Essentially, all metazoan cells can undergo apoptosis, but some cells are more sensitive than others to apoptotic stimuli. To date, it is unclear what determines the apoptotic potential of the cell. ...We set up an in vivo system for monitoring and comparing the activity levels of the two main effector caspases in Drosophila melanogaster, Drice and Dcp-1. Both caspases were activated by the apoptosome after irradiation. However, whereas each caspase alone could induce apoptosis, Drice was a more effective inducer of apoptosis than Dcp-1, which instead had a role in establishing the rate of cell death. These functional differences are attributed to their intrinsic properties rather than merely their tissue specificities. Significantly, the levels of the procaspases are directly proportional to their activity levels and play a key role in determining the cell's sensitivity to apoptosis. Finally, we provide evidence for the existence of a cellular execution threshold of caspase activity, which must be reached to induce apoptosis.
Anat Florentin works in the field of molecular parasitology, studying the cell biology of malaria parasites. In this mSphere of Influence article, she reflects how the book
by Sean B. Carroll (2013) ...made a powerful impact on her by telling scientific stories in the context of dramatic life events.
The infant gut microbiome is impacted by early-life feeding, as human milk oligosaccharides (HMOs) found in breastmilk cannot be digested by infants and serve as nutrients for their gut bacteria. ...While the vast majority of HMO-utilization research has focused on Bifidobacterium species, recent studies have suggested additional HMO-utilizers, mostly Bacteroides, yet their utilization mechanism is poorly characterized. Here, we investigate Bacteroides dorei isolates from breastfed-infants and identify that polysaccharide utilization locus (PUL) 33 enables B. dorei to utilize sialylated HMOs. We perform transcriptional profiling and identity upregulated genes when growing on sialylated HMOs. Using CRISPR-Cas12 to knock-out four PUL33 genes, combined with complementation assays, we identify GH33 as the critical gene in PUL33 for sialylated HMO-utilization. This demonstration of an HMO-utilization system by Bacteroides species isolated from infants opens the way to further characterization of additional such systems, to better understand HMO-utilization in the infant gut.
The deadly malaria parasite,
, contains a unique subcellular organelle termed the apicoplast, which is a clinically-proven antimalarial drug target. The apicoplast is a plastid with essential ...metabolic functions that evolved
secondary endosymbiosis. As an ancient endosymbiont, the apicoplast retained its own genome and it must be inherited by daughter cells during cell division. During the asexual replication of
inside human red blood cells, both the parasite, and the apicoplast inside it, undergo massive morphological changes, including DNA replication and division. The apicoplast is an integral part of the cell and thus its development is tightly synchronized with the cell cycle. At the same time, certain aspects of its dynamics are independent of nuclear division, representing a degree of autonomy in organelle biogenesis. Here, we review the different aspects of organelle dynamics during
intraerythrocytic replication, summarize our current understanding of these processes, and describe the many open questions in this area of parasite basic cell biology.
Maintenance of tissue integrity during development and homeostasis requires the precise coordination of several cell-based processes, including cell death. In animals, the majority of such cell death ...occurs by apoptosis, a process mediated by caspase proteases. To elucidate the role of caspases in tissue integrity, we investigated the behavior of Drosophila epithelial cells that are severely compromised for caspase activity. We show that these cells acquire migratory and invasive capacities, either within 1-2 days following irradiation or spontaneously during development. Importantly, low levels of effector caspase activity, which are far below the threshold required to induce apoptosis, can potently inhibit this process, as well as a distinct, developmental paradigm of primordial germ cell migration. These findings may have implications for radiation therapy in cancer treatment. Furthermore, given the presence of caspases throughout metazoa, our results could imply that preventing unwanted cell migration constitutes an ancient non-apoptotic function of these proteases.
