Long non-coding RNA LINC00930 targeting miR-6792-3p/ZBTB16 regulates the proliferation and EMT of pancreatic cancer.

Emerging evidence suggests the dysregulation of long non-coding RNAs (lncRNAs) involved in pancreatic cancer (PC). However, the function of LINC00930 in PC has not been elaborated. In this study, we found that LINC00930 was significantly down-regulated in PC cell lines and tissues, and associated with tumor size, lymphatic metastasis, TNM stage and poor prognosis. According to the bioinformatics database, the downregulation of LINC00930 was a common event in PC associated with prognosis and EMT. Overexpression of LINC00930 inhibited the aggressive cancer phenotypes including proliferation, metastasis and epithelial-mesenchymal transition (EMT) of PC in vitro and in vivo. Bioinformatics and dual-luciferase reporter assay indicated that miR-6792-3p could directly bind to LINC00930. Additionally, the Zinc finger and BTB domain containing 16 (ZBTB16) was significantly declined in PC, which was predicted to be the downstream gene of miR-6792-3p. MiR-6792-3p mimic rescued the decreased proliferation, metastasis and EMT caused by ZBTB16 in PC cells. The LINC00930/miR-6792-3p/ZBTB16 axis was associated with the malignant progression and process of PC. The relative expression of LINC00930 was negatively correlated with the expression of miR-6792-3p and was closely linked with ZBTB16 levels in PC. LINC00930 might serve as a potential prognostic biomarker and therapeutic target for PC.

Discovering endometriosis biomarkers with multiplex cytokine arrays.

BACKGROUND: Chronic pelvic pain is often overlooked during primary examinations because of the numerous causes of such "vague" symptoms. However, this pain can often mask endometriosis, a smoldering disease that is not easily identified as a cause of the problem. As such, endometriosis has been shown to be a potentially long-term and often undiagnosed disease due to its vague symptoms and lack of any non-invasive testing technique. Only after more severe symptoms arise (severe pelvic pain, excessive vaginal bleeding, or infertility) is the disease finally uncovered by the attending physician. Due to the nature and complexity of endometriosis, high throughput approaches for investigating changes in protein levels may be useful for elucidating novel biomarkers of the disease and to provide clues to help understand its development and progression. METHODS: A large multiplex cytokine array which detects the expression levels of 260 proteins including cytokines, chemokines, growth factors, adhesion molecules, angiogenesis factors and other was used to probe biomarkers in plasma samples from endometriosis patients with the intent of detecting and/or understanding the cause of this disease. The protein levels were then analyzed using K-nearest neighbor and split-point score analysis. RESULTS: This technique identified a 14-marker cytokine profile with the area under the curve of 0.874 under a confidence interval of 0.81-0.94. Our training set further validated the panel for significance, specificity, and sensitivity to the disease samples. CONCLUSIONS: These findings show the utility and reliability of multiplex arrays in deciphering new biomarker panels for disease detection and may offer clues for understanding this mysterious disease.

Optimized Expression and Characterization of a Novel Fully Human Bispecific Single-Chain Diabody Targeting Vascular Endothelial Growth Factor165 and Programmed Death-1 in Pichia pastoris and Evaluation of Antitumor Activity In Vivo.

Bispecific antibodies, which can bind to two different epitopes on the same or different antigens simultaneously, have recently emerged as attractive candidates for study in various diseases. Our present study successfully constructs and expresses a fully human, bispecific, single-chain diabody (BsDb) that can bind to vascular endothelial growth factor 165 (VEGF165) and programmed death-1 (PD-1) in Pichia pastoris. Under the optimal expression conditions (methanol concentration, 1%; pH, 4.0; inoculum density, OD600 = 4, and the induction time, 96 h), the maximum production level of this BsDb is achieved at approximately 20 mg/L. The recombinant BsDb is purified in one step using nickel-nitrilotriacetic acid (Ni-NTA) column chromatography with a purity of more than 95%. Indirect enzyme-linked immune sorbent assay (ELISA) and sandwich ELISA analyses show that purified BsDb can bind specifically to VEGF165 and PD-1 simultaneously with affinities of 124.78 nM and 25.07 nM, respectively. Additionally, the BsDb not only effectively inhibits VEGF165-stimulated proliferation, migration, and tube formation in primary human umbilical vein endothelial cells (HUVECs), but also significantly improves proliferation and INF-γ production of activated T cells by blocking PD-1/PD-L1 co-stimulation. Furthermore, the BsDb displays potent antitumor activity in mice bearing HT29 xenograft tumors by inhibiting tumor angiogenesis and activating immune responses in the tumor microenvironment. Based on these results, we have prepared a potential bispecific antibody drug that can co-target both VEGF165 and PD-1 for the first time. This work provides a stable foundation for the development of new strategies by the combination of an angiogenesis inhibition and immune checkpoint blockade for cancer therapy.

