An integrated microfluidic system for automatic detection of cholangiocarcinoma cells from bile.

Cholangiocarcinoma (CCA) is an aggressive cancer that originates from the epithelial cells lining the bile ducts. Due to its location deep within the body and nonspecific symptoms in the early stages, it is often diagnosed at the advanced stage, thus leading to worse prognosis. Circulating tumor cells within liquid biopsies (i.e. blood) have been considered as promising biomarkers for CCA diagnosis, though current methods for profiling them are not satisfactory in terms of sensitivity and specificity. Herein we developed a new cancer cell probing and immuno-tracking assay known as "CAPTURE", which was performed on an integrated microfluidic system (IMS) to automate CCA diagnosis from bile with a sample amount of only 1 mL. The assay utilized magnetic beads surface-coated with two affinity reagents, a nucleic acid aptamer (HN16) and a glycosaminoglycan (SCH 45-mix), for capturing cancer cells in bile; the "gold standard" anti-epithelial cell adhesion molecule was used as a comparison. In a single-blind test of 54 CCA-positive (+) and 102 CCA-negative (-) clinical samples, sensitivities and specificities of 96 and 80%, respectively, were documented with the CAPTURE assay on-bench. An IMS composed of a centrifugal module for sample pretreatment and a CAPTURE module for cell capture and staining was integrated with a new "vertical integration module" for detecting cancer cells from bile without human intervention. Furthermore, a novel micro-tier structure within the centrifugal module was designed to block passage of gallbladder stones with diameters >1 mm, thereby preventing their interference during the subsequent CAPTURE assay. Improved sensitivity and specificity (100 & 83%, respectively) by using three affinity reagents were achieved on the IMS when using 26 clinical bile samples, confirming its clinical bio-applicability for CCA diagnosis. This approach could be therefore used for early-stage CCA diagnostics, ideally enabling effective treatment, as well as reducing potential for relapse.

A New Phase of Networking: The Molecular Composition and Regulatory Dynamics of Mammalian Stress Granules.

Stress granules (SGs) are cytosolic biomolecular condensates that form in response to cellular stress. Weak, multivalent interactions between their protein and RNA constituents drive their rapid, dynamic assembly through phase separation coupled to percolation. Though a consensus model of SG function has yet to be determined, their perceived implication in cytoprotective processes (e.g., antiviral responses and inhibition of apoptosis) and possible role in the pathogenesis of various neurodegenerative diseases (e.g., amyotrophic lateral sclerosis and frontotemporal dementia) have drawn great interest. Consequently, new studies using numerous cell biological, genetic, and proteomic methods have been performed to unravel the mechanisms underlying SG formation, organization, and function and, with them, a more clearly defined SG proteome. Here, we provide a consensus SG proteome through literature curation and an update of the user-friendly database RNAgranuleDB to version 2.0 (http://rnagranuledb.lunenfeld.ca/). With this updated SG proteome, we use next-generation phase separation prediction tools to assess the predisposition of SG proteins for phase separation and aggregation. Next, we analyze the primary sequence features of intrinsically disordered regions (IDRs) within SG-resident proteins. Finally, we review the protein- and RNA-level determinants, including post-translational modifications (PTMs), that regulate SG composition and assembly/disassembly dynamics.

Zileuton, a 5-Lipoxygenase Inhibitor, Attenuates Haemolysate-Induced BV-2 Cell Activation by Suppressing the MyD88/NF-κB Pathway.

M1 microglia induce neuroinflammation-related neuronal death in animal models of spontaneous subarachnoid haemorrhage. Zileuton is a 5-lipoxygenase inhibitor that reduces the levels of downstream pro-inflammatory cytokines. This study aimed to investigate whether zileuton inhibits microglial activation and describe its underlying mechanisms. BV-2 cells were exposed to 1 mg/mL haemolysate for 30 min, followed by treatment with different concentrations (5, 10, 15, or 20 µM) of zileuton for 24 h. The cells were then assessed for viability, polarisation, and protein expression levels. Haemolysate increases the viability of BV-2 cells and induces M1 polarisation. Subsequent exposure to high concentrations of zileuton decreased the viability of BV-2 cells, shifted the polarisation to the M2 phenotype, suppressed the expression of 5-lipoxygenase, decreased tumour necrosis factor α levels, and increased interleukin-10 levels. Furthermore, high concentrations of zileuton suppressed the expression of myeloid differentiation primary response protein 88 and reduced the phosphorylated-nuclear factor-kappa B (NF-kB)/NF-kB ratio. Therefore, phenotype reversal from M1 to M2 is a possible mechanism by which zileuton attenuates haemolysate-induced neuroinflammation after spontaneous subarachnoid haemorrhage.

