Monitoring of lung malignant epithelial cells by gene methylation analysis in the conditionally reprogrammed cell cultures.

Conditionally reprogrammed cell (CRC) technology is an effective method for culturing primary malignant cells and non-malignant epithelial cells in vitro. This can be useful for precision medicine applications, such as drug sensitivity assays. However, this approach is commonly hindered by the non-specific growth of non-malignant epithelial cells in CRC cultures and the lack of effective biomarkers/assays to distinguish them from primary tumor cells. In this study, we developed a DNA methylation-based, real-time PCR assay to investigate SHOX2 and PTGER4 gene promoters as sensitive markers for human lung cancer. We first found that in formalin-fixed, paraffin-embedded (FFPE) malignant lung samples, 90% (28/31) had increased SHOX2 and/or PTGER4 promoter methylation as compared with their adjacent non-malignant samples. We then applied this assay to fresh surgical tumors and found increased SHOX2 and/or PTGER4 promoter methylation in 80% (20/25) of tumor samples as compared with their corresponding adjacent non-malignant tissues. Increased methylation of SHOX2 or PTGER4 promoter regions was also detected in 52% (13/25) of CRC cultures. The presence of malignant cells was confirmed by growth in soft agar cultures, a hallmark of malignant transformation, as well by EGFR mutation analysis. These results demonstrate that SHOX2 and PTGER4 promoter methylation levels can be used to detect malignant lung epithelial cells in CRC cultures.

Cluster-Based Haldane State in an Edge-Shared Tetrahedral Spin-Cluster Chain: Fedotovite K_{2}Cu_{3}O(SO_{4})_{3}.

Fedotovite K_{2}Cu_{3}O(SO_{4})_{3} is a candidate of new quantum spin systems, in which the edge-shared tetrahedral (EST) spin clusters consisting of Cu^{2+} are connected by weak intercluster couplings forming a one-dimensional array. Comprehensive experimental studies by magnetic susceptibility, magnetization, heat capacity, and inelastic neutron scattering measurements reveal the presence of an effective S=1 Haldane state below T≅4 K. Rigorous theoretical studies provide an insight into the magnetic state of K_{2}Cu_{3}O(SO_{4})_{3}: an EST cluster makes a triplet in the ground state and a one-dimensional chain of the EST induces a cluster-based Haldane state. We predict that the cluster-based Haldane state emerges whenever the number of tetrahedra in the EST is even.

[Application of long term video electroencephalogram and resting-state functional magnetic resonance imaging in detection of cognition in patients with benign epilepsy of childhood with centrotemporal spikes].

Objective: To study the relationship between the changes of brain network and cognition in patients with benign epilepsy of childhood with centrotemporal spikes (BECTS) by using long term video electroencephalogram (VEEG) and resting-state functional magnetic resonance imaging (RS-fMRI) technology. Methods: Eleven patients with right-handed were recruited (from April 2015 to September 2016) from epilepsy specialist outpatients and functional department of neurosurgery of Tianjin Medical University General Hospital. They all underwent the long term VEEG monitoring (one sleep cycle was included at least). According to the spike-wave index (SWI) during slow ware sleep, they were divided into two groups: SWI<50% (5 cases) and SWI≥50% (6 cases). All the patients were assessed with cognitional test including language, execution, memory and attention. They also underwent the head MRI, RS-fMRI examinations. Then the results were comparatively analysed. Results: (1)There were no statisticaly significance in sex, age, age of onset, disease course, total number of seizures, years of education (P>0.05). The Full Intelligence Quotient (FIQ) (87±18), Verbal Intelligence Quotient (VIQ) (88±15) and Performance Intelligence Quotient (PIQ) (89±20) of SWI≥50% group were lower than SWI<50% group(118±8, 114±11, 119±5) and the differences were statistically significant(P<0.05). (2)There was a negative correlation between the FIQ (P=0.002), VIQ (P=0.006), PIQ (P=0.001) and SWI. The FIQ, VIQ and PIQ had no correlation with the sex, age, age of onset, disease course, total number of seizures, years of education (P>0.05). (3)Compared with SWI<50% group, SWI≥50% group showed increased regional homogeneity (ReHo) in the bilateral precentral gyrus, premotor area and the subcortical structure, the right temporal lobe and the bilateral insular lobe(P<0.05); while they showed decreased ReHo in the posterior cingulate gyrus, right posterior inferior temporal lobe and right occipital lobe(P<0.05). Conclusion: The change of the brain network which is caused by the paradoxical and constant discharge during slow ware sleep in patients with BECTS may affect the development of cognition.

