Neutrophil and natural killer cell imbalances prevent muscle stem cell-mediated regeneration following murine volumetric muscle loss.

Volumetric muscle loss (VML) overwhelms the innate regenerative capacity of mammalian skeletal muscle (SkM), leading to numerous disabilities and reduced quality of life. Immune cells are critical responders to muscle injury and guide tissue resident stem cell­ and progenitor-mediated myogenic repair. However, how immune cell infiltration and intercellular communication networks with muscle stem cells are altered following VML and drive pathological outcomes remains underexplored. Herein, we contrast the cellular and molecular mechanisms of VML injuries that result in the fibrotic degeneration or regeneration of SkM. Following degenerative VML injuries, we observed the heightened infiltration of natural killer (NK) cells as well as the persistence of neutrophils beyond 2 wk postinjury. Functional validation of NK cells revealed an antagonistic role in neutrophil accumulation in part via inducing apoptosis and CCR1-mediated chemotaxis. The persistent infiltration of neutrophils in degenerative VML injuries was found to contribute to impairments in muscle stem cell regenerative function, which was also attenuated by transforming growth factor beta 1 (TGFß1). Blocking TGFß signaling reduced neutrophil accumulation and fibrosis and improved muscle-specific force. Collectively, these results enhance our understanding of immune cell­stem cell cross talk that drives regenerative dysfunction and provide further insight into possible avenues for fibrotic therapy exploration.

Discordance between immunofluorescence and immunohistochemistry C4d staining and outcomes following heart transplantation.

BACKGROUND: Capillary deposition of C4d is an important marker of antibody-mediated rejection (AMR) following heart transplantation (HT). There are two immunopathologic assay methods for detecting C4d: frozen-tissue immunofluorescence (IF) and paraffin immunohistochemistry (IHC). The clinical significance of discrepancy between the results of IF and IHC has not been understood. METHODS AND RESULTS: We reviewed 2187 biopsies from 142 HT recipients who had biopsies with assessment of both IF and IHC staining. Among them, 103 (73%) patients had negative IF and IHC C4d staining (Negative Group) and 32 (23%) patients had positive IF but negative IHC staining (Discordant Group). At the time of positive biopsy, 6 (19%) Discordant patients had graft dysfunction, compared to 5 (5%) Negative patients (p = .022). Cumulative incidence of cellular rejection at 1 year was comparable (31% vs. 29%, p = .46); however, cumulative incidence of AMR was significantly higher in the Discordant group (21% vs. 4%, p = .004). Overall 1-year survival was comparable (90% vs. 96%, p = .24); however, freedom from heart failure (HF) was significantly lower in the Discordant group (70% vs. 96%, p < .001). CONCLUSION: The Discordant group showed higher rates of graft dysfunction, AMR and HF admission than the Negative group.

A model for continued LVAD patient success: A 4-year readmission-free HeartMate 3 postoperative course.

Unplanned readmissions frequently occur following the implantation of a durable left ventricular assist device (LVAD) due to complications such as gastrointestinal bleeding and driveline infection. There is a paucity of literature describing the incidence of unplanned readmission in patients with a HeartMate 3 (HM3) Left Ventricular Assist System. In this report, we present the successful outcome of a patient with an HM3 LVAD who has experienced no unplanned readmissions in the 4-year post-implant phase. To our knowledge, this is the longest readmission-free case after HM3 implantation. A successful patient outcome was enabled by the use of the modular HM3 device, the postoperative prescription of beta-blockers and omega-3, the presence of strong social support, and open communication between the patient's caregivers and the LVAD team. Reducing the instance of unplanned readmission confers clinical benefits to the patient, as well as reducing the cost burden on the patient and the healthcare system.

Real-Time Production of PGA, PGV, Intensity, and Sa Shakemaps Using Dense MEMS-Based Sensors in Taiwan.

