loop-mediated isothermal amplification (lamP) as a point-of-care SarS-covdetection method

Pravovremeno testiranje većeg broja ljudi na SARS-CoV-2 virus je povezano sa nižim mortalitetom od COVID-19 oboljenja. Međutim, većina zemalja nema mogućnosti za takvo masivno testiranje putem metode “PCR u realnom vremenu”, zbog visoke cene neophodne opreme i potrebe za stručnim osobljem. Zbog toga se razvijaju brze i ekonomičnije metode, koje se najčešće zasnivaju na izotermalnim metodama amplifikacije DNK. Za ove metode nije potreban ciklični termostat, zbog čega su primenljivije za terensku upotrebu. Fokus je na izotermalnoj amplifikaciji posredovanoj petljom (lamP), zbog njene visoke specifičnosti, mogućnosti za upotrebu neprečišćenih uzoraka i jednostavnosti merenja signala. Autori predstavljaju pregled najvažnijih radova o LAMP metodi za detekciju SARS-CoV-2 virusa objavljenih tokom 2020. godine, kao i opise nekoliko komercijalnih kompleta na bazi LAMP metode za COVID-19 testiranje.Massive testing for SARS-CoV-2 virus is related to lower mortality rates of COVID-19. Most countries face challenges to perform massive testing with the currently available methods (real-time PCR), due to expensive equipment and requirement of highly skilled personnel. To overcome these challenges, faster and cost-effective alternative detection methods are being developed, primarily based on isothermal methods of nucleic acid amplification (isoNAAs). PCR depends on precision instruments, high cleanliness of operating conditions and cannot be easily used on-site, while isoNAAs, which do not require thermal cyclers, are more applicable for point-of-care (PoC) use. Loop-mediated isothermal amplification (LAMP) is one of the isoNAA most in focus for COVID-19 tests due to its high specificity and possibility to use unpurified specimens in combination with simplified detection setup. The article gives a review of the most significant publications on use of LAMP for SARS-CoV-2 detection and of the several commercial LAMP-based COVID-19 testing kits.Ministarstva prosvete, nauke i tehnološkog razvoja Republike Srbije – broj ugovora 451-03-68/2020-14/ 200358 za autore M. Dj, T.K i Lj.J i broj ugovora 451-03-9/2021- 14/200125 za Ž.D.P. Autori zahvaljuju Jeleni Ognjenov na pomoći u pripremi ilustracija za rad

Using Vertebrate Stem and Progenitor Cells for Cellular Agriculture, State-of-the-Art, Challenges, and Future Perspectives

Global food systems are under significant pressure to provide enough food, particularly protein-rich foods whose demand is on the rise in times of crisis and inflation, as presently existing due to post-COVID-19 pandemic effects and ongoing conflict in Ukraine and resulting in looming food insecurity, according to FAO. Cultivated meat (CM) and cultivated seafood (CS) are protein-rich alternatives for traditional meat and fish that are obtained via cellular agriculture (CA) i.e., tissue engineering for food applications. Stem and progenitor cells are the building blocks and starting point for any CA bioprocess. This review presents CA-relevant vertebrate cell types and procedures needed for their myogenic and adipogenic differentiation since muscle and fat tissue are the primary target tissues for CM/CS production. The review also describes existing challenges, such as a need for immortalized cell lines, or physical and biochemical parameters needed for enhanced meat/fat culture efficiency and ways to address them

Insects as a Prospective Source of Biologically Active Molecules and Pharmaceuticals—Biochemical Properties and Cell Toxicity of <i>Tenebrio molitor</i> and <i>Zophobas morio</i> Cell-Free Larval Hemolymph

Insects are of great interest as novel sources of alternative proteins and biologically active compounds, primarily anticancer agents. Protein-rich insect larval hemolymph is a prospective candidate for pharmaceutical and food industry-related research. In this study, selected biochemical properties and cell toxicity of larval hemolymph from two mealworm species, Tenebrio molitor and Zophobas morio, were analyzed. Total proteins and carbohydrates, antioxidant capacity, and the level of lipid peroxidation were determined. Human cancer (U-87) and normometabolic (MRC-5) cells were treated with different concentrations of larval hemolymph proteins, and the effects on cell viability were assayed 24, 48, and 72 h after treatments. Z. morio hemolymph was shown to be richer in total proteins, showing a higher antioxidant capacity and lipid peroxidation level than T. molitor hemolymph, which was richer in total carbohydrates. Cytotoxicity assays showed that T. molitor and Z. morio hemolymphs differently affect the viability of U-87 and MRC-5 cells in cell type-, dose-, and time-dependent manners. Hemolymph from both species was more cytotoxic to U-87 cells than to MRC-5 cells, which was particularly prominent after 48 h. Additionally, a more potent cytotoxic effect of Z. morio hemolymph was observed on both cell lines, likely due to its higher antioxidant capacity, compared to T. molitor hemolymph

Prolonged heat stress during winter diapause alters the expression of stress-response genes in Ostrinia nubilalis (Hbn.)

