[A case of atypical iris corneal endothelial syndrome].

Iridocorneal endothelial syndrome is a rare ophthalmic disease, most of which are unilateral and common in women. The rate of misdiagnosis and missed diagnosis is relatively high due to its various clinical manifestations. In this case, the patient presented uncontrollable high intraocular pressure, corneal edema leading to difficult observation of corneal endothelium morphology, and accompanied by a small amount of iris neovascularization. No clearly diagnosis was made before glaucoma surgery. Further examination was performed after corneal clearance, and the final diagnosis was iris corneal endothelial syndrome (Chandler syndrome).

[Establishment of human colon cancer transplantation tumor model in normal immune mice].

Objective: Establishment of a new model of human primary colon cancer transplantation tumor in normal immune mice and to provide a reliable experimental animal model for studying the pathogenesis of colon cancer under normal immunity. Methods: Human colon cancer cells come from colon cancer patients who underwent surgery in the Affiliated Hospital of Jining Medical College in 2017. The mice in the cell control group were inoculated with phosphate buffered solution (PBS) containing colon cancer cells, the microcarrier control group was inoculated with PBS containing microcarrier 6, and the cell-microcarrier complex group was inoculated with the PBS containing colon cancer cell-microcarrier complex. The cells of each group were inoculated under the skin of the right axilla of mice by subcutaneous injection, and the time, size, tumor formation rate and pathological changes under microscope were recorded. The transplanted tumor tissue was immunohistochemically stained with the EnVisiion two-step method, and the tumor formation rate of the transplanted tumor was judged according to the proportion of positive cells in the visual field. The polymerase chain reaction (PCR) method was used to detect the expression of human-specific Alu sequence in mice tumor tissue. Results: After inoculation with tumor cells, the mice in the cell control group and the microcarrier control group did not die and did not form tumors; the mice in the cell-microcarrier complex group had palpable subcutaneous tumors in the right axillary subcutaneously on the 5th to 7th days after inoculation, and tumor formation rate is 67% (10/15), and the tumor volume can reach about 500 mm(3) 2 to 3 weeks after vaccination. The immunohistochemistry results showed that CK20, CDX-2 and carcinoembryonic antigen were all positively expressed. The PCR results showed that the expression of human-specific Alu sequence can be detected in the transplanted tumor tissue of tumor-bearing mice. Conclusion: Human primary colon cancer cells used microcarrier 6 as a carrier to form tumors in normal immunized mice, and successfully established a new model of human colon cancer transplantation tumor in normal immune mice.

Comparison of vertebral bone marrow fat assessed by 1H MRS and inphase and out-of-phase MRI among family members.

UNLABELLED: Inphase and out-of-phase magnetic resonance imaging is a robust and fast method which can provide similar vertebral bone marrow fat estimation as (1)H proton magnetic resonance spectroscopy, indicating that this technique is a potentially useful tool in both research and clinical practice. INTRODUCTION: The importance of evaluating bone marrow fat lies in the fact that osteoporosis and obesity, two disorders of body composition, are growing in prevalence. Bone fat mass can be reliably assessed using proton magnetic resonance spectroscopy ((1)H MRS), but this method is technically demanding and needs advanced post-processing unlike inphase and out-of-phase magnetic resonance imaging (MRI), which is a robust and fast method. METHODS: We compared vertebral bone marrow fat (BMF) content assessed by inphase and out-of-phase MRI and (1)H MRS using a 1.5-T MRI scanner in mothers (n = 34, aged 49.4 years), fathers (n = 31, aged 53.1 years) and their daughters (n = 40, aged 20.3 years) who participated in the CALEX family study. Signal intensity on the inphase and out-of-phase MRI was analyzed from the same location and size of the single-voxel (1)H MRS measurement. RESULTS: Positive correlations were found between (1)H MRS and inphase and out-of-phase MRI in the axial plane (r = 0.746, p < 0.001) and sagittal plane (r = 0.804, p < 0.001). The mean differences between (1)H MRS and inphase and out-of-phase MRI in the axial and sagittal planes were relatively small, at 4.13 and 2.67 %, and the agreement between techniques was 89.4 and 93.2 %, respectively. Girls had a significantly lower vertebral BMF than mothers and fathers with both methods (for all, p < 0.001). CONCLUSIONS: We conclude that inphase and out-of-phase MRI can provide similar vertebral BMF estimation as (1)H MRS, indicating that this technique is a potentially useful tool in both research and clinical practice.