Using low-cost sensors to build a seismic network for earthquake early warning (EEW) and to generate shakemaps is a cost-effective way in the field of seismology. National Taiwan University (NTU) network employing 748 P-Alert sensors based on micro-electro-mechanical systems (MEMS) technology is operational for almost the last 10 years. This instrumentation is capable of recording the strong ground motions of up to ± 2g and is dense enough to record the near-field ground motion. It has proven effective in generating EEW warnings and delivering real-time shakemaps to the concerned disaster relief agencies to mitigate the earthquake-affected regions. Before 2020, this instrumentation was used to plot peak ground acceleration (PGA) shakemaps only; however, recently it has been upgraded to generate the peak ground velocity (PGV), Central Weather Bureau (CWB) Intensity scale, and spectral acceleration (Sa) shakemaps at different periods as value-added products. After upgradation, the performance of the network was observed using the latest recorded earthquakes in the country. The experimental results in the present work demonstrate that the new parameters shakemaps added in the current work provide promising outputs, and are comparable with the shakemaps given by the official agency CWB. These shakemaps are helpful to delineate the earthquake-hit regions which in turn is required to assist the needy well in time to mitigate the seismic risk.

MAP7 Regulates Axon Collateral Branch Development in Dorsal Root Ganglion Neurons.

Collateral branches from axons are key components of functional neural circuits that allow neurons to connect with multiple synaptic targets. Like axon growth and guidance, formation of collateral branches depends on the regulation of microtubules, but how such regulation is coordinated to ensure proper circuit development is not known. Based on microarray analysis, we have identified a role for microtubule-associated protein 7 (MAP7) during collateral branch development of dorsal root ganglion (DRG) sensory neurons. We show that MAP7 is expressed at the onset of collateral branch formation. Perturbation of its expression by overexpression or shRNA knockdown alters axon branching in cultured DRG neurons. Localization and time-lapse imaging analysis reveals that MAP7 is enriched at branch points and colocalizes with stable microtubules, but enters the new branch with a delay, suggesting a role in branch maturation. We have also investigated a spontaneous mutant mouse that expresses a truncated MAP7 and found a gain-of-function phenotype both in vitro and in vivo Further domain analysis suggests that the amino half of MAP7 is responsible for branch formation, suggesting a mechanism that is independent of its known interaction with kinesin. Moreover, this mouse exhibits increased pain sensitivity, a phenotype that is consistent with increased collateral branch formation. Therefore, our study not only uncovers the first neuronal function of MAP7, but also demonstrates the importance of proper microtubule regulation in neural circuit development. Furthermore, our data provide new insights into microtubule regulation during axonal morphogenesis and may shed light on MAP7 function in neurological disorders.SIGNIFICANCE STATEMENT Neurons communicate with multiple targets by forming axonal branches. In search of intrinsic factors that control collateral branch development, we identified a role for microtubule-associated protein 7 (MAP7) in dorsal root ganglion sensory neurons. We show that MAP7 expression is developmentally regulated and perturbation of this expression alters branch formation. Cell biological analysis indicates that MAP7 promotes branch maturation. Analysis of a spontaneous mouse mutant suggests a molecular mechanism for branch regulation and the potential influence of collateral branches on pain sensitivity. Our studies thus establish the first neuronal function of MAP7 and demonstrate its role in branch morphogenesis and neural circuit function. These findings may help in our understanding of the contribution of MAP7 to neurological disorders and nerve regeneration.

Identification of the murine H-2D(b) and human HLA-A*0201 MHC class I-restricted HPV6 E7-specific cytotoxic T lymphocyte epitopes.

Recurrent respiratory papillomatosis is caused by human papillomavirus (HPV) infection, most commonly types 6 (HPV-6) and 11 (HPV-11). Due to failed host immune responses, HPV is unable to be cleared from the host, resulting in recurrent growth of HPV-related lesions that can obstruct the lumen of the airway within the upper aerodigestive tract. In our murine model, the HPV-6b and HPV-11 E7 antigens are not innately immunogenic. In order to enhance the host immune responses against the HPV E7 antigen, we linked calreticulin (CRT) to HPV-6b E7 and found that vaccinating C57BL/6 mice with the HPV-6b CRT/E7 DNA vaccine is able to induce a CD8+ T cell response that recognizes an H-2D(b)-restricted E7aa21-29 epitope. Additionally, vaccination of HLA-A*0201 transgenic mice with HPV-6b CRT/E7 DNA generated a CD8+ T cell response against the E7aa82-90 epitope that was not observed in the wild-type C57BL/6 mice, indicating this T cell response is restricted to HLA-A*0201. In vivo cytotoxic T cell killing assays demonstrated that the vaccine-induced CD8+ T cells are able to efficiently kill target cells. Interestingly, the H-2D(b)-restricted E7aa21-29 sequence and the HLA-A*0201-restricted E7aa82-90 sequence are conserved between HPV-6b and HPV-11 and may represent shared immunogenic epitopes. The identification of the HPV-6b/HPV-11 CD8+ T cell epitopes facilitates the evaluation of various immunomodulatory strategies in preclinical models. More importantly, the identified HLA-A*0201-restricted T cell epitope may serve as a peptide vaccination strategy, as well as facilitate the monitoring of vaccine-induced HPV-specific immunologic responses in future human clinical trials.

