Impact of protectants and the method of preservation on the stability of potentially probiotic bacteria.

Probiotics offer health advantages when consumed in adequate quantities. As ongoing research identifies promising new strains, ensuring their viability and functionality through simple preservation methods is vital for success within the probiotic industry. This study employed a factorial design to investigate the combined effects of four cryoprotectants [C1: MRS broth + 14 % (w/v) glycerol, C2: Aqueous solution containing 4 % (w/v) trehalose, 6 % (w/v) skimmed milk, and 4 % (w/v) sodium glutamate, C3: Aqueous solution containing 10 % (w/v) skimmed milk and 4 % (w/v) sodium glutamate, C4: Aqueous solution containing 4 % (w/v) sucrose, 6 % (w/v) skimmed milk, and 4 % (w/v) sodium glutamate] and three methods of preservation (P1: -86 °C freezing, P2: -196 °C liquid nitrogen freezing, and P3: storing at 4 °C after lyophilization) on the cell viability of three potentially probiotic strains over 12 months. Pediococcus sp P15 and Weissella cibaria ml6 had the highest viability under treatments C3 and C2, after 12 months of storage, respectively. Meanwhile, Lactococcus lactis ml3 demonstrated the highest viability in both treatments C2 and C4 (P ≤ 0.05). According to the results freezing, either P1 or P2, is the most effective preservation method for P. sp P15 and W. cibaria ml6. Meanwhile, L. lactis ml3 showed the highest colony count under treatment (P1) after 12 months of storage (P ≤ 0.05). Among the tested conditions, P. sp P15 and L. lactis ml3 exhibited the highest viability and bile salt resistance when stored under P1C1. For W. cibaria ml6, the optimal storage condition was P2C2 (frozen in liquid nitrogen with cryoprotectant C2).

Metformin modulates oncogenic expression of HOTAIR gene via promoter methylation and reverses epithelial-mesenchymal transition in MDA-MB-231 cells.

Epithelial-mesenchymal transition (EMT) is a biological event, which critically regulates migration and invasion of cancer cells. EMT is regulated by several protein and nonprotein factors (such as noncoding RNAs). HOTAIR is an oncogenic long noncoding RNA that stimulates EMT in cancers. In the current study, we investigated the effect of metformin on EMT behavior and HOTAIR expression in MDA-MB-231 breast cancer cells. The minimal effective concentrations of metformin (10 and 20 mM) were obtained by the MTT test. Cell migration and invasion in the metformin-containing medium were assayed in the scratch assay and transwell test. Meaningful decreases in both cell migration and invasion were observed in the presence of metformin. Vimentin, snail, ß-catenin, and HOTAIR transcripts were quantified by real-time polymerase chain reaction (PCR). Reduction in the expression of vimentin, ß-catenin, and HOTAIR was detected as the result of metformin treatment, but the snail showed a constant expression. Western blottingrevealed the downregulation of vimentin and ß-catenin proteins. HOTAIR promoter methylation pattern was also investigated in metformin-exposed cells using bisulfite sequencing PCR which the result showed differences in the methylation profile of CpG islands between the treated and untreated cells. In conclusion, metformin modulated oncogenic expression of the HOTAIR gene in the MDA-MB-231 cells. This downregulation was associated with the modification of promoter methylation patterns. Since HOTAIR induces EMT in breast cancer, HOTAIR decline might be one of the mechanisms by which metformin reverses EMT.

Isolation and characterization of breast cancer stem cell-like phenotype by Oct4 promoter-mediated activity.

Cancer stem cells (CSCs) are a small subset of cancer cells responsible for self-renewal activity, drug resistance, and tumor recurrence. CSCs have been derived from diverse tumors and cell lines. The expression of stemness markers has been identified in CSCs. Oct4 is a well-established transcription factor expressed in stem cells and CSCs. In this study, we isolated and characterized breast CSC-like cells from murine MC4-L2 cells by Oct4 promoter-mediated activity. The MC4-L2 cells were electroporated by a plasmid expressing puromycin resistance (PuroR ) gene from the Oct4 promoter and then selected by puromycin. The isolated cells were named as the MC4-L2puro cells and characterized for CSCs properties. Immunostaining indicated CD44high and CD24high phenotype for the MC4-L2 and MC4-L2puro cells. The enhanced expression of stem cell markers was detected in the puromycin-selected cells compared with the parental cells. Moreover, the isolated cells only grew up in sphere-formed shape in low attachment plates. Serial dilution transplantation in syngeneic mouse models showed increased tumorigenicity of the MC4-L2puro cells, as they induced new tumors when injected into the mammary fat pad as few as 104 cells. In conclusion, we designed a novel genetic construct, which allows the isolation of Oct4-positive cells in a cancer population by a simple selection step in a puromycin-containing medium. Transfection of this construct into the MC4-L2 cells resulted in growing a subpopulation of cells having tumor-initiating cell characteristics. To the best of our knowledge, this is the first report on the isolation of CSC-like cells from the mouse breast cancer MC4-L2 cells.

