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Protease-associated cellular networks in malaria parasite Plasmodium falciparum
Timothy G Lilburn1,
Hong Cai2,
Zhan Zhou2,3 &
Yufeng Wang2,4
Malaria continues to be one of the most severe global infectious diseases, responsible for 1-2 million deaths yearly. The rapid evolution and spread of drug resistance in parasites has led to an urgent need for the development of novel antimalarial targets. Proteases are a group of enzymes that play essential roles in parasite growth and invasion. The possibility of designing specific inhibitors for proteases makes them promising drug targets. Previously, combining a comparative genomics approach and a machine learning approach, we identified the complement of proteases (degradome) in the malaria parasite Plasmodium falciparum and its sibling species [1–3], providing a catalog of targets for functional characterization and rational inhibitor design. Network analysis represents another route to revealing the role of proteins in the biology of parasites and we use this approach here to expand our understanding of the systems involving the proteases of P. falciparum.
We investigated the roles of proteases in the parasite life cycle by constructing a network using protein-protein association data from the STRING database [4], and analyzing these data, in conjunction with the data from protein-protein interaction assays using the yeast 2-hybrid (Y2H) system [5], blood stage microarray experiments [6–8], proteomics [9–12], literature text mining, and sequence homology analysis. Seventy-seven (77) out of 124 predicted proteases were associated with at least one other protein, constituting 2,431 protein-protein interactions (PPIs). These proteases appear to play diverse roles in metabolism, cell cycle regulation, invasion and infection. Their degrees of connectivity (i.e., connections to other proteins), range from one to 143. The largest protease-associated sub-network is the ubiquitin-proteasome system which is crucial for protein recycling and stress response. Proteases are also implicated in heat shock response, signal peptide processing, cell cycle progression, transcriptional regulation, and signal transduction networks.
Our network analysis of proteases from P. falciparum uses a so-called guilt-by-association approach to extract sets of proteins from the proteome that are candidates for further study. Novel protease targets and previously unrecognized members of the protease-associated sub-systems provide new insights into the mechanisms underlying parasitism, pathogenesis and virulence.
Malaria remains a major threat to health and economic development in endemic countries, infecting 300-500 million people yearly and claiming 1-2 million deaths, primarily of young children. Symptoms of malaria include high fever, shaking chills, headache, vomiting, and anemia. If left untreated, malaria can quickly become life threatening by disrupting the blood supply to vital organs. Malaria is caused by a group of parasites from the genus Plasmodium. Five species, P. falciparum, P. vivax, P. malariae, P. ovale, and P. knowlesi, are known to cause the disease in humans. P. falciparum is the most devastating and widespread species.
No effective anti-malaria vaccines are available for use in humans [13]. For decades, the management of malaria has relied heavily on chemotherapy, which uses a limited number of drugs. However, the rapid evolution and spread of drug resistance in parasites has led to an increase in morbidity and mortality rates in malaria endemic regions. The development of new drug/vaccine targets is urgently needed.
Thanks to the completion of the genome sequencing projects for P. falciprum and its sibling species [14–19], a novel array of proteins have been proposed as potential drug targets, including (1) proteins like 1-deoxy-D-xylulose 5-phosphate (DOXP) reductoisomerase [20, 21], and apicoplast gyrase [22] that are located in the apicoplast, an organelle with its origin close to the chloroplast; (2) kinases such as cyclin-dependent protein kinases (Pfmrk) [23] and the plant-like calcium-dependent protein kinase (PfCDPK5) [24]; (3) transporters involved in drug resistance and nutrient acquisition from the host [25–30], and (4) proteases.
Proteases are a group of enzymes that degrade proteins by breaking peptide bonds. They are attractive antimalarial targets due to their indispensible roles in parasite development and invasion [31, 32]. Previously we predicted the protease complement (degradome) in the malaria parasite P. falciparum and its four sibling species using a comparative genomics approach and a support vector machine (SVM)-based, supervised machine learning approach [1–3]. This catalog revealed a new line of novel proteases for functional characterization. Studies on malarial proteases have been focused on biochemical and molecular characterization [33–46], structural modeling and analysis [47, 48], and inhibitor design and screening [49–59]. Although significant progress has been made, much remains to be learned about the roles played by these proteins, including how they interact with other proteins in space and time to coordinate important aspects of growth, transmission, invasion, response to drug treatment and pathogenesis of this devastating pathogen.
One approach to gaining wider views on the roles of proteins in biological systems relies on network biology. Known and inferred protein associations are used to build a network of proteins, thus establishing a map of all the associations in the organism and allowing deductions to be made as to the role of proteins that are poorly understood and poorly annotated. Clearly, both proposed and demonstrated protein-protein associations could aid us in understanding the role of a protease in the parasite. Therefore, we constructed a network of P. falciparum proteins using the protein-protein association data from STRING database [4], and analyzed these data, in conjunction with the data from protein-protein interaction assays using the yeast 2-hybrid (Y2H) system [5], blood stage microarray experiments [6–8], proteomics [9–12], literature text mining, and sequence homology analysis. The topology of the protein-protein association network was analyzed and the results examined for information as to how the proteases may function within the parasite. Sets of proteins associated with specific proteases or protease families were extracted from the whole-cell network to create protease-associated subnetworks and five of these subnetworks were examined in detail. Novel protease targets and previously unrecognized members of some sub-systems could be postulated; these insights help us to better understand the mechanisms underlying parasite metabolism, cell cycle regulation, invasion and infection.
Proteases are involved in complex networks
We downloaded and mined the protein-protein association data from the STRING database [4] involving proteins from P. falciparum. Seventy-seven (77) out of 124 predicted proteases were found in this set and were associated with at least one other protein, constituting 2,431 associations (Additional Files 1 and 2). Each association between a pair of proteins has a confidence score (S) ranging from 0.15 to 0.999 that was inferred from the evidence used to establish the association: 221 associations (9.1%) have high confidence scores (S>0.7), 432 associations (17.8%) have medium confidence scores (0.4≤S≤0.7), and strikingly, 1,778 associations (73.1%) have relative low confidence scores (0.15≤S<0.4). The large proportion of low-scored associations arises from the paucity of annotation data. Before the genome of P. falciparum was sequenced, only about 20 proteins had been characterized; after genome sequencing this number increased by two orders of magnitude, but over 60% of the predicted gene products in the genome still had no functional assignment [18] and ten years of subsequent effort have reduced this number to roughly 45% [60]. Consequently, information such as KEGG pathway assignments, PDB protein structures and reactome data, which tend to improve association scores, is scarce for P. falciparum. Therefore, our subsequent analysis will not exclude the associations with low confidence scores as they may well represent associations that have not been previously recognized.
