Shu Chu Shiesh

Integrated microfluidic system for rapid screening of CRP aptamers utilizing systematic evolution of ligands by exponential enrichment (SELEX)

The systematic evolution of ligands by exponential enrichment (SELEX) is an experimental procedure that allows screening of given molecular targets by desired binding affinities from an initial random pool of oligonucleotides and oligomers. The final products of SELEX are usually referred as aptamers, which are recognized as promising molecules for a variety of biomedical applications. However, SELEX is an iterative process requiring multiple rounds of extraction and amplification that demands significant time and labor. Therefore, this study presents a novel, automatic, miniature SELEX platform. As a demonstration, the rapid screening of C-reactive protein (CRP) aptamers was performed. By utilizing microfluidic technologies and magnetic beads conjugated with CRP, aptamers with a high affinity to CRP were extracted from a random single-strand deoxyribonucleic acid (ssDNA) pool. These aptamers were further amplified by an on-chip polymerase chain reaction (PCR) process. After five consecutive extraction and amplification cycles, a specific aptamer with the highest affinity was screened automatically. The screened aptamers were used as a recognition molecule for the detection of CRP. The developed microsystem demonstrated fast screening of CRP aptamers and can be used as a powerful tool to select analyte-specific aptamers for biomedical applications.

Serum total antioxidant capacity reflects severity of illness in patients with severe sepsis.

IntroductionWe conducted the present study to evaluate the changes in serum total antioxidant capacity (TAC) in patients with severe sepsis and to investigate the association between serum TAC and clinical severity.MethodThis was a prospective observational study involving a sample of patients who met established criteria for severe sepsis and were admitted to the emergency department of a university teaching hospital. Serum TAC was determined using the total radical-trapping antioxidant parameter method. The levels of TAC, uric acid, albumin, and bilirubin in sera were obtained in the emergency department and evaluated to determine whether there were any correlations between the major antioxidant biomarkers and clinical severity of sepsis. The Acute Physiology and Chronic Health Evaluation (APACHE) II score was used for clinical evaluation of the severity of sepsis.ResultsA total of 73 patients with sepsis, with a mean (± standard deviation) APACHE II score of 23.2 ± 8.2 and a mortality rate of 26.0%, were included. Seventy-six healthy individuals served as control individuals. Among the patients, serum TAC levels correlated significantly with APACHE II scores. Patients who died also had higher TAC than did those who survived. Serum uric acid levels correlated significantly with serum TAC and APACHE II scores in patients with severe sepsis.ConclusionElevated serum TAC level may reflect clinical severity of sepsis. In addition, serum uric acid levels appear to contribute importantly to the higher TAC levels observed in patients with severe sepsis.

Clinical Significance of Ultrasonography, Computed Tomography, and Biochemical Tests in the Rapid Diagnosis of Gallstone-related Pancreatitis: A Prospective Study

Real-time ultrasonography (US), computed tomography (CT), and biochemical tests were prospectively performed to detect gallstones in 88 consecutive patients immediately after the onset of an attack of acute pancreatitis. The sensitivity of biochemical tests was 84.6% when the patients had three or more positives of five parameters [including serum bilirubin, alkaline phosphatase (AP), gamma-glutamyl transpeptidase (GGT), alanine transaminase (ALT), and alanine transaminase-aspartate transaminase (ALT-AST) ratio]. The sensitivity, specificity, and accuracy were 71.8, 98.0, and 86.4% for US, and 52.9%, 100%, and 79.5% for CT. The sensitivity, specificity, and accuracy were improved to 82.1, 100, and 93.2% by the combination of US and CT, and 94.9, 100, and 97.7% by the combination of US and biochemical tests. Adding CT to the combination of US and biochemical tests resulted in only a slight improvement in sensitivity and accuracy. In conclusion, a combination of US and biochemical tests can provide the best noninvasive method in rapidly detecting gallstones as an etiological factor in acute pancreatitis. Computed tomography is not cost-effective. A positive result of biochemical tests despite a negative finding in US calls for an intensive search for gallstones by further investigation with endoscopic retrograde cholangiography or repeated US examinations.

