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.

TLR3 stimulation improves the migratory potency of adipose-derived mesenchymal stem cells through the stress response pathway in the melanoma mouse model.

BACKGROUND: Mesenchymal stem cells (MSCs) are utilized as a carrier of anti-tumor agents in targeted anti-cancer therapy. Despite the improvements in this area, there are still some unsolved issues in determining the appropriate dose, method of administration and biodistribution of MSCs. The current study aimed to determine the influence of toll-like receptor 3 (TLR3) stimulation on the potential of MSCs migration to the neoplasm environment in the mouse melanoma model. METHODS AND RESULTS: Adipose-derived MSCs (ADMSCs) were isolated from the GFP+ transgenic C57BL/6 mouse and treated with different doses (1 µg/ml and 10 µg/ml) of polyinosinic-polycytidylic acid, the related TLR3 agonist, at various time points (1 and 4 h). Following the treatment, the expression of targeted genes such as α4, α5, and ß1 integrins and TGF-ß and IL-10 anti-inflammatory cytokines was determined using real-time PCR. In vivo live imaging evaluated the migration index of the intraperitoneally (IP) injected treated ADMSCs in a lung tumor-bearing mouse (C57BL/6) melanoma model (n = 5). The presented findings demonstrated that TLR3 stimulation enhanced both migration of ADMSCs to the tumor area compared with control group (n = 5) and expression of α4, α5, and ß1 integrins. It was also detected that the engagement of TLR3 resulted in the anti-inflammatory behavior of the cells, which might influence the directed movement of ADMSCs. CONCLUSION: This research identified that TLR3 activation might improve the migration via the stimulation of stress response in the cells and depending on the agonist concentration and time exposure, this activated pathway drives the migratory behavior of MSCs.

Quantitative measurement of CA 15-3 cancer biomarker using an electrochemical aptasensor based on the electrodeposition of Au thin film on cauliflower-like rGO-MoS2 nanocomposite.

The present research was conducted to design and construct an electrochemical aptasensor for evaluating carbohydrate antigen 15-3 (CA15-3) as a biomarker for breast cancer. The aptasensor has been fabricated by a gold thin film (AuTF) electrodeposited on a cauliflower-like reduced graphene oxide-molybdenum sulfide nanocomposite (rGO-MoS2). The modified electrode's surface was used to immobilize the thiolated aptamer, which was subsequently treated with CA 15-3 antigen. The aptasensor fabrication process was assessed using electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). This research also applied EIS to the quantitative measurement of CA 15-3 antigen by the proposed aptasensor. The interfacial charge transfer resistance (Rct) alteration before and after incubation of CA 15-3 by the immobilized aptamer was considered a signal for the quantitative measurement of CA 15-3. A linear concentration ranging from 5.0 to 200.0 U mL-1 with a detection limit of 3.0 × 10-1 U mL-1 was obtained for CA 15-3 using the EIS method. This designed aptasensor indicates satisfactory repeatability and stability, good selectivity, and high sensitivity. Moreover, clinical samples were assayed by the prepared aptasensor and compared with the ELISA method, yielding acceptable results. The recovery and relative standard deviation (RSD) of CA 15-3 in human serum samples were in the range 95.0 to 107.0% and 3.5 to 7.5%, respectively.

Poly-L-lysine/hyaluronan nanocarriers as a novel nanosystem for gene delivery.

The present research comes up with a novel DNA-loaded poly-L-lysine (PLL)/hyaluronan (HA) nanocarrier (DNA-loaded PLL/HA NCs) for gene delivery applications, as a promising candidate for gene delivery into diverse cells. A straightforward approach was employed to prepare such a nanosystem through masking DNA-loaded PLL molecules by HA. Fourier-transform infrared (FTIR) spectroscopy, dynamic light scattering (DLS), field emission-scanning electron microscopy (FE-SEM) and transmission electron microscopy (TEM) were used to analyse the interaction of the molecules as well as the physicochemical properties of the NCs. The NCs showed a negative charge of -24 ± 3 mV, with an average size of 138 ± 6 nm, in an ellipsoid-shape with smooth surfaces. The DNA loading efficiency (LE) measured by DNA absorbance was around 95 %. The MTT assay showed that the developed NCs are non-toxic to the cells. Furthermore, the uptake of the DNA-loaded PLL/HA NCs by the human embryonic kidney (HEK)-293T cells was evaluated by a flow cytometry method, and demonstrated high potential cellular uptake over 90% for transferring the gene to HEK-293T cells at the optimised conditions. Therefore, the DNA-loaded PLL/HA NCs are the potent strategy for developing nanosystems for gene delivery applications.

