Glioma progression is shaped by genetic evolution and microenvironment interactions.

The factors driving therapy resistance in diffuse glioma remain poorly understood. To identify treatment-associated cellular and genetic changes, we analyzed RNA and/or DNA sequencing data from the temporally separated tumor pairs of 304 adult patients with isocitrate dehydrogenase (IDH)-wild-type and IDH-mutant glioma. Tumors recurred in distinct manners that were dependent on IDH mutation status and attributable to changes in histological feature composition, somatic alterations, and microenvironment interactions. Hypermutation and acquired CDKN2A deletions were associated with an increase in proliferating neoplastic cells at recurrence in both glioma subtypes, reflecting active tumor growth. IDH-wild-type tumors were more invasive at recurrence, and their neoplastic cells exhibited increased expression of neuronal signaling programs that reflected a possible role for neuronal interactions in promoting glioma progression. Mesenchymal transition was associated with the presence of a myeloid cell state defined by specific ligand-receptor interactions with neoplastic cells. Collectively, these recurrence-associated phenotypes represent potential targets to alter disease progression.

DNA-translocation-independent role of INO80 remodeler in DNA damage repairs.

Chromatin remodelers utilize ATP hydrolysis to reposition histone octamers on DNA, facilitating transcription by promoting histone displacements. Although their actions on chromatin with damaged DNA are assumed to be similar, the precise mechanisms by which they modulate damaged nucleosomes and their specific roles in DNA damage response (DDR) remain unclear. INO80-C, a versatile chromatin remodeler, plays a crucial role in the efficient repair of various types of damage. In this study, we have demonstrated that both abasic sites and UV-irradiation damage abolish the DNA translocation activity of INO80-C. Additionally, we have identified compromised ATP hydrolysis within the Ino80 catalytic subunit as the primary cause of the inhibition of DNA translocation, while its binding to damaged nucleosomes remains unaffected. Moreover, we have uncovered a novel function of INO80-C that operates independently of its DNA translocation activity, namely, its facilitation of apurinic/apyrimidinic (AP) site cleavage by the AP-endonuclease 1 (APE1). Our findings provide valuable insights into the role of the INO80-C chromatin remodeler in DDR, thereby advancing our understanding of chromatin remodeling during DNA damage repairs.

Extracellular matrix contribution to disease progression and dysfunction in myopathy.

Myopathic processes affect skeletal muscle and heart. In the muscular dystrophies, which are a subset of myopathies, muscle cells are gradually replaced by fibrosis and fat, impairing muscle function as well as regeneration and repair. In addition to skeletal muscle, these genetic disorders often also affect the heart, where fibrofatty infiltration progressively accumulates in the myocardium, impairing heart function. Although considerable effort has focused on gene-corrective and gene-replacement approaches to stabilize myofibers and cardiomyocytes, the continual and ongoing deposition of extracellular matrix itself contributes to tissue and organ dysfunction. Transcriptomic and proteomic profiling, along with high-resolution imaging and biophysical measurements, have been applied to define extracellular matrix components and their role in contributing to cardiac and skeletal muscle weakness. More recently, decellularization methods have been adapted to an on-slide format to preserve the spatial geography of the extracellular matrix, allowing new insight into matrix remodeling and its direct role in suppressing regeneration in muscle. This review highlights recent literature with focus on the extracellular matrix and molecular mechanisms that contribute to muscle and heart fibrotic disorders. We will also compare how the myopathic matrix differs from healthy matrix, emphasizing how the pathological matrix contributes to disease.

Nanosome-Mediated Delivery Of Hdac Inhibitors and Oxygen Molecules for the Transcriptional Reactivation of Latent Hiv-Infected Cd4+ T Cells.

