Suppressor of Cytokine Signaling (SOCS) 5 Utilises Distinct Domains for Regulation of JAK1 and Interaction with the Adaptor Protein Shc-1

Suppressor of Cytokine Signaling (SOCS)5 is thought to act as a tumour suppressor through negative regulation of JAK/STAT and epidermal growth factor (EGF) signaling. However, the mechanism/s by which SOCS5 acts on these two distinct pathways is unclear. We show for the first time that SOCS5 can interact directly with JAK via a unique, conserved region in its N-terminus, which we have termed the JAK interaction region (JIR). Co-expression of SOCS5 was able to specifically reduce JAK1 and JAK2 (but not JAK3 or TYK2) autophosphorylation and this function required both the conserved JIR and additional sequences within the long SOCS5 N-terminal region. We further demonstrate that SOCS5 can directly inhibit JAK1 kinase activity, although its mechanism of action appears distinct from that of SOCS1 and SOCS3. In addition, we identify phosphoTyr317 in Shc-1 as a high-affinity substrate for the SOCS5-SH2 domain and suggest that SOCS5 may negatively regulate EGF and growth factor-driven Shc-1 signaling by binding to this site. These findings suggest that different domains in SOCS5 contribute to two distinct mechanisms for regulation of cytokine and growth factor signaling.     

Outbreak of Shiga Toxin-Producing Escherichia coli (STEC) O157:H7 Associated with Romaine Lettuce Consumption, 2011

Background

Shiga toxin-producing Escherichia coli (STEC) O157:H7 is the causal agent for more than 96,000 cases of diarrheal illness and 3,200 infection-attributable hospitalizations annually in the United States.

Materials and Methods

We defined a confirmed case as a compatible illness in a person with the outbreak strain during 10/07/2011-11/30/2011. Investigation included hypothesis generation, a case-control study utilizing geographically-matched controls, and a case series investigation. Environmental inspections and tracebacks were conducted.

Results

We identified 58 cases in 10 states; 67% were hospitalized and 6.4% developed hemolytic uremic syndrome. Any romaine consumption was significantly associated with illness (matched Odds Ratio (mOR) = 10.0, 95% Confidence Interval (CI) = 2.1–97.0). Grocery Store Chain A salad bar was significantly associated with illness (mOR = 18.9, 95% CI = 4.5–176.8). Two separate traceback investigations for romaine lettuce converged on Farm A. Case series results indicate that cases (64.9%) were more likely than the FoodNet population (47%) to eat romaine lettuce (p-value = 0.013); 61.3% of cases reported consuming romaine lettuce from the Grocery Store Chain A salad bar.

Conclusions

This multistate outbreak of STEC O157:H7 infections was associated with consumption of romaine lettuce. Traceback analysis determined that a single common lot of romaine lettuce harvested from Farm A was used to supply Grocery Store Chain A and a university campus linked to a case with the outbreak strain. An investigation at Farm A did not identify the source of contamination. Improved ability to trace produce from the growing fields to the point of consumption will allow more timely prevention and control measures to be implemented.     

Auxin-Responsive DR5 Promoter Coupled with Transport Assays Suggest Separate but Linked Routes of Auxin Transport during Woody Stem Development in Populus

