Rational design of unrestricted pRN1 derivatives and their application in the construction of a dual plasmid vector system for Saccharolobus islandicus.

Saccharolobus islandicus REY15A represents one of the very few archaeal models with versatile genetic tools, which include efficient genome editing, gene silencing, and robust protein expression systems. However, plasmid vectors constructed for this crenarchaeon thus far are based solely on the pRN2 cryptic plasmid. Although this plasmid coexists with pRN1 in its original host, early attempts to test pRN1-based vectors consistently failed to yield any stable host-vector system for Sa. islandicus. We hypothesized that this failure could be due to the occurrence of CRISPR immunity against pRN1 in this archaeon. We identified a putative target sequence in orf904 encoding a putative replicase on pRN1 (target N1). Mutated targets (N1a, N1b, and N1c) were then designed and tested for their capability to escape the host CRISPR immunity by using a plasmid interference assay. The results revealed that the original target triggered CRISPR immunity in this archaeon, whereas all three mutated targets did not, indicating that all the designed target mutations evaded host immunity. These mutated targets were then incorporated into orf904 individually, yielding corresponding mutated pRN1 backbones with which shuttle plasmids were constructed (pN1aSD, pN1bSD, and pN1cSD). Sa. islandicus transformation revealed that pN1aSD and pN1bSD were functional shuttle vectors, but pN1cSD lost the capability for replication. These results indicate that the missense mutations in the conserved helicase domain in pN1c inactivated the replicase. We further showed that pRN1-based and pRN2-based vectors were stably maintained in the archaeal cells either alone or in combination, and this yielded a dual plasmid system for genetic study with this important archaeal model.

Machine Learning Reveals Impacts of Smoking on Gene Profiles of Different Cell Types in Lung.

Smoking significantly elevates the risk of lung diseases such as chronic obstructive pulmonary disease (COPD) and lung cancer. This risk is attributed to the harmful chemicals in tobacco smoke that damage lung tissue and impair lung function. Current research on the impact of smoking on gene expression in specific lung cells is limited. This study addresses this gap by analyzing gene expression profiles at the single-cell level from 43,539 lung endothelial cells, 234,349 lung epithelial cells, 189,843 lung immune cells, and 16,031 lung stromal cells using advanced machine learning techniques. The data, categorized by different lung cell types, were classified into three smoking states: active smoker, former smoker, and never smoker. Each cell sample encompassed 28,024 feature genes. Employing an incremental feature selection method within a computational framework, several specific genes have been identified as potential markers of smoking status in different lung cell types. These include B2M, EEF1A1, and TPT1 in lung endothelial cells; FTL and MT-ATP8 in lung epithelial cells; HLA-B and HLA-C in lung immune cells; and HSP90B1 and LCN2 in lung stroma cells. Additionally, this study developed quantitative rules for representing the gene expression patterns related to smoking. This research highlights the potential of machine learning in oncology, enhancing our molecular understanding of smoking's harm and laying the groundwork for future mechanism-based studies.

Identification of potential natural products derived from fungus growing termite, inhibiting Pseudomonas aeruginosa quorum sensing protein LasR using molecular docking and molecular dynamics simulation approach.

Pseudomonas aeruginosa, the most common opportunistic pathogen, is becoming antibiotic-resistant worldwide. The fate of P. aeruginosa, a multidrug-resistant strain, can be determined by multidrug efflux pumps, enzyme synthesis, outer membrane protein depletion, and target alterations. Microbial niches have long used quorum sensing (QS) to synchronize virulence gene expression. Computational methods can aid in the development of novel P. aeruginosa drug-resistant treatments. The tripartite symbiosis in termites that grow fungus may help special microbes find new antimicrobial drugs. To find anti-quorum sensing natural products that could be used as alternative therapies, a library of 376 fungal-growing termite-associated natural products (NPs) was screened for their physicochemical properties, pharmacokinetics, and drug-likeness. Using GOLD, the top 74 NPs were docked to the QS transcriptional regulator LasR protein. The five lead NPs with the highest gold score and drug-like properties were chosen for a 200-ns molecular dynamics simulation to test the competitive activity of different compounds against negative catechin. Fridamycin and Daidzein had stable conformations, with mean RMSDs of 2.48 and 3.67 Å, respectively, which were similar to Catechin's 3.22 Å. Fridamycin and Daidzein had absolute binding energies of -71.186 and -52.013 kcal/mol, respectively, which were higher than the control's -42.75 kcal/mol. All the compounds within the active site of the LasR protein were kept intact by Trp54, Arg55, Asp67, and Ser123. These findings indicate that termite gut and fungus-associated NPs, specifically Fridamycin and Daidzein, are potent QS antagonists that can be used to treat P. aeruginosa's multidrug resistance.Communicated by Ramaswamy H. Sarma.

