bluuebunny/crossref_metadata_2025_split huggingface dataset:
Knockdown of ABCme Impairs Heme Biosynthesis as Revealed by Integrating of RNAi and the LiveCell® Array.
Authors: Haigh Sarah E. , Elorza Alvaro A. , Biran Israel , Zurgil Naomi , Deutsch Motti , Mikkola Hanna , Shirihai Orian S. 
Published: October 2019

Abstract
Heme synthesis begins and ends in the mitochondrion, whilst intermediate steps are performed in the cytosol. Consequently, heme precursors must be transported across the mitochondrial membranes, however, the transporters of these intermediates have not been identified. Conditions such as sideroblastic anemia, porphyria and MDS are all characterized by abnormal heme synthesis and accumulation of heme intermediates and iron. Thus, impaired function of mitochondrial transporters is thought to play a key role in these conditions.
We have previously reported that over-expression of murine ABCme increases hemoglobin production; however the identity of the heme intermediate transported by ABCme is as yet unclear (Shirihai et al. 2000).
We have used lentivirus delivered shRNA, harboring a GFP marker, to knockdown ABCme mRNA in human K562 erythroid cells. Interpreting RNAi experiments in heterogeneous cell populations, such as differentiating cells, can be problematic. Specific challenges which must be addressed are: 1) transgene and RNAi expression is hard to achieve in erythroid cells; 2) cell to cell expression efficiency is variable; 3) differentiation is a stochastic process; 4) erythroid cells are non-adherent rendering imaging of living cells difficult. Taken together, these factors lead to pronounced cellular heterogeneity, which may mask biological effects. Furthermore, the correlation of RNAi expression and heme synthesis requires the use of benzidine staining, which results in protein oxidation and elimination of all fluorescent signals. To address these challenges we developed a new assay that takes advantage of a novel array which corrals living cells into individual wells. The LiveCell® Array is a densely packed array of micron-sized wells, each accommodating an individual cell, embedded within a microscope slide. Sequential measurements of multiple cellular activities can be taken and the images analyzed using Metamorph image analysis software.
Using this system we identified a population of cells which expressed high levels of RNAi by screening for GFP. The stage of differentiation of each cell was determined using antibodies against extra-cellular makers (i.e. Glycophorin A) conjugated to fluorescent moieties thus identifying maturing subpopulations of cells. To measure heme content, benzidine was subsequently applied and light absorbance was quantized at the resolution of the individual cell. Heme content was then cross correlated with the differentiation and expression fluorescent markers. By integrating RNAi with the LiveCell® Array we have found that knockdown of ABCme results in reduced heme production in differentiating erythroid cells. Further analysis of heme intermediates using this model is being pursued in order to identify the substrate of ABCme.
The Co-Recessive Inheritance Model: A Paradigm for Fanconi Anemia and Other Bone Marrow Failure Syndromes.
Authors: Lambert W. Clark , Brown Monique M. , Centurion Santiago A. 
Published: October 2019

Abstract
One of us (WCL) has previously proposed a mathematical model, Co-Recessive Inheritance, for inherited diseases associated with DNA repair deficiencies (Lambert WC, Lambert MW: Mutat. Res., 1985;145:227–234; Lambert WC: Keynote Address, 21st Anniversary Celebration, MRC Cell Mutation Unit, University of Sussex, UK. Mutat. Res., 1992;273:179–102). The model is also applicable to diseases associated with defective cell cycle modulation following specific types of DNA damage, such as Fanconi Anemia, with or without additional defects in DNA repair. The model proposes that in some complementation groups of these diseases defective alleles at more than one locus are required for the disease phenotype to be expressed. It follows from the model (A readily understandable derivation will be presented.) that the carrier frequencies of the genes involved are very much higher than would be predicted based on classical population genetics. This may impact on recent observations of higher than expected co-inheritance of defective alleles of Fanconi Anemia and Bloom Syndrome genes along with BRCA genes in certain populations (e.g., Koren-Michowitz, M, et al.: Am. J. Hematol., 2005;78:203–206), and provides an explanation for the lower than expected incidence of cancer in these individuals. It also provides an explanation for finding biallelic defects in the same DNA repair genes in more than one complementation group of Fanconi Anemia (Howlett NG, et al.: Science, 2002;297:606–609). The Co-Recessive Model predicts that other findings of this nature are to be expected, and provides some guidelines that may be helpful in the process of gene discovery in Fanconi Anemia. Among the more important of these are 1) that the search for defective genes in each complementation group should not cease when one such gene is found, even if one or more patients in the group is homozygous or compound heterozygous for defective alleles of that gene, and 2) that carrier frequencies for some Fanconi Anemia genes may be much higher than would otherwise be anticipated, with a significant proportion of the normal population being carriers. If the latter hypothesis is correct, it follows that the relevance of these rare diseases and their associated genes to disease, including bone marrow failure, in the general population is dramatically greater than has been generally believed.
Serum Cystatin C Is a Better Marker for Renal Function Than Serum Creatinine in Children with Sickle Cell Disease.
Authors: Alvarez Ofelia A. , Wright Dale , Lopez Gabriela , Zilleruelo Gaston 
Published: October 2019

