The Cdc42p GTPase and its regulators, such as the Saccharomyces cerevisiae Cdc24p guanine-nucleotide exchange factor, control signal-transduction pathways in eukaryotic cells leading to actin ...rearrangements. A cross-species genetic screen was initiated based on the ability of negative regulators of Cdc42p to reverse the Schizosaccharomyces pombe Cdc42p suppression of a S. cerevisiae cdc24(ts) mutant. A total of 32 S. pombe nrf (negative regulator of Cdc forty two) cDNAs were isolated that reversed the suppression. One cDNA, nrf1(+), encoded an approximately 15 kD protein with three potential transmembrane domains and 78% amino-acid identity to a S. cerevisiae gene, designated NRF1. A S. pombe Deltanrf1 mutant was viable but overexpression of nrf1(+) in S. pombe resulted in dose-dependent lethality, with cells exhibiting an ellipsoidal morphology indicative of loss of polarized cell growth along with partially delocalized cortical actin and large vacuoles. nrf1(+) also displayed synthetic overdose phenotypes with cdc42 and pak1 alleles. Green fluorescent protein (GFP)-Cdc42p and GFP-Nrf1p colocalized to intracellular membranes, including vacuolar membranes, and to sites of septum formation during cytokinesis. GFP-Nrf1p vacuolar localization depended on the S. pombe Cdc24p homolog Scd1p. Taken together, these data are consistent with Nrf1p functioning as a negative regulator of Cdc42p within the cell polarity pathway.
The Saccharomyces cerevisiae Cdc24p guanine nucleotide exchange factor (GEF) activates the Cdc42p GTPase to a GTP-bound state. Cdc42p and Cdc24p co-localize at polarized growth sites during the cell ...cycle; and analysis of Cdc24p carboxyl-terminal truncation and site-specific mutations identified a 56-amino-acid domain as being necessary and sufficient for localization to these sites. This domain, however, was unable to anchor Cdc24p at these sites. Anchoring was restored by fusing the targeting domain to either the Cdc24p carboxyl-terminal PC domain that interacts with the Bem1p scaffold protein or the Cdc42p KKSKKCTIL membrane-anchoring domain. Mutant analysis and protein solubilization data indicated that anchoring required Bem1p, the Rsr1p/Bud1p GTPase, and the potential transmembrane protein YGR221Cp/Tos2p. These data are consistent with Cdc24p localization being a function of both membrane-specific targeting and subsequent anchoring within a multi-protein complex. Given the highly conserved roles of GEFs in Cdc42p signaling pathways, it is likely that similar targeting and anchoring mechanisms exist for Rho GEFs in other eukaryotes.
Abstract
The Saccharomyces cerevisiae G protein βγ dimer, Ste4p/Ste18p, acts downstream of the a subunit, Gpalp, to activate the pheromone response pathway and therefore must interact with a ...downstream effector. Synthetic sterile mutants that exacerbate the phenotype of ste4-ts mutations were isolated to identify proteins that functionally interact with Ste4p. The identification of a stel8 mutant indicated that this screen could identify proteins that interact directly with Ste4p. The other mutations were in STE5 and the STE20 kinase gene, which act near Ste4p in the pathway, and a new gene called STE21. ste20 null mutants showed residual mating, suggesting that another kinase may provide some function. Overexpression of Ste5p under galactose control activated the pheromone response pathway. This activation was dependent on Ste4p and Ste18p and partially dependent on Ste20p. These results cannot be explained by the linear pathway of Ste4p → Ste20p → Ste5p. Overexpression of Cdc42p resulted in a slight increase in pheromone induction of a reporter gene, and overexpression of activated forms of Cdc42p resulted in a further twofold increase. Mutations in pheromone response pathway components did not suppress the lethality associated with the activated CDC42 mutations, suggesting that this effect is independent of the pheromone response pathway.
Background
Chronic traumatic encephalopathy (CTE) is a neurodegenerative disease thought to be caused, in part, by repetitive head impact (RHI) exposure and characterized by phosphorylated tau ...deposition with varying patterns in the cerebral cortex. Flortaucipir tau PET data from the DIAGNOSE CTE Research Project was examined to investigate tau PET patterns and its relation to RHI exposure in former professional and college American football players.
Method
In this study, 218 DIAGNOSE CTE participants, ages 45‐74, with flortaucipir PET were included; 104 were former National Football League players (PRO), 58 were former college football players (COL), and 56 had no history of contact sports, other exposure to RHI, or traumatic brain injury (UE). Regional flortaucipir uptake was quantified using established procedures for three previously defined statistical regions (superior frontal, left parietal, and medial temporal) and compared across the three groups. Association analyses were also performed to examine the relationship between flortaucipir uptake and exposure to RHI using the cumulative head impact index for linear acceleration g‐force (CHII‐G). The potential clinical utility of flortaucipir PET for CTE was also investigated by examining its ability to differentiate participants meeting criteria for traumatic encephalopathy syndrome (TES) and probable or possible level of certainty for CTE pathology from those who do not meet those criteria.
