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5 Democrats seek Florida Senate 9 seat; Jason Brodeur awaits
mark.harper@news-jrnl.com
Five Democratic candidates are looking to turn a long GOP-held Florida Senate seat blue, while a well-funded former House Republican awaits in the general election.
THE CANDIDATES: All are Seminole County Democrats: Rick Ashby, Alexis Carter, Alexander Duncan, Guerdy Remy and Patricia Sigman. They are seeking a seat that includes all of Seminole and portions of southwest Volusia County, including DeBary, Orange City and Enterprise. The winner will take on Jason Brodeur, a former state representative from Sanford, who has amassed more than $700,000 in direct campaign contributions and has collected nearly $1 million more in political-action committee funds.
[READ ALSO: Florida Legislators' PACs Amass Hundreds of Millions of Dollars]
THE POSITION: There are 40 state senators who make up one of the two legislative bodies. Senators are elected to four-year terms and meet in Tallahassee for 60-day sessions in the winter or spring, plus in committee meetings and special sessions as called. Senators earn $29,697 annually.
NAME: Rick Ashby
RESIDENCE: Chuluota
PROFESSION: Engineer
POLITICAL EXPERIENCE: Community advocacy for environmental and social concerns.
ASHBY'S TOP THREE PRIORITIES: Expand Medicaid and implement a single-payer healthcare system. Raise minimum wage and index to inflation. Clean up Florida's lakes and rivers by implementing known methods of nutrient reduction.
NAME: Alexis Carter
RESIDENCE: Altamonte Springs
PROFESSION: Attorney and adjunct law professor
POLITICAL EXPERIENCE: Experience in advising government policy and budgets at the federal level as an Army lawyer and local experience as a community organizer.
CARTER'S TOP THREE PRIORITIES: Tackling COVID crisis by supporting changes to the unemployment system, economic relief for small business, and Medicaid expansion. Improving Florida schools by increasing salaries for all teacher and ensuring schools are properly funded. Promoting accountability and transparency in local policing and criminal justice reform.
NAME: Alexander Duncan
CITY: Geneva
PROFESSION: Private security contractor
POLITICAL EXPERIENCE: Mullet Lake Water Association, 2008-2019 (President from 2017-2019)
DUNCAN'S TOP THREE PRIORITIES: To establish accountability of elected officials in their service to citizens. To establish accessibility to elected officials and public servants for all citizens. To establish greater transparency in government and expanded open government laws to better serve citizens.
NAME: Guerdy Remy
PROFESSION: Nurse, healthcare case manager
POLITICAL EXPERIENCE: Activist
REMY'S TOP THREE PRIORITIES: Improve health care outcomes, fully fund public schools and protect vulnerable communities.
NAME: Patricia Sigman
RESIDENCE: Longwood
PROFESSION: Florida Bar Board Certified labor and employment lawyer
POLITICAL EXPERIENCE: Voter protection leader in Seminole County since 2004, recruiting, training, organizing and managing volunteer lawyers and non-lawyers in Seminole County to ensure every eligible voter can cast their ballot and have it counted.
SIGMAN'S TOP THREE PRIORITIES: Fully funding and supporting public education, making healthcare more affordable and accessible, protecting our water and environment.
THREE QUESTIONS:
1. The pandemic has exposed Florida's unemployment system as a failure. What should the Legislature do to hold the executive branch accountable and ensure an overhaul?
ASHBY: The Legislature appropriated the money to maintain and upgrade the state unemployment system. The problem is that the Republican Rick Scott administration implemented a system that was designed to fail and deny benefits. This is what we have to help the voters understand: Elections have consequences and if you want your state to function efficiently for your family, you have to elect people that want to do the job. That means electing progressive Democrats. If Andrew Gillum was our governor now, we would not have an unemployment claims issue and we would not lead the country in COVID-19 cases.
CARTER: I have firsthand experience as an attorney helping clients navigate the broken unemployment system. I support legislation outlining specific requirements for accessibility and operability of the online filing portal. I will also demand an increase to the current unemployment benefit amount which is 4th lowest in the country and has not been changed since 1999.
DUNCAN: The Legislature must first be bold enough to call the current unemployment system what it is: a byproduct of open corruption. A task force should be mandated to investigate and bring all persons found involved in any possible wrongdoing to justice. As potentially millions of Floridians have been left to suffer at the expense of the few, the rich and the powerful – when folks start going to jail for such actions, I can ensure citizens will feel reassured by such new leadership and other officials will quickly begin showing much more honesty in their own leadership.
REMY: Florida's Inspector General needs to investigate CONNECT. We need the DEO to get a new system that can handle more than 5,000 people at a time, because this system was designed to make it very difficult for people to apply for benefits. We need to continue payouts due to the system failure. The U.S. Senate Committee on Finance needs to investigate how the CARES Act unemployment payouts are being handled in Florida due to the largely reported deficiencies in the system. Finally the DEO needs to add additional servers, call centers with real staff and reopen the One Stop Centers.
SIGMAN: As a board-certified labor and employment lawyer, I'm uniquely informed, skilled and qualified to help fix our broken and gutted unemployment system. Some highlights include substantially extending the number of weeks and the amount of benefits; restoring the statutory presumption in favor of benefits; opening in-person service centers where people can get real assistance with their benefit claims and obtaining benefits; restoring in-person unemployment appeals; implementing genuine, helpful job training and job placement services; building a completely new, user-friendly and reliable website that serves the people well.
2. Assuming you believe the justice system could be improved for African-Americans in Florida, where would you start?
ASHBY: We need statewide standards for community policing as a baseline. Those would include accountability/liability and training standards. Certainly local communities will want to go above and beyond those and I would encourage them to do so. We have to divert non-violent drug and mental health-related offenses to treatment programs. These programs are generally less expensive than prison and they help our community members instead of needlessly punishing them. Sentencing guidelines need to be updated. These two programs working in tandem would represent significant change and a step forward in criminal justice for our Black communities.
CARTER: As a former public defender, racial inequity in the criminal legal system deeply resonates with me. I support changes to minimum mandatory sentencing, which has been a tool to benefit private prisons and overhaul Florida's direct filing policy which prosecutes more youth as adults than any other state. However, until we are ready to address the systemic impact that unequal access to education, housing, and employment has had on Black communities, we will continue to see disproportional representation of Blacks in the criminal legal system.
DUNCAN: I will fight for the full and complete implementation of Amendment 4, further judicial and criminal justice reforms and more. In my efforts to address racial inequity in Florida's criminal justice system, I will look to introduce from the top down, immediate prison reform — to include greater education, rehabilitation, mental health and family services; to end the use of solitary confinement and other measures. I believe the systematic racism that affects minorities must be addressed in the following areas also but, are not only limited to them: jury selection, mandatory minimum sentencing, drug laws and enforcement and socioeconomic disparities.
REMY: Create community service programs. Ban private prisons and cash bail so that those most affected will have a chance for justice and freedom. Allow our returning citizens the right to register to vote as per Florida's Amendment 4. Dismantle voter suppression and lengthen early voting. Decriminalize cannabis and expunge records for cannabis arrest. Work with organizations and local leaders to heal our Black and Brown communities that have been targeted for years from a racist, white supremacy system that was designed to prevent our Black and Brown communities from advancement. Work on diminishing redlining, rezoning, redistricting and gerrymandering.
SIGMAN: We need to reform our criminal justice system so it provides justice for all. We must listen to the voices and experiences of those who have been impacted by the system. Statistics show there is a disturbing disparity along racial lines when it comes to the enforcement of laws in Florida. Some improvements to start with are increased oversight of law enforcement agencies to ensure the disparate and harmful profiling of people of color is ended, putting discretion back into the hands of our judges by eliminating mandatory minimums for nonviolent and nonsexual crimes, and requiring education regarding implicit bias.
3. Most Democrats in Florida support expanding Medicaid to make more people eligible for federal Affordable Care Act funds. What is your best-case argument to convince Republicans that it's time for the state government to take on this burden?
