Pre-hyperglycemia immune cell trafficking underlies subclinical diabetic cataractogenesis.

BACKGROUND: This work elucidates the first cellular and molecular causes of cataractogenesis. Current paradigm presupposes elevated blood glucose as a prerequisite in diabetic cataractogenesis. Novel evidence in our model of diabetic cataract challenges this notion and introduces immune cell migration to the lens and epithelial-mesenchymal transformation (EMT) of lens epithelial cells (LECs) as underlying causes. METHODS: Paucity of suitable animal models has hampered mechanistic studies of diabetic cataract, as most studies were traditionally carried out in acutely induced hyperglycemic animals. We introduced diabetic cataract in the Nile grass rat (NGR) that spontaneously develops type 2 diabetes (T2D) and showed its closeness to the human condition. Specialized stereo microscopy with dual bright-field illumination revealed novel hyperreflective dot-like microlesions in the inner cortical regions of the lens. To study immune cell migration to the lens, we developed a unique in situ microscopy technique of the inner eye globe in combination with immunohistochemistry. RESULTS: Contrary to the existing paradigm, in about half of the animals, the newly introduced hyper reflective dot-like microlesions preceded hyperglycemia. Even though the animals were normoglycemic, we found significant changes in their oral glucose tolerance test (OGTT), indicative of the prediabetic stage. The microlesions were accompanied with significant immune cell migration from the ciliary bodies to the lens, as revealed in our novel in situ microscopy technique. Immune cells adhered to the lens surface, some traversed the lens capsule, and colocalized with apoptotic nuclei of the lens epithelial cells (LECs). Extracellular degradations, amorphous material accumulations, and changes in E-cadherin expressions showed epithelial-mesenchymal transformation (EMT) in LECs. Subsequently, lens fiber disintegration and cataract progression extended into cortical, posterior, and anterior subcapsular cataracts. CONCLUSIONS: Our results establish a novel role for immune cells in LEC transformation and death. The fact that cataract formation precedes hyperglycemia challenges the prevailing paradigm that glucose initiates or is necessary for initiation of the pathogenesis. Novel evidence shows that molecular and cellular complications of diabetes start during the prediabetic state. These results have foreseeable ramifications for early diagnosis, prevention and development of new treatment strategies in patients with diabetes.

Preventive cancer stem cell-based vaccination modulates tumor development in syngeneic colon adenocarcinoma murine model.

BACKGROUND: Cancer stem cells (CSCs), a rare sub-fraction of tumor cells, with the capability of self-renewal and strong oncogenicity are tightly responsible for chemo and radio resistance and tumor metastasis in colorectal cancer. Hence, CSCs targeting would improve the efficacy of therapeutic strategies and clinical outcomes. METHODS: Here, using three-dimensional CSC spheroids and syngeneic mice model, we evaluated the cancer preventive impact of CSCs-based vaccination. CSCs enrichment was performed via colonosphere formation from CT-26 cell line and CT-26-derived tumor biopsy and characterized by confirming high expression of key stemness genes (OCT4, SOX2, and NANOG) and CSC-related surface biomarkers (CD166, DCLK1, and CD133) via real-time PCR and flow cytometry, respectively. Then, the stemness phenotype and self-renewal in CSC-enriched spheroids were further confirmed by showing serial sphere formation capacity, clonogenicity potential, and enhanced in vivo tumorigenic capacity compared to their parental counterparts. CSCs lysates were used as vaccines in prophylactic settings compared to the parental cell lysate and PBS groups. RESULT: Immunization of syngeneic mice with CSCs lysates was effective in the prevention of tumor establishment and significantly decreased tumor growth rate accompanied by an improvement in survival rate in tumor-bearing mice compared to groups subjected to parental cells lysate and PBS. These results, for the first time, showed that mice immunized with cell lysate from tumor biopsy-derived spheroids are resistant to tumor induction. Immunofluorescence staining indicated that only the serum antibodies from CSC-vaccinated mice reacted with colonospheres. CONCLUSIONS: These findings represent CSCs lysate-based vaccination as a potential approach to hampering immunotherapy failure of colorectal cancer which along with other traditional therapies may effectively apply to prevent the establishment of aggressive tumors harboring stemness features.

Ectopic expression of OCT4B1 Decreases Fertility Rate and Changes Sperm Parameters in Transgenic Mice.

