Etiology of Severe Acute Respiratory Infections, Bangladesh, 2017.

In April 2017, surveillance detected a surge in severe acute respiratory infections (SARI) in Bangladesh. We collected specimens from SARI patients and asymptomatic controls for analysis with multipathogen diagnostic tests. Influenza A(H1N1)pdm09 was associated with the SARI epidemic, suggesting that introducing vaccines and empiric antiviral drugs could be beneficial.

Light Limitation Impacts Growth but Not Constitutive or Jasmonate Induced Defenses Relevant to Emerald Ash Borer (Agrilus planipennis) in White Fringetree (Chionanthus virginicus) or Black Ash (Fraxinus nigra).

White fringetree is a host for the invasive emerald ash borer (EAB) but is of lower quality than the related and highly susceptible black ash. Field observations suggest that host trees grown in full sun are more resistant to EAB than those in shade, however the impact of light limitation on chemical defenses has not been assessed. We quantified constitutive and jasmonate-induced phloem defenses and growth patterns of white fringetree and black ash under differential light conditions and related them to EAB larval performance. White fringetree had significantly lower constitutive and induced activities of peroxidase, polyphenol oxidase, ß-glucosidase, chitinase and lignin content, but significantly higher gallic acid equivalent soluble phenolic, soluble sugar, and oleuropein concentrations compared to black ash. Multivariate analyses based on tissue chemical attributes displayed clear separation of species and induced defense responses. Further, EAB performed significantly worse on white fringetree than black ash, consistent with previous studies. Light limitation did not impact measured defenses or EAB larval performance, but it did decrease current year growth and increase photosynthetic efficiency. Overall our results suggest that phenolic profiles, metabolite abundance, and growth traits are important in mediating white fringetree resistance to EAB, and that short-term light limitation does not influence phloem chemistry or larval success.

Hospital-based surveillance for Japanese encephalitis in Bangladesh, 2007-2016: Implications for introduction of immunization.

BACKGROUND: Japanese encephalitis (JE) virus is recognized as a major cause of encephalitis in Bangladesh. The World Health Organization (WHO) recommends human immunization as the most effective means to control JE. Several WHO-prequalified vaccines are available to prevent JE but no vaccination program has been implemented in Bangladesh. METHODS: We conducted hospital-based surveillance for acute meningitis-encephalitis syndrome (AMES) to describe JE epidemiology and help inform policy decisions about possible immunization strategies for Bangladesh. RESULTS: During 2007-2016, a total of 6543 AMES patients were identified at four tertiary hospitals. Of the 6525 patients tested, 548 (8%) were classified as JE cases. These 548 patients resided in 36 (56%) out of 64 districts of Bangladesh, with the highest proportion of JE cases among AMES patients (12% and 7%) presenting at two hospitals in the northwestern part of the country. The median age of JE cases was 30 years, and 193 (35%) were aged ≤15 years. The majority of JE cases (80%) were identified from July through November. CONCLUSIONS: Surveillance results suggest that JE continues to be an important cause of meningo-encephalitis in Bangladesh. Immunization strategies including JE vaccine introduction into the routine childhood immunization program or mass vaccination in certain age groups or geographic areas need to be examined, taking into consideration the cost-effectiveness ratio of the approach and potential for decreasing disease burden.

The transcription factor osterix (SP7) regulates BMP6-induced human osteoblast differentiation.

The transcription factor osterix (Sp7) is essential for osteoblastogenesis and bone formation in mice. Genome wide association studies have demonstrated that osterix is associated with bone mineral density in humans; however, the molecular significance of osterix in human osteoblast differentiation is poorly described. In this study we have characterized the role of osterix in human mesenchymal progenitor cell (hMSC) differentiation. We first analyzed temporal microarray data of primary hMSC treated with bone morphogenetic protein-6 (BMP6) using clustering to identify genes that are associated with osterix expression. Osterix clusters with a set of osteoblast-associated extracellular matrix (ECM) genes, including bone sialoprotein (BSP) and a novel set of proteoglycans, osteomodulin (OMD), osteoglycin, and asporin. Maximum expression of these genes is dependent upon both the concentration and duration of BMP6 exposure. Next we overexpressed and repressed osterix in primary hMSC using retrovirus. The enforced expression of osterix had relatively minor effects on osteoblastic gene expression independent of exogenous BMP6. However, in the presence of BMP6, osterix overexpression enhanced expression of the aforementioned ECM genes. Additionally, osterix overexpression enhanced BMP6 induced osteoblast mineralization, while inhibiting hMSC proliferation. Conversely, osterix knockdown maintained hMSC in an immature state by decreasing expression of these ECM genes and decreasing mineralization and hMSC proliferation. Overexpression of the osterix regulated gene OMD with retrovirus promoted mineralization of hMSC. These results suggest that osterix is necessary, but not sufficient for hMSC osteoblast differentiation. Osterix regulates the expression of a set of ECM proteins which are involved in terminal osteoblast differentiation.

Time series gene expression profiling and temporal regulatory pathway analysis of BMP6 induced osteoblast differentiation and mineralization.

