Inositol-1-phosphate synthetase mRNA as a new target for antisense inhibition of Mycobacterium tuberculosis

The need for novel antimicrobial agents to combat the emergence of multi-drug-resistant strains of Mycobacterium tuberculosis is a worldwide urgency. This study has investigated the effects on phosphorothioate-modified antisense oligodeoxyribonucleotides (PS-ODNs) against the mRNA of inositol-1-phosphate synthase, the key enzyme in the first step in inositol synthesis. Inositol is utilized by M. tuberculosis in the production of its major thiol, which is an antioxidant that helps M. tuberculosis to get rid of reactive oxygen species and electrophilic toxins. Real-time RT-PCR analysis revealed that mRNA expression of inositol-1-phosphate (I-1-P) synthase was significantly reduced upon addition of 20 microM PS-ODNs. Treatment with antisense PS-ODNs also reduced the level of mycothiol and the proliferation of M. tuberculosis and enhanced susceptibility to antibiotics. The experiments indicated that the antisense PS-ODNs could enter the cytoplasm of M. tuberculosis and inhibit the expression of I-1-P synthase. This study demonstrates that the M. tuberculosis I-1-P synthase is a target for the development of novel antibiotics and PS-ODN to I-1-P synthase is a promising antimycobaterial candidate.     

Divergent terrestrial responses of soil N2O emissions to different levels of elevated CO2 and temperature

Understanding the responses of soil nitrous oxide (N₂O) emissions from terrestrial ecosystems to future CO₂ enrichment and warming is critical for the development of mitigation and adaptation policies. The effects of continuous increase in elevated CO₂ (EC) and elevated temperature (ET) on N₂O emissions are not fully known. We synthesized 209 measurements from 70 published studies and carried out a meta-analysis to examine individual and interactive effects of EC and ET on N₂O emissions from grasslands, croplands and forests. On average, a significant increase of 23% in N₂O emissions was observed under EC across all case studies. EC did not affect N₂O emissions from grasslands or forests, but significantly increased N₂O emissions in croplands by 38%. The extent of ET effects on N₂O emissions was nonsignificant and there was no significant difference in N₂O emission responses among these three terrestrial systems. ET only promoted N₂O emissions in forest by about 32% when ET was less than 2°C. The interactive effect of EC and ET on N₂O emissions was significantly synergistic, showing a greater increase than the sum of the effects caused by EC and ET alone. Our findings indicated that the combination of EC and ET substantially promoted soil N₂O and highlighted the urgent need to explore its mechanisms to better understand N₂O responses under future climate change.     

Acid‐sensing ion channels regulate nucleus pulposus cell inflammation and pyroptosis via the NLRP3 inflammasome in intervertebral disc degeneration

ObjectiveLactate accumulation is an important factor in the intervertebral disc degeneration (IVDD). Currently, the effect and underlying mechanism of action of lactate on nucleus pulposus (NP) cell inflammation during IVDD are unclear. Previous studies have found that the NLRP3 inflammasome plays an important role in the regulation of NP inflammation. This study focused on the regulation of acid‐sensitive ion channels (ASICs) in relation to inflammation and the effect of NLRP3 on pyroptosis levels in NP cells under acidic conditions.DesignFor the in vitro experiments, human NP cells were exposed to 6 mM lactate solution; different groups were either treated with NLRP3 inhibitor or transfected with siRNA against NLRP3, siRNA against ASC or a mix of these, and mRNA and protein expression levels were then assessed. For the in vivo experiment, varying concentrations of lactate were injected into rat intervertebral discs and examined via magnetic resonance imaging (MRI) and histological staining.ResultsExtracellular lactate promoted NLRP3 inflammasome activation and degeneration of the NP extracellular matrix; furthermore, it increased the levels of inflammation and pyroptosis in the NP. Lactate‐induced NLRP3 inflammasome activation was blocked by ASIC inhibitors and NLRP3 siRNA.ConclusionsExtracellular lactate regulates levels of intercellular reactive oxygen species (ROS) through ASIC1 and ASIC3. ROS activate the NF‐κB signalling pathway, thus promoting NLRP3 inflammasome activation and IL‐1β release, both of which promote NP degeneration.     

