The effect of bovine milk lactoferrin-loaded exosomes (exoLF) on human MDA-MB-231 breast cancer cell line.

BACKGROUND: Cancer is still the most challenging disease and is responsible for many deaths worldwide. Considerable research now focuses on targeted therapy in cancer using natural components to improve anti-tumor efficacy and reduce unfavorable effects. Lactoferrin is an iron-binding glycoprotein found in body fluids. Increasing evidence suggests that lactoferrin is a safe agent capable of inducing anti-cancer effects. Therefore, we conducted a study to evaluate the effects of the exosomal form of bovine milk lactoferrin on a human MDA-MB-231 breast cancer cell line. METHODS: The exosomes were isolated from cancer cells by ultracentrifugation and incorporated with bovine milk lactoferrin through the incubation method. The average size of the purified exosome was determined using SEM imaging and DLS analysis. The maximum percentage of lactoferrin-loaded exosomes (exoLF) was achieved by incubating 1 mg/ml of lactoferrin with 30 µg/ml of MDA-MB-231 cells-derived exosomes. Following treatment of MDA-MB-231 cancer cells and normal cells with 1 mg/ml exoLF MTT assay applied to evaluate the cytotoxicity, PI/ annexin V analysis was carried out to illustrate the apoptotic phenotype, and the real-time PCR was performed to assess the pro-apoptotic protein, Bid, and anti-apoptotic protein, Bcl-2. RESULTS: The average size of the purified exosome was about 100 nm. The maximum lactoferrin loading efficiency of exoLF was 29.72%. MTT assay showed that although the 1 mg/ml exoLF treatment of MDA-MB-231 cancer cells induced 50% cell growth inhibition, normal mesenchymal stem cells remained viable. PI/ annexin V analysis revealed that 34% of cancer cells had late apoptotic phenotype after treatment. The real-time PCR showed an elevated expression of pro-apoptotic protein Bid and diminished anti-apoptotic protein Bcl-2 following exoLF treatment. CONCLUSION: These results suggested that exoLF could induce selective cytotoxicity against cancer cells compared to normal cells. Incorporating lactoferrin into the exosome seems an effective agent for cancer therapy. However, further studies are required to evaluate anti-tumor efficacy and the underlying mechanism of exoLF in various cancer cell lines and animal models.

Butyrophilins: Dynamic Regulators of Protective T Cell Immunity in Cancer.

The efficacy of current immunotherapies remains limited in many solid epithelial malignancies. Recent investigations into the biology of butyrophilin (BTN) and butyrophilin-like (BTNL) molecules, however, suggest these molecules are potent immunosuppressors of antigen-specific protective T cell activity in tumor beds. BTN and BTNL molecules also associate with each other dynamically on cellular surfaces in specific contexts, which modulates their biology. At least in the case of BTN3A1, this dynamism drives the immunosuppression of αß T cells or the activation of Vγ9Vδ2 T cells. Clearly, there is much to learn regarding the biology of BTN and BTNL molecules in the context of cancer, where they may represent intriguing immunotherapeutic targets that could potentially synergize with the current class of immune modulators in cancer. Here, we discuss our current understanding of BTN and BTNL biology, with a particular focus on BTN3A1, and potential therapeutic implications for cancer.

Melatonin protects against visible light-induced oxidative stress and promotes the implantation potential of mouse blastocyst in vitro.