Combination of proteasome and HDAC inhibitor enhances HPV16 E7-specific CD8+ T cell immune response and antitumor effects in a preclinical cervical cancer model.

BACKGROUND: Bortezomib, a proteasome inhibitor and suberoylanilide hydroxamic acid (SAHA, also known as Vorinostat), a histone deacetylase inhibitor, have been recognized as potent chemotherapeutic drugs. Bortezomib and SAHA are FDA-approved for the treatment of cutaneous T cell lymphoma and multiple myeloma/mantle cell lymphoma, respectively. Furthermore, the combination of the bortezomib and SAHA has been tested in a variety of preclinical models and in clinical trials and may be ideal for the treatment of cancer. However, it remains unclear how this treatment strategy affects the host immune response against tumors. RESULTS: Here, we used a well-defined E6/E7-expressing tumor model to examine how the immune system can be motivated to act against tumor cells expressing tumor antigens. We demonstrate that the combination of bortezomib and SAHA elicits potent antitumor effects in TC-1 tumor-bearing mice. Additionally, we are the first to show that treatment with bortezomib and SAHA leads to tumor-specific immunity by rendering tumor cells more susceptible to killing by antigen-specific CD8+ T cells than treatment with either drug alone. CONCLUSIONS: The current study serves an important foundation for the future clinical application of both drugs for the treatment of cervical cancer.

Estimation of cardiac output and pulmonary vascular resistance by contrast echocardiography transit time measurement: a prospective pilot study.

BACKGROUND: Studies with other imaging modalities have demonstrated a relationship between contrast transit and cardiac output (CO) and pulmonary vascular resistance (PVR). We tested the hypothesis that the transit time during contrast echocardiography could accurately estimate both CO and PVR compared to right heart catheterization (RHC). METHODS: 27 patients scheduled for RHC had 2D-echocardiogram immediately prior to RHC. 3 ml of DEFINITY contrast followed by a 10 ml saline flush was injected, and a multi-cycle echo clip was acquired from the beginning of injection to opacification of the left ventricle. 2D-echo based calculations of CO and PVR along with the DEFINITY-based transit time calculations were subsequently correlated with the RHC-determined CO and PVR. RESULTS: The transit time from full opacification of the right ventricle to full opacification of the left ventricle inversely correlated with CO (r=-0.61, p<0.001). The transit time from peak opacification of the right ventricle to first appearance in the left ventricle moderately correlated with PVR (r=0.46, p<0.01). Previously described echocardiographic methods for the determination of CO (Huntsman method) and PVR (Abbas and Haddad methods) did not correlate with RHC-determined values (p = 0.20 for CO, p = 0.18 and p = 0.22 for PVR, respectively). The contrast transit time method demonstrated reliable intra- (p<0.0001) and inter-observer correlation (p<0.001). CONCLUSIONS: We describe a novel method for the quantification of CO and estimation of PVR using contrast echocardiography transit time. This technique adds to the methodologies used for noninvasive hemodynamic assessment, but requires further validation to determine overall applicability.

Tracking of Nascent Matrix Deposition during Muscle Stem Cell Activation across Lifespan Using Engineered Hydrogels.

Adult stem cells occupy a niche that contributes to their function, but how stem cells rebuild their microenvironment after injury remains an open-ended question. Herein, biomaterial-based systems and metabolic labeling are utilized to evaluate how skeletal muscle stem cells deposit extracellular matrix. Muscle stem cells and committed myoblasts are observed to generate less nascent matrix than muscle resident fibro-adipogenic progenitors. When cultured on substrates that matched the stiffness of physiological uninjured and injured muscles, muscle stem cells increased nascent matrix deposition with activation kinetics. Reducing the ability to deposit nascent matrix by an inhibitor of vesicle trafficking (Exo-1) attenuated muscle stem cell function and mimicked impairments observed from muscle stem cells isolated from old muscles. Old muscle stem cells are observed to deposit less nascent matrix than young muscle stem cells, which is rescued with therapeutic supplementation of insulin-like growth factors. These results highlight the role of nascent matrix production with muscle stem cell activation.

