Epigenetic variation in maize agronomical traits for breeding and trait improvement.

Epigenetics-mediated breeding (Epibreeding) involves engineering crop traits and stress responses through the targeted manipulation of key epigenetic features to enhance agricultural productivity. While conventional breeding methods raise concerns about reduced genetic diversity, epibreeding propels crop improvement through epigenetic variations that regulate gene expression, ultimately impacting crop yield. Epigenetic regulation in crops encompasses various modes, including histone modification, DNA modification, RNA modification, non-coding RNA, and chromatin remodeling. This review summarizes the epigenetic mechanisms underlying major agronomic traits in maize and identifies candidate epigenetic landmarks in the maize breeding process. We propose a valuable strategy for improving maize yield through epibreeding, combining CRISPR/Cas-based epigenome editing technology and Synthetic Epigenetics (SynEpi). Finally, we discuss the challenges and opportunities associated with maize trait improvement through epibreeding.

Identification of Glandular (Acinar)/Tubule Formation in Invasive Carcinoma of the Breast: A Study to Determine Concordance Using the World Health Organization Definition.

CONTEXT.­: The Nottingham Grading System (NGS) developed by Elston and Ellis is used to grade invasive breast cancer (IBC). Glandular (acinar)/tubule formation is a component of NGS. OBJECTIVE.­: To investigate the ability of pathologists to identify individual structures that should be classified as glandular (acinar)/tubule formation. DESIGN.­: A total of 58 hematoxylin-eosin photographic images of IBC with 1 structure circled were classified as tubules (41 cases) or nontubules (17 cases) by Professor Ellis. Images were sent as a PowerPoint (Microsoft) file to breast pathologists, who were provided with the World Health Organization definition of a tubule and asked to determine if a circled structure represented a tubule. RESULTS.­: Among 35 pathologists, the κ statistic for assessing agreement in evaluating the 58 images was 0.324 (95% CI, 0.314-0.335). The median concordance rate between a participating pathologist and Professor Ellis was 94.1% for evaluating 17 nontubule cases and 53.7% for 41 tubule cases. A total of 41% of the tubule cases were classified correctly by less than 50% of pathologists. Structures classified as tubules by Professor Ellis but often not recognized as tubules by pathologists included glands with complex architecture, mucinous carcinoma, and the "inverted tubule" pattern of micropapillary carcinoma. A total of 80% of participants reported that they did not have clarity on what represented a tubule. CONCLUSIONS.­: We identified structures that should be included as tubules but that were not readily identified by pathologists. Greater concordance for identification of tubules might be obtained by providing more detailed images and descriptions of the types of structures included as tubules.

EZH2 Protein Expression in Estrogen Receptor Positive Invasive Breast Cancer Treated With Neoadjuvant Endocrine Therapy: An Exploratory Study of Association With Tumor Response.

INTRODUCTION: Neoadjuvant endocrine therapy (NET) can be used to treat estrogen receptor positive (ER+) invasive breast cancer (IBC). Tumors with Ki67>10% after 2 to 4 weeks of NET are considered resistant to endocrine therapy. Enhancer of Zeste Homolog 2 (EZH2) is a targetable oncoprotein and overexpression in ER+ IBC has been linked to resistance to endocrine therapy. We examined whether EZH2 expression levels in ER+ IBC could be used to predict response to NET. MATERIALS AND METHODS: We retrospectively identified 46 patients with localized ER+ HER2/neu negative IBC treated with a minimum of 4 weeks of NET. We quantified EZH2 nuclear expression in pretherapy core biopsies using a score that included intensity and percent of cells staining. Ki67 was evaluated in both pretherapy core biopsies and posttherapy tumor resections and scored according to the guidelines of the International Ki67 Working Groups, with a global weighted score. Ki67≤10% after NET was considered endocrine responsive. Logistic regression analysis was performed to determine the association between EZH2 expression and response to NET. RESULTS: We found significant associations of tumor grade ( P =0.011), pretherapy Ki67 ( P =0.003), and EZH2 ( P <0.001), with response to NET. On logistic regression adjusted for tumor grade and pretherapy Ki67, increased EZH2 scores were associated with decreased odds of endocrine responsiveness, defined as posttreatment Ki67≤10% (odds ratio=0.976, 95% CI, 0.956 to 0.997; P =0.026). In addition, with EZH2 score in the model, associations of tumor grade and pretreatment Ki67 with posttreatment Ki67≤10% response to NET became not significant. CONCLUSIONS: Our results suggest that EZH2 might be a useful biomarker to predict response to NET.