Improvement of embryo culture media using antioxidant agents could help to improve embryo quality against environmental factors such as visible light and could overcome implantation failures. The usefulness of the melatonin against the effect of light on the expression of the primary implantation receptors, ErbB1 and ErbB4 on pre-implantation mouse embryo was investigated. Two-cell mouse embryos were exposed to the 1600 LUX light for 30 min then randomly divided into 3 groups including: Melatonin-Treated; Luzindole Treated and Simple media as a Control group. After 72-96  The expanded blastocysts were examined for morphological quality of the embryos by Hoechst and propidium iodide staining and for the expression of ErbB1 and ErbB4 by Real-time PCR and immunocytochemistry. The expression of the Sirt3 gene was also assayed. Furthermore, intracellular reactive oxygen species (ROS) levels and the total antioxidant capacity (TAC) were examined by DCFH-DA fluorescence intensity and radical cation respectively. The number of cells in the inner cell mass (ICM) and outer cell mass (OCM) were elevated significantly in the Melatonin-treated group suggesting increased viability and proliferation. Furthermore, we found that melatonin significantly increased the expression levels of ErbB1, ErbB4, and Sirt3 genes, and the protein expression of ErbB1, ErbB4 correlated with intracellular ROS levels and TAC significantly increased after melatonin treatment. Together, these results demonstrate that melatonin could be helpful to improve preimplantation embryos through its effects in decreasing ROS levels and increasing expression of implantation-related genes.

Characterization of fennel germplasm for physiological persistence and drought recovery: Association with biochemical properties.

In the perennial medicinal plant fennel (Foeniculum vulgare), persistence over years, production stability, and successful post-drought recovery are as important as plant productivity. Characterization of productivity, post-drought recovery, persistency and their association with phytochemical properties has not yet been performed in fennel. In this study, 64 fennel genotypes (from 23 different countries) from four subspecies/varieties including F. vulgare ssp. piperitum, F. vulgare var. vulgare and F. vulgare var. dulce and F. vulgare var. azoricum were evaluated in the field over four years (2015-2018), then was assessed for post-drought recovery over the next two years (2019-2020). High genotypic variation was observed among genotypes and subspecies. Based on the GC-MS analysis, trans-anethole (22.4-90.6%), estragole (2.1-25.8%), fenchone (4.9-19.8%), and limonene (0.5-11.9%) are major components in the essential oils of the studied germplasm. The highest persistence and the lowest average post-drought recovery belonged to ssp. piperitum. This subspecies also had the highest amount of limonene, fenchone, and estragole and the lowest amount of trans-anethole compared to other subspecies. The highest essential oil content and stability was observed in var. dulce. Seed yield and persistence were found to be negatively associated, suggesting that selection for more productivity may indirectly promote less persistent genotypes. Post-drought recovery was negatively associated with two main phytochemical compounds of essential oil, fenchone and estragole, but positively associated with trans-anethole. Persistence had negative correlation with estragole content. Results indicated that indirect selection for post-drought recovery and persistence may be possible through selection for phytochemical properties. The negative correlation between some essential oil components may indicate that some are isomers, which can limit the ability to select for certain combinations. Superior genotypes identified in this study can be used to construct populations for future studies and as parents of crosses to develop new varieties.

Genetic analysis and physiological relationships of drought response in fennel: Interaction with mating system.

The consequences of water deficit and its interaction with pollination system (deliberate selfing compared with open-pollination) on physiological, agronomic and phytochemical traits are not understood in fennel (Foeniculum vulgare Mill.). A research was started by creating selfed (S1) and half-sib (HS) families on a fennel germplasm in 2018. Populations were studied in the field, applying a normal and a water deficit condition during two years (2019-2020). Considerable genotypic variation was observed within S1 and HS families for all of the evaluated traits, demonstrating that selection for these traits would be successful. Consequences of water deficit were manifested as declined most of the traits; and significantly increased essential oil content, harvest index, and proline content, in both populations. Mandatory selfing reduced the performance of genotypes for most of the traits confirming the existence of inbreeding depression (ID) with higher values for plant dry weight, seed yield, essential oil content, and number of umbelets per umbel. In S1 population, some of the studied traits had higher heritability estimates under normal condition and some of them showed higher heritability under water deficit. Positive relationship between GCA and STI in OP population indicated that it is possible to identify genotypes having high values of combining ability and drought tolerance. Results of the present study suggest that physiological traits cannot be used as an indicator to distinguish drought-tolerant genotypes in S1 progenies, whereas in OP progenies Chl a, Chl b, TChl, CAR, PRO, and RWC, which had significant correlations with drought tolerance, may be used for this purpose. Based on the results contrasting genotypes were identified, which can be used to develop mapping populations for genome studies of drought tolerance and physiological traits of this species in future studies.

