuclear ribonucleoprotein L (hnRNPL), providing the first mechanistic insights to explain the CHD8 suppression-derived splicing phenotype, partly implicating SETD2, a H3K36me3 methyltransferase. In summary, our results point toward broad molecular consequences of CHD8 suppression, entailing altered histone deposition/maintenance and RNA processing regulation as important regulatory processes in ASD.

Sentinel Lymph Node Biopsy Is Feasible in Dogs with Scars from Prior Local Excision of Solid Malignancies.

Sentinel lymph node (SLN) biopsy is a well-established staging tool in canine oncology. This study aims to explore the feasibility of SLN biopsy in dogs with scars from prior excised solid malignancies that were referred for further tumor staging and/or adjuvant treatment options. Mapping was either performed using radiopharmaceutical, methylene blue, and/or near-infrared fluorescent (NIRF) imaging. Thirty-three dogs with 34 scars from prior excision of the mast cell tumor (MCT) (n = 29), soft tissue sarcoma (n = 2), oral melanoma (n = 1), subungual melanoma (n = 1), and mammary adenocarcinoma (n = 1) were retrospectively enrolled. Primary treatment consisted of curative intent/wide tumor excisions in 50.0% of dogs and marginal excision in the remaining 50.0%. The median time between tumor excision and SLN biopsy was 50 days (range 17-110 days). The procedure was successful in 31/34 scars, translating to a detection rate of 91.2%. The SLN did not correspond to the regional lymph node in 19/31 scars (61.3%). SLN metastases were histologically identified in 13/31 (41.9%) dogs, all of them affected by MCT. Based on our results, SLN biopsy using lymphoscintigraphy/methylene blue and/or NIRF is feasible in dogs presenting with scars from the prior surgical excision of solid tumors, and should be suggested for accurate nodal staging.

Quantitative Prediction of Drug Interactions Caused by Cytochrome P450 2B6 Inhibition or Induction.

BACKGROUND: Numerous drugs have the potential to be affected by cytochrome P450 (CYP) 2B6-mediated drug-drug interactions (DDIs). OBJECTIVES: In this work, we extend a static approach to the prediction of the extent of pharmacokinetics DDIs between substrates and inhibitors or inducers of CYP2B6. METHODS: This approach is based on the calculation of two parameters (the contribution ratio [CR], representing the fraction of dose of the substrate metabolized via this pathway and the inhibitory or inducing potency of the perpetrator [IR or IC, respectively]) calculated from the area under the concentration-time curve (AUC) ratios obtained in in-vivo DDI studies. RESULTS: Forty-eight studies involving 5 substrates, 11 inhibitors and 18 inducers of CYP2B6 (overall 15 inhibition and 33 induction studies) were divided into test and validation sets and considered for estimation of the parameters. The proposed approach demonstrated a fair accuracy for predicting the extent of DDI related to CYP2B6 inhibition and induction, all predictions related to the validation test (N = 18) being 50-200% of the observed ratios. CONCLUSIONS: This methodology can be used for proposing initial dose adaptations to be adopted, for example in clinical use or for designing DDI studies involving this enzyme.

Visualizing and Annotating Hi-C Data.

Epigenomics studies require the combined analysis and integration of multiple types of data and annotations to extract biologically relevant information. In this context, sophisticated data visualization techniques are fundamental to identify meaningful patterns in the data in relation to the genomic coordinates. Data visualization for Hi-C contact matrices is even more complex as each data point represents the interaction between two distant genomic loci and their three-dimensional positioning must be considered. In this chapter we illustrate how to obtain sophisticated plots showing Hi-C data along with annotations for other genomic features and epigenomics data. For the example code used in this chapter we rely on a Bioconductor package able to handle even high-resolution Hi-C datasets. The provided examples are explained in details and highly customizable, thus facilitating their extension and adoption by end users for other studies.

Outcome of rest with or without bandaging for treatment of carpal flexural contracture deformities in puppies: 47 puppies and 75 joints (2000-2018).

