Engagement of the CrkL adaptor in interferon alpha signalling in BCR-ABL-expressing cells

Interferon alpha (IFNalpha) has significant clinical activity in the treatment of patients with chronic myelogenous leukaemia (CML), but the mechanisms of its selective efficacy in the treatment of the disease are unknown. The CrkL adaptor protein interacts directly with the BCR-ABL fusion protein that causes the malignant transformation and is constitutively phosphorylated in BCR-ABL-expressing cells. In the present study, we provide evidence that CrkL was engaged in IFNalpha-signalling in the CML-derived KT-1 cell line, which expresses BCR-ABL and is sensitive to the growth inhibitory effects of IFNalpha. CrkL is constitutively associated with BCR-ABL in these cells and treatment with IFNalpha had no effect on the BCR-ABL/CrkL interaction. After IFNalpha stimulation, CrkL associated with Stat5, which also underwent phosphorylation in an IFNalpha-dependent manner. The interaction of CrkL with Stat5 was facilitated by the function of both the SH2 and the N-terminus SH3 domains of CrkL. The resulting CrkL-Stat5 complex translocated to the nucleus and could be detected in gel shift assays using elements derived from either the beta-casein promoter or the promoter of the PML gene, an IFNalpha-inducible gene that mediates growth inhibitory responses. In addition to its interaction with Stat5, CrkL interacts with C3G in KT-1 cells and such an interaction regulates the downstream activation of the small GTPase Rap1, which also mediates inhibition of cell proliferation. Thus, despite its engagement by BCR-ABL in CML-derived cells, CrkL mediates activation of downstream signalling pathways in response to the activated type I IFN receptor and such signals may contribute to the generation of the anti-proliferative effects of IFNalpha in CML.     

Toward a disposable low-cost LOC device: heterogeneous polymer micro valve and pump fabricated by UV/ozone-assisted thermal fusion bonding

Herein, a heterogeneous polymer micro valve and pump with a polypropylene (PP) membrane was developed in a low-cost manner via UV/ozone-assisted thermal fusion bonding. The proposed fabrication technique allowed for a geometrically selective bonding; consequently, the membrane was prevented from bonding with the valve seat of the diaphragm micro-valve, without patterning a protection layer or introducing an additional structure. The developed device withstands 480 kPa of static pressure and up to 350 kPa of a vibration pressure, providing sufficient bonding strength for microfluidic actuations. The fabricated micro valve and pump are fully characterized and compared with a poly(dimethylsiloxane) (PDMS) membrane glass device, showing comparable valving and pumping performance. As a result, the robust PP membrane micro valve and pump are simply implemented in a facile manner, and demonstrated excellent performance, which is highly desirable for mass production of disposable lab-on-a-chip (LOC) devices.

Herein, a heterogeneous polymer micro valve and pump with a polypropylene (PP) membrane was developed in a low-cost manner via UV/ozone-assisted thermal fusion bonding.     

Incidence of and Risk Factors for Hospital-Acquired Diarrhea in Three Tertiary Care Public Hospitals in Bangladesh

During April 2007–April 2010, surveillance physicians in adult and pediatric medicine wards of three tertiary public hospitals in Bangladesh identified patients who developed hospital-acquired diarrhea. We calculated incidence of hospital-acquired diarrhea. To identify risk factors, we compared these patients to randomly selected patients from the same wards who were admitted > 72 hours without having diarrhea. The incidence of hospital-acquired diarrhea was 4.8 cases per 1,000 patient-days. Children < 1 year of age were more likely to develop hospital-acquired diarrhea than older children. The risk of developing hospital-acquired diarrhea increased for each additional day of hospitalization beyond 72 hours, whereas exposure to antibiotics within 72 hours of admission decreased the risk. There were three deaths among case-patients; all were infants. Patients, particularly young children, are at risk for hospital-acquired diarrhea and associated deaths in Bangladeshi hospitals. Further research to identify the responsible organisms and transmission routes could inform prevention strategies.     

Overexpression of FoxM1 offers a promising therapeutic target in diffuse large B-cell lymphoma

Background

FoxM1 has been shown to play a critical role in the pathogenesis of various epithelial malignancies. However, its role in lymphoid malignancies has not been fully clarified. We, therefore, investigated the role of FoxM1 expression in a large cohort of diffuse large B-cell lymphoma samples and panel of cell lines.

