Regional Activation of Myosin II in Cancer Cells Drives Tumor Progression via a Secretory Cross-Talk with the Immune Microenvironment.

ROCK-Myosin II drives fast rounded-amoeboid migration in cancer cells during metastatic dissemination. Analysis of human melanoma biopsies revealed that amoeboid melanoma cells with high Myosin II activity are predominant in the invasive fronts of primary tumors in proximity to CD206+CD163+ tumor-associated macrophages and vessels. Proteomic analysis shows that ROCK-Myosin II activity in amoeboid cancer cells controls an immunomodulatory secretome, enabling the recruitment of monocytes and their differentiation into tumor-promoting macrophages. Both amoeboid cancer cells and their associated macrophages support an abnormal vasculature, which ultimately facilitates tumor progression. Mechanistically, amoeboid cancer cells perpetuate their behavior via ROCK-Myosin II-driven IL-1α secretion and NF-κB activation. Using an array of tumor models, we show that high Myosin II activity in tumor cells reprograms the innate immune microenvironment to support tumor growth. We describe an unexpected role for Myosin II dynamics in cancer cells controlling myeloid function via secreted factors.

TIMP-2 secreted by monocyte-like cells is a potent suppressor of invadopodia formation in pancreatic cancer cells.

BACKGROUND: Monocytes are a major component of the tumor microenvironment (TME) in pancreatic ductal adenocarcinoma (PDAC). However, the complex interactions between tumor cells and monocytes and their role in tumor invasion have not been fully established. METHODS: To specifically test the impact of interaction on invasive potential two PDAC cell lines PaTu8902 and CFPAC-1 were selected on their ability to form invasive adhesions, otherwise known as invadopodia and invade in a spheroid invasion assay. RESULTS: Interestingly when the PDAC cells were co-cultured with undifferentiated THP1 monocyte-like cells invadopodia formation was significantly suppressed. Moreover, conditioned media of THP1 cells (CM) was also able to suppress invadopodia formation. Further investigation revealed that both tissue inhibitor of metalloproteinase (TIMP) 1 and 2 were present in the CM. However, suppression of invadopodia formation was found that was specific to TIMP2 activity. CONCLUSIONS: Our findings indicate that TIMP2 levels in the tumour microenvironment may have prognostic value in patients with PDAC. Furthermore, activation of TIMP2 expressing monocytes in the primary tumour could present a potential therapeutic opportunity to suppress cell invasion in PDAC.

Comparative evaluation of a laboratory-developed real-time PCR assay and RealStar® Adenovirus PCR Kit for quantitative detection of human adenovirus.

BACKGROUND: Human adenoviruses are common causes of community-acquired respiratory tract and enteric infections. Severe disseminated infections with high mortality rates may be seen in immunocompromised individuals. An accurate and cost-effective quantitative assay is essential not only for laboratory diagnosis of adenoviral infections, but also for monitoring of response to antiviral treatment. The diagnostic performance of an in-house quantitative polymerase chain reaction assay was compared to a commercial system. METHODS: The analytical sensitivity, specificity, linearity, precision and accuracy of an in-house adenovirus quantitative polymerase chain reaction assay were evaluated against the RealStar® Adenovirus PCR Kit (Altona Diagnostics GmbH, Hamburg, Germany), using 122 clinical specimens and 18 proficiency testing samples. RESULTS: Linear regression analysis of the quantitative results by the in-house assay showed the dynamic range from 2.60 to 9 log10 (plasma) and 2.94 to 9 log10 (viral transport medium) copies/mL, with the coefficient of determination (R2) of 0.996 and 0.998, respectively. A dilution series demonstrated the limits of detection and lower limits of quantification for plasma were 2.06 log10 and 2.60 log10 copies/mL and those for viral transport medium were 2.31 log10 and 2.94 log10 copies/mL respectively. The precision of the in-house assay was highly reproducible among runs with coefficients of variance ranging from 0.07 to 3.21% for plasma and 0.17% to 2.11% for viral transport medium. A comparison of 52 matched samples showed an excellent correlation between the quantitative viral loads measured by the in-house assay and the RealStar® Adenovirus PCR Kit (R2 = 0.984), with an average bias of - 0.16 log10 copies/mL. CONCLUSIONS: The in-house adenovirus assay is a sensitive and reliable assay with lower cost for the detection and quantification of adenoviral DNA when compared to the RealStar® Adenovirus PCR Kit.

