Current Molecular Medicine - Volume 19, Issue 2, 2019

Transplantation of reprogrammed cells from accessible sources and in vivo reprogramming are potential therapies for regenerative medicine. During the last decade, genetic approaches, which mostly involved transcription factors and microRNAs, have been shown to affect cell fates. However, their potential carcinogenicity and other unexpected effects limit their translation into clinical applications. Recently, with the power of modern biology-oriented design and synthetic chemistry, as well as high-throughput screening technology, small molecules have been shown to enhance reprogramming efficiency, replace genetic factors, and help elucidate the molecular mechanisms underlying cellular plasticity and degenerative diseases. As a non-viral and non-integrating approach, small molecules not only show revolutionary capacities in generating desired exogenous cell types but also have potential as drugs that can restore tissues through repairing or reprogramming endogenous cells. Here, we focus on the recent progress made to use small molecules in cell reprogramming along with some related mechanisms to elucidate these issues.

Objective: In the present study, the interaction between drug Tyrphostin AG528 and CNT(6,6-6) nanotube by Density Functional Theory (DFT) calculations in solvent water has been investigated for the first time. Methods and Results: According to the calculations, intermolecular hydrogen bonds take place between an active position of the molecule Tyrphostin AG528 and hydrogen atoms of the nanotube which play an important role in the stability of complex CNT(6,6- 6)/Tyrphostin AG528. The non-bonded interaction effects of the molecule Tyrphostin AG528 with CNT(6,6-6) nanotube on the electronic properties, chemical shift tensors and natural charge have also been detected. The natural bond orbital (NBO) analysis suggested that the molecule Tyrphostin AG528 as an electron donor and the CNT(6,6-6) nanotube play the role of an electron acceptor at the complex CNT(6,6-6)/Tyrphostin AG528. Conclusion: The electronic spectra of the Tyrphostin AG528 drug and complex CNT(6,6-6)/Tyrphostin AG528 in solvent water were calculated by Time-Dependent Density Functional Theory (TD-DFT) for the investigation of adsorption effect of the Tyrphostin AG528 drug over nanotube on maximum wavelength. Then, the possibility of the use of CNT(6,6-6) nanotube for Tyrphostin AG528 delivery to the diseased cells has been established.

Background: Beta thalassemia is a common disorder with autosomal recessive inheritance. The most prenatal diagnostic methods are the invasive techniques that have the risk of miscarriage. Now the non-invasive methods will be gradually alternative for these invasive techniques. Objective: The aim of this study is to evaluate and compare the diagnostic value of two non-invasive diagnostic methods for fetal thalassemia using cell free fetal DNA (cff-DNA) and nucleated RBC (NRBC) in one sampling community. Methods: 10 ml of blood was taken in two k3EDTA tube from 32 pregnant women (mean of gestational age = 11 weeks), who themselves and their husbands had minor thalassemia. One tube was used to enrich NRBC and other was used for cff-DNA extraction. NRBCs were isolated by MACS method and immunohistochemistry; the genome of stained cells was amplified by multiple displacement amplification (MDA) procedure. These products were used as template in b-globin segments PCR. cff-DNA was extracted by THP method and 300 bp areas were recovered from the agarose gel as fetus DNA. These DNA were used as template in touch down PCR to amplify b-globin gen. The amplified b-globin segments were sequenced and the results compared with CVS resul. Results: The data showed that sensitivity and specificity of thalassemia diagnosis by NRBC were 100% and 92% respectively and sensitivity and specificity of thalassemia diagnosis by cff-DNA were 100% and 84% respectively. Conclusion: These methods with high sensitivity can be used as screening test but due to their lower specificity than CVS, they cannot be used as diagnostic test.