Potential novel Colpodella spp. (phylum Apicomplexa) and high prevalence of Colpodella spp. in goat-attached Haemaphysalis longicornis ticks in Shandong province, China.

Tick-borne Apicomplexan parasites pose a significant threat to both public health and animal husbandry. Identifying potential pathogenic parasites and gathering their epidemiological data are essential for prospectively preventing and controlling infections. In the present study, genomic DNA of ticks collected from two goat flocks (Goatflock1 and Goatflock2) and one dog group (Doggroup) were extracted and the 18S rRNA gene of Babesia/Theileria/Colpodella spp. was amplified by PCR and sequenced. Phylogenetic analysis was conducted based on the obtained sequences. The differences in pathogen positive rates between ticks of different groups were statistically analyzed using the Chi-square or continuity-adjusted Chi-square test. As a result, two pathogenic Theileria (T.) luwenshuni genotypes, one novel pathogenic Colpodella sp. HLJ genotype, and two potential novel Colpodella spp. (referred to as Colpodella sp. struthionis and Colpodella sp. yiyuansis in this study) were identified in the Haemaphysalis (H.) longicornis ticks. Ticks of Goatflock2 had a significantly higher positive rate of Colpodella spp. than those from Goatflock1 (χ2=92.10; P = 8.2 × 10-22) and Doggroup (χ2=42.34; P = 7.7 × 10-11), and a significantly higher positive rate of T. luwenshuni than Doggroup (χ2=5.38; P = 0.02). However, the positive rates of T. luwenshuni between Goatflock1 and Goatflock2 were not significantly different (χ2=2.02; P = 0.16), and so as the positive rates of both pathogens between Goatflock1 and Doggroup groups (P > 0.05). For either Colpodella spp. or T. luwenshuni, no significant difference was found in prevalence between male and female ticks. These findings underscore the potential importance of Colpodella spp. in domestic animal-attached ticks, as our study revealed two novel Colpodella spp. and identified Colpodella spp. in H. longicornis for the first time. The study also sheds light on goats' potential roles in the transmission of Colpodella spp. to ticks and provides crucial epidemiological data of pathogenic Theileria and Colpodella. These data may help physicians, veterinarians, and public health officers prepare suitable detection and treatment methods and develop prevention and control strategies.

Development and Comparison of Time Series Models in Predicting Severe Fever with Thrombocytopenia Syndrome Cases - Hubei Province, China, 2013-2020.

Introduction: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by the SFTS virus, which has a high mortality rate. Predicting the number of SFTS cases is essential for early outbreak warning and can offer valuable insights for establishing prevention and control measures. Methods: In this study, data on monthly SFTS cases in Hubei Province, China, from 2013 to 2020 were collected. Various time series models based on seasonal auto-regressive integrated moving average (SARIMA), Prophet, eXtreme Gradient Boosting (XGBoost), and long short-term memory (LSTM) were developed using these historical data to predict SFTS cases. The established models were evaluated and compared using mean absolute error (MAE) and root mean squared error (RMSE). Results: Four models were developed and performed well in predicting the trend of SFTS cases. The XGBoost model outperformed the others, yielding the closest fit to the actual case numbers and exhibiting the smallest MAE (2.54) and RMSE (2.89) in capturing the seasonal trend and predicting the monthly number of SFTS cases in Hubei Province. Conclusion: The developed XGBoost model represents a promising and valuable tool for SFTS prediction and early warning in Hubei Province, China.