Abstract
High serum creatinine is a late manifestation for patients (pts) with sickle cell disease (SCD) who develop severe renal dysfunction, because creatinine is secreted by the renal tubules. Serum cystatin C is a cysteine proteinase inhibitor, that is produced by all nucleated cells in the body, does not undergo tubular secretion, and reflects glomerular filtration rate (GFR)accurately. Normal levels for serum cystatin C are 0.5–1.3 mg/L for children 1–17 years, and 0.5–1 mg/L for adults. The purpose of this study was to compare serum cystatin C and serum creatinine as markers of GFR in children with SCD with different levels of albuminuria. Twenty pts (mean age 16.4, range 9–21 years) had serum creatinine, serum cystatin, and 24-hour urine for creatinine clearance. Pts with normoalbuminuria (N=11) had mean serum creatinine of 0.55±0.14 mg/L, creatinine clearance of 168±36 ml/min/1.73m2, normal serum cystatin C 0.78±0.16 mg/L, and normal estimated GFR derived from cystatin of 106±27 ml/min/1.73m2. In contrast, pts with proteinuria (N=4) had higher or abnormal serum cystatin C (mean 1.25 ±0.34, range 0.9–1.7 mg/L) and reduced estimated GFR (mean 59±21, range 35–85) consistent with poor kidney function; nevertheless, the serum creatinine (0.7±0.2) and creatinine clearance remained normal (125±27). Pts with only microalbuminuria (N=5) maintained normal levels of cystatin C and estimated GFR by cystatin. We conclude that serum cystatin C discriminated better for kidney dysfunction than serum creatinine and creatinine clearance with significantly different values between patients with normoalbuminuria and macroalbuminuria (p=0.038). More studies are warranted in order to investigate further the value of serum cystatin C in the monitoring of patients with SCD and albuminuria.
Reduced Cholesterol Levels in Adults with Sickle Cell Disease.
Authors: Nagajothi Nagaprasad , Sanmugarajah Jasotha 
Published: October 2019

Abstract
Reduced levels of cholesterol was first reported in African children with sickle cell disease (SCD).More recently similar findings were reported in African American adults with SCD in the United States. Epidemiologic studies have identified a relation between low serum levels of total cholesterol (<130 mg/dL) and increased mortality from all causes. We wanted to study the cholesterol levels in adults with SCD especially as their average life expectancy is continuing to increase with advances in medical care.We conducted a retrospective study comparing the serum lipid profile with 15 adults with SCD seen at our hospital with 15 age matched controls who did not have SCD or hyperlipidemia. The mean age in the SCD group was 48±13 years and the mean age in the control group was 46±5years (P=0.73). The mean total cholesterol level in adults with SCD was 135±26 mg/dL and was significantly lower than the mean total cholesterol level in age matched controls which was 169±30 mg/dL (P=0.001). LDL levels were significantly lower in those with SCD compared to controls (67±16 mg/dL versus 90±29mg/dL, respectively, P=0.01). Adults with SCD also had lower HDL levels (45±15 mg/dL) compared with their controls (60±11 mg/dL, P=0.004). There was no difference in the mean triglyceride levels between the two groups. While there was no difference in the mean height between the two groups the SCD group had lower Body Mass Index (BMI) than the control group (23± 4mg/dL versus 27±4mg/dL, respectively, P=0.02). Our results suggest that adults with sickle cell disease have lower serum total cholesterol, LDL, HDL levels as well as lower BMI compared to their age matched controls. Larger studies to validate our observations and to study its clinical implications are warranted.
Lipid profile and Body Mass Index in Sickle Cell Disease and Controls Sickle Cell* Control* Mean(SD) Mean(SD) Pvalue *n=15 Age(years) 48±13 46±5 0.73 Total Cholsterol (mg/dL) 135±26 169±30 0.001 LDL(mg/dL) 67±16 90±29 0.01 HDL(mg/dL) 45±15 60±11 0.004 Triglycerides(mg/dL) 113±51 94±47 0.3 Height(cm) 167±7 167±6 0.7 Weight(lbs) 141±27 178±35 0.007 Body Mass Index 23±4 27±4 0.02
Platelet Binding to Leukocytes as a Determinant of Leukocyte Activation In Vivo.
Authors: Chirinos Julio A. , Jy Wenche , Tiede Maike , Castrellon Aurelio , Soriano Andres O. , Del Carpio Freddy , Arteaga Roque , Jimenez Joaquin J. , Horstman Lawrence , Bidot Carlos , Ahn Yeon S. 
Published: October 2019

