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Antibodies are widely employed as biorecognition elements for the detection of a plethora of compounds including food and environmental contaminants, biomarkers, or illicit drugs. They are also applied in therapeutics for the treatment of several disorders. Recent recommendations from the EU on animal protection and the replacement of animal-derived antibodies by non-animal-derived ones have raised a great controversy in the scientific community. The application of recombinant antibodies is expected to achieve a high growth rate in the years to come thanks to their versatility and beneficial characteristics in comparison to monoclonal and polyclonal antibodies, such as stability in harsh conditions, small size, relatively low production costs, and batch-to-batch reproducibility. This review describes the characteristics, advantages, and disadvantages of recombinant antibodies including antigen-binding fragments (Fab), single-chain fragment variable (scFv), and single-domain antibodies (VHH) and their application in food analysis with especial emphasis on the analysis of biotoxins, antibiotics, pesticides, and foodborne pathogens. Although the wide application of recombinant antibodies has been hampered by a number of challenges, this review demonstrates their potential for the sensitive, selective, and rapid detection of food contaminants.

Recombinant viruses are the workhorse of modern neuroscience. Whether one would like to understand a neuron’s morphology, natural activity patterns, molecular composition, connectivity or behavioural and physiologic function, most studies begin with the injection of an engineered virus, often an adeno-associated virus or herpes simplex virus, among many other types. Recombinant viruses currently enable some combination of cell type-specific, circuit-selective, activity-dependent and spatiotemporally resolved transgene expression. Viruses are now used routinely to study the molecular and cellular functions of a gene within an identified cell type in the brain, and enable the application of optogenetics, chemogenetics, calcium imaging and related approaches. These advantageous properties of engineered viruses thus enable characterization of neuronal function at unprecedented resolution. However, each virus has specific advantages and disadvantages, which makes viral tool selection paramount for properly designing and executing experiments within the central nervous system. In the current Review, we discuss the key principles and uses of engineered viruses and highlight innovations that are needed moving forward.Viral vectors are important tools for neuroscientists. In this Review, Nectow and Nestler discuss state-of-the-art recombinant viral tools, the key principles governing their selection, development and use, and how they could answer some of the most important questions in neuroscience today.

With the advent of highly sensitive technologies such as tandem mass spectrometry and next-generation sequencing, recombinant antibodies are now routinely analyzed for the presence of low-level sequence variants including amino acid misincorporations. During mAb cell culture process development, we found that proline was replaced with the non-canonical amino acid, hydroxyproline, in the protein sequence. We investigated the relationship between proline content in the cell culture media and proline sequence variants and found that the proline concentration was inversely correlated with the amount of sequence variants detected in the protein sequence. Hydroxyproline incorporation has been previously reported in recombinant proteins produced in mammalian expression systems as a post-translational modification. Given the dependency on proline levels, the mechanism was then investigated. To address the possibility of co-translational misincorporation of hydroxyproline, we used tandem mass spectrometry to measure incorporation of stable-isotope labelled hydroxyproline added to the feed of a production bioreactor. We discovered co-translational misincorporation of labelled hydroxyproline in the recombinant antibody. These findings are significant, since they underscore the need to track non-canonical amino acid incorporation as a co-translational event in CHO cells. Understanding the mechanism of hydroxyproline incorporation is crucial in developing an appropriate control strategy during biologics production.

In vitro transcription (IVT) using T7 RNA polymerase (RNAP) is integral to RNA research, yet producing this enzyme in E. coli presents challenges regarding endotoxins and animal-sourced toxins. This study demonstrates the viable production and characterization of T7 RNAP using ClearColi BL21(DE3) (an endotoxin-free E. coli strain) and animal-free media. Compared to BL21(DE3) with animal-free medium, soluble T7 RNAP expression is 50% lower in ClearColi BL21(DE3). Optimal soluble T7 RNAP expression in flask fermentation is achieved through the design of experiments (DoE). Specification and functional testing showed that the endotoxin-free T7 RNAP has comparable activity to conventional T7 RNAP. After Ni-NTA purification, endotoxin levels were approximately 109-fold lower than T7 RNAP from BL21(DE3) with animal-free medium. Furthermore, a full factorial DoE created an optimal IVT system that maximized mRNA yield from the endotoxin-free and animal-free T7 RNAP. This work addresses critical challenges in recombinant T7 RNAP production through innovative host and medium combinations, avoided endotoxin risks and animal-derived toxins. Together with an optimized IVT reaction system, this study represents a significant advance for safe and reliable reagent manufacturing and RNA therapeutics.

