bluuebunny/crossref_metadata_2025 huggingface dataset:
Thermal Drift of Floated Gyroscopes
Authors: Goodman L. E. , Robinson A. R.
Published: June 2021

When a floated gyroscope is subjected to a temperature distribution which is not symmetrical about a plane parallel to the gravitational force, convection currents tend to rotate the gimbal. The rebalance torque and the free drift rate due to thermal effects are first determined for the case of an exactly centered gimbal. It is then shown that moderate gimbal eccentricity has little influence on thermal drift, and, in fact, reduces thermal rebalance torque.
The Forced Lateral Oscillations of Trailers
Authors: Slibar A. , Paslay P. R.
Published: June 2021

This paper gives the mechanical response of a simplified model of a trailer when its tow point moves along a certain prescribed path. The type of path considered here is described by a constant forward velocity on which is superimposed a low-velocity-amplitude, sidewise, periodic motion. In this investigation analytical expressions proposed by Huber (3) were used for the forces and moments on the tires. The equations of motion are studied for small slip angles. The analysis leads to a prediction of the natural frequency of the trailer and shows that the damping of the system due to slip of the tires is inversely proportional to the forward velocity of the trailer.
Stress Concentrations in a Strip Under Tension and Containing Two Pairs of Semicircular Notches Placed on the Edges Symmetrically
Authors: Atsumi A.
Published: June 2021

Distributions of stresses in an infinite strip of finite breadth under tension and containing two pairs of equal semicircular notches placed symmetrically on the edges are studied theoretically. The state of decreasing of stress concentration is studied and compared with those corrected by M. Isida or newly determined by the author in their calculations as the reliable results of Ling’s problem of an infinite strip of finite breadth under tension and containing two semicircular notches placed symmetrically on the edges.
Estimation of Mycoplasma pneumoniae inoculum size by rate of tetrazolium reduction
Authors: Bredt W
Published: June 2021

The rate of appearance of red-colored formazan indicating reduction of 2,3,5,-triphenyltetrazolium chloride by Mycoplasma pneumoniae was dependent on the inoculum size. The test permits estimation of M. pneumoniae cells in preparations that cannot be counted by dilution methods.
Comparison of isolation of Haemophilus vaginalis (Corynebacterium vaginale) from peptone-starch-dextrose agar and Columbia colistin-nalidoxic acid agar
Authors: Golberg R L , Washington JA I I
Published: June 2021

A total of 447 cervical or vaginal specimens were inoculated in parallel onto peptone-starch-dextrose (PSD) and Columbia colistin (10 mg/ml)-nalidixic acid (15 mug/ml) (CNA) agar and were incubated for 48 h at 35 degrees C in an atmosphere with 2 to 10% CO2. One hundred (22.4%) of the cultures were positive for Haemophilus vaginalis. Forty-eight of the isolates were recovered from both PSD and Columbia CNA agar, five from PSD only, and 47 from Columbia CNA agar only (P less than 0.001). On Columbia CNA agar, 76 of the isolates were detected after 24 h of incubation, and the remainder were detected within 4 days of incubation.
Effect of vaccination schedule on immune response of Macaca mulatta to cell culture-grown Rocky Mountain spotted fever vaccine
Authors: Sammons L S , Kenyon R H , Pedersen C E
Published: June 2021

The effect of vaccination schedule on the immune response of Macaca mulatta to formalin-inactivated chicken embryo cell culture (CEC)-grown Rickettsia rickettsii vaccine was studied. Schedules consisted of inoculation on day 1 only, on days 1 and 15, on days 1 and 30, on days 1, 8, and 15, or on days 1, 15, and 45. Humoral antibody measured by microagglutination and indirect immunofluorescence and resistance to challenge with 10(4) plaque-forming units of yolk sac-grown R. rickettsii were assessed. Seroconversion was noted in all monkeys after the first dose of vaccine. A second dose administered 8 or 15 days after the primary infection, or a third given 7 or 30 days after the second, produced no long-term effect on antibody titer. Only monkeys given two doses of vaccine at a 30-day interval showed an increase in antibody titer during the period before challenge. Vaccination with one, two, or three doses of CEC vaccine prevented development of rash and rickettsemia after challenge. The two-dose schedules appeared to induce the highest degree of resistance to challenge, as indicated by unaltered hematological parameters and body temperature in monkeys. The one- and three-dose schedules were somewhat less effective, in that some challenged monkeys within each group displayed febrile and leukocyte responses associated with Rocky Mountain spotted fever infection. Our data suggest that administration of two doses of CEC vaccine at 15- or 30-day intervals is the immunization schedule of choice.
Production of Antibody in Induced Granulomas
Authors: Wolff K. L. , Hudson B. W. , Ormsbee R. A. , Peacock M. G.
Published: June 2021

A method of producing antibodies in artificially induced granulomas with various microbial antigens is examined.
Rubella hemagglutination-inhibition test: false-positive reactions in sera contaminated with bacteria
Authors: Campbell J B , Romach M , Ellins M L
Published: June 2021