Copper-catalyzed azide-alkyne cycloaddition (click chemistry)-based Detection of Global Pathogen-host AMPylation on Self-assembled Human Protein Microarrays

AMPylation (adenylylation) is a recently discovered mechanism employed by infectious bacteria to regulate host cell signaling. However, despite significant effort, only a few host targets have been identified, limiting our understanding of how these pathogens exploit this mechanism to control host cells. Accordingly, we developed a novel nonradioactive AMPylation screening platform using high-density cell-free protein microarrays displaying human proteins produced by human translational machinery. We screened 10,000 unique human proteins with Vibrio parahaemolyticus VopS and Histophilus somni IbpAFic2, and identified many new AMPylation substrates. Two of these, Rac2, and Rac3, were confirmed in vivo as bona fide substrates during infection with Vibrio parahaemolyticus. We also mapped the site of AMPylation of a non-GTPase substrate, LyGDI, to threonine 51, in a region regulated by Src kinase, and demonstrated that AMPylation prevented its phosphorylation by Src. Our results greatly expanded the repertoire of potential host substrates for bacterial AMPylators, determined their recognition motif, and revealed the first pathogen-host interaction AMPylation network. This approach can be extended to identify novel substrates of AMPylators with different domains or in different species and readily adapted for other post-translational modifications.Protein AMPylation (adenylylation) was recently discovered in bacteria-host interactions where virulence factors catalyze AMPylation using either a conserved Fic domain (e.g., VopS, Vibrio parahaemolyticus (V. para) and IbpA, Histophilus somni) or an adenylyl transferase domain (e.g., DrrA, Legionella pneumophila). These bacterial AMPylation enzymes, or AMPylators, are secreted into the host cells by bacterial secretion systems and transfer AMP from ATP to Tyr or Thr residues of their respective substrates (1–3). In the case of VopS and IbpA, several Rho family GTPases (Rac1, RhoA, and Cdc42) are known substrates and AMPylation disrupts the binding of the GTPase to its downstream effectors, for example, PAK1 (2–6). Considering the conservation of AMPylation domains in both prokaryotic and eukaryotic organisms, we expect that AMPylation plays an important role in a wide range of cellular processes (2, 4, 5, 7–9). Nevertheless, our understanding of this post-translational modification (PTM) is still limited to only a handful of known eukaryotic AMPylation substrates, exclusively belonging to the Rho and Rab GTPase families(10–14). Determining the repertoire of substrates modified by AMPylators will help illuminate both the functional consequences of AMPylation and the mechanistic strategies of pathogens that employ them (6).Significant effort has been devoted to identifying AMPylation substrates. Li et al. systematically investigated the fragmentation patterns of chemically synthesized peptides with Thr, Ser, and Tyr AMPylation using matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). They detected AMPylation sites with high confidence and selectively scanned AMPylated peptides in protein mixtures (10). Hao et al. produced a polyclonal antibody that specifically recognized proteins with AMPylation at threonine residues (11). Grammel et al. synthesized an ATP analog, N⁶pATP (N⁶-propargyl adenosine-5′-triphophate), which allows the labeling of AMPylated proteins with azide-functionalized fluorescein or a cleavable biotin enrichment tag (ortho-hydroxy-azidoethoxy-azobiotin) based on copper-catalyzed azide-alkyne cycloaddition (CuAAC)¹. The identification of new substrates for VopS in HeLa cell lysates was explored by a combination of