The purpose of this study was to examine the effect of core technology competence on the competitive advantage of high-tech SMEs . Based on the 379 valid responses collected from a survey, structural equation modeling (SEM) was employed to examine the research model. Infrastructure and technology, state-of-the-art technology, innovative research and development (R&D) capability, and organizational flexibility all have a significant impact on the competitive advantage, while infrastructure and technology, state-of-the-art technology, and innovative R&D capability have a significant effect on organizational flexibility. Organizational flexibility plays a mediating role between innovative R&D capability’s effect and competitive advantage. Under the continuous influence of COVID-19, we should promote development from the perspective of strengthening enterprise infrastructure and technology and improving organizational flexibility to gain a competitive advantage. This study reveals the internal relationship between core technology competence, organizational flexibility, and competitive advantage. The results of this study will help us to fully understand the survival status and competitive advantage of high-tech SMEs under COVID-19. In the modern era of rapid development, information technology has emerged as an essential tool in organizations worldwide. Today, the exponential progress in technological development has enhanced the digital landscape of the leading firms , allowing organizations to discover newer ways of differentiation . High-teHowever, the positive development momentum of high-tech SMEs was broken by the sudden outbreak of COVID-19. From February to August 2020, the scale of revocation of high-tech enterprises in China reached 66,000 and the cancelation rate was 4.5% according to the global patent database of the State Intellectual Property Office, which was higher than the overall level of manufacturing and producer services, such that the number and proportion of revocation in high-tech SMEs were significantly higher than those of high-tech large enterprises.In today’s fast-growing environment, the world’s economies have strived to create significant competitive positioning . HoweverUndoubtedly, extended digitalization has increasingly forced firms to operate in fierce competition. The organizations functioning in such an environment have fundamentally attained a distinctive edge by embracing advanced technological competence. Core technological competence is the foundation and critical part of scientific and technical enterprises. However, in today’s social environment, the technological upgrading faced by high-tech SMEs has been very rapid. As such, high-tech SMEs must maintain one or several differentiated core technologies . A compaA company’s resources can create excellent performance and transform it into a competitive advantage . OrganizChina’s technology R&D and transformation of scientific and technological achievements are guaranteed in a good organizational framework. High-tech SMEs are a group of typical technology-driven enterprises; to create more resource advantages than competitors, there must be a structure with a strategic vision to lead the enterprise to success. A flexible organization, through the accumulation of ability and knowledge, can respond to environmental changes at any time and find the most appropriate strategy to obtain a competitive advantage . TherefoA firm’s technological capabilities strengthen its competence, thus providing a competitive advantage . TherefoThe rest of this review is organized as follows: We propose the research model and hypotheses in section 2. Section 3 reports methodology and data collection. Section 4 reports the finding, and section 5 discusses the findings. We present the study contribution and limitations in section 6.Without a sustainable competitive advantage, enterprises cannot survive continuously. Enterprises with core competitive advantages will be evergreen. Therefore, the research on enterprise competitive advantage has always been the focus of academic interest. However, it has only been in the last decade that China has proposed to revitalize the country through science and technology and has attached importance to the power of science and technology to enhance its comprehensive national strength, with China’s advanced science and technology accounting for only 1.46% of the world. Therefore, there is still a long way to go in exploring the use of technological core competence to enhance the competitive advantage of enterprises in China, especially small- and medium-sized enterprises in science and technology. As the previous research highlights, it is of great significance to find a theory suitable for China’s national conditions that promotes the value of high-tech SMEs.The main theories of competitive advantage covered in the existing literature are resource-based theory, competence-based theory, knowledge-based theory, and dynamic capability theory.The resource school believes that each organization is a combination of unique resources and capabilities, which forms the basis of enterprise competitive strategy . AccordiAccording to the theory of core competence, the core competence of an enterprise is the combination of various skills and the key to obtaining a competitive advantage in the long-term development of the enterprise. However, the limitation of this view is that the organizational process or convention is usually embedded in the enterprise over time . It rearAccording to the resource-based view (RBV) and competence-based view (CBV), the concepts of resource and competence are put forward as the source of competitive advantage. This study agrees with these views, but we also support the complementary evolution process of theories caused by the time and space background of these studies rather than the formation of mutually exclusive theoretical views. These theories emphasize the importance of an industry analysis and argue that the resources and capabilities of enterprises can only be reflected in the industrial competitive environment. Therefore, the strategic management approach of the endogenous theory of competitive advantage can be summarized as industrial environment analysis, enterprise internal resource analysis, formulating competitive strategy, implementing strategy, accumulating strategic resources and establishing core competence matching the industrial environment, winning competitive advantage, and obtaining performance , that isOnce the competitive advantage of knowledge and technology is formed, there will be path dependence and relative stability. This relative stability can easily result in the formation of a rigid core, which causes the enterprise to lose its original competitive advantage. As there is great uncertainty, variability, and complexity in the changing external competitive environment, enterprises are facing confusion and increasing strategic discontinuity, and various factors in the environment are intertwined. Therefore, it is necessary to continuously acquire, integrate, and update the organizational resources and create capabilities that are difficult for competitors to imitate. To achieve this flexible and rapid response, flexible organization and flexible management are needed to promote the renewal of knowledge and technology with the change in environment, so as to obtain a sustainable competitive advantage. As such, according to the theoretical paradigm of resource strategy performance, this study brings the resource-based theory and core competence theory into a theoretical framework.In recent years, emerging technologies have captured the firms’ interest in gaining a competitive advantage. In this regard, the business core competencies have been viewed as the fundamental construct for adapting and renewing business competitiveness. Technology competency accelerates the momentum of a firm’s journey toward achieving competitive advantage . Hence, H1: Core technology competence influences competitive advantage.Core technology competence is a series of knowledge sets used to distinguish the competitive advantage between a specific enterprise and other enterprises. When an enterprise’s core technical competence is regarded as the unique knowledge to identify and solve problems, the core technical competence can often form the most basic sustainable competitive advantage of a specific enterprise and develop more new products to meet various market needs. The research of In this study, “core technology competence” is defined as the key technologies and related technologies that exist in the whole process of core product formation and service and can be widely used, as well as their coordination and combination capability. Among them, infrastructure and technology refer to the company’s supporting capability and essential technology capability when facing competitive pressure. Significantly, technology infrastructure capability plays an integral role in maintaining a firm’s competitiveness. It facilitates the organization’s internal capabilities, thereby upgrading the firm’s strategic position. IT infrastructure enhances the firm’s ability to compete with peers. It improves the organization’s skill set and core competencies by building a foundation of competitive advantages and benefits. In explaining this notion, one study states that IT infrastructure has emerged as a vital tool for differentiation .However, in the rapidly changing business environment, firms have focused on maintaining a competitive edge over their peers. This new technological revolution may see the emergence of technology to advance firms’ innovation and competitiveness. The state-of-the-art technological paradigm reengineers the business processes, thereby enabling firms to improve their competitive position. Based on this statement, one study states that today’s high-tech companies have extensively deployed state-of-the-art technologies to overcome the market competition, inevitably achieving a distinctive competitive advantage .However, the literature shows that a firm’s R&D capabilities also play an integral role in fostering its performance, thus establishing a superior competitive benefit. Innovative R&D capability refers to a company’s capability to develop to maintain its future competitive advantage. The technological R&D capabilities advance the firm’s knowledge and innovation process, thereby attaining a better market position. In explaining this phenomenon, one study states that R&D capabilities enable a firm to gain a distinctive market position, thereby promoting its competitive advantage and sustainability . FurtherH1 (1): Infrastructure and technology have a significant positive impact on sustainable competitive advantage.H1 (2): State-of-the-art technology has a significant positive impact on sustainable competitive advantage.H1 (3): Innovative R&D capability has a significant positive impact on sustainable competitive advantage.Significantly, successful organizations widely adopt flexible means for ensuring a firm’s competitiveness. Organization flexibility elevates the firm’s ability to respond to market uncertainties. It enhances the organizations’ processes by rapidly controlling and maintaining the organizational environment. Business fluctuations are sometimes very chaotic and challenging. As such, one study reveals that organizational flexibility fosters a firm’s processes by effectively managing the market risks . In partBased on contingency theory, scholars have discussed the role of organizational flexibility in the environment organization relationship. On the one hand, when enterprises are in a changing environment and the uncertainty and unpredictability of the environmental change trend of enterprises are increasing, flexibility can play an important role in stabilizing enterprise performance and improving enterprise survival probability . On the Significantly, successful organizations widely adopt flexible means for ensuring a firm’s competitiveness. Organization flexibility elevates the firm’s ability to respond to market uncertainties. It enhances the organizations’ processes by rapidly controlling and maintaining the organizational environment. Business fluctuations are sometimes very chaotic and challenging. As such, one study reveals that organizational flexibility fosters a firm’s processes by effectively managing the market risk . In addiH2: Organizational flexibility influences competitive advantage.A core competence is an action that plays a crucial role in an organization’s “key process,” and organizational flexibility can be used to adjust the allocation of various resources of enterprises. Core competence is a relatively static capability in the process of enterprise development, while organizational flexibility is a relatively dynamic capability. With the development of the market and the progress of science and technology, it is difficult to identify opportunities that cannot be copied, and there will be scarce resources because of technological progress or the need to find alternative resources through artificial synthesis or other factors.Correspondingly, organizational flexibility refers to the driving force in the development of enterprises, and it is a type of flexibility and adaptability to encourage enterprises to obtain a sustainable competitive advantage. In the technological era, achieving a competitive advantage has become the prime concern of today’s firms. In this regard, technological flexibility ensures the implementation of IT infrastructure  and firmFurther, with the increase in globalization, dynamic