Comparison of radiofrequency ablation and antiarrhythmic drug for the treatment of atrial fibrillation: A protocol for systematic review and meta-analysis.

BACKGROUND: Antiarrhythmic drugs and radiofrequency ablation are first-line treatments of atrial fibrillation, however, there exists a paucity of data regarding the potential benefit of different catheter ablation technologies versus antiarrhythmic drugs as an early rhythm strategy. We performed a protocol for systematic review and meta-analysis to compare the efficacy and safety of radiofrequency ablation and antiarrhythmic drugs for the treatment of atrial fibrillation. METHODS: This review protocol is registered in the International Prospective Register of Systematic Reviews (PROSPERO: CRD42022375095). Additionally, this review will adhere to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols 2015 Statement. A computerized literature search will be performed in the following electronic databases from their inceptions to November 2022: PubMed, EMBASE, MEDLINE, Cochrane Central Register of Controlled Clinical Trials, China Knowledge Resource Integrated Database, Wanfang Data Information, and Weipu Database for Chinese Technical Periodicals. The risk of bias will be assessed independently by 2 authors using the Cochrane tool of risk of bias. All statistical analyses will be conducted using the software program Review Manager version 5.3. RESULTS: The results of this systematic review will be published in a peer-reviewed journal. CONCLUSION: This study provides evidence of the comparison of radiofrequency ablation and antiarrhythmic drugs for the treatment of atrial fibrillation.

Clinical outcomes of resecting scarpa's ganglion during vestibular schwannoma surgery.

Vestibular schwannomas are slow-growing tumors arising from the Schwann cells of the vestibular nerve. Scarpa's ganglion, the vestibular nerve ganglion, is located within the internal auditory meatus. Surgical treatment of vestibular schwannomas carries the potential of resecting Scarpa's ganglion along with the tumor. No prior studies have evaluated outcomes based on the presence of Scarpa's ganglion within tumor specimens. The neurosurgery patient records were queried for patients who underwent surgical resection of vestibular schwannomas at the University of Missouri Healthcare between January 1, 2008 and December 31, 2018. Inclusion criteria consisted of minimum age of 18, imaging demonstrating an eighth nerve tumor, surgical resection thereof, and a final pathological diagnosis of WHO grade I schwannoma. Data were collected retrospectively. The histological slides of the tumors were reviewed, and the presence or absence of the ganglion was noted. Outcomes analyzed included postoperative dizziness, hearing, and facial nerve function. Fifty-two patients met inclusion criteria. Ten (19%) resected tumors contained portions of the ganglion. No difference in risk of resection of ganglion occurred based on the surgical approach (p = 0.2454). Mean follow-up duration was 24.6 months ± 26.2 standard deviation. No differences in postoperative hearing or dizziness (p = 0.8483 and p = 0.3190 respectively) were present if Scarpa's ganglion was resected. House-Brackmann classification of facial nerve function at last follow-up was similar (p = 0.9190). Resection of Scarpa's ganglion with vestibular schwannomas does not increase risk of post-operative dizziness, facial nerve weakness, or hearing loss.

The efficacy and safety of endoscopic ultrasound-guided liver biopsy versus percutaneous liver biopsy in patients with chronic liver disease: a retrospective single-center study.

