Exploiting non-permissive CHO cells as a rapid and efficient method for recombinant HSV-1 isolation.

Using herpes simplex virus type 1 (HSV-1) as a therapeutic tool has recently emerged as a promising strategy for enhancing the treatment of various cancers, particularly those associated with the nervous system, which is the virus's natural site of infection. These viruses are specifically engineered to infect and eradicate tumor cells while leaving healthy cells unharmed. To introduce targeted mutations in specific viral genes, gene-modification techniques such as shuttle vector homologous recombination are commonly employed. Plaque purification is then utilized to select and purify the recombinant virus from the parental viruses. However, plaque purification becomes problematic when the insertion of the desired gene at the target site hampers progeny virus replication, resulting in a lower titer of cell-released virus than the parental virus. This necessitates a laborious initial screening process using approximately 10-15 tissue culture dishes (10 cm), making plaque purification time-consuming and demanding. Although the recently developed CRISPR-Cas9 system significantly enhances the efficiency of homologous integration and editing precision in viral genes, the purification of recombinant variants remains a tedious task. In this study, we propose a rapid and innovative method that employs non-permissive Chinese hamster ovary (CHO) cells, representing a remarkable improvement over the aforementioned arduous process. With this approach, only 1-2 rounds of plaque purification are required. Our proposed protocol demonstrates great potential as a viable alternative to current methods for isolating and purifying recombinant HSV-1 variants expressing fluorescent reporter genes using CHO cells and plaque assays.

Neuroprotective Therapeutic Potential of microRNA-149-5p against Murine Ischemic Stroke.

Ischemic stroke resulting from blockade of brain vessels lacks effective treatments, prompting exploration for potential therapies. Among promising candidates, microRNA-149 (miR-149) has been investigated for its role in alleviating oxidative stress, inflammation, and neurodegeneration associated with ischemic conditions. To evaluate its therapeutic effect, male Wistar rats were categorized into five groups, each consisting of 27 rats: sham, MCAO, lentiviral control, lentiviral miR-149, and miR149-5p mimic. Treatments were microinjected intracerebroventricularly (ICV) (right side), and ischemia was induced using middle cerebral artery occlusion (MCAO) procedure. Post-MCAO, neurological function, histopathological changes, blood-brain barrier (BBB) permeability, cerebral edema, and mRNA levels of Fas ligand (Faslg) and glutamate ionotropic NMDA receptor 1 (GRIN1) were assessed, alongside biochemical assays. MiR-149 administration improved neurological function, reduced brain damage, preserved BBB integrity, and attenuated cerebral edema. Upregulation of miR149-5p decreased Faslg and GRIN1 expression in ischemic brain regions. MiR-149 also reduced oxidative stress, enhanced antioxidant activity, decreased caspase-1 and - 3 activity, and modulated inflammatory factors in ischemic brain regions. Moreover, DNA fragmentation as an index of cell death decreased following miR-149 treatment. In conclusion, the study underscores miR-149 potential as a neuroprotective agent against ischemic stroke, showcasing its efficacy in modulating various mechanisms and supporting its candidacy as a promising therapeutic target for innovative strategies in stroke treatment.

Novel neutralizing SARS-CoV-2-specific mAbs offer detection of RBD linear epitopes.

BACKGROUND: To stop the spread of the COVID-19 disease, it is crucial to create molecular tools to investigate and diagnose COVID-19. Current efforts focus on developing specific neutralizing monoclonal antibodies (NmAbs) elicited against the receptor-binding domain (RBD). METHODS: In the present study, recombinant RBD (rRBD) protein was produced in E. coli, followed by immunizing mice with purified rRBD. ELISA was applied to screen the hybridomas for positive reactivity with rRBD protein. The linear and conformational epitopes of the mAbs were subsequently identified using western blot. Finally, the reactivity, affinity, and neutralization activity of the purified mAbs were evaluated using ELISA. RESULTS: All mAbs exhibited similar reactivity trends towards both eukaryotic RBD and prokaryotic rRBD in ELISA. Among them, 2E7-D2 and 2B4-G8 mAbs demonstrated higher reactivity than other mAbs. Additionally, in western blot assays, these two mAbs could detect reducing and non-reducing rRBD, indicating recognition of linear epitopes. Notably, five mAbs effectively blocked rRBD- angiotensin-converting enzyme 2 (ACE2) interaction, while two high-affinity mAbs exhibited potent neutralizing activity against eukaryotic RBD. CONCLUSION: In the current study, we generated and characterized new RBD-specific mAbs using the hybridoma technique that recognized linear and conformational epitopes in RBD with neutralization potency. Our mAbs are novel candidates for diagnosing and treating SARS-CoV-2.