The degrees of connectivity vary among the 77 predicted proteases with protein-protein associations, ranging from one to 143 (Additional File 1). Twenty-four (24) putative proteases have less than five association partners, 13 have 5-10 partners, and 40 are highly connected with more than 11 partners, suggesting that proteases are involved in complex cellular networks. Functional enrichment analysis [61] revealed that 120 Gene Ontology (GO) terms were over-represented in these protease associations (p < 0.05) (Additional File 3). Figure 1 shows the distribution of functional categories in a hierarchical order: proteolysis (GO 6508) is, not surprisingly, enriched (p = 8.29*10-6), while the other most highly represented GO biological processes (p < 10-5) are related to cellular catabolic processes (GO 44248), protein metabolic processes (GO 19538), macromolecule metabolic processes (GO 43170), and cofactor and coenzyme metabolic processes (GO 51186 and 6732). This result reflects the involvement of proteases in fundamental biological processes, many of which have been established in the wet lab. Other processes that are moderately enriched (10-5<p < 0.05) included gene expression and response to endogenous and external stimuli such as heat, abiotic stimulus, organic substances, unfolded proteins, and protein stimuli. Five of the potentially most significant protease-associated sets of proteins are discussed in the following sections. They include the ubiquitin-proteasome system, the stress response system, the regulated intramembrane proteolysis system, the parasite egress network, and the signal peptidase network. These subnetworks were chosen because: (1) proteases are the central players in these networks; (2) These networks play crucial roles in parasite life cycle and are closely associated with adaptive phenotypes such as stress response, transcriptional regulation, pathogenesis, and virulence; (3) These networks are considered to be potential antimalarial targets as their disruption would cause deleterious effects on the growth or infectivity of the parasites.
A graphical representation of the results of a Gene Ontology analysis done using BiNGO. The node size is proportional to the number of proteins represented by that GO term. The color represents the P-value for each enriched GO term as shown in the scale; white nodes are not enriched. The nodes are positioned to approximate their level in the Gene Ontology.
The ubiquitin-proteasome system (UPS)
The largest protease-associated network in P. falciparum is the ubiquitin-proteasome protein degradation system (UPS). The UPS is responsible for degrading unwanted or misfolded proteins and is believed to execute important roles in protein turnover and cell cycle regulation in a wide variety of organisms [62]. We previously identified a group of threonine proteases that form α- and β- subunits of the proteasome complex and two families of ubiquitin-specific hydrolases (C12 and C19) [1, 63] (Additional File 1). The UPS pathway in P. falciparum has been deduced by Dr. Hagai Ginsburg (http://sites.huji.ac.il/malaria/maps/proteaUbiqpath.html), and involves two consecutive steps: (1) tagging the ubiquitin molecules to target proteins and (2) degradation of the tagged protein by the proteasome complex with release and recycling of ubiquitin. The major components of the UPS in P. falciparum are conserved with other eukaryotes. However, a growing body of evidence suggests that the UPS plays a critical role in the parasite-specific life style and it is therefore intriguing to unveil the proteins and pathways that are associated with or regulated by the UPS [64, 65], as they may carry out functions specific to pathogenesis or virulence. We identified 1,148 associations in P. falciparum that involved 11 threonine proteases in the T1 family, two proteases in the C12 ubiquitin C-terminal hydrolase family, and six proteases in the C19 ubiquitin-specific protease family. One hundred and twenty-four (124) associations are protease-protease associations, and the remaining 1,024 associations involve non-protease partners. One hundred and sixty-four (164) of these associations have high confidence scores (S>0.7), the majority of which involve the association between catalytic components and regulatory components in the proteasome complex (Additional File 4).
The protease with the highest connectivity is PF10_0111, a putative 20S proteasome beta subunit, which has 143 association partners (Figure 2). In addition to the proteasome components and ubiquitin conjugation enzymes, the other interacting proteins appear to be involved in a variety of activities (Table 1): (1) a nucleotide binding activity involving a Tat binding protein homolog (PFL2345c) which has an AAA ATPase domain; (2) cell cycle regulation involving MAL13P1.337, which is a putative protein in the Skp1 (S-phase kinase-associated protein 1) family. The Skp1 protein is an important component of the cyclin A-CDK2 S phase kinase complex in baker's yeast (Saccharomyces cerevisiae) [66] and directs cell cycle-regulated proteins to the kinetochore; (3) translation involving a number of ribosomal proteins such as 60S ribosomal proteins L40/UBI (PF13_0346) and L10 (PF14_0141), a putative translation initiation factor eIF-1A (PF11_0447), and a putative elongation factor 1 (EF-1) (PFC0870w); (4) transcriptional regulation involving a putative multiprotein bridging factor type 1 (MBF1) (PF11_0293). MBF1 is a transcriptional cofactor that bridges the TATA box-binding protein (TBP) and its specific regulatory proteins for transcriptional activation [67]; (5) membrane traffic regulation involving a putative rab specific GDP dissociation inhibitor (PFL2060c) [68].
A graph showing the proteins associated with PF10_0111. This protease is the most highly connected member of the set of proteases found in the P. falciparum ubiquitin-proteasome protein degradation system. Square nodes represent proteases. Node size is proportional to the degree of the node. Nodes are colored according to their functional classification in the eggNOG database [122]. The COG categories are [123] (J) Translation, ribosomal structure and biogenesis, (A) RNA processing and modification, (K) Transcription, (L) Replication, recombination and repair, (B) Chromatin structure and dynamics, (D) Cell cycle control, cell division, chromosome partitioning, (Y) Nuclear structure, (V) Defense mechanisms, (T) Signal transduction mechanisms, (M) Cell wall/membrane/envelope biogenesis, (N) Cell motility, (Z) Cytoskeleton, (W) Extracellular structures, (U) Intracellular trafficking, secretion, and vesicular transport, (O) Posttranslational modification, protein turnover, chaperones, (C) Energy production and conversion, (G) Carbohydrate transport and metabolism, (E) Amino acid transport and metabolism, (F) Nucleotide transport and metabolism, (H) Coenzyme transport and metabolism, (I) Lipid transport and metabolism, (P) Inorganic ion transport and metabolism, (Q) Secondary metabolites biosynthesis, transport and catabolism, (R) General function prediction only, and (S) Function unknown. Confidence scores for the interactions among the nodes (S values from STRING) were divided into three groups - low (0.150-0.399), medium (0.400-0.700) and high (0.701-0.999); the groups are represented by thin, medium and heavy lines, respectively.
Table 1 Representative P. falciparum proteins that are associated with PF10_0111, a putative 20S proteasome beta subunit with the highest connectivity. Protein-protein interactions revealed by yeast 2-hybrid assays are italicized.
