Computational Mutagenesis of Antibody Fragments: Disentangling Side Chains from ΔΔG Predictions.

The development of highly potent antibodies and antibody fragments as binding agents holds significant implications in fields such as biosensing and biotherapeutics. Their binding strength is intricately linked to the arrangement and composition of residues at the binding interface. Computational techniques offer a robust means to predict the three-dimensional structure of these complexes and to assess the affinity changes resulting from mutations. Given the interdependence of structure and affinity prediction, our objective here is to disentangle their roles. We aim to evaluate independently six side-chain reconstruction methods and ten binding affinity estimation techniques. This evaluation was pivotal in predicting affinity alterations due to single mutations, a key step in computational affinity maturation protocols. Our analysis focuses on a data set comprising 27 distinct antibody/hen egg white lysozyme complexes, each with crystal structures and experimentally determined binding affinities. Using six different side-chain reconstruction methods, we transformed each structure into its corresponding mutant via in silico single-point mutations. Subsequently, these structures undergo minimization and molecular dynamics simulation. We therefore estimate ΔΔG values based on the original crystal structure, its energy-minimized form, and the ensuing molecular dynamics trajectories. Our research underscores the critical importance of selecting reliable side-chain reconstruction methods and conducting thorough molecular dynamics simulations to accurately predict the impact of mutations. In summary, our study demonstrates that the integration of conformational sampling and scoring is a potent approach to precisely characterizing mutation processes in single-point mutagenesis protocols and crucial for computational antibody design.

Antibody-Antigen Binding Interface Analysis in the Big Data Era.

Antibodies have become the Swiss Army tool for molecular biology and nanotechnology. Their outstanding ability to specifically recognise molecular antigens allows their use in many different applications from medicine to the industry. Moreover, the improvement of conventional structural biology techniques (e.g., X-ray, NMR) as well as the emergence of new ones (e.g., Cryo-EM), have permitted in the last years a notable increase of resolved antibody-antigen structures. This offers a unique opportunity to perform an exhaustive structural analysis of antibody-antigen interfaces by employing the large amount of data available nowadays. To leverage this factor, different geometric as well as chemical descriptors were evaluated to perform a comprehensive characterization.

Clotting factor genes are associated with preeclampsia in high-altitude pregnant women in the Peruvian Andes.

Preeclampsia is a multi-organ complication of pregnancy characterized by sudden hypertension and proteinuria that is among the leading causes of preterm delivery and maternal morbidity and mortality worldwide. The heterogeneity of preeclampsia poses a challenge for understanding its etiology and molecular basis. Intriguingly, risk for the condition increases in high-altitude regions such as the Peruvian Andes. To investigate the genetic basis of preeclampsia in a population living at high altitude, we characterized genome-wide variation in a cohort of preeclamptic and healthy Andean families (n = 883) from Puno, Peru, a city located above 3,800 meters of altitude. Our study collected genomic DNA and medical records from case-control trios and duos in local hospital settings. We generated genotype data for 439,314 SNPs, determined global ancestry patterns, and mapped associations between genetic variants and preeclampsia phenotypes. A transmission disequilibrium test (TDT) revealed variants near genes of biological importance for placental and blood vessel function. The top candidate region was found on chromosome 13 of the fetal genome and contains clotting factor genes PROZ, F7, and F10. These findings provide supporting evidence that common genetic variants within coagulation genes play an important role in preeclampsia. A selection scan revealed a potential adaptive signal around the ADAM12 locus on chromosome 10, implicated in pregnancy disorders. Our discovery of an association in a functional pathway relevant to pregnancy physiology in an understudied population of Native American origin demonstrates the increased power of family-based study design and underscores the importance of conducting genetic research in diverse populations.

Analysis of changes of cavity volumes in predefined directions of protein motions and cavity flexibility.

