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International audience; Scorpion envenomation is a serious health problem in tropical and subtropical zones. The access to scorpion antivenom is sometimes limited in availability and specificity. The classical production process is cumbersome, from the hyper-immunization of the horses to the IgG digestion and purification of the F(ab)'(2) antibody fragments. The production of recombinant antibody fragments in Escherichia coli is a popular trend due to the ability of this microbial host to produce correctly folded proteins. Small recombinant antibody fragments, such as single-chain variable fragments (scFv) and nanobodies (VHH), have been constructed to recognize and neutralize the neurotoxins responsible for the envenomation symptoms in humans. They are the focus of interest of the most recent studies and are proposed as potentially new generation of pharmaceuticals for their use in immunotherapy against scorpion stings of the Buthidae family. This literature review comprises the current status on the scorpion antivenom market and the analyses of cross-reactivity of commercial scorpion anti-serum against non-specific scorpion venoms. Recent studies on the production of new recombinant scFv and nanobodies will be presented, with a focus on the Androctonus and Centruroides scorpion species. Protein engineering-based technology could be the key to obtaining the next generation of therapeutics capable of neutralizing and cross-reacting against several types of scorpion venoms.

The purpose of this study was to determine the phylogenetic relationships among the primary betanodavirus strains circulating in Tunisian coastal waters. A survey was conducted to investigate nodavirus infections at 15 European sea bass Dicentrarchus labrax and gilthead sea bream Sparus aurata farming sites located along the northern and eastern coasts of Tunisia. The primary objective of the study was to create epidemiological awareness of these infections by determining phylogenetic relationships between the main betanodavirus strains circulating during the period 2012-2019, using RNA1 and/or RNA2 genome segments. Approximately 40% (118 of 294) tissue pools tested were positive for betanodavirus. Positive pools were distributed across all of the sampling sites. While fish mortalities were always correlated with the presence of virus in sea bass, a severe outbreak was also identified in sea bream larvae in 2019. Phylogenetic analysis revealed that almost all Tunisian strains from both sea bass and sea bream irrespective of outbreaks clustered within the RGNNV genotype. It is noteworthy that samples collected during the 2019 outbreak from sea bream contained both RNA1 and RNA2 fragments belonging to the RGNNV and SJNNV genotype, respectively, an indication of viral genome reassortment. To our knowledge, this is the first report of reassortant betanodavirus in Tunisia.

R.A73 Enterococcus faecium (HG937697) was tested for investigating anti-nodaviral activity and immunomodulatory properties on E-11 fish cells. Non R.A73-related toxicity was confirmed. Likewise, in-vitro inhibition of nodavirus replication and gene expression of nodavirus capsid protein gene in addition to CCR3, IL-10 and TNFα genes were evaluated using qPCR. A preventive treatment of E-11 cells by the bacterial suspension generated a remarkable reduction in virus loads (RQ values from 0.1 to 0.9) at day 7. Interestingly, R.A73 E. faecium showed a significant gene expression upregulation of proinflammatory IL-10 cytokine whereas it did not significantly enhance CCR3 nor TNF-α immune gene expression in E-11 cells. Therefore, add-on its probiotic potential, R.A73 E. faecium is able to stimulate the immune system in the preventive phase throughout the differential expression of main inflammatory genes. This study provides the first evidence that R.A73 E. faecium strain can prevents effectively nodavirus replication, in vitro.

Tenascin-C (TNC) and tenascin-W (TNW), large hexameric glycoproteins overexpressed in the tumor microenvironment, are useful tumor biomarkers for theranostic applications. For now, polyclonal and monoclonal antibodies, as well as aptamers targeting TNC and TNW have been developed. However, the immunostaining sensitivity of antibodies is very heterogenous. The main aim of this study was to generate antibodies in dromedary that detect TNC and TNW, respectively. We show that immune sera from immunized dromedaries are able to specifically bind native TNC and TNW by ELISA and also to detect TNC and TNW in matrix tracks of mammary tumors by immunostaining. Furthermore, we demonstrate that purified IgG subtypes are able to interact specifically with TNC or TNW by ELISA and immunostaining. These camelid antibodies are a good basis to develop tools for the detection of TNC and TNW in the tumor microenvironment and could potentially have a broader application for early diagnosis of solid cancers.

Effective interventions and treatments for complex diseases have been implemented globally, however, coverage in Africa has been comparatively lower due to lack of capacity, clinical applicability and knowledge on the genetic contribution to disease and treatment. Currently, there is a scarcity of genetic data on African populations, which have enormous genetic diversity. Pharmacogenomics studies have the potential to revolutionise treatment of diseases, therefore, African populations are likely to benefit from these approaches to identify likely responders, reduce adverse side effects and optimise drug dosing. This review discusses clinical pharmacogenetics studies conducted in African populations, focusing on studies that examined drug response in complex diseases relevant to healthcare. Several pharmacogenetics associations have emerged from African studies, as have gaps in knowledge.

