Tumor Promoting Aspects of Senescence in Cancer Progression.

Cancers induced by gene mutation, deletion, and genome instability might be related to aging. With similar pathways of aging but distinct functions, senescence at the cellular level is an irreversible arrest of cell cycle. Senescence has long been believed as a barrier to restrict tumor expansion. However, more and more evidence has been shown that senescence inducers regulate epithelial-mesenchymal transition, stem cell self-renewal, inflammatory response, crosstalk with the oncogenic bypass signaling, and conversion of oncogene to tumor suppressor. Here we will discuss the most recent findings of the oncogenic aspects of senescence which crosstalk with multiple pathways in cancer progression.

High-density lipoprotein-mediated transcellular cholesterol transport in mouse aortic endothelial cells.

Accumulation of unesterified cholesterol-rich lipid vesicles in the subendothelial space contributes to atherogenesis. Transport of cholesterol from the subendothelial intima back to the circulating blood inhibits atherosclerosis development; however, the mechanism for this process has not been fully defined. Using cultured mouse aortic endothelial cells (MAECs), we observed that unesterified cholesterol can be transported across the endothelial cell monolayer from the basolateral to the apical compartment. Administration of high-density lipoprotein (HDL) or apolipoprotein AI (apoAI) to the apical compartment enhanced transendothelial cholesterol transport in a concentration-dependent manner. Knockdown of ATP-binding cassette transporter G1 (ABCG1) or scavenger receptor class B type I (SR-B1), or inhibition of SR-B1 diminished HDL-induced transendothelial cholesterol transport; while knockdown of ABCA1 reduced apoAI-mediated cholesterol transport. HDL enhanced phosphorylation of phosphatidylinositol 3-kinase (PI3K) and Akt in MAECs. However, inhibition of PI3K or Akt did not reduce HDL-induced transendothelial cholesterol transport. These results suggest that HDL enhances transendothelial cholesterol transport by activation of a mechanism involving ABCA1, ABCG1 and SR-B1 but not involving PI3K and Akt.

Study on the influence of syphilis on the outcome of frozen-thawed embryo transfer in infertility patients.

Objective: In this study, the effect of in vitro Fertilization-Embryo Transfer (IVF-ET) on the clinical outcome of patients with syphilis infertility during resuscitation cycle. Methods: A retrospective single-center method was adopted. This study included 4430 pairs of infertile patients who underwent syphilis detection. The influence of the syphilis freeze-thaw embryos transplantation outcome was studied in the patients with infertility by comparing the general clinical characteristics of patients (age, years of infertility, body mass index (BMI), basal follicle stimulating hormone (FSH), serum basal estradiol (Estradiol, E2), transplanted intimal thickness, the number of embryos transferred) and the clinical pregnancy (biochemical pregnancy rate, clinical pregnancy rate, implantation rate, live birth rate and abortion rate). Results: Firstly, in the clinical outcome of one frozen-thawed embryos transfer, the live birth rate of the woman's syphilis-infected group was lower than that of the uninfected group (71.3 % vs. 50.0 %), while the abortion rate was higher than that of the uninfected group (7.8 % vs. 26.7 %), and there was a statistical difference (P < 0.05), and there was no statistical difference in other indicators between other groups (P > 0.05). Secondly, in the clinical outcome of two frozen-thawed embryos transfers, the biochemical pregnancy rate (61.3 % vs. 28.6 %) and clinical pregnancy rate (42.9 % vs. 14.3 %) of the group which was infected with syphilis alone were lower than those of the uninfected group (P < 0.05), and other indicators among the other groups showed no statistical difference (P > 0.05). Thirdly, in the clinical outcomes of frozen-thawed embryos transfer three times or more, there was no significant difference in the clinical indicators between the syphilis infertility patients and the non-infected infertility patients (P > 0.05). Conclusion: When the syphilis infertility patients and the non-infected infertile patients underwent IVF-ET treatment for the first time, the live birth rate and abortion rate of the syphilis group were significantly different (P < 0.05). In the outcome of two transplants, the biochemical pregnancy rate and clinical Pregnancy rates were significantly reduced so patients with syphilis infertility who undergo IVF-ET should be informed about the risk of adverse clinical outcomes.