ASHBY: The real burden is the nine people a day who are dying in Florida due to lack of healthcare. Our current system is not only the most expensive in the world, it is also the most unequal and immoral. It is never OK to deny someone the medical care or drugs that they need. We are sacrificing the lives of Floridians for profit and I will never say that is OK. I support implementation of a single-payer healthcare system that covers all Floridians. Finally, we will be No. 1 in the country instead of 40th in social metrics.
CARTER: In the midst of soaring unemployment and another expected wave of COVID, Medicaid expansion will make affordable healthcare a reality to 830,000 Floridians and support economic recovery. The $500 million dollar price tag for expansion is minimal compared to the $14.3 billion in Affordable Care Act funds that will bolster our economy over the next 5 years.
DUNCAN: That if we work in a bipartisan effort towards being honest in our leadership in the best interest of all citizens; and, are willing to be held accountable in our service to such, both through being accessible to citizens and transparent in the workings of government affairs; and, if as elected officials we are willing to employ simplicity in governance to facilitate greater community understanding of issues such as Medicaid expansion – there is no burden, that together we cannot resolve.
REMY: Medicaid expansion would insure almost 900,000 Floridians and would provide medical coverage that is much needed during this crisis. It would boost our economy by bringing health care jobs and help our communities with health care coverage. We are leaving $65.2 billion of federal Medicaid expansion on the table, while our communities are struggling between paying their utility bills and insulin. That is a shame, and I will make that one of my first bills that I want to revisit and hopefully get passed.
SIGMAN: We must make the economic case. Medicaid expansion is a major economic win for our citizens, bringing an estimated $14.3 billion in new federal dollars over five years against a cost of $517 million, netting our economy over $13 billion over 5 years while creating thousands of good-paying healthcare jobs for Central Floridians recovering from the pandemic. We would also insure nearly 850,000 Floridians, which not only provides better health outcomes and improves financial security, but also helps stabilize insurance premiums for those who have private health insurance.


Americans get fatter every year, and drug companies are racing to treat the resulting health problems--but first they have to prove to the U.S. Food and Drug Administration that their anti-fat drugs don't cause cancer. That hurdle is delaying the development of a particularly promising group of drugs for treating obesity-related health problems. These drugs are the PPAR agonists, which aim to trip master switches in the cell that control the absorption of fat and sugar in the bloodstream. By modifying these switches, called peroxisome proliferator activated receptors (PPARs), these drugs would help treat diabetes and heart disease before they become serious. PPAR agonists could become a major treatment for the bad diets, bad exercise habits and bad genetics that lead to heart attacks and dangerously high levels of blood sugar.


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Stem Cell Research & Therapy
Adipose-derived mesenchymal stem cells from patients with atherosclerotic renovascular disease have increased DNA damage and reduced angiogenesis that can be modified by hypoxia
Ahmed Saad ORCID: orcid.org/0000-0003-1861-45211,
Xiang-Yang Zhu1,
Sandra Herrmann1,
LaTonya Hickson1,
Hui Tang1,
Allan B. Dietz2,
Andre J. van Wijnen3,
Lilach Lerman1 &
Stephen Textor1
Stem Cell Research & Therapy volume 7, Article number: 128 (2016) Cite this article
Adipose-derived MSC (AMSCs) possess angiogenic and immunomodulatory properties that may modulate kidney regeneration. Whether these properties are retained in older patients with atherosclerotic vascular disease is poorly understood. Hypoxic conditions are known to modify properties and growth characteristics of AMSCs. We tested the hypothesis that AMSCs from older patients with atherosclerotic renovascular disease (RVD) differ from normal kidney donors, and whether hypoxia changes their functional and molecular properties to promote angiogenesis.
AMSCs from 11 patients with RVD (mean age =74.5 years) and 10 healthy kidney donors (mean age = 51.2 years) were cultured under normoxia (20 % O2) and hypoxia (1 % O2) for 3–4 days until they reached 80 % confluency. We analyzed expression of genes and microRNAs using RNA sequencing and real-time quantitative rt-PCR. Protein expression of selected angiogenic factors (VEGF, IGF, HGF and EGF) were quantified in conditioned media using ELISAs. Apoptosis was tested using Annexin IV staining.
Normoxic AMSC from RVD patients grew normally, but exhibited increased DNA damage and reduced migration. VEGF protein secretion was significantly lower in the RVD AMSCs (0.08 vs 2.4 ng/mL/ cell, p <0.05) while HGF was higher. Both trends were reversed during growth under hypoxic conditions. Hypoxia upregulated pro-angiogenic mRNAs expression in AMSCs (VEGF, FGF, STC and ANGPTL4), and downregulated expression of many miRNAs (e.g., miR-15a, miR-16, miR-93, miR-424, 126, 132, 221) except miR-210.
Thus, although AMSC from patients with RVD had increased DNA damage and reduced migration, hypoxia stimulated pro-angiogenic responses via increased expression of angiogenic genes, VEGF secretion and induction of the hypoxia-inducible miR-210, while downregulating angiogenesis-related miRNAs.
Occlusive atherosclerotic renovascular disease (RVD) reduces renal blood flow (RBF), eventually producing renal ischemia, tissue hypoxia, and oxidative stress [1, 2]. These changes lead eventually to microvascular dysfunction, activation of inflammatory pathways, and fibrosis [3, 4]. In experimental models, therapies based on adipose-derived mesenchymal stem/stromal cells (AMSCs) enhance microvascular growth and enhance kidney repair and functional recovery, particularly when combined with renal revascularization. Administration of AMSCs in the swine model of RVD is associated with increased vascular endothelial growth factor (VEGF) and suppression of inflammatory cytokines. These changes are accompanied by increased RBF and glomerular filtration rate (GFR), and reduced fibrosis in the post-stenotic kidney [5]. Although the exact mechanisms have not yet been elucidated, regenerative characteristics of MSCs are widely attributed to paracrine effects mediated through secretion of bioactive molecules and chemokines [6].
Potential barriers to clinical application of MSC-based therapy in the kidney include the likelihood for reduced functional capacity related to age, atherosclerotic disease, impaired kidney function and the milieu of diseased tissue. Stem cells are prepared and maintained in vitro with favorable growth conditions and abundant oxygen levels. However, the injured tissue milieu in vivo and surrounding the cells are often hypoxic, have increased levels of inflammatory mediators, and low pH. These parameters collectively may affect the efficacy of cell-based therapy [7–9]. MSC used presently for regenerative medical purposes potentially may undergo apoptosis prior to differentiation into the desired cell type due to these nonideal environmental conditions [10]. Aging, for example, is associated with a progressive decrease in the angiogenic capacities and regenerative potential of MSCs [11].
Strategies designed to modify the functional properties of MSCs may enhance specific features including survival, angiogenesis and anti-inflammatory activity. Through the manipulation of specific genetic targets, many cellular processes can either be enhanced or suppressed. These changes can give the stem cell a powerful advantage in the ability to survive in the face of hostile conditions within the tissue. One approach to improving efficacy of MSCs has been to grow them under hypoxic conditions mimicking the in vivo environment in ischemic tissues like RVD in which oxygen tensions may be 1 % or lower. When cultured under 'hypoxic' conditions (typically 1–4 % oxygen), MSCs demonstrate upregulation of numerous functions, including growth factor receptors and migratory capacity [12]. When infused into acutely infarcted heart experimentally, hypoxic preconditioned MSCs perform better than unconditioned control cells leading to improved cardiac function and reduced infarct size [13, 14]. However, the exact mechanisms by which hypoxia exerts its effects remain unclear and whether hypoxic conditions modify AMSCs from elderly patients with atherosclerosis are poorly understood.
The purpose of this study was to examine the differences between AMSCs from RVD patients compared to healthy subjects (normal kidney donors) and the effects of hypoxia on the functional properties of AMSCs, including survival and angiogenic capacity. Our hypothesis was that functional characteristics of AMSCs from patients with RVD including angiogenic, protein expression, and paracrine effects differ from healthy individuals. We further hypothesized that hypoxic growth conditions could modify the angiogenic and functional characteristics of AMSCs from RVD patients. We addressed these two hypotheses by evaluating the biological properties of AMSCs, as well as by comparing expression of protein-coding mRNAs and regulatory noncoding microRNAs (miRNAs) under normal and hypoxic conditions.