Functional M cells are differentiated by receptor activator of NF-B ligand (RANKL) and capture of luminal antigens to initiate immune responses. We aimed to use postbiotic-based recombinant chicken RANKL (cRANKL) to promote M cell differentiation and test the efficacy of oral vaccines. Chicks were divided into three groups that were administered phosphate-buffered saline (PBS), cell extracts of wild-type Lactococcus lactis subsp. lactis IL1403 (WT_CE), or cell extracts of recombinant L. lactis expressing cRANKL (cRANKL_CE). The expression of the M cell marker was measured, and the gut microbiome was profiled. The efficiency of the infectious bursal disease (IBD) vaccine was tested after 12 consecutive days of administering cRANKL_CE. The chickens that were administered cRANKL_CE (p = 0.038) had significantly higher Annexin A5 (ANXA5) mRNA expression levels than those in the PBS group (PBS vs. WT_CE, p = 0.657). In the gut microbiome analysis, no significant changes were observed. However, the relative abundance of Escherichia-Shigella was negatively correlated (r = - 0.43, p = 0.019) with ANXA5 mRNA expression in Peyer's patches. cRANKL_CE/IBD (p = 0.018) had significantly higher IBD-specific faecal IgA levels than PBS/IBD (PBS/IBD vs. WT_CE/IBD, p = 0.217). Postbiotic-based recombinant cRANKL effectively improved the expression of M cell markers and the efficiency of oral vaccines. No significant changes were observed in the gut microbiome after administration of postbiotic-based recombinant cRANKL. This strategy can be used for the development of feed additives and adjuvants.

Background The sensitivity of parasitological and molecular methods is unsatisfactory for the diagnosis of strongyloidiasis, and serological techniques are remaining as the most effective diagnostic approach. The present study aimed to design and produce a chimeric recombinant antigen from Strongyloides stercoralis immunoreactive antigen (SsIR) and Ss1a antigens, using immune-informatics approaches, and evaluated its diagnostic performance in an ELISA system for the diagnosis of human strongyloidiasis. Methodology/Principal findings The coding sequences for SsIR and Ss1a were selected from GenBank and were gene-optimized. Using bioinformatics analysis, the regions with the highest antigenicity that did not overlap with other parasite antigens were selected. The chimeric recombinant antigen SsIR- Ss1a, was constructed. The solubility and physicochemical properties of the designed construct were analyzed and its tertiary structures were built and evaluated. The construct was expressed into the pET-23a (+) expression vector and the optimized DNA sequences of SsIR-Ss1a (873 bp) were cloned into competent E. coli DH5[alpha] cells. Diagnostic performances of the produced recombinant antigen, along with a commercial kit were evaluated in an indirect ELISA system, using a panel of sera from strongyloidiasis patients and controls. The physicochemical and bioinformatics evaluations revealed that the designed chimeric construct is soluble, has a molecular with of 35 KDa, and is antigenic. Western blotting confirmed the immunoreactivity of the produced chimeric recombinant antigen with the sera of strongyloidiasis patients. The sensitivity and specificity of the indirect ELISA system, using the produced SsIR-Ss1a chimeric antigen, were found to be 93.94% (95% CI, 0.803 to 0.989) and 97.22% (95% CI, 0.921 to 0.992) respectively. Conclusions/Significance The preliminary findings of this study suggest that the produced SsIR-Ss1a chimeric antigen shows promise in the diagnosis of human strongyloidiasis. However, these results are based on a limited panel of samples, and further research with a larger sample size is necessary to confirm its accuracy. The construct has potential as an antigen in the ELISA system for the serological diagnosis of this neglected parasitic infection, but additional validation is required.