Abstract
Background: Platelet binding to leukocytes has been shown to induce leukocyte activation in vitro. It is unknown to what extent platelet binding to leukocytes regulates leukocyte activation in vivo. We examined the correlation between platelet binding to leukocytes (platelet-leukocytes conjugates) and leukocyte activation in various clinical conditions.
Methods: We studied a total of 321 venous blood samples from 291 subjects with acute venous thromboembolism (n=25), atrial fibrillation (n=48), metabolic syndrome (n=37), congestive heart failure (n=44), antiphospholipid syndrome (n=70), early sepsis (n=35), late sepsis (n=30) and normal controls. Using flow cytometry, we measured leukocyte expression of activation marker CD11b. Platelet-leukocyte conjugate measurement was based on co-expression of CD41 and CD45.
Results: Levels of platelet-leukocyte conjugates strongly correlated with CD11b expression. This finding was present in congestive heart failure (r=0.47; p=0.001), atrial fibrillation (r=0.32; p=0.02), metabolic syndrome (r=0.51; p=0.001), venous thromboembolism (r=0.74; p<0.0001), early sepsis (r=0.70; p<0.0001), late sepsis (r=0.66, p=0.0002), and antiphospholipid antibody syndrome (r=0.53; p<0.0001). In normal controls, the relationship between platelet-leukocyte conjugates and CD11b expression was best described by a 2nd-degree polynomial model, which included the quadratic term platelet-leukocyte conjugates squared, suggesting a threshold effect. The quadratic term added significant prediction of CD11b expression, demonstrating a highly significant exponential increase in CD11b expression after a threshold level of platelet-leukocyte conjugates is reached (model R2=0.57; r2 increase from quadratic term=29%; p=0.0001), as shown in Figure 1. This threshold was not seen in any disease state examined (all p for quadratic terms > 0.05). For example, the relation between platelet-leukocyte conjugates and CD11b expression in venous thromboembolism is shown in Figure 2, and is best described by a straight line. The addition of quadratic term did not add any prediction of CD11b expression, as shown.
Conclusions: Our findings strongly suggest that platelet binding to leukocytes is a key determinant of leukocyte activation in vivo. The degree of platelet binding to leukocytes strongly correlates with leukocyte activation after a threshold is reached in normal adults. This threshold is lost in pro-thrombotic and pro-inflammatory disease states, in which a linear increase in leukocyte activation is seen with increasing platelet binding to leukocytes.
Figure 1. Figure 1.
Figure 2. Figure 2.
Use of Recombinant Peptide as Substrate in FRET Based Protease Assays.
Authors: Zhang Lin , Lawson Heather , Harish Vallathucherry , Huff Jason D. , Knovich Mary Ann , Owen John 
Published: October 2019