Efficient three-dimensional (3D) human bone differentiation on quercetin-functionalized isotropic nano-architecture chitinous patterns of cockroach wings.

Developing cost-effective, biocompatible scaffolds with nano-structured surface that truthfully replicate the physico-(bio)chemical and structural properties of bone tissue's extracellular matrix (ECM) is still challenging. In this regard, surface functionalization of natural scaffolds to enhance capability of mimicking 3D niches of the bone tissue has been suggested as a solution. In the current study, we aimed to investigate the potential of chitin-based cockroach wings (CW) as a natural scaffold for bone tissue engineering. To raise the osteogenic differentiation capacity of such a scaffold, a quercetin coating was also applied (hereafter this scaffold is referred as QCW). Moreover, the QCW scaffold exhibited effective antibacterial properties against gram-positive S. aureus bacteria. With respect to bone regeneration, the QCW scaffold optimally induced the differentiation of adipose-derived human mesenchymal stem cells (AD-hMSCs) into osteoblasts, as validated by mineralization assays, alkaline phosphatase (ALP) activity measurements, expression of pre-osteocyte marker genes, and immunocytochemical staining. Confirmation of the potent biocompatibility and physicochemical characteristics of the QCW scaffold through a series of in vitro and in vivo analysis revealed that surface modification had significant effect on multi-purpose features of obtained scaffold. Altogether, surface modification of QCW made it as an affordable bioinspired scaffold for bone tissue engineering.

The chicken chorioallantoic membrane model for isolation of CRISPR/cas9-based HSV-1 mutant expressing tumor suppressor p53.

Oncolytic viruses (OVs) have emerged as a novel cancer treatment modality, which selectively target and kill cancer cells while sparing normal ones. Among them, engineered Herpes simplex virus type 1 (HSV-1) has been proposed as a potential treatment for cancer and was moved to phase III clinical trials. Previous studies showed that design of OV therapy combined with p53 gene therapy increases the anti-cancer activities of OVs. Here, the UL39 gene of the ICP34.5 deleted HSV-1 was manipulated with the insertion of the EGFP-p53 expression cassette utilizing CRISPR/ Cas9 editing approach to enhance oncoselectivity and oncotoxicity capabilities. The ΔUL39/Δγ34.5/HSV1-p53 mutant was isolated using the chorioallantoic membrane (CAM) of fertilized chicken eggs as a complementing membrane to support the growth of the viruses with gene deficiencies. Comparing phenotypic features of ΔUL39/Δγ34.5/HSV1-p53-infected cells with the parent Δγ34.5/HSV-1 in vitro revealed that HSV-1-P53 had cytolytic ability in various cell lines from different origin with different p53 expression rates. Altogether, data presented here illustrate the feasibility of exploiting CAM model as a promising strategy for isolating recombinant viruses such as CRISPR/Cas9 mediated HSV-1-P53 mutant with less virus replication in cell lines due to increased cell mortality induced by exogenous p53.

Efficient CRISPR/Cas9-Mediated BAX Gene Ablation in CHO Cells To Impair Apoptosis and Enhance Recombinant Protein Production.

Background: Despite recent advances in recombinant biotherapeutics production using CHO cells, their productivity remains lower than industrial needs, mainly due to apoptosis. Objectives: Present study aimed to exploit CRISPR/Cas9 technology to specifically disrupt the BAX gene to attenuate apoptosis in recombinant Chinese hamster's ovary cells producing erythropoietin. Materials and Methods: The STRING database was used to identify the key pro-apoptotic genes to be modified by CRISPR/Cas9 technique. The single guide RNAs (sgRNAs) targeting identified gene (BAX) were designed, and CHO cells were then transfected with vectors. Afterward, changes in the expression of the Bax gene and consequent production rates of erythropoietin were investigated in manipulated cells, even in the presence of an apoptosis inducer agent, oleuropein. Results: BAX disruption significantly prolonged cell viability and increased proliferation rate in manipulated clones (152%, P-value = 0.0002). This strategy reduced the levels of Bax protein expression in manipulated cells by more than 4.3-fold (P-value <0.0001). The Bax-8 manipulated cells displayed higher threshold tolerance to the stress and consequence apoptosis compared to the control group. Also, they exhibited a higher IC50 compared to the control in the presence of oleuropein (5095 µM.ml-1 Vs. 2505 µM.ml-1). We found a significant increase in recombinant protein production levels in manipulated cells, even in the presence of 1,000 µM oleuropein compared to the control cell line (p-value=0.0002). Conclusions: CRISPR/Cas9 assisted BAX gene ablation is promising to improve erythropoietin production in CHO cells via engineering anti-apoptotic genes. Therefore, exploiting genome editing tools such as CRISPR/Cas9 has been proposed to develop host cells that result in a safe, feasible, and robust manufacturing operation with a yield that meets the industrial requirements.

