Diagnostic roles of neutrophil-to-lymphocyte ratio, monocyte-to-lymphocyte ratio, platelet-to-lymphocyte ratio, C-reactive protein, and cancer antigen 125 for ovarian cancer.

OBJECTIVE: To compare the diagnostic value of the neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), monocyte-to-lymphocyte ratio (MLR), C-reactive protein (CRP) level, and cancer antigen 125 (CA125) level for ovarian cancer (OC). METHODS: Data of 72 patients with OC, 50 patients with benign ovarian disease, and 46 healthy controls were retrospectively analyzed, and receiver operating characteristic analysis was performed. RESULTS: The platelet count was higher in patients with a tumor diameter of ≥10 vs. <10 cm. The absolute lymphocyte count was significantly higher in patients with stage I/II OC than in those with multiple and stage III/IV OC. The absolute monocyte count, NLR, MLR, and CA125 were significantly higher in patients with multiple and stage III/IV OC than in those with single and stage I/II OC. The NLR, PLR, MLR, fibrinogen, D-dimer, CRP, and CA125 were useful for distinguishing between the OC and healthy control groups. CONCLUSIONS: Our analysis showed that the following combinations have practical diagnostic value in OC: NLR + PLR + MLR + CA125, NLR + PLR + MLR + CA125 + CRP, NLR + MLR +PLR + CA125 + CRP + fibrinogen, and NLR + MLR + PLR + CA125 + CRP + fibrinogen + D-dimer.

Rapid and nondestructive identification of adulterate capsules by NIR spectroscopy combined with chemometrics.

This study aims to develop a rapid and non-destructive method to identify counterfeit and substandard drugs, addressing the critical need for better quality control in drug production. According to the reasons for counterfeit products in actual production, the commonly used solid preparation excipients such as HPMC, MCC, Mg-St and Pregelatinized Starch, as well as three chemical drugs with similar efficacy to Guizhi-Fuling (GZFL) Capsule as adulterants, including Aspirin, Ibuprofen and Sinomenine Hydrochloride were selected and designed as adulteration samples with different levels of adulteration. NIR spectra were collected in a non-invasive mode and analyzed by one-class classification methods. The feasibility of using Near-infrared (NIR) spectroscopy as a detection method to qualitatively identify adulterated samples was explored at three packaging levels of powder, intact capsules and capsules in PVC. The differences between the samples were analyzed by NIR spectra comparison, cluster analysis and principal component analysis. The performance of SVM, OCPLS and DD-SIMCA models in dealing with the authentication of genuine and counterfeit products was established and compared. The results show that the spectra contain sample information and the adulterated samples could be discriminated correctly by established models. Moreover, applying appropriate spectral preprocessing methods can further improve the model's performance. In addition, a PLS regression model was developed to predict the adulteration levels of the three packing level samples, which yielded satisfactory results. This study highlights the potential of NIR spectroscopy combined with Chemometrics as a rapid and non-destructive testing analysis method to accurately identify counterfeit and substandard drugs, thereby ensuring drug quality.

Strategies for the content determination of capsaicin and the identification of adulterated pepper powder using a hand-held near-infrared spectrometer.

To achieve the goals of rapid content determination of capsaicin and adulteration detection of pepper powder. The method based on the hand-held near-infrared spectrometer combined with ensemble preprocessing was proposed. DoE-based ensemble preprocessing technique was utilized to develop the partial least squares regression models of red pepper [Capsicum annuum L. var. conoides (Mill.) Irish] powders. The performance of final models was evaluated using coefficient of determination (R2), root mean square error of prediction (RMSEP) and residual predictive deviation (RPD). Model development using selective ensemble preprocessing gave the best prediction of capsaicin in Yanjiao pepper powder (R2 = 0.9800, RPD = 7.090, RMSEP = 0.00689) and Tianying pepper powder (R2 = 0.8935, RPD = 3.017, RMSEP = 0.06154). Moreover, the potential of grey wolf optimizer-support vector machine (GWO-SVM) to detect adulterated pepper powder was investigated. The samples were composed of two authentic products and three different adulterants with different adulteration levels. The results showed that the classification accuracy of GWO-SVM model for Yanjiao peppers was over 90 %, which realized the adulteration detection of Yanjiao pepper. And GWO-SVM showed better performance in detecting adulterated Tianying pepper compared to hierarchical cluster analysis, orthogonal partial least squares discriminant analysis and random forest. In summary, the quality control strategy established in this paper can provide a solution for the adulteration detection and quality evaluation of pepper powder in a rapid and on-site way.

