A Refined Model of the Prototypical Salmonella SPI-1 T3SS Basal Body Reveals the Molecular Basis for Its Assembly

The T3SS injectisome is a syringe-shaped macromolecular assembly found in pathogenic Gram-negative bacteria that allows for the direct delivery of virulence effectors into host cells. It is composed of a “basal body”, a lock-nut structure spanning both bacterial membranes, and a “needle” that protrudes away from the bacterial surface. A hollow channel spans throughout the apparatus, permitting the translocation of effector proteins from the bacterial cytosol to the host plasma membrane. The basal body is composed largely of three membrane-embedded proteins that form oligomerized concentric rings. Here, we report the crystal structures of three domains of the prototypical Salmonella SPI-1 basal body, and use a new approach incorporating symmetric flexible backbone docking and EM data to produce a model for their oligomeric assembly. The obtained models, validated by biochemical and in vivo assays, reveal the molecular details of the interactions driving basal body assembly, and notably demonstrate a conserved oligomerization mechanism.     

Thymineless death is inhibited by CsrA in Escherichia coli lacking the SOS response

Thymineless death (TLD) is the rapid loss of colony-forming ability in bacterial, yeast and human cells starved for thymine, and is the mechanism of action of common chemotherapeutic drugs. In Escherichia coli, significant loss of viability during TLD requires the SOS replication-stress/DNA-damage response, specifically its role in inducing the inhibitor of cell division, SulA. An independent RecQ- and RecJ-dependent TLD pathway accounts for a similarly large additional component of TLD, and a third SOS- and RecQ/J-independent TLD pathway has also been observed. Although two groups have implicated the SOS-response in TLD, an SOS-deficient mutant strain from an earlier study was found to be sensitive to thymine deprivation. We performed whole-genome resequencing on that SOS-deficient strain and find that, compared with the SOS-proficient control strain, it contains five mutations in addition to the SOS-blocking lexA(Ind⁻) mutation. One of the additional mutations, csrA, confers TLD sensitivity specifically in SOS-defective strains. We find that CsrA, a carbon storage regulator, reduces TLD in SOS- or SulA-defective cells, and that the increased TLD that occurs in csrA⁻ SOS-defective cells is dependent on RecQ. We consider a hypothesis in which the modulation of nucleotide pools by CsrA might inhibit TLD specifically in SOS-deficient (SulA-deficient) cells.     

Gli as a Novel Therapeutic Target in Malignant Pleural Mesothelioma

Malignant pleural mesothelioma (MPM) is a highly aggressive tumor with poor prognosis. Current treatment is rarely curative, thus novel meaningful therapies are urgently needed. Inhibition of Hedgehog (Hh) signaling at the cell membrane level in several cancers has shown anti-cancer activity in recent clinical studies. Evidence of Hh-independent Gli activation suggests Gli as a more potent therapeutic target. The current study is aimed to evaluate the potential of Gli as a therapeutic target to treat MPM. The expression profiles of Gli factors and other Hh signaling components were characterized in 46 MPM patient tissue samples by RT-PCR and immunohistochemistry. Cultured cell lines were employed to investigate the requirement of Gli activation in tumor cell growth by inhibiting Gli through siRNA or a novel small molecule Gli inhibitor (Gli-I). A xenograft model was used to evaluate Gli-I in vivo. In addition, a side by side comparison between Gli and Smoothened (Smo) inhibition was conducted in vitro using siRNA and small molecule inhibitors. Our study reported aberrant Gli1 and Gli2 activation in a large majority of tissues. Inhibition of Gli by siRNAs or Gli-I suppressed cell growth dramatically both in vitro and in vivo. Inhibition of Gli exhibited better cytotoxicity than that of Smo by siRNA and small molecule inhibitors vismodegib and cyclopamine. Combination of Gli-I and pemetrexed, as well as Gli-I and vismodegib demonstrated synergistic effects in suppression of MPM proliferation in vitro. In summary, Gli activation plays a critical role in MPM. Inhibition of Gli function holds strong potential to become a novel, clinically effective approach to treat MPM.     

A mechanism of expansion: Arctic deciduous shrubs capitalize on warming-induced nutrient availability

Warming-induced nutrient enrichment in the Arctic may lead to shifts in leaf-level physiological properties and processes with potential consequences for plant community dynamics and ecosystem function. To explore the physiological responses of Arctic tundra vegetation to increasing nutrient availability, we examined how a set of leaf nutrient and physiological characteristics of eight plant species (representing four plant functional groups) respond to a gradient of experimental nitrogen (N) and phosphorus (P) enrichment. Specifically, we examined a set of chlorophyll fluorescence measures related to photosynthetic efficiency, performance and stress, and two leaf nutrient traits (leaf %C and %N), across an experimental nutrient gradient at the Arctic Long Term Ecological Research site, located in the northern foothills of the Brooks Range, Alaska. In addition, we explicitly assessed the direct relationships between chlorophyll fluorescence and leaf %N. We found significant differences in physiological and nutrient traits between species and plant functional groups, and we found that species within one functional group (deciduous shrubs) have significantly greater leaf %N at high levels of nutrient addition. In addition, we found positive, saturating relationships between leaf %N and chlorophyll fluorescence measures across all species. Our results highlight species-specific differences in leaf nutrient traits and physiology in this ecosystem. In particular, the effects of a gradient of nutrient enrichment were most prominent in deciduous plant species, the plant functional group known to be increasing in relative abundance with warming in this ecosystem.     