Abstract
Proteases are enzymes that catalyze the breakdown of peptide bonds. They are essential to all physiologic and pathological processes. Some protease deficiencies are closely associated with certain disease conditions, such as alpha-1-Antitrypsin deficiency in COPD, Antithrombin III deficiency in clotting disorders, and ADAMTS13 deficiency in thrombotic thrombocytopenic purpura (TTP). Measurement of protease activity is of great interest in research, drug discovery and development, and for use as diagnostic or prognostic markers in disease states.
Traditional protease assay methods involve multiple steps and are very time-consuming. Recently, fluorescence energy transfer (FRET) based protease assays have become popular. These assays eliminate gel electrophoresis, radioactivity, and the need for special skills or complicated instruments. They require only one step incubation and allow high throughput screening of a large number of samples.
Generation of pure, reliable, high quality substrate is essential to the development and improvement of such assays. Most FRET based assays use synthesized peptide substrates, the size of which is restricted by the limitations of chemical synthesis. These substrates are usually labeled with a pair of two different fluor-quencher molecules. However, when two identical fluorescent molecules are in close proximity, their fluorescence emission can also be quenched. This phenomenon of autoquenching has been widely used in protein structural studies and even in single-molecule structural studies, but has never been used to produce FRET substrates. We hypothesized that this mechanism can be applied to the production of recombinant FRET substrates. We used the ADAMTS13 - von Willebrand factor (VWF) system to test this hypothesis. ADAMTS13 is a metalloproteinase that cleaves VWF between Y1605 and M1606. Kokame et al. have shown that the smallest effective substrate is a 73 amino acid (AA) peptide from the VWF A2 domain. A recombinant 73 AA peptide was generated using Qiagen pQE-100 His Tag vector and M15 E. coli expression system. Two native amino acids, Q1599 and P1611, which flank the ADAMTS13 cleavage site, were mutated to cysteines. These two cysteines were subsequently labeled with fluorescein molecules. The separation of these two fluoresceins by ADAMTS13 cleavage generated measurable fluorescence increase. There was a steady increase of fluorescence over time during the incubation of this recombinant peptide with pooled normal plasma (PNP) used as the source of ADAMTS13. There was only minimal change in fluorescence during incubation with heat inactivated PNP and plasma samples from known TTP patients with absent ADAMTS13 activity. The enzyme kinetic assay yielded an excellent linear relationship between the rates of fluorescence increase and ADAMTS13 enzyme concentration. Our study has provided proof of principle that fluorogenic recombinant peptides can be generated for FRET based protease assays.
Comparison of Anti-Angiogenic Activities of Thalidomide and Lenalidomide In Vitro.
Authors: Zhang Ling-Hua , Lu Ling , Wu Lei , Dredge Keith , Bartlett J. Blake , Schafer Peter , Muller George , Dalgleish Angus , Stirling David 
Published: October 2019

Abstract
Inhibition of angiogenesis is currently perceived as one of the promising strategies in the cancer therapy. Thalidomide (Thalomid®) and its Immunomodulatory Drug (IMiD®) analogs have entered into clinical trials for the treatment of various types of cancers. Lenalidomide (Revlimid®, CC-5013) is currently being assessed in oncology clinical trials worldwide. In this study we have compared the anti-angiogenic mechanisms of thalidomide and lenalidomide in a variety of experimental systems representing distinct events of the angiogenic process. Endothelial cell (EC) proliferation is stimulated by growth factors. Neither thalidomide nor lenalidomide have an appreciable inhibitory effect on growth factor-induced proliferation of human umbilical vein endothelial cells (HUVEC). In both the HUVEC and B16-F10 mouse melanoma tube formation assays, lenalidomide appears to be at least 10-fold more potent than thalidomide, the latter indicating that effects extend to vessels lined by tumor cells. However, in endothelial cell migration assays, thalidomide is more active than lenalidomide; in both the HUVEC and B16-F10 monolayer scratch migration assays, thalidomide is clearly more potent than lenalidomide in preventing the migration of cells into the wounded/scratched region. In assays of HUVEC migration towards specific angiogenic factors, such as VEGF, bFGF, and TNF-α, thalidomide is also more potent than lenalidomide. Interestingly, assays where the whole physiological process of angiogenesis can be studied show differential sensitivities. Lenalidomide is more potent in the rat aorta assay, whereas thalidomide is more potent in the human umbilical explant assay. Mechanistic studies on signal transduction events triggered by VEGF show that both thalidomide and lenalidomide partially inhibit Akt phosphorylation in VEGF-induced HUVEC. Furthermore, inhibitory effects on the phosphorylation of Gab1, a scaffolding protein upstream of Akt activation, have been observed. In summary, our data indicate that both thalidomide and lenalidomide interfere with key events in the angiogenic process and that they can be differentiated qualitatively depending on which component part is studied.
Repeat Borderline or Negative Platelet Factor-4 ELISA May Yield Positive Results in Patients with Clinical Suspicion of Heparin-Induced Thrombocytopenia.
Authors: Lakey Meredith A. , Arndt David W. , Marques Marisa B. 
Published: October 2019

