Ratiometric Optical Fiber Dissolved Oxygen Sensor Based on Fluorescence Quenching Principle.

In this study, a ratiometric optical fiber dissolved oxygen sensor based on dynamic quenching of fluorescence from a ruthenium complex is reported. Tris(4,7-diphenyl-1,10-phenanthrolin) ruthenium(II) dichloride complex (Ru(dpp)32+) is used as an oxygen-sensitive dye, and semiconductor nanomaterial CdSe/ZnS quantum dots (QDs) are used as a reference dye by mixing the two substances and coating it on the plastic optical fiber end to form a composite sensitive film. The linear relationship between the relative fluorescence intensity of the ruthenium complex and the oxygen concentration is described using the Stern-Volmer equation, and the ruthenium complex doping concentration in the sol-gel film is tuned. The sensor is tested in gaseous oxygen and aqueous solution. The experimental results indicate that the measurement of dissolved oxygen has a lower sensitivity in an aqueous environment than in a gaseous environment. This is due to the uneven distribution of oxygen in aqueous solution and the low solubility of oxygen in water, which results in a small contact area between the ruthenium complex and oxygen in solution, leading to a less-severe fluorescence quenching effect than that in gaseous oxygen. In detecting dissolved oxygen, the sensor has a good linear Stern-Volmer calibration plot from 0 to 18.25 mg/L, the linearity can reach 99.62%, and the sensitivity can reach 0.0310/[O2] unit. The salinity stability, repeatability, and temperature characteristics of the sensor are characterized. The dissolved oxygen sensor investigated in this research could be used in various marine monitoring and environmental protection applications.

Simultaneous Detection of Six Isoforms of Tau Protein in Human Cerebrospinal Fluid by Multidimensional Mass Spectrometry-Based Targeted Proteomics.

Abnormal expression of Tau protein can cause the development of Alzheimer's disease (AD). So far, much evidence has demonstrated that Tau has multiple isoforms. These isoforms are suggested to have distinct physiological roles and contribute unequally to the progress of AD. Thus, detection of individual Tau isoforms may be helpful to better understand the link between clinical outcome and Tau status and to further improve AD diagnosis and treatment. However, few studies have been conducted on absolute quantification of Tau isoforms, probably due to high sequence homology and also low abundance of these isoforms in biofluids such as cerebrospinal fluid (CSF). Therefore, mass spectrometry-based targeted proteomics was attempted here. This targeted proteomics approach can principally measure a protein of interest at the surrogate peptide level, yet little has been done to detect protein isoforms, probably due to lack of isoform-specific surrogate peptides in mass spectrometry. In this study, separations in more dimensions were added, including immunoprecipitation (IP) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for sample pretreatment and systems of linear equations for post-lab data extraction. Moreover, the reliability of the approach including IP enrichment, gel separation, and linear algebra algorithms was discussed. As a result, each isoform of Tau protein can be individually detected and quantified. Using IP enrichment, ∼250-fold enhancement of sensitivity was achieved. The ultimate LOQ was 0.50 nM. Finally, this multidimensional mass spectrometry-based targeted proteomics assay was validated and applied to simultaneous quantitative analysis of six Tau isoforms in CSF of AD patients.

Blockade of IL-33 signalling attenuates osteoarthritis.