Identification of hub genes correlated with diabetic retinopathy via bioinformatics methods.

OBJECTIVE: The aim of this study was to identify the hub genes and uncover the molecular mechanisms of diabetic retinopathy (DR). MATERIALS AND METHODS: We used the Gene Expression Omnibus (GEO) dataset GSE60436 in our study. After screening for differentially expressed genes (DEGs), we performed gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analysis. Subsequently, a protein-protein interaction (PPI) network was constructed using the Search Tool for Retrieval of Interacting Genes (STRING) database and visualized using the Cytoscape software. Finally, we identified 10 hub genes by cytoHubba plugin. RESULTS: A total of 592 DEGs were identified, including 203 up-regulated genes and 389 downregulated genes. The DEGs were mainly enriched in visual perception, photoreceptor outer segment membrane, retinal binding, and PI3K-Akt signaling pathway. By constructing a protein-protein interaction (PPI) network, 10 central genes were finally identified, including CNGA1, PDE6G, RHO, ABCA4, PDE6A, PDE6B, NRL, RPE65, GUCA1B and AIPL1. CONCLUSIONS: CNGA1, PDE6G, RHO, ABCA4, PDE6A, PDE6B, NRL, RPE65, GUCA1B, and AIPL1 may be potential biomarkers and therapeutic targets for DR.

Lactation is associated with greater maternal bone size and bone strength later in life.

SUMMARY: The association between lactation and bone size and strength was studied in 145 women 16 to 20 years after their last parturition. Longer cumulative duration of lactation was associated with larger bone size and strength later in life. INTRODUCTION: Pregnancy and lactation have no permanent negative effect on maternal bone mineral density but may positively affect bone structure in the long term. We hypothesized that long lactation promotes periosteal bone apposition and hence increasing maternal bone strength. METHODS: Body composition, bone area, bone mineral content, and areal bone mineral density of whole body and left proximal femur were assessed using DXA, and cross-sectional area and volumetric bone mineral density of the left tibia shaft were measured by pQCT in 145 women (mean age 48 years, range 36-60 years) 16 to 20 years after their last parturition. Hip (HSI) and tibia strength indexes (TBSI) were calculated. Medical history and lifestyle factors including breastfeeding patterns and durations were collected via a self-administered questionnaire. Weight change during each pregnancy was collected from personal maternity tracking records. RESULTS: Sixteen to 20 years after the last parturition, women who had breastfed in total more than 33 months in their life, regardless of the number of children, had greater bone strength estimates of the hip (HSI = 1.92 vs. 1.61) and the tibia (TBSI = 5,507 vs. 4,705) owing to their greater bone size than mothers who had breastfed less than 12 months (p < 0.05 for all). The differences in bone strength estimates were independent of body height and weight, menopause status, use of hormone replacement therapy, and present leisure time physical activity level. CONCLUSION: Breastfeeding is beneficial to maternal bone strength in the long run.

LncRNA FAS-AS1 inhibits the progression of non-small cell lung cancer through regulating miR-19a-5p.