Development of coding single nucleotide polymorphic markers in the pearl oyster Pinctada fucata based on next-generation sequencing and high-resolution melting analysis.

The pearl oyster Pinctada fucata is an important commercial marine shellfish that is cultured for producing saltwater pearls. In this study, 468 single nucleotide polymorphisms (SNPs) were screened from P. fucata transcriptome data, and 119 polymorphic SNPs were successfully isolated by a two-step small-amplicon high-resolution melting assay. Of these, 88 were annotated with BLAST in the Nr database and 90 were in the open reading frame, including 16 non-synonymous SNPs and 74 synonymous SNPs; 12 SNPs were in the 3'-untranslated region (UTR) and 1 was in the 5'-UTR. Twenty-five SNPs were randomly chosen to test the genetic diversity of 40 wild individuals from Liusha Bay, China. All of the loci had two alleles. The observed and expected heterozygosities ranged from 0.0417 to 0.6042 and from 0.2945 to 0.5053, respectively. Minor allele frequencies ranged from 0.1771 to 0.5000, and the polymorphism information content ranged from 0.2516 to 0.3750. These novel SNP markers can contribute to P. fucata genetics and breeding studies.

Identification of 15 novel polymorphic microsatellite loci in pearl oyster (Pinctada fucata).

The pearl oyster Pinctada fucata is a commercially important marine shellfish. As a result, genetic improvement and selective-breeding program have been conducted for this species. Polymorphic microsatellites are effective molecular markers to investigate molecular marker-assisted selection and genetic variance. In this study, microsatellite DNAs were screened and characterized based on the partial genome sequence of P. fucata. We identified 111 microsatellite DNA motifs through mining the published draft genome sequence of P. fucata. Forty-two loci were screened with 8 P. fucata individuals, and 15 were found to be polymorphic and were therefore further evaluated using 40 wild individuals from the Daya Bay, Shenzhen City, Guangdong Province, China. The number of alleles per locus ranged from 3 to 8, with an average of 5.2667 for the 15 polymorphic loci. Observed and expected heterozygosities ranged from 0.1154 to 0.6216 (0.3321 on average) and 0.4950 to 0.8491 (0.6768 on average), respectively. Of the 15 polymorphic loci, 12 loci deviated from Hardy-Weinberg equilibrium after Bonferroni correction (P < 0.0033). Polymorphism information content ranged from 0.44 to 0.83 with a mean value of 0.63. The results suggest that the markers isolated in this study can be used for research on molecular marker-assisted selection and genetic variance of P. fucata.

Morphological analysis and muscle-associated gene expression during different muscle growth phases of Megalobrama amblycephala.

Skeletal muscle growth is regulated by both positive and negative factors, such as myogenic regulatory factors (MRFs) and myostatin (MSTN), and involves both hyperplasia and hypertrophy. In the present study, morphological changes during muscle development in Megalobrama amblycephala were characterized and gene expression levels were measured by quantitative real-time polymerase chain reaction (qRT-PCR) analysis in juvenile [60, 90, 120, and 180 days post-hatching (dph)] and adult fish. Our results show that during muscle development, the frequency of muscle fibers with a diameter <20 µm dramatically decreased in both red and white muscles, with a concomitant increase in the frequency of >30 µm fibers in red muscle and >50 µm fibers in white muscle. At 90-120 dph, the ratio of hyperplastic to hypertrophic areas in red and white muscles increased, but later decreased at 120-180 dph. The effect of hypertrophy was significantly larger than hyperplasia during these phases. qRT-PCR indicated MRF and MSTN (MSTNa and MSTNb) genes had similar expression patterns that peaked at 120 dph, with the exception of MSTNa. This new information on the molecular regulation of muscle growth and rapid growth phases will be of value to the cultivation of M. amblycephala.

Co-expression of march5b and tlr7 in large yellow croaker Larimichthys crocea in response to Cryptocaryon irritans infection.