BCG Vaccination: A potential tool against COVID-19 and COVID-19-like Black Swan incidents.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19), and its variants have brought unprecedented impacts to the global public health system, politics, economy, and other fields. Although more than ten COVID-19 specific vaccines have been approved for emergency use, COVID-19 prevention and control still face many challenges. Bacille Calmette-Guérin (BCG) is the only authorized vaccine used to fight against tuberculosis (TB), it has been hypothesized that BCG may prevent and control COVID-19 based on BCG-induced nonspecific immune responses. Herein, we summarized: 1) The nonspecific protection effects of BCG, such as prophylactic protection effects of BCG on nonmycobacterial infections, immunotherapy effects of BCG vaccine, and enhancement effect of BCG vaccine on unrelated vaccines; 2) Recent evidence of BCG's efficacy against SARS-COV-2 infection from ecological studies, analytical analyses, clinical trials, and animal studies; 3) Three possible mechanisms of BCG vaccine and their effects on COVID-19 control including heterologous immunity, trained immunity, and anti-inflammatory effect. We hope that this review will encourage more scientists to investigate further BCG induced non-specific immune responses and explore their mechanisms, which could be a potential tool for addressing the COVID-19 pandemic and COVID-19-like "Black Swan" events to reduce the impacts of infectious disease outbreaks on public health, politics, and economy.

Co-existence of Multiple Anaplasma Species and Variants in Ticks Feeding on Hedgehogs or Cattle Poses Potential Threats of Anaplasmosis to Humans and Livestock in Eastern China.

Background: Anaplasma spp., causative agents of anaplasmosis, pose significant a threat to public health and economic losses in livestock farming. Co-infections/co-existence of various Anaplasma spp. may facilitate pathogen interactions and the emergence of novel variants, represent potential dangers to public health and economic losses from livestock farming, and raise challenges of detection and diagnosis. The information regarding co-infection/co-existence of Anaplasma in their vector ticks and wild animals is limited and needs urgent investigation. Methods: Wild hedgehogs and ticks from hedgehogs and cattle were collected from Jiangsu province, Eastern China, and DNA was extracted from hedgehog organs and tick homogenates. Various genera of species-specific polymerase chain reaction (PCR) or nested PCR amplifications targeting 16S ribosomal RNA (rrs), msp4, or groEL gene coupled with sequencing were conducted to identify Anaplasma spp. Results: Anaplasma phagocytophilum (1, 0.6%), A. marginale (2, 1.2%), A. platys variants xyn10pt-1 (13, 7.7%), xyn21pt-2 (3, 1.8%), and xyn3pt-3 (3, 1.8%), A. bovis variant cwp72bo-1 (12, 7.1%), and a novel Candidatus Cryptoplasma sp. (1, 0.6%) were identified in 168 Haemaphysalis longicornis ticks from cattle. A. platys variant xyn10pt-1 (20, 11.4%) and A. bovis variants cwp72bo-1 (12, 6.9%) and cwp55-36bo-2 (1, 0.6%) were detected in 173 H. flava ticks from hedgehogs. However, only A. bovis variant cwp72bo-1 (15, 46.7%) was identified in 32 Erinaceus amurensis hedgehogs. Various co-existence combinations were found only in ticks. Conclusion: The co-existence of various Anaplasma spp. and variants in H. flava and H. longicornis was detected for the first time in the world. The high infection rate of A. bovis in hedgehogs and its moderate infection rate in their parasitic ticks suggest that Er. amurensis hedgehog could be an important reservoir of A. bovis, rather than A. platys. Horizontal transmission of Anaplasma spp. may exist among different tick species via their shared hosts in the investigated area. This study provided epidemiological data that could be crucial for strategy development for early warning, prevention, and control of potential Anaplasma infections.

Wild Hedgehogs and Their Parasitic Ticks Coinfected with Multiple Tick-Borne Pathogens in Jiangsu Province, Eastern China.