The treatment of human immunodeficiency virus (HIV) infection is notoriously difficult due to the ability of this virus to remain latent in the host's CD4+ T cells. Histone deacetylases (HDACs) interfere with DNA transcription in HIV-infected hosts, resulting in viral latency. Therefore, HDAC inhibitors can be used to activate viral transcription in latently infected cells, after which the virus can be eliminated through a shock-and-kill strategy. Here, a drug delivery system is developed to effectively deliver HDAC inhibitors to latent HIV-infected cells. Given that the efficacy of HDAC inhibitors is reduced under hypoxic conditions, oxygen-containing nanosomes are used as drug carriers. Oxygen-containing nanosomes can improve the efficiency of chemotherapy by delivering essential oxygen to cells. Additionally, their phospholipid bilayer structure makes them uniquely well-suited for drug delivery. In this study, a novel drug delivery system is developed by taking advantage of the oxygen carriers in these oxygen nanosomes, incorporating a multi-drug strategy consisting of HDAC inhibitors and PKA activators, and introducing CXCR4 binding peptides to specifically target CD4+ T cells. Oxygen nanosomes with enhanced targeting capability through the introduction of the CXCR4 binding peptide mitigate drug toxicity and slow down drug release. The observed changes in the expression of p24, a capsid protein of HIV, indirectly confirm that the proposed drug delivery system can effectively induce transcriptional reactivation of HIV in latent HIV-infected cells.

Enhanced homology-directed repair for highly efficient gene editing in hematopoietic stem/progenitor cells.

Lentivector gene therapy for X-linked chronic granulomatous disease (X-CGD) has proven to be a viable approach, but random vector integration and subnormal protein production from exogenous promoters in transduced cells remain concerning for long-term safety and efficacy. A previous genome editing-based approach using Streptococcus pyogenes Cas9 mRNA and an oligodeoxynucleotide donor to repair genetic mutations showed the capability to restore physiological protein expression but lacked sufficient efficiency in quiescent CD34+ hematopoietic cells for clinical translation. Here, we report that transient inhibition of p53-binding protein 1 (53BP1) significantly increased (2.3-fold) long-term homology-directed repair to achieve highly efficient (80% gp91phox+ cells compared with healthy donor control subjects) long-term correction of X-CGD CD34+ cells.

Can CT texture analysis parameters be used as imaging biomarkers for prediction of malignancy in canine splenic tumors?

Splenic hemangiosarcoma has morphological similarities to benign nodular hyperplasia. Computed tomography (CT) texture analysis can analyze the texture of images that the naive human eye cannot detect. Recently, there have been attempts to incorporate CT texture analysis with artificial intelligence in human medicine. This retrospective, analytical design study aimed to assess the feasibility of CT texture analysis in splenic masses and investigate predictive biomarkers of splenic hemangiosarcoma in dogs. Parameters for dogs with hemangiosarcoma and nodular hyperplasia were compared, and an independent parameter that could differentiate between them was selected. Discriminant analysis was performed to assess the ability to discriminate the two splenic masses and compare the relative importance of the parameters. A total of 23 dogs were sampled, including 16 splenic nodular hyperplasia and seven hemangiosarcoma. In each dog, total 38 radiomic parameters were extracted from first-, second-, and higher-order matrices. Thirteen parameters had significant differences between hemangiosarcoma and nodular hyperplasia. Skewness in the first-order matrix and GLRLM_LGRE and GLZLM_ZLNU in the second, higher-order matrix were determined as independent parameters. A discriminant equation consisting of skewness, GLZLM_LGZE, and GLZLM_ZLNU was derived, and the cross-validation verification result showed an accuracy of 95.7%. Skewness was the most influential parameter for the discrimination of the two masses. The study results supported using CT texture analysis to help differentiate hemangiosarcoma from nodular hyperplasia in dogs. This new diagnostic approach can be used for developing future machine learning-based texture analysis tools.

Computed tomographic findings may be useful for differentiating small intestinal adenocarcinomas, lymphomas, and spindle cell sarcomas in dogs.