In this study, molecular characteristics of march5b and co-expression of march5b and tlr7 in response to the infection of Cryptocaryon irritans in the large yellow croaker Larimichthys crocea were investigated. The full-length complementary (c)DNA of march5b was 1314 bp, including an open reading frame of 846 bp encoding a polypeptide of 281 amino acids, and the full-length genomic sequence was composed of 23,577 nucleotides, including six exons and five introns. The putative March5b protein contained a RINGv motif and four transmembrane domains. The march5b transcripts were broadly distributed in all detected tissues, with a strong expression in blood, brain and gills, and a weak expression in kidney by quantitative PCR analysis. The expression of march5b and tlr7 in the skin, gills, spleen and head kidney changed in the same manner at most time points post-primary infection with C. irritans. Significant increase was observed in the skin with march5b at days 2 and 3 by 26.10 and 6.88 fold, respectively, and with tlr7 at day 3 by 57.68 fold, when compared with the control. Their expressions, however, were decreased in the gills, especially at day 3 (march5b by 8.9%, tlr7 by 22.06%). In the spleen and head kidney, march5b and tlr7 transcripts were up-regulated early, then noticeably declined at day 3. These results suggested that march5b and tlr7 are co-expressed in response to parasite infection and March5b probably catalyses ubiquitination of some proteins of TLR7 signalling pathway.

Pharmacokinetics and preliminary safety data of a single oral dose of bosentan, a dual endothelin receptor antagonist, in cats.

The objective of this study was to evaluate the pharmacokinetic properties and adverse effect profile of single-dose oral bosentan, a dual endothelin receptor antagonist, in healthy cats. Pharmacokinetic parameters were determined following a single mean ± SD oral dose of 3.2 ± 0.6 mg/kg of bosentan in 6 adult cats. Blood was collected for quantification of bosentan via high-performance liquid chromatography with ultraviolet detection. Blood and urine were evaluated for CBC, plasma biochemical profile, and urinalysis, and repeat physical examinations were performed to evaluate for adverse effects. The mean terminal half-life of bosentan was 20.4 ± 17.2 h. The mean peak plasma concentration was 0.49 ± 0.24 g/mL, and the mean time to maximum plasma concentration was 6.8 ± 8.6 h. The area under the curve was 5.14 ± 3.81 h·µg/mL. Oral bosentan tablets were absorbed in cats, and no clinically important adverse events were noted. Further evaluation of repeat dosing, investigation into the in vivo efficacy of decreasing endothelin-1 concentrations in cats, as well as safety in conjunction with other medications is warranted.

Competing itinerant and local spin interactions in kagome metal FeGe.

The combination of a geometrically frustrated lattice, and similar energy scales between degrees of freedom endows two-dimensional Kagome metals with a rich array of quantum phases and renders them ideal for studying strong electron correlations and band topology. The Kagome metal, FeGe is a noted example of this, exhibiting A-type collinear antiferromagnetic (AFM) order at TN ≈ 400 K, then establishes a charge density wave (CDW) phase coupled with AFM ordered moment below TCDW ≈ 110 K, and finally forms a c-axis double cone AFM structure around TCanting ≈ 60 K. Here we use neutron scattering to demonstrate the presence of gapless incommensurate spin excitations associated with the double cone AFM structure of FeGe at temperatures well above TCanting and TCDW that merge into gapped commensurate spin waves from the A-type AFM order. Commensurate spin waves follow the Bose factor and fit the Heisenberg Hamiltonian, while the incommensurate spin excitations, emerging below TN where AFM order is commensurate, start to deviate from the Bose factor around TCDW, and peaks at TCanting. This is consistent with a critical scattering of a second order magnetic phase transition with decreasing temperature. By comparing these results with density functional theory calculations, we conclude that the incommensurate magnetic structure arises from the nested Fermi surfaces of itinerant electrons and the formation of a spin density wave order.

Selective anticancer strategies via intervention of the death pathways relevant to cell transformation.

