Effects of blood collection conditions on ovarian cancer serum markers

Background

Evaluating diagnostic and early detection biomarkers requires comparing serum protein concentrations among biosamples ascertained from subjects with and without cancer. Efforts are generally made to standardize blood processing and storage conditions for cases and controls, but blood sample collection conditions cannot be completely controlled. For example, blood samples from cases are often obtained from persons aware of their diagnoses, and collected after fasting or in surgery, whereas blood samples from some controls may be obtained in different conditions, such as a clinic visit. By measuring the effects of differences in collection conditions on three different markers, we investigated the potential of these effects to bias validation studies.

Methodology and Principle Findings

We analyzed serum concentrations of three previously studied putative ovarian cancer serum biomarkers–CA 125, Prolactin and MIF–in healthy women, women with ovarian cancer undergoing gynecologic surgery, women undergoing surgery for benign ovary pathology, and women undergoing surgery with pathologically normal ovaries. For women undergoing surgery, a blood sample was collected either in the clinic 1 to 39 days prior to surgery, or on the day of surgery after anesthesia was administered but prior to the surgical procedure, or both. We found that one marker, prolactin, was dramatically affected by collection conditions, while CA 125 and MIF were unaffected. Prolactin levels were not different between case and control groups after accounting for the conditions of sample collection, suggesting that sample ascertainment could explain some or all of the previously reported results about its potential as a biomarker for ovarian cancer.

Conclusions

Biomarker validation studies should use standardized collection conditions, use multiple control groups, and/or collect samples from cases prior to influence of diagnosis whenever feasible to detect and correct for potential biases associated with sample collection.     

Prevalence and risk factors of overweight and obesity in China

Objective: To examine the prevalence and risk factors of overweight and obesity in China. Research Methods and Procedures: A cross‐sectional survey was conducted in a nationally representative sample of 15,540 Chinese adults in 2000–2001. Body weight, height, and waist circumference were measured by trained observers. Overweight and obesity were defined according to the World Health Organization classification. Central obesity was defined according to guidelines of the International Diabetes Federation. Results: Mean BMI and waist circumference were 23.1 kg/m² and 79.6 cm, respectively, for men and 23.5 kg/m² and 77.2 cm, respectively, for women. The prevalences of overweight and obesity were 24.1% and 2.8% in men and 26.1% and 5.0% in women, respectively. The prevalence of central obesity was 16.1% in men and 37.6% in women. The prevalences of overweight, obesity, and central obesity were higher among residents in northern China compared with their counterparts in southern China and among those in urban areas compared with those in rural areas. Lifestyle factors were the most important risk factors to explain the differences in overweight and central obesity between northern and southern residents. Among women, lifestyle and diet were the most important risk factors to explain the differences between urban and rural residents, whereas socioeconomic status, lifestyle, and diet were all important among men. Discussion: Our study indicates that overweight and obesity have become important public health problems in China. Environmental risk factors may be the main reason for regional differences in the prevalence of overweight and obesity in China.     

Discovery of estrogen receptor α modulators from natural compounds in Si-Wu-Tang series decoctions using estrogen-responsive MCF-7 breast cancer cells

The binding between the estrogen receptor α (ER-α) and a variety of compounds in traditional Chinese formulae, Si-Wu-Tang (SWT) series decoctions, was studied using a stably-transfected human breast cancer cell line (MVLN). In 38 compounds tested from SWT series decoctions, the estrogen-like activity of 22 compounds was above 60% in 20 μg mL(-1). Furthermore, theoretical affinity of these compounds was certificated using the functional virtual screen of ER-α modulators by FlexX-Pharm. The accuracy of functional virtual screening of ER-α modulators could reach to 77.27%. The results showed that some compounds, such as organic acids and flavones in SWT series decoctions could be used as selective estrogen receptor modulators (SERMs) and could be selected for further development as potential agents for estrogen related diseases.     

