Increase In Cell Mass Research Articles

A case of AORPA in an adult presenting as secondary polycythemia

BackgroundPolycythemia refers to a condition of an abnormal increase in the red blood cell (RBC) mass. In men with a hematocrit > 60% or women with a hematocrit > 55%, there is 99% likelihood that the RBC mass is elevated. WHO uses a hemoglobin concentration > 16.5 g/dl in men or 16.0 g/dl in women to define an elevated RBC mass, in its criteria for the diagnosis of polycythemia vera. Congenital cardiac anomalies presenting as polycythemia are very rare in adults.Case presentationIn this case report, the patient presented with non-specific complaint and had an incidental finding of secondary polycythemia, ultimately diagnosed as secondary to AORPA. Our patient, a 43-year-old male, presented to our emergency with focal onset seizures without impairment of consciousness involving left upper and lower limbs. On routine investigations, the patient was found to have grade III clubbing and hemoglobin of 19.8 g/dl with elevated Erythropoietin levels. Further workup of secondary polycythemia revealed anomalous origin of the right pulmonary artery (AORPA) from the ascending aorta with tortuous patent ductus arteriosus (PDA) and pulmonary hypertension. Secondary polycythemia, in our patient, was due to right to left shunt causing Eisenmenger phenomenon, following pulmonary hypertension, making it one of the rarest presentation in adults.ConclusionIn the evaluation of secondary polycythemia, possibility of underlying asymptomatic cardiopulmonary shunt must be considered, though rare.

Targeting pancreatic beta cell death in type 2 diabetes by polyphenols.

Diabetes is a very complex disease which is characterized by the appearance of insulin resistance that is primarily compensated by an increase in pancreatic beta cell mass, generating hyperinsulinemia. After time, pancreatic beta cells die by apoptosis appearing in the second phase of the disease, and characterized by hypoinsulinemia. There are multiple conditions that can alter pancreatic beta cell homeostasis and viability, being the most relevant ones; ER stress, cytotoxicity by amylin, mTORC1 hyperactivity, oxidative stress, mitochondrial dysfunction, inflammation and alterations in autophagy/mitophagy flux. In addition, the possible effects that different polyphenols could exert in the modulation of these mechanisms and regulating pancreatic beta cell viability are analyzed. It is necessary a profound analysis and understanding of all the possible mechanisms involved in the control and maintenance of pancreatic beta cell viability to develop more accurate and target treatments for controlling beta cell homeostasis and preventing or even reversing type 2 diabetes mellitus.

Enhancement of polyhydroxybutyrate production by introduction of heterologous phasin combination in Escherichia coli

Phasin is a surface-binding protein of polyhydroxyalkanoate (PHA) granules that is encoded by the phaP gene. As its expression increases, PHA granules become smaller, to increase their surface area, and are densely packed inside the cell, thereby increasing the PHA content. A wide range of PHA-producing bacteria have phaP genes; however, their PHA productivity differs, although they are derived from the cognate bacterial host cell. Modulating phasin expression could be a new strategy to enhance PHA production. This study aimed to characterize the effect of heterologous phasins on the reconstitution of E. coli BL21(DE3) and determine the best synergistic phaP gene combination to produce polyhydroxybutyrate (PHB). We identified novel phasins from a PHB high-producer strain, Halomonas sp. YLGW01, and introduced a combination of phaP genes into Escherichia coli. The resulting E. coli phaP1,3 strain had enhanced PHB production by 2.9-fold, leading to increased cell mass and increased PHB content from 48 % to 65 %. This strain also showed increased tolerance to inhibitors, such as furfural and vanillin, enabling the utilization of lignocellulose biosugar as a carbon source. These results suggested that the combination of phaP1 and phaP3 genes from H. sp. YLGW01 could increase PHB production and robustness.

