The inner membrane complex (IMC) of Toxoplasma gondii is essential for all phases of the parasite's life cycle. One of its most critical roles is to act as a scaffold for the assembly of daughter ...buds during replication by endodyogeny. While many daughter IMC proteins have been identified, most are recruited after bud initiation and are not essential for parasite fitness. Here, we report the identification of IMC43, a novel daughter IMC protein that is recruited at the earliest stages of daughter bud initiation. Using an auxin-inducible degron system we show that depletion of IMC43 results in aberrant morphology, dysregulation of endodyogeny, and an extreme defect in replication. Deletion analyses reveal a region of IMC43 that plays a role in localization and a C-terminal domain that is essential for the protein's function. TurboID proximity labelling and a yeast two-hybrid screen using IMC43 as bait identify 30 candidate IMC43 binding partners. We investigate two of these: the essential daughter protein IMC32 and a novel daughter IMC protein we named IMC44. We show that IMC43 is responsible for regulating the localization of both IMC32 and IMC44 at specific stages of endodyogeny and that this regulation is dependent on the essential C-terminal domain of IMC43. Using pairwise yeast two-hybrid assays, we determine that this region is also sufficient for binding to both IMC32 and IMC44. As IMC43 and IMC32 are both essential proteins, this work reveals the existence of a bud assembly complex that forms the foundation of the daughter IMC during endodyogeny.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Self-report measures are important in substance use assessment, yet they are susceptible to reporting errors. Urine drug screens (UDS) are often considered a more valid alternative. However, ...collecting in-person UDS may not always be feasible, contributing to the need to understand factors that influence the validity of self-reported substance use.
In this secondary analysis of data from 295 women with co-occurring PTSD and substance use disorders (SUD) who participated in a clinical trial testing behavioral interventions, we examined concordance and discordance between self-reported drug use and associated UDS results. Generalized linear mixed models were used to examine the impact of treatment type and participant characteristics on the associations between self-reported drug use and UDS results.
Findings revealed higher disagreement between self-report and UDS for opioids and sedatives (ranging from.77 to.90) and lower disagreement rates for cannabis and cocaine (ranging from.26 to.33). Treatment type was not a significant moderator of the associations between self-report and UDS across all drugs. Among those with a positive opioid UDS, those who reported employment in the past three years were more likely to self-report no opioid use compared to their counterparts without employment in the past three years.
Findings add to the literature that supports the validity of self-reported cannabis and cocaine use. The greater discrepancies between self-report and UDS test results of opioids and sedatives suggest adjunctive UDS may be required, although a variety of factors other than inaccurate self-report may be associated with this discrepancy.
•We examined the agreement between self-reported drug use and associated UDS results.•There was a higher disagreement between self-report and UDS for opioids and sedatives.•There was lower disagreement between self-report and UDS for cannabis and cocaine.•Employment status influenced agreement among those with a positive opioid UDS.
Melioidosis, caused by the intracellular bacterial pathogen and Tier 1 select agent
(Bp), is a highly fatal disease endemic in tropical areas. No licensed vaccine against melioidosis exists. In ...preclinical vaccine studies, demonstrating protection against respiratory infection in the highly sensitive BALB/c mouse has been especially challenging. To address this challenge, we have used a safe yet potent live attenuated platform vector, LVS Δ
, previously used successfully to develop vaccines against the Tier 1 select agents of tularemia, anthrax, and plague, to develop a melioidosis vaccine. We have engineered melioidosis vaccines (rLVS Δ
/Bp) expressing multiple immunoprotective Bp antigens among type VI secretion system proteins Hcp1, Hcp2, and Hcp6, and membrane protein LolC. Administered intradermally, rLVS Δ
/Bp vaccines strongly protect highly sensitive BALB/c mice against lethal respiratory Bp challenge, but protection is overwhelmed at very high challenge doses. In contrast, administered intranasally, rLVS Δ
/Bp vaccines remain strongly protective against even very high challenge doses. Under some conditions, the LVS Δ
vector itself provides significant protection against Bp challenge, and consistent with this, both the vector and vaccines induce humoral immune responses to Bp antigens. Three-antigen vaccines expressing Hcp6-Hcp1-Hcp2 or Hcp6-Hcp1-LolC are among the most potent and provide long-term protection and protection even with a single intranasal immunization. Protection via the intranasal route was either comparable to or statistically significantly better than the single-deletional Bp mutant Bp82, which served as a positive control. Thus, rLVS Δ
/Bp vaccines are exceptionally promising safe and potent melioidosis vaccines.
