V1 Variant Research Articles

Delphi/AHP-Based Method for Biomass Sustainable Assessment in the Sugar Industry

Multi-criteria methods are highly attractive tools to address the inherent complexity of evaluating problems in various scientific areas. The combination of methods such as Delphi/AHP is emerging as a robust alternative to evaluate the sustainability of renewable energy sources. In this theoretical-descriptive research, the use of the Delphi method is proposed to select criteria and sub-criteria to obtain a high level of reliability, while the AHP method is used to establish an order of preference among the alternatives analyzed. This process requires the support of a committee of experts, whose role is to identify the various biomass alternatives that can be used in the sugar industry, considering aspects related to sustainability. The selected experts have identified energy, exergy, and emergetic indicators, in which economic, environmental, and social aspects are integrated. The multi-criteria analysis shows that the V1 variant was the most satisfactory in terms of biomass sustainability, representing 45% and 53% of the overall priorities in the evaluated case studies. In addition, the sensitivity analysis under an equal-weighted scenario for both study cases evidenced that variant V1 acquired the highest score (38.17%) among all alternatives. Variant V4 achieved the second highest score (31.79%), while alternative V2 achieved only 29.04%, respectively. The integration of Delphi/AHP methods emerges as a novel tool to assess sustainability in different industries of the energy sector.

Influence of Chemical Modifications of the Crystallophore on Protein Nucleating Properties and Supramolecular Interactions Network.

Crystallophores are lanthanide complexes that have demonstrated outstanding induction of crystallization for various proteins. This article explores the effect of tailored modifications of the crystallophore first generation and their impact on the nucleating properties and protein crystal structures. Through high-throughput crystallization experiments and dataset analysis, we evaluated the effectiveness of these variants, in comparison to the first crystallophore generation G1. In particular, the V1 variant, featuring a propanol pendant arm, demonstrated the ability to produce new crystallization conditions for the proteins tested (hen-egg white lysozyme, proteinase K and thaumatin). Structural analysis performed in the case of hen egg-white lysozyme along with Molecular Dynamics simulations, highlights V1's unique behavior, taking advantage of the flexibility of its propanol arm to explore different protein surfaces and form versatile supramolecular interactions.

Abstract 5163: Determining the role of septin expression in response of breast cancer patients to microtubule targeted chemotherapy

Abstract Drugs that disrupt the microtubule cytoskeleton, including both microtubule stabilizing taxanes and destabilizers like eribulin, are regularly used in the treatment of metastatic breast cancer. However, there are currently no molecular biomarkers to guide their use. Septins, a class of small GTPases, interact with both microtubules and actin to functionally link these cytoskeletal components and regulate cell biological processes shown to be critical for the invasion and migration of cancer cells. In particular, septin 9 (SEPT9) directly interacts directly with microtubules through an N-terminal domain on the longest isoform, SEPT9i1, which is sufficient to promote migration and invasion of breast cancer cells in vitro. We hypothesized that the relative expression of septin9 isoforms in breast tumors could play a role in their response to treatment with microtubule-targeted chemotherapy, particularly in the metastatic setting. A retrospective investigation of the relationship between septin expression and response to taxane chemotherapy was conducted utilizing full transcriptome sequencing data from biopsies of 75 breast cancer patients treated at our NCI-Designated Mays Cancer Center who received molecular profiling through Caris Life Sciences. Levels of total SEPT9 expression as well as the expression of specific variants that encode mechanistically distinct septin9 isoforms, SEPT9v1, v2, and v3, were determined for each sample and correlated with demographic, treatment, and outcome data for these patients. The oncogenic sept9_v1 variant (which produces the septin9_i1 isoform) was detected in over half of tumors and those with the highest total septin9 expression also expressed significantly higher levels of this isoform. Strikingly, the expression of total septin9, as well as the v3 variant, was significantly increased in taxane-treated patients who survived over a year after diagnosis with metastatic breast cancer, suggesting that septin9 expression could be a molecular correlate of response to taxane chemotherapy. We found that the expression of total septin9 and its variants was not significantly correlated with race, ethnicity, or cancer type suggesting that our findings would apply to our patient population as a whole, which is over 50% Hispanic. These data were used to support an ongoing prospective study to determine effect of septin expression on response to taxane chemotherapy in the neoadjuvant setting and determine the effect of taxane treatment on septin expression by comparing samples pre and post treatment. Together this work supports our overall goal of identifying predictive biomarkers to facilitate the use of microtubule targeted chemotherapy in a more personalized manner. Citation Format: Tamarah Aldawoodi, Jacob Nathaniel Essif, Amna Naqvi, Lauren D. Boyle, Daniela Urueta Portillo, Kate Lathrop, April L. Risinger. Determining the role of septin expression in response of breast cancer patients to microtubule targeted chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5163.

