KRAS is vulnerable to reversible switch-II pocket engagement in cells.

Current small-molecule inhibitors of KRAS(G12C) bind irreversibly in the switch-II pocket (SII-P), exploiting the strong nucleophilicity of the acquired cysteine as well as the preponderance of the GDP-bound form of this mutant. Nevertheless, many oncogenic KRAS mutants lack these two features, and it remains unknown whether targeting the SII-P is a practical therapeutic approach for KRAS mutants beyond G12C. Here we use NMR spectroscopy and a cellular KRAS engagement assay to address this question by examining a collection of SII-P ligands from the literature and from our own laboratory. We show that the SII-Ps of many KRAS hotspot (G12, G13, Q61) mutants are accessible using noncovalent ligands, and that this accessibility is not necessarily coupled to the GDP state of KRAS. The results we describe here emphasize the SII-P as a privileged drug-binding site on KRAS and unveil new therapeutic opportunities in RAS-driven cancer.

Gender Norms and HIV Testing/Treatment Uptake: Evidence from a Large Population-Based Sample in South Africa.

How does the endorsement of different dimensions of gender norms by men and/or women influence their use of HIV testing and antiretroviral treatment? This question was examined using data from a 2014 population-based survey of 1053 women and 1004 men, ages 18-49, in rural South Africa. We used a global measure for views toward gender norms (the GEM Scale), plus four subsets of scale items (all reliabilities ≥ 0.7). In multivariate analyses using the global measure, endorsement of inequitable gender norms was associated with more testing (AOR 2.47, p < 0.01) and less treatment use (AOR 0.15, p < 0.01) among women but not men. When examining specific subsets of inequitable norms (e.g., endorsing men as the primary decision-maker), decreased odds of treatment use was found for men as well (AOR 0.18, p < 0.01). Careful attention to the role specific gender norms play in HIV service uptake can yield useful programmatic recommendations.

Reductive Elimination from Phosphine-Ligated Alkylpalladium(II) Amido Complexes To Form sp3 Carbon-Nitrogen Bonds.

We report the formation of phosphine-ligated alkylpalladium(II) amido complexes that undergo reductive elimination to form alkyl-nitrogen bonds and a combined experimental and computational investigation of the factors controlling the rates of these reactions. The free-energy barriers to reductive elimination from t-Bu3P-ligated complexes were significantly lower (ca. 3 kcal/mol) than those previously reported from NHC-ligated complexes. The rates of reactions from complexes containing a series of electronically and sterically varied anilido ligands showed that the reductive elimination is slower from complexes of less electron-rich or more sterically hindered anilido ligands than from those containing more electron-rich and less hindered anilido ligands. Reductive elimination of alkylamines also occurred from complexes bearing bidentate P,O ligands. The rates of reactions of these four-coordinate complexes were slower than those for reactions of the three-coordinate, t-Bu3P-ligated complexes. The calculated pathway for reductive elimination from rigid, 2-methoxyarylphosphine-ligated complexes does not involve initial dissociation of the oxygen. Instead, reductive elimination is calculated to occur directly from the four-coordinate complex in concert with a lengthening of the Pd-O bond. To investigate this effect experimentally, a four-coordinate Pd(II) anilido complex containing a flexible, aliphatic linker between the P and O atoms was synthesized. Reductive elimination from this complex was faster than that from the analogous complex containing the more rigid, aryl linker. The flexible linker enables full dissociation of the ether ligand during reductive elimination, leading to the faster reaction of this complex.

Multi-institutional competing risks analysis of distant brain failure and salvage patterns after upfront radiosurgery without whole brain radiotherapy for brain metastasis.