A dietary intervention study to reduce Metabolic Syndrome risks in heart-transplanted patients

Abstract Background Since heart transplantation (HTx) has become the gold standard therapy in end-staged heart failure, many factors, including metabolic syndrome (MS), represent a burden in HTx patients. Considering key role of immunosuppressive therapy and its side effects on the appearance of MS, we focused on modifiable factors including adherence to Mediterranean Diet (MD) and improvement of dietary habits. Methods 21 heart-transplanted patients were enrolled and randomized in a control group (CG; N 10) and an intervention group (IG; N 11). During two meetings (baseline, 6-month follow-up) were administered a validated Food Frequency Questionnaire (FFQ), to assess adherence to MD, and collected clinical and anthropometric parameters, IG were additionally requested to fill a food diary. IG received personalized advices, CG received standard recommendations. Comparison between IG and CG were analyzed, differences into the IG were also assessed. Results The prevalence of MS at baseline was 46% in IG and 20% in CG. During 6-month follow-up, significant lower blood pressure values were observed (median, 25th-75th: systolic 130, 120-130 IG vs 145, 130-147 CG; p = 0.004). Seven patients of IG underwent a 12-month meeting. In this group MD scores increased significantly (7 + 1.3 vs 4 + 1.5, p = 0.001). Furthermore, significant decrease of fat mass percentage (%) (23.3 + 6.3 vs 14.8 + 10.1, p = 0.014), increase of fat free mass % (76 + 6.3 vs 85.2 + 10.1, p = 0.014) and increase of body cell mass % (50.9 + 3.8 vs 53.4 + 3.4, p = 0.031) were observed. Dietary data in IG showed significant decrease of energy from saturated fatty acids % (13.0±2.1 vs 9.6±1.5, p = 0.001), sodium (mg) (2138±359 vs 1822±417, p = 0.045), and decreasing trend for cholesterol (mg) (219±82 vs 171±59, p = 0.082). Conclusions Dietary intervention based on MD perhaps can improve MS risks in heart-transplanted patients. Further investigations may be needed to assess the fundamental role of a structured nutritional follow-up in these patients. Key messages • Personalized nutritional advices based on the MD, compared to general recommendation, can significantly improve health and quality of life in heart-transplanted patients. • A structured nutritional follow-up for heart-transplanted patients may be desirable to prevent risks of Metabolic Syndrome as a public health instrument in selected categories as these patients.

Abstract P2020: A Novel Molecular Mechanism Of Cardiac Hypertrophy

Proliferation of cardiac myocytes is accompanied by increases in cell mass to maintain myocyte size in fetal heart. In adult cardiac myocytes, cell growth becomes uncoupled from proliferation after growth stimuli, resulting in hypertrophic growth. Both proliferation and hypertrophy growth of cardiac myocytes require more protein synthesis. Brf1 (TF IIB-related factor 1) and Pol III genes (RNA Polymerase III-dependent genes) are the dominant factors of protein produce. Brf1 is a key regulator for Pol III genes. Cardiac hypertrophy is a critical risk factor for cardiovascular disease, including heart failure, arrhythmia, and sudden death. Cardiac hypertrophy involves both the processes of embryonic gene expression and transcriptional reprogramming. The processes are tightly modulated by different signal pathways and epigenetic regulation. Diabetic cardiomyopathy is associated with an increased risk for heart failure and death in patients with diabetes. To explore the molecular mechanism of cardiac hypertrophy, we investigate the alterations of Brf1 and Pol III gene transcription under glucose treatment by using cell culture model and animal model. We have found that glucose increases Brf1 expression and Pol III gene transcription. We identified that these genes are mediated by JNK1, MSK1, pAMPK and phosphorylated histone H3. inhibiting the pathways decrease Brf1 expression, reduce Pol III gene transcription, consequently repression of myocytes growth. It implies that developing an effective inhibitor to repress Brf1 expression may provide a potential approach for the therapy of cardiac hypertrophy. More studies on cardiac hypertrophy are going in our laboratory.

Tocopherol Enhances the Antioxidant Defense System and Histomorphometric Parameters in The Gastrointestinal Tract of Rats Treated with Sodium Arsenite.

