GATA-3 regulates the self-renewal of long-term hematopoietic stem cells.

The transcription factor GATA-3 is expressed and required for differentiation and function throughout the T lymphocyte lineage. Despite evidence it may also be expressed in multipotent hematopoietic stem cells (HSCs), any role for GATA-3 in these cells has remained unclear. Here we found GATA-3 was in the cytoplasm in quiescent long-term stem cells from steady-state bone marrow but relocated to the nucleus when HSCs cycled. Relocation depended on signaling via the mitogen-activated protein kinase p38 and was associated with a diminished capacity for long-term reconstitution after transfer into irradiated mice. Deletion of Gata3 enhanced the repopulating capacity and augmented the self-renewal of long-term HSCs in cell-autonomous fashion without affecting the cell cycle. Our observations position GATA-3 as a regulator of the balance between self-renewal and differentiation in HSCs that acts downstream of the p38 signaling pathway.

Dilemma of elevated CA 19-9 in biliary pathology.

Carbohydrate antigen 19-9 (CA 19-9) is a tumor marker which has been extensively evaluated and widely utilized primarily in diagnosing and prognosticating pancreaticobiliary malignancies. Levels may be significantly influenced and elevated in cases of benign biliary conditions however, especially in obstructive jaundice, thereby posing difficulty in distinguishing between benign and malignant cholestasis. A myriad of studies have focused on elucidating proper use and interpretation of CA 19-9 in pancreatic cancer as well as in the setting of cholestasis. These studies have demonstrated that many factors influence CA 19-9 values and various methods for interpreting CA 19-9 in obstructive jaundice have been proposed. With improvements in diagnostic imaging, advancements in endoscopic modalities, and likelihood that management will not change based on the results of the test, clinicians should be cautious when ordering CA 19-9 and consider the reasons for measuring the tumor marker.

Titanium scaffold osteogenesis in healthy and osteoporotic rats is improved by the use of low-level laser therapy (GaAlAs).

The present study aimed to assess the effects of low-level laser therapy (GaAlAs) on the bone repair process within titanium scaffolds in the femurs of healthy and osteoporotic rats. Fifty-six rats were divided into four groups: group Sh: SHAM animals that received scaffolds; group LSh: SHAM animals that received scaffolds and were subjected to laser therapy; group OV: ovarietomized (OVX) animals that received scaffolds; and group LOV: OVX animals that received scaffolds and were subjected to laser therapy. Thirty days following ovariectomy or sham surgery, scaffolds were implanted in the left femurs of all animals in the study. Immediately after opening the surgical site, the inner part of the surgical cavity was stimulated with low-level laser (GaAlAs). In addition to this procedure, the laser group was also subjected to sessions of low-level laser therapy (LLLT) at 48-h intervals, with the first session performed immediately after surgery. The rats were sacrificed at 2 and 6 weeks, time in which femur fragments were submitted for histological and histomorphometric examination, and skin tissue above the scaffold was submitted to histological analysis. At the end of the study, greater bone formation was observed in the animals submitted to LLLT. At 2 and 6 weeks, statistically significant differences were observed between LSh and Sh groups (p = 0.009 and 0.0001) and LOV and OV (p = 0.0001 and 0.0001), respectively. No statistical difference was observed when assessing the estrogen variable. On the basis of our methodology and results, we conclude that LLLT improves and accelerates bone repair within titanium scaffolds in both ovariectomized and healthy rats, when compared to animals not subjected to radiation.

Healing of normal and osteopenic bone with titanium implant and low-level laser therapy (GaAlAs): a histomorphometric study in rats.

The study investigates the influence of low-level laser therapy (LLLT) on bone healing in the femur of osteopenic and normal rats with titanium implants. Ovariectomy and control group were randomly submitted to LLLT, which was applied by gallium-aluminum-arsenium (GaAlAs) laser at the surgical site before and after placing the implant, for seven times. Histomorphometric and statistical analysis were performed. Most irradiated groups showed higher values than the nonirradiated groups. The GaAlAs infrared diode laser may improve the osseointegration process in osteopenic and normal bone, particularly based on its effects in the initial phase of bone formation.