Molecular cloning and characterization of a new cDNA sequence encoding a venom peptide from the centipede Scolopendra subspinipes mutilans.

Many studies have been performed on venomous peptides derived from animals. However, little of this research has focused on peptides from centipede venoms. Here, a venom gland cDNA library was successfully constructed for the centipede Scolopendra subspinipes mutilans. A new cDNA encoding the precursor of a venom peptide, named SsmTx, was cloned from the venomous gland cDNA library of the centipede S. subspinipes mutilans. The full-length SsmTx cDNA sequence is 465 nt, including a 249 nt ORF, a 45 nt 5' UTR and a 171 nt 3' UTR. There is a signal tail AATAAA 31 nt upstream of the poly (A) tail. The precursor nucleotide sequence of SsmTx encodes a signal peptide of 25 residues and a mature peptide of 57 residues, which is bridged by two pairs of disulfide bonds. SsmTx displays a unique cysteine motif that is completely different from that of other venomous animal toxins. This is the first reported cDNA sequence encoding a venom peptide from the centipede S. subspinipes mutilans.

ImKTx96, a peptide blocker of the Kv1.2 ion channel from the venom of the scorpion Isometrus maculates.

Scorpion venom contains diverse bioactive peptides that can recognize and interact with membrane proteins such as ion channels. These natural toxins are believed to be useful tools for exploring the structure and function of ion channels. In this study, we characterized a K+-channel toxin gene, ImKTx96, from the venom gland cDNA library of the scorpion Isometrus maculates. The peptide deduced from the ImKTx96 precursor nucleotide sequence contains a signal peptide of 27 amino acid residues and a mature peptide of 29 residues with three disulfide bridges. Multiple sequence alignment indicated that ImKTx96 is similar with the scorpion toxins that typically target K+-channels. The recombined ImKTx96 peptide (rImKTx96) was expressed in the Escherichia coli system, and purified by GST-affinity chromatography and RP-HPLC. Results from whole-cell patch-clamp experiments revealed that rImKTx96 can inhibit the current of the Kv1.2 ion channel expressed in HEK293 cells. The 3D structure of ImKTx96 was constructed by molecular modeling, and the complex formed by ImKTx96 interacting with the Kv1.2 ion channel was obtained by molecular docking. Based on its structural features and pharmacological functions, ImKTx96 was identified as one member of K+-channel scorpion toxin α-KTx10 group and may be useful as a molecular probe for investigating the structure and function of the Kv1.2 ion channel.

ML-SA1, a selective TRPML agonist, inhibits DENV2 and ZIKV by promoting lysosomal acidification and protease activity.

Arboviruses, especially Dengue virus (DENV) and Zika virus (ZIKV), have been a severe threat to human health in the last few years due to uncontrollable transmission. There are no approved vaccines or clinical drugs available for use to prevent and treat their infections. Transient receptor potential mucolipin 2 and 3 (TRPML2 and TRPML3) were reported to modulate viral entry, but the antiviral function of these modulators was unknown. Here, we reported that ML-SA1, a TRPML agonist, inhibited DENV2 and ZIKV in vitro in a dose-dependent manner. Time-of-drug-addition experiments showed that ML-SA1 mainly restricted viral entry. Moreover, the selective TRPML3 activator SN-2 was found to share a similar antiviral effect against DENV2 and ZIKV, but the specific TRPML1 agonist MK6-83 was not effective. Although ML-SA1 was further revealed to induce autophagy, its antiviral role was independent of autophagy induction. Finally, ML-SA1 was found to inhibit DENV2 and ZIKV by promoting lysosome acidification and protease activity to cause viral degradation. Together, our study identifies two TRPML agonists, ML-SA1 and SN-2, as potent inhibitors of DENV2 and ZIKV, which may lead to the discovery of new candidates against viruses.