Apoptosis is an important physiological process that promotes tissue homeostasis by eliminating unnecessary or malfunctioning cells. Abnormality in this process contributes to tumorigenesis, as well as the resistance to cancer treatment by radiation and chemotherapy. Restoration of normal apoptosis would not only promote cancer cell death and halt tumor progression, but also increase the response to many current cancer therapies. Although apoptosis induction is an important principle of currently used radiation and chemotherapy treatment, uncovering the mechanisms that govern this process, and which are lost during transformation, represents an important direction for realizing improved therapies for the future. This article first briefly reviews aspects of current discovery strategies for new anticancer therapeutics based on intervening in cell death pathways, and then discusses in more detail several cancer-relevant death pathways, which are disabled during transformation and which can be targeted therapeutically. These include anoikis/cell adhesion; energy metabolism and the unfolded protein response. Finally, we introduce a new concept, which utilizes cancer-specific apoptosis induced by oncolytic viruses. The discussion of these topics involves novel targets, compounds and virotherapy.

Efficient tuberculosis treatment in mice using chemotherapy and immunotherapy with the combined DNA vaccine encoding Ag85B, MPT-64 and MPT-83.

Although most cases of tuberculosis (TB) can be cured with antibiotics, relapse is common if patients do not continue chemotherapy for at least 6 months. Thus, improved therapeutic strategies are urgently needed. We previously found that the combined DNA vaccine encoding the Mycobacterium tuberculosis proteins Ag85B, MPT-64 and MPT-83 protected mice from TB following H37Rv challenge and considered whether this combined DNA vaccine has a therapeutic effect. In the present work, we demonstrate that boosting the efficiency of the immune system with the combined DNA vaccine may be a valuable adjunct to shorten the duration of antibacterial chemotherapy. Mice treated with the combined DNA vaccine along with isoniazid and pyrazinamide showed significantly higher interferon-gamma responses to a mixture of the three specific antigens (P<0.001), which were accompanied by a significant reduction in colony-forming unit in H37Rv-infected animals 3-5 months after treatment (P<0.001). These results suggest that the combined DNA vaccine along with conventional TB chemotherapy has strong potential for TB immunotherapy and may provide new alternatives to control the disease.

Direct comparison of optical frequency combs using a comb-injection-lock technique.

This paper demonstrates a direct comparison of optical frequency combs (OFCs) with different repetition rates without a stable intermediate laser using a single-mode comb-injection-lock technique. Two OFCs based on Ti:Sapphire mode-locked lasers were compared utilizing a single-mode diode laser for the selection and the amplification of one mode of an OFC by comb-injection, which makes the direct comb comparison possible. The frequencies of the two combs were found to agree within 0.019 Hz at 352 THz with the uncertainty of 0.25 Hz (7.1 x 10(-16) ). This is one of the best results among the comparisons of combs referenced to a microwave frequency. This technique simplifies the comb comparison utilities and can be applied even when repetition rates differ.

Overexpression of a hematopoietic transcriptional regulator EDAG induces myelopoiesis and suppresses lymphopoiesis in transgenic mice.

Erythroid differentiation-associated gene (EDAG) is a hematopoietic tissue-specific gene that is highly expressed in the earliest CD34+ lin- bone marrow (BM) cells and involved in the proliferation and differentiation of hematopoietic cells. To investigate the role of EDAG in hematopoiesis, we established an EDAG transgenic mouse model driven by human CD11a promoter. The transgenic mice showed increased mortality with severe organ infiltration by neutrophils, and the homeostasis of hematopoiesis was broken. The myelopoiesis was enhanced with expansion of myeloid cells in BM, increased peripheral granulocytes and extramedullary myelopoiesis in spleen. In contrast to myeloid cells, the lymphoid commitment was severely impaired with the B lymphopoiesis blocked at the transition from pro/pre-B I to pre-B II stage in BM and T thymocytes development blocked at the most immature stage (DN I). Moreover, we showed that EDAG was a transcriptional regulator which had transactivation activity and regulated the expression of several key transcription factors such as PU.1 and Pax5 in transgenic hematopoietic stem cells. These data suggested that EDAG was a key transcriptional regulator in maintaining the homeostasis of hematopoietic lineage commitment.

Shp-2 tyrosine phosphatase functions as a negative regulator of the interferon-stimulated Jak/STAT pathway.

