Differences in spending for diabetes and multiple chronic conditions in Michigan Medicare beneficiaries.

OBJECTIVES: To determine which combinations of type 2 diabetes (T2D) and multiple chronic conditions (MCC) contribute to total spending and differences in spending between groups based on sex, race/ethnicity, and rural residency. STUDY DESIGN: Retrospective cohort study using 2012 Medicare claims data from beneficiaries in Michigan with T2D. METHODS: Zero-inflated Poisson regression models to estimate relationships of demographic characteristics and MCC combinations on hospital outpatient, acute inpatient, skilled nursing, hospice, and Part D drug spending. RESULTS: Across most MCC combinations, there are lower odds of no spending, with a concurrent increase in the expected mean of actual spending when payments are made, except for hospital outpatient costs. For hospital outpatient services, we observed lower spending across all MCC combinations. When controlling for MCC, we generally found that compared with White beneficiaries, Black, Asian/Pacific Islander, and Hispanic beneficiaries experience increased odds of no spending, but when payments were made, payments generally increased. American Indian/Alaska Native beneficiaries are the exception; they experience decreased odds of no payments for hospital outpatient and acute inpatient services, with a concurrent decrease in mean expected payments. CONCLUSIONS: When considering a range of MCC combinations, we observed differences in total payments between racial/ethnic minority groups and White beneficiaries. Our results highlight the ongoing need to make changes in the health care system to make the system more accessible to racial/ethnic minority groups.

Activation of bovine somatic cell nuclear transfer embryos by PLCZ cRNA injection.

The production of cloned animals by the transfer of a differentiated somatic cell into an enucleated oocyte circumvents fertilization. During fertilization, the sperm delivers a sperm-specific phospholipase C (PLCZ) that is responsible for triggering Ca(2)(+) oscillations and oocyte activation. During bovine somatic cell nuclear transfer (SCNT), oocyte activation is artificially achieved by combined chemical treatments that induce a monotonic rise in intracellular Ca(2)(+) and inhibit either phosphorylation or protein synthesis. In this study, we tested the hypothesis that activation of bovine nuclear transfer embryos by PLCZ improves nuclear reprogramming. Injection of PLCZ cRNA into bovine SCNT units induced Ca(2)(+) oscillations similar to those observed after fertilization and supported high rates of blastocyst development similar to that seen in embryos produced by IVF. Furthermore, gene expression analysis at the eight-cell and blastocyst stages revealed a similar expression pattern for a number of genes in both groups of embryos. Lastly, levels of trimethylated lysine 27 at histone H3 in blastocysts were higher in bovine nuclear transfer embryos activated using cycloheximide and 6-dimethylaminopurine (DMAP) than in those activated using PLCZ or derived from IVF. These results demonstrate that exogenous PLCZ can be used to activate bovine SCNT-derived embryos and support the hypothesis that a fertilization-like activation response can enhance some aspects of nuclear reprogramming.

Trichostatin A improves histone acetylation in bovine somatic cell nuclear transfer early embryos.

Epigenetic aberrancies likely preclude correct and complete nuclear reprogramming following somatic cell nuclear transfer (SCNT), and may underlie the observed reduced viability of cloned embryos. In the present study, we tested the effects of the histone deacetylase inhibitor (HDACi), trichostatin A (TSA), on development and histone acetylation of cloned bovine preimplantation embryos. Our results indicated that treating activated reconstructed SCNT embryos with 50 nM TSA for 13 h produced eight-cell embryos with levels of acetylation of histone H4 at lysine 5 (AcH4K5) similar to fertilized counterparts and significantly greater than in control NT embryos (p < 0.005). Further, TSA treatment resulted in SCNT embryos with preimplantation developmental potential similar to fertilized counterparts, as no difference was observed in cleavage and blastocyst rates or in blastocyst total cell number (p > 0.05). Measurement of eight selected developmentally important genes in single blastocysts showed a similar expression profile among the three treatment groups, with the exception of Nanog, Cdx2, and DNMT3b, whose expression levels were higher in TSA-treated NT than in in vitro fertilized (IVF) embryos. Data presented herein demonstrate that TSA can improve at least one epigenetic mark in early cloned bovine embryos. However, evaluation of development to full-term is necessary to ascertain whether this effect reflects a true increase in developmental potential.

Polycomb gene expression and histone H3 lysine 27 trimethylation changes during bovine preimplantation development.

