Chemical complementarity of tumor resident, T-cell receptor CDR3s and renalase-1 correlates with increased melanoma survival.

Overexpression of the secretory protein renalase-1 negatively impacts the survival of melanoma and pancreatic cancer patients, while inhibition of renalase-1 signaling drives tumor rejection by promoting T-cell activation. Thus, we investigated the chemical complementarity between melanoma-resident, T-cell receptor (TCR) complementarity-determining region 3 (CDR3) amino acid sequences (AAs) and the renalase-1 protein. Increasing complementarity of TCR CDR3s to renalase-1 AAs, as assessed by a chemical complementarity scoring algorithm, was associated with improved overall survival (OS) in melanoma patients. The expression levels of several immune signature genes were significantly, positively correlated with increasing TCR CDR3-renalase-1 complementarity scores. Additionally, the survival association observed with high complementarity of TCR CDR3s to renalase-1 AAs was more robust in cases with low renalase-1 gene expression levels. Mapping of TCR CDR3-renalase-1 in silico interaction sites identified major epitope candidates including RP220, the signaling module of the renalase-1 protein, consistent with the fact that a monoclonal antibody to RP220 is a potent inhibitor of melanoma growth. These findings indicate that renalase-1 is a potential antigen for TCR recognition in melanoma and could be considered as a target for immunotherapy.

Difficult Airway Assessment Based on Multi-View Metric Learning.

The preoperative assessment of difficult airways is of great significance in the practice of anesthesia intubation. In recent years, although a large number of difficult airway recognition algorithms have been investigated, defects such as low recognition accuracy and poor recognition reliability still exist. In this paper, we propose a Dual-Path Multi-View Fusion Network (DMF-Net) based on multi-view metric learning, which aims to predict difficult airways through multi-view facial images of patients. DMF-Net adopts a dual-path structure to extract features by grouping the frontal and lateral images of the patients. Meanwhile, a Multi-Scale Feature Fusion Module and a Hybrid Co-Attention Module are designed to improve the feature representation ability of the model. Consistency loss and complementarity loss are utilized fully for the complementarity and consistency of information between multi-view data. Combined with Focal Loss, information bias is effectively avoided. Experimental validation illustrates the effectiveness of the proposed method, with the accuracy, specificity, sensitivity, and F1 score reaching 77.92%, 75.62%, 82.50%, and 71.35%, respectively. Compared with methods such as clinical bedside screening tests and existing artificial intelligence-based methods, our method is more accurate and reliable and can provide a reliable auxiliary tool for clinical healthcare personnel to effectively improve the accuracy and reliability of preoperative difficult airway assessments. The proposed network can help to identify and assess the risk of difficult airways in patients before surgery and reduce the incidence of postoperative complications.

Soil conditions modify species diversity effects on tree functional trait expression.

Examples of positive effects of biodiversity on ecosystem functions have kept accumulating in the last two decades, and functional traits are considered suitable tools to explain their underlying mechanisms. However, traits are rarely studied at the scale where these mechanisms (e.g., complementarity) are likely to originate, that is, between two interacting individuals. In an 18-month greenhouse experiment, we investigated how species diversity (i.e., monospecific or heterospecific tree pairs) affects within-individual leaf traits expression and variation and how this effect is modified by soil conditions. While resource addition through phosphorus fertilization partly strengthened the diversity effects, inoculation of soil microbiota (potentially leading to increased resource accessibility) resulted in counter effects. Hence, in contrast to our expectations, we did not find synergistic effects of the two soil treatments, but we found distinct effects on species following an acquisitive or conservative growth strategy. Overall, our study showed that the effect of species diversity on young trees' adaptability and resource-use strategy needs to be considered alongside soil biotic and abiotic aspects. The influence of soil conditions on species diversity effects is essential to understand mechanisms behind complementarity at the individual level, which ultimately translate to the community scale.

CDR grafting and site-directed mutagenesis approach for the generation and affinity maturation of Anti-CD20 nanobody.

