Therapeutic targeting of cellular senescence in diabetic macular edema: preclinical and phase 1 trial results.

Compromised vascular endothelial barrier function is a salient feature of diabetic complications such as sight-threatening diabetic macular edema (DME). Current standards of care for DME manage aspects of the disease, but require frequent intravitreal administration and are poorly effective in large subsets of patients. Here we provide evidence that an elevated burden of senescent cells in the retina triggers cardinal features of DME pathology and conduct an initial test of senolytic therapy in patients with DME. In cell culture models, sustained hyperglycemia provoked cellular senescence in subsets of vascular endothelial cells displaying perturbed transendothelial junctions associated with poor barrier function and leading to micro-inflammation. Pharmacological elimination of senescent cells in a mouse model of DME reduces diabetes-induced retinal vascular leakage and preserves retinal function. We then conducted a phase 1 single ascending dose safety study of UBX1325 (foselutoclax), a senolytic small-molecule inhibitor of BCL-xL, in patients with advanced DME for whom anti-vascular endothelial growth factor therapy was no longer considered beneficial. The primary objective of assessment of safety and tolerability of UBX1325 was achieved. Collectively, our data suggest that therapeutic targeting of senescent cells in the diabetic retina with a BCL-xL inhibitor may provide a long-lasting, disease-modifying intervention for DME. This hypothesis will need to be verified in larger clinical trials. ClinicalTrials.gov identifier: NCT04537884 .

Revealing the mechanism of action of a first-in-class covalent inhibitor of KRASG12C (ON) and other functional properties of oncogenic KRAS by 31P NMR.

Individual oncogenic KRAS mutants confer distinct differences in biochemical properties and signaling for reasons that are not well understood. KRAS activity is closely coupled to protein dynamics and is regulated through two interconverting conformations: state 1 (inactive, effector binding deficient) and state 2 (active, effector binding enabled). Here, we use 31P NMR to delineate the differences in state 1 and state 2 populations present in WT and common KRAS oncogenic mutants (G12C, G12D, G12V, G13D, and Q61L) bound to its natural substrate GTP or a commonly used nonhydrolyzable analog GppNHp (guanosine-5'-[(ß,γ)-imido] triphosphate). Our results show that GppNHp-bound proteins exhibit significant state 1 population, whereas GTP-bound KRAS is primarily (90% or more) in state 2 conformation. This observation suggests that the predominance of state 1 shown here and in other studies is related to GppNHp and is most likely nonexistent in cells. We characterize the impact of this differential conformational equilibrium of oncogenic KRAS on RAF1 kinase effector RAS-binding domain and intrinsic hydrolysis. Through a KRAS G12C drug discovery, we have identified a novel small-molecule inhibitor, BBO-8956, which is effective against both GDP- and GTP-bound KRAS G12C. We show that binding of this inhibitor significantly perturbs state 1-state 2 equilibrium and induces an inactive state 1 conformation in GTP-bound KRAS G12C. In the presence of BBO-8956, RAF1-RAS-binding domain is unable to induce a signaling competent state 2 conformation within the ternary complex, demonstrating the mechanism of action for this novel and active-conformation inhibitor.

Small-molecule inhibition of kinesin KIF18A reveals a mitotic vulnerability enriched in chromosomally unstable cancers.

Chromosomal instability (CIN) is a hallmark of cancer, caused by persistent errors in chromosome segregation during mitosis. Aggressive cancers like high-grade serous ovarian cancer (HGSOC) and triple-negative breast cancer (TNBC) have a high frequency of CIN and TP53 mutations. Here, we show that inhibitors of the KIF18A motor protein activate the mitotic checkpoint and selectively kill chromosomally unstable cancer cells. Sensitivity to KIF18A inhibition is enriched in TP53-mutant HGSOC and TNBC cell lines with CIN features, including in a subset of CCNE1-amplified, CDK4-CDK6-inhibitor-resistant and BRCA1-altered cell line models. Our KIF18A inhibitors have minimal detrimental effects on human bone marrow cells in culture, distinct from other anti-mitotic agents. In mice, inhibition of KIF18A leads to robust anti-cancer effects with tumor regression observed in human HGSOC and TNBC models at well-tolerated doses. Collectively, our results provide a rational therapeutic strategy for selective targeting of CIN cancers via KIF18A inhibition.