BACKGROUND AND AIMS: There is limited literature on endoscopic ultrasound-guided liver biopsy (EUS-LB), a new method of obtaining liver biopsy (LB). METHODS: We conducted a retrospective study of the efficacy and safety of EUS-LB compared to percutaneous liver biopsy (PC-LB) in patients with chronic liver disease at our center between January 2018 and August 2019. RESULTS: Thirty patients underwent EUS-LB and 60 patients underwent PC-LB were identified (median follow-up post-LB was 8 days; interquartile range (IQR), 3-5 days). The median number of portal tracts was significantly higher in the PC-LB group (13 vs. 5; P < 0.0001). A histologic diagnosis was established in 93% of the EUS-LB group, compared to 100% in the PC-LB group (P = 0.841). Patients in EUS-LB group had significantly shorter hospital stay (median time of hospital stay was 3 vs. 4.2 h in the EUS-LB vs. PC-LB group, respectively; P = 0.004) and reported less pain compared to PC-LB group (median pain score was 0 vs. 3.5; P = 0.0009). EUS-LB were performed using a 19-gauge (n = 27) or 22-gauge (n = 3); there was a tendency towards higher number of portal tracts in the 22- vs. the 19-gauge needle group (6 vs. 5; P = 0.501). No patient in either group had significant adverse events such as bleeding or death. CONCLUSION: EUS-LB is safe and is associated with less pain, shorter hospital stay, and high diagnostic yield (93%) compared to PC-LB. Randomized trials are needed to standardize the utility of EUS-LB.

A rare case of plexiform fibromyxoma in stomach: FNA diagnosis with histological correlation and differential diagnoses.

Plexiform angiomyxoma (PF) is a rare benign mesenchymal neoplasm that arises in the antrum and pyloric region of the stomach. To the best of our knowledge, there are only two prior endoscopic ultrasound guided fine needle aspiration cytology examples have been reported. We report a case of PF which was diagnosed via EUS FNA and later confirmed on resection specimen. Differential diagnoses of this tumor are discussed. Although diagnosis of plexiform fibromyxoma on FNA specimen is difficult, a good FNA specimen with subsequent careful morphological evaluation and immunohistochemical staining work-up makes this task possible.

Human GH receptor-IGF-1 receptor interaction: implications for GH signaling.

GH signaling yields multiple anabolic and metabolic effects. GH binds the transmembrane GH receptor (GHR) to activate the intracellular GHR-associated tyrosine kinase, Janus kinase 2 (JAK2), and downstream signals, including signal transducer and activator of transcription 5 (STAT5) activation and IGF-1 gene expression. Some GH effects are partly mediated by GH-induced IGF-1 via IGF-1 receptor (IGF-1R), a tyrosine kinase receptor. We previously demonstrated in non-human cells that GH causes formation of a GHR-JAK2-IGF-1R complex and that presence of IGF-1R (even without IGF-1 binding) augments proximal GH signaling. In this study, we use human LNCaP prostate cancer cells as a model system to further study the IGF-1R's role in GH signaling. GH promoted JAK2 and GHR tyrosine phosphorylation and STAT5 activation in LNCaP cells. By coimmunoprecipitation and a new split luciferase complementation assay, we find that GH augments GHR/IGF-1R complex formation, which is inhibited by a Fab of an antagonistic anti-GHR monoclonal antibody. Short hairpin RNA-mediated IGF-1R silencing in LNCaP cells reduced GH-induced GHR, JAK2, and STAT5 phosphorylation. Similarly, a soluble IGF-1R extracellular domain fragment (sol IGF-1R) interacts with GHR in response to GH and blunts GH signaling. Sol IGF-1R also markedly inhibits GH-induced IGF-1 gene expression in both LNCaP cells and mouse primary osteoblast cells. On the basis of these and other findings, we propose a model in which IGF-1R augments GH signaling by allowing a putative IGF-1R-associated molecule that regulates GH signaling to access the activated GHR/JAK2 complex and envision sol IGF-1R as a dominant-negative inhibitor of this IGF-1R-mediated augmentation. Physiological implications of this new model are discussed.

Functional collaboration of insulin-like growth factor-1 receptor (IGF-1R), but not insulin receptor (IR), with acute GH signaling in mouse calvarial cells.