Arsenic compromises the gastrointestinal integrity and function via the body's anti-oxidative system breakdown. Hence, this study aimed to investigate the effects of tocopherol on redox imbalance and histoarchitectural alterations in rats' gastrointestinal tract exposed to sodium arsenite. Sodium arsenite and graded doses of tocopherol were administered orally into experimental rats assigned to different groups for four weeks concurrently. Redox status assay was done in homogenized samples by spectrophotometry. Parietal cell mass and mucous cell density (stomach), villus height and crypt depth (ileum), goblet cells count, and crypt depth (colon) were evaluated by histomorphometry. Inflammatory cells infiltration was also assessed using a semi-quantitative procedure.Sodium arsenite caused a significant increase in Malondialdehyde and Myeloperoxidase but, decreased Superoxide dismutase, Catalase, Nitric oxide, Glutathione peroxidase, Glutathione, and Glutathione-S-Transferase. Tocopherol treatment reversed the changes (p<0.05) though not largely dose-dependent. Furthermore, tocopherol annulled sodium arsenite-induced increase in parietal cell mass and decrease in mucous cell density in the stomach, decrease in villus height and villus height/crypt depth ratio in the ileum, and decrease in goblets cells and increase in crypt depth in the colon. Moreover, activated inflammatory cell infiltration by sodium arsenite was mitigated by tocopherol. Sodium arsenite provokes not only marked inflammatory cellular infiltration but a focal loss of glands, hyperplasia of crypts, atrophic villi, and hypertrophy of Peyer's patches in the intestines, which are all lessened with tocopherol treatment. These findings underscore the anti-oxidative properties of tocopherol as a potent dietary factor against sodium arsenite toxicity in the gastrointestinal tract. Keywords: Tocopherol, arsenic, stomach, ileum, colon.

P1012: A NUMBER OF CONGENITAL ERYTHROCYTOSIS ARE MULTIGENIC

Background: Imbalance in erythropoiesis and oxygen homeostasis could lead to the onset of Erythrocytosis, a clinical condition characterized by persistently raised hemoglobin (Hb) and hematocrit (Ht) levels. Nowadays, a high number of patients are classified as “Idiopathic Erythrocytosis” (IE) meaning that it is not possible to identify the cause of hematocrit increase. Recently, HFE mutations has been found to be often present in idiopathic erythrocytic patients. The HFE mutations could facilitate the increase in red cell mass, and this indicates the possible involvement of Iron Metabolism in causes of IE. Aims: To identify the possible mutations of IE patients using our gene panel for Next Generation Sequencing (NGS). Methods: In 118 patients with IE, regularly followed in our surgery, we used a gene panel for NGS composed by fifteen genes: JAK2, EGLN1 (PHD2), EPO-R, FTL, FTH, ASXL1, HFE, HFE2, TFR2, HAMP, SLC40A1, SLC11A2, VHL, BPMG, EPAS1 (HIF-2α). We analyzed all the exons parts of these genes. The approach used for the library preparation was a multiplexing PCR and data were analyzed by bioinformatics tools. In all patients, the mutations found were confirmed with Sanger Sequencing. Results: In 78 out of the 118 patients (66%) evaluated, we found one 1 mutated gene in 55 patients (7 EGLN1, 1 VHL, 34 HFE, 6 TFR2, 3 JAK2 and 4 EPO-R), 2 mutated genes in 18 patients (1 EGLN1/JAK2, 4 EGLN1/HFE, 2 EPAS1/HFE, 1 EPAS1/JAK2, 1 EPAS1/EGLN1, 2 EPO-R/HFE, 2 HFE/JAK2, 1 JAK2/TFR2, 2 with TFR2/EGLN1, 1 VHL/HFE, 1 TFR2/HFE) and 3 mutated genes in 5 patients (1 EGLN1/EPO-R/HFE, 1 VHL/HFE/EGLN1, 1 EPAS1/EPO-R/HFE, 1 EPAS1/HFE/EGLN1 and HFE/TFR2/JAK2). All the mutations found were germline. Interestingly, 21 out of the 27 (78%) patients carrying HFE mutation displayed 2 or more mutations. Summary/Conclusion: The use of NGS panels in various clinical conditions has frequently demonstrated that multiple mutations coexist in various patients and that the monogenic diseases are quite rare. This study is the first at our best knowledge that gives evidence of more than one mutated gene in erythrocytotic patients, and this is the results of the use of an appropriate NGS panel. The data here reported suggest that we can confirm that HFE mutations are common in IE patients, in some cases associated with other mutations, but the present data ask some questions: HFE mutations alone can induce erythrocytosis? or other genetic imbalances are needed? The presence of multiple mutations could lead to a greater severity of the disease? A significant number of IE patients displayed TFR2 mutations, giving more relevance to the hypothesis of the iron metabolism implication in IE genesis. Finally, the presence of 11 patients carrying germlines JAK2 mutations is surprising, and we suppose it will be necessary to strictly watch these patients in the suspicion of a future appearance of a polycythemia vera. The present study demonstrates the need to extend the biomolecular knowledge in patients with idiopathic erythrocytosis.