We have shown that bacterial contamination of sera can have a marked influence on the results of the rubella hemagglutination-inhibition (HI) test. In addition to increasing the levels of nonspecific agglutinins, a number of common bacterial species tested had a significant effect on the HI titers. When sera free from rubella-specific antibodies were contaminated with Bacillus subtilis or Pseudomonas fluorescens, HI titers ranged from 16 to 128 (expressed as the reciprocal of the highest serum dilution completely inhibiting hemagglutination) after treatment with heparin/MnCl2. Our observations demonstrate, therefore, that bacterial contamination can be one of the causes of false-positive reactions in this test.
Improved rubella hemagglutination inhibtion test: inactivation of non-immunoglobulin hemagglutination inhibitors by phospholipase C
Authors: Iwasa S , Hori M
Published: June 2021

A method using phospholipase C (PL-C) for removing nonspecific inhibitors (NSI) of rubella virus hemagglutinin is described. PL-C was found to hydrolyze NSI without altering the hemagglutination inhibition (HI) activity of the specific antibody and could be used to remove NSI in the rubella HI test by using formalinized erythrocytes, which resisted the enzymatic action; fresh erythrocytes were lysed by PL-C. The HI test using PL-C treated sera gave true measurements of actual rubella antibody content, and HI titers of PL-C treated sera were identical or equivalent (+/-1 dilution) to those of sera treated with dextran sulfate and CaCl2 (DS-C). Thus, the PL-C method gave results as reproducible and reliable as the DS-C method and was more convenient.
Shared themes of antigenic variation and virulence in bacterial, protozoal, and fungal infections
Authors: Deitsch K W , Moxon E R , Wellems T E
Published: June 2021

Pathogenic microbes have evolved highly sophisticated mechanisms for colonizing host tissues and evading or deflecting assault by the immune response. The ability of these microbes to avoid clearance prolongs infection, thereby promoting their long-term survival within individual hosts and, through transmission, between hosts. Many pathogens are capable of extensive antigenic changes in the face of the multiple constitutive and dynamic components of host immune defenses. As a result, highly diverse populations that have widely different virulence properties can arise from a single infecting organism (clone). In this review, we consider the molecular and genetic features of antigenic variation and corresponding host-parasite interactions of different pathogenic bacterial, fungal, and protozoan microorganisms. The host and microbial molecules involved in these interactions often determine the adhesive, invasive, and antigenic properties of the infecting organisms and can dramatically affect the virulence and pathobiology of individual infections. Pathogens capable of such antigenic variation exhibit mechanisms of rapid mutability in confined chromosomal regions containing specialized genes designated contingency genes. The mechanisms of hypermutability of contingency genes are common to a variety of bacterial and eukaryotic pathogens and include promoter alterations, reading-frame shifts, gene conversion events, genomic rearrangements, and point mutations.
Archaea and the prokaryote-to-eukaryote transition
Authors: Brown J R , Doolittle W F
Published: June 2021

Since the late 1970s, determining the phylogenetic relationships among the contemporary domains of life, the Archaea (archaebacteria), Bacteria (eubacteria), and Eucarya (eukaryotes), has been central to the study of early cellular evolution. The two salient issues surrounding the universal tree of life are whether all three domains are monophyletic (i.e., all equivalent in taxanomic rank) and where the root of the universal tree lies. Evaluation of the status of the Archaea has become key to answering these questions. This review considers our cumulative knowledge about the Archaea in relationship to the Bacteria and Eucarya. Particular attention is paid to the recent use of molecular phylogenetic approaches to reconstructing the tree of life. In this regard, the phylogenetic analyses of more than 60 proteins are reviewed and presented in the context of their participation in major biochemical pathways. Although many gene trees are incongruent, the majority do suggest a sisterhood between Archaea and Eucarya. Altering this general pattern of gene evolution are two kinds of potential interdomain gene transferrals. One horizontal gene exchange might have involved the gram-positive Bacteria and the Archaea, while the other might have occurred between proteobacteria and eukaryotes and might have been mediated by endosymbiosis.
Metabolism of sulfur amino acids in Saccharomyces cerevisiae
Authors: Thomas D , Surdin-Kerjan Y
Published: June 2021

Sulfur amino acid biosynthesis in Saccharomyces cerevisiae involves a large number of enzymes required for the de novo biosynthesis of methionine and cysteine and the recycling of organic sulfur metabolites. This review summarizes the details of these processes and analyzes the molecular data which have been acquired in this metabolic area. Sulfur biochemistry appears not to be unique through terrestrial life, and S. cerevisiae is one of the species of sulfate-assimilatory organisms possessing a larger set of enzymes for sulfur metabolism. The review also deals with several enzyme deficiencies that lead to a nutritional requirement for organic sulfur, although they do not correspond to defects within the biosynthetic pathway. In S. cerevisiae, the sulfur amino acid biosynthetic pathway is tightly controlled: in response to an increase in the amount of intracellular S-adenosylmethionine (AdoMet), transcription of the coregulated genes is turned off. The second part of the review is devoted to the molecular mechanisms underlying this regulation. The coordinated response to AdoMet requires two cis-acting promoter elements. One centers on the sequence TCACGTG, which also constitutes a component of all S. cerevisiae centromeres. Situated upstream of the sulfur genes, this element is the binding site of a transcription activation complex consisting of a basic helix-loop-helix factor, Cbf1p, and two basic leucine zipper factors, Met4p and Met28p. Molecular studies have unraveled the specific functions for each subunit of the Cbf1p-Met4p-Met28p complex as well as the modalities of its assembly on the DNA. The Cbf1p-Met4p-Met28p complex contains only one transcription activation module, the Met4p subunit. Detailed mutational analysis of Met4p has elucidated its functional organization. In addition to its activation and bZIP domains, Met4p contains two regulatory domains, called the inhibitory region and the auxiliary domain. When the level of intracellular AdoMet increases, the transcription activation function of Met4 is prevented by Met30p, which binds to the Met4 inhibitory region. In addition to the Cbf1p-Met4p-Met28p complex, transcriptional regulation involves two zinc finger-containing proteins, Met31p and Met32p. The AdoMet-mediated control of the sulfur amino acid pathway illustrates the molecular strategies used by eucaryotic cells to couple gene expression to metabolic changes.
Comprehensive management of acute pulmonary embolism in primary care using telemedicine in the COVID-era
Authors: Chang Joshua , Isaacs Dayna J , Leung Joseph , Vinson David R
Published: June 2021

