Medicinal herbs for the treatment of anxiety: A systematic review and network meta-analysis.

BACKGROUND: Anxiety disorder is a common psychiatric illness. Medicinal herbs have become a field of interest in the treatment of anxiety. This study aimed to evaluate and compare the efficacy and acceptability of all possible medicinal herbs for the treatment of anxiety. METHODS: A Bayesian network meta-analysis was conducted for adults with diagnosed or subthreshold anxiety in randomized controlled trials identified in PubMed, EMBASE, the Cochrane Library, and Web of Science, searched between Jan 1, 1987, and Dec 31, 2021. The outcomes included efficacy (measured by endpoint Hamilton Anxiety Scale [HAMA] Scores) and acceptability (discontinuation by ineffectiveness, worsening of the symptoms, or adverse events). RESULTS: A total of 29 trials were reviewed, comparing 12 medicinal herbs. Silexan (mean difference [MD]: -3.84, 95% credible interval [CrI]: -6.31 to -1.34) displayed a significant effect on anxiety, and possibly benefitted the treatment of depression (standard mean difference [SMD]: -0.37, 95% confidence interval [CI]: -0.53 to -0.20) and insomnia (SMD: -0.48, 95% CI: -0.76 to -0.21). Kava was found to be an effective anxiolytic (MD: -2.46, 95% CrI: -4.47 to -0.32) but possibly ineffective in patients with generalized anxiety disorder (MD: -0.17, 95% CrI: -2.55 to -1.97). Ginkgo biloba (MD: -4.63, 95% CrI: -9.01 to -0.23) and Withania somnifera (MD: -4.90, 95% CrI: -9.70 to -0.17) were efficacious, as measured by HAMA scores but the trials were limited by their small sample sizes. Galphimia glauca (MD: -1.23, 95% CrI: -4.68 to 2.23) and Manasamitravn Vataka (MD: -1.35, 95% CrI: -7.39 to 4.68) exhibited the same anxiolytic effect as standard treatments, but both were absent from trials that were rated low risk, highlighting that confidence in their ability to provide an anxiolytic effect requires additional study. Conversely, although Passionflower (MD: -4.20, 95% CrI: -8.82 to 0.16) and Saffron (MD: -2.71, 95% CrI: -6.06 to 0.57) did not reduce HAMA scores significantly in the summary network, both were worthy of further study because of support from separate networks. There was insufficient evidence to confirm the effectiveness of Valerian (MD: 0.95, 95% CrI: -6.57 to 8.42) in standard-controlled estimation or the ineffectiveness of Chamomile (MD: 0.54, 95% CrI: -5.13 to 6.25) compared with a placebo for anxiety. Gamisoyo-san (MD: -0.98, 95% CrI: -6.48 to 4.54) and L-theanine (MD: -0.49, 95% CrI: -6.54 to 5.57) did not outperform a placebo for the treatment of anxiety in terms of statistical certainty. All medicinal herbs were well-tolerated and exhibited a good safety profile compared with control groups. When all herbs were compared, there was no statistical evidence to suggest any comparison significantly reduced HAMA scores except Ginkgo biloba vs Kava (MD: -4.41, 95% CrI: -8.32 to -0.35), although Ginkgo biloba was ranked as worst due to its poor tolerability. CONCLUSION: Medicinal herbs may be promising for the treatment of anxiety. However, these results should be considered preliminary because of the unconvincing sample sizes, together with the potential effectiveness of placebos.

Aberrant expression of long noncoding RNA SNHG15 correlates with liver metastasis and poor survival in colorectal cancer.

