Bilateral Implant-based Breast Reconstruction with Unilateral Radiotherapy: A Matched Cohort Study Comparing Nipple-sparing Mastectomy and Skin-sparing Mastectomy.

Background: Nipple-sparing mastectomy (NSM) preserves the natural nipple-areola complex and entire native breast skin, with the goal of better cosmetic outcomes in breast reconstruction. In bilateral TE/implant-based reconstruction requiring unilateral postmastectomy radiotherapy (PMRT), progressive radiation-induced fibrosis can lead to increasing nipple asymmetry with cosmetic dissatisfaction. Thus, PMRT may ultimately negate the intended positive cosmetic value of NSM compared with skin-sparing mastectomy (SSM). This study compares (1) surgical complications, (2) patient satisfaction, and (3) aesthetic outcomes between NSM versus SSM in bilateral implant-based reconstruction with unilateral PMRT. Methods: This retrospective matched cohort study included consecutive NSM patients with bilateral TE/implant breast reconstruction + unilateral PMRT matched 1:2 to SSM group. Patients completed PMRT and TE exchange to implants. Demographics, oncologic stage, comorbidities, and complications were collected. Patient satisfaction was evaluated by BREAST-Q. Aesthetic outcomes were assessed by blinded reviewers with a five-point Likert scale. Results: Among 58 patients who underwent bilateral TE/implant reconstruction with unilateral PMRT, 17 NSM patients were matched to 41 SSM patients by age, body mass index, and comorbidities. No significant differences existed in overall surgical complications and individual BREAST-Q questionnaire scores between cohorts. However, aesthetic outcomes scores were higher in SSM compared with NSM. Conclusions: Although NSM is generally associated with superior cosmetic outcomes compared with SSM, it has far less impact in bilateral implant-based breast reconstruction with unilateral PMRT due to the negative postradiotherapy effect on nipple symmetry.

TCF1-LEF1 co-expression identifies a multipotent progenitor cell (TH2-MPP) across human allergic diseases.

Repetitive exposure to antigen in chronic infection and cancer drives T cell exhaustion, limiting adaptive immunity. In contrast, aberrant, sustained T cell responses can persist over decades in human allergic disease. To understand these divergent outcomes, we employed bioinformatic, immunophenotyping and functional approaches with human diseased tissues, identifying an abundant population of type 2 helper T (TH2) cells with co-expression of TCF7 and LEF1, and features of chronic activation. These cells, which we termed TH2-multipotent progenitors (TH2-MPP) could self-renew and differentiate into cytokine-producing effector cells, regulatory T (Treg) cells and follicular helper T (TFH) cells. Single-cell T-cell-receptor lineage tracing confirmed lineage relationships between TH2-MPP, TH2 effectors, Treg cells and TFH cells. TH2-MPP persisted despite in vivo IL-4 receptor blockade, while thymic stromal lymphopoietin (TSLP) drove selective expansion of progenitor cells and rendered them insensitive to glucocorticoid-induced apoptosis in vitro. Together, our data identify TH2-MPP as an aberrant T cell population with the potential to sustain type 2 inflammation and support the paradigm that chronic T cell responses can be coordinated over time by progenitor cells.

Making the Case for Virtual Surgical Planning: Bilateral Sequential Fibula Flaps with Immediate Dental Implants for Maxillectomy.

SUMMARY: Oncologic maxillectomy defects requiring bony reconstruction are among the most challenging head and neck cases because of the complex three-dimensional geometry of the midface. Virtual surgical planning technology is advantageous in these cases because it provides superior positional precision and accuracy compared with traditional techniques and facilitates prosthodontic rehabilitation. Maxillary cancer recurrence after an initial fibula flap reconstruction presents a unique challenge. The authors report the first two cases of sequential fibula flaps after second or recurrent cancer of the maxilla. Virtual surgical planning facilitated resection with adequate tumor margins, optimized anatomic positioning of the fibula construct with three-dimensional printed plates, and enabled immediate functional dental implant placement.

Plastic Surgical Management of Hidradenitis Suppurativa.