Gram-negative bacteria are attractive hosts for recombinant protein production because they are fast growing, easy to manipulate, and genetically stable in large cultures. However, the utility of these microbes would expand if they also could secrete the product at commercial scales. Secretion of biotechnologically relevant proteins into the extracellular medium increases product purity from cell culture, decreases downstream processing requirements, and reduces overall cost. Thus, researchers are devoting significant attention to engineering Gram-negative bacteria to secrete recombinant proteins to the extracellular medium. Secretion from these bacteria operates through highly specialized systems, which are able to translocate proteins from the cytosol to the extracellular medium in either one or two steps. Building on past successes, researchers continue to increase the secretion efficiency and titer through these systems in an effort to make them viable for industrial production. Efforts include modifying the secretion tags required for recombinant protein secretion, developing methods to screen or select rapidly for clones with higher titer or efficiency, and improving reliability and robustness of high titer secretion through genetic manipulations. An additional focus is the expression of secretion machineries from pathogenic bacteria in the “workhorse” of biotechnology, Escherichia coli , to reduce handling of pathogenic strains. This review will cover recent advances toward the development of high-expressing, high-secreting Gram-negative production strains.

In the evolution landscape of HIV, the coexistence of multiple subtypes has led to new, complex recombinants, posing public health challenges. CRF55₀1B, first identified among MSM in Shenzhen, China, has spread rapidly across China. In this study, 47 plasma samples from newly diagnosed HIV-1 CRF55₀1B patients in Shenzhen, of which the genotype was only identified by the routine HIV drug resistance test, were collected. Multiple gene regions were acquired using Sanger and next-generation sequencing methods, followed by the phylogenetic reconstruction, recombination breakpoint scanning, Bayesian molecular clock, and the prediction of coreceptors. From 47 samples, we found seven new unique recombinants formed by CRF55₀1B and CRF07_(B)C, which shared similar breakpoints in certain gene regions and primarily utilized CCR5 receptors. All of the most recent common ancestors of subregions for these recombinants were estimated to be later than CRF55₀1B and CRF07_(B)C, potentially suggesting they are the third-generation recombinants formed by CRF55₀1B and CRF07_(B)C as parents. The continuous emergence of new recombinants highlights the increasing complexity of circulating strains in Shenzhen, and also suggests that subtype analysis using partial pol gene may lead to an overestimation of the major subtype strains and an underestimation of new complex HIV recombinants. Consequently, to effectively address and mitigate the complex HIV epidemic, there is an urgent need for expanded monitoring and the optimization of testing methodologies.

The anti-cancer drug target poly(ADP-ribose) polymerase 1 (PARP1) and its close homologue, PARP2, are early responders to DNA damage in human cells 1 , 2 . After binding to genomic lesions, these enzymes use NAD + to modify numerous proteins with mono- and poly(ADP-ribose) signals that are important for the subsequent decompaction of chromatin and the recruitment of repair factors 3 , 4 . These post-translational modifications are predominantly serine-linked and require the accessory factor HPF1, which is specific for the DNA damage response and switches the amino acid specificity of PARP1 and PARP2 from aspartate or glutamate to serine residues 5 – 10 . Here we report a co-structure of HPF1 bound to the catalytic domain of PARP2 that, in combination with NMR and biochemical data, reveals a composite active site formed by residues from HPF1 and PARP1 or PARP2 . The assembly of this catalytic centre is essential for the addition of ADP-ribose moieties after DNA damage in human cells. In response to DNA damage and occupancy of the NAD + -binding site, the interaction of HPF1 with PARP1 or PARP2 is enhanced by allosteric networks that operate within the PARP proteins, providing an additional level of regulation in the induction of the DNA damage response. As HPF1 forms a joint active site with PARP1 or PARP2, our data implicate HPF1 as an important determinant of the response to clinical PARP inhibitors. Assembly of a catalytic centre formed by HPF1 bound to PARP1 or PARP2 is essential for protein ADP-ribosylation after DNA damage in human cells.