Energy Sustainability Indicators for the Use of Biomass as Fuel for the Sugar Industry

There are numerous analytical and/or computational tools for evaluating the energetic sustainability of biomass in the sugar industry. However, the simultaneous integration of the energetic–exergetic and emergetic criteria for such evaluation is still insufficient. The objective of the present work is to propose a range of indicators to evaluate the sustainability of the use of biomass as fuel in the sugar industry. For this purpose, energy, exergy, and emergy evaluation tools were theoretically used as sustainability indicators. They were validated in five variants of different biomass and their mixtures in two studies of technologies used in Cuba for the sugar industry. As a result, the energy method showed, for all variants, an increase in efficiency of about 5% in the VU-40 technology compared to the Retal technology. There is an increase in energy efficiency when considering AHRs of 2.8% or Marabu (Dichrostachys cinerea) (5.3%) compared to the V1 variant. Through the study of the exergetic efficiency, an increase of 2% was determined in both technologies for the case of the V1 variant, and an increase in efficiency is observed in the V2 variant of 5% and the V3 variant (5.6%) over the V1 variant. The emergetic method showed superior results for the VU-40 technology over the Retal technology due to higher fuel utilization. In the case of the V1 variant, there was a 7% increase in the renewability ratio and an 11.07% increase in the sustainability index. This is because more energy is produced per unit of environmental load.

Effect of refined multi-scale microstructures on the ultra-high impact toughness of bainitic steel austempered below Ms temperature

In this study, the multi-scale microstructures of bainitic steel were refined to different degrees by austempering below Ms (martensite start temperature). The influence of multi-scale microstructures and their boundaries on impact toughness was analyzed. The results revealed that the impact toughness of bainitic steel significantly improved as the austempering temperature decreased. The block structure and Bain groups are refined by austempering below Ms, thereby introducing high-density HAGBs (high-angle grain boundaries) and increasing crack propagation resistance. The proportion of V2, V3, and V5 variants increased and formed HAGBs with the V1 variant, which regulated phase transformation strain and delayed crack formation. The improvement in impact toughness can be attributed to the formation of more HAGBs. This is mainly caused by the refinement of the block structure. Therefore, the block structure can be considered as the dominating and effective controlling unit of impact toughness.

Microstructural evolution and ultra-high impact toughness of austempered lamellar bainitic steel far below Ms temperature

The present study investigated the effects of the austempering process far below Ms (martensite start temperature) on the microstructural evolution and impact toughness of bainitic steel with ultrahigh impact toughness and compared the results with the tempered martensitic steel. The results revealed that the lamellar bainitic steel composed of bainitic ferrite laths and retained austenite films is obtained by austempering far below Ms temperature. The obtained steel exhibited the Charpy V-notch impact absorbed energy of up to 131 ± 6 J, which was 2.7 times higher than that of the tempered martensitic steel (49 ± 1 J). The ultra-high impact toughness of bainitic steel was attributed to the deflection of the crack propagation direction by the refined bainitic ferrite laths and the passivation of the crack tip by the retained austenite films. Furthermore, the proportion of V2, V3, and V5 variants increased and formed HAGBs (high-angle grain boundaries) with the V1 variant in the austempered bainitic steel B-32. These HAGBs inhibited crack propagation effectively, thereby significantly improving the impact toughness of the austempered bainitic steel.