GH signals through the GH receptor (GHR), a cytokine receptor linked to Janus kinase 2 (JAK2). GH activates signal transducer and activator of transcription 5 (STAT5), causing expression of genes including IGF-I. IGF-I binds IGF-I receptor (IGF-IR), a heterotetrameric (α2-ß2) tyrosine kinase growth factor receptor similar to insulin receptor (IR). In addition to this GH -> GHR -> IGF-I -> IGF-IR pathway, GH induces a complex including GHR, JAK2, and IGF-IR and deletion of floxed IGF-1R in primary murine calvarial cells with Cre-recombinase-expressing adenovirus (Ad-Cre) desensitizes cells to GH for STAT5 activation and IGF-I mRNA accumulation. Diminished GH-induced STAT5 phosphorylation in Ad-Cre-treated cells is rescued by adenoviruses encoding either IGF-IR or IGF-IR lacking the ß-chain intracellular domain. Reasoning that IGF-IR's extracellular portion (α or extracellular ß) mediates functional interaction with GH signaling, we pursued reconstitution studies. Although structurally related to IGF-IR, IR expressed adenovirally did not rescue GH-induced STAT5 phosphorylation in Ad-Cre-treated cells. We thus created chimeras, swapping homologous IR extracellular regions into IGF-IR. IR and IGF-IR possess N-terminal L1, cysteine-rich (CR), and L2 α-chain domains. We created Ad-IGF-IR/IR-L1 and Ad-IGF-IR/IR-L1-CR-L2, in which L1 alone or L1, CR, and L2 of IR replace corresponding IGF-IR regions, respectively. Ad-IGF-IR/IR-L1, but not Ad-IGF-IR/IR-L1-CR-L2, rescued GH-induced STAT5 phosphorylation in Ad-Cre-treated cells. Additionally, medium containing a soluble IGF-IR (including only L1-CR-L2) dampened GH-induced STAT5 phosphorylation in calvarial cells and two other GH-responsive cell lines. Thus, an extracellular determinant(s), likely in CR-L2, specifically allows IGF-IR to collaborate with GHR and JAK2 for robust GH-induced acute STAT5 phosphorylation.

IGF-1R modulation of acute GH-induced STAT5 signaling: role of protein tyrosine phosphatase activity.

GH is a potent anabolic and metabolic factor that binds its cell surface receptor (GHR), activating the GHR-associated tyrosine kinase, Janus kinase 2, which phosphorylates and activates the latent transcription factor, signal transducer and activator of transcription 5 (STAT5). Some GH actions are mediated by the elaboration of IGF-1, which exerts effects by binding and activating the heterotetrameric tyrosine kinase growth factor receptor, IGF-1R. In addition to this GH-GHR-IGF-1-IGF-1R scheme, we have demonstrated in primary osteoblasts and in islet ß-cells that then deletion or silencing of IGF-1R results in diminished GH-induced STAT5 phosphorylation, suggesting that the presence of IGF-1R may facilitate GH signaling. In this study, we explore potential roles for protein tyrosine phosphatase activity in modulating GH-induced signaling, comparing conditions in which IGF-1R is present or diminished. We confirm that in mouse primary osteoblasts harboring loxP sites flanking the IGF-1R gene, infection with an adenovirus that expresses the Cre recombinase results in IGF-1R deletion and diminished acute GH-induced STAT5 phosphorylation. Furthermore, we present a new model of IGF-1R silencing, in which expression of short hairpin RNA directed at IGF-1R greatly reduces IGF-1R abundance in LNCaP human prostate cancer cells. In both models, treatment with a chemical inhibitor of protein tyrosine phosphatase-1B (PTP-1B), but not one of src homology region 2 domain-containing phosphotase-1 (SHP-1) and SHP-2, reverses the loss of GH-induced STAT5 phosphorylation in cells lacking IGF-1R but has no effect in cells with intact IGF-1R. Furthermore, expression of either a dominant-negative PTP-1B or the PTP-1B-interacting inhibitory protein, constitutive photomorphogenesis 1, also rescues acute GH-induced STAT5 signaling in IGF-1R-deficient cells but has no effect in IGF-1R replete cells. By expressing a substrate-trapping mutant PTP-1B, we demonstrate that tyrosine phosphorylated Janus kinase-2 is a PTP-1B substrate only in cells lacking IGF-1R. Collectively, our data suggest that IGF-1R positively regulates acute GH signaling by preventing access of PTP-1B activity to Janus kinase 2 and thereby preventing PTP-1B-mediated suppression of GH-induced STAT5 activation.

