Upregulated circZMIZ1 promotes the proliferation of prostate cancer cells and is a valuable marker in plasma.

Increasing evidences have proved that circular RNAs (circRNAs), identified as a specific kind of non-coding RNAs, play a potential critical role in tumorigenesis including prostate cancer. However, the function of circRNAs in human prostate cancer remain largely unknown. In this study, we demonstrated that the expression of circZMIZ1 was higher in plasma of human prostate cancer than the paired benign prostatic hyperplasia (BPH) patients' plasma. Moreover, in cultured prostate cancer cells, knockdown of circZMIZ1 inhibited cell proliferation and caused cell cycle arrest at G1. Mechanistically, we also showed that circZMIZ1 could increase the expression of androgen receptor (AR) and androgen receptor splice variant 7 (AR-V7), which may be partly contributed to the occurrence and development of prostate cancer. In conclusion, these results revealed that circZMIZ1 might serve as a novel biomarker and a treatment target for prostate cancer treatment.

[Osseointegrated implant supported prostheses in the rehabilitation of severe orbital deformity in 6 cases].

Objective: To assess the effectiveness of osseointegrated implant supported prostheses in the rehabilitation of severe orbital deformity. Methods: Retrospective case series. The study collected 6 patients (6 eyes) with severe orbital deformity, who were treated with osseointegrated implant supported prostheses between 2010 and 2016 in Beijing Tongren Eye Center, Beijing Tongren Hospital. Data included demographic characteristics, causes of the deformity, the history of radiotherapy, the site, number and survival of implants, and the ability to wear prostheses. Results: Among the 6 patients, 4 were males, and 2 were females, with a mean age of 27 (16-44) years. The deformity resulted from evisceration or enucleation and radiotherapy for malignancies in 4 patients, from evisceration because of inflammatory pseudotumor in 1 patient, and from enucleation and debridement because of explosion injury and secondary infection in 1 patient. Each patient received 3 implants at the first operation. A total of 18 implants were installed, including 9 placed into the lateral aspect of the supraorbital rim, 6 into the lateral aspect of the infraorbital rim, 1 into the medial aspect of the supraorbital rim, and 2 into the medial aspect of the infraorbital rim. One superior lateral implant failed half a year after implantation, and an additional implant was implanted into the medial aspect of the inferior medial orbital rim for prostheses retention. All the patients were followed up for more than 2 years. No other failures were observed. The soft tissue reaction was acceptable in all patients. All of them were able to wear prostheses with satisfying appearance. Conclusions: Osseointegrated implants provid excellent retention for orbital prostheses. This technique could be used in patients with severe orbital deformity to improve their life quality. (Chin J Ophthalmol, 2019, 55: 665-669).

Differences in expression of YKL-40 and TLR4 in nasal sinus mucosa of chronic sinusitis patients with and without nasal polyps.

This work studies the expression differences of YKL-40 and TLR4 in nasal sinus mucosa of chronic sinusitis patients with and without nasal polyps and its clinical significances. Fifty chronic sinusitis patients with nasal polyps and 50 chronic sinusitis patients without, accepted by our hospital during February 2016-February 2017, were included and taken as group A and group B, respectively. In addition, another 50 patients with nasal deviation were taken as group C (control group). The ostiomeatal complex mucosa of group A and B and the inferior turbinate mucosa of group C were taken and the fluorescence quantitative PCR method was applied to detect the expression of YKL-40, TLR4 and NF- κB of the mucosa and explore and influence of YKL-40 and TLR4 on NF-κB. There was a negative correlation between YKL-40 and TLR4 in group A, and the difference was statistically significant (P less than 0.05) while there was no relationship between YKL-40 and TLR4 expression in group B. The level of YKL-40 protein in group A was higher than that in group B, which was statistically significant (P less than 0.05). YKL-40 and TLR4 were positively correlated in group A while there was no correlation between YKL- 40 and TLR4 expression in group B. The expression of YKL-40, TLR4 and NF-κB in chronic sinusitis patients with nasal polyps was high. In addition, there was a negative correlation between YKL-40 and TLR4 expression in chronic sinusitis patients with nasal polyps. YKL-40 and TLR4 interacted with each other to activate NF-κB and promote disease progression.