AMP-specific pull-down and LC-MS (12). Using the same approach, Lewallen et al. tried to identify the substrates of VopS in MCF7 cell extracts by employing a commercial N⁶-(6-amino)hexyl-ATP-5-carboxyl-fluorescein (F1-ATP) and anti-fluorescein antibody(13). With these efforts combined, four potential new VopS substrates have been identified (SCCA2, NAGK, NME1, and PFKP), though not yet confirmed. These approaches might miss substrates because of temporal and spatial expression or low abundance in cell lysate, poor recognition by the capture molecules or loss during pull-down procedures (12, 14).Protein microarrays offer a promising approach to identify candidate substrates because they display thousands of unique proteins in a high-throughput and reproducible format (15–17). However, producing arrays with consistent levels of well-folded proteins is challenging because of limitations of protein production, purification, and storage, particularly for mammalian proteins (18).To circumvent these limitations, cell-free protein arrays, which do not require protein purification, have been developed over the past decade (19–22). These methods provide rapid and economical approaches of fabricating protein arrays in terms of cost, shelf life, and storage (23, 24). In cell-free protein arrays, a nucleotide template is printed on the slide and used to produce proteins in vitro with cell-free expression systems from several organisms such as E. coli, wheat germ, and rabbit reticulocyte lysate, etc. (24, 25). These proteins can be engineered to contain fusion tags that enable their capture to the array surface with an appropriate agent. Of these cell-free protein array methods, the Nucleic Acid Programmable Protein Array (NAPPA) is the most advanced, having achieved both high-density and high content containing ∼2300–8000 proteins per slide (20, 26, 27). In NAPPA, a plasmid-based cDNA configured to include an epitope tag is printed on a microscope slide along with the corresponding tag-specific binding reagent, such as an anti-tag antibody, and stored. At the time of experimentation, the cDNA is transcribed/translated into recombinant protein and captured/displayed in situ by the binding reagent. Using a rabbit reticulocyte lysate-based cell-free expression system, NAPPA has been applied toward the identification of novel protein-protein interactions and disease-related antibody biomarkers (20, 26, 28, 29). However, cell-free protein arrays have yet to be employed in the study of PTMs.In this work, we established a novel, nonradioactive unbiased AMPylation screening platform by developing a novel click chemistry-based detection assay for use on high-density cell-free protein microarrays displaying human proteins. Labeling AMP-modified substrates covalently with a fluorophore coupled with the use of human ribosomal machinery and chaperones to produce proteins achieved much higher sensitivity and signal to noise (S/N) ratio compared with previous studies. We screened 10,000 human proteins with two bacterial pathogen AMPylators, VopS and IbpAFic2, identifying more than twenty new substrates each. Two novel Rho GTPases (Rac2 and Rac3) were validated in vivo as substrates of the virulence factor VopS in HEK293T cells during V. para infection. Using mass spectrometry, we verified that a non-GTPase protein, ARHGDIB/LyGDI, was AMPylated by VopS on its threonine 51, which is located in a highly regulated part of this protein. This modification inhibited phosphorylation of LyGDI by Src kinase in vitro. Finally, the identification of these new targets allowed us to build the first bacteria-host interaction AMPylation network and may reveal signaling interactions that could potentially be important for bacterial pathogenesis in the future functional studies.     