Quantification of local matrix deposition during muscle stem cell activation using engineered hydrogels.

Adult stem cells occupy a niche that contributes to their function, but how stem cells remodel their microenvironment remains an open-ended question. Herein, biomaterials-based systems and metabolic labeling were utilized to evaluate how skeletal muscle stem cells deposit extracellular matrix. Muscle stem cells and committed myoblasts were observed to generate less nascent matrix than muscle resident fibro-adipogenic progenitors. When cultured on substrates that matched the stiffness of physiological uninjured and injured muscles, the increased nascent matrix deposition was associated with stem cell activation. Reducing the ability to deposit nascent matrix in muscle stem cells attenuated function and mimicked impairments observed from muscle stem cells isolated from old aged muscles, which could be rescued with therapeutic supplementation of insulin-like growth factors. These results highlight how nascent matrix production is critical for maintaining healthy stem cell function.

Cellular mechanotransduction of human osteoblasts in microgravity.

Astronauts experience significant and rapid bone loss as a result of an extended stay in space, making the International Space Station (ISS) the perfect laboratory for studying osteoporosis due to the accelerated nature of bone loss on the ISS. This prompts the question, how does the lack of load due to zero-gravity propagate to bone-forming cells, human fetal osteoblasts (hFOBs), altering their maturation to mineralization? Here, we aim to study the mechanotransduction mechanisms by which bone loss occurs in microgravity. Two automated experiments, 4 microfluidic chips capable of measuring single-cell mechanics of hFOBs via aspiration and cell spheroids incubated in pressure-controlled chambers, were each integrated into a CubeLab deployed to the ISS National Laboratory. For the first experiment, we report protrusion measurements of aspirated cells after exposure to microgravity at the ISS and compare these results to ground control conducted inside the CubeLab. Our analysis revealed slightly elongated protrusions for space samples compared to ground samples indicating softening of hFOB cells in microgravity. In the second experiment, we encapsulated osteoblast spheroids in collagen gel and incubated the samples in pressure-controlled chambers. We found that microgravity significantly reduced filamentous actin levels in the hFOB spheroids. When subjected to pressure, the spheroids exhibited increased pSMAD1/5/9 expression, regardless of the microgravity condition. Moreover, microgravity reduced YAP expression, while pressure increased YAP levels, thus restoring YAP expression for spheroids in microgravity. Our study provides insights into the influence of microgravity on the mechanical properties of bone cells and the impact of compressive pressure on cell behavior and signaling in space.

Cellular mechanotransduction of human osteoblasts in microgravity.

Astronauts experience significant and rapid bone loss as a result of an extended stay in space, making the International Space Station (ISS) the perfect laboratory for studying osteoporosis due to the accelerated nature of bone loss on the ISS. This prompts the question, how does the lack of load due to zero-gravity propagate to bone-forming cells, human fetal osteoblasts (hFOBs), altering their maturation to mineralization? Here, we aim to study the mechanotransduction mechanisms by which bone loss occurs in microgravity. Two automated experiments, microfluidic chips capable of measuring single-cell mechanics via aspiration and cell spheroids incubated in pressure-controlled chambers, were each integrated into a CubeLab deployed to the ISS National Laboratory. For the first experiment, we report protrusion measurements of aspirated cells after exposure to microgravity at the ISS and compare these results to ground control conducted inside the CubeLab. We found slightly elongated protrusions for space samples compared to ground samples indicating softening of hFOB cells in microgravity. In the second experiment, we encapsulated osteoblast spheroids in collagen gel and incubated the samples in pressure-controlled chambers. We found that microgravity significantly reduced filamentous actin levels in the hFOB spheroids. When subjected to pressure, the spheroids exhibited increased pSMAD1/5/9 expression, regardless of the microgravity condition. Moreover, microgravity reduced YAP expression, while pressure increased YAP levels, thus restoring YAP expression for spheroids in microgravity. Our study provides insights into the influence of microgravity on the mechanical properties of bone cells and the impact of compressive pressure on cell signaling in space.

Clinical characteristics and optical coherence tomography of concomitant macular hole and rhgematogenous retinal detachment.