Shp-2 is an SH2 domain-containing protein tyrosine phosphatase. Although the mechanism remains to be defined, substantial experimental data suggest that Shp-2 is primarily a positive regulator in cell growth and development. We present evidence here that Shp-2, while acting to promote mitogenic signals, also functions as a negative effector in interferon (IFN)-induced growth-inhibitory and apoptotic pathways. Treatment of mouse fibroblast cells lacking a functional Shp-2 with IFN-alpha or IFN-gamma resulted in an augmented suppression of cell viability compared to that of wild-type cells. To dissect the molecular mechanism, we examined IFN-induced activation of signal transducers and activators of transcription (STATs) by electrophoretic mobility shift assay, using a specific DNA probe (hSIE). The amounts of STAT proteins bound to hSIE upon IFN-alpha or IFN-gamma stimulation were significantly increased in Shp-2(-/-) cells. Consistently, tyrosine phosphorylation levels of Stat1 upon IFN-gamma treatment and, to a lesser extent, upon IFN-alpha stimulation were markedly elevated in mutant cells. Furthermore, IFN-gamma induced a higher level of caspase 1 expression in Shp-2(-/-) cells than in wild-type cells. Reintroduction of wild-type Shp-2 protein reversed the hypersensitivity of Shp-2(-/-) fibroblasts to the cytotoxic effect of IFN-alpha and IFN-gamma. Excessive activation of STATs by IFNs was also diminished in mutant cells in which Shp-2 had been reintroduced. Together, these results establish that Shp-2 functions as a negative regulator of the Jak/STAT pathway. We propose that Shp-2 acts to promote cell growth and survival through two mechanisms, i.e., the stimulation of growth factor-initiated mitogenic pathways and the suppression of cytotoxic effect elicited by cytokines, such as IFNs.

Adenovirus type 5 E1A gene products act as transformation suppressors of the neu oncogene.

The adenovirus type 5 early region 1A (E1A) gene was introduced into neu-transformed B104-1-1 cells. Cells that expressed E1A possessed reduced transforming activity in vitro and reduced tumorigenicity in nude mice. These results demonstrate that the E1A gene products can act negatively to suppress the transformed phenotype in neu-transformed cells.

Molecular mechanism for the Shp-2 tyrosine phosphatase function in promoting growth factor stimulation of Erk activity.

We have previously shown that activation of extracellular signal-regulated kinase (Erk) by epidermal growth factor (EGF) treatment was significantly decreased in mouse fibroblast cells expressing a mutant Shp-2 molecule lacking 65 amino acids in the SH2-N domain, Shp-2(Delta46-110). To address the molecular mechanism for the positive role of Shp-2 in mediating Erk induction, we evaluated the activation of signaling components upstream of Erk in Shp-2 mutant cells. EGF-stimulated Ras, Raf, and Mek activation was significantly attenuated in Shp-2 mutant cells, suggesting that Shp-2 acts to promote Ras activation or to suppress the down-regulation of activated Ras. Biochemical analyses indicate that upon EGF stimulation, Shp-2 is recruited into a multiprotein complex assembled on the Gab1 docking molecule and that Shp-2 seems to exert its biological function by specifically dephosphorylating an unidentified molecule of 90 kDa in the complex. The mutant Shp-2(Delta46-110) molecule failed to participate in the Gab1-organized complex for dephosphorylation of p90, correlating with a defective activation of the Ras-Raf-Mek-Erk cascade in EGF-treated Shp-2 mutant cells. Evidence is also presented that Shp-2 does not appear to modulate the signal relay from EGF receptor to Ras through the Shc, Grb2, and Sos proteins. These results begin to elucidate the mechanism of Shp-2 function downstream of a receptor tyrosine kinase to promote the activation of the Ras-Erk pathway, with potential therapeutic applications in cancer treatment.

Understanding the Unusual Response to High Pressure in KBe2BO3F2.

Strong anisotropic compression with pressure on the remarkable non-linear optical material KBe2BO3F2 has been observed with the linear compression coefficient along the c axis found to be about 40 times larger than that along the a axis. An unusual non-monotonic pressure response was observed for the a lattice parameter. The derived bulk modulus of 31 ± 1 GPa indicates that KBe2BO3F2 is a very soft oxide material yet with stable structure up to 45 GPa. A combination of high-pressure synchrotron powder X-ray diffraction, high-pressure Raman spectroscopy, and Density Functional Theory calculations points to the mechanism for the unusual pressure response being due to the competition between the K-F bond length and K-F-K bond angle and the coupling between the stretching and twisting vibration modes.