The increasing awareness of emerging tickborne pathogens (TBPs) has inspired much research. In the present study, the coinfections of TBPs both in ticks and their wild hedgehog hosts in Jiangsu province, Eastern China were determined by metagenome next-generation sequencing and nested PCR. As a result, Rickettsia japonica (81.1%), novel Rickettsia sp. SFGR-1 (5.1%), Anaplasma bovis (12%), A. platys (6.3%), novel Ehrlichia spp. Ehr-1 (16%) and Ehr-2 (0.6%), E. ewingii-like strain (0.6%), Coxiella burnetii (10.9%), and a novel Coxiella-like endosymbiont (CLE) strain (61.1%) were detected in Haemaphysalis flava ticks. A. bovis (43.8%), Ehrlichia sp. Ehr-1 (83.3%), and C. burnetii (80%) were detected in Erinaceus amurensis hedgehogs. Coinfection rates with various TBPs were 71.5% and 83.3% in ticks and hedgehogs, respectively, both with double-pathogen/endosymbiont coinfection rates over 50%. We found the following. (i) Er. amurensis hedgehogs seem to contribute to the natural cycles of R. japonica, A. bovis, Ehrlichia sp., and C. burnetii and may be reservoirs of them except for R. japonica, and A. bovis is proved to infect hedgehogs for the first time. (ii) H. flava is proved to harbor various TBPs as a reservoir host, including CLE identified for the first time, which could inhibit coinfection of C. burnetii while promoting that of Rickettsia spp. in H. flava. (iii) Four novel TBP species were identified. This study provides useful epidemiological information crucial for assessing the potential infection risks to humans, thus benefiting the development of strategies to prevent and control tick-borne diseases. IMPORTANCE In the present study, we found the following. (i) Er. amurensis hedgehogs seem to contribute to the natural cycles of R. japonica, A. bovis, Ehrlichia sp., and C. burnetii and may be reservoirs of them except for R. japonica, and A. bovis is proved to infect hedgehogs for the first time. (ii) H. flava is proved to harbor various tickborne pathogens (TBPs) as a reservoir host, including Coxiella-like endosymbiont (CLE) identified for the first time, which could inhibit coinfection of C. burnetii while promoting that of Rickettsia spp. in H. flava. (iii) Four novel TBP species were identified. This study provides useful epidemiological information on TBPs harbored and transmitted by ticks and their hosts, for assessing the potential infection risks to humans, thus benefiting the developing strategies for tick-borne diseases prevention and control.

A potential tool for predicting epidemic trends and outbreaks of scrub typhus based on Internet search big data analysis in Yunnan Province, China.

Introduction: Scrub typhus, caused by Orientia tsutsugamushi, is a neglected tropical disease. The southern part of China is considered an important epidemic and conserved area of scrub typhus. Although a surveillance system has been established, the surveillance of scrub typhus is typically delayed or incomplete and cannot predict trends in morbidity. Internet search data intuitively expose the public's attention to certain diseases when used in the public health area, thus reflecting the prevalence of the diseases. Methods: In this study, based on the Internet search big data and historical scrub typhus incidence data in Yunnan Province of China, the autoregressive integrated moving average (ARIMA) model and ARIMA with external variables (ARIMAX) model were constructed and compared to predict the scrub typhus incidence. Results: The results showed that the ARIMAX model produced a better outcome than the ARIMA model evaluated by various indexes and comparisons with the actual data. Conclusions: The study demonstrates that Internet search big data can enhance the traditional surveillance system in monitoring and predicting the prevalence of scrub typhus and provides a potential tool for monitoring epidemic trends of scrub typhus and early warning of its outbreaks.

High prevalence of Rickettsia spp. in ticks from wild hedgehogs rather than domestic bovine in Jiangsu province, Eastern China.

Background: Spotted fever group Rickettsia (SFGR), containing various pathogenic Rickettsia spp., poses remarkable negative influences to public health by causing various severe or mild diseases. Information regarding prevalence of SFGR in ticks in Jiangsu province, Eastern China, is still limited and needs urgent investigations. Methods: Hedgehog- and bovine-attached ticks were collected from Jiangsu province, Eastern China. DNA of individual ticks was extracted for nested polymerase chain reaction amplifications targeting gltA, 16S ribosomal RNA (rrs), ompA, ompB, and sca4 genes following with sequencing. SFGR-specific IgG antibodies in sera of local donators were evaluated using ELISA. Results: Overall, 144 (83.2%) of the 173 ticks from hedgehogs and 2 (1.2%) of the 168 ticks from bovine were positive for one of the three identified Rickettsia spp., with significant difference between the two groups (P = 3.6e-52). Candidatus Rickettsia principis (9; 5.2%) and R. heilongjiangensis (135; 78.0%) were detected in Haemaphysalis flava rather than in H. longicornis ticks from hedgehogs. R. heilongjiangensis (1; 0.6%) and Candidatus R. jingxinensis (or Candidatus R. longicornii) (1; 0.6%) were identified in H. longicornis and Rhipicephalus microplus ticks from bovine, respectively. Phylogenetic analysis indicated Candidatus R. jingxinensis belonged to R. japonica subgroup, whereas Candidatus R. principis belonged to a novel subgroup. Higher serological prevalence of spotted fever and SFGR-specific IgG antibody level in humans were observed around the investigated area than in urban areas, without significant difference. Conclusion: Candidatus R. principis and Candidatus R. jingxinensis were identified in Jiangsu province, Eastern China, and fully genetically characterized for the first time. The higher prevalence of SFGR in hedgehog-attached ticks as well as the higher SFGR-specific IgG antibody level and seropositive rate in humans around the investigated area suggested that more attention should be paid to SFGR. This pathogen is usually transmitted or harbored by wild animals and ticks. This study provides important epidemiological data for both physicians and public health officers in developing early prevention and control strategies against potential Rickettsia infections and in the preparation of suitable testing and treatment needs for rickettsiosis in the endemic areas.