AMSCs were obtained from patients with atherosclerotic RVD (n = 11) and healthy volunteers (n = 10) (Table 1). The RVD patients were enrolled for autologous AMSCs infusion as a part of separate treatment protocol under an Investigational New Drug study (FDA 15082, clinicaltrials.gov). Inclusion criteria for RVD were ages between 40 and 80 years, advanced vascular occlusive disease affecting one or both kidneys defined as loss of parenchymal volume and severe renal artery stenosis measured by duplex ultrasound velocity above 300 cm/sec to the affected kidney and serum creatinine below 2.5 mg/dL. The healthy volunteers were kidney donors undergoing laparoscopic donor nephrectomy with age ranging from 20 to 40 years, as young healthy and another group from 40 to 80 years old considered as old healthy control, all of them without chronic health problems.
Table 1 Clinical and demographic data of Donors and RVD patients
Isolation and culture of AMSCs
A subcutaneous fat biopsy (approximately 2–4 g) was obtained under sterile conditions from RVD patients in an outpatient surgical suite or during the surgical nephrectomy from kidney donors. Cells were expanded ex vivo as follows. The fat tissues were minced with surgical scalpels and incubated in 2 % collagenase type H (Roche, Mannheim, Germany) for 90 min at 37 °C. Digested tissue was centrifuged at 400 g for 5 min with the pellet washed in PBS, passed through a 70-μm cell strainer (BD Biosciences, San Jose, CA, USA), and incubated in red blood cell lysis buffer (154 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA). Cells were grown in T-75 cm2 flasks at a concentration of 1.0–2.5 × 103 cells/cm2 in Advanced MEM with 5 % PLTmax (Mill Creek Life Sciences, Rochester, MN, USA) and 2 mM L-glutamine (Invitrogen, Carlsbad, CA, USA) in a 37 °C 5 % CO2 incubator for 3–4 days. When cells were 60–80 % confluent, they were passaged using TrypLE (Trypsin-like Enzyme, Invitrogen) [15]. Cell yield was quantified with a Countess hemocytometer (Thermo Fisher Scientific, Inc., NY, USA).
Experimental design of the hypoxia protocol
Cultured AMSCs were maintained (passage 3–5) under normoxic (20 % O2) or hypoxic conditions (1 % O2) for 3–5 days. Hypoxia was achieved by placing cells in a Modular Incubator Chamber (Billumps-Rothenberg; Del Mar, CA, USA) that was flushed with a mixture of 1 % O2, 5 % CO2, and 94 % N2, confirmed by an infrared gas analyzer (Novametrics, Wallingford, CT, USA).
Flow cytometry characterization of AMSCs
Isolated AMSCs were characterized by immunostaining and fluorescence-activated cell sorting (FACS) analysis to determine cellular phenotype for the MSC markers CD34, CD14, CD45, CD44, CD73, CD90, CD105 (1:100; Abcam, Cambridge, MA, USA and BD Pharmigen, San Jose, CA, USA). Population-doubling time (PDT) was computed by linear regression of log2 values of cell number.
High-throughput RNA sequencing
Total RNA was isolated from confluent AMSCs of three RVD patients (RVD-1, -2 and -4) and three healthy controls (Healthy-1, -5 and -7) both under normoxia and hypoxia, to characterize the basal state of expression by high-throughput RNA sequencing using the TruSeq method ("poly A RNA Seq") as previously described [16]. RNA libraries were prepared according to the manufacturer's instructions for the TruSeq RNA Sample Prep Kit v2 (Illumina, San Diego, CA, USA). The miRNA-Seq data were analyzed using CAP-miRSeq v1.1 [17]. Normalization and differential expression analysis were performed using edgeR 2.6.2 [18].
mRNA expression analysis
Expression values for each gene were normalized to 1 million reads and corrected for gene length (reads per kilo base pair per million mapped reads, RPKM). Genes with RPKM >0.1, fold change (hypoxia/normoxia) >1.4 and p values <0.05 (hypoxia vs. normoxia, Student's t test) were classified as genes enriched in hypoxia [16].
miRNA expression analysis
miRNA expression levels (normalized total reads) of AMSCs were examined under normoxic and hypoxic conditions, and the fold change enrichment was calculated. We used miRDB (Version 6.2) to predict target genes for miRNA with fold change >1.4 and p values <0.05 (Student's t test) [19].
Measurement of miRNAs by quantitative real-time PCR
To confirm the results from miRNA-Seq, we selected some miRNAs of interest to measure using PCR. Total RNA was isolated from AMSCs by the mirVana PARIS total RNA isolation kit (Life Technologies, Carlsbad, CA, USA, Cat# AM1556) according to the kit protocol. RNA concentrations were measured by a NanoDrop Spectrophotometer (NanoDrop, Thermo Fisher Scientific, Inc.). A fixed volume of 5 μL of RNA elute at 1 ng/uL was reverse transcribed by using the TaqMan MicroRNA Reverse Transcription Kit (Life Technologies, Cat# 4366596). For PCR, 1.33 μL of RT product was combined with 10 μL of TaqMan Universal Master Mix (Cat# 4440038), 7.67 μL of H2O and 1 μL of primers, including miR-21, miR-146a, miR-155, and miR-210 (Life Technologies, Cat# 000397, 001097, 002623, and 000512 respectively) to make up a 20 μL reaction. RNU6B (Life Technology Cat# 001093) was included in the assay as reference control. Real-time PCR was carried out on an Applied Biosystems (Foster City, CA, USA) ViiA7 Real-Time PCR system at 50 °C for 2 min, 95 °C for 10 min and 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Fold changes of miRNA levels in hypoxia relative to normoxia were calculated using the 2−ΔΔCt method.
Conditioned media aliquots were collected before harvesting the cells and stored at −80 °C until they were assayed. Levels of VEGF (Life Technology KHG0111), epidermal growth factor (EGF R&D Systems, Minneapolis, MN, USA, DEG00), hepatocyte growth factor (HGF, R&D Systems, DHG00) and insulin-like growth factor 1 (IGF-1, R&D Systems, DG100) were measured by enzyme-linked immunosorbent assays (ELISAs) according to manufacturer protocols.
Cell viability, migration, and apoptosis
Cell viability was detected by trypan blue staining [20] that identifies cells in which the cell membrane is compromised. Cells were diluted 1:100 in 0.25 % trypan blue solution (Invitrogen) and counted in a hemocytometer to assess the number of dead blue cells from the total number of cells counted. An additional measure of cell viability was obtained using FACS for Annexin V. After harvesting the AMSCs, cells were centrifuged for 5 min at 1200 rpm.
Apoptotic cells were detected using APC Annexin V/Dead Cell Apoptosis Kit with APC Annexin V and SYTOX® Green (Invitrogen, Eugene, OR, USA). Cells were resuspended at a concentration of 1 × 106 cells/mL in 1X Annexin-binding buffer; 5 μL APC-Annexin V and 1 μL of the 1 μM SYTOX® Green stain working solution were added to each 100 μL cell suspension. The cells were incubated at 37 °C in an atmosphere of 5 % CO2 for 15 min, and then analyzed by flow cytometry [21]. DNA damage that is associated with cellular senescence and migration were each measured by western blot using, respectively, H2AX antibodies that detect nuclear sites with telomere loss or other DNA damage, and the EMD Millipore (Billerica, MA, USA) QCM™ 24-well chemotaxis cell migration assay (Cat# ECM508) as described [22, 23].
Student's t test was used to compare data between the two experimental groups. Genes with RPKM >0.1, fold change (hypoxia/normoxia) >1.4 and p values <0.05 (hypoxia vs. normoxia, Student's t test) were classified as genes enriched in hypoxia. MiRDB (Version 6.2) was used to predict target genes of miRNA with fold change >1.4 and p values <0.05 (Student's t test). Data are expressed as the mean ± SD. p <0.05 was considered statistically significant.
Patient demographics: data obtained from AMSCs from 11 patients with RVD and 10 healthy volunteers are summarized in Table 1.