structural flexibilities seek to provide firms with a distinctive competitive advantage. Significantly, structural flexibility plays an incalculable role in facilitating corporate functions. Organizations enjoy favorable market positions due to changes in their structure . TherefoHowever, in the competitive global environment, companies have strongly adopted core competencies to enhance their market position. In this regard, culture has also become prominent as a factor providing firms with a superior competitive advantage. Cultural flexibility influences the organizational environment by transforming and enhancing the firm’s competitiveness . It encoH2 (1): Technological flexibility has a significant positive impact on the sustainable competitive advantage.H2 (2): Structural flexibility has a significant positive impact on the sustainable competitive advantage.H2 (3): Cultural flexibility has a significant positive impact on the sustainable competitive advantage.For small- and medium-sized scientific and technological enterprises, technological innovation is the basis for their survival. An enterprise needs a matching management model to update its products and technologies. While continuously innovating and upgrading its core competencies, it will bring a series of good chain reactions to the organization: First, it triggered the adjustment and upgrading of the industrial structure of enterprises: (1) the direction of industrial adjustment shifted toward the top of the industrial chain—bringing high added value and high profits and (2) promoting productivity, thereby promoting production efficiency.Second, it promoted the rational distribution of human resources. Employees must strive to learn professional knowledge while the products or technologies of the enterprise are upgrading in the international market. This process can form a virtuous circle of new technological innovation. Third, it broke the rigid management inherent at the department level and made the strategic adjustment of the enterprise timelier.Based on the above analysis, this study proposes the first hypothesis:H3 (1): Infrastructure and technology have a significant positive impact on technological flexibility.H3 (2): State-of-the-art technology has a significant positive impact on technical flexibility.H3 (3): Innovative R&D capability has a significant positive impact on technological flexibility.H3 (4): Infrastructure and technology have a significant positive impact on structural flexibility.H3 (5): State-of-the-art technology has a significant positive impact on structural flexibility.H3 (6): Innovative R&D capability has a significant positive impact on structural flexibility.H3 (7): Infrastructure and technology have a significant positive impact on cultural flexibility.H3 (8): State-of-the-art technology has a significant positive impact on cultural flexibility.H3 (9): Innovative R&D capability has a significant positive impact on cultural flexibility.Core competence will be transformed into core rigidity, that is, if managers respond to the current situation with a fixed pattern, it will reduce the enterprises’ competitive advantage. Therefore, it cannot be assumed that a core technological competence will bring a competitive advantage. A mediator is needed to enhance the relationship between core technology competence and competitive advantage . It is cUndoubtedly, flexibility is central to achieving successful development. In recent years, structural flexibility has helped companies tap new business opportunities, thus enhancing their operations. A firm’s structural flexibility enables it to expand its operations by strengthening its infrastructure development. In an uncertain environment, a firm’s technological infrastructure allows it to change its structural processes, thus adjusting to the new environment. It makes the firms anticipate the changes needed in the business operations. In explaining this notion, the prior research states that the flexibility system increases a firm’s value by managing unknown business scenarios . MoreoveH4 (1): Technology flexibility mediates the relationship between infrastructure and technology and competitive advantage.H4 (2): Technology flexibility mediates the relationship between state-of-the-art technology and competitive advantage.H4 (3): Technology flexibility mediates the relationship between innovative R&D capability and competitive advantage.H4 (4): Structural flexibility mediates the relationship between infrastructure and technology and competitive advantage.H4 (5): Structural flexibility mediates the relationship between state-of-the-art technology and competitive advantage.H4 (6): Structural flexibility mediates the relationship between innovative R&D capability and competitive advantage.H4 (7): Cultural flexibility mediates the relationship between infrastructure and technology and competitive advantage.H4 (8): Cultural flexibility mediates the relationship between state-of-the-art technology and competitive advantage.H4 (9): Cultural flexibility mediates the relationship between innovative R&D capability and competitive advantage.This study adopts the empirical research method, and the required data were collected by questionnaire. In the process of questionnaire analysis, we deleted the unreasonable options and improved the questionnaire. A seven-point Likert scale was selected for the questionnaire items measurement ranging from 1 to 7 . The formal questionnaire consists of three parts: the first part is the background of the questionnaire—this part mainly explains the rationality of the survey purpose; the second part is the basic information of the survey enterprise and the personal information of the participants—to help confirm whether the received questionnaire is representative, and the third part is the main part of the questionnaire, with a total of 37 items, including core technology, organizational flexibility environmental uncertainty, enterprise performance, and four categories.Core technical competence is the independent variable in this study. The measurement items of core technical competence are based on the studies of The sample size should be kept at more than 150 in the data analysis with the structural equation model , and theHere are two ways to distribute the questionnaire: The first way is to distribute and recycle the questionnaire according to the pre-determined research objects, mainly select specific MBA students from universities and qualified middle and senior managers of enterprises selected through personal relations and distribute the questionnaire by directly receiving the enterprise site or e-mail. The second is to entrust the contact person in the organization for questionnaire distribution and collect the questionnaire. In this study, we have adopted an electronic questionnaire, which is more convenient in the current circumstances.A total of 410 questionnaires were distributed, and 380 questionnaires were received from the participants. We have excluded 66 incomplete questionnaires from the received questionnaires. Finally, 314 valid questionnaires were kept for data analysis. The sample size met the requirements of the data analysis method for large sample data .The current study data analysis includes two steps. First, we examined the measurement model to test reliability and validity. Then, we examined the structural model to test the research hypotheses. We examined data normality before data analysis. First, we checked the reliability through Cronbach’s α coefficient method. The composite reliability (CR) method should be used for measurement testing. In terms of validity tests, this study mainly adopts convergent validity and differential validity  to measure the validity of the questionnaire. Each AVE (the average variance extracted) is greater than 0.5, reaching the acceptance standard; the CR value meets the threshold requirement of greater than 0.7, indicating that the questionnaire has good convergent validity. The test results of discriminant validity are shown in Second, the main variables included in this study passed the test of reliability and validity. Structural equation modeling was used to analyze the assumed relationship between core technical competence, organizational flexibility, and sustainable competitive advantage. Based on the conceptual model constructed in this study and the corresponding research assumptions, the initial structural equation model’s path diagram is constructed using AMOS22.0. F1 represents infrastructure and technology, F2 represents state-of-the-art technology, F3 represents innovative R&D capability, F4 represents technological flexibility, F5 represents structural flexibility, F6 represents cultural flexibility, and F7 represents sustained competitive advantage. There are seven latent variables in the study. The study’s independent variables are infrastructure and technology, state-of-the-art technology, and innovative R&D capability. The four internal-derived latent variables (dependent variables) of enterprises’ are technological, structural, cultural, and sustainable competitive advantages. The model has residuals of e1–e33 and e34–e37, and their path coefficients are one by default. This study will verify the 15 impact paths set in the initial structural equation model. AMOS22.0 analyzed the initial structural equation model. p < 0.001). The standardized path coefficients of state-of-the-art technology and innovative R&D capability to sustainable competitive advantage are 0.044 (p = 0.460) and 0.075 (p = 0.277), respectively. This shows that the infrastructure and technology of core technology competence have a significant positive effect on sustainable competitive advantage. In contrast, state-of-the-art technology and innovative R&D capabilities have no direct effect on sustainable competitive advantage.The empirical results show that the standardized path coefficient of infrastructure and technology and sustainable competitive advantage is 0.243 , 0.290 (p < 0.001), and 0.288 (p < 0.01), respectively. The standardized path coefficients between state-of-the-art technology and technical flexibility, structural flexibility, and cultural flexibility are 0.185 (p = 0.004 < 0.01), 0.276 (p < 0.001), and 0.178 (p = 0.005 < 0.01), respectively. The standardized path coefficients between innovative R&D capability and technological flexibility, structural flexibility, and cultural flexibility are 0.292 (P < 0.001), 0.293 (P < 0.001), and 0.264 (P < 0.001), respectively. The results show that actively strengthening the core technology construction of SMEs can help cultivate and enhance enterprises’ organizational capacity.Improving enterprise core technology competence can help enhance organizational flexibility, which is an effective way to improve core technology competence. The empirical analysis by constructing a structural equation model shows that the standardized path coefficients between infrastructure and technology and technology flexibility, structural flexibility, and cultural flexibility are 0.334 (P = 0.008 < 0.01). The standardized path coefficients between cultural flexibility and firm performance are 0.215 and 0.215, respectively (p < 0.001). Thus, the dimensions of organizational flexibility have a significant positive impact on sustainable competitive advantage.The empirical results show that all dimensions of organizational flexibility positively affect sustainable competitive advantage, which strongly proves the importance of organizational flexibility in enhancing SMEs’ competitive advantage. By constructing a structural equation model for empirical analysis, we draw the following conclusions: The standardized path coefficient between technological flexibility and competitive advantage is 0.279. The standardized path coefficients between structural flexibility and competitive advantage were 0.179  to attain improvised organizational outcomes. As such, the prior research states that a firm’s structural flexibility improves its technology and infrastructure, thus allowing the organization to operate in a turbulent business environment . SimilarThis research has a few limitations. First, although the topic selection and related research design are scientific and objective and the expected research objectives and some valuable conclusions are achieved, due to the limitation of the specific background, time, funding, and personal knowledge of COVID-19, the compositional dimensions and measurement of organizational flexibility of high-tech SMEs are still in the exploratory stage. Second, to narrow the scope of the study and make the research more accurate and targeted, this study takes high-tech SMEs as the main research object. To study the accuracy of the results, we used the size of the enterprise and the years of its establishment as a control variable, but there are other control variables that may also have an impact on the results of the study, which could be explored in future research.The results of this study demonstrated that, during COVID-19, the only factors that could directly affect the sustained competitive advantage of high-tech SMEs were infrastructure and technology. During COVID-19, SMEs have been eager to seize market share through transformation and innovation; however, many enterprises neither are ready for transformation nor have the ability for cross-domain development, because new products and new technologies are updated too fast, and management cannot keep up with development. Management is busy creating momentum and making money, relaxing the cultivation and transformation of basic business and basic technology, resulting in enterprises not only failing to gain a competitive advantage but also failing to hold their position in the COVID-19 pandemic situation. Overall, with the impact of the global pandemic, the serious recession of the world economy, and the decline in domestic consumption, investment, and export, we should pay more attention to the cultivation of basic technology and skills.The empirical results show that technological flexibility and structural flexibility have a significant positive impact on sustainable competitive advantage. Therefore, organizational flexibility with high flexibility and agility is an important basis for the formation of competitive advantage and plays a key role in the promotion of sustainable competitive advantage. Under the continuous influence of COVID-19, we should attach great importance to the organization from a strategic perspective. The promotion of flexibility can enhance a high-tech SME’s ability to resist environmental uncertainty and maintain a sustainable competitive advantage. In the COVID-19 pandemic context, high-tech SMEs should form an alliance under the consideration of the strategic goal between the individual enterprise and individuals, exchange complementary resources independently, reach the phased goal of the target product, respectively, obtain the long-term market competitive advantage, and then form a lasting and formal relationship.The original contributions presented in this study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.TT wrote the main manuscript. KI proofread and revised the manuscript. AR gave the core idea. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Cement augmentation of pedicle screws is one of the most promising approaches to enhance the anchoring of screws in the osteoporotic spine. To date, there is no ideal cement for pedicle screw augmentation. The purpose of this study was to investigate whether an injectable, bioactive, and degradable calcium sulfate/hydroxyapatite (CaS/HA) cement could increase the maximum pull-out force of pedicle screws in osteoporotic vertebrae. Herein, 17 osteoporotic thoracic and lumbar vertebrae were obtained from a single fresh-frozen human cadaver and instrumented with fenestrated pedicle screws. The right screw in each vertebra was augmented with CaS/HA cement and the un-augmented left side served as a paired control. The cement distribution, interdigitation ability, and cement leakage were evaluated using radiographs. Furthermore, pull-out testing was used to evaluate the immediate mechanical effect of CaS/HA augmentation on the pedicle screws. The CaS/HA cement presented good distribution and interdigitation ability without leakage into the spinal canal. Augmentation significantly enhanced the maximum pull-out force of the pedicle screw in which the augmented side was 39.0% higher than the pedicle-screw-alone side. Therefore, the novel biodegradable biphasic CaS/HA cement could be a promising material for pedicle screw augmentation in the osteoporotic spine. Osteoporosis is the most common age-related bone disease. Over 200 million people worldwide suffer from osteoporosis . The preOver the past few decades, numerous techniques have been developed to improve the anchorage of pedicle screws in order to reduce the risk of screw failure. These techniques primarily suggested larger outer diameters or an extended length of screws, screws with different thread profiles, a cylindrical or conical core shape, expanding screws, bi-cortical pedicle screws, coated pedicle screws, or cement augmentation screws ,9. The cIn this study, we aim to show that a recently established injectable calcium sulfate/hydroxyapatite (CaS/HA) biomaterial, which has been approved for human use both in Europe and North America , may meeWe hypothesized that CaS/HA cement could strengthen the interface between the pedicle screw and the vertebral trabecular bone with the potential to increase the pull-out force of the pedicle screw in the osteoporotic vertebrae in a preliminary proof-of-principle study.A male human cadaver was used in this study as a model of osteoporotic vertebrae to test the hypothesis that the CaS/HA cement could augment the pedicle screws with a potential improvement of the immediate anchorage in the osteoporotic vertebrae. The computed tomography (CT) scan was performed to measure the Hounsfield Units (HU). The fenestrated pedicle screws were implanted into the osteoporotic vertebrae, in which the right side of the vertebra was augmented with the CaS/HA cement and the left side of the vertebra was not augmented. During the procedure, the C-arm X-ray was used to evaluate cement distribution and cement leakage. After the pedicle screw augmentation, the specimens were embedded in epoxy resin for pull-out testing to evaluate the pull-out force. The maximum pull-out force was used to quantify the effect of cement augmentation by comparison with the non-augmented side. . A total of 17 vertebrae (12 thoracic and 5 lumbar) were obtained from a fresh-frozen cadaver of an 81-year-old male . All soft tissues on the vertebral body were completely removed and CT scans of all vertebral bodies were performed . The diameter of the pedicle and the length of the vertebral body were measured to determine the respective size of each pedicle screw. The radiodensity in Hounsfield Units (HU) of the vertebral body, screw trajectory, and augmentation area were also measured using AGFA HealthCare software . The HU of vertebral bodies was measured according to a method described by Li et al. , and theThe cortex and hyperplastic osteophytes of the lamina were removed with a rongeur, so that the polyaxial tulip-head of the pedicle screw could be rotated freely to prevent it from blocking the pedicle screw. A pilot hole was made by an awl oriented forward and inward along the pedicle axis. Then we checked the hole with a probe to ensure that the pedicle was not damaged. Each hole was tapped using a 4.5 mm tap, then each fenestrated pedicle screw  was implanted with a ratchet screwdriver using a free-hand technique until the hub of the screw had been firmly fixed on the posterior cortex. Because the trajectory of the screw during implantation affects the required extraction force later, the classical trajectory of passing the center of the pedicle parallel to the superior and inferior endplates was adopted in this study . All insAccording to the preparation protocol, the CaS/HA cement , which consists of a premixed powder of 60 weight% (wt%) CaS and 40 wt% HA, was mixed with iohexol (1 g CaS/HA:0.43 mL iohexol) in a 6-well plate for 30 s. The CaS/HA slurry was then transferred into the syringe used for injection. This process allowed the cement to cure for 4 min prior to injection ,19. DuriA customized metal container was used to hold the vertebra, while a band was used to fix the vertebra by insertion through the spinal canal. The vertebrae were then embedded in epoxy resin  with the embedded line of each vertebra under the pedicle to avoid interference with mechanical testing A,B. Thisp < 0.05 (*).Data were presented as mean ± standard deviation (SD). Statistical analysis was performed by using GraphPad Prism 8 . Significant differences between the groups were evaluated using the paired t-test. The level of significance was chosen at This study involved cadaveric specimens voluntarily donated by a donor to the Institute of Anatomy, University Hospital Carl Gustav Carus at Technische Universität Dresden for the advancement of science. This study has been carried out in accordance with the Code of Ethics of the World Medical Association (WMA Statement on Organ and Tissue Donation) and the local ethics policy of Dresden University Hospital for experiments.For this cadaveric study, all thoracic and lumbar vertebrae from one cadaveric donor were collected. The T7 height was reduced which may have suffered a previous osteoporotic vertebral compression fracture, without previous cement-related surgical treatment. The applied individual, vertebra-specific pedicle screw sizes are listed in All pedicle screws were successfully implanted into the vertebrae. After pedicle screw implantation, X-ray images were taken in order to demonstrate that the placement of all pedicle screws was accurate, and that no pedicle or vertebral body was damaged during the operation .The CaS/HA cement was completely and successfully injected in all vertebrae (1 mL in the thoracic vertebrae and 2 mL in the lumbar vertebrae) under fluoroscopic guidance. After cement injection, X-ray images demonstrated that the CaS/HA cement was evenly distributed around the anterior hole of the fenestrated pedicle screws both in the coronal plane from the anteroposterior radiograph and in the sagittal plane from the lateral radiograph in all vertebrae. No obvious cement leakage was seen into the spinal canal. In addition, the irregular shape of the cement edges also indicated the adequate cement-bone interdigitation ability in the trabecular bone .Further visual observation of the vertebral appearance after cement injection revealed that the cement was exposed in the superior endplate plane of four vertebrae  and in the inferior endplate plane of three vertebrae  .All specimens were stabilized in the customized metal container to minimize variation. Regarding the maximum pull-out force, the determined force for the augmented screw was higher than the pull-out force of the pedicle screw without augmentation among 14 vertebrae. Whereas test outliers on the unenhanced side of T3 may be due to the destruction of the trabecular microstructure of the pedicle and vertebral body, cement augmentation significantly increased its pull-out resistance although the pedicle trabecular microarchitecture on the augmented side was also destroyed from the analysis of CT scans  . The maxPedicle screw failure occurred at the screw-bone interface in both the screw-alone and the augmented screw sides. After testing, 10 specimens could be retrieved from the epoxy resin while the other vertebrae were damaged during removal of the osteoporotic vertebral body from the container due to the tight adhesion of the vertebral body and the epoxy resin. The vertebral cross-section photographs along the midline of the pedicle demonstrated that the cement was evenly distributed within the trabecular network in the vertebral body, which densified the loose architecture of the osteoporotic vertebral body. At the screw trajectory area, a local cavity was formed inside the vertebrae after screw extraction at the screw-only side; cement was still filled within the screw trajectory at the augmented side A,B. AfteIn this study, we described the novel application of a recently established CaS/HA biomaterial cement for the augmentation of pedicle screws in the osteoporotic spine. By injecting CaS/HA cement into the internal channel of the fenestrated pedicle screws, the cement can be evenly distributed into the screw threads and into the surrounding trabecular in order to strengthen the interlocking effect at the bone-screw interface . Biomech3, the stability of the pedicle screws may be insufficient so that additional stabilization should be considered [Anchoring of pedicle screws in the spine with poor bone quality remains challenging. The success of a pedicle screw instrumentation depends on the implant design as well as on the quality of the bone which could provide adequate compression between the threads and surrounding bone; this proves to be a critical aspect of fixation stability . Previounsidered . Dual ennsidered . Howevernsidered . Some stnsidered . Two recnsidered ,32. The nsidered . There wAt present, pedicle screw augmentation techniques mainly include prefilling with cement prior to solid screw insertion and fenestrated pedicle screws injected with cement through the inner channel, but the biomechanical testing results of PMMA augmentation are controversial . CompareBiphasic CaS/HA biomaterials have different rates of degradation, as the CaS phase is rapidly absorbed within 6–8 weeks in vivo while the HA phase persists for 9–12 months, integrating with new bone ingrowth over time . The undBesides a successful outcome of the study, certain technical limitations need to be considered: the damaged cartilage endplate during specimen preparation due to the tight connection between the endplate and intervertebral disc in this study may affect the quantification of absolute pull-out force results. The anatomical variation of the pedicle in the available cadaveric vertebrae may also influence the biomechanical results. However, these limitations are not expected to affect a pairwise side-to-side comparison of individual vertebrae since specimen handling did not influence the morphology and macroscopic anatomy of the bone specimens . AnotherThis study demonstrates a novel application of the recently established injectable, biodegradable biphasic CaS/HA cement for augmentation of fenestrated pedicle screws in osteoporotic spine. In a cadaveric model, the CaS/HA augmentation was proven as an effective technique which significantly enhanced the required pull-out force of fenestrated pedicle screws in osteoporotic vertebrae in comparison with non-augmented screws. The technique allowed adequate cement distribution and interdigital ability to the surrounding trabecular bone. In addition, the CaS/HA material has been approved for clinical utilization, which could allow for an easier translation to the clinical application of this technique. In conclusion, this biodegradable biphasic CaS/HA cement could be a promising material for pedicle screw augmentation in the osteoporotic spine.