A healthy, active woman in her 70s reported intermittent exertional dyspnoea for 2 months, notable during frequent open-water swimming. Symptoms were similar to an episode of travel-provoked pulmonary embolism 3 years prior. She denied chest pain, cough, fever, extremity complaints and symptoms at rest. Due to the COVID-19 pandemic, her healthcare system was using secure telemedicine to evaluate non-critical complaints. During the initial video visit, she appeared well, conversing normally without laboured breathing. An elevated serum D-dimer prompted CT pulmonary angiography, which identified acute lobar pulmonary embolism. After haematology consultation and telephone conversation with the patient, her physician prescribed rivaroxaban. Her symptoms rapidly improved. She had an uneventful course and is continuing anticoagulation indefinitely. The pandemic has increased the application of telemedicine for acute care complaints. This case illustrates its safe and effective use for comprehensive management of acute pulmonary embolism in the primary care setting.
Short-term intensified training temporarily impairs mitochondrial respiratory capacity in elite endurance athletes
Authors: Cardinale Daniele A. , Gejl Kasper D. , Petersen Kristine G. , Nielsen Joachim , Ørtenblad Niels , Larsen Filip J.
Published: June 2021

We show that mitochondrial respiration is temporarily impaired after a period of intensified exercise training in elite athletes. In parallel, proteins involved in the antioxidative response including SOD2, UCP3, and ANT2 were upregulated, whereas mitochondrial biogenesis was slightly activated. Despite the mitochondrial respiratory impairments, physical performance was improved a few days after the intense training period.
Altered Gene Expression in the Testis of Infertile Patients with Nonobstructive Azoospermia
Authors: Wang Zhiqiang , Ding Zhongjun , Guan Yan , Liu Chunhui , Wang Linjun , Shan Wensheng , Yang Jie
Published: June 2021

Background. The molecular mechanism of nonobstructive azoospermia (NOA) remains unclear. The aim of this study was to identify gene expression changes in NOA patients and to explore potential biomarkers and therapeutic targets. Methods. The gene expression profiles of GSE45885 and GSE145467 were collected from the Gene Expression Omnibus (GEO) database, and the differences between NOA and normal spermatogenesis were analyzed. Enrichment analysis was performed to explore biological functions for common differentially expressed genes (DEGs) in GSE45885 and GSE145467. Coexpression analysis of DEGs in GSE45885 was performed, and two modules with the highest correlation with NOA were screened. Key genes were then screened from the intersection genes of the two modules and common DEGs and PPI network. The expression of key genes was validated by quantitative real-time polymerase chain reaction (qRT-PCR) experiments. Finally, through miRTarBase, miRDB, and RAID, the miRNAs were predicted to regulate key genes, respectively. Results. A total of 345 common DEGs were identified and they were mainly related to spermatogenesis, insulin signaling pathway. Coexpression analysis of DEGs in GSE45885 yielded eight modules; MEblack and MEturquoise had the highest correlation with NOA. Six genes in MEturquoise and RNF141 in MEblack were identified as key genes. qRT-PCR experiments validated the differential expression of key genes between NOA and control. Furthermore, RNF141 was regulated by the largest number of miRNAs. Conclusion. Our findings suggest that the significant change expression of key genes may be potential markers and therapeutic targets of NOA and may have some impact on the development of NOA.
GCWOAS2: Multiobjective Task Scheduling Strategy Based on Gaussian Cloud‐Whale Optimization in Cloud Computing
Authors: Ni Lina , Sun Xiaoting , Li Xincheng , Zhang Jinquan
Published: June 2021

An important challenge facing cloud computing is how to correctly and effectively handle and serve millions of users’ requests. Efficient task scheduling in cloud computing can intuitively affect the resource configuration and operating cost of the entire system. However, task and resource scheduling in a cloud computing environment is an NP‐hard problem. In this paper, we propose a three‐layer scheduling model based on whale‐Gaussian cloud. In the second layer of the model, a whale optimization strategy based on the Gaussian cloud model (GCWOAS2) is used for multiobjective task scheduling in a cloud computing which is to minimize the completion time of the task via effectively utilizing the virtual machine resources and to keep the load balancing of each virtual machine, reducing the operating cost of the system. In the GCWOAS2 strategy, an opposition‐based learning mechanism is first used to initialize the scheduling strategy to generate the optimal scheduling scheme. Then, an adaptive mobility factor is proposed to dynamically expand the search range. The whale optimization algorithm based on the Gaussian cloud model is proposed to enhance the randomness of search. Finally, a multiobjective task scheduling algorithm based on Gaussian whale‐cloud optimization (GCWOA) is presented, so that the entire scheduling strategy can not only expand the search range but also jump out of the local maximum and obtain the global optimal scheduling strategy. Experimental results show that compared with other existing metaheuristic algorithms, our strategy can not only shorten the task completion time but also balance the load of virtual machine resources, and at the same time, it also has a better performance in resource utilization.
Exploration of Productivity, Chemical Composition, and Antioxidant Potential of Origanum vulgare L. Grown at Different Geographical Locations of Western Himalaya, India
Authors: Goyal Shweta , Tewari Geeta , Pandey H. K , Kumari Anjali
Published: June 2021

