Cantubernard7444

Endocytic internalization of extracellular proteins plays roles in signaling, nutrient uptake, immunity, and extracellular protein quality control. However, there are few protocols for analyzing the lysosomal degradation of extracellular protein. Here, we purified secreted proteins fused with pH-sensitive GFP and acid- and protease-resistant RFP from mammalian cells and describe an internalization assay for mammalian cells. This protocol enables quantification of cellular uptake and lysosomal degradation of protein-of-interest (POI) via cell biological and biochemical analyses. For full details on the use and execution of this protocol, please refer to Itakura et al. (2020).CausalPath (causalpath.org) evaluates proteomic measurements against prior knowledge of biological pathways and infers causality between changes in measured features, such as global protein and phospho-protein levels. It uses pathway resources to determine potential causality between observable omic features, which are called prior relations. The subset of the prior relations that are supported by the proteomic profiles are reported and evaluated for statistical significance. The end result is a network model of signaling that explains the patterns observed in the experimental dataset. For complete details on the use and execution of this protocol, please refer to Babur et al. (2021).This protocol provides an improved pipeline for dissociating intact projection neurons from adult mouse cortex for applications including droplet and plate-based single-cell RNA sequencing, qPCR, immunocytochemistry, and long-term in vitro cell culture. Atuveciclib This protocol provides a robust and reproducible dissociation pipeline that uses exclusively off-the-shelf reagents, not requiring the use of expensive dissociation kits. The unique incubation steps, in combination with the FACS gating strategy, results in unparalleled enrichment for intact cortical neurons from the adult brain. For complete details on the use and execution of this protocol, please refer to Golan et al. (2021).Here we describe a protocol for identifying metabolites in respiratory specimens of patients that are SARS-CoV-2 positive, SARS-CoV-2 negative, or H1N1 positive. This protocol provides step-by-step instructions on sample collection from patients, followed by metabolite extraction. We use ultra-high-pressure liquid chromatography (UHPLC) coupled with high-resolution mass spectrometry (HRMS) for data acquisition and describe the steps for data analysis. The protocol was standardized with specific customization for SARS-CoV-2-containing respiratory specimens. For complete details on the use and execution of this protocol, please refer to Maras et al. (2021).Procyanidin is an important polyphenol for its health-promoting properties, however, the study of procyanidin in sea buckthorn was limited. In this paper, sea buckthorn procyanidin (SBP) was obtained through a green isolation and enrichment technique with an extraction rate and purity of 9.1% and 91.5%. The structure of SBP was analyzed using Ultraviolet-visible spectroscopy (UV-vis), Fourier-transform infrared spectroscopy (FT-IR), and liquid chromatography-mass spectrometry (LC-MS/MS). The results show that SBP is an oligomeric procyanidin, mainly composed of (-)-epicatechin gallate, procyanidin B, (+)-gallocatechin-(+)-catechin, and (+)-gallocatechin dimer. SBP showed superior scavenging capacity on free radicals. Furthermore, the cleaning rate of the ABTS radical was 4.8 times higher than vitamin C at the same concentration. Moreover, SBP combined with vitamin C presented potent synergistic antioxidants with combined index values below 0.3 with concentration rates from 55 to 28. SBP also provided significant protection against oxidative stress caused by hydrogen peroxide (H2O2) on RAW264.7 cells. These findings prove the potential of SBP as a natural antioxidant in food additives and support the in-depth development of sea buckthorn resources.Lactating medical residents face unique barriers due to intense clinical work schedules, limited support in the clinic and hospital workspaces, and competing pressures between career development and childcare. The objective of this project was to explore the perceived culture of breastfeeding support among medical trainees and design an action plan to improve support for lactating residents in Hawai'i. Resident and faculty representatives from the Hawai'i Residency Programs and the University of Hawai'i John A Burns School of Medicine participated in an 8 month national learning collaborative to review the existing resident lactation policy and resident perception of lactation support. In a pre-survey, the majority of residents (88%) agreed that 20-30 minutes every 2-3 hours should be allowed to express milk but only 18% felt comfortable asking for a change in schedule to accommodate time to pump. An action plan was created with the following objectives (1) revamping the existing policy to address protected pumping times, lactation spaces, and responsibilities of administration, faculty, and residents; (2) improving lactation space through uniform provision of educational material on available facilities and efficiency tips for new parents, and (3) improving awareness of the unique challenges lactating residents face and empowering faculty and trainees to advocate for lactating residents through department and educational presentations. Medical residents in Hawai'i recognize the importance of breastfeeding but perceive a lack of support in the workplace. A comprehensive action plan to revamp the resident lactation policy and improve faculty and resident education may