Influences of low level of dietary calcium on bone characters in laying hens.

Cage layer fatigue (CLF), which is commonly caused by calcium deficiency in the feed, leads to loss of structural bone and increase of bone fragility. In order to investigate the influence of low-calcium diets on bone quality and strength, histopathology, and egg quality, 72 laying hens were randomly allocated to 2 groups at 22 wk of age and received low calcium and control calcium until 34 wk, respectively. Egg production, feed consumption, BW, and egg quality were measured throughout. Bone mineral density, bone biomechanical properties, and histomorphology of femurs and tibias were assessed after birds were sacrificed in 26, 30, and 34 wk. The results showed that low-calcium (1.5%) diets decreased BW, feed consumption, and egg production. The broken eggs rate increased, and the eggshell strength and thickness were lower in treated birds than those in control birds at 30 wk and 34 wk. Femoral and tibial bone index and bone mineral density were lower, cortical thicknesses were thinner, and bone length were shorter over time when birds are in a low-calcium diet than those in control birds. In biomechanical properties, the values of stiffness, Young's modulus, and breaking strength were lower in both femurs and tibias in low-calcium hens at 30 wk and 34 wk than those in bones of control hens. In histomorphology of bone, the cortex turned thinner and there were more cavities in cortex and cancellous bone; the trabecular bone network was fewer, thinner, less cohesive, and generally fragmented; and trabeculae were less well-connected in low-calcium birds. Some cell nuclei in cancellous bone disappeared, and vacuolation was observed in bone cells. There appeared osteoid in cortex bone and cancellous bone in tibias. It was concluded that low-calcium diets could facilitate the development of osteoporosis characterized by an increase of osteoid and loss of structural bone and decrease the values of bone quality and strength, accompanied with a decrease in egg production and egg qualities, which may elucidate the developing mechanism of CLF.

The single nucleotide polymorphism rs700518 is an independent risk factor for metabolic syndrome and benign prostatic hyperplasia (MetS-BPH).

Studies have shown that 48.59% of benign prostate hyperplasia (BPH) is combined with metabolic syndrome (MetS). The mainstream view supports the correlation between MetS and BPH, but the pathogenesis of MetS-BPH is not fully understood. Four hundred and seventy-four men, aged 47 years or older, were recruited into this study by consecutive routine physical examination programs, and several parameters were obtained from each participant. Based on the diagnosis of BPH, MetS, and MetS-BPH, the participants were divided into BPH and Non-BPH groups, MetS and Non-MetS groups, as well as MetS-BPH and Non-MetS-BPH groups. The values of the obtained parameters were evaluated using Student's t-test, chi-square test, and logistic regression analysis. The value of estradiol (E2) was higher in the diseased groups (BPH, MetS, and MetS-BPH groups) compared with the corresponding control groups (Non-BPH, Non-MetS, and Non-MetS-BPH groups), and the differences were statistically significant. Also, E2 had an independent association with BPH (OR = 2.286, 95% CI: 1.723-3.593, p < 0.001), MetS (OR = 1.406, 95% CI: 0.585-2.315, p < 0.001), and MetS-BPH (OR = 1.249, 95% CI: 0.795-1.962, p < 0.001). Regarding SNPs of CYP19A1 gene, both the rs4646 genotypes (CC, CA, and AA) and the rs700518 genotypes (CC, CT, and TT) were present in every group, and all genotypes had statistically significant differences between the diseased and corresponding control groups. However, only the TT genotype of rs700518 was independently associated with BPH, MetS, and MetS-BPH after adjusting for age. The TT genotype of rs700518 is an independent risk factor for the MetS-BPH populations, and the CYP19A1 gene regulation of estrogen leads to MetS-BPH.

Role of E2F-1 in chemosensitivity.