BACKGROUND: BMP6 mediated osteoblast differentiation plays a key role in skeletal development and bone disease. Unfortunately, the signaling pathways regulated by BMP6 are largely uncharacterized due to both a lack of data and the complexity of the response. RESULTS: To better characterize the signaling pathways responsive to BMP6, we conducted a time series microarray study to track BMP6 induced osteoblast differentiation and mineralization. These temporal data were analyzed using a customized gene set analysis approach to identify temporally coherent sets of genes that act downstream of BMP6. Our analysis identified BMP6 regulation of previously reported pathways, such as the TGF-beta pathway. We also identified previously unknown connections between BMP6 and pathways such as Notch signaling and the MYB and BAF57 regulatory modules. In addition, we identify a super-network of pathways that are sequentially activated following BMP6 induction. CONCLUSION: In this work, we carried out a microarray-based temporal regulatory pathway analysis of BMP6 induced osteoblast differentiation and mineralization using GAGE method. This novel temporal analysis is more informative and powerful than the classical static pathway analysis in that: (1) it captures the interconnections between signaling pathways or functional modules and demonstrates the even higher level organization of molecular biological systems; (2) it describes the temporal perturbation patterns of each pathway or module and their dynamic roles in osteoblast differentiation. The same set of experimental and computational strategies employed in our work could be useful for studying other complex biological processes.

Clinicopathologic findings following intra-articular injection of autologous and allogeneic placentally derived equine mesenchymal stem cells in horses.

BACKGROUND AIMS: The development of an allogeneic mesenchymal stem cell (MSC) product to treat equine disorders would be useful; however, there are limited in vivo safety data for horses. We hypothesized that the injection of self (autologous) and non-self (related allogeneic or allogeneic) MSC would not elicit significant alterations in physical examination, gait or synovial fluid parameters when injected into the joints of healthy horses. METHODS: Sixteen healthy horses were used in this study. Group 1 consisted of foals (n = 6), group 2 consisted of their dams (n = 5) and group 3 consisted of half-siblings (n = 5) to group 1 foals. Prior to injection, MSC were phenotyped. Placentally derived MSC were injected into contralateral joints and MSC diluent was injected into a separate joint (control). An examination, including lameness evaluation and synovial fluid analysis, was performed at 0, 24, 48 and 72 h post-injection. RESULTS: MSC were major histocompatibility complex (MHC) I positive, MHC II negative and CD86 negative. Injection of allogeneic MSC did not elicit a systemic response. Local responses such as joint swelling or lameness were minimal and variable. Intra-articular MSC injection elicited marked inflammation within the synovial fluid (as measured by nucleated cell count, neutrophil number and total protein concentration). However, there were no significant differences between the degree and type of inflammation elicited by self and non-self-MSC. CONCLUSIONS: The healthy equine joint responds similarly to a single intra-articular injection of autologous and allogeneic MSC. This pre-clinical safety study is an important first step in the development of equine allogeneic stem cell therapies.

Identification of variables that optimize isolation and culture of multipotent mesenchymal stem cells from equine umbilical-cord blood.

OBJECTIVE-To optimize the isolation and culture of mesenchymal stem cells (MSCs) from umbilical-cord blood (UCB), identify variables that predicted successful MSC isolation, and determine whether shipping, processing, and cryopreservation altered MSC viability, recovery rates, and expansion kinetics. SAMPLE POPULATION-UCB samples from 79 Thoroughbred and Quarter Horse mares. PROCEDURES-UCB samples were processed to reduce volume and remove RBCs. Nucleated cells (NCs) were cryopreserved or grown in various culture conditions to optimize MSC monolayer expansion and proliferation. Donor and UCB-sample factors were analyzed to determine their influence on the success of MSC isolation and monolayer expansion. RESULTS-MSCs capable of multilineage in vitro differentiation were expanded from > 80% of UCB samples. Automated UCB processing and temperature-controlled shipping facilitated sterile and standardized RBC reduction and NC enrichment from UCB samples. The number of NCs after UCB samples were processed was the sole variable that predicted successful MSC expansion. The UCB-derived MSCs and NCs were successfully cryopreserved and thawed with no decrease in cell recovery, viability, or MSC proliferation. The use of fibronectin-coated culture plates and reduction of incubator oxygen tension from 20% to 5% improved the MSC isolation rate. Some UCB-derived MSC clones proliferated for > 20 passages before senescence. Onset of senescence was associated with specific immunocytochemical changes. CONCLUSIONS AND CLINICAL RELEVANCE-Equine UCB samples appeared to be a rich source of readily obtainable, highly proliferative MSCs that could be banked for therapeutic use.

GAGE: generally applicable gene set enrichment for pathway analysis.