Dynamics of protein cavities associated with protein fluctuations and conformational plasticity is essential for their biological function. NMR ensembles, molecular dynamics (MD) simulations, and normal mode analysis (NMA) provide appropriate frameworks to explore functionally relevant protein dynamics and cavity changes relationships. Within this context, we have recently developed analysis of null areas (ANA), an efficient method to calculate cavity volumes. ANA is based on a combination of algorithms that guarantees its robustness against numerical differentiations. This is a unique feature with respect to other methods. Herein, we present an updated and improved version that expands it use to quantify changes in cavity features, like volume and flexibility, due to protein structural distortions performed on predefined biologically relevant directions, for example, directions of largest contribution to protein fluctuations (principal component analysis [PCA modes]) obtained by MD simulations or ensembles of NMR structures, collective NMA modes or any other direction of motion associated with specific conformational changes. A web page has been developed where its facilities are explained in detail. First, we show that ANA can be useful to explore gradual changes of cavity volume and flexibility associated with protein ligand binding. Secondly, we perform a comparison study of the extent of variability between protein backbone structural distortions, and changes in cavity volumes and flexibilities evaluated for an ensemble of NMR active and inactive conformers of the epidermal growth factor receptor structures. Finally, we compare changes in size and flexibility between sets of NMR structures for different homologous chains of dynein.

Baseline and 1-year follow-up differences between hip-fracture patients admitted from nursing homes and the community. A cohort study on 509 consecutive patients (FONDA Cohort).

OBJECTIVE: To determine the clinical and functional differences at hospital admission and at 1 year after a hip fracture (HF) in nursing homes (NH) and community-dwelling (CD) patients. METHODS: All patients with HF admitted to the orthogeriatric unit at a university hospital between January 2013 and February 2014 were prospectively included. Clinical and functional variables, and mortality were recorded during the hospital admission. The patients were contacted by telephone at 1 year to determine their vital condition and functional status. RESULTS: A total of 509 patients were included, 116 (22.8%) of whom came from NH. Compared with the CD patients, the NH patients had higher surgical risk (ASA ≥3: 83.6% vs. 66.4%, P<.001), poorer theoretical vital prognosis (Nottingham Profile ≥5: 98.3% vs. 56.6%, P<.001), higher rate of previous functional status (median Barthel index: 55 [IQR, 36-80] vs. 90 [IQR, 75-100], P<.001), poorer mental status (Pfeiffer's SPMSQ>2: 74.1% vs. 40.2%, P<.001), and a higher rate of sarcopenia (24.3% vs. 15.2%, P<.05). There were no differences in in-hospital or at 1-year mortality. At 1 year, NH patients recovered their previous walking capacity at a lower rate (38.5% vs. 56.2%, P<.001). CONCLUSIONS: Among the patients with HF treated in an orthogeriatric unit, NH patients had higher, surgical risk, functional and mental impairment, and a higher rate of sarcopenia than CD patients. At 1 year of follow-up, NH patients did not have higher mortality, but they recovered their previous capacity for walking less frequently.

Dynamics fingerprints of active conformers of epidermal growth factor receptor kinase.

Epidermal growth factor receptor (EGFR) is a prototypical cell-surface receptor that plays a key role in the regulation of cellular signaling, proliferation and differentiation. Mutations of its kinase domain have been associated with the development of a variety of cancers and, therefore, it has been the target of drug design. Single amino acid substitutions (SASs) in this domain have been proven to alter the equilibrium of pre-existing conformer populations. Despite the advances in structural descriptions of its so-called active and inactive conformations, the associated dynamics aspects that characterize them have not been thoroughly studied yet. As the dynamic behaviors and molecular motions of proteins are important for a complete understanding of their structure-function relationships we present a novel procedure, using (or based on) normal mode analysis, to identify the collective dynamics shared among different conformers in EGFR kinase. The method allows the comparison of patterns of low-frequency vibrational modes defining representative directions of motions. Our procedure is able to emphasize the main similarities and differences between the collective dynamics of different conformers. In the case of EGFR kinase, two representative directions of motions have been found as dynamics fingerprints of the active conformers. Protein motion along both directions reveals to have a significant impact on the cavity volume of the main pocket of the active site. Otherwise, the inactive conformers exhibit a more heterogeneous distribution of collective motions. © 2018 Wiley Periodicals, Inc.

Low intensity sonosynthesis of iron carbide@iron oxide core-shell nanoparticles.

Here we demonstrate a simple method for the organic sonosynthesis of stable Iron Carbide@Iron Oxide core-shell nanoparticles (ICIONPs) stabilized by oleic acid surface modification. This robust synthesis route is based on the sonochemistry reaction of organometallic precursor like Fe(CO)5 in octanol using low intensity ultrasonic bath. As obtained, nanoparticles diameter sizes were measured around 6.38 nm ±â€¯1.34 with a hydrodynamic diameter around 25 nm and an estimated polydispersity of 0.27. Core-Shell structure of nanoparticles was confirmed using HR-TEM and XPS characterization tools in which a core made up of iron carbide (Fe3C) and a shell of magnetite (γ-Fe2O3) was found. The overall nanoparticle presented ferromagnetic behavior at 4 K by SQUID. With these characteristics, the ICIONPs can be potentially used in various applications such as theranostic agent due to their properties obtained from the iron oxides and iron carbide phases.