LEARNING OBJECTIVES: After studying this article, the participant should be able to: 1. Describe the pathogenesis of hidradenitis suppurativa. 2. Discuss perioperative multimodal therapy of hidradenitis suppurativa, including medical optimization. 3. Determine an appropriate surgical plan with excision and reconstruction based on hidradenitis suppurativa severity, size, and anatomical location. SUMMARY: Successful treatment of hidradenitis suppurativa requires a multidisciplinary team approach and multimodal therapy.

Digital Transfer for Hand Reconstruction in Cleft Hand and Foot Differences.

BACKGROUND: Digital transfer for hand reconstruction in children with cleft hand and foot differences present unique challenges with anomalous anatomy and rare opportunities to dramatically improve function of one- or two-digit hands. METHODS: Medical records were reviewed for patients with cleft hand and foot treated at two pediatric institutions between 1996 and 2018. Hospital records, clinical photographs, radiographs, and alginate molds were available on all patients. Patient characteristics, indications for transfer, associated syndromes, donor and recipient anatomy, and complications were examined. RESULTS: Twenty digital transfers were identified in 16 patients. The mean age at time of transfer was 6 years (range: 3-18 years). Associated syndromes in this study included ectrodactyly ectodermal dysplasia clefting (EEC) syndrome and Goltz's syndrome. Recipient sites included the thumb (n = 17) and index ray (n = 3) in 10 hands with monodactyly, 6 hands with a two-digit ulnar syndactyly, and 3 hands with central deficiency and associated polydactyly or other anomalies. Donor sites included the great toe (n = 7), fifth toe (n = 9), great toe polydactyly (n = 2), thumb polydactyly (n = 1), and second toe (n = 1). All transfers survived. Revisions included tenolysis (n = 2), repeat fixation for nonunion or malunion (n = 2), and fusion for instability (n = 3). CONCLUSION: Digital transfer in cleft hand and foot patients is a functional endeavor. The transferred digits provide sensation, mobility, and stability for opposition. Technically challenging due to small structures and atypical anatomy, these rare cases represent unique opportunities to improve function and appearance in the pediatric hand. This is a therapeutic study and reflects level of evidence IV.

The effects of human following behaviours on decision making during aperture crossing scenarios.

BACKGROUND: Everyday locomotion often requires that we navigate crowded and cluttered environments. Individuals navigating through nonconfined space will require a deviation from the straight path in order to avoid apertures smaller than 1.4 times their shoulder width. When in a crowd, humans will follow the behaviours of those directly in front of them, making changes to their walking speed and direction heading based on the changes made by the people they are following. RESEARCH QUESTION: The current study aimed to discover whether the decisions made by young adults regarding the passability of an aperture would be influenced by the presence of a leader completing the same nonconfined aperture crossing task. METHODS: Participants (N = 24) walked in a virtual reality environment along a 6.5 m pathway towards a goal while avoiding two virtual poles which created an aperture (0.8-1.8 times the participants' shoulder widths). For some trials, a sex-matched avatar (shoulder width of 0.8, 1.0, or 1.2 times the participants' shoulder widths) completed the aperture crossing task, using its own body-scaled information, ahead of the participant. RESULTS: Participants walked through apertures smaller than 1.4 times their shoulder width (i.e. critical point) regardless of avatars' independent behaviours. Participants began to deviate 3.69 m from the aperture on all trials that required a deviation and approached their goal at a slower speed when the avatar was present. SIGNIFICANCE: This study demonstrates that during a nonconfined aperture crossing task, individuals are not influenced by human following behaviours and will continue to make decisions based on their own body-scaled information.

Epigenetic Control of Cdkn2a.Arf Protects Tumor-Infiltrating Lymphocytes from Metabolic Exhaustion.

T-cell exhaustion in cancer is linked to poor clinical outcomes, where evidence suggests T-cell metabolic changes precede functional exhaustion. Direct competition between tumor-infiltrating lymphocytes (TIL) and cancer cells for metabolic resources often renders T cells dysfunctional. Environmental stress produces epigenome remodeling events within TIL resulting from loss of the histone methyltransferase EZH2. Here, we report an epigenetic mechanism contributing to the development of metabolic exhaustion in TIL. A multiomics approach revealed a Cdkn2a.Arf-mediated, p53-independent mechanism by which EZH2 inhibition leads to mitochondrial dysfunction and the resultant exhaustion. Reprogramming T cells to express a gain-of-function EZH2 mutant resulted in an enhanced ability of T cells to inhibit tumor growth in vitro and in vivo. Our data suggest that manipulation of T-cell EZH2 within the context of cellular therapies may yield lymphocytes that are able to withstand harsh tumor metabolic environments and collateral pharmacologic insults. SIGNIFICANCE: These findings demonstrate that manipulation of T-cell EZH2 in cellular therapies may yield cellular products able to withstand solid tumor metabolic-deficient environments. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/21/4707/F1.large.jpg.