Saponin and fluorine-modified polycation as a versatile gene delivery system.

Despite the development of many novel carriers for the delivery of various types of genetic material, the lack of a delivery system with high efficiency and low cytotoxicity is a major bottleneck. Herein, low molecular weight polyethylenimine (PEI1.8k) was functionalized with saponin residues using phenylboronic acid (PBA) as an ATP-responsive cross-linker, and a fluorinated side chain to construct PEI-PBA-SAP-F polycation as a highly efficient delivery vector. This vehicle could transfect small plasmid DNA (∼3 kb) with outstanding efficiency into various cells, including HEK 293T, NIH3T3, A549, PC12, MCF7 and HT-29, as well as robust transfection of a large plasmid (∼9 kb) into HEK 293T cells. The carrier indicated good transfection efficacy even at high concentration of serum and low doses of plasmid. The use of green fluorescent protein (GFP) knock-out analysis demonstrated transfection of different types of CRISPR/Cas9 complexes (Cas9/sgRNA ribonucleoproteins RNP, plasmid encoding Cas9 plus sgRNA targeting GFP, Cas9 expression plasmid plusin vitro-prepared sgRNA). In summary, we report an effective PEI-PBA-SAP-F gene carrier with the appropriate lipophilic/cationic balance for biomedical applications.

Bioengineering of fibroblast-conditioned polycaprolactone/gelatin electrospun scaffold for skin tissue engineering.

BACKGROUND: Synthetic tissue engineering scaffolds has poor biocompatiblity with very low angiogenic properties. Conditioning the scaffolds with functional groups, coating with biological components, especially extracellular matrix (ECM), is an excellent strategy for improving their biomechanical and biological properties. METHODS: In the current study, a composite of polycaprolactone and gelatin (PCL/Gel) was electrospun in the ratio of 70/30 and surface modified with 1% gelatin-coating (G-PCL/Gel) or plasma treatment (P-PCL/Gel). The surface modification was determined by SEM and ATR-FTIR spectroscopy, respectively. The scaffolds were cultured with fibroblast 3T3, then decellularized during freeze-thawing process to fabricate a fibroblast ECM-conditioned PCL/Gel scaffold (FC-PCL/Gel). The swelling and degaradtion as well as in vitro and in vivo biocompatibility and angiogenic properties of the scaffolds were evaluated. RESULTS: The structure of the surface-modified G-PCL/Gel and P-PCL/Gel were unique and not changed compared with the PCL/Gel scaffolds. ATR-FTIR analysis admitted the formation of oxygen-containing groups, hydroxyl and carboxyl, on the surface of the P-PCL/Gel scaffold. The SEM micrographs and DAPI staining confirmed the cell attachment and the ECM deposition on the platform and successful removal of the cells after decellularization. P-PCL/Gel showed better cell attachment, ECM secretion and deposition after decellularization compared with G-PCL/Gel. The FC-PCL/Gel was considered as an optimized scaffold for further assays in this study. The FC-PCL/Gel showed increased hydrophilic behavior and cytobiocompatibility compared with P-PCL/Gel. The ECM on the FC-PCL/Gel scaffold showed a gradual degradation during 30 days of degradation time, as a small amount of ECM remained over the FC-PCL/Gel scaffold at day 30. The FC-PCL/Gel showed significant biocompatibility and improved angiogenic property compared with P-PCL/Gel when subcutaneously implanted in a mouse animal model for 7 and 28 days. CONCLUSIONS: Our findings suggest FC-PCL/Gel as an excellent biomimetic construct with high angiogenic properties. This bioengineered construct can serve as a possible application in our future pre-clinical and clinical studies for skin regeneration.

Production and characterization of a new specific monoclonal antibody against A-isoform of SALL4: A novel emerging testicular cancer marker.

