PMI 5011 regulates the ubiquitin proteasome system in skeletal muscle

Insulin resistance in type 2 diabetes is associated with impaired glucose and protein metabolism in skeletal muscle. The impaired insulin signaling in skeletal muscle affects muscle mass by tilting the balance between skeletal muscle protein synthesis and degradation toward degradation, a process that is primarily regulated by the ubiquitin-proteasome system. Studies have shown that an extensively characterized ethanol extract of Artemisia dracunculus L (Russian Tarragon), termed PMI 5011, enhances insulin signaling in human primary skeletal muscle cells and in a rodent model of insulin resistance. The aim of this project is to determine if the effect of PMI 5011 on insulin signaling extends to regulation of ubiquitin-proteasome activity in skeletal muscle. To evaluate the effect of PMI 5011 on the ubiquitin-proteasome system, we used two in vitro models of insulin resistance in C2C12 myotubes and the KKAy mouse model of insulin resistance in vivo. Our studies show that PMI 5011 enhances the inhibitory effect of insulin on proteasome activity and ubiquitylation in skeletal muscle in vitro and in vivo. In addition, PMI 5011 inhibits non-proteasomal protein degradation in vivo, indicating that PMI 5011 is a potent inhibitor of skeletal muscle protein degradation. PMI 5011 also regulates the expression of Atrogin-1 and MuRF-1, muscle-specific ubiquitin ligases that are required for ubiquitin-dependent protein degradation in skeletal muscle. Both Atrogin-1 and MuRF-1 gene and protein expression is elevated with impaired insulin signaling and our studies show that PMI 5011 reduces the expression of these ligases while enhancing Akt phosphorylation. In summary, these studies demonstrate that PMI 5011 regulates the ubiquitin-proteasome in insulin resistant states in vitro and in vivo. PMI 5011 may therefore be a therapeutic target for enhancing insulin sensitivity leading to conservation of muscle mass in type 2 diabetes

Flow cytometric and immunohistochemical detection of in vivo BrdU-labeled cells in mouse fat depots

This study has determined the natural frequency and localization of progenitor/stem cells within fat depots in situ based on their ability to retain DNA nucleotide label (BrdU). Neonate and mature male C57BL6/J mice were injected intraperitoneally with BrdU- and label-retaining cells (LRC) were quantified in fat depots by immunohistochemical, immunofluorescent, and flow cytometric methods. In neonates, LRC constituted 27% of the cells in inguinal fat (iWAT) and 65% in interscapular brown fat (BAT) after Day 10 and 26% of the cells in epididymal fat (eWAT) after Day 28. After 52 days, the LRC accounted for 0.72% of iWAT, 0.53% of eWAT and 1.05% of BAT, respectively. The BrdU-labeled cells localized to two areas: single cells distributed among adipocytes or those adjacent to the blood vessels wall. In mature C57BL6/J mice, flow cytometric analysis determined that a majority of the LRC were also positive for stem cell antigen-1 (Sca-1). © 2008 Elsevier Inc. All rights reserved

Dynamic Volunteer Engagement and Impactful Educational Outreach Taking Us into the Next 50 Years of the Extension Master Gardener Program

According to the 2021 Extension Master Gardener (EMG) National Summary, the EMG Volunteer Program had an estimated 84,700 volunteers throughout the United States. These volunteers helped communities garden and grow food, provided opportunities to engage in activities that improved physical and mental health, and worked on projects that addressed environmental issues. In total, these programs contributed 3.1 million hours of education to local communities and $88 million dollars in value to the public. However, the COVID-19 pandemic presented challenges for the program, with many states implementing reduced requirements and increased flexibility for volunteers. The workshop “Dynamic Volunteer Engagement and Impactful Educational Outreach Taking Us Into the Next 50 Years of the EMG Program” at the 2022 ASHS conference discussed how to engage EMG volunteers despite the limitations of limited in-person contact. The workshop featured three Extension educators and EMG coordinators who shared their experiences and strategies for engaging volunteers during the pandemic. Topics discussed included engaging volunteers in local food systems and community gardens, engaging students in horticulture at an earlier age, and digital volunteer opportunities. Overall, the workshop provided valuable insights and facilitated discussions on how to adapt and continue the EMG program during challenging times

Multiorgan MRI findings after hospitalisation with COVID-19 in the UK (C-MORE): a prospective, multicentre, observational cohort study