Proteogenomic analysis of melanoma brain metastases from distinct anatomical sites identifies pathways of metastatic progression.

Melanoma brain metastases (MBM) portend a grim prognosis and can occur in up to 40% of melanoma patients. Genomic characterization of brain metastases has been previously carried out to identify potential mutational drivers. However, to date a comprehensive multi-omics approach has yet to be used to analyze brain metastases. In this case report, we present an unbiased proteogenomics analyses of a patient's primary skin cancer and three brain metastases from distinct anatomic locations. We performed molecular profiling comprised of a targeted DNA panel and full transcriptome as well as proteomics using mass spectrometry. Phylogeny demonstrated that all MBMs shared a SMARCA4 mutation and deletion of 12q. Proteogenomics identified multiple pathways upregulated in the MBMs compared to the primary tumor. The protein, PIK3CG, was present in many of these pathways and had increased gene expression in metastatic melanoma tissue from the cancer genome atlas data. Proteomics demonstrated PIK3CG levels were significantly increased in all 3 MBMs and this finding was further validated by immunohistochemistry. In summary, this case report highlights the potential role of proteogenomics in identifying pathways involved in metastatic tumor progression. Furthermore, our multi-omics approach can be considered to aid in precision oncology efforts and provide avenues for therapeutic innovation.

Recent Advances in Implant-Based Breast Reconstruction.

LEARNING OBJECTIVES: After reading this article, the participant should be able to: 1. Understand the indications for implant-based breast reconstruction and the indications for nipple preservation compared to skin-sparing or skin-reducing patterns. 2. Understand the indications for direct-to-implant breast reconstruction versus tissue expander/implant breast reconstruction and the advantages and disadvantages of total, partial, or no muscle coverage. 3. Understand the role of acellular dermal matrix or mesh in reconstruction. 4. Learn the advantages and disadvantages of different types and styles of implants and develop a postoperative plan for care and pain management. SUMMARY: Breast reconstruction with implants has seen a decade of advances leading to more natural breast reconstructions and lower rates of complications.

Histone Modifications as Biomarkers for Immunotherapy.

Recent advances in immunotherapy have revolutionized the treatment of certain cancers. Some patients show a durable response to these immunotherapies, while others show little benefit or develop resistance. Identification of biomarkers to predict responsiveness will be helpful for informing treatment strategies; and would furthermore lead to the identification of molecular pathways dysregulated in nonresponding patients that could be targeted for therapeutic development. Pathways of epigenetic modification, such as histone posttranslational modifications (PTMs), have been shown to be dysregulated in certain cancer and immune cells. Histones are abundant cellular proteins readily assayed with high-throughput technologies, making them attractive targets as biomarkers. We explore promising advancements for using histone PTMs as immunotherapy responsiveness biomarkers in both cancer and immune cells, and provide a methodological workflow for assaying histone PTMs in relevant samples.

Coronary Artery Disease Association With Arterial Calcifications on Routine Hand Radiographs.