OBJECTIVES: Osteoarthritis (OA) is the most common form of arthritis characterised by cartilage degradation, synovitis and pain. Disease modifying treatments for OA are not available. The critical unmet need is to find therapeutic targets to reduce both disease progression and pain. The cytokine IL-33 and its receptor ST2 have been shown to play a role in immune and inflammatory diseases, but their role in osteoarthritis is unknown. METHODS: Non-OA and OA human chondrocytes samples were examined for IL-33 and ST2 expression. Novel inducible cartilage specific knockout mice (IL-33Acan CreERT2) and inducible fibroblast-like synoviocyte knockout mice (IL-33Col1a2 CreERT2) were generated and subjected to an experimental OA model. In addition, wild-type mice were intra-articularly administered with either IL-33- or ST2-neutralising antibodies during experimental OA studies. RESULTS: IL-33 and its receptor ST2 have increased expression in OA patients and a murine disease model. Administering recombinant IL-33 increased OA and pain in vivo. Synovial fibroblast-specific deletion of IL-33 decreased synovitis but did not impact disease outcomes, whilst cartilage-specific deletion of IL-33 improved disease outcomes in vivo. Blocking IL-33 signalling also reduced the release of cartilage-degrading enzymes in human and mouse chondrocytes. Most importantly, we show the use of monoclonal antibodies against IL-33 and ST2 attenuates both OA and pain in vivo. CONCLUSION: Overall, our data reveal blockade of IL-33 signalling as a viable therapeutic target for OA.

Liquid Lens with Large Focal Length Tunability Fabricated in a Polyvinyl Chloride/Dibutyl Phthalate Gel Tube.

Usually, an adaptive liquid lens only has a positive focal length, which severely limits its application in imaging and other fields. Therefore, a liquid lens consisting of polyvinyl chloride/dibutyl phthalate (PVC/DBP) gel, glycerol solution, and a glass substrate is proposed to extend the dynamic focal length range. A spherical tube is formed by the PVC/DBP gel under the effect of hydrostatic and surface tensions, which is used to restrict the glycerol solution. The PVC/DBP gel does not deform under the effect of an electric field, so the tangent line at the three-phase junction changes with the change of contact angle, which leads to an enlargement of the dynamic focal length range. At different voltage values, the proposed lens can be configured to work in three different schemes, namely, converging light, nondeflecting light, and diverging light. Here, the proposed lens has high imaging quality; the resolution is better than 114 lp/mm. A lens with a reconfigurable focal length holds great promise in diverse applications such as fluorescence detection, beam shaping, and adaptive optics.

A novel WARS mutation (p.Asp314Gly) identified in a Chinese distal hereditary motor neuropathy family.

Distal hereditary motor neuropathy (dHMN) is a clinically and genetically heterogeneous group of inherited neuropathies characterized by distal limb muscle wasting and weakness with no or minimal sensory abnormalities. To investigate the clinical and genetic features of dHMN caused by WARS mutations in mainland China, we performed Sanger sequencing of the coding and untranslated region (UTR) regions of WARS in 160 unresolved dHMN and Charcot-Marie-Tooth (CMT) index patients. We detected a novel heterozygous variant c.941A>G (p.Asp314Gly) of WARS in an index patient from an autosomal dominant dHMN family including five affected members over three generations. The variant completely co-segregates with the dHMN phenotype in the family, and it was classified as likely pathogenic according to the American College of Medical Genetics and Genomics standards and guidelines. The clinical features included juvenile to adult onset (15-23 years), distal wasting and weakness, minimal sensory disturbance and length-dependent motor axonal degeneration with CMT examination score ranging from 6 to 10. Our report further confirms the role of WARS in dHMN and indicates that the variant c.941A>G (p.Asp314Gly) of WARS is related to a mild to moderate affected and later onset phenotype of dHMN.

A novel AIFM1 mutation in a Chinese family with X-linked Charcot-Marie-Tooth disease type 4.

X-linked Charcot-Marie-Tooth disease type 4 (CMTX4), caused by AIFM1 (Apoptosis-Inducing Factor, Mitochondrion associated 1) mutations and associated with deafness and cognitive impairment, is a rare subtype of Charcot-Marie-Tooth disease. Here, we report a novel missense variant of AIFM1 in a X-linked recessive Chinese family with childhood-onset, slowly progressive, isolated axonal motor and sensory neuropathy. Calf magnetic resonance imaging revealed fatty infiltration and atrophy severely involving the muscles of peroneal compartment. Pathologies exhibited abnormal mitochondrial morphology and accumulation in axoplasm of nerve fiber and subsarcolemmal area of muscle. A hemizygous variant (c.513G>A, p.Met171Ile) in the family was identified and was classified as likely pathogenic according to the standards and guidelines of the American College of Medical Genetics and Genomics. Our report expands the genetic spectrum of diseases related to AIFM1 mutations and indicates that fatty infiltration and atrophy of muscles in the peroneal compartment may be a feature of CMTX4 in early stage.