Prediction of Acute Kidney Injury in Intracerebral Hemorrhage Patients Using Machine Learning.

Background: Acute kidney injury (AKI) is prevalent in patients with intracerebral hemorrhage (ICH) and is associated with mortality. This study aimed to verify the predictive accuracy of different machine learning algorithms for AKI in patients with ICH using a large dataset. Methods: A total of 1366 ICH patients received treatments between 2001 and 2012 from the Medical Information Mart for Intensive Care-III (MIMIC-III) database were identified based on the ICD-9 code: 431. The main outcome of AKI during hospitalizations was confirmed based on the KDIGO criteria. Overall, ICH patients were randomly divided into the training cohort and validation cohort with the ratio of 7:3. Six machine learning algorithms including extreme gradient boosting, logistic, light gradient boosting machine, random forest, adaptive boosting, support vector machine were trained in the training cohort with the 5-fold cross-validation method to predict the AKI. The predictive accuracy of those algorithms was compared by area under the receiver operating characteristics curve (AUC). Results: A total of 1213 ICH patients were included with the incidence of AKI being 29.3%. The incidence of AKI was 29.3% among the 1213 patients with ICH. The AKI group had higher 30-day mortality (p<0.001), longer ICU stay (p<0.001), and longer hospital stay (p<0.001). Among the six machine learning algorithms, the random forest performed the best in predicting AKI in both the training cohort (AUC=1.000) and the validation cohort (AUC=0.698). The top five features in the random forest algorithm-based model were platelets, serum creatinine, vancomycin, hemoglobin, and hematocrit. Conclusion: The random forest algorithm-based predictive model we developed incorporating important features, including platelet count, serum creatinine level, vancomycin level, hemoglobin level, and hematocrit level, performed the best in predicting AKI among patients with ICH.

The canonical single-stranded DNA-binding protein is not an essential replication factor but an RNA chaperon in Saccharolobus islandicus.

Single-stranded DNA-binding proteins (SSBs) have been regarded as indispensable replication factors. Herein, we report that the genes encoding the canonical SSB (SisSSB) and the non-canonical SSB (SisDBP) in Saccharolobus islandicus REY15A are not essential for cell viability. Interestingly, at a lower temperature (55°C), the protein level of SisSSB increases and the growth of ΔSisssb and ΔSisssbΔSisdbp is retarded. SisSSB exhibits melting activity on dsRNA and DNA/RNA hybrid in vitro and is able to melt RNA hairpin in Escherichia coli. Furthermore, the core SisSSB domain is able to complement the absence of cold-shock proteins in E. coli. Importantly, these activities are conserved in the canonical SSBs from Crenarchaeota species that lack bacterial Csp homologs. Overall, our study has clarified the function of the archaeal canonical SSBs which do not function as a DNA-processing factor, but play a role in the processes requiring melting of dsRNA or DNA/RNA hybrid.

The FHA domain protein ArnA functions as a global DNA damage response repressor in the hyperthermophilic archaeon Saccharolobus islandicus.

