In this work, double-layer heterogeneous calcium alginate scaffolds were designed for alveolar bone defects where the outer layer featured high hardness and slow degradation, and the inner layer characterized by large pores and rapid degradation. The morphology of the calcium alginate scaffold was akin to that of bone defects, and its direct implantation reduced the operation time. A higher concentration of calcium alginate resulted in smaller pores and slower degradation. Calcium alginate can promote the formation of mineralized nodules and the expression of genes related to mineralization without inducing cytotoxic effects. It also promoted the expression of cellular inflammatory factors, potentially through the TLR4 pathway. In vivo studies confirmed that calcium alginate did not promote the aggregation of inflammatory cells nor the expression of inflammatory factors. In conclusion, the scaffold's characteristics of high surface hardness and slow degradation were beneficial for surface osteogenesis and maintaining the defect's shape and osteogenic space. Conversely, rapid internal degradation favors the formation of bone tissue.

Studying neurological disorders in vitro is still challenging due to the human brain’s complexity and the difficulty of obtaining primary neural cells. However, tissue engineering and tridimensional (3D) cell culture have become increasingly important tools for disease modeling. By providing an extracellular matrix (ECM) substrate that closely resembles physiological conditions, 3D cell culture offers several advantages over standard monolayer cell culture, including enhanced cell-cell and cell-matrix interactions. This results in a microenvironment that more accurately reflects in vivo biology. In this study, we performed an in-depth analysis of the proteome and matrisome of 3D tissue-engineered dermis, made from human primary dermal fibroblasts cultivated in 3D and embedded in a self-produced ECM. Interestingly, in silico analysis revealed that neurogenesis and associated functions were predicted to be strongly activated in this tissue-engineered 3D model. Indeed, we showed that ECM proteins involved in neuronal development and maintenance, typically produced by cerebral cells, were also expressed by dermal fibroblasts. Of particular interest, the 3D co-cultivation of dermal fibroblasts with iPSC-derived motor neurons readily enabled long-lasting culture periods without costly media supplementation with exogenous additives. Patient-derived dermal fibroblasts, cultivated in 3D, could therefore become valuable models for the study of neurological diseases. This approach offers a cost-effective and a less invasive alternative to brain biopsies for modeling complex neurological disorders in vitro.

Laboratory automation in the biopharmaceutical industry as a rule requires contracted service from highly professional automation solution provider, at times involving the purchase and development of specialized or customized hardware and software, which can be proprietary and expensive. Alternatively, with the availability of open-source software customized for automation, it is possible to automate existing laboratory instruments in a do-it-yourself (DIY), low-cost, and flexible fashion. In this work, we used an open-source scripting language, AutoIt, to integrate an existing microplate imager into an existing automation platform that is already equipped with a 4-axis robotic arm and an automated incubator, to achieve automation of the imaging procedure in our cell line development workflow. Furthermore, optimizations were performed using AutoIt to improve the overall automated imaging process, namely i) incorporating an automated scan profile selection step, ii) setting up automated handling of system errors, and iii) setting up remote handling of system errors. In summary, the use of AutoIt for DIY instrument integration proves to be cost-saving, versatile, and able to enhance the efficiency of automation workflows in the laboratory.

While it is well understood that muscle tissue generates contractile forces, it is less appreciated that muscle also dynamically responds to applied forces during development as well as in regular motion. We previously fabricated tissue engineered muscle comprising skeletal myocytes in co-culture with spinal motor neurons on aligned nanofiber poly-caprolactone scaffolding, demonstrating that innervation elicited more robust myofibers and formation of neuromuscular junctions. The current study utilized custom mechanobioreactors to apply tensile elongation to this engineered muscle platform to explore the effects of exogenous forces and scaffold topology on innervated versus non-innervated myocytes. We found that nanofiber scaffold alignment played a significant role in myocyte thickness, width, and fusion under both innervated and non-innervated conditions. We observed that a combination of tensile loading and nanofiber alignment increased myocyte fusion, suggesting these parameters work together to expedite and enhance myofiber formation and maturation. Overall, this multi-faceted paradigm featuring biomechanical loading, substrate topology, and innervation mimics key features of the developmental microenvironment experienced by myocytes in vivo. Future work may further apply this biofidelic paradigm to study muscle development, function, and responses to trauma, as well as explore the utility of fabricating large-scale engineered muscle for repair of major muscle defects.