OBJECTIVE: To review outcome of dogs with carpal flexural contracture deformities treated with rest alone or with rest and bandaging. ANIMALS: 47 dogs (75 joints). PROCEDURES: Medical records of dogs with unilateral or bilateral carpal flexural contracture deformities were reviewed, and dogs were grouped according to deformity severity grade (graded on a scale from 1 to 3) at the time of diagnosis. Two treatment groups were compared: rest only and rest with a modified Robert-Jones bandage. All dogs were reevaluated weekly until recovery (ie, resolution of the deformity and lameness). RESULTS: All dogs responded to conservative management, with all dogs regaining full extension of the antebrachiocarpal joint and ambulating normally at the time of the final visit. Mean ± SD time from initial diagnosis to recovery (ie, resolution of the deformity and lameness) was 2.9 ± 2.2 weeks (median, 2 weeks; range, 1 to 9 weeks). For dogs with grade 1 or 2 severity, mean time to recovery did not differ significantly between treatment groups. For dogs with grade 3 severity, however, mean time to recovery was significantly shorter for dogs treated with rest and bandaging than for dogs treated with rest alone. CLINICAL RELEVANCE: Results suggested that conservative management (rest alone or rest and bandaging) was a successful treatment option for puppies with carpal flexural contracture deformity and that bandaging resulted in a shorter time to recovery for dogs that were severely affected.

A Quantitative Approach to the Prediction of Drug-Drug Interactions Mediated by Cytochrome P450 2C8 Inhibition.

BACKGROUND: Ohno and Colleagues proposed an approach for predicting drug-drug interactions (DDIs) mediated by cytochrome P450 (CYP) 3A4 based on the use of the ratio of the inhibited to non-inhibited area under the plasma concentration time curve (AUC) of substrates to estimate the fraction of the dose metabolized via CYP3A4 (contribution ratio, CR) and the in vivo inhibitory potency of a perpetrator (inhibition ratio, IR). This study evaluated the performance of this approach on DDIs mediated by CYP2C8 inhibitors. RESEARCH DESIGN AND METHODS: Initial estimates of CR and IR of CYP2C8 substrates and inhibitors were calculated for 33 DDI in vivo studies. The approach was externally validated with 17 additional studies. Bayesian orthogonal regression was used to refine the estimates of the parameters. Assessment of prediction success was conducted by plotting observed versus predicted AUC ratios. RESULTS: Final estimates of CRs and IRs were obtained for 19 CYP2C8 substrates and 23 inhibitors, respectively. The method demonstrated good predictive capacity, with only two values outside of the prespecified limits. CONCLUSIONS: The approach may help to adapt dose regimens for CYP2C8 substrates when given in combination with CYP2C8 inhibitors and to map the potential DDIs of new molecular entities.

Endoscopic Pilonidal Sinus Treatment: A Tertiary Care Academic Center Experience.

Background: Pilonidal disease (PD) represents one of the most common proctological diseases in young adults. Although several approaches to treating PD have been described, there is still a lack of agreement on which is the best. The aim of this study was to evaluate the long-term efficacy of endoscopic pilonidal sinus treatment (EPSiT) at a tertiary care academic center. Methods: Between June 2017 and January 2021, a total of 32 patients [12 women (37.5%) and 20 men (62.5%)] with a mean age of 29.22 ± 12.98 years were treated with EPSiT. Pre- and post-operative symptoms were assessed with a score of 0-5. Success was defined as the absence of any subjective symptoms, as well as by complete post-operative wound healing. Results: Most of the patients had a midline external opening (17/32; 53.1%), with a mean number of external openings of 2.41 (1-4) ± 1.04. The median post-operative pain score was 0, and the mean follow-up period was 22 (4-42) ± 11.49 months. The time to wound healing was reduced in patients with one opening (28.14 ± 4.06 days) compared to patients with two or more openings (33.64 ± 7.3 days) (p = 0.067). The mean operative time was longer in patients who subsequently had a recurrence (41.75 ± 6.24 vs. 34.18 ± 6.24 min; p = 0.031). The overall success rate was 87.5% (28/32), and the mean time to recurrence was 3.25 (2-5) ± 1.26 months. Conclusions: EPSiT represents a viable option for the treatment of PD. More evidence and a longer follow-up period are needed to validate the results.

Leveraging three-dimensional chromatin architecture for effective reconstruction of enhancer-target gene regulatory interactions.

