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Snakebite envenoming (SBE) remains a severely neglected public health issue, particularly affecting tropical and subtropical regions, with Africa experiencing an estimated 435,000 to 580,000 snakebites annually, leading to high morbidity and mortality rates, especially across Africa and Asia. Recognized as a Neglected Tropical Disease, SBE management is further complicated by the inadequate efficacy of current antivenom treatments. Of particular concern are cobras (Naja sp.), whose neurotoxins can induce rapid fatal respiratory paralysis. In this study, we investigate the potential of nanobodies as a promising next-generation of immunotherapeutics against cobra venoms. Through a dual strategy of the characterization of venom toxic fractions from cobras captured for the first time in Algeria and Tunisia biotopes, coupled with in vitro assays to evaluate their interactions with acetylcholine receptors, and subsequent immunization of dromedaries to produce specific nanobodies, we identified two lethal fractions, F5 and F6, from each venom, and selected five nanobodies with significant binding and neutralizing of 3DL50 (0.74 mg/kg). The combination of these nanobodies demonstrated a synergistic effect, reaching 100% neutralizing efficacy of 2DL50 lethal venom fraction (0.88 mg/kg) doses in mice. Additionally, our findings highlighted the complex mechanism of cobra venom action through the lethal synergism among its major toxins.

The injudicious usage of antibiotics during infections caused by Gram-negative bacteria leads to the emergence of β-lactamases. Among them, the NDM-1 enzyme poses a serious threat to human health. Developing new antibiotics or inhibiting β-lactamases might become essential to reduce and prevent bacterial infections. Nanobodies (Nbs), the smallest antigen-binding single-domain fragments derived from Camelidae heavy-chain-only antibodies, targeting enzymes, are innovative alternatives to develop effective inhibitors. The biopanning of an immune VHH library after phage display has helped to retrieve recombinant antibody fragments with high inhibitory activity against recombinant-NDM-1 enzyme. Nb02NDM-1, Nb12NDM-1, and Nb17NDM-1 behaved as uncompetitive inhibitors against NDM-1 with Ki values in the nM range. Remarkably, IC50 values of 25.0 nM and 8.5 nM were noted for Nb02NDM-1 and Nb17NDM-1, respectively. The promising inhibition of NDM-1 by Nbs highlights their potential application in combating particular Gram-negative infections.

Essential oils (Eos) have demonstrated antiviral activity, but their toxicity can hinder their use as therapeutic agents. Recently, some essential oil components have been used within safe levels of acceptable daily intake limits without causing toxicity. The “ImmunoDefender,” a novel antiviral compound made from a well-known mixture of essential oils, is considered highly effective in treating SARS-CoV-2 infections. The components and doses were chosen based on existing information about their structure and toxicity. Blocking the main protease (Mpro) of SARS-CoV-2 with high affinity and capacity is critical for inhibiting the virus’s pathogenesis and transmission. In silico studies were conducted to examine the molecular interactions between the main essential oil components in “ImmunoDefender” and SARS-CoV-2 Mpro. The screening results showed that six key components of ImmunoDefender formed stable complexes with Mpro via its active catalytic site with binding energies ranging from −8.75 to −10.30 kcal/mol, respectively for Cinnamtannin B1, Cinnamtannin B2, Pavetannin C1, Syzyginin B, Procyanidin C1, and Tenuifolin. Furthermore, three essential oil bioactive inhibitors, Cinnamtannin B1, Cinnamtannin B2, and Pavetannin C, had significant ability to bind to the allosteric site of the main protease with binding energies of −11.12, −10.74, and −10.79 kcal/mol; these results suggest that these essential oil bioactive compounds may play a role in preventing the attachment of the translated polyprotein to Mpro, inhibiting the virus’s pathogenesis and transmission. These components also had drug-like characteristics similar to approved and effective drugs, suggesting that further pre-clinical and clinical studies are needed to confirm the generated in silico outcomes.

Rapid and specific detection of pathogenic bacteria in fecal samples is of critical importance for the diagnosis of neonatal diarrhea in veterinary clinics. Nanobodies are a promising tool for the treatment and diagnosis of infectious diseases due to their unique recognition properties. In this study, we report the design of a nanobody-based magnetofluorescent immunoassay for the sensitive detection of pathogenic Escherichia coli F17-positive strains (E. coli F17). For this, a camel was immunized with purified F17A protein from F17 fimbriae and a nanobody library was constructed by phage display. Two specific anti-F17A nanobodies (Nbs) were selected to design the bioassay. The first one (Nb1) was conjugated to magnetic beads (MBs) to form a complex capable of efficiently capturing the target bacteria. A second horseradish peroxidase (HRP)-conjugated nanobody (Nb4) was used for detection by oxidizing o-phenylenediamine (OPD) to fluorescent 2,3-diaminophenazine (DAP). Our results show that the immunoassay recognizes E. coli F17 with high specificity and sensitivity, with a detection limit of 1.8 CFU/mL in only 90 min. Furthermore, we showed that the immunoassay can be applied to fecal samples without pretreatment and remains stable for at least one month when stored at 4 °C.

