Analysis of CDPK1 targets identifies a trafficking adaptor complex that regulates microneme exocytosis in Toxoplasma.

Apicomplexan parasites use Ca2+-regulated exocytosis to secrete essential virulence factors from specialized organelles called micronemes. Ca2+-dependent protein kinases (CDPKs) are required for microneme exocytosis; however, the molecular events that regulate trafficking and fusion of micronemes with the plasma membrane remain unresolved. Here, we combine sub-minute resolution phosphoproteomics and bio-orthogonal labeling of kinase substrates in Toxoplasma gondii to identify 163 proteins phosphorylated in a CDPK1-dependent manner. In addition to known regulators of secretion, we identify uncharacterized targets with predicted functions across signaling, gene expression, trafficking, metabolism, and ion homeostasis. One of the CDPK1 targets is a putative HOOK activating adaptor. In other eukaryotes, HOOK homologs form the FHF complex with FTS and FHIP to activate dynein-mediated trafficking of endosomes along microtubules. We show the FHF complex is partially conserved in T. gondii, consisting of HOOK, an FTS homolog, and two parasite-specific proteins (TGGT1_306920 and TGGT1_316650). CDPK1 kinase activity and HOOK are required for the rapid apical trafficking of micronemes as parasites initiate motility. Moreover, parasites lacking HOOK or FTS display impaired microneme protein secretion, leading to a block in the invasion of host cells. Taken together, our work provides a comprehensive catalog of CDPK1 targets and reveals how vesicular trafficking has been tuned to support a parasitic lifestyle.

A positive feedback loop controls Toxoplasma chronic differentiation.

Successful infection strategies must balance pathogen amplification and persistence. In the obligate intracellular parasite Toxoplasma gondii this is accomplished through differentiation into dedicated cyst-forming chronic stages that avoid clearance by the host immune system. The transcription factor BFD1 is both necessary and sufficient for stage conversion; however, its regulation is not understood. In this study we examine five factors that are transcriptionally activated by BFD1. One of these is a cytosolic RNA-binding protein of the CCCH-type zinc-finger family, which we name bradyzoite formation deficient 2 (BFD2). Parasites lacking BFD2 fail to induce BFD1 and are consequently unable to fully differentiate in culture or in mice. BFD2 interacts with the BFD1 transcript under stress, and deletion of BFD2 reduces BFD1 protein levels but not messenger RNA abundance. The reciprocal effects on BFD2 transcription and BFD1 translation outline a positive feedback loop that enforces the chronic-stage gene-expression programme. Thus, our findings help explain how parasites both initiate and commit to chronic differentiation. This work provides new mechanistic insight into the regulation of T. gondii persistence, and can be exploited in the design of strategies to prevent and treat these key reservoirs of human infection.

Analysis of CDPK1 targets identifies a trafficking adaptor complex that regulates microneme exocytosis in Toxoplasma.

Apicomplexan parasites use Ca2+-regulated exocytosis to secrete essential virulence factors from specialized organelles called micronemes. Ca2+-dependent protein kinases (CDPKs) are required for microneme exocytosis; however, the molecular events that regulate trafficking and fusion of micronemes with the plasma membrane remain unresolved. Here, we combine sub-minute resolution phosphoproteomics and bio-orthogonal labeling of kinase substrates in Toxoplasma gondii to identify 163 proteins phosphorylated in a CDPK1-dependent manner. In addition to known regulators of secretion, we identify uncharacterized targets with predicted functions across signaling, gene expression, trafficking, metabolism, and ion homeostasis. One of the CDPK1 targets is a putative HOOK activating adaptor. In other eukaryotes, HOOK homologs form the FHF complex with FTS and FHIP to activate dynein-mediated trafficking of endosomes along microtubules. We show the FHF complex is partially conserved in T. gondii, consisting of HOOK, an FTS homolog, and two parasite-specific proteins (TGGT1_306920 and TGGT1_316650). CDPK1 kinase activity and HOOK are required for the rapid apical trafficking of micronemes as parasites initiate motility. Moreover, parasites lacking HOOK or FTS display impaired microneme protein secretion, leading to a block in the invasion of host cells. Taken together, our work provides a comprehensive catalog of CDPK1 targets and reveals how vesicular trafficking has been tuned to support a parasitic lifestyle.

Temporal and thermal profiling of the Toxoplasma proteome implicates parasite Protein Phosphatase 1 in the regulation of Ca2+-responsive pathways.