Melioidosis, a major neglected disease caused by the intracellular bacterial pathogen
, is endemic in many tropical areas of the world and causes an estimated 165,000 cases and 89,000 deaths in humans annually. Moreover,
is categorized as a Tier 1 select agent of bioterrorism, largely because inhalation of low doses can cause rapidly fatal pneumonia. No licensed vaccine is available to prevent melioidosis. Here, we describe a safe and potent melioidosis vaccine that protects against lethal respiratory challenge with
in a highly sensitive small animal model-even a single immunization is highly protective, and the vaccine gives long-term protection. The vaccine utilizes a highly attenuated replicating intracellular bacterium as a vector to express multiple key proteins of
; this vector platform has previously been used successfully to develop potent vaccines against other Tier 1 select agent diseases including tularemia, anthrax, and plague.
Diabetes is an epidemic of worldwide proportions caused by β-cell failure. Nutrient fluctuations and insulin resistance drive β-cells to synthesize insulin beyond their capacity for protein folding ...and secretion and thereby activate the unfolded protein response (UPR), an adaptive signalling pathway to promote cell survival upon accumulation of unfolded protein in the endoplasmic reticulum (ER). Protein kinase-like endoplasmic reticulum kinase (PERK) signals one component of the UPR through phosphorylation of eukaryotic initiation factor 2 on the α-subunit (eIF2α) to attenuate protein synthesis, thereby reducing the biosynthetic burden. β-Cells uniquely require PERK-mediated phosphorylation of eIF2α to preserve cell function. Unabated protein synthesis in β-cells is sufficient to initiate a cascade of events, including oxidative stress, that are characteristic of β-cell failure observed in type 2 diabetes. In contrast to acute adaptive UPR activation, chronic activation increases expression of the proapoptotic transcription factor CAAT/enhancer-binding protein homologous protein (CHOP). Chop deletion in insulin-resistant mice profoundly increases β-cell mass and prevents β-cell failure to forestall the progression of diabetes. The findings suggest an unprecedented link by which protein synthesis and/or misfolding in the ER causes oxidative stress and should encourage the development of novel strategies to treat diabetes.
The
inner membrane complex (IMC) is a specialized organelle that is crucial for the parasite to establish an intracellular lifestyle and ultimately cause disease. The IMC is composed of both membrane ...and cytoskeletal components, further delineated into the apical cap, body, and basal subcompartments. The apical cap cytoskeleton was recently demonstrated to govern the stability of the apical complex, which controls parasite motility, invasion, and egress. While this role was determined by individually assessing the apical cap proteins AC9, AC10, and the mitogen-activated protein kinase ERK7, how the three proteins collaborate to stabilize the apical complex is unknown. In this study, we use a combination of deletion analyses and yeast two-hybrid experiments to establish that these proteins form an essential complex in the apical cap. We show that AC10 is a foundational component of the AC9:AC10:ERK7 complex and demonstrate that the interactions among them are critical to maintaining the apical complex. Importantly, we identify multiple independent regions of pairwise interaction between each of the three proteins, suggesting that the AC9:AC10:ERK7 complex is organized by multivalent interactions. Together, these data support a model in which multiple interacting domains enable the oligomerization of the AC9:AC10:ERK7 complex and its assembly into the cytoskeletal IMC, which serves as a structural scaffold that concentrates ERK7 kinase activity in the apical cap.