A combined DNA vaccine-prime, BCG-boost strategy results in better protection against Mycobacterium bovis challenge.

In this study, we demonstrated that calves vaccinated with a combined DNA vaccine encoding Ag85B, MPT- 64, and MPT-83 antigens from the Mycobacterium tuberculosis for the priming and subsequently boosting with BCG prior to experimental challenge with virulent Mycobacterium bovis (M. bovis) resulted in improved immune responses over immunizing. Vaccination with the combined DNA/BCG induced higher levels of antigen- specific gamma interferon (IFN-gamma) in whole-blood cultures 4 weeks after final vaccination and the level of antigen-specific IFN-gamma in response to Ag85, MPT-64, and MPT-83 were still higher 4 weeks after challenge when compared to the combined DNA group. There was a significant bias toward induction of CD4+ T cells rather than CD8+ T cells responses, and the mean percentage of CD4+ T cells was increased about 2.6-fold in peripheral blood mononuclear cells (PBMC) cultures in DNA prime-BCG boost vaccination when compared to the nonvaccinated group. In addition, DNA prime-BCG boost vaccination resulted in stronger humoral immune responses, and the levels of the specific antibodies to three antigens were increased two- to 32- fold when compared to the combined DNA group. Vaccination with the combined DNA/BCG induced a high level of protection against an intratracheal challenge with virulent M. bovis, based on a significant enhancement of six pathological and microbiological parameters of protection compared to the nonvaccinated group. Finally, the combined DNA/BCG increased the protective efficacy by more than 10-100-fold as measured by reduced CFU counts in the lungs from calves challenged with M. bovis compared to the combined DNA and BCG groups. These results suggest that use of the prime-boost strategy offers better protection against bovine tuberculosis than does the combined DNA vaccines and BCG.

Fyn is a redox sensor involved in solar ultraviolet light-induced signal transduction in skin carcinogenesis.

Solar ultraviolet (UV) light is a major etiological factor in skin carcinogenesis, with solar UV-stimulated signal transduction inducing pathological changes and skin damage. The primary sensor of solar UV-induced cellular signaling has not been identified. We use an experimental system of solar simulated light (SSL) to mimic solar UV and we demonstrate that Fyn is a primary redox sensor involved in SSL-induced signal transduction. Reactive oxygen species (ROS) generated by SSL exposure directly oxidize Cys488 of Fyn, resulting in increased Fyn kinase activity. Fyn oxidation was increased in mouse skin after SSL exposure and Fyn-knockout mice formed larger and more tumors compared with Fyn wild-type mice when exposed to SSL for an extended period of time. Murine embryonic fibroblasts (MEFs) lacking Fyn and cells in which Fyn expression was knocked down were resistant to SSL-induced apoptosis. Furthermore, cells expressing mutant Fyn (C448A) were resistant to SSL-induced apoptosis. These findings suggest that Fyn acts as a regulatory nexus between solar UV, ROS and signal transduction during skin carcinogenesis.

Expression of activated rat neu oncogene is sufficient to induce experimental metastasis in 3T3 cells.

Amplification or overexpression of the human neu oncogene has been shown to correlate with the number of lymph node metastases in breast cancer patients, suggesting that expression of the neu oncogene may be associated with increased metastatic potential. However, there has been no systematic study on the role of the neu oncogene in metastasis to support this correlation. In our study, mouse embryo fibroblast 3T3 cells transformed by the mutation-activated rat neu oncogene exhibited metastatic properties both in vitro and in vivo, while parental 3T3 cells did not. Monoclonal antibodies capable of inducing down-regulation of the neu-encoded p185 protein reduced the metastatic potential induced by neu. These data provide strong experimental evidence that neu oncogene expression is sufficient for the induction of metastasis in the 3T3 cell system and supply a molecular basis supporting the correlations found in clinical observation. The results also suggest that neu-specific monoclonal antibodies may have preventative or therapeutic potential for neu-induced metastasis.