Long noncoding RNAs (lncRNAs) play a critical role in the initiation and progression of colorectal cancer (CRC), but little is known about the function of lncRNAs in the colorectal liver metastasis (CLM). This study was designed to identify specific lncRNAs correlating to liver metastasis of CRC, and to further assess their clinical value. Seventeen patients with primary CRC lesions, adjacent normal mucosa, and synchronous liver metastases lesions were divided into discovery set (six patients) and test set (11 patients). Transcriptome sequencing (RNAseq) was used to screen differential expression of lncRNAs in the discovery set. Based on bioinformatics data, quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was used to verify the target lncRNA in test set. The relationships between target lncRNA and clinical values were analysed in an expanded validation set of additional 91 patients. 23 upregulated and 14 downregulated lncRNAs were detected for distinguishing synchronous liver metastases, primary CRC lesions from adjacent normal mucosa in the RNAseq set. The expression levels of four lncRNAs in the 37 lncRNA signature were verified by qRT-PCR in the test set. Compared with the paired normal mucosa, high expression levels of lnc-small-nucleolar RNA host gene 15 (SNHG15) were detected not only in primary CRC lesions but also in liver metastases lesions in the test set. Furthermore, in the expanded validation set, high expression of lnc-SNHG15 was significantly associated with lymph-node metastasis and liver metastasis (p < 0.05), and patients displaying high lncRNA-SNHG15 expression exhibited a shorter median overall survival duration than those displaying low expression (30.7 vs. 35.2 months; p = 0.003). Multivariate analyses demonstrated that lncRNA-SNHG15 overexpression may serve as a poor prognostic biomarker for CRC patients (p = 0.049; Cox's regression: 2.731). Lnc-SNHG15 overexpression was significantly associated with CLM and high-expression of lnc-SNHG15 in CRC was an independent predictor of poor survival.

Associations between the cyclooxygenase-2 expression in circulating tumor cells and the clinicopathological features of patients with colorectal cancer.

While previous studies have shown that the number of circulating tumor cells (CTCs) alone is not sufficient to reflect tumor progression and that cyclooxygenase-2 (COX-2) expression is correlated with colorectal cancer (CRC) metastasis, COX-2 expression status and its potential functions in CTCs of CRC patients are unknown. Here, epithelial-mesenchymal transition (EMT) phenotype-based subsets of CTCs and the COX-2 expression status in CTCs were identified and their potential clinical values were assessed in 91 CRC patients. CTCs were enumerated in peripheral blood and subsets of CTCs (epithelial [eCTCs], mesenchymal [mCTCs], and biophenotypic [bCTCs]) and the COX-2 expression status were determined using the RNA in situ hybridization method. CTCs were detected in 80.2% (73 of 91) patients. Neither the total CTC nor eCTC numbers were found to significantly associate with any of the clinicopathological features. However, the number of mCTCs was significantly associated with distance metastasis (P = 0.035) and had a trend of being associated with lymph node metastasis ( P = 0.055). Among the 73 patients enrolled for evaluating COX-2 expression, 52.5% (38 of 73) were found to express COX-2 in CTCs, and COX-2 expression in CTCs was not found to associate with the clinicopathological factors. However, COX-2 expression in mCTCs tended to have a higher rate in patients with metastasis compared with those without metastasis (72.0% vs 42.8%; P = 0.072). Furthermore, COX-2 expression and mCTC marker expression correlated positively ( R = 0.287; P = 0.017). Further studies are required to investigate the clinical value of the expression of COX-2 in mCTCs, especially in CRC patients with the advanced tumor stage and distant metastasis.

Blood-based biopsies-clinical utility beyond circulating tumor cells.

Circulating tumor cells (CTCs), epithelial-mesenchymal transition (EMT) cells, as well as a number of circulating cancer stromal cells (CStCs) are known to shed into the blood of cancer patients. Individually, and together, these cells provide biological and clinical information about the cancers. Filtration is a method used to isolate all of these cells, while eliminating red and white blood cells from whole peripheral blood. We have previously shown that accurate identification of these cell types is paramount to proper clinical assessment by describing the overlapping phenotypes of CTCs to one such CStC, the cancer-associated macrophage-like cell (CAML). We report that CAMLs possess a number of parallel applications to CTCs but have a broader range of clinical utility, including cancer screening, companion diagnostics, diagnosis, prognosis, monitoring of treatment response, and detection of recurrence. © 2018 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of ISAC.

Detection of tumor-associated cells in cryopreserved peripheral blood mononuclear cell samples for retrospective analysis.