Trimethylation of histone H3 at lysine 27 (H3K27me3) is established by polycomb group genes and is associated with stable and heritable gene silencing. The aim of this study was to characterize the expression of polycomb genes and the dynamics of H3K27me3 during bovine oocyte maturation and preimplantation development. Oocytes and in vitro-produced embryos were collected at different stages of development. Polycomb gene expression was analyzed by real-time quantitative RT-PCR and immunofluorescence. Global H3K27me3 levels were determined by semiquantitative immunofluorescence. Transcripts for EZH2, EED, and SUZ12 were detected at all stages analyzed, with EZH2 levels being the highest of the three at early stages of development. By the time the embryo reached the blastocyst stage, the level of PcG gene mRNA levels significantly increased. Immunofluorescence staining indicated nuclear expression of EZH2 at all stages while nuclear localized EED and SUZ12 were only evident at the morula and blastocyst stages. Semiquantitative analysis of H3K27me3 levels showed that nuclear fluorescence intensity was the highest in immature oocytes, which steadily decreased after fertilization to reach a nadir at the eight-cell stage, and then increased at the blastocyst stage. These results suggest that the absence of polycomb repressive complex 2 proteins localized to the nucleus of early embryos could be responsible for the gradual decrease in H3K27me3 during early preimplantation development.

Factors affecting cardiovascular and cerebrovascular complications of carotid artery stenting in Northern Michigan: A retrospective study.

BACKGROUND: This study seeks to identify factors associated with periprocedural complications of carotid artery stenting (CAS) to best understand CAS complication rates and optimize patient outcomes. Periprocedural complications include major adverse cardiovascular and cerebrovascular events (MACCE) that include myocardial infarction (MI), stroke, or death. METHODS: We retrospectively analyzed 181 patients from Northern Michigan who underwent CAS. Rates of stroke, MI, and death occurring within 30days post-procedure were examined. Associations of open vs. closed cell stent type, demographics, comorbidities, and symptomatic carotid stenosis were compared to determine significance. All patients had three NIH Stroke Scale (NIHSS) exams: at baseline, 24h post-procedure, and at the one-month visit. Cardiac enzymes were measured twice in all patients, within 24h post-procedure. All patients were treated with dual anti-platelet therapy for at least 6months post-procedure. RESULTS: Three patients (1.66%) experienced a major complication within one-month post-procedure. These complications included one MI (0.55%), one stroke (0.55%), and one death (0.55%). The following variable factors were not associated with the occurrence of MACCE complications within 30days post-procedure: stent design (open vs. closed cell) (p=1.000), age ≥80 (p=0.559), smoking history (p=0.569), hypertension (p=1.000), diabetes (p=1.000), and symptomatic carotid stenosis (p=0.254). CONCLUSIONS: Age of 80years old or above, symptomatic carotid stenosis, open-cell stent design, and history of diabetes, smoking, or hypertension were not found to have an association with MACCE within 1month after CAS. Future studies using a greater sample size will be beneficial to better assess periprocedural complication risks of CAS, while also considering the effect of operator experience and technological advancements on decreasing periprocedural complication rates.

Is arginine/asymetric dimethylarginine ratio depletion an indicator of insufficient resuscitation in a porcine model of hemorrhage-reperfusion?

BACKGROUND: Hemorrhagic shock leads to a complex cascade of metabolic and hormonal processes that may result in hypoperfusion, end organ damage, and death even when blood pressure is restored. Studies have shown that morbidity and mortality could be attributable to a diminished availability of endothelial-derived nitric oxide (eNO). It is unclear whether adequate levels of citrulline (CIT) and arginine (ARG)--the precursors of eNO synthesis--are available to sustain the eNO needed to maintain adequate perfusion in severe shock. An indirect measure of eNO is the ratio between the levels of ARG and its inhibitor asymmetric dimethylarginine (ARG/ADMA). The purpose of the study was to identify the temporal impact of the ARG/ADMA ratio, ARG, CIT, and ADMA in response to hemorrhage and crystalloid fluid resuscitation by the use of a porcine model of severe hemorrhagic shock. METHODS: Hemorrhagic shock was induced in Yorkshire cross pigs by mimicking a bleeding pattern of rapid uncontrolled hemorrhage to achieve a shed volume of 30 mL/kg, a 50% decrease in mean arterial pressure, and an oxygen debt of >60 mL/kg. Normal saline, up to 2 times the shed blood volume, was started 1 hour after the start of hemorrhage with the goal of restoring mean arterial pressure to >50 mm Hg. Hemodynamics, blood gas measurements, and plasma samples were obtained at baseline, 1 hour after the start of hemorrhage, and 1 hour after resuscitation. Amino acids were measured by liquid chromatography coupled to mass spectrometry. RESULTS: During hemorrhage, a distinct subset of pigs was better able to tolerate ischemia than the rest. These pigs required less resuscitation, had evidence of better organ perfusion, and exhibited less of an increase in interleukin-6 (IL-6) after resuscitation. Compared with their less-tolerant counterparts, this group had a greater increase in CIT above baseline (analysis of variance, P < .05) with hemorrhage. ARG levels were similar and remained stable with hemorrhage, which indicated the similar availability of substrate for eNO synthesis but differences in the quantity produced in response to the blood volume loss. With crystalloid fluid resuscitation, ARG levels and ARG/ADMA decreased (analysis of variance, P < .05), whereas CIT remained increased in the group less able to tolerate hemorrhage. ARG/ADMA decreased proportional to greater oxygen debt during hemorrhage and greater IL-6 levels with fluid resuscitation. CONCLUSION: Our results suggest that a sufficient decrease in MAP during hemorrhagic shock is associated with a subsequent increase in IL-6, persisting impairment of end organ perfusion, and evidence of ongoing eNO deficit and an increase in ADMA despite resuscitation. The ARG/ADMA ratio reflects both of these parameters and corresponds to the increase in IL-6 and persistent ischemia after resuscitation. We propose that the mechanism of IL-6 increase in trauma derives from eNO deficiency, and the ARG/ADMA ratio more accurately depicts the pathologic mechanism responsible for increased morbidity and mortality in trauma.

