Affinity maturation and characterization of the ofatumumab monoclonal antibody

CD20 molecule, a phosphoprotein with 297 amino acids and four transmembrane domains, is a member of MS4A protein family. Anti-CD20 antibodies such as ofatumumab, which have been developed for cancer treatment and has demonstrated efficacy in relapsed/refractory chronic lymphocytic leukemia, are among the most successful therapies to date. Rational engineering methods can be applied with reasonable success to improve functional characteristics of antibodies. Considering the importance of this issue, we have used in silico modeling approach for the improvement of ofatumumab monoclonal antibody. Four mutated variants of ofatumumab were developed and expressed in Chinese hamster ovary (CHO) cells along with the unmodified antibody. Analysis of affinity of the purified antibodies with CD20 showed significant improvement in antigen-binding characteristics of one of the variants compared with the control antibody. This study represents the first step toward development of the second generation ofatumumab antibody with improved affinity.     

Dysregulated expression of STAT1, miR-150, and miR-223 in peripheral blood mononuclear cells of coronary artery disease patients with significant or insignificant stenosis

Coronary artery disease (CAD) is a multicellular disease characterized by chronic inflammation. Peripheral blood-mononuclear cells (PBMCs), as a critical component of immune system, actively cross-talk with pathophysiological conditions induced by endothelial cell injury, reflecting in perturbed PBMC expression. STAT1 is believed to be relevant to CAD pathogenesis through regulating key inflammatory processes and modulating STAT1 expression play key roles in fine-tuning CAD-related inflammatory processes. This study evaluated PBMC expressions of STAT1, and its regulators (miR-150 and miR-223) in a cohort including 72 patients with CAD with significant ( ≥ 50%) stenosis, 30 patients with insignificant ( < 50%) coronary stenosis (ICAD), and 74 healthy controls, and assessed potential of PBMC expressions to discriminate between patients and controls. We designed quantitative real-time polymerase chain reaction (RT-qPCR) assays and identified stable reference genes for normalizing PBMC quantities of miR-150, miR-223, and STAT1 applying geNorm algorithm to six small RNAs and five mRNAs. There was no significant difference between CAD and ICAD patients regarding STAT1 expression. However, both groups of patients had higher levels of STAT1 than healthy controls. miR-150 and miR-223 were differently expressed across three groups of subjects and were downregulated in patients compared with healthy controls, with the lowest expression levels being observed in patients with ICAD. ROC curves suggested that PBMC expressions may separate between different groups of study subjects. PBMC expressions also discriminated different clinical manifestations of CAD from ICADs or healthy controls. In conclusion, the present study reported PBMC dysregulations of STAT1, miR-150, and miR-223, in patients with significant or insignificant coronary stenosis and suggested that these changes may have diagnostic implications.     

Development of α4 integrin DNA aptamer as a potential therapeutic tool for multiple sclerosis

One of the most important molecules for multiple sclerosis pathogenesis is α4 integrin, which is responsible for autoreactive leukocytes migration into the brain. The monoclonal antibody, natalizumab, was introduced to market for blocking the extravasation of autoreactive leukocytes via inhibition of α4 integrin. However, the disadvantages of antibodies provided a suitable background for other agents to be replaced with antibodies. Considering the profound advantages of aptamers over antibodies, aptamer isolation against α4 integrin was intended in the current study. The α4 integrin-specific aptamers were selected using cell-systematic evolution of ligands by exponential enrichment (SELEX) method with human embryonic kidney (HEK)-293T overexpressing α4 integrin and HEK-293T as target and control cells, respectively. Evaluation of selected aptamer was performed through flow cytometric analysis. The selected clones were then sequenced and analyzed for any possible secondary structure and affinity. The results of this study led to isolation of 13 different single-stranded DNA clones in 11 rounds of selection which were categorized to three clusters based on common structural motifs and the equilibrium dissociation constant (K _d) of the most stable structure was calculated. The evaluation of SELEX progress showed growth in aptamer affinity with increasing of the number of cycles. Taken together, the findings of this study demonstrated the isolation of α4-specific single-stranded DNA aptamers with suitable affinity for ligand, which can further be replaced with natalizumab.     