BACKGROUND: Cryopreserved peripheral blood mononuclear cells (PBMCs) are commonly collected in biobanks. However, little data exist regarding the preservation of tumor-associated cells in cryopreserved collections. The objective of this study was to determine the feasibility of using the CellSieve™ microfiltration assay for the isolation of circulating tumor cells (CTCs) and circulating cancer-associated macrophage-like cells (CAMLs) from cryopreserved PBMC samples. METHODS: Blood samples spiked with breast (MCF-7), prostate (PC-3), and renal (786-O) cancer cell lines were used to establish analytical accuracy, efficiency, and reproducibility after cryopreservation. The spiked samples were processed through Ficoll separation, and cryopreservation was followed by thawing and microfiltration. RESULTS: MCF-7 cells were successfully retrieved with recovery efficiencies of 90.5 % without cryopreservation and 87.8 and 89.0 %, respectively, on day 7 and day 66 following cryopreservation. The corresponding recovery efficiencies of PC-3 cells were 83.3 % without cryopreservation and 85.3 and 84.7 %, respectively, after cryopreservation. Recovery efficiencies of 786-O cells were 92.7 % without cryopreservation, and 82.7 and 81.3 %, respectively, after cryopreservation. The recovered cells retained the morphologic characteristics and immunohistochemical markers that had been observed before freezing. The protocols were further validated by quantitation of CAMLs in blood samples from two patients with renal cell carcinoma (RCC). The recovery rates of CTCs and CAMLs from cryopreserved samples were not statistically significant different (P > 0.05) from matched fresh samples. CONCLUSIONS: To our knowledge, this is the first report that CAMLs could be cryopreserved and analyzed after thawing with microfiltration technology. The application of microfiltration technology to cryopreserved samples will enable much greater retrospective study of cancer patients in relation to long-term outcomes.

Correlation between motor behavior and age-related intervertebral disc degeneration in cynomolgus monkeys.

Background: The motor behavior in patients with lumbar intervertebral disc degeneration (IDD) and animal models should be changed due to pain. However, there does not seem to be a strong correlation between IDD and motor behavior. Therefore, it is necessary to understand the correlation between motor behavior and age-related IDD. Methods: Twenty-one healthy male cynomolgus monkeys (Macaca fascicularis) distributed across the age range were included in this study. The experimental animals were divided into two groups: caged group (n = 14) and free-range group (n = 7). The data of IDD and motor behavior were obtained through magnetic resonance imaging (MRI) and PrimateScan Automatic Behavior Analysis System. More than 20 basic motor behaviors could be recorded and quantified, and then reclassified into 9 combined categories. We defined the sum of the duration of activity-related combined categories as the total duration of activity in 3 hours. The activity zone of the cynomolgus monkeys in the cage could be divided into top and bottom zones. Analyze the correlation between motor behavior and IDD. Results: Age was correlated with both Pfirrmann grades (r = .700; P < .001) and T2 values (r = -.369; P < .001). The T2 value in the caged group was 45.97 ± 8.35 ms, which was significantly lower than the 55.90 ± 8.73 ms in the free-range group (P < .001). The mean T2 values were positively correlated with hanging duration (r = .548, P < .05), the total duration of activity (r = .496, P < .05), and top zone duration (r = .541, P < .05). Conclusions: There is an interactional relationship between IDD and motor behavior. Motor behavior could be used as one of the diagnostic indicators of IDD. It could also be used to infer the presence or extent of IDD in animal models. Avoiding a sedentary lifestyle and engaging in exercise in daily life could alleviate IDD.

lncRNA UCA1 Contributes to 5-Fluorouracil Resistance of Colorectal Cancer Cells Through miR-23b-3p/ZNF281 Axis.

PURPOSE: The chemoresistance of 5-fluorouracil (5-FU) limited the application of chemotherapy in colorectal cancer (CRC) treatment. Herein, we aimed to uncover the potential mechanism behind the 5-FU resistance of CRC cells. METHODS: The abundance of long noncoding RNA urothelial carcinoma associated 1 (lncRNA UCA1), microRNA-23b-3p (miR-23b-3p) and zinc finger protein 281 (ZNF281) was measured by quantitative real-time polymerase chain reaction (qRT-PCR) in CRC tissues and cells. Western blot was conducted to examine autophagy-related proteins, apoptosis-associated proteins and ZNF281 in CRC tissues and cells. Cell counting kit-8 (CCK8) assay was performed to detect the viability and inhibitory concentration 50% (IC50) value of 5-FU of CRC cells. The apoptosis of CRC cells was measured by flow cytometry. The binding sites between miR-23b-3p and UCA1 or ZNF281 were predicted by miRcode and Starbase software, respectively, and the combination was confirmed by dual-luciferase reporter assay and RIP assay. Murine xenograft model was established to verify the role of UCA1 on the 5-FU resistance of CRC in vivo. RESULTS: The 5-FU resistance of CRC was positively related to the level of UCA1 and autophagy. UCA1 accelerated the 5-FU resistance of CRC cells through facilitating autophagy and suppressing apoptosis. MiR-23b-3p was a target of UCA1 in 293T and CRC cells. The knockdown of miR-23b-3p reversed the inhibitory effects of UCA1 interference on the 5-FU resistance and autophagy and the promoting impact on the apoptosis of CRC cells. ZNF281 could bind to miR-23b-3p in 293T cells. MiR-23b-3p elevated the 5-FU sensitivity through down-regulating ZNF281 in CRC cells. UCA1 interference enhanced the 5-FU sensitivity of CRC through miR-23b-3p/ZNF281 axis in vivo. CONCLUSION: UCA1 mediated 5-FU resistance of CRC cells through facilitating autophagy and inhibiting apoptosis via miR-23b-3p/ZNF281 axis in vivo and in vitro.