PURPOSE: Arterial calcifications in the lower extremity, chest, and cardiac vessels have been linked to coronary artery disease (CAD). However, the relation between arterial calcifications observed on routine hand and upper-extremity radiographs and atherosclerosis has not been determined. This study examined whether arterial calcifications found on hand radiographs are associated with CAD. METHODS: A record review from a single institution identified 345 patients with both hand radiographs and CAD screening with cardiac stress testing or coronary angiography. Patients with chronic kidney disease, end-stage renal disease, or incomplete hand films were excluded. We reviewed x-rays for findings of arterial calcifications. Cardiac testing results were used to establish a baseline diagnosis of CAD. We made group comparisons and employed multivariable logistic regression to evaluate the association between upper-extremity calcification and CAD. RESULTS: A total of 210 patients met inclusion criteria: 155 with CAD and 55 without it. Mean age was 72 years, body mass index was 28.8, and 54% were male. Patients had comorbidities of hypertension (91%), hyperlipidemia (87%), diabetes (39%), cerebrovascular accident (9%), and a history of tobacco use (53%). Of 155 CAD patients, 67 had arterial calcifications on hand radiographs (43%), compared with 6 of 55 without it (11%). In a multivariable model controlling for sex, hyperlipidemia, and diabetes, the presence of arterial calcifications on hand plain films indicated a 6.2-fold increased odds of CAD. CONCLUSIONS: The current data demonstrate that arterial calcifications on hand radiographs are independently associated with CAD. This may represent an opportunity to the treating physician as a point of referral or investigation for underlying or occult CAD. TYPE OF STUDY/LEVEL OF EVIDENCE: Prevalence III.

Impact of Unilateral versus Bilateral Breast Reconstruction on Procedure Choices and Outcomes.

BACKGROUND: In choosing between implant-based and autologous breast reconstruction, surgeons and patients must weigh relative risks and benefits. However, differences in outcomes across procedure types may vary between unilateral versus bilateral reconstructions. Procedure-related differences in complications and patient-reported outcomes were evaluated for unilateral and bilateral reconstruction. METHODS: Complications and patient-reported outcomes (BREAST-Q and Patient-Reported Outcomes measurement Information System surveys) were assessed at 2 years for patients undergoing autologous or implant-based reconstructions. Stratified regression models compared outcomes between autologous and implant-based reconstructions, separately for unilateral and bilateral cohorts. RESULTS: Among 2125 patients, 917 underwent unilateral (600 implant and 317 autologous) and 1208 underwent bilateral (994 implant and 214 autologous) reconstructions. Complication rates were significantly higher in the autologous versus implant-based group for both unilateral (overall: OR, 2.50, p < 0.001; major: OR, 2.19, p = 0.001) and bilateral (overall: OR, 2.13, p < 0.001; major: OR, 1.69, p = 0.014) cohorts. In unilateral reconstruction, the autologous group demonstrated significantly better patient-reported outcomes versus implant-based group in satisfaction with breast (mean difference, 9.85; p < 0.001), psychosocial well-being (mean difference, 4.84; p = 0.006), and sexual well-being (mean difference, 11.42; p < 0.001). In bilateral reconstruction, the autologous group demonstrated significantly higher patient-reported outcomes only for satisfaction with breast (mean difference, 5.13; p = 0.001). CONCLUSIONS: Although autologous reconstruction is associated with significantly better patient-reported outcomes compared to implant-based techniques in unilateral reconstruction, procedure choice has far less impact in bilateral reconstruction. Autologous procedures have higher complications rates in both unilateral and bilateral settings. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, III.

Loss of E-Cadherin Inhibits CD103 Antitumor Activity and Reduces Checkpoint Blockade Responsiveness in Melanoma.

Identifying controlling features of responsiveness to checkpoint blockade therapies is an urgent goal in oncology research. Our group and others have previously shown melanoma tumors resistant to checkpoint blockade display features of mesenchymal transition, including E-cadherin loss. Here, we present the first in vivo evidence that E-cadherin from tumor cells facilitate immune attack, using a B16F10 melanoma mouse model in which E-cadherin is exogenously expressed (B16.Ecad). We find, compared with vector control, B16.Ecad exhibits delayed tumor growth, reduced metastatic potential, and increased overall survival in vivo. Transplantation of B16.Ecad into Rag1-/- and CD103-/- mice abrogated the tumor growth delay. This indicates the anti-melanoma response against B16.Ecad is both immune and CD103+ mediated. Moreover, B16.Ecad showed increased responsiveness to combination immune checkpoint blockade (ICB) compared with vector control. This work establishes a rationale for ICB responses observed in high E-cadherin-expressing tumors and suggests therapeutic advancement through amplifying CD103+ immune cell subsets.Significance: These findings identify the mechanism behind checkpoint blockade resistance observed in melanoma that has undergone mesenchymal transition and suggest activation of CD103+ immune cells as a therapeutic strategy against other E-cadherin-expressing malignancies.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/6/1113/F1.large.jpg.

Quantitative microinjection using fluorescence calibration of streaming microdroplets on a superhydrophobic surface.