Origanum vulgare L., the most complex and variable species of the genus Origanum, is used to cure various diseases in the traditional medicinal system. In the current investigation, O. vulgare was collected from its natural origin and further multiplied for two years at the experimental farm of DIBER field station, Auli, and afterwards, it was grown at three different altitudes, i.e., DIBER field station of Auli (2744 m), Pithoragarh (1524 m), and Haldwani (412 m), under natural field conditions. The essential oil was extracted using the Clevenger apparatus and analyzed by GC/MS and GC/FID. The total phenolic, flavonoid, and tannin content in the methanolic extracts of all the cultivations were estimated by using Folin–Ciocalteu reagent, aluminium chloride reagent, and Folin–Denis methods, respectively. The antioxidant activity of the essential oils and extracts of all the cultivations was also estimated by DPPH, ABTS, and reducing power methods. The highest amount of phenolics, flavonoids, and tannins was obtained in the Pithoragarh cultivation. Thymol was the major component in all the essential oils contributing 38.81% (Auli), 52.83% (Pithoragarh), and 31.86% (Haldwani) of the total oil. The highest antioxidant activity was exhibited by oregano oil of Pithoragarh cultivation, followed by Auli and Haldwani, respectively. The alcoholic extract of Pithoragarh cultivation had the highest antioxidant activity. The study infers that the plant demonstrated good results in terms of fresh weight, oil yield, antioxidant potential, and EO composition when grown at higher altitudes.
Medical Image Fusion Based on Low-Level Features
Authors: Zhang Yongxin , Guo Chenrui , Zhao Peng
Published: June 2021

Medical image fusion is an important technique to address the limited depth of the optical lens for a completely informative focused image. It can well improve the accuracy of diagnosis and assessment of medical problems. However, the difficulty of many traditional fusion methods in preserving all the significant features of the source images compromises the clinical accuracy of medical problems. Thus, we propose a novel medical image fusion method with a low-level feature to deal with the problem. We decompose the source images into base layers and detail layers with local binary pattern operators for obtaining low-level features. The low-level features of the base and detail layers are applied to construct weight maps by using saliency detection. The weight map optimized by fast guided filtering guides the fusion of base and detail layers to maintain the spatial consistency between the source images and their corresponding layers. The recombination of the fused base and detail layers constructs the final fused image. The experimental results demonstrated that the proposed method achieved a state-of-the-art performance for multifocus images.
Outcome Reporting Variability in Trials of Chinese Medicine for Hyperlipidemia: A Systematic Review for Developing a Core Outcome Set
Authors: Li Geng , Han Ruxue , Cao Wencong , Wen Zehuai , Chen Xiankun
Published: June 2021

Introduction. Hyperlipidemia is an underlying process behind cardiovascular disease. Chinese medicine (CM) may be effective in treating hyperlipidemia, but there is a lack of studies with high methodological quality. A major reason for this is heterogeneity in outcome reporting. Therefore, this study explores the degree of outcome reporting variation in CM trials for hyperlipidemia. It then generates a list of potentially important outcomes for developing a core outcome set (COS). Methods. A systematic review of literature focusing on studies of CM for hyperlipidemia was conducted. Outcomes were listed verbatim and grouped into 8 domains. Outcome frequency and definition uniformity were analyzed. Results. 3,702 studies and 452 individual outcomes were identified. These outcomes were reported 27,328 times, of which 1.6% were reported as primary outcomes, and 13.3% were defined. The most frequent outcome was total triglyceride, represented in 86.7% of the studies, followed by total cholesterol (86.0%), total effective rate (75.1%), high-density lipoprotein cholesterol (73.2%), and low-density lipoprotein cholesterol (60.5%). However, 43.6% of outcomes were reported only once. The largest outcome domain was “pathological or pathophysiological outcomes,” which included 67.0% of outcomes. Of the “response rate related outcomes” domain, total effective rate was the most frequently reported outcome (n = 2,780), and 95.3% of the studies gave a clear definition. However, these definitions were often contradictory. Only 10 papers reported cardiovascular events, 3 of which referred to them as primary outcomes. Moreover, ten patient-reported outcomes were reported in the retrieved literature 19 times in total. The majority of the outcomes did not report measurement instruments (MIs) (269/453, 59.4%). MIs of the surrogate outcomes were reported more frequently. Conclusion. Outcome reporting in CM trials for hyperlipidemia is inconsistent and ill-defined, creating barriers to data synthesis and comparison. Thus, we propose and are developing a COS for CM trials for hyperlipidemia.
Remote Fault-Tolerant Control for Industrial Smart Surveillance System
Authors: Mahmood Atif , Khan Abdul Qayyum , Mustafa Ghulam , Ullah Nasim , Abid Muhammad , Khan Aadil Sarwar
Published: June 2021