The phylum Apicomplexa consists of obligate, intracellular parasites, including the causative agents of toxoplasmosis, malaria, and cryptosporidiosis. Hallmarks of these parasites are the IMC and the apical complex, both of which are unique structures that are conserved throughout the phylum and required for parasite survival. The apical cap portion of the IMC has previously been shown to stabilize the apical complex. Here, we expand on those studies to determine the precise protein-protein interactions of the apical cap complex that confer this essential function. We describe the multivalent nature of these interactions and show that the resulting protein oligomers likely tether ERK7 in the apical cap. This study represents the first description of the architecture of the apical cap at a molecular level, expanding our understanding of the unique cell biology that drives
infections.
The inner membrane complex (IMC) is a unique organelle of apicomplexan parasites that plays critical roles in parasite motility, host cell invasion, and replication. Despite the common functions of ...the organelle, relatively few IMC proteins are conserved across the phylum and the precise roles of many IMC components remain to be characterized. Here, we identify a novel component of the
IMC (IMC32) that localizes to the body portion of the IMC and is recruited to developing daughter buds early during endodyogeny. IMC32 is essential for parasite survival, as its conditional depletion results in a complete collapse of the IMC that is lethal to the parasite. We demonstrate that localization of IMC32 is dependent on both an N-terminal palmitoylation site and a series of C-terminal coiled-coil domains. Using deletion analyses and functional complementation, we show that two conserved regions within the C-terminal coiled-coil domains play critical roles in protein function during replication. Together, this work reveals an essential component of parasite replication that provides a novel target for therapeutic intervention of
and related apicomplexan parasites.
The IMC is an important organelle that apicomplexan parasites use to maintain their intracellular lifestyle. While many IMC proteins have been identified, only a few central players that are essential for internal budding have been described and even fewer are conserved across the phylum. Here, we identify IMC32, a novel component of the
IMC that localizes to very early daughter buds, indicating a role in the early stages of parasite replication. We then demonstrate that IMC32 is essential for parasite survival and pinpoint conserved regions within the protein that are important for membrane association and daughter cell formation. As IMC32 is unique to these parasites and not present in their mammalian hosts, it serves as a new target for the development of drugs that exclusively affect these important intracellular pathogens.
The
inner membrane complex (IMC) is a unique organelle that plays critical roles in parasite motility, invasion, egress, and replication. The IMC is delineated into the apical, body, and basal ...regions, defined by proteins that localize to these distinct subcompartments. The IMC can be further segregated by proteins that localize specifically to the maternal IMC, the daughter bud IMC, or both. While the function of the maternal IMC has been better characterized, the precise roles of most daughter IMC components remain poorly understood. Here, we demonstrate that the daughter protein IMC29 plays an important role in parasite replication. We show that Δ
parasites exhibit severe replication defects, resulting in substantial growth defects and loss of virulence. Deletion analyses revealed that IMC29 localization is largely dependent on the N-terminal half of the protein containing four predicted coiled-coil domains while IMC29 function requires a short C-terminal helical region. Using proximity labeling, we identify eight novel IMC proteins enriched in daughter buds, significantly expanding the daughter IMC proteome. We additionally report four novel proteins with unique localizations to the interface between two parasites or to the outer face of the IMC, exposing new subregions of the organelle. Together, this work establishes IMC29 as an important early daughter bud component of replication and uncovers an array of new IMC proteins that provides important insights into this organelle.
The inner membrane complex (IMC) is a conserved structure across the Apicomplexa phylum, which includes obligate intracellular parasites that cause toxoplasmosis, malaria, and cryptosporidiosis. The IMC is critical for the parasite to maintain its intracellular lifestyle, particularly in providing a scaffold for daughter bud formation during parasite replication. While many IMC proteins in the later stages of division have been identified, components of the early stages of division remain unknown. Here, we focus on the early daughter protein IMC29, demonstrating that it is crucial for faithful parasite replication and identifying specific regions of the protein that are important for its localization and function. We additionally use proximity labeling to reveal a suite of daughter-enriched IMC proteins, which represent promising candidates to further explore this IMC subcompartment.