Moreover, the yeast 2-hybrid assay using PF10_0111 as a bait revealed 15 PPI preys (Table 1), confirming that it is associated with (1) transcriptional regulation involving a CCAAT-box DNA binding protein subunit B (PF11_0477) containing a histone-like transcription factor domain, and (2) translation involving a putative translation elongation factor EF-1 subunit alpha (PF11_0245), a putative 60S ribosomal protein L4 (PFE0350c), and a putative ribosomal protein L15 precursor predicted to localize to the apicoplast (PF14_0270), a specific organelle of prokaryotic origin found in Apicomplexa parasites. PF10_0111 may also be associated with protein modifications involving a putative ubiquitin transferase (MAL7P1.19) [69] and chromatin fluidity involving a putative nucleosome assembly protein (PFI0930c).
Interestingly, PF10_0111 is shown to have PPI with three predicted surface antigens: (1) merozoite surface protein 3 (PF10_0345), which was shown by global RNA decay and nuclear run-on assays to serve a role in transcriptional regulation and RNA stabilization [70, 71]; (2) a merozoite surface protein (PF10_0348). Domain analysis revealed a N-terminus Duffy binding domain that is present in the Duffy receptors expressing blood group surface determinants and a C-terminus SPAM (secreted polymorphic antigen associated with merozoites) domain, both of which have been implicated in parasite immune evasion, cytoadherence and pathogenesis [72, 73]; (3) a parasite-infected erythrocyte surface protein (PFE0060w). The microarray and proteomics assays show that these three surface proteins are expressed at the invasive merozoite stage [6, 8, 10, 11].
These results reflect much that is known about the UPS, but also suggest that it may also be associated with a variety of processes ranging from transcriptional regulation, translation, cell cycle progression, invasion, protein trafficking, and immune evasion. Not surprisingly, the UPS has become a promising antimalarial target. Various independent studies have shown that inhibition of proteasome activity can arrest parasite growth, and yet show limited toxicity to human cell lines [64, 74, 75].
Stress response network
The adaptation of the malaria parasite to the host environment requires a rapid and effective response to diverse physiological signals and stress conditions, such as changes in temperature within hosts, nutritional challenges, host immune responses, antimalarial administration, and so on. One such adaptive network in the malaria parasite is the robust heat shock response system. During its life cycle, the parasite is transmitted from the mosquito vector (~25°C) to the human host (37°C), resulting in heat shock. Periodic fever, the patient's response to infection, also presents recurrent heat shock to the parasite. A comprehensive chaperone system has been identified in P. falciparum genome, accounting for 2% of the open reading frames (ORFs) [76]. The system is comprised of various chaperone proteins [77] and includes proteases that degrade misfolded proteins. We identified 344 associations involving five putative proteases in the ClpP endopeptidase family (S14), a lon protease PF14_0147 (S16), and an hslV protease PFL1465c (T1B). As shown in Figure 3, these proteases are associated with a large number of heat shock proteins (HSPs) including Hsp90, Hsp70, Hsp40, and DnaJ proteins. The protease having the highest degree of connectivity (80) in the heat shock response network is PFL1465c, a threonine protease hslV. In addition to the classical HSPs, it is associated with a wide variety of enzymes such as ubiE/COQ5 methyltransferase, rRNA methyltransferase, multiple tRNA synthetases, various phosphate isomerases, amino transferase, aldolase, and a number of kinases, suggesting it may have an important role in parasite metabolism. It is also associated with three other heat-shock response proteases (PFC0310c and PF08_0063 in the S14 ClpP endopeptidase family, and PF14_0147 in the S16 ATP-dependent protease lon family), an organelle processing peptidase in the M16 pitrilysin family (PFI1625c), a leucyl aminopeptidase in the M17 family (PF14_0439), and aminopeptidase P in the M24 family (PF14_0517); together, they form a complex protease degradation network. It is also interesting to note that this putative hslV protease appears to be linked to a second very important stress system in the malaria parasite that acts against oxidative challenges: Protease hslV is associated with the thioredoxin 1 protein (PF14_0545), a member of the thioredoxin system which controls cell redox homeostasis, and a putative Fe-superoxide dismutase (Fe-SOD, PF08_0071) which is critical for antioxidant defense. Because the malaria parasite is sensitive to oxidative stress, both the thioredoxin system and SOD have been considered as potential antimalaria targets [78]. Finally, our phylogenetic analysis revealed that this hslV protease (PFL1465) is of prokaryotic origin and there is no homolog in the human host, a desirable feature for drug targets [79–81]. A second heat shock response protease PfClpP (PFC0310c) was recently characterized [82, 83]; protease inhibition assays have shown that it, along with other ATP-dependent chaperones, plays a crucial role in parasite growth and development. Furthermore, PfClpP is localized to the apicoplast, which is of cyanobacterial origin, making this protein an apicoplast-targeting antimalarial candidate. This protein is indeed highly connected with 69 association partners, including Hsp70, Hsp60, Hsp40, co-chaperones, and proteins involved in proteasome acitivities, replication, translation, protein biosynthesis, metabolism, and heat shock response, implying that its inactivation would have devastating consequences for the parasite.
The graph shows a subnetwork of proteins linked to stress responses in P. falciparum. It was detected using the MINE plug in for Cytoscape, which uses an agglomerative approach to search the topology of large networks for significant clusters. The visualization is as for Figure 2.
Signal transduction via the regulated intramembrane proteolysis (RIP) network
The common belief that proteases cleave peptide bonds in a water environment was challenged by the discovery of a set of proteases that conduct hydrolysis in the hydrophobic environment of cellular membranes [84]. During RIP, intramembrane proteases cleave transmembrane-spanning helical (TMH) segments of the substrates and release soluble effectors, many of which are signaling molecules, thereby triggering cascades of signal transduction pathways [85, 86]. RIP is now believed to be a ubiquitous signaling mechanism in a wide variety of organisms from bacteria to humans [87]. The roles of RIP in the parasite life cycle have begun to be unraveled. Three families of membrane-tethered proteases involved in RIP have been identified in P. falciparum, including an aspartic signal peptide peptidase (PfAPP, PF14_0543) in the A22 presenilin family, eight rhomboid serine proteases (PfROMs) in the S54 family, and two putative Site-2 metallo proteases (S2Ps, PF13_0028 and PF10_0317) in the M50 family[1, 88–93].