Deletion of IGF-I receptor (IGF-IR) in primary osteoblasts reduces GH-induced STAT5 signaling.

GH promotes longitudinal growth and regulates multiple cellular functions in humans and animals. GH signals by binding to GH receptor (GHR) to activate the tyrosine kinase, Janus kinase 2 (JAK2), and downstream pathways including signal transducer and activator of transcription 5 (STAT5), thereby regulating expression of genes including IGF-I. GH exerts effects both directly and via IGF-I, which signals by activating the IGF-I receptor (IGF-IR). IGF-IR is a cell surface receptor that contains intrinsic tyrosine kinase activity within its intracellular domain. In this study, we examined the potential role of IGF-IR in facilitating GH-induced signal transduction, using mouse primary calvarial osteoblasts with Lox-P sites flanking both IGF-IR alleles. These cells respond to both GH and IGF-I and in vitro infection with an adenovirus that drives expression of Cre recombinase (Ad-Cre) dramatically reduces IGF-IR abundance without affecting the abundance of GHR, JAK2, STAT5, or ERK. Notably, infection with Ad-Cre, but not a control adenovirus, markedly inhibited acute GH-induced STAT5 activity (more than doubling the ED(50) and reducing the maximum activity by nearly 50%), while sparing GH-induced ERK activity, and markedly inhibited GH-induced transactivation of a STAT5-dependent luciferase reporter. The effect of Ad-Cre on GH signaling was specific, as platelet-derived growth factor-induced signaling was unaffected by Ad-Cre-mediated reduction of IGF-IR. Ad-Cre-mediated inhibition of GH signaling was reversed by adenoviral reexpression of IGF-IR, but not by infection with an adenovirus that drives expression of a hemagglutination-tagged somatostatin receptor, which drives expression of the unrelated somatostatin receptor, and Ad-Cre infection of nonfloxed osteoblasts did not affect GH signaling. Notably, infection with an adenovirus encoding a C-terminally truncated IGF-IR that lacks the tyrosine kinase domain partially rescued both acute GH-induced STAT5 activity and GH-induced IGF-I gene expression in cells in which endogenous IGF-IR was reduced. These data, in concert with our earlier findings that GH induces a GHR-JAK2-IGF-IR complex, suggest a novel function for IGF-IR. In addition to its role as a key IGF-I signal transducer, this receptor may directly facilitate acute GH signaling. The implications of these findings are discussed.

Interruption of growth hormone signaling via SHC and ERK in 3T3-F442A preadipocytes upon knockdown of insulin receptor substrate-1.

Insulin receptor substrate-1 (IRS-1) is a docking protein tyrosine phosphorylated in response to insulin, IGF-1, GH, and other cytokines. IRS-1 has an N-terminal plekstrin homology domain (which facilitates membrane localization), a phosphotyrosine-binding domain [which associates with tyrosine-phosphorylated insulin receptor or IGF-1 receptor (IGF-1R)], and tyrosine residues that, when phosphorylated, bind signaling molecules. The role of IRS-1 in GH signaling is uncertain. We previously reported that IRS-1 and Janus kinase 2 associate independently of tyrosine phosphorylation via IRS-1's N terminus and that IRS-1 reconstitution greatly enhances GH-induced ERK, but not STAT5, activation. We now use GH-responsive 3T3-F442A preadipocytes to study the influence of IRS-1 on GH action. We stably transfected cells with vector only (Control) or a vector encoding IRS-1 short hairpin RNA [knockdown (KD)] and compared representative clones. Immunoblotting confirmed more than 80% knockdown of IRS-1 in KD cells. GH caused characteristic Janus kinase 2 and STAT5 activation in both Control and KD cells, but ERK activation was dramatically reduced in KD cells in GH time course and dose-response experiments. Notably, GH-induced Src homology collagen (SHC) activation and SHC-Grb2 association in KD cells were also markedly diminished compared with Control cells. Subcellular fractionation revealed that IRS-1 in Control cells was largely cytosolic, but the component isolated with plasma membranes was highly enriched in lipid raft membranes (LR). In KD cells, GH-induced ERK activation in the LR fraction was particularly diminished compared with Control cells. These data suggest that LR-enriched IRS-1 contributes substantially to GH-induced ERK activation in LR in 3T3-F442A fibroblasts. Furthermore, our results are consistent with IRS-1 residing upstream of SHC in the GH-induced ERK-signaling pathway.

