The coronavirus disease 2019 (COVID‐19) pandemic caused by SARS‐CoV‐2 infections has led to a substantial unmet need for treatments, many of which will require testing in appropriate animal models of this disease. Vaccine trials are already underway, but there remains an urgent need to find other therapeutic approaches to either target SARS‐CoV‐2 or the complications arising from viral infection, particularly the dysregulated immune response and systemic complications which have been associated with progression to severe COVID‐19. At the time of writing, in vivo studies of SARS‐CoV‐2 infection have been described using macaques, cats, ferrets, hamsters, and transgenic mice expressing human angiotensin I converting enzyme 2 (ACE2). These infection models have already been useful for studies of transmission and immunity, but to date only partly model the mechanisms involved in human severe COVID‐19. There is therefore an urgent need for development of animal models for improved evaluation of efficacy of drugs identified as having potential in the treatment of severe COVID‐19. These models need to reproduce the key mechanisms of COVID‐19 severe acute respiratory distress syndrome and the immunopathology and systemic sequelae associated with this disease. Here, we review the current models of SARS‐CoV‐2 infection and COVID‐19‐related disease mechanisms and suggest ways in which animal models can be adapted to increase their usefulness in research into COVID‐19 pathogenesis and for assessing potential treatments.http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.21/issuetocThis article is part of a themed issue on The Pharmacology of COVID‐19. To view the other articles in this section visit  ARDSacute respiratory distress syndromeDICdisseminated intravascular coagulationNETsneutrophil extracellular trapsSARSsevere acute respiratory syndrome1SARS‐CoV‐2 infection, has led to the rapid development of animal models of SARS‐CoV‐2 infection which have already provided insights into the natural history of the disease and, together with data from previous studies, have permitted identification of potential antiviral approaches identified in studies of other viral infections , the disease manifestation of ACE2 in enabling infection , studies using animal models have already provided some evidence that immunity might confer protection from reinfection , no reported animal model of SARS‐CoV‐2 infection fully reproduces every key feature of severe COVID‐19  to extents that differ between patients  to intratracheal inoculation with SARS‐CoV‐2 has also been compared with that of younger controls (3–5 years old)  of sub‐adult (aged 6–9 months) and three pairs of juvenile cats (aged 10–14 weeks) were housed together, with faecal samples analysed for the presence of viral RNA to confirm successful infection. In both the sub‐adult and juvenile cats, only one out of the three naïve cats had viral RNA detected in the faecal samples, suggestive of some transmission, albeit more limited than in ferrets. It is notable here that the researchers were unable to perform nasal washes on the sub‐adult cats due to the aggression of the cats.Also notable was the reported death of one of the inoculated juvenile cats at 3 days post infection during this study, suggestive of the provocation of severe disease in a young animal. Lung histology on autopsy showed pronounced alveolar flooding, suggesting pulmonary oedema development. Further study of whether severe disease can be reproduced in cats may be useful for severe COVID‐19 efficacy testing or for veterinary medicine development.3.4Hamsters have been used for a range of medical research studies, and importantly show a relatively high degree of homology with humans within the region of ACE2, which is involved in interaction with the ACE2‐binding domain of SARS‐CoV‐2 Spike  which did not induce pathology in mice without transgene expression. The transgenic mice replicated virus in their lungs, experienced weight loss, and developed severe histological evidence of lung inflammation and mortality at around 4 days post infection  expression is driven in mouse epithelial cells under the control of the human cytokeratin 18 (K18) promoter .Histological analysis, superficial visual inspection, and radiological imaging have been the predominant approaches used to assess the development of lung inflammation and injury following experimental SARS‐CoV‐2 infection. However, these pathological observations have so far often been reported in only some animals under study and only some regions of the lungs of those animals Table . This isSo far, repeated quantifications relevant to ARDS have not been demonstrated in animal models of SARS‐CoV‐2 infection, with the exception of one macaque study where pulmonary oedema was measured consistently in infected animals using the index of lung weight as a percentage of body weight  compared to younger animals (5 years old) , diabetes , the innate immune response and hyperinflammation, their influence on the patency of the alveolar capillary unit, and the association of low blood platelet counts with severe disease; the manipulation of platelets might be a useful experimental tool to replicate these pathological events in animal models of SARs‐CoV‐2 infection. Our previous findings that experimental thrombocytopenia can convert a mild self‐resolving bacterial lung infection to a more severe form of systemic infection, with extra‐pulmonary organ involvement and death  and Persistence of infection and inflammation due to ineffective viral clearance is a key feature of severe COVID‐19, although maintenance of infection and systemic cytokine release has not yet been reported in animal models of SARS‐CoV‐2 infection. A potential approach to model this feature of severe disease is the experimental disruption of important antiviral host defence pathways. A key mediator of defensive signalling might be the transcription factor STAT1, the genetic deletion of which can convert a mild SARS‐CoV mouse infection model into one resulting in 100% mortality  when such approaches would not be realistic for clinical treatments , and are permanently archived in the Concise Guide to PHARMACOLOGY 2019/20 (Alexander et al., Key protein targets and ligands in this article are hyperlinked to corresponding entries in The authors declare no conflicts of interest.