Diagnóstico de vértigo periférico para el médico de atención primaria / Diagnosis of peripheral vertigo for primary care physicians / Diagnóstico de vertigem periférica para o médico de atenção primária

Resumen Para los médicos de atención primaria, constituye un desafío el paciente con sintomatologia compatible con síndromes vestibulares, debido a su amplia variedad de presentaciones y la dificultad en la diferenciación de las diversas patologías que ocasionan los síntomas. La necesidad de tener conceptos claros y actualizados en cuanto a los mecanismos patológicos del vértigo surge en el momento de evaluar al paciente. Para esto, es necesario realizar un adecuado interrogatorio y un examen físico neurotológico completo que permita un abordaje diagnóstico certero. El objetivo del presente artículo es presentar una revisión de la literatura actual, de las diferentes etiologías desencadenantes del vértigo periférico y, a partir de sus características clínicas, desarrollar un algoritmo diagnóstico que permita al profesional médico entender el mecanismo patológico del vértigo y proporcionar el tratamiento idóneo.

Summary For primary care physicians, a patient with symptoms compatible with vestibular syndromes is a challenge, due to its wide variety of presentations and the difficulty in differentiating the various pathologies that cause the symptoms. The need to have clear and up-to-date concepts regarding the pathological mechanisms of vertigo arises when evaluating the patient; for this, it is necessary to carry out an adequate interrogation and a complete neurological physical examination that allows for an accurate diagnostic approach. The objective of this article is to present a review of the current literature and of the different etiologies that trigger peripheral vertigo, as well as, based on the clinical characteristics, develop a diagnostic algorithm that allows the medical professional to understand the pathological mechanism of vertigo and provide appropriate treatment.

Resumo Para os médicos de atenção primária, constitui um desafio o paciente com sintomatologia compatível com síndromes vestibulares, devido a sua ampla variedade de apresentaçöes e a dificuldade na diferenciação das diversas patologias que ocasionam os sintomas. A necessidade de ter conceitos claros e atualizados em quanto aos mecanismos patológicos da vertigem surge no momento de avaliar ao paciente. Para isto, é necessário realizar um adequado interrogatório e um exame físico neurotológico completo que permita uma abordagem diagnóstica certeira. O objetivo do presente artigo é apresentar uma revisão da literatura atual, das diferentes etiologias desencadeantes da vertigem periférica e, a partir de sus características clínicas, desenvolver um algoritmo diagnóstico que permita ao profissional médico entender o mecanismo patológico da vertigem e proporcionar o tratamento idôneo.

Clinical Practice Guideline: Hoarseness (Dysphonia) (Update) Executive Summary.