Uncertainty associated with ice sheet motion plagues sea level rise predictions. Much of this uncertainty arises from imperfect representations of physical processes including basal slip and internal ice deformation, with ice sheet models largely incapable of reproducing borehole-based observations. Here, we model isolated three-dimensional domains from fast-moving  and slow-moving (Isunnguata Sermia) ice sheet settings in Greenland. By incorporating realistic geostatistically simulated topography, we show that a spatially highly variable layer of temperate ice (much softer ice at the pressure-melting point) forms naturally in both settings, alongside ice motion patterns which diverge substantially from those obtained using smoothly varying BedMachine topography. Temperate ice is vertically extensive (>100 meters) in deep troughs but thins notably (<5 meters) over bedrock highs, with basal slip rates reaching >90 or <5% of surface velocity dependent on topography and temperate layer thickness. Developing parameterizations of the net effect of this complex motion can improve the realism of predictive ice sheet models. Geostatistically realistic topography forces complex patterns of basal slip and deformation in Greenland outlet glaciers. State-of-the-art GrIS models run with BedMachine, the most advanced gridded data product of GrIS basal topography, which is relatively smooth compared to deglaciated terrain  and impr−1) Sermeq Kujalleq  land-terminating Isunnguata Sermia , where g= 9.81 m s−2, to remove the requirement for vertical displacement of periodic inflow-outflow boundaries. Boundary conditions are specified for FS and TC runs separately. Stress is related to strain using the Nye-Glen isotropic flow law (−1) is the strain rate tensor, n is the flow exponent assumed to equal 3, and A (MPa−3 a−1) is the creep parameter. A is calculated from the homologous temperature, Th (in kelvin), if below the pressure-dependent melting point, Tm, or water content, ω (proportion), if above asTm(p) = Ttr − γ(p − ptr), γ (K MPa−1) is the Clausius-Clapeyron constant, Ttr is the triple point water temperature, and ptr is the triple point water pressure. A1 and A2 (MPa a−1) are rate factors, and Q1 and Q2 (J mol−1) are activation energies for T ≤ Tlim and Tlim < T < Tm, respectively, where Tlim= 263.2 K is the limit temperature. R (J mol−1 K−1) is the gas constant, and W1, W2, and W3  are water viscosity factors, with default values taken from the linear fit of Duval , is used as the state variable with the Elmer/Ice EnthalpySolver (T and ω asCa (J kg−1 K−2) and Cb (J kg−1 K−1) are enthalpy heat capacity constants, L (J kg-1) is the latent heat capacity of ice, Tref = 200 K is the reference temperature.Specific enthalpy, b (MPa), is given asC (dimensionless) is a parameter that depends on basal morphology and cannot be readily estimated from irregular topographies but must be less than the maximum up-slope gradient of the bed  tangential to the ice-bed interface, and n = 3 is the same exponent used in the flow relationship. As (m a−1 MPa−3) depends on ice rheology and morphology of the bed and is used in the case of hard-bed sliding with no cavitation (Ne = pi − pw (MPa) is the effective pressure at the bed where pi (MPa) is the ice overburden pressure and pw (MPa) is the subglacial water pressure. Helanow et al.  via a proportionality parameter, O (dimensionless), as Ne = − ρzOg(s − b) where b (m) is the elevation of the glacier base and s (m) is the surface elevation. An O of 1 is equivalent to a water pressure of 0, while an O of 0 means water pressure effectively balances ice overburden pressure. However, the upper limit of basal traction tangential to the bed under maxb = NeC, which can lead to model instability if the areally averaged basal traction cannot support the driving stress, τd = ρxg(s − b), across the domain. Setting τmaxb ≥ τd and expanding xg and zg then give the inequalityF is a parameter we introduce to account for the resistive influence of intermediate-scale topographic obstacles, or lack thereof (further description in Supplementary Text). For the range of slopes and F values covered and C= 0.1617, this gives a range in O from 0.0874 to 0.240, comparable to values in other studies (O) to obtain the best match between modeled surface velocity and satellite measurements. Surface slopes in the regions studied are 1° to 2° with variation in g away from the long-wavelength borehole site value expected, as g in our model will also be accounting for longitudinal and transverse stresses in the ice. Altering the basal topography slope will slightly alter the relative angle of obstacle stoss and lee sides, but we view this change as small relative to the absolute angle of these obstacles (up to 40°).We make the simplifying assumption that A are calculated as a function of normalized depth, d (m) is depth, asaE, bE, and cE are quadratic curve parameters. aE, bE, and cE are obtained via a second-order polynomial fitting procedure (MATLAB polyfit) of the borehole temperature record at the center of the domain, converted to enthalpy with minor adjustments to only remove the temperate ice layer (fig. S7). This approach ensures consistent rheology at the periodic inflow-outflow boundaries. These profiles are also used for the input enthalpy field in TC runs.In FS runs, enthalpy and xu, yu, and zu are components of the surface velocity vector u. No accumulation or ablation is accounted for in FS runs, as this would require a corresponding removal of mass from elsewhere in the model in order for mass to remain constant. Outstanding FS boundary conditions areσ is the Cauchy stress tensor, n is the normal vector, and tl  are tangent vectors at the specified surface, with tangential stress at the bed specified in The free surface is computed with the Elmer/Ice FreeSurfaceSolver as−1 s−1) is the enthalpy diffusivity defined asThe specific enthalpy field is set at the inflow boundary based on depth and the central borehole record following c and κt are enthalpy diffusivities for cold and temperate ice, respectively, meaning that water movement within the temperate ice is assumed to be a diffusive process. H is limited by ωmax set at 2.5%, around the level of field observations of water content [, are then obtained from specific enthalpy loads, Hloads, and element weighting, Hweights, asz coordinate of the CTS. More advanced drainage formulations exist (where κMb (kg m−2 a−1), is calculated asFb = ubτb is the frictional heating at the bed, Gb is the geothermal heat flux, q = −k ∇ T is the energy flux into the ice where k is the thermal conductivity of the ice, and Qb is the rate at which hydrological storage and transport mechanisms deliver latent heat to the base of the ice that we set as 0 across all of our model domains. When a temperate layer is present, q is a small negative value (flux directed toward the base) determined by the local pressure gradient. If no temperate layer is present, then q is a small positive value (directed away from the base). Comparisons of inflowing and outflowing ice volumes for the RESPONDER BedMachine run, where mass loss is greatest, show that basal melting has a negligible effect on overall ice flow volumes (0.3%). This leaves outstanding TC boundary conditions asFor the purposes of basal sliding, we make no adjustment for the small sections where basal temperatures are below the pressure melting point, effectively assuming that the bed is sufficiently well-hydrologically connected to provide water to these regions for freeze-on. Considerations for basal freeze-on rates required to pin the basal temperature at the pressure melting point are presented in the Supplementary Materials, showing that the water required is negligible in context. The basal mass balance, Elmer/Ice solver input files and postprocessing scripts are available in the Supplementary Materials.We use conditional geostatistical simulations , and (iib(αx) is measured bed topography, xα is a spatial location, and N is the number of point pairs for a given lag distance, h (m). Each empirical variogram was calculated with different azimuthal directions to capture any roughness anisotropy. A variogram model is manually fitted to the empirical variogram. For S5 the modeled variogram ish, c) is the exponential variogram function with a range c (m)  is the spherical variogram function   topographic features that can cause unrealistic model behavior. The simulation was implemented using the GeostatsPy software package (


It was shown that D-lipophilic nucleoside derivatives manifested higher inhibition activity than their L-analogs, and configuration of the carbohydrate moiety can influence the mechanism of Tdp1 inhibition.Inhibition of human DNA repair enzyme tyrosyl-DNA phosphodiesterase 1 (Tdp1) by different chiral lipophilic nucleoside derivatives was studied. New Tdp1 inhibitors were found in the series of the studied compounds with IC R, 2R) isomer of chloramphenicol (1-(4-nitrophenyl)-2-dichloroacetylaminopropan-1,3-diol) is used in medical practice for the treatment of infections caused by Gram-positive and Gram-negative bacteria and acts by binding to the 70S ribosome and thus inhibiting the peptidyl transferase reaction, whereas the L-erythro , D-erythro , and L-threo  isomers are all inactive as antibacterial agents [R-thalidomide is a potent sedative drug while S-thalidomide can cause adverse effects [Chirality plays an important role in the development of novel drugs, improvement of their selectivity, and characterization of receptors ,2. Most Since the 1980s, more than 450 chiral drugs have been developed from natural and synthetic products. Even though the proportion of synthetic drugs in the form of single isomers has increased over the past decades, mixtures of stereoisomers are found in the majority of therapeutic groups [The design of novel drugs based on nucleoside structure is one of the most fruitful areas of medicinal chemistry. Approximately 100 drugs were developed based on nucleosides, half of them being antiviral, and a quarter of them being antitumor drugs ,7,8,9. N50 values in the range 0.015–10 µM have been described [50 values in the submicromolar range have been found [O-benzoylpentafuranose residue is essential for Tdp1 inhibition [A DNA repair enzyme, tyrosyl-DNA phosphodiesterase 1 (Tdp1), represents an important target for the development of novel antitumor therapeutics based on various