Downregulation of LINC00515 in high-grade serous ovarian cancer and its relationship with platinum resistance.

Aim: To investigate the expression of long intergenic noncoding RNA 00515 (LINC00515) in high-grade serous ovarian cancer (HGSOC) and its potential correlation with platinum resistance. Patients & methods: Expression of LINC00515 in HGSOC (n = 115) and normal (n = 19) tissues was detected via quantitative real-time PCR (qRT-PCR). We further explored the statistical significance of the relationship between LINC00515 expression and platinum resistance in HGSOC. Results: LINC00515 was gradually downregulated in the order of normal > platinum-sensitive > platinum-resistant tissue (p < 0.05). Results demonstrated that LINC00515 downregulation was correlated with platinum resistance and relapse-free survival (RFS) of HGSOC (p < 0.05). Conclusion: LINC00515 downregulation is correlated with HGSOC development, platinum resistance and RFS, supporting its utility as a potential biomarker to predict platinum resistance and prognosis of RFS.

The aryl hydrocarbon receptor is a novel negative regulator of interleukin-17-mediated signaling and inflammation in vitro.

Interleukin (IL)-17 plays a critical role in the pathogenesis of inflammation and autoimmune diseases. The aryl hydrocarbon receptor (AHR) is a transcription factor responsible for the elimination of xenobiotic chemicals. However, it remains unknown whether AHR is involved in IL-17 signaling. Here, we demonstrate that knockdown of AHR significantly enhances, while overexpression or activation of AHR inhibits IL-17-induced inflammation in Hela cells. AHR specifically suppresses IL-17-induced p38 activation, and inhibition of p38 activity markedly reverses the effect of AHR knockdown on IL-17-induced inflammation. Mechanistically, AHR physically interacts with TAK1 and mitogen-activated protein kinase kinase 3/6 (MKK3/6) and disrupts TAK1-MKK3/6 interaction, leading to impaired IL-17 signaling. Thus, our study indicates that AHR negatively regulates IL-17-mediated signaling and inflammation at least partially through interfering with the interaction between TAK1 and MKK3/6.

Apatinib for chemotherapy-refractory extensive-stage SCLC: a retrospective study.

PURPOSE: There is no standard treatment strategy for patients with extensive-stage small cell lung cancer (SCLC) who have failed two or more prior chemotherapeutic regimens. In this study, we retrospectively evaluated the efficacy and safety of apatinib in patients with extensive-stage SCLC after failure of more than second-line chemotherapy. METHODS: A study group comprised of 22 patients with extensive-stage SCLC after failure of more than two prior chemotherapeutic regimens was given apatinib orally at an initial dose of 500 mg daily until disease progression or unacceptable toxicity. This study was analyzed according to the National Cancer Institute Common Toxicity Criteria for adverse events (AEs) and Response Evaluation Criteria in Solid Tumors (RECIST) for response assessment. RESULTS: Between August 30, 2015, and May 26, 2017, 22 patients were enrolled for evaluating the efficacy and safety of apatinib. Among them, 12/22 (54.5%) underwent dose reduction during treatment. Up to July 31, 2018, the median progression-free survival rate was 135.0 days [95% confidence interval (CI) 63.8-206.2]. According to the RECIST criteria, the disease control rate (DCR) was 86.4%, 19/22 [comprised of partial response (PR) 18.2%, 4/22; and stable disease (SD) 68.2%, 15/22 patients]. The most frequent AEs were hand-foot syndrome (45.5%, 10/22), secondary hypertension (45.5%, 10/22) and fatigue (40.9%, 9/22). The primary grade 3 or 4 toxicities were hypertension (22.7%, 5/22), hand-foot syndrome (13.6%, 3/22), and proteinuria (9.1%, 2/22). CONCLUSIONS: Apatinib exhibits modest activity and acceptable toxicity for patients with heavily pretreated extensive-stage SCLC.