Effects of Different Concentrations of Reversine on Plasticity of Mesenchymal Stem Cells.

Dedifferentiation can be induced by small molecules. One of these small molecules used in this study in order to increase the plasticity of differentiation of stem cells was reversine. The objective of present study was to investigate the effect of different concentrations of reversine on the plasticity of ovine fetal bone-marrow mesenchymal stem cells (BM-MSCs). BM-MSCs were extracted from ovine fetal and cultured. Passaged-3 cells were evaluated for their differentiation potential into osteocytes and adipocytes cells. In the present study, BM-MSCs were culture plated in the presence of 0, 300, 600, 900 and 1200 nM of reversine. The number of viable cells was determined by MTT test after addition of different concentrations of reversine. Furthermore, expression of the nanog gene was evaluated. The culture without reversine was taken as the control group. Expression of nanog was analysed by immunocytochemistry. Multi-lineage differentiation showed that the BM-MSCs could be differentiated into adipose cells and osteocytes. Our results indicated that the addition of 1200 nM of reversine to medium significantly decreased overall proliferation compared to the other treatment groups (p > 0.05). Real-time PCR analysis showed that after addition of 600 nM of reversine significantly increased nanog expression compared to other treatments. All treatments received reversine were seen to be relative expression of nanog. Our findings confirm that low concentrations reversine increases the plasticity of ovine BM-MSCs.

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.

Cell type-dependent functions of microRNA-92a.

The role of miR-17/92 family in development and progression of various cancers has been established. The members of this miRNA family have been shown to be over expressed and target various genes within proliferation, metastasis and angiogenesis pathways. Although all members might be overexpressed in a certain cancer type, only certain members of the family may have roles in progression of that cancer. In this study, we have chosen miR-92a, a member of the miR-17/92 family to compare its function in three different cancer cell lines. HL60, MCF7, and Jurkat cell lines were transduced with miR-92a and proliferation and apoptosis was measured in these cells by cell count, MTT, and caspase assays. Although in comparison to pre-miR-17/92, the level of miR-92a is higher in Jurkat cells compared to MCF7 and HL60 cells, here we have shown that increasing miR-92a levels results in apoptosis in Jurkat cells and proliferation in MCF7 and HL60 cells. miR-92a was also microinjected into mice fertilized eggs and after dissection, apoptosis was only observed in white pulp of spleen that is mainly made up of white blood cells. Our results show that miR-92a possesses a cell-type dependent function.

Pancreatic Endoderm-Derived From Diabetic Patient-Specific Induced Pluripotent Stem Cell Generates Glucose-Responsive Insulin-Secreting Cells.

Human-induced pluripotent stem cells (hiPSCs) can potentially serve as an invaluable source for cell replacement therapy and allow the creation of patient- and disease-specific stem cells without the controversial use of embryos and avoids any immunological incompatibility. The generation of insulin-producing pancreatic ß-cells from pluripotent stem cells in vitro provides an unprecedented cell source for personal drug discovery and cell transplantation therapy in diabetes. A new five-step protocol was introduced in this study, effectively induced hiPSCs to differentiate into glucose-responsive insulin-producing cells. This process mimics in vivo pancreatic organogenesis by directing cells through stages resembling definitive endoderm, primitive gut-tube endoderm, posterior foregut, pancreatic endoderm, and endocrine precursor. Each stage of differentiation were characterized by stage-specific markers. The produced cells exhibited many properties of functional ß-cells, including expression of critical ß-cells transcription factors, the potency to secrete C-peptide in response to high levels of glucose and the presence of mature endocrine secretory granules. This high efficient differentiation protocol, established in this study, yielded 79.18% insulin-secreting cells which were responsive to glucose five times higher than the basal level. These hiPSCs-derived glucose-responsive insulin-secreting cells might provide a promising approach for the treatment of type I diabetes mellitus. J. Cell. Physiol. 232: 2616-2625, 2017. © 2016 Wiley Periodicals, Inc.