Scorpion α-toxins are neurotoxins that target the fast inactivation mechanism of voltage-gated sodium (Na

The extracellular matrix molecule Tenascin-C (TNC) promotes cancer and chronic inflammation by multiple mechanisms. Recently, TNC was shown to promote an immune suppressive tumor microenvironment (TME) through binding soluble chemoattracting factors, thus retaining leukocytes in the stroma. TNC also binds to fibronectin (FN) and other molecules, raising the question of a potential common TNC binding mechanism. By sequence comparison of two TNC-interacting domains in FN, the fifth (FN5) and thirteenth (FN13) fibronectin type III domains we identified a MAtrix REgulating MOtif "MAREMO" or M-motif that is highly conserved amongst vertebrates. By sequence analysis, structural modeling and functional analysis we found also putative M-motifs in TNC itself. We showed by negative staining electron microscopic imaging that the M-motif in FN mediates interactions with FN as well as with TNC. We generated two M-motif mimetic peptides P5 and P13 resembling the M-motif in FN5 and FN13, respectively. By using structural information we modelled binding of these M-motif mimetics revealing a putative MAREMO binding site MBS in FN5 and TN3, respectively overlapping with the M-motif. We further demonstrated that the M-motif mimetic peptides blocked several functions of TNC, such as binding of TNC to FN, cell rounding on a mixed FN/TNC substratum, FN matrix expression and subsequent assembly, TNC-induced signaling and gene expression, TNC chemokine binding and dendritic cell retention, thus providing novel opportunities to inhibit TNC actions. Our results suggest that targeting the MAREMO/MBS interaction could be exploited for reducing inflammation and matrix functions in cancer and fibrosis.

We describe the design of a simple and highly sensitive electrochemical bioanalytical method enabling the direct detection of a conserved RNA region within the capsid protein gene of a fish nodavirus, making use of nanostructured disposable electrodes. To achieve this goal, we select a conserved region within the nodavirus RNA2 segment to design a DNA probe that is tethered to the surface of nanostructured disposable screen-printed electrodes. In a proof-of-principle test, a synthetic RNA sequence is detected based on competitive hybridization between two oligonucleotides (biotinylated reporter DNA and target RNA) complimentary to a thiolated DNA capture probe. The method is further validated using extracted RNA samples obtained from healthy carrier Sparus aurata and clinically infected Dicentrarchus labrax fish specimens. In parallel, the sensitivity of the newly described biosensor is compared with a new real-time RT-PCR protocol. The current differences measured in the negative control and in presence of each concentration of target RNA are used to determine the dynamic range of the assay. We obtain a linear response (R2 = 0.995) over a range of RNA concentrations from 0.1 to 25 pM with a detection limit of 20 fM. The results are in good agreement with the results found by the RT-qPCR. This method provides a promising approach toward a more effective diagnosis and risk assessment of viral diseases in aquaculture.

Heterotrophic bacteria associated with the green alga Ulva rigida, collected from the coast of Tunisia, were isolated and subsequently identified by their 16S rRNA gene sequences and by phylogenetic analysis. The 71 isolates belong to four phyla: Proteobacteria (Alpha-and Gamma- subclasses), Bacteroidetes, Firmicutes, and Actinobacteria. Most of the isolates belong to Proteobacteria. The Gram-positive Firmicutes and especially the genus Bacillus were well-represented at the surface of U. rigida, collected from the coast as well as from the lagoon, while Actinobacteria were represented only at the surface of algae collected from the coast of Cap Zebib. Bacteroidetes were more represented at the surface of algae collected from the Ghar El Melh lagoon. The bacterial community of the water surrounding the algae was different from that associated with the surface of the algae. Moreover, the abundance of bacteria in the surrounding water was much lower compared to the density of bacteria associated with the surface of the algae. Bacteria isolated from the algal surface were tested for their antimicrobial potential. The results show that ~ 36% of the algae-associated bacterial isolates possess antibacterial activity whereas free-living bacteria, isolated from the surrounding water, did not show such activity. The surface of U. rigida was colonized by a high diversity of culturable and possibly novel epiphytic bacteria that may be an important source of antimicrobial compounds and are therefore of biotechnological interest.