The cis-polyisoprenoid lipids namely polyprenols, dolichols and their derivatives are linear polymers of several isoprene units. In eukaryotes, polyprenols and dolichols are synthesized as a mixture ...of four or more homologues of different length with one or two predominant species with sizes varying among organisms. Interestingly, co-occurrence of polyprenols and dolichols, i.e. detection of a dolichol along with significant levels of its precursor polyprenol, are unusual in eukaryotic cells. Our metabolomics studies revealed that cis-polyisoprenoids are more diverse in the malaria parasite Plasmodium falciparum than previously postulated as we uncovered active de novo biosynthesis and substantial levels of accumulation of polyprenols and dolichols of 15 to 19 isoprene units. A distinctive polyprenol and dolichol profile both within the intraerythrocytic asexual cycle and between asexual and gametocyte stages was observed suggesting that cis-polyisoprenoid biosynthesis changes throughout parasite's development. Moreover, we confirmed the presence of an active cis-prenyltransferase (PfCPT) and that dolichol biosynthesis occurs via reduction of the polyprenol to dolichol by an active polyprenol reductase (PfPPRD) in the malaria parasite.
The deadly malaria parasite Plasmodium falciparum contains a nonphotosynthetic plastid, known as the apicoplast, that functions to produce essential metabolites, and drugs that target the apicoplast ...are clinically effective. Several prokaryotic caseinolytic protease (Clp) genes have been identified in the Plasmodium genome. Using phylogenetic analysis, we focused on the Clp members that may form a regulated proteolytic complex in the apicoplast. We genetically targeted members of this complex and generated conditional mutants of the apicoplast-localized PfClpC chaperone and PfClpP protease. Conditional inhibition of the PfClpC chaperone resulted in growth arrest and apicoplast loss and was rescued by addition of the essential apicoplast-derived metabolite IPP. Using a double-conditional mutant parasite line, we discovered that the chaperone activity is required to stabilize the mature protease, revealing functional interactions. These data demonstrate the essential function of PfClpC in maintaining apicoplast integrity and its role in regulating the proteolytic activity of the Clp complex.
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•PfClpC and PfClpP are Clp orthologs in the apicoplast of Plasmodium falciparum•Conditional inhibition of the PfClpC results in growth arrest and apicoplast loss•Addition of apicoplast-derived metabolite IPP rescues the growth phenotype•PfClpC chaperone activity is required to stabilize the PfClpP protease
Plasmodium falciparum contains a unique organelle, the apicoplast. Using genetic and phenotypic assays, Florentin et al. characterize the apicoplast Clp chaperone and protease. They find that the chaperone is essential for protease stability and that together they function to maintain organelle integrity and segregation into daughter cells.
Export of parasite proteins into the host erythrocyte is essential for survival of
during its asexual life cycle. While several studies described key factors within the parasite that are involved in ...protein export, the mechanisms employed to traffic exported proteins within the host cell are currently unknown. Members of the Hsp70 family of chaperones, together with their Hsp40 cochaperones, facilitate protein trafficking in other organisms, and are thus likely used by
in the trafficking of its exported proteins. A large group of Hsp40 proteins is encoded by the parasite and exported to the host cell, but only one Hsp70,
Hsp70x (PfHsp70x), is exported with them. PfHsp70x is absent in most
species and is found only in
and closely related species that infect apes. Herein, we have utilized clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 genome editing in
to investigate the essentiality of PfHsp70x. We show that parasitic growth was unaffected by knockdown of PfHsp70x using both the dihydrofolate reductase (DHFR)-based destabilization domain and the
ribozyme system. Similarly, a complete gene knockout of PfHsp70x did not affect the ability of
to proceed through its intraerythrocytic life cycle. The effect of PfHsp70x knockdown/knockout on the export of proteins to the host red blood cell (RBC), including the critical virulence factor
erythrocyte membrane protein 1 (PfEMP1), was tested, and we found that this process was unaffected. These data show that although PfHsp70x is the sole exported Hsp70, it is not essential for the asexual development of
.