Aptamer selection against alpha-defensin human neutrophil peptide 1 on an integrated microfluidic system for diagnosis of periprosthetic joint infections.

Periprosthetic joint infections (PJIs) arising from joint arthroplasty are dreadful, yet difficult to diagnose in subtle cases. Definite diagnosis requires microbiological culture to confirm the causative pathogens. However, up to 40% of culture-negative PJI needs other surrogate biomarkers such as human neutrophil peptide 1 (HNP 1) to improve diagnostic accuracy or gauge therapeutic responses. To devise a diagnostic method, systematic evolution of ligands by exponential enrichment (SELEX) (five rounds) was used to screen PJI biomarkers on a compact (20 × 20 × 35 cm), integrated microfluidic system equipped with two separate Peltier devices and one magnetic control module where an aptamer with high affinity and specificity for HNP 1, which has been used as one of the synovial fluid (SF) biomarkers for detecting PJI, was identified for the first time. Two rounds of negative selection (with immunoglobulin G & human serum album) on-chip followed by one round of unique "competitive selection" with SF extracted from PJI patients validated the specificity of the HNP 1 aptamer. The dissociation constant was measured to be 19 nM. The applicability of SF HNP 1 levels for diagnosing PJI was then verified by a new aptamer-based enzyme-linked immunosorbent assay (ELISA)-like assay. It is envisioned that this new aptamer and the associated assay could be used in future clinical applications.

Experiment level curation of transcriptional regulatory interactions in neurodevelopment.

To facilitate the development of large-scale transcriptional regulatory networks (TRNs) that may enable in-silico analyses of disease mechanisms, a reliable catalogue of experimentally verified direct transcriptional regulatory interactions (DTRIs) is needed for training and validation. There has been a long history of using low-throughput experiments to validate single DTRIs. Therefore, we reason that a reliable set of DTRIs could be produced by curating the published literature for such evidence. In our survey of previous curation efforts, we identified the lack of details about the quantity and the types of experimental evidence to be a major gap, despite the theoretical importance of such details for the identification of bona fide DTRIs. We developed a curation protocol to inspect the published literature for support of DTRIs at the experiment level, focusing on genes important to the development of the mammalian nervous system. We sought to record three types of low-throughput experiments: Transcription factor (TF) perturbation, TF-DNA binding, and TF-reporter assays. Using this protocol, we examined a total of 1,310 papers to assemble a collection of 1,499 unique DTRIs, involving 251 TFs and 825 target genes, many of which were not reported in any other DTRI resource. The majority of DTRIs (965; 64%) were supported by two or more types of experimental evidence and 27% were supported by all three. Of the DTRIs with all three types of evidence, 170 had been tested using primary tissues or cells and 44 had been tested directly in the central nervous system. We used our resource to document research biases among reports towards a small number of well-studied TFs. To demonstrate a use case for this resource, we compared our curation to a previously published high-throughput perturbation screen and found significant enrichment of the curated targets among genes differentially expressed in the developing brain in response to Pax6 deletion. This study demonstrates a proof-of-concept for the assembly of a high resolution DTRI resource to support the development of large-scale TRNs.

Valproic Acid Reduces Vasospasm through Modulation of Akt Phosphorylation and Attenuates Neuronal Apoptosis in Subarachnoid Hemorrhage Rats.