We design a remote fault-tolerant control for an industrial surveillance system. The designed controller simultaneously tolerates the effects of local faults of a node, the propagated undesired effects of neighboring connected nodes, and the effects of network-induced uncertainties from a remote location. The uncertain network-induced time delays of communication links from the sensor to the controller and from the controller to the actuator are modeled using two separate Markov chains and packet dropouts using the Bernoulli process. Based on linear matrix inequalities, we derive sufficient conditions for output feedback-based control law, such that the controller does not directly depend on output, for stochastic stability of the system. The simulation study shows the effectiveness of the proposed approach.
N-Acetylcysteine: A Review of Clinical Usefulness (an Old Drug with New Tricks)
Authors: Schwalfenberg Gerry K.
Published: June 2021

Objective. To review the clinical usefulness of N-acetylcysteine (NAC) as treatment or adjunctive therapy in a number of medical conditions. Use in Tylenol overdose, cystic fibrosis, and chronic obstructive lung disease has been well documented, but there is emerging evidence many other conditions would benefit from this safe, simple, and inexpensive intervention. Quality of Evidence. PubMed, several books, and conference proceedings were searched for articles on NAC and health conditions listed above reviewing supportive evidence. This study uses a traditional integrated review format, and clinically relevant information is assessed using the American Family Physician Evidence-Based Medicine Toolkit. A table summarizing the potential mechanisms of action for N-acetylcysteine in these conditions is presented. Main Message. N-acetylcysteine may be useful as an adjuvant in treating various medical conditions, especially chronic diseases. These conditions include polycystic ovary disease, male infertility, sleep apnea, acquired immune deficiency syndrome, influenza, parkinsonism, multiple sclerosis, peripheral neuropathy, stroke outcomes, diabetic neuropathy, Crohn’s disease, ulcerative colitis, schizophrenia, bipolar illness, and obsessive compulsive disorder; it can also be useful as a chelator for heavy metals and nanoparticles. There are also a number of other conditions that may show benefit; however, the evidence is not as robust. Conclusion. The use of N-acetylcysteine should be considered in a number of conditions as our population ages and levels of glutathione drop. Supplementation may contribute to reducing morbidity and mortality in some chronic conditions as outlined in the article.
Blood Biomarker Landscape in Patients with High-risk Nonmetastatic Castration-Resistant Prostate Cancer Treated with Apalutamide and Androgen-Deprivation Therapy as They Progress to Metastatic Disease
Authors: Smith Matthew R. , Thomas Shibu , Gormley Michael , Chowdhury Simon , Olmos David , Oudard Stéphane , Feng Felix Y. , Rajpurohit Yashoda , Urtishak Karen , Ricci Deborah S. , Rooney Brendan , Lopez-Gitlitz Angela , Yu Margaret , Wyatt Alexander W. , Li Mark , Attard Gerhardt , Small Eric J.
Published: June 2021

Purpose:

In the placebo-controlled SPARTAN study, apalutamide added to androgen-deprivation therapy (ADT) improved metastasis-free survival, second progression-free survival (PFS2), and overall survival (OS) in patients with nonmetastatic castration-resistant prostate cancer (nmCRPC). Mechanisms of resistance to apalutamide in nmCRPC require evaluation.

Patients and Methods:

In a subset of patients from SPARTAN, aberrations were assessed at baseline and end of study treatment (EOST) using targeted next-generation sequencing or qRT-PCR. Circulating-tumor DNA (ctDNA) levels were assessed qualitatively. Select aberrations in androgen receptor (AR) and other common PC-driving genes were detected and summarized by the treatment group; genomic aberrations were summarized in ctDNA-positive samples. Association between detection of aberrations in all patients and outcomes was assessed using Cox proportional-hazards models and multivariate analysis.

Results:

In 247 patients, the overall prevalence of ctDNA, AR aberrations, and TP53 inactivation increased from baseline (40.6%, 13.6%, and 22.2%) to EOST (57.1%, 25.4%, and 35.0%) and was comparable between treatment groups at EOST. In patients who received subsequent androgen signaling inhibition after study treatment, detectable biomarkers at EOST were significantly associated with poor outcomes: ctDNA with PFS2 or OS (HR, 2.01 or 2.17, respectively; P < 0.0001 for both), any AR aberration with PFS2 (1.74; P = 0.024), and TP53 or BRCA2 inactivation with OS (2.06; P = 0.003; or 3.1; P < 0.0001).

