Targeting high affinity and epitope-distinct oligoclonal nanobodies to HER2 over-expressing tumor cells

Modern anti-HER2 antibody therapy tends to exploit a panel of different antibodies against different epitopes on the antigen. For this aim, nanobodies are very striking targeting agents and can be easily produced against any cell-specific membrane antigen. The oligoclonal nanobodies can be used to block more than one functional epitope on a target antigen and inhibit the generation of escape variants associated with cancer therapy. In this study, 12 nanobody clones selected from an immune camel library were examined for their ability to differ between tumor markers. These oligoclonal nanobodies targeted breast cancer cells better than each individual nanobody. In epitope mapping, several nanobodies overlapped in the epitope recognized by trastuzumab and some of the non-overlapping nanobodies could affect the binding of trastuzumab to HER2. This study demonstrates that the oligoclonal nanobodies are potential therapeutic tools that can be used instead of, or in combination with trastuzumab to assess tumor viability during treatment.     

Plasmonic Circular Dichroism Study of DNA–Gold Nanoparticles Bioconjugates

Plasmonic circular dichroism (CD) responses of hybrid nanostructures containing noble metal nanoparticles and chiral molecules have received increasing interest with various applications in nanophotonics. Chiral biomolecules show strong CD signals typically found in the ultraviolet region, whereas, in the visible range, they produce a weak signal. Strengthening the CD signal in the visible region is of high importance, which could be achieved through fabrication of novel hybrid nanostructures. Herein, gold nanoparticles (GNPs) have been assembled via DNA linker to investigate the possibility of enhancing plasmonic CD signal in the visible range. DNA-linked assemblies with pre- and postannealed conditions were characterized by ultraviolet–visible spectroscopy, dynamic light scattering (DLS), and CD spectropolarimetry. In the presence of DNA linker with sticky ends, the aggregation phenomenon was traced by red shifts of surface plasmon resonance of nanoparticles. Time-dependent hybridization of single-stranded “sticky ends” with DNA-conjugated GNPs and increased probability of hydrogen bond formation lead to enhancement of CD signals in the ultraviolet region. Complexation of biomolecule and nanoparticle assemblies induced enhanced CD signals in the visible range, which was noticed both before and after purification. DLS characterization of the assemblies also confirmed the difference in the size of aggregates, which could be controlled by the linker molecules. This investigation encourages possibility of utilizing plasmonic CD technique as a tool for tracing fabricated nanostructure assemblies with enhanced characterization possibility.     

The association between fat mass and obesity-associated (FTO) genotype and serum vitamin D level in breast cancer patients

The preventive effect of vitamin D against breast cancer can be influenced by gene polymorphisms. This study aimed to investigate the association between serum level of 25(OH) vitamin D and FTO genotype in breast cancer patients. A cross-sectional study was carried out on 180 newly diagnosed patients with breast cancer in Tehran, Iran. The blood samples were collected from the participants in order to assess the FTO gene rs9939609 polymorphism by the tetra-primer amplification refractory mutation system (Tetra-ARMS) PCR method. The serum level of 25(OH) vitamin D was measured using the direct competitive enzyme-linked immunosorbent assay (ELISA) method. The association between vitamin D and the FTO genotype in patients with breast cancer was assessed after adjustment for cofounders. The frequency of TT, AT and AA genotypes in the breast cancer patients were 43% (n = 77), 49% (n = 89) and 8% (n = 14), respectively. All patients with higher than 40 ng/dl of serum 25(OH) vitamin D had one or two copies of FTO rs9939609 risk allele (p = 0.019). No linear association was found between the number of FTO risk allele and the level of serum vitamin D. All patients with high serum level of 25(OH) vitamin D had one or two copies of FTO rs9939609 risk allele. FTO gene polymorphisms may counteract the beneficial effects of vitamin D in breast cancer prevention. Further studies can help to better understand the genetic factors predisposing to breast cancer and their effect on the association between vitamin D and breast cancer.     