Result
Significantly higher flortaucipir uptake was observed in former football players than in participants without RHI exposure, controlling for age and race for the superior frontal region (Fig. 1A), left parietal, and medial temporal regions. Association between regional flortaucipir uptake and CHII‐G was only observed in the superior frontal region in former players (PRO+COL) 60 years and older (Fig. 1B) and no association was observed in the full PRO+COL group. Flortaucipir PET was not able to differentiate TES groups in former football players (Fig. 1C).
Conclusion
Elevated flortaucipir uptake was observed in former American football players to a group of unexposed men. The association between flortaucipir uptake and exposure to RHI may require a long latent period to manifest. Further investigation of more sensitive tau PET tracers and quantification techniques is warranted to better characterize CTE related tau pathology.
Background
Chronic traumatic encephalopathy (CTE) is a neurodegenerative disease that is characterized by perivascular foci of phosphorylated tau (p‐tau) notably in the depths of sulci, with varying ...patterns in the cerebral cortex. Prior studies show elevated flortaucipir (FTP) PET binding in former American professional football players(PRO). However, the association of p‐tau with clinical measures remains unclear using regional FTP SUVR as early signs of CTE related tau pathology. Graph theory (GT) takes advantages of the spatial variability in p‐tau load and allows the integration of regional measures to define GT based measures. Here we examine the GT based measures in two CTE cohorts to evaluate their utility in assessing p‐tau related pathology in CTE.
Method
The graph procedure was optimized in FTP PET images in a group of 26 PRO with reported cognitive and/or behavorial symptoms and 30 asymptomatic controls without a history of traumatic brain injury (the DETECT Study) and identified the GT measure, entorhinal tau strength, as the target metric with the best group separation between PRO and controls. The optimized graph procedures were then applied to the DIAGNOSE CTE Research Project dataset (99 former PRO, 51 former college football players COL, and 52 controls without exposure to repetitive head impacts CTL) and we examined entorhinal tau strength’s ability to differentiate the exposure groups and relationship to consensus based diagnoses of Traumatic Encephalopathy Syndrome (TES), the clinical syndrome associated with CTE.
Result
Entorhinal tau strength was significantly different among the three exposure groups (p=0.0001) similar to regional SUVR measures (Figure 1). Regional SUVR did not differentiate players (PRO+COL) with and without TES, while entorhinal tau strength approached although did not reach significance (Figure 2). Entorhinal tau strength is the only FTP measure that showed significant differences (p=0.006) between those players with cognitive impairment and players without (Figure 3).
Conclusion
While there is still considerable overlap in between‐group measurements, GT based entorhinal tau strength, is potentially more sensitive in detecting tau changes in players with and without TES. Additional studies are necessary to clarify the value of this new measure in detecting underlying CTE pathology.
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3099
Background: FGFR2 genomic alterations (GA) have been described in a variety of solid tumors and emerged as biomarkers for investigational agents undergoing clinical trials. ...Methods: 201,766 primarily relapsed/refractory malignancies were evaluated with a hybrid-capture based sequencing assay Tumor mutational burden (TMB) was determined on 0.8-1.1 Mbp of sequenced DNA and reported as mut/Mb. Microsatellite instability (MSI) was determined on 114 loci. PD-L1 expression was determined by IHC (Dako 22C3 antibody). Results: FGFR2 GA were detected in 2,993 (1.5%) cases featuring short variant (SV) mut (42%), copy number changes (27%), rearrangements/fusions (28%) and multiple GA (3%). The most frequent SV GA were S252W, N549K, C382R, P253R, Y375C, K659E and R664W. A small cohort (2%) of tumors featured the V564I and V564L GA that are associated with resistance to TKI drugs. The FGFR2-altered cases were 69% female/31% male with median age of 61 yrs. Most frequent GA in FGFR2 altered cancers: TP53 (47%), PIK3CA (22%), PTEN (20%), ARID1A (18%), CDKN2A/2B (18/14%) and MYC (12%). FGFR2 SVs most common in endometrial, breast carcinomas (ca) and CUP. FGFR2 amplification most common in breast, gastroesophageal and lung ca. FGFR rearrangement/fusions most common in cholangioca (37%), CUP (15%), pancreatobiliary (12%) and breast ca (6%). The FGFR2- BICC1 was the most frequent fusion followed by fusions with TACC2, AHCYL1, CCDC6, VCL, and KIAA1217. MSI-High status present in 6.8% of evaluable FGFR2 altered cases (63% in endometrial ca). Median TMB was 3.5 mut/Mb with 21.8% featuring ≥ 10 mut/Mb and 12.0% featuring ≥ 20 mut/Mb. Only 63% of MSI-High FGFR2 mut tumors had TMB ≥ 20 mut/Mb. 12.7% FGFR2 mut+ had > 1% PD-L1 staining with 3.4% > 50% staining. 29% of PD-L1 IHC+ cases in NSCLC. FGFR mut ca’s responding to anti-FGFR2 therapies will be presented. Conclusions: FGFR2 GA are most frequent in cholangioca, breast, GI tract, lung ca and CUP, with enrichment of FGFR2 fusions in biliary tract ca. Cases with FGFR2 GA typically do not feature other kinase driver GA and are associated with mut in the MTOR/PIK3CA/AKT pathways. Finally, in contrast with RTK driver GA in EGFR (5.7%) and ERBB2 (7.9%), at 12.0%, across all tumor types, FGFR2 mut cancers may have higher frequency of TMB ≥ 20 mut/Mb suggesting potential immunotherapy responsiveness.