scaffolds ,16,17,18escribed ,29,30,31en found . It was hibition . This stO-benzoyl-protection of a nucleoside or a carbohydrate by the previously described procedures  was slightly higher than the corresponding L-isomer (2L). The same trend was observed for the pair 3D/3L. For the 4D/4L pair, the difference between the D- and L-isomers was one order of magnitude . Thus, the configuration at the C-2 carbon center of the carbohydrate moiety can affect the binding affinity and inhibition of the enzyme. The replacement of the acetoxy group (OAc) in position 1 of 2D and 2L with a uracil base did not lead to a significant difference in Tdp1 inhibition, though 3D appeared to be a slightly more potent inhibitor than 3L. The introduction of a thymine pyrimidine base (Thy) to the 2,3,5-tri-O-benzoyl-β-D/L-ribofuranose residue (4D and 4L) led to a significant difference in Tdp1 inhibition by the D- and L-isomers that was missing from the initial ribofuranoses, 2D and 2L. At the same time, the inhibition activity of 3D and 4D was comparable with the initial D-ribofuranose derivative 2D and D-arabinofuranose derivative 1D, respectively. The removal of the benzoyl groups from 3D and 3L nucleoside derivatives led to a significant decrease in Tdp1 inhibition by both isomers (IC50 > 50 μM for 8D and 8L). Inhibition by 1-thymine 4D (IC50 = 2.7 μM) was comparable with its 6 positional isomer 6-methyl-1-uracil 4′D (IC50 = 3.4 μM). Therefore, the presence of a methyl group in the heterocyclic moiety  slightly influences the activity of the D-ribofuranosyl derivatives and significantly influences the activity of the L-ribofuranosyl derivatives.According to the data given in C-methylnucleosides [C-methyl nucleosides on Tdp1 inhibition, 6-deoxy-D-allofuranosylnucleosides and 6-deoxy-L-talofuranosylnucleosides of both purine and pyrimidine were obtained. The 6-Deoxy-D-allofuranosylnucleosides correspond to 5′(R)-C-methylribonucleosides 5R-7R and the 6-deoxy-L-talofuranosylnucleosides correspond to 5′(S)-C-methylribonucleosides 5S-7S -C-methyl-2′,3′,5′-tri-O-benzoyl-N6-benzoyladenosine (7S) was comparable with the corresponding R-isomer 7R.According to the data given in 3D and 3L tend to bind to a cavity adjacent to the active site of Tdp1 .For the first time, we identified a binding cavity for nucleoside and carbohydrate series by superposition of their conformers complexed with Tdp1 . It was  of Tdp1 A. The puresidues , 7R.The presence of an inhibitor in the reaction mixture affects the kinetic parameters of the enzymatic reaction. The determination of the enzyme behavior in the presence of inhibitors and the estimation of the kinetic parameters for this process can reveal significant insight into the mechanisms of inhibition. Depending on the binding site, as well as what stage of the catalytic process it occurs, there are four main types of inhibition: competitive (the inhibitor competes with the substrate for binding at the active site), non-competitive , mixed , and uncompetitive (inhibitor binds to the enzyme-substrate complex). The type of inhibition is one of the important factors that determine the pharmacodynamic properties of the compound and, in turn, the possible side effects of therapy.3D, 3L, 7R, and 7S was determined by a steady-state kinetic assay. During the experiment, dependence curves of the enzymatic reaction rate (V) on substrate concentration (S) in the presence of different concentrations of the inhibitors were obtained (M) and the maximum reaction rate (Vmax) were calculated according to the Michaelis–Menten equation. Conclusions about the type of inhibition were made from the characteristics of the dependence of Vmax and KM on the concentration of the inhibitor.To confirm the interaction model obtained by molecular docking, the type of inhibition by compounds obtained . The app3D and 3L, both Vmax and KM values decreased as the concentration of the inhibitor increased, which is typical for an uncompetitive type of inhibition (3D and 3L bind to the enzyme near the DNA binding site (substrate).For compounds hibition . Such in7S, KM increased but Vmax remained nearly constant -configuration at the 5′ carbon atom of the ribofuranosyl moiety manifested slightly lower inhibition of Tdp1 than the 5′(S)-isomers (50 between the (R) and (S)-isomers . According to the calculations for the distribution coefficient (clogP) between 1-octanol-water phases carried out with Instant JChem software, the value of the Tdp1 inhibition effect for the D-nucleoside derivatives 1D-4D, 4′D and the 5′(R)-stereoisomers 5R-7R depends upon the lipophilicity associated with a benzoyl moiety as a part of the modified carbohydrate moiety . Isomeric pairs 2D/2L and 3D/3L were characterized by comparable ER values. Thus, the replacement of the α-methoxide group at position C-1 of the D-2,3,5-tri-O-benzoylarabinofuranosyl moiety with a uracil base at the β-configuration and simultaneous inversion of the configuration at position C-2 of the D-2,3,5-tri-O-benzoylarabinofuranosyl moiety with the formation of a D-2,3,5-tri-O-benzoylribofuranosyl moiety leads to comparable selectivity for the D- and L-derivatives. The lowest ER value (highest pER) was calculated for the thymine derivatives 4D and 4L  by a hydrophobic interaction of the phenyl residue from the 5′-O-benzoyl residue with Trp590 , and Tyr204 associates with the carbonyl group of 2′-O-benzoyl residue  that is a specific feature of the docking procedure. These conditions differ substantially from inhibition in an aqueous solution, where the interaction of 4D with the enzyme can be energetically preferable. For example, it may be proposed that the interaction of 4D with the enzyme strongly increases the entropy factor (ΔS) upon interaction in aqueous solution, and this lowers the system free energy leading to an increase in Tdp1 inhibition by 4D. Therefore, the docking method allows us to visualize ligand-enzyme interactions and evaluate the binding energy with a docking score index for ligand-enzyme interactions in a crystal structure, which is a potent instrument for various mechanistic studies. The docking score index can also generally indicate differences in inhibition by different stereoisomers in solution, but it cannot reliably evaluate these differences as it does not correlate well with IC50 values because of the complex intermolecular interactions in aqueous solutions, which are difficult to predict by computer simulations.The experimental results for the mechanism of action agreed with the molecular modeling data for intermolecular enzyme-ligand interactions. Compounds h Trp590 A,B. A hyd Cys205 B. The urd Tyr204 . The urae enzyme B. Compour Asp230 , Table 2 residue , Table 2C-methylribofuranosyl derivatives, compounds of this type occupied the active site, preventing the formation of the DNA-Tdp1 complex. The 5′ (S)-stereoisomer  7S forms three hydrogen bonds between benzoyl residues and amino acid residues: one between the 2′-O-carbonyl group and Thr466, another between the 5′-O-carbonyl group and Trp590, and the last between the N6-carbonyl group and Tyr204 (R)-stereoisomer  7R favored the other spatial occupation in the apo-form of Tdp1 due to the formation of four hydrogen bonds between benzoyl residues and amino acid residues: one between the 3′-O-carbonyl group and Ser462, two between the 2′-O-carbonyl and 5′-O-carbonyl groups and associated aqueous molecules, while the hydrogen bond between the N6-carbonyl group and Tyr204 remains unchanged (R)-stereoisomer  7R with Tdp1. When binding with the allosteric site of Tdp1 (Tdp1-DNA), 7R formed a hydrogen bond with Gly458, mediated by one aqueous molecule and two stacking interactions between Trp590, the phenyl ring of 5′-O-benzoyl residue, and the purine moiety (7S seems to be a more potent competitive inhibitor (docking score −2.506) than 7R (docking score −1.299), while 7R tends mostly towards uncompetitive inhibition by interacting with the allosteric site of Tdp1. The formation of several hydrogen bonds and two stacking interactions leads to the lowest docking score for 7R (−6.490) among the series of studied stereoisomers . Fully benzoylated purine 5′(S)-C-methylribonucleoside  (7S) tends mostly towards competitive inhibition, but the corresponding D-allo-stereoisomer (7R) can display mixed inhibition by several possibilities of binding, both to an allosteric site and the active center of Tdp1. These experimental results are in agreement with the computational data and indicate a cooperative role for the benzoyl groups and the nucleic base in different modes of interaction with the enzyme. A mode of interaction is defined by a structure of a carbohydrate moiety that participates as a core for spatial location of benzoyl residues and nucleic bases.The value of Tdp1 inhibition by lipophilic nucleoside derivatives increased with an increase in the quantity of ate core . Non-proate core , Table 21H and 13C (with complete proton decoupling) NMR spectra were recorded on a Bruker AMX 300 NMR instrument and are given in the 1H-NMR-spectra were recorded at 300 MHz and 13C-NMR-spectra at 75 MHz. Chemical shifts in ppm were measured relative to the residual solvent signals as internal standards . Spin-spin coupling constants (J) are given in Hz. Carbohydrate derivatives 1(D/L)-2(D/L) were obtained according to the procedures described in [3D, 8D) and D-ribothymidine derivatives (4D) were obtained according to the procedures described in [C-methylribonucleosides were obtained according to the procedures described in [®, Budapest, Hungary).The solvents and materials were reagent grade and were used without additional purification. Column chromatography was performed on silica gel , 0.063–0.200 mm). TLC was performed on a Alugram SIL G/UV254  with UV visualization. ribed in . D-Uridiribed in ,41,42. T50) were determined using a six-point concentration response curve in three independent experiments and calculated using MARS Data Analysis 2.0 .Real-time detection of Tdp1 activity was reported in our previous work . This apmax), Michaelis constant (KM), and possible inhibition mechanism, steady-state kinetic experiments were carried out at five fixed concentrations for the substrate with various inhibitor concentrations. The standard reaction mixtures (200 μL) contained 50 nM, 100 nM, 200 nM, 500 nM, or 1000 nM substrate, testable inhibitor, 1.5 nM recombinant human Tdp1, and reaction buffer components . After the addition of the enzyme, the