Dynamics of transparent exopolymeric particles (TEP) production by Phaeocystis globosa under N- or P-limitation: a controlling factor of the retention/export balance

The concentration of transparent exopolymeric particles (TEP) was monitored during Phaeocystis globosa blooms that developed in mesocosms under different initial N:P ratios (from N- to P-limited conditions). TEP concentration was measured using the microscopic (TEP_micro, ppm) and the colorimetric (TEP_color, Xanthan equiv. L⁻¹) methods. TEP concentrations varied from 5 to >75 ppm and from 60 to >1500 μg Xanthan equiv. L⁻¹, and were relatively low until the mesocosms reached nutrient (either N or P) depletion and then increased abruptly. From the TEP_micro versus TEP_color concentrations comparison and from their relation to chlorophyll a concentrations, two phases for the dynamics of TEP production were identified: (1) production through active release of precursors during the growth phase of P. globosa — defined as TEP₁ — and their integration into the TEP pool through coagulation processes; (2) release of large TEP from the mucilaginous matrix of P. globosa colonies subsequent to disruption caused by nutrient depletion — defined as TEP₂ — and their direct integration into the TEP pool outside the constraint of coagulation. The formation of a multiorigin TEP pool during P. globosa blooms may have implications for the fate of the blooms, due to difference in TEP bioreactivity according to their source and to difference in timing and intensity of TEP₁ versus TEP₂ production according to N- or P-depletion. For P. globosa blooms developing under N-limiting conditions, the transition from the first source (i.e. TEP₁) to the second one (i.e. TEP₂) was a slow and continuous process. In contrast, the P. globosa bloom developing under P-limiting conditions showed the sudden formation of heavy mucous aggregates when P became depleted, that may have been caused by a massive release of TEP₂. Our study suggests that the nutrient regime may control the export vs. retention balance during P. globosa blooms, via production of a multiorigin TEP pool.     

Designed Amino Acid Feed in Improvement of Production and Quality Targets of a Therapeutic Monoclonal Antibody

Cell culture feeds optimization is a critical step in process development of pharmaceutical recombinant protein production. Amino acids are the basic supplements of mammalian cell culture feeds with known effect on their growth promotion and productivity. In this study, we reported the implementation of the Plackett-Burman (PB) multifactorial design to screen the effects of amino acids on the growth promotion and productivity of a Chinese hamster ovary DG-44 (CHO-DG44) cell line producing bevacizumab. After this screening, the amino acid combinations were optimized by the response surface methodology (RSM) to determine the most effective concentration in feeds. Through this strategy, the final monoclonal antibody (mAb) titre was enhanced by 70%, compared to the control group. For this particular cell line, aspartic acid, glutamic acid, arginine and glycine had the highest positive effects on the final mAb titre. Simultaneously, the impact of the designed amino acid feed on some critical quality attributes of bevacizumab was examined in the group with highest productivity. The product was analysed for N-glycan profiles, charge variant distribution, and low molecular weight forms. The results showed that the target product quality has been improved using this feeding strategy. It was shown how this strategy could significantly diminish the time and number of experiments in identifying the most effective amino acids and related concentrations in target product enhancement. This model could be successfully applied to other components of culture media and feeds.     

Effects of trehalose and sorbitol on the activity and structure of Pseudomonas cepacia lipase: spectroscopic insight

The stability of enzymes with no reduction in their catalytic activity still remains a critical issue in industrial applications. Naturally occurring osmolytes are commonly used as protein stabilizers. In this study we have investigated the effects of sorbitol and trehalose on the structural stability and activity of Pseudomonas cepacia lipase (PCL), using UV-visible, circular dichroism (CD) and fluorescence spectroscopy. Surface plasmon resonance (SPR) technique was used to trace changes in the refractive index and dielectric constant of the environment. The results revealed that catalytic activity and intrinsic fluorescence intensity of PCL increased in the presence of both osmolytes. Far-UV CD spectra indicated that the protein has undergone some conformational changes upon interacting with these osmolytes. Increasing the concentration of sorbitol led to changes in the refractive index and consequently the dielectric constant of environment; whereas in the case of trehalose, such changes were not significant. Unfavorable interactions of trehalose with protein surface induced higher preferential exclusion from the enzyme-water interface than that of sorbitol. Results of this report could give further insights about the stabilization mechanism of osmolytes.     

Characterization of a branched lipopeptide candidate vaccine against influenza A/Puerto Rico 8/34 which is recognized by human B and T-cell immune responses

Background

Rotavirus NSP4 localizes to multiple intracellular sites and is multifunctional, contributing to RV morphogenesis, replication and pathogenesis. One function of NSP4 is the induction of early secretory diarrhea by binding surface receptors to initiate signaling events. The aims of this study were to determine the transport kinetics of NSP4 to the exofacial plasma membrane (PM), the subsequent release from intact infected cells, and rebinding to naïve and/or neighboring cells in two cell types.