SALL4 transcription factor plays an important role to maintain the pluripotent and self-renewal of embryonic stem cells. It contributes to the growth of many cancers and embryonic development. With the exception of spermatogonia, SALL4 expression is silenced in most adult tissues after birth; nevertheless, it is re-expressed in a subset of different solid malignancies. SALL4 is a new, precise biomarker for testicular germ cell cancers that was just introduced. The whole isoform of SALL4 is called SALL4-A. Regarding the lack of antibody against human SALL4 isoforms, the pattern of expression, the role of each isoform remain unknown. Furthermore, in isoform specific evaluations, we aimed, for the first time, to produce and characterize mAb against human SALL4-A. Immunization of mice were performed with a selected 33-mer synthetic peptide of SALL4-A conjugated with KLH. Hybridoma cells were screened by ELISA for positive reactivity with SALL4-A peptide. From the ascites fluid of mice that had been injected with hybridoma cells, anti-SALL4-A mAbs were isolated using a protein G column. Reactivity of the mAbs was evaluated using the peptide and SALL4-A recombinant protein by ELISA and IHC on testicular cancer tissue as positive control, and normal kidney, stomach and prostate tissues as negative control. The produced mAb could well detect SALL4-A in testicular cancer tissues using IHC, while the reactivity was negative in normal kidney, stomach and prostate tissues. Using ELISA, the mAb affinity for the peptide and SALL4-A recombinant protein was assessed, and it was shown to be reasonably high. The mAb detected SALL4-A in nucleus and cytoplasm of several cancer cells and spermatogonia in testicular cancer tissue. In addition, it could recognize SALL4-A recombinant protein. Our produced monoclonal antibody against isoform-A of human SALL4 can specifically recognize SALL4-A using either IHC or ELISA. We hope that this mAb could help researchers in isoform-specific study of human SALL4.

Dendritic cells loaded with exosomes derived from cancer stem cell-enriched spheroids as a potential immunotherapeutic option.

Cancer stem cells (CSCs) are responsible for therapeutic resistance and recurrence in colorectal cancer. Despite advances in immunotherapy, the inability to specifically eradicate CSCs has led to treatment failure. Hence, identification of appropriate antigen sources is a major challenge in designing dendritic cell (DC)-based therapeutic strategies against CSCs. Here, in an in vitro model using the HT-29 colon cancer cell line, we explored the efficacy of DCs loaded with exosomes derived from CSC-enriched colonospheres (CSCenr -EXOs) as an antigen source in activating CSC-specific T-cell responses. HT-29 lysate, HT-29-EXOs and CSCenr lysate were independently assessed as separate antigen sources. Having confirmed CSCs enrichment in spheroids, CSCenr -EXOs were purified and characterized, and their impact on DC maturation was investigated. Finally, the impact of the antigen-pulsed DCs on the proliferation rate and also spheroid destructive capacity of autologous T cells was assessed. CSCenr -EXOs similar to other antigen groups had no suppressive/negative impacts on phenotypic maturation of DCs as judged by the expression level of costimulatory molecules. Notably, similar to CSCenr lysate, CSCenr -EXOs significantly increased the IL-12/IL-10 ratio in supernatants of mature DCs. CSCenr -EXO-loaded DCs effectively promoted T-cell proliferation. Importantly, T cells stimulated with CSCenr -EXOs disrupted spheroids' structure. Thus, CSCenr -EXOs present a novel and promising antigen source that in combination with conventional tumour bulk-derived antigens should be further explored in pre-clinical immunotherapeutic settings for the efficacy in hampering recurrence and metastatic spread.

MSC-derived exosomes carrying a cocktail of exogenous interfering RNAs an unprecedented therapy in era of COVID-19 outbreak.

BACKGROUND: The onset of the SARS-CoV-2 pandemic has resulted in ever-increasing casualties worldwide, and after 15 months, standard therapeutic regimens are yet to be discovered. MAIN BODY: Due to the regenerative and immunomodulatory function of MSCs, they can serve as a suitable therapeutic option in alleviating major COVID-19 complications like acute respiratory distress syndrome. However, the superior properties of their cognate exosomes as a cell-free product make them preferable in the clinic. Herein, we discuss the current clinical status of these novel therapeutic strategies in COVID-19 treatment. We then delve into the potential of interfering RNAs incorporation as COVID-19 gene therapy and introduce targets involved in SARS-CoV-2 pathogenesis. Further, we present miRNAs and siRNAs candidates with promising results in targeting the mentioned targets. CONCLUSION: Finally, we present a therapeutic platform of mesenchymal stem cell-derived exosomes equipped with exogenous iRNAs, that can be employed as a novel therapeutic modality in COVID-19 management aiming to prevent further viral spread within the lung, hinder the virus life cycle and pathogenesis such as immune suppression, and ultimately, enhance the antiviral immune response.

Highly Photoluminescent Nitrogen- and Zinc-Doped Carbon Dots for Efficient Delivery of CRISPR/Cas9 and mRNA.