Aneurysmal subarachnoid hemorrhage (SAH) is a devastating emergent event associated with high mortality and morbidity. Survivors usually experience functional neurological sequelae caused by vasospasm-related delayed ischemia. In this study, male Sprague-Dawley rats were randomly assigned to five groups: sham (non-SAH) group, SAH group, and three groups with SAH treated with different doses of valproic acid (VPA) (10, 20, 40 mg/kg, once-daily, for 7 days). The severity of vasospasm was determined by the ratio of cross-sectional areas to intima-media thickness of the basilar arteries (BA) on the seventh day after SAH. The BA showed decreased expression of phospho-Akt proteins. The dentate gyrus showed increased expression of cleaved caspase-3 and Bax proteins and decreased expression of Bcl-2, phospho-ERK 1/2, phospho-Akt and acetyl-histone H3 proteins. The incidence of SAH-induced vasospasm was significantly lower in the SAH group treated with VPA 40 mg/kg (p < 0.001). Moreover, all groups treated with VPA showed reversal of the above-mentioned protein expression in BA and the dentate gyrus. Treatment with VPA upregulated histone H3 acetylation and conferred anti-vasospastic and neuro-protective effects by enhancing Akt and/or ERK phosphorylation. This study demonstrated that VPA could alleviate delayed cerebral vasospasm induced neuro-apoptosis after SAH.

Therapeutic effect of and mechanisms underlying the effect of miR-195-5p on subarachnoid hemorrhage-induced vasospasm and brain injury in rats.

OBJECTIVES: There is much evidence suggesting that inflammation contributes majorly to subarachnoid hemorrhage (SAH)-induced cerebral vasospasm and brain injury. miRNAs have been found to modulate inflammation in several neurological disorders. This study investigated the effect of miR-195-5p on SAH-induced vasospasm and early brain injury in experimental rats. METHODS: Ninety-six Sprague-Dawley male rats were randomly and evenly divided into a control group (no SAH, sham surgery), a SAH only group, a SAH + NC-mimic group, and a SAH + miR-195-5p group. SAH was induced using a single injection of blood into the cisterna magna. Suspensions containing NC-mimic and miR-195-5p were intravenously injected into rat tail 30 mins after SAH was induced. We determined degree of vasospasm by averaging areas of cross-sections the basilar artery 24h after SAH. We measured basilar artery endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κ B), phosphorylated NF-κ B (p-NF-κ B), inhibitor of NF-κ B (Iκ Bα) and phosphorylated-Iκ Bα (p-Iκ Bα). Cell death assay was used to quantify the DNA fragmentation, an indicator of apoptotic cell death, in the cortex, hippocampus, and dentate gyrus. Tumor necrosis factor alpha (TNF-α) levels were measured using sample protein obtained from the cerebral cortex, hippocampus and dentate gyrus. RESULTS: Prior to fixation by perfusion, there were no significant physiological differences among the control and treatment groups. SAH successfully induced vasospasm and early brain injury. MiR-195-5p attenuated vasospasam-induced changes in morphology, reversed SAH-induced elevation of iNOS, p-NF-κ B, NF-κ B, and p-Iκ Bα and reversed SAH-induced suppression of eNOS in the basilar artery. Cell death assay revealed that MiR-195-5p significantly decreased SAH-induced DNA fragmentation (apoptosis) and restored TNF-α level in the dentate gyrus. CONCLUSION: In conclusion, MiRNA-195-5p attenuated SAH-induced vasospasm by up-regulating eNOS, down-regulating iNOS and inhibiting the NF-κ B signaling pathway. It also protected neurons by decreasing SAH-induced apoptosis-related cytokine TNF-α expression in the dentate gyrus. Further study is needed to elucidate the detail mechanism underlying miR-195-5p effect on SAH-induced vasospasm and cerebral injury. We believe that MiR-195-5p can potentially be used to manage SAH-induced cerebral vasospasm and brain injury.

Nr4A1 modulates inflammation-associated intestinal fibrosis and dampens fibrogenic signaling in myofibroblasts.