Clinical Evaluation of the New High-Throughput Luminex NxTAG Respiratory Pathogen Panel Assay for Multiplex Respiratory Pathogen Detection.

A broad range of viral and bacterial pathogens can cause acute respiratory tract infection. For rapid detection of a broad respiratory pathogen spectrum, multiplex real-time PCR is ideal. This study evaluated the performance of the new Luminex NxTAG Respiratory Pathogen Panel (NxTAG-RPP) in comparison with the BioFire FilmArray Respiratory Panel (FA-RP) or singleplex real-time PCR as reference. A total of 284 clinical respiratory specimens and 3 influenza A/H7N9 viral culture samples were tested. All clinical specimens were processed and analyzed in parallel using NxTAG-RPP and the reference standard method. The H7N9 viral culture samples were tested using NxTAG-RPP only. Overall, the NxTAG-RPP demonstrated ≥93% sensitivity and specificity for all respiratory targets except human coronavirus OC43 (HCoV-OC43) and HCoV-HKU1. The H7N9 virus was detected by the influenza A virus matrix gene target, while other influenza A virus subtyping gene targets in the panel remained negative. Complete concordance between NxTAG-RPP and FA-RP was observed in 98.8% (318/322) of positive results (kappa = 0.92). Substantial agreement was found for most respiratory targets, but significant differences were observed in human metapneumovirus (P = 0.001) and parainfluenza virus type 3 (P = 0.031). NxTAG-RPP has a higher sample throughput than FA-RP (96 samples versus 1 sample per run) while the turnaround times for NxTAG-RPP and FA-RP were 5 h (up to 96 samples) and 1 h (for one sample), respectively. Overall, NxTAG-RPP demonstrated good diagnostic performance for most respiratory pathogens. The high sample throughput with reasonable turnaround time of this new assay makes it a suitable multiplex platform for routine screening of respiratory specimens in hospital-based laboratories.

Development and Evaluation of Novel Real-Time Reverse Transcription-PCR Assays with Locked Nucleic Acid Probes Targeting Leader Sequences of Human-Pathogenic Coronaviruses.

Based on findings in small RNA-sequencing (Seq) data analysis, we developed highly sensitive and specific real-time reverse transcription (RT)-PCR assays with locked nucleic acid probes targeting the abundantly expressed leader sequences of Middle East respiratory syndrome coronavirus (MERS-CoV) and other human coronaviruses. Analytical and clinical evaluations showed their noninferiority to a commercial multiplex PCR test for the detection of these coronaviruses.

Bridging the sanitation gap between disaster relief and development.

By interpreting disasters as opportunities to initiate the fulfilment of development needs, realise the vulnerability of the affected community and environment, and extend the legacy of relief funds and effort, this paper builds upon the concept linking relief, rehabilitation and development (LRRD) in the sanitation sector. It aims to use a composite of case studies to devise a framework for a semi-hypothetical scenario to identify critical components and generic processes for a LRRD action plan. The scenario is based on a latrine wetland sanitation system in a Muslim community. Several sub-frameworks are developed: (i) latrine design; (ii) assessment of human waste treatment; (iii) connective sanitation promotion strategy; and (iv) ecological systems and environmental services for sanitation and development. This scenario illustrates the complex issues involved in LRRD in sanitation work and provides technical notes and references for a legacy plan for disaster relief and development.

Human cytomegalovirus immediate early proteins promote degradation of connexin 43 and disrupt gap junction communication: implications for a role in gliomagenesis.

A lack of gap junctional intercellular communication (GJIC) is common in cancer. Many oncogenic viruses have been shown to downregulate the junctional protein connexin 43 (Cx43) and reduce GJIC. Human cytomegalovirus (HCMV) is a ubiquitous, species-specific betaherpesvirus that establishes life-long latency after primary infection. It encodes two viral gene products, immediate early (IE) proteins IE1 and IE2, which are crucial in viral replication and pathogenesis of many diseases. Emerging evidence demonstrates that HCMV DNA and proteins are highly prevalent in glioblastoma multiforme (GBM) and in other tumors, but HCMV's role in tumorigenesis remains obscure. In the present study, we examined the effects of HCMV infection on Cx43 expression and GJIC as well as the viral mechanism mediating the effects in human GBM cells and tissue samples. We found that HCMV downregulated Cx43 protein, resulting in disruption of functional GJIC as assayed by fluorescent dye transfer assay. We show that both HCMV-IE72 and IE86 mediate downregulation of Cx43 by silencing RNA targeting either IE72 or IE86 coupled with ganciclovir. This finding was further validated by transfection with expression vectors encoding IE72 or IE86, and we show that viral-mediated Cx43 depletion involved proteasomal degradation. Importantly, we also observed that the Cx43 protein levels and IE staining correlated inversely in 10 human GBM tissue specimens. Thus, HCMV regulates Cx43 expression and GJIC, which may contribute to gliomagenesis.