Apicomplexan parasites cause persistent mortality and morbidity worldwide through diseases including malaria, toxoplasmosis, and cryptosporidiosis. Ca2+ signaling pathways have been repurposed in these eukaryotic pathogens to regulate parasite-specific cellular processes governing the replicative and lytic phases of the infectious cycle, as well as the transition between them. Despite the presence of conserved Ca2+-responsive proteins, little is known about how specific signaling elements interact to impact pathogenesis. We mapped the Ca2+-responsive proteome of the model apicomplexan Taxoplasma gondii via time-resolved phosphoproteomics and thermal proteome profiling. The waves of phosphoregulation following PKG activation and stimulated Ca2+ release corroborate known physiological changes but identify specific proteins operating in these pathways. Thermal profiling of parasite extracts identified many expected Ca2+-responsive proteins, such as parasite Ca2+-dependent protein kinases. Our approach also identified numerous Ca2+-responsive proteins that are not predicted to bind Ca2+, yet are critical components of the parasite signaling network. We characterized protein phosphatase 1 (PP1) as a Ca2+-responsive enzyme that relocalized to the parasite apex upon Ca2+ store release. Conditional depletion of PP1 revealed that the phosphatase regulates Ca2+ uptake to promote parasite motility. PP1 may thus be partly responsible for Ca2+-regulated serine/threonine phosphatase activity in apicomplexan parasites.

A functional genomics screen identifying blood cell development genes in Drosophila by undergraduates participating in a course-based research experience.

Undergraduate students participating in the UCLA Undergraduate Research Consortium for Functional Genomics (URCFG) have conducted a two-phased screen using RNA interference (RNAi) in combination with fluorescent reporter proteins to identify genes important for hematopoiesis in Drosophila. This screen disrupted the function of approximately 3500 genes and identified 137 candidate genes for which loss of function leads to observable changes in the hematopoietic development. Targeting RNAi to maturing, progenitor, and regulatory cell types identified key subsets that either limit or promote blood cell maturation. Bioinformatic analysis reveals gene enrichment in several previously uncharacterized areas, including RNA processing and export and vesicular trafficking. Lastly, the participation of students in this course-based undergraduate research experience (CURE) correlated with increased learning gains across several areas, as well as increased STEM retention, indicating that authentic, student-driven research in the form of a CURE represents an impactful and enriching pedagogical approach.

Zebrafish expression reporters and mutants reveal that the IgSF cell adhesion molecule Dscamb is required for feeding and survival.

Down syndrome cell adhesion molecules (DSCAMs) are broadly expressed in nervous systems and play conserved roles in programmed cell death, neuronal migration, axon guidance, neurite branching and spacing, and synaptic targeting. However, DSCAMs appear to have distinct functions in different vertebrate animals, and little is known about their functions outside the retina. We leveraged the genetic tractability and optical accessibility of larval zebrafish to investigate the expression and function of a DSCAM family member, dscamb. Using targeted genome editing to create transgenic reporters and loss-of-function mutant alleles, we discovered that dscamb is expressed broadly throughout the brain, spinal cord, and peripheral nervous system, but is not required for overall structural organization of the brain. Despite the absence of obvious anatomical defects, homozygous dscamb mutants were deficient in their ability to ingest food and rarely survived to adulthood. Thus, we have discovered a novel function for dscamb in feeding behavior. The mutant and transgenic lines generated in these studies will provide valuable tools for identifying the molecular and cellular bases of these behaviors.

Long-term evaluation of cantilevered versus fixed-fixed resin-bonded fixed partial dentures for missing maxillary incisors.