Forkhead-associated (FHA) domain proteins specifically recognize phosphorylated threonine via the FHA domain and are involved in signal transduction in various processes especially DNA damage response (DDR) and cell cycle regulation in eukaryotes. Although FHA domain proteins are found in prokaryotes, archaea, and bacteria, their functions are far less clear as compared to the eukaryotic counterparts, and it has not been studied whether archaeal FHA proteins play a role in DDR. Here, we have characterized an FHA protein from the hyperthermophilic Crenarchaeon Saccharolobus islandicus (SisArnA) by genetic, biochemical, and transcriptomic approaches. We find that ΔSisarnA exhibits higher resistance to DNA damage agent 4-nitroquinoline 1-oxide (NQO). The transcription of ups genes, encoding the proteins for pili-mediated cell aggregation and cell survival after DDR, is elevated in ΔSisarnA. The interactions of SisArnA with two predicted partners, SisvWA1 (SisArnB) and SisvWA2 (designated as SisArnE), were enhanced by phosphorylation in vitro. ΔSisarnB displays higher resistance to NQO than the wild type. In addition, the interaction between SisArnA and SisArnB, which is reduced in the NQO-treated cells, is indispensable for DNA binding in vitro. These indicate that SisArnA and SisArnB work together to inhibit the expression of ups genes in vivo. Interestingly, ΔSisarnE is more sensitive to NQO than the wild type, and the interaction between SisArnA and SisArnE is strengthened after NQO treatment, suggesting a positive role of SisArnE in DDR. Finally, transcriptomic analysis reveals that SisArnA represses a number of genes, implying that archaea apply the FHA/phospho-peptide recognition module for extensive transcriptional regulation. IMPORTANCE Cellular adaption to diverse environmental stresses requires a signal sensor and transducer for cell survival. Protein phosphorylation and its recognition by forkhead-associated (FHA) domain proteins are widely used for signal transduction in eukaryotes. Although FHA proteins exist in archaea and bacteria, investigation of their functions, especially those in DNA damage response (DDR), is limited. Therefore, the evolution and functional conservation of FHA proteins in the three domains of life is still a mystery. Here, we find that an FHA protein from the hyperthermophilic Crenarchaeon Saccharolobus islandicus (SisArnA) represses the transcription of pili genes together with its phosphorylated partner SisArnB. SisArnA derepression facilitates DNA exchange and repair in the presence of DNA damage. The fact that more genes including a dozen of those involved in DDR are found to be regulated by SisArnA implies that the FHA/phosphorylation module may serve as an important signal transduction pathway for transcriptional regulation in archaeal DDR.

Identification of fungus-growing termite-associated halogenated-PKS maduralactomycin a as a potential inhibitor of MurF protein of multidrug-resistant Acinetobacter baumannii.

Multidrug-resistant Acinetobacter baumannii infections have become a major public health concern globally. Inhibition of its essential MurF protein has been proposed as a potential target for broad-spectrum drugs. This study aimed to evaluate the potential of a novel ecological niche of 374 fungus-growing termite associated Natural Products (NPs). The molecular docking and computational pharmacokinetics screened four compounds, i.e., Termstrin B, Fridamycin A, Maduralactomycin A, and Natalenamide C, as potential compounds that have higher binding affinities and favourable protein-ligand interactions. The compound Maduralactomycin A induced more stability based on its lowest average RMSD value (2.31 Å) and low standard deviation (0.35) supported by the consistent flexibility and ß-factor during the protein's time-dependent motion. While hydrogen bond analysis indicated that Termstrin B has formed the strongest intra-protein interaction, solvent accessibility was in good agreement with Maduralactomycin A compactness. Maduralactomycin A has the strongest binding energy among all the compounds (-348.48 kcal/mol) followed by Termstrin B (-321.19 kcal/mol). Since these findings suggest Maduralactomycin A and Termstrin B as promising candidates for inhibition of MurF protein, the favourable binding energies of Maduralactomycin A make it a more important compound to warrant further investigation. However, experimental validation using animal models and clinical trials is recommended before reaching any final conclusions.

A novel RHH family transcription factor aCcr1 and its viral homologs dictate cell cycle progression in archaea.