Abstract
Heparin-induced thrombocytopenia (HIT) is mediated by antibodies to heparin-platelet factor 4 (PF4) complexes which lead to platelet activation and aggregation with or without thrombosis. HIT diagnosis relies on clinical and serologic grounds. Although the detection of PF4 antibodies alone is not diagnostic of HIT without thrombocytopenia (relative or absolute) and/or thrombosis, a negative result does not exclude the diagnosis. Thus, it is expected that initially negative ELISAs may turn positive on repeat testing. We reviewed all PF4 ELISA (GTI Diagnostics, Waukesha, WI) assay reports generated at our institution from January 2003 to May 2005 in search of patients who had more than one test performed in this time-period. Of 1,128 tests performed, 181 (16%) were positive, 95 (8%) were borderline, and 852 (76%) were negative. Borderline results were considered those with an OD405 between 0.3 and 0.4 (cutoff value for positive) or two times that of the negative control of each run. No patients were evaluated using the serotonin-release assay. Nine of 70 patients (13%) with initially negative or borderline ELISAs had a positive result upon repeat testing from 2 to 63 days later (Table). All were inpatients, and 6 of 9 were from surgical specialties, mostly cardiac (5 of 6). Five of 8 patients (62.5%) with an initial positive PF4 ELISA result had either a negative or a borderline subsequent test from 7 to 225 days later (Table). The mean platelet count of the patients that had a nondiagnostic ELISA followed by a positive result was 98 x 109/L (N = 9) at the time of the initial test compared with 137 x 109/L at subsequent testing (p = 0.43 by paired Student’s t test). Of note, 2 patients had a normal platelet count when the ELISA result was positive. None had evidence of thrombosis. Borderline test results tended to be repeated sooner (mean of 16 days) than negative ones (mean of 21 days). We suspect that this difference reflects the clinicians’ high index of suspicion for HIT. Our findings suggest that in patients with a high pre-test probability for HIT (status post cardiac surgery with thrombocytopenia), there should be a low threshold for repeating an initial negative or borderline ELISA assay, thereby increasing detection and minimizing complications from this potentially fatal, yet common clinical entity.
Test Results of the 78 Patients With Repeat HIT ELISAs. Initial Test Result Repeat Test Result Number of Patients (% of Total) Mean Number of Days Between Tests (Range) NA: Not applicable Negative Positive 7 (13) 17 (2–63) (N=56) Negative 45 (80) 26 (1–216) Borderline 4 (7) 22 (8–36) Borderline Positive 2 (14) 21.5 (10–33) (N=14) Negative 6 (43) 17.5 (3–42) Borderline 6 (43) 9 (1–23) Positive Positive 3 (37.5) 34 (4–88) (N=8) Negative 4 (50) 26 (7–80) Borderline 1 (12.5) 225 (NA) Total 78
Case Report of Temozolamide Associated Steroid-Refractory ITP and Successful Treatment with Rituximab.
Authors: Reddy Sandeep , Janis Mark , Curti Mario , Minow Robert 
Published: October 2019

Abstract
We report the first case of temozolamide associated ITP and successful treatment with Rituximab. A 59 year-old male with grade III anaplastic astrocytoma is treated with resection followed by adjuvant temozolamide and gamma knife radiation therapy (XRT). He had been taking ramipril for hypertension, and atorvastatin for hyperlipidemia for greater than 1 year. He began taking phenytoin for seizure prophylaxis at the time of his original diagnosis in December of 2004. His CBC is normal at baseline. He recieves XRT from 2/1/05 until 2/15/05 with a normal platelet count of 142 on 4/21/05. He restarts temozolamide from 4/22 to 4/26 and presents with petechiae and platelet count 0. He receives platelet transfusion but platelet count remians at 3. A bone marrow biopsy confirms adequate megakaryoctes. He develops subcutaneous hemorrhage in the neck and is hospitalized for airway precautions. Prednisone at 2mg/kg and IVIG are given with no response. Rituximab is given weekly x 4 weeks at 375 mg/m2 with response by day 22 to platelet count of 123. The response is durable. See table 1.
Response to Rituxan DATE PLATELET COUNT 6/1/05 3 6/16/05 9 6/23/05 104 6/30/05 123 7/21/05 118