Intestinal fibrosis is a common complication of the inflammatory bowel diseases (IBDs), contributing to tissue stiffening and luminal narrowing. Human nuclear receptor 4A 1 (NR4A1) was previously reported to regulate mesenchymal cell function and dampen fibrogenic signaling. NR4A1 gene variants are associated with IBD risk, and it has been shown to regulate intestinal inflammation. Here, we tested the hypothesis that NR4A1 acts as a negative regulator of intestinal fibrosis through regulating myofibroblast function. Using the SAMP1/YitFc mouse, we tested whether two pharmacological agents known to enhance NR4A1 signaling, cytosporone B (Csn-B) or 6-mercaptopurine (6-MP), could reduce fibrosis. We also used the dextran sulfate sodium (DSS) model of colitis and assessed the magnitude of colonic fibrosis in mouse nuclear receptor 4A 1 (Nr4a1-/-) and their wild-type littermates (Nr4a1+/+). Lastly, intestinal myofibroblasts isolated from Nr4a1-/- and Nr4a1+/+ mice or primary human intestinal myofibroblasts were stimulated with transforming growth factor-ß1 (TGF-ß1), in the presence or absence of Csn-B or 6-MP, and proliferation and ECM gene expression assessed. Csn-B or 6-MP treatment significantly reduced ileal thickness, collagen, and overall ECM content in SAMP1/YitFc mice. This was associated with a reduction in proliferative markers within the mesenchymal compartment. Nr4a1-/- mice exposed to DSS exhibited increased colonic thickening and ECM content. Nr4a1-/- myofibroblasts displayed enhanced TGF-ß1-induced proliferation. Furthermore, Csn-B or 6-MP treatment was antiproliferative in Nr4a1+/+ but not Nr4a1-/- cells. Lastly, activating NR4A1 in human myofibroblasts reduced TGF-ß1-induced collagen deposition and fibrosis-related gene expression. Our data suggest that NR4A1 can attenuate fibrotic processes in intestinal myofibroblasts and could provide a valuable clinical target to treat inflammation-associated intestinal fibrosis.NEW & NOTEWORTHY Fibrosis and increased muscle thickening contribute to stricture formation and intestinal obstruction, a complication that occurs in 30%-50% of patients with CD within 10 yr of disease onset. More than 50% of those who undergo surgery to remove the obstructed bowel will experience stricture recurrence. To date, there are no drug-based approaches approved to treat intestinal strictures. In the current submission, we identify NR4A1 as a novel target to treat inflammation-associated intestinal fibrosis.

An aptamer interacting with heat shock protein 70 shows therapeutic effects and prognostic ability in serous ovarian cancer.

Ovarian cancer (OvCa) is the most lethal gynecologic malignancy owing to its high chemoresistance and late diagnosis, which lead to a poor prognosis. Hence, developing new therapeutic modalities is important for OvCa patient treatment. Our previous results indicated that a novel aptamer, Tx-01, can specifically recognize serous carcinoma cells and tissues. Here, we aim to clarify the clinical role and possible molecular mechanisms of Tx-01 in OvCa. Immunostaining and statistical analysis were performed to detect the interaction of Tx-01 and heat shock protein 70/Notch1 intracellular domain (HSP70/NICD) in OvCa. The in vitro and in vivo experiments were carried out to demonstrate the potential mechanisms of Tx-01. Results show that Tx-01 reduced serous OvCa OVCAR3 cell migration and invasion and inhibited HSP70 nuclear translocation by interrupting the intracellular HSP70/NICD interaction. Furthermore, Tx-01 suppressed serous-type OVCAR3 cell tumor growth in vivo. Tx-01 acts as a prognostic factor through its interaction with membrane-bound HSP70 (mHSP70 that locates on the cell surface without direct interaction to NICD) on ascitic circulating tumor cells (CTCs) and is reported to be involved in natural killer (NK) cell recognition and activation. Our data demonstrated that Tx-01 interacted with HSP70 and showed therapeutic and prognostic effects in serous OvCa. Tx-01 might be a potential inhibitor for use in serous OvCa treatment.

Optimization of aptamer selection on an automated microfluidic system with cancer tissues.