Endotoxin-induced proteolytic reduction in hepatic growth hormone (GH) receptor: a novel mechanism for GH insensitivity.

GH is an important anabolic hormone. We previously demonstrated in cell culture that the cell surface GH receptor (GHR) is susceptible to inducible metalloproteolytic cleavage that yields the shed receptor extracellular domain (called GH binding protein) and renders the cells desensitized to subsequent GH stimulation. Sepsis and inflammatory states are associated with hepatic desensitization to GH, although disparate mechanisms have been postulated in various animal models. Using C3H/HeJ mice, we now demonstrate that administration of lipopolysaccharide (LPS) causes marked hepatic desensitization to GH, assessed by monitoring signal transducer and activator of transcription 5 tyrosine phosphorylation and nuclear accumulation and with a novel noninvasive bioluminescence imaging system to track in vivo hepatic GH signaling serially in individual mice. This endotoxin-induced desensitization was accompanied by marked loss of hepatic GHR, which was not explained by changes in GHR mRNA abundance. Furthermore, we observe that LPS causes GH-binding protein shedding of a hepatically expressed wild-type GHR but not a GHR with a mutation in the metalloprotease cleavage site. These data suggest that in this model system, LPS-induced desensitization to GH is associated with proteolytic GHR cleavage. These data are the first to demonstrate inducible in vivo GHR proteolysis and suggest this is a mechanism to regulate GH sensitivity and its anabolic effects during sepsis or inflammation.

Mode of growth hormone action in osteoblasts.

Growth hormone (GH) affects bone size and mass in part through stimulating insulin-like growth factor type 1 (IGF-1) production in liver and bone. Whether GH acts independent of IGF-1 in bone remains unclear. To define the mode of GH action in bone, we have used a Cre/loxP system in which the type 1 IGF-1 receptor (Igf1r) has been disrupted specifically in osteoblasts in vitro and in vivo. Calvarial osteoblasts from mice homozygous for the floxed IGF-1R allele (IGF-1R(flox/flox)) were infected with adenoviral vectors expressing Cre. Disruption of IGF-1R mRNA (>90%) was accompanied by near elimination of IGF-1R protein but retention of GHR protein. GH-induced STAT5 activation was consistently greater in osteoblasts with an intact IGF-1R. Osteoblasts lacking IGF-1R retained GH-induced ERK and Akt phosphorylation and GH-stimulated IGF-1 and IGFBP-3 mRNA expression. GH-induced osteoblast proliferation was abolished by Cre-mediated disruption of the IGF-1R or co-incubation of cells with an IGF-1-neutralizing antibody. By contrast, GH inhibited apoptosis in osteoblasts lacking the IGF-1R. To examine the effects of GH on osteoblasts in vivo, mice wild type for the IGF-1R treated with GH subcutaneously for 7 days showed a doubling in the number of osteoblasts lining trabecular bone, whereas osteoblast numbers in similarly treated mice lacking the IGF-1R in osteoblasts were not significantly affected. These results indicate that although direct IGF-1R-independent actions of GH on osteoblast apoptosis can be demonstrated in vitro, IGF-1R is required for anabolic effects of GH in osteoblasts in vivo.