Characteristics of human AMSCs in RVD patients under normoxic conditions
Human AMSCs from adipose tissue from both healthy and RVD patients retained characteristic plastic-adherent, fibroblast-like morphology, expressed HLA-ABC, CD44, CD90, CD29, CD73, and CD105 markers, and did not express HLA-DR, CD45, CD14 or CD34 markers either under normoxia or hypoxia (Additional file 1: Figure S1). The morphology of cells grown under hypoxia differed slightly in some individuals (both healthy and RVD), insofar as the cells appeared larger and more granular (Fig. 1). VEGF secretion in the supernatant of RVD AMSC at normoxic conditions was markedly lower compared to healthy controls (p <0.05), whereas HGF levels were slightly higher (Fig. 2a). These changes were mainly due to age rather than vascular disease, as cytokine secretion (IGF, HGF and EGF) was not different in RVD compared to older healthy subjects except for VEGF (Fig. 7). Compared to healthy volunteers (p <0.05), AMSCs from patients with RVD had higher levels of nuclear foci for the phosphorylated form of histone variant H2AX (Fig. 2b). This protein is recruited to sites of DNA damage and phosphorylated by DNA repair kinases, which is particularly evident in aging cells that lose telomeres prior to becoming fully senescent. Under normoxia, cell viability (measured with trypan blue) and percentage of apoptotic cells (measured by Annexin V) were similar in both groups (Fig. 2c). The AMSCs from patients with RVD had lower migration capacity compared to healthy volunteers (Fig. 2d).
Morphologic appearance of AMSCs grown under normoxic and hypoxic conditions from healthy kidney donors (top row) and older individuals with atherosclerotic RVD (lower rows). Although growth rates were not changed under hypoxic conditions, the morphological appearance of AMSCs was different after hypoxia compared with normoxia (left, normoxia; right, hypoxia). The hypoxic AMSCs appeared bigger, denser and more granular (higher magnification - bottom row). AMSCs adipose-derived mesenchymal stem/stromal cells, RVD renovascular disease
Functional differences between RVD AMSC expression and normal donors: VEGF secretion from AMSCs was low in RVD while HGF secretion is a higher compared to healthy controls (a). Senescence-associated DNA damage was increased in RVD patients compared to healthy individuals (b). The percentage of apoptosis was similar between RVD and healthy donors (c). AMSCS from RVD patient had lower migratory capacity compared to healthy control (d). HGF hepatocyte growth factor, RVD renovascular disease, VEGF vascular endothelial growth factor
Hypoxia upregulated survival and pro-angiogenic gene expression of RVD AMSCs
Expression of more than 3050 mRNAs (out of 23,398 mRNAs) was different in RVD AMSCs vs. healthy volunteers (fold change >1.4; p values <0.05; RVD vs. healthy volunteers). Expression of more than 460 genes with adequate basal levels (RPKM >0.5) was increased under hypoxic conditions (fold change >1.4; p values <0.05; hypoxia vs. normoxia) This set of mRNAs encode regulators of metabolism, DNA repair, cell cycle regulation, and proteins involved in related functions associated with cell survival (Additional file 2: Figure S2). Among these were Cyclin-dependent kinase inhibitor 1A (CDKN1A), TMEM45A, Nox4, BNIP3, SLC2A1 or glucose transporter 1 (GLUT1), carbonic anhydrase 9 (CA 9) and heme oxygenase 1 (HMOX1) genes associated with increased cell survival and anti-apoptotic roles that were highly expressed under hypoxia in both healthy and RVD AMSCs (p <0.05) (Fig. 3a). Hypoxia decreased the expression of protein tyrosine phosphatase, non-receptor type 2 (PTPN2) that negatively regulates numerous signaling pathways and biological processes including cell proliferation and cell growth. Hypoxia increased the expression of BNIP3 and ATG9b, which are known to induce mitochondrial autophagy as well as the pro-angiogenic genes VEGF, FGF, Stanniocalcin-1, -2 (STC 1, 2) and angiopoietin-like 4 (ANGPTL4) (p <0.05) (Fig. 3b,c) both in RVD and healthy donors but to a higher degree in RVD. HGF expression levels were lower under hypoxia in RVD and healthy AMSCs. These data indicate that hypoxia modulates the biological functions of AMSCs and expression of a number of paracrine factors.
Upregulation of gene expression of AMSCs under hypoxia in both RVD and healthy individuals: hypoxia upregulated pro-survival genes (CDKN1A, HMOX1, TMEM45A, CA9, SLC2A1/ GLUT1, and the autophagy regulatory gene BNIP3) (a). Hypoxia upregulated pro-angiogenic genes (ANGPTL4 and VEGF-a) (b) and (STC1, STC2, and FGF2) (c). RVD renovascular disease
Hypoxia maintained AMSCs cell viability and proliferation without affecting apoptosis
Levels of viable cells from donors/RVD patients were above 90 % as measured with trypan blue. Although, the percentage of apoptosis (measured by Annexin V) in both groups did not change with hypoxia, the individual differences between donors and/or RVD AMSCs were significant (Fig. 4). While it was minimal, the differential effects of hypoxia on cell viability for AMSCs from RVD patients indicate that the diseased state may have favored adaptation of AMSCs to low oxygen tolerance.
Differential effects of hypoxic conditions on RVD versus normal kidney donors. Graphs showing within-individual differences and changes in percent of apoptosis and death under hypoxia in RVD (a) and healthy control (b). The percentage of apoptosis (measured by Annexin V) in RVD and healthy controls did not change under hypoxia. However, hypoxia increased cell death levels only in the healthy group (c and d). AMSCs adipose-derived mesenchymal stem/stromal cells, RVD renovascular disease
miRNAs regulating survival and pro-angiogenic genes were downregulated under hypoxic conditions
There were 415 out of 2253 miRNAs that were different in RVD from healthy donors under normal conditions (FC >1.4). We detected more than 360 miRNAs with acceptable basal levels (normalized reads >0.2) and differentially expressed in hypoxia (fold change >1.4; p values <0.05; hypoxia vs. normoxia). The majority of these miRNAs (approximately 85 %) were downregulated under hypoxia, with the most notable exception being the pro-angiogenic miR-210 (that increases VEGF levels), which was highly upregulated (fold change FC >65, in RVD cells). miRNA-210 was also increased in healthy control AMSCs under hypoxia (2.9 FC) but to a lesser degree than was seen in RVD. Baseline miR-210 was low in RVD compared to healthy control AMSCs (Fig. 5a). The miRNAs that exhibited reduced expression under hypoxia, included pro-apoptotic miRNAs (miR-34a, miR-15 and miR-16), senescence-associated miRNAs (miR-10a and miR-21) and anti-angiogenic miRNAs (miR-221/222, miR-24, miR-29, miR-15, miR-16, miR-17, miR-424, miR-20b, miR-218, miR-132, miR-92a, and miR-101) (Fig. 5b). The latter miRNAs may negatively modulate angiogenesis by repressing VEGF expression under normoxic conditions. For example, reduction of miR-15, miR-16, and miR-424 contributes to increased VEGF expression [24–26]. Downregulation of miR-221 correlates with an increase in its target gene expression CDKN1A in our study (Fig. 3a), consistent with previous observations [27]. There were not similar effects of hypoxia on most miRNAs from healthy donors AMSCs 'both young and old'.
Hypoxia changed the expression of miRNAs regulating survival and pro-angiogenic genes: hypoxia upregulated the pro-angiogenic miRNA-210 (65 FC in RVD, but to a lesser degree in healthy control 2.9 FC) (a), whereas downregulated pro-apoptotic miRNAs (miR-34a, miR-15, and miR-16) and also downregulated antiangiogenic miRNAs (miR-221/222 and miR-424) only in RVD (b). AMSCs adipose-derived mesenchymal stem/stromal cells, RVD renovascular disease
Hypoxia increased the posttranscriptional protein secretion of VEGF but decreased HGF
Hypoxia increased the secretion levels of VEGF in the supernatant of RVD AMSCs (p < 0.0001) and AMSCs from old healthy individuals (p = 0.007) to levels seen in young healthy cells from donors. There was no such effect of hypoxia in young donor AMSCs (p = 0.35). Conversely, hypoxia decreased HGF protein levels in the supernatant both in RVD and healthy donors (Fig. 6), again, this effect was predominately in old individuals (p = 0.01 vs. p = 0.06, old vs. young). IGF and EGF secretions tended to increase under hypoxia. Hypoxia increased secretions of IGF, EFG, and VEGF and decreased HGF from old healthy AMSCs to normal levels seen in young healthy cells (data not shown).