Landscape of potentially targetable receptor tyrosine kinase fusions in diverse cancers by DNA-based profiling

Recurrent fusions of receptor tyrosine kinases (RTKs) are often driving events in tumorigenesis that carry important diagnostic value and are potentially targetable by the increasing number of tyrosine kinase inhibitors (TKIs). Here, we characterized the spectrum of 1324 RTK fusions with intact kinase domains in solid tumors by DNA-based high-throughput sequencing. Overall, the prevalence of RTK fusions were 4.7%, with variable frequencies and diverse genomic structures and fusion partners across cancer types. Cancer types, such as thyroid cancers, urological cancers and neuroendocrine tumors are selective in the RTK fusions they carry, while others exhibit highly complex spectra of fusion events. Notably, most RTKs were promiscuous in terms of the partner genes they recombine with. A large proportion of RTK fusions had one of the breakpoints localized to intergenic regions. Comprehensive genomic profiling revealed differences in co-mutational patterns pre- and post-TKI treatments across various RTK fusions. At baseline, multiple cases were detected with co-occurring RTK fusions or concomitant oncogenic mutations in driver genes, such as KRAS and EGFR. Following TKI resistance, we observed differences in potential on- and off-target resistance mutations among fusion variants. For example, the EML4-ALK v3 variant displayed more complex on-target resistance mechanisms, which might explain the reduced survival outcome compared with the v1 variant. Finally, we identified two lung cancer patients with MET+ and NTRK1+ tumors, respectively, who responded well to crizotinib treatment. Taken together, our findings demonstrate the diagnostic and prognostic values of screening for RTK fusions using DNA-based sequencing in solid tumors.

Detection of ALK fusion variants by RNA-based NGS and clinical outcome correlation in NSCLC patients treated with ALK-TKI sequences

IntroductionAnaplastic lymphoma kinase (ALK) fusions identify a limited subset of non–small cell lung cancer (NSCLC) patients, whose therapeutic approach have been radically changed in recent years. However, diagnostic procedures and clinical-radiological responses to specific targeted therapies remain heterogeneous and intrinsically resistant or poor responder patients exist. MethodsA total of 290 patients with advanced NSCLC defined as ALK+ by immunohistochemistry (IHC) and/or fluorescent in situ hybridisation (FISH) test and treated with single or sequential multiple ALK inhibitors (ALKi) from 2011 to 2017 have been retrospectively retrieved from a multicentre Italian cancer network database. In 55 patients with enough leftover tumour tissue, specimens were analysed with both targeted and customised next generation sequencing panels. Identified fusion variants have been correlated with clinical outcomes. ResultsOf the 55 patients, 24 received crizotinib as first-line therapy, 1 received ceritinib, while 30 received chemotherapy. Most of the patients (64%) received ALKi in sequence. An ALK fusion variant was identified in 73% of the cases, being V3 variant (E6A20) the most frequent, followed by V1 (E13A20) and more rare ones (e.g. E6A19). In three specimens, four new EML4-ALK fusion breakpoints have been reported. Neither fusion variants nor brain metastases were significantly associated with overall survival (OS), while it was predictably longer in patients receiving a sequence of ALKi. The presence of V1 variant was associated with progression-free survival (PFS) improvement when crizotinib was used (p = 0.0073), while it did not affect cumulative PFS to multiple ALKi. ConclusionOutcomes to sequential ALKi administration were not influenced by fusion variants. Nevertheless, in V1+ patients a prolonged clinical benefit was observed. Fusion variant identification by NGS technology may add relevant information about rare chromosomal events that could be potentially correlated to worse outcomes.

Functional characterization of two variants of mitochondrial topoisomerase TOP1MT that impact regulation of the mitochondrial genome

TOP1MT encodes a mitochondrial topoisomerase that is important for mtDNA regulation and is involved in mitochondrial replication, transcription, and translation. Two variants predicted to affect TOP1MT function (V1 - R198C and V2 - V338L) were identified by exome sequencing of a newborn with hypertrophic cardiomyopathy. As no pathogenic TOP1MT variants had been confirmed previously, we characterized these variants for their ability to rescue several TOP1MT functions in KO cells. Consistent with these TOP1MT variants contributing to the patient phenotype, our comprehensive characterization suggests that both variants had impaired activity. Critically, we determined neither variant was able to restore steady state levels of mitochondrial-encoded proteins nor to rescue oxidative phosphorylation when re-expressed in TOP1MT KO cells. However, we found the two variants behaved differently in some respects; while the V1 variant was more efficient in restoring transcript levels, the V2 variant showed better rescue of mtDNA copy number and replication. These findings suggest that the different TOP1MT variants affect distinct TOP1MT functions. Altogether, these findings begin to provide insight into the many roles that TOP1MT plays in the maintenance and expression of the mitochondrial genome and how impairments in this important protein may lead to human pathology.