Longitudinal clinical phenotyping of post COVID condition in Mexican adults recovering from severe COVID-19: a prospective cohort study.

Introduction: Few studies have evaluated the presence of Post COVID-19 conditions (PCC) in people from Latin America, a region that has been heavily afflicted by the COVID-19 pandemic. In this study, we describe the frequency, co-occurrence, predictors, and duration of 23 symptoms in a cohort of Mexican patients with PCC. Methods: We prospectively enrolled and followed adult patients hospitalized for severe COVID-19 at a tertiary care centre in Mexico City. The incidence of PCC symptoms was determined using questionnaires. Unsupervised clustering of PCC symptom co-occurrence and Kaplan-Meier analyses of symptom persistence were performed. The effect of baseline clinical characteristics was evaluated using Cox regression models and reported with hazard ratios (HR). Results: We found that amongst 192 patients with PCC, respiratory problems were the most prevalent and commonly co-occurred with functional activity impairment. 56% had ≥5 persistent symptoms. Symptom persistence probability at 360 days 0.78. Prior SARS-CoV-2 vaccination and infection during the Delta variant wave were associated with a shorter duration of PCC. Male sex was associated with a shorter duration of functional activity impairment and respiratory symptoms. Hypertension and diabetes were associated with a longer duration of functional impairment. Previous vaccination accelerated PCC recovery. Discussion: In our cohort, PCC symptoms were frequent (particularly respiratory and neurocognitive ones) and persistent. Importantly, prior SARS-CoV-2 vaccination resulted in a shorter duration of PCC.

OncoVEXmGM-CSFexpands tumor antigen-specific CD8+ T-cell response in preclinical models.

BACKGROUND: Checkpoint inhibitors targeting cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1) have demonstrated clinical efficacy in advanced melanoma, but only a subset of patients with inflamed tumors are responsive. Talimogene laherparepvec (T-VEC), a modified herpes simplex virus type 1 (HSV-1) expressing granulocyte-macrophage colony-stimulating factor (GM-CSF), is a first-in-class oncolytic immunotherapy approved for the treatment of melanoma and has been shown to inflame the tumor microenvironment. To evaluate the potential and mechanisms of T-VEC to elicit systemic antitumor immunity and overcome resistance to checkpoint inhibitors in murine tumor models, OncoVEXmGM-CSF was developed similarly to T-VEC, except the human GM-CSF transgene was replaced with murine GM-CSF. Previous work had demonstrated that OncoVEXmGM-CSF generated systemic antitumor immunity dependent on CD8+ T cells in an immune checkpoint-sensitive tumor cell model. METHODS: A novel B16F10 syngeneic tumor model with both HSV-1-permissive subcutaneous tumors and HSV-1-refractory experimental lung metastasis was used to study the local and systemic effects of OncoVEXmGM-CSF treatment alone or in combination with checkpoint inhibitors. RESULTS: Intratumoral injection of OncoVEXmGM-CSF in combination with an anti-CTLA-4 or anti-PD-1 blocking antibody led to increased tumor growth inhibition, a reduction in the number of lung metastases, and prolonged animal survival. OncoVEXmGM-CSF induced both neoantigen-specific and tumor antigen-specific T-cell responses. Furthermore, cured mice from the combination treatment of OncoVEXmGM-CSF and anti-CTLA-4 antibody rejected tumor rechallenges. CONCLUSIONS: These data support the concept that T-VEC and checkpoint inhibition may be an effective combination to treat patients with advanced melanoma.

Modulation of Glucose Production by Central Insulin Requires IGF-1 Receptors in AgRP Neurons.