Hypoxia increased the posttranscriptional protein secretion of VEGF (a) but decreased protein secretion of HGF (b) in RVD AMSCs. In AMSCs from healthy controls, hypoxia did not change VEGF (c) but decreased HGF (d). Graphs showing within-individual changes in VEGF and HGF secretions under hypoxia in RVD, healthy control. HGF hepatocyte growth factor, RVD renovascular disease, VEGF vascular endothelial growth factor
The results of this study identify considerable differences between AMSCs from patients with atherosclerotic RVD and those from healthy controls. Some of these differences were related to older age, but were magnified by disease. Platelet lysate-expanded AMSC from RVD patients exhibited greater levels of senescence-associated DNA damage and reduced migration capacity as compared to AMSC from healthy individuals. Importantly, expression of angiogenic proteins such as VEGF and EGF was reduced in RVD patients, although of HGF levels were higher. Expansion of AMSC from RVD subjects under hypoxic conditions largely reversed the differences seen under normoxic conditions. Hypoxic conditions resulted in a more robust increase in VEGF, which is a key stimulant for angiogenesis. Senescence-associated DNA damage was increased in elderly patients with RVD compared to healthy individuals. Hypoxia enhanced AMSC survival as reflected by a reduced fraction of dead cells in some patients. The angiogenic functions of RVD AMSCs increased to normal levels during hypoxic expansion as reflected by enhanced expression of angiogenesis-related mRNAs (Fig. 3b), alterations in relevant miRNAs and increased secretion of VEGF, IGF and EGF cytokines.
Hypoxic growth has been employed as a strategy to enhance stem cell in vivo survival and tissue regeneration [10]. Our results indicate that these conditions upregulate angiogenic factors and downregulate apoptotic effector pathways. MicroRNAs regulate cellular functions by translational repression and/or mRNA degradation. We focused on common miRNAs known to regulate survival and angiogenesis and their target gene and posttranscriptional protein expressions. Under hypoxia, miR-210 is most consistently and robustly induced [28]. MiR-210 mediates the proliferation and migration under chronic hypoxia and ultimately leads to downregulation of PTPN2. Consistent with previous studies, which showed that inhibition of PTPN2 (a direct target gene of miR-210) lead to increased proliferation and migration of AMSCs [29], the AMSC from RVD patients under hypoxia in our study highly expressed miR-210 (FC > 65) and downregulated PTPN2.
Our results demonstrate further that hypoxia reduced the expression of miR-34a (FC > -3). Enhanced expression of miR-34a induces apoptosis, cell cycle arrest and differentiation, or reduces migration [30, 31]. Reducing the expression of the pro-apoptotic miR-34a improves survival of bone marrow stem cells in vitro and enhances the therapeutic benefit of cell therapy in mice after acute myocardial infarction [32]. Previous studies have shown that suppression of miR-10a and miR-21 in aged endothelial progenitor cell (EPCs) increase HMGA2 expression, rejuvenate EPCs, resulting in decreased senescence [33]. Hypoxia downregulated both miR-10a (FC > -2) and miR-21 (FC > -3), but without much change in HMGA2 in RVD. CDKN1A is a gene known to regulate cell survival and growth [34] and is regulated by miR-221-3p and miR-93 [35]. Similarly, our results showed that hypoxia downregulated miR-221 (FC > -2) and miR-93 (FC > -2), CDKN1A was upregulated, thereby potentially promoting survival of AMSCs.
Another important gene overexpressed under hypoxia was HMOX1, which is known to have anti-inflammatory effects and may improve survival in the ischemia/reperfusion-acute kidney injury microenvironment. Liprin-α4, which is required for maintenance of cell-cell contacts [36], is a hypoxia-inducible gene that was highly expressed in AMSCs under hypoxia (FC >100). TMEM45A has anti-apoptotic functions and is essential for hypoxia-induced protection against apoptosis [37]. We found that TMEM45A was overexpressed under hypoxia in RVD AMSC. Taken together, these changes in mRNA and miRNA indicate that hypoxic conditions boosted cell survival and amplified angiogenic and anti-inflammatory functions of AMSC from atherosclerotic patients.
Autophagy modulates homeostatic and cytoprotective physiological cellular functions, such as degradation of long-lived proteins, organelle turnover, adaptation to stress, extension of lifespan, and cellular development [38]. Recent studies identify crucial functions of BNIP3 and BNIP3L in hypoxia-induced autophagy and indicate that hypoxia impacts cell survival. BNIP3 family disrupts the Bcl-2/Beclin1 complex and induces autophagy, especially when Bcl-2 or Bcl-XL is weakly expressed [39, 40]. In our study, BNIP3 was highly expressed under hypoxia (FC >7), while Bcl-2, Bcl-KL and Beclin1 were downregulated. We suggest that hypoxia-induced autophagy may promote survival, block induction of apoptosis and reduce hypoxic cellular injury, because survival rates between cells grown under normoxia versus hypoxia were comparable, while hypoxia reduced the percentage of dead cells in most RVD patients.
Angiogenesis potentially includes restoration of normal vascular function and structure, and the reversal of vascular senescence. Growth of new blood vessels is crucial in the treatment of RVD and other ischemic diseases [41]. Recent experimental studies indicate that renal VEGF levels are altered in pathologic situations, such as chronic and acute renal ischemia [42]. Our results indicate that AMSC from patients with RVD secreted less VEGF compared to healthy individuals in the AMSC supernatant (Fig. 7). Increased expression of angiogenic factors, including the upregulation of VEGF, has previously been shown to increase blood vessel formation in vivo [43]. Elevated expression of miR-210 induces angiogenesis and is associated with local increased VEGF levels [44]. Reduction of miR-15b and miR-16 contributes to an increase in VEGF and improve angiogenesis [45, 46]. Stanniocalcin-1 and -2 (STC 1, 2) promote angiogenic sprouting in human umbilical vein endothelial cells (HUVECs) via VEGF/VEGFR2 and angiopoietin signaling pathways [47]. Consistent with these data, we observed that hypoxia downregulated miR-101b and miR-29a and increased the expression of their target genes STC1 and STC2 in AMSCs.
AMSCs from older healthy donors and RVD patients both showed lower secretion of VEGF, IGF and EGF compared to AMSCs from young healthy donors. These data suggest that at least some of the differential effects between healthy donor subjects and RVD are related primarily to differences in age (see text). EGF epidermal growth factor, IGF insulin-like growth factor; HGF hepatocyte growth factor, RVD renovascular disease, VEGF vascular endothelial growth factor
Recent studies show that human miR-221 and miR-222 are important in vascular biology and exhibit marked antiangiogenic properties. They are upregulated in early atherosclerosis, causing inhibition of angiogenic recruitment of endothelial cells (ECs). These miRNAs increase endothelial dysfunction and EC apoptosis and negatively regulate VEGF [48, 49]. We showed in this study that hypoxia decreased the expression of miR-221 (FC approximately -2) and miR-222 (FC > -1.4) and increased expression of their known common target gene CDKN1.
HGF is an angiogenic growth factor that plays an important role in angiogenesis. Its level was increased in AMSCs from RVD compared to healthy donors. HGF is thought to increase the collateral vascular growth (arteriogenesis) [50], although its role is insufficient to protect vascular integrity in RVD. VEGF alone seems to affect capillary angiogenesis more efficiently than collateral growth [51]. Regulation of local HGF production under hypoxia is unclear, with most reports suggesting that it increases under hypoxia [12, 52]. However, our results indicate that HGF was downregulated under hypoxia both relative to gene expression and protein secretion. Our results are supported by the studies by Hayashi and colleagues [53] who demonstrated that hypoxic treatment of vascular cells downregulated HGF production due to decreased cAMP, consistent with their potential role in the pathophysiology of ischemic diseases.