Enhanced osteogenesis on proantocyanidin-loaded date palm endocarp cellulosic matrices: A novel sustainable approach for guided bone regeneration.

Developing inexpensive, biocompatible natural scaffolds that can support the differentiation and proliferation of stem cells has been recently emphasized by the research community to faster obtain the FDA approvals for regenerative medicine. In this regard, plant-derived cellulose materials are a novel class of sustainable scaffolding materials with high potentials for bone tissue engineering (BTE). However, low bioactivity of the plant-derived cellulose scaffolds restricts cell proliferation and cell differentiation. This limitation can be addressed though surface-functionalization of cellulose scaffolds with natural antioxidant polyphenols, e.g., grape seed proanthocyanidin (PCA)-rich extract (GSPE). Despite the various merits of GSPE as a natural antioxidant, its impact on the proliferation and adhesion of osteoblast precursor cells, and on their osteogenic differentiation is an as-yet unknown issue. Here, we investigated the effects of GSPE surface functionalization on the physicochemical properties of decellularized date (Phoenix dactyliferous) fruit inner layer (endocarp) (DE) scaffold. In this regard, various physiochemical characteristics of the DE-GSPE scaffold such as hydrophilicity, surface roughness, mechanical stiffness, porosity, and swelling, and biodegradation behavior were compared with those of the DE scaffold. Additionally, the impact of the GSPE treatment of the DE scaffold on the osteogenic response of human mesenchymal stem cells (hMSCs) was thoroughly studied. For this purpose, cellular activities including cell adhesion, calcium deposition and mineralization, alkaline phosphatase (ALP) activity, and expression levels of bone-related genes were monitored. Taken together, the GSPE treatment enhanced the physicochemical and biological properties of the DE-GSPE scaffold, thereby raising its potentials as a promising candidate for guided bone regeneration.

In vitro modeling of hepatocellular carcinoma niche on decellularized tomato thorny leaves: a novel natural three-dimensional (3D) scaffold for liver cancer therapeutics.

Liver cancer is now one of the main causes leading to death worldwide. To achieve reliable therapeutic effects, it is crucial to develop efficient approaches to test novel anticancer drugs. Considering the significant contribution of tumor microenvironment to cell's response to medications, in vitro 3D bioinspiration of cancer cell niches can be regarded as an advanced strategy to improve the accuracy and reliability of the drug-based treatment. In this regard, decellularized plant tissues can perform as suitable 3D scaffolds for mammalian cell culture to create a near-to-real condition to test drug efficacy. Here, we developed a novel 3D natural scaffold made from decellularized tomato hairy leaves (hereafter called as DTL) to mimic the microenvironment of human hepatocellular carcinoma (HCC) for pharmaceutical purposes. The surface hydrophilicity, mechanical properties, and topography measurement and molecular analyses revealed that the 3D DTL scaffold is an ideal candidate for liver cancer modeling. The cells exhibited a higher growth and proliferation rate within the DTL scaffold, as verified by quantifying the expression of related genes, DAPI staining, and SEM imaging of the cells. Moreover, prilocaine, an anticancer drug, showed a higher effectiveness against the cancer cells cultured on the 3D DTL scaffold, compared to a 2D platform. Taken together, this new cellulosic 3D scaffold can be confidently proposed for chemotherapeutic testing of drugs on hepatocellular carcinoma.

Clinical perspective: Antibody-drug conjugates for the treatment of HER2-positive breast cancer.