Cell cycle regulation is of paramount importance for all forms of life. Here, we report that a conserved and essential cell cycle-specific transcription factor (designated as aCcr1) and its viral homologs control cell division in Sulfolobales. We show that the transcription level of accr1 reaches peak during active cell division (D-phase) subsequent to the expression of CdvA, an archaea-specific cell division protein. Cells over-expressing the 58-aa-long RHH (ribbon-helix-helix) family cellular transcription factor as well as the homologs encoded by large spindle-shaped viruses Acidianus two-tailed virus (ATV) and Sulfolobus monocaudavirus 3 (SMV3) display significant growth retardation and cell division failure, manifesting as enlarged cells with multiple chromosomes. aCcr1 over-expression results in downregulation of 17 genes (>4-fold), including cdvA. A conserved motif, aCcr1-box, located between the TATA-binding box and the translation initiation site of 13 out of the 17 highly repressed genes, is critical for aCcr1 binding. The aCcr1-box is present in the promoters and 5' UTRs of cdvA genes across Sulfolobales, suggesting that aCcr1-mediated cdvA repression is an evolutionarily conserved mechanism by which archaeal cells dictate cytokinesis progression, whereas their viruses take advantage of this mechanism to manipulate the host cell cycle.

The archaeal KEOPS complex possesses a functional Gon7 homolog and has an essential function independent of the cellular t6A modification level.

Kinase, putative Endopeptidase, and Other Proteins of Small size (KEOPS) is a multisubunit protein complex conserved in eukaryotes and archaea. It is composed of Pcc1, Kae1, Bud32, Cgi121, and Gon7 in eukaryotes and is primarily involved in N6-threonylcarbamoyl adenosine (t6A) modification of transfer RNAs (tRNAs). Recently, it was reported that KEOPS participates in homologous recombination (HR) repair in yeast. To characterize the KEOPS in archaea (aKEOPS), we conducted genetic and biochemical analyses of its encoding genes in the hyperthermophilic archaeon Saccharolobus islandicus. We show that aKEOPS also possesses five subunits, Pcc1, Kae1, Bud32, Cgi121, and Pcc1-like (or Gon7-like), just like eukaryotic KEOPS. Pcc1-like has physical interactions with Kae1 and Pcc1 and can mediate the monomerization of the dimeric subcomplex (Kae1-Pcc1-Pcc1-Kae1), suggesting that Pcc1-like is a functional homolog of the eukaryotic Gon7 subunit. Strikingly, none of the genes encoding aKEOPS subunits, including Pcc1 and Pcc1-like, can be deleted in the wild type and in a t6A modification complementary strain named TsaKI, implying that the aKEOPS complex is essential for an additional cellular process in this archaeon. Knock-down of the Cgi121 subunit leads to severe growth retardance in the wild type that is partially rescued in TsaKI. These results suggest that aKEOPS plays an essential role independent of the cellular t6A modification level. In addition, archaeal Cgi121 possesses dsDNA-binding activity that relies on its tRNA 3' CCA tail binding module. Our study clarifies the subunit organization of archaeal KEOPS and suggests an origin of eukaryotic Gon7. The study also reveals a possible link between the function in t6A modification and the additional function, presumably HR.

Dissection of Functional Domains of Orc1-2, the Archaeal Global DNA Damage-Responsive Regulator.