The first family, PfAPP (PF14_0543), has 54 association partners (Figure 4 andTable 2). The association partner with the highest confidence score is a putative Rer1 (retrieval receptor for endoplasmic reticulum (ER) membrane proteins, PFI0150c) that is important for localizing proteins to the ER. Another related partner for PfAPP is a putative ER lumen protein retaining receptor (PF13_0280), which contains a signal sequence that facilitates the protein transport between the cis side of the Golgi apparatus and the ER [94]. It is believed that parasite invasion of erythrocytes requires the export of proteins to the ER and the cell surface. They then traverse the parasitophorous vacuole membrane (PVM) into the erythrocyte or parasite-derived membranous structures known as Maurer's clefts. PfAPP, along with the ER-localization proteins, may play a role in protein trafficking, cell-cell communication and remodeling of the host erythrocyte for parasite entry. Other proteins that are associated with PfAPP include secretory proteins, translation initiation and elongation factors, splicing factors and the spliceosome unit, peptide chain release factor, and various enzymes, suggesting it is involved in diverse networks related to transport, translation, posttranslational processing and metabolism. Recent gene disruption assays showed that PfAPP is essential for merozoite invasion and parasite growth [92, 93]; the versatile associations of this protease underscore its potential as a drug target.
The set of proteins associated with proteases that carry out regulated intramembrane proteolysis (RIP). This novel form of proteolysis is linked to signaling and the associated proteins may be targets for, or mediators of, this system. The red edges indicate experimentally validated interactions; other details of the visualization are as for Figure 2.
Table 2 Representative P. falciparum proteins that are associated with the regulated intramembrane proteolysis (RIP) network.
The second family, PfROM, includes a group of serine proteins with demonstrated roles in parasite invasion [90, 91, 95, 96]. Only one out of the ten rhomboid protease homologs in P. falciparum, PfRom1 (PF11_0150), was predicted to have protein-protein associations. Most interestingly, all the six proteins associated with it are antigens that have been considered as vaccine candidates; they belong to three families of adhesins that are essential for parasite invasion (Figure 4 andTable 2): (1) the apical membrane antigen 1 (AMA1, PF11_0344) is an adhesin required for merozoite invasion and it plays an indispensible role in the proliferation and survival of the malaria parasite [97]. PfRom1 was shown to be able to cleave AMA1 [88]; (2) the erythrocyte binding-like (EBL) family is involved in binding to a host chemokine receptor, the Duffy antigen [98]. Among the four EBAs with predicted association with PfRom1, EBA-175(MAL7P1.176) is proven a natural substrate for PfRom1 [88], but it remains unclear whether PfRom1 can cleave EBA-140 (MAL13P1.60), EBA-181 (MAL1P1.16), and a putative merozoite adhesive erythrocytic binding protein (PF11_0486); (3) a reticulocyte binding protein 2 homolog b protein (MAL13P1.176) in the reticulocyte binding-like (RBL) family. PfRom1 is able to cleave the RBL proteins [88]. Apparently, PfRom1 plays a central role in the RIP network that is tightly linked to the invasion process [86] and as such merits further investigation as a drug target.
S2Ps in the third family, PF10_0317 and PF13_0028, have two and one associations, respectively (Figure 4 andTable 2). PF10_0317 is associated with a proteasome 26S regulatory subunit and a cell division cycle (CDC) protein 48 homolog, which is implicated by GO analysis in ER localization and cell cycle regulation. Our previous domain analysis showed that PF10_0317 contains a Der-1 like domain, which was implicated in proteolysis associated with the ER [99–102]. PF13_0028 is associated with an adenylosuccinate synthetase AdsS (PF13_0287), which is important for the de novo biosynthesis of purine nucleotides. This association was predicted based on the genome synteny analysis, which revealed that the homologs of S2P and AdsS are located in the same chromosomal neighborhood in a variety of Actinobacteria. The functions of these S2Ps in malaria parasites are yet to be defined.
Parasite egress mediated by proteolysis
Egress, the parasite's emergence from host erythrocytes, is a well-coordinated process involving the rupture of the parasitophorous membrane (PVM) and the erythrocyte membrane (EM). Proteases that have been implicated in parasite egress [31, 36] include (1) aspartic proteases (plasmepsins PMI, PMII, and PMIII, also known as histo-aspartic protease (HAP)) in the A1 family, (2) cystein proteases in the A1 papain family including falcipain 2a, 2b, and 3, dipeptidyl peptidase 3 (PfDPAP3), and a series of Serine Repeat Antigens (SERAs), and (3) a serine protease subtilase 1 (PfSUB1) in the subtilisin S8 family. We analyzed the protein-association network (Figure 5) involving proteases mediating egress and found that a central player in the network is SERA5 (PFB0340c), which has 28 associations. SERA5 is associated with PfSUB1 (PFE0370c) and PfDPAP3 (PFD0230c). Both these proteases can proteolytically activate SERA5, which triggers downstream processing of cellular substrates [103, 104]. SERA5 is also associated with several erythrocyte membrane antigens such as PfEMP2 and EBA-175. It is abundantly expressed in the blood stage, especially in the schizont stage, as revealed by microarray and proteomic analysis. SERA5 has an in vitro catalytic activity and it is refractory to gene disruption [105], suggesting its vital role in the parasite life cycle.
The protein associations of proteases involved in egress (exit from the erythrocyte). SERA5 (PFB0340c) is the most highly connected protease and appears to be a key player. It is linked with two proteases known to activate it, as well as potential substrates (see text). The red edges indicate experimentally validated interactions; other details of the visualization are as for Figure 2.
Signal peptidase network
As an adaptive survival strategy, the malaria parasite harbors a powerful secretion system that transports parasite-encoded virulence proteins to their subcellular locations. The central players in this secretion system are a group of signal peptidases that are capable of cleaving signal sequences from the target proteins that can then be routed to their destinations. Five signal peptidases have been predicted and characterized, constituting the signal peptidase complex (SPC) in P. falciparum [3, 106, 107]. Three of these peptidases have association partners: PfSPC21 (MAL13P1.167) has 120 associations; the putative microsomal signal peptidase (PF14_0317) has five associations; and the putative SPC22 (PFI0215c) has five associations (Figure 6). The associated proteins are part of the secretion pathway and include secretory complex protein 61 (Pf61) alpha and gamma subunits, a signal recognition particle (SRP) and an SRP receptor, an ER lumen protein retaining receptor, and a transport protein particle (TRAPP) component. These signal peptidases are also associated with members of the ubiquitin-proteasome system and the heat shock response system, with the translational machinery, and with metabolic networks.
Proteins associated with three signal peptidases. As these proteases process signaling sequences on proteins, it is not surprising that they have a wide array of associations. PfSPC21 (MAL13P1.167) is associated with a large number of proteins from COG category O (Posttranslational modification, protein turnover, chaperones). The visualization is as for Figure 2.