Antibody-drug conjugates (ADCs) are a promising class of cancer biopharmaceuticals that exploit the specificity of a monoclonal antibody (mAb) to selectively deliver highly cytotoxic small molecules to targeted cancer cells, leading to an enhanced therapeutic index through increased antitumor activity and decreased off-target toxicity. ADCs hold great promise for the treatment of patients with human epidermal growth factor receptor 2 (HER2)-positive breast cancer after the approval and tremendous success of trastuzumab emtansine and trastuzumab deruxtecan, representing a turning point in both HER2-positive breast cancer treatment and ADC technology. Additionally and importantly, a total of 29 ADC candidates are now being investigated in different stages of clinical development for the treatment of HER2-positive breast cancer. The purpose of this review is to provide an insight into the ADC field in cancer treatment and present a comprehensive overview of ADCs approved or under clinical investigation for the treatment of HER2-positive breast cancer.

Urtica dioica agglutinin (UDA) as a potential candidate for inhibition of SARS-CoV-2 Omicron variants: In silico prediction and experimental validation.

BACKGROUND: The high number of mutations and consequent structure modifications in a Receptor-Binding Domain (RBD) of the spike protein of the Omicron variant of SARS-CoV-2 increased concerns about evading neutralization by antibodies induced by previous infection or vaccination. Thus, developing novel drugs with potent inhibitory activity can be considered an alternative for treating this highly transmissible variant. Considering that Urtica dioica agglutinin (UDA) displays antiviral activity against SARS-CoV-2, the potency of this lectin to inhibit the Receptor Binding Domain of the Omicron variant (RBDOmic) was examined in this study. PURPOSE: This study examines how UDA inhibits the Omicron variant of SARS-CoV-2 by blocking its RBD, using a combination of in silico and experimental methods. METHODS: To investigate the interaction between UDA and RBDOmic, the CLUSPRO 2.0 web server was used to dock the RBDOmic-UDA complex, and molecular dynamics simulations were performed by the Gromacs 2020.2 software to confirm the stability of the selected docked complex. Finally, the binding affinity (ΔG) of the simulation was calculated using MM-PBSA. In addition, ELISA and Western blot tests were used to examine UDA's binding to RBDOmic. RESULTS: Based on the docking results, UDA forms five hydrogen bonds with the RBDOmic active site, which contains mutated residues Tyr501, Arg498, Arg493, and His505. According to MD simulations, the UDA-RBDOmic complex is stable over 100 ns, and its average binding energy during the simulation is -87.201 kJ/mol. Also, the ELISA test showed that UDA significantly binds to RBDOmic, and by increasing the concentration of UDA protein, the attachment to RBDOmic became stronger. In Western blotting, RBDOmic was able to attach to and detect UDA. CONCLUSION: This study indicates that UDA interaction with RBDOmic prevents virus attachment to Angiotensin-converting enzyme 2 (ACE2) and, therefore, its entry into the host cell. Altogether, UDA exhibited a significant suppression effect on the Omicron variant and can be considered a new candidate to improve protection against severe infection of this variant.

Defined Physicochemical Cues Steering Direct Neuronal Reprogramming on Colloidal Self-Assembled Patterns (cSAPs).

Direct neuronal reprogramming of somatic cells into induced neurons (iNs) has been recently established as a promising approach to generating neuron cells. Previous studies have reported that the biophysical cues of the in vitro microenvironment are potent modulators in the cell fate decision; thus, the present study explores the effects of a customized pattern (named colloidal self-assembled patterns, cSAPs) on iN generation from human fibroblasts using small molecules. The result revealed that the cSAP, composed of binary particles in a hexagonal-close-packed (hcp) geometry, is capable of improving neuronal reprogramming efficiency and steering the ratio of the iN subtypes. Cells exhibited distinct cell morphology, upregulated cell adhesion markers (i.e., SDC1 and ITGAV), enriched signaling pathways (i.e., Hippo and Wnt), and chromatin remodeling on the cSAP compared to those on the control substrates. The result also showed that the iN subtype specification on cSAP was surface-dependent; therefore, the defined physicochemical cue from each cSAP is exclusive. Our findings show that direct cell reprogramming can be manipulated through specific biophysical cues on the artificial matrix, which is significant in cell transdifferentiation and lineage conversion.

Rapid, label-free and low-cost diagnostic kit for COVID-19 based on liquid crystals and machine learning.