Serum molecular biomarkers in neuromyelitis optica and multiple sclerosis.

BACKGROUND: Neuromyelitis optica spectrum disorder (NMOSD) is a rare and severe inflammatory demyelinating disorder of the central nervous system (CNS), which mainly affects the optic nerves and spinal cord. The aims of this study were to determine whether the expression levels of serological cytokines could distinguish 1) NMOSD from healthy controls (HCs); and 2) NMOSD patients with and without the aquaporin-4 (AQP4) antibody biomarker from each other; and 3) NMOSD patients without the antibody to AQP4 from MS patients. METHODS: The expression levels of 200 proteins in serum from 41 NMOSD (32 with antibodies to AQP4, 9 without antibodies to AQP4), 12 MS patients, and 34 HCs were measured using glass-based antibody arrays. None of the patients received any immunosuppressive treatment. In parallel, the correlation between protein expression in NMOSD/MS patients and clinical traits was determined with Weighted Gene Co-expression Network Analysis (WGCNA). RESULTS: Thirty-nine serological proteins were differentially expressed in NMOSD patients compared to HCs, with 29 of these proteins not observed in MS patients. In addition, the data reveal 15 differentially-expression proteins (DEPs) between AQP4-IgG seronegative and AQP4-IgG seropositive NMOSD patients, and 9 DEPs between NMOSD and MS patients who did not have AQP4-IgG. CONCLUSION: Serological IL-17B is significantly upregulated in both NMOSD and MS patients compared to HCs, and could be a key biomarker of NMOSD and MS. Serological VEGF, MPIF-1 and NrCAM were positively associated with AQP4-IgG titer. We also demonstrate that EGF may be involved in the breakdown of the BBB by downregulating Claudin-5.

A Case-Control Study of Follicular Fluid Cytokine Profiles in Women with Diminished Ovarian Reserve.

Ovarian reserve is an important determinant of a woman's reproductive potential, and women with diminished ovarian reserve (DOR) often seek in vitro fertilization (IVF). The underlying etiology of DOR is unknown, but follicular fluid cytokine concentrations likely play a role in follicular development and maturation. The present study seeks to investigate the expression of cytokines in follicular fluid (FF) of women with DOR undergoing IVF and explore correlated functional pathways. One hundred ninety-four women undergoing ovarian stimulation were recruited at the time of oocyte retrieval. Women were classified as having DOR if they met one or more of the following criteria: AMH < 1 ng/ml, FSH > 10 mIU/ml, and/or AFC < 10. Controls included women undergoing IVF for male factor, tubal factor due to tubal ligation, or planned oocyte cryopreservation (non-oncologic). The concentrations of 480 cytokines and related growth factors in follicular fluid were determined using a multiplex immunoassay. Fifty-nine cytokines had significantly different concentrations (53 higher and 6 lower) in the DOR relative to the control group after adjusting for age and body mass index (BMI) (false discovery rate; FDR < 0.1). Using the most informative 44 biomarkers as indicated by a random forest (RF) model, an area under the curve (AUC) of 0.78 was obtained. Thus, follicular microenvironment differs between women with DOR and normal ovarian reserve. The differentially expressed cytokines belong to diverse processes that are primarily involved in follicular maturation and ovulation. These changes may play an important role in treatment outcomes in women with DOR.

Exosomal circ_0030167 derived from BM-MSCs inhibits the invasion, migration, proliferation and stemness of pancreatic cancer cells by sponging miR-338-5p and targeting the Wif1/Wnt8/ß-catenin axis.