Background: The octamer-binding transcription factor-4 (OCT4) is known as an established important regulator of pluripotency, as well as a genetic "master switch" in the self-renewal of embryonic stem and germ cells. OCT4B1, one of the three spliced variants of human OCT4, plays crucial roles in the regulation of pluripotency and stemness. Objectives: The present study developed a transgenic mouse model containing an OCT4B1-expressing construct under the transcriptional direction of mouse mammary tumor virus promoter (pMMTV) to evaluate the role of OCT4B1 in the function of male germ cells in terms of fertility potential. Additionally, the effect of ectopic OCT4B1 overexpression on endogenous OCT4 expression was examined in mouse embryonic stem cells (mESCs). Material and Methods: The pMMTV-OCT4B1cDNA construct was injected into the pronuclei of 0.5-day NMRI embryos. Transgenic mice were identified based on the PCR analysis of tail DNA. Further, Diff-Quik staining was applied to assess sperm morphology, while the other sperm parameters were analyzed through a conventional light microscopic evaluation according to World Health Organization (WHO) criteria. The fertility rate was scored by using in vitro frtilization (IVF) method. Furthermore, mESCs was electroporated with the OCT4B1cDNA-containing constructs, followed by analyzing through employing semi-quantitative RT-PCR and western blotting. Results: The results demonstrated the changes in sperm morphology, as well as a statistically significant decrease in the other sperm parameters (count, viability, and motility) and fertility rate (p<0.05) in the transgenic mice compared with the control group. The assessment of the cause of the embryonic stem cell (ESC) death following transfection revealed a significant reduction in the endogenous OCT4 expression at both mRNA and protein levels in the transfected mESCs compared to the control ones. Conclusion: In general, the in vivo results suggested a potential role of OCT4B1 in the spermatogenesis process. These results represented that the overexpression of OCT4B1 may induce its role in spermatogenesis and fertility rate by interfering endogenous OCT4 expression. However, further studies are required to clarify the mechanisms underlying OCT4B1 function.

Nanoarchitectonics for Photo-Controlled Intracellular Drug Release in Immune Modulation.

Local stimuli differentiate monocytes into M2-like macrophages that mechanistically drive the pathologies in cancer and age-related macular degeneration (AMD). A photo-controlled nanodrug that halts macrophage polarization through Rho-associated kinase (ROCK) inhibition was developed. A small-molecule ROCK inhibitor, fasudil, was conjugated to a photo-responsive group and a short poly(ethylene glycol) (PEG) chain. This resulted in the novel amphiphilic prodrug, PEG-2-(4'-(di(prop-2-yn-1-yl)amino)-4-nitro-[1,1'-biphenyl]-yl)propan-1-ol (PANBP)-Fasudil, that spontaneously formed micelles. Ultraviolet (UV) irradiation of PEG-PANBP-Fasudil nanoparticles rapidly released fasudil. For visualization of linker degradation, a reporter nanoprobe was synthesized, in which 2-Me-4-OMe TokyoGreen (TG), a fluorophore that does not fluoresce in conjugation, was incorporated. Irradiation of nanoprobe-laden monocytes activated the reporter fluorophore. Cytokine stimulation differentiated monocytes into macrophages, while UV irradiation prevented polarization of PEG-PANBP-Fasudil nanoparticle-laden monocytes. Nanoarchitectonics-based design opens new possibilities for intracellular drug delivery and precise spatiotemporal immune cell modulation toward the development of new therapies.

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.

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.

Increased cytoplasmic expression of DLL4 is associated with favorable prognosis in colorectal cancer.

Aims: DLL4 of the Notch pathway is a key regulator of VEGF expression, which mediates tumor neovascularization and stem cell self-renewal in colorectal cancer (CRC). The authors investigated the association of DLL4 expression with the clinicopathological characteristics and survival outcomes of CRC patients. Methods: DLL4 expression level was evaluated in 199 CRC samples using immunohistochemistry analysis of tissue microarrays. Results: The high expression of DLL4 was inversely associated with distant metastasis (p < 0.029), tumor recurrence (p < 0.04) and longer overall survival following curative surgery compared with those with low DLL4 expression with 95% CI (log-rank test: p = 0.050). In univariate analysis, histological grade (hazard ratio: 3.859; 95% CI: 1.081-13.784; p = 0.038) was a strong prognostic risk factor, affecting the overall survival of CRC patients. Conclusion: The authors' results demonstrate that DLL4 expression might be considered a favorable prognostic factor for overall survival in CRC patients.