OBJECTIVES: To evaluate the long-term longevity and patient-reported outcomes of two-unit cantilevered (CL2) and three-unit fixed-fixed (FF3) resin-bonded fixed partial dentures (RBFPDs) for the replacement of a maxillary permanent incisor. MATERIALS AND METHODS: Twenty-eight subjects were randomly assigned to receive either a CL2 or FF3 RBFPD placed by one operator. Prosthesis longevity was determined by clinical examination and history. Success was defined as absence of complications requiring intervention and survival as retention of the original prosthesis in mouth. Subjects' satisfaction was assessed using visual analogue scale (VAS) and oral health-related quality of life (OHRQoL) using Oral Health Impact Profile (OHIP-49). Outcomes were analysed with t-test/Mann-Whitney U test, chi-square and log-rank test at significance level α=0.05. RESULTS: Twenty-two subjects were reviewed. Thirteen of fifteen CL2 and ten of fourteen FF3 RBFPDs were examined (79.3 percent response rate) with a mean service life of 216.5±20.8months. All CL2 RBFPDs survived with no complications while only 10 percent of FF3 experienced no complications and only 50 percent of them survived (both P=0.000). CL2 had a significantly better success and survival rate than FF3 (P=0.000 and P=0.009, respectively). There was no significant difference in subjects' satisfaction and OHRQoL apart from CL2 group subjects had a higher satisfaction in cleaning of the prosthesis (84.1±13.6) than FF3 group (72.6±11.7) (P=0.05). CONCLUSIONS: Two-unit cantilevered RBFPDs were observed to have a significantly better success and survival than the FF3 design for the replacement of a maxillary incisor. Good patient-reported outcomes have been found for RBFPDs in single-tooth replacement in aesthetic zone.

Unusual maxillary lateral incisors: case reports.

Root canal morphology is often complex and the number of root canals may vary for any type of tooth. Abnormalities in the root canal morphology of maxillary lateral inciors are rare. Maxillary lateral incisors can have two root canals, even though the dental literature supports their 100% single-canal anatomy. It is vital to consider the possibility of extra root canal(s), even in teeth with a low frequency of abnormal root canal anatomy. This report presents two cases of maxillary lateral incisors with two root canals.

Micro-tensile bond testing of resin cements to dentin and an indirect resin composite.

OBJECTIVES: Micro-tensile bond strength (microTBS) evaluation and fractographic analysis were used to compare four resin cement systems (AC: All-Bond 2/Choice; RX: Single Bond/RelyX ARC; SB: Super-Bond C & B; and PF: Panavia F) in indirect composite/dentin adhesive joints. METHODS: Flat dentin surfaces were created on extracted human third molars. The resin cements were used according to the manufacturers' instructions for bonding silanized composite overlays to deep coronal dentin. 0.9x0.9 composite-dentin beams prepared from the luted specimens were stressed to failure in tension. Dentin sides of all fractured specimens were examined by scanning electron microscopy (SEM) to examine the failure modes. In group PF, morphologic features that could not be resolved at the SEM level were further validated by transmission electron microscopy (TEM) examination of the SEM specimens. RESULTS: Statistical analyses revealed significant difference (p<0.05) among microTBS and failure modes in the resin cement groups. The two groups (AC and RX) with highest microTBS failed predominantly along the composite overlay/cement interface. Cohesive failure in resin cement was primarily observed in group SB that exhibited intermediate microTBS values. In group PF with the lowest microTBS, failure occurred mostly along the dentin surface. Globular resin agglomerates seen by SEM on PF-treated dentin were distinguished from silica fillers by TEM. SIGNIFICANCE: The bond between the processed composite and the luting resin cement was the weak link in indirect composite restorations cemented with AC or RX. Super-Bond C&B exhibited intermediate tensile strength and Panavia F is less reliable when used in conjunction with a self-etching primer for bonding indirect restorations to dentin.

Longevity of two-unit cantilevered resin-bonded fixed partial dentures.

PURPOSE: To examine the clinical performance of two-unit cantilevered resin-bonded fixed partial dentures that were inserted at The Prince Philip Dental Hospital, Hong Kong by students and staff between 1992 and 1998. MATERIALS AND METHODS: A search of the Hospital computer records system revealed a list of 130 patients who had received two-unit cantilevered resin-bonded fixed partial dentures (RBFPD) placed more than 24 months previous to the review date. For each patient clinically examined, the following data were recorded: gender, age, operator, cementation date, endodontic treatment if performed, bone support, tooth mobility, the presence of shimstock contacts on abutment or pontic in intercuspal position, the presence of interproximal contacts adjacent to the prosthesis assessed by dental floss. Date of any debonds with subsequent treatment was recorded and the patient was asked qualitative questions about their prosthesis. RESULTS: 82 prostheses were placed in 69 patients and were found to have a mean service life of 36.7 +/- 15.4 months and a range of 4.3 months to 95.4 months. In total, four prostheses were reported to have debonded resulting in a clinical retention rate of 95.1%. No rotation, drifting or tipping was observed for any of the prostheses during the time of this study. Overall patients' satisfaction with RBFPDs was good with an average assessment rating of 8.2.