The E2F family of transcription factors, in partnership with DP proteins, is thought to regulate transcription of genes that encode protein products that are required for DNA synthesis, which include important cancer chemotherapeutic targets such as thymidylate synthase and dihydrofolate reductase. This study was conducted to investigate the effects of overexpression of human E2F-1 cDNA on chemosensitivity in a human fibrosarcoma cell line, HT-1080. The E2F-1-overexpressing HT-1080 cells had a shorter doubling time both in vitro and in vivo. Associated with an up-regulation of TS, E2F-1-transfected cells were more resistant to 5-fluorouracil than were untransfected cells. These E2F-1 transfectants, although resistant to fluoropyrimidines and serum deprivation, were more sensitive to etoposide, doxorubicin, and SN38 (the active metabolite of irinotecan) but not to Taxol.

Post-transplant methotrexate administration leads to improved curability of mice bearing a mammary tumor transplanted with marrow transduced with a mutant human dihydrofolate reductase cDNA.

To test the concept that protection of bone marrow progenitor cells via introduction of a drug resistance gene would allow larger and curative doses of chemotherapy to be administered, we used mice bearing a transplanted breast cancer as a model system. Mice bearing the E0771 tumor were treated with lethal doses of cyclophosphamide (CPA) and rescued from toxicity by administration of bone marrow transduced with a mutant dihydrofolate reductase (DHFR) cDNA (Ser-31) in a retroviral construct. Animals receiving marrow not transduced with mutant DHFR cDNA died from methotrexate (MTX) toxicity, whereas mice transduced with mutant DHFR cDNA containing marrow were able to tolerate MTX treatment post-transplant; 44% of the mice had no demonstrable tumor when sacrificed on day 52. Another control group of mice treated with CPA and transplanted but not treated with MTX post-transplant succumbed to tumor regrowth. These data provide a strong rationale for the use of drug resistance genes to protect marrow from drug toxicity because the increase in dose tolerated may result in an improved cure rate of chemosensitive tumors.

Expression of a novel double-mutant dihydrofolate reductase-cytidine deaminase fusion gene confers resistance to both methotrexate and cytosine arabinoside.

A novel fusion gene consisting of the open reading frame of a double-mutant (Phe22-Ser31) dihydrofolate reductase (dmDHFR) cDNA fused to the open reading frame of cytidine deaminase (CD) was constructed and characterized for the purpose of conferring simultaneous resistance to methotrexate (MTX) and cytosine arabinoside (ara-C). The kinetic properties of purified recombinant dmDHFR-CD fusion protein were compared with those of purified CD and dmDHFR. The fusion protein was found to retain enzymatic properties of both dmDHFR and CD, in that the Km and Kcat values of purified dmDHFR-CD protein were found to be virtually identical to those of CD and dmDHFR alone. Retrovirus-mediated expression of dmDHFR-CD in NIH 3T3 cells conferred significant resistance (10- to 12-fold) against MTX and ara-C, compared with mock- and single gene-infected cells and the level of resistance obtained was similar to that of cells expressing both CD and dmDHFR from a retroviral bicistronic vector. Infection of mouse bone marrow cells with the dmDHFR-CD construct also showed high levels of resistance to MTX and ara-C in a CFU-GM assay. This fusion protein confers resistance to two antineoplastic agents that differ in their mechanism of action, and may be useful in the design of gene transfer strategies for protection of target cells against multiple drugs. Since high-dose ara-C and MTX are used in the treatment of lymphomas, this vector may be of value in protecting human hematopoietic progenitor cells from the toxicity of these antimetabolites.

Comparison of the expression of a mutant dihydrofolate reductase under control of different internal promoters in retroviral vectors.

To determine the effect of different promoters on the expression of an altered dihydrofolate reductase (DHFR) gene conferring methotrexate (MTX) resistance in different cell types, double-copy retroviral vectors were constructed carrying a murine mutant DHFR under the control of five different promoters, i.e., human adenosine deaminase (ADA), simian virus 40 (SV40), thymidine kinase (TK), human beta-actin, and cytomegalovirus (CMV). Their expression was compared in NIH-3T3 cells, three human leukemia cell lines, and mouse bone marrow. The variant DHFR is readily expressed from these various promoters in retroviral vectors at a selectable level. In 3T3 cells, the DHFR constructs containing the SV40 promoter conferred the highest levels of resistance to MTX. In K562 and Raji cells, the construct with the TK promoter produced the highest level of resistance. However granulocyte-macrophage colony-forming unit (CFU-GM) colonies from mouse marrow were more resistant to MTX when infected with vectors containing the SV40 promoter and ADA promoter as compared to the other promoter constructs. These studies show that mouse fibroblast cell lines such as NIH-3T3 do not predict the effectiveness of retroviral-mediated gene transfer for marrow progenitor cells, and that the activity of retroviral vector-encoded promoters vary in an unpredictable manner from cell type to cell type. Possible implications for basic gene transfer studies and clinical applications are discussed.