Evaluation of sars Cov -2 variants among RT-PCR tested positive COVID-19 patients at our teriary care hospital in Telangana State, India

Background: The initial reports of coronavirus disease 2019 (COVID-19) came out with an outbreak in Wuhan, China on 30 December 2019 [1,2], SARS-CoV-2 has been spreading worldwide and, as of 15 July 2021, there have been 188.13 million confirmed infections and 4.06 million deaths [3]. New variants are thought to be responsible for re-infections, either after natural infection or after vaccination, as observed in Brazil and the United States, respectively. There is evidence that re-infections are already happening in India; a recent survey identified a re-infection proportion of 4.5% from a pool of 1,300 participants infected between January 2020 and October 2020. Another concern about the emergence of new variants is the potential failure of RT-PCR tests for diagnostics. Failure to target the gene encoding the spike protein was observed during detection of the 501Y.V1 variant in the UK2. Aim and Objectives of the study: The aim and objective of the current study are to identify the lineages and calculate the prevalence among our population. Methodology: The work was initiated following approvals from the Institutional Ethical Committee.

Impact of Deposition of the (TiBx/TiSiyCz) x3 Multilayer on M2 HSS on the Cutting Force Components and Temperature Generated in the Machined Area during the Milling of 316L Steel.

High-speed steel (HSS) tools account for 20 percent of the cutting tools materials’ global market. This is due to both their significant toughness and resistance to cracking, compared to cemented carbides. Covering steel tools with hard coatings clearly improves their mechanical properties, wear resistance, and significantly increases their durability. Physical vapor deposition methods are preferred for coating metal substrates, as they allow low temperature deposition. The most widely deposited coating materials are carbides, nitrides, and borides. They are combined with softer ones in the multilayer structure to promote increased resistance to cracking and delamination in comparison to monolayered structures. In this paper, the M2 steel end mills were coated by (TiBx/TiSiyCz) x3 multilayer by the pulsed laser deposition method. Coated and uncoated tools were tested in the cylindrical down milling of AISI 316L steel. Components of the cutting force and temperature generated in the machined area during dry milling were measured under two variants of operating conditions: V1 and V2. Tool wear mechanism was examined using scanning electron microscopy (SEM), accompanied by EDS analysis of worn areas. It was found that milling with higher speed (variant V2) is accompanied by lower cutting force components and a lower temperature generated in cutting area. The presence of the coating allowed lower cutting forces and temperature in the case of variant V1. The temperature measured during milling did not exceed 200 °C. The SEM observation of the edges of cutting tools indicated that the main mechanism of wear for both types of tools was abrasion. The built-up edge formation was observed in the case of tools tested at the V1 cutting parameters variant. It was assumed that it was the reason for higher cutting forces measured during milling according to this variant. The chemical composition of built-up edges was different for coated and uncoated tools. Tribo-chemical reactions were responsible for the reduction of the cutting force and temperature components observed during milling with a coated tool at V1 variant. Boron and titanium were the elements of the coating that enabled the tribo-oxidation reactions thanks to which friction was reduced. Our results show that this beneficial effect occurs with (TiBx/TiSiyCz) x3 coated tools, but can easily be lost with inadequately selected cutting parameters.

Evolution, Mode of Transmission, and Mutational Landscape of Newly Emerging SARS-CoV-2 Variants.