A growing amount of evidence in literature suggests that germline sequence variants and somatic mutations in non-coding distal regulatory elements may be crucial for defining disease risk and prognostic stratification of patients, in genetic disorders as well as in cancer. Their functional interpretation is challenging because genome-wide enhancer-target gene (ETG) pairing is an open problem in genomics. The solutions proposed so far do not account for the hierarchy of structural domains which define chromatin three-dimensional (3D) architecture. Here we introduce a change of perspective based on the definition of multi-scale structural chromatin domains, integrated in a statistical framework to define ETG pairs. In this work (i) we develop a computational and statistical framework to reconstruct a comprehensive map of ETG pairs leveraging functional genomics data; (ii) we demonstrate that the incorporation of chromatin 3D architecture information improves ETG pairing accuracy and (iii) we use multiple experimental datasets to extensively benchmark our method against previous solutions for the genome-wide reconstruction of ETG pairs. This solution will facilitate the annotation and interpretation of sequence variants in distal non-coding regulatory elements. We expect this to be especially helpful in clinically oriented applications of whole genome sequencing in cancer and undiagnosed genetic diseases research.

T Cells Expressing Receptor Recombination/Revision Machinery Are Detected in the Tumor Microenvironment and Expanded in Genomically Over-unstable Models.

Tumors undergo dynamic immunoediting as part of a process that balances immunologic sensing of emerging neoantigens and evasion from immune responses. Tumor-infiltrating lymphocytes (TIL) comprise heterogeneous subsets of peripheral T cells characterized by diverse functional differentiation states and dependence on T-cell receptor (TCR) specificity gained through recombination events during their development. We hypothesized that within the tumor microenvironment (TME), an antigenic milieu and immunologic interface, tumor-infiltrating peripheral T cells could reexpress key elements of the TCR recombination machinery, namely, Rag1 and Rag2 recombinases and Tdt polymerase, as a potential mechanism involved in the revision of TCR specificity. Using two syngeneic invasive breast cancer transplantable models, 4T1 and TS/A, we observed that Rag1, Rag2, and Dntt in situ mRNA expression characterized rare tumor-infiltrating T cells. In situ expression of the transcripts was increased in coisogenic Mlh1-deficient tumors, characterized by genomic overinstability, and was also modulated by PD-1 immune-checkpoint blockade. Through immunolocalization and mRNA hybridization analyses, we detected the presence of rare TDT+RAG1/2+ cells populating primary tumors and draining lymph nodes in human invasive breast cancer. Analysis of harmonized single-cell RNA-sequencing data sets of human cancers identified a very small fraction of tumor-associated T cells, characterized by the expression of recombination/revision machinery transcripts, which on pseudotemporal ordering corresponded to differentiated effector T cells. We offer thought-provoking evidence of a TIL microniche marked by rare transcripts involved in TCR shaping.

Oncolytic adenovirus and gene therapy with EphA2-BiTE for the treatment of pediatric high-grade gliomas.

BACKGROUND: Pediatric high-grade gliomas (pHGGs) are among the most common and incurable malignant neoplasms of childhood. Despite aggressive, multimodal treatment, the outcome of children with high-grade gliomas has not significantly improved over the past decades, prompting the development of innovative approaches. METHODS: To develop an effective treatment, we aimed at improving the suboptimal antitumor efficacy of oncolytic adenoviruses (OAs) by testing the combination with a gene-therapy approach using a bispecific T-cell engager (BiTE) directed towards the erythropoietin-producing human hepatocellular carcinoma A2 receptor (EphA2), conveyed by a replication-incompetent adenoviral vector (EphA2 adenovirus (EAd)). The combinatorial approach was tested in vitro, in vivo and thoroughly characterized at a molecular level. RESULTS: After confirming the relevance of EphA2 as target in pHGGs, documenting a significant correlation with worse clinical outcome of the patients, we showed that the proposed strategy provides significant EphA2-BiTE amplification and enhanced tumor cell apoptosis, on coculture with T cells. Moreover, T-cell activation through an agonistic anti-CD28 antibody further increased the activation/proliferation profiles and functional response against infected tumor cells, inducing eradication of highly resistant, primary pHGG cells. The gene-expression analysis of tumor cells and T cells, after coculture, revealed the importance of both EphA2-BiTE and costimulation in the proposed system. These in vitro observations translated into significant tumor control in vivo, in both subcutaneous and a more challenging orthotopic model. CONCLUSIONS: The combination of OA and EphA2-BiTE gene therapy strongly enhances the antitumor activity of OA, inducing the eradication of highly resistant tumor cells, thus supporting the clinical translation of the approach.