Original Line Research Articles

Comparison of Different Methods for Ancient Ship Calm Water Resistance Estimation

The remains of ancient ships from various time periods are commonly found on land and under the sea in conditions that make it difficult to reconstruct their original form and structure. For this reason, the reconstruction should be supported by other data, such as data on similar ships, but also by certain assumptions. The results of the reconstruction are significant not only in a historical sense but are of exceptional importance when building floating replicas. Two ships, Nin 1 and Nin 2, today for promotional purposes known as Condurae Croaticae, were found in Nin (Croatia) at the end of the 1960s. They are about 8 to 10 m long, and tentatively dated to the 11th century AD, although there are indications that they could be dated two centuries later. Based on archaeological finds exhibited in the Museum of Nin Antiquities, hull line drawings were created, according to which two floating replicas were made at the end of the 1990s. Considering the problem of hogging that appeared in both ships, a new proposal for the reconstruction of the original hull lines was performed based on the available documentation. The aim of this paper is a systematic analysis of its calm water resistance. Based on the established credibility of experimental testing, a scale model (1:4 ratio) of the Nin 1 vessel is constructed and evaluated through towing tank experiments. The second approach, the CFD method, is a reliable numerical method for calm resistance estimation, but it is rarely used in the analysis of ancient ships. Finally, the widely used empirical Holtrop method is also applied, but it was developed for ships of larger dimensions and with large parts of flat bottoms and, therefore, the more appropriate Delft Hull Yacht Series method is also tested. The results obtained by applying the four mentioned methods are compared and discussed.

Intraoperative reliability of the tibial anteroposterior axis "Akagi's Line" in total knee arthroplasty.

The tibial anatomical anteroposterior (AP) axis "Akagi's line" was originally defined on computed tomography (CT) in total knee arthroplasty (TKA); however, its intraoperative reproducibility remains unknown. This study aimed to evaluate the intraoperative reproducibility of the Akagi's line and its effect on postoperative clinical outcomes. This prospective study included 171 TKAs. The rotational angle of the intraoperative Akagi's line relative to the original Akagi's line (RAA) defined on CT was measured. The RAA was calculated based on the tibial component rotational angles relative to the intraoperative Akagi's line measured using the navigation system and CT. The effects of RAA on postoperative clinical outcomes and rotational alignments of components were also evaluated. The mean absolute RAA (standard deviation) value was 5.5° (3.9°). The range of RAA was 22° internal rotation to 16° external rotation. Intraoperative Akagi's line outliers (RAA > 10°) were observed in 14% of the knees (24 knees). In outlier analysis, the tibial component rotation angle was externally rotated 6.5° (5.6°) in the outlier group and externally rotated 3.7° (4.2°) in the nonoutlier group (≤10°), with a significant difference between the two groups. Additionally, the outlier group (RAA > 10°) showed lower postoperative clinical outcomes. The original Akagi's line defined on CT showed insufficient reproducibility intraoperatively. The poor intraoperative detection of Akagi's line could be the reason for the tibial component rotational error and worse postoperative clinical outcomes. Level IV, case series.

Real-Time Observation of Germinal Vesicle Migration During Oocyte Meiotic Cell Division Using Ovarian Fluorescent Transgenic Zebrafish.

The transgenic (TG) zebrafish allows researchers to bio-image specific biological phenomena in cells and tissues in vivo. We established TG lines to monitor changes in the ovaries of live fish. The original TG line with ovarian fluorescence was occasionally established. Although the cDNA integrated into the line was constructed for the expression of enhanced green fluorescent protein (EGFP) driven by the medaka β-actin promoter, the expression of EGFP is restricted to the oocytes and gills in adult fish. Furthermore, we found that germinal vesicles (GVs) in oocytes of the established line can be observed by relatively strong fluorescence around the GV. In this study, we tried to capture the dynamic processes of germinal vesicle breakdown (GVBD) during meiotic cell division using the GV fluorescent oocytes. As a result, GV migration and GVBD could be monitored in real time. We also succeeded in observing actin filaments involved in the migration of GV to the animal pole. This strain can be used for education in the process of oocyte meiotic cell division.