Molecular Analysis-Based Genetic Characterization of a Cohort of Patients with Duchenne and Becker Muscular Dystrophy in Eastern China.

BACKGROUND: Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are common X-linked recessive neuromuscular disorders caused by mutations in dystrophin gene. Multiplex polymerase chain reaction (multiplex PCR) and multiplex ligation-dependent probe amplification (MLPA) are the most common methods for detecting dystrophin gene mutations. This study aimed to contrast the two methods and discern the genetic characterization of patients with DMD/BMD in Eastern China. METHODS: We collected 121 probands, 64 mothers of probands, and 15 fetuses in our study. The dystrophin gene was detected by multiplex PCR primarily in 28 probands, and MLPA was used in multiplex PCR-negative cases subsequently. The dystrophin gene of the remaining 93 probands and 62 female potential carriers was tested by MLPA directly. In fetuses, multiplex PCR and MLPA were performed on 4 fetuses and 10 fetuses, respectively. In addition, sequencing was also performed in 4 probands with negative MLPA. RESULTS: We found that 61.98% of the subjects had genetic mutations including deletions (50.41%) and duplications (11.57%). There were 43.75% of mothers as carriers of the mutation. In 15 fetuses, 2 out of 7 male fetuses were found to be unhealthy and 2 out of 8 female fetuses were found to be carriers. Exons 3-26 and 45-52 have the maximum frequency in mutation regions. In the frequency of exons individually, exon 47 and exon 50 were the most common in deleted regions and exons 5, 6, and 7 were found most frequently in duplicated regions. CONCLUSIONS: MLPA has better productivity and sensitivity than multiplex PCR. Prenatal diagnosis should be applied in DMD high-risk fetuses to reduce the disease incidence. Furthermore, it is the responsibility of physicians to inform female carriers the importance of prenatal diagnosis.

Novel Mutation of the NOTCH3 Gene in a Chinese Pedigree with CADASIL.

BACKGROUND: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) results from NOTCH3 gene mutations, which lead to the degeneration of vascular smooth muscle cells (VSMCs). The clinical presentation of CADASIL patients is dependent on the impact of other vascular risk factors and the type of NOTCH3 mutation present. METHODS: Here, we report a rare pathogenic mutation on exon 14 of the NOTCH3 gene in a Chinese family affected by CADASIL with phenotypic peculiarities. We performed genetic testing, clinical and neuropsychological examination, brain magnetic resonance images (MRI), and electron microscopy (EM) in skin biopsies. RESULTS: NOTCH3 gene analysis revealed a c.2182CT substitution on exon 14, which is the first example of this mutation in a Chinese individual from the Han ancestry. Granular osmiophilic material (GOM) was found in the proband, and all patients had migraine, subcortical ischemic events, and mood disturbances, without progressive cognitive impairment. Cranial MRI further showed white matter hyperintensity, involving bilateral basal ganglia and multiple microbleeds (MBs), in the thalamus and brain stem. CONCLUSIONS: This study suggests that different missense mutations in NOTCH3 might contribute to atypical clinical features of CADASIL. This report also indicates that for individuals with a positive family history having clinical and neuroradiological findings suggestive of CADASIL, genetic testing and GOM detection should be performed.

[Study of a CADASIL family with migraine as the presenting symptom].

OBJECTIVE: To analyze the clinical features and genetic cause for a family affected with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). METHODS: Clinical manifestations, neuroimaging, and genetic analysis were performed. RESULTS: The main clinical features have included stroke, emotional disturbance and history of migraine without progressive memory impairment. A positive family history was confirmed. Cranial MRI has revealed multi-infarct lesions and white matter hyperintensity involving bilateral basal ganglia, subcortex and brain stem. All such features were in keeping with the diagnosis of CADASIL. A rare 2182C>T mutation in exon 14 of the NOTCH3 gene was identified in all available cases. CONCLUSION: Both clinical and molecular features suggested that the family has been affected with CADASIL.