Explore pre-hospital emergency challenges in the face of the COVID-19 pandemic: A quality content analysis in the Iranian context.

Background: pre-hospital emergency is a community-oriented system that responds to the medical needs of the injured or patients with acute and emergency illnesses outside of health care facilities until they are transferred to a medical center. This study aimed to explore pre-hospital emergency challenges in the face of the COVID-19 pandemic. Material and methods: This study was conducted as a qualitative content analysis in Iran. Using the purposive sampling method, data were collected through in-depth individual interviews with 28 prehospital paramedic personnel from November 2020 to November 2021. Graneheim and Lundman's conventional content analysis methods were used to analyze the data and for the trustworthiness of the data, this study used Lincoln and Guba's recommendations. Results: After multiple rounds of analyzing and summarizing the data and taking into consideration similarities and differences, four main categories and 10 subcategories were created based on the results of the data analysis and including (1) Culture and Community. (2) Service delivery (3) Human resources; (4) Medical supplies and equipment. Conclusion: According to the findings of this study emergency medical system employees are suffering from a range of psychiatric problems as a result of a lack of equipment and job overload, which has a detrimental impact on the quality of pre-hospital emergency care. Therefore, emergency care senior management should develop comprehensive guidelines, provide more equipment and minimize professional challenges to improve the quality and safety of pre-hospital emergency care services.

The Frequency and Typing of HPV Virus Among Suspected Women Referred to HPV Genotyping Test in Mazandaran, Northern Iran.

BACKGROUND: Human papilloma viruses are a group of the Papillomaviridae family (ds DNA viruses), which infect basal epithelial cells. So far, 228 types of HPV have been identified, in which about 40 types infect the genital mucosa. In Iran, cervical cancer has been reported as the second most common malignancy in women which is approximately 8.8% of all cancers in women. HPV genotypes are classified as high-risk and low-risk according to cervical cancer. According to previous reports, nearly 70% of cervical cancers occur by HPV genotypes 16 and 18, of which genotype 16 is known as the most prevalent type. The main goal of this study was determining the frequency of HPV virus and its genotypes in the female population of Mazandaran. METHODS: This study was performed on 91 pathological samples. DNA was extracted from 500 µL of liquid-based cytology samples and PCR was performed for all of the samples. Genotyping step was performed based on strip assay method. RESULTS: HPV 39 (6.55%), 56 (3.27%), 51 (3.27%), and 68 (3.27%) were the most frequent types respectively. Also, HPV 11 (8.19%) and 6 (3.27%) show the most frequency among LR-HPV genotypes. HPV type 6 (16.39%), 56 (14.75%), 11 (14.75%), 16 (13.11%), and 66 (11.47%) were the four most common types seen in mixed infection samples. CONCLUSIONS: Differences among the types of HPV can be due to various geographical distributions of HPV. Our results revealed HPV 39 (6.55%) is the most common type among of HR-HPV followed by HPV 56, 51, 68 (3.27%); however, HPV 16 and HPV 18 were seen in just one case. HPV 11 (8.19%) and HPV 6 (3.27%) were the most common type among of LR-HPV.

Exploring the challenges of Iranian government hospitals related to Covid-19 pandemic management: a qualitative content analysis research from the nurses perspective.

BACKGROUND: The COVID-19 pandemic poses a major threat to global public health. As a result, to prepare healthcare systems for this unprecedented threat, a coordinated worldwide response is required. This study aimed to explore the hospitals challenges related to covid-19 pandemic management from the iranian nurses perspective. METHODS: This study was conducted as a qualitative content analysis in Iran. Using the purposive sampling method, data were collected through in-depth individual interviews with 35 nurse personnel. Graneheim and Lundman's conventional content analysis methods were used to analyze the data and for the trustworthiness of the data, this study used Lincoln and Guba's recommendations. RESULTS: After multiple rounds of analyzing and summarizing the data and taking into consideration similarities and differences, 5 main categories and 14 subcategories created based on the results of data analysis and including1) Leadership and management 2) Service delivery management 3) Human resources management 4) Equipment and Supplies Management and 5) Economic resources management. CONCLUSION: Identifying the most important challenges of nursing can play an important role in improving the management of COVID-19pandemic. The analysis of the challenges by managers at local, provincial and national levels can lead to the presentation of effective solutions to address these challenges and improve the pandemic management process in the country.

