Apoptosis,DAP-Kinase1 Expression and the Influences of Cytokine Milieu and Mesenchymal Stromal Cells on Ex Vivo Expansion of Umbilical Cord Blood-Derived Hematopoietic Stem Cells

Expansion of umbilical cord blood-derived CD34⁺ cells can potentially provide them in numbers sufficient for clinical applications in adult humans. In this study apoptosis rate of expanded cells, mRNA expression and promoter methylation status of DAPK1 were evaluated during cord blood hematopoietic stem cell (CB-HSC) ex vivo expansion using cytokines and a co-culture system with mesenchymal stromal cells (MSCs). Ex vivo cultures of CB-HSCs were performed in three culture conditions for 14 days: cytokines with MSCs feeder layer, cytokines without MSCs feeder layer and co-culture with MSCs feeder layer without cytokine. Total number of cells, CD34⁺ cells and colony forming unit assay were performed during expansion. Flow cytometric analysis by propidium iodide was performed to detection of apoptosis rate in expanded cells. Methylation status of the DAPK1 gene promoter was analyzed using methylation specific PCR, and DAPK1 mRNA expression was evaluated by real time-PCR. Maximum CB-CD34⁺ cells expansion was observed in day 10 of expansion. The highest apoptosis rate was observed in cytokine culture without feeder layer that showed significant difference with co-culture condition. The data showed that ex vivo expansion of CD34⁺ cells in all three culture conditions after 10 days resulted in, significant increased expression of DAPK1, also a significant difference between co-culture without cytokine and two other cytokine culture was observed (p < 0.01). DAPK1gene promoter of expanded CD34⁺ cells at days 5, 10 and 14 of culture remained in unmethylated form similar to fresh CD34⁺ cells. Expression of DAPK1 in hematopoietic cells was increased during 10 days expansion of CD34⁺ cells. Also no methylation of DAPK1 promoter was observed; otherwise it would be capable of initiating some leukemic cell progression or disruption in hematopoietic regeneration.     

Recombinant Production and One Step Purification of IL-1Ra in Escherichia coli and Evaluation its IL-1 Antagonizing Efficacy

Background: Anakinra (Kineret®), an IL-1 receptor antagonist, is the first FDA approved biologic drug for antagonizing IL-1 effects in patients with Rheumatoid arthritis. Notably, the less expensive production of this drug might help reduce the final therapeutic costs. Objectives: This study aimed to evaluate the possibility of producing biologically active recombinant IL-1Ra by a single step purification procedure mediated by a self-cleavable intein. Methods: Soluble expression of the rIL-1Ra was performed in E. coli BL21 (DE3) in fusion to intein1 of pTWIN-1 vector and its cleavage induction using an elution buffer (pH 6.8) at room temperature. Evaluation of the antagonizing efficacy of this protein in various concentrations, was performed on A375 and HEK293 cells treated by a constant concentration of IL-1β (2ng/mL). Results: IPTG induction of E. coli BL21 (DE3) transformed with the recombinant pTWIN-1, revealed a band approximately in 45 kDa, which is related to the intein1-rIL-1Ra fusion protein in the SDS-PAGE. Moreover, protein purification was confirmed by observing a band in 18 kDa. Finally, the percentage of inhibition effects of rIL-1Ra and Kineret® against IL-1β was not statistically significant in IL-1-responsive A375 cells. The inhibition percentage was calculated as 86% in cells treated with 15µg/mL of rIL-1Ra, which it was 96% for the inhibitory effects of the standard drug. Conclusion: In this study, biologically active soluble rIL1-Ra was successfully produced with high purity through a one-step procedure. This method can reduce the cost and time of production for this protein and might be applicable for producing other biologics.     