[Clinical features and risk factors of anastomotic leakage after radical esophagectomy].

OBJECTIVE: To analyze the clinical features and risk factors of anastomotic leakage after radical esophagectomy of esophageal carcinoma. METHODS: The clinical data of 547 esophageal cancer patients underwent radical esophagectomy in Tianjin Medical University Cancer Hospital from January 2012 to December 2013 was analyzed retrospectively. There were 421 male and 126 female patients, with a median age of 65 years (ranging from 29 to 82 years). There were 155 cases of upper esophageal carcinoma, 340 cases of middle esophageal carcinoma and 52 cases of lower esophageal carcinoma. The surgical procedures included 41 cases completed through Sweet, 145 cases completed through McKeown, 279 cases completed through Ivor Lewis, 82 cases completed through minimally invasive esophagectomy. Moreover, 24 of 547 cases underwent preoperative neoadjuvant radiochemotherapy. χ² test and Cox's proportional hazards regression model were used for univariate analysis and multivariate analysis of the risk factors of postoperative anastomotic leakage. RESULTS: Twenty-seven of 547 cases with esophagectomy occurred anastomotic leakage and the incidence rate was 4.94% (27/547). One of 27 cases died and the mortality rate was 3.70% (1/27). The time of anastomotic leakage found was 4 to 45 days, with a median time of 10 days. There were 0 case of early leakage, 20 cases of mid-term leakage, 7 cases of late leakage. Three of 27 cases with anastomotic leakage had tracheoesophageal fistula, while 3 cases had contralateral pleural fistula. As to the incidence rate of anastomotic leakage, there was statistically significant difference between cervical anastomotic leakage (8.14%, 18/221) and intrathoracic anastomotic leakage (2.76%, 9/326) (χ² =7.41, P=0.000), among Sweet (4.88%, 2/41), McKeown (9.66%, 14/145), Ivor Lewis (2.51%, 7/279) and MIE (4.88%, 4/82) (χ² =21.48, P=0.000), and between with (16.67%, 4/24) and without (4.40%, 23/523) neoadjuvant radiochemotherapy (χ² =9.20, P=0.000). The multivariate analysis showed that anastomotic site (HR=2.594, P=0.048), surgical approach (HR=5.689, P=0.003) and preoperative neoadjuvant radiochemotherapy (HR=3.604, P=0.027) are independent risk factors for anastomotic leakage after esophagectomy. CONCLUSIONS: The mid-term anastomotic leakage after esophagectomy occurs higher. McKeown is a main surgical procedure and neoadjuvant radiochemotherapy is an important factor for the anastomotic leakage.

Is tumor length a prognostic indicator for esophageal squamous cell carcinoma? A single larger study among Chinese patients.

OBJECTIVE: In esophageal cancer, depth of wall penetration, reflected by T classification, represents the most important prognostic variable. Our study aimed to investigate the impact of tumor length, measured as the longitudinal length, on the outcome of esophageal squamous cell carcinoma (ESCC) patients. METHODS: The survival data of 362 ESCC patients who underwent surgical resection as the primary treatment between 1999 and 2007 were collected retrospectively. Receiver-operator characteristic analysis was applied to identify the optimal cut-off values. RESULTS: 4.0 cm was identified as the optimal cut-off value within the whole group. Tumor length greater than 4.0 cm was associated with increasing T stage (P=0.001), N stage (P=0.046), and tumor differentiation (P=0.033). Univariate analysis and multivariate analysis both found that tumor length greater than 4.0 cm was associated with worse overall survival compared with shorter tumors (P<0.001). It appeared to have a greater impact on N0-N1 (P<0.001, P=0.026, respectively) than N2-N3 and appeared to have a higher impact on the lower-stage patients than the higher-stage patients. CONCLUSIONS: Tumor length proved to be an independent prognostic parameter for ESCC patients, especially for node-negative and lower-stage patients. More attention should be paid to its role in the management of ESCC.