ABSTRACTThe recent emergence of multiple variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a significant concern for public health worldwide. New variants have been classified either as variants of concern (VOCs) or variants of interest (VOIs) by the CDC (USA) and WHO. The VOCs include lineages such as B.1.1.7 (20I/501Y.V1 variant), P.1 (20J/501Y.V3 variant), B.1.351 (20H/501Y.V2 variant), and B.1.617.2. In contrast, the VOI category includes B.1.525, B.1.526, P.2, and B.1.427/B.1.429. The WHO provided the alert for last two variants (P.2 and B.1.427/B.1.429) and labeled them for further monitoring. As per the WHO, these variants can be reclassified due to their status at a particular time. At the same time, the CDC (USA) has marked these two variants as VOIs up through today. This article analyzes the evolutionary patterns of all these emerging variants, as well as their geographical distributions and transmission patterns, including the circulating frequency, entropy diversity, and mutational event diversity throughout the genomes of all SARS-CoV-2 lineages. The transmission pattern was observed highest in the B.1.1.7 lineage. Our frequency evaluation found that this lineage achieved 100% frequency in early October 2020. We also critically evaluated the above emerging variants mutational landscape and significant spike protein mutations (E484K, K417T/N, N501Y, and D614G) impacting public health. Finally, the effectiveness of vaccines against newly SARS-CoV-2 variants was also analyzed.

A Possible Role of Remdesivir and Plasma Therapy in the Selective Sweep and Emergence of New SARS-CoV-2 Variants.

Since summer 2020, SARS-CoV-2 strains at the origin of the COVID-19 pandemic have suddenly been replaced by new SARS-CoV-2 variants, some of which are highly transmissible and spread at a high rate. These variants include the Marseille-4 lineage (Nextclade 20A.EU2) in Europe, the 20I/501Y.V1 variant first detected in the UK, the 20H/501Y.V2 variant first detected in South Africa, and the 20J/501Y.V3 variant first detected in Brazil. These variants are characterized by multiple mutations in the viral spike protein that is targeted by neutralizing antibodies elicited in response to infection or vaccine immunization. The usual coronavirus mutation rate through genetic drift alone cannot account for such rapid changes. Recent reports of the occurrence of such mutations in immunocompromised patients who received remdesivir and/or convalescent plasma or monoclonal antibodies to treat prolonged SARS-CoV-2 infections led us to hypothesize that experimental therapies that fail to cure the patients from COVID-19 could favor the emergence of immune escape SARS-CoV-2 variants. We review here the data that support this hypothesis and urge physicians and clinical trial promoters to systematically monitor viral mutations by whole-genome sequencing for patients who are administered these treatments.

On the Association between SARS-CoV-2 Variants and COVID-19 Mortality During the Second Wave of the Pandemic in Europe

BACKGROUND: Preliminary clinical evidence suggests an increased COVID-19 mortality associated with the variant of concern 20I/501Y.V1. The evidence outside the UK and a real-world comparison of variants spread and mortality is sparse. This study aims at investigating the association between COVID-19 mortality and SARS-COV-2 variants spread during the second wave of the COVID-19 pandemic in Europe.METHODS: For 38 European countries, publicly available data were collected on numbers of COVID-19 deaths, SARS-COV-2 variants spread through time using Nextstrain classification and countries’ demographic and health characteristics. The cumulative number of COVID-19 deaths and the height of COVID-19 daily deaths peak during the second wave of the pandemic were considered as outcomes. Pearson correlations and multivariate generalized linear models with selection algorithms were used.FINDINGS: The average proportion of 20I/501Y.V1 variant (B.1.1.7) was found to be a significant predictor of cumulative number of COVID-19 deaths within two months before the deaths peak and between 1 January – 25 February 2021, as well as of the deaths' peak height when calculating the proportion during the second wave and the pre-peak period. The average proportion of 20A.EU2 variant (S:477N) was a significant predictor of cumulative COVID-19 deaths in the pre-peak period.INTERPRETATION: Our findings suggest that the spread of a new variant of concern 20I/501Y.V1 had a significant impact on the mortality during the second wave of COVID-19 pandemic in Europe and that proportions of 20A.EU2 and 20I/501Y.V1 variants were associated with increased mortality in the initial phase of that wave.FUNDING: None.DECLARATION OF INTERESTS: None.ETHICS APPROVAL STATEMENT: Not required. This study does not require ethical approval as it was conducted on country-level data and involved information freely available in the public domain.