Objective This guideline provides evidence-based recommendations on treating patients presenting with dysphonia, which is characterized by altered vocal quality, pitch, loudness, or vocal effort that impairs communication and/or quality of life. Dysphonia affects nearly one-third of the population at some point in its life. This guideline applies to all age groups evaluated in a setting where dysphonia would be identified or managed. It is intended for all clinicians who are likely to diagnose and treat patients with dysphonia. Purpose The primary purpose of this guideline is to improve the quality of care for patients with dysphonia, based on current best evidence. Expert consensus to fill evidence gaps, when used, is explicitly stated and supported with a detailed evidence profile for transparency. Specific objectives of the guideline are to reduce inappropriate variations in care, produce optimal health outcomes, and minimize harm. For this guideline update, the American Academy of Otolaryngology-Head and Neck Surgery Foundation selected a panel representing the fields of advanced practice nursing, bronchoesophagology, consumer advocacy, family medicine, geriatric medicine, internal medicine, laryngology, neurology, otolaryngology-head and neck surgery, pediatrics, professional voice, pulmonology, and speech-language pathology. Action Statements The guideline update group made strong recommendations for the following key action statements (KASs): (1) Clinicians should assess the patient with dysphonia by history and physical examination to identify factors where expedited laryngeal evaluation is indicated. These include but are not limited to recent surgical procedures involving the head, neck, or chest; recent endotracheal intubation; presence of concomitant neck mass; respiratory distress or stridor; history of tobacco abuse; and whether the patient is a professional voice user. (2) Clinicians should advocate voice therapy for patients with dysphonia from a cause amenable to voice therapy. The guideline update group made recommendations for the following KASs: (1) Clinicians should identify dysphonia in a patient with altered voice quality, pitch, loudness, or vocal effort that impairs communication or reduces quality of life (QOL). (2) Clinicians should assess the patient with dysphonia by history and physical examination for underlying causes of dysphonia and factors that modify management. (3) Clinicians should perform laryngoscopy, or refer to a clinician who can perform laryngoscopy, when dysphonia fails to resolve or improve within 4 weeks or irrespective of duration if a serious underlying cause is suspected. (4) Clinicians should perform diagnostic laryngoscopy, or refer to a clinician who can perform diagnostic laryngoscopy, before prescribing voice therapy and document/communicate the results to the speech-language pathologist (SLP). (5) Clinicians should advocate for surgery as a therapeutic option for patients with dysphonia with conditions amenable to surgical intervention, such as suspected malignancy, symptomatic benign vocal fold lesions that do not respond to conservative management, or glottic insufficiency. (6) Clinicians should offer, or refer to a clinician who can offer, botulinum toxin injections for the treatment of dysphonia caused by spasmodic dysphonia and other types of laryngeal dystonia. (7) Clinicians should inform patients with dysphonia about control/preventive measures. (8) Clinicians should document resolution, improvement or worsened symptoms of dysphonia, or change in QOL of patients with dysphonia after treatment or observation. The guideline update group made a strong recommendation against 1 action: (1) Clinicians should not routinely prescribe antibiotics to treat dysphonia. The guideline update group made recommendations against other actions: (1) Clinicians should not obtain computed tomography (CT) or magnetic resonance imaging (MRI) for patients with a primary voice complaint prior to visualization of the larynx. (2) Clinicians should not prescribe antireflux medications to treat isolated dysphonia, based on symptoms alone attributed to suspected gastroesophageal reflux disease (GERD) or laryngopharyngeal reflux (LPR), without visualization of the larynx. (3) Clinicians should not routinely prescribe corticosteroids in patients with dysphonia prior to visualization of the larynx. The policy level for the following recommendation about laryngoscopy at any time was an option: (1) Clinicians may perform diagnostic laryngoscopy at any time in a patient with dysphonia. Differences from Prior Guideline (1) Incorporating new evidence profiles to include the role of patient preferences, confidence in the evidence, differences of opinion, quality improvement opportunities, and any exclusion to which the action statement does not apply (2) Inclusion of 3 new guidelines, 16 new systematic reviews, and 4 new randomized controlled trials (3) Inclusion of a consumer advocate on the guideline update group (4) Changes to 9 KASs from the original guideline (5) New KAS 3 (escalation of care) and KAS 13 (outcomes) (6) Addition of an algorithm outlining KASs for patients with dysphonia.

Clinical Practice Guideline: Hoarseness (Dysphonia) (Update).