Cancer is among the world's most deadly inflictions, and early diagnosis is critical. Aptamers have shown utility as cancer probes since they can be screened rapidly in vitro against cancer tissues using systematic evolution of ligands by exponential enrichment (SELEX) process. However, bench-top SELEX procedures are relatively labor-intensive and time-consuming; ideally, they could instead be carried out on microfluidic devices, yet this requires optimization of buffer and reaction conditions. Herein an integrated microfluidic system (IMS) was established to automatically carry out the optimization of aptamer selection. A "formulation chip" was developed that could mix salt solutions at differing final concentrations, and the resulting optimal binding buffer was transferred to another "optimization-SELEX chip" for the following tissue-SELEX. Two aptamers were successfully screened; one of which, H-45, exhibited high specificity and affinity towards ovarian cancer tissue samples, suggesting that this IMS might be a promising device for screening of cancer associated aptamers for cancer diagnosis.

Mechanisms and therapeutic implications of RTA 408, an activator of Nrf2, in subarachnoid hemorrhage-induced delayed cerebral vasospasm and secondary brain injury.

OBJECTIVES: More and more evidence suggests oxidative stress and inflammation contribute importantly to subarachnoid hemorrhage (SAH)-induced cerebral vasospasm and secondary brain injury. Recent evidence indicates Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) increases the expression of antioxidant genes and decreases the expression of pro-inflammatory genes. This study examines the effects of an activator of Nfr2, RTA 408, on SAH-induced cerebral vasospasm and possible mechanism underlying its effect in a two-hemorrhage rodent model of SAH. METHODS: We randomly assigned 60 Sprague-Dawley male rats (350 to 420g) to five groups twelve rats each: one control group (no SAH), one untreated SAH only group and three RTA-408 treatment groups (SAH+ RTA 408 0.5 mg/kg/day, SAH+RTA 408 1 mg/kg/day and a SAH+RTA 408 1.5 mg/kg/day). The treatment groups were administered RTA 408 by intraperitoneal injection thirty min following first induction of SAH for seven days starting with first hemorrhage. Cerebral vasospasm was determined by averaging the cross-sectional areas of basilar artery 7 days after first SAH. Expressions of Nrf2, NF-κB and iNOS in basilar artery and expressions of Nrf2, HO-1, NQO1 and Cleaved caspase-3 were evaluated. Tissue TNF-alpha was assessed by ELISA using the protein sampled from the dentate gyrus, cerebral cortex, and hippocampus. RESULTS: Prior to perfusion fixation, there were no significant physiological differences among the control and treated groups. RTA 408 treatment attenuated the morphological changes caused by cerebral vasospasm. It mitigated SAH-induced suppression of Nrf2 and increased expression of NF-κB and iNOS in the basilar artery. In dentate gyrus, it reversed SAH-decreases in Nrf2, HO-1, NQO-1 and cleaved caspase-3 and RTA 408 1.5 mg/kg/day reversed SAH increases in TNF-alpha. CONCLUSION: It was concluded that RTA 408 reversal vasospasm was achieved via increases in Nrf2 and decreases in NF-κB and iNOS. It exerted a neuron-protection effect by decreasing the apoptosis-related protein cleaved caspase-3 and decreasing the information cytokine TNF-alpha expression, which it achieved by increasing HO-1 and NQO-1 protein found downstream from Nrf2 and Nrf2. We believe that RTA 408 can potentially be used to manage of cerebral vasospasm and secondary brain injury following SAH.

An integrated microfluidic system for rapid, automatic and high-throughput staining of clinical tissue samples for diagnosis of ovarian cancer.

Accurate cancer diagnostic methods are of urgent need. Since traditional immunohistochemistry (IHC)-based approaches, while reliable, are labor-intensive and require well-trained technicians, we developed an integrated microfluidic platform capable of labeling ovarian cancer biomarkers (i.e. aptamer) within formalin-fixed, paraffin embedded tissues via molecular probes. Both aptamer-based 1) fluorescent staining and 2) IHC staining of clinical tissue samples could be automated in the microfluidic system in only 2-3 h (40-50% faster than conventional approaches) with <0.5 mL of reagents, signifying that this device could serve as a promising diagnostic tool for ovarian cancer.

