Wandi Zhang

Fulminant and fatal gas gangrene of the stomach in a healthy live liver donor

A 57‐year‐old male with a history of hypercholesterolemia and anxiety but otherwise in good health volunteered to donate the right lobe of his liver to his brother. The operation was performed uneventfully, without transfusion. Postoperatively he did well, until he developed tachycardia, profound hypotension, and coffee ground emesis on postoperative day 3. Despite resuscitative measures, he arrested and expired. Autopsy demonstrated gas gangrene of the stomach as the underlying cause of the hemorrhage and numerous colonies of Gram‐positive bacilli were identified. Subsequent polymerase chain reaction (PCR) analysis identified these bacteria to be Clostridium perfringens (C. perfringens) type D. This patients death was devastating, both to his family and his medical team. The impact of his death has transcended that of an individual occurrence. In conclusion, herein we present the facts and discuss this extraordinary example of florid clostridial infection and toxin‐mediated shock. It was completely unexpected and probably unpreventable, and its cause was almost inconceivable. (Liver Transpl 2004;10:1315–1319.)

Anticancer Activity of Scutellaria baicalensis and Its Potential Mechanism

OBJECTIVE Scutellaria baicalensis is a widely used Chinese herbal medicine that historically is used in anti-inflammatory and anticancer therapy. The aim of the study is to determine its ability to inhibit human cancer cells in vitro and to determine whether its anticancer activity is because of the inhibition of prostaglandin E(2) (PGE(2)) production that is derived from arachidonic acid through cyclooxygenase-2 (COX-2) pathway. METHODS Cell lines from the most common human cancers, including squamous cell carcinoma (SCC-25, KB), breast cancer (MCF-7), hepatocellular carcinoma (HepG2), prostate carcinoma (PC-3 and LNCaP), and colon cancer (KM-12 and HCT-15) were tested. The cells were treated with various concentrations of Scutellaria baicalensis (0.1-100 mg/mL) for 72 hours. Percentage of viable cells after treatment was assessed using a trypan blue dye exclusion assay and the level of PGE(2) production was determined by enzyme immunoassay (EIA). RESULTS Scutellaria baicalensis demonstrated a strong dose-dependent growth inhibition in all cell lines. Inhibition concentration at 50% (IC(50)) for HepG2, MCF-7, PC-3, LNCaP, KM-12, HCT-15, KB and SCC-25 cells was 1.1, 0.9, 0.52, 0.82, 1.1, 1.5, 1.0, and 1.2 mg/mL, respectively. Three cell lines (KB, SCC-25, and HepG2) were assessed for the production of PGE(2) and a high level of extracellular (KB and SCC-25) and intracellular PGE(2) (HepG2) was noted. In the presence of Scutellaria baicalensis extract, there was a significant decrease of PGE(2) in a dose-dependent fashion. CONCLUSIONS Scutellaria baicalensis strongly inhibits cell growth in all cancer cell lines tested. However, prostate and breast cancer cells (PC-3, LNCaP, and MCF-7) are slightly more sensitive than other type of cancer cells. It also inhibits PGE(2) production, indicating that suppression of tumor cell growth may be due to its ability to inhibit COX-2 activity. This study supports the notion of using Scutellaria baicalensis as a novel anticancer agent to treat various cancers.

Molecular Zipper: a fluorescent probe for real-time isothermal DNA amplification

Rolling-circle amplification (RCA) and ramification amplification (RAM, also known as hyperbranched RCA) are isothermal nucleic acid amplification technologies that have gained a great application in in situ signal amplification, DNA and protein microarray assays, single nucleotide polymorphism detection, as well as clinical diagnosis. Real-time detection of RCA or RAM products has been a challenge because of most real-time detection systems, including Taqman and Molecular Beacon, are designed for thermal cycling-based DNA amplification technology. In the present study, we describe a novel fluorescent probe construct, termed molecular zipper, which is specially designed for quantifying target DNA by real-time monitoring RAM reactions. Our results showed that the molecular zipper has very low background fluorescence due to the strong interaction between two strands. Once it is incorporated into the RAM products its double strand region is opened by displacement, therefore, its fluorophore releases a fluorescent signal. Applying the molecular zipper in RAM assay, we were able to detect as few as 10 molecules within 90 min reaction. A linear relationship was observed between initial input of targets and threshold time (R2 = 0.985). These results indicate that molecular zipper can be applied to real-time monitoring and qualification of RAM reaction, implying an amenable method for automatic RAM-based diagnostic assays.