The coronavirus disease (COVID‐19) outbreak forced an emergency transition to online classes across the world with little warning or instruction for faculty and students. The goal of this research was to document how this response impacted undergraduate students studying the principles of evolution in an introductory organismal biology class over time; specifically, how their study habits for exams differed (a) one week and (b) one month after a university's decision to transition to emergency remote instruction. We asked students about the extent to which COVID‐19 impacted their study habits, and we categorized students’ responses using open coding. We identified a number of consistent similarities—as well as dramatic differences—in their responses as the time away from campus increased. The report that follows is a summary of the documented barriers and recommendations based on literature concerning crises and equitable practices. The goal of this research was to document how the response to COVID‐19 impacted undergraduate students studying the principles of evolution in an introductory organismal biology class over time; specifically, how their study habits for exams differed (a) one week and (b) one month after a university's decision to transition to emergency remote instruction. The report that follows is a summary of the documented barriers and recommendations based on literature concerning crises and equitable practices. These incidences have strong emotional and cognitive impacts on students—even those who are not directly affected  one week and (b) one month after our institution's decision to transition to remote instruction. Our study took place in the context of a large, organismal biology class with an explicit focus on organismal evolution and classification. This course was taught at a southeast university in the United States (U.S.), so it is important to note the conclusions of this study may not be generalizable outside of this particular region or country. By using a repeated measures design, we captured collective snapshots of our students’ condition as they forcibly adapted to remote learning during this global emergency. We hypothesized that student challenges would markedly differ at these two time points, but some challenges would persist throughout the semester. For example, one week after the transition online we hypothesized students would report challenges related to the chaos of moving off campus and adjusting to online content delivery. This is because at that time, they had not grappled with learning content online for an extended time. In contrast, we expected that one month after the transition online, students may report persistent barriers they faced while adapting to the new norm of online coursework. By conducting exploratory studies that document the student experience, we contribute to a knowledge base that can inform institutional policy‐makers as well as instructors as we prepare for the future.22.1We collected data in an Organismal Biology course from spring 2020, with a total enrollment of 415 students across two class sections. This class is the second in an introductory biology sequence taught at a research intensive, land‐grant university in the southeast region of the United States, with two of the paper's authors as the instructors of record (AB and CJB). The principle objective of the course is for students to develop an understanding of the evolution, classification, structure, and spectacular diversity of living organisms, focusing on plants and animals. This course is designed for those in biology‐related majors to prepare them for future coursework. In this course, students were assigned to small groups (ranging from 4 to 6 members) at the beginning of the semester and they sat and worked together for every class. It is a three‐credit class that includes two 75‐min class sessions each week and is offered every semester. The course initially met in a large auditorium, with seating for 270 students.Each class period included lecture and group activities to reinforce the lecture material. For example, a typical class period may include some lecture, with 3–4 iClicker questions/group discussions every 15 min, as well as a 20‐min group activity. Students also took a group exam following individual examinations, allowing for students to confer with their group members over exam materials.During the first ten weeks of the semester, students completed the first unit, including both the lessons and the first examination as well as the lesson material for the second examination. The students were then released for spring break with the plan to take the second examination in person