Topology, Antiviral Functional Residues and Mechanism of IFITM1.

Interferon-inducible transmembrane proteins (IFITM1/2/3) have been reported to suppress the entry of a wide range of viruses. However, their antiviral functional residues and specific mechanisms are still unclear. Here, we firstly resolved the topology of IFITM1 on the plasma membrane where N-terminus points into the cytoplasm and C-terminus resides extracellularly. Further, KRRK basic residues of IFITM1 locating at 62-67 of the conserved intracellular loop (CIL) were found to play a key role in the restriction on the Zika virus (ZIKV) and dengue virus (DENV). Similarly, KRRK basic residues of IFITM2/3 also contributed to suppressing ZIKV replication. Finally, IFITM1 was revealed to be capable of restricting the release of ZIKV particles from endosome to cytosol so as to impede the entry of ZIKV into host cells, which was tightly related with the inhibition of IFITM1 on the acidification of organelles. Overall, our study provided topology, antiviral functional residues and the mechanism of interferon-inducible transmembrane proteins.

Establishment and characterization of three new human breast cancer cell lines derived from Chinese breast cancer tissues.

BACKGROUND: Breast cancer is a major malignancy affecting females worldwide. It is the most common cause of death from cancer in women. Cell lines are widely used in laboratory research and particularly as in vitro models in cancer research. But we found that the routinely used breast cancer cell lines were mostly derived from Caucasians or African-Americans. There were few standard models to study the pathogenic mechanism at molecular level and cell signaling pathway of breast cancer for Asian patients. It is quite necessary to establish new breast cancer cell lines from xanthoderm to study the pathogenic mechanism and therapeutic methods. RESULTS: Three new breast cancer cell lines, designated BC-019, BC-020 and BC-021, were successfully established and characterized from breast invasive ductal carcinoma tissues of three Chinese female patients. These new cell lines growing as adherent monolayer with characteristic epithelial morphology could be maintained continuously in vitro, and they were ER-, PR- and C-erbB-2-positive. Their chromosomes showed high hyperdiploidy and complex rearrangements, and they displayed aggressive tumorigencity in tumorigenesis test. CONCLUSION: The three newly established breast cancer cell lines from Chinese patients were tested for a number of, and the results indicate that the cell lines were in good quality and could be served as new cell models in breast cancer study.

[Design and application of retention enema device].

OBJECTIVE: Drug retention enema is a common therapy for various illnesses. However, it is impossible to keep the drug in the colon for a long time, due to the limitation of the current equipment, and it is unable to achieve the purpose of retention enema. A retention enema device was designed by the department of intensive care unit (ICU) of Dongfeng Hospital Affiliated to Hubei University of Medicine. The retention enema device adds a spindle shaped inflatable air bag on the basis of the traditional enema device, which not only fix on the anus, but also prevent the leakage of enema fluid. It can achieve retention enema, play the enema drug effect fully, and significantly reduce the nursing workload, in addition, the silica gel material of the retention enema device ensures the comfort of the patients, the decompression air bag also avoids the damage of the high pressure of the spindle fixed air bag for the patients, which is worthy of clinical use.

Peptidylarginine deiminase 4 overexpression resensitizes MCF-7/ADR breast cancer cells to adriamycin via GSK3ß/p53 activation.

