N = 195 households) with extensive qualitative data from focus group discussions (N = 55 participants). Results document limited awareness of AMR but also find that knowledge and attitudes are not correlated with practise. Thematic analysis of qualitative data pointed to three reasons behind this disconnect, including (1) Maasai self-perceptions as veterinary experts, (2) the central role of livestock in Maasai culture, and (3) the use of ethnoveterinary knowledge in animal health treatment. We argue that mixed-method approaches will be critical to developing the targeted awareness campaigns needed to limit the emergence and transmission of AMR.Global, national, and local efforts to limit antimicrobial resistance (AMR) often stress the importance of raising awareness among users, sellers, and prescribers of antimicrobial drugs. This emphasis is founded upon two assumptions. First, awareness is limited, particularly concerning the links between antimicrobial use (AMU) and AMR. Second, “filling the awareness gaps” will motivate practises that will limit AMR. The first assumption is supported by knowledge, attitudes, and practises (KAP) surveys but these same studies provide mixed support for the second, with several studies finding that knowledge and attitudes are not correlated with related practises. This disconnect may arise as these surveys typically do not collect data on the cultural or historical contexts that pattern AMU. To explore how these contexts impact KAP related to AMU and AMR, we use a mixed-methods approach to examine veterinary practises among Maasai pastoralists in Tanzania. We combine a quantitative KAP survey ( The health and economic threat posed by antimicrobial resistance (AMR) globally has motivated international agencies, including the WHO and the Food and Agriculture of the United Nations (FAO) to develop and implement global action plans and strategies to address AMR –3. TheseThe assumptions supporting AMR awareness efforts can be assessed through results of knowledge, attitudes, and practises (KAP) surveys –13. KAP Results of KAP studies provide support for the first assumption behind awareness-raising campaigns—that gaps exist in awareness and knowledge of AMR and prudent AMU—but provide more mixed support for the second assumption—that increases in awareness will promote behaviour change. In terms of the former, KAP surveys across public health and agricultural sectors find that awareness of AMR is often limited and non-prudent practises common, particularly in LMICs where antimicrobials are often bought over the counter without a prescription –13. HoweAlthough limited in number, results of KAP studies suggest that awareness is not the primary driver of practises related to AMU and AMR. This disconnect between awareness and behaviour has been well-documented in areas across public health and is driven by factors that lie both within and outside the individual , 17. Psybomas (enkang in Maa) organised along patrilineal lines. Polygamy remains common, especially in rural areas, and marriages are structured by patrilocal residence and clan exogamy. Bride price, usually paid in livestock, is still required. Livestock is grazed over a large area during the daytime and brought back home in the evening where they are kept inside the boma with smallstock (sheep and goats) and large stock (cattle) kept separately. Livestock remains important for the Maasai, both economically and culturally, and livestock care begins at a young age within Maasai communities. Children begin tending livestock as early as 5 or 6 years, initially by tending kids/calves, which stay around the boma. Boys progress to herding adult goats, sheep, and cattle as they age and become moran (warriors)  –28 whilearriors) –31.bomas. Given we used purposive sampling, KAP results should not be considered representative of the Maasai generally or Maasai living within the four wards. See The mixed-methods study, informed by the exploratory sequential study design , was carQualitative data were collected on themes related to AMU and AMR including farm management and economic practises, livestock disease histories, and KAP relating to AMU and AMR, including use, governance, regulations, policies, and enforcement. FGDs were conducted with livestock keepers, who were mostly heads of households, both male and female. See Kiswahili and Maa . After transcription into text, documents were translated to English following a meaning-based translation approach  in Tanzania and certificate clearance no. NIMR/HQ/R.8a/Vol.IX/2926 was issued. Prior to requesting for consent, an information sheet containing a detailed narrative of the study and its aims was provided to potential participants. Participants were informed of the benefits and risks of participation and assured of their right to withdraw from study participation at any point. Written informed consent was sought from all study participants who could write. For those who could not write, a thumbprint signature was requested.A total of six FGDs were held with 55 Maasai participants. A slight majority of participants were 33 males (60%) vs. 22 (40%) females. Most participants were married 47 (84%) with a majority (53%) falling into the 41–80 age bracket. About half of participants had no education 27 (48%) with fewer having a primary education 25 (45%) and only three participants (5%) having a secondary education. See A total of 195 Maasai were interviewed for the KAP survey. Most participants were male 182 (93%), with a majority 120 (62%) having no education, 58 (30%) having some/completed primary, 13 (7%) some/completed secondary, and 4 (2%) some/completed college. The average household was composed of 8–9 people and owned an average of 146 cattle and 230 small stock (sheep and goats), three donkeys, and three chickens. Households cultivated an average of 18 hectares of farmed land. Twelve percent  said they stop treatment when the animal looks better. Even fewer responded that they consult an animal health professional 19%  or the bp = 0.012) to not use the information on bottle/sachet labels or advice from animal health professionals for administration instructions (79.67%) compared to those who did use this information and advice (62.50%). Likewise, those who agreed using antimicrobials inappropriately could limit effectiveness were more likely to report treating an entire group when one animal falls ill (94.04%) compared to those who never or rarely reported engaging in this practise (63.36%). More consistent with expectations was that respondents who knew about AMR were significantly less likely (p = 0.009) to underdose (68%) compared to those who did not know about AMR (43%). Likewise, those who knew about withdrawal were significantly more likely (p = 0.00001) to report observing withdrawal.Fisher's exact tests showed that most AMU and AMR-related practises were not significantly associated with related knowledge or agreement measures  responded that they use antimicrobials as growth promoters, which contrasts with other studies on pastoralists  who reported to “sometimes” or “always” get prescriptions for antimicrobials. Prescriptions may be perceived as an unnecessary cost if information from the veterinarian is not perceived as more correct.Interestingly, we found that those Maasai who agreed that medicines used inappropriately limited drug effectiveness were Finally, the central role of livestock in the Maasai culture served as a backdrop for rationalising their animal health practises. Livestock, particularly cattle, are perceived as gifts from God and, as such, are bestowed with a sense of purity. Wasting the products provided by these gifts (meat and milk) violates social norms among the Maasai –43. GiveThis mixed-methods study provides important insights into future investigations of AMU and AMR-related KAP as well as efforts to design AMR awareness campaigns upon these investigations. First, survey instruments that quantify knowledge and attitudes solely based upon “Western” biomedical understandings of antimicrobials and AMR are unlikely to identify the myriad of social, cultural, and historical reasons patterning AMU practises. As this study demonstrates, some of these reasons, including the role of livestock in cultural continuity, are likely to account for a considerable amount of behavioural variance within a community. These reasons, as our results suggest, may supersede any knowledge of the biomedical realities of AMR. Even when this biomedical knowledge is significantly related practises, as we documented with knowledge of withdrawal, the impact on behaviour may be limited. For example, only about 26% of Maasai who reported knowing about withdrawal also reported observing withdrawal. Acknowledging these discrepancies is vitally important in efforts to limit AMR as awareness campaigns and other behavioural change interventions are often based on the KAP study results. These interventions, by not assessing the broader structural issues underlying animal health practises, risk creating better-informed communities that are still engaging in the same non-prudent antimicrobial practises.The original contributions presented in the study are included in the article/The study was reviewed and approved by Medical Research Coordinating Committee of the National Institute for Medical Research (NIMR) in Tanzania (clearance no. NIMR/HQ/R.8a/Vol.IX/2926). Written informed consent was not provided because illiteracy among the Maasai community means written consent could not be secured from all participants. If written consent could be not secured, a thumbprint was secured.PM, MC, EM, MO-N, AD-G, and EK conceived the study and developed the methodology. PM, MC, EM, and MO-N conducted the fieldwork. PM and MC conducted formal analysis. PM and MC prepared the original draught. EM, MO-N, TK, AD-G, and EM reviewed and edited the manuscript. All authors have read and agreed to the published version of the manuscript.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.