In the pharmaceutical industry, nanobodies show promising properties for its application in serotherapy targeting the highly diffusible scorpion toxins. The production of recombinant nanobodies in Escherichia coli has been widely studied in shake flask cultures in rich medium. However, there are no upstream bioprocess studies of nanobody production in defined minimal medium and the effect of the induction temperature on the production kinetics. In this work, the effect of the temperature during the expression of the chimeric bispecific nanobody CH10-12 form, showing high scorpion antivenom potential, was studied in bioreactor cultures of E. coli. High biomass concentrations (25 g cdw/L) were achieved in fed-batch mode, and the expression of the CH10-12 nanobody was induced at temperatures 28, 29, 30, 33, and 37°C with a constant glucose feed. For the bispecific form NbF12-10, the induction was performed at 29°C. Biomass and carbon dioxide yields were reported for each culture phase, and the maintenance coefficient was obtained for each strain. Nanobody production in the CH10-12 strain was higher at low temperatures (lower than 30°C) and declined with the increase of the temperature. At 29°C, the CH10-12, NbF12-10, and WK6 strains were compared. Strains CH10-12 and NbF12-10 had a productivity of 0.052 and 0.021 mg/L/h of nanobody, respectively, after 13 h of induction. The specific productivity of the nanobodies was modeled as a function of the induction temperature and the specific growth rates. Experimental results confirm that low temperatures increase the productivity of the nanobody.Key points• Nanobodies with scorpion antivenom activity produced using two recombinant strains.• Nanobodies production was achieved in fed-batch cultures at different induction temperatures.• Low induction temperatures result in high volumetric productivities of the nanobody CH10-12.

Background Gastric and colorectal cancers are the most common malignant tumours, leading to a significant number of cancer-related deaths worldwide. Recently, increasing evidence has demonstrated that cancer cells exhibit a differential expression of potassium channels and this can contribute to cancer progression. However, their expression and localisation at the somatic level remains uncertain. In this study, we have investigated the expression levels of KCNB1 and KCNA5 genes encoding ubiquitous Kv2.1 and Kv1.5 potassium channels in gastric and colorectal tumours. Methods Gastric and colorectal tumoral and peritumoral tissues were collected to evaluate the expression of KCNB1 and KCNA5 mRNA by quantitative PCR. Moreover, the immunohistochemical staining profile of Kv2.1 and Kv1.5 was assessed on 40 Formalin-Fixed and Paraffin-Embedded (FFPE) gastric carcinoma tissues. Differences in gene expression between tumoral and peritumoral tissues were compared statistically with the Mann-Whitney U test. The association between the clinicopathological features of the GC patients and the expression of both Kv proteins was investigated with χ2 and Fisher’s exact tests. Results The mRNA fold expression of KCNB1 and KCNA5 genes showed a lower mean in the tumoral tissues (0.06 ± 0.17, 0.006 ± 0.009) compared to peritumoral tissues (0.08 ± 0.16, 0.16 ± 0.48, respectively) without reaching the significance rate (p = 0.861, p = 0.152, respectively). Interestingly, Kv2.1 and Kv1.5 immunostaining was detectable and characterised by a large distribution in peritumoral and tumoral epithelial cells. More interestingly, inflammatory cells were also stained. Surprisingly, Kv2.1 and Kv1.5 staining was undoubtedly and predominantly detected in the cytoplasm compartment of tumour cells. Indeed, the expression of Kv2.1 in tumour cells revealed a significant association with the early gastric cancer clinical stage (p = 0.026). Conclusion The data highlight, for the first time, the potential role of Kv1.5 and Kv2.1 in gastrointestinal-related cancers and suggests they may be promising prognostic markers for these tumours.

Background Colorectal cancer (CRC) is the third most diagnosed malignancy worldwide. The global burden is expected to increase along with ongoing westernized behaviors and lifestyle. The etiology of CRC remains elusive and most likely combines environmental and genetic factors. The Kv2.1 potassium channel encoded by KCNB1 plays a collection of roles in malignancy of cancer and may be a key factor of CRC susceptibility. Our study provides baseline association between Tunisian CRC and interactions between KCNB1 variants and lifestyle factors. Methods A case-control study involving 300 CRC patients, and 300 controls was conducted Patients were carefully phenotyped and followed till the end of study. KCNB1 genotyping was confirmed by Sanger sequencing. Bivariate and multivariable logistic regression analyses were used to assess the clinical status, lifestyle and study polymorphisms association with CRC. Results We noted significant gender association with CRC occurrence. Moreover, CRC risk increases with high meat and fat consumption, alcohol use and physical activity (PA). Carriage of rs1051296 A/G and both rs11468831 ins/del and del/del genotypes (p p = 0.01) with CRC susceptibility regardless of patients’ gender while rs3331 T/C (p = 0.012) was associated with females. Stratification study according to malignancy site; Rectal Cancer (RC) and Colon Cancer (CC), reveals increasing RC risk by gender and high meat and fat consumption, alcohol use and PA. However, additional association with high brine consumption was noted for CC. The rs1051295 A/G (p = 0.01) was associated with RC risk. Increased CC risk was associated with carriage of rs1051295 A/G, rs11168831 (del/del) and (ins/del) genotypes. Conclusion The risk of CRC increases with modifiable factors by Western influences on Tunisian lifestyle such as alcohol use, high fat consumption and possibly inadequate intake of vegetables. In addition, KCNB1 polymorphisms also markedly influence CRC susceptibility. Our study establishes key elements of a baseline characterization of clinical state, Western influenced lifestyle and KCNB1 variants associated with Tunisian CRC.