Introduction: Multiple myeloma (MM) cells accumulate in the bone marrow and produce enormous quantities of immunoglobulins, causing endoplasmatic reticulum stress and activation of protein handling machinery, such as heat shock protein response, autophagy and unfolded protein response (UPR). Methods: We evaluated cell lines viability after treatment with bortezomib (B) in combination with HSP70 (VER-15508) and autophagy (SBI-0206965) or UPR (STF- 083010) inhibitors. Results: For RPMI-8226, after 72 hours of treatment with B+VER+STF or B+VER+SBI, we observed 15% of viable cells, but treatment with B alone was better (90% of cell death). For U266, treatment with B+VER+STF or with B+VER+SBI for 72 hours resulted in 20% of cell viability and both treatments were better than treatment with B alone (40% of cell death). After both triplet combinations, RPMI-8226 and U266 presented the overexpression of XBP-1 UPR protein, suggesting that it is acting as a compensatory mechanism, in an attempt of the cell to handle the otherwise lethal large amount of immunoglobulin overload. Conclusion: Our in vitro results provide additional evidence that combinations of protein homeostasis inhibitors might be explored as treatment options for MM.

Background: Homocysteine (Hcy) has been suggested as an independent risk factor for atherosclerosis. Apolipoprotein M (apoM) is a constituent of the HDL particles. The goal of this study was to examine the serum levels of homocysteine and apoM and to determine whether homocysteine influences apoM synthesis. Methods: Serum levels of apoM and Hcy in 17 hyperhomocysteinemia (HHcy) patients and 19 controls were measured and their correlations were analyzed. Different concentrations of homocysteine (Hcy) and LY294002, a specific phosphoinositide 3- kinase (PI3K) inhibitor, were used to treat HepG2 cells. The mRNA levels were determined by RT-PCR and the apoM protein mass was measured by western blot. Results: We found that decreased serum apoM levels corresponded with serum HDL levels in HHcy patients, while the serum apoM levels showed a statistically significant negative correlation with the serum Hcy levels. Moreover, apoM mRNA and protein levels were significantly decreased after the administration of Hcy in HepG2 cells, and this effect could be abolished by addition of LY294002. Conclusions: Present study demonstrates that Hcy downregulates the expression of apoM by mechanisms involving the PI3K signal pathway.

Background: Despite intensive investigation, effective therapeutic procedures for myocardial I/R injury are still in demand. Objective: To explore the effect of adeno-associated virus 9 (AAV9)-mediated small interfering RNA targeting TLR4 in the treatment of myocardial ischemia and reperfusion (I/R) injury and its influence on the NF-ΚB and MAPK signaling pathways. Methods: Rats were divided into 3 groups, namely, the sham, AAV9-siRNA control, and AAV9-TLR4 siRNA groups. siRNA solution or normal saline was injected through the tail vain. The rat myocardial I/R injury model was then established. HE staining and TUNEL staining were applied to compare the pathological changes in cardiomyocytes in the three groups. Immunohistochemical staining and western blotting were utilized to detect TLR4 expression under siRNA interference. Serum inflammatory factor (IL-1β, TNF-α) expression was determined by an ELISA commercial kit. Key proteins in the MAPK (p38, JNK 1/2) and NF-ΚB (p65) signaling pathways were determined to identify the TLR4 siRNA functional mechanism. Results: Fluorescence microscopic images of the myocardium indicated that AAV9- mediated siRNA was efficiently transfected into the myocardium, and the infarcted size after I/R injury was decreased by AAV9-TLR4 siRNA when compared with negative control rats (P<0.05). TLR4 protein expression was significantly decreased by siRNA interference (P<0.001). Apoptosis-related factor BCL-2 expression was increased in the TLR4 gene silencing group, whereas Bax expression was decreased. The Bax/BCL-2 ratio was also decreased, demonstrating a protective effect for cardiomyocytes. Inflammatory factors were lower in the TLR4 gene silencing group than in the siRNA control group (P<0.001). The MAPK and NF-ΚB signaling pathways were activated in myocardial I/R injury; however, the primary proteins in these two signaling pathways were downregulated upon interference of TLR4 siRNA, with significant differences (P<0.05). Conclusion: AAV9-TLR4 siRNA has a positive effect on myocardial I/R injury by inhibiting the MAPK and NF-ΚB signaling pathways and can be used as a potential therapeutic method for myocardial I/R injury.