Human Serum Albumin Nanoparticles as a Novel Delivery System for Cabazitaxel.

BACKGROUND: Due to use of Tween-80 as an enhancer of solubility, the current clinical formulation of cabazitaxel (CBT) (Jevtana®) causes hypersensitivity, neurotoxicity and other severe side-effects. To reduce these vehicle-related effects, a suitable nanocarrier is needed. MATERIALS AND METHODS: Human serum albumin (HSA) was used to encapsulate CBT by a simple self-assembly method. Physicochemical properties of HSA-CBT nanoparticles were characterized. In vitro release property and cytotoxicity were also determined. In vivo imaging system was used to study nanocarrier distribution in vivo. The safety profile was assessed by hemolysis and acute-toxicity study. Finally, the antitumor efficacy in vivo was investigated in tumor-bearing mice. RESULTS: The average size of HSA-CBT nanoparticles was about 240 nm and the encapsulation efficiency reached 97%. The hemolysis and acute-toxicity experiments confirmed biocompatibility of HSA-CBT nanoparticles. CONCLUSION: HSA nanoparticles are a safe and effective drug delivery system for hydrophobic anticancer drugs such as CBT.

Estrogen therapy to treat retinopathy in newborn mice.

The aim of the present study was to treat retinopathy of prematurity (ROP) with estrogen (E2) so as to elucidate the role of E2 in the pathogenesis of ROP. A total of 120 postnatal 7-day-old (P7) C57BL/6J mice were selected and raised in a high-oxygen environment (75% oxygen) for 5 days, followed by 5 days in normal room air. Different doses of E2 or normal saline (NS) were injected intraperitoneally during different time-periods, and the mice were divided into 14 groups according dose of E2 injection (0.5-1.5 µg/0.05 ml) and dosing time. Blood vessel changes and hyperplasia were evaluated in flat-mounted retina and retinal slices. All mice that were exposed to room air, whether they were administered E2 or NS, showed good vascular development in the flat-mounted retina at P17. No increase in the number of endothelial cell nuclei in the new blood vessels was observed. In ascending order of E2 dose the numbers of cell nuclei were as follows: 0.18±0.129, 0.28±0.086 and 0.55±0.110. The number in the NS group was 2.12±0.373. When the results of the room-air groups were compared with those of the hyperoxia groups, a highly significant difference was found in each comparison (P<0.0001). All mice showed varying degrees of neovascularization and vascular obstruction in the flat-mounted retina at P17, and it was difficult to compare the blood vessels morphologically among these groups. The number of endothelial cell nuclei decreased following E2 injection, and the difference from the NS group exposed to hyperoxia was highly significant (P<0.0001). For all dose levels, the number of cell nuclei was the lowest when the drug was administered during P7-16, and the difference from the other two time-periods was statistically significant (P<0.05). When E2 was administered during P7-16, the number of cell nuclei was 15.5±1.993 in the 0.5-µg group, 14.23±2.49 in the 1.0-µg group and 18.05±1.62 in the 1.5-µg group. No significant difference was found among these three groups (P>0.05). In conclusion, E2 treatment during the development of retinopathy can improve symptoms in neonatal mice, suggesting that E2 plays an important role at the two initial stages in the pathogenesis of ROP. This may indicate new pharmacological measures to prevent and treat ROP.

Application of fetal neural stem cells transplantation in delaying denervated muscle atrophy in rats with peripheral nerve injury.