Doctor of Pharmacy (PharmD) programs continually engage in curricular redesign to ensure practice readiness of graduates. With ever-increasing demands on clinical competency and curricular time, it is important to be intentional when determining curricular priorities and prioritize contemporary pharmacist practice. This paper describes how to adapt a national framework for pharmacotherapy curricula to emphasize the pharmacist’s role within a given topic area in order to facilitate conversations about allotting curricular time during a curricular redesign. Customized Tier descriptions based on various factors expected of student pharmacists during Advanced Pharmacy Practice Experiences (APPEs) were developed . Topics were also reprioritized to address regional variations in practice and ideologies. Customizing a national framework to determine program-specific considerations for prioritizing topics within the pre-APPE curriculum can help faculty and students alike maintain focus on highly critical and foundational concepts, while also making sure not to completely disregard topics of lower priority. We have proposed such a framework for programs to utilize when facilitating conversations surrounding curricular reforms and topic prioritization. A multitude of Doctor of Pharmacy (PharmD) programs across the United States are engaging in curricular redesigns as a result of several factors, including advancements in pharmaceutical research and development, increased reliance on medications for managing chronic conditions, continual evolution of pharmacy practice standards and scope of practice, and the corresponding implementation of new accreditation standards from the Accreditation Council for Pharmacy Education (ACPE) in 2016, among numerous other factors ,4,5. TheThe existing literature provides several examples of strategies to evaluate curricular content and make critical decisions regarding content prioritization. Within pharmacy education, Longyhore, Dixon and Noel propose using a modified Ebel grid for setting curricular boundaries in pharmacy school by evaluating topics on a scale from “need to know” to “nice to know”, taking into consideration a topic’s criticality and relevance . Outsidehttps://www.accp.com/docs/positions/Other_Documents_of_Interest/Flannery_jac5.1178.pdf (Accessed on 21 April 2021).Whenever making decisions about the curriculum, either during a complete redesign or a routine self-assessment for quality improvement, it is helpful to have external references to provide a “standard” against which to benchmark. The ACPE Standards provide that resource for the overall PharmD, while also affording programs a certain degree of flexibility to meet the standards . To provAlthough the design of the toolkit is helpful for discerning between high and low priority topics to be emphasized within PharmD curricula, subsequent application to inform the appropriate breadth and depth of content coverage necessary to achieve clinical competence for each individual topic is difficult. This is especially true for programs that integrate the foundational and clinical sciences throughout their coursework—how might these tier descriptions help inform faculty within the pharmaceutical sciences? A modified Ebel grid as proposed by Longyhore and colleagues can help, but incorporating an additional level of detail beyond what the ACCP Pharmacotherapy Toolkit provides can ensure these decisions are more consistently applied across the wide range of topics . The aimPacific University School of Pharmacy (SoP), a 3-year PharmD program, initiated the process of revising the PharmD curriculum in 2017 for implementation in Fall 2021, partly in response to the dramatic changes in contemporary pharmacy practice since the curriculum was originally adopted in 2006. The most significant change includes a shift from a modified 2-week block curriculum to an integrated, semester-based one. This shift towards integrated coursework is helping the faculty break through the silos that traditionally exist in pharmacy curricula, especially those related to organ system/specialty area and discipline. The steering group responsible for overseeing the curricular revision consists of eleven faculty and staff members (which includes representatives from the curriculum committee): two co-chairs, four pharmacy practice representatives, three pharmaceutical science representatives, the Director of Curricular Operations, and the Assistant Dean for Educational Outcomes.The ACCP Pharmacotherapy Didactic Curriculum Toolkit was certainly helpful for framing the initial conversations during the process by serving as a national standard for what to prioritize and provided a framework to consider when prioritizing topics. Although the methods regarding topic prioritization were appropriate, institution-specific customization was warranted to account for state/region-specific scope of practice expectations, local prevalence of certain conditions, and populations served at available experiential settings for students, amongst other factors. Therefore, a small number of topics were reprioritized based on faculty feedback to accommodate these variations. During these conversations, it was generally understood by all that Tier 1 topics are high priority and Tier 3 topics likely would not be covered in our curriculum, but the 133 topics designated as Tier 2 were more difficult to conceptualize because they fell somewhere in between the two extremes. In addition, it was difficult for many to conceptualize how each topic’s prioritization translated into teaching time and content coverage, recommendations that the ACCP Toolkit intentionally avoided by design. With definitions geared towards practice expectations after graduation, an adaptation of the framework was needed.The importance of developing clear guidance to faculty for what each final Tier placement would mean for individual topics was identified to be essential. The most common assumption was that higher priority topics automatically warranted more time, which was not inherently true. A topic of higher priority may actually need less time to achieve the desired level of competence, while a topic of lower priority may actually take more time, even if the expected level of competence may be lesser. Therefore, the steering group brainstormed the various factors, including the knowledge, skills, and abilities expected of student pharmacists during Advanced Pharmacy Practice Experiences (APPEs) through the lens of the various topics/conditions within each tier. Factors that were identified during the initial brainstorm include the relative autonomy of the pharmacist in managing the topic/condition, the emphasis of the topic/condition on licensing exams, the frequency with which students can expect to encounter the topic/condition at our school-specific experiential placements, and condition-specific information including treatment guidelines, goals of therapy, and medication-specific knowledge . The steering group used these criteria to develop program-specific descriptions for each tier.To address the need for better differentiation between the 133 different Tier 2 topics, each member of the steering group was invited to participate in an anonymous survey to re-prioritize each topic with three options for each Tier 2 topic—“Higher priority (Tier 2A)”, “Lower Priority (Tier 2B)”, and “Not a priority (move to Tier 3)”. After approximately one week, results were tabulated. Items with ≥50% agreement for topic prioritization were considered as having achieved consensus and were declared final. Items with 50% agreement for multiple tiers or not achieving 50% agreement were reconciled through discussion among the steering group and group consensus. A similar process was utilized for differentiating between the Tier 1 topics to further stratify topics (if necessary), and although Tier 1A topics were identified as having the highest priority, this level of stratification did not result in any meaningful degree of differentiation in criteria for the purposes of the curricular redesign.Lastly, the original ACCP Toolkit is designed around a combination of major organ systems , specialty practice areas , and individual populations . Although this approach works well when thinking as a clinical practitioner, it does not necessarily work as well when thinking about a student pharmacist learning about how to use these medications for the first time . For example, although “psoriasis” is appropriately categorized as a dermatologic condition and “rheumatoid arthritis” is appropriately categorized as a musculoskeletal condition, the treatments are similar so these topics make sense to be co-located near each other.Once the program-specific tier descriptions were developed and all topics were prioritized by the steering group, the final results were disseminated to the entire faculty and staff for a two-week comment period. Comments related to the program-specific tier descriptions, the prioritization of individual topics, and the categorization of individual topics were solicited via email and a series of five one-hour town hall forums. All comments received were subsequently considered by the steering group and addressed as appropriate prior to declaring the documents final.The final program-specific descriptions for each Tier are provided in Over the course of the two-week commentary period, the steering group received 44 unique comments on topic prioritization , an additional 59 unique comments related to topic categorization , and no comments or suggestions for the program-specific Tier descriptions. All comments were reconciled by the steering group with a response formulated for each comment, regardless of outcome (accepted vs. rejected). The original ACCP Pharmacotherapy Toolkit identified 94 Tier 1, 133 Tier 2, and 75 Tier 3 topics . The finOften, in an attempt to have a comprehensive curriculum that attempts to emphasize all topics, students risk becoming overloaded and unable to apply the material within a broader clinical context. Rather, the end result is a brain full of unintegrated knowledge and facts. In order to ensure clinical competency, a methodical approach to topic prioritization is necessary to help enhance student pharmacists’ clinical competency upon graduation.Applying the concept of “understanding by design” to