Similar to insulin, central administration of IGF-1 can suppress hepatic glucose production (HGP), but it is unclear whether this effect is mediated via insulin receptors (InsRs) or IGF-1 receptors (IGF-1Rs) in the brain. To this end, we used pharmacologic and genetic approaches in combination with hyperinsulinemic-euglycemic clamps to decipher the role of these receptors in mediating central effects of IGF-1 and insulin on HGP. In rats, we observed that intracerebroventricular (ICV) administration of IGF-1 or insulin markedly increased the glucose infusion rate (GIR) by >50% and suppressed HGP (P < 0.001). However, these effects were completely prevented by preemptive ICV infusion with an IGF-1R and InsR/IGF-1R hybrid (HybridR) blocking antibody. Likewise, ICV infusion of the InsR antagonist, S961, which also can bind HybridRs, interfered with the ability of central insulin, but not IGF-1, to increase the GIR. Furthermore, hyperinsulinemic clamps in mice lacking IGF-1Rs in AgRP neurons revealed ∼30% reduction in the GIR in knockout animals, which was explained by an impaired ability of peripheral insulin to completely suppress HGP (P < 0.05). Signaling studies further revealed an impaired ability of peripheral insulin to trigger ribosomal S6 phosphorylation or phosphatidylinositol (3,4,5)-trisphosphate production in AgRP neurons lacking IGF-1Rs. In summary, these data suggest that attenuation of IGF-1R signaling in the mediobasal hypothalamus, and specifically in AgRP neurons, can phenocopy impaired regulation of HGP as previously demonstrated in mice lacking InsRs in these cells, suggesting a previously unappreciated role for IGF-1Rs and/or HybridRs in the regulation of central insulin/IGF-1 signaling in glucose metabolism.

Long-Term V-EEG in Epilepsy: Chronological Distribution of Recorded Events Focused on the Differential Diagnosis of Epileptic Seizures and Psychogenic Non-Epileptic Seizures.

Differential diagnosis in epilepsy is sometimes challenging. Video-electroencephalography (V-EEG) is an essential tool in the diagnosis and management of epilepsy. The prolonged duration of V-EEG recording increases the diagnostic yield of a conventional V-EEG. The right length of monitoring for different indications is still to be established. We present a retrospective descriptive study with a sample of 50 patients with long-term V-EEG monitoring, with a mean age of 36.1 years, monitored from 2013 to 2019 at the Burgos University Hospital. The mean monitoring time was 3.6 days. Events were obtained in 76% of the patients, corresponding to epileptic seizures (ES) in 57.9% of them, with psychogenic non-epileptic seizures (PNES) in 39.5%, and with episodes of both pathologies in 2.6% of the patients. We found that the first event was highly representative, and it correlated with the rest of the events that would be recorded. Moreover, 92% of the first PNES had been captured at the end of the second day, and 89% of the first ES by the end of the third day. V-EEG for differential diagnosis between ES and PNES can be performed in hospitals without specialized epilepsy surgery units. For this indication, the duration of long-term V-EEG can be adjusted individually depending on the nature of the first event.

Pathological angiogenesis in retinopathy engages cellular senescence and is amenable to therapeutic elimination via BCL-xL inhibition.

Attenuating pathological angiogenesis in diseases characterized by neovascularization such as diabetic retinopathy has transformed standards of care. Yet little is known about the molecular signatures discriminating physiological blood vessels from their diseased counterparts, leading to off-target effects of therapy. We demonstrate that in contrast to healthy blood vessels, pathological vessels engage pathways of cellular senescence. Senescent (p16INK4A-expressing) cells accumulate in retinas of patients with diabetic retinopathy and during peak destructive neovascularization in a mouse model of retinopathy. Using either genetic approaches that clear p16INK4A-expressing cells or small molecule inhibitors of the anti-apoptotic protein BCL-xL, we show that senolysis suppresses pathological angiogenesis. Single-cell analysis revealed that subsets of endothelial cells with senescence signatures and expressing Col1a1 are no longer detected in BCL-xL-inhibitor-treated retinas, yielding a retina conducive to physiological vascular repair. These findings provide mechanistic evidence supporting the development of BCL-xL inhibitors as potential treatments for neovascular retinal disease.

AMG 757, a Half-Life Extended, DLL3-Targeted Bispecific T-Cell Engager, Shows High Potency and Sensitivity in Preclinical Models of Small-Cell Lung Cancer.