Injury to peripheral nerves always results in progressive skeletal muscle atrophy and poor functional recovery. Previous studies have demonstrated that transplanting neural stem cells (NSCs) into peripheral nerve can differentiate into neurons and delay muscle atrophy. However, the mechanism was not very clear. In this study, we transplanted the fetal NSCs to the injured nerve and examined new formed neuromuscular junctions (NMJs) in the denervated muscle and arrest of muscle atrophy. In our study, two pregnant Fischer rats were used to harvest fetal NSCs, 70 rats were randomly divided into NSC-transplanted and control groups, five rats without surgery were used as the normal control. A volume of 5 microl culture media with or without fetal NSCs (5 x 10(6)) were transplanted into distal tibial nerve stump after the nerve was transected in two groups, respectively. Three, five, and seven months after denervation, the dry weight of gastrocnemius muscle was found significant heavier, and the fiber area was more retained in NSC-transplanted group comparing to the control group (P < 0.05). Neurons were found in the distal tibial nerves even 7 months after fetal NSCs transplantation. Newly formed NMJs were detected by immunohistochemistry. In addition, the results of electrophysiological analysis and retrograde tracing manifested that the neural pathway between muscle and differentiated neurons was integrity. In conclusions, our study demonstrated that fetal NSCs transplanted into peripheral nerves could differentiate into neurons and form functional NMJs with denervated muscle, which may be beneficial for the treatment of muscle atrophy after peripheral nerve injury.

The structural and biological significance of the EAAEAE insert in the alpha-domain of human neuronal growth inhibitory factor.

Human neuronal growth inhibitory factor (hGIF) is able to inhibit the outgrowth of neurons. As compared with the amino acid sequences of metallothionein 1/2, hGIF contains two insertions: a Thr at position 5 and an acidic hexapeptide EAAEAE(55-60) close to the C-terminus. Moreover, all mammalian growth inhibitory factor sequences contain a conserved CPCP(6-9) motif. Previous studies have demonstrated that the TCPCP(5-9) motif is pivotal to its bioactivity, but no specific role has been assigned to the unique EAAEAE(55-60) insert. To investigate the potential structural and biological significance of the EAAEAE(55-60) insert, several mutants were constructed and investigated in detail. Notably, deletion of the acidic insert (the Delta55-60 mutant) reduced the inhibitory activity, whereas the bioactivities of other mutants did not change much. Then, spectroscopic characterization and molecular dynamics simulation were performed to investigate the potential causes of the reduced bioactivity of the Delta55-60 mutant. It was found that the domain-domain interaction mechanism of hGIF was different from that of metallothionein 2. It was also shown that the acidic insert could regulate the interdomain interactions in hGIF, leading to the structural change in the beta-domain, which resulted in the alteration of the solvent accessibility and metal release ability, thus playing an important role in the biological activity of hGIF. Our studies provided useful information on the domain-domain interaction at the molecular level for the first time, and shed new light on the mechanism of the bioactivity of hGIF.

Important roles of the conserved linker-KKS in human neuronal growth inhibitory factor.

Metallothinein-3 (MT3), also named neuronal growth inhibitory factor (GIF), is attractive by its distinct neuronal growth inhibitory activity, which is not shared by other MT isoforms. The polypeptide chain of GIF is folded into two individual domains, which are connected by a highly conserved linker, KKS. In order to figure out the significance of the conserved segment, we constructed several mutants of human GIF (hGIF), including the K31/32A mutant, the K31/32E mutant and the KKS-SP mutant by site-directed mutagenesis. pH titration and DTNB reaction exhibited that all the three mutations made the beta-domain lower in stability and looser. More significantly, change of KKS to SP also altered the general backbone conformation and metal-thiolate cluster geometry. Notably, bioassay results showed that the bioactivity of the K31/32A mutant and the K31/32E mutant decreased obviously, while the KKS-SP mutant lost inhibitory activity completely. Based on these results, we proposed that the KKS linker was a crucial factor in modulating the stability and the solvent accessibility of the Cd(3)S(9) cluster in the beta-domain through domain-domain interactions, thus was indispensable to the biological activity of hGIF.

Incorporation of a glycine within the conserved TCPCP motif of human neuronal growth inhibitory factor significantly reduces its bioactivity.