To study the clinical characteristics of macula off rhegmatogenous retinal detachment (RRD) with peripheral causative breaks and concomitant macular hole (RRD+MH). This is a bi-center study. Consecutive eyes of macula off RRD with or without macular hole (MH) were collected. Eyes in these two groups were compared with best corrected visual acuity in logarithm of minimal angle of resolution (logMAR BCVA), the presence of choroidal detachment (CD), proliferative vitreoretinopathy (PVR) and the extent of RRD. In the group of RRD+MH, regression analysis was used to evaluate the correlation of clinical factors and final logMar BCVA. In addition, optical coherence tomography was performed both pre-and post-operatively if possible. There were 40 eyes in the RRD+MH group and 80 eyes in the control group. Eyes with RRD+MH had worse initial and final logMar BCVA (p < 0.001), higher incidence of CD (p < 0.001), PVR and extensive RRD at baseline (p < 0.001). Among the eyes with RRD+MH, final BCVA was correlated with initial BCVA (p < 0.001, CI 0.637 to 0.837), recurrent RRD (p = 0.004, CI - 0.661 to - 0.126), duration of RRD (p = 0.021, CI - 0.576 to - 0.048) and presence of PVR (p = 0.001, CI - 0.131 to - 0.035). The hole closure rate at final follow up is 87.5%.11 of the 17 eyes had preoperative optical coherence tomography (OCT) obtained had ellipsoid zone lining the bottom of MH. CD, PVR and extensive RRD were more commonly observed in RRD+MH. The morphology of MH may suggest the pathogenesis of MH in RRD+MH include mechanism different from that of idiopathic MH.

Epigenetic erosion of H4K20me1 induced by inflammation drives aged stem cell ferroptosis.

Aging is associated with a decline in stem cell functionality and number across the organism. In this study, we aimed to further unravel Muscle Stem Cells (MuSCs) aging by assessing how systemic factors influence MuSC fate decisions through long-term epigenetic landscape remodelling. As aging is intricately linked to a pro-inflammatory shift, we studied the epigenetic effects of inflammatory signals in MuSCs and measured decreased H4K20me1 levels. This loss disrupts MuSC quiescence, largely through epigenetic silencing of Notch target genes. In the setting of inflammatory signals or aging, the lack of Kmt5a and the subsequent absence of de novoH4K20me1 culminate in cell death by ferroptosis. Aged MuSCs manifest abnormal iron metabolism and reduced Gpx4 levels, resulting in the accumulation of intracellular iron, increased reactive oxygen species, genomic instability, and lipid peroxidation. We showed that ferroptosis is the predominant mode of cell death in aged MuSCs, with remarkably high levels of lipid peroxidation; a phenomenon we also observed in aged hematopoietic stem cells. Implementing preventative strategies to inhibit systemic inflammation prevented aged MuSC ferroptosis, preserving their numbers and regenerative capabilities. This intervention significantly enhanced aged muscle regeneration and strength recovery and extended both lifespan and healthspan in mice. This study delineates a previously underappreciated fate trajectory for stem cell aging, and offers meaningful insights into the treatment of age-related disorders.

Three-dimensional chromatin re-organization during muscle stem cell aging.

Age-related skeletal muscle atrophy or sarcopenia is a significant societal problem that is becoming amplified as the world's population continues to increase. The regeneration of damaged skeletal muscle is mediated by muscle stem cells, but in old age muscle stem cells become functionally attenuated. The molecular mechanisms that govern muscle stem cell aging encompass changes across multiple regulatory layers and are integrated by the three-dimensional organization of the genome. To quantitatively understand how hierarchical chromatin architecture changes during muscle stem cell aging, we generated 3D chromatin conformation maps (Hi-C) and integrated these datasets with multi-omic (chromatin accessibility and transcriptome) profiles from bulk populations and single cells. We observed that muscle stem cells display static behavior at global scales of chromatin organization during aging and extensive rewiring of local contacts at finer scales that were associated with variations in transcription factor binding and aberrant gene expression. These data provide insights into genome topology as a regulator of molecular function in stem cell aging.

Manipulation of the nucleoscaffold potentiates cellular reprogramming kinetics.

Somatic cell fate is an outcome set by the activities of specific transcription factors and the chromatin landscape and is maintained by gene silencing of alternate cell fates through physical interactions with the nuclear scaffold. Here, we evaluate the role of the nuclear scaffold as a guardian of cell fate in human fibroblasts by comparing the effects of transient loss (knockdown) and mutation (progeria) of functional Lamin A/C, a core component of the nuclear scaffold. We observed that Lamin A/C deficiency or mutation disrupts nuclear morphology, heterochromatin levels, and increases access to DNA in lamina-associated domains. Changes in Lamin A/C were also found to impact the mechanical properties of the nucleus when measured by a microfluidic cellular squeezing device. We also show that transient loss of Lamin A/C accelerates the kinetics of cellular reprogramming to pluripotency through opening of previously silenced heterochromatin domains while genetic mutation of Lamin A/C into progerin induces a senescent phenotype that inhibits the induction of reprogramming genes. Our results highlight the physical role of the nuclear scaffold in safeguarding cellular fate.