Binding affinity and mechanisms of SARS-CoV-2 variants

During the rapid worldwide spread of SARS-CoV-2, the viral genome has been undergoing numerous mutations, especially in the spike (S) glycoprotein gene that encode a type-I fusion protein, which plays an important role in the infectivity and transmissibility of the virus into the host cell. In this work, we studied the effect of S glycoprotein residue mutations on the binding affinity and mechanisms of SARS-CoV-2 using molecular dynamics simulations and sequence analysis. We quantitatively determined the degrees of binding affinity caused by different S glycoprotein mutations, and the result indicated that the 501Y.V1 variant yielded the highest enhancements in binding affinity (increased by 36.8%), followed by the N439K variant (increased by 29.5%) and the 501Y.V2 variant (increased by 19.6%). We further studied the structures, chemical bonds, binding free energies (enthalpy and entropy), and residue contribution decompositions of these variants to provide physical explanations for the changes in SARS-CoV-2 binding affinity caused by these residue mutations. This research identified the binding affinity differences of the SARS-CoV-2 variants and provides a basis for further surveillance, diagnosis, and evaluation of mutated viruses.

Complex CSF3R Protein Isoform Interactions Dictate Cytokine Responsiveness

CSF3R, the receptors for granulocyte colony stimulating factor, is a critical regulator of neutrophil production. Multiple CSF3R mRNA transcripts have been identified and are annotated in Genbank. The expression and function of the different CSF3R proteins have not been fully elucidated. We generated antibodies specific for two of the identified and annotated isoforms, V3 and V4. CSF3R-V4 is a truncated variant of V1 with a unique C-terminal 34 amino acids and this variant confers enhanced growth signals. Changes in the ratio of V1:V4 isoforms have been implicated in chemotherapy resistance and relapse of AML. CSF3R-V3 is a variant of V1 with a 27 amino acid insertion between two conserved domains in the cytoplasmic portion of the receptor involved in JAK/STAT activation, termed the box 1 and box 2. CSF3R-V3 produces reduced proliferative signaling in response to G-CSF. When V3 is co-expressed with V1, proliferative signaling is reduced in a concentration dependent manner. In order to generate custom rabbit polyclonal antibodies specific for CSF3R-V3 and CSF3R-V4 we used either a peptide that corresponds to a unique amino acid sequence present only in CSF3R-V3 or a peptide specific for a portion of the C-terminal amino acid sequence unique to the CSF3R-V4 isoform conjugated to an immunogenic carrier protein. These immunogens both produced robust immune responses, and the polyclonal antibodies were subsequently purified from bulk sera. Immunoblot analysis of lysates from Ba/F3 cells expressing CSF3R-V1 (V1), CSF3R-V3 (V3), or CSF3R-V4 (V4) demonstrated that both the custom generated anti-CSF3R-V3 and anti-CSF3R-V4 antibodies were very specific, recognizing only the appropriate CSF3R receptor isoform. All three CSF3R splice variants are recognized by commercially available anti-CSF3R (clone LMM741 to CD114), while the anti-CSF3R-V4 custom antibody and the custom anti-CSF3R-V3 antibody recognizes only the CSF3R-V4 and CSF3R-V3 isoforms, respectively. We next sought to detect the CSF3R receptor isoforms in primary human cells. Using our custom antibodies, we detected for the first time, both the CSF3R-V3 and CSF3R-V4 receptor forms in primary neutrophils isolated from healthy donors. Each of the CSF3R isoforms produce unique signaling, and we hypothesized that the observed differences in G-CSF-dependent signaling is produced by the expression level of each receptor isoform via both homodimerization and by heterodimerization of the receptor splice variant proteins. To investigate the potential for heterodimerization of the CSF3R-V1 with the V3 and V4 isoforms, we generated a CSF3R-V1 with a c-terminal epitope tag and co-expressed this construct with both CSF3R-V3 or CSF3R-V4. Immunoprecipitation with an antibody to the epitope tag (recognizing the V1 variant) followed by immunoblotting with the custom anti-V3 or anti-V4 antibodies demonstrated that both CSF3R-V3 and CSF3R-V4 co-immunoprecipitated with CSF3R-V1, in agreement with our hypothesis that the splice variants form receptor heterodimers. Of note, the CSF3R receptor heterodimers are detected even in the absence of G-CSF, thus demonstrating that CSF3R exist as a preformed receptor dimer in an inactive state. In conclusion, we have generated antibodies that specifically detect the CSF3R-V3 and the CSF3R-V4 receptor proteins. These are the first studies to demonstrate the expression of the CSF3R splice variants at the protein level, in both cell lines and primary human cells. In addition, these are the first studies to demonstrate the formation of heterodimers of the CSF3R splice variants, providing a mechanism for the observed alteration in ligand-dependent signaling produced under conditions of altered splice variant expression. Disclosures Avalos: Juno: Membership on an entity's Board of Directors or advisory committees; Best Practice-Br Med J: Patents & Royalties: receives royalties from a coauthored article on evaluation of neutropenia.