Background: Mutations in mitochondrial tRNA (mt-tRNA) genes have been found to be associated with both syndromic and non-syndromic hearing impairment. However, the pathophysiology underlying mt-tRNA mutations in clinical expression of hearing loss remains poorly understood. Objective: The aim of this study was to explore the potential association between mttRNA mutations and hearing loss. Methods and Results: We reported here the molecular features of a pedigree with maternally transmitted non-syndromic hearing loss. Among 12 matrilineal relatives, five of them suffered variable degree of hearing impairment, but none of them had any medical history of using aminoglycosides antibiotics (AmAn). Genetic screening of the complete mitochondrial genomes from the matrilineal relatives identified the coexistence of mt-tRNAHis G12192A and mt-tRNAThr G15927A mutations, together with a set of polymorphisms belonging to human mitochondrial haplogroup B5b1b. Interestingly, the G12192A mutation occurred 2-bp from the 3’ end of the TψC loop of mt-tRNAHis, which was evolutionarily conserved from various species. In addition, the well-known G15927A mutation, which disrupted the highly conserved C-G base-pairing at the anticodon stem of mt-tRNAThr, may lead to the failure in mt-tRNA metabolism. Furthermore, a significant decreased in ATP production and an increased ROS generation were observed in polymononuclear leukocytes (PMNs) which were isolated from the deaf patients carrying these mt-tRNA mutations, suggested that the G12192A and G15927A mutations may cause mitochondrial dysfunction that was responsible for deafness. However, the absence of any functional mutations/variants in GJB2, GJB3, GJB6 and TRMU genes suggested that the nuclear genes may not play important roles in the clinical expression of non-syndromic hearing loss in this family. Conclusion: Our data indicated that mt-tRNAHis G12192A mutation may increase the penetrance and expressivity of deafness-associated m-tRNAThr G15927A mutation in this family.

Purpose: Breast cancer is the most prevalent malignancy and the leading cause of death among women. Triple-negative breast cancer (TNBC) is a subtype of breast cancer and shows a distinctly aggressive nature with higher rates of relapse and shorter overall survival in the metastatic setting compared to other subtypes of breast cancer. This study aimed to assess the effect of KIF15 on various clinicopathological characteristics, survival analysis, and cell proliferation in triple-negative breast cancer, which has not been reported to our knowledge. Methods: A total of 165 patients with triple-negative breast cancer were enrolled and clinical data were obtained, Mann-Whitney U analysis was performed to assess the correlation between the expression of KIF15 and clinical pathological characteristics of TNBC patients. Survival analysis was performed by Kaplan-Meier analysis and Log-rank test. The expression levels of KIF15 in cancer tissues and adjacent tissues were evaluated via Sign test. Lentivirus was used to down-regulate the expression of KIF15 in TNBC cells. The cell proliferation, colony formation capacity and apoptosis were examined by MTT, Giemsa staining and flow cytometry assay, respectively. Results: Our results showed that, among the 165 TNBC patients, the expression of KIF15 was positive correlation with clinicopathological features of TNBC. In addition, KIF15 low-expression group showed higher disease-free survival than KIF15 highexpression group and univariate analysis showed that KIF15 high-expression group appeared higher mortality than KIF low-expression group (P ≤ 0.05). Meanwhile, the expression levels of KIF15 in cancer tissue notably up-regulated in comparison with adjacent tissue. In vitro, knockdown of KIF15 significantly promoted cell apoptosis and suppressed cell proliferation and colony formation of TNBC cells. Conclusion: By utilizing survival analysis, we found that high-expression of KIF15 in the TNBC samples were associated with poorer overall survival, while the anti-tumor effect of KIF15 knockdown was also confirmed at the cellular level in vitro. Taken together, KIF15 can be applied as a potential diagnostic and therapeutic target in TNBC.