Objective This guideline provides evidence-based recommendations on treating patients who present with dysphonia, which is characterized by altered vocal quality, pitch, loudness, or vocal effort that impairs communication and/or quality of life. Dysphonia affects nearly one-third of the population at some point in its life. This guideline applies to all age groups evaluated in a setting where dysphonia would be identified or managed. It is intended for all clinicians who are likely to diagnose and treat patients with dysphonia. Purpose The primary purpose of this guideline is to improve the quality of care for patients with dysphonia, based on current best evidence. Expert consensus to fill evidence gaps, when used, is explicitly stated and supported with a detailed evidence profile for transparency. Specific objectives of the guideline are to reduce inappropriate variations in care, produce optimal health outcomes, and minimize harm. For this guideline update, the American Academy of Otolaryngology-Head and Neck Surgery Foundation selected a panel representing the fields of advanced practice nursing, bronchoesophagology, consumer advocacy, family medicine, geriatric medicine, internal medicine, laryngology, neurology, otolaryngology-head and neck surgery, pediatrics, professional voice, pulmonology, and speech-language pathology. Action Statements The guideline update group made strong recommendations for the following key action statements (KASs): (1) Clinicians should assess the patient with dysphonia by history and physical examination to identify factors where expedited laryngeal evaluation is indicated. These include, but are not limited to, recent surgical procedures involving the head, neck, or chest; recent endotracheal intubation; presence of concomitant neck mass; respiratory distress or stridor; history of tobacco abuse; and whether the patient is a professional voice user. (2) Clinicians should advocate voice therapy for patients with dysphonia from a cause amenable to voice therapy. The guideline update group made recommendations for the following KASs: (1) Clinicians should identify dysphonia in a patient with altered voice quality, pitch, loudness, or vocal effort that impairs communication or reduces quality of life (QOL). (2) Clinicians should assess the patient with dysphonia by history and physical examination for underlying causes of dysphonia and factors that modify management. (3) Clinicians should perform laryngoscopy, or refer to a clinician who can perform laryngoscopy, when dysphonia fails to resolve or improve within 4 weeks or irrespective of duration if a serious underlying cause is suspected. (4) Clinicians should perform diagnostic laryngoscopy, or refer to a clinician who can perform diagnostic laryngoscopy, before prescribing voice therapy and document/communicate the results to the speech-language pathologist (SLP). (5) Clinicians should advocate for surgery as a therapeutic option for patients with dysphonia with conditions amenable to surgical intervention, such as suspected malignancy, symptomatic benign vocal fold lesions that do not respond to conservative management, or glottic insufficiency. (6) Clinicians should offer, or refer to a clinician who can offer, botulinum toxin injections for the treatment of dysphonia caused by spasmodic dysphonia and other types of laryngeal dystonia. (7) Clinicians should inform patients with dysphonia about control/preventive measures. (8) Clinicians should document resolution, improvement or worsened symptoms of dysphonia, or change in QOL of patients with dysphonia after treatment or observation. The guideline update group made a strong recommendation against 1 action: (1) Clinicians should not routinely prescribe antibiotics to treat dysphonia. The guideline update group made recommendations against other actions: (1) Clinicians should not obtain computed tomography (CT) or magnetic resonance imaging (MRI) for patients with a primary voice complaint prior to visualization of the larynx. (2) Clinicians should not prescribe antireflux medications to treat isolated dysphonia, based on symptoms alone attributed to suspected gastroesophageal reflux disease (GERD) or laryngopharyngeal reflux (LPR), without visualization of the larynx. (3) Clinicians should not routinely prescribe corticosteroids for patients with dysphonia prior to visualization of the larynx. The policy level for the following recommendation about laryngoscopy at any time was an option: (1) Clinicians may perform diagnostic laryngoscopy at any time in a patient with dysphonia. Disclaimer This clinical practice guideline is not intended as an exhaustive source of guidance for managing dysphonia (hoarseness). Rather, it is designed to assist clinicians by providing an evidence-based framework for decision-making strategies. The guideline is not intended to replace clinical judgment or establish a protocol for all individuals with this condition, and it may not provide the only appropriate approach to diagnosing and managing this problem. Differences from Prior Guideline (1) Incorporation of new evidence profiles to include the role of patient preferences, confidence in the evidence, differences of opinion, quality improvement opportunities, and any exclusion to which the action statement does not apply (2) Inclusion of 3 new guidelines, 16 new systematic reviews, and 4 new randomized controlled trials (3) Inclusion of a consumer advocate on the guideline update group (4) Changes to 9 KASs from the original guideline (5) New KAS 3 (escalation of care) and KAS 13 (outcomes) (6) Addition of an algorithm outlining KASs for patients with dysphonia.

Protein Fluctuations and Cavity Changes Relationship.

Protein cavities and tunnels are critical for function. Ligand recognition and binding, transport, and enzyme catalysis require cavities rearrangements. Therefore, the flexibility of cavities should be guaranteed by protein vibrational dynamics. Molecular dynamics simulations provide a framework to explore conformational plasticity of protein cavities. Herein, we present a novel procedure to characterize the dynamics of protein cavities in terms of their volume gradient vector. For this purpose, we make use of algorithms for calculation of the cavity volume that result robust for numerical differentiations. Volume gradient vector is expressed in terms of principal component analysis obtained from equilibrated molecular dynamics simulations. We analyze contributions of principal component modes to the volume gradient vector according to their frequency and degree of delocalization. In all our test cases, we find that low frequency modes play a critical role together with minor contributions of high frequency modes. These modes involve concerted motions of significant fractions of the total residues lining the cavities. We make use of variations of the potential energy of a protein in the direction of the volume gradient vector as a measure of flexibility of the cavity. We show that proteins whose collective low frequency fluctuations contribute the most to changes of cavity volume exhibit more flexible cavities.

Vaterite coatings on electrospun polymeric fibers for biomedical applications.