When secondary comes first – The importance of non-canonical DNA structures

Secondary structure-forming DNA motifs have achieved infamy because of their association with a variety of human diseases and cancer. The 3rd FASEB summer conference on dynamic DNA structures focused on the mechanisms responsible for the instabilities inherent to repetitive DNA and presented many exciting and novel aspects related to the metabolism of secondary structures. In addition, the meeting encompassed talks and posters on the dynamic structures that are generated during DNA metabolism including nicked DNA, Holliday junctions and RNA:DNA hybrids. New approaches for analysis and sequencing technologies put forth secondary structures and other DNA intermediates as vital regulators of a variety of cellular processes that contribute to evolution, polymorphisms and diseases.     

Selective inhibition of ATPase activity during contraction alters the activation of p38 MAP kinase isoforms in skeletal muscle

Muscle contractions strongly activate p38 MAP kinases, but the precise contraction‐associated sarcoplasmic event(s) (e.g., force production, energetic demands, and/or calcium cycling) that activate these kinases are still unclear. We tested the hypothesis that during contraction the phosphorylation of p38 isoforms is sensitive to the increase in ATP demand relative to ATP supply. Energetic demands were inhibited using N‐benzyl‐p‐toluene sulphonamide (BTS, type II actomyosin) and cyclopiazonic acid (CPA, SERCA). Extensor digitorum longus muscles from Swiss Webster mice were incubated in Ringer's solution (37°C) with or without inhibitors and then stimulated at 10 Hz for 15 min. Muscles were immediately freeze‐clamped for metabolite and Western blot analysis. BTS and BTS + CPA treatment decreased force production by 85%, as measured by the tension time integral, while CPA alone potentiated force by 310%. In control muscles, contractions resulted in a 73% loss of ATP content and a concomitant sevenfold increase in IMP content, a measure of sustained energetic imbalance. BTS or CPA treatment lessened the loss of ATP, but BTS + CPA treatment completely eliminated the energetic imbalance since ATP and IMP levels were nearly equal to those of non‐stimulated muscles. The independent inhibition of cytosolic ATPase activities had no effect on contraction‐induced p38 MAPK phosphorylation, but combined treatment prevented the increase in phosphorylation of the γ isoform while the α/β isoforms unaffected. These observations suggest that an energetic signal may trigger phosphorylation of the p38γ isoform and also may explain how contractions differentially activate signaling pathways. J. Cell. Biochem. 114: 1445–1455, 2013. © 2013 Wiley Periodicals, Inc.     

An Inactivated Cell Culture Japanese Encephalitis Vaccine (JE-ADVAX) Formulated with Delta Inulin Adjuvant Provides Robust Heterologous Protection against West Nile Encephalitis via Cross-Protective Memory B Cells and Neutralizing Antibody

West Nile virus (WNV), currently the cause of a serious U.S. epidemic, is a mosquito-borne flavivirus and member of the Japanese encephalitis (JE) serocomplex. There is currently no approved human WNV vaccine, and treatment options remain limited, resulting in significant mortality and morbidity from human infection. Given the availability of approved human JE vaccines, this study asked whether the JE-ADVAX vaccine, which contains an inactivated cell culture JE virus antigen formulated with Advax delta inulin adjuvant, could provide heterologous protection against WNV infection in wild-type and β2-microglobulin-deficient (β2m^−/−) murine models. Mice immunized twice or even once with JE-ADVAX were protected against lethal WNV challenge even when mice had low or absent serum cross-neutralizing WNV titers prior to challenge. Similarly, β2m^−/− mice immunized with JE-ADVAX were protected against lethal WNV challenge in the absence of CD8⁺ T cells and prechallenge WNV antibody titers. Protection against WNV could be adoptively transferred to naive mice by memory B cells from JE-ADVAX-immunized animals. Hence, in addition to increasing serum cross-neutralizing antibody titers, JE-ADVAX induced a memory B-cell population able to provide heterologous protection against WNV challenge. Heterologous protection was reduced when JE vaccine antigen was administered alone without Advax, confirming the importance of the adjuvant to induction of cross-protective immunity. In the absence of an approved human WNV vaccine, JE-ADVAX could provide an alternative approach for control of a major human WNV epidemic.