Background Whole-genome sequencing using high throughput technologies has revolutionized and speeded up the scientific investigation of bacterial genetics, biochemistry, and molecular biology. Lactic acid bacteria (LABs) have been extensively used in fermentation and more recently as probiotics in food products that promote health. Genome sequencing and functional genomics investigations of LABs varieties provide rapid and important information about their diversity and their evolution, revealing a significant molecular basis. This study investigated the whole genome sequences of the Enterococcus faecium strain (HG937697), isolated from the mucus of freshwater fish in Tunisian dams. Genomic DNA was extracted using the Quick-GDNA kit and sequenced using the Illumina HiSeq2500 system. Sequences quality assessment was performed using FastQC software. The complete genome annotation was carried out with the Rapid Annotation using Subsystem Technology (RAST) web server then NCBI PGAAP. Results The Enterococcus faecium R.A73 assembled in 28 contigs consisting of 2,935,283 bps. The genome annotation revealed 2884 genes in total including 2834 coding sequences and 50 RNAs containing 3 rRNAs (one rRNA 16 s, one rRNA 23 s and one rRNA 5 s) and 47 tRNAs. Twenty-two genes implicated in bacteriocin production are identified within the Enterococcus faecium R.A73 strain. Conclusion Data obtained provide insights to further investigate the effective strategy for testing this Enterococcus faecium R.A73 strain in the industrial manufacturing process. Studying their metabolism with bioinformatics tools represents the future challenge and contribution to improving the utilization of the multi-purpose bacteria in food.

Background: Whole-genome sequencing using high throughput technologies has revolutionized and speeded up the scientific investigation of bacterial genetics, biochemistry, and molecular biology. Lactic acid bacteria (LABs) have been extensively used in fermentation and more recently as probiotics in food products that promote health. Genome sequencing and functional genomics investigations of LABs varieties provide rapid and important information about their diversity and their evolution, revealing a significant molecular basis.This study investigates the whole genome sequences of the Enterococcus faecium genome (HG937697), isolated from the mucus of freshwater fish in Tunisian dams. Genomic DNA was extracted using the Quick-GDNA kit (Zymoresearch) and sequenced using the Illumina HiSeq2500 system. Sequences quality assessment was performed using FastQC software. The complete genome annotation was carried out with the RAST web server then NCBI PGAAP. Results: The Enterococcus faecium R.A73 assembled in 28 contigs consisting of 2,935,283 bps. The genome annotation revealed 2,884 genes in total including 2,834 coding sequences and 50 RNAs containing 3 rRNAs (one rRNA 16s, one rRNA 23s and one rRNA 5s) and 47 tRNAs. Twenty-two genes implicated in bacteriocin production are identified within the Enterococcus faecium R.A73 genome.Conclusions: Data obtained provide insights to further investigate the effective strategy for testing this Enterococcus faecium R.A73 strain in the industrial manufacturing process. Studying their metabolism with bioinformatics tools represents the future challenge and contribution to improving the utilization of the multi-purpose bacteria in food.

New Delhi Metallo-&beta, lactamase-1 (NDM-1) is the most prevalent type of metallo-&beta, lactamase, able to hydrolyze almost all antibiotics of the &beta, lactam group, leading to multidrug-resistant bacteria. To date, there are no clinically relevant inhibitors to fight NDM-1. The use of dromedary polyclonal antibody inhibitors against NDM-1 represents a promising new class of molecules with inhibitory activity. In the current study, immunoreactivities of dromedary Immunoglobulin G (IgG) isotypes containing heavy-chain and conventional antibodies were tested after successful immunization of dromedary using increasing amounts of the recombinant NDM-1 enzyme. Inhibition kinetic assays, performed using a spectrophotometric method with nitrocefin as a reporter substrate, demonstrated that IgG1, IgG2, and IgG3 were able to inhibit not only the hydrolytic activity of NDM-1 but also Verona integron-encoded metallo-&beta, lactamase (VIM-1) (subclass B1) and L1 metallo-&beta, lactamase (L1) (subclass B3) with inhibitory concentration (IC50) values ranging from 100 to 0.04 &mu, M. Investigations on the ability of IgG subclasses to reduce the growth of recombinant Escherichia coli BL21(DE3)/codon plus cells containing the recombinant plasmid expressing NDM-1, L1, or VIM-1 showed that the addition of IgGs (4 and 8 mg/L) to the cell culture was unable to restore the susceptibility of carbapenems. Interestingly, IgGs were able to interact with NDM-1, L1, and VIM-1 when tested on the periplasm extract of each cultured strain. The inhibitory concentration was in the micromolar range for all &beta, lactams tested. A visualization of the 3D structural basis using the three enzyme Protein Data Bank (PDB) files supports preliminarily the recorded inhibition of the three MBLs.