ALK resistance mutations (muts) developed in different ALK fusion types.

e14741 Background: Patients (pts) treated with ALK inhibitors (ALKi) inevitably develops resistance. Diverse mutations (muts) confer resistance to ALKis, which may be related to the diverse types of ALK fusion. The objective of this study is to see whether the resistance mechanisms developed during ALKi therapy are ALK fusion types dependent. Methods: The mutation profiles of 38 pts with known ALK fusion partners and positions, who ordered NGS test in our institution after progressed on ≥ 1 ALKis, were analyzed. Results: Resistant muts in ALK kinase domain ( ALK resistant muts) were detected in 53% (20/38) of the resistant pts. 100% (1/1) of the EML4-ALK V5’ variant (V5’), 81% (13/16) of the EML4-ALK V3 variant (V3), 60% (3/5) of the non- EML4-ALK, 23% (3/13) of EML4-ALK V1 variant (V1), and 0% (0/3) of the EML4-ALK V2 variant (V2) had ALK resistant muts. ALK G1202R was detected in 29% of the pts. 100% (1/1) of the V5’, 50% (8/16) of the V3, 20% (1/5, HIP1-ALK) of the non- EML4-ALK, and none of the V1 (0/13) and V2 (0/3) had G1202R. Amplification and gain of function muts in oncogenes and loss of function muts in tumor suppressor gene were detected in 16% (6/38) of the pts, namely 4 V1, 1 V2, and 1 V3. On average, pts treated with 3rd generation ALKis had significantly more muts in ALK (7.5 vs. 3.6, p = 0.0008) and in all targeted regions (9.1 vs. 4.3, p = 0.0005) than pts treated with only 1st and 2nd generation ALKis. Conclusions: The type of resistance mechanisms developed during ALKi therapy may depend on the ALK fusion type of the pt. ALK resistant muts, especially G1202R, developed most frequently in V3/V5’, followed by non- EML4-ALK, and V1/V2, while amplification and grain of function muts in oncogenes and loss of function muts in tumor suppressor genes developed more often in V1/V2 than in V3/V5’. The 3rd generation ALKi may increase the genomic alterations in treated pts.

High-level expression of PRSS3 correlates with metastasis and poor prognosis in patients with gastric cancer.

Serine protease-3 (PRSS3) is a known contributor to the genesis and development of malignant tumors, although its role in gastric cancer (GC) is still unclear. PRSS3 expression in GC tissue samples and its relationship with clinicopathological features were analyzed. Effects of GC cellular responses to the introduction of small interfering RNA (siRNA)-mediated and short hairpin RNA (shRNA)-mediated interference with tumor PRSS3 expression were also assessed. PRSS3 was significantly upregulated in GC tissues, and PRSS3 protein levels were higher in tumors that developed metastases soon after the surgery compared with those that remained metastasis-free. High expression of PRSS3 was associated with tumor N staging and independently predictive of postoperative prognosis in patients with GC. The V1 variant of PRSS3 was primarily detected in GC tissue and cell lines, the others (V2-V4) being scarcely detectable. Methylation and demethylation drugs had no impact on expression levels of any PRSS3 transcriptional variant. The downregulated PRSS3 expression suppressed GC cell growth, migration, and invasion in vitro and in vivo. PRSS3 appears to act as an oncogene of GC. High PRSS3 expression portends postoperative metastasis, serving as an effective biomarker of poor therapeutic outcomes.