Half of the world's population lives at risk for malaria. The intraerythrocytic life cycle of
spp. is responsible for clinical manifestations of malaria; therefore, knowledge of the parasite's ability to survive within the erythrocyte is needed to combat the deadliest agent of malaria,
. An outstanding question in the field is how
undertakes the essential process of trafficking its proteins within the host cell. In most organisms, chaperones such as Hsp70 are employed in protein trafficking. Of the
species causing human disease, the chaperone PfHsp70x is unique to
, and it is the only parasite protein of its kind exported to the host (S. Külzer et al., Cell Microbiol 14:1784-1795, 2012). This has placed PfHsp70x as an ideal target to inhibit protein trafficking and kill the parasite. However, we show that PfHsp70x is not required for export of parasite effectors and it is not essential for parasite survival inside the RBC.
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•In attempt to upscale NRWI measurements two turbulence-based.•All methods were examined recorded similar diurnal dynamics of λE.•The eddy-covariance underestimated λE and the ...scintillometer (SLS) overestimated λE.•The SLS underestimation was either due to miscalculations of G or mismeasurement of H.
Non-rainfall water inputs (NRWI) are a significant water source in drylands. Small latent heat flux (λE) involved in the formation and evaporation of NRWI presents measurement challenges. Microlysimeters (MLs) are a point measurement that was previously shown to be accurate for detecting NRWI. In an attempt to upscale these measurements, two turbulence-based methods were examined. Their larger spatial extent is potentially an important improvement over the ML point measurements. However, accumulation of NRWI largely occurs during the night, during which strong stable conditions are typical for many drylands. This may challenge turbulence-based methods.Therefore the applicability of such methods for monitoring NRWI needs to be carefully examined before being disseminated for use. In this research, NRWI-derived λE obtained by an eddy-covariance (EC) and a surface layer scintillometer (SLS) using the energy balance approach were tested against ML measurements over a bare soil in the Negev Desert during the dry summer season. The microlysimeter, the EC, and the SLS all recorded similar diurnal dynamics of λE but, compared to the ML measurements, the EC tended to underestimate the λE flux while the SLS (with ancillary measurements) over estimated λE. Closures of 93% and 89% for the ML and the EC respectively are indicative of the EC underestimation. In the case of the SLS, under the research conditions, the large magnitude of soil heat flux (G) and the divergence of its calculation by two different methods, make G a prime suspect. However, a question still remains as to the accuracy of the scintillometer-derived H.
Lyophilized unilamellar liposomes (ULV), the dosage form of choice for shelf-life, revert upon reconstitution to the larger multilamellar liposomes (MLV), which is detrimental to the many ...carrier-mediated therapies that require small particles. High doses of sugars such as trehalose, sucrose and others, included in the original formulations for cryoprotection, were shown to prevent the conversion to MLV. In this study we set out to test whether hyaluronan (HA), the surface-bound ligand in our previously developed targeted bioadhesive liposomes (BAL), can also act as a cryoprotectant. The studies included structural and physicochemical characterization of original and reconstituted hyaluronan-ULV (HA-ULV). For each HA-ULV, similar regular ULV (RL-ULV) served as controls. Four properties were tested: particle size, zeta potential, encapsulation efficiency and half-life of drug release (τ1/2), for three drugs—chloramphenicol (CAM), vinblastine (VIN) and mitomycin C (MMC). Encapsulation efficiencies of the original systems were quite alike for similar RL-ULV and HA-ULV ranging from 25% to 70%. All systems acted as sustained-release drug depots, τ1/2 ranging from 1.3 to 5.3 days. Drug species and lipid composition were the major determinants of encapsulation and release magnitudes. By all tests, as anticipated, lyophilization generated significant changes in the reconstituted RL-ULV: 17-fold increase in diameter; tripling of zeta potential; 25–60% drop in encapsulation efficiencies; 25–30% decrease in τ1/2. In contrast, the reconstituted HA-ULV retained the same dimensions, zeta potentials, encapsulation efficiencies and τ1/2 of the original systems. These data clearly show HA to be a cryoprotectant, adding another clinically relevant advantage to HA-BAL. We propose that, like the sugars, HA cryoprotects by providing substitute structure-stabilizing H-bonds.
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