Effect of blastocoel fluid reduction before vitrification on gene expression in mouse blastocysts.

Artificial collapse of the blastocoel cavity before vitrification can improve the quality of warmed embryos, yet how reduction of blastocoel fluid impacts formation of the blastocyst cell lineages is not clear. The present study assessed the effect of pre-vitrification blastocoel fluid reduction on the survival, hatching rate, and the expression of genes related to apoptosis (Tp53), pluripotency (Pou5f1, Nanog), and differentiation (Cdx2, Eomes, Gata6) in mouse blastocysts. In vivo-produced blastocysts were randomly divided into three groups: The first group was vitrified and warmed; the second group underwent artificial collapse of the blastocoel cavity prior to vitrification and warming; the third group served as the control, in which neither vitrification or artificial collapse was performed. The survival rate of treatment groups was similar to the control group, whereas the hatching rate of artificial collapse/vitrified blastocysts was significantly higher than vitrified blastocysts. Quantitative reverse-transcription PCR analysis revealed a considerable reduction in the expression of Cdx2, Eomes, Gata6, Grb2, and Tp53 transcripts following artificial collapse/vitrification in comparison to the vitrification-alone group; the abundance of Pou5f1 and Nanog, however, did not change. These results suggest that artificial collapse of the blastocoel cavity before vitrification leads to relatively normal expression of apoptosis and development-related genes plus higher hatching rates. Mol. Reprod. Dev. 83: 735-742, 2016 © 2016 Wiley Periodicals, Inc.

Toward Chemical Perfection of Graphene-Based Gene Carrier via Ugi Multicomponent Assembly Process.

The graphene-based materials with unique, versatile, and tunable properties have brought new opportunities for the leading edge of advanced nanobiotechnology. In this regard, the use of graphene in gene delivery applications is still at early stages. In this study, we successfully designed a new complex of carboxylated-graphene (G-COOH) with ethidium bromide (EtBr) and used it as a nanovector for efficient gene delivery into the AGS cells. G-COOH, with carboxyl functions on its surface, in the presence of EtBr, formaldehyde, and cyclohexylisocyanide were participated in Ugi four component reaction to fabricate a stable amphiphilic graphene-EtBr (AG-EtBr) composite. The coupling reaction was confirmed by further analyses with FT-IR, AFM, UV-vis, Raman, photoluminescence, EDS, and XPS. The AG-EtBr nanocomposite was able to interact with a plasmid DNA (pDNA). This nanocomposite has been applied for transfection of cultured mammalian cells successfully. Moreover, the AG-EtBr composites showed a remarkable decreased cytotoxicity in compared to EtBr. Interestingly, the advantages of AG-EtBr in cell transfection are more dramatic (3-fold higher) than Lipofectamine2000 as a commercial nonviral vector. To the best of our knowledge, this is the first report in which EtBr is used as an intercalating agent along with graphene to serve as a new vehicle for gene delivery application.

Ovine fetal mesenchymal stem cell differentiation to cardiomyocytes, effects of co-culture, role of small molecules; reversine and 5-azacytidine.

