Combined PI3K and MAPK inhibition synergizes to suppress PDAC.

Oncogenic KRAS mutations are nearly ubiquitous in pancreatic ductal adenocarcinoma (PDAC), yet therapeutic attempts to target KRAS as well as its target MAPK pathway effectors have shown limited success due to the difficulty to pharmacologically target KRAS, inherent drug resistance in PDAC cells, and acquired resistance through activation of alternative mitogenic pathways such JAK-STAT and PI3K-AKT. While KRAS canonically drives the MAPK signaling pathway via RAF-MEK-ERK, it is also known to play a role in PI3K-AKT signaling. Our therapeutic study targeted the PI3K-AKT pathway with the drug Omipalisib (p110α/ß/δ/γ and mTORC1/2 inhibitor) in combination with MAPK pathway targeting drug Trametinib (MEK1/2 inhibitor) or SHP099-HCL (SHP099), which is an inhibitor of the KRAS effector SHP2. Western blot analysis demonstrated that application of Trametinib or SHP099 alone selectively blocked ERK phosphorylation (pERK) but failed to suppress phosphorylated AKT (pAKT) and in some instances increased pAKT levels. Conversely, Omipalisib alone successfully inhibited pAKT but failed to suppress pERK. Therefore, we hypothesized that a combination therapeutic comprised of Omipalisib with either Trametinib or SHP099 would inhibit two prominent mitogenic pathways, MEK and PI3K-AKT, to more effectively suppress pancreatic cancer. In vitro studies demonstrated that both Omipalisib/Trametinib and Omipalisib/SHP099 combination therapeutic strategies were generally more effective than treatment with each drug individually at reducing proliferation, colony formation, and cell migration compared to vehicle controls. Additionally, we found that while combination Omipalisib/SHP099 treatment reduced implanted tumor growth in vivo , the Omipalisib/Trametinib treatment was significantly more effective. Therefore, we additionally tested the Omipalisib/Trametinib combination therapeutic in the highly aggressive PKT (Ptf1a cre , LSL-Kras G12D , TGFbR2 fl/fl ) spontaneous mouse model of PDAC. We subsequently found that PKT mice treated with the Omipalisib/Trametinib combination therapeutic survived significantly longer than mice treated with either drug alone, and more than doubled the mean survival time of vehicle control mice. Altogether, our data support the importance of a dual treatment strategy targeting both MAPK and PI3K-AKT pathways.

Conformational dynamics of complement protease C1r inhibitor proteins from Lyme disease- and relapsing fever-causing spirochetes.

Borrelial pathogens are vector-borne etiological agents known to cause Lyme disease, relapsing fever, and Borrelia miyamotoi disease. These spirochetes each encode several surface-localized lipoproteins that bind components of the human complement system to evade host immunity. One borrelial lipoprotein, BBK32, protects the Lyme disease spirochete from complement-mediated attack via an alpha helical C-terminal domain that interacts directly with the initiating protease of the classical complement pathway, C1r. In addition, the B. miyamotoi BBK32 orthologs FbpA and FbpB also inhibit C1r, albeit via distinct recognition mechanisms. The C1r-inhibitory activities of a third ortholog termed FbpC, which is found exclusively in relapsing fever-causing spirochetes, remains unknown. Here, we report the crystal structure of the C-terminal domain of Borrelia hermsii FbpC to a limiting resolution of 1.5 Å. We used surface plasmon resonance and assays of complement function to demonstrate that FbpC retains potent BBK32-like anticomplement activities. Based on the structure of FbpC, we hypothesized that conformational dynamics of the complement inhibitory domains of borrelial C1r inhibitors may differ. To test this, we utilized the crystal structures of the C-terminal domains of BBK32, FbpA, FbpB, and FbpC to carry out molecular dynamics simulations, which revealed borrelial C1r inhibitors adopt energetically favored open and closed states defined by two functionally critical regions. Taken together, these results advance our understanding of how protein dynamics contribute to the function of bacterial immune evasion proteins and reveal a surprising plasticity in the structures of borrelial C1r inhibitors.