Other potentially important protease-associated networks
Proteases in P. falciparum may play other roles important for parasite biology. We previously identified a single copy of calpain PfCalp (MAL13P1.310) in P. falciparum genome [3, 106, 107]. Calpain is crucial for signal transduction, cell cycle regulation, differentiation, development, and cell-cell communication from bacteria to humans. Very little is known about its role in P. falciparum. Only four proteins seemed to be associated with calpain: including a putative protein with a C3HC4 type zinc finger, the motif commonly present in transcriptional regulators, a ribosomal protein, and two proteins with unknown function. However, partial knockdown assays recently suggested that PfCalp is essential for the parasite's optimal growth and cell cycle progression [108]. Phylogenetic analysis revealed that PfCalp is a unique type of calpain confined to alveolates (a group of protists) with distant relatedness to human calpains [63, 108], adding it to a new line of promising drug target. Another class of proteases that mediate cell cycle regulation and programmed cell death is comprised of the three metacaspases from the C14 protease family [63, 109]. Only one association partner was identified for PF13_0289 and PF14_0363, (polyubiquitin and a hypothetical protein with unknown function respectively), and no associations were found for PF14_0160, reflecting our limited knowledge about their functions in malaria parasite.
Our network analysis of proteases from P. falciparum uses a so-called guilt-by-association approach to extract sets of proteins from the proteome that are candidates for further study. The network biology approach is readily adapted to any system for which a genome sequence exists and for which some type of protein-protein association is available, although there are limitations. Some of these stem from missing data, and/or noisy data, which lead to underestimation of the S value for a pair of associated proteins, but this problem becomes less significant with each release of data. A second problem is the lack of any dynamic element in evaluating the associations. A more formal integration of expression data could help to ameliorate this situation, especially expression data sets gathered under different conditions. Despite these limitations, our results produced known associations, which serve as positive controls such as the ubiquitin-proteasome system (UPS). It also indicated that proteases are playing previously unrecognized role in the biology of the parasite, such as the proteases that mediate the stress responses. Our results also imply that certain of these proteases, such as the proteases that mediate regulated intramembrane proteolysis, parasite egress, and signal peptide processing and protein secretion, may be good candidates for antimalarial targeting, as they are highly connected in the network. Furthermore, some of these candidates are known to have no or only distantly related homologs in humans, which reduces the probability of adverse effects resulting from their inactivation. Finally, our analysis has identified new components of previously recognized systems in the parasite, such as the protein(s) involved in transcriptional regulation, cell cycle progression, invasion, protein trafficking, and immune evasion in the UPS, or the antioxidant defense proteins associated with the heat shock response systems.
The protease data
The proteases in P. falciparum were predicted using a comparative genomics approach and a support vector machine (SVM)-based, supervised machine learning approach [1–3]. The classification and annotation were according to the MEROPS protease nomenclature, which is based on intrinsic evolutionary and structural relationships [110].
Network data and analysis
The complete set of protein-protein associations for P. falciparum was extracted from the downloaded STRING database [4]; each association between a pair of proteins has a confidence score (S) ranging from 0.15 to 0.999 that was inferred from the evidence used to establish the association, such as homology transfer, KEGG pathway assignments, conserved chromosome synteny, phylogenetic co-occurence, and literature co-occurence [111]. This set of associations was visualized in Cytoscape [112] and converted to an undirected weighted graph, where there is a single edge between any pair of proteins and the S value is used as the weight. The network was characterized using NetworkAnalyzer [113] and significant modules were detected using MINE [114] and MCODE [115]. The default values were used for all three plugins. The set of proteins directly associated with the 77 proteases in the association set were screened using BiNGO [116] to determine if any categories of proteins, as identified by their Gene Ontology terms, were over-represented. The hypergeometric test was used with the Benjamini and Hochberg false discovery date correction. A significance level of 0.05 was selected.
The omics data mining
We downloaded the P. falciparum genomic sequence and annotation data [18], transcriptomic microarray data [6–8], mass-spectrometry proteomic data [9–12], and protein-protein interactome [5] data for network associated proteins from PlasmoDB, the Plasmodium Genome resource center (http://www.plasmodb.org) [117]. Conserved domains/motifs in P. falciparum sequences were identified by searching InterPro [118]. Multiple alignments were obtained using the ClustalX program [119] and T-coffee [120], followed by manual inspection and editing. Phylogenetic trees were inferred by the neighbor-joining, maximum-parsimony and maximum-likelihood methods, using MEGA5 [121].
apical membrane antigen
CDPK:
calcium-dependent protein kinase
DOXP:
1-deoxy-D-xylulose 5-phosphate
EBL:
erythrocyte binding-like
EF:
elongation factor
EM:
erythrocyte membrane
ER:
endoplasmic reticulum
HAP:
histo-aspartic protease
HSP:
MBF:
multiprotein bridging factor
PPI:
protein-protein interaction
PVM:
parasitophorous vacuole membrane
RBL:
reticulocyte binding-like
RIP:
regulated intramembrane proteolysis
SERA:
Serine Repeat Antigen
SKP:
S-phase kinase-associated protein
SOD:
superoxide dismutase
SPAM:
secreted polymorphic antigen associated with merozoites
signal peptidase complex
SVM:
TBP:
TATA box-binding protein
TMH:
transmembrane-spanning helical
UPS:
ubiquitin-proteasome system
Y2H:
yeast 2-hybrid
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von Mering C, Jensen LJ, Snel B, Hooper SD, Krupp M, Foglierini M, Jouffre N, Huynen MA, Bork P: STRING: known and predicted protein-protein associations, integrated and transferred across organisms. Nucleic Acids Res. 2005, D433-437. 33 Database
Smoot ME, Ono K, Ruscheinski J, Wang PL, Ideker T: Cytoscape 2.8: new features for data integration and network visualization. Bioinformatics. 2010, 27 (3): 431-432.
Assenov Y, Ramirez F, Schelhorn SE, Lengauer T, Albrecht M: Computing topological parameters of biological networks. Bioinformatics. 2008, 24 (2): 282-284. 10.1093/bioinformatics/btm554.
Rhrissorrakrai K, Gunsalus KC: MINE: Module Identification in Networks. BMC Bioinformatics. 2011, 12: 192-10.1186/1471-2105-12-192.
Bader GD, Hogue CW: An automated method for finding molecular complexes in large protein interaction networks. BMC Bioinformatics. 2003, 4: 2-10.1186/1471-2105-4-2.
Maere S, Heymans K, Kuiper M: BiNGO: a Cytoscape plugin to assess overrepresentation of gene ontology categories in biological networks. Bioinformatics. 2005, 21 (16): 3448-3449. 10.1093/bioinformatics/bti551.
Aurrecoechea C, Brestelli J, Brunk BP, Dommer J, Fischer S, Gajria B, Gao X, Gingle A, Grant G, Harb OS: PlasmoDB: a functional genomic database for malaria parasites. Nucleic Acids Res. 2009, D539-543. 37 Database
Hunter S, Apweiler R, Attwood TK, Bairoch A, Bateman A, Binns D, Bork P, Das U, Daugherty L, Duquenne L, et al: InterPro: the integrative protein signature database. Nucleic Acids Res. 2009, 37: D211-D215. 10.1093/nar/gkn785.