Transfection with a cDNA encoding a Ser31 or Ser34 mutant human dihydrofolate reductase into Chinese hamster ovary and mouse marrow progenitor cells confers methotrexate resistance.

Chinese hamster ovary (CHO) DHFR- cells were converted into the DHFR+ phenotype when they were transfected with a mammalian expression vector carrying human dihydrofolate reductase-encoding cDNAs (DHFR) containing a Ser31 or a Ser34 mutation. Furthermore, transfection of these mutants into wild-type CHO cells resulted in resistance to high levels of methotrexate (MTX), indicating that these human variants can act as dominant selectable markers. Southern blot analysis and polymerase chain reaction amplifications confirmed that the transfected plasmids were integrated into the CHO DNA. Gene copy number analysis revealed that both the Ser3 1 and the Ser3.4 mutants amplifiable when grown in increasing concentrations of MTX. Retrovirus-mediated gene transfer of the Ser31 mutant into mouse marrow progenitor cells also resulted in MTX-resistant CFU-GM (colony-forming unit-granulocyte macrophage) cells.

Transfection of a nonactive site mutant murine DHFR cDNA (the tryptophan 15 mutant) into Chinese hamster ovary and mouse marrow progenitor cells imparts MTX resistance in vitro.

Chinese hamster ovary (CHO) DHFR-cells were converted to the DHFR+ (dihydrofolate reductase) phenotype when transfected with a mammalian expression vector containing the murine mutant Trp15 DHFR cDNA. Transfection of the Trp15 DHFR cDNA into wild-type CHO cells resulted in resistance to high levels of methotrexate (MTX) in vitro indicating that this mutant DHFR cDNA can act as a dominant marker. Southern and Northern blot analyses of transfected cells indicated that the transfected mutant DHFR cDNA was integrated and expressed. Gene copy number analysis showed that the Trp15 cDNA was amplifiable in increasing concentrations of MTX. Transfection of murine bone marrow progenitor cells with the Trp15 mutant DHFR cDNA resulted in MTX resistant colony forming units-granulocyte macrophage.

Purine salvage rescue by xanthine-guanine phosphoribosyltransferase (XGPRT) potentiates methotrexate resistance conferred by transfer of a mutated dihydrofolate reductase gene.

We have previously shown that successful gene transfer of a mutated dihydrofolate reductase (DHFR) cDNA confers resistance to methotrexate (MTX) upon infected cells. We constructed a retrovirus vector, DC/SV6S31GPT, which carries both the Escherichia coli xanthine-guanine phosphoribosyltransferase gene and the mutated Serine 31 DHFR gene. Mouse fibroblast NIH3T3 cells infected with DC/SV6S31 GPT are more resistant to MTX than cells infected with DC/SV6S31, which carries the Serine 31 DHFR and the neomycin resistance gene cDNA. The mechanism of this augmented resistance is the increased salvaging of purines due to expression of xanthine-guanine phosphoribosyltransferase, as the augmentation does not occur when dialyzed serum, containing little xanthine or guanine, is used for cytotoxicity assays. These results indicate that coexpression of a metabolically related gene can potentiate the resistance carried by a drug resistance gene. This vector may be useful in clinical gene therapy to protect bone marrow from the toxic effects of MTX.

Retroviral transduction of human dihydropyrimidine dehydrogenase cDNA confers resistance to 5-fluorouracil in murine hematopoietic progenitor cells and human CD34+-enriched peripheral blood progenitor cells.