Many novel modulated radiation treatment techniques are sensitive to patient motion which may degrade the dose distribution considerably. As there may be a simultaneous movement of the tumour and ...treatment machine, undesired heterogeneities in the dose distribution can be resulted. Methods to estimate the dosimetric effect of motion and treatment deliveries for both photons and protons are needed. We have recently studied Hodgkin's lymphoma, liver and left sided breast cancer cases and developed tools to be able to simulate simultaneous organ movement and treatment delivery. Furthermore, it is of great importance to validate potential simulations in a realistic quality control set-up, ideally including a complete dosimetry volume and movement/deformation (4D). Radiation sensitive deformable gels have the potential to meet this dosimetry challenge owing to the unique 3D characteristic to form both phantom and detector in one volume. Multi-array detectors together with a moving platform and a realistic object trajectory is an alternative to evaluate the clinical setting. The evaluation could then in principle be done on-line. Gel/plastic 3D dosimeters have the potential to also be irradiated during motion in a similar matter but have to be read-out post irradiation.
The deepwater fold and thrust belt offshore Sabah, NW Borneo, is located on a continental margin that hosts a number of proven hydrocarbon accumulations. Reprocessed and depth-migrated regional 2D ...seismic data reveal the occurrence of a wide range of compressional, syn-depositional deformation features including large-scale thrust faults and prominent thrust-hangingwall anticlines. This paper focuses on the documentation and detailed description of the style of deepwater folds and the associated stratal patterns within a study area of ca. 3000
km
2 offshore Sabah, NW Borneo. This region exhibits a variety of fold systems including (1) anticlines with large interlimb angles that exhibit no seafloor expression; (2) anticlines with medium interlimb angles, seafloor relief and a faulted anticline crest; (3) anticlines with small to medium interlimb angles, a seafloor expression and flanks associated with slides and slumps, and finally; (4) anticlines with small interlimb angles that are commonly buried by a thick sedimentary overburden and, in places, modified by superimposed syn-sedimentary, deltaic normal faults. If analysed section per section perpendicular to the strike of the fold and thrust belt, the widest anticlines occur in the most seaward parts of the fold belt near the present-day thrust front, whereas the narrower anticlines are located further landward. However, if viewed along the strike of the deepwater fold belt, there are significant variations in the geometry of laterally corresponding, contemporaneously developing fold systems. To explain these differences, factors such as rock inhomogeneities in the deformation wedge, variable surface slope, irregular basement topography, differential sediment input or the presence or absence of subsurface gas hydrates need to be taken into account. These factors vary significantly within the studied system, and are therefore difficult to generalise and transfer to other submarine fold and thrust belts.
Respiratory motion during dynamic radiotherapy may affect the absorbed dose distribution both by dose-reducing smoothing and by more complicated interplay effects. In this study we present a novel ...method to determine the relative importance of these two effects. For the two dynamic deliveries studied in this work, the expected target dose reduction due to the smoothing effect was estimated by measurements convolved by the motion function. Remaining absorbed dose differences were attributed to interplay effects between the motion of the gel phantom and the movement of the modulating MLC leaves during modulated arc radiotherapy. The total dosimetric effect due to breathing motion and dynamic MLC motion during VMAT delivery resulted in an average of about 4% target dose reduction. Comparing with only the smoothing effect, the average difference was decreased to around 1%, and the remaining distribution was attributed to interplay effects. Although the interplay effects were small compared to the smoothing effect, the standard deviations of 1.4–2.3% (1SD) were larger than the narrow distribution for repeated stationary measurement with a standard deviation between 0.5–0.9% (1SD).
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