Designing a novel tetradentate polyoxometalate eco-catalyst for the synthesis of β-aminocyclohexanone derivatives in water

The synthesis of a series of known β-aminocyclohexanones has been accomplished using pentaerythrityl tetramethyl imidazolium phosphotungstate (C(MIM-PTA)₄) as a new tetradentate acidic catalyst. It was prepared via condensation of pentaerythrityl tetrabromide with methyl imidazole. Then, bulky anion H₂PW₁₂O₄₀¹⁻ was substituted with Br⁻ in the structure. This tetradentate catalyst provides designable cations and anions. Anions have two types of acids, acidic protons, and metals with Lewis acidity. In order to test the efficient catalytic behavior of the tetradentate catalyst, a controlled reaction was performed using benzaldehyde, aniline and cyclohexanone. Imine from the condensation of benzaldehyde and aniline was observed in the absence of ionic catalyst instead of desired products. Thus, this reaction would be attractive because of the time, energy, and raw material saving considerations because of the absence of isolation of intermediates and stereospecificity. The catalyst shows high catalytic activity such that after four recycles the product was obtained with high yield and purity. This reaction was performed at room temperature. Although high temperature could improve the reaction rate, it contributes to side reactions and oxidation of aldehyde and amine. The catalyst was characterized by elemental analysis, FT-IR spectroscopy, ¹H NMR, ¹³C NMR, and TGA.

The synthesis of a series of known β-aminocyclohexanones has been accomplished using pentaerythrityl tetramethyl imidazolium phosphotungstate (C(MIM-PTA)₄) as a new tetradentate acidic catalyst.     

From the Cover: Herded and hunted goat genomes from the dawn of domestication in the Zagros Mountains

The Aceramic Neolithic (∼9600 to 7000 cal BC) period in the Zagros Mountains, western Iran, provides some of the earliest archaeological evidence of goat (Capra hircus) management and husbandry by circa 8200 cal BC, with detectable morphological change appearing ∼1,000 y later. To examine the genomic imprint of initial management and its implications for the goat domestication process, we analyzed 14 novel nuclear genomes (mean coverage 1.13X) and 32 mitochondrial (mtDNA) genomes (mean coverage 143X) from two such sites, Ganj Dareh and Tepe Abdul Hosein. These genomes show two distinct clusters: those with domestic affinity and a minority group with stronger wild affinity, indicating that managed goats were genetically distinct from wild goats at this early horizon. This genetic duality, the presence of long runs of homozygosity, shared ancestry with later Neolithic populations, a sex bias in archaeozoological remains, and demographic profiles from across all layers of Ganj Dareh support management of genetically domestic goat by circa 8200 cal BC, and represent the oldest to-this-date reported livestock genomes. In these sites a combination of high autosomal and mtDNA diversity, contrasting limited Y chromosomal lineage diversity, an absence of reported selection signatures for pigmentation, and the wild morphology of bone remains illustrates domestication as an extended process lacking a strong initial bottleneck, beginning with spatial control, demographic manipulation via biased male culling, captive breeding, and subsequently phenotypic and genomic selection.

The initial domestication of southwest Asian crops and livestock species unfolded across the Fertile Crescent after the end of the Younger Dryas climatic downturn circa 9600 BC and coalesced into fully integrated agricultural economies by 7500 cal BC (1). Until recently, the western part of this region was cast as the epicenter of this transition, while the Zagros Mountains, at the eastern end of the Fertile Crescent, was considered a backwater, slow to receive and embrace domesticates and food-producing technologies from farther west (2, 3). However, this region of western Iran has been recently postulated as a primary center for the domestication of a number of plant and animal species (4), including barley (5, 6), possibly emmer wheat (7), several pulse species (8), and most notably, goats (9). This latter hypothesis has been supported by ancient genomic data, which indicate that the eastern Fertile Crescent was one of three regions key in shaping the Neolithic goat gene pool (10).Here we explore the transition from the hunting of wild bezoar goats (Capra aegagrus) to their initial management and subsequent domestication in the eastern Fertile Crescent by combining ancient genome sequencing and archaeozoological evidence from Ganj Dareh and Tepe Abdul Hosein, two sites in the central Zagros dating to the early or Aceramic Neolithic (AN) circa 8000 cal BC. These genomic data present a singular opportunity to deepen our understanding of the consequences of goat management at the dawn of domestication.     