Jeanmougin F, Thompson JD, Gouy M, Higgins DG, Gibson TJ: Multiple sequence alignment with Clustal x. Trends Biochem Sci. 1998, 23 (10): 403-405. 10.1016/S0968-0004(98)01285-7.
Notredame C, Higgins DG, Heringa J: T-Coffee: a novel method for fast and accurate multiple sequence alignment. J Mol Biol. 2000, 302 (1): 205-217. 10.1006/jmbi.2000.4042.
Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S: MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. 2011
Muller J, Szklarczyk D, Julien P, Letunic I, Roth A, Kuhn M, Powell S, von Mering C, Doerks T, Jensen LJ: eggNOG v2.0: extending the evolutionary genealogy of genes with enhanced non-supervised orthologous groups, species and functional annotations. Nucleic Acids Res. 2010, D190-195. 38 Database
Tatusov RL, Koonin EV, Lipman DJ: A genomic perspective on protein families. Science. 1997, 278 (5338): 631-637. 10.1126/science.278.5338.631.
We thank PlasmoDB for providing an all-in-one portal for malaria omic data. This work is supported by NIH grants GM081068 and AI080579 to Y. Wang. ZZ is supported by the government scholarship from China Scholarship Council. YW is also supported by NIH grant RR013646. We thank the Computational Biology Initiative at UTSA for providing computational support. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of General Medical Sciences, National Institute of Allergy and Infectious Diseases, National Center for Research Resources, or the National Institutes of Health.
Department of Bacteriology, American Type Culture Collection, Manassas, VA, 20110, USA
Timothy G Lilburn
Department of Biology, University of Texas at San Antonio, San Antonio, TX, 78249, USA
Hong Cai, Zhan Zhou & Yufeng Wang
College of Life Sciences, Zhejiang University, Hangzhou, 310058, PR China
Zhan Zhou
South Texas Center for Emerging Infectious Diseases, University of Texas at San Antonio, San Antonio, TX, 78249, USA
Yufeng Wang
Hong Cai
Correspondence to Yufeng Wang.
TGL and YW conceived and designed the study, performed bioinformatics data analysis, and drafted the manuscript. HC wrote scripts, and ZZ helped with data analysis. All authors read and approved the final manuscript.
Additional file 1: P. falciparum proteases and their degrees of connectivity in protein association networks. (DOCX 24 KB)
Additional file 2: The protein-protein associations involving proteases in Plasmodium falciparum. (XLSX 327 KB)
12864_2011_3768_MOESM3_ESM.xls
Additional file 3: Functional categories involving protease associations identified by Gene Ontology enrichment analysis. (XLS 57 KB)
Additional file 4: The graph shows the set of proteins associated with the proteases thought to be part of the P. falciparum ubiquitin-proteasome protein degradation system. Nodes are colored according to their functional classification in the eggNOG database [122] (key is shown). Node size is proportional to the degree of the node. Confidence scores for the interactions among the nodes (S values from STRING) were divided into three groups - low (0.150-0.399), medium (0.400-0.700) and high (0.701-0.999); the groups are represented by thin, medium and heavy lines, respectively. (PDF 441 KB)
Lilburn, T.G., Cai, H., Zhou, Z. et al. Protease-associated cellular networks in malaria parasite Plasmodium falciparum. BMC Genomics 12 (Suppl 5), S9 (2011). https://doi.org/10.1186/1471-2164-12-S5-S9
Heat Shock Response
Merozoite Surface Protein
Malaria Parasite Plasmodium Falciparum
High Confidence Score


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text:
Victorian England & Wales Map 1897: Central South 8
Old House Books Victorian England & Wales Map 1897: Central South 8
Now £4.49£4.49
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Covers the area from Barnstaple, Okehampton and Exmouth in the West to Maidenhead, Wokingham and Chichester in the East, with Bristol and Reading in the north, down to the south coast including the Isle of Wight.
Early coloured Ordnance Survey Maps 4 miles to the inch in nine sheets.1897, the year in which these maps first appeared in The Royal English Atlas, was one of great celebration for it marked the Diamond Jubilee of Britain�s longest reigning monarch, Queen Victoria. But there was much more to celebrate than her continuing occupancy of the throne as the previous sixty years had seen the greatest ever period of industrial development. The rapidly expanding industrial towns and cities were now the homes of workers who once laboured on the land but were now in the factories and mines creating and processing the needs of the mother country and the Empire. To record these changes the Victorians had become great map makers and the Ordnance Survey had already been publishing maps for a century. This series, created from Ordnance Survey maps, were coloured and published in book form. They are now available in sheets for the first time.The construction of the railway network, commenced a mere 72 years before, was now virtually complete with over 20, 000 miles in use. They are all marked, as are their �predecessors� the canals. But, despite all the changes brought about by the Industrial Revolution, the great conurbations we know today were still surprisingly small in 1897. The countryside had changed little for centuries. There were still scores of aristocratic homes surrounded by park land where now the urban sprawl, new towns and industrial estates may be found.Just as interesting is what is not shown. Notice how small the towns and scattered villages are, indeed there is a village just to the west of London on sheet 9 which shows the three or four houses that made up the village of Heathrow. There are no by-passes and all the major trunk roads pass along the high streets of country towns. Motorways had, of course, not even been dreamt about.These maps, now available in nine sheets, provide a fascinating insight into the past and will be a delight for local historians
Old House Books
Old House Folded Map
630x950mm (unfolded)
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text:
David Pearce, charged in deaths of Christy Giles, Hilda Marcela Cabrales-Arzola, held on bail for sex assaults
Arrests made in death of model and her friend in LA County, suspects bragged about deaths
Three men have been arrested in connection with the deaths of two women who were allegedly drugged during a night out in Los Angeles, and later dumped at separate hospitals, police announced Thursday morning.
LOS ANGELES - A judge Thursday ordered a Beverly Hills man who was arrested in connection with the deaths of two women last month and subsequently charged with sexually assaulting four women in alleged attacks dating back as far as 2010 to remain jailed in lieu of $3.4 million bail.
Superior Court Judge Victoria B. Wilson ordered David Brian Pearce, 39, to return to a downtown Los Angeles courtroom for arraignment Jan. 12 on two counts each of forcible rape and one count each of rape of an unconscious or asleep person and sexual penetration by a foreign object stemming from alleged attacks in August 2010, February 2019, February 2020 and October 2020.
Prosecutors have "asked for further investigation" into the Nov. 13 death of Christy Giles and the subsequent death Nov. 27 of Hilda Marcela Cabrales-Arzola, according to Greg Risling, a spokesman for the Los Angeles County District Attorney's Office.