OBJECTIVE: Non-small cell lung cancer (NSCLC) is one of the most common and deadly tumors in the world. LncRNA FAS-AS1 was abnormally expressed in various cancers, such as non-small cell lung cancer. However, the underlying mechanism of FAS-AS1 in NSCLC remains to be elucidated. MATERIALS AND METHODS: The levels of FAS-AS1 and miR-19a-5p were measured using qRT-PCR in NSCLC cells. MTT and cell colony formation assays were performed to detect cell proliferative capacity. Transwell assay was carried out to measure cell migration and invasion. The relationship between FAS-AS1 and miR-19a-5p was confirmed using Luciferase reporter assay. Xenograft tumor experiment was conducted to detect the tumor growth in vivo. RESULTS: FAS-AS1 was remarkably down-regulated in NSCLC cells. FAS-AS1 inhibited cell proliferation, migration, and invasion in NSCLC cells. Additionally, FAS-AS1 directly targeted miR-19a-5p and negatively regulated the expression of miR-19a-5p in NSCLC cells. Furthermore, FAS-AS1 overexpression restored the promotion of miR-19a-5p overexpression on proliferation, migration, and invasion of NSCLC cells. Additionally, suppression of FAS-AS1 abrogated the inhibitory effects of miR-19a-5p knockdown on the progression of NSCLC. FAS-AS1 suppressed the tumor growth in vivo. CONCLUSIONS: FAS-AS1 suppressed cell proliferation, migration, and invasion by sponging miR-19a-5p in NSCLC, indicating that FAS-AS1 might be a potential biomarker and therapeutic target for NSCLC.

Effects of a low-frequency sound wave therapy programme on functional capacity, blood circulation and bone metabolism in frail old men and women.

OBJECTIVE: To evaluate the effects of a low-frequency sound wave therapy programme on functional capacity, blood circulation and bone metabolism of the frail elderly. DESIGN: Single-blind, randomized, controlled trial. SETTING: Two senior service centres. SUBJECTS: Forty-nine volunteers (14 males and 35 females) aged 62-93 years with up to 12 diagnosed diseases were allocated in either the intervention group (n = 30) or control group (n = 19). INTERVENTION: The intervention group underwent sound wave therapy, 3-5 times a week for 30 minutes per session over a period of 6 months. The control group received no intervention. MAIN MEASUREMENTS: Blood pressure, functional capacity, mobility, bone density, biochemical markers, isometric muscle strength, balance, and skin surface temperature. RESULTS: Compared with the control group, the intervention group's mobility and the amount of self-reported kilometres walked per week increased by 3 km (P<0.05), while levels of cholesterol (4.97 (0.72) to 4.52 (0.65) mmol/L, P =0.019), low-density lipoprotein (2.82 (0.72) to 2.45 (0.61) mmol/L, P =0.022), bone markers of total osteocalcin (11.0 (6.5) to 10.3 (5.9) ng/mL, P =0.048)) and tartrate-resistant acid phosphatase isoform 5b (2.50 (1.0) to 2.41 (1.1) IU/L, P =0.021)) decreased. The average skin surface temperature was significantly higher during active sessions at the end of the intervention than in the beginning (P = 0.004). No change was found during placebo sessions. CONCLUSIONS: Low-frequency sound wave therapy may have the potential to promote well-being of frail elderly subjects via improved functional capacity, especially in subjects who are too frail to undertake exercise.

Increasing tuberculosis case detection through intensive referral and tracing in Hunan, China.

OBJECTIVE: To explore a feasible approach to increase case finding of tuberculosis (TB) through intensive referral and tracing of TB suspects and patients. METHODS: A quasi-experimental study was conducted in three Chinese cities. A strategic referral and tracing system was developed for the local situation in Hunan, China. Data from a 1-year monitoring of referral, tracing and diagnosis of TB suspects/cases were used to assess outcomes. RESULTS: Among 126 public general hospitals and clinics in 38 project counties, the 124 (98.4%) health facilities that participated referred an average of 10 TB suspects and cases to the TB dispensary every month. A total of 6364 suspects and 5759 cases were referred. Compared to the previous year, the number of TB suspects increased by 102.1%, from 25 719 to 51 967; the referral of TB suspects increased five-fold; 10 596 new smear-positive pulmonary tuberculosis (PTB) cases were identified; and the notification of new smear-positive PTB increased by 112.9%, from 27.1/100 000 before the project year to 57.7/100 000, a significantly higher percentage than that of non-project areas, which had a notification rate of 38.8/100 000. CONCLUSION: Intensive referral and tracing of TB suspects/patients is a feasible and effective method of increasing case finding. Strengthening administrative interventions and incentives is essential to achieve project objectives.

Trends in drug-resistant tuberculosis after the implementation of the DOTS strategy in Shenzhen, China, 2000-2013.