The aim of the present study was to investigate the effect of small molecules: Reversine and 5-azacytidine (5-AC), in an indirect co-culture condition with the cardiac fibroblasts as well as non co-culture condition, in order to explore the effect of such molecules in the process of differentiation of the ovine bone-marrow mesenchymal stem cells (BM-MSCs) towards cardiomyocytes. Surface antigens of the isolated cells were analysed using flow-cytometry. In addition, following to three passages cells were examined for their differentiation capacity into osteocytes and adipose cells, in order to ensure the mesenchymal origin of the stem cells. Six types of treatments were carried out in the present investigation, such that, in the first treatment BM-MSCs were cultured for 28 days as control group; the second treatment was composed of culturing ovine fetal cardiac fibroblasts on inserts, aiming to use these inserts for culturing plates which were seeded with BM-MSCs (Chamber group). As the third treatment, BM-MSCs were supplemented with 10-µM 5-AC and incubated for 48 h. The fourth treatment was composed of supplementing BM-MSCs with the 600-nM reversine, incubated for 48 h, and subsequently the incubation was further extended for another 48 h in the presence of 5-AC. The fifth treatment was composed of supplementing the chamber group with 10-µM 5-AC and incubation for 48 h, and the last or the sixth treatment was such that chamber group was supplemented with 600-nM reversine and an incubation period of 48 h. Following to the incubation, medium was replaced with 10-µM 5-AC and further incubated for another round of 48 h. In all treatments, following to addition of the small molecules incubations were carried out for 28 days; same as controls. Expression of cardiac alpha-actinin was analysed by immunocytochemistry. BM-MSCs have shown to express CD44 and CD166 along with a weak expression of the CD90, CD34, in addition to CD45. Multilineage differentiation has indicated that BM-MSCs could differentiate into adipose and osteocytes cells as well. In the treatment 4 it was observed that FGF signalling involved genes and all cardiac-related genes (ANP, MYH6 and Troponin I) were significantly expressed, except connexin 43 compared to other treatments. All treatments received small molecules, either alone or as a co-culture were seen to express sarcomeric alpha-actinin. This finding was partially supported by immunocytochemistry. These results validate that reversine and 5-AC have an effect on ovine BM-MSC differentiation into cardiomyocytes. Copyright © 2016 John Wiley & Sons, Ltd.

Human Dental Pulp Stem Cells Differentiate into Oligodendrocyte Progenitors Using the Expression of Olig2 Transcription Factor.

The helix-loop-helix transcription factor Olig2 is essential for lineage determination of oligodendrocytes. Differentiation of stem cells into oligodendrocytes and transplanting them is a novel strategy for the repair of different demyelination diseases. Dental pulp stem cells (DPSCs) are of great interest in regenerative medicine due to their potential for repairing damaged tissues. In this study, DPSCs were isolated from human third molars and transfected with the human Olig2 gene as a differentiation inducer for the oligodendrogenic pathway. Following the differentiation procedure, the expression of Sox2, NG2, PDGFRα, Nestin, MBP, Olig2, Oct4, glial fibrillary acidic protein and A2B5 as stage-specific markers was studied by real-time RT-qPCR, immunocytochemistry and Western blot analysis. The cells were transplanted into a mouse model of local sciatic damage by lysolecithin as a model for demyelination. Oligodendrocyte progenitor cells (OPCs) actively remyelinated and recovered the lysolecithin-induced damages in the sciatic nerve as revealed by treadmill exercise, the von Frey filament test and hind paw withdrawal in response to a thermal stimulus. Recovery of behavioral reflexes occurred 2-6 weeks after OPC transplantation. The results demonstrate that the expression of Olig2 in DPSCs reduces the expression of stem cell markers and induces the development of oligodendrocyte progenitors as revealed by the emergence of oligodendrocyte markers. DPSCs could be programmed into oligodendrocyte progenitors and considered as a simple and valuable source for the cell therapy of neurodegenerative diseases.

Study of the effect of F17A mutation on characteristics of Bacillus thermocatenulatus lipase expressed in Pichia pastoris using in silico and experimental methods.

Bacillus thermocatenulatus lipase 2 (BTL2), a thermoalkalophilic lipase, is the best studied enzyme for its particular properties, which make it useful in different industries. Displacement of conserved phenylalanine 17 (Phe-17) residue in the active site of BTL2 has a critical role in oxyanion hole formation, which is important for enzyme activity. In this study, to facilitate oxyanion hole formation, Phe-17 was substituted with Alanine residue (F17A). The best structures of the opened form of the native and mutated lipases were garnered based on the crystal structures of 2W22. To evaluate catalytic activity, both lipases were docked to a set of ligands using Hex 6.3 software. Following in silico study, both native and mutant btl2 genes were cloned and expressed in Pichia pastoris. Based on the results obtained, the mutation increased lipase lipolytic activity against most of the applied substrates, especially for tributyrin and tricaprylin, by 1.9 and 2.15 fold, respectively. However, optimum temperature and pH were the same for both lipases (60 °C and pH 8.0). As previously reported, it is believed that F17A mutation simplifies oxyanion hole formation and declines steric hindrance in the enzyme active site, which might ultimately lead to more efficient accessibility of substrates.

TBHP-induced oxidative stress alters microRNAs expression in mouse testis.