upon returning from break. However, unpredictably, the university canceled in‐class meetings four days before students were to return from spring break due to the COVID‐19 pandemic. For that reason, class instruction became fully online four days after the cancelation of in‐class meetings, and instruction was to remain online for the rest of the semester . Students then took their second examination 1 week after the transition online and took their third examination 1 month following the transition to the online format. Following the transition to online teaching, the instructors made all prerecorded “mini‐lectures” available, via Canvas, to students for the rest of the semester and required students complete a 10‐question “quarantine quiz” that corresponded to each class period. Of note, neither instructor had formal experience teaching online, but both had some experience with taking online classes as students.2.2Immediately after students completed exams, both one week and one month after the transition online, we encouraged students to take a voluntary postexamination survey online via Qualtrics in exchange for a small amount of extra credit (awarded for clicking on the link to the survey). We selected an open‐ended survey question that could not be answered with a simple yes or no. Rather, we probed for elaboration concerning changes in student study habits: “to what extent did the Coronavirus disease (COVID‐2019) impact your study habits?” Our study offered an unusual opportunity to examine students’ responses after quickly transitioning from one type of instruction to another, rather than opting‐in to a particular mode of content delivery. In this repeated measures study, we measured the same variable(s) from the same population at two or more time points .2.3We downloaded survey responses one week following implementation. We found 330 students responded to both survey items (80% of students). For the first dataset, two of the authors (CJB and EPD) created categories through first‐ and second‐cycle analyses Saldana,  using opIn response to the one month survey, we found 296 students responded to both survey items (71% of students). Four of the authors  created an additional 5 codes to account for new emergent themes: (a) routine change, (b) suboptimal learning environment, (c) more difficult to learn online, (d) illness, and (e) easier to learn online. The same two coders coded to consensus. After the 1‐month data were coded, the two coders reanalyzed the 1‐week dataset with the additional 5 categories and coded to consensus again. Then, we calculated percentages for each code.3Results consisted of one survey question answered at two time points: one week and one month after the university's transition to online instruction in response to the COVID‐19 pandemic. We received a total of 330 survey responses one week after the transition (80% of students) and 296 survey responses one month after the transition (71% of students). We asked “to what extent did the Coronavirus disease (COVID‐2019) impact your study habits?” and developed categories that encompassed most student responses  and their focus   institutional and (b) instructional policies in times of crisis.First, institutions that seek feedback in the wake of COVID‐19 can develop an informed response in the case of future emergencies. For example, students in our sample reported COVID‐19 negatively impacted their emotional well‐being, which in turn affected their study habits, and this persisted over time. Under normal circumstances—those uncomplicated by a global pandemic—23.2% and 15.4% of American college students’ academic performances were negatively impacted by anxiety and depression, respectively ; Data curation ; Formal analysis (supporting); Methodology ; Writing‐original draft (lead); Writing‐review & editing (lead). Abby Beatty: Writing‐original draft (supporting); Writing‐review & editing (supporting). Alexis Stokes: Formal analysis (lead). Sara Wood: Formal analysis (lead). Cissy Ballen: Conceptualization (lead); Data curation ; Investigation (supporting); Methodology (supporting); Writing‐original draft ; Writing‐review & editing (supporting).


The abscopal effect is a rare phenomenon that describes the regression of distant lesions outside the treatment field of radiation therapy (RT). So far, this rare effect has been reported for several cancers which are considered to be immunogenic, including renal cell carcinoma (RCC).et al. reported that the patient with advanced RCC who showed the tumor reduction in non‐irradiated metastases at 9 months after nivolumab discontinuation.The article by Nakajima The author declares no conflict of interest.