Pancreatic cancer (PC) is one of the most lethal malignant tumors and has the lowest survival rate due to early metastasis and drug resistance. Exosomes derived from bone marrow mesenchymal stem cells (BM-MSCs) have emerged as crucial regulators of the progression of various tumors. These vesicles contain abundant circRNAs that have important biological functions. This study aimed to elucidate the role of exosomal circRNAs in PC progression. In this study, we successfully isolated BM-MSCs from human bone marrow based on their surface marker expression and osteogenic and adipogenic differentiation potential. We found that BM-MSC-derived exosomes significantly reduced the invasion, migration, and proliferation of PC cells, as well as tumor stemness. According to whole-transcriptome resequencing and clustering heat map analysis, we identified the key molecule circ_0030167 and miR-338-5p, its downstream target. We revealed that circ_0030167 mainly regulates miR-338-5p, enhances Wif1 expression, and inhibits the Wnt8/ß-catenin pathway, thereby inhibiting the stemness of PC cells and tumor progression. Overall, BM-MSC exosomal circ_0030167 contributes to the progression and stemness of PC cells via the miR-338-5p/wif1/Wnt 8/ß-catenin axis. Our study provides a new perspective for the treatment of PC.

Dried blood sample analysis by antibody array across the total testing process.

Dried blood samples (DBSs) have many advantages; yet, impediments have limited the clinical utilization of DBSs. We developed a novel volumetric sampling device that collects a precise volume of blood, which overcomes the heterogeneity and hematocrit issues commonly encountered in a traditional DBS card collection as well as allowing for more efficient extraction and processing procedures and thus, more efficient quantitation, by using the entire sample. We also provided a thorough procedure validation using this volumetric DBS collection device with an established quantitative proteomics analysis method, and then analyzed 1000 proteins using this approach in DBSs concomitantly with serum for future consideration of utility in clinical applications. Our data provide a first step in the establishment of a DBS database for the broad application of this sample type for widespread use in clinical proteomic and other analyses applications.

Three subtypes of lung cancer fibroblasts define distinct therapeutic paradigms.

Cancer-associated fibroblasts (CAFs) are highly heterogeneous. With the lack of a comprehensive understanding of CAFs' functional distinctions, it remains unclear how cancer treatments could be personalized based on CAFs in a patient's tumor. We have established a living biobank of CAFs derived from biopsies of patients' non-small lung cancer (NSCLC) that encompasses a broad molecular spectrum of CAFs in clinical NSCLC. By functionally interrogating CAF heterogeneity using the same therapeutics received by patients, we identify three functional subtypes: (1) robustly protective of cancers and highly expressing HGF and FGF7; (2) moderately protective of cancers and highly expressing FGF7; and (3) those providing minimal protection. These functional differences among CAFs are governed by their intrinsic TGF-ß signaling, which suppresses HGF and FGF7 expression. This CAF functional classification correlates with patients' clinical response to targeted therapies and also associates with the tumor immune microenvironment, therefore providing an avenue to guide personalized treatment.

Developing a periodontal disease antibody array for the prediction of severe periodontal disease using machine learning classifiers.

BACKGROUND: The aim of this study was to simultaneously and quantitatively assess the expression levels of 20 periodontal disease-related proteins in gingival crevicular fluid (GCF) from normal controls (NOR) and severe periodontitis (SP) patients with an antibody array. METHODS: Antibodies against 20 periodontal disease-related proteins were spotted onto a glass slide to create a periodontal disease antibody array (PDD). The array was then incubated with GCF samples collected from 25 NOR and 25 SP patients. Differentially expressed proteins between NOR and SP patients were then used to build receiver operator characteristic (ROC) curves and compare five classification models, including support vector machine, random forest, k nearest neighbor, linear discriminant analysis, and Classification and Regression Trees. RESULTS: Seven proteins (C-reactive protein, interleukin [IL]-1α, interleukin-1ß, interleukin-8, matrix metalloproteinase-13, osteoprotegerin, and osteoactivin) were significantly upregulated in SP patients compared with NOR, while receptor activator of nuclear factor-kappa was significantly downregulated. The highest diagnostic accuracy using a ROC curve was observed for IL-1ß with an area under the curve of 0.984. Five of the proteins (IL-1ß, IL-8, MMP-13, osteoprotegerin, and osteoactivin) were identified as important features for classification. Linear discriminant analysis had the highest classification accuracy across the five classification models that were tested. CONCLUSION: This study highlights the potential of antibody arrays to diagnose periodontal disease.

Antibody arrays in biomarker discovery.