It has been reported that the (6)CPCP(9) motif near the N-terminus is pivotal to the inhibitory activity of human neuronal growth inhibitory factor (hGIF). In order to better understand the biological significance of this region on the structure, property and function of hGIF, we introduced a highly flexible residue, Gly, either in front of the (6)CPCP(9) motif (the IG6 mutant, TGCPCP) or in the middle of it (the IG8 mutant, TCPGCP) and investigated their structural and metal binding properties in detail. The results showed that the overall structure and the stability of the metal-thiolate clusters of the two mutants were comparable to that of hGIF. However, the bioassay results showed that the bioactivity of the IG6 mutant decreased significantly, while the bioactivity of the IG8 mutant was almost abolished. Molecular dynamics simulation results showed that the backbone of the IG6 mutant exhibited high similarity to that of hGIF, and the two prolines could still induce structural constraints on the (6)CPCP(9) tetrapeptide and form a similar conformation with that of hGIF, however, the conformation of the first five amino acid residues in the N-terminus was quite different. In hGIF, the five residues are twisted and form a restricted conformation, while in the IG6 mutant this peptide extends more naturally and smoothly, which is similar to that of MT2. As to the IG8 mutant, the Gly insertion broke the (6)CPCP(9) motif, thus probably abolishing the interactions with other molecules and eliminating its inhibitory activity. Based on these results, we suggested that although the structure adopted by the (6)CPCP(9) motif is the determinant factor of the inhibitory bioactivity of hGIF, other residues within the N-terminal fragment (residue 1-13) may also influence the peptide conformation and contribute to the protein's bioactivity.

Effect of alpha-domain substitution on the structure, property and function of human neuronal growth inhibitory factor.

Human metallothionein-3 (hMT3), also named human neuronal growth inhibitory factor (hGIF), is attractive due to its distinct neuronal growth inhibitory activity, which is not shown by other human MT isoforms. It has been reported that the neuronal growth inhibitory activity arises from the N-terminal beta-domain rather than its C-terminal alpha-domain. However, previous bioassay results have shown that the single beta-domain is less effective at inhibiting the neuron growth than that in intact hMT3 on a molar basis, which suggests that the alpha-domain is indispensable to the neuronal growth inhibitory activity of hMT3. In order to confirm this assumption, we constructed two domain-hybrid mutants, the beta(MT3)-beta(MT3) mutant and the beta(MT3)-alpha(MT1) mutant, and investigated their structural and metal binding properties by UV-vis spectroscopy, CD spectroscopy, pH titration, DTNB reaction, EDTA reaction, etc. The results showed that stability of the Cd(3)S(9) cluster of the beta(MT3)-beta(MT3) mutant decreased significantly while the Cd(3)S(9) cluster of the beta(MT3)-alpha(MT1) mutant had a similar stability and solvent accessibility to that of hMT3. Interestingly, the bioassay results showed that the neuronal growth inhibitory activity of the beta(MT3)-beta(MT3) mutant decreased significantly, while the beta(MT3)-alpha(MT1) mutant showed similar inhibitory activity to hMT3. Based on these results, we conclude that the alpha-domain is indispensable and plays an important role in modulating the stability of the metal cluster in the beta-domain by domain-domain interactions, thus influencing the bioactivity of hMT3.

Mutation at Glu23 eliminates the neuron growth inhibitory activity of human metallothionein-3.

Human metallothionein-3 (hMT3), first isolated and identified as a neuronal growth inhibitory factor (GIF), is a metalloprotein expressed predominantly in brain. However, until now, the exact mechanism of the bioactivity of hMT3 is still unknown. In order to study the influence of acid-base catalysis on S-nitrosylation of hMT3, we constructed the E23K mutant of hMT3. During the course of bioassay, we found out unexpectedly that mutation at E23 of hMT3 eliminates the neuronal growth inhibitory activity completely. To the best of our knowledge, it is the first report that other residues, besides the TCPCP motif, in the beta-domain can alter the bioactivity of hMT3. In order to figure out the causes for the loss of bioactivity of the E23K mutant, the biochemical properties were characterized by UV-vis spectroscopy, CD spectroscopy, pH titration, DTNB reaction, EDTA reaction, and SNOC reaction. All data demonstrated that stability of the metal-thiolate cluster and overall structure of the E23K mutant were not altered too much. However, the reaction of the E23K mutant with SNOC exhibited biphasic kinetics and the mutant protein released zinc ions much faster than hMT3 in the initial step, while hMT3 exhibited single kinetic process. The 2D [1H-15N] HSQC was also employed to characterize structural changes during the reaction of hMT3 with varying mounts of nitric oxide. It was shown that the resonance of Glu23 disappeared at a molar ratio of NO to protein of 4. Based on these results, we suggest that mutation at Glu23 may alter the NO metabolism and/or affect zinc homeostasis in brain, thus altering the neuronal growth inhibitory activity.

