Recombinant expression and purification of human placental growth factor 1 and specific camel heavy chain polyclonal antibody preparation

Placental growth factor (PlGF) is a member of the vascular endothelial growth factor (VEGF) family. Unlike VEGF, PlGF is dispensable for normal cell development as well as playing various roles in pathological angiogenesis which occurs in tissue ischemia, inflammation, and malignancy. The PlGF-1 has been considered as a potential candidate for the diagnosis and targeting of pathological angiogenesis. Camelidae serum contains an important fraction of functional antibodies, called heavy-chain antibodies (HcAbs) that are naturally devoid of light chains. Camelid HcAbs recognize their cognate antigens by a single variable-domain, referred to as VHH or Nanobody.Here, we describe the expression and purification of recombinant human PlGF-1 (rhPlGF-1). This protein was subsequently used for the preparation of camel heavy chain polyclonal antibody against rhPlGF-1.The recombinant expression plasmid pET-26b-hPlGF-1 was introduced into Escherichia coli BL21 cells to express the rhPlGF-1 protein. Purified rhPlGF-1 was used to immunize camel, the specific reactivity of HcAb was determined with ELISA and western blot. Western blot analysis indicated that the antiserum specifically reacted to the recombinant protein. The rhPlGF-1 protein and its antibody may be used for the development of detection assays needed for clinical research.     

Strategies toward rheumatoid arthritis therapy; the old and the new

Currently, medications used to treat rheumatoid arthritis (RA) are glucocorticoids (GCs) and nonsteroidal anti-inflammatory drugs (NSAIDs), predominantly used for controlling the pain and inflammation, disease-modifying antirheumatic drugs (DMARDs), administered as first-line medication for newly diagnosed RA cases, and biological therapies, used to target and inhibit specific molecules of the immune and inflammatory responses. NSAIDs and other GCs are effective in alleviating the pain, inflammation, and stiffness due to RA. DMARDs that are used for RA therapy are hydroxychloroquine, methotrexate, leflunomide, and sulfasalazine. The biological therapies, on the contrary, are chimeric anti-CD20 monoclonal antibody, rituximab, inhibitors of tumor necrosis factor-α (TNF-α) like etanercept, infliximab, and adalimumab, a recombinant inhibitor of interleukin-1 (IL-1), anakinra, and costimulation blocker, abatacept. Moreover, newly under evaluation biological therapies include new TNF-α inhibitors, JAK inhibitors, anti-interleukin-6-receptor monoclonal antibodies (mABs), and antibodies against vital molecules involved in the survival and development of functional B cells. The new strategies to treat RA has improved the course of the disease and most of the patients are successful in remission of the clinical manifestations if the diagnosis of the disease occur early. The probability of remission increase if the diagnosis happens rapidly and treat-to-target approach are implemented. In this review article, we have attempted to go through the treatment strategies for RA therapy both the routine ones and those which have been developed over the past few years and currently under investigation.     

Two Novel Mutations in LAMC2 Gene in Iranian Families Affected by Junctional Epidermolysis Bullosa

Background:Junctional epidermolysis bullosa (JEB) is an autosomal recessive skin disorder with defective adhesion of dermal- epidermal within the lamina lucida region of the basement membrane zone. The main characterization of JEB is blistering and fragile skin and mucous membrane. Laminins are noncollagenous part of basement membrane and classified as a family of extracellular matrix glycoprotein. Laminins contain three chains: Laminin α, Laminin β and Laminin γ. LAMC2 (laminin subunit gamma 2) gene encodes γ subunit of laminin and its mutation contributes to JEB. Here, we report a disease-causing nonsense mutation and a large deletion mutation in LAMC2 gene in two families affected by JEB.Methods:Whole exome sequencing (WES) was carried out on the mother of patient in family I and the patient himself in family II to detect the underlying mutations. Then, sanger sequencing was performed to confirm the identified mutations.Results:Next generation sequencing (NGS) data analysis of the first family showed a novel, nonsense mutation in LAMC2 gene (LAMC2: {"type":"entrez-nucleotide","attrs":{"text":"NM_005562","term_id":"1653960840"}}NM_005562: exon14:c.C2143T: p.R715X). The heterozygous state of the mutation was confirmed by sanger sequencing in the parents and unaffected brother. In Family II, NGS data had no coverage in the large area of LAMC2 gene. Thus, to confirm the possible deletion sanger sequencing was done and blasting of sequence showed the deleted region of 9.4 kb (exon10-17) in LAMC2 gene.Conclusion:In summary, current study reported a novel disease-causing premature termination codon (PTC) mutation in LAMC2 gene and a large deletion mutation in patients affected by JEB.Key Words: Junctional Epidermolysis Bullosa, LAMC2 gene, Novel mutation, Skin disorder     

Mass production of bacterial communities adapted to the degradation of volatile organic compounds (TEX)