Complications of nonionizing radiofrequency on divided attention

Exposure to electromagnetic fields is considered as a potential hazard for biological systems. The objective of our investigation is the study of probable consequences of radiofrequency electromagnetic fields from Wi-Fi router devices on the short-term memory, and attention's levels. A population consisting of 312 female college students (14 to 17 years old) was elected by cluster random sampling. Teenagers were divided into two groups of control group (Wi-Fi nonusers; n = 138), and experiment group (Wi-Fi users; n = 174). Both groups have been examined using short-term memory tests; selective attention, and also divided attention tests. According to the results, there was no significant difference between using Wi-Fi router devices on levels of selective attentions and short-term memory of the sample students with the control group. However, analyses revealed that there is a significant correlation between the use of Wi-Fi routers and declining levels of divided attentions. Our investigation has demonstrated the adverse consequences of 2.4-2.48 GHz radiofrequency electromagnetic fields of Wi-Fi router devices on divided attention levels of female university students that should be mentioned as a technological risk factor and taken into account by healthcare organizations.     

First report of molecular diagnosis of Tunisian hemophiliacs A: identification of 8 novel causative mutations

ABSTRACT: INTRODUCTION: Hemophilia A is an X linked recessive hemorrhagic disorder caused by mutations in the F8 gene that lead to qualitative and/or quantitative deficiencies of coagulation factor VIII (FVIII). Molecular diagnosis of hemophilia A is challenging because of the high number of different causative mutations that are distributed throughout the large F8 gene. Molecular studies of these mutations are essential in order to reinforce our understanding of their pathogenic effect responsible for the disorder. Aim In this study we have performed molecular analysis of 28 Tunisian hemophilia A patients and analyzed the F8 mutation spectrum. METHODS: We screened the presence of intron 22 and intron 1 inversion in severe hemophilia A patients by southern blotting and polymerase chain reaction (PCR). Detection of point mutations was performed by dHPLC/sequencing of the coding F8 gene region. We predict the potential functional consequences of novel missense mutations with bioinformatics approaches and mapping of their spatial positions on the available FVIII 3D structure. RESULTS: We identified 23 different mutations in 28 Tunisian hemophilia A patients belonging to 22 unrelated families. The identified mutations included 5 intron 22 inversions, 7 insertions, 4 deletions and 7 substitutions. In total 18 point mutations were identified, of which 9 are located in exon 14, the most mutated exonic sequence in the F8 gene. Among the 23 mutations, 8 are novel and not deposited in the HAMSTeRS database nor described in recently published articles. CONCLUSION: The mutation spectrum of Tunisian hemophilia A patients is heterogeneous with the presence of some characteristic features. Virtual slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1693269827490715.     

First Report of Northern Root-Knot Nematode,Meloidogyne hapla,Parasitic on Oaks,Quercus brantii and Q. infectoria in Iran

