Phage display – a tool for detection and prevention against pathogens. A review

In this article are reviewed the promising uses of phage display in the areas such as microbial pathogens detection of and vaccination. Phage display is a molecular technique by which foreign proteins are expressed at the surface of phage particles. Such phages thereby become vehicles for expression that not only carry within them the nucleotide sequence encoding expressed proteins, but have also the capability to replicate. Recent data acquired from genome sequencing and advances in phage biology research have aided the development of phage-derived bacterial detection and treatment strategies.

Review of testing for foreign horse and pig DNA in meats in Croatia

Several years after the food industry scandal when horsemeat was found in products sold in Europe as beef products in 2013, Croatia began testing food for the presence of foreign protein. For the time being, these tests are not part of routine monitoring, but the result of examining the situation on the market in the city of Zagreb. Namely, in recent years, central Croatia has been trying to establish itself as a tourist destination, and Zagreb hosted hundreds of thousands of tourists from all over the world before the COVID-19 pandemic. The eating habits of the various groups that came to Zagreb were different, and the larger hotel chains recognized the seriousness of the services and sought help to ensure that the food offered was consistent with their declarations and would not conflict with religious requirements. One of these requirements was the testing for foreign proteins such as horse and pork in foods where they were not declared. Although horse and pork are safe for human consumption, they are not part of the eating habits in all countries. The Dr. Andrija Štampar Teaching Institute for Public Health introduced methods for detection of horse and pig DNA in food samples.

Vectored Immunotherapeutics for Infectious Diseases: Can rAAVs Be The Game Changers for Fighting Transmissible Pathogens?

Conventional vaccinations and immunotherapies have encountered major roadblocks in preventing infectious diseases like HIV, influenza, and malaria. These challenges are due to the high genomic variation and immunomodulatory mechanisms inherent to these diseases. Passive transfer of broadly neutralizing antibodies may offer partial protection, but these treatments require repeated dosing. Some recombinant viral vectors, such as those based on lentiviruses and adeno-associated viruses (AAVs), can confer long-term transgene expression in the host after a single dose. Particularly, recombinant (r)AAVs have emerged as favorable vectors, given their high in vivo transduction efficiency, proven clinical efficacy, and low immunogenicity profiles. Hence, rAAVs are being explored to deliver recombinant antibodies to confer immunity against infections or to diminish the severity of disease. When used as a vaccination vector for the delivery of antigens, rAAVs enable de novo synthesis of foreign proteins with the conformation and topology that resemble those of natural pathogens. However, technical hurdles like pre-existing immunity to the rAAV capsid and production of anti-drug antibodies can reduce the efficacy of rAAV-vectored immunotherapies. This review summarizes rAAV-based prophylactic and therapeutic strategies developed against infectious diseases that are currently being tested in pre-clinical and clinical studies. Technical challenges and potential solutions will also be discussed.

Detailed metabolic phenotyping of four tissue specific Cas9 transgenic mouse lines

CRISPR/Cas9 technology has revolutionized gene editing and fast tracked our capacity to manipulate genes of interest for the benefit of both research and therapeutic applications. Whilst many advances have, and continue to be made in this area, perhaps the most utilized technology to date has been the generation of knockout cells, tissues and animals by taking advantage of Cas9 function to promote indels in precise locations in the genome. Whilst the advantages of this technology are many fold, some questions still remain regarding the effects that long term expression of foreign proteins such as Cas9, have on mammalian cell function. Several studies have proposed that chronic overexpression of Cas9, with or without its accompanying guide RNAs, may have deleterious effects on cell function and health. This is of particular concern when applying this technology in vivo, where chronic expression of Cas9 in tissues of interest may promote disease-like phenotypes and thus confound the investigation of the effects of the gene of interest. Although these concerns remain valid, no study to our knowledge has yet to demonstrate this directly. Thus, in this study we used the lox-stop-lox (LSL) spCas9 ROSA26 transgenic (Tg) mouse line to generate four tissue-specific Cas9-Tg models with expression in the heart, liver, skeletal muscle and adipose tissue. We performed comprehensive phenotyping of these mice up to 20-weeks of age and subsequently performed molecular analysis of their organs. We demonstrated that Cas9 expression in these tissues had no detrimental effect on whole body health of the animals, nor did it induce any tissue-specific effects on energy metabolism, liver health, inflammation, fibrosis, heart function or muscle mass. Thus, our data suggests that these models are suitable for studying the tissue specific effects of gene deletion using the LSL-Cas9-Tg model, and that phenotypes observed utilizing these models can be confidently interpreted as being gene specific, and not confounded by the chronic overexpression of Cas9.

Expression of Foreign Proteins in Escherichia coli

BackgroundOptimization of conditions for the recombinant production of proteins in a prokaryotic expression system is essential as the recombinant proteins impose a metabolic burden on cell's growth leading to low protein yield and low protein expression resulting from cell death.Main textThe concentration of media components is optimized to accommodate for depleted nutrients due to foreign protein expression. The temperature is optimized to reduce proteolytic degradation and accumulation of protein as inclusion bodies in Escherichia coli. The concentration of inducer and time of induction for high protein yield is also optimized. These optimization conditions depend on the promoter under which the gene of interest is present and the characteristics of the target protein.ConclusionIn the past few years, many optimization conditions for the production of recombinant proteins in Escherichia coli have been studied. These conditions depend mainly upon the promoter used to produce protein and the type of protein produced. Optimizing the expression parameters of protein produced in Escherichia coli ensures maximum yield of the desired protein.