A simple and reproducible procedure was developed to measure the volume of liquid microinjected into cells. A calibration curve of droplet fluorescence intensity versus volume was constructed by injecting a fluorescent dextran solution through a 125-150 µm diameter micropipette into an oil-filled culture dish to create a spray of varied-sized droplets. The droplets retained a spherical shape because they were in an oil medium and they settled onto a glass surface coated with a superhydrophobic surface. Fluorescent micrographs of the droplets were obtained and analyzed with Image-J software to quantify the fluorescence intensity and radius of each spherical droplet to produce the calibration curve. Subsequently, Dut-145 human prostate carcinoma cells were microinjected with the same fluorescent dextran solution and fluorescent micrographs of the cells were obtained using the identical exposure conditions used to photograph the droplets. The measured fluorescence intensity of the microinjected cells was entered into the formula for the regression line that was fit to the calibration curve allowing determination of the volume of solution injected into each cell. Thus, a mixture consisting of known concentrations of a test material of test material (macromolecules, drugs, etc.) and a fluorescent dextran, volumetric, tracer can be used to quantify the relationship between the amount of a microinjected material and subsequent effects on cells.

Indicators of responsiveness to immune checkpoint inhibitors.

Modulation of the immune system can produce anti-tumor responses in various cancer types, including melanoma. Recently, immune checkpoint inhibitors (ICI), in single agent and combination regimens, have produced durable and long-lasting clinical responses in a subset of metastatic melanoma patients. These monoclonal antibodies, developed against CTLA-4 and PD-1, block immune-inhibitory receptors on activated T-cells, amplifying the immune response. However, even when using anti-CTLA-4 and anti-PD-1 in combination, approximately half of patients exhibit innate resistance and suffer from disease progression. Currently, it is impossible to predict therapeutic response. Here, we report the first proteomic and histone epigenetic analysis of patient metastatic melanoma tumors taken prior to checkpoint blockade, which revealed biological signatures that can stratify patients as responders or non-responders. Furthermore, our findings provide evidence of mesenchymal transition, a known mechanism of immune-escape, in non-responding melanoma tumors. We identified elevated histone H3 lysine (27) trimethylation (H3K27me3), decreased E-cadherin, and other protein features indicating a more mesenchymal phenotype in non-responding tumors. Our results have implications for checkpoint inhibitor therapy as patient specific responsiveness can be predicted through readily assayable proteins and histone epigenetic marks, and pathways activated in non-responders have been identified for therapeutic development to enhance responsiveness.

Reconstruction of quadriceps function with composite free tissue transfers following sarcoma resection.

BACKGROUND AND OBJECTIVES Wide margin resection of a soft tissue sarcoma (STS) may require extensive removal of quadriceps muscle with or without the knee extensor mechanism. The objective of this study is to present present the use of a combined functional muscle transfer and soft tissue coverage through the use of chimeric anterolateral thigh flaps. METHODS: Patients were retrospectively reviewed who underwent deep STS resection of the anterior compartment of the thigh with functional reconstruction of knee extension using a contralateral free anterolateral thigh (ALT) flap. RESULTS: Three patients with an average age 53.6 years (range: 33-66) were included. Average follow-up was 82 weeks (76-92 months). Full active extension was regained in 66% of patients with all patients regaining active extensor capabilities beyond 100°. The mean Knee Society Score was 83.3 (range; 76-92) and Musculoskeletal Tumor Society Score 21.6 (range; 19-21). Isometric knee extensor strength exceeded 4+/5 in all patients. CONCLUSION: Following soft tissue sarcoma resections of the lower extremity, chimeric anterolateral thigh flaps for restoration of knee extension can provide significant improvements in the potential for ambulation and regaining quadriceps function.

Skeletal Muscle PGC1α -1 Nucleosome Position and -260 nt DNA Methylation Determine Exercise Response and Prevent Ectopic Lipid Accumulation in Men.