Root-knot nematodes (RKN) are the most serious plant parasitic nematodes having a broad host range exceeding 2,000 plant species. Quercus brantii Lindl. and Q. infectoria Oliv are the most important woody species of Zagros forests in west of Iran where favors sub-Mediterranean climate. National Botanical Garden of Iran (NBGI) is scheduled to be the basic center for research and education of botany in Iran. This garden, located in west of Tehran, was established in 1968 with an area of about 150 ha at altitude of 1,320 m. The Zagros collection has about 3-ha area and it has been designed for showing a small pattern of natural Zagros forests in west of Iran. Brant’s oak (Q. brantii) and oak manna tree (Q. infectoria) are the main woody species in Zagros collection, which have been planted in 1989. A nematological survey on Zagros forest collection in NBGI revealed heavily infection of 24-yr-old Q. brantii and Q. infectoria to RKN, Meloidogyne hapla. The roots contained prominent galls along with egg sac on the surface of each gall. The galls were relatively small and in some parts of root several galls were conjugated, and all galls contained large transparent egg masses. The identification of M. hapla was confirmed by morphological and morphometric characters and amplification of D2-D3 expansion segments of 28S rRNA gene. The obtained sequences of large-subunit rRNA gene from M. hapla was submitted to the GenBank database under the accession number {"type":"entrez-nucleotide","attrs":{"text":"KP319025","term_id":"852381377","term_text":"KP319025"}}KP319025. The sequence was compared with those of M. hapla deposited in GenBank using the BLAST homology search program and showed 99% similarity with those {"type":"entrez-nucleotide","attrs":{"text":"KJ755183","term_id":"667714404","term_text":"KJ755183"}}KJ755183, {"type":"entrez-nucleotide","attrs":{"text":"GQ130139","term_id":"239819330","term_text":"GQ130139"}}GQ130139, {"type":"entrez-nucleotide","attrs":{"text":"DQ328685","term_id":"84794916","term_text":"DQ328685"}}DQ328685, and {"type":"entrez-nucleotide","attrs":{"text":"KJ645428","term_id":"657709843","term_text":"KJ645428"}}KJ645428. The second stage juveniles of M. hapla isolated from Brant’s oak (Q. Brantii) showed the following morphometric characters: (n = 12), L = 394 ± 39.3 (348 to 450) µm; a = 30.9 ± 4 (24.4 to 37.6); b = 4.6 ± 0.44 (4 to 5.1); b΄ = 3.3 ± 0.3 (2.7 to 3.7), c = 8.0 ± 1 (6.2 to 10.3), ć = 5.3 ± 0.8 (3.5 to 6.3); Stylet = 12.1 ± 0.8 (11 to 13) µm; Tail = 50 ± 5.6 (42 to 57) µm; Hyaline 15 ± 1.8 (12 to 18) µm. Oak manna, Q. infectoria population of second stage juveniles clearly possessed short body length and consequently other morphometric features were less than those determined for Q. brantii population, and these features were: (n = 12), L = 359.0 ± 17.3 (319 to 372) µm; a = 28.6 ± 3 (22.8 to 31); b = 5.0 ± 0.3 (4.8 to 5.2); b΄ = 3.3 ± 0.2 (3 to 3.6), c = 8.1 ± 0.5 (7.4 to 8.8), ć = 4.7 ± 0.5 (3.9 to 5.2); Stylet = 11.4 ± 0.7 (10 to 12) µm; Tail = 44 ± 1.8 (42 to 47) µm; Hyaline 12 ± 1.7 (10 to 15) µm. To date two species of Meloidogyne, M. querciana Golden, 1979 and M. christiei Golden and Kaplan, 1986 have been reported to parasitize oaks (Quercus spp.) from the United States of America. M. querciana was found on pin oak Quercus palustris in Virginia. The oak RKN infected pine oak, red oak, and American chestnut heavily in greenhouse tests (Golden, 1979). The other species M. christiei was described from turkey oak and Q. laevis in Florida, which has monospecific host range (Golden and Kaplan, 1986). Both of these RKN species seem to be restricted to the United States of America and have not been reported from other place. According to our knowledge this is the first report of occurrence of M. hapla on Q. brantii and Q. infectoria in the world. This study includes these two oak species to the host range of RKN, M. hapla for the world and expands the information of RKN, M. hapla host ranges on oaks.     