this context, the most logical consideration when prioritizing a topic is its prevalence within the scope of practice of the generalist pharmacist . The lenAnother area that provides guidance is the prevalence of a topic in experiential education. As the experiential placements for each PharmD program are highly varied, it is important to take this into consideration when designing the pre-APPE curriculum. What types of practice settings and patient populations will students encounter during this highly formative portion of the curriculum? As an example, if all students are required to complete some or all of their experiential education within a specific health system, it makes sense to prioritize the medical conditions encountered there and emphasize topic areas within their specific pharmacist scope of practice to best prepare the students to be ready to apply their knowledge and skills in an authentic practice setting. Similarly, focusing on topics frequently encountered on national licensing exams can be used as another source of validation for a topic’s prioritization.Lastly, the various Tiers were differentiated by the breadth and depth of information necessary to cover within the curriculum. This is one of the most valuable steps since it speaks on a more granular level than the prior factors, which focused on a topic’s presence or absence in various places . When decisions need to be made about allocating time within the curriculum for both the foundational and clinical sciences, these are the factors that can inform those decisions. From the perspective of managing a specific condition, familiarity with clinical guidelines and goals of therapy are useful measures to apply—the higher priority topics simply require a stronger working knowledge of these items while topics of a lower priority more appropriately warrant awareness or ability to search for this information. It is important to emphasize that guidelines do change; some more often than others. Memorization of a guideline and/or goals of therapy will only serve students well in the short term, while more extensive training about how guidelines are made, how to critically evaluate the recommendations, and how to stay up to date with changes to the guidelines are also critical for assuring clinical competence. Therefore, although one component for the definition of each Tier 1 topic is “recall specific goals of therapy from memory”, it must be emphasized that additional coursework has been added to the curriculum in order to provide this more extensive training outside of the “pharmacotherapy curriculum”.From the perspective of covering medication-specific information within the curriculum, the depth of content was based on place in therapy. Tier 1 topics require an emphasis on common first- and second-line agents, Tier 2A topics only require an emphasis on common first-line agents, and Tier 2B topics require a focus on names, clinical pearls, and major safety concerns. The Tier 1 and 2A distinctions focus on clinical management of the conditions, while the Tier 2B distinction was focused on individual medications for topics that may not otherwise be covered rather than clinical management of each topic. This was an important element to emphasize as medications that fall in this category may be those that are less commonly seen in practice or managed by pharmacists, but still come with significant risk of adverse effects, drug interactions, or toxicity that a pharmacist would be expected to know.Although there is no significant role for most Tier 3 topics within the PharmD curriculum due to space constraints, these may be excellent topics that can be sprinkled into the curriculum, especially if the topic warrants a modification to the traditional management approach of a topic/condition within a higher Tier. For example, a student may not need to know how to actively manage or otherwise treat nephrolithiasis, but relevant clinical pearls for nephrolithiasis may be introduced briefly when deciding between medication options for another medical condition, and a history of nephrolithiasis may be the deciding factor for selecting one agent over another. In addition, there were a few Tier 3 topics that were deemed particularly pertinent to clinical practice in the Northwest region of the United States, which were allocated specific curricular time . Some Tier 3 topics will be incorporated into clinical cases as opportunities to apply core concepts and problem-solving during recitation.Creating descriptions for the revised Tier structure was something the steering group originally tried to avoid doing, as there was concern that it would get faculty bogged down in too much detail and specificity. However, these descriptions have been extremely helpful for framing conversations about topic prioritization and allocating curricular time. Rather than focus on topic “importance”, these descriptions helped faculty keep their focus on identifying how much time and energy it would take to make a student proficient in a specific topic, how valuable that information will be to practice as an entry-level pharmacist, and whether or not that time is worthwhile relative to all the other curricular needs/demands. This was especially true for faculty who have a strong connection to a specific topic area and may have felt slighted by having their clinical area perceived as “less important”. Similarly, these definitions have also been helpful for faculty in the pharmaceutical sciences by providing application guidance for their content to prioritize the most relevant content for maximal learning, thereby enhancing clinical competence.The process of recategorizing individual topics was also an extremely important step in this curricular design. Therefore, the comment period was essential for allowing faculty to identify topics that needed to be moved for a variety of reasons. For example, certain topics within the OB/GYN category were more appropriately covered in closer proximity to other topics . Similarly, other topics outside of the OB/GYN category had correlations to topics within the category . Therefore, the OB/GYN category was broadened into a more inclusive “Sexual and Reproductive Health” category. Beyond identifying where topics are placed within the curriculum and how much time to dedicate to each, this schematic also informs the development of classroom exercises that intentionally integrate content from across the curriculum rather than operating within the silo of a specific disease state or topic area, especially if the focus is on helping assure clinical competence of students for generalist-pharmacist focus.As previously mentioned, the memorization of drug facts and current guidelines will not assist in assuring clinical competence of current student pharmacists. Although the focus of this manuscript has been on prioritizing topics more traditionally characterized as the pharmacotherapy curriculum, it is important to note that additional elements have been implemented within our revised curriculum to support students’ development of their clinical competence. The redesigned, integrated, semester-based curriculum includes several course streams. These streams will support the scaffolding of topics across courses and longitudinally throughout the didactic curriculum. Course stream directors will be responsible for continually ensuring faculty adherence to the topic prioritization schematic when allocating content time, reviewing assessment items, and determining course policies each academic year.The major limitation of this work is the institution-specific nature of the end results . Although the end results may differ by institution, our intention in presenting this work is to share the process and concepts utilized to inform our curricular topic prioritization. Because the curriculum has not officially launched, data evaluating the effectiveness of these methods are not yet available. However, each curriculum needs to start somewhere, and undergoing this process has helped provide a solid foundation with core philosophies that can be applied to future iterations when necessary.Several areas in need of further research have been identified during this curricular redesign process. Students will be provided with information about the curricular Tier levels and associated program-specific Tier descriptions. The team intends to evaluate whether providing this level of curricular information to students translates into enhancing their ability to better allocate study time and energy—and in turn, does this level of curricular focus help students become more APPE ready, practice ready, or even clinically competent?The need for a corollary foundational science tier-system toolkit has also been identified, which might, perhaps, form the basis for national discussion and adoption. Furthermore, the intention to include some of the Tier 3 topics into the recitation courses, designed for integration and application of knowledge, provides an opportunity to study this approach as curriculum expanding without reducing the time allocated to priority core topics. The effectiveness of using Tier 3 topics as opportunities for foundational principle review and critical thinking also warrants research. Additionally, the role of elective coursework for covering niche practice areas and/or emerging therapy, and how to best incorporate this alongside a core-curriculum deserves investigation.With ever-increasing demands on clinical competency, it is important to be intentional when determining curricular priorities. As pharmacists continue to increase their autonomy and scopes of practice within the healthcare arena, they are expected to acquire more knowledge, and there is the temptation to continually add to the curriculum without critically analyzing curricular content. Customizing a national framework to determine program-specific considerations for prioritizing topics within the pre-APPE curriculum can help faculty, and students alike, maintain focus on highly critical and foundational concepts, while also making sure not to completely disregard topics of lower priority. We have proposed such a framework for programs to utilize when facilitating conversations surrounding curricular reforms and topic prioritization and advocate for its use to inform competency-based curricular reform.