Moving microcapillary antibiotic susceptibility testing (mcAST) towards the clinic: unravelling kinetics of detection of uropathogenic E. coli, mass-manufacturing and usability for detection of urinary tract infections in human urine.

Innovation in infection based point-of-care (PoC) diagnostics is vital to avoid unnecessary use of antibiotics and the development of antimicrobial resistance. Several groups including our research team have in recent years successfully miniaturised phenotypic antibiotic susceptibility tests (AST) of isolated bacterial strains, providing validation that miniaturised AST can match conventional microbiological methods. Some studies have also shown the feasibility of direct testing (without isolation or purification), specifically for urinary tract infections, paving the way for direct microfluidic AST systems at PoC. As rate of bacteria growth is intrinsically linked to the temperature of incubation, transferring miniaturised AST nearer the patient requires building new capabilities in terms of temperature control at PoC, furthermore widespread clinical use will require mass-manufacturing of microfluidic test strips and direct testing of urine samples. This study shows for the first-time application of microcapillary antibiotic susceptibility testing (mcAST) directly from clinical samples, using minimal equipment and simple liquid handling, and with kinetics of growth recorded using a smartphone camera. A complete PoC-mcAST system was presented and tested using 12 clinical samples sent to a clinical laboratory for microbiological analysis. The test showed 100% accuracy for determining bacteria in urine above the clinical threshold (5 out of 12 positive) and achieved 95% categorical agreement for 5 positive urines tested with 4 antibiotics (nitrofurantoin, ciprofloxacin, trimethoprim and cephalexin) within 6 h compared to the reference standard overnight AST method. A kinetic model is presented for metabolization of resazurin, demonstrating kinetics of degradation of resazurin in microcapillaries follow those observed for a microtiter plate, with time for AST dependent on the initial CFU ml-1 of uropathogenic bacteria in the urine sample. In addition, we show for the first time that use of air-drying for mass-manufacturing and deposition of AST reagents within the inner surface of mcAST strips matches results obtained with standard AST methods. These results take mcAST a step closer to clinical application, for example as PoC support for antibiotic prescription decisions within a day.

CD73 controls Myosin II-driven invasion, metastasis, and immunosuppression in amoeboid pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) has a very poor prognosis because of its high propensity to metastasize and its immunosuppressive microenvironment. Using a panel of pancreatic cancer cell lines, three-dimensional (3D) invasion systems, microarray gene signatures, microfluidic devices, mouse models, and intravital imaging, we demonstrate that ROCK-Myosin II activity in PDAC cells supports a transcriptional program conferring amoeboid invasive and immunosuppressive traits and in vivo metastatic abilities. Moreover, we find that immune checkpoint CD73 is highly expressed in amoeboid PDAC cells and drives their invasive, metastatic, and immunomodulatory traits. Mechanistically, CD73 activates RhoA-ROCK-Myosin II downstream of PI3K. Tissue microarrays of human PDAC biopsies combined with bioinformatic analysis reveal that rounded-amoeboid invasive cells with high CD73-ROCK-Myosin II activity and their immunosuppressive microenvironment confer poor prognosis to patients. We propose targeting amoeboid PDAC cells as a therapeutic strategy.

Deciphering the link between PI3K and PAK: An opportunity to target key pathways in pancreatic cancer?

The development of personalised therapies has ushered in a new and exciting era of cancer treatment for a variety of solid malignancies. Yet pancreatic ductal adenocarcinoma (PDAC) has failed to benefit from this paradigm shift, remaining notoriously refractory to targeted therapies. Chemotherapy is the cornerstone of management but can offer only modest survival benefits of a few months with 5-year survival rates rarely exceeding 3%. Despite these disappointing statistics, significant strides have been made towards understanding the complex biology of pancreatic cancer, with deep genomic sequencing identifying novel genetic aberrations and key signalling pathways. The PI3K-PDK1-AKT pathway has received great attention due to its prominence in carcinogenesis. However, efforts to target several components of this network have resulted in only a handful of drugs demonstrating any survival benefit in solid tumors; despite promising pre-clinical results. p-21 activated kinase 4 (PAK4) is a gene that is recurrently amplified or overexpressed in PDAC and both PAK4 and related family member PAK1, have been linked to aberrant RAS activity, a common feature in pancreatic cancer. As regulators of PI3K, PAKs have been highlighted as a potential prognostic marker and therapeutic target. In this review, we discuss the biology of pancreatic cancer and the close interaction between PAKs and the PI3K pathway. We also suggest proposals for future research that may see the development of effective targeted therapies that could finally improve outcomes for this disease.