Diabetic cataract in the Nile grass rat: A longitudinal phenotypic study of pathology formation.

Diabetes is a major risk factor for cataract, the leading cause of blindness worldwide. There is an unmet need for a realistic model of diabetic cataract for mechanistic and longitudinal studies, as existing models do not reflect key aspects of the complex human disease. Here, we introduce and characterize diabetic cataract in the Nile grass rat (NGR, Arvicanthis niloticus), an established model of metabolic syndrome and type 2 diabetes (T2D). We conducted a longitudinal study of cataract in over 88 NGRs in their non-diabetic, pre-diabetic, and diabetic stages of metabolism. Oral glucose tolerance test (OGTT) results distinguished the metabolic stages. Diverse cataract types were observed in the course of diabetes, including cortical, posterior subcapsular (PSC), and anterior subcapsular (ASC), all of which succeeded a characteristic dotted ring stage in all animals. The onset ages of diabetes and cataract were 44 ± 3 vs 29 ± 1 (P < .001) and 66 ± 5 vs 58 ± 6 (not significant) weeks in females and males, respectively. Histological analysis revealed fiber disorganization, vacuolar structures, and cellular proliferation and migration in cataractous lenses. The lens epithelial cells (LECs) in non-diabetic young NGRs expressed the stress marker GRP78, as did LECs and migrated cells in the lenses of diabetic animals. Elucidating mechanisms underlying LEC proliferation and migration will be clinically valuable in prevention and treatment of posterior capsule opacification, a dreaded complication of cataract surgery. Marked changes in N-cadherin expression emphasized a role for LEC integrity in cataractogenesis. Apoptotic cells were dispersed in the equatorial areas in early cataractogenesis. Our study reveals diverse cataract types that spontaneously develop in the diabetic NGR, and which uniquely mirror the cataract and its chronic course of development in individuals with diabetes. We provide mechanistic insights into early stages of diabetic cataract. These unique characteristics make NGR highly suited for mechanistic studies, especially in the context of metabolism, diabetes, and aging.

Morphological and molecular characteristics of spheroid formation in HT-29 and Caco-2 colorectal cancer cell lines.

BACKGROUND: Relapse and metastasis in colorectal cancer (CRC) are often attributed to cancer stem-like cells (CSCs), as small sub-population of tumor cells with ability of drug resistance. Accordingly, development of appropriate models to investigate CSCs biology and establishment of effective therapeutic strategies is warranted. Hence, we aimed to assess the capability of two widely used and important colorectal cancer cell lines, HT-29 and Caco-2, in generating spheroids and their detailed morphological and molecular characteristics. METHODS: CRC spheroids were developed using hanging drop and forced floating in serum-free and non-attachment conditions and their morphological features were evaluated by scanning electron microscopy (SEM). Then, the potential of CSCs enrichment in spheroids was compared to their adherent counterparts by analysis of serial sphere formation capacity, real-time PCR of key stemness genes (KLF4, OCT4, SOX2, NANOG, C-MYC) and the expression of potential CRC-CSCs surface markers (CD166, CD44, and CD133) by flow cytometry. Finally, the expression level of some EMT-related (Vimentin, SNAIL1, TWIST1, N-cadherin, E-cadherin, ZEB1) and multi-drug resistant (ABCB1, ABCC1, ABCG2) genes was evaluated. RESULTS: Although with different morphological features, both cell lines were formed CSCs-enriched spheroids, indicated by ability to serial sphere formation, significant up-regulation of stemness genes, SOX2, C-MYC, NANOG and OCT4 in HT-29 and SOX2, C-MYC and KLF4 in Caco-2 spheroids (p-value < 0.05) and increased expression of CRC-CSC markers compared to parental cells (p-value < 0.05). Additionally, HT-29 spheroids exhibited a significant higher expression of both ABCB1 and ABCG2 (p-value = 0.02). The significant up-regulation of promoting EMT genes, ZEB1, TWIST1, E-cadherin and SNAIL1 in HT-29 spheroids (p-value = 0.03), SNAIL1 and Vimentin in Caco-2 spheroids (p-value < 0.05) and N-cadherin down-regulation in both spheroids were observed. CONCLUSION: Enrichment of CSC-related features in HT-29 and Caco-2 (for the first time without applying special scaffold/biochemical) spheroids, suggests spheroid culture as robust, reproducible, simple and cost-effective model to imitate the complexity of in vivo tumors including self-renewal, drug resistance and invasion for in vitro research of CRC-CSCs.