A strong interaction between colistin, a last‐resort antibiotic of the polymyxin family, and free lipopolysaccharide , released from the Gram‐negative bacterial (GNB) outer membrane (OM), has been identified that can decrease the antibacterial efficacy of colistin, potentially increasing the dose of this antibiotic required for treatment. The competition between LPS in the GNB OM and free LPS for the interaction with colistin was prevented by using a supramolecular trap to capture free LPS. The supramolecular trap, fabricated from a subnanometer gold nanosheet with methyl motifs (SAuM), blocks lipid A, preventing the interaction between lipid A and colistin. This can minimize endotoxemia and maximize the antibacterial efficacy of colistin, enabling colistin to be used at lower doses. Thus, the potential crisis of colistin resistance could be avoided. Caught in a trap: The antibiotic colistin targets lipopolysaccharide (LPS) in the Gram‐negative bacterial (GNB) membrane. This interaction is disrupted by free LPS released during infection (path a). A methylated gold nanosheet (SAuM) binds to free LPS, preventing free LPS from binding colistin and reducing endotoxemia (path b). This increases the antibacterial efficacy of colistin, decreasing both the required dose and the risk of resistance. Antibiotic resistance has become an emerging challenge to global public health that should encourage people in general and health care providers in particular to realize the severe dangers stemming from antibiotic misuse.Figure −1 and 2.0 μg mL−1) failed to effectively kill Escherichia coli (a GNB) in the presence of even a low concentration of only 0.1 nm LPS. Surprisingly, however, the antibacterial activity of colistin (at doses of 1.0 μg mL−1 and 2.0 μg mL−1) was restored in the absence of LPS . These results suggest that colistin can associate with LPS to suppress LPS self‐assembly. Notably, LPS is prone to self‐assembly, forming various aggregates that are directly implicated in its bioactivity.−1 and are similar to LPS‐alone . This clearly illustrates that only colistin strongly interacts with LPS, while the other antibiotics tested do not. Thus, it is essential to further study how it might be possible to intervene in the competition between free LPS and bacteria for interacting with colistin. Recently, some supramolecules have been reported to directly target antibiotics and thereby switch‐on/off their activity,−1  and therefore may act as a supramolecular trap to capture free LPS, thereby disrupting the interaction between free LPS and colistin. The standard test of minimal inhibitory concentration (MIC) for antimicrobial susceptibility was conducted.−1  with typical excitation and emission peaks at approximately 390 nm and approximately 460 nm,The binding specificity assay results showed that the interaction between LPS and SAuM is actually stronger than that between LPS and colistin  and this survival rate is only slightly lower than that of SAuM‐treated mice that were not challenged with LPS (100 %). The results illustrated that SAuM is an excellent LPS antagonist and also possesses excellent biocompatibility. The hydrodynamic size of SAuM of around 2 nmIn summary, we can prevent the interaction between the antibiotic colistin and free LPS by using a supramolecular trap fabricated from subnanometer gold nanosheets. The capture and deactivation of free LPS not only minimizes the degree of endotoxemia but also boosts the low‐dose antibacterial activity of colistin which, in turn, reduced the degree of death due to GNB bacteremia. The LPS‐antagonistic activity of this supramolecular trap provides an effective steric hindrance by sealing off lipid A of free LPS to prevent the interaction between colistin and free LPS. Without the interference of free LPS, colistin can maintain its maximum antibacterial activity at a low dose, minimizing both endotoxemia and bacteremia. The supramolecular trap thus offers a potential strategy for avoiding the risk of colistin resistance.The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


Artificial intelligence technology has advanced rapidly in recent years and has the potential to improve healthcare outcomes. However, technology uptake will be largely driven by clinicians, and there is a paucity of data regarding the attitude that clinicians have to this new technology. In June–August 2019 we conducted an online survey of fellows and trainees of three specialty colleges  in Australia and New Zealand on artificial intelligence. There were 632 complete responses , equating to a response rate of 20.4%, 5.1%, and 13.2% for the above colleges, respectively. The majority  believed artificial intelligence would improve their field of medicine, and that medical workforce needs would be impacted by the technology within the next decade . Improved disease screening and streamlining of monotonous tasks were identified as key benefits of artificial intelligence. The divestment of healthcare to technology companies and medical liability implications were the greatest concerns. Education was identified as a priority to prepare clinicians for the implementation of artificial intelligence in healthcare. This survey highlights parallels between the perceptions of different clinician groups in Australia and New Zealand about artificial intelligence in medicine. Artificial intelligence was recognized as valuable technology that will have wide-ranging impacts on healthcare. This technology is now poised for clinical application. In order for AI