An improved understanding of the CT characteristics for histologically confirmed primary intestinal tumors would be helpful for guiding prognosis and treatment plans in affected dogs. This retrospective, multi-center, analytical study aimed to evaluate the CT characteristics for the differentiation of adenocarcinoma, lymphoma, and spindle cell sarcoma (SCS) in dogs. Thirty-seven dogs who underwent contrast CT and histopathological examinations were included (adenocarcinomas, n = 11; lymphomas, n = 12; SCS, n = 14). Quantitative and qualitative CT parameters, including tumor morphology, contrast enhancement pattern, Hounsfield unit (HU) value, and presence or absence of intraabdominal lymphadenopathy, were evaluated for each included small intestine tumor CT case. Adenocarcinomas tended to show endophytic growth, intestinal obstruction, and a heterogeneous enhancement pattern. Lymphomas tended to show exophytic growth, contrast enhancement of the intestinal tumor mucosal layer, a homogeneous enhancement pattern, and the presence of lymphadenopathies in the abdominal cavity. SCSs tended to show lobulated growth, a large cystic portion within the tumor, a heterogeneous enhancement pattern, a large size with fat stranding sign, and lower HU values in postcontrast images. Cut-off values of the minimum diameter/fifth lumbar vertebral mid-body height (≥5.80; area under the curve [AUC] = 0.97, P < 0.001) and minimum HU value/HU value of the aorta (≤0.26; AUC = 0.96, P < 0.001) were derived to discriminate SCS from the two other tumor types. In conclusion, contrast CT characteristics may be useful in differentiating small intestinal adenocarcinomas, lymphomas, and SCSs in dogs.

CT features of malignant and benign oral tumors in 28 dogs.

An improved understanding of the computed tomographic (CT) features for malignant versus benign oral tumors would be helpful for guiding prognosis and treatment planning decisions in dogs. This retrospective, multi-center, observational study compared the CT features of malignant and benign tumors in 28 dogs with 31 oral masses. Malignant tumors were present in 20 dogs, including malignant melanoma (n = 14), squamous cell carcinoma (SCC, n = 4), adenocarcinoma (n = 1), and fibrosarcoma (n = 1). Eight dogs had benign tumors, including giant cell granuloma (n = 2), peripheral odontogenic fibroma (n = 2), acanthomatous ameloblastoma (n = 2), plasmacytoma (n = 1), and oncocytoma (n = 1). Common CT features of malignant tumors included heterogeneous enhancement, tumor invasion into the adjacent bone, tooth loss, and ipsilateral mandibular lymphadenopathy. Malignant tumors were significantly larger than benign tumors. Bone lysis was found in benign tumors (n = 4) such as acanthomatous ameloblastoma, giant cell granuloma, and plasmacytoma. The bone lysis was a well-defined geographic area regardless of malignancy and tumor type. In periosteal reactions, amorphous patterns were seen in both malignant (n = 2) and benign tumors (n = 2); the latter subgroup also showed solid patterns. Bone expansion (n = 2) was identified in malignant melanoma and acanthomatous ameloblastoma. Findings supported a diagnosis of possible malignancy for dogs with oral tumors having the following CT characteristics: large size, heterogeneous contrast enhancement pattern, bone lysis, tooth loss, and ipsilateral lymphadenopathy. However, there was a considerable overlap of CT findings among the different types of oral tumors and between benign and malignant tumors. Histological evaluation therefore remains necessary for definitive diagnosis.

Effect of Steamed Onion (ONIRO) Consumption on Body Fat and Metabolic Profiles in Overweight Subjects: A 12-Week Randomized, Double-Blind, Placebo-Controlled Clinical Trial.

Objective: The aim of the study is to investigate the effect of Jeju steamed onion (ONIRO) on body fat and metabolic profiles in overweight subjects.Methods: This randomized, double-blind, placebo-controlled clinical intervention was conducted and completed at one clinical research site. The subjects (n = 70) were randomly divided into placebo or test group and were instructed to take before each meal either the placebo or ONIRO capsule for 12 weeks. Anthropometric as well as serum and metabolic parameters, including triglycerides, cholesterol, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, leptin, adiponectin, C-peptide, and aspartate aminotransferase (AST) were measured at baseline and after 12 weeks. Body composition was also measured by dual-energy x-ray absorptiometry (DEXA) and computed tomography (CT). This trial is registered under the trial registration code clinicaltrials.gov: NCT03645382 (https://register.clinicaltrials.gov).Results: Compared to the placebo, ONIRO supplementation for significantly reduced the percentage of body fat and fat mass as measured by DEXA (p = 0.028 and 0.022, respectively) with no significant effects on lean body mass. CT analyses at the L1 level showed a significant decrease in the areas of whole fat, visceral fat, and subcutaneous fat (p = 0.009, p = 0.039, p = 0.020, respectively), while CT scan of L4 resulted in a significant reduction of whole fat area and subcutaneous area (p = 0.006 and p = 0.012, respectively). The levels of triglycerides (TG) and C-peptide were significantly lower after 12 weeks of ONIRO treatment.Conclusions: These findings suggest that ONIRO supplementation reduces total body fat, notably abdominal visceral fat, with positive changes of the clinically relevant metabolic parameters serum TG and C-peptide.