Integrating waveguide biosensor.

The Integrating Waveguide Biosensor was developed for rapid and sensitive detection of bacterial cells, spores, and toxins. A sandwich format of immunoassay was employed using Salmonella as model. The analyte was immunocaptured on the inner surface of the waveguide and then detected by the antibody conjugated with fluorescent dye. The waveguide was illuminated by an excitation light at a 90 degrees angle. The emitted light from fluorescent labels on the surface of the waveguide was efficiently collected and channeled to a detector at the end of the waveguide, while minimizing interference from the excitation light. Utilizing fluorescent dye Cy5, a 635-nm diode laser for excitation, and a photomultiplier tube detector, the Integrating Waveguide Sensor System was able to detect approximately ten captured cells of Salmonella.

Influence and mechanism of miR-99a suppressing development of colorectal cancer (CRC) with diabetes mellitus (DM).

OBJECTIVE: This study aimed to identify the changes of miRNAs in colorectal cancer (CRC) complicated with diabetes mellitus (DM) (CRC + DM) tissues and their potential effects. METHODS: The changes of miRNAs in CRC + DM tissues were determined by miRNA microarray. The expression levels of miR-99a in 40 clinical specimens and 6 CRC cell lines were determined by qRT-PCR. The capacity for miR-99a to induce cell proliferation and invasion was examined with miR-99a-overexpressing HCT-116 cells. The relative mTOR mRNA and protein levels were determined by qRT-PCR and Western blotting, respectively, in HCT-116 cells transfected with miR-99a. The dual luciferase assay was performed to confirm the direct regulation of miR-99a on mTOR 3'-UTR. The HCT-116 cells were treated with 100 mg/L advanced glycation end products (AGEs); then, the mTOR expression levels were determined by qRT-PCR, Western blotting, and immunohistochemistry. RESULTS: Seventeen miRNAs were found to be differentially expressed among normal tissue, CRC tissue, and CRC with DM tissue, including 15 upregulated and 2 downregulated with fold changs of more than 2 times. qRT-PCR confirmed that miR-99a was downregulated in CRC and CRC + DM tissues. In addition, miR-99a overexpression remarkably impaired CRC cell proliferation and metastasis, and negatively regulated mTOR signaling through direct binding to the 3'-UTR of mTOR. AGEs could suppress miR-99a and stimulate mTOR signaling in CRC cells. Increased mTOR was also identified in CRC with DM tissues. CONCLUSION: Our findings indicate that miR-99a is a potential marker and therapeutic target of CRC complicated with DM, and that AGEs impair miR-99a-overactivated mTOR signaling in CRC with DM patients, which promotes CRC development.

Development of a rapid and sensitive immunoassay for detection and subsequent recovery of Bacillus anthracis spores in environmental samples.

Bacillus anthracis is considered a major threat as an agent of bioterrorism. B. anthracis spores are readily dispersed as aerosols, are very persistent, and are resistant to normal disinfection treatments. Immunoassays have been developed to rapidly detect B. anthracis spores at high concentrations. However, detection of B. anthracis spores at lower concentrations is problematic due to the fact that closely related Bacillus species (e.g., B. thuringiensis) can cross-react with anti-B. anthracis antibodies, resulting in false positive detections. Subsequent polymerase chain reaction (PCR) analysis is required to differentiate virulent strains. We report here on a protocol for the rapid, sensitive detection of B. anthracis spore using the Integrating Waveguide Biosensor followed by a method for the rapid release and germination of immunocaptured spores. A detection limit of ca. 10(3) spores was achieved by incubating spores simultaneously with capture and detection antibodies ("liquid-phase" assay) prior to capture on capillary tubes/waveguides. Subsequent incubation with BHI broth directly in capillary tubes allowed for rapid germination, outgrowth, and release of spores, resulting in vegetative cells for PCR analysis.