Orc1-2 is a non-initiator ortholog of archaeal/eukaryotic Orc1 proteins, which functions as a global regulator in DNA damage-responsive (DDR) expression. As for Orc1 initiators, the DDR regulator harbors an AAA+ ATPase domain, an Initiator-Specific Motif (ISM) and a winged-helix (wH) DNA-binding domain, which are also organized in a similar fashion. To investigate how Orc1-2 mediates the DDR regulation, the orc1-2 mutants inactivating each of these functional domains were constructed with Saccharolobus islandicus and genetically characterized. We found that disruption of each functional domain completely abolished the DDR regulation in these orc1-2 mutants. Strikingly, inactivation of ATP hydrolysis of Orc1-2 rendered an inviable mutant. However, the cell lethality can be suppressed by the deficiency of the DNA binding in the same protein, and it occurs independent of any DNA damage signal. Mutant Orc1-2 proteins were then obtained and investigated for DNA-binding in vitro. This revealed that both the AAA+ ATPase and the wH domains are involved in DNA-binding, where ISM and R381R383 in wH are responsible for specific DNA binding. We further show that Orc1-2 regulation occurs in two distinct steps: (a) eliciting cell division inhibition at a low Orc1-2 content, and this regulation is switched on by ATP binding and turned off by ATP hydrolysis; any failure in turning off the regulation leads to growth inhibition and cell death; (b) activation of the expression of DDR gene encoding DNA repair proteins at an elevated level of Orc1-2.

A Well-Conserved Archaeal B-Family Polymerase Functions as an Extender in Translesion Synthesis.

B-family DNA polymerases (PolBs) of different groups are widespread in Archaea, and different PolBs often coexist in the same organism. Many of these PolB enzymes remain to be investigated. One of the main groups that is poorly characterized is PolB2, whose members occur in many archaea but are predicted to be inactivated forms of DNA polymerase. Here, Sulfolobus islandicus DNA polymerase 2 (Dpo2), a PolB2 enzyme, was expressed in its native host and purified. Characterization of the purified enzyme revealed that the polymerase possesses a robust nucleotide incorporation activity but is devoid of the 3'-5' exonuclease activity. Enzyme kinetics analyses showed that Dpo2 replicates undamaged DNA templates with high fidelity, which is consistent with its inefficient nucleotide insertion activity opposite different DNA lesions. Strikingly, the polymerase is highly efficient in extending mismatches and mispaired primer termini once a nucleotide is placed opposite a damaged site. This extender polymerase represents a novel type of prokaryotic PolB specialized for DNA damage repair in Archaea. IMPORTANCE In this work, we report that Sulfolobus islandicus Dpo2, a B-family DNA polymerase once predicted to be an inactive form, is a bona fide DNA polymerase functioning in translesion synthesis. S. islandicus Dpo2 is a member of a large group of B-family DNA polymerases (PolB2) that are present in many archaea and some bacteria, and they carry variations in well-conserved amino acids in the functional domains responsible for polymerization and proofreading. However, we found that this prokaryotic B-family DNA polymerase not only replicates undamaged DNA with high fidelity but also extends mismatch and DNA lesion-containing substrates with high efficiencies. With these data, we propose this enzyme functions as an extender polymerase, the first prokaryotic enzyme of this type. Our data also suggest this PolB2 enzyme represents a functional counterpart of the eukaryotic DNA polymerase Pol zeta, an enzyme that is devoted to DNA damage repair.

TET3-mediated accumulation of DNA hydroxymethylation contributes to the activity-dependent gene expression of Rab3a in post-mitotic neurons.

Rab3a, a subtype protein in the Rab3 family amongst the small G proteins, is closely associated with the learning and memory formation process. Various neuronal stimuli can induce the expression of Rab3a; however, how DNA modification is involved in regulating its expression is not fully understood. Ten-eleven translocation (TET) proteins can oxidate methylcytosine to hydroxymethylcytosine, which can further activate gene expression. Previous studies reported that TET-mediated regulation of 5hmC induced by learning is involved in neuronal activation. However, whether Tet protein regulates Rab3a is unknown. To understand the role of TET-mediated 5hmC on Rab3a in neuronal activation, we adopted a KCl-induced depolarization protocol in cultured primary cortical neurons to mimic neuronal activity in vitro. After KCl treatment, Rab3a and Tet3 mRNA expression were induced. Moreover, we observed a decrease in the methylation level and an increase of hydroxymethylation level surrounding the CpG island near the transcription start site of Rab3a. Furthermore, recently, Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) has proven powerful in identifying open chromatin in the genome of various eukaryotes. Using FAIRE-qPCR, we observed a euchromatin state and the increased occupancy of Tet3, H3K4me3, and H3K27ac at the promoter region of Rab3a after KCl treatment. Finally, by using shRNA to knockdown Tet3 prior KCl treatment, all changes mentioned above vanished. Thus, our findings elucidated that the neuronal activity-induced accumulation of hydroxymethylation, which Tet3 mediates, can introduce an active and permissive chromatin structure at Rab3a promoter and lead to the induction of Rab3a mRNA expression.