Safe and efficient delivery of CRISPR/Cas9 systems is still a challenge. Here we report the development of fluorescent nitrogen- and zinc-doped carbon dots (N-Zn-doped CDs) using one-step microwave-aided pyrolysis based on citric acid, branched PEI25k, and different zinc salts. These versatile nanovectors with a quantum yield of around 60% could not only transfect large CRISPR plasmids (∼9 kb) with higher efficiency (80%) compared to PEI25k and lipofectamine 2000 (Lipo 2K), but they also delivered mRNA into HEK 293T cells with the efficiency 20 times greater than and equal to that of PEI25k and Lipo 2K, respectively. Unlike PEI25k, N-Zn-doped CDs exhibited good transfection efficiency even at low plasmid doses and in the presence of 10% fetal bovine serum (FBS). Moreover, these nanovectors demonstrated excellent efficiency in GFP gene disruption by transferring plasmid encoding Cas9 and sgRNA targeting GFP as well as Cas9/sgRNA ribonucleoproteins into HEK 293T-GFP cells. Hence, N-Zn-doped CDs with remarkable photoluminescence properties and high transfection efficiency in the delivery of both CRISPR complexes and mRNA provide a promising platform for developing safe, efficient, and traceable delivery systems for biological research.

Overexpression of DDIT4 and TPTEP1 are associated with metastasis and advanced stages in colorectal cancer patients: a study utilizing bioinformatics prediction and experimental validation.

BACKGROUND: Various diagnostic and prognostic tools exist in colorectal cancer (CRC) due to multiple genetic and epigenetic alterations causing the disease. Today, the expression of RNAs is being used as prognostic markers for cancer. METHODS: In the current study, various dysregulated RNAs in CRC were identified via bioinformatics prediction. Expression of several of these RNAs were measured by RT-qPCR in 48 tissues from CRC patients as well as in colorectal cancer stem cell-enriched spheroids derived from the HT-29 cell line. The relationships between the expression levels of these RNAs and clinicopathological features were analyzed. RESULTS: Our bioinformatics analysis determined 11 key mRNAs, 9 hub miRNAs, and 18 lncRNAs which among them 2 coding RNA genes including DDIT4 and SULF1 as well as 3 non-coding RNA genes including TPTEP1, miR-181d-5p, and miR-148b-3p were selected for the further investigations. Expression of DDIT4, TPTEP1, and miR-181d-5p showed significantly increased levels while SULF1 and miR-148b-3p showed decreased levels in CRC tissues compared to the adjacent normal tissues. Positive relationships between DDIT4, SULF1, and TPTEP1 expression and metastasis and advanced stages of CRC were observed. Additionally, our results showed significant correlations between expression of TPTEP1 with DDIT4 and SULF1. CONCLUSIONS: Our findings demonstrated increased expression levels of DDIT4 and TPTEP1 in CRC were associated with more aggressive tumor behavior and more advanced stages of the disease. The positive correlations between TPTEP1 as non-coding RNA and both DDIT4 and SULF1 suggest a regulatory effect of TPTEP1 on these genes.

Smart arginine-equipped polycationic nanoparticles for p/CRISPR delivery into cells.

An efficient and safe delivery system for the transfection of CRISPR plasmid (p/CRISPR) into target cells can open new avenues for the treatment of various diseases. Herein, we design a novel nonvehicle by integrating an arginine-disulfide linker with low-molecular-weight PEI (PEI1.8k) for the delivery of p/CRISPR. These PEI1.8k-Arg nanoparticles facilitate the plasmid release and improve both membrane permeability and nuclear localization, thereby exhibiting higher transfection efficiency compared to native PEI1.8kin the delivery of nanocomplexes composed of PEI1.8k-Arg and p/CRISPR into conventional cells (HEK 293T). This nanovehicle is also able to transfect p/CRISPR in a wide variety of cells, including hard-to-transfect primary cells (HUVECs), cancer cells (HeLa), and neuronal cells (PC-12) with nearly 5-10 times higher efficiency compared to the polymeric gold standard transfection agent. Furthermore, the PEI1.8k-Arg nanoparticles can edit the GFP gene in the HEK 293T-GFP reporter cell line by delivering all possible forms of CRISPR/Cas9 system (e.g. plasmid encoding Cas9 and sgRNA targeting GFP, and Cas9/sgRNA ribonucleoproteins (RNPs) as well as Cas9 expression plasmid andin vitro-prepared sgRNA) into HEK 293T-GFP cells. The successful delivery of p/CRISPR into local brain tissue is also another remarkable capability of these nanoparticles. In view of all the exceptional benefits of this safe nanocarrier, it is expected to break new ground in the field of gene editing, particularly for therapeutic purposes.

