Diagnostic Accuracy of Lipopolysaccharide-Binding Protein as Biomarker for Sepsis in Adult Patients: A Systematic Review and Meta-Analysis

IntroductionLipopolysaccharide-binding protein (LBP) is widely reported as a biomarker to differentiate infected from non-infected patients. The diagnostic use of LBP for sepsis remains a matter of debate. We aimed to perform a systematic review and meta-analysis to assess the diagnostic accuracy of serum LBP for sepsis in adult patients.MethodsWe performed a systematic review and meta-analysis to assess the accuracy of LBP for sepsis diagnosis. A systematic search in PubMed and EMBASE for studies that evaluated the diagnostic role of LBP for sepsis through December 2015 was conducted. We searched these databases for original, English language, research articles that studied the diagnostic accuracy between septic and non-septic adult patients. Sensitivity, specificity, and other measures of accuracy, such as diagnostic odds ratio (DOR) and area under the receiver operating characteristic curve (AUC) of LBP were pooled using the Hierarchical Summary Receiver Operating Characteristic (HSROC) method.ResultsOur search returned 53 reports, of which 8 fulfilled the inclusion criteria, accounting for 1684 patients. The pooled sensitivity and specificity of LBP for diagnosis of sepsis by the HSROC method were 0.64 (95% CI: 0.56–0.72) and 0.63 (95% CI: 0.53–0.73), respectively. The value of the DOR was 3.0 (95% CI: 2.0–4.0) and the AUC was 0.68 (95% CI: 0.64–0.72). Meta-regression analysis revealed that cut-off values accounted for the heterogeneity of sensitivity and sample size (> = 150) accounted for the heterogeneity of specificity.ConclusionsBased on the results of our meta-analysis, LBP had weak sensitivity and specificity in the detection of sepsis. LBP may not be practically recommended for clinical utilization as a single biomarker.     

Spectroscopic and Functional Characterizations of Cyanobacterium Synechocystis PCC 6803 Mutants on and near the Heme Axial Ligand of Cytochrome b559 in Photosystem II

The functional role of cytochrome (cyt) b₅₅₉ in photosystem II (PSII) was investigated in H22Kα and Y18Sα cyt b₅₅₉ mutants of the cyanobacterium Synechocystis sp. PCC6803. H22Kα and Y18Sα cyt b₅₅₉ mutant carries one amino acid substitution on and near one of heme axial ligands of cyt b₅₅₉ in PSII, respectively. Both mutants grew photoautotrophically, assembled stable PSII, and exhibited the normal period-four oscillation in oxygen yield. However, both mutants showed several distinct chlorophyll a fluorescence properties and were more susceptible to photoinhibition than wild type. EPR results indicated the displacement of one of the two axial ligands to the heme of cyt b₅₅₉ in H22Kα mutant reaction centers, at least in isolated reaction centers. The maximum absorption of cyt b₅₅₉ in Y18Sα mutant PSII core complexes was shifted to 561 nm. Y18Sα and H22Kα mutant PSII core complexes contained predominately the low potential form of cyt b₅₅₉. The findings lend support to the concept that the redox properties of cyt b₅₅₉ are strongly influenced by the hydrophobicity and ligation environment of the heme. When the cyt b₅₅₉ mutations placed in a D1-D170A genetic background that prevents assembly of the manganese cluster, accumulation of PSII is almost completely abolished. Overall, our data support a functional role of cyt b₅₅₉ in protection of PSII under photoinhibition conditions in vivo.     