Knockdown of long non-coding RNA AGAP2-AS1 suppresses the proliferation and metastasis of glioma by targeting microRNA-497-5p.

Long non-coding RNA (LncRNA) AGAP2-AS1 has been demonstrated to involve in various malignancies. However, the expression and biological effect of AGAP2-AS1 on glioma remain enigmatic. We aimed to explore the effects of AGAP2-AS1 on glioma. Expressions and relationship of AGAP2-AS1 and microRNA-497-5p (miR-497-5p) in different grades of glioma tissues and cell lines as well as normal ones were assessed by quantitative real-time polymerase chain reaction (qRT-PCR), starBase, and dual-luciferase reporter assay. In addition, the effect of AGAP2-AS1 on the cell biological behaviors, epithelial-mesenchymal transition (EMT)-related markers, and miR-497-5p level was detected by cell functional experiments, western blot, and qRT-PCR. After transfection with miR-497-5p mimic (M), inhibitor (I), and AGAP2-AS1 knockdown, miR-497-5p level, cell biological behaviors, and EMT-related markers were detected again. AGAP2-AS1 expression was increased while miR-497-5p expression was decreased in glioma tissues and cell lines, and increase of AGAP2-AS1 expression or reduction of miR-497-5p expression was also correlated with clinicopathological grades of glioma. Furthermore, AGAP2-AS1 knockdown repressed cell biological behaviors and EMT-related markers expressions. Mechanically, AGAP2-AS1 targeted miR-497-5p and AGAP2-AS1 knockdown led to elevation of miR-497-5p expression. In addition, rescue experiments were conducted to validate the vital influence of miR-497-5p on the AGAP2-AS1-regulated proliferation and metastasis of glioma. AGAP2-AS1 may serve as an oncogene in the tumorigenesis of glioma by inhibiting miR-497-5p expression, showing its potential as a prognostic biomarker and a therapeutic target for glioma.

A type III-A CRISPR-Cas system mediates co-transcriptional DNA cleavage at the transcriptional bubbles in close proximity to active effectors.

Many type III CRISPR-Cas systems rely on the cyclic oligoadenylate (cOA) signaling pathway to exert immunization. However, LdCsm, a type III-A lactobacilli immune system mediates efficient plasmid clearance in spite of lacking cOA signaling. Thus, the system provides a good model for detailed characterization of the RNA-activated DNase in vitro and in vivo. We found ATP functions as a ligand to enhance the LdCsm ssDNase, and the ATP enhancement is essential for in vivo plasmid clearance. In vitro assays demonstrated LdCsm cleaved transcriptional bubbles at any positions in non-template strand, suggesting that DNA cleavage may occur for transcribing DNA. Destiny of target plasmid versus nontarget plasmid in Escherichia coli cells was investigated, and this revealed that the LdCsm effectors mediated co-transcriptional DNA cleavage to both target and nontarget plasmids, suggesting LdCsm effectors can mediate DNA cleavage to any transcriptional bubbles in close proximity upon activation. Subcellular locations of active LdCsm effectors were then manipulated by differential expression of LdCsm and CTR, and the data supported the hypothesis. Strikingly, stepwise induction experiments indicated allowing diffusion of LdCsm effector led to massive chromosomal DNA degradation, suggesting this unique IIIA system can facilitate infection abortion to eliminate virus-infected cells.

Polyene-Producing Streptomyces spp. From the Fungus-Growing Termite Macrotermes barneyi Exhibit High Inhibitory Activity Against the Antagonistic Fungus Xylaria.