Abstract 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.

Abstract 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

Scorpion envenoming is a severe health problem in many regions causing significant clinical toxic effects and fatalities. In the Middle East/North Africa (MENA) region, Buthidae scorpion stings are responsible for devastating toxic outcomes in human. The only available specific immunotherapeutic treatment is based on IgG fragments of animal origin. To overcome the limitations of classical immunotherapy, we have demonstrated the in vivo efficacy of NbF12-10 bispecific nanobody at preclinical level. Nanobodies were developed against BotI analogues belonging to a distinct structural and antigenic group of scorpion toxins, occurring in the MENA region. From Buthus occitanus tunetanus venom, BotI-like toxin was purified. The 41 N-terminal amino acid residues were sequenced, and the LD50 was estimated at 40 ng/mouse. The BotI-like toxin was used for dromedary immunization. An immune VHH library was constructed, and after screening, two nanobodies were selected with nanomolar and sub-nanomolar affinity and recognizing an overlapping epitope. NbBotI-01 was able to neutralize 50% of the lethal effect of 13 LD50 BotI-like toxins in mice when injected by i.c.v route, whereas NbBotI-17 neutralized 50% of the lethal effect of 7 LD50. Interestingly, NbBotI-01 completely reduced the lethal effect of the 2 LD50 of BotG50 when injected at 1:4 molar ratio excess. More interestingly, an equimolar mixture of NbBotI-01 with NbF12-10 neutralized completely the lethal effect of 7 and 5 LD50 of BotG50 or AahG50, at 1:4 and 1:2 molar ratio, respectively. Hence, NbBotI-01 and NbF12-10 display synergic effects, leading to a novel therapeutic candidate for treating Buthus occitanus scorpion stings in the MENA region.

Scorpion α-toxins are neurotoxins that target the fast inactivation mechanism of voltage-gated sodium (NaV) channels leading to several neuro- and cardiotoxic effects in mammals. The toxin AahII is the most active α-toxin from the North African scorpion Androctonus australis Hector that slows the fast inactivation of NaV channels. To fight scorpion envenomation, an anti-AahII nanobody named NbAahII10 (Nb10) was developed. The efficiency of this nanobody has been evaluated in vivo on mice, but its mechanism of action at the cellular level remains unknown. Here we have shown that AahII toxin slows the fast inactivation of the adult cardiac NaV1.5 channels, expressed in HEK293 cells, in a dose-dependent manner, while current amplitude was not affected. The inactivation of NaV1.5 is slower by a factor of 4, 7, and 35 in the presence of [AahII] at 75, 150, and 300 nM, respectively. The washout partially reversed the toxin effect on inactivation from 8.3 ± 0.9 ms to 5.2 ± 1.2 ms at 75 nM. We have also demonstrated that the highly neutralizing Nb10 can fully reverse the effect of AahII toxin on the channel inactivation kinetics even at the 1:1 M ratio. However, the 1:0.5 M ratio is not able to neutralize completely the AahII effect. Therefore, the application of Nb10 promotes a partial abolishment of AahII action. Bioinformatic analysis and prediction of NaV1.5-driven docking with AahII show that Ala39 and Arg62 of AahII play a crucial role to establish a stable interaction through H-bound interactions with Gln1615 and Lys1616 (S3–S4 extracellular loop) and Asp1553 (S1–S2 loop) from the voltage-sensing domain IV (VSD4) of NaV1.5, respectively. From this, we notice that AahII shares the same contact surface with Nb10. This strongly suggests that Nb10 dynamically replaces AahII toxin from its binding site on the NaV1.5 channel. At the physiopathological level, Nb10 completely neutralized the enhancement of breast cancer cell invasion induced by AahII. In summary, for the first time, we made an electrophysiological and structural characterization of the neutralization potent of Nb10 against the α-scorpion toxin AahII in a cellular model overexpressing NaV1.5 channels.

Abstract 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.

Abstract 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.