Soil fungal diversity in three nature reserves of Jiuzhaigou County,Sichuan Province,China

Jiuzhaigou County is located at the southern transition zone of Sichuan Basin and the Qinghai–Tibet Plateau and is the site of three famous nature reserves, namely, the Jiuzhaigou Nature Reserve (JZNR), Baihe Nature Reserve (BHNR) and Wujiao Nature Reserve (WJNR). The soil fungal diversity in this region has not yet been investigated. In this study, we collected 25 soil samples from these three nature reserves. Soil fungi were isolated using the soil dilution plate technique and Rose Bengal agar medium. The culturable soil fungal density based on analysis of the 25 samples ranged from 2.18 log to 4.38 log CFU g^?1 dry weight soil, with the fungal density being highest in samples from JZNR and lowest in those from BHNR. Based on morphological characters and the results of phylogenetic analysis of the internal transcribed spacer (ITS) of the rDNA operon, we identified 38 genera (two genera could not be identified) belonging to Ascomycota, Zygomycota and Basidiomycota. The dominant genera were Penicillium, Humicola, Aspergillus and Trichoderma. The species richness index S, biodiversity index H′ and evenness index E of the 25 sampling sites were in the range 10–29, 1.96–3.05 and 0.74–0.95, respectively. The highest mean values of the S, H′ and E indices were in soil samples from BHNR, where the values of these indices were 20.00, 2.66 and 0.90, respectively. These results indicate that the diversity of culturable fungi in these three nature reserves was high. Furthermore, a total 14 Trichoderma isolates were tested for their antagonism activity against mycelium growth of three pathogens: Bipolaris maydis, Curvularia lunata, Rhizoctonia solani. The results showed that six Trichoderma isolates had good antagonistic effects on the three pathogenic fungi.     

Mitotic stability of small supernumerary marker chromosomes depends on their shape and telomeres — A long term in vitro study

Mosaicism is present in more than 50% of the cases with small supernumerary marker chromosomes (sSMCs) and karyotype 47,XX,+mar/46,XX or 47,XY,+mar/46,XY. Recently we provided first evidence that the mitotic stability of sSMC is dependent on their structure, i.e. their shape. Thus, here we performed a long term in vitro study on 12 selected cell lines from the Else Kröner–Fresenius-sSMC-cellbank (http://ssmc-tl.com/ekf-cellbank.html) to test mitotic sSMC stability systematically. The obtained results showed that inverted duplicated shaped and also the so-called complex sSMCs (group 1) are by far more stable, than centric-minute- or ring-shaped sSMCs (groups 2). Generally speaking, the percentage of cells with group-1-sSMCs remained stable over 90 days of cell culture, while that of group-2-sSMCs in parts dramatically decreased. In one group-2-cell line the sSMC was even lost completely after 30 days of in vitro culture, in others the sSMC was depleted in up to 40% of the cells. Still the highest rate of sSMC loss was recorded during EBV-transformation. Overall, the major difference between groups 1 and 2 was the number of telomeres per sSMC. In group 1 the sSMCs had “original” telomeres at both of their ends; in group 2 the sSMCs had either no, possibly secondary acquired and/or only one original telomere. This absence of protective telomeric sequences in group 2 seems to make sSMC more susceptible for loss during cell division. Still, also a growth advantage of cells without sSMC cannot be neglected entirely.     

Predicting Local Dengue Transmission in Guangzhou,China, through the Influence of Imported Cases,Mosquito Density and Climate Variability

Introduction

Each year there are approximately 390 million dengue infections worldwide. Weather variables have a significant impact on the transmission of Dengue Fever (DF), a mosquito borne viral disease. DF in mainland China is characterized as an imported disease. Hence it is necessary to explore the roles of imported cases, mosquito density and climate variability in dengue transmission in China. The study was to identify the relationship between dengue occurrence and possible risk factors and to develop a predicting model for dengue’s control and prevention purpose.

Methodology and Principal Findings

Three traditional suburbs and one district with an international airport in Guangzhou city were selected as the study areas. Autocorrelation and cross-correlation analysis were used to perform univariate analysis to identify possible risk factors, with relevant lagged effects, associated with local dengue cases. Principal component analysis (PCA) was applied to extract principal components and PCA score was used to represent the original variables to reduce multi-collinearity. Combining the univariate analysis and prior knowledge, time-series Poisson regression analysis was conducted to quantify the relationship between weather variables, Breteau Index, imported DF cases and the local dengue transmission in Guangzhou, China. The goodness-of-fit of the constructed model was determined by pseudo-R², Akaike information criterion (AIC) and residual test. There were a total of 707 notified local DF cases from March 2006 to December 2012, with a seasonal distribution from August to November. There were a total of 65 notified imported DF cases from 20 countries, with forty-six cases (70.8%) imported from Southeast Asia. The model showed that local DF cases were positively associated with mosquito density, imported cases, temperature, precipitation, vapour pressure and minimum relative humidity, whilst being negatively associated with air pressure, with different time lags.