Signal transduction through the Rho family GTPases requires regulated cycling of the GTPases between the active GTP-bound
state and the inactive GDP-bound state. Rho family members containing an ...arginine residue at position 186 in the C-terminal
polybasic region were found to possess a self-stimulatory GTPase-activating protein (GAP) activity through homophilic interaction,
resulting in significantly enhanced intrinsic GTPase activities. This arginine residue functions effectively as an âarginine
fingerâ in the GTPase activating reaction to confer the catalytic GAP activity but is not essential for the homophilic binding
interactions of Rho family proteins. The arginine 186-mediated negative regulation seems to be absent from Cdc42, a Rho family
member important for cell-division cycle regulation, of lower eukaryotes, yet appears to be a part of the turn-off machinery
of Cdc42 from higher eukaryotes. Introduction of the arginine 186 mutation into S. cerevisiae CDC42 led to phenotypes consistent with down-regulated CDC42 function. Thus, specific Rho family GTPases may utilize a built-in arginine finger, in addition to RhoGAPs, for negative
regulation.
To evaluate the impact of active screening for methicillin-resistant Staphylococcus aureus (MRSA) on MRSA infection rates and cost avoidance in units where the risk of MRSA transmission is high.
...During a 15-month period, all patients admitted to our adult medical and surgical intensive care units (ICUs) were screened for MRSA nasal carriage on admission and weekly thereafter. The overall rates of all MRSA infections and of nosocomial MRSA infection in the 2 adult ICUs and the general wards were compared with rates during the 15-month period prior to the start of routine screening. The percentage of patients colonized or infected with MRSA on admission and the cost avoidance of the surveillance program were also assessed.
The overall rate of MRSA infections for all 3 areas combined decreased from 6.1 infections per 1,000 census-days in the preintervention period to 4.1 infections per 1,000 census-days in the postintervention period (P = .01). The decrease remained statistically significant when only nosocomial MRSA infections were examined (4.5 vs 2.8 infections per 1,000 census-days; P < .01), despite a corresponding increase during the postintervention period in the percentage of patients with onset of MRSA infection in the first 72 hours after admission to the general wards (46% to 81%; P < .005). A total of 3.7% of ICU patients were colonized or infected with MRSA on admission; MRSA would not have been detected in 91% of these patients if screening had not been performed. At a cost of Dollars 3,475/month for the program, we averted a mean of 2.5 MRSA infections/month for the ICUs combined, avoiding Dollars 19,714/month in excess cost in the ICUs.
Even in a setting of increasing community-associated MRSA, active MRSA screening as part of a multi-factorial intervention targeted to high-risk units may be an effective and cost-avoidant strategy for achieving a sustained decrease of MRSA infections throughout the hospital.
The Cdc42p GTPase regulates multiple signal transduction pathways through its interactions with downstream effectors. Specific functional domains within Cdc42p are required for guanine-nucleotide ...binding, interactions with downstream effectors, and membrane localization. However, little is known about how Cdc42p is clustered at polarized growth sites or is extracted from membranes by Rho guanine-nucleotide dissociation inhibitors (RhoGDIs) at specific times in the cell cycle. To address these points, localization studies were performed in Saccharomyces cerevisiae using green fluorescent protein (GFP)-tagged Cdc42p and the RhoGDI Rdi1p. GFP-Rdi1p localized to polarized growth sites at specific times of the cell cycle but not to other sites of Cdc42p localization. Overexpression of Rdi1p led to loss of GFP-Cdc42p from internal and plasma membranes. This effect was mediated through the Cdc42p Rho-insert domain, which was also implicated in interactions with the Bni1p scaffold protein. These data suggested that Rdi1p functions in cell cycle-specific Cdc42p membrane detachment. Additional genetic and time-lapse microscopy analyses implicated nucleotide binding in the clustering of Cdc42p. Taken together, these results provide insight into the complicated nature of the relationships between Cdc42p localization, nucleotide binding, and protein-protein interactions.
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