Expressions of TWIST1 and CD105 markers in colorectal cancer patients and their association with metastatic potential and prognosis.

BACKGROUND: TWIST1 and CD105, which contribute to tumor malignancy, are overexpressed in cancers. Accordingly, TWIST1 enhances epithelial-to-mesenchymal transition (EMT) and promotes the formation of cancer stem cells (CSCs). Also, CD105 is a neoangiogenesis marker in endothelial cells, which is introduced as a CSC marker in tumoral epithelial cells in several types of cancers. The present study was aimed to investigate expressions of TWIST1 and CD105 in colorectal cancer (CRC) patients. METHODS: Expressions of TWIST1 and CD105 in 250 CRC tissue samples were evaluated using immunohistochemistry on tissue microarrays (TMAs). In this regard, TWIST1 expression was investigated in the subcellular locations (cytoplasm and nucleus), while CD105 was mapped in endothelial cells and cytoplasmic tumor cells of CRC tissues. The association between the expression of these markers and clinicopathological parameters, as well as survival outcomes were analyzed. RESULTS: Results indicate a statistically significant association between higher nuclear expression levels of TWIST1 and distant metastases in CRC (P = 0.040) patients. In addition, it was shown that the increased nuclear expression of TWIST1 had a poor prognostic value for disease-specific survival (DSS) and progression-free survival (PFS) (P = 0.042, P = 0.043, respectively) in patients with CRC. Moreover, analysis of CD105 expression level has revealed that there is a statistically significant association between the increased expression of CD105 in tumoral epithelial cells and more advanced TNM stage (P = 0.050). CONCLUSIONS: Our results demonstrate that nuclear TWIST1 and cytoplasmic CD105 expressions in tumor cells had associations with more aggressive tumor behavior and more advanced diseases in CRC cases.

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.

High expression of tumor susceptibility gene 101 (TSG101) is associated with more aggressive behavior in colorectal carcinoma.

INTRODUCTION: Identification of genetic determinants such as exosomal content that drives progression and metastasis of colorectal cancer (CRC) has received considerable attention. The present study aims to identify a suitable biomarker in CRC tissues and exosomes based on bioinformatics data to evaluate its expression patterns in CRC tissues as well as its clinicopathological significance. MATERIALS AND METHODS: Protein-protein interaction (PPI) network and enrichment analysis were applied to identify up-regulated genes that contributed in CRC exosomes to select the marker. The expression patterns and clinical significance of selected exosomal marker were evaluated in tissue microarrays (TMAs) of 445 CRC tumors and 39 adjacent normal tissues using immunohistochemistry method. RESULTS: Based on bioinformatics data, TSG101 gene was prominent amongst the tumor tissues and exosomes. Expression of TSG101 was significantly up-regulated in tumor cells compared to adjacent normal tissues (p-value = 0.04). Moreover, higher expressions of TSG101 (cytoplasmic and nuclear) were significantly associated with tumor differentiation (p-value = 0.042) and distant metastasis (p-value = 0.027). A significant association was found in the cytoplasmic expression of TSG101 between well and moderate tumor differentiation (p-value = 0.005) as well as moderate and poor differentiation (p-value = 0.050). CONCLUSION: These findings indicate that the exploration of crosstalk between exosome content and CRC may be valuable for the development of novel exosomal biomarkers. Increased expression of TSG101, as a promising exosome marker, is more associated with more aggressive tumor behaviors, metastasis, and progression of CRC, which paves the way for therapeutic strategies and CRC management. However, further investigations are warranted to clarify the molecular mechanisms of TSG101 in CRC.

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.

Low expression of Talin1 is associated with advanced pathological features in colorectal cancer patients.