Detection of Chlamydia trachomatis by Isothermal Ramification Amplification Method: a Feasibility Study

ABSTRACT Chlamydia trachomatis is the leading cause of sexually transmitted disease in the United States. Effective screening for this agent can facilitate prompt treatment and prevent its sequelae. The recent introduction of liquid-based cytology has made possible the simultaneous screening of cervical intraepithelial lesions and detection of C. trachomatis in a single collection vial. In this study we determined whether cytological fluid could support DNA-based amplification for the detection of C. trachomatis. Three methods were compared, including ramification amplification (RAM), real-time PCR with molecular beacon, and Abbott’s ligase chain reaction (LCx). RAM is a novel, recently introduced, isothermal DNA amplification technique that utilizes a circular probe for target detection and achieves exponential amplification through the mechanism of primer extension, strand displacement, and ramification. Our results show that RAM can detect as few as 10 C. trachomatis elementary bodies in less than 2 h, comparable to results with real-time PCR. Thirty clinical specimens collected in PreservCyt solution were tested by LCx, real-time PCR, and RAM. Among 30 specimens, 15 were positive by PCR and LCx and 14 were positive by RAM. One specimen missed by RAM had an inadequate amount of residual cellular material. Our results show that nucleic acid amplification methods can serve to detect C. trachomatis and presumably other sexually transmitted agents in cytological fluid and that the RAM assay can be an alternative to PCR and LCx because of its simplicity and isothermal amplification.

Use of Ramification Amplification Assay for Detection of Escherichia coli O157:H7 and Other E. coli Shiga Toxin-Producing Strains

ABSTRACT Escherichia coli O157:H7 and other Shiga toxin-producing E. coli (STEC) strains are important human pathogens that are mainly transmitted through the food chain. These pathogens have a low infectious dose and may cause life-threatening illnesses. However, detection of this microorganism in contaminated food or a patients stool specimens presents a diagnostic challenge because of the low copy number in the sample. Often, a more sensitive nucleic acid amplification method, such as PCR, is required for rapid detection of this microorganism. Ramification amplification (RAM) is a recently introduced isothermal DNA amplification technique that utilizes a circular probe for target detection and achieves exponential amplification through the mechanism of primer extension, strand displacement, and ramification. In this study, we synthesized a circular probe specific for the Shiga toxin 2 gene (stx2). Our results showed that as few as 10 copies of stx2 could be detected, indicating that the RAM assay was as sensitive as conventional PCR. We further tested 33 isolates of E coli O157:H7, STEC, Shigella dysenteriae, and nonpathogenic E. coli by RAM assay. Results showed that all 27 STEC isolates containing the stx2 gene were identified by RAM assay, while S. dysenteriae and nonpathogenic E. coli isolates were undetected. The RAM results were 100% in concordance with those of PCR. Because of its simplicity and isothermal amplification, the RAM assay could be a useful method for detecting STEC in food and human specimens.

Detection and Toxin Typing of Clostridium perfringens in Formalin-Fixed, Paraffin-Embedded Tissue Samples by PCR

Since current microbiology methods are not suitable to detect Clostridium perfringens in formalin-fixed, paraffin-embedded tissue samples, we developed a PCR assay to detect toxin-encoding genes and the 16S rRNA gene of C. perfringens. We successfully detected and genotyped C. perfringens in tissue sections from two autopsy cases.