Fungus-growing termites are engaged in a tripartite mutualism with intestinal microbes and a monocultivar (Termitomyces sp.) in the fungus garden. The termites are often plagued by entomopathogen (Metarhizium anisopliae) and fungus garden is always threatened by competitors (Xylaria spp.). Here, we aim to understand the defensive role of intestinal microbes, the actinomycetes which were isolated from the gut of Macrotermes barneyi. We obtained 44 antifungal isolates, which showed moderate to strong inhibition to Xylaria sp. HPLC analysis indicated that different types of polyenes (tetraene, pentene, and heptaene) existed in the metabolites of 10 strong antifungal Streptomyces strains. Two pentene macrolides (pentamycin and 1'14-dihydroxyisochainin) were firstly purified from Streptomyces strain HF10, both exhibiting higher activity against Xylaria sp. and M. anisopliae than cultivar Termitomyces. Subsequently, tetraene and heptaene related gene disruption assay showed that the mutant strains lost the ability to produce corresponding polyenes, and they also had significantly decreased activities against Xylaria sp. and M. anisopliae compared to that of wild type strains. These results indicate that polyene-producing Streptomyces from the guts of M. barneyi have strong inhibition to competitor fungus and polyenes contribute to inhibitory effects on Xylaria sp.

Mobility-Aware Privacy-Preserving Mobile Crowdsourcing.

The increasing popularity of smartphones and location-based service (LBS) has brought us a new experience of mobile crowdsourcing marked by the characteristics of network-interconnection and information-sharing. However, these mobile crowdsourcing applications suffer from various inferential attacks based on mobile behavioral factors, such as location semantic, spatiotemporal correlation, etc. Unfortunately, most of the existing techniques protect the participant's location-privacy according to actual trajectories. Once the protection fails, data leakage will directly threaten the participant's location-related private information. It open the issue of participating in mobile crowdsourcing service without actual locations. In this paper, we propose a mobility-aware trajectory-prediction solution, TMarkov, for achieving privacy-preserving mobile crowdsourcing. Specifically, we introduce a time-partitioning concept into the Markov model to overcome its traditional limitations. A new transfer model is constructed to record the mobile user's time-varying behavioral patterns. Then, an unbiased estimation is conducted according to Gibbs Sampling method, because of the data incompleteness. Finally, we have the TMarkov model which characterizes the participant's dynamic mobile behaviors. With TMarkov in place, a mobility-aware spatiotemporal trajectory is predicted for the mobile user to participate in the crowdsourcing application. Extensive experiments with real-world dataset demonstrate that TMarkov well balances the trade-off between privacy preservation and data usability.

Archaeal extracellular vesicles are produced in an ESCRT-dependent manner and promote gene transfer and nutrient cycling in extreme environments.

Membrane-bound extracellular vesicles (EVs), secreted by cells from all three domains of life, transport various molecules and act as agents of intercellular communication in diverse environments. Here we demonstrate that EVs produced by a hyperthermophilic and acidophilic archaeon Sulfolobus islandicus carry not only a diverse proteome, enriched in membrane proteins, but also chromosomal and plasmid DNA, and can transfer this DNA to recipient cells. Furthermore, we show that EVs can support the heterotrophic growth of Sulfolobus in minimal medium, implicating EVs in carbon and nitrogen fluxes in extreme environments. Finally, our results indicate that, similar to eukaryotes, production of EVs in S. islandicus depends on the archaeal ESCRT machinery. We find that all components of the ESCRT apparatus are encapsidated into EVs. Using synchronized S. islandicus cultures, we show that EV production is linked to cell division and appears to be triggered by increased expression of ESCRT proteins during this cell cycle phase. Using a CRISPR-based knockdown system, we show that archaeal ESCRT-III and AAA+ ATPase Vps4 are required for EV production, whereas archaea-specific component CdvA appears to be dispensable. In particular, the active EV production appears to coincide with the expression patterns of ESCRT-III-1 and ESCRT-III-2, rather than ESCRT-III, suggesting a prime role of these proteins in EV budding. Collectively, our results suggest that ESCRT-mediated EV biogenesis has deep evolutionary roots, likely predating the divergence of eukaryotes and archaea, and that EVs play an important role in horizontal gene transfer and nutrient cycling in extreme environments.