Introduction: The multiorgan impact of moderate to severe coronavirus infections in the post-acute phase is still poorly understood. We aimed to evaluate the excess burden of multiorgan abnormalities after hospitalisation with COVID-19, evaluate their determinants, and explore associations with patient-related outcome measures. Methods: In a prospective, UK-wide, multicentre MRI follow-up study (C-MORE), adults (aged ≥18 years) discharged from hospital following COVID-19 who were included in Tier 2 of the Post-hospitalisation COVID-19 study (PHOSP-COVID) and contemporary controls with no evidence of previous COVID-19 (SARS-CoV-2 nucleocapsid antibody negative) underwent multiorgan MRI (lungs, heart, brain, liver, and kidneys) with quantitative and qualitative assessment of images and clinical adjudication when relevant. Individuals with end-stage renal failure or contraindications to MRI were excluded. Participants also underwent detailed recording of symptoms, and physiological and biochemical tests. The primary outcome was the excess burden of multiorgan abnormalities (two or more organs) relative to controls, with further adjustments for potential confounders. The C-MORE study is ongoing and is registered with ClinicalTrials.gov, NCT04510025. Findings: Of 2710 participants in Tier 2 of PHOSP-COVID, 531 were recruited across 13 UK-wide C-MORE sites. After exclusions, 259 C-MORE patients (mean age 57 years [SD 12]; 158 [61%] male and 101 [39%] female) who were discharged from hospital with PCR-confirmed or clinically diagnosed COVID-19 between March 1, 2020, and Nov 1, 2021, and 52 non-COVID-19 controls from the community (mean age 49 years [SD 14]; 30 [58%] male and 22 [42%] female) were included in the analysis. Patients were assessed at a median of 5·0 months (IQR 4·2–6·3) after hospital discharge. Compared with non-COVID-19 controls, patients were older, living with more obesity, and had more comorbidities. Multiorgan abnormalities on MRI were more frequent in patients than in controls (157 [61%] of 259 vs 14 [27%] of 52; p<0·0001) and independently associated with COVID-19 status (odds ratio [OR] 2·9 [95% CI 1·5–5·8]; padjusted=0·0023) after adjusting for relevant confounders. Compared with controls, patients were more likely to have MRI evidence of lung abnormalities (p=0·0001; parenchymal abnormalities), brain abnormalities (p<0·0001; more white matter hyperintensities and regional brain volume reduction), and kidney abnormalities (p=0·014; lower medullary T1 and loss of corticomedullary differentiation), whereas cardiac and liver MRI abnormalities were similar between patients and controls. Patients with multiorgan abnormalities were older (difference in mean age 7 years [95% CI 4–10]; mean age of 59·8 years [SD 11·7] with multiorgan abnormalities vs mean age of 52·8 years [11·9] without multiorgan abnormalities; p<0·0001), more likely to have three or more comorbidities (OR 2·47 [1·32–4·82]; padjusted=0·0059), and more likely to have a more severe acute infection (acute CRP >5mg/L, OR 3·55 [1·23–11·88]; padjusted=0·025) than those without multiorgan abnormalities. Presence of lung MRI abnormalities was associated with a two-fold higher risk of chest tightness, and multiorgan MRI abnormalities were associated with severe and very severe persistent physical and mental health impairment (PHOSP-COVID symptom clusters) after hospitalisation. Interpretation: After hospitalisation for COVID-19, people are at risk of multiorgan abnormalities in the medium term. Our findings emphasise the need for proactive multidisciplinary care pathways, with the potential for imaging to guide surveillance frequency and therapeutic stratification

Convalescent plasma in patients admitted to hospital with COVID-19 (RECOVERY): a randomised controlled, open-label, platform trial