The protein purity is generally checked using SDS‐PAGE, where densitometry could be used to quantify the protein bands. In literature, few studies have been reported using image analysis for the quantification of protein in SDS‐PAGE: that is, imaged with Stain‐Free™ technology. This study presents a protocol of image analysis for electrophoresis gels that allows the quantification of unknown proteins using the molecular weight markers as protein standards. Escherichia coli WK6/pHEN6 encoding the bispecific nanobody CH10‐12 engineered by the Pasteur Institute of Tunisia was cultured in a bioreactor and induced with isopropyl β‐D‐1‐thiogalactopyranoside (IPTG) at 28°C for 12 hr. Periplasmic proteins extracted by osmotic shock were purified by immobilized metal affinity chromatography (IMAC). Images of the SDS‐PAGE gels were analyzed using ImageJ, and the lane profiles were obtained in grayscale and uncalibrated optical density. Protein load and peak area were linearly correlated, and optimal image processing was then performed by background subtraction using the rolling ball algorithm with radius size 250 pixels. No brightness and contrast adjustment was applied. The production of the nanobody CH10‐12 was obtained through a fed‐batch strategy and quantified using the band of 50 kDa in the marker as reference for 750 ng of recombinant protein. The molecular weight marker was used as a sole protein standard for protein quantification in SDS‐PAGE gel images., This article proposes a protocol for image analysis of electrophoresis gels that allows the quantification of unknown proteins using the molecular weight markers in SDS‐PAGE gels as protein standards. The periplasmic production of the nanobody CH10‐12 was obtained through fed‐batch strategy and quantified using the band of 50 kDa in the marker as a reference for 750 ng of recombinant protein. The molecular weight marker was used as a sole protein standard for protein quantification in SDS‐PAGE gel images.

Lactic acid bacteria (LAB) are traditionally used in food and feed bio-industries as they have health-promoting probiotic effect for their host and currently considered safe for human and animal consumption. Recently, we isolated 177 strains from freshwater fishes of dams of Tunisia exhibiting antimicrobial activities against various food-borne pathogens and generated a laboratory biobank to be characterized. Herein, we investigated whether MALDI-TOF could assist rapid identification of LAB genus. First, a lactic microflora-selective Man Rogosa Sharpe medium was used. Isolates were further screened according to the antimicrobial activity, using well-difusion agar. In total, four major genera have been well identified and species frequency has been estimated (74% Enterococcus, 24% Leuconostoc, 3% Lactococcus, and 2% Vagococcus). Eighteen isolates was further analyzed using MALDI-TOF. Comparative analysis of spectral fingerprints to six referenced MALDI-TOF fingerprints was carried out from the recently developed USC library ( www.spectrabank.org ). The intra- and inter-specific phyloproteomic relationships among strains were compared to available phylogenetic data based on 16S rDNA genes. This study showed that MALDI-TOF MS is a potent and reliable rapid method for both discrimination and identification of LABs. This highlights the insight of proteomic approach into the screening of food-derived beneficial microorganisms.

International audience; Scorpion envenoming and its treatment is a public health problem in many parts of the world due to highly toxic venom polypeptides diffusing rapidly within the body of severely envenomed victims. Recently, 38 AahII-specific Nanobody sequences (Nbs) were retrieved from which the performance of NbAahII10 nanobody candidate, to neutralize the most poisonous venom compound namely AahII acting on sodium channels, was established. Herein, structural computational approach is conducted to elucidate the Nb-AahII interactions that support the biological characteristics, using Nb multiple sequence alignment (MSA) followed by modeling and molecular docking investigations (RosettaAntibody, ZDOCK software tools). Sequence and structural analysis showed two dissimilar residues of NbAahII10 CDR1 (Tyr27 and Tyr29) and an inserted polar residue Ser30 that appear to play an important role. Indeed, CDR3 region of NbAahII10 is characterized by a specific Met104 and two negatively charged residues Asp115 and Asp117. Complex dockings reveal that NbAahII17 and NbAahII38 share one common binding site on the surface of the AahII toxin divergent from the NbAahII10 one's. At least, a couple of NbAahII10 - AahII residue interactions (Gln38 - Asn44 and Arg62, His64, respectively) are mainly involved in the toxic AahII binding site. Altogether, this study gives valuable insights in the design and development of next generation of antivenom.