Polar auxin transport (PAT) is a major determinant of plant morphology and internal anatomy with important roles in vascular patterning, tropic growth responses, apical dominance and phyllotactic arrangement. Woody plants present a highly complex system of vascular development in which isolated bundles of xylem and phloem gradually unite to form concentric rings of conductive tissue. We generated several transgenic lines of hybrid poplar (Populus tremula x alba) with the auxin-responsive DR5 promoter driving GUS expression in order to visualize an auxin response during the establishment of secondary growth. Distinct GUS expression in the cambial zone and developing xylem-side derivatives supports the current view of this tissue as a major stream of basipetal PAT. However, we also found novel sites of GUS expression in the primary xylem parenchyma lining the outer perimeter of the pith. Strands of primary xylem parenchyma depart the stem as a leaf trace, and showed GUS expression as long as the leaves to which they were connected remained attached (i.e., until just prior to leaf abscission). Tissue composed of primary xylem parenchyma strands contained measurable levels of free indole-3-acetic acid (IAA) and showed basipetal transport of radiolabeled auxin (³H-IAA) that was both significantly faster than diffusion and highly sensitive to the PAT inhibitor NPA. Radiolabeled auxin was also able to move between the primary xylem parenchyma in the interior of the stem and the basipetal stream in the cambial zone, an exchange that was likely mediated by ray parenchyma cells. Our results suggest that (a) channeling of leaf-derived IAA first delineates isolated strands of pre-procambial tissue but then later shifts to include basipetal transport through the rapidly expanding xylem elements, and (b) the transition from primary to secondary vascular development is gradual, with an auxin response preceding the appearance of a unified and radially-organized vascular cambium.     

Reduced Crowding and Poor Contour Detection in Schizophrenia Are Consistent with Weak Surround Inhibition

Background

Detection of visual contours (strings of small oriented elements) is markedly poor in schizophrenia. This has previously been attributed to an inability to group local information across space into a global percept. Here, we show that this failure actually originates from a combination of poor encoding of local orientation and abnormal processing of visual context.

Methods

We measured the ability of observers with schizophrenia to localise contours embedded in backgrounds of differently oriented elements (either randomly oriented, near-parallel or near-perpendicular to the contour). In addition, we measured patients’ ability to process local orientation information (i.e., report the orientation of an individual element) for both isolated and crowded elements (i.e., presented with nearby distractors).

Results

While patients are poor at detecting contours amongst randomly oriented elements, they are proportionally less disrupted (compared to unaffected controls) when contour and surrounding elements have similar orientations (near-parallel condition). In addition, patients are poor at reporting the orientation of an individual element but, again, are less prone to interference from nearby distractors, a phenomenon known as visual crowding.

Conclusions

We suggest that patients’ poor performance at contour perception arises not as a consequence of an “integration deficit” but from a combination of reduced sensitivity to local orientation and abnormalities in contextual processing. We propose that this is a consequence of abnormal gain control, a phenomenon that has been implicated in orientation-selectivity as well as surround suppression.     

Reexamination of the Species Assignment of Diacavolinia Pteropods Using DNA Barcoding

Thecosome pteropods (Mollusca, Gastropoda) are an ecologically important, diverse, and ubiquitous group of holoplanktonic animals that are the focus of intense research interest due to their external aragonite shell and vulnerability to ocean acidification. Characterizing the response of these animals to low pH and other environmental stressors has been hampered by continued uncertainty in their taxonomic identification. An example of this confusion in species assignment is found in the genus Diacavolinia. All members of this genus were originally indentified as a single species, Cavolinia longirostris, but over the past fifty years the taxonomy has been revisited multiple times; currently the genus comprises 22 different species. This study examines five species of Diacavolinia, including four sampled in the Northeast Atlantic (78 individuals) and one from the Eastern tropical North Pacific (15 individuals). Diacavolina were identified to species based on morphological characteristics according to the current taxonomy, photographed, and then used to determine the sequence of the “DNA barcoding” region of the cytochrome c oxidase subunit I (COI). Specimens from the Atlantic, despite distinct differences in shell morphology, showed polyphyly and a genetic divergence of <3% (K2P distance) whereas the Pacific and Atlantic samples were more distant (∼19%). Comparisons of Diacavolinia spp. with other Cavolinia spp. reveal larger distances (∼24%). These results indicate that specimens from the Atlantic comprise a single monophyletic species and suggest possible species-level divergence between Atlantic and Pacific populations. The findings support the maintenance of Diacavolinia as a separate genus, yet emphasize the inadequacy of our current taxonomic understanding of pteropods. They highlight the need for accurate species identifications to support estimates of biodiversity, range extent and natural exposure of these planktonic calcifiers to environmental variability; furthermore, the apparent variation of the pteropods shell may have implications for our understanding of the species’ sensitivity to ocean acidification.     