Influence of the Mother Bulb Size on the Growth and Development of Allium ‘Purple Rain’

The experiment aims was to study the influences of the mother bulb weight on the growth and development, of the plants of ornamental onion. The study was conducted during October 2015 – June 2016 and the biologic material was represented by Allium ‘Purple Rain’. The bulbs were divided in three different weight groups: W1 (15.1-30 g), W2 (5.1-15 g), W3 (< 5 g), which were reprezented the three variants (V1, V2, respectively V3). The bulbs were planted in the field and the plants were studied thrue the biometric determinations. The results were compared with the average of the experiment. The research showed that the all the analyzed characters (number and length of leaves, number and weight of new formed bulbs, flowers yield, diameter of the inflorescence) were decreasing from V1 to the V3 variant. The highest number of the flowering plants (98.9 %) was resulted from V1 variant. From V2, bloomed only 20% of the bulbs and from V3 the bulbs did not got flowers. The flowering time was earlier for the plants resulted from the biggest bulbs (V1), with around 3 days, then the V2 and around 7 days then V3. The capacity to form new bulbs, expressed in bulb number or bulb weight per plant, was increased with the increasing of the mother bulb weight. The number of leaves per plant and the length of the leaves were higher for V1, as against the other two variants

Abstract 4123: Investigation of growth factor signaling through macrophage colony stimulating factor (CSF-1) and its receptor (FMS) in the bone marrow stromal microenvironment in acute myeloid leukemia (AML).

Abstract Acute myeloid leukemia (AML) is a highly heterogeneous and growth factor-dependent disease of the bone marrow. The bone marrow stromal microenvironment plays a critical role in providing growth and survival signals. hFMS, the human homologue of the Feline (McDonough strain) Sarcoma virus oncogene, also known as macrophage colony stimulating factor 1 receptor (CSF1R, FMS, CD115) is a tyrosine kinase receptor found on mononuclear phagocytes that promotes their growth and differentiation. The human macrophage CSF-1 ligand (MCSF-1, CSF-1) produced by supporting cells in the bone marrow acts on FMS. CSF-1 has a full length secreted isoform v1 and a membrane-bound isoform v3. Upon activation, FMS modulates the activity of important effectors of growth and survival pathways such as the mitogen-activated protein kinase (MAPK)/ERK and phosphoinositol-3 kinase (PI3K)/Akt pathways, respectively. There is a variable association of Fms with the stem cell marker CD34 on AML cells and this suggests different structures within an AML hierarchy. To better understand stromal cell interactions mediated by the CSF-1/FMS axis and examine the high engraftment failures of AML human samples in mice, AML cells were grown on murine stromal cells that were ‘humanized’ through lentiviral-mediated introduction of human CSF-1. The soluble v1 and membrane-bound v3 isoforms of CSF-1 were investigated. We found that stroma-presented CSF-1 effectively supports the growth of leukemic cells in short and long-term growth cultures, with the v3 CSF-1 variant sustaining better growth than v1. Interestingly the FMS-(low)/CD34+ subpopulation displayed significantly better growth on stroma compared to the FMS+ populations. Eventually this subpopulation gave rise to FMS+ cells, suggesting that it may harbor stem or initiating properties. In interrogating molecular signaling events induced by the CSF-1/FMS interaction, we found that the membrane-bound v3 isoform leads to increases in phosphorylation of effectors of the MAPK and PI3k/Akt pathways, namely Akt, p70S6K and 4E-BP1, whereas the v1 variant does not elicit such signals as robustly in leukemic cells grown on stroma. Taken together, this indicates that the manner in which the ligand is presented (soluble vs. membrane-bound) impacts the growth, survival and molecular signaling arising from the leukemic-stromal cell interaction. Future work will involve generation of gene and protein expression profiles of leukemic and stromal cells after grown in co-culture in aims of identifying targets induced by the soluble vs. membrane-bound forms of CSF-1. Particular identification of druggable targets in both leukemic and stromal cell compartments will allow for the design of therapeutic strategies to interfere with leukemic-stromal cell interactions driven by the CSF-1/FMS axis. Citation Format: Ayesha Rashid, Mohamed Fateen, Aaron Schimmer, Mark Minden. Investigation of growth factor signaling through macrophage colony stimulating factor (CSF-1) and its receptor (FMS) in the bone marrow stromal microenvironment in acute myeloid leukemia (AML). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4123. doi:10.1158/1538-7445.AM2013-4123