Endurance exercise has been shown to improve lipid oxidation and increase mitochondrial content in skeletal muscle, two features that have shown dependence on increased expression of the peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1α). It is also hypothesized that exercise-related alterations in PGC1α expression occur through epigenetic regulation of nucleosome positioning in association with differential DNA methylation status within the PGC1α promoter. In this study, we show that when primary human myotubes from obese patients with type 2 diabetes are exposed to lipolytic stimulus (palmitate, forskolin, inomycin) in vitro, nucleosome occupancy surrounding the -260 nucleotide (nt) region, a known regulatory DNA methylation site, is reduced. This finding is reproduced in vivo in the vastus lateralis from 11 healthy males after a single, long endurance exercise bout in which participants expended 650 kcal. Additionally, we show a significant positive correlation between fold change of PGC1α messenger RNA expression and -1 nucleosome repositioning away from the -260 nt methylation site in skeletal muscle tissue following exercise. Finally, we found that when exercise participants are divided into high and low responders based on the -260 nt methylation status, the -1 nucleosome is repositioned away from the regulatory -260 nt methylation site in high responders, those exhibiting a significant decrease in -260 nt methylation, but not in low responders. Additionally, high but not low responders showed a significant decrease in intramyocellular lipid content after exercise. These findings suggest a potential target for epigenetic modification of the PGC1α promoter to stimulate the therapeutic effects of endurance exercise in skeletal muscle.

Surgeon-Based 3D Printing for Microvascular Bone Flaps.

Background Three-dimensional (3D) printing has developed as a revolutionary technology with the capacity to design accurate physical models in preoperative planning. We present our experience in surgeon-based design of 3D models, using home 3D software and printing technology for use as an adjunct in vascularized bone transfer. Methods Home 3D printing techniques were used in the design and execution of vascularized bone flap transfers to the upper extremity. Open source imaging software was used to convert preoperative computed tomography scans and create 3D models. These were printed in the surgeon's office as 3D models for the planned reconstruction. Vascularized bone flaps were designed intraoperatively based on the 3D printed models. Results Three-dimensional models were created for intraoperative use in vascularized bone flaps, including (1) medial femoral trochlea (MFT) flap for scaphoid avascular necrosis and nonunion, (2) MFT flap for lunate avascular necrosis and nonunion, (3) medial femoral condyle (MFC) flap for wrist arthrodesis, and (4) free fibula osteocutaneous flap for distal radius septic nonunion. Templates based on the 3D models allowed for the precise and rapid contouring of well-vascularized bone flaps in situ, prior to ligating the donor pedicle. Conclusions Surgeon-based 3D printing is a feasible, innovative technology that allows for the precise and rapid contouring of models that can be created in various configurations for pre- and intraoperative planning. The technology is easy to use, convenient, and highly economical as compared with traditional send-out manufacturing. Surgeon-based 3D printing is a useful adjunct in vascularized bone transfer. Level of Evidence Level IV.

Quercetin and Quercetin-Rich Red Onion Extract Alter Pgc-1α Promoter Methylation and Splice Variant Expression.

Pgc-1α and its various isoforms may play a role in determining skeletal muscle mitochondrial adaptations in response to diet. 8 wks of dietary supplementation with the flavonoid quercetin (Q) or red onion extract (ROE) in a high fat diet (HFD) ameliorates HFD-induced obesity and insulin resistance in C57BL/J mice while upregulating Pgc-1α and increasing skeletal muscle mitochondrial number and function. Here, mice were fed a low fat (LF), high fat (HF), high fat plus quercetin (HF + Q), or high fat plus red onion extract (HF + RO) diet for 9 wks and skeletal muscle Pgc-1α isoform expression and DNA methylation were determined. Quantification of various Pgc-1α isoforms, including isoforms Pgc-1α-a, Pgc-1α-b, Pgc-1α-c, Pgc-1α4, total NT-Pgc-1α, and FL-Pgc-1α, showed that only total NT-Pgc-1α expression was increased in LF, HF + Q, and HF + RO compared to HF. Furthermore, Q supplementation decreased Pgc-1α-a expression compared to LF and HF, and ROE decreased Pgc-1α-a expression compared to LF. FL-Pgc-1α was decreased in HF + Q and HF + RO compared to LF and HF. HF exhibited hypermethylation at the -260 nucleotide (nt) in the Pgc-1α promoter. Q and ROE prevented HFD-induced hypermethylation. -260 nt methylation levels were associated with NT-Pgc-1α expression only. Pgc-1α isoform expression may be epigenetically regulated by Q and ROE through DNA methylation.