365-OR: Interruption of Glucagon Signaling Increases Delta-Cell Proliferation in Mouse and Human Islets

The determinants of delta cell mass are not known. As part of studies showing interruption of glucagon signaling (IGS) stimulates alpha cell proliferation in an amino acid (AA) -dependent manner, we noted an increase in delta cells. To investigate, we examined delta cell proliferation and mass in 3 mouse models and a human islet xenograft model. Adult mice with deletion of glucagon (Gcg-/- ) had a 4.7-fold increase in Ki67-positive delta cells (1.50±0.14% vs. 0.32±0.12%, P&amp;lt;0.001, n=8) and a 3.1-fold increase in delta cell mass (0.49±0.vs. 0.16±0.mg, P=0.003, n=10) . Treatment of adult C57BL/6J mice with a Gcg receptor antibody (GCGR Ab) elicited a 6.3-fold increase in delta cell proliferation (2.01±0.4% vs. 0.32±0.07%, P=0.002, n=10) and a 2.6-fold increase in delta cell mass (0.20±0.03 vs. 0.078±0.mg, P=0.001, n=10) , respectively, compared to IgG-treated controls. Since hyperaminoacidemia is necessary for alpha cell proliferation in response to IGS, we investigated the requirement for Slc7a2, a cationic AA transporter, in delta cell growth. Indeed, Slc7a2+/+ mice, but not Slc7a2-/- mice, treated with GCGR Ab had a 3.8-fold increase in delta cell proliferation (3.29±0.14% vs. 0.86±0.05%, P&amp;lt;0.001) and 1.6-fold increase in delta cell mass (0.20±0.02 vs. 0.126±0.mg, P=0.016, n=8) . Given that mTOR is a known intracellular mediator of AA-regulated cellular growth, we evaluated phospho-ribosomal protein S6 (pS6) expression in situ and found a marked increase in pS6 labeling of delta cells in Gcg-/- , but not Gcg+/+ , mice. We also observed a 3.6-fold increase in delta cell proliferation (1.75±0.25% vs. 0.49±0.06%, P=0.004, n=3 adult normal donors) in human islets transplanted into immunodeficient mice treated with GCGR Ab. Taken together, these data indicate that IGS promotes delta cell proliferation and mass through mechanisms involving Slc7a2 and the mTOR signaling pathway and that AAs may be a determinant of delta cell mass. Disclosure C.Dai: None. A.Bradley: None. E.Spears: None. A.K.Singh: None. F.O.Oladipupo: None. R.Jenkins: None. K.C.Coate: None. A.C.Powers: None.

Investigation of the Therapeutic Potential of New Antidiabetic Compounds Using Islet-on-a-Chip Microfluidic Model.

Nowadays, diabetes mellitus is one of the most common chronic diseases in the world. Current research on the treatment of diabetes combines many fields of science, such as biotechnology, transplantology or engineering. Therefore, it is necessary to develop new therapeutic strategies and preventive methods. A newly discovered class of lipids—Palmitic Acid Hydroxy Stearic Acid (PAHSA) has recently been proposed as an agent with potential therapeutic properties. In this research, we used an islet-on-a-chip microfluidic 3D model of pancreatic islets (pseudoislets) to study two isomers of PAHSA: 5-PAHSA and 9-PAHSA as potential regulators of proliferation, viability, insulin and glucagon expression, and glucose-stimulated insulin and glucagon secretion. Due to the use of the Lab-on-a-chip systems and flow conditions, we were able to reflect conditions similar to in vivo. In addition, we significantly shortened the time of pseudoislet production, and we were able to carry out cell culture, microscopic analysis and measurements using a multi-well plate reader at the same time on one device. In this report we showed that under microfluidic conditions PAHSA, especially 5-PAHSA, has a positive effect on pseudoislet proliferation, increase in cell number and mass, and glucose-stimulated insulin secretion, which may qualify it as a compound with potential therapeutic properties.

Myeloproliferative neoplasms – a global view

Myeloproliferative neoplasms – a global view

Hemochromatosis Gene Mutation in Persons Developing Erythrocytosis on Combined Testosterone and SGLT-2 Inhibitor Therapy.