BACKGROUND: Recently, new and advanced techniques have been adopted to design and produce nanobodies, which are used in diagnostic and immunotherapy treatments. Traditionally, nanobodies are prepared from camelid immune libraries that require animal treatments. However, such approaches require large library sizes and complicated selection procedures. The current study has employed CDR grafting and site-directed mutagenesis techniques to create genetically engineered nanobodies against the tumor marker CD20 (anti-CD20 nanobodies) used in leukemia treatment. METHODS AND RESULTS: In this study, we utilized the swapping method to graft CDRs from the VH Rituximab antibody to VHH CDRs. We aimed to enhance the binding affinity of the nanobodies by substituting the amino acids (Y101R-Y102R-Y107R) in the VHH-CDR3. To assess the binding capacity of the mutated nanobodies, we conducted an ELISA test. Moreover, through flow cytometry analysis, we compared the fluorescence intensity of the grafted CD20 and mutant nanobodies with that of the commercially available human anti-CD20 in Raji cells. The results showed a significant difference in the fluorescence intensity of the grafted nanobodies and mutant nanobodies when compared to the commercially available human anti-CD20. CONCLUSION: The approach we followed in this study makes it possible to create multiple anti-CD20 nanobodies with varying affinities without the need for extensive selection efforts. Additionally, our research has demonstrated that computational tools are highly reliable in designing functional nanobodies.

What happens when the tasks dry up? Exploring the impact of medical technology on workforce planning.

Increasing evidence suggests that new technologies tend to substitute for low skilled labour and complement highly skilled labour. This paper considers the manner in which new technology impacts on two distinct groups of highly skilled health care labour, cardiologists and cardiac surgeons. We consider the diffusion impact of PCI as it replaces CABG in the treatment of cardiovascular disease in the English NHS, and explicitly estimate the degree to which the cardiac surgical workforce reacts to this newer technology. Using administrative data we trace the complementarity between CABG and PCI during the mature phase of technology adoption, mapped against an increasing employment of cardiologists as they replace cardiothoracic surgeons. Our findings show evidence of growing employment of cardiologists, as PCI is increasingly expanded to older and sicker patients. While in cardiothoracic surgery, surgeons compensate falling CABG rates in a manner consistent with undertaking replacement activity and redeployment. While for cardiologists this reflects the general findings in the literature, that new technology enhances rather than substitutes for skilled labour, for the surgeons the new technology leads to redeployment rather than a downsizing of their labour.

IGH Complementarity Determining Region-3-Cytomegalovirus Protein Chemical Complementarity Linked to Better Overall Survival Probabilities for Glioblastoma.

Cytomegalovirus (CMV) has long been thought to have an association with glioblastoma multiforme (GBM), although the exact role of CMV and any subsequent implications for treatment have yet to be fully understood. This study addressed whether IGH complementarity determining region-3 (CDR3)-CMV protein chemical complementarity, with IGH CDR3s representing both tumor resident and blood-sourced IGH recombinations, was associated with overall survival (OS) distinctions. IGH recombination sequencing reads were obtained from (a) the Clinical Proteomic Tumor Analysis Consortium, tumor RNAseq files; and (b) the cancer genome atlas, blood exome-derived files. The Adaptive Match web tool was used to calculate chemical complementarity scores (CSs) based on hydrophobic interactions, and those scores were used to group GBM cases and assess survival probabilities. We found a higher OS probability for cases whose hydrophobic IGH CDR3-CMV protein chemical complementarity scores (Hydro CSs) were in the upper 50th percentile for several CMV proteins, including UL99 and UL123, as well as for CSs based on known B cell epitopes representing these proteins. We also identified multiple immune signature genes, including CD79A and TNFRSF17, for which higher RNA expression was associated with higher Hydro CSs. Results were consistent with the idea that stronger immunoglobulin responses to CMV are associated with better OS probabilities for GBM.

Mycorrhizal associations modify tree diversity-productivity relationships across experimental tree plantations.

Decades of studies have demonstrated links between biodiversity and ecosystem functioning, yet the generality of the relationships and the underlying mechanisms remain unclear, especially for forest ecosystems. Using 11 tree-diversity experiments, we tested tree species richness-community productivity relationships and the role of arbuscular (AM) or ectomycorrhizal (ECM) fungal-associated tree species in these relationships. Tree species richness had a positive effect on community productivity across experiments, modified by the diversity of tree mycorrhizal associations. In communities with both AM and ECM trees, species richness showed positive effects on community productivity, which could have resulted from complementarity between AM and ECM trees. Moreover, both AM and ECM trees were more productive in mixed communities with both AM and ECM trees than in communities assembled by their own mycorrhizal type of trees. In communities containing only ECM trees, species richness had a significant positive effect on productivity, whereas species richness did not show any significant effects on productivity in communities containing only AM trees. Our study provides novel explanations for variations in diversity-productivity relationships by suggesting that tree-mycorrhiza interactions can shape productivity in mixed-species forest ecosystems.