MiR-449a Affects Epithelial Proliferation during the Pseudoglandular and Canalicular Phases of Avian and Mammal Lung Development

Congenital diaphragmatic hernia is associated with pulmonary hypoplasia and respiratory distress, which result in high mortality and morbidity. Although several transgenic mouse models of lung hypoplasia exist, the role of miRNAs in this phenotype is incompletely characterized. In this study, we assessed microRNA expression levels during the pseudoglandular to canalicular phase transition of normal human fetal lung development. At this critical time, when the distal respiratory portion of the airways begins to form, microarray analysis showed that the most significantly differentially expressed miRNA was miR-449a. Prediction algorithms determined that N-myc is a target of miR-449a and identified the likely miR-449a:N-myc binding sites, confirmed by luciferase assays and targeted mutagenesis. Functional ex vivo knock-down in organ cultures of murine embryonic lungs, as well as in ovo overexpression in avian embryonic lungs, suggested a role for miR-449a in distal epithelial proliferation. Finally, miR-449a expression was found to be abnormal in rare pulmonary specimens of human fetuses with Congenital Diaphragmatic Hernia in the pseudoglandular or canalicular phase. This study confirms the conserved role of miR-449a for proper pulmonary organogenesis, supporting the delicate balance between expansion of progenitor cells and their terminal differentiation, and proposes the potential involvement of this miRNA in human pulmonary hypoplasia.     

An Examination of the Association between FOXA1 Staining Level and Biochemical Recurrence following Salvage Radiation Therapy for Recurrent Prostate Cancer

Background

Standardly collected clinical and pathological patient information has demonstrated only moderate ability to predict risk of biochemical recurrence (BCR) of prostate cancer in men undergoing salvage radiation therapy (SRT) for a rising PSA after radical prostatectomy (RP). Although elevated FOXA1 staining has been associated with poor patient outcomes following RP, it has not been studied in the specific setting of SRT after RP. The aim of this study was to evaluate the association between FOXA1 staining level and BCR after SRT for recurrent prostate cancer.

Methods

A total of 141 men who underwent SRT at our institution were included. FOXA1 staining levels in primary tumor samples were detected using immunohistochemistry. FOXA1 staining percentage and intensity were measured and multiplied together to obtain a FOXA1 H-score (range 0–12) which was our primary staining measure. P-values ≤ 0.0056 were considered as statistically significant after applying a Bonferroni correction for multiple comparisons.

Results

There was not a significant association between FOXA1 H-score and risk of BCR when considering H-score as an ordinal variable or as a categorical variable (all P≥0.090). Similarly, no significant associations with BCR were observed for FOXA1 staining percentage or staining intensity (all P≥0.14).

Conclusions

FOXA1 staining level does not appear to have a major impact on risk of BCR after SRT.     

Generation and preclinical characterization of an antibody specific for SEMA4D

Semaphorin 4D (SEMA4D or CD100) is a member of the semaphorin family of proteins and an important mediator of the movement and differentiation of multiple cell types, including those of the immune, vascular, and nervous systems. Blocking the binding of SEMA4D to its receptors can result in physiologic changes that may have implications in cancer, autoimmune, and neurological disease. To study the effects of blocking SEMA4D, we generated, in SEMA4D-deficient mice, a panel of SEMA4D-specific hybridomas that react with murine, primate, and human SEMA4D. Utilizing the complementarity-determining regions from one of these hybridomas (mAb 67-2), we generated VX15/2503, a humanized IgG4 monoclonal antibody that is currently in clinical development for the potential treatment of various malignancies and neurodegenerative disorders, including multiple sclerosis and Huntington's disease. This work describes the generation and characterization of VX15/2503, including in vitro functional testing, epitope mapping, and an in vivo demonstration of efficacy in an animal model of rheumatoid arthritis.