Taken together, our results demonstrated that growing AMSCs from RVD patients under hypoxic conditions (1 % O2) altered their cellular characteristics, mRNA and protein expression and increased both survival and angiogenic potential. Oxygen concentration in medullary compartments of the kidney is normally low [30], and in kidneys with severe RVD accompanied by inflammation, the degree and extent of hypoxia increase [6, 31]. These studies suggest that functional responses of AMSC under hypoxic conditions may favorably influence their efficacy for renal repair. These effects were associated with increased secretion of several cytokines crucial for angiogenesis and survival such as VEGF.
AMSCs obtained from patients with atherosclerotic RVD are different and less angiogenic from those obtained from healthy individuals. Our data support the concept that age and/or disease-related disturbances in AMSC function may be modifiable. Physiological approaches like hypoxia that normalize the biological properties of stem cells that were harvested under pathophysiological conditions (e.g., elderly RVD patients) will be important to improve cell survival and functionality, as well as permit their adaptation for novel therapies for occlusive vascular disease.
AMSCs:
adipose-derived mesenchymal stem/stromal cells
ECs:
EGF:
epidermal growth factor
GFR:
HGF:
hepatocyte growth factor
IGF-1:
insulin-like growth factor 1
PDT:
population-doubling time
RBF:
renal blood flow
RPKM:
reads per kilo base pair per million mapped reads
RVD:
renovascular disease
VEGF:
vascular endothelial growth factor
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This project was partly supported by National Institutes of Health (NIH) grants, including P01 HL85307 from the National Heart, Lung and Blood Institute (NHLBI), R01 DK100081, DK10232, and R01 DK 73608 from the National Institute for Digestive, Diabetic and Kidney Diseases (NIDDK), as well as Clinical and Translational Science Award (CTSA) Grant UL1 RR024150 from NIH/ National Center for Research Resources (NCRR). Our studies were also supported by funds from the Center of Regenerative Medicine at Mayo Clinic. Furthermore, we appreciate the generous philanthropic support of William and Karen Eby, as well as the charitable foundation in their names. The content is solely the responsibility of the authors and does not represent the official views of the NHLBI, NIDDK or the National Institutes of Health.
AS designed all experiments, was responsible for the collection, assembly, and management of data, performed the experiments, wrote the manuscript draft and contributed to the final approval of the submitted manuscript. SCT was involved in the conception and the design of the study, and the analysis and interpretation of the data, contributed to the manuscript draft, and contributed to the final approval of the submitted manuscript. HT supported the laboratory work, supervised the sample analysis, was responsible for sample storage, revised the manuscript draft critically, and approved the final version of the manuscript. LL was involved in the design of the study and contributed to the manuscript draft, and contributed to the final approval of the submitted manuscript. XYZ, SH and LH revised the manuscript draft critically, and approved the final version of the manuscript. AD was responsible for the conception and the design of the study, revised the manuscript draft critically, and approved the final version of the manuscript. AvW involved in the conception of the study, was responsible for analysis of the mRNA data, revised the manuscript draft critically, and approved the final version of the manuscript. All authors read, critically revised and approved the final manuscript.
Ethical approval and consent to participate
The study involving human material was approved by the Ethics Committee and the institutional review board of the Mayo Clinic. Written informed consent was obtained from all participants prior to the study.
Division of Nephrology and Hypertension, Mayo Clinic, Rochester Minnesota, 200 First Street SW, Rochester, MN, USA
Ahmed Saad, Xiang-Yang Zhu, Sandra Herrmann, LaTonya Hickson, Hui Tang, Lilach Lerman & Stephen Textor
Division of Transfusion Medicine, Mayo Clinic, Rochester Minnesota, 200 First Street SW, Rochester, MN, USA
Allan B. Dietz
Department of Orthopedic Surgery, Biochemistry and Molecular Biology, Mayo Clinic, Rochester Minnesota, 200 First Street SW, Rochester, MN, USA
Andre J. van Wijnen
Ahmed Saad
Xiang-Yang Zhu
Sandra Herrmann
LaTonya Hickson
Lilach Lerman
Stephen Textor
Correspondence to Stephen Textor.
Additional file 1:
Representative flow cytometric plots (FACS) showing that AMSCs expressed HLA-ABC, CD44, CD90, CD29, CD73, and CD105 markers, and did not express HLA-DR, CD45, CD14 or CD34 markers. (JPG 233 kb)
Hypoxia increased the expression of more than 460 genes. This set mRNAs encode regulators of metabolism, DNA repair, cell cycle regulation, and proteins involved in related functions associated with cell survival. (JPG 68 kb)
Saad, A., Zhu, XY., Herrmann, S. et al. Adipose-derived mesenchymal stem cells from patients with atherosclerotic renovascular disease have increased DNA damage and reduced angiogenesis that can be modified by hypoxia. Stem Cell Res Ther 7, 128 (2016). https://doi.org/10.1186/s13287-016-0389-x
Revised: 27 July 2016
DOI: https://doi.org/10.1186/s13287-016-0389-x
VEGF and MicroRNAs

redpajama_set_name: RedPajamaCommonCrawl


Using the traditional "Rice Krispie Treats" recipe, substitute corn flakes for Rice Krispies, add 2 tablespoons of green food coloring and mix thoroughly.
Place tablespoon sized portions of your "wreaths" on waxed paper. Sprinkle with red sprinkles OR cinnamon candies. Chill for 1 hour and they're ready to serve!
It looks like you have Christmas wreaths... but they're yummy treats!
What a great idea! It sounds so easy!


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ESCBubble – Eurovision news, straight from the bubble! - Eurovision news, straight from the bubble!
Gliwice-Silesia 2019
Bulgaria 2015
Semi Final 1
Daniel Kajmakoski releases the first single from his first album!
Source: TAA
ImageEurostarsEurovision 2015Media
By Daria Syrkova	 Last updated Jul 23, 2015
Daniel Kajmakoski, who represented Macedonia in Eurovision Song Contest 2015, released his first single from his upcoming first album. The song is called "Decenije" (in Serbian/Croatian) or "10 Leta" (in Macedonian).
This is a summer tune with a nice guitar sound, that you can expect from a Balkan singer, but as in every Daniel's song you can hear something "extra Balkan". The song was written in collaboration with Aleksandra Milutinovic (music, lyrics and guitars), Darko Dimitrov (arrangement and production, back vocals); translation & adaptation of Macedonian lyrics was performed by Ognen Nedelkovski.
You can also watch the video, which was filmed a couple of weeks ago. Feel a bit of sunny and warm summer. And love, of course! This is what you need for a perfect July!
Daniel Kajmakoski is one of the most popular Balkan singers nowadays – thanks to X Factor Adria! In the spring of 2014 Daniel won the first series of the show, and a few moths later in November 2014 he won the ticket to Vienna (which is his home city as well) to represent his country – Macedonia – in Eurovision 2015! Although he didn't qualify to the final, an army of his fans has become bigger. Balkan charm mixed with the European background has made him quite a unique singer, who can sing both typical Balkan tunes and international ones. You can hear it yourself in the video below, which was taken at his audition for the above mentioned show:
We also found Daniel as a very nice person, when we took an interview with him in Vienna 2 months ago. Let's watch it once again!
The summer is still on so we expect more new songs from the Eurovision artists. Stay tuned!
Daniel KajmakoskiEurovision Song Contest 2015Macedoniasummer hitДаниэл Каjмакоскимакедониjа
Daria Syrkova 70 posts 1 comments
I've been into Eurovision since 2000 when Alsou made a huge breakthrough for Russia on Eurovision stage. Since 2008 I started to follow Eurovision national selections as well, and it was a point of no return. Thanks to this Contest I discovered a lot of good music and also many (if not most) of my friends. It's difficult to name my favorite song but I prefer guitar or ethnic music. But I can definitely name the capital city, flag colors and best Eurovision entry for each country!
Kate Ryan is back with 'Runaway'
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JP Morgan held the top rank in the latest M&A league table of the top 10 financial advisers for the information and communication technology (ICT) sector for FY2018, compiled by GlobalData, a leading data analytics company.