Ginsenoside Rc Is a New Selective UGT1A9 Inhibitor in Human Liver Microsomes and Recombinant Human UGT Isoforms.

Ginseng is known to have inhibitory effects on UGT1A9 activity. However, little is known about the inhibitory effects of ginsenosides, the major active compounds in ginseng, on UGT1A9 activity. In vitro investigation of UGT1A9 inhibition by ginsenosides was carried out using human liver microsomes (HLMs). Among 10 ginsenosides, ginsenoside Rc was the strongest inhibitor of UGT1A9-mediated mycophenolic acid glucuronidase activity. Further inhibition kinetic studies using HLMs suggested that ginsenoside Rc competitively and noncompetitively inhibited UGT1A9-mediated propofol and mycophenolic acid glucuronidation activities, with K i values of 2.83 and 3.31 µM, respectively. Next, to investigate whether the inhibitory effect of ginsenoside Rc is specific to the UGT1A9 isoform, we studied the inhibitory potency of ginsenoside Rc on nine human uridine diphospho-glucuronosyltransferase (UGT) activities using recombinant human UGT isoforms. Ginsenoside Rc exhibited a 12.9-fold selectivity (which was similar to niflumic acid at 12.5-fold) for UGT1A9 inhibition. Ginsenoside Rc at 50 µM also inhibited none of the other UGT isoform-specific activities above 12.0%, except for UGT1A9 (>91.5%) in HLMs, indicating that ginsenoside Rc might be used as a selective UGT1A9 inhibitor in reaction phenotyping studies of new chemical entities. Considering lower plasma concentrations (0.01 µM) of ginsenoside Rc in healthy subjects and no induction potential on UGT isoforms, ginsenoside Rc does not cause pharmacokinetic drug interactions with other coadministered drugs metabolized by UGT1A9. SIGNIFICANCE STATEMENT: Ginsenoside Rc selectively inhibited UGT1A9-mediated propofol and mycophenolic acid glucuronidation activities in human liver microsomes and recombinant uridine diphospho-glucuronosyltransferase (UGT) isoforms. It exhibited a 12.9-fold selectivity for UGT1A9 inhibition. Therefore, ginsenoside Rc might be used as a selective UGT1A9 inhibitor in reaction phenotyping studies of new chemical entities, such as niflumic acid.

Effects of multipurpose solutions on the adhesion of Acanthamoeba to rigid gas permeable contact lenses.

PURPOSE: To evaluate the effect of multipurpose contact lens care solutions (MPSs) on the adhesion of Acanthamoeba to rigid gas permeable (RGP) contact lenses. METHODS: Acanthamoeba castellanii (AC) trophozoites were inoculated onto untreated RGP contact lenses (FP, Extra, or Menicon Z), and numbers of trophozoites adhering to lenses were counted under a phase contrast microscope at 18 h post-inoculation (controls). Similarly, adhering trophozoites were counted at 6 h post-inoculation on each of the three RGP lens types with one of three MPSs (Boston Simplus, Menicare Plus, and O2 Care). Scanning electron microscopic examinations were performed to compare lens surfaces. RESULTS: Adhesion of AC trophozoites to untreated FP was greater than to untreated Extra or Menicon Z. Surfaces of Extra and Menicon Z lenses were waxier, smoother, and more homogeneous than those of FP lenses. After treatment with Boston Simplus, adhesion of AC trophozoites was significantly reduced for all lens types as compared with controls (p < 0.0001). Treatments with Menicare Plus or O2 Care reduced the number of adherent AC trophozoites significantly on FP lenses only as compared with controls (p < 0.0001). CONCLUSIONS: The adhesion rates of AC trophozoites to RGP lenses depended on lens surfaces. Boston Simplus reduced the adhesion rate of AC trophozoites more than Menicare Plus or O2 Care. Appropriate RGP lens and MPS selection could decrease the prevalence of Acanthamoeba keratitis.