Background: In this study, we use a competing risks analysis to assess factors predictive of early-salvage whole brain radiotherapy (WBRT) and early death after upfront stereotactic radiosurgery (SRS) alone for brain metastases in an attempt to identify populations that benefit less from upfront SRS. Patients and methods: Patients from eight academic centers were treated with SRS for brain metastasis. Competing risks analysis was carried out for distant brain failure (DBF) versus death prior to DBF as well as for salvage SRS versus salvage WBRT versus death prior to salvage. Linear regression was used to determine predictors of the number of brain metastases at initial DBF (nDBF). Results: A total of 2657 patients were treated with upfront SRS alone. Multivariate analysis (MVA) identified an increased hazard of DBF associated with increasing number of brain metastases (P < 0.001), lowest SRS dose received (P < 0.001), and melanoma histology (P < 0.001), while there was a decreased hazard of DBF associated with increasing age (P < 0.001), KPS < 70 (P < 0.001), and progressive systemic disease (P = 0.004). MVA for first salvage SRS versus WBRT versus death prior to salvage revealed an increased hazard of first salvage WBRT seen with increasing number of brain metastases (P < 0.001) and a decreased hazard with widespread systemic disease (P = 0.002) and increasing age (P < 0.001). Variables associated with nDBF included age (P = 0.02), systemic disease status (P = 0.03), melanoma histology (P = 0.05), and initial number of brain metastases (P < 0.001). Conclusions: Patients with a higher initial number of brain metastases were more likely to experience DBF, have a higher nDBF, and receive early-salvage WBRT, while patients who were older, had lower KPS, or had more systemic disease were more likely to experience death prior to DBF or salvage WBRT.

Use of Mohr diagrams to predict fracturing in rock masses, with applications for predicting sub-surface behavior.

Mohr diagrams are a simple and effective method that can help geoscientists consider, model, and predict the ranges of mechanical properties of rock, stresses, fluid pressures, and the resultant fractures that are likely to occur in the sub-surface. Mohr diagrams can be used to make predictions about how rocks may respond to change, with a transition from a stable state to fracturing occurring if there are changes in (1) the failure envelope, (2) stresses, and/or (3) fluid pressure. This article uses Mohr diagrams to address two questions of significance to the energy transition. First, how will metasedimentary rocks, which are potential geothermal reservoir rocks, respond to thermal stimulation? Second, will fractures that may influence the underground storage of radioactive waste develop in a clay sequence during exhumation? Mohr diagrams are shown to be useful for highlighting misconceptions and input data problems, leading to improved understanding of how structures develop.

Drugging the Next Undruggable KRAS Allele-Gly12Asp.

Since its discovery as the first human oncogene in 1983, the small GTPase KRAS has been a major target of cancer drug discovery. The paper reported in this issue describes a long-awaited small molecule drug candidate of the oncogenic KRAS (G12D) mutant for the treatment of currently incurable pancreatic cancer.

Probing the KRas Switch II Groove by Fluorine NMR Spectroscopy.

While there has been recent success in the development of KRasG12C inhibitors, unmet needs for selective inhibitors of KRasG12D and the remaining oncogenic KRas proteins remain. Here, we applied trifluoromethyl-containing ligands of KRas proteins as competitive probe ligands to assay the occupancy of the switch II pocket by 19F NMR spectroscopy. Structure-activity-relationship studies of probe ligands increased the sensitivity of the assay and identified structures that differentially detected each nucleotide state of KRasG12D. These differences in selectivity, combined with the high resolution of 19F NMR spectroscopy, enabled this method to be expanded to assay both nucleotide states of the protein simultaneously.

Angiogenesis inhibition suppresses collagen arthritis.

Neovascularization is observed in a spectrum of diseases such as solid tumors, diabetic retinopathy, and rheumatoid arthritis. It is also evident in rat collage-induced arthritis (CIA), an animal model with histologic, clinical, and radiographic manifestations resembling rheumatoid arthritis. To evaluate the effects of angioinhibition in CIA, Louvain rats were immunized with type II collagen to induce arthritis and then administered an angiogenesis inhibitor, AGM-1470, in an attempt to either prevent arthritis or suppress established disease. Using clinical and radiographic criteria, AGM-1470 prevented CIA and significantly suppressed established disease without evidence of immunosuppression. Histologic sections from control ankle joints manifested pannus and neovascularization, which were absent in experimental animals. This is the first study to investigate this novel agent in an autoimmune disease, and additional evaluation of this promising compound in other diseases that are potentially angiogenesis dependent, such as rheumatoid arthritis, might be warranted.

Evidence of infection with Toxoplasma gondii and Neospora caninum in South Australia: using wild rabbits as a sentinel species.