Filtration and Analysis of Circulating Cancer Associated Cells from the Blood of Cancer Patients.

Filtration is one of the most efficient methods to remove red and white blood cells from whole blood, while retaining larger cells on the surface of the filter. Precision pore microfilters, such as the CellSieve™ microfilters, are ideally suited for this purpose, as they are strong, with uniform pore size and distribution, and have low fluorescent background required for microscopic image analysis. We present a system to implement the filtration of whole blood in combination with CellSieve™ microfilters that is simple and straightforward to use. Being that the blood of cancer patients often contains both tumor cells and stromal cells associated with cancer that are larger than normal blood cells, microfiltration shows great promise in better understanding these cell types. Accurate identification and characterization of cancer associated cells has led to increased specificity as it relates to CTCs and epithelial-mesenchymal transition cells (EMTs) and enabled the identification of previously unknown cell types, such as cancer associated macrophage-like cells (CAMLs). Using a system that isolates both CTCs and circulating stromal cells, clinicians can better diagnose cancer patients to determine therapy, monitor treatment, and watch for recurrence.

Size-based detection of sarcoma circulating tumor cells and cell clusters.

Metastatic disease is the most important factor in determining the survival of sarcoma patients. Since sarcoma metastasis is predominantly hematogenous, we hypothesized that detection and quantification of circulating tumor cells (CTCs) could reflect response to therapy and risk of metastatic relapse. We evaluated the presence of CTCs using a novel animal model and in the blood of patients with high grade sarcomas utilizing the CellSieve™ size-based low pressure microfiltration system. Sarcoma CTCs were identified based on antibody staining patterns and nuclear morphology. Additionally, RNA was extracted from the CTCs for molecular analysis including demonstration of an EWS-FLI1 translocation, identification of a previously unrecognized p53 mutation in a patient with Ewing sarcoma, and single cell RNA sequencing of CTC from a child with alveolar rhabdomyosarcoma. In mouse xenograft models, the presence of CTC correlates with disease burden and with clinically silent metastases. In human patients, CTCs were readily detected at diagnosis, decreased with successful treatment, and were detectable in the blood of patients with no radiographic evidence of disease prior to the development of overt metastasis. Although evaluation of CTC is established in the care of patients with carcinomas, this technology has yet to be effectively applied to the evaluation and treatment of sarcoma patients. Our work demonstrates that the CellSieve™ microfiltration system can be used to study the biology of CTC in both mouse models and human sarcoma patients, with the potential for application to the monitoring of disease response and prediction of metastatic relapse.

Enrichment and Molecular Analysis of Breast Cancer Disseminated Tumor Cells from Bone Marrow Using Microfiltration.

PURPOSE: Molecular characterization of disseminated tumor cells (DTCs) in the bone marrow (BM) of breast cancer (BC) patients has been hindered by their rarity. To enrich for these cells using an antigen-independent methodology, we have evaluated a size-based microfiltration device in combination with several downstream biomarker assays. METHODS: BM aspirates were collected from healthy volunteers or BC patients. Healthy BM was mixed with a specified number of BC cells to calculate recovery and fold enrichment by microfiltration. Specimens were pre-filtered using a 70 µm mesh sieve and the effluent filtered through CellSieve microfilters. Captured cells were analyzed by immunocytochemistry (ICC), FISH for HER-2/neu gene amplification status, and RNA in situ hybridization (RISH). Cells eluted from the filter were used for RNA isolation and subsequent qRT-PCR analysis for DTC biomarker gene expression. RESULTS: Filtering an average of 14×106 nucleated BM cells yielded approximately 17-21×103 residual BM cells. In the BC cell spiking experiments, an average of 87% (range 84-92%) of tumor cells were recovered with approximately 170- to 400-fold enrichment. Captured BC cells from patients co-stained for cytokeratin and EpCAM, but not CD45 by ICC. RNA yields from 4 ml of patient BM after filtration averaged 135ng per 10 million BM cells filtered with an average RNA Integrity Number (RIN) of 5.3. DTC-associated gene expression was detected by both qRT-PCR and RISH in filtered spiked or BC patient specimens but, not in control filtered normal BM. CONCLUSIONS: We have tested a microfiltration technique for enrichment of BM DTCs. DTC capture efficiency was shown to range from 84.3% to 92.1% with up to 400-fold enrichment using model BC cell lines. In patients, recovered DTCs can be identified and distinguished from normal BM cells using multiple antibody-, DNA-, and RNA-based biomarker assays.