SETTING: The DOTS strategy has been regarded as the most cost-effective way to stop the spread of tuberculosis (TB) since its launch by the World Health Organization. OBJECTIVE: To estimate the effects of DOTS by tracking long-term trends in multidrug-resistant TB (MDR-TB). DESIGN: A retrospective cohort study was conducted from 2000 to 2013 to analyse trends in resistance to anti-tuberculosis drugs and the effect of DOTS-based treatment in Shenzhen, China, using the χ2 test. RESULTS: An overall MDR-TB rate of 4.2% was observed between 2000 and 2013, with an annual reduction of 0.16%. From 2000 to 2013, trends in resistance to isoniazid (INH), rifampicin (RMP) and MDR-TB declined significantly in new TB patients (P < 0.01), but not in retreatment cases. Sputum smear conversion rates after 2 months of treatment decreased significantly, in particular after 2007, in new and retreatment cases. CONCLUSION: INH and RMP resistance and MDR-TB rates declined significantly, suggesting that DOTS-based programmes were successful in reducing drug resistance in new cases but not in retreatment cases. The decreasing sputum smear conversion rates may have been due to an increase in the number of migrants. These two findings suggest that TB is unlikely to be completely eliminated by 2050 in Shenzhen.

Genetic and phylogenetic analysis of multi-continent human influenza A(H1N2) reassortant viruses isolated in 2001 through 2003.

Genetic analyses were performed on 228 influenza A(H1) viruses derived from clinical subjects participating in an experimental vaccine trial conducted in 20 countries on four continents between 2001 and 2003. HA1 phylogenetic analysis of these viruses showed multiple clades circulated around the world with regional prevalence patterns. Sixty-five of the A(H1) viruses were identified as A(H1N2), 40 of which were isolated from South Africa. The A(H1) sequences of these viruses cluster with published H1N2 viruses phylogenetically and share with them diagnostic signature V169A and A193T changes. The results also showed for the first time that H1N2 viruses were prominent in South Africa during the 2001-2002 influenza season, accounting for over 90% of the A(H1) cases in our study, and infecting both children (29/31) and the elderly (11/13). Phylogenetic analysis of the 65 H1N2 viruses we identified, in conjunction with the 56 recent H1N2 viruses currently available in the database, provided a comprehensive view of the circulation and evolution of distinct clades of H1N2 viruses in a temporal manner between early 2001 and mid-2003, shortly after the appearance of these recent reassortant viruses in or near year 2000.

Constitutive overexpression of DNA binding activity to the distal CCAAT box of human thymidine kinase promoter in human tumor cell lines.

We have used the sequence of the cell-cycle regulatory region of human thymidine kinase promoter to study the DNA-protein interaction in human tumor and normal cells. By performing band-shift assays and DNase I footprint analysis, we have demonstrated that human tumor cells exhibited an elevated level of binding activity to the distal CCAAT box of human thymidine kinase promoter. The expression of human thymidine kinase CCAAT-binding activity was serum or independent in human tumor cells but serum dependent in normal human diploid fibroblasts. Our results present the fact that the constitutive interaction of CCAAT-binding factor with the promoter of a cell growth-controlled gene, such as thymidine kinase, is consistent with the loss of stringent cell growth regulation associated with tumorigenic phenotype.

Development and evaluation of a secondary reference panel for BCR-ABL1 quantification on the International Scale.

Molecular monitoring of chronic myeloid leukemia patients using robust BCR-ABL1 tests standardized to the International Scale (IS) is key to proper disease management, especially when treatment cessation is considered. Most laboratories currently use a time-consuming sample exchange process with reference laboratories for IS calibration. A World Health Organization (WHO) BCR-ABL1 reference panel was developed (MR(1)-MR(4)), but access to the material is limited. In this study, we describe the development of the first cell-based secondary reference panel that is traceable to and faithfully replicates the WHO panel, with an additional MR(4.5) level. The secondary panel was calibrated to IS using digital PCR with ABL1, BCR and GUSB as reference genes and evaluated by 44 laboratories worldwide. Interestingly, we found that >40% of BCR-ABL1 assays showed signs of inadequate optimization such as poor linearity and suboptimal PCR efficiency. Nonetheless, when optimized sample inputs were used, >60% demonstrated satisfactory IS accuracy, precision and/or MR(4.5) sensitivity, and 58% obtained IS conversion factors from the secondary reference concordant with their current values. Correlation analysis indicated no significant alterations in %BCR-ABL1 results caused by different assay configurations. More assays achieved good precision and/or sensitivity than IS accuracy, indicating the need for better IS calibration mechanisms.