The process of porous calcium carbonate (CaCO3 ) covering on electrospun poly(ε-caprolactone) (PCL) fibers is described in this study. Uniform CaCO3 coatings, composed of vaterite microparticles and its aggregates, were formed on PCL fibers by mineral precipitation from solution under ultrasonic treatment. The porous structure of CaCO3 in vaterite polymorphic form is useful for loading of various substances (drugs and nanoparticles), and this property makes vaterite an appropriate material for design of drug delivery systems. Such mineralization was implemented to attain therapeutic and/or biological activity of tissue engineering scaffolds based on electrospun PCL, by means of CaCO3 coatings. Various structures and polymorphs of CaCO3 coatings were obtained by variation of growth conditions (time of fiber incubation in work solution, ultrasonic treatment of this system). Coating homogeneity, CaCO3 polymorphic form, morphology, and CaCO3 mass can be controlled by number of successive stages of fibrous material treatment. Cytotoxicity tests showed that PCL fibers mineralized with CaCO3 did not release substances toxic for cells. SEM images of PCL/CaCO3 scaffolds cultured with cells demonstrate that scaffolds supported cell adhesion and spreading. The presented results show the new technique of controlled PCL scaffold mineralization with vaterite, and an opportunity of using PCL/CaCO3 as scaffolds for tissue engineering. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 94-103, 2017.

Phosphorylation-dependent cleavage regulates von Hippel Lindau proteostasis and function.

Loss of von Hippel Lindau (VHL) protein function is a key driver of VHL diseases, including sporadic and inherited clear cell renal cell carcinoma. Modulation of the proteostasis of VHL, especially missense point-mutated VHL, is a promising approach to augmenting VHL levels and function. VHL proteostasis is regulated by multiple mechanisms including folding, chaperone binding, complex formation and phosphorylation. Nevertheless, many details underlying the regulations of VHL proteostasis are unknown. VHL is expressed as two variants, VHL30 and VHL19. Furthermore, the long-form variant of VHL was often detected as multiple bands by western blotting. However, how these multiple species of VHL are generated and whether the process regulates VHL proteostasis and function are unknown. We hypothesized that the two major species are generated by VHL protein cleavage, and the cleavage regulates VHL proteostasis and subsequent function. We characterized VHL species using genetical and pharmacological approaches and showed that VHL was first cleaved at the N-terminus by chymotrypsin C before being directed for proteasomal degradation. Casein kinase 2-mediated phosphorylation at VHL N-terminus was required for the cleavage. Furthermore, inhibition of cleavage stabilized VHL protein and thereby promoted HIF downregulation. Our study reveals a novel mechanism regulating VHL proteostasis and function, which is significant for identifying new drug targets and developing new therapeutic approaches targeting VHL deficiency in VHL diseases.

Targeting MET and AXL overcomes resistance to sunitinib therapy in renal cell carcinoma.

Antiangiogenic therapy resistance occurs frequently in patients with metastatic renal cell carcinoma (RCC). The purpose of this study was to understand the mechanism of resistance to sunitinib, an antiangiogenic small molecule, and to exploit this mechanism therapeutically. We hypothesized that sunitinib-induced upregulation of the prometastatic MET and AXL receptors is associated with resistance to sunitinib and with more aggressive tumor behavior. In the present study, tissue microarrays containing sunitinib-treated and untreated RCC tissues were stained with MET and AXL antibodies. The low malignant RCC cell line 786-O was chronically treated with sunitinib and assayed for AXL, MET, epithelial-mesenchymal transition (EMT) protein expression and activation. Co-culture experiments were used to examine the effect of sunitinib pretreatment on endothelial cell growth. The effects of AXL and MET were evaluated in various cell-based models by short hairpin RNA or inhibition by cabozantinib, the multi-tyrosine kinases inhibitor that targets vascular endothelial growth factor receptor, MET and AXL. Xenograft mouse models tested the ability of cabozantinib to rescue sunitinib resistance. We demonstrated that increased AXL and MET expression was associated with inferior clinical outcome in patients. Chronic sunitinib treatment of RCC cell lines activated both AXL and MET, induced EMT-associated gene expression changes, including upregulation of Snail and ß-catenin, and increased cell migration and invasion. Pretreatment with sunitinib enhanced angiogenesis in 786-0/human umbilical vein endothelial cell co-culture models. The suppression of AXL or MET expression and the inhibition of AXL and MET activation using cabozantinib both impaired chronic sunitinib treatment-induced prometastatic behavior in cell culture and rescued acquired resistance to sunitinib in xenograft models. In summary, chronic sunitinib treatment induces the activation of AXL and MET signaling and promotes prometastatic behavior and angiogenesis. The inhibition of AXL and MET activity may overcome resistance induced by prolonged sunitinib therapy in metastatic RCC.