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.

TRAIL/S-layer/graphene quantum dot nanohybrid enhanced stability and anticancer activity of TRAIL on colon cancer cells.

Tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL), known as a cytokine of the TNF superfamily, is considered a promising antitumor agent due to its ability to selectively induce apoptosis in a wide variety of cancer cells. However, failure of its successful translation into clinic has led to development of nano-based platforms aiming to improve TRAIL therapeutic efficacy. In this regard, we fabricated a novel TRAIL-S-layer fusion protein (S-TRAIL) conjugated with graphene quantum dots (GQDs) to benefit both the self-assembly of S-layer proteins, which leads to elevated TRAIL functional stability, and unique optical properties of GQDs. Noncovalent conjugation of biocompatible GQDs and soluble fusion protein was verified via UV-visible and fluorescence spectroscopy, size and ζ-potential measurements and transmission electron microscopy. The potential anticancer efficacy of the nanohybrid system on intrinsically resistant cells to TRAIL (HT-29 human colon carcinoma cells) was investigated by MTT assay and flow cytometry, which indicated about 80% apoptosis in cancer cells. These results highlight the potential of TRAIL as a therapeutic protein that can be extensively improved by taking advantage of nanotechnology and introduce S-TRAIL/GQD complex as a promising nanohybrid system in cancer treatment.

Frequency of PAH Mutations Among Classic Phenylketon Urea Patients in Mazandaran and Golestan Provinces, North of Iran.

BACKGROUND: Phenylketonuria (PKU) is the most common aminoacidopathy with an autosomal recessive inheritance pattern. A global PKU prevalence is estimated about 6.002 in 100,000 newborns. In Iran, the prevalence of PKU is estimated at about 1 in 4,698, and it shows an increasing trend from north (0.0015%) to south (0.02%) of the country. Untreated PKU causes mental retardation, microcephaly, and seizure. PAH gene mutations located at chromosome 12q23 are responsible for the classical type of this disease. The spectrum of PAH mutations is varied in different ethnicities and different parts of the world. The aim of this study was to investigate the frequency of PAH mutation in the Mazandaran province, which could be useful for genetic counseling and prenatal diagnosis. METHODS: A total of 66 individuals from 33 families from two provinces (9 families from Golestan and 24 families from Mazandaran) from north of Iran participated in this study. After genomic DNA extraction, PAH gene analysis was carried out using DNA sequencing of both coding and non-coding regions by ABI 3130XL genetic analyzer. RESULTS: Twenty-six different mutations were identified in the PAH gene in this study. Four mutations including IVS10-11 (c.1066-11G>A), c.727C>T (p.Arg243X), c.898G>T (p.Ala300Ser), and c.601C>T (p.His201Tyr) were the most common mutations with 37.48% frequency in Mazandaran province. Most frequent mutations in Golestan province were IVSI0-11 (c.1066-11G>A), c.722delG (p.Arg241fs), c.842C>T (p.Pro281Leu), and IVSII+5 (G>A) with frequency 58.57%. CONCLUSIONS: The results from the present study verify heterogeneity of the PAH gene and may help to diagnose tests for carrier detection and prenatal diagnosis of the PKU disease in Iranian population.

New Insights into the Biosensing of Parkinson's Disease Biomarkers: A Concise Review.