BACKGROUND: Gene set analysis (GSA) is a widely used strategy for gene expression data analysis based on pathway knowledge. GSA focuses on sets of related genes and has established major advantages over individual gene analyses, including greater robustness, sensitivity and biological relevance. However, previous GSA methods have limited usage as they cannot handle datasets of different sample sizes or experimental designs. RESULTS: To address these limitations, we present a new GSA method called Generally Applicable Gene-set Enrichment (GAGE). We successfully apply GAGE to multiple microarray datasets with different sample sizes, experimental designs and profiling techniques. GAGE shows significantly better results when compared to two other commonly used GSA methods of GSEA and PAGE. We demonstrate this improvement in the following three aspects: (1) consistency across repeated studies/experiments; (2) sensitivity and specificity; (3) biological relevance of the regulatory mechanisms inferred.GAGE reveals novel and relevant regulatory mechanisms from both published and previously unpublished microarray studies. From two published lung cancer data sets, GAGE derived a more cohesive and predictive mechanistic scheme underlying lung cancer progress and metastasis. For a previously unpublished BMP6 study, GAGE predicted novel regulatory mechanisms for BMP6 induced osteoblast differentiation, including the canonical BMP-TGF beta signaling, JAK-STAT signaling, Wnt signaling, and estrogen signaling pathways-all of which are supported by the experimental literature. CONCLUSION: GAGE is generally applicable to gene expression datasets with different sample sizes and experimental designs. GAGE consistently outperformed two most frequently used GSA methods and inferred statistically and biologically more relevant regulatory pathways. The GAGE method is implemented in R in the "gage" package, available under the GNU GPL from http://sysbio.engin.umich.edu/~luow/downloads.php.

Wnt11 promotes osteoblast maturation and mineralization through R-spondin 2.

Wnt11 signals through both canonical (beta-catenin) and non-canonical pathways and is up-regulated during osteoblast differentiation and fracture healing. In these studies, we evaluated the role of Wnt11 during osteoblastogenesis. Wnt11 overexpression in MC3T3E1 pre-osteoblasts increases beta-catenin accumulation and promotes bone morphogenetic protein (BMP)-induced expression of alkaline phosphatase and mineralization. Wnt11 dramatically increases expression of the osteoblast-associated genes Dmp1 (dentin matrix protein 1), Phex (phosphate-regulating endopeptidase homolog), and Bsp (bone sialoprotein). Wnt11 also increases expression of Rspo2 (R-spondin 2), a secreted factor known to enhance Wnt signaling. Overexpression of Rspo2 is sufficient for increasing Dmp1, Phex, and Bsp expression and promotes bone morphogenetic protein-induced mineralization. Knockdown of Rspo2 abrogates Wnt11-mediated osteoblast maturation. Antagonism of T-cell factor (Tcf)/beta-catenin signaling with dominant negative Tcf blocks Wnt11-mediated expression of Dmp1, Phex, and Rspo2 and decreases mineralization. However, dominant negative Tcf fails to block the osteogenic effects of Rspo2 overexpression. These studies show that Wnt11 signals through beta-catenin, activating Rspo2 expression, which is then required for Wnt11-mediated osteoblast maturation.

Osteogenic differentiation of human mesenchymal stem cells is regulated by bone morphogenetic protein-6.

Bone marrow-derived mesenchymal stem cells (MSC) are multipotent, self-renewing, mesodermal-origin stem cells that are sequestered in the endosteal compartment. MSC are maintained in a relative state of quiescence in vivo but in response to a variety of physiological and pathological stimuli, proliferate and differentiate into osteoblasts, chondrocytes, adipocytes, or hematopoiesis-supporting stromal cells. Little is understood regarding the cellular or molecular events underlying MSC fate decisions. We report that human MSC (hMSC) cultured in defined, serum-free conditions respond to a narrow spectrum of growth factors with osteogenic commitment, differentiation, and hydroxyapatite deposition. Of the osteogenic factors we examined, only treatment with bone morphogenetic protein (BMP) results in osteoinduction under defined serum-free conditions. Among BMP-2, 4, 6, and 7, BMP-6 is the most consistent and potent regulator of osteoblast differentiation and, of these BMPs, only BMP-6 gene expression is detected prior to hMSC osteoblast differentiation. Addition of exogenous BMP-6 to hMSC induces the expression or upregulation of a repertoire of osteoblast-related genes including type I collagen, osteocalcin, bone sialoprotein, and their regulatory transcription factors Cbfa1/Runx2, and Osterix. This translates into increased production of osteogenic extracellular matrix (ECM) with subsequent hydroxyapatite deposition.

Highly efficient genetic transduction of primary human synoviocytes with concentrated retroviral supernatant.

We are developing retroviral-mediated gene transfer to human fibroblast-like synovial cells (FLS) as one approach to characterizing genetic pathways involved in synoviocyte pathophysiology. Prior work has suggested that FLS are relatively refractory to infection by Moloney murine leukemia virus based vectors. To determine if viral titer influenced the transduction efficiency of FLS, we optimized a rapid, efficient, and inexpensive centrifugation method to concentrate recombinant retroviral supernatant. The technique was evaluated by measurement of the expression of a viral enhanced green fluorescent protein transgene in transduced cells, and by analysis of viral RNA in retroviral supernatant. Concentration (100-fold) was achieved by centrifugation of viral supernatant for four hours, with 100% recovery of viral particles. The transduction of FLS increased from approximately 15% with unconcentrated supernatant, to nearly 50% using concentrated supernatant. This protocol will be useful for investigators with applications that require efficient, stable, high level transgene expression in primary FLS.