reaction mixtures were incubated at a constant temperature of 26 °C and measured in a POLARstar OPTIMA fluorimeter  set to measure fluorescence every 55 s (Ex485/Em520 nm) during the linear phase (0 to 8 min). The initial data (kinetic curves) were obtained in three independent experiments and statistically processed with OriginPro 8.6.0 software.To determine the apparent maximum rate of enzymatic reaction . The structure of model B was prepared for docking using Schrödinger molecular modeling suite (version 2020-4) by Protein Preparation Wizard  and optimized by the Loop Refinement module in Prime. The quality of the structure was checked using the Protein Health tool and was confirmed by a free energy calculation with OPLS-3ext force field. This made this model appropriate for conducting molecular docking studies. Model A was obtained by the addition of a DNA molecule from crystal structure 5NW9  into the active center using a geometry superposition followed by energy minimization of the complex. The calculations were carried out using Schrodinger 2020-4 software. Molecular docking was performed using Glide, and the precision of docking was XP. Ligands were built in a 2D visualizer before modeling, and then optimized using ligand preparation with the generation of conformers. The LigPrep procedure was to keep the chirality as it was specified in 2D Sketcher. A maximum of 22 conformers per ligand was specified in the docking studies. The active site of the enzymatic target was identified, and a grid generation was carried out. The quantity of positions for each conformer when docking with Tdp1-DNA (model A) was 61 for 3D-3L, 4L, 7R and 60 for 4L. The quantity of positions for each conformer when docking with Tdp1 (model B) was 106 for 7R/7S. The binding energy of ligands was estimated by sorting the docking scores for each position of the conformers in complex with the enzyme. Binding propensity with the enzyme was estimated by sorting glide emodel and Glide score parameters.Two Tdp1 models were used in our docking studies, (A) and enzyme−substrate complex (Tdp1-DNA), and (B) an apo-form. Model B was based on the crystal structure 6MJ5 (resolution 1.85 Å) from the Protein Data Bank . For the series of synthesized compounds, O-benzoylated, D-nucleoside, and D-carbohydrate derivatives manifested greater Tdp1 inhibition than their L-analogs. It was shown that O-benzoylated D-lipophilic nucleoside derivatives manifested greater inhibition activity than their L-analogs, but no significant difference in Tdp1 inhibition was observed for the O-benzoylated 5′(R)-C-methylribonucleosides compared to the 5′(S)-forms.In conclusion, we have studied the inhibition of Tdp1, an important human DNA repair enzyme and a target of anticancer therapy, by different chiral lipophilic nucleoside derivatives and carbohydrate stereoisomers. New efficient Tdp1 inhibitors were found in a series of studied compounds with IC3D and 3L bind to the cavity adjacent to the Tdp1 active center, as confirmed by kinetic experiments. The purine 5′-C-methylribofuranose derivatives 7R and 7S bind to the active site for DNA, thus preventing the formation of the DNA-Tdp1 complex. Their ability to compete with DNA for the active site of the enzyme is also supported by kinetic data.According to the computer modeling data, nucleosides It can be concluded from the obtained results that the stereochemistry of the carbohydrate moiety determines the spatial location of the benzoyl groups and the heterocyclic base, which favors binding to different sites of Tdp1 and thereby affects a mechanism of Tdp1 inhibition.The structures of the described lipophilic nucleosides as Tdp1 inhibitors are protected by Russian Federation Patent RU№ 2748103 C1 (priority from 13.12.2019).


Ordered cell cycle progression is coordinated by cyclin dependent kinases (CDKs). CDKs often phosphorylate substrates at multiple sites clustered within disordered regions. However, for most substrates, it is not known which phosphosites are functionally important. We developed a high-throughput approach, Phosphosite Scanning, that tests the importance of each phosphosite within a multisite phosphorylated domain. We show that Phosphosite Scanning identifies multiple combinations of phosphosites that can regulate protein function and reveals specific phosphorylations that are required for phosphorylation at additional sites within a domain. We applied this approach to the yeast transcription factor Hcm1, a conserved regulator of mitotic genes that is critical for accurate chromosome segregation. Phosphosite Scanning revealed a complex CDK-regulatory circuit that mediates Cks1-dependent phosphorylation of key activating sites in vivo. These results illuminate the mechanism of Hcm1 activation by CDK and establish Phosphosite Scanning as a powerful tool for decoding multisite phosphorylated domains. Many kinases phosphorylate proteins on multiple sites, however in most cases it is not known which sites are functionally important. Here, the authors describe a high-throughput approach to quantitatively evaluate the contribution of each phosphosite within a multisite phosphorylated domain. This critical mechanism of cell cycle control is conserved from yeast to humans7. Phosphorylation of CDK substrates can trigger a variety of actions, including substrate degradation or activation, interactions of substrates with other proteins, and re-localization of substrates to different sub-cellular compartments. These changes have widespread implications in cell cycle control, including the regulation of key phase transitions and the expression of cyclically transcribed genes.Progression through the cell division cycle requires the coordinated function of hundreds of proteins. This coordination is achieved in part through the phosphorylation of numerous substrates by cyclin-dependent kinase (CDK) complexes10. However, a minimal consensus motif (S/T-P) is frequently phosphorylated in vivo. These consensus motifs often occur in clusters and are enriched in intrinsically disordered regions (IDRs)12. Despite these common features, the numbers and/or positions of phosphates required to elicit functional changes vary considerably among CDK substrates. Some substrates contain many CDK phosphorylation sites, but functional changes depend on the phosphorylation of only one or a few specific sites. Phosphorylation at a specific site can be required to trigger structural changes that impact protein function15, or to generate a linear binding epitope17. Conversely, in a different group of substrates, phosphorylation at many sites is required to reduce the overall charge of a domain and elicit functional changes18. For most CDK substrates, the CDK-regulatory circuitry remains poorly understood, including (1) the fraction of phosphosites required to regulate protein function and (2) which particular sites, among many that are phosphorylated by CDKs, have regulatory function.A common feature among CDK substrates is that they are often phosphorylated on multiple sites. CDKs are serine and threonine-directed kinases that efficiently phosphorylate an optimal consensus motif (S/T-P-x-K/R) in vitro22. Most deep mutational scanning approaches analyze all possible amino acid changes at each residue within a domain, which provides high-resolution information about protein structure. Here, we modified deep mutational scanning to focus specifically on individual phosphosites within a multiphosphorylated domain, and to determine how phosphorylation at each site impacts protein function in vivo. Our modified approach, called Phosphosite Scanning, uses bulk competition assays to measure the cellular fitness outcomes caused by the mutation of phosphorylation sites to unphosphorylatable or phosphomimetic residues, in all possible combinations. By comparing the fitness effects of proteins with different combinations of wild-type (WT), unphosphorylatable and phosphomimetic residues, it is possible to determine which phosphosites contribute to protein regulation, the combinations of phosphosites that are necessary and sufficient for normal activity, and how phosphorylation at a particular site impacts phosphorylation at additional sites within a domain.Determining how individual phosphosites contribute to protein regulation has remained a significant challenge because it requires generating large numbers of mutant proteins for any given substrate and evaluating the effects of each mutant one by one. Although it may be feasible to generate a series of mutants in which each phosphosite is mutated individually, this strategy only examines the importance of each phosphosite in an otherwise wild-type context, potentially missing effects observed when combinations of phosphosites are mutated. One streamlined approach that has been developed to systematically examine the effects of mutations on protein function in a pooled format is deep mutational scanning24. Notably, mutations in Hcm1 impact cellular fitness. Mutation of all CDK phosphoacceptor sites in Hcm1 to alanine inactivates the protein and reduces cellular fitness25. Conversely, mutation of all eight phosphoacceptor sites within the transactivation domain (TAD) to two glutamic acid residues—mimicking the charge of a phosphate—results in elevated activity and increased fitness compared to WT26. These phosphorylation-dependent fitness effects make the Hcm1 TAD an ideal model to investigate the mechanism of phosphoregulation by CDK.To examine how CDK can regulate protein function through multisite phosphorylation, we applied Phosphosite Scanning to the yeast forkhead family transcription factor Hcm1. Like its human homolog, FoxM1, Hcm1 activates expression of genes that are required for mitotic spindle function and accurate chromosome segregationPhosphosite Scanning of Hcm1 revealed several features of activation that highlight the complex regulation of multisite phosphorylated domains. Remarkably, we found that non-overlapping combinations of phosphomimetic mutations were able to activate Hcm1. Phosphomimetic mutations at two central phosphosites, T460 and S471, had the greatest impact on Hcm1 activity and, on their own, could restore WT activity when all other phosphosites were changed to alanine. However, phosphomimetic mutations at all sites except T460 and S471 led to similar activation, indicating a mechanism in which both the position and the number of CDK sites are important for