"That's currently an ongoing investigation so I can't say too much about that, but that investigation is absolutely still ongoing," Deputy District Attorney Catherine Mariano told reporters outside court.
Giles, a 24-year-old model and aspiring actress, and Cabrales-Arzola were last seen at an apartment in the Pico-Robertson neighborhood before their bodies were dumped outside Southern California Hospital in Culver City and Kaiser Permanente West Los Angeles Hospital.
Giles was already dead when she was found outside the hospital on Nov. 13. Cabrales-Arzola, an architect, was in critical condition and her family took her off life support a day before her 27th birthday.
RELATED: 3 arrested in deaths of 2 women who were drugged during night out in Los Angeles and later died: Police
"It is believed that both women were given drugs and overdosed at a residence in the 8600 block of Olympic Boulevard in the city of Los Angeles," police said in a statement earlier this week.
"Three suspects were identified and arrested with the assistance of the LAPD-FBI Fugitive Task Force and Metropolitan Division. Based on the investigation, the LAPD is concerned that there could be other victims in our community who could have been drugged by one or more of these men," police said, calling it an "active and ongoing investigation."
RELATED: The final hours before 2 women were drugged during night out in Los Angeles before ending up dead
Pearce — who was booked on suspicion of manslaughter — has remained behind bars since his arrest Dec. 15 by the Los Angeles Police Department's West Bureau, jail records show.
Prosecutors have asked for further investigation involving the two other men, Michael Ansbach, 47, and Brandt Osborn, 42, whom police said were each booked on suspicion of being an accessory to manslaughter.
Anspach was released last Thursday on $100,000 bond, while Osborn was released from custody late Friday afternoon, according to jail records.
In a statement announcing the sexual assault charges against Pearce, District Attorney George Gascon said, "This case is evolving and we continue to work with law enforcement in developing evidence of other possible crimes."
Gascon urged anyone with any information to immediately contact Los Angeles police.
RELATED: Man arrested in deaths of model, her friend charged in series of sexual assaults
"We have a good faith belief there may be other victims, but again, based on the ongoing investigation of this case I can't comment as to whether there are, in fact, additional victims at this point," Mariano told reporters outside the courtroom after Pearce's second court appearance on the case.
Giles' husband, Jan Cilliers, said earlier that police contacted him about the arrests last week.
"Even if they get charged to the fullest extent of the law, no amount of legal justice will bring back Christy and Hilda," Cilliers told Fox 11.
Cilliers had been quoted in previous published reports as saying video exists of three masked men dressed in black who pulled up to the hospital and pushed the model's body out of a Toyota Prius without license plates.
Giles and Cabrales-Arzola exchanged text messages hours before they were found, appearing to have called an Uber to leave the apartment.
The women's family members have claimed they were possibly drugged against their will at some point before they were found.
International criminals targeting some of California's poorest families, stealing state benefits
LA County eviction protections extended
Eviction protections extended


"He broke the power of death and illuminated the way to life and immortality through the Good News." This verse came to life at Del Oro's Say Summit as many youth drew closer to God and made decisions all throughout the weekend. It was an awesome sight to see!
With the change in name came some new ideas that were implemented in our division to promote spiritual growth and discipleship as well as reaching out to those who have not made a decision to follow Jesus. We had opportunities throughout the weekend for connecting with God, learning about him and His Word and connecting with peers and leaders. We created Summit groups to meet 3 times throughout the weekend. They were broken up by age to do interactive activities that enhanced the message. These were a hit! They did things like making LED lights, performing acts of kindness throughout the retreat, art war and more. We also heard great conversations happening during evening Cabin talks and Corps Morning devotions. We created special Pastoral groups for the Officers and leaders during the weekend led by DHQ and THQ staff. We tried to provide them with some encouragement, support and treats! Our motto is Take care of your leaders and they'll take care of the youth! This was a new item that was well received in addition to the leader lounge provided once again this year.
AMAZING testimony this morning from one of the youth about her trip to her 1st ever Say Summit. She didn't know what to expect except that it would be fun and: "I knew that God was going to change my life. I knew that God was going to work in my life whether I accepted it or not…I knew that he was going to make my achievements bigger and my sins smaller! I accepted Christ…It wasn't me – it was God transforming me because he knew [where I had been and what I have experienced and that] I needed a change in my life." She's already impacting people by illuminating in the world around her!
And we keep hearing of others testifying at their Corps about what God did in their hearts.
We had a good selection of workshops. They included learning to perform spoken word or rap, using clean comedy to be a light, being a missionary in your world, finding God's path for you, how to deal with difficult circumstances of life, painting, digging deeper into God's Word, performing a Polynesian dance, and a leader workshop on helping kids through dark times in life. They were able to attend two of their choice.
Of course the weekend wouldn't be complete without fun activities to enjoy. On Saturday we had a lawn party, 4 different tournaments, Hungry Human hippos, Human bowling, live DJ, Crafts and other great things available at the camp. Saturday evening we had a talent program with participants from several corps and guests we brought in to do hula, spoken word, rap and comedy. It was a great night! Afterwards we had Glow games, a campfire with s'mores, and a Tahitian dance and fire dance presentation. The times spent enjoying fellowship were some of the best of the weekend.
To God be the glory! He did amazing things during Say Summit. We trust he will continue to work in the lives that he touched this weekend and we'll see revival at work in the Corps of the Del Oro division.


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Ever since hulking lawman Hobbs (Johnson), a loyal agent of America's Diplomatic Security Service, and lawless outcast Shaw (Statham), a former British military elite operative, first faced off in 2015's Furious 7, the duo have swapped smack talk and body blows as they've tried to take each other down. But when cyber-genetically enhanced anarchist Brixton (Idris Elba) gains control of an insidious bio-threat that could alter humanity forever — and bests a brilliant and fearless rogue MI6 agent (The Crown's Vanessa Kirby), who just happens to be Shaw's sister — these two sworn enemies will have to partner up to bring down the only guy who might be badder than themselves.
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"Our intention was to have Molly quickly dive into the shark cage with the camera and begin broadcasting as lemon sharks circled nearby," Camsoda vice president Dar Ren Press said.
"From what we gathered, lemon sharks are predominantly docile and relatively harmless.
Transform the terrace to ceremony space, and speak your vows with the Freedom Tower at your side.
Young actors will learn how to nail cold read commercial auditions.