Severe 5-fluorouracil (5-FU) toxicity has been reported among patients lacking dihydropyrimidine dehydrogenase (DPD) enzymatic activity. DPD is the principal enzyme involved in the degradation of 5-FU to 5'-6'-dihydrofluorouracil, which is further metabolized to fluoro-beta-alanine. We demonstrate here that overexpression of human DPD confers resistance to 5-FU in NIH3T3 cells, mouse bone marrow cells, and in human CD34+-enriched hematopoietic progenitor cells. An SFG-based dicistronic retroviral vector containing human DPD cDNA, an internal ribosomal entry site (IRES), and the neomycin phosphotransferase (Neo) gene was constructed (SFG-DPD-IRES-Neo). Transduced NIH3T3 cells demonstrated a 2-fold (ED50) increase in resistance to a 4-hour exposure of 5-FU in comparison to nontransduced cells. Expression of DPD was confirmed by Northern and Western blot analyses, and DPD enzyme activity was detectable only in transduced cells. Infection of mouse bone marrow cells with this retroviral construct resulted in an increased number of 5-FU-resistant CFU-GM colonies, compared to mock-transduced bone marrow in both 4-hour and 12- to 14-day exposures. Infection of human CD34+-enriched cells with this construct and incubation with 5-FU (10(-6) M) for 14 days also resulted in an increased number of 5-FU-resistant colonies. Retroviral transduction of human hematopoietic progenitor cells with a cDNA-expressing human DPD conferred resistance to 5-FU in NIH3T3 cells, mouse bone marrow cells, and human CD34+-enriched cells. These results encourage the use of this gene as a method to protect patients from 5-FU myelotoxicity.

Long-term protection of recipient mice from lethal doses of methotrexate by marrow infected with a double-copy vector retrovirus containing a mutant dihydrofolate reductase.

A double-copy Moloney murine leukemia virus-based retroviral construct containing both the NEOr gene and a mutated dihydrofolate reductase cDNA (Leu 22-->Arg) was used to infect mouse bone marrow cells. The infected mouse marrow was returned to lethally irradiated mice. Primary, secondary, and even tertiary recipients transplanted with bone marrow cells infected with the recombinant virus showed protection from lethal methotrexate toxicity. The viral construct containing a SV-40 promoter in the U3 region of the 3' long terminal repeat appeared to be more effective than a similar construct containing the adenosine deaminase promoter, although both afforded protection. Evidence for integration into blood cells of both the NEOr gene and the mutated dihydrofolate reductase gene was obtained by polymerase chain reaction; sequencing of the amplified dihydrofolate reductase cDNA showed the presence of the point mutation. These results indicate that early hematopoietic progenitor cells in the mouse can be successfully transduced with a drug resistance gene.

Comparison of methotrexate resistance conferred by a mutated dihydrofolate reductase (DHFR) cDNA in two different retroviral vectors.

We previously reported the protection of hematopoietic cells from methotrexate (MTX) toxicity using an N2-based double copy vector containing serine 31 (S31)-mutated dihydrofolate reductase (DHFR) (DC/SV6S31). To examine whether the use of SFG-based dicistronic vectors will lead to improvement in gene transfer over the DC/SV6 vector, we compared the protection provided by MTX to NIH3T3 cells and hematopoietic progenitor cells infected with these retroviral constructs containing the S31 variant DHFR cDNA. In NIH3T3 cells, the 50% effective dose values of MTX conferred by the SFG vector were 8-fold higher than those obtained with the DC/SV6 vector. DHFR mRNA levels were 22-fold and 38-fold higher than that seen for the DC/SV6 vector according to Northern blot and real-time polymerase chain reaction analysis, respectively. However, DHFR protein expression and DHFR enzyme activity were only 1.5-fold and 2-fold higher in the SFG vector, respectively, indicating that the mRNA from the SFG vector is translated less efficiently than the mRNA generated from the DC/SV6 vector. Furthermore, the degree of MTX protection conferred by each vector in both mouse and human hematopoietic cells was the same. These results indicate that the in vitro transduction efficiency and transgene expression of human DHFR in hematopoietic progenitor cells is equally conferred by both vectors.

Antitumor activity of cytotropic heterogeneous molecular lipids (CHML) on human breast cancer xenograft in nude mice.