PURPOSE: Small-cell lung cancer (SCLC) is an aggressive neuroendocrine tumor with a high relapse rate, limited therapeutic options, and poor prognosis. We investigated the antitumor activity of AMG 757, a half-life extended bispecific T-cell engager molecule targeting delta-like ligand 3 (DLL3)-a target that is selectively expressed in SCLC tumors, but with minimal normal tissue expression. EXPERIMENTAL DESIGN: AMG 757 efficacy was evaluated in SCLC cell lines and in orthotopic and patient-derived xenograft (PDX) mouse SCLC models. Following AMG 757 administration, changes in tumor volume, pharmacodynamic changes in tumor-infiltrating T cells (TILs), and the spatial relationship between the appearance of TILs and tumor histology were examined. Tolerability was assessed in nonhuman primates (NHPs). RESULTS: AMG 757 showed potent and specific killing of even those SCLC cell lines with very low DLL3 expression (<1,000 molecules per cell). AMG 757 effectively engaged systemically administered human T cells, induced T-cell activation, and redirected T cells to lyse tumor cells to promote significant tumor regression and complete responses in PDX models of SCLC and in orthotopic models of established primary lung SCLC and metastatic liver lesions. AMG 757 was well tolerated with no AMG 757-related adverse findings up to the highest tested dose (4.5 mg/kg weekly) in NHP. AMG 757 exhibits an extended half-life in NHP, which is projected to enable intermittent administration in patients. CONCLUSIONS: AMG 757 has a compelling safety and efficacy profile in preclinical studies making it a viable option for targeting DLL3-expressing SCLC tumors in the clinical setting.

Thermal Shock Response of Yeast Cells Characterised by Dielectrophoresis Force Measurement.

Dielectrophoresis is an electric force experienced by particles subjected to non-uniform electric fields. Recently, several technologies have been developed focused on the use of dielectrophoretic force (DEP) to manipulate and detect cells. On the other hand, there is no such great development in the field of DEP-based cell discrimination methods. Despite the demand for methods to differentiate biological cell states, most DEP developed methods have been focused on differentiation through geometric parameters. The novelty of the present work relies upon the point that a DEP force cell measurement is used as a discrimination method, capable of detecting heat killed yeast cells from the alive ones. Thermal treatment is used as an example of different biological state of cells. It comes from the fact that biological properties have their reflection in the electric properties of the particle, in this case a yeast cell. To demonstrate such capability of the method, 279 heat-killed cells were measured and compared with alive cells data from the literature. For each cell, six speeds were taken at different points in its trajectory inside a variable non-uniform electric field. The electric parameters in cell wall conductivity, cell membrane conductivity, cell membrane permittivity of the yeast cell from bibliography explains the DEP experimental force measured. Finally, alive and heat-treated cells were distinguished based on that measure. Our results can be explained through the well-known damage of cell structure characteristics of heat-killed cells.

Characterization of Simple and Double Yeast Cells Using Dielectrophoretic Force Measurement.

Dielectrophoretic force is an electric force experienced by particles subjected to non-uniform electric fields. In recent years, plenty of dielectrophoretic force (DEP) applications have been developed. Most of these works have been centered on particle positioning and manipulation. DEP particle characterization has been left in the background. Likewise, these characterizations have studied the electric properties of particles from a qualitative point of view. This article focuses on the quantitative measurement of cells' dielectric force, specifically yeast cells. The measures are obtained as the results of a theoretical model and an instrumental method, both of which are developed and described in the present article, based on a dielectrophoretic chamber made of two V-shaped placed electrodes. In this study, 845 cells were measured. For each one, six speeds were taken at different points in its trajectory. Furthermore, the chamber design is repeatable, and this was the first time that measurements of dielectrophoretic force and cell velocity for double yeast cells were accomplished. To validate the results obtained in the present research, the results have been compared with the dielectric properties of yeast cells collected in the pre-existing literature.

AMG 176, a Selective MCL1 Inhibitor, Is Effective in Hematologic Cancer Models Alone and in Combination with Established Therapies.