The Effects of the Recombinant CCR5 T4 Lysozyme Fusion Protein on HIV-1 Infection.

BACKGROUND: Insertion of T4 lysozyme (T4L) into the GPCR successfully enhanced GPCR protein stability and solubilization. However, the biological functions of the recombinant GPCR protein have not been analyzed. METHODS: We engineered the CCR5-T4L mutant and expressed and purified the soluble recombinant protein using an E.coli expression system. The antiviral effects of this recombinant protein in THP-1 cell lines, primary human macrophages, and PBMCs from different donors were investigated. We also explored the possible mechanisms underlying the observed antiviral effects. RESULTS: Our data showed the biphasic inhibitory and promotion effects of different concentrations of soluble recombinant CCR5-T4L protein on R5 tropic human immunodeficiency virus-1 (HIV-1) infection in THP-1 cell lines, human macrophages, and PBMCs from clinical isolates. We demonstrated that soluble recombinant CCR5-T4L acts as a HIV-1 co-receptor, interacts with wild type CCR5, down-regulates the surface CCR5 expression in human macrophages, and interacts with CCL5 to inhibit macrophage migration. Using binding assays, we further determined that recombinant CCR5-T4L and [125I]-CCL5 compete for the same binding site on wild type CCR5. CONCLUSIONS: Our results suggest that recombinant CCR5-T4L protein marginally promotes HIV-1 infection at low concentrations and markedly inhibits infection at higher concentrations. This recombinant protein may be helpful in the future development of anti-HIV-1 therapeutic agents.

A novel DYNC1H1 mutation causing spinal muscular atrophy with lower extremity predominance.

Recent studies have identified mutations in the dynein heavy chain gene (DYNC1H1), which lead to 2 closely related human motor neuropathies: a dominant spinal muscular atrophy with lower extremity predominance (SMALED) and axonal Charcot-Marie-Tooth (CMT) disease.(1,2) We describe the identification of a novel mutation (p.G807S) in DYNC1H1 as the cause of SMALED.

Role for the conserved N-terminal cysteines in the anti-chemokine activities by the chemokine-like protein MC148R1 encoded by Molluscum contagiosum virus.

Molluscum contagiosum poxvirus (MCV) type 1 and type 2 encode two chemokine-like proteins MC148R1 and MC148R2. It is believed that MC148R proteins function by blocking the inflammatory response. However, the mechanism of the proposed biological activities of MC148R proteins and the role of the additional C-terminal cysteines that do not exist in other chemokines are not understood. Here, we demonstrated in two different assay systems that His-tagged MC148R1 displaces the interaction between CXCL12α and CXCR4. The N-terminal cysteines but not the additional C-terminal cysteines modulate this displacement. His-tagged MC148R1 blocked both CXCL12α-mediated and MIP-1α-mediated chemotaxis. In contrast, MC148R2 blocked MIP-1α-mediated but not CXCL12α-mediated chemotaxis. Immunoprecipitation by antibodies to MC148R1 or CXCL12α followed by immunoblotting and detection by antibodies to the other protein demonstrated physical interaction of His-tagged CXCL12α and His-tagged MC148R1. Interaction with chemokines might mask the receptor interaction site resulting in decreased binding and impairment of the biological activities.

Alternate receptor usage of neuropilin-1 and glucose transporter protein 1 by the human T cell leukemia virus type 1.