Essential role for Ptpn11 in survival of hematopoietic stem and progenitor cells.

Src homology 2 domain-containing phosphatase 2 (Shp2), encoded by Ptpn11, is a member of the nonreceptor protein-tyrosine phosphatase family, and functions in cell survival, proliferation, migration, and differentiation in many tissues. Here we report that loss of Ptpn11 in murine hematopoietic cells leads to bone marrow aplasia and lethality. Mutant mice show rapid loss of hematopoietic stem cells (HSCs) and immature progenitors of all hematopoietic lineages in a gene dosage-dependent and cell-autonomous manner. Ptpn11-deficient HSCs and progenitors undergo apoptosis concomitant with increased Noxa expression. Mutant HSCs/progenitors also show defective Erk and Akt activation in response to stem cell factor and diminished thrombopoietin-evoked Erk activation. Activated Kras alleviates the Ptpn11 requirement for colony formation by progenitors and cytokine/growth factor responsiveness of HSCs, indicating that Ras is functionally downstream of Shp2 in these cells. Thus, Shp2 plays a critical role in controlling the survival and maintenance of HSCs and immature progenitors in vivo.

Targeted deletion of the tachykinin 4 gene (TAC4-/-) influences the early stages of B lymphocyte development.

Hemokinin-1 (HK-1), encoded by the TAC4 gene, is a tachykinin peptide that is predominantly expressed in non-neuronal cells, such as immune cells. We have disrupted the mouse TAC4 gene to obtain a better understanding of the actions of HK-1 during hematopoiesis. We demonstrate here that TAC4(-/-) mice exhibit an increase of CD19(+)CD117(+)HSA(+)BP.1(-) "fraction B" pro-B cells in the bone marrow, whereas pre-B, immature, and mature B cells are within the normal range. We show that in vitro cultures derived from TAC4(-/-) bone marrow, sorted "fraction B" pro-B cells or purified long-term reconstituting stem cells, contain significantly higher numbers of pro-B cells compared with controls, suggesting an inhibitory role for HK-1 on developing B cells. Supporting this idea, we show that addition of HK-1 to cultures established from long-term reconstituting stem cells and the newly described intermediate-term reconstituting stem cells leads to a significant decrease of de novo generated pro-B cells. Based on our studies, we postulate that HK-1 plays an inhibitory role in hematopoiesis, and we hypothesize that it may be part of the bone marrow microenvironment that supports and regulates the proliferation and differentiation of hematopoietic cells.

Capturing, sharing and analysing biophysical data from protein engineering and protein characterization studies.

Large amounts of data are being generated annually on the connection between the sequence, structure and function of proteins using site-directed mutagenesis, protein design and directed evolution techniques. These data provide the fundamental building blocks for our understanding of protein function, molecular biology and living organisms in general. However, much experimental data are never deposited in databases and is thus 'lost' in journal publications or in PhD theses. At the same time theoretical scientists are in need of large amounts of experimental data for benchmarking and calibrating novel predictive algorithms, and theoretical progress is therefore often hampered by the lack of suitable data to validate or disprove a theoretical assumption. We present PEAT (Protein Engineering Analysis Tool), an application that integrates data deposition, storage and analysis for researchers carrying out protein engineering projects or biophysical characterization of proteins. PEAT contains modules for DNA sequence manipulation, primer design, fitting of biophysical characterization data (enzyme kinetics, circular dichroism spectroscopy, NMR titration data, etc.), and facilitates sharing of experimental data and analyses for a typical university-based research group. PEAT is freely available to academic researchers at http://enzyme.ucd.ie/PEAT.

Remeasuring HEWL pK(a) values by NMR spectroscopy: methods, analysis, accuracy, and implications for theoretical pK(a) calculations.