Combining Complementarity and Binding Energetics in the Assessment of Protein Interactions: EnCPdock-A Practical Manual.

The combined effect of shape and electrostatic complementarities (Sc, EC) at the interface of the interacting protein partners (PPI) serves as the physical basis for such associations and is a strong determinant of their binding energetics. EnCPdock (https://www.scinetmol.in/EnCPdock/) presents a comprehensive web platform for the direct conjoint comparative analyses of complementarity and binding energetics in PPIs. It elegantly interlinks the dual nature of local (Sc) and nonlocal complementarity (EC) in PPIs using the complementarity plot. It further derives an AI-based ΔGbinding with a prediction accuracy comparable to the state of the art. This book chapter presents a practical manual to conceptualize and implement EnCPdock with its various features and functionalities, collectively having the potential to serve as a valuable protein engineering tool in the design of novel protein interfaces.

Dysfunction of Complementarity Determining Region 1 Encoded by T Cell Receptor Beta Variable Gene Is Potentially Associated with African Swine Fever Virus Infection in Pigs.

The beta T-cell receptor (TRB) expressed by beta T cells is essential for foreign antigen recognition. The TRB locus contains a TRBV family that encodes three complementarity determining regions (CDRs). CDR1 is associated with antigen recognition and interactions with MHC molecules. In contrast to domestic pigs, African suids lack a 284-bp segment spanning exons 1 and 2 of the TRBV27 gene that contains a sequence encoding CDR1. In this study, we used the African swine fever virus (ASFV) as an example to investigate the effect of deleting the TRBV27-encoded CDR1 on the resistance of domestic pigs to exotic pathogens. We first successfully generated TRBV27-edited fibroblasts with disruption of the CDR1 sequence using CRISPR/Cas9 technology and used them as donor cells to generate gene-edited pigs via somatic cell nuclear transfer. The TRBV-edited and wild-type pigs were selected for synchronous ASFV infection. White blood cells were significantly reduced in the genetically modified pigs before ASFV infection. The genetically modified and wild-type pigs were susceptible to ASFV and exhibited typical fevers (>40 °C). However, the TRBV27-edited pigs had a higher viral load than the wild-type pigs. Consistent with this, the gene-edited pigs showed more clinical signs than the wild-type pigs. In addition, both groups of pigs died within 10 days and showed similar severe lesions in organs and tissues. Future studies using lower virulence ASFV isolates are needed to determine the relationship between the TRBV27 gene and ASFV infection in pigs over a relatively long period.

Aerobic anoxygenic phototrophs play important roles in nutrient cycling within cyanobacterial Microcystis bloom microbiomes.

BACKGROUND: During the bloom season, the colonial cyanobacterium Microcystis forms complex aggregates which include a diverse microbiome within an exopolymer matrix. Early research postulated a simple mutualism existing with bacteria benefitting from the rich source of fixed carbon and Microcystis receiving recycled nutrients. Researchers have since hypothesized that Microcystis aggregates represent a community of synergistic and interacting species, an interactome, each with unique metabolic capabilities that are critical to the growth, maintenance, and demise of Microcystis blooms. Research has also shown that aggregate-associated bacteria are taxonomically different from free-living bacteria in the surrounding water. Moreover, research has identified little overlap in functional potential between Microcystis and members of its microbiome, further supporting the interactome concept. However, we still lack verification of general interaction and know little about the taxa and metabolic pathways supporting nutrient and metabolite cycling within Microcystis aggregates. RESULTS: During a 7-month study of bacterial communities comparing free-living and aggregate-associated bacteria in Lake Taihu, China, we found that aerobic anoxygenic phototrophic (AAP) bacteria were significantly more abundant within Microcystis aggregates than in free-living samples, suggesting a possible functional role for AAP bacteria in overall aggregate community function. We then analyzed gene composition in 102 high-quality metagenome-assembled genomes (MAGs) of bloom-microbiome bacteria from 10 lakes spanning four continents, compared with 12 complete Microcystis genomes which revealed that microbiome bacteria and Microcystis possessed complementary biochemical pathways that could serve in C, N, S, and P cycling. Mapping published transcripts from Microcystis blooms onto a comprehensive AAP and non-AAP bacteria MAG database (226 MAGs) indicated that observed high levels of expression of genes involved in nutrient cycling pathways were in AAP bacteria. CONCLUSIONS: Our results provide strong corroboration of the hypothesized Microcystis interactome and the first evidence that AAP bacteria may play an important role in nutrient cycling within Microcystis aggregate microbiomes. Video Abstract.