ULTRASONOGRAPHIC ELASTOGRAPHY OF THE LIVER, SPLEEN, KIDNEYS, AND PROSTATE IN CLINICALLY NOR-MAL BEAGLE DOGS [corrected].

Standard ultrasonography is often insensitive for distinguishing normal vs. diseased states for canine abdominal organs. Ultrasonographic elastography is a new technique that is becoming increasingly available and may help to improve sensitivity. This study evaluated the feasibility, repeatability, and reproducibility of strain elastography of the liver, spleen, kidneys, and prostate in healthy dogs and described the elasticity of each organ using strain values and strain ratios. The reproducibility of strain elastography was excellent, and intraobserver repeatability was moderate to excellent. The strain value of each organ was not significantly different among dogs (liver = 143.38 ± 7.41, spleen = 141.04 ± 9.03, left renal cortex = 141.26 ± 7.50, right renal cortex = 145.80 ± 7.79, and prostate = 135.46 ± 5.80), except for the renal medulla (left = 51.19 ± 4.54 and right = 51.93 ± 5.09) (P < 0.05). The strain ratios for the liver, spleen, renal cortex, and prostate were similar with no significant difference (liver = 10.20 ± 1.47, spleen = 8.40 ± 1.53, left renal cortex = 9.62 ± 1.56, right renal cortex = 8.29 ± 1.63, and prostate = 8.20 ± 1.21), except for the renal medulla (left = 3.48 ± 0.68 and right = 2.95 ± 0.63) (P < 0.05). Our results indicated that strain elastography was feasible for estimating tissue stiffness in the canine liver, spleen, kidneys, and prostate. This study provides basic information for strain values and strain ratios for the liver, spleen, kidneys, and prostate in clinically normal dogs.

STRAIN ELASTOGRAPHY USING DOBUTAMINE-INDUCED CAROTID ARTERY PULSATION IN CANINE THYROID GLAND.

Thyroid disease is common in dogs and conventional ultrasonography is a standard diagnostic test for diagnosis and treatment planning. Strain elastography can provide additional information about tissue stiffness noninvasively after applying external or internal compression. However, natural carotid artery pulsations in the canine thyroid gland are too weak to maintain sufficient internal compression force. The objective of the present study was to describe the feasibility of strain elastography for evaluating the canine thyroid gland and the repeatability of dobutamine-induced carotid artery pulsation as an internal compression method. In seven healthy Beagle dogs, strain on each thyroid lobe was induced by external compression using the ultrasound probe and internal compression using carotid artery pulsation after dobutamine infusion. The thyroid appeared homogeneously green and the subcutaneous fat superficial to the thyroid lobe appeared blue. Strain values and strain ratios did not differ among dogs or between the left and right lobes. Interobserver repeatability was excellent for both compression methods. Intraobserver repeatability of the strain ratio measured using the carotid artery pulsation method (intraclass coefficient correlation = 0.933) was higher than that measured using the external compression method (0.760). Mean strain values of thyroid lobes for the external compression method (142.93 ± 6.67) differed from the internal method (147.31 ± 8.24; P < 0.05). Strain ratios between the two methods did not differ. Strain elastography was feasible for estimating thyroid stiffness in dogs. Carotid artery pulsation induced by dobutamine infusion can be used for canine thyroid strain elastography with excellent repeatability.

Measurements of the pulmonary vasculature on thoracic radiographs in healthy dogs compared to dogs with mitral regurgitation.