BACKGROUND: Adriamycin (ADR) is widely used in the clinical chemotherapy against breast cancer. But its efficacy is strongly limited due to the acquisition of multidrug resistance (MDR). Therefore, acquisition of the resistance to ADR is still a major cause of chemotherapy failure in breast cancer patients. Peptidylarginine deiminase IV (PAD4) is reported to target non-histone proteins for citrullination, regulate their substrate activities, and thereby play critical roles in maintaining cell phenotype in breast cancer cells. However, whether PAD4 is involved in the development of MDR in breast cancer is poorly understood. MATERIALS AND METHODS: We examined the expression of PAD family members, including PAD4 in ADR-resistant MCF-7 cells compared with the parental control cells by real-time PCR and Western blotting analyses. Rescue of PAD4 expression in MCF-7/ADR cells was performed to assess whether PAD4 could restore the sensitivity of MCF-7/ADR cells to ADR treatment with cell counting kit-8, flow cytometry, TUNEL, nuclear and cytoplasmic extract preparations, and immunofluorescence staining analyses. RESULTS: Both PAD2 and PAD4 were significantly decreased in ADR-resistant cells. However, only PAD4 overexpression can increase the sensitivity of MCF-7/ADR cells to ADR treatment and decrease MDR1 gene expression. Overexpression of PAD4 in MCF-7/ADR cells inhibited cell proliferation by inducing cell apoptosis. Under ADR treatment, overexpression of PAD4 promoted nuclear accumulation of glycogen synthase kinase-3ß and p53, which further activated proapoptotic gene expression and downregulated MDR1 expression. Moreover, PAD4 activity was required for activating proapoptotic gene transcripts. CONCLUSION: We demonstrate the previously unappreciated role of PAD4 in reversing ADR resistance in MCF-7/ADR cells and help establish PAD4 as a candidate biomarker of prognosis and chemotherapy target for MDR in breast cancers.

Inhibiting PAD2 enhances the anti-tumor effect of docetaxel in tamoxifen-resistant breast cancer cells.

BACKGROUND: Tamoxifen resistance presents a huge clinical challenge for breast cancer patients. An understanding of the mechanisms of tamoxifen resistance can guide development of efficient therapies to prevent drug resistance. METHODS: We first tested whether peptidylarginine deiminase 2 (PAD2) may be involved in tamoxifen-resistance in breast cancer cells. The effect of depleting or inhibiting PAD2 in tamoxifen-resistant MCF-7 (MCF7/TamR) cells was evaluated both in vitro and in vivo. We then investigated the potential of Cl-amidine, a PAD inhibitor, to be used in combination with tamoxifen or docetaxel, and further explored the mechanism of the synergistic and effective drug regimen of PADs inhibitor and docetaxel on tamoxifen-resistant breast cancer cells. RESULTS: We report that PAD2 is dramatically upregulated in tamoxifen-resistant breast cancer. Depletion of PAD2 in MCF7/TamR cells facilitated the sensitivity of MCF7/TamR cells to tamoxifen. Moreover, miRNA-125b-5p negatively regulated PAD2 expression in MCF7/TamR cells, therefore overexpression of miR-125b-5p also increased the cell sensitivity to tamoxifen. Furthermore, inhibiting PAD2 with Cl-amidine not only partially restored the sensitivity of MCF7/TamR cells to tamoxifen, but also more efficiently enhanced the efficacy of docetaxel on MCF7/TamR cells with lower doses of Cl-amidine and docetaxel both in vivo and in vivo. We then showed that combination treatment with Cl-amidine and docetaxel enhanced p53 nuclear accumulation, which synergistically induced cell cycle arrest and apoptosis. Meanwhile, p53 activation in the combination treatment also accelerated autophagy processes by synergistically decreasing the activation of Akt/mTOR signaling, thus enhancing the inhibition of proliferation. CONCLUSION: Our results suggest that PAD2 functions as an important new biomarker for tamoxifen-resistant breast cancers and that inhibiting PAD2 combined with docetaxel may offer a new approach to treatment of tamoxifen-resistant breast cancers.

A novel human foamy virus mediated gene transfer of GAD67 reduces neuropathic pain following spinal cord injury.