Site-specific pK(a) values measured by NMR spectroscopy provide essential information on protein electrostatics, the pH-dependence of protein structure, dynamics and function, and constitute an important benchmark for protein pK(a) calculation algorithms. Titration curves can be measured by tracking the NMR chemical shifts of several reporter nuclei versus sample pH. However, careful analysis of these curves is needed to extract residue-specific pK(a) values since pH-dependent chemical shift changes can arise from many sources, including through-bond inductive effects, through-space electric field effects, and conformational changes. We have re-measured titration curves for all carboxylates and His 15 in Hen Egg White Lysozyme (HEWL) by recording the pH-dependent chemical shifts of all backbone amide nitrogens and protons, Asp/Glu side chain protons and carboxyl carbons, and imidazole protonated carbons and protons in this protein. We extracted pK(a) values from the resulting titration curves using standard fitting methods, and compared these values to each other, and with those measured previously by ¹H NMR (Bartik et al., Biophys J 1994;66:1180­1184). This analysis gives insights into the true accuracy associated with experimentally measured pK(a) values. We find that apparent pK(a) values frequently differ by 0.5­1.0 units depending upon the nuclei monitored, and that larger differences occasionally can be observed. The variation in measured pK(a) values, which reflects the difficulty in fitting and assigning pH-dependent chemical shifts to specific ionization equilibria, has significant implications for the experimental procedures used for measuring protein pK(a) values, for the benchmarking of protein pK(a) calculation algorithms, and for the understanding of protein electrostatics in general.

Negative effects of alcohol intake and estrogen deficiency combination on osseointegration in a rat model.

Alcohol intake and estrogen deficiency can both affect bone physiology and have shown to have an adverse effect on dental implant therapy. However, the combination of both factors on osseointegration is unknown. The aim of this study was to evaluate osseointegration in rats fed with alcohol and presenting induced estrogen deficiency. Ninety-six female rats were divided according to diet and hormonal condition into 6 groups as follows: group Sh-W: sham (simulated ovariectomy) control, food and water ad libitum; group Sh-Et: sham, food and 20% ethanol solution ad libitum; group Sh-Su: sham, food and sucrose solution controlled to ensure an isocaloric diet in relation to Sh-Et; group Ov-W: ovariectomy, food and water ad libitum; group Ov-Et: ovariectomy, food and 20% ethanol solution ad libitum; and group Ov-Su: ovariectomy, food and sucrose solution controlled to ensure an isocaloric diet as Ov-Et. The groups were subdivided according to time of euthanasia: 30 and 45 days after placement of implants. Implant surgery was performed 1 month after ovariectomy or sham. After euthanasia, the femurs were removed and evaluated by histomorphometry. Groups Ov-Et and Ov-Su showed the lowest percentage of bone-to-implant contact. The combination of alcohol intake and estrogen deficiency, and the combination of estrogen deficiency and reduced ingestion of food can negatively affect osseointegration in rats.

The role of zinc in the prevention and treatment of nonalcoholic fatty liver disease.

The zinc element is an essential nutrient for human health. Zinc is involved in the glucose, lipid, and protein metabolism and antioxidant processes in biological pathways. Zinc deficiency can lead to several chronic liver diseases. Nonalcoholic fatty liver disease (NAFLD) is one of the most common liver diseases where zinc deficiency plays a critical role in pathogenesis. Human and animal studies showed that both NAFLD risk factors (i.e., insulin resistance, diabetes mellitus, dyslipidemia, obesity, hypertension) and NAFLD itself are associated with decreased blood levels of zinc. Additionally, endoplasmic reticulum stress and inflammation due to unfolded protein response, inadequate dietary zinc intake, and decreased zinc absorption from the gastrointestinal tract can result in zinc deficiency leading to NAFLD. Herein, we reviewed the mechanistic links between zinc deficiency and NAFLD development and the role of zinc in the prevention and treatment of NAFLD.

Drug-Induced Liver Injury Associated With Alpha Bolic (RAD-140) and Alpha Elite (RAD-140 and LGD-4033).

We report a 52-year-old man who developed drug-induced liver injury after taking Alpha Bolic (contains RAD-140) and Alpha Elite (contains both RAD-140 and LGD-4033) supplements. Liver biopsy demonstrated diffuse centrilobular canalicular cholestasis, prominent ductular reaction, and mild lobular inflammation with rare non-necrotizing epithelioid granuloma suggestive of drug-induced liver injury. Liver enzymes returned to normal levels approximately 3 months after the patient stopped both supplements. We present the mechanism of drug-induced liver injury associated with 2 selective androgen receptor modulators, including RAD-140 and LGD 4033.