Оцінка товарних кондицій помісей першого покоління антонінсько-зозуленецьких та галицьких рамчастих коропів

Purpose. To characterize age-1+ hybrids obtained from the crossing of Galician framed carp (GFC) and Antoniny-Zozulenets framed carp (AZFC) according to a set of commercial conditions in comparison with parental lines. Methodology. The objects of the study were age-1+ hybrids of the reciprocal crossing of Galician and Antoniny-Zozulenets carps grown in pond conditions of the “Karpatskyi Vodogray” LLC. The study was conducted at the end of the growing season according to methodological recommendations. The body of the experimental carp for analysis was divided into its constituent parts: scales, fillet with skin, entrails, fins and bones. In the process of work, blood was removed, its weight was attributed to the entrails. The head was separated from the carcass by breaking it off between the occipital bone and the first vertebra.Each part was weighed separately and presented as a percentage of the total body weight. Findings. The obtained results showed that the yield of the main part of the fillet with skin in crossbred groups of age-1+ carp was 48.7–52.0% and was higher compared to purebred offspring.In comparison with the original maternal line, crossbred age-1+ ♀GFC ×♂AZFC had a higher index of relative fillet weight by 3.15%, and the similar 1.6% superiority was observed in crossbreds ♀AZFC × ♂GFC. At the same time, with an increase in the average weight of age-1+ by 28.9–68.6% in the 2023 studies, the relative weight of the carcass increased by 2.4–11.0%. At the same time, the relative weight of the head decreased by 5.3-6.1%, and the relative weight of the entrails increased by 0.7-4.7%. In general, the inedible part of the studied groups of fish was in the range of 27.2–32.4%. In terms of the yield of fillets per 1 ha of fattening pond, the superiority of interbreeding groups of age-1+ was 31.5–62.7%. The obtained results indicate the expediency of their use as new highly productive commercial lines in pond farms of the Polissiya zone. Originality. For the first time, a comparative analysis of the nutritional value parameters of age-1+ carp from reciprocal crossings of Galician and Antoniny-Zozulenet framed carp and the original pure lines cultivated in the conditions of Ukrainian Polissiya was carried out. Practical value. The obtained results will complement the studies of previous years and will allow providing recommendations on the use of these hybrids for commercial cultivation in the Polissiya zone of Ukraine. Keywords: carp, weight, market conditions, edible and inedible body parts.

The multidepot drone general routing problem with duration and capacity constraints

AbstractThis paper studies the multidepot drone general routing problem with duration and capacity constraints (MDdGRP), an extension of the classical general routing problem with several depots. A fleet of drones with limited flight time and payload, each one located in a different depot, must jointly perform the service. In this continuous optimization problem, a set of lines of a network has to be serviced and a set of nodes has to be visited to deliver a given commodity. The aim is to find a set of drone routes performing the complete service with the shortest total duration. Aerial drones can enter a line at any point, service a portion of that line, and exit through another point, without following the lines of the network. While this flexibility allows for potentially better solutions, it also adds complexity to the problem. To deal with this, we digitize the MDdGRP instances by approximating each line with a polygonal chain containing a finite number of points that can be used to enter and exit each line. Thus, an instance of a discrete multidepot general routing problem (MDGRP) is obtained. This paper proposes an integer formulation for the MDGRP, some families of valid inequalities, and a branch‐and‐cut algorithm based on the strengthened formulation. Two matheuristic algorithms are presented, focused on sequentially selecting (and adding) some promising intermediate points to enter and exit a line. Instances involving up to six depots, 22 delivery points, and 88 original lines have been generated to test the performance of the methods proposed.

Line graph contrastive learning for node classification

Existing graph contrastive learning methods often rely on differences in node features within subgraphs, lacking effective capture of the global structural information of the graph. To address this issue, we propose a novel graph contrastive learning method, Line Graph Contrastive Learning (LineGCL), aimed at overcoming the deficiencies in understanding the overall characteristics and topological structures of graphs found in current methods. LineGCL utilizes the characteristics of line graphs to transform the original graph into corresponding line graphs, presenting edge information in the form of node features, effectively capturing the global structural information of the graph. Additionally, LineGCL adopts a novel multi-view contrastive learning approach, characterizing the similarity and differences between the original graph and line graph comprehensively from the perspectives of node features and spectral features, further enhancing the model's understanding and learning capabilities of the global structure of graphs. Experimental results on public datasets demonstrate that LineGCL outperforms baseline models, achieving performance improvements ranging from 0.5% to 28.5%. These results validate the effectiveness and superiority of LineGCL in capturing and understanding global structural information, surpassing the limitations of baseline methods in graph contrastive learning.