BACKGROUND: Parkinson's disease (PD) is a long-term, degenerative, and neurological disease in which a person loses control of certain body functions. The formulation of novel effective therapeutics for PD as a neurodegenerative disease requires accurate and efficient diagnosis at the early stages. OBJECTIVE: Analyzing data gathered by measurable signals converted from biological reactions allows for qualitative and quantitative evaluations. Among various approaches reported so far, biosensors are powerful analytical tools that have been used in detecting the biomarkers of PD. METHODS: Biosensor's biological recognition components include antibodies, receptors, microorganisms, nucleic acids, enzymes, cells and tissues, and biomimetic structures. This review introduces electrochemical, optical, and optochemical detection of PD biomarkers based on recent advances in nanotechnology and material science, which resulted in the development of high-performance biosensors in this field. RESULTS: PD biomarkers such as α-synuclein protein, dopamine (DA), urate, ascorbic acid, miRNAs, and their biological roles are summarized. Additionally, the advantages and disadvantages of the usual standard methods are reviewed. We compared electrochemical, optical, and optochemical biosensors' properties and novel strategies for higher sensitivity and selectivity. CONCLUSION: The development of novel biosensors is required for the early diagnosis of PD as sensitive, rapid, reliable, and cost-effective systems.

Antibacterial, antibiofilm, anti-inflammatory, and wound healing effects of nanoscale multifunctional cationic alternating copolymers.

Infectious diseases caused by new or unknown bacteria and viruses, such as anthrax, cholera, tuberculosis and even COVID-19, are a major threat to humanity. Thus, the development of new synthetic compounds with efficient antimicrobial activity is a necessity. Herein, rationally designed novel multifunctional cationic alternating copolymers were directly synthesized through a step-growth polymerization reaction using a bivalent electrophilic cross-linker containing disulfide bonds and a diamine heterocyclic ring. To optimize the activity of these alternating copolymers, several different diamines and cross-linkers were explored to find the highest antibacterial effects. The synthesized nanopolymers not only displayed good to excellent antibacterial activity as judged by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, and Escherichia coli, but also reduced the number of biofilm cells even at low concentrations, without killing mammalian cells. Furthermore, in vivo experiments using infected burn wounds in mice demonstrated good antibacterial activity and stimulated wound healing, without causing systemic inflammation. These findings suggest that the multifunctional cationic nanopolymers have potential as a novel antibacterial agent for eradication of multidrug resistant bacterial infections.

Frequencies of CYP2B6∗4,∗5, and ∗6 Alleles within an Iranian Population (Mazandaran).

Background: The human CYP2B subfamily consists of one functional gene (CYP2B6) and one pseudogene (CYP2B7P). Cytochrome P450 2B6 (CYP2B6) is a highly polymorphic enzyme that shows marked interindividual and interethnic variations. Currently, 38 alleles have been described, and some of the allelic variants have been associated with low enzyme activity. The aim of this study was to investigate the frequencies of CYP2B6∗4, CYP2B6∗5, and CYP2B6∗6 alleles in the Mazani ethnic group among Iranian Population. Methods: The study was conducted in 289 unrelated healthy volunteers. DNA was extracted from peripheral blood and analyzed by the PCR-RFLP protocol. The PCR product was digested with restriction enzymes and then separated using agarose gel electrophoresis. Results: The frequency of CYP2B6∗4, CYP2B6∗5, and CYP2B6∗6 in this study was 34.60%, 7.26%, and 34.54%, respectively. Conclusion: The frequency of the CYP2B6∗4 allele in the Mazani ethnic group was much higher (34.60%) than other population. The frequency of CYP2B6∗6 (34.54%) also was higher than its frequency in other previously reported population. But the frequency of CYP2B6∗5 in this study was lower than expected. These results will be useful in understanding the ethnic diversity in Iranian population and offer a preliminary basis for more rational use of drugs that are substrates for CYP2B6 in this population.

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.

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.

Synthesis and characterization of vitamin D3-functionalized carbon dots for CRISPR/Cas9 delivery.

Aim: To develop a novel nanovector for the delivery of genetic fragments and CRISPR/Cas9 systems in particular. Materials & methods: Vitamin D3-functionalized carbon dots (D/CDs) fabricated using one-step microwave-aided methods were characterized by different microscopic and spectroscopic techniques. The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide assay and flow cytometry were employed to determine the cell viability and transfection efficiency. Results: D/CDs transfected CRISPR plasmid in various cell lines with high efficiency while maintaining their remarkable efficacy at high serum concentration and low plasmid doses. They also showed great potential for the green fluorescent protein disruption by delivering two different types of CRISPR/Cas9 systems. Conclusion: Given their high efficiency and safety, D/CDs provide a versatile gene-delivery vector for clinical applications.

