Rapid de-localization of actin leading edge components with BDM treatment

Background

2,3-Butanedione monoxime (BDM) has been widely used as a non-muscle myosin inhibitor to investigate the role of non-muscle myosinII in the process of actin retrograde flow and other actin cytoskeletal processes. Recent reports show that BDM does not inhibit any non-muscle myosins so far tested, including nm-myosinII, prompting the question, how were these process affected in BDM studies?

Results

We have found that treatment of mammalian cells with BDM for only 1 min blocks actin incorporation at the leading edge in a permeabilized cell system. We show that inhibition of actin incorporation occurs through de-localization of leading edge proteins involved in actin polymerization – the Arp2/3 complex, WAVE, and VASP – that de-localize concomitantly with the leading edge actin network.

Conclusion

De-localization of actin leading edge components by BDM treatment is a newly described effect of this compound. It may explain many of the results previously ascribed to inhibition of non-muscle myosinII by BDM, particularly in studies of leading edge dynamics. Though this effect of BDM is intriguing, future studies probing actin dynamics at the leading edge should use more potent and specific inhibitors.

     

Leptin response to short-term fasting in sympathectomized men: role of the SNS

We studied plasma leptin levels in six people with high-lesion spinal cord injury [SCI; body mass index (BMI) 25.9 +/- 1.5 kg/m(2), age 37 +/- 3.0 yr] and six able-bodied (AB) controls (BMI 29.1 +/- 1.9 kg/m(2), age 35 +/- 3.5 yr) before and after 12, 24, and 36 h of fasting. The plasma leptin levels significantly decreased during 36 h fasting by 48.8 +/- 4.5% (pre: 11.3 +/- 2.3, post: 6.2 +/- 1.5 ng/ml) and 38.6 +/- 7.9% (pre: 7.6 +/- 5.0, post: 4.2 +/- 1.0 ng/ml) in SCI and AB, respectively. Plasma leptin started to decrease at 24 h of fasting in the SCI group, whereas plasma leptin started to decrease at 12 h of fasting in the AB group. The current study demonstrated that plasma leptin decreased with fasting in both SCI and AB groups, with the leptin decrease being delayed in the SCI group. The delayed leptin response to fasting in the SCI group may be because of increased fat mass (%body fat, SCI: 33.8 +/- 3.0, AB: 24.1 +/- 2.9) and sympathetic nervous system dysfunction.     

Differences in immunogenicity and protection in mice and guinea pigs following intranasal immunization with Helicobacter pylori outer membrane antigens

Mice and guinea pigs were intranasally immunized with either recombinant lipoprotein 20 or Helicobacter pylori outer membrane vesicles (OMV). Cholera toxin was used as mucosal adjuvant. In mice, both vaccines elicited systemic and local IgG responses, which correlated with significantly lower levels of H. pylori colonization. In contrast, only OMV proved immunogenic in guinea pigs, with the development of both systemic and local immune responses. These antibodies did not, however, correlate with protection in these animals, which suggests that vaccine formulation is as important as choice of antigen in the development of an H. pylori vaccine.     

A plasmid-encoded nicotinamidase (PncA) is essential for infectivity of Borrelia burgdorferi in a mammalian host

Borrelia burgdorferi, a spirochaete that causes Lyme borreliosis, contains 21 linear and circular plasmids thought to be important for survival in mammals or ticks. Our results demonstrate that the gene BBE22 encoding a nicotinamidase is capable of replacing the requirement for the 25 kb linear plasmid lp25 during mammalian infection. Transformation of B. burgdorferi lacking lp25 with a shuttle vector containing the lp25 gene BBE22 (pBBE22) restored infectivity in mice to a level comparable to that of wild-type Borrelia. This complementation also restored the growth and host adaptation of lp25-B. burgdorferi in dialysis membrane chambers (DMCs) implanted in rats. A single Cys to Ala conversion at the putative active site of BBE22 abrogated the ability of pBBE22 to re-establish infectivity or growth in DMCs. Additional Salmonella typhimurium complementation studies and enzymatic analysis demonstrated that the BBE22 gene product has nicotinamidase activity and is most probably required for the biosynthesis of NAD. These results indicate that some plasmid-encoded products fulfil physiological functions required in the enzootic cycle of pathogenic Borrelia.     