Text Summarization for Personalized Movie Review

The aim of this project is to create a personalised review creation system for movies using a sequence-to-sequence model and abstractive text summary techniques. The idea is to developinsightful and personalised evaluations that capture the spirit and sentiment of a film.Abstractive text summarization is an Natural language processing (NLP) techniqueof generating new and brief summary of source text data. It generates new lines in summary of text data which are relevant to the original lines. Abstractive summarization yields a numberof applications in different domains, from books and literature, to scienceand R&D, to financial research and legal documents analysis. This project introduces a fresh method for generating personalized movie reviews using abstractive text summarization. The approach employs a BERT-based encoder-decoder model, trained onuser reviews enriched with movieratings. This enables the model to create reviews that are not only informative and captivating but also match the user's preferences. By merging NLP and user preferences, we provide summaries that offer insights into reviewswhile aligning with the user's cinematic likes. In the era of abundant online content, our personalized summarization technique is a valuable tool for helping users navigate movie-related information. Keywords : Transformers, Bert model, Bert for Sequence Classification, Bert Tokenizer, Pegasus model,Pegasus tokenizer, Data Loader, Pandas.

Comparison of Blast, Bacterial Leaf Blight and Brown Plant Hopper Introgressed Lines for Yield and Morphological Traits Over Original Line

Rice as a staple food for very large population suffers from various biotic and abiotic stresses. Among the biotic stresses, Blast, Bacterial leaf blight and Brown plant hopper (BPH) are considered to most potential threats that significantly affect the rice productivity. The use of chemical usage for controlling these diseases and pest attacks is not environmentally friendly and is expensive. Using Molecular Marker assisted backcross breeding program we have improved one of our hybrid parental line with 5 genes (xa13, Xa21, Pi54, Bph20 and Bph21). We have developed 15 near isogenic lines having similar agronomical characters as of recipient parent, they are promising for their direct induction in breeding program. Our results indicated that out of fifteen lines, Six lines i.e. GK 101-12, GK 101-15, GK 101-9, GK 101-5 and GK 101-2 out-performed in all the three locations tested, they had not only have very high level of resistance to BLB, Blast and BPH but also showed significantly higher yield compared to susceptible check as well as recipient parent. These lines may be used for improvement of our existing hybrid and also for developing of new hybrids with other CMS lines. These lines are also useful for developing new set of restorer lines through (R x R) and also useful for introgression of wide spectrum of resistance to the varieties/hybrid.

Variability of broomrape parasitizing on sunflower during its intensive cultivation in the regions of the Russian Federation

Annual monitoring of broomrape seed infestation of fields over the last 15 years shows that despite the widespread distribution of race G, seeds of other, weaker races E and F remain in many fields. The aim of the research was to identify the racial belonging of broomrape seeds from fields in sunflower-growing regions of the Russian Federation and to describe new botanical characteristics in representatives of the most widespread race G of the parasite. To identify the races, there were used the internationally accepted sunflower differential lines LC 1093, P 96, resistant to races A to F, as well as the original line RG, which is immune to race G and all previous races, hybrid NK Brio (resistant to races A to E) and hybrid Tunka (resistant to races A to G). A highly aggressive parasite biotype (future race H) has so far been identified in small numbers in some fields in the Krasnodar, Stavropol, Rostov, Voronezh, Samara and Orenburg regions. Obviously, the continuation of intensive sunflower cultivation in these fields will lead to a rapid spread of race H in these regions. In addition to the racial diversity, frequent changes in plant habitus (bushy forms) were observed in some representatives of race G. For the first time, there has been demonstrated an overgrowth of the haustorial-tubercle area of the parasite and the formation of multiple stems from a single tubercle. This creates an advantage in the competition for food between adjacent broomrape individuals on the same sunflower root and ultimately accelerates and increases the seed production of the plant. The rapid evolution of Orobanche cumana Wallr. during the intensification of sunflower cultivation is expressed not only in the formation of new physiological races, but also in an accelerated increase in the seed productivity of the parasite by changing the habitus of the plants, including the haustorial-tubercle area.