Polyethylenimine-Functionalized Carbon Dots for Delivery of CRISPR/Cas9 Complexes.

Carbon dots (CDs) have become the focus of many studies due to their outstanding optical properties and good biocompatibility. We investigated their potential application to produce a smart and highly efficient yet nontoxic nanovector for gene delivery. This was achieved by conjugating PEI1.8k-functionalized CDs (synthesized by one-step microwave-assisted pyrolysis) with arginine-disulfide linkers to produce CD-PEI1.8k-Arg nanoparticles. This nanovector could deliver p-CRISPR (9.3 kb) into different types of cell lines with higher efficiency compared to native PEI1.8k or PEI25k. CD-PEI1.8k-Arg also maintained its outstanding transfection efficiency at a high serum concentration and low p-CRISPR dose, compared to PEI25k, which was ineffective under those conditions. Additionally, CD-PEI1.8k-Arg could knock out the GFP gene with great efficiency by delivering the required components of CRISPR/Cas9, including a plasmid encoding Cas9, sgRNA targeting GFP, and Cas9/sgRNA ribonucleoproteins (RNPs) into the HEK 293T-GFP cells. Moreover, the nanoparticles showed potential for the local delivery of p-CRISPR into brain tissue. The remarkable properties of CD-PEI1.8k-Arg could enable the development of a safe, highly efficient gene-delivery nanovector for the treatment of various diseases in the near future.

The Lumiptosome, an engineered luminescent form of the apoptosome can report cell death by using the same Apaf-1 dependent pathway.

Detection of the apoptosis signature becomes central in understanding cell death modes. We present here a whole-cell biosensor that detects Apaf-1 association and apoptosome formation using a split-luciferase complementary assay. Fusion of N-terminal (Nluc) and C-terminal (Cluc)-fragments of firefly luciferase to the N-terminus of human Apaf-1 was performed in HEK293 cells by using CRISPR-Cas9 technology. This resulted in a luminescent form of the apoptosome that we named 'Lumiptosome'. During Apaf-1 gene editing, a high number of knock-in events were observed without selection, suggesting that the Apaf-1 locus is important for the integration of exogenous transgenes. Since activation of caspase-9 is directly dependent on the apoptosome formation, measured reconstitution of luciferase activity should result from the cooperative association of Nluc-Apaf-1 and Cluc-Apaf-1. Time-response measurements also confirmed that formation of the apoptosome occurs prior to activation of caspase-3. Additionally, overexpression of the Bcl2 apoptosis regulator in transgenic and normal HEK293 cells confirmed that formation of the Lumiptosome depends on release of cytochrome c Thus, HEK293 cells that stably express the Lumiptosome can be utilized to screen pro- and anti-apoptotic drugs, and to examine Apaf-1-dependent cellular pathways.

Development and Clinical Translation of Approved Gene Therapy Products for Genetic Disorders.

The field of gene therapy is striving more than ever to define a path to the clinic and the market. Twenty gene therapy products have already been approved and over two thousand human gene therapy clinical trials have been reported worldwide. These advances raise great hope to treat devastating rare and inherited diseases as well as incurable illnesses. Understanding of the precise pathomechanisms of diseases as well as the development of efficient and specific gene targeting and delivery tools are revolutionizing the global market. Currently, human cancers and monogenic disorders are indications number one. The elevated prevalence of genetic disorders and cancers, clear gene manipulation guidelines and increasing financial support for gene therapy in clinical trials are major trends. Gene therapy is presently starting to become commercially profitable as a number of gene and cell-based gene therapy products have entered the market and the clinic. This article reviews the history and development of twenty approved human gene and cell-based gene therapy products that have been approved up-to-now in clinic and markets of mainly North America, Europe and Asia.