Discovery of novel P3-oxo inhibitor of hepatitis C virus NS3/4A serine protease

A novel series of P3 oxo-modified macrocyclic hepatitis C virus NS3/4A serine protease inhibitor was designed, synthesized and biologically evaluated. The hydroxy-substituted inhibitor 10 demonstrated high potency in genotype 1a and 1b replicon and in the panel of HCV protease mutants. Interestingly, the t-butyl carbonate analog 9c, while not the most potent one in this series, exhibited a virtually flat potency profile in the panel of HCV protease mutants, thus providing opportunity for further optimization.     

Shh signaling guides spatial pathfinding of raphespinal tract axons by multidirectional repulsion

Relatively little is known about the molecular mechanisms underlying spatial pathfinding in the descending serotonergic raphespinal tract (RST) in the developing spinal cord, one of the most important nerve pathways for pain, sensory and motor functions. We provide evidence that ventral floor plate-secreted Sonic hedgehog (Shh) is responsible for the establishment of decreasing gradients in both the anterior-to-posterior (A-P) and the medial-to-lateral (M-L) directions in the ventral spinal cord during serotonergic RST axon projection. Downstream components of the Shh pathway, Patched 1 (Ptch1) and Smoothened (Smo), were expressed in the serotonergic caudal raphe nuclei and enriched in the descending serotonergic RST axons. Diffusible Shh repulsion of serotonergic RST axons was shown to be mediated by Shh-Ptch1 interactions and derepression of Smo. Using a co-culture assay, we showed that A-P graded repulsion mediated by Shh signaling pushed the serotonergic axons caudally through the ventral spinal cord and M-L graded repulsion mediated by Shh signaling simultaneously restricted the serotonergic axons to the ventral and ventral-lateral funiculus. Prominent pathfinding errors of serotonergic RST axons were observed in various Shh, Ptch1 and Smo mutants. We conclude that Shh signaling-mediated multidirectional repulsion is required to push descending serotonergic RST axons in the A-P direction, and to restrict these axons to the ventral and ventral-lateral funiculus in the M-L direction. This is the first demonstration that Shh signaling-mediated multidirectional repulsion of serotonergic RST axons maintains spatial axon pathfinding in the developing spinal cord.     

Simulated microgravity affects semicarbazide-sensitive amine oxidase expression in recombinant Escherichia coli by HPLC-ESI-QQQ analysis

Simulated microgravity has been reported to affect the gene, protein expression, and its function in the cells. Semicarbazide-sensitive amine oxidase (SSAO; E.C.1.4.3.6.) is widely distributed in vascular cells, smooth muscle cells, and adipocytes. It is noteworthy whether the expression of SSAO is affected under simulated microgravity or not. In this study, an SSAO-transformed Escherichia coli BL21 was constructed firstly. Then, a sensitive, selective, and accurate method based on high-performance liquid chromatography electrospray ionization triple quadrupole (HPLC-ESI-QQQ) was developed to determine the amount of SSAO in the E. coli BL21. The limit of detection and limit of quantification were 5.0 and 10 fmol, respectively. Finally, SSAO expression in the recombinant E. coli BL21 was evaluated with various gravity and temperature conditions by HPLC-ESI-QQQ analysis. It is interesting that the tendency in the alteration of SSAO under simulated microgravity showed temperature difference. At 18 °C, the amount of SSAO in the inclusion bodies and soluble fractions under the simulated microgravity increased by 83% and 116%, respectively, compared with normal gravity. However, the decrease by 38% and 49% in the inclusion bodies and soluble fractions under the simulated microgravity was observed at 37 °C. Results obtained here indicate that the SSAO expression under simulated microgravity is dramatically sensitive to the temperature. On the other hand, a novel bioreactor from this study may also be useful for the recombinant protein expression in the field of gene engineering.     