foster an increased culture of lactation support and healthy development of the physician workforce.Seasonal affective disorder (SAD) is a prevalent and potentially serious medical condition. Young adults are at particularly high risk. However, it is unknown if college students whose hometowns are in geographic areas with less seasonal variability, such as in the state of Hawai'i, are particularly vulnerable if they attend schools in areas with seasonal variability. An adapted version of the Seasonal Patterns Assessment Questionnaire (SPAQ) was administered to students at 3 universities to test this hypothesis. Surveys were administered twice a baseline (T0) assessment in the fall and a follow-up (T1) assessment in the winter and were administered in the second month of each semester. A linear regression model was constructed to identify potential risk factors for developing seasonal fluctuations in mood (SPAQ scores T1-T0). Study subjects (n=115) from non-seasonal hometowns had a 1.6-point greater increase in SPAQ score than students from seasonal hometowns (-0.26 ± 3.88 vs 1.35 ± 3.03; P =.01). This difference is independent of demographic and lifestyle predictors (linear regression coefficient β = 1.73; standard error = 0.68; P =.012). Interestingly, SPAQ score changes of students from seasonal hometowns did not differ significantly from 0 (t = -0.97; P =.33), indicating that they did not generally experience seasonal shifts in depressive symptoms. Students from less seasonal hometowns and counselors at seasonal institutions should be aware that these students could be more at risk of developing depressive symptoms and address these concerns before interfering with students' daily and academic lives.Both Hodgkin's lymphoma and testicular cancers can present in young men; however, concurrent Hodgkin's lymphoma with seminoma is very rare. When they do coexist, careful consideration must be made to avoid missing new cancer by assuming the presence of primary metastatic disease when lymphadenopathy presents. Here we present a rare case of coexistence of seminoma and Hodgkin's lymphoma and the staging and treatment challenges associated with a 2-cancer diagnosis.Mycotic coronary aneurysms are rare, with potentially fatal complications. The treatment of choice is surgical intervention. We present a case of a mycotic coronary aneurysm secondary to a catheter-related bloodstream infection, failed surgical treatment, and eventual treatment with percutaneous coil embolization.Isostructural MOFs with similar crystallographic parameter are easily available for MOF-on-MOF growth and possible to form core-shell structure by isotropic growth. However, due to well-matched cell lattice, selective growth in isostructural MOF heterostructures remains a great challenge for engineering atypical MOF heterostructures. Herein, an anisotropic MOF-on-MOF growth strategy was developed to structure a range of multilayer sandwich-like ZIF-L heterostructures via stacking isostructural ZIF-L-Zn and ZIF-L-Co alternately with three-, five-, seven-, and more layer structures. Moreover, these heterostructures with highly designable feature were fantastic precursors for fabricating derivatives with tunable magnetic and catalytic properties. Such strategy explores a novel way of achieving anisotropic MOF-on-MOF growth between isostructural MOFs and opens up new horizons for regulating the properties by MOF modular assembly in versatile functional nanocomposites.Controlled polypeptide synthesis via α-amino acid N-carboxylic anhydride (NCA) polymerization using conventional primary amine initiators encounters two major obstacles (i) normal amine mechanism (NAM) and activated monomer mechanism (AMM) coexist due to amine basicity and nucleophilicity and (ii) NCA is notoriously sensitive towards moisture and heat and unstable upon storage. We serendipitously discover that N-phenoxycarbonyl-functionalized α-amino acid (NPCA), a latent NCA precursor, could be polymerized solely based on NAM with high initiating efficiency by using primary amine hydrochloride as an initiator. The polymerization affords well-defined polypeptides with narrow polydispersity and high-fidelity terminal functionalities, as revealed by the clean set of MALDI-TOF MS patterns. We further demonstrate successful syntheses of random and block copolypeptides, even under open-vessel conditions. Overall, the integration of moisture-insensitive and air-tolerant NPCA precursors with stable primary amine hydrochloride initiators represents a general strategy for controlled synthesis of high-fidelity polypeptides with sophisticated functions.Remote dysconnectivity following cerebellar ischaemic stroke may have a negative impact on supratentorial brain tissue. Since the cerebellum is connected to the individual cerebral lobes via contralateral tracts, cerebellar lesion topography might determine the distribution of contralateral supratentorial brain tissue changes. We investigated (i) the occurrence of delayed cerebral atrophy after cerebellar ischaemic stroke and its relationship to infarct volume; (ii) whether cerebellar stroke topography determines supratentorial atrophy location; and (iii) how cortical atrophy after cerebellar stroke impacts clinical outcome. We performed longitudinal volumetric MRI analysis of patients with isolated cerebellar stroke from the Swiss Stroke Registry database. Stroke location and volume were determined at baseline MRI. Delayed cerebral atrophy was measured as supratentorial cortical volumetric change at follow-up, in contralateral target as compared to ipsilateral reference-areas. In patients with bilateral stroke, both hemispheres were analysed separately.