4-Fluoro-N-butylphenylacetamide (H6) inhibits cell growth via cell-cycle arrest and apoptosis in human cervical cancer cells

Phenylacetate induced tumor cytostasis and differentiation. The chemotherapeutic function of the compound in lung cancer has been previously reported, however, whether or not phenylacetate performs other activities is not known. In this study, the potential usage of synthetic phenylacetate derivatives, 4-fluoro-N-butylphenylacetamides (H6) was investigated in human cervical cancer cells. H6 displayed anti-proliferative and apoptosis effects, with an IC₅₀ of 1.0–1.5 mM and an ID₅₀ of about 3 days. Moreover, it significantly induced apoptosis as evidenced by morphological changes, DAPI and TUNEL staining and DNA fragmentation. H6 increased the expression of Bax protein, whereas it decreased the expression of Bcl-2 protein. H6 also induced accumulation of cytosolic cytochrome c and activation of caspase cascade (caspase-9 and -3), and then DNA fragmentation and apoptosis occurred. The underlying anti-proliferative mechanism for H6 is likely due to the down-regulation of G2/M-phase association cdks and cyclins and up-regulation of p53 to mediate G2/M-phase arrest. Furthermore, the decrease of Bcl-2 and activation of Bax, caspase-9/caspase-3 may be the effectors of H6-induced apoptosis.     

Leukemia inhibitory factor antisense oligonucleotide inhibits the development of murine embryos at preimplantation stages

Leukemia inhibitory factor (LIF) is an essential factor for implantation and establishment of pregnancy. However, its role in the development of preimplantation embryos remains controversial. In this study, changes in preimplantation embryos were determined after microinjection of LIF antisense oligonucleotide at the two-pronucleus stage. Although no significant differences were found in the percentages between the untreated group and the 0.25-fmol-treated group, the 0.5- or 1.0-fmol-treated groups had significantly lower percentages of embryos developed to the morula or blastocyst stage and the 2.0-fmol-treated group had significantly lower percentages of embryos developed to the four-cell, morula, or blastocyst stage. No embryos developed to the four-cell stage in the 4.0-fmol-treated group. Moreover, there was a decreasing trend in the levels of LIF immunoactivity with the increasing amount of LIF antisense oligonucleotide injected. The diameter of blastocysts in the 2.0-fmol-treated group was significantly smaller than that in the untreated group. The blastocysts in this group had significantly lower numbers of blastomeres and cells in the inner cell mass (ICM) or trophectoderm (TE) and ICM:TE ratio. The 1.0- or 2.0-fmol-treated groups had significantly lower implantation rates than their corresponding control groups. In the 2.0-fmol groups with supplementing exogenous LIF, significantly lower percentages were also observed in the four-cell, morula, and blastocyst stages. However, blastocysts treated with 50 ng/ml LIF had a significantly higher percentage than those in the LIF gene-impaired group without LIF supplement. These results indicate that LIF is a critical factor for the normal development of embryos at the preimplantation stages.     

Site-directed mutagenesis on the heme axial-ligands of cytochrome b559 in photosystem II by using cyanobacteria Synechocystis PCC 6803

Cytochrome (cyt) b559 has been proposed to play an important role in the cyclic electron flow processes that protect photosystem II (PSII) from light-induced damage during photoinhibitory conditions. However, the exact role(s) of cyt b559 in the cyclic electron transfer pathway(s) in PSII remains unclear. To study the exact role(s) of cyt b559, we have constructed a series of site-directed mutants, each carrying a single amino acid substitution of one of the heme axial-ligands, in the cyanobacterium Synechocystis sp. PCC6803. In these mutants, His-22 of the α or the β subunit of cyt b559 was replaced with either Met, Glu, Tyr, Lys, Arg, Cys or Gln. On the basis of oxygen-evolution and chlorophyll a fluorescence measurements, we found that, among all mutants that were constructed, only the H22Kα mutant grew photoautotrophically, and accumulated stable PSII reaction centers (∼ 81% compared to wild-type cells). In addition, we isolated one pseudorevertant of the H22Yβ mutant that regained the ability to grow photoautotrophically and to assemble stable PSII reaction centers (∼ 79% compared to wild-type cells). On the basis of 77 K fluorescence emission measurements, we found that energy transfer from the phycobilisomes to PSII reaction centers was uncoupled in those cyt b559 mutants that assembled little or no stable PSII. Furthermore, on the basis of immunoblot analyses, we found that in thylakoid membranes of cyt b559 mutants that assembled little or no PSII, the amounts of the D1, D2, cyt b559α and β polypeptides were very low or undetectable but their CP47 and PsaC polypeptides were accumulated to the wild-type level. We also found that the amounts of cyt b559β polypeptide were significantly increased (larger than two folds) in thylakoid membranes of cyt b559 H22YβPS+ mutant cells. We suspected that the increase in the amounts of cyt b559 H22YβPS+ mutant polypeptides in thylakoid membranes might facilitate the assembly of functional PSII in cyt b559 H22YβPS+ mutant cells. Moreover, we found that isolated His-tagged PSII particles from H22Kα mutant cells gave rise to redox-induced optical absorption difference spectra of cyt b559. Therefore, our results concluded that significant fractions of H22Kα mutant PSII particles retained the heme of cyt b559. Finally, this work is the first report of cyt b559 mutants having substitutions of an axial heme-ligands that retain the ability to grow photoautotrophically and to assemble stable PSII reaction centers. These two cyt b559 mutants (H22Kα and H22YβPS+) and their PSII reaction centers will be very suitable for further biophysical and biochemical studies of the functional role(s) of cyt b559 in PSII.     