Virus-induced cell gigantism and asymmetric cell division in archaea.

Archaeal viruses represent one of the most mysterious parts of the global virosphere, with many virus groups sharing no evolutionary relationship to viruses of bacteria or eukaryotes. How these viruses interact with their hosts remains largely unexplored. Here we show that nonlytic lemon-shaped virus STSV2 interferes with the cell cycle control of its host, hyperthermophilic and acidophilic archaeon Sulfolobus islandicus, arresting the cell cycle in the S phase. STSV2 infection leads to transcriptional repression of the cell division machinery, which is homologous to the eukaryotic endosomal sorting complexes required for transport (ESCRT) system. The infected cells grow up to 20-fold larger in size, have 8,000-fold larger volume compared to noninfected cells, and accumulate massive amounts of viral and cellular DNA. Whereas noninfected Sulfolobus cells divide symmetrically by binary fission, the STSV2-infected cells undergo asymmetric division, whereby giant cells release normal-sized cells by budding, resembling the division of budding yeast. Reinfection of the normal-sized cells produces a new generation of giant cells. If the CRISPR-Cas system is present, the giant cells acquire virus-derived spacers and terminate the virus spread, whereas in its absence, the cycle continues, suggesting that CRISPR-Cas is the primary defense system in Sulfolobus against STSV2. Collectively, our results show how an archaeal virus manipulates the cell cycle, transforming the cell into a giant virion-producing factory.

An alkaline thermostable laccase from termite gut associated strain of Bacillus stratosphericus.

Laccase, an important oxidoreductase, is widely distributed in various organisms. Termites are known to decompose lignocellulose efficiently with the aid of gut microorganisms. However, few laccases have been characterized from termite or its gut microbes. We aimed to screen the strain capable of degrading lignocellulose from fungus-growing termites. In this study, Bacillus stratosphericus BCMC2 with lignocellulolytic activity was firstly isolated from the hindgut of fungus-growing termite Macrotermes barneyi. The laccase gene (BaCotA) was cloned both from the BCMC2 strain and termite intestinal metagenomic DNA. BaCotA was overexpressed in E. coli, and the recombinant BaCotA showed high specific activity (554.1 U/mg). BaCotA was thermostable with an optimum temperature of 70 °C, pH 5.0. Furthermore, BaCotA was resistant to alkali and organic solvents. The enzyme remained more than 70% residual activity at pH 8.0 for 120 min; and the organic solvents such as methanol, ethanol and acetone (10%) had no inhibitory effect on laccase activity. Additionally, BaCotA exhibited efficient decolorization ability towards indigo and crystal violet. The multiple enzymatic properties suggested the presented laccase as a potential candidate for industrial applications. Moreover, this study highlighted that termite intestine is a good resource for either new strains or enzymes.

First record of gregarine protists (Apicomplexa: Sporozoa) in Asian fungus-growing termite Macrotermes barneyi (Blattaria: Termitidae).

Macrotermes barneyi, widely distributed in southern China, is the major fungus-growing termite in the subfamily Macrotermitinae. It has no flagellated protists in the guts. Here, we report occurrence of gregarine, a protozoan parasite in the digestive tract of M. barneyi. The general morphology and ultrastructure of the gregarine gamonts and syzygies by light micrograph and scanning electron micrograph are presented. SSU rDNA sequence analysis showed that the termite gregarine has the highest identity (90.10%) to that of Gregarina blattarum from cockroaches. Phylogenetic analysis based on the SSU rDNA sequences from diverse insect eugregarines indicated that the gregarine from M. barneyi is phylogenetically close to G. blattarus, L. erratica and G. tropica from Gregarinidae and Leidyanidae families, and may represent a novel species. This study expands our knowledge about the diversity of terrestrial eugregarines parasitizing in termites.