OBJECTIVE: The aim of this study was to utilise wild rabbits (Oryctolagus cuniculus) as a sentinel species to study levels of environmental contamination with N. caninum and T. gondii in South Australia, and to examine associations with rainfall, climate and land use. DESIGN: Toxoplasma gondii (T. gondii), an apicomplexan parasite, causes the clinical disease toxoplasmosis, which can affect sheep as well as humans and many other animals. Neosporosis, the clinical disease caused by closely related Neospora caninum (N. caninum), causes abortions in cattle, with large economic impacts to cattle industries. METHODS: Aliquots of wild rabbit (Oryctolagus cuniculus) serum were obtained from twelve sites across South Australia over a period of eighteen years, with a total of 2114 samples. An in-house Modified Agglutination Test (MAT) was developed, and samples were screened for the specific antibodies against both T.gondii and N. caninum. RESULTS: Overall, 9.9% of samples screened for T. gondii tested positive and 6.1% of samples screened for N. caninum tested positive. There was no difference observed in seroprevalence of T.gondii specific antibodies between samples collected throughout summer, autumn, winter or spring. By contrast, a significantly higher (p=0.030) seroprevalence of N. caninum specific antibodies was observed in spring than any other season. T. gondii and N. caninum antibodies were discovered at sites across a broad area of South Australia, indicating these environments maybe infected with both parasites. CONCLUSION: Results provide evidence that suggests N. caninum oocysts may have different survival characteristics, such as varying tolerances to low relative humidity, than T. gondii oocysts.

GTP-State-Selective Cyclic Peptide Ligands of K-Ras(G12D) Block Its Interaction with Raf.

We report the identification of three cyclic peptide ligands of K-Ras(G12D) using an integrated in vitro translation-mRNA display selection platform. These cyclic peptides show preferential binding to the GTP-bound state of K-Ras(G12D) over the GDP-bound state and block Ras-Raf interaction. A co-crystal structure of peptide KD2 with K-Ras(G12D)·GppNHp reveals that this peptide binds in the Switch II groove region with concomitant opening of the Switch II loop and a 40° rotation of the α2 helix, and that a threonine residue (Thr10) on KD2 has direct access to the mutant aspartate (Asp12) on K-Ras. Replacing this threonine with non-natural amino acids afforded peptides with improved potency at inhibiting the interaction between Raf1-RBD and K-Ras(G12D) but not wildtype K-Ras. The union of G12D over wildtype selectivity and GTP state/GDP state selectivity is particularly desirable, considering that oncogenic K-Ras(G12D) exists predominantly in the GTP state in cancer cells, and wildtype K-Ras signaling is important for the maintenance of healthy cells.

No Evidence of Toxoplasma Gondii Exposure in South Australian Koalas (Phascolarctos cinereus).

Infection with the cat-borne parasite Toxoplasma gondii has been detected in numerous Australian marsupials and can lead to severe disease (toxoplasmosis) in some cases. The seroprevalence of Toxoplasma on Kangaroo Island, South Australia has been reported to be higher than the South Australian mainland in macropods, cats, and sheep, suggesting an increased risk of infection on this island. However, Toxoplasma seroprevalence in small- and medium-sized terrestrial mammals was almost zero on the island and did not differ from that on the mainland. We surveyed Toxoplasma seroprevalence in koala (Phascolarctos cinereus) populations on the island and on the mainland and assessed their risk of infection and their role in the life cycle of Toxoplasma. All screened koalas from the island (n = 94) and the mainland (n = 63) were seronegative. This represents the largest Toxoplasma seroprevalence survey in this species and provided sufficient evidence to confidently demonstrate freedom from parasite exposure in both island and mainland populations at the time of the survey. Because koalas are extensively arboreal and predominately consume tree foliage, they appear to be at negligible risk of Toxoplasma infection. Furthermore, as koalas are rarely consumed by cats, we suggest that they have a minor role in the parasite's life cycle.

Velocity of radial expansion of contrast-enhancing gliomas and the effectiveness of radiotherapy in individual patients: a proof of principle.

AIMS: The initial aims were to use recently available observations of glioblastomas (as part of a previous study) that had been imaged twice without intervening treatment before receiving radiotherapy in order to obtain quantitative measures of glioma growth and invasion according to a new bio-mathematical model. The results were so interesting as to raise the question whether the degree of radio-sensitivity of each tumour could be estimated by comparing the model-predicted and actual durations of survival and total numbers of glioma cells after radiotherapy. MATERIALS AND METHODS: The gadolinium-enhanced T1-weighted and T2-weighted magnetic resonance imaging volumes were segmented and used to calculate the velocity of radial expansion (v) and the net rates of proliferation (rho) and invasion/dispersal (D) for each patient according to the bio-mathematical model. RESULTS: The ranges of the values of v, D and rho show that glioblastomas, although clustering at the high end of rates, vary widely one from the other. The effects of X-ray therapy varied from patient to patient. About half survived as predicted without treatment, indicating radio-resistance of these tumours. The other half survived up to about twice as long as predicted without treatment and could have had a corresponding loss of glioma cells, indicating some degree of radio-sensitivity. These results approach the historical estimates that radiotherapy can double survival of the average patient with a glioblastoma. CONCLUSIONS: These cases are among the first for which values of v, D and rho have been calculated for glioblastomas. The results constitute a 'proof of principle' by combining our bio-mathematical model for glioma growth and invasion with pre-treatment imaging observations to provide a new tool showing that individual glioblastomas may be identified as having been radio-resistant or radio-sensitive.