Ligandrol (LGD-4033)-Induced Liver Injury.

We described a 32-year-old man who developed severe drug-induced liver injury after using Ligandrol (LGD-4033). The diagnosis was confirmed by a liver biopsy that showed cholestatic hepatitis with a mild portal, periportal, and perisinusoidal fibrosis. Ligandrol is a selective androgen receptor modulator that is available over the counter and via the internet.

Idiosyncratic Liver Injury Due to Levocetirizine.

Levocetirizine is an over-the-counter nonsedating antihistaminic agent commonly used to treat allergic diseases. Clinically significant acute liver injury has been very rarely associated with its use. Only 2 cases of acute liver injury associated with levocetirizine have been reported in the literature. We describe the case of a 67-year-old man who developed clinically significant acute hepatic injury after using levocetirizine. On the liver biopsy, there were histological findings of periportal inflammation and prominent cholestatic injury; such findings are consistent with those previously described in another similar case.

Talking Genes in Breast and Pancreatic Malignancies.

INTRODUCTION: Both breast and pancreatic cancers have high mortality rates. Breast cancer is the second leading cause of cancer death in females, while pancreatic ductal adenocarcinoma (PDAC) is the fourth most common cause of cancer death. Almost 4-16 % of individuals with pancreatic cancer have a family history of the disease. Intra-ductal papillary mucinous neoplasms (IPMNs) are cystic lesions that received more attention lately due to their associations with PDAC and other solid organ tumors, such as breast cancer. AIM: The purpose of this article is to discuss the association of the familiar pancreatic cancer (FPC), sporadic pancreatic cancer, and IPMNs with the breast cancer. RESULTS: Mutations in BRCA2, BRCA1, p16 and PALB2 play a major role in the genetic etiologies of familial pancreatic cancer. In familial and sporadic pancreatic cancers, mutations in BRCA2 are associated with a high incidence of PDAC, while mutations in BRCA1have shown inconsistent results. Data is insufficient to prove an association between IPMNs and breast cancer. CONCLUSION: The familial clustering of PDAC is not well understood. Further studies are required for greater comprehension of the genetic basis of PDAC and the association between IPMNs and breast cancer.

New insights into genetic predisposition and novel therapeutic targets for nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disease in the United States affecting 80-100 million Americans. NAFLD encompasses a spectrum of diseases ranging from excess liver fat (nonalcoholic fatty liver or NAFL), to necro-inflammation (nonalcoholic steatohepatitis or NASH), to fibrosis/ cirrhosis, and malignant transformation (hepatocellular carcinoma). Susceptibility to NAFLD is highly variable and it remains unclear why some patients with NAFLD exhibit NASH, whereas patients with known risk factors have NAFL only. The reasons for this variability can be a partially attributed to differences in genetic background. In the last decade, there have been multiple genome wide association studies, which have enriched our understanding of the genetic basis of NAFLD. The I148M PNPLA3 (patatin-like phospholipase domain-containing protein 3) variant has been identified as the major common genetic determinant of NAFLD. Variants with moderate effect size like TM6SF2, MBOAT7 and GCKR have also been shown to have a significant contribution. New research has uncovered major pathways leading to disease development and progression; therefore, multiple medications are being developed and tested for the treatment of advanced NAFLD. These agents target metabolic mechanisms as well as inflammation and fibrosis pathways. Several randomized clinical trials (RCTs) are evaluating the efficacy of these novel agents on histological improvement of disease severity and decreasing liver-related outcomes. FDA-approved medications for NASH and NASH-related fibrosis are expected by 2020.

Acute Pancreatitis in Chronic Dialysis Patients.