activation. We also found that phosphorylation of three N-terminal threonine residues within the TAD  was necessary for the phosphorylation of T460 and S471. Our results suggest that CDK sites within the TAD are efficiently phosphorylated via the CDK phosphoadaptor subunit Cks1, which interacts specifically with phosphothreonines to promote increased phosphorylation. In sum, our results reveal the mechanism of Hcm1 activation by CDK and establish Phosphosite Scanning as a powerful approach that can be used to interrogate multisite phosphorylated domains.27 that is highly phosphorylated as cells enter S phase  and assayed the fitness of this strain compared to wild type (WT) in a co-culture assay. Low copy centromeric plasmids expressing WT HCM1 or hcm1-8A from its endogenous promoter were transformed into strains expressing either mutant (non-fluorescent) or WT GFP, respectively, and in which the galactose-inducible GAL1 promoter was integrated upstream of the genomic copy of HCM1. At the start of the assay, equivalent amounts of each strain were diluted into dextrose-containing medium to shut off the expression of endogenous HCM1  was previously shown to result in a gain of function allele that exhibited increased recruitment to target gene promoters and elevated expression of target genes25. This effect was observed when all eight S/T-Ps were replaced with E-Es, but not when S/T-Ps were mutated to E-Ps, suggesting that it is the charge of the phosphate that is important for Hcm1 activation. Interestingly, the phosphomimetic mutant, hcm1-8E, showed the opposite phenotype as the hcm1-8A mutant and conferred a fitness benefit relative to WT, similar to previous results (hcm1-8E was previously referred to as hcm1-16E26)  Fig. . We soug19 this method focuses on amino acid substitutions that mimic or prevent phosphorylation. The fitness of strains expressing phosphomutant proteins can be measured by monitoring the frequency of all mutants in the population over time using deep sequencing. By using fitness as a proxy for protein activity, the impact of phosphorylation at each site on Hcm1 activity can be quantified. Screens utilizing only unphosphorylatable and phosphomimetic mutations quantify the activity of proteins with a fixed phosphorylation state. In addition, the inclusion of WT sites along with phosphosite mutations allows us to infer which sites are phosphorylated in vivo (described below).We developed an approach, called Phosphosite Scanning, in which bulk fitness screens are used to interrogate all possible combinations of phosphorylated sites within a multiphosphorylated domain. In contrast to saturating mutagenesis approachesHCM1 promoter. Despite variation in codon usage, phosphosite mutations did not affect Hcm1 protein levels  but includes all eight C-terminal phosphosites of interest30. We fused WT or phosphomutant TAD regions to the C-terminus of the LexA-DBD and expressed these proteins in a reporter strain that contains eight LexA binding sites integrated upstream of the lacZ gene32. β-galactosidase activity assays confirmed that the WT Hcm1 TAD strongly activates transcription  restored low levels of activity, and phosphomimetic mutations at both key sites (AAAEEAAA) was sufficient to restore WT-like activity. In addition, the mutant that only lacks phosphomimetic mutations at sites T460 and S471 (EEEAAEEE) displayed activity similar to WT, consistent with its ability to restore near-WT fitness  showed intermediate sensitivity, correlating with the increased fitness imparted by T460 or S471 phosphomimetic mutations. Moreover, mimicking phosphorylation at both T460 and S471 (AAAEEAAA), or at all sites except T460 and S471 (EEEAAEEE), was sufficient to restore nocodazole sensitivity to WT levels. Together, these data demonstrate that the fitness phenotypes of Hcm1 unphosphorylatable and phosphomimetic mutants correlate with their cell cycle regulatory functions in vivo, and that both the position and number of phosphorylations contribute to Hcm1 activity.Next, we determined if the observed fitness phenotypes correlated with Hcm1 function in vivo. Hcm1 regulates several genes that are required for mitotic spindle function and as a result els Fig. . Doublinels Fig. . This reels Fig. . Althoug25. Blocking phosphorylation of these sites with alanine substitutions (hcm1-3N) stabilizes Hcm1 . We found that in the presence of stabilizing mutations, fitness also increased with the number of phosphomimetic mutations in the TAD  would allow us to infer which sites are phosphorylated in vivo.In the first screen, we constructed a library in which each phosphoacceptor site was mutated to alanine or left as the WT S-P or T-P site in all possible combinations  was sufficient to restore WT fitness  displayed the largest fitness increase. Again, if T460 and S471 are phosphorylated in vivo, we would expect that a mutant with WT sequence at these positions (EEEWWEEE) would display similar fitness and activity. However, we found that this mutant exhibited reduced fitness compared to hcm1-8E and was identical to the corresponding alanine mutant (EEEAAEE)  can also support transcriptional activity, since an increased amount of negative charge that is less optimally placed could be sufficient for cofactor recruitment. We anticipate that this mechanism of regulation may be common among multisite phosphorylated substrates: that regulation can occur via different combinations of phosphosites, but that evolution has selected for sequence contexts that promote rapid phosphorylation and switch-like regulation.Phosphorylation of the Hcm1 TAD activates the expression of Hcm1 target genes in vivo. Our data indicate that TAD phosphorylation stimulates transcriptional activation through a charge-based mechanism that is independent of DNA binding Fig. . A likelnce Fig. . The TADnce Fig. . However40.Historically, determining which phosphosites within a multiphosphorylated domain impact protein function has been challenging, since it requires making large numbers of mutant alleles and screening them individually. Phosphosite Scanning offers several benefits over traditional approaches. Since all possible combinations of alleles can be generated and screened in bulk, it is both comprehensive and rapid. Moreover, as is evident from our data, fitness readouts are very sensitive and can detect small differences in activity among mutants. The ideal substrate for this type of screen is a protein like Hcm1, in which eliminating and mimicking phosphorylation lead to opposing effects on protein function and cellular fitness. This approach can easily be applied to any phosphorylated protein that is regulated by multisite phosphorylation and demonstrates phosphorylation-dependent fitness effects. Phosphosite Scanning could also be adapted for substrates that do not meet these criteria. Instead of measuring cellular fitness as a readout, protein function could be measured in another way. For example, screens could be developed that utilize GFP as a readout, and mutants with different effects on activity determined by sorting cells based on GFP expression. This type of approach has been successful in combination with traditional deep mutational scanning18. However, there are instances when the addition of a phosphate group is required, and the effect cannot be replicated by charge alone. This is often the case when phosphorylation regulates a protein-protein interaction. For example, the degradation of many substrates of SCF ubiquitin ligases depends on substrate phosphorylation to form a phosphodegron motif that can interact with an F-box protein17. Since glutamic acid substitutions are structurally dissimilar to a phosphate, they are unable to form an interaction motif. Similarly, interaction between a CDK substrate and the processivity factor Cks1 depends on a phosphorylated threonine priming site within the substrate38. As our data shows, phosphomimetic substitutions at the most N-terminal threonine residues in the Hcm1 TAD prevented phosphorylation of more C-terminal WT sites, suggesting that they do not interact with Cks1. Despite these limitations, screens of phosphomimetic mutations can be informative for many targets. Alternatively, variations on Phosphosite Scanning that only interrogate unphosphorylatable substitutions could also be used to determine which sites are required for a functional output.One consideration when designing a Phosphosite Scanning screen is that the functional consequence of phosphorylation is not always replicated by phosphomimetic mutations. Substitution of phosphoacceptor sites with two glutamic acids replicates the change of charge caused by phosphorylation. This can mimic the outcome of phosphorylation when the change of charge of the substrate region is important for protein function3, and its phosphorylation leads to clear fitness phenotypes that correlate extremely well with protein function. However, the fact that cells expressing the fully phosphomimetic mutant (hcm1-8E) are more fit than WT cells suggests that not all sites are phosphorylated at any one time and raises the interesting question of why Hcm1 phosphoregulation has evolved. A likely possibility is that in some conditions having too much Hcm1 activity is detrimental, so it is beneficial to keep its levels low and rapidly toggle activity on and off via phosphorylation, as needed. In support of this model, activating phosphates in the TAD are specifically removed by the phosphatase calcineurin in response to stress26. We show here that phosphorylation-mediated degradation is important to sensitize Hcm1 to changes in TAD phosphorylation, since phosphomimetic mutations in the TAD do not provide the same fitness advantage when N-terminal phosphosites are mutated and the protein is stabilized  or synthetic medium lacking a single amino acid with 2% dextrose or galactose, where indicated, at 30 °C. Strains with HCM1 mutations integrated into the genome were generated from synthesized gene fragments (Invitrogen) that were cloned into pFA6a-3V5-KanMX6 using the NEBuilder HiFi DNA Assembly kit (New England Biolabs). All genomic mutations were confirmed by sequencing. GAL1p-HCM1 was introduced into strains for pairwise competition assays by genetic cross with previously characterized strains expressing WT or non-fluorescent GFP43.All strains are described in Supplementary Table HCM1 expression plasmids, the HCM1 open reading frame and endogenous promoter