Neuropathic pain is a long-lasting clinical problem that is often refractory to medical management. Gene transfer of specific genes for therapeutic benefit offers a novel approach to the treatment of neuropathic pain. In this study, we tested whether the transfer of the glutamic acid decarboxylase (GAD) gene to dorsal root ganglion (DRG) cells would attenuate below-injury level central neuropathic pain after spinal cord injury (SCI) by using a novel human foamy virus (HFV) vector to achieve release of gamma-aminobutyric acid (GABA). Subcutaneous inoculation of a replication-defective HFV vector, which expresses GAD (vector rdvGAD67) for 7days after T13 spinal cord hemisection, reversed mechanical allodynia and thermal hyperalgesia evoked by SCI. The antiallodynic effect lasted 6 weeks and was reestablished by reinoculation. We also found that subcutaneous inoculation of rdvGAD67 resulted in enhanced production of GAD and tonical GABA release from transduced DRG neurons. These results suggest that HFV-mediated gene transfer to DRG could be employed to treat below-injury level central neuropathic pain after incomplete SCI.

Cloning and functional identification of two novel BRCA1 splicing variants.

BRCA1 is an important tumor suppressor gene associated with inherited breast and ovarian cancers. In this investigation, two novel BRCA1 splicing variants were cloned from breast cancer cell line ZR-75-30. These transcripts, named BRCA1-PI21-Delta2-21 and BRCA1-Delta2-14, lacked most exons of full length BRCA1 gene, but maintained the original reading frame. We also demonstrated the presence of BRCA1-PI21-Delta2-21 in several human cell lines. Expression of both variants fused with green fluorescent protein (GFP) showed that they targeted different subcellular compartments in the transfected cells. Viability of the cells expressing both fusion proteins decreased notably compared with the viability of cells expressing only GFP. Fluorescence activated cell sorting assay confirmed that the overexpression of two splicing variants resulted in cell apoptosis. Taken together, the different subcellular localization and cell effects of two BRCA1 splicing variants imply that they can have different biological functions in breast cancer cells. Elucidating the functions of BRCA1 splicing variants would help to understand the exact roles of the BRCA1 gene in tumor suppression.

PAD1 promotes epithelial-mesenchymal transition and metastasis in triple-negative breast cancer cells by regulating MEK1-ERK1/2-MMP2 signaling.

Peptidylargininedeiminase 1 (PAD1) catalyzes protein for citrullination, and this activity has been linked to the epidermal cornification. However, a role for PAD1 in tumorigenesis, including breast cancers has not been previously explored. Here we first showed that PAD1 is overexpressed in human triple negative breast cancer (TNBC). In cultured cells and xenograft mouse models, PAD1 depletion or inhibition reduced cell proliferation, suppressed epithelial-mesenchymal transition, and prevented metastasis of MDA-MB-231 cells. These changes were correlated with a dramatic decrease in MMP2/9 expression. Furthermore, ERK1/2 and P38 MAPK signaling pathways are activated upon PAD1 silencing. Treatment with MEK1/2 inhibitor in PAD1 knockdown cells significantly recovered MMP2 expression, while inhibiting P38 activation only slightly elevated MMP9 levels. We then showed that PAD1 interacts with and citrullinates MEK1 thereby disrupting MEK1-catalyzed ERK1/2 phosphorylation, thus leading to the MMP2 overexpression. Collectively, our data indicate that PAD1 appears to promote tumorigenesis by regulating MEK1-ERK1/2-MMP2 signaling in TNBC. These results also raise the possibility that PAD1 may function as an important new biomarker for TNBC tumors and suggest that PAD1-specific inhibitors could potentially be utilized to treat metastatic breast cancer.

PSA and Prostate Health Index based prostate cancer screening in a hereditary migration complicated population: implications in precision diagnosis.