Leishmaniasis is the 3rd most challenging vector-borne disease after malaria and lymphatic filariasis. Currently, no vaccine candidate is approved or marketed against leishmaniasis due to difficulties in eliciting broad immune responses when using sub-unit vaccines. The aim of this work was the design of a particulate sub-unit vaccine for vaccination against leishmaniasis. The poly (D,L-lactide) nanoparticles (PLA-NPs) were developed in order to efficiently adsorb a recombinant L. major histone H2B (L. major H2B) and to boost its immunogenicity. Firstly, a study was focused on the production of well-formed nanoparticles by the nanoprecipitation method without using a surfactant and on the antigen adsorption process under mild conditions. The set-up preparation method permitted to obtain H2B-adsorbed nanoparticles H2B/PLA (adsorption capacity of about 2.8% (w/w)) with a narrow size distribution (287 nm) and a positive zeta potential (30.9 mV). Secondly, an in vitro release assay performed at 37 °C, pH 7.4, showed a continuous release of the adsorbed H2B for almost 21 days (30%) from day 7. The immune response of H2B/PLA was investigated and compared to H2B + CpG7909 as a standard adjuvant. The humoral response intensity (IgG) was substantially similar between both formulations. Interestingly, when challenged with the standard parasite strain (GLC94) isolated from a human lesion of cutaneous leishmaniasis, mice showed a significant reduction in footpad swelling compared to unvaccinated ones, and no deaths occurred until week 17th. Taken together, these results demonstrate that PLA-NPs represent a stable, cost-effective delivery system adjuvant for use in vaccination against leishmaniasis.

Coronavirus illness (COVID-19) is an infectious pathology generated by intense severe respiratory syndrome coronavirus 2 (SARS-CoV-2). This infectious disease has emerged in 2019. The COVID-19-associated pandemic has considerably affected the way of life and the economy in the world. It is consequently crucial to find solutions allowing remedying or alleviating the effects of this infectious disease. Natural products have been in perpetual application from immemorial time given that they are attested to be efficient towards several illnesses without major side effects. Various studies have shown that plant extracts or purified molecules have a promising inhibiting impact towards coronavirus. In addition, it is substantial to understand the characteristics, susceptibility and impact of diet on patients infected with COVID-19. In this review, we recapitulate the influence of extracts or pure molecules from medicinal plants on COVID-19. We approach the possibilities of plant treatment/co-treatment and feeding applied to COVID-19. We also show coronavirus susceptibility and complications associated with nutrient deficiencies and then discuss the major food groups efficient on COVID-19 pathogenesis. Then, we covered emerging technologies using plant-based SARS-CoV-2 vaccine. We conclude by giving nutrient and plants curative therapy recommendations which are of potential interest in the COVID-19 infection and could pave the way for pharmacological treatments or co-treatments of COVID-19.

The extracellular matrix (ECM) molecule Tenascin-C (TNC) is well-known to promote tumor progression by multiple mechanisms. However, reliable TNC detection in tissues of tumor banks remains limited. Therefore, we generated dromedary single-domain nanobodies Nb3 and Nb4 highly specific for human TNC (hTNC) and characterized the interaction with TNC by several approaches including ELISA, western blot, isothermal fluorescence titration and negative electron microscopic imaging. Our results revealed binding of both nanobodies to distinct sequences within fibronectin type III repeats of hTNC. By immunofluroescence and immunohistochemical imaging we observed that both nanobodies detected TNC expression in PFA and paraffin embedded human tissue from ulcerative colitis, solid tumors and liver metastasis. As TNC impairs cell adhesion to fibronectin we determined whether the nanobodies abolished this TNC function. Indeed, Nb3 and Nb4 restored adhesion of tumor and mesangial cells on a fibronectin/TNC substratum. We recently showed that TNC orchestrates the immune-suppressive tumor microenvironment involving chemoretention, causing tethering of CD11c+ myeloid/dendritic cells in the stroma. Here, we document that immobilization of DC2.4 dendritic cells by a CCL21 adsorbed TNC substratum was blocked by both nanobodies. Altogether, our novel TNC specific nanobodies could offer valuable tools for detection of TNC in the clinical practice and may be useful to inhibit the immune-suppressive and other functions of TNC in cancer and other diseases.

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

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.

Aging is often associated with a loss of function. We believe aging to be more an adaptation to the various, and often continuous, stressors encountered during life in order to maintain overall functionality of the systems. The maladaptation of a system during aging may increase the susceptibility to diseases. There are basic cellular functions that may influence and/or are influenced by aging. Mitochondrial function is amongst these. Their presence in almost all cell types makes of these valuable targets for interventions to slow down or even reserve signs of aging. In this review, the role of mitochondria and essential physiological regulators of mitochondria and cellular functions, ion channels, will be discussed in the context of human aging. The origins of inflamm-aging, associated with poor clinical outcomes, will be linked to mitochondria and ion channel biology.

New Delhi Metallo-β-lactamase-1 (NDM-1) is the most prevalent type of metallo-β-lactamase, able to hydrolyze almost all antibiotics of the β-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-β-lactamase (VIM-1) (subclass B1) and L1 metallo-β-lactamase (L1) (subclass B3) with inhibitory concentration (IC50) values ranging from 100 to 0.04 μM. 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 β-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.

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’ 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 µg, 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 µg inhibits xINa in the same way as ATX 0.1 µg does. When ATX was added to the entire extract at 10 µg, inhibition reached that induced with ATX 1 µg. 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.