In clinical trials, sodium-glucose cotransporter-2 inhibitors (SGLT-2i) use alone in persons with type 2 diabetes (T2D) or testosterone replacement therapy (TRT) prescription alone in men with hypogonadism was shown to lead to a modest but significant increase in red blood cell mass. Recent evidence indicates that combined use of TRT and SGLT-2i in persons with T2D may be associated with risk of erythrocytosis. However, factor(s) that may lead to the development of erythrocytosis in these patients is unknown. We describe here 5 consecutive patients with hypogonadism on chronic TRT who developed erythrocytosis following addition of SGLT-2i empagliflozin for optimization of T2D management. In addition to the careful review of medical history, all patients underwent genetic screening for hereditary hemochromatosis. We have found that none of the patients had C282Y mutation in the HFE (Homeostatic Iron Regulator) gene and 4 out of 5 patients had heterozygosity in the H63D allele. Upon TRT discontinuation or its dose reduction or referral for scheduled phlebotomy, patients showed resolution of erythrocytosis. Our study reaffirms that practitioners should monitor for changes in hematocrit following the initiation of SGLT-2i in persons with T2D and hypogonadism on chronic TRT. Also, for the first time, we showed that in some of the patients receiving combined TRT and SGLT-2i H63D heterozygosity in the HFE gene may mediate the development of new-onset erythrocytosis.

Changes in body composition depending on the rs1801282 PPARG polymorphism in patients with different variants of treatment of early carbohydrate metabolism disorders

Objective: To study the effect of the rs1801282 PPARG polymorphism on changes in the body composition of patients with early carbohydrate metabolism disorders in groups with different variants of treatment.Materials and Methods: The study involved 64 patients (8 men and 56 women) with early carbohydrate metabolism disorders. At baseline, all patients underwent genotyping for the rs1801282 PPARG polymorphism and body composition determination with bioelectrical impedance analysis (BIA). Then, the patients were divided into two groups depending on the type of therapy. The patients from Group 1 (40 subjects, mean age 45.2±15.4 years) kept a generally accepted diet with the exclusion of simple carbohydrates and limitation of complex carbohydrates and fats. The patients from Group 2 (24 subjects, mean age 51.2±14.5 years) took metformin in addition to the diet therapy. The effects of different types of treatment on body composition changes were assessed with follow-up BIA 3 months after the start of treatment.Results: Carriers of the mutant G allele of rs1801282 PPARG in the metformin and diet therapy group showed a significant increase in the content of body cell mass (1.28±0.51% vs 0.36±0.37%; P = 0.021) compared with CC homozygotes in the absence of differences in body weight changes (P &gt; 0.05).Conclusions: The presence of the mutant allele G of rs1801282 PPARG promotes the increase in body cell mass in case of adding metformin to the diet therapy in patients with early carbohydrate metabolism disorders.

Graphene Oxide Nanoparticels Interaction with Jurkat Cell Line in Cell-IQ System.

In recent years, materials based on graphene oxide (GO) have been actively studied for their use in biomedicine. The aim of our study was to investigate the increase in cell mass and viability of Jurkat tumor line T cells during 24 h of contact with GO nanoparticles in the Cell-IQ system of intravital observation. We used nanoparticles of different sizes coated with linear or branched polyethylene glycol (PEG) at concentrations of 5 and 25 μg/mL. It was shown for the first time that direct contact with GO nanoparticles reduced the growth in cell mass at the visualization points by more than twofold, regardless of nanoparticle size and concentration. Moreover, the number of live cells in the culture decreased by 5-9% after 24 h of monitoring. Thus, PEG-coated GO nanoparticles were found to suppress the proliferation and viability of Jurkat cell line T lymphocytes.

Non-Photosynthetic CO2 Utilization to Increase Fatty Acid Production in Yarrowia lipolytica.