We report a label-free method for detection of the SARS-CoV-2 virus in nasopharyngeal swab samples without purification steps and multiplication of the target which simplifies and expedites the analysis process. The kit consists of a textile grid on which liquid crystals (LC) are deposited and the grid is placed in a crossed polarized microscopy. The swab samples are subsequently placed on the LCs. In the presence of a particular biomolecule, the direction of LCs changes locally based on the properties of the biomolecule and forms a particular pattern. As the swab samples are not perfectly purified, image processing and machine learning techniques are employed to detect the presence of specific molecules or quantify their concentrations in the medium. The method can differentiate negative and positive COVID-19 samples with an accuracy of 96% and also differentiate COVID-19 from influenza types A and B with an accuracy of 93%. The kit is portable, simple to manufacture, convenient to operate, cost effective, rapid and sensitive. The simplicity of the specimen processing, the speed of image acquisition, and fast diagnostic operations enable the deployment of the proposed technique for performing extensive on-spot screening of COVID-19 in public places.

Polydopamine-Mediated Protein Adsorption Alters the Epigenetic Status and Differentiation of Primary Human Adipose-Derived Stem Cells (hASCs).

Polydopamine (PDA) is a biocompatible cell-adhesive polymer with versatile applications in biomedical devices. Previous studies have shown that PDA coating could improve cell adhesion and differentiation of human mesenchymal stem cells (hMSCs). However, there is still a knowledge gap in the effect of PDA-mediated protein adsorption on the epigenetic status of MSCs. This work used gelatin-coated cell culture surfaces with and without PDA underlayer (Gel and PDA-Gel) to culture and differentiate primary human adipose-derived stem cells (hASCs). The properties of these two substrates were significantly different, which, in combination with a variation in extracellular matrix (ECM) protein bioactivity, regulated cell adhesion and migration. hASCs reduced focal adhesions by downregulating the expression of integrins such as αV, α1, α2, and ß1 on the PDA-Gel compared to the Gel substrate. Interestingly, the ratio of H3K27me3 to H3K27me3+H3K4me3 was decreased, but this only occurred for upregulation of AGG and BMP4 genes during chondrogenic differentiation. This result implies that the PDA-Gel surface positively affects the chondrogenic, but not adipogenic and osteogenic, differentiation. In conclusion, for the first time, this study demonstrates the sequential effects of PDA coating on the biophysical property of adsorbed protein and then focal adhesions and differentiation of hMSCs through epigenetic regulation. This study sheds light on PDA-mediated mechanotransduction.

Urtica dioica Agglutinin: A plant protein candidate for inhibition of SARS-COV-2 receptor-binding domain for control of Covid19 Infection.

Despite using effective drugs and vaccines for Covid 19, due to some limitations of current strategies and the high rate of coronavirus mutation, the development of medicines with effective inhibitory activity against this infection is essential. The SARS-CoV-2 enters the cell by attaching its receptor-binding domain (RBD) of Spike to angiotensin-converting enzyme-2 (ACE2). According to previous studies, the natural peptide Urtica dioica agglutinin (UDA) exhibited an antiviral effect on SARS-CoV, but its mechanism has not precisely been elucidated. Here, we studied the interaction between UDA and RBD of Spike protein of SARS-CoV-2. So, protein-protein docking of RBD-UDA was performed using Cluspro 2.0. To further confirm the stability of the complex, the RBD-UDA docked complex with higher binding affinity was studied using Molecular Dynamic simulation (via Gromacs 2020.2), and MM-PBSA calculated the binding free energy of the system. In addition, ELISA assay was used to examine the binding of UDA with RBD protein. Results were compared to ELISA of RBD-bound samples of convalescent serum IgG (from donors who recovered from Covid 19). Finally, the toxicity of UDA is assessed by using MTT assay. The docking results show UDA binds to the RBD binding site. MD simulation illustrates the UDA-RBD complex is stable during 100 ns of simulation, and the average binding energy was calculated to be -47.505 kJ/mol. ELISA and, MTT results show that UDA binds to RBD like IgG-RBD binding and may be safe in human cells. Data presented here indicate UDA interaction with S-protein inhibits the binding sites of RBD, it can prevent the virus from attaching to ACE2 and entering the host cell.

In silico prediction and in vitro validation of the effect of pH on adhesive behaviour of the fused CsgA-MFP3 protein.