Precision diagnosis requires specific markers for differential ethnic populations. Prostate-Specific Antigen (PSA) level (threshold of 4ng/ml) has been widely used to screen prostate cancer and as reference of pro-biopsy but false diagnosis frequently occurs. Prostate health Index (PHI) is a new diagnosis marker which combines PSA, free PSA and p2PSA4. Overall the PCa screening database is lacking in Kazakhstani patients. We analyzed the PSA levels and Gleason scores of 222 biopsies collected in 2015 in Almaty area, Kazakhstan approved by institutional ethics board. We found using PSA of 4ng/ml as threshold, only 25.68% of patients have cancer with Gleason score ranged 6-8 and 65.77% of patients have no character of cancer. Moreover, there is no significant correlation between PSA and cancerous (P=0.266) or Gleason grade (P=0.3046) based on pathological biopsy. In addition, PHI is not correlated to prostate cancer (P=0.4301). Our data suggest that false-positive rate is much higher than the correct-positive diagnosis when using PSA as the first screening. Thus in this cohort study, most patients can not get benefit from the PSA screening for precision PCa diagnosis. As Kazakhstani family trees are unique and complicated because of history and migration, the high rate of over diagnosis might be due to the hyperexpression of PSA via heterosis in Eurasian men. Therefore we should be cautious when using pro-biopsy in precision diagnosis for Eurasian prostate cancer patients.

Nuclear matrix metalloproteinases: functions resemble the evolution from the intracellular to the extracellular compartment.

Matrix metalloproteinase (MMP) is defined as an endopeptidase in the extracellular matrix (ECM), which plays essential roles in physiological processes such as organogenesis, wound healing, angiogenesis, apoptosis and motility. MMPs are produced and assembled in the cytoplasm as proenzymes with a cytoplasmic domain and require extracellular activation. MMPs can degrade receptors, extracellular matrix proteins, PARPs and release apoptotic substances. MMPs have been found in the cytosol, organelles and extracellular compartments and recently many types of MMPs have been found in the nucleus. However, the mechanisms and roles of MMPs inside the cell nucleus are still poorly understood. Here we summarized the nuclear localization mechanisms of MMPs and their functions in the nucleus such as apoptosis, tissue remodeling upon injury and cancer progression. Most importantly, we found that nuclear MMPs have evolved to translocate to membrane and target ECM possibly through evolution of nuclear localization signal (NLS), natural selection and anti-apoptotic survival. Thus, the knowledge about the evolution and regulation of nuclear MMPs appears to be essential in understanding a variety of cellular processes along with the development of MMP-targeted therapeutic drugs against the progression of certain diseases.

Analysis of origin and protein-protein interaction maps suggests distinct oncogenic role of nuclear EGFR during cancer evolution.

Receptor tyrosine kinase EGFR usually is localized on plasma membrane to induce progression of many cancers including cancers in children (Bodey et al. In Vivo. 2005, 19:931-41), but it contains a nuclear localization signal (NLS) that mediates EGFR nuclear translocation (Lin et al. Nat Cell Biol. 2001, 3:802-8). Here we report that NLS of EGFR has its old evolutionary origin. Protein-protein interaction maps suggests that nEGFR pathways are different from membrane EGFR and EGF is not found in nEGFR network while androgen receptor (AR) is found, which suggests the evolution of prostate cancer, a well-known AR driven cancer, through changes in androgen- or EGF-dependence. Database analysis suggests that nEGFR correlates with the tumor grades especially in prostate cancer patients. Structural predication analysis suggests that NLS can compromise the differential protein binding to EGFR through stretch linkers with evolutionary mutation from N to V. In experiment, elevation of nEGFR but not membrane EGFR was found in castration resistant prostate cancer cells. Finally, systems analysis of NLS and transmembrane domain (TM) suggests that NLS has old origin while NLS neighboring domain of TM has been undergone accelerated evolution. Thus nEGFR has an old origin resembling the cancer evolution but TM may interfere with NLS driven signaling for natural selection of survival to evade NLS induced aggressive cancers. Our data suggest NLS is a dynamic inducer of EGFR oncogenesis during evolution for advanced cancers. Our model provides novel insights into the evolutionary role of NLS of oncogenic kinases in cancers.

[An experimental study on destruction of K562 and HL60 induced by 5-aminolaevulinic acid-based photodynamic therapy].