The rise of antibiotic resistance has become a major threat to human health and it is spreading globally. It can cause common infectious diseases to be difficult to treat and leads to higher medical costs and increased mortality. Hence, multifunctional polymeric nanofibers with distinctive structures and unique physiochemical properties have emerged as a neo-tool to target biofilm and overcome deadly bacterial infections. This review emphasizes electrospun nanofibers’ design criteria and properties that can be utilized to enhance their therapeutic activity for antimicrobial therapy. Also, we present recent progress in designing the surface functionalization of antimicrobial nanofibers with non-antibiotic agents for effective antibacterial therapy. Lastly, we discuss the future trends and remaining challenges for polymeric nanofibers. Clostridioides difficile arose in 2017 and caused mortality in 12,800 people [Staphylococcus aureus strains collected are resistant to methicillin and even vancomycin and carbapenems [In recent decades, the inception of multidrug-resistant (MDR) bacteria or “superbugs” has become a global threat due to the resistance of bacteria to antibiotics. The treatment of MDR bacteria with ineffective antibiotics has formed new resistances that have spread remarkably across continents through the environment, people, and animals. The 2019 Antibiotic Resistance Threats report from the Centers for Disease Control and Prevention (CDC) classified a few multidrug-resistant bacteria and fungi based on their threat levels to human health, and reported that in the US, more than 2.8 million antibiotic-resistant infections occur yearly, resulting in the death of more than 35,000 people. In addition, 223,900 cases of bapenems ,5. NotabAntibiotics act on bacteria by inhibiting their cell walls and interfering with DNA, RNA, or essential proteins. However, bacteria innately have the ability to alter their structural properties and characteristics to reduce the efficacy of antibiotics ,7,8,9,10There is increasing interest in utilizing polymeric nanofibers with a drug cargo of antibiotics in killing bacteria. Most studies have only focused on intrinsic structure and tunable structure, components, and properties of nanofibers which enable the generation of drug-loaded nanofibers with a sustained release pattern for drug delivery application ,20,21. NIn recent years, electrospun nanofiber scaffolds have been demonstrated to be effective nano-scale therapeutic devices, as their physicochemical properties can be tailored to several applications requiring necessary antimicrobial capabilities ,28,29. Iw/w concentration. The antibiotic-loaded nanofibers also showed good antimicrobial activity against both Escherichia coli and S. aureus [Due to their small size, nanofibers possess a very large surface area-to-volume ratio along with interconnectivity and microscale interstitial space, rendering them more effective than their bulk form. The high surface area of nanofibers can promote the hemostasis of injured tissues and fluid absorption ,43,44 an. aureus .The performance of nanofibers is influenced highly by their porosity 60–90%) ,52,53, w–90% ,52,S. aureus, E. coli, Listeria innocua and Salmonella typhymurium [Natural and synthetic polymers are widely used to fabricate nanofiber matrices due to their processability, biocompatibility, and biodegradability ,78,79,80hymurium ,86. In chymurium ,87. The hymurium ,89,90,91hymurium ,95,96,97hymurium ,98,99. Thymurium .The electrospinning technique is a straightforward process to fabricate polymeric nanostructures and thus offers the versatility of structure, morphology, and spatial distribution of electrospun nanofibers to achieve specific mechanical properties ,106,107.A typical electrospinning setup consists of a high-voltage power supply, a ground collector, a syringe pump, and a syringe with a capillary needle. The polymer solution is loaded into a syringe attached to the needle at a controlled flow rate. The repulsive electrical force is applied to overcome the surface tension of the polymer solution, resulting in the formation of a Taylor cone. The polymer solution will stretch and evaporate, and the fibers will be deposited on the metal-conductive rotating ground collector ,114,115.The unique characteristics of electrospun nanofibers, such as high-surface-to-volume ratio, controllable fibers orientation and diameters, high porosity, and modulated surface roughness, are greatly influenced by the electrospinning process. Thus, the morphological features of electrospun nanofibers can be altered by tuning parameters such as the polymer solution properties  or processing parameters such as applied voltage, solution flow rate, tip-to-collector distance, and collector speed ,127,128.Nanofibers have been used as a drug cargo delivery vehicle for therapeutic agents such as antibiotics, metal nanoparticles, carbon materials, peptides, and natural extracts . These tS. aureus and Streptococcus pyogenes are the dominant organisms involved, while gram-negative organisms like E. coli and P. aeruginosa are only found in later stages of the process, i.e., when a chronic wound has developed [In the initial stage of the infectious process, gram-positive microbes such as eveloped ,142,143.eveloped ,146,147.eveloped ,149,150.eveloped . Furthereveloped ,153,154.S. aureus, E. coli, L. innocua, and S. typhymurium [Clostridium difficile isolates with tetracycline and chloramphenicol resistance genes. The successful bacterial inhibition activity suggested that protein synthesis disruption is not the mechanism of the antibacterial action of chitosan nanofibers [The inherent antimicrobial activity and mechanism of nanofibers alone have not been widely explored. Nanofibers’ most well-documented antimicrobial activity revolves only around electrospun chitosan nanofibers—chitosan being a natural polymer with antimicrobial properties ,147,149.hymurium . An antiFunctionalized nanofibers can express various bactericidal pathways depending on the core material used, the morphology of the material, and the surface chemistry of the scaffold ,155. TheThe bactericidal effects of nanofibers depend on their size, diameter, shape, and surface chemistry. Three-dimensional nanofiber scaffolds with hierarchical structures as small as a few microns to a few hundred nanometers provide a high surface area and thus enhanced therapeutic efficacy against bacterial infection ,106. A rE. coli cells had also significantly decreased after attachment onto the diethylamine surface, but remained high on sodium sulfite surface [The bacterial adhesion and attachment surface interaction is essential for biofilm control, and is influenced by the surface chemistry such as the surface charge, roughness, topography, and wettability ,159,160. surface . Despite surface . For exa surface . Hence, Meanwhile, rough surface nanofibers with large surface areas promote bacterial contact and attachment due to higher bacterial contact are ,163. HowSurface wettability plays a crucial role in the attachment or detachment of the biofilm from the surface. The surface wettability can be influenced by the degree of hydrophobicity of the nanofibers . HydrophThe porosity, which is three-dimensional (3D) holes formed on the nanofibers, can influence bacterial attachment by affecting surface wettability . Porous To sum up, functionalized nanofibers should be designed to have: (1) small diameters (300–1000 nm), (2) positively charged surfaces, (3) rough surfaces with high surface area, (4) hydrophobic surfaces, and also (5) large pore diameters to ensure better adhesion  to improVarious nanomaterials such as metal nanoparticles, nanodots , nano blades  and nano spikes  showed the effectiveness of the mechano-bactericidal mechanism in penetrating and rupturing bacterial cell walls, eventually causing cell death ,153,154.Metal and metal oxide nanoparticles are known for their ability to penetrate bacterial cells and disrupt cellular activity by generating reactive oxygen species (ROS) such as hydrogen peroxide or superoxide anions. Excessive ROS production will cause severe oxidative stress that will damage the bacterial cellular components, disrupt protein synthesis, inhibit enzymatic action, and cause cell membrane disruption and site-specific DNA damage, ultimately leading to cell lysis ,177,178.E. coli, S. aureus and P. aeruginosa [E. coli even at low Ag concentrations (0.5% w/v). The bacteria inhibition zones observed were increased as the concentration of Ag increased due to the increase of metal toxicity [S. aureus and E. coli [S. aureus and E. coli [Metals like silver (Ag), gold (Au), copper (Cu), zinc (Zn) and their corresponding oxides are commonly used to design potent antimicrobial nanomaterials . Among truginosa . Chitosatoxicity . Moon et E. coli . In a st E. coli . It is w E. coli . The viaE. coli, P. aeruginosa, Bacillus subtilis and S. aureus compared to chitosan/PVA nanofibers. In vivo wound healing analysis also revealed that the chitosan/PVA/ZnO nanofibers accelerated the wound healing of subcutaneous wounds in induced diabetic rabbits [Unlike Ag, metal ions like zinc ions, Zn+ are essential for bacteria to regulate several metabolic pathways such as