Recombinant production of mussel foot proteins among marine-inspired proteinaceous adhesive materials has been attracted high attention for medical applications, due to their exceptional versatility potential of hierarchically arranged nanostructures. Various biochemical and proteinous factors such as amyloid CsgA curli protein have been used as a synergistic factor to enhance the constancy of obtained bio-adhesion but their mechanistic interactions have not yet been deeply investigated widely in different pH conditions. To this end, the present study has first sought to assess molecular simulation and prediction by using RosettaFold to predict the 3-dimensional structure of the fused CsgA subunit and the MFP3 protein followed by in vitro verification. It was developed an ensemble of quantitative structure-activity relationship models relying on simulations according to the surface area and molecular weight values of the fused proteins in acidic to basic situations using PlayMolecule (protein preparation app for MD simulations) online databases followed by molecular dynamic simulation at different pHs. It was found that acidic conditions positively affect adhesive strength throughout the chimeric structure based on comparative structure-based analyses along with those obtained in prevailing literature. Atomic force microscopy analysis was confirmed obtained in silico data which showed enhanced adhesive properties of fused protein after self-assembly in low pH conditions. In conclusion, the augmented model for reactivity predictions not only unravels the performance and explain ability of the adhesive proteins but in turn paves the way for the decision-making process for chimeric subunits modifications needed for future industrial production.

Crossing Phylums: Butterfly Wing as a Natural Perfusable Three-Dimensional (3D) Bioconstruct for Bone Tissue Engineering.

Despite the advent of promising technologies in tissue engineering, finding a biomimetic 3D bio-construct capable of enhancing cell attachment, maintenance, and function is still a challenge in producing tailorable scaffolds for bone regeneration. Here, osteostimulatory effects of the butterfly wings as a naturally porous and non-toxic chitinous scaffold on mesenchymal stromal cells are assessed. The topographical characterization of the butterfly wings implied their ability to mimic bone tissue microenvironment, whereas their regenerative potential was validated after a 14-day cell culture. In vivo analysis showed that the scaffold induced no major inflammatory response in Wistar rats. Topographical features of the bioconstruct upregulated the osteogenic genes, including COL1A1, ALP, BGLAP, SPP1, SP7, and AML3 in differentiated cells compared to the cells cultured in the culture plate. However, butterfly wings were shown to provide a biomimetic microstructure and proper bone regenerative capacity through a unique combination of various structural and material properties. Therefore, this novel platform can be confidently recommended for bone tissue engineering applications.

Optimization of a PDMS-Based Cell Culture Substrate for High-Density Human-Induced Pluripotent Stem Cell Adhesion and Long-Term Differentiation into Cardiomyocytes under a Xeno-Free Condition.

Despite the numerous advantages of PDMS-based substrates in various biomedical applications, they are limited by their highly hydrophobic surface that does not optimally interact with cells for attachment and growth. Hence, the lack of lengthy and straightforward procedures for high-density cell production on the PDMS-based substrate is one of the significant challenges in cell production in the cell therapy field. In this study, we found that the PDMS substrate coated with a combination of polydopamine (PDA) and laminin-511 E8 fragments (PDA + LME8-coated PDMS) can support human-induced pluripotent stem cell (hiPSC) attachment and growth for the long term and satisfy their demands of differentiation into cardiomyocytes (iCMs). Compared with prior studies, the density of hiPSCs and their adhesion time on the PDMS surface were increased during iCM production. Although the differentiated iCMs beat and produce mechanical forces, which disturb cellular attachments, the iCMs on the PDA + LME8-coated PDMS substrate showed dramatically better attachment than the control condition. Further, the substrate required less manipulation by enabling one-step seeding throughout the process in iCM formation from hiPSCs under animal-free conditions. In light of the results achieved, the PDA + LME8-coated PDMS substrate will be an up-and-coming tool for cardiomyocyte production for cell therapy and tissue engineering, microfluidics, and organ-on-chip platforms.

Recent Advances in Development of Natural Cellulosic Non-Woven Scaffolds for Tissue Engineering.