State-dependent conformations of the translocation pathway in the tyrosine transporter Tyt1, a novel neurotransmitter:sodium symporter from Fusobacterium nucleatum

The gene of a novel prokaryotic member (Tyt1) of the neurotransmitter:sodium symporter (NSS) family has been cloned from Fusobacterium nucleatum. In contrast to eukaryotic and some prokaryotic NSSs, which contain 12 transmembrane domains (TMs), Tyt1 contains only 11 TMs, a characteristic shared by approximately 70% of prokaryotic NSS homologues. Nonetheless upon heterologous expression in an engineered Escherichia coli host, Tyt1 catalyzes robust Na+-dependent, highly selective l-tyrosine transport. Genetic engineering of Tyt1 variants devoid of cysteines or with individually retained endogenous cysteines at positions 18 or 238, at the cytoplasmic ends of TM1 and TM6, respectively, preserved normal transport activity. Whereas cysteine-less Tyt1 was resistant to the inhibitory effect of sulfhydryl-alkylating reagents, N-ethylmaleimide inhibited transport by Tyt1 variants containing either one or both of the endogenous cysteines, and this inhibition was altered by the substrates sodium and tyrosine, consistent with substrate-induced dynamics in the transport pathway. Our findings support a binding model of Tyt1 function in which an ordered sequence of substrate-induced structural changes reflects distinct conformational states of the transporter. This work identifies Tyt1 as the first functional bacterial NSS member putatively consisting of only 11 TMs and shows that Tyt1 is a suitable model for the study of NSS dynamics with relevance to structure/function relationships of human NSSs, including the dopamine, norepinephrine, serotonin, and gamma-aminobutyric acid transporters.     

Structure and function of glucose-6-phosphate dehydrogenase-deficient variants in Chinese population

A systematic study on the structure and function of Glucose-6-phosphate dehydrogenase (G6PD) variations was carried out in China. A total of 155,879 participants were screened for G6PD deficiency by the G6PD/6PGD ratio method and 6,683 cases have been found. The prevalence of G6PD deficiency ranged from 0 to 17.4%. With informed consent, 1,004 cases from 11 ethnic-based groups were subjected to molecular analysis. Our results showed the followings: (1) The G6PD variants are consistent across traditional ethnic boundaries, but vary in frequencies across ethnic-based groups in Chinese population, (2) The G6PD variants in Chinese population are different from those in African, European, and Indian populations, (3) A novel G6PD-deficiency mutation, 274C→T, has been found, and (4) Denaturing high performance liquid chromatography is of great advantage to detecting G6PD-deficient mutations for diagnosis and genetic counseling. Moreover, functional analysis of the human G6PD variants showed the following: (1) The charge property, polarity, pK-radical and side-chain radical of the substituting amino acid have an effect on G6PD activity, (2) The G6PDArg459 and Arg463 play important roles in anchoring NADP⁺ to the catalytic domain to maintain the enzymatic activity, and (3) The sequence from codon 459 to the carboxyl terminal is essential for the enzymatic function.     

Alkaline sphingomyelinase: an old enzyme with novel implications

Alkaline sphingomyelinase (alk-SMase) is present in the intestinal tract and additionally human bile. It hydrolyses sphingomyelin in both intestinal lumen and the mucosal membrane in a specific bile salt dependent manner. The enzyme was discovered 36 years ago but got real attention only in the last decade, when sphingomyelin metabolism was realized to be a source of multiple lipid messengers, and when dietary sphingomyelin was found to inhibit colonic tumorigenesis in animals. The enzyme shares no structural similarity with other SMases and belongs to the nucleotide pyrophosphatase/phosphodiesterase family. The enzyme is of specific properties, such as bile salt dependency, trypsin resistance, high stability, and tissue specific expression. In the colon, the enzyme may play antiproliferative and antiinflammatory roles through generating ceramide, reducing the formation of lysophosphatidic acid, and inactivating platelet-activating factor. The enzyme is down regulated in human long-standing ulcerative colitis and colonic adenocarcinoma, and mutation of the enzyme has been found in colon cancer cells. In the small intestine, alk-SMase is the key enzyme for sphingomyelin digestion. The hydrolysis of sphingomyelin may affect the cholesterol uptake and have impact on sphingomyelin levels in plasma lipoproteins. The review summarizes the new information of alk-SMase from biochemical, cell and molecular biological studies in the last decade and evaluates its potential implications in development of colon cancer, inflammatory bowel diseases, and atherosclerosis.