Metabolic engineering of non-photosynthetic microorganisms to increase the utilization of CO2 has been focused on as a green strategy to convert CO2 into valuable products such as fatty acids. In this study, a CO2 utilization pathway involving carbonic anhydrase and biotin carboxylase was formed to recycle CO2 in the oleaginous yeast Yarrowia lipolytica, thereby increasing the production of fatty acids. In the recombinant strain in which the CO2 utilization pathway was introduced, the production of fatty acids was 10.7 g/L, which was 1.5-fold higher than that of the wild-type strain. The resulting strain had a 1.4-fold increase in dry cell mass compared to the wild-type strain. In addition, linoleic acid was 47.7% in the fatty acid composition of the final strain, which was increased by 11.6% compared to the wild-type strain. These results can be applied as an essential technology for developing efficient and eco-friendly processes by directly utilizing CO2.

Nutrigenomics in Regulating the Expression of Genes Related to Type 2 Diabetes Mellitus.

Nutrigenomics is the study of the gene-nutrient interaction and it indicates that some nutrients, called bioactive compounds, can mold the genetic expression or change the nucleotide chain. Polyphenols are secondary metabolites found in plants that are regularly consumed in functional foods and help prevent or delay the onset of type 2 diabetes mellitus (T2DM) and its complications. This article objected to review studies about the interaction of diet with polyphenols and Mediterranean diet in the expression of human genes related to T2DM. Resveratrol acts as an antioxidant, anti-inflammatory, and increases mitochondrial function. Regular consumption of quercetin resulted in improvement of hypertension and suppression of diabetes-induced vasoconstriction. Genistein also showed positive results in T2DM, such as increased cell mass and improved glucose tolerance and insulin levels. Catechins showed efficiency in inducing genes in triacylglycerol biosynthesis, inhibition of fatty acids and cholesterol, and resulting in their participation in mitigating complications of diabetes. Lastly, curcumin was demonstrated to be a protector of the pancreatic islets against streptozotocin-induced oxidative stress. Growing evidence suggest that bioactive compounds such as polyphenols have an important role in T2DM and the prevention and treatment of its complication, as they cause activation or inhibition of related genes.

Relation of Intravascular Volume Profiles to Heart Failure Progression and Clinical Outcomes

Heart failure (HF) commonly progresses over time and identifying differences in volume profiles may help stratify risk and guide therapy. The aim of this study was to assess the pathophysiologic and prognostic roles of volume profiles for HF progression in stable ambulatory and hospitalized patients. HF patients who had undergone quantitative intravascular volume analysis (185 outpatients and 139 inpatients) were retrospectively assessed for the combined end point of HF-related hospital admissions (outpatients), HF-readmissions (inpatients), and overall all-cause mortality. After multivariate Cox regression analysis, greater total blood volume expansion was associated with higher risk of HF-admission in previously stable outpatients (HR: 1.023, CI 1.005 to 1.043; p=0.013) while in more advanced HF (inpatients) total blood volume expansion was associated with lower risk for HF-readmission and mortality (HR: 0.982, CI 0.967 to 0.997; p=0.017). Secondary analysis suggests that subclinical plasma volume expansion was a driving factor for the detrimental association in outpatients (HR: 1.018, CI 0.997 to 1.036; p=0.054), while an increase in red blood cell mass was central to the beneficial association in advanced HF (HR: 0.979, CI 0.968 to 0.991; p <0.001). In conclusion, understanding differences in plasma volume and red blood cell mass profiles can provide insight into the pathophysiology and progression of HF.

Elucidation of Mechanical, Physical, Chemical and Thermal Properties of Microbial Composite Films by Integrating Sodium Alginate with Bacillus subtilis sp.

Materials are the foundation in human development for improving human standards of life. This research aimed to develop microbial composite films by integrating sodium alginate with Bacillus subtilis. Sodium alginate film was fabricated as control. The microbial composite films were fabricated by integrating 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 g of Bacillus subtilis into the sodium alginate. Evaluations were performed on the mechanical, physical, chemical and thermal properties of the films. It was found that films reinforced with Bacillus subtilis significantly improved all the mentioned properties. Results show that 0.5 g microbial composite films had the highest tensile strength, breaking strain and toughness, which were 0.858 MPa, 87.406% and 0.045 MJ/m3, respectively. The thickness of the film was 1.057 mm. White light opacity, black light opacity and brightness values were 13.65%, 40.55% and 8.19%, respectively. It also had the highest conductivity, which was 37 mV, while its water absorption ability was 300.93%. Furthermore, it had a higher melting point of 218.94 °C and higher decomposition temperature of 252.69 °C. SEM also showed that it had filled cross-sectional structure and smoother surface compared to the sodium alginate film. Additionally, FTIR showed that 0.5 g microbial composite films possessed more functional groups at 800 and 662 cm−1 wavenumbers that referred to C–C, C–OH, C–H ring and side group vibrations and C–OH out-of-plane bending, respectively, which contributed to the stronger bonds in the microbial composite film. Initial conclusions depict the potential of Bacillus subtilis to be used as reinforcing material in the development of microbial composite films, which also have the prospect to be used in electronic applications. This is due to the conductivity of the films increasing as Bacillus subtilis cell mass increases.