The pregnane X receptor and its microbiota-derived ligand indole 3-propionic acid regulate endothelium-dependent vasodilation.

We proposed that circulating metabolites generated by the intestinal microbiota can affect vascular function. One such metabolite, indole 3-propionic acid (IPA), can activate the pregnane X receptor(PXR), a xenobiotic-activated nuclear receptor present in many tissues, including the vascular endothelium. We hypothesized that IPA could regulate vascular function by modulating PXR activity. To test this, Pxr+/+ mice were administered broad-spectrum antibiotics for 2 wk with IPA supplementation. Vascular function was evaluated by bioassay using aorta and pulmonary artery ring tissue from antibiotic-treated Pxr+/+ and Pxr-/-mice, supplemented with IPA, and using aorta tissue maintained in organ culture for 24 h in the presence of IPA. Endothelium-dependent, nitric oxide(NO)-mediated muscarinic and proteinase-activated receptor 2(PAR2)-stimulated vasodilation was assessed. Endothelial nitric oxide synthase (eNOS) abundance was evaluated in intact tissue or in aorta-derived endothelial cell cultures from Pxr+/+ and Pxr-/- mice, and vascular Pxr levels were assessed in tissues obtained from Pxr+/+ mice treated with antibiotics and supplemented with IPA. Antibiotic-treated Pxr+/+ mice exhibited enhanced agonist-induced endothelium-dependent vasodilation, which was phenocopied by tissues from either Pxr-/- or germ-free mice. IPA exposure reduced the vasodilatory responses in isolated and cultured vessels. No effects of IPA were observed for tissues obtained from Pxr-/- mice. Serum nitrate levels were increased in antibiotic-treated Pxr+/+and Pxr-/- mice. eNOS abundance was increased in aorta tissues and cultured endothelium from Pxr-/- mice. PXR stimulation reduced eNOS expression in cultured endothelial cells from Pxr+/+ but not Pxr-/- mice. The microbial metabolite IPA, via the PXR, plays a key role in regulating endothelial function. Furthermore, antibiotic treatment changes PXR-mediated vascular endothelial responsiveness by upregulating eNOS.

An automated microfluidic system for selection of aptamer probes against ovarian cancer tissues.

Because of the difficulty of treatment in its latest stages, cancer is among the leading causes of death worldwide. Therefore, high-affinity and specificity biomarkers are still in demand for many cancer types, and the utility of aptamers to serve in this regard has been explored recently. Although a process known as "systematic evolution of ligands by exponential enrichment" (SELEX) has been used to generate aptamer-based cancer biomarkers, this approach is complicated, time-consuming, and labor-intensive. An automated microfluidic system was consequently developed herein to screen ovarian cancer-specific aptamers via on-chip SELEX with clinical cancer tissue samples. The integrated microfluidic system consisted of an integrated microfluidic chip, a temperature control module equipped with 12 thermoelectric coolers, and a flow control module for controlling 36 electromagnetic valves such that the entire, tissue-based SELEX process could be fully automated and carried out within 15 h. Highly specific ovarian cancer aptamers with high affinity (dissociation constant of 129 nM) to their cellular targets were screened with this system. Given the comparable specificity to their much more expensive antibody counterparts, these aptamers, when used in conjunction with the developed microfluidic system, may be used to diagnose ovarian cancer in its earliest stages.

Absorption coefficient of water vapor across atmospheric troposphere layer.