Graphical abstract, 7-Ketocholesterol, which is one of the earliest cholesterol oxidization products identified, is essentially formed by the auto-oxidation of cholesterol. In the body, 7-ketocholesterol is both provided by food and produced endogenously. This pro-oxidant and pro-inflammatory molecule, which can activate apoptosis and autophagy at high concentrations, is an abundant component of oxidized Low Density Lipoproteins. 7-Ketocholesterol appears to significantly contribute to the development of age-related diseases (cardiovascular diseases, age-related macular degeneration, and Alzheimer's disease), chronic inflammatory bowel diseases and to certain cancers. Recent studies have also shown that 7-ketocholesterol has anti-viral activities, including on SARS-CoV-2, which are, however, lower than those of oxysterols resulting from the oxidation of cholesterol on the side chain. Furthermore, 7-ketocholesterol is increased in the serum of moderately and severely affected COVID-19 patients. In the case of COVID-19, it can be assumed that the antiviral activity of 7-ketocholesterol could be counterbalanced by its toxic effects, including pro-oxidant, pro-inflammatory and pro-coagulant activities that might promote the induction of cell death in alveolar cells. It is therefore suggested that this oxysterol might be involved in the pathophysiology of COVID-19 by contributing to the acute respiratory distress syndrome and promoting a deleterious, even fatal outcome. Thus, 7-ketocholesterol could possibly constitute a lipid biomarker of COVID-19 outcome and counteracting its toxic effects with adjuvant therapies might have beneficial effects in COVID-19 patients.

International audience; Voltage-gated potassium (Kv) channels form cells repolarizing power and are commonly expressed in excitable cells. In non-excitable cells, Kv channels such as Kv2.1 are involved in cell differentiation and growth. Due to the involvement of Kv2.1 in several physiological processes, these channels are promising therapeutic targets. To develop Kv2.1 specific antibody-based channel modulators, we applied a novel approach and immunized a dromedary with heterologous Ltk- cells that overexpress the mouse Kv2.1 channel instead of immunizing with channel protein fragments. The advantage of this approach is that the channel is presented in its native tetrameric configuration. Using a Cell-ELISA, we demonstrated the ability of the immune serum to detect Kv2.1 channels on the surface of cells that express the channel. Then, using a Patch Clamp electrophysiology assay we explored the capability of the dromedary serum in modulating Kv2.1 currents. Cells that were incubated for 3h with serum taken at Day 51 from the start of the immunization displayed a statistically significant 2-fold reduction in current density compared to control conditions as well as cells incubated with serum from Day 0. Here we show that an immunization approach with cells overexpressing the Kv2.1 channel yields immune serum with Kv2.1 specific antibodies.

Stability and quality control of therapeutic protein formulations is a substantial part of drug development process. The objective of this study is to obtain information about stability of a recombinant human granulocyte colony stimulating factor (rhG-CSF) against various stress factors. This will play a crucial role in the finished product formulation development. In this study, rhG-CSF was exposed to various chemical and physical stress conditions at different levels in order to identify degradation pathways and the nature of impurities generated. Experiments were performed by a combination of orthogonal analytical techniques (reversed phase chromatography (RP-HPLC), size exclusion chromatography (SEC-HPLC), polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF)) to set and characterize the different degraded samples. The SEC-HPLC results suggest that the major degradation factors generating aggregated forms of the protein are basically thermal stress, freeze-thaw cycles and vortexing. Meanwhile, deamidated rhG-CSF was induced by basic pH as shown by IEF electrophoregram. As well, oxidized forms were generated increasingly with the time of exposure to hydrogen peroxide as outlined by RP-HPLC analysis. Based on these results, it was possible to define the storage and handling conditions of rhG-CSF finished product during its shelf life.

Background: Astaxanthin (ATX) is a lipophilic compound found in many marine organisms. Studies have shown that ATX has many strong biological properties, including antioxidant, antiviral, anticancer, cardiovascular, anti-inflammatory, neuro-protective and anti-diabetic activities. However, no research has elucidated the effect of ATX on ionic channels. ATX can be extracted from shrimp by-products. Our work aims to characterize ATX cell targets to lend value to marine by-products. Methods: We used the Xenopus oocytes cell model to characterize the pharmacological target of ATX among endogenous Xenopus oocytes&rsquo, ionic channels and to analyze the effects of all carotenoid-extract samples prepared from shrimp by-products using a supercritical fluid extraction (SFE) method. Results: ATX inhibits amiloride-sensitive sodium conductance, xINa, in a dose-dependent manner with an IC50 of 0.14 &micro, g, a maximum inhibition of 75% and a Hill coefficient of 0.68. It does not affect the potential of half activation, but significantly changes the kinetics, according to the slope factor values. The marine extract prepared from shrimp waste at 10 &micro, g inhibits xINa in the same way as ATX 0.1 &micro, g does. When ATX was added to the entire extract at 10 &micro, g, inhibition reached that induced with ATX 1 &micro, g. Conclusions: ATX and the shrimp Extract inhibit amiloride-sensitive sodium channels in Xenopus oocytes and the TEVC method makes it possible to measure the ATX inhibitory effect in bioactive SFE-Extract samples.