"Conformational dynamics of C1r inhibitor proteins from Lyme disease and relapsing fever spirochetes".

Borrelial pathogens are vector-borne etiological agents of Lyme disease, relapsing fever, and Borrelia miyamotoi disease. These spirochetes each encode several surface-localized lipoproteins that bind to components of the human complement system. BBK32 is an example of a borrelial lipoprotein that protects the Lyme disease spirochete from complement-mediated attack. The complement inhibitory activity of BBK32 arises from an alpha helical C-terminal domain that interacts directly with the initiating protease of the classical pathway, C1r. Borrelia miyamotoi spirochetes encode BBK32 orthologs termed FbpA and FbpB, and these proteins also inhibit C1r, albeit via distinct recognition mechanisms. The C1r-inhibitory activities of a third ortholog termed FbpC, which is found exclusively in relapsing fever spirochetes, remains unknown. Here we report the crystal structure of the C-terminal domain of B. hermsii FbpC to a limiting resolution of 1.5 Å. Surface plasmon resonance studies and assays of complement function demonstrate that FbpC retains potent BBK32-like anti-complement activities. Based on the structure of FbpC, we hypothesized that conformational dynamics of the complement inhibitory domains of borrelial C1r inhibitors may differ. To test this, we utilized the crystal structures of the C-terminal domains of BBK32, FbpA, FbpB, and FbpC to carry out 1 µs molecular dynamics simulations, which revealed borrelial C1r inhibitors adopt energetically favored open and closed states defined by two functionally critical regions. This study advances our understanding of how protein dynamics contribute to the function of bacterial immune evasion proteins and reveals a surprising plasticity in the structures of borrelial C1r inhibitors.

Borrelia miyamotoi FbpA and FbpB Are Immunomodulatory Outer Surface Lipoproteins With Distinct Structures and Functions.

Pathogens that traffic in the blood of their hosts must employ mechanisms to evade the host innate immune system, including the complement cascade. The Lyme disease spirochete, Borreliella burgdorferi, has evolved numerous outer membrane lipoproteins that interact directly with host proteins. Compared to Lyme disease-associated spirochetes, relatively little is known about how an emerging tick-borne spirochetal pathogen, Borrelia miyamotoi, utilizes surface lipoproteins to interact with a human host. B. burgdorferi expresses the multifunctional lipoprotein, BBK32, that inhibits the classical pathway of complement through interaction with the initiating protease C1r, and also interacts with fibronectin using a separate intrinsically disordered domain. B. miyamotoi encodes two separate bbk32 orthologs denoted fbpA and fbpB; however, the activities of these proteins are unknown. Here, we show that B. miyamotoi FbpA binds human fibronectin in a manner similar to B. burgdorferi BBK32, whereas FbpB does not. FbpA and FbpB both bind human complement C1r and protect a serum-sensitive B. burgdorferi strain from complement-mediated killing, but surprisingly, differ in their ability to recognize activated C1r versus zymogen states of C1r. To better understand the observed differences in C1r recognition and inhibition properties, high-resolution X-ray crystallography structures were solved of the C1r-binding regions of B. miyamotoi FbpA and FbpB at 1.9Å and 2.1Å, respectively. Collectively, these data suggest that FbpA and FbpB have partially overlapping functions but are functionally and structurally distinct. The data presented herein enhances our overall understanding of how bloodborne pathogens interact with fibronectin and modulate the complement system.

Scale-out of a Total Worker Health® approach for designing interventions to reduce teacher stress: pilot implementation evaluation.

