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Membranes and membrane proteins from undifferentiated cells and torpedo-stage embryos were compared. A comparison of marker enzyme profiles on linear sucrose gradients showed that the membrane vesicles obtained from 14-day-old embryos were consistently less dense than those obtained from undifferentiated carrot cells. The density of the endoplasmic reticulum, for instance, was 1.10g/cm in embryos and 1.12g/cm in undifferentiated cells. Proteins and glycoproteins from endoplasmic reticulum-, Golgi apparatus-, and plasma membrane-enriched fractions were compared from undifferentiated carrot cells with 14-day-old embryos by 2D SDS-PAGE. When these two tissues were compared, extensive qualitative and quantitative changes in the steady-state endomembrane and plasma membrane proteins were observed. The plasma membrane was examined further by labeling the plasma membrane proteins with sulfosuccinimidylbiotin. Using this specific label, plasma membrane proteins of 54 kD, 41 kD, 16 kD, and 15 kD were found to be uniquely associated with the embryonic state. Conversely, a 70 kD protein and a 45 kD glycoprotein were found to be associated with only undifferentiated cells. These results demonstrate that proteins of the plasma membrane exhibit distinct changes as a result of somatic embryogenesis in carrot. [ABSTRACT FROM AUTHOR]

Background: Only 1% of Americans eligible for metabolic and bariatric surgery (MBS) receive MBS. Prior studies have analyzed primary care provider (PCP) referral patterns and perceptions of MBS as a potential barrier to increasing MBS. However, less data exists regarding PCP knowledge of MBS indications and outcomes. Following the 2022 update to the indications for MBS by the ASMBS and IFSO, the number of eligible patients is only expected to increase. We evaluated PCP knowledge regarding the existence of the 2022 ASMBS and IFSO updated guidelines, MBS indications, and MBS outcomes., Methods: An 11-question survey was emailed to primary care residents, advanced practice providers, and faculty at a single institution., Results: Of 151 surveys distributed, 39.7% responded (n = 60). 95% were unaware of the 2022 updated guidelines. On multiple choice questions, 16.3% correctly identified the average weight loss from MBS, and 46.8% correctly answered the diabetes remission rate following MBS. Trainee answers were not statistically significant from practicing PCPs. Fifteen respondents had referred a patient for MBS, but this subgroup did not perform significantly better on the assessment. A total of 72.3% of respondents reported inadequate MBS education during their training, and 85.1% were interested in additional education., Conclusions: We present the first assessment of PCP MBS knowledge since the release of the 2022 updated ASMBS and IFSO guidelines. This study indicates a gap in PCPs' knowledge regarding the updated guidelines and represents an opportunity for collaboration with our primary care colleagues to provide further MBS education., (© 2024. The Author(s).)

Radiotherapy (RT) of colorectal cancer (CRC) can prime adaptive immunity against tumor-associated antigen (TAA)-expressing CRC cells systemically. However, abscopal tumor remissions are extremely rare, and the postirradiation immune escape mechanisms in CRC remain elusive. Here, we found that irradiated CRC cells used ATR-mediated DNA repair signaling pathway to up-regulate both CD47 and PD-L1, which through engagement of SIRPα and PD-1, respectively, prevented phagocytosis by antigen-presenting cells and thereby limited TAA cross-presentation and innate immune activation. This postirradiation CD47 and PD-L1 up-regulation was observed across various human solid tumor cells. Concordantly, rectal cancer patients with poor responses to neoadjuvant RT exhibited significantly elevated postirradiation CD47 levels. The combination of RT, anti-SIRPα, and anti-PD-1 reversed adaptive immune resistance and drove efficient TAA cross-presentation, resulting in robust TAA-specific CD8 T cell priming, functional activation of T effectors, and increased T cell clonality and clonal diversity. We observed significantly higher complete response rates to RT/anti-SIRPα/anti-PD-1 in both irradiated and abscopal tumors and prolonged survival in three distinct murine CRC models, including a cecal orthotopic model. The efficacy of triple combination therapy was STING dependent as knockout animals lost most benefit of adding anti-SIRPα and anti-PD-1 to RT. Despite activation across the myeloid stroma, the enhanced dendritic cell function accounts for most improvements in CD8 T cell priming. These data suggest ATR-mediated CD47 and PD-L1 up-regulation as a key mechanism restraining radiation-induced immune priming. RT combined with SIRPα and PD-1 blockade promotes robust antitumor immune priming, leading to systemic tumor regressions.

The ability of two biotinylating reagents, sulfosuccinimidobiotin and sulfosuccinimidyl 2-(biotinamido)ethyl-1,3'-dithiopropionate, to label plasma membrane proteins was examined. These compounds form covalent bonds with the free amino groups of proteins and label the proteins with biotin. Biotinylated proteins can be detected with avidin-peroxidase staining. Protoplasts isolated from embryogenic Daucus carota suspension cells were labeled with biotin and the membranes were separated on linear sucrose gradients. The conditions used for labeling the protoplasts did not cause protoplast rupture or loss of viability. The distribution of the biotin label in these linear sucrose gradients was analyzed and compared to the distribution of vanadate-sensitive ATPase activity, a marker for the plasma membrane. Both the biotin label and the vanadate-sensitive ATPase activity were strongly localized in the gradient at peak density of 1.16 gram per cubic centimeter. When the protoplast surface was labeled, biotinylated polypeptides were detected after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and polypeptides of 153, 94, 51, 30, 20, 17, and 14 kilodaltons were shown to be plasma membrane in origin. When a crude membrane pellet was labeled, numerous biotinylated polypeptides were distributed throughout the gradient. Because the position of the biotin label in the gradient is strongly correlated with the distribution of vanadate-sensitive ATPase, it is concluded that these biotinylating reagents are effective and reliable labels for proteins of the plant plasma membrane. Furthermore, these labels permit the positive identification of plasma membrane proteins after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and can serve as convenient markers for solubilization and purification of these proteins.

In a 52-year-old man, a spontaneous right tension pneumothorax was associated with transient bradycardia, hypotension and electrocardiogram (ECG) changes consisting of precordial ST elevation suggestive of acute myocardial injury. Immediately after chest tube placement, the ECG reverted to near normal. Serial cardiac enzymes and isoenzymes showed no evidence of myocardial infarction. To our knowledge, this is the first reported case of this association. The ECG changes may be related to the hypotensive state induced by the tension pneumothorax with resulting decreased coronary artery blood flow and myocardial ischemia that resolved without infarction.