SummaryBackground Azithromycin has been proposed as a treatment for COVID-19 on the basis of its immunomodulatoryactions. We aimed to evaluate the safety and efficacy of azithromycin in patients admitted to hospital with COVID-19.Methods In this randomised, controlled, open-label, adaptive platform trial (Randomised Evaluation of COVID-19Therapy [RECOVERY]), several possible treatments were compared with usual care in patients admitted to hospitalwith COVID-19 in the UK. The trial is underway at 176 hospitals in the UK. Eligible and consenting patients wererandomly allocated to either usual standard of care alone or usual standard of care plus azithromycin 500 mg once perday by mouth or intravenously for 10 days or until discharge (or allocation to one of the other RECOVERY treatmentgroups). Patients were assigned via web-based simple (unstratified) randomisation with allocation concealment andwere twice as likely to be randomly assigned to usual care than to any of the active treatment groups. Participants andlocal study staff were not masked to the allocated treatment, but all others involved in the trial were masked to theoutcome data during the trial. The primary outcome was 28-day all-cause mortality, assessed in the intention-to-treatpopulation. The trial is registered with ISRCTN, 50189673, and ClinicalTrials.gov, NCT04381936.Findings Between April 7 and Nov 27, 2020, of 16 442 patients enrolled in the RECOVERY trial, 9433 (57%) wereeligible and 7763 were included in the assessment of azithromycin. The mean age of these study participants was65·3 years (SD 15·7) and approximately a third were women (2944 [38%] of 7763). 2582 patients were randomlyallocated to receive azithromycin and 5181 patients were randomly allocated to usual care alone. Overall,561 (22%) patients allocated to azithromycin and 1162 (22%) patients allocated to usual care died within 28 days(rate ratio 0·97, 95% CI 0·87–1·07; p=0·50). No significant difference was seen in duration of hospital stay (median10 days [IQR 5 to >28] vs 11 days [5 to >28]) or the proportion of patients discharged from hospital alive within 28 days(rate ratio 1·04, 95% CI 0·98–1·10; p=0·19). Among those not on invasive mechanical ventilation at baseline, nosignificant difference was seen in the proportion meeting the composite endpoint of invasive mechanical ventilationor death (risk ratio 0·95, 95% CI 0·87–1·03; p=0·24).Interpretation In patients admitted to hospital with COVID-19, azithromycin did not improve survival or otherprespecified clinical outcomes. Azithromycin use in patients admitted to hospital with COVID-19 should be restrictedto patients in whom there is a clear antimicrobial indication

Assessing Biodegradable Mulch Duration and Nutsedge Suppression during Late Summer Cucumber Production in Mississippi and Louisiana

Environmental concerns as well as labor costs associated with the use of polyethylene plastic mulch have turned producers’ focus to alternative mulch treatments. A preliminary study was conducted to evaluate nutsedge control, mulch degradation, and cucumber yields on biodegradable mulches at two locations (Louisiana and Mississippi). Mulch treatments included two paper-based mulch products, two biodegradable plastic mulches, the industry standard non-biodegradable black plastic mulch, and an unmulched control. The heavy weight paper-based mulch and light weight paper mulches in the Louisiana location were reduced to 50% or less coverage at the end of the study. Similarly, the paper-based mulches both degraded below 40% at the conclusion of the study at the Mississippi location. The heavy weight paper mulch was able to hold back nutsedge (p ≤ 0.05) at similar rates as the two biodegradable plastic mulches and industry standard plastic mulch, while the light weight paper mulch and unmulched plots were ineffective at reducing emerged sedges. There were no statistical differences in nutsedge control (averaging < 14 emerged plants per 1.5 m subplots) between all mulch materials at the conclusion of the study in the Mississippi location. Combining both states yield data, the heavyweight mulch (8.7 fruit, 5.3 lb) performed comparably to the industry standard plastic mulch (5.8 fruit, 3.5 lb) in terms of both average fruit number and average weights harvested off 9.1 m rows at each harvest date

Paclitaxel-induced apoptosis is blocked by camptothecin in human breast and pancreatic cancer cells

The combination of paclitaxel (PTX) and topoisomerase I inhibitors such as camptothecin (CPT) constitutes a therapeutic strategy based on anticipated synergism. However, previous in vitro studies have generated contradictory findings for this strategy. The interaction between these drugs can be synergistic or antagonistic, depending on the cell type examined. To gain additional insight into this promising yet controversial strategy, we investigated the interaction between PTX and CPT in three different cell lines (PANC-1, MDA-MB-231 and HL-60) and explored possible underlying mechanisms of synergy or antagonism. Using a novel solubilizing natural compound, rubusoside, water-insoluble PTX and CPT were solubilized to enable the comparison of the effects of single drugs and their combination on cell viability. Intracellular drug concentrations were quantified to examine the effect of CPT on cellular uptake and accumulation of PTX. Flow cytometry and quantitative real-time PCR gene array analyses were used to explore the mechanisms behind the interaction between PTX and CPT. Our studies confirmed that rubusoside-solubilized PTX or CPT maintained cytotoxicity, causing significant reductions in cell viability. However, the efficacy of the combination of PTX and CPT produced varied results based on the cell line tested. CPT antagonistically reduced the cytotoxic activity of PTX in PANC-1 and MDA-MB-231 cells. The effect of CPT on the cytotoxicity of PTX was less pronounced in HL-60 cells, showing neither synergy nor antagonism. Analysis of apoptosis by flow cytometry revealed that upon co-treatment with CPT, apoptosis induced by PTX was attenuated in PANC-1 and MDA-MB-231 cells. In agreement with our cytotoxicity findings, no synergistic or antagonistic effects on apoptosis were observed in HL-60 cells. The antagonism in PANC-1 and MDA-MB-231 cells was not a result of reduced PTX uptake and accumulation because the amount of intracellular PTX was not altered upon co-treatment with CPT. Moreover, higher expression of anti-apoptosis-related transcripts (BCL2L10, CFLAR, HIP1 and TRADD) in PANC-1 cells was observed upon combination treatment over PTX treatment alone. Although exact underlying mechanisms are unknown, the suspected CPT-dependent reduction of intracellular PTX accumulation was ruled out. The findings of antagonism and increased anti-apoptotic gene transcription serve as a precaution to the design of combination drug strategies where a synergistic interaction may not exist