Methods

Transport kinetics was evaluated using surface-specific biotinylation/streptavidin pull-downs and exofacial exposure of NSP4 was confirmed by antibody binding to intact cells, and fluorescent resonant energy transfer. Transfected cells similarly were monitored to discern NSP4 movement in the absence of infection or other viral proteins. Endoglycosidase H digestions, preparation of CY3- or CY5- labeled F(ab)₂ fragments, confocal imaging, and determination of preferential polarized transport employed standard laboratory techniques. Mock-infected, mock-biotinylated and non-specific antibodies served as controls.

Results

Only full-length (FL), endoglycosidase-sensitive NSP4 was detected on the exofacial surface of two cell types, whereas the corresponding cell lysates showed multiple glycosylated forms. The C-terminus of FL NSP4 was detected on exofacial-membrane surfaces at different times in different cell types prior to its release into culture media. Transport to the PM was rapid and distinct yet FL NSP4 was secreted from both cell types at a time similar to the release of virus. NSP4-containing, clarified media from both cells bound surface molecules of naïve cells, and imaging showed secreted NSP4 from one or more infected cells bound neighboring cell membranes in culture. Preferential sorting to apical or basolateral membranes also was distinct in different polarized cells.

Conclusions

The intracellular transport of NSP4 to the PM, translocation across the PM, exposure of the C-terminus on the cell surface and subsequent secretion occurs via an unusual, complex and likely cell-dependent process. The exofacial exposure of the C-terminus poses several questions and suggests an atypical mechanism by which NSP4 traverses the PM and interacts with membrane lipids. Mechanistic details of the unconventional trafficking of NSP4, interactions with host-cell specific molecules and subsequent release require additional study.     

A novel variant of t-PA resistant to plasminogen activator inhibitor-1; expression in CHO cells based on in silico experiments

Resistance to PAI-1 is a factor which confers clinical benefits in thrombolytic therapy. The only US FDA approved PAI-1 resistant drug is Tenecteplase?. Deletion variants of t-PA have the advantage of fewer disulfide bonds in addition to higher plasma half lives. A new variant was developed by deletion of the first three domains in t-PA in addition to substitution of KHRR 128-131 amino acids with AAAA in truncated t-PA. The specific activity of this new variant, 570 IU/μg, was found to be similar to those found in full length t-PA (Alteplase?), 580 IU/μg. A 65% and 85% residual activity after inhibition by rPAI-1 was observed for full length and truncated-mutant form, respectively. This new variant as the first PAI-1 resistant truncated t-PA may offer more advantages in clinical conditions in which high PAI-1 levels makes the thrombolytic system prone to re-occlusion.     

Overexpression of reactive oxygen species modulator 1 is associated with advanced grades of bladder cancer

Molecular Biology Reports - Reactive Oxygen Species Modulator 1 (ROMO1) plays a pivotal role in the regulation of mitochondrial structure integrity, and the production of reactive oxygen species...     

Synthesis of folic acid functionalized terbium‐doped dendritic fibrous nano‐silica and Interaction with HEK 293 normal,MDA breast cancer and HT 29 colon cancer cells

A novel folic acid functionalized terbium‐doped dendritic fibrous nanoparticle (Tb@KCC‐1‐NH₂‐FA) with high surface area was synthesized using a novel hydrothermal protocol. In the present work, we report the fluorescent Tb‐doted nanomaterial with emission wavelength at 497 nm which confirms the formation of Tb@KCC‐1‐NH₂‐FA. Synthesized nanoparticles were investigated through transmission electron microscope, field emission scanning electron Microscopy, Fourier transform infrared spectra, Brunauer‐Emmett‐Teller, energy dispersive X‐ray, Zeta potential and particle size distribution values and AFM (Atomic force microscopy) techniques. Specially, our desired nanomaterial which has FA moieties on the surface of Tb@KCC‐1‐NH2‐FA where interact with folate receptor (FR) which there is on the surface of the various cancer cells. For this purpose, fluorescence microscopy images were used to prove the uptake of FA based nanomaterial with FR‐positive MDA breast cancer and HT 29 colon cancer cells. Also HEK 293 normal cells as FR‐negative cells verified the specificity of our desired nanomaterial toward the FR‐positive cells. The cytotoxicity survey of Tb@KCC‐1‐NH₂‐FA was examined by MTT assays against MDA breast cancer, HT 29 colon cancer and HEK 293 Normal cell lines which confirmed their biocompatible nature with any significant cytotoxic effects even for concentration higher than 900 μg/mL which could be used as a non‐toxic catalyst or carrier in biological ambient. Hence, Tb@KCC‐1‐NH₂‐FA were synthesized using green and hydrothermal method; the process was simple with good productivity and desired nanocomposite was non‐toxic.