Absorption coefficient of water vapor proposed to be responsible for an increase in temperature in the troposphere layer with altitude less than 10 km is systematically presented in this work. Since global warming plays an important role in affecting the human life, a confirmative and detailed study of global warming is essentially need. Solar irradiation within short wavelength range can be extinguished from absorption and scattering by the atmosphere, and absorbed and reflected by the Earth's surface. Radiative within high wavelength range from the Earth's surface can be absorbed by atmospheric water vapor, carbon dioxide and other gases. The difference in solar irradiation and energy escaped to the space from the atmosphere results in the atmosphere acting as the glass of a greenhouse and increase atmospheric temperature. Extending the previous work [1] for predicting absorption coefficient of carbon dioxide through the troposphere, this work further determines absorption coefficients of water vapor in different wavelength bands centered at 71, 6.3, 2.7, 1.87 and 1.38 µ m across the temperature, pressure and concentration-dependent troposphere layer. Solving one-dimensional unsteady heat conduction-radiation equation with the COMSOL computer code, the predicted temperature together with water vapor density for different optical path lengths can be used to interpret in details absorption coefficient or the ratio between band intensity and effective band width by using the exponential wide band model. The results show that absorption coefficients are strongly affected by water vapor concentration. For example, absorption coefficients in the band centered at 71 µ m increases from 0.3 to 1.2 m - 1 at the tropopause and 0.6 to 3.1 m - 1 at the Earth's surface as mole fraction of water vapor increases from 0.005 to 0.02. The predicted absorption coefficients agree with experimental and theoretical results in the literature. A more detailed and realistic temperature profile through the troposphere with optical path length of 10 4 m is presented.

Microfluidic platforms for rapid screening of cancer affinity reagents by using tissue samples.

Cancer is the most serious disease worldwide, and ovarian cancer (OvCa) is the second most common type of gynecological cancer. There is consequently an urgent need for early-stage detection of OvCa, which requires affinity reagent biomarkers for OvCa. Systematic evolution of ligands by exponential enrichment (SELEX) and phage display technology are two powerful technologies for identifying affinity reagent biomarkers. However, the benchtop protocols for both screening technologies are relatively lengthy and require well-trained personnel. We therefore developed a novel, integrated microfluidic system capable of automating SELEX and phage display technology. Instead of using cancer cell lines, it is the first work which used tissue slides as screening targets, which possess more complicated and uncovered information for affinity reagents to recognize. This allowed for the identification of aptamer (nucleic acid) and peptide probes specific to OvCa cells and tissues. Furthermore, this developed system could be readily modified to uncover affinity reagents for diagnostics or even target therapy of other cancer cell types in the future.

Absorption coefficient of carbon dioxide across atmospheric troposphere layer.

Absorption coefficient affected by carbon dioxide concentration and optical path length responsible for temperature or global warming across the troposphere layer, which is less than the altitude of 10 km in the atmosphere, is systematically presented in this work. Solar irradiation within a short wavelength range can be absorbed, scattered and transmitted by the atmosphere, and absorbed and reflected by the Earth's surface. Radiative emission in high wavelength ranges from the Earth's surface at low temperature can be absorbed by atmospheric water vapor, carbon dioxide and other gases. Unbalance of radiation thus results in the atmosphere to act as the glass of a greenhouse and increase atmospheric temperature. Even though global warming strongly affects the life of the human being, the cause of global warming is still controversial. This work thus proposes a fundamental and systematical unsteady one-dimensional heat conduction-radiation model together with exponential wide band model to predict absorption coefficients affected by concentration, temperature, optical path lengths and radiation correlated parameters in different bands centered at 15, 4.3, 2.7, and 2 µm of carbon dioxide across the troposphere layer. It shows that absorption coefficient required for calculating heat transfer is strongly affected by carbon dioxide concentration and optical path length across the troposphere. Relevant values of the latter should be greater than 5,000 m. Absorption coefficients in the band centered at 4.3 µm subject to a chosen optical path length of 10 4 m increase from 0.04 m-1 and 0.165 m-1at the tropopause to 0.11 m-1 and 0.44 m-1 at the Earth's surface for carbon dioxide concentrations of 100 and 400 ppm, respectively. A more relevant and detailed temperature profile across the troposphere is presented.

Overexpression of Fli-1 in astrocytoma is associated with poor prognosis.