These findings suggest a dynamic coupling and uncoupling of calcium-dependent signal transduction pathways to a stably expressed surface adhesion molecule, whose function in a given neuron may therefore evolve with cellular maturation.
div.hero-slide" data-cycle-swipe=true data-cycle-swipe-fx=scroll Horz data-cycle-prev="#hero-prev" data-cycle-next="#hero-next" data-cycle-pager="#hero-current" data-cycle-pager="#hero-current" data-cycle-paused="true" " data-alt="W New York - Downtown Terrace" data-vu="terrace" data-org-size="1600x900" data-org=" data-bg=" style="background: transparent no-repeat center; background-size: cover"Guests will enjoy a cocktail hour overlooking the stunning downtown NYC views." data-alt="W New York - Downtown Terrace North" data-vu="terrace_1" data-org-size="1600x900" data-org=" North.jpg" data-bg=" North.jpg" style="background: transparent no-repeat center; background-size: cover"Bold design, stunning views, curated nightly musical programming, & spectacular cocktails await at our Financial District Bar: The Living Room Bar Terrace, located on the fifth floor at W New York - Downtown." data-alt="Living Room Bar and Terrace | W New York - Downtown" data-vu="living-room" data-org-size="1600x900" data-org=" Room Bar Terrace.jpg" data-bg=" Room Bar Terrace.jpg" style="background: transparent no-repeat center; background-size: cover"W New York - Downtown provides you with a front row seat to the Big Apple.
The owner of Florida Shark Diving said Cavalli wanted to make a video of a shark bite that would go "viral," and wanted his help, according to the newspaper.
It was nothing but a "PR stunt." Shark experts are taking bites out of an adult entertainer's claim that she was attacked while doing a photo shoot underwater.Budding thespians will learn how to quickly analyze commercial copy and find the acting beats within the text to make a lasting impression on both casting directors and on ad agency executives! Where: NYC Location TBD (Pearl Studios – 500/519 8th Ave. Where: NYC Location TBD (Pearl Studios – 500/519 8th Ave.Improvisation also plays a crucial role in commercial auditions. or Ripley-Grier Studios – 520 8th Ave.) Cost: 575.00 Video Audition Instructions: Please perform a 1 minute monologue that is age appropriate. Students will also learn to identify clues within the text as well as physical techniques to deliver the material that will help set them apart in their auditions. or Ripley-Grier Studios – 520 8th Ave.) Cost: 575.00 Video Audition Instructions: Please perform a 1 minute monologue that is age appropriate."We are pro-shark, pro-wildlife, and want to show the importance of sharks, not villainize them." "For a wound that deep, one would expect to see multiple parallel slashes from teeth, not a single slash," Kajiura told the newspaper.Woman 'fighting for her life' after losing part of leg to shark "The wound didn't look like a shark bite to me, either," R.
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Did Pitt give us a hit? – World War Z
24th June 2013 Max Freeman-Mills
At one point, World War Z looked set to join the pantheon of great Hollywood failures, with a budget of anywhere between $200 million and $400 million wasted on an overlong and trite mess. After dedicated reshoots and editing, however, Marc Forster's film has instead emerged as a predictable, but nonetheless effective and tense action-thriller.
The film's pandemic outbreak does away with the over-used trope of a character waking up from a coma/surgery/sleep to a changed world, instead opening with the strongest portion of the film, a day and night in New Jersey, as Brad Pitt's Gerry marshals his family towards a UN extraction. The section is taut and nervous, and the sense of vulnerability and panic is at its height; it is also the only part of the film which feels true in a significant way to the tone of Max Brooks' novel of the same name, which inspired the screen version.
Here we get short glimpses into other people's stories, such as the Hispanic family who briefly shelter Gerry, his wife and his children. There are clues that this multiplicity could have been a calculated direction for the film before the studio or producers got a hold of it, such as when a threatening police officer beelines past the family to hastily grab childcare supplies from a looted shop, rather than arrest the gun-wielding Pitt. The subtlety of such hints at everyone having desperate stories to tell is lost as the film progresses, growing into a more predictable and safe affair.
Gerry, a UN investigator, is despatched to investigate the origin of the disease, in the hope of eventually finding a cure, and his globe-trotting adventure features stints in Korea, Israel and, somewhat amazingly, Wales. Each effectively boils down to a pattern of exposition, set-piece, then progress onwards, but this is not necessarily a problem. The dank rain of the Korean night is foreboding, and the finale in a Welsh World Health Organisation outpost is full of tension; the attack from hordes of zombies moving with almost hive-mind-like efficacy in Israel strains the borders of belief, though, and ends up feeling too action-packed and unbelievable. That this is the only point when such an issue arises is thanks to the reshoots – the initial ending featured Pitt escaping Israel for Moscow, and eventually participating in a multi-level pitched battle in the Red Square, in what sounds like a very literal interpretation of the title. Instead, the plane he boards heads for Wales, and a less bleak but more effective conclusion.
The film's main issues are that, as a trade-off of the reshoot, the last forty minutes are overly predictable, and more consistently there is an odd lack of gore for a film in the zombie tradition. We see Pitt creep around, ending undead lives with axe, crowbar and gun, but whenever he delivers a killing blow, the camera is pointedly not looking. While too much gore can be comedic, as AMC's The Walking Dead has evidenced at times on television, the lack of intimate violence leads to the zombies as individuals feeling too weak. This is only exacerbated by the extras playing zombies' clacking of jaws and overly jerky movements – the audience I watched with laughed at multiple points when the film-makers clearly wanted the opposite reaction.
These flaws aside, however, World War Z does not do nearly enough wrong to earn itself the status of 'flop', and has jump-scared aplenty to keep you on edge – unless the intelligence of the source material is sacrosanct to you to the point where deviation will cause distress, there is nothing that much wrong with World War Z, if nothing outstandingly right either.
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You need some honey in your life! ASAP!
Happy New year! I would just like to take this time to thank you for visiting today and every other time. I hope this post meets you well and I pray you will have an amazing year!
This blog post is dedicated to honey. I have been so astounded by how well it works, I had to share how I felt with someone! I have been using honey to cleanse my face for the last two months or so. It is hands down the best facial cleaner I have EVER used! It is amazing because it really cleanses your face without leaving your skin feeling stripped. It is very mild but works. I can attest to this fact after not having any (or significantly less) t zone spots since around when I started using honey. BTW I would normally get t zone spots once every week or two weeks.
At present, I use honey once a day, in the morning. I apply and massage it into my face and let it sit while I shower. After I finish, I rinse it with very lukewarm water and voila! I'll then moisturise etc as usual.
As you may or may not know I favour natural products. I have used Ose Dudu osun (which actually contains honey!) for the longest time to bathe and wash my face (I reviewed it here! http://wp.me/p2a4MJ-2F ). I was happy and I still use it to bathe but I was aware the Ose Dudu Osun was really drying my face out. I had tried a number of other facial cleansers that I felt they were still drying out my face and leaving it too tight.
A post or something out of a book by Dephne Madyra (check her out on YouTube) is what prompted my decision to try some honey.
I stand by this as being the best ever facial cleaner I have EVER used! I hope you will feel the same.