All of life is regulated by complex and organized chemical reactions that help dictate when to grow, to move, to reproduce, and to die. When these processes go awry, or are interrupted by pathological agents, diseases such as cancer, autoimmunity, or infections can result. Cytokines, chemokines, growth factors, adipokines, and other chemical moieties make up a vast subset of these chemical reactions that are altered in disease states, and monitoring changes in these molecules could provide for the identification of disease biomarkers. From the first identification of carcinoembryonic antigen, to the discovery of prostate-specific antigen, to numerous others described within, biomarkers of disease are detectable in a plethora of sample types. The growing number of biomarkers for infection, autoimmunity, and cancer allow for increasingly early detection, to identification of novel drug targets, to prognostic indicators of disease outcome. However, more and more studies are finding that a single cytokine or growth factor is insufficient as a true disease biomarker and that a more global perspective is needed to understand true disease biology. Such a broad view requires a multiplexed platform for chemical detection, and antibody arrays meet and exceed this need by performing this detection in a high-throughput fashion. Herein, we will discuss how antibody arrays have evolved, and how they have helped direct new drug target design, helped identify therapeutic disease markers, and helped in earlier disease detection. From asthma to renal disease, and neurological dysfunction to immunologic disorders, antibody arrays afford a bright future for new biomarkers discovery.

Development of IGF signaling antibody arrays for the identification of hepatocellular carcinoma biomarkers.

PURPOSE: Our objective was to develop a system to simultaneously and quantitatively measure the expression levels of the insulin-like growth factor (IGF) family proteins in numerous samples and to apply this approach to profile the IGF family proteins levels in cancer and adjacent tissues from patients with hepatocellular carcinoma (HCC). EXPERIMENTAL DESIGN: Antibodies against ten IGF family proteins (IGF-1, IGF-1R, IGF-2, IGF-2R, IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-6, and Insulin) were immobilized on the surface of a glass slide in an array format to create an IGF signaling antibody array. Tissue lysates prepared from patient's liver cancer tissues and adjacent tissues were then applied to the arrays. The proteins captured by antibodies on the arrays were then incubated with a cocktail of biotinylated detection antibodies and visualized with a fluorescence detection system. By comparison with standard protein amount, the exact protein concentrations in the samples can be determined. The expression levels of the ten IGF family proteins in 25 pairs of HCC and adjacent tissues were quantitatively measured using this novel antibody array technology. The differential expression levels between cancer tissues and adjacent tissues were statistically analyzed. RESULTS: A novel IGF signaling antibody array was developed which allows the researcher to simultaneously detect ten proteins involved in IGF signal pathway with high sensitivity and specificity. Using this approach, we found that the levels of IGF-2R and IGFBP-2 in HCC tissues were higher than those in adjacent tissues. CONCLUSION: Our IGF signaling antibody array which can detect the expression of ten IGF family members with high sensitivity and specificity will undoubtedly prove a powerful tool for drug and biomarker discovery.

Hydroxyquinone O-methylation in mitomycin biosynthesis.

Mitomycins are bioreductively activated DNA-alkylating agents. One member of this family, mitomycin C, is in clinical use as part of combination therapy for certain solid tumors. The cytotoxicity displayed by mitomycins is dependent on their electrochemical potential which, in turn, is governed in part by the substituents of the quinone moiety. In this paper we describe studies on the biogenesis of the quinone methoxy group present in mitomycins A and B. An engineered Streptomyces lavendulae strain in which the mmcR methyltransferase gene had been deleted failed to produce the three mitomycins (A, B, and C) that are typically isolated from the wild type organism. Analysis of the culture extracts from the mmcR-deletion mutant strain revealed that two new metabolites, 7-demethylmitomycin A and 7-demethylmitomycin B, had accumulated instead. Production of mitomycins A and C or mitomycin B was selectively restored upon supplementing the culture medium of a S. lavendulae strain unable to produce the key precursor 3-amino-5-hydroxybenzoate with either 7-demethylmitomycin A or 7-demethylmitomycin B, respectively. MmcR methyltransferase obtained by cloning and overexpression of the corresponding mmcR gene was shown to catalyze the 7-O-methylation of both C9beta- and C9alpha-configured 7-hydroxymitomycins in vitro. This study provides direct evidence for the catalytic role of MmcR in formation of the 7-OMe group that is characteristic of mitomycins A and B and demonstrates the prerequisite of 7-O-methylation for the production of the clinical agent mitomycin C.