International audience; Rapid and sensitive detection of low levels of scorpion venom toxins in biological fluids is of tremendous importance for decision-taking in cases of envenomation by scorpions stings. In Tunisia, at least 1200 severe envenomation cases by Androctonus australis hector (Aah) scorpion stings were reported annually. In this work, we report on a novel electrochemical immuno-sandwich to detect the Aah50 toxic fraction within the Aah scorpion venom using the bispecific nanobody format specially designed to highly recognize and neutralize the two most toxic molecules in the AahG50 venom fraction (i.e. AahI and AahII toxins), graphene quantum dots (GQDs) constructed on the surface carbon screen-printed electrodes. Hydroquinone/H2O2/peroxidase system was used to amplify the current in order to achieve the detection of low levels of AahG50. Electrochemical studies revealed a high sensitivity toward the AahG50 with a sensitivity of 18.2 nA mL pg-1 and a picomolar limit of detection as low as 0.55 pg mL-1. The platform exhibits very good metrological performances such as repeatability, reproducibility, selectivity and long storage stability. Matrix effect was found to be insignificant as demonstrated by assays performed in human blood serum and urine.

A central role for advanced glycation end products (AGE) and their receptor (RAGE) in the pathogenesis of multiple cancer types, including colorectal cancer (CRC) was reported. We investigated the association between CRC and rs2853807, rs77170610, rs184003, rs1035798, rs2070600, rs1800684, rs1800624, and rs1800625 RAGE gene (AGER) polymorphic variants. Study subjects comprised 293 CRC patients [186 colon cancer (CC) and 107 rectal cancer (RC)] patients), and 264 age-, gender-, BMI-, and ethnicity-matched controls. Minor allele frequency (MAF) of rs77170610 and rs1800625 were significantly lower, while MAF of rs1035798 was significantly higher in CRC patients compared to control subjects, which was associated with reduced and increased risk of CRC, respectively; MAF of the remaining variants was comparable between CRC patients and controls. Significant difference in the distribution of rs2853807 and rs77170610 genotypes was seen between CRC patients and controls, with both variants associated with decreased risk of CRC. Comparison of the distribution of minor allele-carrying genotypes in CC and RC patient subgroups revealed lack of significant difference in the distribution of these genotypes between the patient subgroups. In view of the lack of LD between rs2853807 and rs77170610 with other variants, six-locus (rs184003, rs1035798, rs2070600, rs1800684, rs1800624, rs1800625) haplotypes were constructed. Haplotype analysis did not identify any specific 6-locus AGER haplotype associated with CRC. In conclusion, AGER gene rs2853807 and rs77170610 variants rs77170610 are associated with altered risk of CRC in Tunisians, but with no discrimination between CC and RC types.

The KCNB1 gene variants were differentially associated with cancers. However, their association with colorectal cancer has not yet been explored. We investigated the contribution of the KCNB1 gene variants rs3331, rs1051295, and indel (insertion/deletion) rs11468831 Polymorphism as predictors of the treatment response in colorectal cancer patients. A retrospective study, which involved 291 Tunisian colorectal cancer patients (aged 60.0 ± 13.1 years), who were stratified into responder and non-responder groups, according to TNM stages and their responsiveness to chemotherapy based on fluorouracil. KCNB1 genotyping was performed with amplification-refractory mutation system–polymerase chain reaction, and was confirmed by Sanger sequencing. Sex-specific response was found and colorectal cancer females are less likely to achieve a positive response during the chemotherapy strategy, compared to males. Weight and body mass index, tumor size, and tumor localization are considered as predictive factors to treatment responsiveness. Carriage of rs11468831 Ins allele was significantly associated with successful therapy achievement ( p adjusted