High Oxidative Stress Is Correlated with Frailty in Elderly Chinese

OBJECTIVES: To evaluate the relationship between oxidative stress and frailty in elderly people.

Bacterial density of Helicobacter pylori predicts the success of triple therapy in bleeding duodenal ulcer

BACKGROUNDnWe studied whether different initial bacterial densities of Helicobacter pylori would alter the eradication rate of H. pylori by triple therapy (amoxicillin 500 mg t.i.d. and metronidazole 500 mg t.i.d. for 14 days; bismuth subcitrate 120 mg t.i.d. for 28 days) in patients with duodenal ulcer bleeding.nnnMETHODnOne hundred thirty-six cases with duodenal ulcer bleeding and H. pylori infection (proved by rapid urease test and histology during emergency endoscopy) were studied. One hundred twenty-seven of these patients completed a course of triple therapy. In each case, anti-H. pylori IgG titer, gastric biopsies for H. pylori density (score 1 to 5), and evaluation of severity of gastritis were collected at the first endoscopy and 1 month after completion of the triple therapy.nnnRESULTSnThe ulcer healing rate was 84.3% (107 of 127) at the time of the second evaluation. The eradication rate of H. pylori was 76.4% (97 or 127). Eradication for H. pylori failed in 30 cases. In these eradication failure cases, initial serologic titer and density of H. pylori were higher than those of eradication success cases. The eradication rate of H. pylori decreased as the initial density of H. pylori increased (density of H. pylori: 1, 88.3%; 2, 83.8%; 3, 74.2%; 4, 68%; 5, 50%). At the second evaluation, the serologic titer was lower and continued to decline in eradication success cases whose mean residual titer ratio (100% x follow-up titer/initial titer) was lower than that of eradication failure cases (57.1% +/- 14.6% vs 107.1% +/- 24.1%, p < 0.001). The mean residual titer ratio also disclosed an upward trend as the density of H. pylori increased (density of H. pylori 1 to 5: 57.5%, 66.6%, 73.5%, 75.3%, 81.8%, respectively).nnnCONCLUSIONSnWe suggest routine gastric biopsy to detect both the presence of H. pylori and its density inasmuch as quantitative results may predict the usefulness of triple therapy. The higher the H. pylori density, the less effective triple therapy will be at successful eradication of H. pylori.

An integrated microfluidic system for C-reactive protein measurement

This study presents a new microfluidic chip integrated with pneumatic micropumps, normally close microvalves and vortex-type micromixers for C-reactive protein (CRP) measurement. CRP is a protein produced during the inflammation process. It has been reported that CRP in serum can be used for risk assessment of cardiovascular diseases. In this study, CRP measurements were performed by using the integrated microfluidic chip incorporated with magnetic beads. The magnetic beads coated with CRP-specific DNA aptamers were used to recognize, purify and enrich the target CRP. The entire process including sample pre-treatment, and the interaction between the target CRP and anti-CRP antibody was automatically performed on a single chip. The chemiluminescence signal was measured using a luminometer to detect the concentration of CRP afterwards. The entire reaction time is less then 25 min, which is only about 20% of the time required when compared to using traditional bench-top machines (150 min). More importantly, the detection limit has been improved from 0.125 to 0.0125 mg/L with only half the amount of reagent consumption. The development of this microfluidic system is promising for fast, accurate, and sensitive detection of CRP.

An integrated microfluidic system for rapid screening of alpha-fetoprotein-specific aptamers.

The systematic evolution of ligands by exponential enrichment (SELEX) is a screening technique that involves the progressive selection of highly specific ligands via repeated rounds of partition and amplification from a large random pool of nucleic acid sequences. The products of this selection process are called aptamers and are either short single-stranded deoxyribonucleic acid (ssDNA) or ribonucleic acid (RNA) molecules with a high binding affinity to a large variety of target analytes. However, SELEX is a lengthy, labor-intensive, iterative process requiring multiple rounds of extraction and polymerase chain reaction (PCR) amplification. In order to address these problems, this study presents a new integrated microfluidic system consisting of a magnetic bead-based microfluidic SELEX chip and a competitive assay chip to automate the aptamer screening process. More importantly, the selected ssDNA sequences were confirmed to have a high affinity and specificity to the target molecules, using the developed competitive assay chip. With this approach, an aptamer specific to alpha-fetoprotein (AFP), which is a biomarker for liver cancers, has been successfully selected. The screened aptamer was used as a recognition molecule for AFP and has a linear detection range from 12.5 to 800 ng/mL, which was suitable for rapid clinical applications.