Generation of an in vitro model of ß-thalassemia using the CRISPR/Cas9 genome editing system.

ß-Thalassemia is a common monogenic disease characterized by defective ß-globin chains synthesis. In vitro ß-thalassemia-related research on increasing ß-like globin genes or identification of factors reducing the severity of the disease, has been performed on mouse erythroleukaemia or K562 cell lines. The aim of this study was the production of an in vitro model of ß-thalassemia using the highly efficient CRISPR-Cas9 system. Embryonic stem (ES) cells were nucleofected with guide RNA (gRNA)-Cas9 expression vectors. Molecular testing was done on extracted DNA to assess Hbb-b1 mutation. Analysis of transcription factors and hemoglobin genes were evaluated using quantitative reverse transcription-polymerase chain reaction following erythroid differentiation of ES cells. Sequencing data confirmed Hbb-b1 knockout alleles. Significant expression of erythroid transcription factors was observed in wild-type, Hbb-b1+/- and Hbb-b1-/- groups (P < .001). Compared with the wild-type group, the absolute number of Hbb-b1 mRNA in Hbb-b1+/- group significantly decreased from 6.44 × 106 to 3.23 × 106 copy number (P < .01), whereas in Hbb-b1-/- group had zero expression. The CRISPR/Cas9-mediated Hbb-b1 knockout in ES cells provides accessibility to an in vitro thalassemia model following erythroid differentiation. Considering the need for in vitro and mouse models to investigate the molecular basis of ß-thalassemia which also enables testing of therapeutic approaches, this method can be utilized to produce a mouse model of ß-thalassemia intermedia (Hbbth1/th1).

Effects of human placenta-derived mesenchymal stem cells with NK4 gene expression on glioblastoma multiforme cell lines.

Poor prognosis and low survival are commonly seen in patients with glioblastoma multiforme (GBM). Due to the specific nature of solid tumors such as GBM, delivery of therapeutic agents to the tumor sites is difficult. So, one of the major challenges in the treatment of these tumors is a selection of appropriate method for drug delivery. Mesenchymal stem cells (MSCs) have a unique characteristic in migration toward the tumor tissue. In this regard, the present study examined the antitumor effects of manipulating human placenta-derived mesenchymal stem cells (PDMSCs) with NK4 expression (PDMSC-NK4) on GBM cells. After separation and characterization of PDMSCs, these cells were transduced with NK4 which was known as the antagonist of hepatocyte growth factor (HGF). The results indicated that engineered PDMSCs preferably migrate into GBM cells by transwell coculture system. In addition, the proliferation of the GBM cells significantly reduced after coculture with these cells. In fact, manipulated PDMSCs inhibited growth of tumor cells by induction of apoptosis. Our findings suggested that besides having antitumor effects, PDMSCs can also be applied as an ideal cellular vehicle to target the glioblastoma multiforme.

Decellularized amniotic membrane Scaffolds improve differentiation of iPSCs to functional hepatocyte-like cells.

Human-induced pluripotent stem cells-derived hepatocyte-like cells (hiPSCs-HLCs) holds considerable promise for future clinical personalized therapy of liver disease. However, the low engraftment of these cells in the damaged liver microenvironment is still an obstacle for potential application. In this study, we explored the effectiveness of decellularized amniotic membrane (dAM) matrices for culturing of iPSCs and promoting their differentiation into HLCs. The DNA content assay and histological evaluation indicated that cellular and nuclear residues were efficiently eliminated and the AM extracellular matrix component was maintained during decelluarization. DAM matrices were developed as three-dimensional scaffolds and hiPSCs were seeded into these scaffolds in defined induction media. In dAM scaffolds, hiPSCs-HLCs gradually took a typical shape of hepatocytes (polygonal morphology). HiPSCs-HLCs that were cultured into dAM scaffolds showed a higher level of hepatic markers than those cultured in tissue culture plates (TCPs). Moreover, functional activities in term of albumin and urea synthesis and CYP3A activity were significantly higher in dAM scaffolds than TCPs over the same differentiation period. Thus, based on our results, dAM scaffold might have a considerable potential in liver tissue engineering, because it can improve hepatic differentiation of hiPSCs which exhibited higher level of the hepatic marker and more stable metabolic functions.