Palladium-Catalyzed Cross Coupling of Secondary and Tertiary Alkyl Bromides with a Nitrogen Nucleophile.

We report a new class of catalytic reaction: the thermal substitution of a secondary and or tertiary alkyl halide with a nitrogen nucleophile. The alkylation of a nitrogen nucleophile with an alkyl halide is a classical method for the construction of C-N bonds, but traditional substitution reactions are challenging to achieve with a secondary and or tertiary alkyl electrophile due to competing elimination reactions. A catalytic process could address this limitation, but thermal, catalytic coupling of alkyl halides with a nitrogen nucleophile and any type of catalytic coupling of an unactivated tertiary alkyl halide with a nitrogen nucleophile are unknown. We report the coupling of unactivated secondary and tertiary alkyl bromides with benzophenone imines to produce protected primary amines in the presence of palladium ligated by the hindered trialkylphosphine Cy2t-BuP. Mechanistic studies indicate that this amination of alkyl halides occurs by a reversible reaction to form a free alkyl radical.

The horizontal equity of health care in New Zealand.

OBJECTIVE: Given that 'equal access for equal need' is a clearly articulated goal of the New Zealand public health system, this study is an attempt to determine if access to public health care services in New Zealand is, for people of equal health need, independent of income. METHOD: Information on health status, income and health service utilisation for just over 6,000 New Zealanders was obtained from the national Household Health Survey 1992-93. Using standardised expenditure concentration curves and a concentration index, the distribution of health service use by individuals in different income groups, as a proxy for access, was illustrated and quantified. RESULTS: The results suggest either appropriate or slightly excess use of services by the poor given their estimated health need. Due to analytical problems caused by data deficiencies, these results must be regarded as tentative. CONCLUSION: For the period under study, no evidence was found to indicate significant access barriers to publicly funded health care for people on different incomes. This study has served to demonstrate one approach to measuring inequality and analysing the relationship between inequality and inequity. Given the reforms to the health sector since 1993, ongoing monitoring of equity of access to health care services is essential. IMPLICATIONS: Given the income-related disparities in health that do exist, the public health community should endeavour to develop techniques to monitor the delivery of publicly funded health care to ensure that further inequity is not borne by the poor.

Were NZ's structural changes to the welfare state in the early 1990s associated with a measurable increase in oral health inequalities among children?

OBJECTIVE: To test the hypothesis that the 1990-91 social and economic policy changes in New Zealand were associated with a subsequent increase in socioeconomic and ethnic inequalities in the dental caries experience of five-year-old children. METHOD: Dental caries data from the School Dental Service treating the greater Wellington area were analysed for the period 1995-2000. Multivariate models were developed for deciduous caries prevalence (logistic regression) and severity (negative binomial regression). RESULTS: In the years 1995, 1996, 1997, 1998, 1999 and 2000, complete data were available for 2,627, 3,335, 4,404, 4,155, 3,154 and 2,804 children, respectively. Ethnic and socio-economic differences in caries prevalence and severity were substantial and persistent during the observation period. Where caries severity was concerned, there was a significant interaction between time and Maori ethnicity, indicating that (on average) the oral health of Maori children deteriorated in comparison to their European counterparts. CONCLUSIONS: The early-1990s social and economic policy changes were associated with an apparent widening of ethnic inequalities in caries severity among five-year-old children. IMPLICATIONS: Economic rationalism appears to have oral health disadvantages for non-European children. Before implementation of proposed major social and economic policy changes, policymakers should consider their health implications.

Plesiomonas shigelloides septicemia and meningitis in a neonate.

A newborn infant is described who presented with septicemia and meningoencephalitis caused by Plesiomonas shigelloides, a Gram-negative rod belonging to the family Vibrionaceae. This appears to be the first documented case in a neonate in Canada. Despite prompt treatment with appropriate antibiotics, he developed endophthalmitis and lytic brain lesions.