The New York-based financial institution took the top spot with a total advised deal value of $236.4bn for advising on 62 deals—the second highest in the sector in 2018.
According to GlobalData, which tracks all merger and acquisition, private equity/venture capital and asset transaction activity around the world to compile the league table, Morgan Stanley and Goldman Sachs stood second and third, advising on deals worth a total of $232.8bn and $206.9bn, respectively.
The ICT sector witnessed a surge of over 69.38% in value from $400.9bn in 2017 to $679.1bn in 2018. Volumes too rose by 31.12% from 10,302 in 2017 to 13,508 in 2018.
JP Morgan, which topped the ICT Deals league table, occupied second in the global league table of top 20 M&A financial advisers released by GlobalData recently.
Goldman Sachs, which is placed third in the ICT Deals league table, ranked first in the global list.
US-based law firm Skadden Arps Slate Meagher & Flom led the top 10 legal advisers table for FY2018 with a value of $141.8bn. Despite advising on the highest number of deals at 77, Goodwin Procter occupied tenth position with a value of $81.4bn. Skadden Arps Slate Meagher & Flom also topped the global league table of top 20 legal advisers for FY2018.


text:
How Chateau Tumbleweed Makes Such Eye-Catching Labels
Kris Pothier
Robert Isenberg | May 12, 2017 | 6:00am
When you see a bottle of Chateau Tumbleweed in the wine aisle, you might do a double-take. Whoa! you think. Those people have no heads!
Well, not no heads. Their crania have been replaced with a scraggly bunch of tumbleweed, drawn in intricate pen-and-ink. It's creepy and funny at the same time, and you have to wonder where such a surreal idea came from.
It's Wine O'Clock: 48 Hours in the Verde Valley Is an Intoxicating Experience
"We started to develop these wines as people, as family members," says Kris Pothier, co-owner of Chateau Tumbleweed and the artist behind its eccentric labels. "We came up with images that feel like the wines feel. It's fun to come up with fresh content all the time. This is a pretty competitive industry, and you want to create something that everyone is talking about."
The winery is a small business based in Cottonwood. Pothier started Chateau Tumbleweed in 2011 with her husband, Joe Bechard, and fellow couple Kim Koistinen and Jeff Hendricks. In the half-decade since, their unlikely startup has become something of a local legend. The Arizona wine industry is still pretty modest, despite its own Wine Growers Association. Chateau Tumbleweed has gained some attention as a startup run by Gen Xers, and the labels have caught extra attention.
"The four of us riff really well together," says Pothier. "That's what makes our business really tight. We sit together and drink wine and come up with these funny names. I used to make silly line drawings of things Joe said when he was drunk. Suddenly, we were making wine, and we needed to come up with a label."
Now 42, Pothier hails from the Northwest and has a lively bohemian personality to match. After growing up in Seattle, Pothier and Bechard lived in Eugene, Oregon, where they enjoyed the college town's rich and offbeat culture.
"We used to have this group of friends to party with," she recalls. "I would go to bookstores, sit at a typewriter. Eugene was such a different life. I lived for five years with an artist, and he taught me how to draw. He had an old-school method. He was very underground, unexposed to normal life."
This unconventional creativity stuck with her, even when Pothier and Bechard moved to Arizona. Pothier worked briefly in the film industry, then shifted her attention to Chateau Tumbleweed, but the desire to compose drawings by hand has never waned. Here was a chance to put that talent to good use, by creating labels that look like they come out of high-concept comic books.
The drawings may looks simple and free, but they're actually the result of an elaborate digital process.
"[First] I draw them, then reduce them to basically cookie-cutters so we can put them into Photoshop," Pothier explains by e-mail. "Jeff [Hendricks] takes control of the process from there, then I come back in to decide on colors. Then the image goes into [Adobe] Illustrator for the background and font. Also, our labels are super nerdy on the back, so Joe collects all of the pertinent info and passes it to Jeff. Then we edit a billion times and send to the federal government for approval, then they go to the Paragon label company for printing."
While many of their labels are merely descriptive, several have distinct characters, such as "Cousin Id," "Will E. Cox," "Lil Frankie," and "Earth Cuckoo." Each portrait incorporates the same tumbleweed image, usually replacing the figure's face, although the tumbleweed is often just a presence: Rolling View Vineyard shows an elephant balancing on the tumbleweed just like a circus ball. The figures are drawn without heads, and only two different tumbleweed drawings are used; one or the other is digitally inserted into the composition.
Given the growing popularity of the images, it's easy to imagine a gallery show of those original drawings. But the quartet is kept busy crafting and bottling their stock, and they're not sure exactly what to do with the artwork.
"There's just a huge folder [of sketches] in my drawing area," Pothier says. "We talk about making coloring books. They are really neat on their own. But they're headless, so they're kind of weird."
Follow Pothier on Instagram @chateautumbleweed to learn more about the wine and to see more labels.


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Published on August 30, 2019 September 10, 2019 by abbiemeehan
You already know what's coming, you've been craving it since last month. It is time for the monthly round up of our top ten up and coming tracks, curated by a new face this time. I'm Abbie, and I'll be standing in for Chloe this month as she gallivants around the Fringe and gets up to more musical mischief.
'Nobody' – Roman Rogue
To start us off, Texan-born R&B singer and songwriter Roman Rogue takes us on a fiery journey with highly-anticipated new song 'Nobody'.
Following the release of his debut EP 'Voicemails', Roman drops 'Nobody', which tells the story of wanting your feelings to be reciprocated from your love interest. An all too familiar feeling for many!
The songs intense drum beats match well with the sensitivity of the lyrics, a good mix of light and heavy. This is something that Roman does well in his other tracks, and is only improving upon in his new music.
'Go Time' – TAYLO
A little closer to home now with British rapper TAYLO who has just dropped his debut single
'Go Time', a funky, fresh hit with all the distinctive sounds to make it perfect for nightclubs up and down the country.
The music video is very Fast and Furious-esque with shots of racing supercars trailed by a helicopter and TAYLO in the garage geared up in racing attire grooving alongside some amazing dancers.
This song pairs the likes of hip hop, trap and R&B to make it the perfect blend of all the genres that the UK do so well. TAYLO may just be starting out in the British scene but he is a name to watch out for in the months to come.
'Running Free' – Fox and Bones
Time to go off the beaten track slightly and check out folk-pop due Fox and Bones who recently released their new single 'Running Free'.
As the title of the track suggests, this song is all about freedom and allowing mistakes to happen as they only further progress and help you create better memories with the good times.
Scott Gilmore and Sarah Vitort both decided to create alternate personas of 'Fox' and 'Bones' in order to have a more narrative approach to writing and tour life. Vitort is 'Fox' and Gilmore is 'Bones', and together they travel the country, perform and push their relationship to the extremes.
Therefore, a song about free flowing whimsical adventure is exactly the type of song what duo can produce and perform.
'Hold on for the Night' – WOLVES
Another switch of genres here for LA-based, fresh twosome Marc Avery and Sean Carney who make up the band WOLVES. Their new single, 'Hold on for the Night', breaks the boundaries of pop music, into something they have dubbed 'Surf Trap'.
The vocals in 'Hold on for the Night' are like velvet on the ear and the addictive beat will have everybody's head bopping and toes tapping to this track. It is almost infectious to sing along to, even if you barely know the lyrics, and those are the best types of songs.
WOLVES will be a name to keep in mind, as I don't doubt we will be seeing these American boys at a few UK festivals next year.
'Crush' – Tyreese ft. Zion B
Everybody had that high-school crush that you couldn't even look in the eye, right? Well,
Tyreese has and he makes his feelings known alongside fellow artist Zion B in 'Crush'. A laidback, summer anthem, Tyreese takes us back to the days of crushing on a cutie at school and vying for their attention.
In the music video, both Tyreese and Zion B have used their history of dancing to their advantage here, switching from shots of them chilling with their crushes to solo shots of them showing off their best moves and jamming along to the beat.
These two are set to only go up from here, and I am so excited to hear the next collaboration.