Lack of Bdnf and TrkB signalling in the postnatal cochlea leads to a spatial reshaping of innervation along the tonotopic axis and hearing loss

Members of the neurotrophin gene family and their high-affinity Trk receptors control innervation of the cochlea during embryonic development. Lack of neurotrophin signalling in the cochlea has been well documented for early postnatal animals, resulting in a loss of cochlear sensory neurones and a region-specific reduction of target innervation along the tonotopic axis. However, how reduced neurotrophin signalling affects the innervation of the mature cochlea is currently unknown. Here, we have analysed the consequences of a lack of the TrkB receptor and its ligand, the neurotrophin brain-derived neurotrophic factor (Bdnf), in the late postnatal or adult cochlea using mouse mutants. During early postnatal development, mutant animals show a lack of afferent innervation of outer hair cells in the apical part of the cochlea, whereas nerve fibres in the basal part are maintained. Strikingly, this phenotype is reversed during subsequent maturation of the cochlea, which results in a normal pattern of outer hair cell innervation in the apex and loss of nerve fibres at the base in adult mutants. Measurements of auditory brain stem responses of these mice revealed a significant hearing loss. The observed innervation patterns correlate with opposing gradients of Bdnf and Nt3 expression in cochlear neurones along the tonotopic axis. Thus, the reshaping of innervation may be controlled by autocrine signalling between neurotrophins and their receptors in cochlear neurones. Our results indicate a substantial potential for re-innervation processes in the mature cochlea, which may also be of relevance for treatment of hearing loss in humans.     

Mouse Mesenchyme forkhead 2 (Mf2): expression, DNA binding and induction by sonic hedgehog during somitogenesis

Cloning and sequencing of mouse Mf2 (mesoderm/mesenchyme forkhead 2) cDNAs revealed an open reading frame encoding a putative protein of 492 amino acids which, after in vitro translation, binds to a DNA consensus sequence. Mf2 is expressed at high levels in the ventral region of newly formed somites, in sclerotomal derivatives, in lateral plate and cephalic mesoderm and in the first and second branchial arches. Other regions of mesodermal expression include the developing tongue, meninges, nose, whiskers, kidney, genital tubercule and limb joints. In the nervous system Mf2 is transcribed in restricted regions of the mid- and forebrain. In several tissues, including the early somite, Mf2 is expressed in cell populations adjacent to regions expressing sonic hedgehog (Shh) and in explant cultures of presomitic mesoderm Mf2 is induced by Shh secreted by COS cells. These results suggest that Mf2, like other murine forkhead genes, has multiple roles in embryogenesis, possibly mediating the response of cells to signaling molecules such as SHH.     

Knowledge,Attitudes, and Practices (KAP) Relating to Avian Influenza (H10N8) among Farmers’ Markets Workers in Nanchang,China

Three cases of avian influenza virus H10N8 were reported in Nanchang, China, as of April 2014. To identify the knowledge, attitudes, and practices (KAP) related to H10N8 among farmers’ market workers, a cross-sectional survey was conducted in 63 farmers’ markets in Nanchang. Using the resulting data, characteristics of poultry and non-poultry workers’ knowledge, attitudes, and practice were described. Results suggest that interventions targeting high-risk workers should be developed and implemented by public health agencies to prevent the spread of H10N8. Additionally policies that encourage farmers’ market workers to receive influenza vaccine should be developed, adopted, and enforced.     

Blockade of PD-1/PD-L1 Promotes Adoptive T-Cell Immunotherapy in a Tolerogenic Environment

Adoptive cellular immunotherapy using in vitro expanded CD8⁺ T cells shows promise for tumour immunotherapy but is limited by eventual loss of function of the transferred T cells through factors that likely include inactivation by tolerogenic dendritic cells (DC). The co-inhibitory receptor programmed death-1 (PD-1), in addition to controlling T-cell responsiveness at effector sites in malignancies and chronic viral diseases is an important modulator of dendritic cell-induced tolerance in naive T cell populations. The most potent therapeutic capacity amongst CD8⁺ T cells appears to lie within Tcm or Tcm-like cells but memory T cells express elevated levels of PD-1. Based on established trafficking patterns for Tcm it is likely Tcm-like cells interact with lymphoid-tissue DC that present tumour-derived antigens and may be inherently tolerogenic to develop therapeutic effector function. As little is understood of the effect of PD-1/PD-L1 blockade on Tcm-like CD8⁺ T cells, particularly in relation to inactivation by DC, we explored the effects of PD-1/PD-L1 blockade in a mouse model where resting DC tolerise effector and memory CD8⁺ T cells. Blockade of PD-1/PD-L1 promoted effector differentiation of adoptively-transferred Tcm-phenotype cells interacting with tolerising DC. In tumour-bearing mice with tolerising DC, effector activity was increased in both lymphoid tissues and the tumour-site and anti-tumour activity was promoted. Our findings suggest PD-1/PD-L1 blockade may be a useful adjunct for adoptive immunotherapy by promoting effector differentiation in the host of transferred Tcm-like cells.