International audience; PEGylation is one of the strategies used for enhancing in vivo residence time of recombinant human Granulocyte Colony-Stimulating Factor (rhG-CSF) and therefore reducing in dose frequency to better fit with patient comfort treatment. In this study, three methoxy polyethylene glycol propionaldehydes (mPEG- ALD) of 10, 20 and 30kDa MW were utilized to produce biologically active monoPEGylated rhG-CSF with enhanced molecular weight. PEGylation reactions were carried out at room temperature and pH 5.0 in the presence of cyanoborohydride and two mPEG-ALD: protein molar ratios (3:1 and 5:1). The reactions were monitored with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion chromatography (SEC-HPLC). The results showed that a 2h reaction with 5:1 mPEG-ALD: protein molar ratio was sufficient to direct the reaction toward optimal yields of monoPEGylated protein (86%). Subsequently, isolation of the monoPEGylated forms was successfully achieved. The purified products were compared with respect to their purity (≥95%), identity and isoelectric focusing parameter characteristics. Biological potencies were measured by cell proliferation assay and showed 20.80-42.73% retention of bioactivities. This study highlights the possible improvement of rhG-CSF efficiency by PEGylation. Further studies will investigate in vitro and in vivo immunogenicity and toxicity of monoPEGylated conjugates.

The potency of a recombinant human erythropoietin (rhEPO) pharmaceutical preparation was evaluated by the normocythaemic mice bioassay using standardized and randomized protocol of European Pharmacopeia 5.0. Eight-week-old normocythaemic B6D2F1/c mice received single daily injections of test solutions (EPOTIN recombinant human erythropoietin, batch preparation) at different concentrations. Erythropoietin biological reference product (batch 3.3) was used as standard. The suitability of the bioassay was recorded according to gender and age. The blood sampling was performed 4 days after the last injection and reticulocytes were counted by manual and automated methods. Intra-assay precision was analyzed and Bland-Altman plot was obtained. The effect of rhEPO administration doses on the hemoglobin concentration, hematocrit and number of red blood cells in the blood stream were investigated. The dose response curve was linear in the concentration range of 20–80 IU/ml. The relative potency was estimated to 13.78 and 71.53% of the stated potency, for male and female mice, respectively. The recorded limits were compared to the required ones. This study gives valuable insights for better control of biosimilars and bioproducts.

International audience; Enoxaparin is a low-molecular-weight heparin widely used for the prevention and treatment of thromboembolism. With the development of several enoxaparin biosimilars, real medical concerns about their safety and efficacy have been raised. This repeated dose toxicity study consists of preclinical toxicological evaluation of a biosimilar biological version of enoxaparin, the drug product ``Enoxa'', compared to the enoxaparin reference drug product, ``Lovenox''. Eighty white Wistar rats were treated with ``Enoxa'' versus the reference product, using subcutaneous therapeutic and toxic doses, varying from 3.5 to 100 mg/kg/day. Dose levels were adjusted and ultimately fixed at 3.5 and 20 mg/kg/day as therapeutic and toxic doses, respectively. A sodium chloride solution (0.9%) was used as the control, and the comparative study was conducted over periods of 14 and 28 days. Comparable effects were observed with few adverse effects at the administration dose of 20 mg/kg/day, for both enoxaparin biosimilar and reference products. Interestingly, mortality started only at high doses of 40 mg/kg/day and reached 25% at 100 mg/kg/day for both products. These results, as part of the recommended biosimilarity monitoring, demonstrated comparable toxicity profiles of ``Enoxa'' and ``Lovenox'' products in rats. Continuing investigation of biosimilarity on humans to confirm safety and efficacy is suggested. (C) 2016 Elsevier Inc. All rights reserved.

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) has been increasingly recognized from among one of the most abundant families of biosimilars. Upon long-term storage, the rhG-CSF is subject to subtle chemical modifications that rapidly occur and, in particular, produce deaminated variants with divergent charge. Indeed, changes in charge from glutamine deamination may alter the way rhG-SCF will refold and the structure of resulting molecule. To assess this charge heterogeneity, 2-D gel electrophoresis has limited application. Recent micro-fluidic- based technical advances offer a great alternative method to better control liquid volumes on a minute scale. Here, we used IEF OFFGEL–lab-on-chip electrophoresis for 2-D separation of the rhG-CSF peptides according to their isoelectric point (pI) and molecular weight (kDa). We used an rhG-CSF commercial therapeutic formulation, kept refrigerated 24 months after expiry. The samples were analyzed for particulate matter and charge variants. Subsequently, the secondary structure was assessed by FTIR spectroscopy and residual biological activity was recorded. Interestingly, we showed an additional band in the acidic gel area above and below the most intense protein band (fractions 10, 11, and 12 at 22.84s). This observation reveals the presence of the rhG-CSF variant charges without any additional high molecular weight impurity or biological activity decrease. We conclude that after two years of storage, the rhG-CSF solution maintained its native secondary structure with little $\beta $ -sheet deviation, as reflected in the 1622 cm-1 and 1695 cm-1. These data demonstrated that a combined strategy is a more suitable and accurate analytical assessment of the rhG-CSF and recombinant protein-based biosimilars.