Polymer microfilters with nanostructured surfaces for the culture of circulating cancer cells.

There is a critical need to improve the accuracy of drug screening and testing through the development of in vitro culture systems that more effectively mimic the in vivo environment. Surface topographical features on the nanoscale level, in short nanotopography, effect the cell growth patterns, and hence affect cell function in culture. We report the preliminary results on the fabrication, and subsequent cellular growth, of nanoscale surface topography on polymer microfilters using cell lines as a precursor to circulating tumor cells (CTCs). To create various nanoscale features on the microfilter surface, we used reactive ion etching (RIE) with and without an etching mask. An anodized aluminum oxide (AAO) membrane fabricated directly on the polymer surface served as an etching mask. Polymer filters with a variety of modified surfaces were used to compare the effects on the culture of cancer cell lines in blank culture wells, with untreated microfilters or with RIE-treated microfilters. We then report the differences of cell shape, phenotype and growth patterns of bladder and glioblastoma cancer cell lines after isolation on the various types of material modifications. Our data suggest that RIE modified polymer filters can isolate model cell lines while retaining ell viability, and that the RIE filter modification allows T24 monolayering cells to proliferate as a structured cluster.

Precision Microfilters as an all in one System for Multiplex Analysis of Circulating Tumor Cells.

Enumeration of circulating tumor cells (CTCs) from cancer patient blood is an established diagnostic assay used to evaluate patient status as a singleplex test. However, in the coming age of personalized medicine, multiplex analysis of patient CTCs, including proteomic and genomic techniques, will have to be integrated with CTC isolation platform technologies. Advancements in microfabrication have demonstrated that CTCs can be isolated and analyzed using microfluidic lab-on-a-chip devices. However, to date, most microfluidic devices are either still in the development phase, not applicable to all clinical tests, or are not commercially available. To overcome these discrepancies, we describe an all-in-one device for the isolation and multiplexing of clinically applicable CTC assays. Microfilters present an ideal lab-on-a-chip platform for analysis of CTCs as non-toxic and inert materials allow for a multitude of tests from cell growth through clinical staining techniques, all without background interference. Lithographically fabricated microfilters, can be made with high porosity, precise pore dimensions, arrayed pore distribution, and optimized for CTC size-based isolation. In this study we describe microfilter use in isolation and in situ analysis of CTCs using multiple sequential techniques including culture, FISH, histopathological analysis, H&E staining, photobleaching and re-staining. Further, as a proof of principle, we then describe the ability to quantitatively release patient derived CTCS from the microfilters for potential use in downstream genomic/proteomic analysis.

The design and application of DNA chips for early detection of SARS-CoV from clinical samples.

BACKGROUND: SARS coronavirus has been identified as the cause of severe acute respiratory syndrome (SARS). Few tests allow confirmation or exclusion of SARS within the first few days of infection. A gene chip is a useful tool for the study of microbial infections mainly for its capability of performing multi-target analysis in a single test. OBJECTIVES: Investigate the possibility of early detection of SARS virus from clinical samples using the gene chip-based method. STUDY DESIGN: We purified RNA from SARS-CoV obtained from routinely collected peripheral blood and sputum samples of 34 patients who had been identified as probable SARS patients by following the interim U.S. case definition. Four segments of the SARS-CoV were amplified using reverse transcription-nested PCR and the products examined using the 70-mer gene chips for SARS-CoV detection. RESULTS: A blind-test of both peripheral blood and sputum specimens lead to the positive detection of SARS-CoV in 31 out of 34 patients. SARS-CoV was not found in peripheral blood or sputum specimens from three patients. Two of the 34 patients were only 3 days post-onset of symptoms and were subsequently confirmed to be SARS positive. Our results indicate that the gene chip-based molecular test is specific for SARS-CoV and allows early detection of patients with SARS with detection rate about 8% higher than the single PCR test when the sputum sample is available.

Detection of water-borne E. coli O157 using the integrating waveguide biosensor.