Characterization of a complementary deoxyribonucleic acid for the coagulogen of Limulus polyphemus.

An 869-nucleotide-long cDNA clone for the coagulogen from Limulus amebocyte has been isolated and its nucleotide sequence has been determined. The deduced amino-acid sequence revealed a signal peptide, 20 amino acids long, and a mature protein of 175 amino acids. The amino-acid sequence of the coagulogen was compared to all known proteins by two computer programs. Using these programs, Limulus coagulogen showed 70% homology with the coagulogen of Tachypleus tridentatus (Japanese horseshoe crab). Further computer analysis showed no statistically significant homology to support an evolutionary origin of the horseshoe crab coagulogen common to other protein families. These results place horseshoe crab coagulogen in a new superfamily unrelated to any other proteins investigated. RNA blot analysis of Limulus RNA indicated that the coagulogen mRNA was about 900 bases long and represented an abundant species in the amebocyte while detected only in small quantities in the hepatopancreas. Besides mature RNA, high-molecular-weight forms of coagulogen RNA were also observed. Southern blot analysis of Limulus DNA digested with restriction endonucleases suggested that the Limulus coagulogen gene contains at least three introns, or belongs to a multigene family.

YM155 down-regulates survivin and XIAP, modulates autophagy and induces autophagy-dependent DNA damage in breast cancer cells.

BACKGROUND AND PURPOSE: The aim of this study was to determine the potency and molecular mechanism of action of YM155, a first-in-class survivin inhibitor that is currently under phase I/II clinical investigations, in various drug-resistant breast cancers including the oestrogen receptor positive (ER(+) ) tamoxifen-resistant breast cancer and the caspase-3-deficient breast cancer. EXPERIMENTAL APPROACH: The potency of YM155 in SK-BR-3, MDA-MB-231, MCF7 and its tamoxifen-resistant sublines, TamR6, TamR7, TamR8, TamC3 and TamC6, were determined by MTT assay. Western blot analysis, flow cytometric analysis, reverse transcription-PCR, fluorescent microscopy and comet assay were used to determine the molecular mechanism of action of YM155 in different breast cancer cell lines. KEY RESULTS: YM155 was equally potent towards the parental ER(+) /caspase-3-deficient MCF7 breast cancer cells and its tamoxifen-resistant sublines in vitro. The ER(-) /HER2(+) SK-BR-3 breast cancer cells and the triple-negative/caspase-3-expressing metastatic aggressive MDA-MB-231 breast cancer cells were also sensitive to YM155 with IC50 values in the low nanomolar range. Targeting survivin by YM155 modulated autophagy, induced autophagy-dependent caspase-7 activation and autophagy-dependent DNA damage in breast cancer cells. Interestingly, YM155 also induced XIAP degradation and the degradation of XIAP might play an important role in YM155-induced autophagy in breast cancer cells. CONCLUSIONS AND IMPLICATIONS: YM155 is a potent survivin inhibitor that has potential for the management of various breast cancer subtypes regardless of the expression of ER, HER2 and caspase-3. Importantly, this study provides new insights into YM155's molecular mechanism of action and therapeutic potential in the treatment of tamoxifen-resistant breast cancer.

Tissue plasminogen activator (tPA) as a reporter gene in transient gene expression.

Using the gene coding for tissue plasminogen activator (tPA) as a reporter gene, a transient gene expression system has been established. Vectors containing the full-length cDNA of tPA with its signal sequences were introduced into mammalian recipient cells by a modified gene transfer procedure. Thirty hours after transfection, the secreted tPA was found in serum-free medium and measured by a fibrin-agarose plate assay (FAPA). In this assay, tPA converts plasminogen into plasmin which then degrades high-Mr fibrin to produce cleared zones. The sizes of these zones correspond to quantities of tPA. The combination of transient tPA expression system and the FAPA provides a quick, sensitive, quantitative and non-destructive method to examine the strength of eukaryotic regulatory elements in tissue-culture cells.