The prosurvival BCL2 family member MCL1 is frequently dysregulated in cancer. To overcome the significant challenges associated with inhibition of MCL1 protein-protein interactions, we rigorously applied small-molecule conformational restriction, which culminated in the discovery of AMG 176, the first selective MCL1 inhibitor to be studied in humans. We demonstrate that MCL1 inhibition induces a rapid and committed step toward apoptosis in subsets of hematologic cancer cell lines, tumor xenograft models, and primary patient samples. With the use of a human MCL1 knock-in mouse, we demonstrate that MCL1 inhibition at active doses of AMG 176 is tolerated and correlates with clear pharmacodynamic effects, demonstrated by reductions in B cells, monocytes, and neutrophils. Furthermore, the combination of AMG 176 and venetoclax is synergistic in acute myeloid leukemia (AML) tumor models and in primary patient samples at tolerated doses. These results highlight the therapeutic promise of AMG 176 and the potential for combinations with other BH3 mimetics. SIGNIFICANCE: AMG 176 is a potent, selective, and orally bioavailable MCL1 inhibitor that induces a rapid commitment to apoptosis in models of hematologic malignancies. The synergistic combination of AMG 176 and venetoclax demonstrates robust activity in models of AML at tolerated doses, highlighting the promise of BH3-mimetic combinations in hematologic cancers.See related commentary by Leber et al., p. 1511.This article is highlighted in the In This Issue feature, p. 1494.

Late-life targeting of the IGF-1 receptor improves healthspan and lifespan in female mice.

Diminished growth factor signaling improves longevity in laboratory models, while a reduction in the somatotropic axis is favorably linked to human aging and longevity. Given the conserved role of this pathway on lifespan, therapeutic strategies, such as insulin-like growth factor-1 receptor (IGF-1R) monoclonal antibodies (mAb), represent a promising translational tool to target human aging. To this end, we performed a preclinical study in 18-mo-old male and female mice treated with vehicle or an IGF-1R mAb (L2-Cmu, Amgen Inc), and determined effects on aging outcomes. Here we show that L2-Cmu preferentially improves female healthspan and increases median lifespan by 9% (P = 0.03) in females, along with a reduction in neoplasms and inflammation (P ≤ 0.05). Thus, consistent with other models, targeting IGF-1R signaling appears to be most beneficial to females. Importantly, these effects could be achieved at advanced ages, suggesting that IGF-1R mAbs could represent a promising therapeutic candidate to delay aging.

The spanish versión of the job crafting scale / Versión española de la Escala Job Crafting

Background: A literature review reveals that there is no measure of job crafting available in Spanish. This paper presents the translation, adaptation and validation of a scale to measure job crafting behaviors (i.e. the Spanish Job Crafting Scale - SJCS; Tims, Bakker, & Derks, 2012). Methods: The scale was applied to a sample of 896 employees in Spain (52.26% women and 47.4% men). We tested the reliability and factorial validity of the 21-item instrument. Results: After confirmatory factor analysis (CFI=.858, TLI= .838, IFI= .860, RMSEA= .067), the results show a structure consisting of four factors: Increasing structural job resources; Decreasing hindering job demands; Increasing social job resources; Increasing challenging job demands. These four factors demonstrate adequate reliability and evidence of validity with others scales that refer to Engagement at Work and Proactivity. Conclusion: The questionnaire may be a useful tool for the assessment of job crafting and for future research in Spanish speaking countries (AU)

Antecedentes: una revisión de la literatura revela que no existen instrumentos de medida del job crafting en lengua española. Este artículo presenta la traducción, adaptación y validación de una escala para medir el comportamiento del job crafting (Spanish Job Crafting Scale -SJCS; Tims, Bakker, & Derks, 2012). Método: la escala fue aplicada a una muestra de 896 empleados (52,6% mujeres y 47,4% hombres). Se han testeado la fiabilidad y la validez factorial de un instrumento con 21 ítems. Resultados: después de realizar análisis confirmatorio (CFI= .858, TLI= .838, IFI= .860, RMSEA= .067), los resultados obtenidos confirman una estructura constituida por cuatro factores: Aumento de los recursos estructurales del empleo, Disminución de las demandas de trabajo, Aumento de los recursos sociales del empleo, Creciente demanda de desafíos en el trabajo. Estos cuatro factores tienen una fiabilidad adecuada y se constatan evidencias de validez con otras escalas que hacen referencia al Engagement en el trabajo y la Proactividad. Conclusiones: el cuestionario puede ser una herramienta útil para la evaluación del job crafting y para su uso en la investigación en el contexto de los países de lengua española (AU)

The Spanish version of the Job Crafting Scale.