Line simplification with sidedness relationship consistency using the constrained total least squares method

AbstractThe simplification of lines plays a crucial role in map generalization and multiscale representation; however, addressing inconsistent sidedness relationships between the simplified lines and their neighboring features poses a persistent challenge. In order to preserve correct and consistent sidedness relationships following simplification, this study introduces a novel line simplification method. This method incorporates topological constraints pertaining to left–right sidedness relationships, formulated as an optimization procedure based on the proposed partial total least squares method with constraints. The primary objective is to minimize the positional difference between the line features before and after simplification, with the sidedness relationship represented as an inequality constraint. Moreover, an optimization algorithm is derived to address the simplification problem effectively while adhering to the specified constraints. The proposed method is then applied to simplify the lines within three distinct datasets. Experimental results validate the efficacy of the proposed method in maintaining sidedness consistency across all tested datasets. In comparison to the Douglas–Peucker (DP) method, the proposed method exhibits minimal vertical displacement variance in the line feature points post‐simplification. Additionally, it achieves a smaller overall positional difference between the simplified and original lines compared to the DP method. These findings underscore the superior performance of the proposed method in maintaining sidedness relationships and minimizing positional differences during the line simplification process.

Selfing revealed potential for higher yield performance than backcrossing among tomato segregating populations of Solanum lycopersicum × S. pimpinellifolium crosses under tropical humid climate

The objectives of this study were to assess and identify new source of phenotypic variability among F3 and BC1F2 tomato populations, and apply genotype by yield*trait (GYT) biplots for population and line selection based on multiple traits. Four diverse cultivated parents (‘CLN2498D’ [D] and ‘CLN2417H’ [H] from Ethiopia; ‘UC Dan INDIA’ [U] and ‘Tima’ [T] from Nigeria), and wild parent ‘LA2093’ [W] were used to generate 276 potential breeding lines. The lines were categorized into eight populations (‘pop_1_W/H1’, ‘pop_2_W/H2’, ‘pop_3_W/D1’, ‘pop_4_W/D2’, ‘pop_5_W/T1’, ‘pop_6_W/T2’, ‘pop_7_W/U1’, and ‘pop_8_W/U2’), and evaluated twice in the field using 19 × 15 alpha-lattice design with two replicates. Significant differences were observed among lines and populations for all yield enhancing traits. ‘Pop_1_W/H1’, ‘pop_4_W/D2’ and ‘pop_6_W/T2’ expressed the highest genetic divergence for plant height, number of leaves, total flower and fruit number, and fruit weight. GYT biplots revealed that all yield*trait interactions had a positive correlation with each other. F3 populations, ‘pop_5_W/T1’ and ‘pop_1_W/H1’ exhibited the best performance for majority of the yield*trait combinations. Hierarchical clustering on principal components (HCPC) revealed overlapping lines (70.58% of Cluster D lines) and (54.05% of Cluster U lines) from the two F3 populations. In BC1F2 population, 32.35% of the 34 original lines of Cluster D and 48.48% of Cluster T lines overlapped between Clusters D and T, while 18.18% of Cluster T lines and 8.82% of Cluster H lines were transgressive between Clusters T and H. Transgressive segregants ‘0210U1’, ‘0211U1’, and ‘0171T1’ of selfed population using multivariate analysis were believed to represent potential sources of novel genetic variation for future tomato breeding.

ViralWasm: a client-side user-friendly web application suite for viral genomics.

The genomic surveillance of viral pathogens such as SARS-CoV-2 and HIV-1 has been critical to modern epidemiology and public health, but the use of sequence analysis pipelines requires computational expertise, and web-based platforms require sending potentially sensitive raw sequence data to remote servers. We introduce ViralWasm, a user-friendly graphical web application suite for viral genomics. All ViralWasm tools utilize WebAssembly to execute the original command line tools client-side directly in the web browser without any user setup, with a cost of just 2-3x slowdown with respect to their command line counterparts. The ViralWasm tool suite can be accessed at: https://niema-lab.github.io/ViralWasm.