Design and Methods

FoxM1 expression was investigated in a large series of diffuse large B-cell lymphoma tissues in a tissue microarray format by immunohistochemistry. Apoptosis was measured by flow cytometry and protein expression was detected by immunoblotting using diffuse large B-cell lymphoma cell lines following treatment with either pharmacological inhibitor of FoxM1 or small interference RNA knockdown strategy. Invasion/migration and soft agar colony assays were also performed following treatment with FoxM1 inhibitor.

Results

FoxM1 expression was detected in 84.6% of diffuse large B-cell lymphoma tumors and found to be significantly associated with proliferative tumor marker Ki67 (P<0.0001), matrix metalloproteinases-2 (P=0.0008), matrix metalloproteinases-9 (P=0.0002), S-phase kinase associated protein-2 (P<0.0001) and inversely associated with p27 expression (P=0.0215). Expression of small interference RNA targeted against FoxM1 or treatment of diffuse large B-cell lymphoma cells with thiostrepton caused its downregulation accompanied by decreased expression of matrix metalloproteinases-2 and matrix metalloproteinases-9. Inhibition of FoxM1 in diffuse large B-cell lymphoma cells also decreased invasive and migratory capability, and induced caspase dependent apoptosis via activation of the mitochondrial apoptotic pathway. Finally, combined thiostrepton and bortezomib at sub-toxic doses led to efficient apoptosis in diffuse large B-cell lymphoma cells.

Conclusions

Altogether, these results suggest that FoxM1 is over-expressed in the majority of diffuse large B-cell lymphoma samples. These data also indicate that targeting FoxM1 signaling can serve as a potential therapeutic modality in the management of diffuse large B-cell lymphoma.     

SEX DIFFERENCES IN DNA METHYLATION MAY CONTRIBUTE TO RISK OF PTSD AND DEPRESSION: A REVIEW OF EXISTING EVIDENCE

There are well‐established sex differences in the prevalence of certain mental disorders. Work in animal models has provided us with an emerging understanding of the role that epigenetic factors play in establishing sex differences in the brain during development. Similarly, work in animal models, and a more limited but growing literature based on human studies, has demonstrated that DNA methylation (DNAm) changes occur in response to environmental stress, with some of these occurring in a sex‐specific manner. In this review, we explore whether DNAm plays a role in contributing to the observed sex differences in prevalence of mental disorders in which stress contributes significantly to their etiologies, specifically posttraumatic stress disorder (PTSD) and depression. We propose that investigating sex differences in DNAm among genes known to influence brain development may help to shed light on the sexually dimorphic risk for, or resilience to, developing PTSD and depression.     

Immune Responses to the O-Specific Polysaccharide Antigen in Children Who Received a Killed Oral Cholera Vaccine Compared to Responses following Natural Cholera Infection in Bangladesh

Current oral cholera vaccines induce lower levels of protective efficacy and shorter durations of protection in young children than in adults. Immunity against cholera is serogroup specific, and immune responses to Vibrio cholerae lipopolysaccharide (LPS), the antigen that mediates serogroup-specific responses, are associated with protection against disease. Despite this, responses against V. cholerae O-specific polysaccharide (OSP), a key component of the LPS responsible for specificity, have not been characterized in children. Here, we report a comparison of polysaccharide antibody responses in children from a region in Bangladesh where cholera is endemic, including infants (6 to 23 months, n = 15), young children (24 to 59 months, n = 14), and older children (5 to 15 years, n = 23) who received two doses of a killed oral cholera vaccine 14 days apart. We found that infants and young children receiving the vaccine did not mount an IgG, IgA, or IgM antibody response to V. cholerae OSP or LPS, whereas older children showed significant responses. In comparison to the vaccinees, young children with wild-type V. cholerae O1 Ogawa infection did mount significant antibody responses against OSP and LPS. We also demonstrated that OSP responses correlated with age in vaccinees, but not in cholera patients, reflecting the ability of even young children with wild-type cholera to develop OSP responses. These differences might contribute to the lower efficacy of protection rendered by vaccination than by wild-type disease in young children and suggest that efforts to improve lipopolysaccharide-specific responses might be critical for achieving optimal cholera vaccine efficacy in this younger age group.