Monotcytes/Macrophages Are Involved in Erythrocytosis Caused By Both EPO-Dependent and -Independent Activation of EPOR

Excess activation of EPO receptor (EPOR) signaling leads to erythrocytosis, which is defined as an absolute increase of red cell mass. EPO-independent and erythrocytes-intrinsic EPOR activation is induced by gain-of-function EPOR or JAK2 mutations, resulting in “primary” erythrocytosis. Erythrocytes-extrinsic EPOR activation is called “secondary” erythrocytosis, which is caused by EPOR stimulation with high level of EPO.In order to investigate primary and secondary erythrocytosis in detail, we generated two unique mouse models. LysM -Cre; Jak2 V617F knock-in heterozygous mice express constitutively active JAK2 in LysM-expressing cells, which include a small portion of HSCs and their downstream progenitors. We also generated a Alb -Cre; Rosa26 LoxP-Stop-LoxP-rtTA (LSL-rtTA);human EPAS1 double-point-mutant (DPM) mice (Alb -Cre;DPM). EPAS1 is a master regulator of EPO gene. The EPAS1-DPM has mutations in P531 and N847 sites, which are critical sites for stability and transcriptional inactivation in normoxia. This mouse expresses the DPM mutant (P531A and N847A) specifically in hepatocytes in a doxycycline-inducible manner, resulting in high EPO production in the liver. We used LysM -Cre; Jak2 V617F as an erythrocytes-intrinsic “primary” erythrocytosis model, and Alb -Cre;DPM as an erythrocytes-extrinsic “secondary” erythrocytosis model. Interestingly, both of them developed comparable degree of erythrocytosis quickly. The plasma EPO level in LysM -Cre; Jak2 V617F was significantly decreased, while that in Alb -Cre/DPM was significantly increased in comparison to the control and also to LysM -Cre; Jak2 V617F. They both exhibited splenomegaly, but spleens of Lys M-Cre; Jak2 V617F mice were significantly larger than those of Alb -Cre;DPM mice. In Alb -Cre;DPM mice, CD45- CD71+ erythroblasts were detected in the liver, whereas there were almost no erythroblasts in the liver of the control mice, suggesting that extremely high level of EPO expression in the liver causes erythropoiesis in the liver of Alb -Cre;DPM mice.Increased monocyte count has been reported in patients with erythrocytosis. Consistent with the observation in the patients, total CD11b+ CD115+ monocytes were significantly increased in PB from both LysM -Cre; Jak2 V617F and Alb -Cre;DPM mice compared to those in PB from the wild-type mice. It is also well known that macrophages play a crucial role in erythropoiesis, supplying the iron and engulfing the protruded nuclei from erythroblasts in the terminal maturation process. The complex of erythroblasts and macrophages directly interacting each other is called as “erythroblastic island (EBI)”. Thus, we next analyzed macrophages in bone marrow (BM) and spleen (SP) in our erythrocytosis models. Macrophages in SP were dramatically increased in the both models, while those in BM were not significantly altered. The percentage of EBI (multiplet F4/80+ Ter119+) in BM and SP were increased in both models.EPOR expression on the cell surface is required for EPO-EPOR signaling. To confirm the expression of EPOR by genetic approach, we combined EPOR -Cre knock-in allele and Rosa26LSL-GFP reporter allele to generate EPOR -Cre;GFP reporter mice. In this model, pro-erythroblasts and basophilic erythroblasts in BM expressed GFP as expected. We did not find GFP expression on the Macrophage and dendritic cell precursors (MDPs), common monocyte progenitors (cMoPs), mature monocytes in BM and PB monocytes. In a single-cell-gated macrophage population, we found a small fraction expressing GFP. However, FACS sorting revealed that those GFP+ cells are not macrophages alone, but macrophages with erythroblasts adhered to them. Even after adding EDTA, some of those macrophages still retain bound erythroblasts. It is possible that they could acquire activated EPOR signaling from erythroblasts within EBI through direct interaction. We are currently investigating the mechanisms for expansion of monocytes/macrophages in erythrocytosis.Collectively, here we demonstrate that both Jak2V617F and high EPO could activate EPOR signaling and expand erythroblasts and monocytes/macrophages, which might contribute to erythrocytosis phenotype in our primary and secondary erythrocytosis models. Further exploration of the roles of monocytes/macrophages in erythrocytosis could lead to development of novel therapeutic strategy for erythrocytosis by modulating their functions in EBI. DisclosuresNo relevant conflicts of interest to declare.