Conclusions:

Apalutamide plus ADT did not increase detectable AR/non-AR aberrations over ADT alone. Detectable ctDNA, AR aberrations, and TP53/BRCA2 inactivation at EOST were associated with poor outcomes in patients treated with first subsequent androgen signaling inhibitor.
Abstract A02: Probing amino acids residues chemical reactivity of KRAS 4b using N-hydroxysuccinimide esters
Authors: Chertov Oleg , Ye Xiaoying , Waybright Timothy , Stephen Andrew G.
Published: June 2021

RAS oncoproteins are frequently mutated in pancreatic, colon and lung cancers and play a causal role in human carcinogenesis. Although it is a potentially good target for anticancer drugs development, it has been challenging to specifically target different forms of RAS using small molecules because of absence of evident target binding sites. Chemical reactivity of protein functional groups depends on the environment of a particular residue and reagent accessibility due to protein structure. We wanted to explore chemical reactivity of KRAS 4b functional groups using chemical reagents to identify novel sites for covalent and noncovalent drug development. NHS esters have been used for years for chemical modification of proteins. They are believed to selectively react with primary amino groups. However, there are reports in the literature that other nucleophilic groups such as sulfhydryl group of Cys can react with N-hydroxysuccinimide esters. It is possible that other functional groups may also react. Sulfo-NHS-acetate, sulfo-NHS-biotin and NHS-biotin were used for modification of K, HRAS proteins in GDP and GppNHp-loaded forms. To follow the extent of protein modification, MALDI-TOF MS was used. Generally, the reaction was performed until almost no unmodified protein was left. After modification the protein was digested by Glu-C and peptides were analyzed by MALDI-TOF MS and ES-MS/MS. A “modification index” (ratio of intensities of modified and unmodified peptide forms in MALDI-TOF MS spectra) was calculated to enable relative quantitation for all modified peptides. The comparison of the modification indexes for peptides derived from NHS-biotin modified KRAS and HRAS in GDP or GppNHp form suggested that GppNHp-loaded protein in general allows higher degree of modification. This suggests that there are inducible and expandable binding sites for small organic molecules that could be exploited for design of covalent and noncovalent drug leads. Acknowledgments: Supported by NCI contract HHSN261200800001E.

Citation Format: Oleg Chertov, Xiaoying Ye, Timothy Waybright, Andrew G. Stephen. Probing amino acids residues chemical reactivity of KRAS 4b using N-hydroxysuccinimide esters [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A02.
Abstract A04: Biophysical and biochemical characterization of Src-phosphorylated KRas
Authors: Gebregiworgis Teklab , Marshall Christopher B. , Kano Yoshihito , Ohh Michael , Ikura Mitsuhiko
Published: June 2021

Tyrosine phosphorylation of several small GTPase proteins including Ras has been reported; however, investigations to understand the biologic role of tyrosine phosphorylated small GTPases have been limited. We will present the detailed characterization of the impact of tyrosyl phosphorylation of KRas on each stage of the GTPase cycle, and on the binding to the RBD of BRaf. Using NMR and MS analysis we demonstrate that a catalytic amount of Src (1 mole of Src into 250 moles of KRas) specifically phosphorylates KRas at two of the eight tyrosine residues (Y32 and Y64) in vitro. In HEK293 cells, ectopic expression of Src with KRas results in tyrosyl phosphorylation of the same sites in KRas. Tyrosyl phosphorylation of KRas perturbs the chemical shifts of residues in the two switch regions. Using our real-time NMR-based small GTPase assay, we illustrate that tyrosyl phosphorylation alters regulation of the KRas GTPase cycle by guanine exchange factor (SOScat) and GTPase activating protein (p120GAP), rendering KRas resistant to both activities. Furthermore, using an Octet BLI binding assay, we demonstrated that tyrosyl phosphorylation of KRas strongly impairs BRaf-RBD binding. In vitro, Src-phosphorylated KRas can be completely dephosphorylated by the phosphatase SHP2. In cells, we were able to detect endogenous tyrosyl phosphorylated Ras upon SHP2 CRISPR knockout. Together, our findings demonstrate that the GTPase cycle and effector binding affinity of KRas can be regulated by the balance of Src and SHP2 activities.

Citation Format: Teklab Gebregiworgis, Christopher B. Marshall, Yoshihito Kano, Michael Ohh, Mitsuhiko Ikura. Biophysical and biochemical characterization of Src-phosphorylated KRas [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A04.
Abstract A09: Cooperative membrane interaction between G-domain and HVR defines unique diffusion behavior of KRAS4b
Authors: Goswami Debanjan , Chen De , Columbus John , Turbyville Thomas
Published: June 2021

Rat sarcoma (RAS) family of proteins function as GTP/GDP-dependent control switch to regulate Raf-MAPK effector pathway for cell proliferation and are frequently mutated in human cancer. RAS is anchored to the inner leaflet of the plasma membrane (PM) via C-terminal lipid-tether(s) and hypothesized to concentrate in distinct locations in membrane nano-domains to control the activation step. However, very little is known about the molecular dynamics of RAS on PM and the precise mechanism of activation. Here we characterize the molecular mobility of RAS variants by single-molecule tracking (SMT) method and estimate underlying mobility states. KRAS4b exhibit confined mobility with three diffusive states, a unique characteristic compared to all the other RAS isoforms (KRAS4a, NRAS, and HRAS). Although the major difference across RAS isoforms lies within the C-terminus HVR (hypervariable region) and its post-translational lipid modifications, the additional confinement for KRAS4b is contributed by the G-domain (globular domain) of the protein. Simulation of KRAS4b on membrane revealed a detailed atomistic mechanism that corroborates with the experimental results. Importantly, the oncogenic mutant of KRAS4b shows an altered mode of diffusion, implicating a plausible attribution to/from enhanced effector interaction. This study uncovers a novel underlying principle of KRAS4b functionality on living cell membrane with a potential for innovation in therapeutic strategies against its oncogenic variant.