This study reassessed the previously reported radiographic method of comparing pulmonary vessels versus rib diameter for differentiating healthy dogs and dogs with mitral regurgitation. The width of the right cranial pulmonary artery and vein at the fourth rib level, right caudal pulmonary artery and vein at the ninth rib level, and the diameters of the fourth rib and ninth rib were measured in prospectively recruited healthy dogs (n = 40) and retrospectively recruited dogs with mitral regurgitation (n = 58). In healthy dogs, the pulmonary arteries and accompanying veins were similar in size. The cranial lobar vessels were smaller than the fourth rib. However, 67.5% of right caudal pulmonary artery diameters and 65% of vein diameters were larger than the ninth rib in healthy dogs. The right caudal pulmonary vein diameter in dogs with mitral regurgitation, particularly those within moderate and severe grades, was significantly larger than that in healthy dogs (P < 0.001). The comparative method used to detect enlargement of the right caudal pulmonary vein relative to the accompanying pulmonary artery had the highest sensitivity (80.2%) and specificity (82.5%) for predicting mitral regurgitation. A cut-off of 1.22 when applying the ninth rib criterion had better specificity (73%) than the most used value ≤ 1 (89.7% sensitivity and 63.8% specificity), although it has less sensitivity (73%). We recommend using the accompanying pulmonary artery and 1.22 × the diameter of the ninth rib as a radiographic criterion for assessing the size of the right caudal pulmonary vein and differentiating healthy dogs from those with mitral regurgitation.

Treatment of an Infrabony Defect of a Maxillary Canine Tooth Using Enamel Matrix Derivatives with Allogeneic Bone Augmentation in a Dog.

A 5-year-old Miniature Dachshund was presented having an infrabony pocket on the palatal aspect of the right maxillary canine tooth. The bony defect had worsened despite previous closed root planing and administration of a perioceutic agent. A second surgery using an allogeneic cancellous bone augmentation with an enamel matrix derivative was performed in the infrabony defect following open root planing. Eight months after the periodontal surgery, the osseous defect showed healing by improved periodontal probing measurements and increased radiopacity using dental radiography and computed tomography.

Structural and Biochemical Characterization of the Nucleosome Containing Variants H3.3 and H2A.Z.

Variant H3.3, along with H2A.Z, is notably enriched at promoter regions and is commonly associated with transcriptional activation. However, the specific molecular mechanisms through which H3.3 influences chromatin dynamics at transcription start sites, and its role in gene regulation, remain elusive. Using a combination of biochemistry and cryo-electron microscopy (cryo-EM), we show that the inclusion of H3.3 alone does not induce discernible changes in nucleosome DNA dynamics. Conversely, the presence of both H3.3 and H2A.Z enhances DNA's flexibility similarly to H2A.Z alone. Interestingly, our findings suggest that the presence of H3.3 in the H2A.Z nucleosome provides slightly increased protection to DNA at internal sites within the nucleosome. These results imply that while H2A.Z at active promoters promotes the formation of more accessible nucleosomes with increased DNA accessibility to facilitate transcription, the simultaneous presence of H3.3 offers an additional mechanism to fine-tune nucleosome accessibility and the chromatin environment.

A comparative analysis of canine pancreatic lipase tests for diagnosing pancreatitis in dogs.

IMPORTANCE: Early diagnosis of canine pancreatitis is challenging due to non-specific clinical signs. Currently, abdominal ultrasonography and measurement of canine pancreatic lipase (cPL) have been employed for the diagnosis of pancreatitis. OBJECTIVE: Many qualitative and quantitative commercial cPL tests have been developed and used in veterinary clinics. This study aimed to compare three different methodologies SNAP cPL, Spec cPL, and Vcheck cPL tests to assess the concordance of these assays. METHODS: Fifty serum samples were collected from 36 dogs with or without pancreatitis and subjected to SNAP cPL, Spec cPL, and Vcheck cPL tests. Agreement and correlation coefficients were calculated between the test results, and correlations were determined during the management of the patients. RESULTS: The results of the three cPL assays were strongly correlated in 47/50 serum samples (94%). Cohen's kappa analysis between the Spec cPL and Vcheck cPL showed near perfect agreement (κ = 0.960, p < 0.001), SNAP cPL and Vcheck cPL (κ = 0.920, p < 0.001), and Spec cPL and SNAP cPL (κ = 0.880, p < 0.001). The correlation coefficients (r) between data from Spec cPL and Vcheck cPL tests was calculated by Spearman's correlation test (r = 0.958, p < 0.001). Furthermore, the patterns of change in serum cPL concentrations determined using Spec cPL and Vcheck cPL were significantly consistent during the monitoring period in 11 patients. CONCLUSIONS AND RELEVANCE: Our data illustrated that Spec cPL and Vcheck cPL tests are compatible for clinical use in the diagnosis and monitoring of canine pancreatitis.