222-LB: Canonical Wnt Signaling Regulates Beta-Cell Neogenesis from Pancreatic Duct Cells

Diabetes results from loss or insufficient numbers of insulin producing beta cells. Thus, restoration of beta cell mass can offer a lasting cure for diabetes. Physiological conditions such as obesity and pregnancy are known to induce compensatory increase in beta cell mass; suggesting the presence of an endogenous mechanism for beta cell regeneration. Significant progress has been made in understanding the regeneration of beta-cells through proliferation of existing beta-cells. In addition to proliferation of existing beta-cells, there is mounting evidence, from both animal models and human tissue, indicating that adult pancreatic duct cells can regenerate endocrine cells, including insulin producing cells. However, the molecular mechanisms governing the regeneration of pancreatic endocrine cells, especially beta cell from pancreatic duct cells remain to be defined. We have found that adult pancreatic duct cells are active in both Notch and Wnt/beta-catenin signaling. Transcriptional profiling revealed Hes1 as the predominant Notch target gene in adult mouse pancreatic duct cells. Using lineage-tracing mouse models, we found that duct-specific deletion of Hes1 in vivo resulted in the conversion of pancreatic duct cells into predominantly somatostatin+ cells. Conversely, pancreatic duct-specific deletion of the canonical Wnt signaling effector, beta-catenin, in vivo resulted in downregulation of Hes1 expression, and the conversion of pancreatic duct cells into predominantly insulin producing cells. Taken together our results demonstrate that Notch and Wnt signaling maintain the adult pancreatic duct state, with duct-derived beta-cell neogenesis requiring the loss of Wnt/beta-catenin signaling. Disclosure S. Afelik: None. Z. Yu: None.

1203-P: High-Dose Erythropoietin and Cardiac Function in Mice

Erythropoietin (EPO) is the cytokine required for erythropoiesis. EPO stimulated increase in hematocrit elevates blood viscosity that may cause hemodynamic risks in the cardiovascular system, promote ischemic heart disease, and stroke. Herein, we studied the physiological effect of high dose exogenous EPO (i.p. 3000 U/kg, 3 times/week for three weeks) with high fat diet (HFD) on cardiac function using echocardiographic measurements in young wild-type (WT) male and female mice and older obese WT male mice with fat mass &amp;gt;45%. In young male mice, but not in female and older obese male mice, EPO treatment with HFD decreased body weight and fat mass accumulation compared with saline, although all groups showed similar EPO stimulated increases in hematocrit. EPO reduced ejection fraction and fractional shortening of left ventricle (LV) in both young male and female mice. This is consistent with the previous report that mice with chronic overexpression of human EPO have decreased fractional shortening of LV. In contrast, older obese male mice have reduced basal ejection fraction and fractional shortening that remain unchanged with EPO treatment indicating that the changes in basal ejection fraction and fractional shortening with EPO treatment in younger mice were not due to EPO stimulated increase in red cell mass. EPO decreased peak velocity of blood flow in the pulmonary artery in all groups indicative of increased hematocrit promoting higher blood viscosity. In addition, EPO increased LV posterior wall thickness in diastole in older male obese mice. This is consonant with observations that high hematocrit is related to concentric left ventricular hypertrophy in human. These data suggest that high dose EPO treatment may be associated with possible cardiac dysfunction with decreased ejection fraction and fractional shortening of LV, and alteration of left ventricular geometry. Disclosure J. Lee: None. C. T. Noguchi: None.