'If You Go I Go' – India Newland
Another home-grown artist here as London-based India Newland drops her new EP 'Fighter', which features the single 'If You Go I Go'.
The easy-listening, smooth track centres around needing a certain person in your life and following them wherever life takes you. The singer, despite her age, has been performing and writing music for five years and has just recently done a stint at the Edinburgh Fringe Festival.
Other tracks on the EP include 'Fire' and 'Midnight Thoughts'. It sheds more of a generational light on mental health issues and is definitely one to slot into your daily playlist.
'We Could Be' – Chelsea Blues
If you were looking for the song of the summer, look no further than jazz singer and soul artist Chelsea Blues and her new song 'We Could Be'.
The song tells the perfect tale of hoping for a summer romance, that could be so much more than just a casual fling. Paired with sultry vocals and winding rhythms, it is the perfect tune to get down to with your mates in a party setting.
For more from Chelsea Blues be sure to check out 'Emotional Availability' and 'Waves'.
'Brand New Day' – Sidiq
If you need a song that is both sad and uplifting in the same way, then Sidiq's new acoustic single 'Brand New Day' is the one for you
Telling the tale of leaving the past behind and opening your eyes to a brand-new day, it is the perfect mix of sadness and happiness. With heavenly harmonies and soft guitar, it is one of the chillest acoustic songs I have ever listened to.
With the promise of more songs coming in late 2019 and early 2020, Sidiq is not an artist you'd want to miss!
'Rushing Remix' – Alicai Harley x Kojo Funds
No summer would be completed without a remix of a song, and this summer isn't any different. Jamaican-born Alicai Harley, also known as 'Hot Shot', has collaborated with well-known British rapper Kojo Funds to remake her already smash-hit single 'Rushing'.
A unique song with the premise of 'rushing' for someone's love and affection, both Alicai and Kojo's home-grown roots are shown through this song. Kojo's West African background blends perfectly with Alicai's Jamaican roots to create a diverse song.
With both BBC 1Xtra and Capital Xtra listing Alicai as one to watch in 2019, it is only sensible to keep an eye on what she has coming up next.
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I worked the night shift at the Mead factory for three summers in college, paying for my tuition with paper cuts, hard work and sexual harassment. (Ahh, memories...) I learned a lot of valuable life lessons for sure -- but as I packed box after box of freshly minted notebooks and stacked them onto splintery pallets, never once did I consider the pallet's potential as home decor. Until now.
Pallets have been making the rounds on design blogs lately, and because I'm cheap and don't mind a little sanding, this is a fad I can get behind.
Before I try any of this myself I want make sure the pallets I use are free from any of the unsafe chemicals that are typically used to treat them. Anyone have any ideas about where I can find safe or otherwise untreated pallets?
You could have your parents check at that blue building along 522, right before Tops. Aren't they a pallet place?
How about some of that old fencing material?
Good points, guys. That old fencing is definitely going to be put to good use somehow! And Lace - I forgot about that pallet place. I'm not sure how that's possible since I used to drive past it EVERY DAY and even worked next door... haha sad. I'll have to get my dad to do some investigating there.


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Celtic v Rangers: Chance to make history not weighing the Hoops down
Brendan Rodgers tells his players to seize their opportunity in Old Firm clash
Michael Gannon
Celtic manager Brendan Rodgers
Brendan Rodgers insists his men won't get weighed down by history as the Hoops hone in on Scottish football's first double Treble.
The Celtic boss has shut his squad away from the mayhem in the build-up to Sunday's Old Firm Scottish Cup showdown.
Rodgers' men are just one point away from claiming the Premiership title but are determined to dump their old foes at Hampden to set up the chance to pen a stunning chapter in the record books.
Rodgers – who is unbeaten in all nine of his Old Firm encounters – said: "The history is not weighing on us. We just have to do our job. We set our targets and goals and one of them is to win the league. The Scottish Cup is another.
"We wanted to not just defend the trophy but to win it again.
"But it doesn't cloud our thinking. We still come in and enjoy our work. I tend not to think about the other stuff too much.
"If you do, and got wrapped up in the whole history or the expectation, then you put yourself in a pressure trap. You don't need that. There's enough pressure in football."
Rodgers bristled at suggestion only Rangers stand in his side's way of destiny. The Hoops boss insisted there is much more to Scottish football than the Old Firm showdown and he doesn't care if there's a strong challenge from the other side of the city.
He said: "It doesn't make any difference to me. I came up here and the standard I wanted to make was for Celtic. Rangers as a competitor in the league, of course, it pushes you but no more so than what Hibs or Hearts will do.
"You look at these teams, and Aberdeen, clubs with great histories.
"I think the league is very competitive but at Celtic we try to set our own standard and that's to be the best that we can be."
Celtic v Rangers title clash needs a Firm response says Brendan Rodgers
The Hoops boss is preparing for his fourth Hampden cup semi-final and admitted he has stuck to the tried and tested routine that has paid dividends so spectacularly in the last two seasons.
Rodgers said: "We got the recovery done after Sunday and then we've had a clear run at it. Nothing changes.
"We will be relaxed and focused. We enjoy the occasions at Hampden and we have done well in the games there, so hopefully will again this time.
"It's important the players relax and stay focused. Avoidance is a good way to do it. Avoid all the newspapers and media. There's always plenty of hype in these games but no matter the game or the occasion, we don't need to magnify a Celtic versus Rangers game, whatever the competition.
Celtic boss Brendan Rodgers sees 'a lot of envy' towards captain Scott Brown
"They are always big games. The key thing for a manager is to manage the players and that is every week, no matter the opponent or occasion.
"There's no more or less. There's no travelling away, unless it's the preparation for every game.
"We'll do what we do and look forward to it - and hopefully grasp the opportunity."


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(770) 609-5640 derrick@dmospeaks.com
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Gamecocks officially tab Moore as Executive Director of Character and Player Development
by dmospeaks | Jan 22, 2021 | General | 0 comments
University of South Carolina head football coach Shane Beamer has named Derrick Moore as the program's Executive Director of Character and Player Development.
Moore brings his leadership, passion, energy, experience and 24 years of inspiring student-athletes, coaches, and administrators to the University South Carolina and Gamecock Football.
Moore comes to Columbia after working with the Georgia Tech football program since 2001, assisting head coaches Chan Gailey, Paul Johnson and Geoff Collins. He originally was on staff with the Fellowship of Christian Athletes at Georgia Tech, tasked with heading up Yellow Jackets spiritual emphasis program, and later became the Director of Character Development for Georgia Tech Athletics.
From USC Athletics Communications and Public Relations
He helped inspire the Georgia Tech squad to reach four ACC title games, including an ACC title in 2009, and making three Orange Bowl appearances while seeing the Yellow Jackets ranked in the top-25 multiple years, ranking as high as seventh during the 2014 season. He was a key member of the renowned Georgia Tech Total Person Program, which is a program designed to provide development opportunities for student-athletes.
In addition, Moore is one of the most sought-after keynote speakers in the country. Speaking to over 100,000 people annually and over 50 corporate organizations per year such as FedEx, Coca-Cola, The DEA, WellStar Health Systems, and Chick-fil-A. He is well recognized as a motivational speaker and video voice-over artist as he has voiced over videos for the likes of Georgia Tech, the Atlanta Falcons, the Ohio State Buckeyes, and several other top sports organizations and corporations.
A native of Albany, Ga., Moore is a 1992 graduate of Northeastern State University Oklahoma, where he was an All-American running back. He was selected in the eighth round of the 1992 NFL draft by the Atlanta Falcons and would go on to play six years in the NFL. He had stops with the Detroit Lions, where he played alongside Hall of Famer, Barry Sanders and was the starting running back in the Carolina Panthers' inaugural season, where he set an expansion rushing record. He finished his playing career with the Arizona Cardinals.
After his pro career, he joined the coaching staff at Morehouse College in Atlanta, Ga., where he coached running backs and was the Special Teams Coordinator for three years.
Moore and his wife, Stephanie Perry Moore, have three adult children, Dustyn, Sydni and Sheldyn.
Article from 247sports.com.
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