Citation Format: Debanjan Goswami, De Chen, John Columbus, Thomas Turbyville. Cooperative membrane interaction between G-domain and HVR defines unique diffusion behavior of KRAS4b [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A09.
Abstract A14: Mutations in the ubiquitin ligase adaptor LZTR1 drive human disease by dysregulating RAS ubiquitination and signaling
Authors: Steklov Mikhail , Pandolfi Silvia , Baietti Maria Francesca , Carai Paolo , Zhang Mingzhen , Jang Hyunbum , Cai Yanyan , Legius Eric , Tavernier Jan , Gevaert Kris , Impens Francis , Heymans Stephane , Messiaen Ludwine , Nussimov Ruth , Eyckerman Sven , Sablina Anna
Published: June 2021

Genetic evidence explicitly points out a role for LZTR1, an adaptor for cullin 3 (CUL3) ubiquitin ligase complex, in human disease. Mutations in LZTR1 concurrent with loss of heterozygosity have been associated with glioblastoma and schwannomatosis. LZTR1 emerges as a predisposition gene for paediatric neoplasms. LZTR1 mutations are significantly higher than the background in liver cancers and testicular germ cell tumours. LZTR1 has been also recently added to the list of genes causing Noonan Syndrome, a RASopathy disorder. However, how LZTR1 contributes to human disease and whether it regulates the RAS/MAPK pathway are not known. We found that whereas Lztr1 knockout in mice is embryonically lethal, Lztr1 haploinsufficiency partially recapitulates Noonan syndrome phenotypes, including growth delay, craniofacial dysmorphism, and congenital heart defects. On the other hand, LZTR1 loss in primary Schwann cells drives their dedifferentiation into proliferating cells. By trapping LZTR1 complexes from intact mammalian cells, we identified RAS as a substrate for the LZTR1/CUL3 ubiquitin ligase complex. Immunoprecipitation experiments confirmed that endogenous LZTR1 is associated with endogenous RAS. Ubiquitome and proximity ligation analyses showed that Lztr1 loss abrogates ubiquitination of endogenous RAS specifically at lysine 170. Molecular simulations together with the analysis of Hras-K170R knock-in cells demonstrated that LZTR1-mediated ubiquitination at lysine 170 inhibits RAS signaling by attenuating its association with the membrane. Disease-associated LZTR1 mutations abolish RAS ubiquitination due to disruption of either the LZTR1/CUL3 complex formation, or the interaction with RAS proteins. In line with the work of Bigenzahn et al., Giulio Superti-Furga lab, CeMM (see separate abstract), our study implies LZTR1-mediated RAS ubiquitination as a key step in the control of the RAS GTPases. The discovered molecular mechanism provides a fundamental explanation for the role of LZTR1 in human disease.

Citation Format: Mikhail Steklov, Silvia Pandolfi, Maria Francesca Baietti, Paolo Carai, Mingzhen Zhang, Hyunbum Jang, Yanyan Cai, Eric Legius, Jan Tavernier, Kris Gevaert, Francis Impens, Stephane Heymans, Ludwine Messiaen, Ruth Nussimov, Sven Eyckerman, Anna Sablina. Mutations in the ubiquitin ligase adaptor LZTR1 drive human disease by dysregulating RAS ubiquitination and signaling [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A14.
Abstract A18: Characterization of K-RasG13D as a unique activating allele in a mouse model of colorectal cancer
Authors: Lin Yi-Jang , Dischinger Patrick , Graveel Carrie , Steensma Matthew , Haigis Kevin M.
Published: June 2021

The K-RAS (Kirsten rat sarcoma) oncoprotein is mutationally activated in ~40% of colorectal cancers (CRCs), and K-RAS mutation state is an excellent predictor of the failure of a cancer to respond to conventional chemotherapy or targeted therapy. The colonic epithelium provides a biologically interesting context in which to study the allele-specific characteristics of K-RAS, because CRC displays a broad spectrum of activating mutations that occur at lower frequencies than K-RAS-mutant tumors of other organs. Of particular interest to us, activating G>D mutations in codon 13 of K-RAS occur in 10-20% of K-RAS-mutant CRC, are biochemically distinct from the G12D mutation in mechanism of RAS activation, and have a different epidemiologic profile in patient survival. We established a novel mouse model to compare the colon phenotype of the K-RasG13D allele to that of the previously characterized K-RasG12D allele. In the context of homeostasis and colorectal cancer, we characterized the murine colonic epithelium at histologic, proteomic and transcriptomic levels. Ultimately, we aim to elucidate mutant-specific drug sensitivities by deeply understanding K-Ras biology in the genetically controlled setting of our mouse models.