Prevalence and phylogenetic characterization of human enterovirus D68 among children with respiratory infection in Hong Kong.

This is the first report on the prevalence of human enterovirus D68 (EV-D68) among children with respiratory infection in Hong Kong. Among 1461 respiratory samples taken in 2014, EV-D68 was identified in 24 (1.64%) of them with a unusual seasonal pattern. Phylogenetic analysis indicated that all EV-D68 detected in this study belong to clade B.

Human cytomegalovirus may promote tumour progression by upregulating arginase-2.

BACKGROUND: Both arginase (ARG2) and human cytomegalovirus (HCMV) have been implicated in tumorigenesis. However, the role of ARG2 in the pathogenesis of glioblastoma (GBM) and the HCMV effects on ARG2 are unknown. We hypothesize that HCMV may contribute to tumorigenesis by increasing ARG2 expression. RESULTS: ARG2 promotes tumorigenesis by increasing cellular proliferation, migration, invasion and vasculogenic mimicry in GBM cells, at least in part due to overexpression of MMP2/9. The nor-NOHA significantly reduced migration and tube formation of ARG2-overexpressing cells. HCMV immediate-early proteins (IE1/2) or its downstream pathways upregulated the expression of ARG2 in U-251 MG cells. Immunostaining of GBM tissue sections confirmed the overexpression of ARG2, consistent with data from subsets of Gene Expression Omnibus. Moreover, higher levels of ARG2 expression tended to be associated with poorer survival in GBM patient by analyzing data from TCGA. METHODS: The role of ARG2 in tumorigenesis was examined by proliferation-, migration-, invasion-, wound healing- and tube formation assays using an ARG2-overexpressing cell line and ARG inhibitor, N (omega)-hydroxy-nor-L-arginine (nor-NOHA) and siRNA against ARG2 coupled with functional assays measuring MMP2/9 activity, VEGF levels and nitric oxide synthase activity. Association between HCMV and ARG2 were examined in vitro with 3 different GBM cell lines, and ex vivo with immunostaining on GBM tissue sections. The viral mechanism mediating ARG2 induction was examined by siRNA approach. Correlation between ARG2 expression and patient survival was extrapolated from bioinformatics analysis on data from The Cancer Genome Atlas (TCGA). CONCLUSIONS: ARG2 promotes tumorigenesis, and HCMV may contribute to GBM pathogenesis by upregulating ARG2.

Increased genetic diversity of HIV-1 circulating in Hong Kong.

HIV-1 group M strains are characterized into 9 pure subtypes and 48 circulating recombinant forms (CRFs). Recent studies have identified the presence of new HIV-1 recombinants in Hong Kong and their complexity continues to increase. This study aims to characterize the HIV-1 genetic diversity in Hong Kong. Phylogenetic analyses were performed by using HIV-1 pol sequences including protease and partial reverse transcriptase isolated from 1045 local patients in Hong Kong from 2003 to 2008. For the pol sequences with unassigned genotype, the evidence of recombination was determined by using sliding-window based bootscan plots and their env C2V3 region were also sequenced. Epidemiological background of these patients was further collected. The pol phylogenetic analyses highlighted the extent of HIV-1 genetic diversity in Hong Kong. Subtype B (450/1045; 43.1%) and CRF01_AE (469/1045; 44.9%) variants were clearly predominant. Other genotypes (126/1045; 12.1%) including 3 defined subtypes, 10 CRFs, 1 unassigned subtype and 33 recombinants with 11 different mosaic patterns were observed. Recombinants of subtype B and CRF01_AE were mainly found among local Chinese MSM throughout 2004 to 2008, while the CRF02_AG and subtype G recombinants were circulating among non-Chinese Asian population in Hong Kong through heterosexual transmission starting from 2008. Our study demonstrated the complex recombination of HIV-1 in Hong Kong and the need in developing surveillance system for tracking the distribution of new HIV-1 genetic variants.