An extract of Artemisia dracunculus L. inhibits ubiquitin-proteasome activity and preserves skeletal muscle mass in a murine model of diabetes.

Impaired insulin signaling is a key feature of type 2 diabetes and is associated with increased ubiquitin-proteasome-dependent protein degradation in skeletal muscle. An extract of Artemisia dracunculus L. (termed PMI5011) improves insulin action by increasing insulin signaling in skeletal muscle. We sought to determine if the effect of PMI5011 on insulin signaling extends to regulation of the ubiquitin-proteasome system. C2C12 myotubes and the KK-A(y) murine model of type 2 diabetes were used to evaluate the effect of PMI5011 on steady-state levels of ubiquitylation, proteasome activity and expression of Atrogin-1 and MuRF-1, muscle-specific ubiquitin ligases that are upregulated with impaired insulin signaling. Our results show that PMI5011 inhibits proteasome activity and steady-state ubiquitylation levels in vitro and in vivo. The effect of PMI5011 is mediated by PI3K/Akt signaling and correlates with decreased expression of Atrogin-1 and MuRF-1. Under in vitro conditions of hormonal or fatty acid-induced insulin resistance, PMI5011 improves insulin signaling and reduces Atrogin-1 and MuRF-1 protein levels. In the KK-A(y) murine model of type 2 diabetes, skeletal muscle ubiquitylation and proteasome activity is inhibited and Atrogin-1 and MuRF-1 expression is decreased by PMI5011. PMI5011-mediated changes in the ubiquitin-proteasome system in vivo correlate with increased phosphorylation of Akt and FoxO3a and increased myofiber size. The changes in Atrogin-1 and MuRF-1 expression, ubiquitin-proteasome activity and myofiber size modulated by PMI5011 in the presence of insulin resistance indicate the botanical extract PMI5011 may have therapeutic potential in the preservation of muscle mass in type 2 diabetes

PMI5011 enhances the effect of insulin on proteasome activity and inhibits ubiquitylation in skeletal muscle.

<p>C2C12 myotubes were incubated with 10 µg/ml PMI5011 for 16 hours. The cells were subsequently incubated with wortmannin (200 nM) for 1 hour prior to the addition of insulin (100 nM) for 2 hours as indicated. (A) The cells were harvested and assayed for the chymotrysin-like protease activity of the proteasome. Proteasome activity is reported as Relative Fluorescence Units (RFU) RFU/µg protein/hr. The data are reported as the mean −/+ standard deviation from triplicate measurements and are representative of three independent experiments. a = compared to control; b = compared to related treatment (−) wortmannin; *<i>p</i><0.05, **<i>p</i><0.01 (B) Whole cell extracts were also subjected to SDS-PAGE followed by western blot analysis using an anti-ubiquitin antibody to assay steady-state ubiquitylation levels. The data are representative of three independent experiments.</p

PMI5011 alters ubiquitin conjugation patterns in skeletal muscle.

<p>Steady-state ubiquitylation were measured in (A) control (N = 4) and PMI5011 supplemented (N = 4) KK-A^y mice or (B) control (N = 4) and PMI5011 supplemented (N = 4) KK-A^y mice administered insulin (1.5 U/kg IP) at the end of the study with tissue harvested 90 minutes thereafter. Whole cell extracts were subjected to SDS-PAGE followed by western blot analysis using an anti-ubiquitin antibody. β-actin is included as a loading control. Statistical significance is compared to insulin-treated animals in (B). *p<0.05.</p