The α2 isoform of the Na,K-pump is important for intercellular communication, agonist-induced contraction, and EDHF-like response in rat mesenteric arteries

The specific role of different isoforms of the Na,K-pump in the vascular wall is still under debate. We have previously suggested that the α(2) isoform of the Na,K-pump (α(2)), Na(+), Ca(2+)-exchange (NCX), and connexin43 form a regulatory microdomain in smooth muscle cells (SMCs), which controls intercellular communication and contractile properties of the vascular wall. We have tested this hypothesis by downregulating α(2) in cultured SMCs and in small arteries with siRNA in vivo. Intercellular communication was assessed by using membrane capacitance measurements. Arteries transfected in vivo were tested for isometric and isobaric force development in vitro; [Ca(2+)](i) was measured simultaneously. Cultured rat SMCs were well-coupled electrically, but 10 μM ouabain uncoupled them. Downregulation of α(2) reduced electrical coupling between SMCs and made them insensitive to ouabain. Downregulation of α(2) in small arteries was accompanied with significant reduction in NCX expression. Acetylcholine-induced relaxation was not different between the groups, but the endothelium-dependent hyperpolarizing factor-like component of the response was significantly diminished in α(2)-downregulated arteries. Micromolar ouabain reduced in a concentration-dependent manner the amplitude of norepinephrine (NE)-induced vasomotion. Sixty percent of the α(2)-downregulated arteries did not have vasomotion, and vasomotion in the remaining 40% was ouabain insensitive. Although ouabain increased the sensitivity to NE in the control arteries, it had no effect on α(2)-downregulated arteries. In the presence of a low NE concentration the α(2)-downregulated arteries had higher [Ca(2+)](i) and tone. However, the NE EC50 was reduced under isometric conditions, and maximal contraction was reduced under isometric and isobaric conditions. The latter was caused by a reduced Ca(2+)-sensitivity. The α(2)-downregulated arteries also had reduced contraction to vasopressin, whereas the contractile response to high K(+) was not affected. Our results demonstrate the importance of α(2) for intercellular coupling in the vascular wall and its involvement in the regulation of vascular tone.     

Protective Ventilation of Preterm Lambs Exposed to Acute Chorioamnionitis Does Not Reduce Ventilation-Induced Lung or Brain Injury

Background

The onset of mechanical ventilation is a critical time for the initiation of cerebral white matter (WM) injury in preterm neonates, particularly if they are inadvertently exposed to high tidal volumes (V_T) in the delivery room. Protective ventilation strategies at birth reduce ventilation-induced lung and brain inflammation and injury, however its efficacy in a compromised newborn is not known. Chorioamnionitis is a common antecedent of preterm birth, and increases the risk and severity of WM injury. We investigated the effects of high V_T ventilation, after chorioamnionitis, on preterm lung and WM inflammation and injury, and whether a protective ventilation strategy could mitigate the response.

Methods

Pregnant ewes (n = 18) received intra-amniotic lipopolysaccharide (LPS) 2 days before delivery, instrumentation and ventilation at 127±1 days gestation. Lambs were either immediately euthanased and used as unventilated controls (LPS_UVC; n = 6), or were ventilated using an injurious high V_T strategy (LPS_INJ; n = 5) or a protective ventilation strategy (LPS_PROT; n = 7) for a total of 90 min. Mean arterial pressure, heart rate and cerebral haemodynamics and oxygenation were measured continuously. Lungs and brains underwent molecular and histological assessment of inflammation and injury.

Results

LPS_INJ lambs had poorer oxygenation than LPS_PROT lambs. Ventilation requirements and cardiopulmonary and systemic haemodynamics were not different between ventilation strategies. Compared to unventilated lambs, LPS_INJ and LPS_PROT lambs had increases in pro-inflammatory cytokine expression within the lungs and brain, and increased astrogliosis (p<0.02) and cell death (p<0.05) in the WM, which were equivalent in magnitude between groups.

Conclusions

Ventilation after acute chorioamnionitis, irrespective of strategy used, increases haemodynamic instability and lung and cerebral inflammation and injury. Mechanical ventilation is a potential contributor to WM injury in infants exposed to chorioamnionitis.     

Characterization of genetic diversity in chickpea using SSR markers,Start Codon Targeted Polymorphism (SCoT) and Conserved DNA-Derived Polymorphism (CDDP)

Physiology and Molecular Biology of Plants - To evaluate the genetic diversity among 48 genotypes of chickpea comprising cultivars, landraces and internationally developed improved lines genetic...