BACKGROUND: Astrocytoma, a common and highly malignant type of brain tumor, is associated with poor overall survival despite advances in surgical treatment, radiotherapy, and chemotherapy. The nuclear transcription factor Fli-1 has been shown to increase cellular proliferation and tumorigenesis in many types of cancer; however, previous reports have not described a correlation between clinical outcomes and Fli-1 in astrocytoma patients. The present study aimed to elucidate the clinical role of Fli-1 in astrocytoma. RESULTS: High-level of Fli-1 protein expression was significantly association with World Health Organization (WHO) high grade and poor prognosis. A multivariate analysis revealed that the WHO grade and Fli-1 protein expression were independent factor of prognostic factors of patients with astrocytoma. In addition, Fli-1 silencing inhibited proliferation, migration, and invasion and led to the downregulation of Ki-67, VEGF, and cyclin D1 expression in the astrocytoma cells. MATERIALS AND METHODS: Fli-1 protein expression in astrocytoma tissue samples were detected via immunohistochemistry, and potential correlations between clinical parameters and Fli-1 expression were assessed in patients with astrocytoma. Additionally, proliferation, invasion, and migration assays of astrocytoma cell lines were conducted to evaluate the effects of short interfering RNA (siRNA) on these processes; in addition, these cells were subjected to western blotting to detect the expression levels of Fli-1, Ki-67, VEGF, and Cyclin D1. CONCLUSION: Fli-1 shows promise as a potential prognostic biomarker and therapeutic molecular target for astrocytoma patients.

Regulation of CTP Synthase Filament Formation During DNA Endoreplication in Drosophila.

CTP synthase (CTPsyn) plays an essential role in DNA, RNA, and lipid synthesis. Recent studies in bacteria, yeast, and Drosophila all reveal a polymeric CTPsyn structure, which dynamically regulates its enzymatic activity. However, the molecular mechanism underlying the formation of CTPsyn polymers is not completely understood. In this study, we found that reversible ubiquitination regulates the dynamic assembly of the filamentous structures of Drosophila CTPsyn. We further determined that the proto-oncogene Cbl, an E3 ubiquitin ligase, controls CTPsyn filament formation in endocycles. While the E3 ligase activity of Cbl is required for CTPsyn filament formation, Cbl does not affect the protein levels of CTPsyn. It remains unclear whether the regulation of CTPsyn filaments by Cbl is through direct ubiquitination of CTPsyn. In the absence of Cbl or with knockdown of CTPsyn, the progression of the endocycle-associated S phase was impaired. Furthermore, overexpression of wild-type, but not enzymatically inactive CTPsyn, rescued the endocycle defect in Cbl mutant cells. Together, these results suggest that Cbl influences the nucleotide pool balance and controls CTPsyn filament formation in endocycles. This study links Cbl-mediated ubiquitination to the polymerization of a metabolic enzyme and reveals a role for Cbl in endocycles during Drosophila development.

Endophilin B is required for the Drosophila oocyte to endocytose yolk downstream of Oskar.

The nutritional environment is crucial for Drosophila oogenesis in terms of controlling hormonal conditions that regulate yolk production and the progress of vitellogenesis. Here, we discovered that Drosophila Endophilin B (D-EndoB), a member of the endophilin family, is required for yolk endocytosis as it regulates membrane dynamics in developing egg chambers. Loss of D-EndoB leads to yolk content reduction, similar to that seen in yolkless mutants, and also causes poor fecundity. In addition, mutant egg chambers exhibit an arrest at the previtellogenic stage. D-EndoB displayed a crescent localization at the oocyte posterior pole in an Oskar-dependent manner; however, it did not contribute to pole plasm assembly. D-EndoB was found to partially colocalize with Long Oskar and Yolkless at the endocytic membranes in ultrastructure analysis. Using an FM4-64 dye incorporation assay, D-EndoB was also found to promote endocytosis in the oocyte. When expressing the full-length D-endoB(FL) or D-endoB(ΔSH3) mutant transgenes in oocytes, the blockage of vitellogenesis and the defect in fecundity in D-endoB mutants was restored. By contrast, a truncated N-BAR domain of the D-EndoB only partially rescued these defects. Taken together, these results allow us to conclude that D-EndoB contributes to the endocytic activity downstream of Oskar by facilitating membrane dynamics through its N-BAR domain in the yolk uptake process, thereby leading to normal progression of vitellogenesis.