In recent years, tissue engineering researchers have exploited a variety of biomaterials that can potentially mimic the extracellular matrix (ECM) for tissue regeneration. Natural cellulose, mainly obtained from bacterial (BC) and plant-based (PC) sources, can serve as a high-potential scaffold material for different regenerative purposes. Natural cellulose has drawn the attention of researchers due to its advantages over synthetic cellulose including its availability, cost effectiveness, perfusability, biocompatibility, negligible toxicity, mild immune response, and imitation of native tissues. In this article, we review recent in vivo and in vitro studies which aimed to assess the potential of natural cellulose for the purpose of soft (skin, heart, vein, nerve, etc.) and hard (bone and tooth) tissue engineering. Based on the current research progress report, it is sensible to conclude that this emerging field of study is yet to satisfy the clinical translation criteria, though reaching that level of application does not seem far-fetched.

A novel recombinant chimeric bio-adhesive protein consisting of mussel foot protein 3, 5, gas vesicle protein A, and CsgA curli protein expressed in Pichia pastoris.

Despite various efforts to produce potent recombinant bio-adhesive proteins for medical purposes, efficient production of a safe and feasible bio-glue is not yet a commercial reality due to the weak properties or low expression levels. Here, a feasible expression system has been developed to produce strong recombinant fusion bioinspired protein using mussel foot protein 3 and 5 (Mfps) along with gas vesicle protein A (GvpA) of Anabaena flos-aquae, and a curli protein CsgA from E. coli, expressed under the control of alcohol oxidase (AOX1) promoter for high-level production in yeast P. pastoris using pPICZα vector. Purified chimeric proteins were first evaluated using western blotting, and their remaining dihydroxyphenylalanine (DOPA) was measured in the modified proteins by NBT assay. We further elucidated the mechanistic properties of obtained adhesive protein assembly in various pH levels based on its different subunits using atomic force microscopy (AFM) when adsorbed onto the mica surface. We found that both combinational structural features of subunits and post-translational changes during expression in yeast host have led to potent adherence due to higher DOPA residues specially in acidic condition and tetrad complex which is higher than that of earlier reports in prokaryotic systems. We believe that our obtained chimeric protein resulted from the fusion of GvpA and CsgA proteins with DOPA-containing Mfp proteins, expressed in the methylotrophic yeast, P. pastoris, not only presents a candidate for future biomedical applications but also provides novel biological clues used for high-performance bioinspired biomaterial designation.

Targeting Caspase-3 Gene in rCHO Cell Line by CRISPR/Cas9 Editing Tool and Its Effect on Protein Production in Manipulated Cell Line.

Background: Chinese hamster ovary (CHO) cells are the widely used mammalian cell host for biopharmaceutical manufacturing. During cell cultures, CHO cells lose viability mainly from apoptosis. Inhibiting cell death is useful because prolonging cell lifespans can direct to more productive cell culture systems for biotechnology requests. Objectives: This study exploited a CRISPR/Cas9 technology to generate site-specific gene disruptions in the caspase-3 gene in the apoptosis pathway, which acts as an apoptotic regulator to extend cell viability in the CHO cell line. Methods: The STRING database was used to identify the key pro-apoptotic genes to be modified by CRISPR/Cas9 system. The guide RNAs targeting the caspase-3 gene were designed, and vectors containing sgRNA and Cas9 were transfected into CHO cells that expressed erythropoietin as a heterologous protein. Indel formation was investigated by DNA sequencing. Caspase-3 expression was quantified by real-time PCR and western blot. The effect of editing the caspase-3 gene on the inhibition of apoptosis was also investigated by induction of apoptosis in manipulated cell lines by oleuropein. Finally, the erythropoietin production in the edited cells was compared to the control cells. Results: The caspase-3 manipulation significantly prolongation of the cell viability and decreased the caspase-3 expression level of protein in manipulated CHO cells (more than 6-fold, P-value < 0.0001). Manipulated cells displayed higher threshold tolerance to apoptosis compared to the control cells when they were induced by oleuropein. They show a higher IC50 than the control ones (7271 µM/mL Vs. 5741 µM/mL). They also show a higher proliferation rate than the control cells in the presence of an apoptosis inducer (P-value < 0.0001). Furthermore, manipulated cell lines significantly produce more recombinant protein in the presence of 2,000 µM oleuropein compared to the control ones (P-value = 0.0021). Conclusions: We understood that CRISPR/Cas9 could be effectively applied to suppress the expression of the caspase-3 gene and rescue CHO cells from apoptosis induced by cell stress and metabolites. The CRISPR/Cas9 system-assisted caspase-3 gene ablation can potentially increase erythropoietin yield in CHO cells.