sugar, lipid, and protein degradation . However rabbits .E. coli and gram-positive S. aureus [Similar to Zn, copper ions, Cu2+ are also essential for biological processes such as the enzymatic reactions and protein interactions of bacteria. However, excess Cu2+ concentrations may lead to cell membrane and DNA disruptions . The Cu2. aureus . The ion. aureus ,196. Carbon materials such as carbon dots, fullerene, graphite, graphite oxide, graphene oxide and reduced graphene oxide can cause bactericidal effects towards bacteria via several mechanisms: (1) membrane stress induced by the sharp edges of carbon material nanosheets, which can lead to membrane damage and results in the leakage of RNA and other intracellular electrolytes ,198,199,2 [2. Smaller GO sheets have a higher capacity to induce oxidative stress in bacterial cells, leading to membrane damage and cell death [E. coli and S. aureus [The effect of graphene oxide (GO) size evaluated from GO in bacterial suspension showed that the antimicrobial effect of GO increases as its sheet area increases from 0.01 to 0.65 µm2 . This is2 . In contll death . These f. aureus . The inh. aureus . Despite. aureus ,204. How. aureus . In addi. aureus . These rE. coli, S. aureus, Klebsiella pneumoniae, and multidrug-resistant Acinetobacter baumannii [E. coli. The small size of the CQDs allowed it to penetrate bacteria cells and disrupt the cell wall [Apart from graphene-based materials, carbon quantum dots or carbon dots (CQDs) have been extensively studied for their antimicrobial activity. Nie et al., (2020) found that synthesized CQDs had generated ROS, leading to cell membrane damage, thus inhibiting aumannii . In anotell wall .60) have shown antimicrobial activity against S. aureus, E. coli, and Shewanella oneidensis. Fullerene can inhibit the energy metabolism of the bacteria, impair respiratory action, and induce disruption of the cell membrane [L-lactide) (PLA) nanofibers and simultaneously electrosprayed them with a zinc oxide/fullerene (ZnO/C60) hybrid. The fiber mats exhibited excellent antimicrobial activity against S. aureus at a low fullerene concentration (0.5 and 1.0% w/w) [Soccer ball-shaped fullerenes (Cmembrane . Virovsk.0% w/w) . The outOther than metal and carbon materials, nanofiber-loaded antimicrobial peptides have also shown a bactericidal effect. Antimicrobial peptides (AMPs) are positively charged peptides with broad-spectrum antimicrobial activity found in various life forms including humans and microorganism . Since mS. aureus, Staphylococcus epidermidis, E. coli and P. aeruginosa. Interestingly, the antimicrobial activity of CYs-KR12 was maintained after three weeks [Lactococcus lactis showed remarkable antimicrobial activity against S. aureus, and interestingly, the antimicrobial effect lasted for 14 days [As a part of the innate immune response, antimicrobial peptides have a broad-spectrum activity against bacterial infection and demonstrate a potent therapeutic agent ,213. Theee weeks . The nan 14 days . Taken tBiden Pilosa, Hibiscus sabdariffa, Rosmarinus officinalis, and Thymus vulgaris extracts have been used widely as antimicrobial agents. Due to alkaline stress, these extracts exhibit bactericidal effects through cell membrane hyperpolarisation [Natural plant or herbal extracts like aloe vera, chamomile, curcumin, propolis, risation  and cytorisation . Bacteririsation . The disrisation ,220. On risation .S. aureus and S. epidermidis. No inhibition was observed for pure PLGA nanofibers [S. aureus and E. coli, as well as promoting rapid proliferation rate for fibroblast cells [Recently, PLGA nanofibers fabricated with aloe vera extract having an average diameter of 356 nm with 87.92% porosity were shown to inhibit the growth of nofibers . A similst cells . An in vS. aureus and E. coli was observed, as the concentration of extract loaded was increased from 0.1 up to 0.5 g [Listeria monocytogenes and S. aureus [Moringa (MR) extract incorporated into polyacrylonitrile (PAN) nanofibers showed a concentration-dependent antimicrobial activity whereby increased inhibition of to 0.5 g . In anot. aureus . E. coli and 51.7% of S. aureus growth. In comparison, the fabricated nanofibers showed higher antimicrobial activity with 75.4% E. coli and 91% S. aureus inhibition [On the other hand, Kegere et al., (2019) analyzed the effect of PVA/chitosan nanofibers blended with Biden Pilosa (BP) crude extract. BP crude extract alone can inhibit 64% of hibition . The anthibition  and the The surface of nanofibers can be functionalized using different molecular moieties, during or after the treatment electrospinning process ,227,228.E. coli attachment. The highest amount of E. coli attached was observed on the PS surface with ppAAm. Although allylamine is a hydrophilic monomer, its positive charge surface attracts the bacteria and encourages their attachment and proliferation on the surface of nanofibers. Similar to allylamine, acrylic acid is also a hydrophilic monomer. However, only a small proportion of bacteria cells are attached to the ppAAc due to its negatively charged surface. The electrostatic repulsion between the ppAAc nanofibers and E. coli resulted in low attachment of bacteria cells.Abrigo et al., (2015) studied the influence of the fiber wettability, surface charge and surface chemistry of polystyrene (PS) nanofibers functionalized with acrylic acid (ppAAc), allylamine (ppAAm), 1,7-octadiene (ppOct), and 1,8-octadiene (ppCo), using the plasma treatment method, on In contrast, a significant amount of the bacteria was found on the ppOct, attributed to the hydrophobicity of the surface. Although the functionalized nanofibers attracted the bacteria to stick onto the surface, ppAAm and ppOct did not induce bacteria inhibition. Instead, the bacteria proliferated around the nanofibers . The obsE. coli, P. aeruginosa and S. aureus, the cells became flattened and wrinkled, causing conformational changes and leading to cell death [In another study, upon exposure of PLGA/chitosan nanofibers functionalized with GO-Ag to the attached ll death . The bacll death . In contll death . The functionalization of electrospun nanofibers with antimicrobial agents is a promising strategy to combat bacterial infection and resistance. Different functionalization methods and materials used will provide different interactions and mechanism of actions in killing the bacteria. Therefore, the nanofiber criteria and designs discussed above can provide a basic guideline to further understand the relationship between functionalized nanofibers and bacteria cells.At present, smart antimicrobial nanofibers have been developed in different fields, including wound dressing, tissue repair and regeneration, nanomedicine, air, and water filtering. Nanomaterial-based antimicrobials can be used as an alternative to antibiotics to achieve an effective therapeutic effect, especially in wound dressing applications. The design of fabricated nanofibers plays an important role to ensure antimicrobial effectiveness. Therefore, we have discussed the properties that can influence nanofibers’ bactericidal effects, such as its (1) morphology including size, diameter and porosity, (2) the surface charge of the nanofibers, and (3) surface wettability. However, it has been proven that nanomaterials or nanofibers are not able to fight bacteria alone. The addition of antimicrobial agents is strongly required to enhance the antimicrobial activity of nanofibers. Additional studies will be required to enable a deeper understanding on the interactions of these nanomaterials with the target bacteria as bacteria are complex microorganisms that can easily adapt to their surroundings for survival. Additionally, interdisciplinary research involving the chemical, biological, and pharmacological fields is necessary to translate these nanofiber designs clinically.However, electrospinning is still not ready for the large-scale industrialization of antibacterial fiber production as required from the market. The optimization of nanofibers with highly complex morphologies  still present some difficulties in terms of large-scale feasibility, and further studies to improve entrapment mechanisms and fabrication processes are needed. For this purpose, different manufacturing methods—i.e., the simultaneous or sequential deposition of fibers and/or nanoparticles ,239 have