A case study of whale optimization algorithm for scheduling in C2M model

With the continuous upgrading of industrial technology and information technology, consumers can deeply participate in the whole life cycle of products and realize customized production. These unprecedented changes have brought consumers and manufacturers closer together, resulting in the intelligent business model of "Internet + Customized Production" and "Customer to Manufacturer (C2M)". C2M has been adopted by more and more companies. However, the transition from traditional business models to C2M is a problem that every company must face and solve. Personalized orders of many varieties and small lots put enormous pressure on the production of mainly labor-intensive electronic assembly companies. The theoretical findings of Industry 4.0 and Lean Manufacturing show that people play a central role in assembly operations. As an important element of the production system, worker scheduling has a direct impact on delivery time and cost. Worker scheduling requires not only matching people to jobs, but also considering flexible employment. According to the "Learning Curve" theory, workers with learning potential can continuously enrich their skills and work efficiency will show dynamic changes. Therefore, under the condition of shortest order delivery time and lowest cost, worker scheduling considering the learning effect becomes a challenge for enterprise decision makers. Firstly, the production method of manufacturing industry in C2M environment is studied. Then, based on single-skill task and multi-skill task, respectively, a learning curve-based model of dynamic change in worker skill level is constructed. And this model is used as the input of the assembly line worker scheduling model. Secondly, an Elite Non-dominant Sorting Whale Optimization Algorithm (ENS-WOA) is designed for this multi-objective optimization problem. The correctness and feasibility of the proposed algorithm are verified by selecting classical arithmetic cases for experimental comparison with other algorithms. Finally, the established worker efficiency change model, worker scheduling model and the proposed algorithm are applied to optimize the assembly line of water pump products of Company B, which is being transformed to C2M, and solved by MATLAB software. The results show that the model proposed in this paper is effective, stable and practical compared with the worker costs and delivery period required to complete the order in the original assembly line. Worker costs were reduced by 29.02% and orders were completed approximately 10 days earlier.

Analysis of the Load‐Bearing Capacity of the Steel‐Shell Concrete Support Structure Formed by the Construction of the Pipe Curtain Freezing Method

The Sanya estuary passage project is the latest and highly technical underground project after the large shield underground construction, which is of great significance to the construction of the Hainan free trade port and underground space construction innovation. In this paper, we use finite element software to analyze the deformation, stress, and surface settlement of the pipe curtain structure during construction under two conditions (original riverbed line and maximum scour riverbed line) in order to provide guidance for the actual project. Numerical simulations show that due to excavation and unloading, the bottom and top of the pipe curtain structure are subject to tensile stress; the bottom rises, the top settles, and converges in the horizontal direction; under the original riverbed line condition, the displacement values of each index are larger; changing the riverbed line does not change the maximum and minimum main stress of the pipe curtain structure significantly, and the stiffness of the pipe curtain structure can meet the maximum water level and the maximum scour in two unfavorable conditions; the pipe curtain structure can meet the structural. The pipe screen structure can meet the structural load‐bearing requirements. Suggestions for the design and construction of the project: strengthen the thickness of the pipe curtain at the side wall position, that is, use larger diameter steel pipes; do a good job of antiseepage and anticracking of the concrete at the corner of the pipe curtain structure and the side wall position during the construction process.

Optimizing the integration of express bus services with micro-mobility: a case study

Considering a bus line, the problem of defining an express bus line consists in determining which stops of the original line will belong to the express line and the frequency of the new line. The objective of the optimization is a trade-off between the advantages of the passengers that benefit from the new service and the disadvantages of the passengers that cannot exploit the improved service. The advantages and disadvantages are measured in terms of total travel and waiting time.In this work we consider the introduction of micro-mobility, such as shared electric scooters or bikes, to mitigate the disadvantages and to increase the ridership of the express line. The contribution of the paper is twofold. On the one hand, we propose an improved mathematical model for defining an express bus line. The model is based on a longest path problem on an acyclic graph; thus, it is very efficient with respect to the other models in the literature. On the other hand, we consider the real case of two urban bus lines in the city of Milan and present the results computed on real passenger data. The results provide evidence that implementing an express line can improve total travel and waiting time. The time wasted by switching lines is in general higher than the time gained by the passengers. However, this can be compensated by the introduction of micro-mobility means of transportation.

Long-term passages of Plantago asiatica mosaic virus alter virulence and multiplication in Nicotiana benthamiana plants.