To explore the proper prognostic markers for the likelihood of metastasis in CRC patients. Seventy-seven fresh CRC samples were collected to evaluate the mRNA level of the selected marker using Real-time PCR. Moreover, 648 formalin-fixed paraffin-embedded CRC tissues were gathered to evaluate protein expression by immunohistochemistry (IHC) on tissue microarrays. The results of Real-Time PCR showed that low expression of Talin1 was significantly associated with advanced TNM stage (p = 0.034) as well as gender (p = 0.029) in mRNA levels. Similarly, IHC results indicated that a low level of cytoplasmic expression of Talin1 was significantly associated with advanced TNM stage (p = 0.028) as well as gender (p = 0.009) in CRC patients. Moreover, decreased expression of cytoplasmic Talin1 protein was found to be a significant predictor of worse disease-specific survival (DSS) (p = 0.011) in the univariate analysis. In addition, a significant difference was achieved (p = 0.039) in 5-year survival rates of DSS: 65% for low, 72% for moderate, and 88% for high Talin1 protein expression. Observations showed that lower expression of Talin1 at both the gene and protein level may drive the disparity of CRC patients' outcomes via worse DSS and provide new insights into the development of progression indicators because of its correlation with increased tumor aggressiveness.

Tumor-derived exosomes: the next generation of promising cell-free vaccines in cancer immunotherapy.

Identification of immunogenic tumor antigens that are efficiently processed and delivered by dendritic cells to prime the immune system and to induce an appropriate immune response is a research hotspot in the field of cancer vaccine development. High biosafety is an additional demand. Tumor-derived exosomes (TEXs) are nanosized lipid bilayer encapsulated vesicles that shuttle bioactive information to the tumor microenvironment facilitating tumor progression. However, accumulating evidence points toward the capacity of TEXs to efficiently stimulate immune responses against tumors provided they are appropriately administered. After briefly describing the function of exosomes in cancer biology and their communication with immune cells, we summarize in this review in vitro and preclinical studies eliciting the potency of TEXs in inducing effective anti-tumor responses and recently modified strategies further improving TEX-vaccination efficacy. We interpret the available data as TEXs becoming a lead in cancer vaccination based on tumor antigen-selective high immunogenicity.

Primary colonospheres maintain stem cell-like key features after cryopreservation.

The development of efficient and repeatable protocols for biobanking and prolonged storage of cancer stem cells (CSCs), with minimum alterations in biological function, is valuable and desired, particularly for retrospective analysis and clinical applications. In particular, data regarding the effect of cryopreservation on CSCs's functional features is scarce. In this regard, few studies have been shown that 3D spheroid structures, which enriched for CSCs, can keep their biological phenotype and genetic profiles. Here, for the first time, we present data on cryopreservation of CT-26 colonospheres, with the focus on essential stem cell-like properties after thawing. Tumor biopsy-derived colonospheres were frozen in standard freezing media (90% fetal bovine serum + 10% dimethyl sulfoxide) and stored in liquid nitrogen for 10 months. Then, cryopreservation effect on preservation of CSCs-related features was verified using real-time polymerase chain reaction for evaluation of stemness genes and flow cytometry for the putative colorectal CSC surface biomarkers. The self-renewal capacity of thawed spheres was also compared with their fresh counterparts using serial formation assay. Finally, tumorigenic capacity of both groups was evaluated in immunocompetence mouse model. Our data indicated that postthawed colonospheres had high viability without drastic alteration in biological and structural features and maintained self-renewal potential after sequential passages. Real-time analysis showed that both fresh and frozen colonospheres displayed similar expression pattern for key stemness genes: SOX2 and OCT4. Cryopreserved spheroids expressed CD133, CD166, and DCLK1 CSCs surface biomarkers at elevated levels when compared with parental as non-cryopreserved counterparts. Our electron scanning microscopy micrographs clearly demonstrated that postthawed colonospheres retain their integrity and cell surface morphology and characteristics. We also found that both fresh and frozen spheroids were equally tumorigenic. This study represented an effective strategy for reliable storage of intact CT-26 colonospheres; this can provide researchers with a functionally reliable repository of murine colorectal CSCs for their future CSCs projects.

Effects of topical sesame (Sesamum indicum) oil on the pain severity of chemotherapy-induced phlebitis in patients with colorectal cancer: A randomized controlled trial.