To compare overall number of downstream tests and total costs between negative exercise stress echocardiograms (ESE) or cardiac computed tomography angiography scans (CCTA) in symptomatic Tricare beneficiaries suspected of having coronary artery disease (CAD). This is a retrospective cohort study examining 651 propensity-matched patients who underwent ESE or CCTA with normal results between 2008 and 2014 at the United States’ largest Department of Defense hospital. The total number of additional downstream tests over the next five years was determined. The total costs associated with each arm, inclusive of the initial test and all subsequent tests, were calculated using the 2018 Medicare Physician Fee Schedule. 18.5 percent of patients with a normal ESE result underwent some additional form of cardiac testing over the five years after initial testing compared to 12.8 percent of patients with a normal CCTA. The absolute difference in total number of downstream tests between both study groups was 5.7 percent (p = 0.03). When factoring the costs of the initial test as well as the downstream tests, the ESE group was associated with overall lower costs compared to the CCTA group, 351 United States Dollars (USD) versus 496 USD (p < 0.0001). This study demonstrates that, when compared to CCTA, ESE is associated with a higher total number of downstream tests, but overall lower total costs when chosen as initial testing strategy for suspected CAD. Coronary artery disease (CAD) remains a leading cause of death around the world. The clinician evaluating the patient suspected of having symptoms attributable to stable obstructive CAD is confronted with the decision of choosing the most appropriate test. Exercise stress echocardiography (ESE) is a well-validated form of functional testing boasting a high sensitivity and specificity for the detection of obstructive CAD with the notable benefits of widespread availability and the absence of ionizing radiation exposure. Cardiac computed tomography angiography (CCTA) has established itself as a commonly utilized noninvasive alternative to invasive coronary angiography for anatomic evaluation of coronary anatomy. Multiple randomized controlled trials and subsequent meta-analyses have demonstrated that CCTA can reliably exclude obstructive CAD with a very high sensitivity and negative predictive value (NPV) –4. FurthThis is a retrospective study of symptomatic patients without known CAD who underwent noninvasive testing for CAD between the January 1, 2008 and June 31, 2014 at Brooke Army Medical Center, the largest hospital in the Department of Defense. The aim of the study was to compare the downstream consequences between those with a negative test result when initial testing was ESE or CCTA. The patient population includes active-duty personnel, their family members, and retired military beneficiaries. The electronic medical record was queried to identify individuals who underwent cardiac stress testing during the study period. To be included in the study, patients required symptoms potentially attributable to myocardial ischemia  software, including two-sided Chi-squared testing of categorical variables, analysis of variance (ANOVA) of continuous variables with normal distribution, and Wilcoxon signed-rank test and the Kruskal–Wallis Test for non-normally distributed continuous variables. Univariate and multivariate regression were completed to determine factors that might lead to increased testing. Propensity matching using the CAD Consortium score was used to compare CCTA with ESE.In the specified six-and-a-half-year time interval, a total of 11,636 patients undergoing any form of noninvasive cardiac testing were screened. Of those screened, 2,864 patients underwent CCTA and 1,285 of those were included after excluding patients who did not meet study parameters described above. A total of 985 patients who underwent ESE were screened. Of these, 327 were eligible for inclusion. The initial patient demographics are displayed in Table According to the 2018 Final Medicare Physician Fee Schedule, the price of ESE was 239 United States Dollars (USD) whereas the cost of CCTA was 432 USD [This study set out to examine the downstream effects of negative ESE or CCTA when chosen as the initial noninvasive test for suspected CAD. As described in detail above, the choice of ESE was found to have a greater number of downstream tests, which is in line with the “warranty period” of CCTA. However, when accounting for the cost of the initial negative test, ESE was associated with a lower total cost. We originally hypothesized that CCTA would be associated with both total lower number of downstream tests and total cost due to the previously published negative predictive value of CCTA in RCTs.In the past few years, two large RCTs, PROMISE and SCOT-HEART, have compared clinical outcomes between groups randomized to functional or anatomic testing with CCTA. As a result of these two and other studies, the European Society of Cardiology (ESC) guidelines for chronic coronary endorsed CCTA as a Class I recommendation for initial test to diagnose CAD . As founNotably, ESE was underrepresented in the functional imaging control group in both landmark studies. Specifically, only 22% of patients underwent ESE compared to 67% undergoing nuclear testing in the PROMISE trial . FurtherMoreover, ESE possesses a few clear advantages when compared to CCTA. Despite the growing use of CCTA, ESE remains widely available in the clinic and emergency department settings with minimal equipment requirements. In addition to wall motion analysis, additional prognostic data can be derived, such as metabolic equivalents, heart rate response, and exercise induced hypertension. For patients being referred for cardiac testing with chief complaint of dyspnea, ESE can provide diagnostic information in nonischemic etiologies, including exercise-induced diastolic dysfunction, pulmonary hypertension, and severity of mitral valve disease. Importantly, ESE does not expose the patient to ionizing radiation. These advantages are in addition to the post cost savings discussed above.Our study has several strengths. First, as fewer downstream tests were performed in the CCTA arm, it shows that providers likely act in light of the perceived “warranty period” of a normal CCTA. Additionally, our study benefits from the comprehensive electronic medical record available through the Department of Defense health system. As a result, over 11,000 patients were screened. Lastly, propensity matching was completed as outlined above, further strengthening the comparison between the two arms .Our trial has some limitations. Most importantly, it should be noted that the cost-analysis cannot be used to compare CCTA and ESE as a whole because our analysis does not include patients with positive results. Further, while the studied populations underwent propensity matching, this cannot eliminate all potential cofounders present in this retrospective analysis. Second, the results may lack generalizability given the population studied was Tricare beneficiaries. This is exemplified by the notable low pre-test probability noted in our study even after excluding very-low risk patients with a CAD consortium pretest probability of less than five percent. Last, it is retrospective and thus it should principally be viewed as hypothesis-generating Fig. .Fig. 1ScThis study demonstrates an association between ESE with higher total number of downstream tests as well as lower costs when compared to CCTA. These findings are unexpected and potentially worthwhile as the cardiology community moves towards CCTA as a first line test for suspected CAD. As these findings are retrospective, future RCTs specifically examining the financial and clinical outcomes of CCTA compared with ESE would bring further clarity to these questions.


In this paper, we developed a reactive molecular dynamics (RMD) scheme to simulate the Self-Stable Precipitation (SP) polymerization of 1-pentene and cyclopentene (C5) with maleic anhydride (MAn) in an all-atom resolution. We studied the chain propagation mechanism by tracking the changes in molecular conformation and analyzing end-to-end distance and radius of gyration. The results show that the main reason of chain termination in the reaction process was due to intramolecular cyclic entanglement, which made the active center wrapped in the center of the globular chain. After conducting the experiment in the same condition with the simulation, we found that the distribution trend and peak value of the molecular-weight-distribution curve in the simulation were consistent with experimental results. The simulated number average molecular weight (Mn) and weight average molecular weight (Mw) were in good agreement with the experiment. Moreover, the simulated molecular polydispersity index (PDI) for cyclopentene reaction with maleic anhydride was accurate, differing by 0.04 from the experimental value. These show that this model is suitable for C5–maleic anhydride self-stable precipitation polymerization and is expected to be used as a molecular weight prediction tool for other maleic anhydride self-stable precipitation polymerization system. Our group proposed that α-olefin and maleic anhydride can undergo a self-stable precipitation polymerization reaction in a specific solvent environment ,2,3. A sI and rP. The reaction can take place whenever a free monomer bead M comes into the interaction sphere rI of an initiator I*or rP of a terminal reactive chain monomer P*. Torres et al. judged the termination of the reaction by introducing a gel point in the model [However, the influence of the entanglement/aggregation process of molecular chains in the solution phase in the early stage of the reaction has not been investigated, which is critical for revealing the mechanism in the molecular-level microscope. The main reason is that the size of molecules generated in the early stage of the reaction is very small, which is difficult to observe with current observation methods, and the chain growth process is extremely fast, which is difficult to research by sampling