Recent studies have demonstrated that neuropilin 1 (NP-1) is involved in HTLV-1 entry; however, the role NP-1 plays in this process is not understood. We demonstrated that ectopic expression of human NP-1 but not NP-2 cDNA increased susceptibility to HTLV-1. SiRNA-mediated inhibition of NP-1 expression correlated with significant reduction of HTLV-1 Env-mediated fusion. The vascular endothelial growth factor (VEGF(165)) caused downmodulation of surface NP-1 and inhibited HTLV-1 infection of U87 cells. In contrast, VEGF(165) partially inhibited infection of primary astrocytes and had no significant effect on infection of HeLa cells. VEGF(165) and antibodies to the glucose transporter protein 1 (anti-GLUT-1) were both needed to block infection of primary astrocytes, however, only anti-GLUT-1 antibodies were sufficient to block infection of HeLa cells. HTLV-1 Env forms complexes with both NP-1 and GLUT-1 in primary human astrocytes. The alternate usage of these two cellular receptors may have important implications regarding HTLV-1 neuro-tropism.

The potent anti-HIV activity of CXCL12gamma correlates with efficient CXCR4 binding and internalization.

We previously demonstrated that the naturally occurring splice variant stromal cell-derived factor 1gamma/CXCL12gamma is the most potent CXCL12 isoform in blocking X4 HIV-1, with weak chemotactic activity. A conserved BBXB domain (B for basic and X for any residue) located in the N terminus ((24)KHLK(27)) is found in all six isoforms of CXCL12. To determine whether the potent antiviral activity of CXCL12gamma is due to the presence of the extra C-terminal BBXB domains, we mutated each domain individually as well as in combination. Although binding of CXCL12gamma to heparan sulfate proteoglycan (HSPG) was 10-fold higher than that observed with CXCL12alpha, the results did not demonstrate a direct correlation between HSPG binding and the potent antiviral activity. CXCL12gamma mutants lacking the conserved BBXB domain (designated gammaB1) showed increased binding to HSPG but reduced anti-HIV activity. In contrast, the mutants lacking the C-terminal second and/or third BBXB domain but retaining the conserved domain (designated B2, B3, and B23) showed decreased binding to HSPG but increased anti-HIV activity. The B2, B3, and B23 mutants were associated with enhanced CXCR4 binding, receptor internalization, and restored chemotaxis. Internalization of CXCR4 was more potent with CXCL12gamma than with CXCL12alpha and was significantly reduced when the conserved BBXB domain was mutated. We concluded that the observed potent anti-HIV-1 activity of CXCL12gamma is due to increased affinity for CXCR4 and to efficient receptor internalization.

Resistance to human immunodeficiency virus type 1 (HIV-1) generated by lentivirus vector-mediated delivery of the CCR5{Delta}32 gene despite detectable expression of the HIV-1 co-receptors.

It has previously been demonstrated that there are two distinct mechanisms for genetic resistance to human immunodeficiency virus type 1 (HIV-1) conferred by the CCR5Delta32 gene: the loss of wild-type CCR5 surface expression and the generation of CCR5Delta32 protein, which interacts with CXCR4. To analyse the protective effects of long-term expression of the CCR5Delta32 protein, recombinant lentiviral vectors were used to deliver the CCR5Delta32 gene into human cell lines and primary peripheral blood mononuclear cells that had been immortalized by human T-cell leukemia virus type 1. Blasticidin S-resistant cell lines expressing the lentivirus-encoded CCR5Delta32 showed a significant reduction in HIV-1 Env-mediated fusion assays. It was shown that CD4(+) T lymphocytes expressing the lentivirus-encoded CCR5Delta32 gene were highly resistant to infection by a primary but not by a laboratory-adapted X4 strain, suggesting different infectivity requirements. In contrast to previous studies that analysed the CCR5Delta32 protective effects in a transient expression system, this study showed that long-term expression of CCR5Delta32 conferred resistance to HIV-1 despite cell-surface expression of the HIV co-receptors. The results suggest an additional unknown mechanism for generating the CCR5Delta32 resistance phenotype and support the hypothesis that the CCR5Delta32 protein acts as an HIV-suppressive factor by altering the stoichiometry of the molecules involved in HIV-1 entry. The lentiviral-CCR5Delta32 vectors offer a method of generating HIV-resistant cells by delivery of the CCR5Delta32 gene that may be useful for stem cell- or T-cell-based gene therapy for HIV-1 infection.