PURPOSE: Reactive oxygen species (ROS) and oxidative stress is one of the main reasons of male infertility. MicroRNAs (miRNAs) regulate multiple intracellular processes. Alterations in miRNAs expression may occur in different conditions and diseases. In this study, the effect of oxidative stress induced by tertiary-butyl hydroperoxide (TBHP) on the expression of candidate miRNAs in mouse testis was investigated. METHODS: After determining median lethal dose (LD50), TBHP was intraperitoneally (ip) injected at the dilution of 1:10 LD50 into the adult male mice for 2 weeks, and then testis tissues were removed in order to assay the ROS level. Total RNA was extracted and the expression of five miRNAs was quantified by reverse transcription-real time polymerase chain reaction (RT-qPCR). RESULTS: The flow cytometry analysis showed a significant increase in ROS level in testis. The expression of three out of five selected miRNAs, including miR-34a, miR-181b and miR-122a, showed some degrees of changes following exposure to oxidative stress. These miRNAs are involved in antioxidant responses, inflammation pathway and spermatogenesis arrest. CONCLUSIONS: In conclusion, TBHP alters the miRNA expression profile of testis which might play a potential role in oxidative and antioxidative responses and spermatogenesis.

Induction of protective T-helper 1 immune responses against Echinococcus granulosus in mice by a multi-T-cell epitope antigen based on five proteins.

In this study, we designed an experiment to predict a potential immunodominant T-cell epitope and evaluate the protectivity of this antigen in immunised mice. The T-cell epitopes of the candidate proteins (EgGST, EgA31, Eg95, EgTrp and P14-3-3) were detected using available web-based databases. The synthesised DNA was subcloned into the pET41a+ vector and expressed in Escherichia coli as a fusion to glutathione-S-transferase protein (GST). The resulting chimeric protein was then purified by affinity chromatography. Twenty female C57BL/6 mice were immunised with the antigen emulsified in Freund's adjuvant. Mouse splenocytes were then cultured in Dulbecco's Modified Eagle's Medium in the presence of the antigen. The production of interferon-γ was significantly higher in the immunised mice than in the control mice (> 1,300 pg/mL), but interleukin (IL)-10 and IL-4 production was not statistically different between the two groups. In a challenge study in which mice were infected with 500 live protoscolices, a high protectivity level (99.6%) was demonstrated in immunised BALB/C mice compared to the findings in the control groups [GST and adjuvant (Adj)]. These results demonstrate the successful application of the predicted T-cell epitope in designing a vaccine against Echinococcus granulosus in a mouse model.

Induction of prominent Th1 response in C57Bl/6 mice immunized with an E. coli-expressed multi T-cell epitope EgA31 antigen against Echinococcus granulosus.

First step in developing an epitope-based vaccine is to predict peptide binding to the major histocompatibility complex (MHC) molecules. We performed computational analysis of unique available EgA31 sequence to locate appropriate antigenic propensity positions. T-cell epitopes with best binding affinity values of < 50% inhibitory concentration were selected using different available servers (Propred and IEDB). Peptides with 100% population coverage were selected. A DNA fragment corresponding to the furin linker enriched in Golgi apparatus was inserted sequentially between each epitope sequences in a synthetic DNA in order to cleave the chimeric protein into four separated peptides. Subsequently, the synthetic DNA was cloned into the pGEX4T-1 and pEGFP-N1 vectors and GST-ChEgA31 was expressed in E. coli strain BL21-DE3. The recombinant protein was detected by western blotting using an HRP-conjugated polyclonal anti-GST antibody. Fusion protein purified by affinity chromatography was used to raise antisera in rabbits. Results in agar gel immunodiffusion assay indicated induction of specific antibodies against multiepitope antigen in the tested rabbits. Cytokine assay was carried out in C57Bl/6 mice and the levels of cytokines were analyzed by sandwich ELISA. Interestingly, production of specific IFN-gamma was prominently higher in mice immunized with GST-ChEgA31 and pEGFP-ChEgA31 (650-1300 pg/ml) compared to control groups. No difference was observed in the level of IL-10 and IL-4 in immunized and GST control group. Challenge study with 500 live protoscolices of Echinococcus granulosus on immunized mice demonstrated protectivity level (50-60%). Based on our results, it appeared that the chimeric protein in the study was able to stimulate T-helper cell-1 (Th1) development and high level of cell mediated immunity in mice.

The effects of PFT-µ on spermatogonial stem cell viability and Pluripotency.