Three-component synthesis of pyrano[2,3-d]-pyrimidine dione derivatives facilitated by sulfonic acid nanoporous silica (SBA-Pr-SO3H) and their docking and urease inhibitory activity

Background

A straightforward and efficient method for the synthesis of pyrano[2,3-d]pyrimidine diones derivatives from the reaction of barbituric acid, malononitrile and various aromatic aldehydes using SBA-Pr-SO₃H as a nanocatalyst is reported.

Results

Reactions proceed with high efficiency under solvent free conditions. Urease inhibitory activity of pyrano[2,3-d]pyrimidine diones derivatives were tested against Jack bean urease using phenol red method. Three compounds of 4a, 4d and 4l were not active in urease inhibition test, but compound 4a displayed slight urease activation properties. Compounds 4b, 4k, 4f, 4e, 4j, 4g and 4c with hydrophobic substitutes on phenyl ring, showed good inhibitory activity (19.45-279.14 μM).

Discussion

The compounds with electron donating group and higher hydrophobic interaction with active site of enzyme prevents hydrolysis of substrate. Electron withdrawing groups such as nitro at different position and meta-methoxy reduced urease inhibitory activity. Substitution of both hydrogen of barbituric acid with methyl group will convert inhibitor to activator.     

Rehabilitation of tibial eminence fracture

Tibial eminence fractures occur as a result of high amounts of tension placed upon the anterior cruciate ligament (ACL). The incidence of these fractures is higher among adolescent girls due to their inherent skeletal immaturity. In such an injury, direct trauma causes an avulsion fracture occurring at the tibial eminence while the ACL is spared. Imaging is used to confirm the diagnosis of a tibial eminence fracture and regardless of the extent of injury, rehabilitation is crucial for a full recovery. The following is a case study of a 17-year-old girl who was involved in a motor vehicle accident. In the accident, she sustained a left lateral tibial eminence fracture, along with soft tissue injuries at the cervical and lumbar spine. Her treatment included passive and active range of motion (ROM), strength training, physical modalities, and proprioceptive training of the injured areas. An improvement was noted post-treatment and after a 5-month follow-up according to subjective reports and objective assessments (ROM and girth measurements).     

Comparative analysis of cerebrospinal fluid adenosine deaminase in tuberculous and non-tuberculous meningitis

Objective

To calculate cut-off point for the adenosine deaminase (ADA) activity in the CSF of patients with tuberculous meningitis (TBM).

Patients and methods

The ADA assay was based on the automatic indirect method in which ADA catalyzes adenosine to inosine. ADA activity in the CSF was calculated based on ammonia liberated from adenosine and quantified spectrophotometrically. Arithmetic mean values and standard deviation of each variable were measured. Mann–Whitney U and Fisher exact tests were applied to compare continuous and dichotomous variables between tuberculous and non-tuberculous groups. A receiver operating characteristic curve was plotted to identify various cut-off points to determine the best level for ADA activity.

Results

Totally 42 patients were enrolled into the study. The median of ADA activity in the TBM group was 22 and in the non-TBM group was 8.0. The mean CSF-ADA activity was found to be significantly higher in TBM group (23.05 ± 13.1 IU/L) than in the CSF from non-TBM patients (9.39 ± 5.18 IU/L). The highest accuracy is at the cut-off value of 10.5 IU/L. The sensitivity and specificity of the test at this cut-off to differentiate TBM from non-tuberculous meningitis is 81% and 86% respectively.

Conclusion

Considering that a high positive value of ADA activity cannot confirm TBM, however, in suspected patients it may lead the physician to treat patient earlier before the confirmatory diagnostic reports will be received. The suggested cut-off value in this pilot study is 10.5 IU/L with high sensitivity and specificity.