BACKGROUND: Teachers have high rates of daily stress and the majority of available interventions are focused at the teacher-level. Yet, best practices in Total Worker Health® approaches indicate organization-level interventions identified using a participatory approach are most effective. We conducted an exploratory scale-out pilot study to examine the adoption of the Healthy Workplace Participatory Program (HWPP), an evidence-based, Total Worker Health approach to engage employees (e.g., teachers) and supervisory personnel (e.g., administrators) in the design and implementation of workplace well-being interventions within two elementary schools. METHODS: We evaluated the program both quantitatively and qualitatively collecting implementation outcome data (i.e., fidelity, acceptability, understanding, feasibility, system alignment) as well as data-driven adaptations using the Framework for Reporting Adaptations and Modifications-Expanded. Data from the first school informed scale-out adaptation of the HWPP intervention, HWPP-Education, within the second school. We compared implementation outcomes between Pilot Schools 1 and 2 to evaluate improvements in the adapted HWPP. RESULTS: Adaptations to HWPP program content and process were suggested to increase feasibility and contextual fit. Acceptability, understanding, and feasibility ratings showed statistically significant improvements comparing School 1 to School 2 which implemented the improved HWPP-Education. Furthermore, users reported adaptations including shorter meeting design and faster process were feasible within their work context. CONCLUSION: This pilot study is the first attempt to scale out the HWPP to educators, and while not intended to confirm efficacy, it showed promising results for scale-out. Results from Pilot Schools 1 and 2 suggest systematic use of quantitative and qualitative implementation data can effectively inform scale-out efforts that increase critical outcomes such as fidelity, acceptability, understanding, feasibility, system alignment, and leader engagement as well as decrease the extent of system resources needed. As such, this scale-out process may be a feasible approach on which to base large-scale implementation efforts of the HWPP among educators.

A Structural Basis for Inhibition of the Complement Initiator Protease C1r by Lyme Disease Spirochetes.

Complement evasion is a hallmark of extracellular microbial pathogens such as Borrelia burgdorferi, the causative agent of Lyme disease. Lyme disease spirochetes express nearly a dozen outer surface lipoproteins that bind complement components and interfere with their native activities. Among these, BBK32 is unique in its selective inhibition of the classical pathway. BBK32 blocks activation of this pathway by selectively binding and inhibiting the C1r serine protease of the first component of complement, C1. To understand the structural basis for BBK32-mediated C1r inhibition, we performed crystallography and size-exclusion chromatography-coupled small angle X-ray scattering experiments, which revealed a molecular model of BBK32-C in complex with activated human C1r. Structure-guided site-directed mutagenesis was combined with surface plasmon resonance binding experiments and assays of complement function to validate the predicted molecular interface. Analysis of the structures shows that BBK32 inhibits activated forms of C1r by occluding substrate interaction subsites (i.e., S1 and S1') and reveals a surprising role for C1r B loop-interacting residues for full inhibitory activity of BBK32. The studies reported in this article provide for the first time (to our knowledge) a structural basis for classical pathway-specific inhibition by a human pathogen.

Recrystallization and damage of ice in winter sports.

Ice samples, after sliding against a steel runner, show evidence of recrystallization and microcracking under the runner, as well as macroscopic cracking throughout the ice. The experiments that produced these ice samples are designed to be analogous to sliding in the winter sport of skeleton. Changes in the ice fabric are shown using thick and thin sections under both diffuse and polarized light. Ice drag is estimated as 40-50% of total energy dissipation in a skeleton run. The experimental results are compared with visual inspections of skeleton tracks, and to similar behaviour in rocks during sliding on earthquake faults. The results presented may be useful to athletes and designers of winter sports equipment.This article is part of the themed issue 'Microdynamics of ice'.

American Indian adolescent girls: vulnerability to sex trafficking, intervention strategies.

The Minnesota Indian Women's Resource Center offers harm reduction programming to at-risk adolescent American Indian girls, including outreach, case management, advocacy, healthy sexuality education, and support groups. To evaluate program impact, participants are assessed at intake and every 6 months afterward for current vulnerability to commercial sexual exploitation, violence, and addiction. Evaluation results indicate frequent exposure to sex traffickers and suggest that harm reduction methods can help girls reduce risk of commercial sexual exploitation.