This study focuses on the mass cultivation of bacteria adapted to the degradation of a mixture composed of toluene, ethylbenzene, o-, m- and p-xylenes (TEX). For the cultivation process Substrate Pulse Batch (SPB) technique was adapted under well-automated conditions. The key parameters to be monitored were handled by LabVIEW software including, temperature, pH, dissolved oxygen and turbidity. Other parameters, such as biomass, ammonium or residual substrate concentrations needed offline measurements. SPB technique has been successfully tested experimentally on TEX. The overall behavior of the mixed bacterial population was observed and discussed along the cultivation process. Carbon and nitrogen limitations were shown to affect the integrity of the bacterial cells as well as their production of exopolymeric substances (EPS). Average productivity and yield values successfully reached the industrial specifications, which were 0.45 kg_DW m⁻³ d⁻¹ and 0.59 g_DW g_C⁻¹, respectively. Accuracy and reproducibility of the obtained results present the controlled SPB process as a feasible technique.     

Nanoparticles of Selenium as Species with Stronger Physiological Effects in Sheep in Comparison with Sodium Selenite

The present study was designed to compare the effects of nano red selenium and sodium selenite on the antioxidative activities of neutrophils and the hematological parameters in sheep. Fifteen sheep were randomly allocated into three groups. Groups 1 and 2 received selenium nanoparticles orally at 1 mg/kg and sodium selenite at 1 mg Se/kg for 10 consecutive days; group 3 served as the control. To assess the degrees of oxidative stress and of lipid peroxidation of the cellular membranes, the levels of thiobarbituric acid reactive substances (TBARS) were determined in serum samples that were collected at different supplementation intervals, i.e., after 0, 10, 20, and 30 days. In addition, hematological parameters in the serum samples were measured by routine procedures. It was found that TBARS levels in groups 1 and 2 were significantly higher on days 20 and 30 compared to the basal level on day 0. It was also found that on day 30, the TBARS activities in both treated groups were significantly higher than those of the controls (P < 0.05). These findings may explain the seemingly paradoxical effects of supplemental selenium on the indicators of oxidative stress, as the levels of TBARS were generally expected to decrease in the presence of selenium. There were no significant differences between the PCV and RBC values in the three groups. The white blood cell count (WBC) in group 1 showed a significant increase on days 20 and 30 in comparison with the control group. However, in group 2, there was a significant increase of the WBC value just on day 20 in comparison with the control group. Also, there were significant increases of the neutrophil counts and significant decreases of the lymphocyte counts on day 10 in group 1, in comparison with those in group 2 and controls, and on days 20 and 30 in groups 1 and 2 in comparison with those in the control group.     

In vitro study of DNA interaction with clodinafop-propargyl herbicide

The interaction of native calf thymus DNA with clodinafop-propargyl (CP), in 10 mM HEPES aqueous solutions at neutral pH 7.2, has been investigated by spectrophotometric, circular dichroism (CD), spectrofluorometric, melting temperature (Tm), and viscosimetric techniques. It was found that CP molecules could intercalate between base pairs of DNA as evidenced by hyperchromism in UV absorption band of DNA, an increase in melting temperature, a sharp increase in specific viscosity of DNA, induced CD spectral changes, and increase in the fluorescence of methylene blue (MB)-DNA solutions in the presence of increasing amounts of CP, which indicates that it is able to release the intercalated MB completely. All results suggest that the CP interacts with calf thymus DNA by an intercalative mode of binding.     

Stabilization of firefly luciferase against thermal stress by osmolytes

The effects of osmolytes, including sucrose, sorbitol and proline on the remaining activity of firefly luciferase were measured. Heat inactivation studies showed that these osmolytes maintain the remaining activity of enzyme and increase activation energy of thermal unfolding reaction. Fluorescence and circular dichroism (CD) experiments showed changes in secondary and tertiary structure of firefly luciferase, in the presence of sucrose, sorbitol and proline. The unfolding curves of luciferase (obtained by far-UV CD spectra), indicated an irreversible thermal denaturation and raising of the midpoint of the unfolding transition temperature (T(m)) in the presence of osmolytes.     

Severely damaged kidneys possess multipotent renoprotective stem cells

Background aimsTissue-specific stem cells are a promising target for kidney regeneration, because it has been shown that they play a primary role in kidney repair. Several methods have been developed for the isolation of stem/progenitor cells from healthy kidneys but the existence of these cells in chronically damaged kidneys has not been noticed so far.MethodsA mouse model of chronic kidney failure was developed by ligation of the left ureter for 5 months, and then isolation of stem cells from this tissue as well as normal kidneys was attempted.ResultsWe found that multipotent stem cells could be isolated from both types of tissue. In addition, the cells isolated from damaged kidneys showed potential for homing to the site of injury and a renoprotective effect in an animal model of cisplatin-induced nephropathy.ConclusionsThese results show that multipotent renoprotective stem cells exist in severely damaged kidneys, which could be a target for designing new therapies.