Novel biomarkers of arterial and venous ischemia in microvascular flaps.

The field of reconstructive microsurgery is experiencing tremendous growth, as evidenced by recent advances in face and hand transplantation, lower limb salvage after trauma, and breast reconstruction. Common to all of these procedures is the creation of a nutrient vascular supply by microsurgical anastomosis between a single artery and vein. Complications related to occluded arterial inflow and obstructed venous outflow are not uncommon, and can result in irreversible tissue injury, necrosis, and flap loss. At times, these complications are challenging to clinically determine. Since early intervention with return to the operating room to re-establish arterial inflow or venous outflow is key to flap salvage, the accurate diagnosis of early stage complications is essential. To date, there are no biochemical markers or serum assays that can predict these complications. In this study, we utilized a rat model of flap ischemia in order to identify the transcriptional signatures of venous congestion and arterial ischemia. We found that the critical ischemia time for the superficial inferior epigastric fasciocutaneus flap was four hours and therefore performed detailed analyses at this time point. Histolgical analysis confirmed significant differences between arterial and venous ischemia. The transcriptome of ischemic, congested, and control flap tissues was deciphered by performing Affymetrix microarray analysis and verified by qRT-PCR. Principal component analysis revealed that arterial ischemia and venous congestion were characterized by distinct transcriptomes. Arterial ischemia and venous congestion was characterized by 408 and 1536>2-fold differentially expressed genes, respectively. qRT-PCR was used to identify five candidate genes Prol1, Muc1, Fcnb, Il1b, and Vcsa1 to serve as biomarkers for flap failure in both arterial ischemia and venous congestion. Our data suggests that Prol1 and Vcsa1 may be specific indicators of venous congestion and allow clinicians to both diagnose and successfully treat microvascular complications before irreversible tissue damage and flap loss occurs.

Downregulation of H19 improves the differentiation potential of mouse parthenogenetic embryonic stem cells.

Parthenogenetic embryonic stem cells (P-ESCs) offer an alternative source of pluripotent cells, which hold great promise for autologous transplantation and regenerative medicine. P-ESCs have been successfully derived from blastocysts of several mammalian species. However, compared with biparental embryonic stem cells (B-ESCs), P-ESCs are limited in their ability to fully differentiate into all 3 germ layers. For example, it has been observed that there is a differentiation bias toward ectoderm derivatives at the expense of endoderm and mesoderm derivatives-muscle in particular-in chimeric embryos, teratomas, and embryoid bodies. In the present study we found that H19 expression was highly upregulated in P-ESCs with more than 6-fold overexpression compared with B-ESCs. Thus, we hypothesized that manipulation of the H19 gene in P-ESCs would alleviate their limitations and allow them to function like B-ESCs. To test this hypothesis we employed a small hairpin RNA approach to reduce the amount of H19 transcripts in mouse P-ESCs. We found that downregulation of H19 led to an increase of mesoderm-derived muscle and endoderm in P-ESCs teratomas similar to that observed in B-ESCs teratomas. This phenomenon coincided with upregulation of mesoderm-specific genes such as Myf5, Myf6, and MyoD. Moreover, H19 downregulated P-ESCs differentiated into a higher percentage of beating cardiomyocytes compared with control P-ESCs. Collectively, these results suggest that P-ESCs are amenable to molecular modifications that bring them functionally closer to true ESCs.