Increased Aortic Stiffness and Attenuated Lysyl Oxidase Activity in Obesity

Objective—One potential mechanism through which obesity exerts adverse effects on the vascular system is by increasing aortic stiffness, a change known to be predictive of increased cardiovascular mortality. The aim of this study was to investigate the pathophysiology that links obesity to aortic stiffening. Approach and Results—Obese (ob/ob) mice were used to examine physical, morphological, and molecular changes in the aorta in response to obesity. ob/ob mice had increased aortic pulse wave velocity and tissue rigidity. ob/ob aorta exhibited decreases of lysyl oxidase (LOX) activity and cross-linked elastin, and increases of elastin fragmentation and elastolytic activity. The aortas of ob/ob mice were surrounded by a significant amount of proinflammatory and pro-oxidative perivascular adipose tissue. In vitro studies revealed that the conditioned medium from differentiated adipocytes or the perivascular adipose tissue of ob/ob mice attenuated LOX activity. Furthermore, inhibition of LOX in wild-type lean mice caused elastin fragmentation and induced a significant increase in pulse wave velocity. Finally, we found that obese humans had stiffer arteries and lower serum LOX levels than do normal-weight humans. Conclusion—Our results demonstrated that obesity resulted in aortic stiffening in both humans and mice, and established a causal relationship between LOX downregulation and aortic stiffening in obesity.

Serum amylase, isoamylase, and lipase in the acute abdomen: their diagnostic value for acute pancreatitis

We evaluated the diagnostic value of serum amylase, isoamylase, and lipase for the diagnosis of acute pancreatitis from sera of patients with acute abdominal pain. Comparison was first made in condition A between 32 patients with image-proven pancreatitis and 414 patients with nonpancreatic acute abdomen (the control group), then in condition B, between 62 pancreatitis patients with or without image proof and the control group. We found (a) that patients with image-proven pancreatitis suffer a more severe clinical course than those without; (b) that the sensitivity, positive predictive value, and accuracy in condition B are higher than in condition A at any cutoff level; (c) that none of the enzyme assays is specific at the upper reference limit, but their diagnostic yields are much improved by raising cutoff levels to about three or four times the upper limit; and (d) that at these selected cutoff levels, amylase had a diagnostic value similar to p-isoamylase or lipase in both conditions (sensitivity 84% and 92% for amylase in conditions A and B, respectively; specificity 98% and 98%; positive predictive value 75% and 90%; negative predictive value 99% and 99%; accuracy 91% and 97%). In conclusion, at an appropriately selected cutoff level, amylase can be effectively used as the first-line test and isoamylase or lipase as adjunct tests for acute abdominal conditions.

Expression of oncogene products HER2/Neu and Ras and fibrosis-related growth factors bFGF, TGF-beta, and PDGF in bile from biliary malignancies and inflammatory disorders.

The expression of several growth factors and K-ras gene mutation in bile were studied to better understand the pathogenesis and improve early diagnosis of bile duct cancers. Bile samples were collected from 12 cholangiocarcinomas (CLC), 10 ampullary cancers (APC), 3 gallbladder cancers (GBC), 7 pancreatic cancers (PNC), 9 biliary tract infection (BTI), 8 biliary stone disease (ST), and 5 normal controls (NC). The highest mean value of TGF-β in bile was in patients with BTI; the mean levels of bFGF and PDGF were highest in CLC, and patients with APC and CLC had higher expression of HER2/Neu than other groups. In bile, a K-ras gene codon 12 mutation was found in 5 of 6 (83%) cases of CLC by the PCR-RFLP method. The results suggest overexpression of bFGF, PDGF, and HER2/Neu and the presence of K-ras mutation are important for carcinogenesis of bile duct cancers, and detection of the above abnormalities in bile is helpful for early diagnosis.