INTRODUCTION: Spermatogonial stem cells (SSCs) offer remarkable competencies for animal reproduction and overcoming human disease as a result of their differentiation capability. We evaluated the effect of small molecule pifithrin-mu (PFT-µ) as a well-known inhibitor of P53 on SSC biological processes such as viability, apoptosis, and gene expression pattern. METHODS: The SSCs were isolated from the testes of adult NMRI mice and then cultured in DMEM / F12 medium containing 10% FBS. Then, they were characterized by the immunocytochemistry (ICC) technique by high PLZF and low c-Kit expressions. SSCs colony formation assay was carried out and their viability was estimated by MTT (Methylthiazolyldiphenyl-tetrazolium bromide, or 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay upon exposure to PFT-µ (0, 0.6, 1.2, 2.5, and 5µM). The apoptosis percentages also were measured using FACS analysis, and finally, Oct4 and Stra8 expression at mRNA levels was assessed using real-time quantitative PCR. RESULTS: The 0.6 and 1.2µM PFT-µ improved the viability of SSC based on MTT assay results; however, 2.5 and 5µM PFT-µ reduced SSC viability compared with the control group. Moreover, PFT-µ at lower concentration enhanced the colony size of SSCs and diminished their apoptosis. As well, as exposure to PFT-µ up-regulated Oct4 expression, while down-regulating the meiotic entry marker, Stra8. CONCLUSION: Based on findings, optimized concentrations of PFT-µ can decrease SSCs apoptosis, and conversely potentiate their pluripotency and self-renewal capacities in vitro.

Generation of Mouse Model of Hemophilia A by Introducing Novel Mutations, Using CRISPR/Nickase Gene Targeting System.

Developing mouse models of hemophilia A has been shown to facilitate in vivo studies to explore the probable mechanism(s) underlying the disease and to examine the efficiency of the relevant potential therapeutics. This study aimed to knockout (KO) the coagulation factor viii (fviii) gene in NMRI mice, using CRISPR/Cas9 (D10A/nickase) system, to generate a mouse model of hemophilia A. Two single guide RNAs (sgRNAs), designed from two distinct regions on NMRI mouse FVIII (mFVIII) exon 3, were designed and inserted in the pX335 vector, expressing both sgRNAs and nickase. The recombinant construct was delivered into mouse zygotes and implanted into the pseudopregnant female mice's uterus. Mutant mice were identified by genotyping, genomic sequencing, and mFVIII activity assessment. Two separate lines of hemophilia A were obtained through interbreeding the offspring of the female mice receiving potential CRISPR-Cas9-edited zygotes. Genomic DNA analysis revealed disruptions of the mfviii gene reading frame through a 22-bp deletion and a 23-bp insertion in two separate founder mice. The founder mice showed all the clinical signs of hemophilia A including; excessive bleeding after injuries, and spontaneous bleeding in joints and other organs. Coagulation test data showed that mFVIII coagulation activity was significantly diminished in the mFVIII knockout (FVIIIKO) mice compared to normal mice. The CRISPR/nickase system was successfully applied to generate mouse lines with the knockout fviii gene. The two novel FVIIIKO mice demonstrated all clinical symptoms of hemophilia A, which could be successfully inherited. Therefore, both of the developed FVIIIKO mouse lines are eligible for being considered as proper mouse models of hemophilia A for in vivo therapeutic studies.

Different Effects of Low-Level Laser Therapy on the Proliferation of HT29 Cells in Culture and Xenograft Models.

Introduction: Different kinds of treatments have been developed to fight cancers. Low-level laser therapy (LLLT), also known as photobiomodulation therapy (PBMT), is a low-power monochromatic and coherent light that has been used successfully for healing injuries and combating malignancies. However, there are concerns about the application of LLLT to cancers due to the increased proliferation of some cancer cells after LLLT. Methods: This study investigated the effects of 650 nm and 870 nm lasers on the proliferation of HT29 colorectal cancer cell lines in vitro and in vivo. Results: The results showed that the laser with a wavelength of 870 nm did not meaningfully alter the proliferation of cultured cells. However, cell proliferation was promoted when the laser was applied within a wavelength of 650 nm. Treatment of HT29-derived tumors in nude mice with the 650 nm laser resulted in the decline of the tumor progression rate compared to controls. This result was inconsistent with the proliferative effects of the laser on the cultured cells. Conclusion: Cell behavior in response to LLLT might be different between cell culture and xenograft models.