Local chicken breeds exhibit abundant TCR-V segments but similar repertoire diversity.

The thymus-derived lymphocytes of jawed vertebrates have four T-cell receptor (TCR) chains that play a significant role in immunity. As chickens have commercial value, their immune systems require a great deal of attention. Local chicken breeds are an essential part of poultry genetic resources in China. Here, we used high-throughput sequencing to analyze the TCRα and TCRß repertoires and their relative expression levels in the native chicken breeds Baier Buff, Longyou Partridge, Xiaoshan, and Xianju. We found that TCR Vα and TCR Vß were expressed and included 17, 19, 17, and six segments of the Vα2, Vα3, Vß1, and Vß2 subgroups, respectively. V-J pairing was biased; Jα11 was utilized by nearly all Vα segments and was the most commonly used. Breed-specific V segments and V-J pairings were detected as well. The results of the principal coordinate analysis (PCoA) as well as the V-J pairing and CDR3 diversity analyses suggested that the four local chicken breeds did not significantly differ in terms of TCR diversity. Hence, they expressed not significant differentiation, and they are rich genetic resources for the development and utilization of immune-related poultry breeding.

Initial Validation of the Coach-Athlete Relationship Questionnaire in a Sample of Portuguese Athletes.

Our primary objectives in this study were to translate and provide psychometric support for the Coach Athlete Relationship Questionnaire (CART-Q) Portuguese version, assess its invariance across sex, and explore its nomological validity in relation to enjoyment. Our sample participants were 470 athletes (226 females, 244 males) aged between 16 to 39 years from various individual and team sports. We found that the translated Portuguese version of the CART-Q exhibited satisfactory test-retest reliability and can serve as a reliable tool for evaluating the core constructs of the coach-athlete relationship - closeness, commitment, and complementarity. Moreover, this instrument showed evidence of nomological validity through significant positive correlations between its underlying factors and athletes' enjoyment with their sport. The proposed model for explaining item variance was also found to be invariant between male and female respondents. We recommend further use of this instrument in research and practical applications.

Loss of TET2 increases B-1 cell number and IgM production while limiting CDR3 diversity.

Recent studies have demonstrated a role for Ten-Eleven Translocation-2 (TET2), an epigenetic modulator, in regulating germinal center formation and plasma cell differentiation in B-2 cells, yet the role of TET2 in regulating B-1 cells is largely unknown. Here, B-1 cell subset numbers, IgM production, and gene expression were analyzed in mice with global knockout of TET2 compared to wildtype (WT) controls. Results revealed that TET2-KO mice had elevated numbers of B-1a and B-1b cells in their primary niche, the peritoneal cavity, as well as in the bone marrow (B-1a) and spleen (B-1b). Consistent with this finding, circulating IgM, but not IgG, was elevated in TET2-KO mice compared to WT. Analysis of bulk RNASeq of sort purified peritoneal B-1a and B-1b cells revealed reduced expression of heavy and light chain immunoglobulin genes, predominantly in B-1a cells from TET2-KO mice compared to WT controls. As expected, the expression of IgM transcripts was the most abundant isotype in B-1 cells. Yet, only in B-1a cells there was a significant increase in the proportion of IgM transcripts in TET2-KO mice compared to WT. Analysis of the CDR3 of the BCR revealed an increased abundance of replicated CDR3 sequences in B-1 cells from TET2-KO mice, which was more clearly pronounced in B-1a compared to B-1b cells. V-D-J usage and circos plot analysis of V-J combinations showed enhanced usage of VH11 and VH12 pairings. Taken together, our study is the first to demonstrate that global loss of TET2 increases B-1 cell number and IgM production and reduces CDR3 diversity, which could impact many biological processes and disease states that are regulated by IgM.

Environmental corporate social responsibility practices and firm innovation: Complementarities and empirical evidence from Spanish firms.