Background: The functions of GOLPH3, STIP1, and the STAT3 signaling pathway in the invasion and migration of CC cells were examined in this study. Method: High-speed centrifugation was used to collect the exosomes. The expression of GOLPH3, STIP1, and epithelial-mesenchymal transition (EMT)-related proteins in CC tissues, cells, and exosomes were analyzed using Western blotting (WB) experiments. The abilities of CC cell invasion and migration were evaluated by the Transwell assay. The binding relationship between GOLPH3 and STIP1 was validated through Co-immunoprecipitation (Co-IP), and their sublocalization in CC cells was determined by immunofluorescence detection under laser confocal microscopy. Immunohistochemistry (IHC) experiments detected the expression levels of each protein in the transplanted tumor mass. Animal experiments confirmed the impact of the GOLPH3/STIP1/STAT3 regulatory axis on the CC growth. Results: In CC tissues and cells, GOLPH3 was highly expressed, and silencing GOLPH3 not only greatly reduced CC cell invasion and migration but also prevented EMT. Furthermore, GOLPH3 and STIP1 interacted in CC cells, and the GOLPH3-STIP1 complex affected the capacity for cell invasion and migration by triggering the STAT3 signaling pathway. Noteworthily, GOLPH3, and STIP1 could also be detected in CC cell exosomes, and the exosomes carried the GOLPH3-ST1P1 complex to act on CC cells to activate intracellular STAT3 signaling, ultimately affecting the cancer cell migration and invasion. The above molecular regulatory mechanisms have also been validated in mice. Conclusion: The GOLPH3-STIP1 complex acted on surrounding CC cells through exosomes and activated the STAT3 signaling pathway to stimulate CC cell invasion and migration

Chinese hamster ovary (CHO) cells are widely used to produce recombinant proteins, including monoclonal antibodies (mAbs), through various process modes. Traditionally, fed batch (FB) processes have been the standard. However, the shift towards high-density perfusion processes is driven by increased productivity, flexible facility footprints, and lower costs. Ensuring the clearance of process-related impurities, such as host cell proteins (HCPs), is crucial in biologics manufacturing. While purification processes remove most impurities, integrated strategies are being developed to enhance biologics purity and address high-risk HCPs. Current understanding of HCP expression dynamics in cell culture is limited. This study utilized data-independent acquisition (DIA) proteomics to compare the proteomic profiles of cell culture supernatants from 14 FB clones and 3 perfusion clones, all expressing the same mAb from the same host cell line. Results showed that perfusion processes enhance cell growth and productivity, exhibiting distinct proteomic profiles compared to FB processes. Perfusion processes also maintain a more stable HCP profile across clones, especially for 46 problematic HCPs monitored. Cluster analysis of FB proteomics revealed distinct abundance patterns and correlations with process parameters. Differential abundance analysis identified significant protein differences between the two processes. Compared to FB, the perfusion process may provide a less stressful cellular environment. This is the first extensive study characterizing HCPs expressed by different clones under different process modes. Further research could lead to strategies for preventing or managing problematic HCPs in biologics manufacturing.

Ultrasound (US) can easily penetrate media with excellent spatial precision corresponding to its wavelength. Naturally, US plays a pivotal role in the echolocation abilities of certain mammals such as bats and dolphins. In addition, medical US generated by transducers interact with tissues via delivering ultrasonic energy in the modes of heat generation, exertion of acoustic radiation force (ARF), and acoustic cavitation. Based on the principle of echolocation, various assistive devices for visual impairment people have been developed. High-Intensity Focused Ultrasound (HIFU) are developed for targeted ablation and tissue destruction. Besides thermal ablation, histotripsy with US is designed to damage tissue purely via mechanical effect without thermal coagulation. Low-Intensity Focused Ultrasound (LIFU) has been proven to be an effective stimulation method for neuromodulation. Furthermore, US has been reported to transiently increase permeability of biological membranes, enabling acoustic transfection and blood-brain barrier open. All of these advances in US are changing the clinic. This review mainly introduces the advances in these aspects, focusing on the physical and biological principles, challenges and future direction.

javascript:void(0) Antioxidants are known for their health beneficial effects by reducing oxidative damage, increasing free radical scavenging rate, blocking and terminating oxidation reactions. They have been widely used as supplements in food, cosmetics, nutraceuticals and medicines. Emerging researches increasingly indicate that the development of diverse research methodologies and the exploration of new microorganisms for heterogeneous synthesis of antioxidants play an indispensable role in socioeconomic development. In this study, we engineered Saccharomyces boulardii for probiotic supplementation of antioxidants such as L-ergothioneine (EGT). We first constructed double knockout of ura3 and trp1 gene in S. boulardii, to facilitate plasmid-based expressions. To further enable effective genome editing of S. boulardii, we implemented the PiggyBac system to transpose the heterologous gene expression cassettes into the chromosomes of S. boulardii. By using enhanced green fluorescent protein (EGFP) as the reporter gene, we achieved random chromosomal integration of EGFP expression cassette. EGT-producing strains were subsequently obtained via the PiggyBac transposon system. One isolated S. boulardii mutant produced EGT at 17.50 mg/L after 120 h cultivation. In summary, we have applied the PiggyBac transposon system to S. boulardii for the first time for genetic engineering. The engineered probiotic yeast S. boulardii has been endowed with new antioxidant properties and produces EGT. It has potential applications in developing novel therapeutics and dietary supplements for the prevention and treatment of gastrointestinal disorders.