We demonstrated the infectivity and host adaptation of a viola isolate of Plantago asiatica mosaic virus (PlAMV-Vi) in an asymptomatic host, Nicotiana benthamiana, through long-term serial passages. Serial passaging of a green fluorescent protein-tagged full-length cDNA clone of PlAMV-Vi (PlAMV-ViGFP) in N. benthamiana plants resulted in the appearance of a new virus line inducing leaf-crinkle symptoms, the Leaf Crinkle (LC) line. Virus titers were higher for both in the LC and the 14th passage line(s) of PlAMV-ViGFP compared with the original line. The LC line was found to have seven unique nucleotide mutations that may have contributed to its higher virulence and multiplication rate in N. benthamiana.

Sources of valuable traits for the selection of varieties and hybrids of agricultural crops on the Yaltushkiv EBS

Purpose. To identify sources of economic and valuable traits for the breeding process of industrial, grain and bioenergy crops at the Yaltushkivska Experimental Breeding Station of the Institute of Bioenergy Crops and Sugar Beet of the National Academy of Agrarian Sciences.
 Methods. Hybridization, selection, field tests, analytical, mathematical and statistical.
 Results. The best breeding genotypes of beets of foreign and domestic selection that are the donors of valuable traits, are concentrated at the Yaltushkivska EBS. The best hybrid combinations created on the basis of the Yaltushkivska EBS materials and evaluated in the variety test under the Beta cross program significantly exceeded the group standard: in yield (103.6–114.9%), sugar content (96.4–105.7%), sugar yield (110.1–110.8%). A highly productive buckwheat variety ‘Yuliana’ with a yield of 2.2 t/ha and a protein content of 15.0% was created and submitted for state variety testing. The best highly productive and energy valuable varieties of switchgrass were selected for cultivation in areas of insufficient and unstable soil moisture, which ensure the yield of dry biomass at the level of 7.5–16.2 t/ha, the yield of solid biofuel of 8.2–17.2 t/ha, and energy of 112.5–243 GJ/ha. Genotypes from the Miscanthus genus have been identified, which combine signs of tolerance to osmotic and salt stress and high energy yield.
 Conclusions. Selected sources of breeding-valuable traits became the basis for creating varieties and hybrids of agricultural crops (sugar beet ‘Barskyi’, ‘Chereshnevyi’, ‘Ruthenia 11’, ‘Ruthenia 12’, ‘Ruthenia 13’, etc., buckwheat ‘Yuliana’, switchgrass ‘Morozko’ and ‘Liadovske’ and varieties of miscanthus resistant to abiotic factors of the environment). Breeding genotypes and original lines of these crops were transferred to the National Center of Plant Genetic Resources of Ukraine.

Schrödinger’s yeast: the challenge of using transformation to compare fitness among Saccharomyces cerevisiae that differ in ploidy or zygosity

How the number of genome copies modifies the effect of random mutations remains poorly known. In yeast, researchers have investigated these effects for knock-out or other large-effect mutations, but have not accounted for differences at the mating-type locus. We set out to compare fitness differences among strains that differ in ploidy and/or zygosity using a panel of spontaneously arising mutations acquired in haploid yeast from a previous study. To ensure no genetic differences, even at the mating-type locus, we embarked on a series of transformations, which first sterilized and then temporarily introduced plasmid-borne mating types. Despite these attempts to equalize the haplotypes, fitness variation introduced during transformation swamped the differences among the original mutation-accumulation lines. While colony size looked normal, we observed a bi-modality in the maximum growth rate of our transformed yeast and determined that many of the slow growing lines were respiratory deficient (“petite”). Not previously reported, we found that yeast that were TID1/RDH54 knockouts were less likely to become petite. Even for lines with the same petite status, however, we found no correlation in fitness between the two replicate transformations performed. These results pose a challenge for any study using transformation to measure the fitness effect of genetic differences among strains. By attempting to hold haplotypes constant, we introduced more mutations that overwhelmed our ability to measure fitness differences between the genetic states. In this study, we transformed over one hundred different lines of yeast, using two independent transformations, and found that this common laboratory procedure can cause large changes to the microbe studied. Our study provides a cautionary tale of the need to use multiple transformants in fitness assays.