BACKGROUND: A literature review reveals that there is no measure of job crafting available in Spanish. This paper presents the translation, adaptation and validation of a scale to measure job crafting behaviors (i.e. the Spanish Job Crafting Scale – SJCS; Tims, Bakker, & Derks, 2012). METHODS: The scale was applied to a sample of 896 employees in Spain (52.26% women and 47.4% men). We tested the reliability and factorial validity of the 21-item instrument. RESULTS: After confirmatory factor analysis (CFI=.858, TLI= .838, IFI= .860, RMSEA= .067), the results show a structure consisting of four factors: Increasing structural job resources; Decreasing hindering job demands; Increasing social job resources; Increasing challenging job demands. These four factors demonstrate adequate reliability and evidence of validity with others scales that refer to Engagement at Work and Proactivity. CONCLUSION: The questionnaire may be a useful tool for the assessment of job crafting and for future research in Spanish speaking countries.

High Frequency Data Acquisition System for Modelling the Impact of Visitors on the Thermo-Hygrometric Conditions of Archaeological Sites: A Casa di Diana (Ostia Antica, Italy) Case Study.

The characterization of the microclimatic conditions is fundamental for the preventive conservation of archaeological sites. In this context, the identification of the factors that influence the thermo-hygrometric equilibrium is key to determine the causes of cultural heritage deterioration. In this work, a characterization of the thermo-hygrometric conditions of Casa di Diana (Ostia Antica, Italy) is carried out analyzing the data of temperature and relative humidity recorded by a system of sensors with high monitoring frequency. Sensors are installed in parallel, calibrated and synchronized with a microcontroller. A data set of 793,620 data, arranged in a matrix with 66,135 rows and 12 columns, was used. Furthermore, the influence of human impact (visitors) is evaluated through a multiple linear regression model and a logistic regression model. The visitors do not affect the environmental humidity as it is very high and constant all the year. The results show a significant influence of the visitors in the upset of the thermal balance. When a tourist guide takes place, the probability that the hourly temperature variation reaches values higher than its monthly average is 10.64 times higher than it remains equal or less to its monthly average. The analysis of the regression residuals shows the influence of outdoor climatic variables in the thermal balance, such as solar radiation or ventilation.

Local Delivery of OncoVEXmGM-CSF Generates Systemic Antitumor Immune Responses Enhanced by Cytotoxic T-Lymphocyte-Associated Protein Blockade.

Purpose: Talimogene laherparepvec, a new oncolytic immunotherapy, has been recently approved for the treatment of melanoma. Using a murine version of the virus, we characterized local and systemic antitumor immune responses driving efficacy in murine syngeneic models.Experimental Design: The activity of talimogene laherparepvec was characterized against melanoma cell lines using an in vitro viability assay. Efficacy of OncoVEXmGM-CSF (talimogene laherparepvec with the mouse granulocyte-macrophage colony-stimulating factor transgene) alone or in combination with checkpoint blockade was characterized in A20 and CT-26 contralateral murine tumor models. CD8+ depletion, adoptive T-cell transfers, and Enzyme-Linked ImmunoSpot assays were used to study the mechanism of action (MOA) of systemic immune responses.Results: Treatment with OncoVEXmGM-CSF cured all injected A20 tumors and half of contralateral tumors. Viral presence was limited to injected tumors and was not responsible for systemic efficacy. A significant increase in T cells (CD3+/CD8+) was observed in injected and contralateral tumors at 168 hours. Ex vivo analyses showed these cytotoxic T lymphocytes were tumor-specific. Increased neutrophils, monocytes, and chemokines were observed in injected tumors only. Importantly, depletion of CD8+ T cells abolished all systemic efficacy and significantly decreased local efficacy. In addition, immune cell transfer from OncoVEXmGM-CSF-cured mice significantly protected from tumor challenge. Finally, combination of OncoVEXmGM-CSF and checkpoint blockade resulted in increased tumor-specific CD8+ anti-AH1 T cells and systemic efficacy.Conclusions: The data support a dual MOA for OncoVEXmGM-CSF that involves direct oncolysis of injected tumors and activation of a CD8+-dependent systemic response that clears injected and contralateral tumors when combined with checkpoint inhibition. Clin Cancer Res; 23(20); 6190-202. ©2017 AACR.

MAPK pathway inhibition induces MET and GAB1 levels, priming BRAF mutant melanoma for rescue by hepatocyte growth factor.