Characterization of the simian adenovirus type 30 inverted terminal repeat.

The presence of an inverted terminal repeat (ITR), which plays an important role in the initiation of DNA replication, is one of the characteristic properties of adenoviruses (Ads). We have established the nucleotide (nt) sequences for the ITR of simian adenovirus type 30 (SV30), a subgroup-III oncogenic virus. This repeat consists of 185 nt, representing the longest ITR found in an Ad so far. It contains multiple copies of internal repeats, as well as the consensus sequences of the putative binding sites for replication and transcription factors. The conserved features of the known ITRs are also found in SV30. Interestingly, the ITR of SV30 is more closely related to that of Ad5 (human), than to that of SA7 (simian).

Construction and expression of a hybrid plasminogen activator gene with sequences from non-protease region of tissue-type plasminogen activator (t-PA) and protease region of urokinase (u-PA).

There are two physiological plasminogen activators (PAs), tissue-type PA (t-PA) and urokinase (u-PA) which possess distinct immunological and biochemical characteristics. Using genetic engineering techniques a hybrid t:u-PA cDNA, comprised of amino acid (aa) sequences corresponding to the non-protease region (aa 1-261) of t-PA and the protease region (aa 132-411) of u-PA, was constructed. The t:u-PA gene after insertion into the SV40 expression vector was expressed in monkey Cos-1 cells. The 66-67 kDa t:u-PA was produced in an enzymatically active form. The fibrinolytic activity of the t:u-PA could be quenched by anti-urokinase as well as by anti-t-PA sera. Like urokinase, the t:u-PA showed a high intrinsic plasminogen activation. This activity, as in the case of t-PA, was stimulated by fibrin. The u-PA, on the other hand, stimulated plasminogen activation marginally in the presence of fibrin. Both the t:u-PA and t-PA showed binding affinity for fibrin clot. This study strongly suggests the autonomous nature of the structural domains in PA and also demonstrates the feasibility of shuffling these domains without loss of their functional activities.

Isolation of a human cDNA of urokinase and its expression in COS-1 cells.

The cDNA encoding human urokinase (UK) has been isolated from a cDNA library prepared from human normal fibroblast (WI38) cells, which had been stimulated by endothelial cell growth factor and heparin. This cDNA was sequenced and found to contain a few silent substitutions, thus encoding the same amino acids as deduced from the published genomic sequence of UK. After modification, the cDNA of UK was inserted into a transient expression vector and used to transfect COS-1 cells. The recombinant UK protein (rUK) in the serum-free medium of transfected COS-1 cells was characterized by biochemical and functional assays. These studies indicated that rUK from COS-1 cells is glycosylated, enzymatically active, and very similar to native single-chain plasminogen activator (scuPA). Therefore, such rUK can be a convenient source of scuPA for any further studies.

Conservation of essential sequences in the major late promoter and tripartite leader of the simian adenovirus type 30.

We report here the cloning and sequencing of the major late promoter (MLP) and the tripartite leader (TPL) from simian adenovirus type 30 (sAd30) and the comparison of the sAd30 nucleotide (nt) sequence with that of human adenoviruses (hAd). The nt sequence homology between sAd30 and hAd2 is 75% from -66 to +190 relative to the cap site. This sAd30 MLP segment contains the upstream regulatory sequence element, TATA box, and downstream regulatory sequence elements that are homologous to hAd MLP. The sAd30 upstream regulatory sequence has a small palindromic DNA sequence GTCACGTGAC, and the TATA box contains the sequence of ATAAA instead of TATAAA. The sAd30 TPL was located on the sAd30 genome as identified by sequence homology with the hAd counterpart. The splice sites of TPL introns were confirmed by sequence analysis of cDNAs synthesized from sAd30-infected cells. There is a 74.2% nt sequence homology between the TPL of sAd30 and hAd2. The conservation of these sequence elements during evolution of Ad suggests that they are essential for the transcription and translation of Ad ML transcripts.