The annual incidence of acute pancreatitis (AP) ranges from 4.9 to 73.4 cases per 100,000 worldwide. Patients with end-stage renal disease on dialysis have an increased risk for developing AP compared with patients without renal disease. In addition to the general population risk factors, there are factors related to renal insufficiency and dialysis process that might predispose to AP in this population. Clinical features and diagnosis are the same as those in patients without renal failure; however, amylase and lipase levels should be interpreted cautiously as they might be falsely elevated in renal failure. In this article, we will describe the risk factors that are exclusive to this population. In addition, we will also focus on the laboratory indices and clinical features that are unique to this population with patients with end-stage renal disease.

Redesigning protein pKa values.

The ability to re-engineer enzymatic pH-activity profiles is of importance for industrial applications of enzymes. We theoretically explore the feasibility of re-engineering enzymatic pH-activity profiles by changing active site pK(a) values using point mutations. We calculate the maximum achievable DeltapK(a) values for 141 target titratable groups in seven enzymes by introducing conservative net-charge altering point mutations. We examine the importance of the number of mutations introduced, their distance from the target titratable group, and the characteristics of the target group itself. The results show that multiple mutations at 10A can change pK(a) values up to two units, but that the introduction of a requirement to keep other pK(a) values constant reduces the magnitude of the achievable DeltapK(a). The algorithm presented shows a good correlation with existing experimental data and is available for download and via a web server at http://enzyme.ucd.ie/pKD.

Representation is faithfully preserved in global cDNA amplified exponentially from sub-picogram quantities of mRNA.

Analysis of transcript representation on gene microarrays requires microgram amounts of total RNA or DNA. Without amplification, such amounts are obtainable only from millions of cells. However, it may be desirable to determine transcript representation in few or even single cells in aspiration biopsies, rare population subsets isolated by cell sorting or laser capture, or micromanipulated single cells. Nucleic-acid amplification methods could be used in these cases, but it is difficult to amplify different transcripts in a sample without distorting quantitative relationships between them. Linear isothermal RNA amplification has been used to amplify as little as 10 ng of total cellular RNA, corresponding to the amount obtainable from thousands of cells, while still preserving the original abundance relationships. However, the available procedures require multiple steps, are labor intensive and time consuming, and have not been shown to preserve abundance information from smaller starting amounts. Exponential amplification, on the other hand, is a relatively simple technology, but is generally considered to bias abundance relationships unacceptably. These constraints have placed beyond current reach the secure and routine application of microarray analysis to single or small numbers of cells. Here we describe results obtained with a rapid and highly optimized global reverse transcription#150;PCR (RT-PCR) procedure. Contrary to prevalent expectations, the exponential approach preserves abundance relationships through amplification as high as 3 x 10(11)-fold. Further, it reduces by a million-fold the input amount of RNA needed for microarray analysis, and yields reproducible results from the picogram range of total RNA obtainable from single cells.

Trajectories of recovery of quality of life in women after an acute cardiac event.

OBJECTIVES: Female cardiac patients' health-related quality of life (HRQoL) during the first year after an acute cardiac event was compared with age-weighted Australian population norms. The impact of age, event type and cardiac rehabilitation (CR) programme attendance on recovery was assessed. METHODS: The short form 36-item health survey (SF-36) was administered to 229 women aged from 36 to 84 years consecutively admitted to 4 hospitals after acute myocardial infarction (AMI) or to undergo coronary artery bypass graft surgery (CABGS). Data were collected at 4 time points over 12 months. SF-36 subscale scores were compared with age-weighted norms for Australian women. Mplus was used to analyse growth trajectories for SF-36 subscales. RESULTS: Patients had impaired HRQoL at baseline (except in general health), with progressive improvement over time. Recovery to normative levels was fastest in the areas of bodily pain and mental health (by 2 months) and slowest in the area of physical functioning, and physical and emotional role limitations (by 12 months). By 4 months, general health scores had surpassed population norms. For all scales, most improvement occurred in the first 2 months, with little subsequent improvement. CABGS patients showed significantly more improvement than AMI patients in several areas, partly due to the poorer functioning of CABGS patients at baseline. Rate of improvement was not influenced by patients' age or frequency of CR attendance. CONCLUSIONS: Impairment of HRQoL in female cardiac patients is most pronounced at the time of the event, with most recovery occurring during early convalescence and full recovery in all domains by 12 months post-event.