Therapeutic resistance is a major obstacle to achieving durable clinical responses with targeted therapies, highlighting a need to elucidate the underlying mechanisms responsible for resistance and identify strategies to overcome this challenge. An emerging body of data implicates the tyrosine kinase MET in mediating resistance to BRAF inhibitors in BRAFV600E mutant melanoma. In this study we observed a dominant role for the HGF/MET axis in mediating resistance to BRAF and MEK inhibitors in models of BRAFV600E and NRAS mutant melanoma. In addition, we showed that MAPK pathway inhibition induced rapid increases in MET and GAB1 levels, providing novel mechanistic insight into how BRAFV600E mutant melanoma is primed for HGF-mediated rescue. We also determined that tumor-derived HGF, not systemic HGF, may be required to convey resistance to BRAF inhibition in vivo and that resistance could be reversed following treatment with AMG 337, a selective MET inhibitor. In summary, these findings support the clinical evaluation of MET-directed targeted therapy to circumvent resistance to BRAF and MEK inhibitors in BRAFV600E mutant melanoma. In addition, the induction of MET following treatment with BRAF and MEK inhibitors has the potential to serve as a predictive biomarker for identifying patients best suited for MET inhibitor combination therapy.

Assessment of the Minimum Sampling Frequency to Avoid Measurement Redundancy in Microclimate Field Surveys in Museum Buildings.

Monitoring temperature and relative humidity of the environment to which artefacts are exposed is fundamental in preventive conservation studies. The common approach in setting measuring instruments is the choice of a high sampling rate to detect short fluctuations and increase the accuracy of statistical analysis. However, in recent cultural heritage standards the evaluation of variability is based on moving average and short fluctuations and therefore massive acquisition of data in slowly-changing indoor environments could end up being redundant. In this research, the sampling frequency to set a datalogger in a museum room and inside a microclimate frame is investigated by comparing the outcomes obtained from datasheets associated with different sampling conditions. Thermo-hygrometric data collected in the Sorolla room of the Pio V Museum of Valencia (Spain) were used and the widely consulted recommendations issued in UNI 10829:1999 and EN 15757:2010 standards and in the American Society of Heating, Air-Conditioning and Refrigerating Engineers (ASHRAE) guidelines were applied. Hourly sampling proved effective in obtaining highly reliable results. Furthermore, it was found that in some instances daily means of data sampled every hour can lead to the same conclusions as those of high frequency. This allows us to improve data logging design and manageability of the resulting datasheets.

Antibody-mediated neutralization of autocrine Gas6 inhibits the growth of pancreatic ductal adenocarcinoma tumors in vivo.

Gas6 and its receptors Axl, Mer and Tyro-3 (TAM) are highly expressed in human malignancy suggesting that signaling through this axis may be tumor-promoting. In pancreatic ductal adenocarcinoma (PDAC), Gas6 and the TAM receptor Axl are frequently co-expressed and their co-expression correlates with poor survival. A strategy was devised to generate fully human neutralizing antibodies against Gas6 using XenoMouse® technology. Hybridoma supernatants were selected based on their ability to inhibit Gas6 binding to the receptor Axl and block Gas6-induced Axl phosphorylation in human cells. Two purified antibodies isolated from the screened hybridomas, GMAB1 and GMAB2, displayed optimal cellular potency which was comparable to that of the soluble extracellular domain of the receptor Axl (Axl-Fc). In vivo characterization of GMAB1 was conducted using a pharmacodynamic assay that measured inhibition of Gas6-induced Akt activation in the mouse spleen. Treatment of mice with a single dose (100-1000 µg) of GMAB1 led to greater than 90% inhibition of Gas6-induced phosphorylated Akt (pAkt) for up to 72 hr. Based on the target coverage observed in the PD assay, the efficacy of GMAB1 was tested against human pancreatic adenocarcinoma xenografts. At doses of 50 µg and 150 µg, twice weekly, GMAB1 was able to inhibit 55% and 76% of tumor growth, respectively (p < 0.001 for both treatments vs. control Ig). When combined with gemcitabine, GMAB1 significantly inhibited tumor growth compared to either agent alone (p < 0.001). Together, the data suggest that Gas6 neutralization may be important as a potential strategy for the treatment of PDAC.