Escherichia coli O157:H7, the most common serotype of enterohemorrhagic E. coli (EHEC), is responsible for numerous food-borne and water-borne infections worldwide. An integrating waveguide biosensor is described for the detection of water-borne E. coli O157, based on a fluorescent sandwich immunoassay performed inside a glass capillary waveguide. The genomic DNA of captured E. coli O157 cells was extracted and quantitative real-time PCR subsequently performed to assess biosensor-capture efficiency. In vitro microbial growth in capillary waveguide is also documented. The biosensor allows for quantitative detection of as few as 10 cells per capillary (0.075 ml volume) and can be used in conjunction with cell amplification, PCR and microarray technologies to positively identify a pathogen.

Identification of opcA gene in Neisseria polysaccharea: interspecies diversity of Opc protein family.

The gene encoding the outer membrane adhesin/invasin protein OpcA was previously described in the genomes of two pathogenic Neisseria species, N. meningitidis (Nm) and N. gonorrhoeae (Ng). In order to understand the presence or absence of opcA in nonpathogenic Neisseria species, 13 strains of N. polysaccharea (Np), four strains of N. lactamica, three strains of N. subflava and nine strains of other species were examined by DNA hybridization, polymerase chain reaction (PCR) and nucleotide sequencing. The opcA gene was found in two Np strains (85322 and 89357). The Np-opcA gene is a novel member of this gene family with 93% homology to Ng-opcA. Comparison of opcA-surrounding regions among eight Neisseria strains revealed five types of genetic organization at the opcA locus in Neisseria, which result from insertion or deletion of genetic elements at the upstream region of opcA. Comparison of the deduced peptide sequences from two Np strains, two representative Ng strains, two representative Nm strains and 13 Nm sequence variants demonstrates interspecies diversity of the OpcA protein family with conserved transmembrane regions and species-specific polymorphism at the surface-exposed loops and periplasmic turns. Reverse transcription-PCR analysis and Northern blotting showed that Np-opcA was transcribable. From an alignment of the Np-OpcA and Ng-OpcA sequences against the three-dimensional crystal structure of Nm-OpcA we conclude that there is no obvious structural reason why these proteins would not be able to form stable, folded, outer membrane proteins. The data presented here provide additional information for understanding the distribution, variation and expression of opcA in Neisseria.

Immunologic and genetic characterization of lipooligosaccharide variants in a Neisseria meningitidis serogroup C strain.

Neisseria meningitidis shows great variation in expression of structurally different lipooligosaccharides (LOS) on its cell surface. To better understand the LOS diversity that may occur within an individual strain, a group C wild-type strain, BB305-Tr4, and two stable isogenic LOS variants, Tr5 and Tr7, were selected for this study. SDS-PAGE analysis showed a size reduction of Tr5 and Tr7 LOS compared to that of Tr4. Immunoblotting showed that parental Tr4 LOS reacted with L1, L2 and L3,7 antibodies, variant Tr5 LOS with L1 and L6 antibodies, while Tr7 LOS was non-typeable. Genetic analysis showed that the gene organization at the lgt-1 locus in the three strains was lgtZ,C,A,B,H4 in Tr4, lgtZ,C,A,H4 in Tr5 and lgtZ,C,A,H9 in Tr7. The genetic differences in the three strains were consistent with their phenotypic changes. Sequence comparison revealed two independent recombination events. The first was the recombination of repeated DNA fragments in the flanking regions to delete lgtB in Tr5. The second was the recombination of a fragment of two genes, lgtB and lgtH4, to create an inactive lgtH9 allele with a mosaic structure in Tr7. These findings suggest that besides phase variation, homologous recombination can contribute to the genetic diversity of the lgt locus and to the generation of LOS variation in N. meningitidis.

The systematic study of circulating tumor cell isolation using lithographic microfilters.

Circulating tumor cells (CTCs) disseminated into peripheral blood from a primary, or metastatic, tumor can be used for early detection, diagnosis and monitoring of solid malignancies. CTC isolation by size exclusion techniques have long interested researchers as a simple broad based approach, which is methodologically diverse for use in both genomic and protein detection platforms. Though a variety of these microfiltration systems are employed academically and commercially, the limited ability to easily alter microfilter designs has hindered the optimization for CTC capture. To overcome this problem, we studied a unique photo-definable material with a scalable and mass producible photolithographic fabrication method. We use this fabrication method to systematically study and optimize the parameters necessary for CTC isolation using a microfiltration approach, followed by a comparison to a "standard" filtration membrane. We demonstrate that properly designed microfilters can capture MCF-7 cancer cells at rate of 98 ± 2% if they consist of uniform patterned distributions, ≥160 000 pores, and 7 µm pore diameters.