The role of Thr5 in human neuron growth inhibitory factor.

GIF, a member of the metallothionein (MT) family (assigned as MT3), is a neuron growth inhibitory factor that inhibits neuron outgrowth in Alzheimer's disease. The conserved Thr5 is one of the main differences between GIF and other members in the MT family. However, natural sheep GIF has an unusual Ala5, casting doubt on the role of common Thr5. We constructed a series of human GIF mutants at site 5, and characterized their biochemical properties by UV spectroscopy, circular dichroism spectroscopy, EDTA reaction, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) reaction, and pH titration. Their inhibitory activity toward neuron survival and neurite extension was also examined. Interestingly, the T5A mutant exhibited distinct metal thiolate activity in the EDTA and DTNB reactions, and also lost its bioactivity. Meanwhile, the T5S mutant had similar biochemical properties and biological activity as wild-type human GIF, indicating the hydroxyl group on the Thr5 was critical to the bioactivity of human GIF. We suggest the hydroxyl group in human GIF may help stabilize the biologically active conformation. On the other hand, lack of the hydroxyl group in sheep GIF may be partially compensated by its abnormal structure.

Synthesis of Human Proinsulin C-peptide and Preparation of Specific Antibody to It.

A HPLC and CE pure human proinsulin C-peptide was synthesized by solid-phase method and TSK column purification. Its amino acid sequence and MS were consistent with theoretical values. In comparison with the formly reported chemical synthesis of C-peptide, this method has the advantage of simplicity and higher overall yield (41%). To improve the immunogenicity and specificity of oligopeptide antibody, the acrylyl-C-peptides were transformed into a polymer the product had a poly-propionyl-core matrix with C-peptide branches. This treatment gave a macromolecule with a M(r) about 25 kD. By using the polymer to immunize New Zealand rabbits for 30 days, specific antiserum was obtained with titer of 2.5x10(4) (by ELISA), which did not cross react with BSA. Thus, the poly-propionyl-peptide system provided a new approach for preparing synthetic peptide antibody and therefore is promising for the preparation of synthetic peptide-based vaccine.

Synthesis and Biological Activity of Human Calcitonin Analogue.

The potency of salmon calcitonin (sCT) is higher but the structural homology between sCT and human calcition (hCT) is only 50%. Based on the comparison of the structure between sCT and hCT, we have designed and synthesized a hCT analogue (mhCT-2) by solid phase method, using air oxidation in diluted solution to obtain a peptide with intramolecular disulfide bond. Through HPLC purification, we obtained a capillary electrophoresis-homogeneous mhCT, results of analysis of its mass spectrum and N-terminal sequencing were in accordance with the theoretical values. The results of calcitonin bioassay by estimating the blood calcium levels in rats showed that the potency of mhCT-2 was around 2 000 IU/mg, one order of magnitude higher than that of hCT. In RIA assay, we have found that the immunoactivity of mhCT-2 and hCT was much different because of their different binding abilities to anti-hCT antibody. This indicated that the conformation of mhCT-2 was changed as compared with hCT. In rat osteoporosis model, the results showed that pharmacologic effects of mhCT-2 was the same as that of sCT. The synthetic mhCT-2 seems promising to be a clinically useful peptide with high potential in osteoporosis therapy, because it is similar in biological properties to, but less immunogenic than sCT.