CHANGE-seq-BE enables simultaneously sensitive and unbiased in vitro profiling of base editor genome-wide activity.

Base editors ( BE ) enable programmable conversion of nucleotides in genomic DNA without double-stranded breaks and have substantial promise to become new transformative genome editing medicines. Sensitive and unbiased detection of base editor off-target effects is important for identifying safety risks unique to base editors and translation to human therapeutics, as well as accurate use in life sciences research. However, current methods for understanding the global activities of base editors have limitations in terms of sensitivity or bias. Here we present CHANGE-seq-BE, a novel method to directly assess the off-target profile of base editors that is simultaneously sensitive and unbiased. CHANGE-seq-BE is based on the principle of selective sequencing of adenine base editor modified genomic DNA in vitro , and provides an accessible, rapid, and comprehensive method for identifying genome-wide off-target mutations of base editors.

The extracellular matrix differentially directs myoblast motility and differentiation in distinct forms of muscular dystrophy: Dystrophic matrices alter myoblast motility.

Extracellular matrix (ECM) pathologic remodeling underlies many disorders, including muscular dystrophy. Tissue decellularization removes cellular components while leaving behind ECM components. We generated "on-slide" decellularized tissue slices from genetically distinct dystrophic mouse models. The ECM of dystrophin- and sarcoglycan-deficient muscles had marked thrombospondin 4 deposition, while dysferlin-deficient muscle had excess decorin. Annexins A2 and A6 were present on all dystrophic decellularized ECMs, but annexin matrix deposition was excessive in dysferlin-deficient muscular dystrophy. Muscle-directed viral expression of annexin A6 resulted in annexin A6 in the ECM. C2C12 myoblasts seeded onto decellularized matrices displayed differential myoblast mobility and fusion. Dystrophin-deficient decellularized matrices inhibited myoblast mobility, while dysferlin-deficient decellularized matrices enhanced myoblast movement and differentiation. Myoblasts treated with recombinant annexin A6 increased mobility and fusion like that seen on dysferlin-deficient decellularized matrix and demonstrated upregulation of ECM and muscle cell differentiation genes. These findings demonstrate specific fibrotic signatures elicit effects on myoblast activity.

The super-healing MRL strain promotes muscle growth in muscular dystrophy through a regenerative extracellular matrix.

The Murphy Roths Large (MRL) mouse strain has "super-healing" properties that enhance recovery from injury. In mice, the DBA/2J strain intensifies many aspects of muscular dystrophy, so we evaluated the ability of the MRL strain to suppress muscular dystrophy in the Sgcg-null mouse model of limb girdle muscular dystrophy. A comparative analysis of Sgcg-null mice in the DBA/2J versus MRL strains showed greater myofiber regeneration, with reduced structural degradation of muscle in the MRL strain. Transcriptomic profiling of dystrophic muscle indicated strain-dependent expression of extracellular matrix (ECM) and TGF-ß signaling genes. To investigate the MRL ECM, cellular components were removed from dystrophic muscle sections to generate decellularized myoscaffolds. Decellularized myoscaffolds from dystrophic mice in the protective MRL strain had significantly less deposition of collagen and matrix-bound TGF-ß1 and TGF-ß3 throughout the matrix. Dystrophic myoscaffolds from the MRL background, but not the DBA/2J background, were enriched in myokines like IGF-1 and IL-6. C2C12 myoblasts seeded onto decellularized matrices from Sgcg-/- MRL and Sgcg-/- DBA/2J muscles showed the MRL background induced greater myoblast differentiation compared with dystrophic DBA/2J myoscaffolds. Thus, the MRL background imparts its effect through a highly regenerative ECM, which is active even in muscular dystrophy.