BACKGROUND AND PURPOSE: Chemotherapy-induced phlebitis (CIP) is one of the most important and common complications in patients with cancer. Currently, the use of complementary methods to prevent or alleviate phlebitis symptoms has attracted great attention. In this study, we aimed to assess the effects of topical sesame oil in reducing the pain severity of CIP. MATERIALS AND METHODS: This randomized clinical trial was conducted on 60 patients with colorectal cancer afflicted with CIP. Patients received, twice a day for seven consecutive days, a 5-min massage solely (as the control group) or with 10 drops of sesame oil (as the experimental group) within the 10 cm radius of the affected site. The pain severity was evaluated by the visual analog scale on the first, third, fifth, and seventh days of the intervention. RESULTS: Mean changes of the pain severity compared to the baseline were significant on the third (P = 0.009), fifth (P < 0.001), and seventh (P < 0.001) days of the intervention in favor of the experimental group. Also, a significant reduction in the pain severity both in the experimental and control groups was observed during the seven days (F = 720.66, Ptime < 0.001); however, the decrease was more significant in the experimental group (F = 21.46, Pgroup < 0.001). CONCLUSION: Application of massage with sesame oil as a complementary method is effective in reducing the pain severity of patients with CIP.

In Vivo Evaluation of PAX6 Overexpression and NMDA Cytotoxicity to Stimulate Proliferation in the Mouse Retina.

Retinal degenerative diseases, due to the lack of regeneration systems and self-renewable cells, often lead to visual impairment. Pax6 is a pleiotropic transcription factor and its expression level determines self-renewal status or differentiation of retinal cells. Here, we investigated the fate of simultaneous induction of retinal ganglion cell death and Pax6 overexpression in retro-differentiation of retinal cells and their commitment to re-enter into the cell cycle. Induction of acute retinal ganglion cell death and generation of mouse experimental model was performed by N-methyl D-aspartic acid (NMDA) injection. Recombinant AAV2 virus harboring PAX6 cDNA and reporter gene was injected into untreated and model mouse eyes. Histological analyses, including IHC and retinal flatmounts immunostaining were performed. The number of Ki67+ cells was clearly increased in model mice, presumably due to NMDA treatment and regardless of Pax6 over-expression. Unlike previous studies, Ki67+ cells were found in GCL layer and interestingly ONL cells expressed Sox2 stemness marker after NMDA cytotoxicity. The potential of retinal cells for robust Ki67 expression, after injury, and expression of Sox2, confirmed their intrinsic plasticity and made a vivid prospect for retinal regenerative medicine.

Genetic Linkage Analysis of DFNB4, DFNB28, DFNB93 Loci in Autosomal Recessive Non-syndromic Hearing Loss: Evidence for Digenic Inheritance in GJB2 and GJB3 Mutations.

BACKGROUND: Autosomal recessive non-syndromic hearing loss (ARNSHL) a most frequent hereditary type of hearing impairment, exhibit tremendous genetic heterogeneity. We aimed to determine the contribution of three common DFNB loci (DFNB4, DFNB28, and DFNB93), and mutation analysis of Gap Junction Beta-2 gene (GJB2) and GJB3 genes in ARNSHL subjects in southern Iran. METHODS: Thirty-six large ARNSHL pedigrees (167 individuals) with at least two affected subjects (72 patients) were included in this descriptive study from Hormozgan Province of Iran, during 2014 - 2015. The variation of GJB2 and GJB3 genes were screened using direct sequencing method. The negative samples for GJB2 and GJB3 genes mutations were analyzed for the linkage to DFNB4, DFNB28, and DFNB93 loci by genotyping the corresponding short tandem repeat (STR) markers using polymerase chain reaction (PCR) and polyacrylamide gel electrophoresis (PAGE) methods. RESULTS: DNA sequencing of GJB2 were identified heterozygous mutation (964 C/T) in 13.88% of the studied families. Three missense mutations (788G/A, 284C/T and 973G/C) were also detected in coding region of the GJB3 gene. The 284C/T mutation in the GJB3 occurs in compound heterozygosity along with the 964T/C mutation in the GJB2 in one family. Finally, we found no evidence of linkage to either of DFNB4, DFNB93 and DFNB28 loci. CONCLUSION: Highlighting the hypothesis that a genetic interaction between GJB2 and GJB3 genes could be lead to ARNSHL, however, no evidence of linkage to the DFNB loci was found. 284C/T variant in GJB3 gene might be pathogenic when accompanied by variant in GJB2 in a digenic pattern. However, further large-scale familial and functional studies are required to challenge this hypothesis.