Biomarker Driven Strategies for Targeted Elimination of Hypoxia Adapted Lymphoma Cells

Large body of evidence suggests that hypoxia drives aggressive molecular features irrespective of cancer type. Non-Hodgkin lymphomas (NHL) are the most common hematologic malignancies characterized by widespread disease and frequent involvement of diverse hypoxic microenvironments (e.g., bone marrow, malignant effusions, large lymphoma masses). Impact of long-term hypoxia on the biology of lymphoma cells and its potential role in mediating drug resistance and disease relapses due to adaptive changes shaped by the hypoxic microenvironment remain only partially understood. In this study we analyzed impact of short- and long-term hypoxia on a panel of six lymphoma cell lines including diffuse large B-cell lymphoma (UPF4D, UPF8D), Burkitt lymphoma (Ramos, UPF9T), and mantle cell lymphoma (HBL2, MINO). Only 2 out of 8 tested lymphoma cell lines (Ramos, and HBL2) survived longer than 4 weeks under deep hypoxia (1% O2). The remaining tested lymphoma cell lines died by hypoxia-induced apoptosis (which could be inhibited by co-cultivation with pan-caspase inhibitor Z-VAD-FMK). The hypoxia-adapted (HA) Ramos and HBL2 cells had severely decreased proliferation rate accompanied by complex changes of the transcriptome, proteome, and metabolome. Seahorse analysis revealed significant inhibition of both oxidative phosphorylation and glycolytic pathways ( Figure 1). Of note, the whole-proteome profiling confirmed significant downregulation of proteins regulating oxidative phosphorylation, but significant upregulation of proteins regulating glycolysis. Sensitivity of HA cells to 2-deoxyglucose, an inhibitor of glycolysis was markedly increased ( Figure 1). The data suggest that under long-term deep hypoxia, lymphoma cells try to compensate for the decrease in ATP production from the oxidative phosphorylation process by boosting structural machinery of glycolysis, on which they became vitally dependent for survival. Despite the increased glycolytic machinary, however, the level of glycolysis decreases due to severe lack of oxygen. In our opinion, this is a clear representation of the Warburg effect, a hallmark of cancer based on the metabolic reprogramming of the cancer cells under hypoxia. Sensitivity of HA cells to a panel of the tested cytotoxic drugs (cytarabin, cisplatin, bortezomib) and targeted agents (venetoclax, S63545, A1155463, TRAIL, polatuzumab vedotin, IM-156, copanlisib) was either unchanged or increased (compared to sensitivity of the original cell lines cultured under normoxia). In contrast to so far published data, long-term hypoxia was not associated with acquired drug resistance in case of any of the tested agent. Of note, HA cells became significantly more sensitive to A1155463, an inhibitor of anti-apoptotic protein BCL-XL ( Figure 1). Unchanged levels of BCL-XL protein in HA cells and the respective normoxic cell lines (detected by western blotting) suggest that more complex mechanisms plausibly underlie the observed markedly increased sensitivity to A1155463. The whole-transcriptome and proteome analyses of both HA cell lines (compared to the parental cell lines cultured under normoxic conditions) detected among other complex changes signficant upregulation of P4HA1 (both mRNA, and protein), a prolyl hydroxylase involved in the stabilization of hypoxia-induced factor (HIF) 1 alpha, and putative oncogene associated with tumor aggressiveness. Transgenic (over)expression of P4HA1 in hypoxia-sensitive MINO cells effectively inhibited the hypoxia-induced apoptosis. Western blot analysis implemented on a panel of primary lymphoma cells revealed markedly higher expression of P4HA1 protein in the lymphoma cells obtained from malignant effusions (putative hypoxic microenvironment) compared to lymphoma cells obtained from leukemized blood (putative normoxic microenvironment). Our data suggest that P4HA1 positively impacts survival of lymphoma cells under hypoxia. In translation, P4HA1, BCL-XL, and structural proteins of the glycolytic pathway may represent novel drugable targets for more effective elimination of hypoxia-adapted lymphoma cells. Financial Support: Ministry of Health of the Czech Republic AZV NU23-03-00172, Grant Agency of the Czech Republic GA23-05377S, and National Institute for Cancer Research (EXCELES) LX22NPO5102.