Blood-brain barrier remodeling in an organ-on-a-chip device shows Dkk1 to be a regulator of early metastasis.

Brain metastases are the most lethal progression event, in part because the biological processes underpinning brain metastases are poorly understood. There is a paucity of realistic models of metastasis, as current in vivo murine models are slow to manifest metastasis. We set out to delineate metabolic and secretory modulators of brain metastases by utilizing two models consisting of in vitro microfluidic devices: 1) a blood brain niche (BBN) chip that recapitulates the blood-brain-barrier and niche; and 2) a migration chip that assesses cell migration. We report secretory cues provided by the brain niche that attract metastatic cancer cells to colonize the brain niche region. Astrocytic Dkk-1 is increased in response to brain-seeking breast cancer cells and stimulates cancer cell migration. Brain-metastatic cancer cells under Dkk-1 stimulation increase gene expression of FGF-13 and PLCB1. Further, extracellular Dkk-1 modulates cancer cell migration upon entering the brain niche.

Maresin 1 repletion improves muscle regeneration after volumetric muscle loss.

The acute traumatic or surgical loss of skeletal muscle, known as volumetric muscle loss (VML), is a devastating type of injury that results in exacerbated and persistent inflammation followed by fibrosis. The mechanisms that mediate the magnitude and duration of the inflammatory response and ensuing fibrosis after VML remain understudied, and as such, the development of regenerative therapies has been limited. To address this need, we profiled how lipid mediators, which are potent regulators of the immune response after injury, varied with VML injuries that heal or result in fibrosis. We observed that non-healing VML injuries displayed increased pro-inflammatory eicosanoids and a lack of pro-resolving lipid mediators. Treatment of VML with a pro-resolving lipid mediator synthesized from docosahexaenoic acid, called Maresin 1, ameliorated fibrosis through reduction of neutrophils and macrophages and enhanced recovery of muscle strength. These results expand our knowledge of the dysregulated immune response that develops after VML and identify a novel immuno-regenerative therapeutic modality in Maresin 1.

Transit Time Flow Measurement in Robotic Totally Endoscopic Coronary Artery Bypass: What Do the Numbers Mean?

OBJECTIVE: Transit time flow measurement (TTFM) is valuable for assessing intraoperative graft patency in coronary artery bypass surgery (CAB). The significance of competitive native coronary flow on patency, as predicted by percentage of backflow (%BF) on TTFM, is unknown. This study aims to evaluate intraoperative TTFM parameters, and specifically %BF, in predicting graft patency in robotic totally endoscopic CAB (TECAB). METHODS: We reviewed TTFM parameters in 311 patients undergoing robotic off-pump TECAB at our institution between February 2016 and January 2020. Patients with sequential or Y grafts were excluded, leaving 277 patients with a total of 387 isolated end-to-side grafts (248 left internal mammary artery [LIMA], 149 right IMA [RIMA]). Mean graft flow, diastolic flow, pulsatility index, and %BF were measured intraoperatively. Early postoperative angiograms were obtained in 83 patients undergoing percutaneous coronary intervention for hybrid revascularization, with a total of 125 grafts. Angiograms were independently analyzed and separated into 2 groups based on IMA graft patency, which were patent (FitzGibbon A/B) and nonpatent (FitzGibbon O) groups. RESULTS: Early angiographic patency at a median of 31.0 days after surgery showed 123 (97.1%) patent grafts and 3 (2.9%) occluded grafts in both LIMA and RIMA grafts to both left anterior descending (LAD) and non-LAD targets. Mean graft flow was 77.4 ± 41.6 mL/min. There was no difference in mean flow, pulsatility index, or %BF between the patent and occluded grafts. CONCLUSIONS: Excellent intraoperative flow parameters and early angiographic patency can be obtained via robotic, off-pump TECAB. Our data did not demonstrate an association between intraoperative TTFM evidence of competitive native coronary flow and early angiographic graft outcomes.