During diapause, a state of temporarily arrested development, insects require low winter temperatures to suppress their metabolism, conserve energy stores and acquire cold hardiness. A warmer winter could, thus, reduce diapause incidence and duration in many species, prematurely deplete their energy reserves and compromise post-diapause fitness. In this study, we investigated the combined effects of thermal stress and the diapause program on the expression of selected genes involved in antioxidant defense and heat shock response in the European corn borer Ostrinia nubilalis. By using qRT-PCR, it has been shown that response to chronic heat stress is characterized by raised mRNA levels of grx and trx, two important genes of the antioxidant defense system, as well as of hsp70 and, somewhat, of hsp90, two major heat shock response proteins. On the other hand, the expression of hsc70, hsp20.4 and hsp20.1 was discontinuous in the latter part of diapause, or was strongly controlled by the diapause program and refractory to heat stress, as was the case for mtn and fer, genes encoding two metal storage proteins crucial for metal ion homeostasis. This is the first time that the effects of high winter temperatures have been assessed on cold-hardy diapausing larvae and pupae of this important corn pest

Molecular Approaches for Detection of <i>Trichoderma</i> Green Mold Disease in Edible Mushroom Production

Due to the evident aggressive nature of green mold and the consequently huge economic damage it causes for producers of edible mushrooms, there is an urgent need for prevention and infection control measures, which should be based on the early detection of various Trichoderma spp. as green mold causative agents. The most promising current diagnostic tools are based on molecular methods, although additional optimization for real-time, in-field detection is still required. In the first part of this review, we briefly discuss cultivation-based methods and continue with the secondary metabolite-based methods. Furthermore, we present an overview of the commonly used molecular methods for Trichoderma species/strain detection. Additionally, we also comment on the potential of genomic approaches for green mold detection. In the last part, we discuss fast screening molecular methods for the early detection of Trichoderma infestation with the potential for in-field, point-of-need (PON) application, focusing on isothermal amplification methods. Finally, current challenges and future perspectives in Trichoderma diagnostics are summarized in the conclusions

Bioengineering Outlook on Cultivated Meat Production

Cultured meat (also referred to as cultivated meat or cell-based meat)—CM—is fabricated through the process of cellular agriculture (CA), which entails application of bioengineering, i.e., tissue engineering (TE) principles to the production of food. The main TE principles include usage of cells, grown in a controlled environment provided by bioreactors and cultivation media supplemented with growth factors and other needed nutrients and signaling molecules, and seeded onto the immobilization elements—microcarriers and scaffolds that provide the adhesion surfaces necessary for anchor-dependent cells and offer 3D organization for multiple cell types. Theoretically, many solutions from regenerative medicine and biomedical engineering can be applied in CM-TE, i.e., CA. However, in practice, there are a number of specificities regarding fabrication of a CM product that needs to fulfill not only the majority of functional criteria of muscle and fat TE, but also has to possess the sensory and nutritional qualities of a traditional food component, i.e., the meat it aims to replace. This is the reason that bioengineering aimed at CM production needs to be regarded as a specific scientific discipline of a multidisciplinary nature, integrating principles from biomedical engineering as well as from food manufacturing, design and development, i.e., food engineering. An important requirement is also the need to use as little as possible of animal-derived components in the whole CM bioprocess. In this review, we aim to present the current knowledge on different bioengineering aspects, pertinent to different current scientific disciplines but all relevant for CM engineering, relevant for muscle TE, including different cell sources, bioreactor types, media requirements, bioprocess monitoring and kinetics and their modifications for use in CA, all in view of their potential for efficient CM bioprocess scale-up. We believe such a review will offer a good overview of different bioengineering strategies for CM production and will be useful to a range of interested stakeholders, from students just entering the CA field to experienced researchers looking for the latest innovations in the field

Mammalian Cell-Growth Monitoring Based on an Impedimetric Sensor and Image Processing within a Microfluidic Platform

In recent years, advancements in microfluidic and sensor technologies have led to the development of new methods for monitoring cell growth both in macro- and micro-systems. In this paper, a microfluidic (MF) platform with a microbioreactor and integrated impedimetric sensor is proposed for cell growth monitoring during the cell cultivation process in a scaled-down simulator. The impedimetric sensor with an interdigitated electrode (IDE) design was realized with inkjet printing and integrated into the custom-made MF platform, i.e., the scaled-down simulator. The proposed method, which was integrated into a simple and rapid fabrication MF system, presents an excellent candidate for the scaled-down analyses of cell growths that can be of use in, e.g., optimization of the cultivated meat bioprocess. When applied to MRC-5 cells as a model of adherent mammalian cells, the proposed sensor was able to precisely detect all phases of cell growth (the lag, exponential, stationary, and dying phases) during a 96-h cultivation period with limited available nutrients. By combining the impedimetric approach with image processing, the platform enables the real-time monitoring of biomasses and advanced control of cell growth progress in microbioreactors and scaled-down simulator systems