Dynamic Signature of tRNA-Derived Small RNAs in Cancer Pathogenesis as a Promising Valuable Approach.

AIM: Noncoding RNAs are a cluster of RNAs that do not encode functional proteins. Instead, they are incorporated into DNA structure and regulate gene expression. Of these two classes, transfer RNAs (tRNAs) belong to the former, and small RNAs (sRNAs) belong to the latter. Recently, tRNA-derived small RNAs (tsRNAs/tDRs) were discovered among small noncoding RNAs, as the newly discovered regulatory small RNA. They play a role in pathological and physiological processes, in which gene expression is frequently dysregulated. TsRNAs can be bound to Argonaute proteins and Piwi proteins, such as miRNAs and piRNAs sequentially. METHODS: In initial searches, 2,744 articles were identified with the following literature databases, up to February 25, 2020: PubMed, Embase, Web of Science, Scopus, and Google Scholar. Finally, after full-text assessment, 48 articles were identified that are related to gene expression profiling of tsRNA in cancer. RESULTS: The development of cancer biomarkers based on noncoding RNAs is a thriving area of biomedical research that has expanded significantly. Currently, several groups of tsRNA/tDR biomarkers should be considered in updating the latest findings. CONCLUSION: In this systematic review, we summarized the most recent findings related to the expression of tsRNAs in 17 cancer types. We suggested that use of tsRNAs in the cancer field attracted researcher focus and facilitated diagnostic and therapeutic approaches.

Pipeline for the generation of gene knockout mice using dual sgRNA CRISPR/Cas9-mediated gene editing.

Animal models possess undeniable utility for progress on biomedical research projects and developmental and disease studies. Transgenic mouse models recreating specific disease phenotypes associated with ß-hemoglobinopathies have been developed previously. However, traditional methods for gene targeting in mouse using embryonic stem cells (ESCs) are laborious and time consuming. Recently, CRISPR has been developed to facilitate and improve genomic modifications in mouse or isogenic cell lines. Applying CRISPR to gene modification eliminates the time consuming steps of traditional approach including selection of targeted ESC clones and production of chimeric mouse. This study shows that microinjection of a plasmid DNA encoding Cas9 protein along with dual sgRNAs specific to Hbb-bs gene (hemoglobin, beta adult s chain) enables breaking target sequences at exons 2 and 3 positions. The injections led to a knockout allele with efficiency around 10% for deletion of exons 2 and 3 and 20% for indel mutation.

Photoluminescent functionalized carbon dots for CRISPR delivery: synthesis, optimization and cellular investigation.

Gene therapy using clustered regularly interspaced short palindromic repeat plasmids (pCRISPR) reduces mistakes in gene editing and prevents engendering integrational mutagenesis that has been seen in available genome engineering technologies. Developing an ideal and traceable nanocarrier, which can accurately and efficiently transfer this complex into the cytosol and which facilitates the journey towards the nucleus, is a fascinating area of research. Polyethylenimine (PEI) functionalized carbon dots (CD-PEI) were fabricated by one-step microwave assisted pyrolysis with an average size around 3 nm. This CD-PEI showed good potential for intracellular delivery of genetic materials (∼70%). Also, this CD-PEI with passive surface modification with low molecular PEI (2 kDa) has a very high quantum yield, as high as 40% with low cytotoxicity. The expression rate of the pCRISPR was around 15% in the HEK-293 cell which is comparable with the pristine PEI. Furthermore, the CD-PEI demonstrated good properties, such as high quantum yield, biocompatibility and tunable emission wavelengths, suggesting the potential application of photoluminescent functionalized CDs as a suitable, traceable nanocarrier for CRISPR delivery.

Addressing cancer immunotherapy research in Iran: adoptive cell therapy on the horizon.

Recent advances in immunotherapeutic modalities have profoundly changed the prospect of cancer treatment. These modalities mainly focus on modulating the immune response toward tumor cells by using monoclonal antibodies, cancer vaccines, adoptive cell transfer or combination of these methods. In the last few years, Iranian scientists have conducted several projects in these arenas. Here, we provide an overview of these studies and analyze the quality and trend of publications in each sub-specialty of the field. In addition, the contribution of different universities and scientific institutes is assessed. This study may benefit scientific community and policymakers to plan future cancer immunotherapies in Iran and other countries.