Impact of female age and male infertility on ovarian reserve markers to predict outcome of assisted reproduction technology cycles

Background  

This study was designed to assess the capability of ovarian reserve markers, including baseline FSH levels, baseline anti-Müllerian hormone (AMH) levels, and antral follicle count (AFC), as predictors of live births during IVF cycles, especially for infertile couples with advanced maternal age and/or male factors.     

Effects of ethylene glycol and methanol on ammonia-induced structural changes of the oxygen-evolving complex in photosystem II

Ammonia is an inhibitor of water oxidation and a structural analogue for substrate water, making it a valuable probe for the structural properties of the possible substrate-binding site on the oxygen-evolving complex (OEC) in photosystem II (PSII). By using the NH(3)-induced upshift of the 1365 cm(-)(1) IR mode in the S(2)Q(A)(-)/S(1)Q(A) spectrum and the NH(3)-modified S(2) state EPR signals of PSII as spectral probes, we found that ethylene glycol has clear effects on the binding properties of the NH(3)-specific site on the OEC. Our results show that in PSII samples containing 30% (v/v) ethylene glycol, the affinity of the NH(3)-specific binding site on the OEC is estimated to be more than 10 times lower than that in PSII samples containing 0.4 M sucrose. In addition, our results show that the NH(3)-induced upshift of the 1365 cm(-)(1) IR mode in the S(2)Q(A)(-)/S(1)Q(A) spectrum is dependent on the concentration of ethylene glycol, but not dependent on the concentration of sucrose (up to 1.5 M) or methanol (up to 5.4 M). By comparing the concentration dependence of sucrose and ethylene glycol on NH(3)-induced spectral change and also by comparing the sucrose and ethylene glycol data at similar concentrations ( approximately 1 M), we conclude that ethylene glycol has a clear effect on the NH(3)-induced spectral changes. Furthermore, our results also show that ethylene glycol alters the steric requirement of the amine effect on the upshift of the 1365 cm(-)(1) mode in the S(2)Q(A)(-)/S(1)Q(A) spectrum. In PSII samples containing 30% (v/v) ethylene glycol, only NH(3), not other bulkier amines (e.g., Tris, AEPD, and CH(3)NH(2)), has a clear effect on the upshift of the 1365 cm(-)(1) mode in the S(2)Q(A)(-)/S(1)Q(A) spectrum; in contrast, in PSII samples containing 0.4 M sucrose, both NH(3) and CH(3)NH(2) have a clear effect. On the basis of the results mentioned above, we propose that ethylene glycol acts directly or indirectly to decrease the affinity or limit the accessibility of NH(3) and CH(3)NH(2) to the NH(3)-specific binding site on the OEC in PSII. Finally, we also applied the same approach to test whether methanol is able to compete with ammonia on its binding site on the OEC. We found that 4% (v/v) methanol does not have any significant effect on the NH(3)-induced upshift of the 1365 cm(-)(1) mode in the S(2)Q(A)(-)/S(1)Q(A) spectrum and the NH(3)-modified S(2) state g = 2 multiline EPR signal. Our results suggest that methanol is unable to compete with NH(3) upon binding to the Mn site of the OEC that gives rise to the altered S(2) state g = 2 multiline EPR signal.     