PD-1 Expression in Lymphoma Cells Mediates Cellular Proliferation By Engaging Phosphatase SHP-1/SHP-2

Introduction: Despite the improved clinical outcomes with novel therapies in patients with chronic lymphocytic leukemia (CLL), Richter transformation (RT) of CLL remains a major clinical challenge. Our group showed a selective response (~40%) with the PD-1 blocking antibody pembrolizumab in patients with RT, particularly after prior exposure to ibrutinib (Ding et al, Blood, 2017). It has been established that PD-1 inhibits cytotoxic T-cell functions via the interaction with its ligands, PD-L1 and PD-L2. However, the role of PD-1 in tumor B-cells is understudied. Here we show the expression and functional implications of the PD-1 signaling axis in B-cell pathobiology. Methods: CLL-involved lymph node (LN) (n=26) and RT (with diffuse large B-cell lymphoma [DLBCL] histology) -involved LN (n=20) samples were tested for PD-1 expression by immunohistochemistry (IHC) (mouse clone NAT105, Abcam). Then, PD-1 expression was examined in 10 lymphoma cell lines and 1 CLL-like cell line (MEC-1) by both flow cytometry (FC) and Western blot (WB) analysis. PD-1 overexpression using pLEX-lentiviral packaging system (Thermo Scientific) was evaluated for its impact on in vitro cell cycle regulation and in vivo tumor growth. CRISPR/CAS9 mediated PD-1 knockdown evaluated on cell growth in MEC-1 and Mino cells. Role of PD-1 was further examined on the regulation of cell signaling pathways using human phospho kinase array kit (R&D) and WB analysis. Additionally, gene expression signatures were evaluated by RNA-seq using pLEX-lentiviral-PD-1 transfected DLBCL cell lines OCI-LY7 and OCI-LY19. Results: PD-1 expression was significantly increased in RT-LN compared to CLL-LN (mean ± SEM in RT vs. CLL, 30.6% ± 4.7% vs. 11.5% ± 2.8%, p<0.001) as determined by IHC. PD-1 expression was highest in patients with RT where the last prior CLL therapy was ibrutinib. To test the role of PD-1 in tumor B-cells, its expression was assessed in lymphoma and CLL cell lines. The expression of PD-1 was found to be variable by both FC and WB analysis, but at very low-levels in DLBCL cell lines OCI-LY7 and OCI-LY19. Constitutive lentiviral (pLEX-PD-1)-mediated overexpression of PD-1 in OCI-LY7 and OCI-LY19 cells led to increased cell growth (1.4 and 1.9 fold, respectively, compared to original lines after 4 days of culturing of equal number of cells), which was further confirmed by cell cycle analysis which showed an increase in S phase by 20.4% (p<0.05) and 24.5% (p<0.05) in OCI-LY7 and OCI-LY19 cells, respectively. When the luciferase+ PD-1 overexpressing OCI-LY7 cells (1X10 6) were injected intravenously into NSG mice, increased tumor growth (p<0.01) was observed by bioluminescence imaging after 22 days of follow-up. PD-1 knockdown in MEC-1 and Mino cells (high basal PD-1 expression) led to significant inhibition of cell growth (85% and 79.5%, respectively, p<0.05). Using phospho-kinase array, an overall decrease of phosphorylation was detected on multiple sites of p53 (S392, S15, S46) and Chk-2 (T68) in PD-1 overexpressing OCI-LY7 and OCI-LY19 cell lines. This finding was further confirmed by WB analyses. In addition, decreased total p53 and increased phosphorylation on both SHP-1 and SHP-2 were found in PD-1 overexpressing OCI-LY7 and OCI-LY19 cell lines. SHP-1 (TPI-1) and SHP-1+SHP-2 (TPI-1+TNO155) inhibitor treatment promoted OCI-LY19 cell death and led to recovery of phosphorylation on ATM, Chk-2 and p53 in these cells. RNA sequencing data in PD-1 overexpressing OCI-LY7 and OCI-LY19 cells revealed increased expression of genes that promote cell cycle progression and DNA replication. Conclusion: Our data showed robust PD-1 expression in patients with Richter transformation from CLL to DLBCL. PD-1 overexpression in DLBCL lymphoma cell lines enhanced cell proliferation in vitro and in vivo. Further investigation identified that PD-1 modified the phosphorylation/function of SHP phosphatases and thereby regulated p53 pathways ( Figure 1). These findings indicate that RT with DLBCL cells have the ability to “hijack” the PD-1 pathway which results in downregulation of p53 mediated DNA repair. This novel finding provides for new strategies to further evaluate the interaction of PD-1 signals with DNA repair pathways and suggest potential novel targets for RT.