Conclusions

Imported DF cases and mosquito density play a critical role in local DF transmission, together with weather variables. The establishment of an early warning system, using existing surveillance datasets will help to control and prevent dengue in Guangzhou, China.     

HIV Pre-Exposure Prophylaxis Interest among Female Sex Workers in Guangxi,China

Objectives

Acceptability of pre-exposure prophylaxis (PrEP) and willingness to participate in a clinical trial for both safety and efficacy of PrEP were investigated among female sex workers (FSWs) in Guangxi, China.

Methods

A cross-sectional study was performed in three cities in Guangxi. Structured, self-administered questionnaires were used to assess the acceptability of PrEP and the willingness to participate in a clinical trial. Multivariable logistic regression models were fitted to identify predictors.

Results

Among 405 participants, 15.1% had heard of PrEP. If PrEP was deemed to be effective, safe and provided for free, 85.9% reported that they would accept it, and 54.3% of those who accepted PrEP said that they would participate in a clinical trial. The increased acceptability of PrEP was associated with working in male dominated venues, higher income, a poor family relationship, better HIV/AIDS knowledge, not realizing HIV risk from unfamiliar clients, not being forced to use condoms by the gatekeepers, consistent use of condoms, and use of drugs to prevent STD infection. The increased willingness to participate in a clinical trial was associated with a poor family relationship, better HIV/AIDS knowledge, not realizing HIV risk from unfamiliar clients, a willingness to adhere to daily PreP use, and not being concerned about discrimination by others. The main reason for rejecting PrEP or participating in a clinical trial was the concern about the side effects of PrEP.

Conclusions

Acceptability of PrEP among Guangxi FSWs is relatively high, indicating that PrEP intervention programs may be feasible for Chinese FSWs. Given the fact that most of the participants had never heard of PrEP before, and that family, gatekeepers, and social discrimination could significantly affect its acceptability, a comprehensive mix of multiple interventions is necessary for the successful implementation of a PrEP program among this population in Guangxi.     

Cytosolic CARP Promotes Angiotensin II- or Pressure Overload-Induced Cardiomyocyte Hypertrophy through Calcineurin Accumulation

The gene ankyrin repeat domain 1 (Ankrd1) is an enigmatic gene and may exert pleiotropic function dependent on its expression level, subcellular localization and even types of pathological stress, but it remains unclear how these factors influence the fate of cardiomyocytes. Here we attempted to investigate the role of CARP on cardiomyocyte hypertrophy. In neonatal rat ventricular cardiomyocytes (NRVCs), angiotensin II (Ang II) increased the expression of both calpain 1 and CARP, and also induced cytosolic translocation of CARP, which was abrogated by a calpain inhibitor. In the presence of Ang-II in NRVCs, infection with a recombinant adenovirus containing rat Ankrd1 cDNA (Ad-Ankrd1) enhanced myocyte hypertrophy, the upregulation of atrial natriuretic peptide and β-myosin heavy chain genes and calcineurin proteins as well as nuclear translocation of nuclear factor of activated T cells. Cyclosporin A attenuated Ad-Ankrd1-enhanced cardiomyocyte hypertrophy. Intra-myocardial injection of Ad-Ankrd1 in mice with transverse aortic constriction (TAC) markedly increased the cytosolic CARP level, the heart weight/body weight ratio, while short hairpin RNA targeting Ankrd1 inhibited TAC-induced hypertrophy. The expression of calcineurin was also significantly increased in Ad-Ankrd1-infected TAC mice. Olmesartan (an Ang II receptor antagonist) prevented the upregulation of CARP in both Ang II-stimulated NRVCs and hearts with pressure overload. These findings indicate that overexpression of Ankrd1 exacerbates pathological cardiac remodeling through the enhancement of cytosolic translocation of CARP and upregulation of calcineurin.