A phase 1, randomized, dose-ranging study of GS-5816, a once-daily NS5A inhibitor, in patients with genotype 1-4 hepatitis C virus.

GS-5816 is an inhibitor of the hepatitis C virus (HCV) NS5A protein that has demonstrated pan-genotypic activity and a high barrier to resistance in HCV replicon assays. The aim of this study was to evaluate the safety, antiviral activity and pharmacokinetics of once-daily doses of GS-5816 in patients with genotype 1-4 HCV infection. Patients with genotype 1-4 HCV infection were randomized to 3 days of GS-5816 at doses ranging from 5 to 150 mg or placebo. Adverse events were recorded, and plasma samples obtained for analysis of pharmacokinetics, HCV RNA and NS5A sequencing studies. GS-5816 5-150 mg for 3 days was well tolerated and resulted in rapid declines in HCV RNA that were sustained over the dosing period. In patients treated with the 150 mg dose of GS-5816, the mean maximal HCV RNA declines were 4.0, 4.0, 4.4, 3.3 and 3.5 log10 IU/mL in patients with genotype 1a, 1b, 2, 3 and 4 HCV infection, respectively. Pretreatment NS5A resistance-associated polymorphisms were detected in 31% (22/70) of patients. Genotype 1 and 3 HCV-infected patients without pretreatment NS5A resistance-associated polymorphisms had greater declines in HCV RNA than patients with resistance-associated polymorphisms. Plasma pharmacokinetics were supportive of once-daily dosing. GS-5816 demonstrated pangenotypic antiviral activity in patients with genotype 1-4 HCV infection. It will be further evaluated in combination with other pangenotypic direct-acting antivirals to achieve the goal of developing a well-tolerated, highly effective treatment for all HCV genotypes.

Autophagy mediates HIF2α degradation and suppresses renal tumorigenesis.

Autophagy is a conserved process involved in lysosomal degradation of protein aggregates and damaged organelles. The role of autophagy in cancer is a topic of intense debate, and the underlying mechanism is still not clear. The hypoxia-inducible factor 2α (HIF2α), an oncogenic transcription factor implicated in renal tumorigenesis, is known to be degraded by the ubiquitin-proteasome system (UPS). Here, we report that HIF2α is in part constitutively degraded by autophagy. HIF2α interacts with autophagy-lysosome system components. Inhibition of autophagy increases HIF2α, whereas induction of autophagy decreases HIF2α. The E3 ligase von Hippel-Lindau and autophagy receptor protein p62 are required for autophagic degradation of HIF2α. There is a compensatory interaction between the UPS and autophagy in HIF2α degradation. Autophagy inactivation redirects HIF2α to proteasomal degradation, whereas proteasome inhibition induces autophagy and increases the HIF2α-p62 interaction. Importantly, clear-cell renal cell carcinoma (ccRCC) is frequently associated with monoallelic loss and/or mutation of autophagy-related gene ATG7, and the low expression level of autophagy genes correlates with ccRCC progression. The protein levels of ATG7 and beclin 1 are also reduced in ccRCC tumors. This study indicates that autophagy has an anticancer role in ccRCC tumorigenesis, and suggests that constitutive autophagic degradation of HIF2α is a novel tumor suppression mechanism.

Lack of a clinically important pharmacokinetic interaction between sofosbuvir or ledipasvir and hormonal oral contraceptives norgestimate/ethinyl estradiol in HCV-uninfected female subjects.