This study examines the complementarity effect of combining different types of environmental corporate social responsibility (ECSR) practices on firm innovation. We apply the complementarity approach to test whether the adoption of different ECSR practices (i.e. practices for fewer materials per unit produced [materials], less energy per unit produced [energy], or decreasing environmental impact [impact]) generates super-additive effects on firms' innovation, measured by innovations type: adoption, new-to-the-market, and new-to-the-firm innovation. We use data from the Spanish Community Innovation Survey for the period 2009-2014. The results show that the best combination of ECSR practices depends on the innovation type. For innovation adoption, all possible combinations of the three practices produce super-additive effects; however, the complementarity patterns differ for new-to-the-market and new-to-the-firm innovations. For new-to-the-market innovation, energy practices appear to be a key factor in fostering innovation when combined with materials or impact practices. For new-to-the-firm innovation, the combination of these three ECSR practices shows complementarity effects. These findings provide useful insights for the design of corporate social responsibility strategies.

Multi-Time-Scale Optimal Scheduling Strategy for Marine Renewable Energy Based on Deep Reinforcement Learning Algorithm.

Surrounded by the Shandong Peninsula, the Bohai Sea and Yellow Sea possess vast marine energy resources. An analysis of actual meteorological data from these regions indicates significant seasonality and intra-day uncertainty in wind and photovoltaic power generation. The challenge of scheduling to leverage the complementary characteristics of various renewable energy sources for maintaining grid stability is substantial. In response, we have integrated wave energy with offshore photovoltaic and wind power generation and propose a day-ahead and intra-day multi-time-scale rolling optimization scheduling strategy for the complementary dispatch of these three energy sources. Using real meteorological data from this maritime area, we employed a CNN-LSTM neural network to predict the power generation and load demand of the area on both day-ahead 24 h and intra-day 1 h time scales, with the DDPG algorithm applied for refined electricity management through rolling optimization scheduling of the forecast data. Simulation results demonstrate that the proposed strategy effectively meets load demands through complementary scheduling of wave power, wind power, and photovoltaic power generation based on the climatic characteristics of the Bohai and Yellow Sea regions, reducing the negative impacts of the seasonality and intra-day uncertainty of these three energy sources on the grid. Additionally, compared to the day-ahead scheduling strategy alone, the day-ahead and intra-day rolling optimization scheduling strategy achieved a reduction in system costs by 16.1% and 22% for a typical winter day and a typical summer day, respectively.

Boosted Enzyme Activity via Encapsulation within Metal-Organic Frameworks with Pores Matching Enzyme Size and Shape.

A novel and versatile approach called "physical imprinting" is introduced to modulate enzyme conformation using mesoporous materials, addressing challenges in achieving improved enzyme activity and stability. Metal-organic frameworks with tailored mesopores, precisely matching enzyme size and shape, are synthesized. Remarkably, enzymes encapsulated within these customized mesopores exhibit over 1670% relative activity compared to free enzymes, maintaining outstanding efficiency even under harsh conditions such as heat, exposure to organic solvents, wide-ranging pH extremes from acidic to alkaline, and exposure to a digestion cocktail. After 18 consecutive cycles of use, the immobilized enzymes retain 80% of their initial activity. Additionally, the encapsulated enzymes exhibit a substantial increase in catalytic efficiency, with a 14.1-fold enhancement in kcat/KM compared to native enzymes. This enhancement is among the highest reported for immobilized enzymes. The improved enzyme activity and stability are corroborated by solid-state UV-vis, electron paramagnetic resonance, Fourier-transform infrared spectroscopy, and solid-state NMR spectroscopy. The findings not only offer valuable insights into the crucial role of size and shape complementarity within confined microenvironments but also establish a new pathway for developing solid carriers capable of enhancing enzyme activity and stability.

A Concept of Complementarity Between Complexity and Redundancy can Account for Kant's Biological Teleology and Unify Mechanistic and Finalistic Biology.

Over 160 years after Darwin and 70 years after the discovery of DNA, two fundamental questions of biology remain unanswered: What differentiates the living from the nonliving? How can mechanistic and finalistic or holistic biology be unified? Niels Bohr introduced a concept of complementarity in quantum physics and based on the paradox of light as a simultaneous wave and particle, conjectured that a similar concept might exist in biology that would solve the paradox of life originating from the nonliving. Bohr proposed that two mutually exclusive-independent observations may be necessary to explain a phenomenon and provided support to Immanuel Kant's idea that the "purposive" behaviour of organisms could only be explained in teleological terms and that mechanical and teleological approaches were necessary and complementary to explain biology. We present a concept of complementarity whereby biochemical pathways or cellular channels for the flow of information are simultaneously complex and redundant and complexity and redundancy complement each other. The postulates of biological complementarity are that (1) it was an essential condition in the origin of life; (2) it provided physiological flexibility that allowed organisms to mount self-protection response and complexity to evolve in the face of deleterious mutations before the evolution of bi-parental sex; (3) it laid the foundation for the evolution of a choice of response when confronted with threat; and (4) it applies to all levels of biological organizations and, thus, can serve as a basis for the unification of mechanistic and holistic biology. It is proposed that teleology is simultaneously constitutive and heuristic: constitutive because organisms' "purposive" behaviours are adaptive and are grounded in mechanism (complexity and redundancy), and heuristic because with our finite cognition and our goal-oriented (humans alone are aware of "tomorrow") and anthropomorphic pre-disposition, teleology will remain useful as a guide to our making sense of the world, even how to ask a meaningful question.