Cytotropic heterogeneous molecular lipid (CHML), which is a new anticancer agent with US patent number 5,260,067, has recently been shown to suppress tumor cell growth in multiple tumor lines and induce apoptosis in vitro (1). These results indicate that CHML may be an effective antitumor agent. In the present study, using both local injection and intravenous injection, we have investigated the suppressive effect of CHML on human breast caner cells MCF-7 xenograft in nude mice. In the local injection, CHML was introduced into nude mice implanted with human breast cancer xenograft at doses of 25 mg/tumor area (cm2), 35 mg/tumor area (cm2), or 50 mg/tumor area (cm2), once every two days, total 3 times. The inhibition of tumor growth was 81.3%, 93.8% and 100%, respectively. In the intravenous injection, the nude mice bearing MCF-7 xenografts were treated with CHML at 10 mg/kg/day, or 15 mg/kg/day, or 20 mg/kg/day, once a day, total 7 days, the growth inhibition of tumor area was 58.1%, 77.4%, and 83.9%, respectively. At the same time, the toxicity of CHML was determined through examining the number of the white blood cell (WBC) and the activity of the serum glutamic-pyruvic transaminase (SGPT). However, no evident alterations of WBC and SGPT were detected in all animals treated with CHML, suggesting that CHML has little toxicity on nude mice. Taken together, these results indicate that CHML is an effective agent that suppresses breast tumor growth and suggest the possibility of using CHML in the clinical trial in the near future.

Vas deferens occlusion by percutaneous injection of polyurethane elastomer plugs: clinical experience and reversibility.

A non-incision method of vas occlusion based on the percutaneous injection of polyurethane elastomer solution to form plugs is described. The results are based on clinical experience in 12,000 men in which only 56 cases of minor complications were recorded. Follow-up of 500 men for up to 3 years demonstrated an azoospermia rate of 98%. Plugs have been removed from 86 men and, to date, 51 have made their wives pregnant. In those from which the plugs have been removed for more than 1 year (n = 31), the pregnancy rate is 100%.

PIP: Between 1983 and 1987, clinicians have occluded the vas deferens of 12,000 men living in China with polyurethane elastomer plugs via percutaneous injection. 87% of these men were agricultural workers. Once they had secured the vas and anesthetized the area with procaine, they inserted a 6 gauge hypodermic needle to deliver 0.16-0.22 ml of the elastomer into the vas. Even though polymerization occurred in 1-3 minutes, the men had to rest for 24 hours and they could not engage in sexual intercourse for 1 month. After 1,2, and 3 years, the azoospermia rate stood at 98%. The rate of complications was .47% (56 cases). 84% of men with complications (47) suffered from local infection and 16% (9) from local hematoma. 2600 men followed for 3 years and 1000 men followed for 5 years did not experience any residual problems. This technique did not cause any changes in libido or sexual function. Clinicians have removed the plugs from 86 men and 51 of them (59%) have impregnated their wives. The duration of occluded vas deferens was 4 years. Of the 31 men whose length of time since removal was 1-2 years, the pregnant rate of their wives was 100%. The pregnancy rate for the remaining 55 men was 36%. All infants born (38 of 51 pregnancies as of mid-1989) were healthy. As of mid-2989, the oldest child was 5 years, 8 months old. This method of vasectomy is safe and effective and has high rate of reversibility. Its advantages (low complication rate, high azoospermia rate, minimal trauma, high reversibility) make it a potentially acceptable method in many cultures in the world.

Curative effects of highly active atrial natriuretic peptide on severe pregnancy-induced hypertension.

A new highly active atrial natriuretic peptide (haANP), synthesized by a solid phase technique, was given by intravenous infusion to 20 patients with severe pregnancy-induced hypertension (PIH) and the curative result of haANP was observed. Compared with basal values, supine systolic and diastolic BP was lowered significantly (P < 0.01), which may be related to the specific receptor of hANP and inhibition of renin-angiotensin-aldosterone system (RAAS). The haANP was found to possess significant effects of antispasm, detumescence and reducing proteinuria, probably by repairing mildly injured glomerulae, strong effects of diuresis and improving heart function with no side effects. Auto-antibody of hANP was found in patients with severe PIH, which affected the function of target cells of highly concentrated endogenous hANP. This auto-antibody might be one of the causes for PIH.