Site-directed mutagenesis on the heme axial-ligands of cytochrome b559 in photosystem II by using cyanobacteria Synechocystis PCC 6803

Cytochrome (cyt) b559 has been proposed to play an important role in the cyclic electron flow processes that protect photosystem II (PSII) from light-induced damage during photoinhibitory conditions. However, the exact role(s) of cyt b559 in the cyclic electron transfer pathway(s) in PSII remains unclear. To study the exact role(s) of cyt b559, we have constructed a series of site-directed mutants, each carrying a single amino acid substitution of one of the heme axial-ligands, in the cyanobacterium Synechocystis sp. PCC6803. In these mutants, His-22 of the alpha or the beta subunit of cyt b559 was replaced with either Met, Glu, Tyr, Lys, Arg, Cys or Gln. On the basis of oxygen-evolution and chlorophyll a fluorescence measurements, we found that, among all mutants that were constructed, only the H22Kalpha mutant grew photoautotrophically, and accumulated stable PSII reaction centers ( approximately 81% compared to wild-type cells). In addition, we isolated one pseudorevertant of the H22Ybeta mutant that regained the ability to grow photoautotrophically and to assemble stable PSII reaction centers ( approximately 79% compared to wild-type cells). On the basis of 77 K fluorescence emission measurements, we found that energy transfer from the phycobilisomes to PSII reaction centers was uncoupled in those cyt b559 mutants that assembled little or no stable PSII. Furthermore, on the basis of immunoblot analyses, we found that in thylakoid membranes of cyt b559 mutants that assembled little or no PSII, the amounts of the D1, D2, cyt b559alpha and beta polypeptides were very low or undetectable but their CP47 and PsaC polypeptides were accumulated to the wild-type level. We also found that the amounts of cyt b559beta polypeptide were significantly increased (larger than two folds) in thylakoid membranes of cyt b559 H22YbetaPS+ mutant cells. We suspected that the increase in the amounts of cyt b559 H22YbetaPS+ mutant polypeptides in thylakoid membranes might facilitate the assembly of functional PSII in cyt b559 H22YbetaPS+ mutant cells. Moreover, we found that isolated His-tagged PSII particles from H22Kalpha mutant cells gave rise to redox-induced optical absorption difference spectra of cyt b559. Therefore, our results concluded that significant fractions of H22Kalpha mutant PSII particles retained the heme of cyt b559. Finally, this work is the first report of cyt b559 mutants having substitutions of an axial heme-ligands that retain the ability to grow photoautotrophically and to assemble stable PSII reaction centers. These two cyt b559 mutants (H22Kalpha and H22YbetaPS+) and their PSII reaction centers will be very suitable for further biophysical and biochemical studies of the functional role(s) of cyt b559 in PSII.     

Protein solubility and differential proteomic profiling of recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> overexpressing double-tagged fusion proteins

Background  

Overexpression of recombinant proteins usually triggers the induction of heat shock proteins that regulate aggregation and solubility of the overexpressed protein. The two-dimensional gel electrophoresis (2-DE)-mass spectrometry approach was used to profile the proteome of Escherichia coli overexpressing N-acetyl-D-glucosamine 2-epimerase (GlcNAc 2-epimerase) and N -acetyl-D-neuraminic acid aldolase (Neu5Ac aldolase), both fused to glutathione S-transferase (GST) and polyionic peptide (5D or 5R).