or in situ observation. However, MD simulation has great advantages in observing the small size and ultra-fast evolution process. It has been proved to be one of the most important tools to explore the mechanism in the field of self-assembly ,10. As the model . Karim ehe model . Jiwon ehe model , which pThrough the prior experiments of our group, we found that the molecular weight of the copolymerization product of the C5–maleic anhydride reaction was much lower than that of common free radical polymerization. The degree of polymerization (DP) of the product was less than 100. In this study, the RMD simulation method was used to study two representative molecules 1-pentane and cyclopentene reaction with maleic anhydride. We investigated the process of chain growth and conformation evolution of the alternating copolymerization of pentene and cyclopentene with maleic anhydride, respectively, finding the mechanism for generating lower molecular weight product in this kind of SP polymerization. Correspondingly, we experiment with the same conditions as simulation in order to compare the simulated and experimental number average molecular weight (Mn) and weight average molecular weight (Mw) and the molecular weight distribution trend, which can validate the model we prepared. Since the number of free radicals in the early stage of the reaction is rare, the probability of free radicals connecting is small. To simplify the model and reduce the amount of calculation, only one active center was introduced in the model to study the evolution of molecular conformation during single-chain growth, the way of termination, and molecular weight distribution. Based on the algorithms proposed by Jiwon et al. , we deveSince our simulation focuses on the early stage of the polymerization, the molecular weight is not high, DP < 100, and precipitation has not formed yet in solution. There is a different termination method from Jiwon’s in our algorithm. Jiwon’s termination condition is whether the conversion rate reaches a target value. Ours is whether the “no” loop cycles five times in a row; that is, if there are no head atoms within 1000 ps.Construction of atomic model and RMD simulations were carried out with Materials Studio 7.0 software, Accelrys Company, .p-xylene molecules (a non-polar solvent) were constructed in the Amorphous Cell modules of Materials studio 7.0 software pack. The density was set to be 0.86 g/cm3 (measured in the experiment). The cell size was 64.7 Å × 64.7 Å × 64.7 Å. Two nanoseconds of NVT dynamic equilibrium was employed to the cell. The integration time step was 1 fs, the temperature T = 500 K, and the COMPASS force field was adopted, which was validated on alkane and phenyl liquid substances in the literature by Sun [First, the double bonds in maleic anhydride and 1-pentene (cyclopentene) were changed to single bonds and linked to form a composite monomer, as shown in e by Sun . The temThen the carbon atom on the side of maleic anhydride in a composite monomer away from pentane was defined as the active center  tests. GPC 1515-2414 detector produced by Waters company  was used for the test, polystyrene (PS) as the standards, and tetrahydrofuran as the eluent. The samples were filtered through a 0.45 μm pore size syringe filter before sample injection. None of polymers were detected from the sample within the first 4 min, and there was polymer in the sample at the 5th minute. The polymer obtained at this time was used as the product of the early stage of the reaction for subsequent studies.MAH is the mass fraction of MAn %; COH is the KOH–ethanol standard solution concentration, mol/L; VOH is the amount of excess KOH–ethanol standard solution, mL; CH is the concentration of HCl–isopropanol standard solution, mol/L; VH is the amount of HCl-isopropanol standard solution in back titration, mL; and m_ is the mass of the copolymer sample, g. To verify the composition ratio of the copolymer, the acid–base titration method was used to determine the content of acid anhydride in the copolymer . We tookThe data obtained by GPC were relative values to the molecular weight of the standard PS, which can be corrected by the universal calibration method  to obtaiFrom the distribution trend of the histogram (simulation) and the curve (experiment) in This paper proposed a new algorithm for simulating low-molecular-weight (degree of polymerization (DP) < 100) polymers in the early stage of Self-Stable Precipitation (SP) polymerization. Using this algorithm, the chain growth process of the reaction of 1-pentene and cyclopentene with maleic anhydride was simulated under the same conditions as the experiment. The process of the chain growth was stated by the conformation image and the changing of molecular end-to-end distance and the radius of gyration. It was found that the reason for the termination of the two chains 1-pentene–maleic anhydride (1PM) and cyclopentene–maleic anhydride (CPM) was the intramolecular cyclic entanglement; the active centers were wrapped in the center of the globular chains, which cannot make contact with other monomers; and, finally, the chains terminate at a low DP. This investigation revealed the mechanism of entanglement/aggregation of SP polymer chains on a microscopic scale. Furthermore, we simulated more molecular chains to obtain the molecular weight distribution. We compared the molecular-weight-distribution curve with that of the experiment. It was found that the simulated value in a limited sample can be consistent with most of the experimental curves. The experimental and simulated values of number average molecular weight (Mn), weight average molecular weight (Mw), and PDI were in good agreement between experiment and simulation. It was validated that the model is suitable for simulating the alternating copolymerization of C5–maleic anhydride, and the chain growth mechanism is reliable. This model may be suitable to a wider range of self-stable precipitation polymerization systems, and we will further explore it in the future research.


Class switch recombination (CSR) is the process by which B cells switch production from IgM/IgD to other immunoglobulin isotypes, enabling them to mount an effective immune response against pathogens. Timely resolution of CSR prevents damage due to an uncontrolled and prolonged immune response. While many positive regulators of CSR have been described, negative regulators of CSR are relatively unknown. Using an shRNA library screen targeting more than 28,000 genes in a mouse B cell line, we have identified a novel, uncharacterized protein of 82kD , which we have named SANBR (SANT and BTB domain regulator of CSR), as a negative regulator of CSR. The purified, recombinant BTB domain of SANBR exhibited characteristic properties such as homodimerization and interaction with corepressor proteins, including HDAC and SMRT. Overexpression of SANBR inhibited CSR in primary mouse splenic B cells, and inhibition of CSR is dependent on the BTB domain while the SANT domain is largely dispensable. Thus, we have identified a new member of the BTB family that serves as a negative regulator of CSR. Future investigations to identify transcriptional targets of SANBR in B cells will reveal further insights into the specific mechanisms by which SANBR regulates CSR as well as fundamental gene regulatory activities of this protein. Immunoglobulin (Ig) production by B cells is an essential component of the immune response against pathogens and malignancies. Upon encounter with antigen, mature B cells are activated and undergo class switch recombination (CSR) , 2, 3. TFollowing AID deamination of S region DNA, two complementary pathways, base excision repair (BER) and mismatch repair (MMR), generate the DSBs that are necessary for CSR. In BER, uracil DNA glycosylase (UNG) removes the AID-generated uracil base to create an abasic site, which is processed into a DNA break by apurinic/apyrimidinic endonucleases (APE) , 7. DuriSuv4-20h1 and Suv4-20h2), which modify histone H4, in B cells significantly impairs CSR  ribose polymerase Parp3  and the A). CH12 cells are a well-established model system for the study of CSR, as they can be induced to class switch from IgM to IgA in culture when stimulated with anti-CD40, interleukin 4 (IL4), and transforming growth factor β (TGF-β), which is henceforth referred to as CIT (A). As depletion of negative regulators would likely promote CSR, we expected shRNAs that targeted them to be enriched in the IgA+ population as compared with the IgA– population. Genes with shRNAs showing at least twofold enrichment in IgA+ compared with IgA– samples, i.e., log2(Cy3/Cy5) > 1, were identified as negative regulators (2(Cy3/Cy5) values, indicating that shRNAs for these genes are enriched in the IgA– population and their knockdown prevented CSR (2(Cy3/Cy5) of -1.42, demonstrating the validity of the shRNA screen .To identify novel negative regulators of CSR, we performed an shRNA screen using the CH12 mouse B lymphoma cell line A. CH12 co as CIT , 26, 27.o as CIT , 29. Trao as CIT A. As depgulators . As contnted CSR . For ins2(Cy3/Cy5) values for the two shRNAs in the screen targeting NM_027860 were 3.09 and –0.92. NM_027860 encodes for a well-conserved protein KIAA1841 , vertebrates , and single-cell organisms  express an ortholog of KIAA1841. The mouse KIAA1841 gene is located on chromosome 11 and consists of 28 exons. While a potential splice isoform missing exons 3, 4, and 5, which encodes the first 146 amino acids, has been reported (B). Amino acid sequence alignment by BLAST and structure prediction by Phyre2 (B). Although SANBR was not detected at S regions by chromatin immunoprecipitation , as a candidate negative regulator of CSR based on our selection criteria of at least one targeting shRNA with log2(Cy3/Cy5) > 1 at 10% FDR . The logKIAA1841  that is KIAA1841 , 31. An reported , 33, we reported B. Amino y Phyre2  further y Phyre2 B. Althoupitation , SANBR epitation . Wild-tyt of AID .C). Both the untagged and Xpress-tagged SANBR significantly reduced CSR to IgG1 (D and E), reinforcing the role of SANBR as a negative regulator of CSR. Overexpression of SANBR did not affect germline transcription (1C), or B cell proliferation  and germinal center B cells (A). As full-length SANBR is poorly expressed and aggregated in Escherichia coli (data not shown), a fragment containing the putative BTB domain, SANBR(BTB), was expressed as a His6-tagged recombinant protein and purified (B) before analysis on a size-exclusion chromatography (SEC) column (C). Comparison with protein molecular weight standards separated on the column under the same conditions indicated that SANBR(BTB) migrates as a dimer (C). To further verify that SANBR(BTB) dimerizes, we cross-linked purified SANBR(BTB) with glutaraldehyde before analysis on a denaturing SDS-PAGE (D). When cross-linked, the 15kD fragment of SANBR(BTB) formed a species approximately 30kD in size, corresponding to the expected molecular weight of a dimer (D). Thus, SANBR contains a BTB domain that dimerizes in vitro.We next investigated the function of the putative BTB domain of SANBR. Many cellular processes require BTB domain-containing proteins, including transcription and protein degradation . In the  B cells , 39. The B cells . The put B cells A. As fulpurified B before ) column C. Compar a dimer C. To furSDS-PAGE D. When c a dimer D. Thus, in vivo, dimerization assays were performed in 293T cells. Full-length Flag-StrepII-tagged wild-type (WT) or deletion mutants of SANBR, which lacked the BTB domain , or helix 3 of the SANT domain . These proteins were then used in binding assays with purified His6-tagged SANBR(BTB), where a His6-tagged BTB domain of PLZF was used as a positive control. Consistent with a previous report (6-PLZF(BTB) interacted with HDAC1 and the SMRT fragment . Similarly, His6-SANBR(BTB) also bound to these corepressor proteins . Taken together with the earlier dimerization findings, these data suggest that SANBR contains a functional BTB domain that mediates dimer formation and binding to corepressors.BTB-containing proteins commonly function by interacting with corepressors, such as histone deacetylases (HDACs), nuclear corepressors (N-CoR), and silencing mediator of retinoic acid and thyroid hormone receptor (SMRT), B domain . To deteB domain  , retroviral expression of WT SANBR protein (A) reduced CSR to IgG1 in splenic B cells (B and C). While the ΔSANT mutant inhibited CSR to similar levels as WT SANBR, the ΔBTB mutant only partially suppressed CSR (B and C). This suggests that the BTB domain of SANBR plays an important role in the negative regulation of CSR, while the SANT domain is largely dispensable. As WT levels of CSR were not achieved with ΔBTB mutant expression, other regions of SANBR likely function in tandem with the BTB domain to mediate the suppression of CSR.To evaluate the role of the SANBR BTB and SANT domains in the regulation of CSR, Flag-StrepII-tagged ΔBTB or ΔSANT deletion mutants were expressed in mouse splenic B cells that were stimulated for CSR to IgG1 by LPS plus IL4. As shown earlier , D and E protein A reduced B cells , B and Cssed CSR , B and Cin vitro . BTB domains can also mediate heterodimerization  and stimulated with LPS and IL4 to induce CSR to IgG1 as described . RetroviThe polyclonal SANBR antibody was produced by immunizing rabbits with the C-terminal SANBR peptide (RSKSRFGQGRPA) and isolating reactive antibodies from sera (Covance). Antibodies for flow cytometry include: anti-IgG1-APC , anti-IgA-FITC . Antibodies for immunoblot include: anti-AID , anti-tu2(Cy3/Cy5) > 1.The lentiviral shRNA library targeting the mouse genome was a kind gift from S.J. Elledge , 29. Scr2(Cy3/Cy5) ratios for the probes. Probes with a signal < twofold above background and a false discovery rate (FDR) of more than 10% were removed. Genes with at least one targeting shRNA that satisfy these criteria were identified as candidates.Statistical analysis was performed as described previously . Briefly3, 0.5% SDS) for 10 min at room temperature. Cells were isolated by centrifugation at 135g for 6 min and sonicated in IP buffer  using Bioruptor Pico (Diagenode) for ten cycles . Cell debris was removed by high-speed centrifugation, and lysates were precleared with Protein A agarose/Salmon Sperm beads . Cells (2.5 − 106) were immunoprecipitated with each antibody at 4 °C overnight, and 5% was taken as input prior to immunoprecipitation. Immuno-complexes were captured with ProteinA agarose/Salmon Sperm beads and washed with wash buffer , followed by reversal of cross-links at 65 °C overnight. DNA fragments were purified and quantified by qPCR using iQ SYBR Green Supermix (BioRad) with primers as described  and stimulated with either anti-CD40 and IL4 or LPS plus IL4. Cells (1 − 106) were harvested at 24 h, 48 h, 72 h or 96 h poststimulation. Unstimulated B cells (10 − 106) were harvested at the time of isolation as 0 h. RNA was extracted from cells using TRIzol reagent (Invitrogen) and cDNA generated by reverse transcription (RT) using SuperScript III first-strand synthesis system (Invitrogen). SANBR mRNA level was determined by qPCR using cDNA and iQ SYBR Green Supermix (BioRad); SANBR RT-qPCR Cq values were normalized to β-actin mRNA Cq values and to the respective 0 h controls. qPCR primers used were as follows: SANBR , β-actin .WT and AID6-tag in BL21DE3(RIPL) E. coli and induced with 0.2 mM IPTG at 16 °C for 17–20 h. Cells were lysed in lysis buffer  using the LM20 Microfluidizer (Microfluidics). Lysates were clarified by centrifugation, and His6-SANBR(BTB) was isolated on a HisTrap FF column . The column was washed with ten column volumes (CVs) of lysis buffer, and bound His6-SANBR(BTB) protein was eluted with five CVs of elution buffer . Fractions containing His6-SANBR(BTB) were pooled, concentrated, and further purified by SEC using Superdex 200 10/300 GL  equilibrated and eluted with SEC buffer . The BTB domain of PLZF (6-PLZF(BTB), was expressed and purified as described above.Residues 144–261 of SANBR containing the BTB domain was expressed with an N-terminal His of PLZF , 54, HisE. coli and induced with 0.2 mM IPTG at 16 °C for 17–20 h. This region corresponds to region 1414–1498 of human SMRT, which was previously reported to interact with BTB domain of PLZF (GST-tagged mouse HDAC1 and residues 1383–1467 of mouse SMRT were expressed in BL21DE3(RIPL) 6-SANBR(BTB) protein at 0.1 mg/ml in sample buffer  was cross-linked with fivefold increasing concentration of glutaraldehyde ) in a total volume of 10 μl at 37 °C for 5 min. The reaction was quenched by adding 1 μl of 1 M Tris, pH 8.0 and boiled in 1× SDS loading buffer, before separation on a 12% SDS-PAGE denaturing gel and stained with Coomassie blue to visualize the migration of the resultant protein species.Purified His6-SANBR(BTB) or His6-PLZF(BTB) at 200 nM was mixed with equimolar amounts of purified GST, GST-HDAC1, or GST-SMRT(1383–1467) fragment in binding buffer , in a total volume of 400 μl, at 4 °C for 2 h. Glutathione sepharose 4B resin , which was prewashed thrice with binding buffer, was added and mixed with the proteins at 4 °C for an additional 2 h, then washed thrice with binding buffer. Bound complexes were released by boiling in 2× SDS loading buffer and analyzed by immunoblot with anti-His antibody. In total, 30 ng His6-SANBR(BTB) or 33 ng His6-PLZF(BTB)  were loaded as inputs.Purified Hisg for 15 min at 4 °C and incubated with 20 μl MagStrep “type 3” XT beads (IBA) for 2 h at 4 °C to pull down Flag-Strep-tagged SANBR and mutant proteins. The beads were washed thrice with lysis buffer and bound proteins were recovered by boiling in 1× SDS loading dye and analyzed by immunoblot.293T cells were cotransfected with GFP-tagged SANBR and either Flag-Strep-tagged WT KIAA; SANBRΔBTB, which lacks amino acids 147–255 or SANBRΔSANT, which lacks amino acids 48–59. After transfection (48 h), cells from two confluent wells of a six-well plate were washed with PBS and lysed with 200 μl lysis buffer  supplemented with 1 mM PMSF, 1 mM DTT, and cOmplete mini protease inhibitor cocktail (Roche). Lysates were clarified by centrifugation at 21,000bvuong@ccny.cuny.edu). All remaining data are contained within the article.Raw Cy3/Cy5 fluorescence data obtained from the custom microarray are available upon request to Bao Q. Vuong at The City College of New York (This article contains The authors declare that they have no conflicts of interest with the contents of this article.