Citation Format: Yi-Jang Lin, Patrick Dischinger, Carrie Graveel, Matthew Steensma, Kevin M. Haigis. Characterization of K-RasG13D as a unique activating allele in a mouse model of colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A18.
Abstract A41: Nucleotide metabolism heterogeneity in mutant KRAS pancreatic cancer
Authors: Le Thuc M. , Kim Woosuk , Capri Joseph R. , Cabebe Anthony E. , Armstrong Wes , Abt Evan , Poddar Soumya , Xu Shili , Dawson Dave , Donahue Timothy R. , Radu Caius G.
Published: June 2021

Mutant KRAS orchestrates major metabolic adaptations critical for pancreatic ductal adenocarcinoma (PDAC) growth and survival, including changes in glucose, amino acid, lipid and energy metabolism. However, how nucleotide metabolism in PDAC is impacted by mutant KRAS has received much less attention, even though nucleotides play critical roles in major biologic processes including nucleic acid synthesis, phospholipid biosynthesis and protein glycosylation. Pyrimidine nucleotide metabolism is a highly complex and regulated system with multiple pathways contributing to its plasticity and robustness. Pyrimidine nucleotides can be produced via the de novo pathway (DNP) that utilizes glucose and amino acids, and the nucleoside scavenging pathway (NSP) that utilizes extracellular uridine and cytidine. We hypothesize that nucleotide products of RNA turnover can fuel a third route for pyrimidine biosynthesis, which we term the nucleotide recycling pathway (NRP). Among these three major pyrimidine nucleotide biosynthetic pathways, the DNP has been studied most extensively. The biologic and therapeutic significance of the NSP and NRP in PDAC, as well as their regulation and coordination with DNP in the context of mutant KRAS signaling, are poorly understood. We further hypothesize that since PDAC tumor microenvironment is characterized by insufficient supply of nutrients and oxygen, these tumors have limited DNP capacity and increased reliance on alternate pyrimidine biosynthetic pathways. Here, we describe a novel mass spectrometric (MS) method to measure the contributions of pyrimidine nucleotide convergent pathways to RNA and DNA synthesis in PDAC models. Applying this method to a panel of PDAC models, including patient-derived primary lines, revealed previously unappreciated pyrimidine biosynthetic heterogeneity, suggesting the existence of distinct pyrimidine nucleotide metabolic subtypes. Identifying and characterizing these subtypes in the context of oncogenic KRAS signaling may enable stratification of PDAC tumors and guide the development of novel therapeutic approaches.

Citation Format: Thuc M. Le, Woosuk Kim, Joseph R. Capri, Anthony E. Cabebe, Wes Armstrong, Evan Abt, Soumya Poddar, Shili Xu, Dave Dawson, Timothy R. Donahue, Caius G. Radu. Nucleotide metabolism heterogeneity in mutant KRAS pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A41.
Abstract B21: Cancer-specific intracellular delivery of therapeutic antibodies against KRAS
Authors: McGuire Michael J. , Li Susan , Venugopal Indu , Gormley Claire , Luebke Kevin J. , Allred Curtis A. , Brown Kathlynn C.
Published: June 2021

Antibodies and peptides directed against the switch regions of RAS can disrupt interactions with its downstream effector proteins, blocking downstream protumorigenic signaling. However, these biologic therapies are unable to reach the intracellular environment. We have overcome this problem using peptidic molecular guidance system (MGSs) that have the unique ability to bind specifically to epithelial-derived cancer cells, and upon binding, trigger rapid internalization and trafficking to a specific subcellular location. Using an unbiased phage display approach, we have identified a series of cancer-specific MGSs that deliver cargo to discrete subcellular locations. Chemical optimization results in MGSs with 1-10 nM affinity for their cellular target, serum stability >48 hours, and 50- to 1000-fold specificity for cancer cells compared to normal control cells. The rate of MGS internalization is rapid with t1/2 of 10-30 minutes, achieving intracellular concentrations up to 1.5 μM. Using these MGSs as delivery agents, we have delivered a function-blocking RAS antibody (Y13-259), resulting in a reduction of p-ERK and apoptosis in KRAS mutant cell lines. The effect is MGS dependent, and confocal microscopy indicates co-localization of KRAS and the antibody indicating target engagement. The MGS-antibody homes to tumors in an animal model, resulting in significant suppression in tumor growth compared to untreated animals. Free MAb does not impact tumor growth. Using MGSs to delivery MAb for intracellular targets is a paradigm change for immunotherapy and represents a new approach for controlling intracellular protein-protein interactions. Combination of these cancer-specific delivery agents with effectors of RAS signaling, such as antibodies and other biologics, has therapeutic potential for KRAS-driven cancers.

Citation Format: Michael J. McGuire, Susan Li, Indu Venugopal, Claire Gormley, Kevin J. Luebke, Curtis A. Allred, Kathlynn C. Brown. Cancer-specific intracellular delivery of therapeutic antibodies against KRAS [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B21.
Abstract B34: Targeted destruction of endogenous K-RAS using an Affinity directed PROtein Missile (AdPROM)
Authors: Roth Sascha O. M. , Macartney Thomas J. , Konopacka Agnieszka , Queisser Markus , Sapkota Gopal P.
Published: June 2021

K-RAS is one of the most frequently mutated oncogenes in cancer. Yet there are no effective strategies to target K-RAS for inhibition. Our laboratory has developed an Affinity-directed PROtein Missile (AdPROM) system for targeted proteolysis of endogenous proteins. Here, we show that we can achieve a robust degradation of endogenous K-RAS and mutant K-RAS from different cell lines in a targeted manner using our AdPROM system. We are currently assessing the impact of the targeted destruction of K-RAS and mutant K-RAS in cell signaling and proliferation.

Citation Format: Sascha O. M. Roth, Thomas J. Macartney, Agnieszka Konopacka, Markus Queisser, Gopal P. Sapkota. Targeted destruction of endogenous K-RAS using an Affinity directed PROtein Missile (AdPROM) [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B34.