Optimisation of isoamylase production in Escherichia coli under different nutrient limitation conditions

Protein farming, or molecular farming, is an innovative approach that utilizes plants/animals/microorganisms as biofactories to produce valuable pharmaceutical proteins, antibodies, vaccines, and other high-value bio-active molecules. This emerging field has gained significant attention due to its potential to address the growing demand for affordable and scalable biopharmaceutical production platform. Correspondingly, plants offer several advantages over traditional expression systems, including low production costs, scalability, ease of manipulation, and reduced risk of contamination by pathogens. This review provides an overview of principles underlying protein farming in plants (P-farm), including the identification and selection of host, optimization of transgene expression, and downstream processing techniques for protein purification. It also discusses recent advancements in plant biotechnology and the development of transient expression systems to use genetically modified plants (transgenic plants) in the enhancement of protein production. Additionally, the potential applications and challenges associated with molecular farming in plants are considered, including prospects of commercialization and novel opportunities to meet the growing demands of global healthcare markets.

After the COVID-19 pandemic, several companies and organisations are actively developing, scaling up and optimising mRNA manufacturing processes to produce their vaccines and therapeutics. Herein, we evaluated tangential flow filtration (TFF) for high-recovery, and high-purity separation of mRNA from unreacted NTPs in the IVT reaction mixture. For the first time, the membrane fouling behaviour caused by mRNA and separation of nucleoside triphosphates (NTPs) was mathematically modelled. The mRNA membrane fouling model is necessary for optimising mRNA separation processes, designing a suitable strategy for membrane clean-up, estimating the end of production life and reducing the process cost. Recovery greater than 70% mRNA without degradation was obtained by implementing a capacity load of ~19 g/m2, <2.5 psi transmembrane pressure (TMP) and feed flux of 300 LMH. This approach enables the purification of multiple RNA drug substance sequences for the prevention and treatment of a wide range of diseases.

Plant gene editing technology has significantly advanced in recent years, thereby transforming both biotechnological research and agricultural practices. This review provides a comprehensive summary of recent advancements in this rapidly evolving field, showcasing significant discoveries from improved transformation efficiency to advanced construct design. The primary focus is on the maturation of the CRISPR-Cas9 system, which has emerged as a powerful tool for precise gene editing in plants. Through a detailed exploration, we elucidate the intricacies of integrating genetic modifications into plant genomes, shedding light on transport mechanisms, transformation techniques, and optimization strategies specific to CRISPR constructs. Furthermore, we explore the initiatives aimed at extending the frontiers of gene editing to non-model plant species, showcasing the growing scope of this technology. Overall, this comprehensive review highlights the significant impact of recent advancements in plant gene editing, illuminating its transformative potential in driving agricultural innovation and biotechnological progress.

Kombucha is one of the most popular fermented beverages, made from sweetened tea that is fermented by bacteria, and yeast. These microorganisms produce a variety of bioactive compounds. Researchers continue to look for new base drinks for making kombucha. In this study, we tested herbs characterized by a high content of health-promoting substances: field horsetail, linden, chamomile, nettle, St. John’s wort and rockrose. The aim of study was to analyze the qualitative and quantitative composition as well as the antioxidant and antibiotic properties of kombucha prepared on the basis of herbs. The analyzed herbs turned out to be a good substrate for the production of a fermented beverage containing a number of both antibacterial and antioxidant compounds. Particularly noteworthy was the rockrose kombucha, which had the best ability to scavenge the free hydroxyl radicals, showed high antioxidant and antibiotic activity. Moreover, this drink had the highest concentration of succinic acid.

Plants are naturally affected against wide range of human diseases. They were used from ancient times for several purposes including cooking, preserving food, for treating a wide range of health conditions including cold, dysentery, anemia, wound heeling, curing insect bites and injuries. Nowadays people prefer products derived from natural sources more then products that are made from chemical methods. The plant Ocimum basilicum has a very range of health benefits including its potency to cure several skin conditions. This study aims to develop ,evaluate ,and determine the effectiveness of the herbal ointment made from Ocimum basilicum without alcohol and bees wax, using chea butter as thickening agent with the main focus on determining its outcome on common dermatological conditions such as acne(bacterial infection),eczema, inflammation, minor cuts, scrape wound. The prepared ointment was then evaluated for physiochemical characteristics including safety assessment of skin and stability tests. Then In Vivo experiments were carried in a group of volunteers including teenage and adults(two boys and three girls) skin, it reduced redness, inflammation and cured acne. The results showed the wound heeling ,anti-inflammatory and anti-bacterial activity of this ointment .In conclusion ,no adverse reaction of this ointment was observed on skin , The thorough evaluation of this ointment has confirmed its anti-bacterial activity because of its ability to kill bacterial strains causing acne. Further research is needed for its commercial usage and clinical trials to use for Post surgery wounds, serious bacterial and fungal infections.