Advances in Genetic Engineering Technology and Its Application in the Industrial Fungus Aspergillus oryzae

The filamentous fungus Aspergillus oryzae is an important strain in the traditional fermentation and food processing industries and is often used in the production of soy sauce, soybean paste, and liquor-making. In addition, A. oryzae has a strong capacity to secrete large amounts of hydrolytic enzymes; therefore, it has also been used in the enzyme industry as a cell factory for the production of numerous native and heterologous enzymes. However, the production and secretion of foreign proteins by A. oryzae are often limited by numerous bottlenecks that occur during transcription, translation, protein folding, translocation, degradation, transport, secretion, etc. The existence of these problems makes it difficult to achieve the desired target in the production of foreign proteins by A. oryzae. In recent years, with the decipherment of the whole genome sequence, basic research and genetic engineering technologies related to the production and utilization of A. oryzae have been well developed, such as the improvement of homologous recombination efficiency, application of selectable marker genes, development of large chromosome deletion technology, utilization of hyphal fusion techniques, and application of CRISPR/Cas9 genome editing systems. The development and establishment of these genetic engineering technologies provided a great deal of technical support for the industrial production and application of A. oryzae. This paper reviews the advances in basic research and genetic engineering technologies of the fermentation strain A. oryzae mentioned above to open up more effective ways and research space for the breeding of A. oryzae production strains in the future.

Recombinant Silk Proteins with Additional Polyalanine Have Excellent Mechanical Properties

This paper explores the structures of exogenous protein molecules that can effectively improve the mechanical properties of silkworm silk. Several transgenic vectors fused with the silkworm fibroin light chain and type 3 repeats in different multiples of the ampullate dragline silk protein 1 (MaSp1) from black widow spider with different lengths of the polyalanine motifs were constructed for this study. Transgenic silkworms were successfully obtained by piggyBac-mediated microinjection. Molecular detection showed that foreign proteins were successfully secreted and contained within the cocoon shells. According to the prediction of PONDR® VSL2 and PONDR® VL-XT, the type 3 repeats and the polyalanine motif of the MaSp1 protein were amorphous. The results of FTIR analysis showed that the content of β-sheets in the silk of transgenic silkworms engineered with transgenic vectors with additional polyalanine was significantly higher than that of wild-type silkworm silk. Additionally, silk with a higher β-sheet content had better fracture strength and Young’s modulus. The mechanical properties of silk with longer chains of exogenous proteins were improved. In general, our results provide theoretical guidance and technical support for the large-scale production of excellent bionic silk.

Allergy and its treatment with antihistamines

Allergy is a disease of the 20th century. 30% - 40% of the world's population is allergic to various irritants. "Dangerous" agents invading the human body: bacteria, viruses, airborne dust, animal fur, foreign proteins, etc. cause various allergic diseases and can often lead to serious complications (including lethal outcome). This article discusses the causes and characteristic symptoms of allergies, modern methods of diagnosis and treatment with effective and safe antihistamines - Polesin, Lotegra, Loranex, Erius, Eslotin, Telfast, Allerfast, Nixar, Loratadine, Claritine, Cetek, Alertek , Zetrin, Parlazin, Kestin, Zirtek, Terrix, etc. Immunotherapy - or specific hyposensitization - is also characterized as a modern and effective method of treatment, the principle of which lies in the production of specific tolerance (indiscretion) towards the causative allergen.

Adulteration of Milk: A Review

Milk is a good nutritious drink and is eaten for drinking as such, as well as by milk products, by a majority of the population worldwide. The practice of milk adulteration, however, constantly reduces its quality and can introduce hazardous substances into the dairy supply chain which endangers the health of consumers. Various cases of milk adulteration have been reported globally, in which substances such as extraneous water, foreign proteins, whey proteins, melamine and urea, vegetable or animal fats, plus many minor constituents of milk fat were added as possible adulterants in milk and milk products. This article provides some information about adulteration of milk and their detection process.

Human Embryonic-Derived Mesenchymal Progenitor Cells (hES-MP Cells) are Fully Supported in Culture with Human Platelet Lysates

Human embryonic stem cell-derived mesenchymal progenitor (hES-MP) cells are mesenchymal-like cells, derived from human embryonic stem cells without the aid of feeder cells. They have been suggested as a potential alternative to mesenchymal stromal cells (MSCs) in regenerative medicine due to their mesenchymal-like proliferation and differentiation characteristics. Cells and cell products intended for regenerative medicine in humans should be derived, expanded and differentiated using conditions free of animal-derived products to minimize risk of animal-transmitted disease and immune reactions to foreign proteins. Human platelets are rich in growth factors needed for cell culture and have been used successfully as an animal serum replacement for MSC expansion and differentiation. In this study, we compared the proliferation of hES-MP cells and MSCs; the hES-MP cell growth was sustained for longer than that of MSCs. Growth factors, gene expression, and surface marker expression in hES-MP cells cultured with either human platelet lysate (hPL) or fetal bovine serum (FBS) supplementation were compared, along with differentiation to osteogenic and chondrogenic lineages. Despite some differences between hES-MP cells grown in hPL- and FBS-supplemented media, hPL was found to be a suitable replacement for FBS. In this paper, we demonstrate for the first time that hES-MP cells can be grown using platelet lysates from expired platelet concentrates (hPL).