This experiment was designed to explore the pattern of K562 and HL60 leukemia cells death, the effects on their cell cycle and the cytoplasmic free calcium concentration ([Ca2+]i) induced by 5-aminolaevulinic acid-based photodynamic therapy (ALA-PDT). Under the transmission electron microscope (TEM), two kinds of leukemia cells' ultrastructure were observed. Flow cytometry combined with Annexin V-FITC/PI labeling was used to detect the pattern of K562 and HL60 cells' death induced by ALA-PDT. Flow cytometry combined with PI labeling was used to analyze the change in the cell cycle induced by ALA-PDT, and confocal laser scanning microscopy (CLSM) combining with calcium fluorescence probe was used to detect the change in the cytoplasmic free calcium concentration ([Ca2+]i). Immediately after irradiation, many typical apoptotic bodies were seen in the cells treated. Most of the cells treated were necrotic at 24 hours following irradiation. Flow cytometry analysis suggested that the main patterns of the cells' death were apoptosis immediately after irradiation and necrosis post-apoptosis at 24 hours post irradiation. Immediately and 24 hours after irradiation, the proportion of S phase of K562 was 57. 67% +/- 1.13% and 84.77% +/- 6.20% respectively, and the proportion of S phase of HL60 was 74.60% +/- 7.27% and 84.60% 1.74% respectively. Both [Ca+]i of the treated K562 and HL60 were increased obviously. In the best experiment condition, the initial pattern of the K562 and HL60 leukemia cells' death induced by PDT was apoptosis and the main pattern was necrosis post apoptosis. The two kinds of cells were arrested at S phase by ALA-PDT. During the death of the leukemia cells, the increase in intracellular free calcium concentration could be responsible for the ALA photodynamically induced damage to K562 and HL60 cells.

[Cloning and sequencing of the proBA gene from the selected mutant resistant to proline analogue from Bacillus subtilis].

NTG was used to make chemical mutation for Bacillus subtilis 93151. An enhanced osmotolerant mutant was obtained, which could grow in minimal medium containing 14% NaCl (w/v) and was not subject to proline-mediated feedback repression. The content of the intracellular free proline from the mutant increased rapidly with the rising of NaCl concentration. A 2.3 kb DNA fragment from the mutant was amplified using PCR method. Sequence analysis indicated that three bases changed within the proB gene, compared with the wild-type strain. One of the mutations was substitution of an A for a T at nt position 781, leading to a change of a Ser to a Thr at amino acid residue 261 of the deduced protein product, while other two were silent mutations. The recombinant vector pBE2-proB could functionally complement the proline auxotrophy E. coli 1.1252. Sequence analysis of proA showed that proA and proB overlapped by 4 nt, and there was a SD sequence at nt 14 upstream of the start codon of proA. The deduced amino acid of proA gene shared a high similarity with that of Bacillus subtilis 168 (77%).

Plasma microRNA-320, microRNA-let-7e and microRNA-21 as novel potential biomarkers for the detection of retinoblastoma.

Retinoblastoma (RB) is a childhood malignancy caused by inactivation of the RB gene, with neuron-specific enolase (NSE) levels considered as its diagnostic marker. MicroRNAs (miRNAs) have been proven to play a significant role in multiple physiological and pathological processes and several miRNAs were identified as tumor biomarkers in recent studies. In the present study, 65 plasma samples were collected from RB patients and 65 samples from healthy individuals to serve as controls. The miRNA levels were measured via quantitative reverse transcription-polymerase chain reaction and their association with RB was assessed by statistical data analysis and receiver operating characteristic curves. Plasma miRNA (miR)-320, miR-let-7e and miR-21 levels were downregulated in the patient samples, the areas under the curves (AUCs) were 0.548-0.660, whereas the AUCs of combined classifiers were ≥0.990. The plasma miRNA levels, particularly of miR-320, were found to be of value in RB diagnosis and may be considered as novel diagnostic biomarkers.