This study evaluated the potential for a drug-drug interaction between HCV direct-acting antivirals sofosbuvir or ledipasvir and oral hormonal contraceptive (OC) norgestimate/ethinyl estradiol (norgestimate 0.18/0.215/0.25 mg with ethinyl estradiol 25 µg). This was a 112-day, open-label, fixed-sequence pharmacokinetic (PK) study in healthy female subjects that included a lead-in cycle (OC only; N = 21), cycle 1 (OC only; N = 15), cycle 2 (OC + sofosbuvir; N = 15), and cycle 3 (OC + ledipasvir; N = 15). Administration of sofosbuvir with OC did not alter PK of norelgestromin (primary norgestimate metabolite) or ethinyl estradiol. Small increases in norgestrel (secondary norgestimate metabolite) AUC(tau) (19%) and C(tau) (23%) with sofosbuvir were noted. Ledipasvir did not impact PK of norelgestromin or norgestrel but modestly increased ethinyl estradiol C(max) (40%). Sofosbuvir, GS- 331007 (predominant circulating metabolite of SOF), and ledipasvir PK were similar to historical data. Pharmacodynamic markers luteinizing hormone, follicle-stimulating hormone, and progesterone values were generally comparable in all cycles. No loss in contraceptive efficacy is expected upon administration of sofosbuvir or ledipasvir/sofosbuvir with oral contraceptives containing norgestimate and ethinyl estradiol. The use of sofosbuvir or ledipasvir/sofosbuvir FDC with oral contraceptives is permitted.

Site-specific activation of AKT protects cells from death induced by glucose deprivation.

The serine/threonine kinase AKT is a key mediator of cancer cell survival. We demonstrate that transient glucose deprivation modestly induces AKT phosphorylation at both Thr308 and Ser473. In contrast, prolonged glucose deprivation induces selective AKTThr308 phosphorylation and phosphorylation of a distinct subset of AKT downstream targets leading to cell survival under metabolic stress. Glucose-deprivation-induced AKTThr308 phosphorylation is dependent on PDK1 and PI3K but not EGF receptor or IGF1R. Prolonged glucose deprivation induces the formation of a complex of AKT, PDK1 and the GRP78 chaperone protein, directing phosphorylation of AKTThr308 but not AKTSer473. Our results reveal a novel mechanism of AKT activation under prolonged glucose deprivation that protects cells from metabolic stress. The selective activation of AKTThr308 phosphorylation that occurs during prolonged nutrient deprivation may provide an unexpected opportunity for the development and implementation of drugs targeting cell metabolism and aberrant AKT signaling.

Anti-CRLF2 Antibody-Armored Biodegradable Nanoparticles for Childhood B-ALL.

B-precursor acute lymphoblastic leukemia (B-ALL) lymphoblast (blast) internalization of anti-cytokine receptor-like factor 2 (CRLF2) antibody-armored biodegradable nanoparticles (AbBNPs) are investigated. First, AbBNPsaere synthesized by adsorbing anti-CRLF2 antibodies to poly(D,L-lactide- co -glycolide) (PLGA) nanoparticles of various sizes and antibody surface density (Ab/BNP) ratios. Second, AbBNPs are incubated with CRLF2-overexpressing (CRLF2+) or control blasts. Third, internalization of AbBNPs by blasts is evaluated by multicolor flow cytometry as a function of receptor expression, AbBNP size, and Ab/BNP ratio. Results from these experiments are con-firmed by electron microscopy, fluorescence microscopy, and Western blotting. The optimal size and Ab/BNP for internalization of AbBNPs by CRLF2+ blasts is 50 nm with 10 Ab/BNP and 100 nm with 25 Ab/BNP. These studies show that internalization of AbBNPs in childhood B-ALL blasts is AbBNP size-and Ab/BNP ratio-dependent. All AbBNP combinations are non-cytotoxic. It is also shown that CD47 is very slightly up-regulated by blasts exposed to AbBNPs. CD47 is "the marker of self" overexpressed by blasts to escape phagocytosis, or "cellular devouring", by beneficial macrophages. The results indicate that precise engineering of AbBNPs by size and Ab/BNP ratio may improve the internalization and selectivity of future biodegradable nanoparticles for the treatment of leukemia patients, including drug-resistant minority children and Down's syndrome patients with CRLF2+B-ALL.

Awake hippocampal sharp-wave ripples support spatial memory.

The hippocampus is critical for spatial learning and memory. Hippocampal neurons in awake animals exhibit place field activity that encodes current location, as well as sharp-wave ripple (SWR) activity during which representations based on past experiences are often replayed. The relationship between these patterns of activity and the memory functions of the hippocampus is poorly understood. We interrupted awake SWRs in animals learning a spatial alternation task. We observed a specific learning and performance deficit that persisted throughout training. This deficit was associated with awake SWR activity, as SWR interruption left place field activity and post-experience SWR reactivation intact. These results provide a link between awake SWRs and hippocampal memory processes, which suggests that awake replay of memory-related information during SWRs supports learning and memory-guided decision-making.