were amplified from the genome and cloned into pRS316. Plasmids for β-galactosidase transcriptional activation assays were generated using the pBTM116 backbone. The mutant or WT HCM1 TAD region (amino acids 306-511) was amplified by PCR from genomic DNA. The C-terminal 3V5 tag was PCR amplified from the pFA6a-3V5-KanMX6 vector. Both products were purified and cloned pBTM116 using the NEBuilder HiFi DNA assembly kit (New England Biolabs). All plasmids were verified by sequencing. Phosphomutant libraries used for Phosphosite Scanning screens were cloned into a pRS316 vector containing the endogenous HCM1 promoter upstream of the HCM1 open reading frame (WT or hcm1-3N) in which the region to be mutated had been deleted and replaced by an SphI restriction enzyme site (pRS316-HCM1*SphI or pRS316-hcm1-3N*SphI), to facilitate HiFi DNA assembly. All strains and plasmids generated during this study are available from the corresponding author upon reasonable request.All plasmids are listed in Supplementary Table GAL1p-HCM1 strains expressing WT GFP or non-fluorescent GFP-Y66F were transformed with pRS316 plasmids expressing the indicated HCM1 alleles and cultured in synthetic media lacking uracil with 2% galactose. Logarithmic phase cells were mixed in equal proportions by diluting 1 optical density (OD600) of each strain into 10 mL media with galactose. In total, 0.15 optical densities of the mixed culture were then collected, pelleted by centrifugation, resuspended in 2 mL sodium citrate buffer  and stored at 4 °C, for analysis by flow cytometry at the conclusion of the experiment. Mixed cultures were diluted to 0.006 optical densities in synthetic media lacking uracil with 2% dextrose and allowed to grow for 12 h, during which time the cultures did not reach saturation. Cultures were diluted in this manner every 12 h for a total of 72 h. At each timepoint, 0.15 optical densities were collected and resuspended in 2 mL sodium citrate buffer and stored at 4 °C for subsequent analysis. At the conclusion of the time course, the percentage of each strain in each sample were determined by quantifying the percentage of GFP-positive cells with a Guava EasyCyte HT flow cytometer and GuavaSoft software. In total, 5000 cells were measured in each sample. Data were analyzed with FlowJo software. N = 3 technical replicates were performed in each time course and percentages at each timepoint were averaged together. The data presented is an average of n = 3 biological replicates.For pairwise co-culture assays, g for 10 min at 4 °C. The supernatant was removed by aspiration and pellets were resuspended in 75 µL resuspension solution . Samples were then incubated at 95 °C for 5 min, then allowed to cool to room temperature. Lysates were then clarified by centrifugation at 16,000×g for 30 sec. The supernatant was collected and added to 25 µL 4× SDS-PAGE sample buffer  and heated to 95 °C for 5 min. Samples were then stored at −80 °C.Cell pellets  were collected for western blotting. Cells were lysed by incubation with 300 µL cold TCA buffer  for 10 min on ice. Lysates were then vortexed and pelleted by centrifugation at 16,000×Standard resolving gels contain 10% acrylamide/bis solution 37.5:1, 0.375 M Tris pH 8.8, 0.1% SDS, 0.1% ammonium persulfate (APS), 0.04% tetremethylethylenediamine (TEMED). Phos-tag gels contain 6% or 8% acrylamide/bis solution 29:1, 386 mM Tris pH 8.8, 0.1% SDS, 0.2% APS, 25 µM Phos-tag acrylamide (Wako), 50 µM manganese chloride and 0.17% TEMED. All stacking gels contain 5% acrylamide/bis solution 37.5:1, 126 mM Tris pH 6.8, 0.1% SDS, 0.1% APS and 0.1% TEMED. Gels were run in 1× running buffer  at 140 V (standard) or 150 V (Phos-tag) for two hours. Prior to transferring to nitrocellulose, Phos-tag gels were washed twice with 100 mL 1× transfer buffer with 10 mM EDTA for 15 min, and once with 1× transfer buffer for 10 min. Protein was transferred to nitrocellulose in cold 1× transfer buffer  at 0.45 A for 2 h. Nitrocellulose was blocked in 4% milk for 30 min. Proteins were visualized using antibodies that recognize PSTAIRE , V5 , and HA . The specificity of antibodies against V5 and HA epitope tags were validated by comparing lysates from yeast strains with each tag to a yeast strain lacking the tags as a negative control. Note that molecular weights are not shown alongside Phos-tag gels since they do not accurately reflect the molecular weights of the proteins.E. coli (High Efficiency) (New England Biolabs). All plasmid libraries were constructed in triplicate and mixed prior to transformation to maximize coverage of phosphomutants and avoid bias in oligo mixture or annealing steps.Phosphomutant plasmids were generated by annealing and extending overlapping mutagenic oligonucleotides spanning a 270 base pair region that encompasses all phosphosites, as detailed in Supplementary Fig. GAL1p-HCM1 strain grown in YM-1 containing 2% galactose, to maintain the expression of endogenous HCM1. For each screen, wild-type or Hcm1-3N expressing plasmids were added to each library as a control. Transformed cells were grown overnight in 5 mL C-Ura with 2% galactose at 23 °C. After approximately 16 h an aliquot of cells was removed and plated on C-Ura to confirm sufficient transformation efficiency (10X library coverage). The remaining cells were pelleted and washed five times with 15 mL C-Ura with 2% galactose to remove any untransformed plasmid and resuspended in 50 mL C-Ura with 2% galactose and allowed to grow to log phase (~48 h) at 30 °C. Cells were then diluted to an optical density of 0.008 in 75 mL C-Ura with 2% galactose and grown overnight at 30 °C. At the start of the experiment, the population was sampled to determine the representation of each plasmid in the population before selection. Twenty optical densities were collected, frozen on dry ice and stored at −80 °C for subsequent recovery of plasmids and preparation of sequencing libraries. One optical density of cells was harvested for western blotting to confirm levels of plasmid and endogenous Hcm1. Cells were then diluted to an optical density of 0.008–0.016 into C-Ura with 2% dextrose to repress expression of endogenous HCM1 and allowed to grow for 12 h at 30 °C to mid-logarithmic phase. The population was sampled as above and diluted into C-Ura with 2% dextrose every 12 h for a total of 72 h.Plasmid libraries that were constructed in triplicate were mixed in equal proportion for transformation into cells. Plasmid libraries were transformed into a hcm1 sequence was amplified by PCR for 21 cycles with vector-specific primers using Phusion High-Fidelity DNA polymerase (New England Biolabs). Products were extracted from a 1% agarose gel using the QIAquick Gel Extraction Kit (Qiagen). TruSeq sequencing adapters were added to mutant fragments by PCR using HCM1 specific primers fused to the TruSeq universal adapter or unique TruSeq indexed adapters. Sequences of oligonucleotides used for library construction are included in Supplementary Table Plasmids were recovered from frozen cell pellets using the YeaStar Genomic DNA Kit (Zymo Research). Plasmid HCM1 alleles were counted for each paired-end (PE) sequencing fragment that had an exact sequence match in both reads, using a custom python script. Count tables for all screens are included in Supplementary Data hcm1-3N for the 3N A/E screen). Selection coefficients for all screens are included in Supplementary Data boxplot in R. In all cases, values from all three replicates of each screen are included in the medians. Bar graphs comparing selection coefficients of select mutants show average values from replicate screens.Cells were grown to mid-logarithmic phase, then diluted to 0.1 optical densities in YM-1 with or without 5 µg/mL nocodazole. Cells were transferred to 96 well plates in triplicate and grown at 30 °C with shaking in a Tecan Infinite M Nano plate reader. Optical densities at 600 nm were measured every 20 min for a total of 33 h. Doubling times were quantified by fitting data points between 0.2 and 0.5 optical densities to an exponential growth equation using GraphPad Prism software. Multiple isolates were tested for each genotype listed.lacZ reporter and carrying plasmids expressing LexA-DBD-Hcm1 fusion proteins were grown in synthetic media lacking tryptophan. For each strain, the optical density at 600 nm was measured and 1 mL of cells were collected. Cells were pelleted, and the media was removed. Cells were then resuspended in 0.5 mL Z-buffer  and permeabilized through the addition 50 µL chloroform and 10 µL 0.4% SDS followed by vortexing. 300 µL Z-buffer containing 2.4 mg/mL o-nitrophenyl-β-d-galactopyranoside and 6 µL/mL β-mercaptoethanol was then added and the time recorded. Reactions were mixed, moved to 30 °C and monitored for a colorimetric change for a maximum of 120 min. Reactions were stopped either when a yellow color change was observed or after 120 min. To stop reactions, 0.5 mL 1 M Na2CO3 was added with vortexing and the time recorded. Completed reactions were stored on ice until all were finished. Chloroform was removed by centrifugation and the optical density of 1 mL of aqueous supernatant was measured at 405 nm. β-galactosidase units were calculated as (1000*OD405)/(OD600*1 mL*reaction time [min]). All reactions were carried out in triplicate and averaged together. Average values from biological replicates were then averaged together and plotted where indicated.Cells with an integrated Cells were grown to mid-log phase and arrested in G1 phase with 10 µg/mL alpha-factor and released into media without alpha-factor to resume cell cycle progression. Every 15 min, samples were collected for Western blotting and flow cytometry to confirm cell cycle position. Alpha-factor was added back to the synchronized population 45 min after release to prevent cells from entering a second cycle. Cells  were collected at the indicated timepoints, fixed in 70% ethanol, and stored at 4 °C for flow cytometry. Fixed cells were sonicated, treated with 0.25 mg/mL RNase A at 50 °C for an hour, then subsequently treated with 0.125 mg/mL Proteinase K at 50 °C for one hour. Cells were then labeled with 1 µM Sytox Green (Invitrogen). Flow cytometry data were collected using a Millipore Guava easyCyte 5HT with GuavaSoft (v3.3) software. In total, 5000 cells were measured at each timepoint. Data were analyzed using FlowJo software (v10.8.1).Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Reporting Summary