Synthetic biology is contributing to the advancement of the global net-negative carbon economy, with emphasis on formate as a member of the one-carbon substrate garnering substantial attention. In this study, we employed base editing tools to facilitate adaptive evolution, achieving a formate tolerance of Yarrowia lipolytica to 1 M within two months. This effort resulted in two mutant strains, designated as M25-70 and M25-14, both exhibiting significantly enhanced formate utilization capabilities. Transcriptomic analysis revealed the up-regulation of nine endogenous genes encoding formate dehydrogenases when cultivated utilizing formate as the sole carbon source. Furthermore, we uncovered the pivotal role of the glyoxylate and threonine-based serine pathway in enhancing glycine supply to promote formate assimilation. The full potential of Yarrowia lipolytica to tolerate and utilize formate establishing the foundation for pyruvate carboxylase-based carbon sequestration pathways. Importantly, this study highlights the existence of a natural formate metabolic pathway in Yarrowia lipolytica.

Genetic diseases can be caused by monogenic diseases, which result from a single gene mutation in the DNA sequence. Many innovative approaches have been developed to cure monogenic genetic diseases, namely by genome editing. A specific type of genomic editing, prime editing, has the potential advantage to edit the human genome without requiring double-strand breaks or donor DNA templates for editing. Additionally, prime editing does not require a precisely positioned protospacer adjacent motif (PAM) sequence, which offers flexible target and more precise genomic editing. Here we detail a novel construction of a prime editing extended guide RNA (pegRNA) to target mutated leptin receptors in B6.BKS(D)-Leprdb/J mice (db/db mice). The pegRNA was then injected into the flexor digitorum brevis (FDB) muscle of db/db mice to demonstrate in vivo efficacy, which resulted in pegRNA mediated base transversion at endogenous base transversion. Genomic DNA sequencing confirmed that prime editing could correct the mutation of leptin receptor gene in db/db mice. Furthermore, prime editing treated skeletal muscle exhibited enhanced leptin receptor signals. Thus, the current study showed in vivo efficacy of prime editing to correct mutant protein and rescue the physiology associated with functional protein.

During plant-pathogen interaction, plant exhibits a strong defense system utilizing diverse groups of proteins to suppress the infection and subsequent establishment of the pathogen. However, in response, pathogens trigger an anti-silencing mechanism to overcome the host defense machinery. Among plant viruses, geminiviruses are the second largest virus family with a worldwide distribution and continue to be production constraints to food, feed, and fiber crops. These viruses are spread by a diverse group of insects, predominantly by whiteflies, and are characterized by a single-stranded DNA (ssDNA) genome coding for four to eight proteins that facilitate viral infection. The most effective means to managing these viruses is through an integrated disease management strategy that includes virus-resistant cultivars, vector management, and cultural practices. Dynamic changes in this virus family enable the species to manipulate their genome organization to respond to external changes in the environment. Therefore, the evolutionary nature of geminiviruses leads to new and novel approaches for developing virus-resistant cultivars and it is essential to study molecular ecology and evolution of geminiviruses. This review summarizes the multifunctionality of each geminivirus-encoded protein. These protein-based interactions trigger the abrupt changes in the host methyl cycle and signaling pathways that turn over protein normal production and impair the plant antiviral defense system. Studying these geminivirus interactions localized at cytoplasm-nucleus could reveal a more clear picture of host-pathogen relation. Data collected from this antagonistic relationship among geminivirus, vector, and its host, will provide extensive knowledge on their virulence mode and diversity with climate change

The liver is one of the most important organs in the human body. It performs many important functions, including being responsible for the metabolism of most drugs, which is often associated with its drug-induced damage. Currently, there are no ideal pharmacological models that would allow the evaluation of the effect of newly tested drugs on the liver in preclinical studies. Moreover, the influence of hepatic metabolism on the effectiveness of the tested drugs is rarely evaluated. Therefore, in this work we present an advanced model of the liver, which reflects most of the morphologically and metabolically important features of the liver in vivo, namely: three-dimensionality, cellular composition, presence of extracellular matrix, distribution of individual cell types in the structure of the liver model, high urea and albumin synthesis efficiency, high cytochrome p450 activity. In addition, the work, based on the example of commonly used anticancer drugs, shows how important it is to take into account hepatic metabolism in the effective assessment of their impact on the target organ, in this case cancer.