High-throughput quantitative assessments of the chemical complementarity of celiac disease-related IGH CDR3s and a gliadin epitope.

The long-term value of efficient antigen discovery includes gaining insights into the variety of potential cancer neoantigens, effective vaccines lacking adverse effects, and adaptive immune receptor (IR) targets for blocking adaptive IR-antigen interactions in autoimmunity. While the preceding goals have been partially addressed via big data approaches to HLA (human leukocyte antigen)-epitope binding, there has been little such progress in the big data setting for adaptive IR-epitope binding. This delay in progress for the latter is likely due to, among other things, the much more complicated adaptive IR repertoire in an individual compared to individual HLA alleles. Thus, results described here represent the application of an algorithm for efficient assessment of immunoglobulin heavy chain complementarity determining region-3 (IGH CDR3)-gliadin epitope interactions, with a focus on epitopes known to be associated with an immune response in celiac disease. The hydrophobic, chemical complementarity between celiac case IGH CDR3s and known celiac epitopes was found to be greater in comparison to the hydrophobic, chemical complementarity between the same celiac case IGH CDR3s and a series of control epitopes. Thus, the approaches indicated here likely offer guidance for the development of conveniently applied algorithms for antigen verification and discovery.

Intraspecific chemical variation of Tanacetum vulgare affects plant growth and reproductive traits in field plant communities.

The study investigated the impact of intraspecific plant chemodiversity on plant growth and reproductive traits at both the plant and plot levels. It also aimed to understand how chemodiversity at stand level affects ecosystem functioning and plant-plant interactions. We describe a biodiversity experiment in which we manipulated intraspecific plant chemodiversity at the plot level using six different chemotypes of common tansy (Tanacetum vulgare L., Asteraceae). We tested the effects of chemotype identity and plot-level chemotype richness on plant growth and reproductive traits and plot-level headspace emissions. The study found that plant chemotypes differed in growth and reproductive traits and that traits were affected by the chemotype richness of the plots. Although morphological differences among chemotypes became less pronounced over time, reproductive phenology patterns persisted. Plot-level trait means were also affected by the presence or absence of certain chemotypes in a plot, and the direction of the effect depended on the specific chemotype. However, chemotype richness did not lead to overyielding effects. Lastly, chemotype blends released from plant communities were neither richer nor more diverse with increasing plot-level chemotype richness, but became more dissimilar as they became more dissimilar in their leaf terpenoid profiles. We found that intraspecific plant chemodiversity is crucial in plant-plant interactions. We also found that the effects of chemodiversity on plant growth and reproductive traits were complex and varied depending on the chemotype richness of the plots. This long-term field experiment will allow further investigation into plant-insect interactions and insect community assembly in response to intraspecific chemodiversity.

From symbiosis to independence: Investigating changes in the relationship between general practitioners' presence and pharmacies' market size in Slovakia.

Using the Slovak pharmacy retail market case, this study examines the evolving interdependency between general practitioners (GPs) and pharmacies. Traditionally, they have operated symbiotically, with pharmacy revenues heavily reliant on prescriptions. However, the development of the market structures of these providers after the liberalization of the pharmacy retail market in 2005 raises a question about the stability of this relationship. By analyzing entry thresholds as a measure of the market size required for pharmacies to cover their entry costs, the study reveals that the dependency of pharmacies on the presence of GPs has diminished over time. In the initial year following the liberalization, the presence of a GP decreased the market size sufficient to cover entry costs for the first pharmacy by about 83% compared to a market without a GP. However, in 2019, this effect decreased to approximately 65%. This could imply worsened coverage of pharmaceutical services in small and rural areas with GPs as the entry decision of pharmacies is less elastic towards their presence.