to fully deliver its potential benefits for healthcare, medical practitioners need to understand and embrace the technology. Equally, patients need to entrust aspects of their healthcare to AI systems.Nomograms and clinical algorithms have long played a role in supporting clinical decision-making in medicine. More recently, major advances in research on artificial intelligence (AI) have been applied to medical image analysis with promising results5, radiology8, dermatology10, and in other fields of medicine. A significant milestone in the clinical application of AI came in 2018 with the US Food and Drug Administration approval of the IDx-DR system for the autonomous detection of referable diabetic retinopathy—the first such approval in any field of medicine2. Although the application of this technology is nascent, it has the potential to transform aspects of healthcare11.Numerous AI image analysis algorithms have been shown to achieve high level performance, in some instances comparable to human experts, in the detection of a range of diseases in ophthalmology14, radiology trainees15, radiologists18, pathologists19, psychiatrists20, general practitioners (GP)21, as well as general physicians, surgeons, and trainees23, with contrasting results. Most surveys indicate that clinicians believe that AI will have a positive impact on their profession22. Psychiatrists and general practitioners indicated that AI would not affect their profession21. In contrast, surveys of medical students12 and radiology trainees15 have highlighted concerns about the implications of AI for training and employment prospects27.As is true for any new medical technology, the extent to which AI algorithms for screening, diagnosis or prognosis are adopted in medicine will be dependent upon the attitudes of clinicians and patients. Consideration of these attitudes and knowledge gaps will therefore be essential for health systems, medical educators, professional bodies, AI developers and regulators. Few studies have examined clinician perceptions of new AI technologies on healthcare provision and the clinical workforce. Those that have been conducted have surveyed the potential impacts of AI in samples of medical students28. These include ophthalmology, radiology/radiation oncology and dermatology. In this study we conducted a survey of fellows and trainees of these specialty colleges in Australia and New Zealand in order to ascertain their current use, understanding and perceptions of AI.Areas of medicine that are most reliant on imaging will be amongst the first to be impacted by advances in AI technologiesA total of 632 trainees and fellows from three specialty colleges  completed the survey (Supplementary Tables p = 0.064). Respondents from each group had similar years of professional experience (p = 0.608) and almost half of all respondents had been in practice for 20 years or more  , followed by a combination of metropolitan and rural practice  and rural practice only . Ophthalmologists were more likely to spend at least part of their time in a rural setting than radiologists/radiation oncologists or dermatologists . Most respondents indicated that they had never used AI applications in their work as a clinician . Ophthalmologists were more than twice as likely to use AI in their daily clinical practice than radiologists/radiation oncologists or dermatologists  .Almost half of respondents  rated their knowledge of AI as average relative to their peers, with few rating their knowledge as excellent n = 35, 5.5%) or very poor  .Most respondents predicted that the introduction of AI would improve their field of practice   reported that AI will impact workforce needs ‘somewhat’ or ‘to a great extent’ in these specialty areas of medicine within the next decade. Most  respondents believed that AI will impact workforce needs ‘somewhat’ or ‘to a great extent’ beyond the next decade. Radiologists/radiation oncologists were more likely to report workforce needs will be impacted ‘to a great extent’ within the next decade than ophthalmologists or dermatologists .The majority of radiologists/radiation oncologists  and dermatologists  considered that their medical specialist workforce would be impacted by AI to a greater extent than other health professionals in their field of practice . In contrast, ophthalmologists considered that the impact of AI on the workforce would be greater for optometrists, than for general practitioners or ophthalmologists. Respondents who reported using AI were more likely to report that the introduction of AI would lead to the need for an increase in workforce numbers than did those who did not use AI .A hypothetical clinical workflow was proposed as follows: “patient clinical images undergo artificial intelligence analysis. A specialist subsequently reviews both the image and the artificial intelligence findings”. Most respondents would consider using this clinical workflow, however significantly more ophthalmologists and radiologists/radiation oncologists were willing to consider this than dermatologists  improved patient access to disease screening, (2) improved diagnostic confidence, and (3) reduced time spent by specialists on monotonous tasks. The top ranked advantage for ophthalmologists and dermatologists was ‘improved patient access to disease screening’ and the top ranked advantage for radiologists/radiation oncologists was ‘reduced time spent on monotonous tasks’ Fig. .Figure 7The top three potential concerns about the use of AI were (1) concerns over the divestment of healthcare to large technology and data companies, (2) concerns over medical liability due to machine error, and (3) decreasing reliance on medical specialists for diagnosis and treatment advice. The top ranked concern for ophthalmologists and radiologists/radiation oncologists was ‘concerns over the divestment of healthcare to large technology and data companies.’ The top ranked concern for dermatologists was ‘concerns over medical liability due to machine error’ Fig. .Figure 8. Some respondents highlighted the need for the colleges to be proactive in their approach in order to safeguard member interests (n = 99), with one participant requesting the college “make sure any changes are in the best interests of patients and [the] profession”. Others endorsed the development of frameworks and/or guidelines for AI implementation by the colleges (n = 30). The formation of sub-committees and working groups with expertise in AI to develop position statements (n = 46) was seen by some as a priority. Other response themes related to colleges taking leadership in relation to AI policy development and licensing (n = 74) and to be intimately involved in the trial and application of emerging AI systems (n = 69). Others called for the development of measures to ensure patient safety (n = 16) and the optimisation of clinical outcomes (n = 17).A minority of respondents  felt that the specialist training colleges were adequately prepared for the introduction of AI into clinical practice. Qualitative analysis of a total of 632 open-ended responses was performed, with similar responses grouped according to theme. Examples of verbatim responses are provided for each of the top five themes in Supplementary Table Ensuring that patient care remains in the realm of  (n = 39) was recognized as a priority. In addition, the implementation of measures to assist clinicians in adapting to changes in care delivery (n = 17) was reported as a goal, with one respondent stating that this will “assist trainees to prepare for the AI era”. Some respondents highlighted a need for colleges to support more AI research (n = 69) and engage with industry, regulators and Government (n = 74). A need for clarity regarding the responsibilities of clinicians who will use the technology was also emphasised. Several respondents highlighted the need for a redefinition of the professional scope of specialists and related healthcare provider groups as AI is deployed more widely in clinical practice.p > 0.149, Supplementary Table p < 0.001), despite no differences in self-reported knowledge of AI and its applications between these groups. Similarly, those still in training  or with less than 5 years of experience as a specialist  were more likely to believe that AI would impact practice ‘to a great extent’ beyond the next decade than were those with 20–30 years of experience or more than 30 years of experience (p < 0.001). Those with more than 30 years of experience were less likely to believe that workforce needs would decrease compared to those with fewer years of practice (p = 0.007).No statistical differences were found for responses based on location of practice , with the exception of knowledge of AI  and the number of years before AI has a noticeable effect on their field . It is possible that responses from a larger sample of specialists in these fields may identify group differences.Despite the distinct clinical roles of radiologists and radiation oncologists, no meaningful differences were detected in responses between the two professional groups  and was conducted in accordance with the doctrines of the Declaration of Helsinki and its subsequent revisions. Electronic informed consent was obtained from each participant online prior to survey commencement.Study data were collected and managed using REDCap electronic data capture tools hosted at the Centre for Eye Research Australia. Survey questions were developed after a review of previous survey literature and in consultation with ophthalmologists, radiologists and dermatologists to ensure face validity. Example scenarios and related occupational fields differed between surveys designed for each specialty. Draft surveys were circulated to executives from the RANZCO, the RANZCR and the ACD for testing. The survey consisted of 18 multiple choice and open-ended questions  generated a list of initial codes after reviewing responses. Codes were built into broader categories and recurring themes were developed. Thematic discrepancies were resolved via discussion. Responses to questions regarding advantages and disadvantages of AI were analysed as follows: the first preference of each respondent received a score of 3, the second preference a score of 2 and the third preference a score of 1. Scores were tallied for each response and divided by the number of respondents to provide an overall score for each response. Higher scores indicate higher rankings. Scores were displayed as radar plots . A complete-case analysis was conducted (i.e., incomplete surveys were excluded from the analyses, see Supplementary Table ts Figs. , 8.Supplementary Tables.


The targeted recognition, penetration of blood vessels, intercellular penetration, special intracellular distribution (escaping from lysosomes and accumulating in Golgi and nucleus), 3D multicellular tumor spheroids (3D MTSs) penetration, degradation of tumor extracellular matrix (ECM), and reversal of MDR based on the synergistic effects of the motion ability and sustained NO release performance of the NO‐driven nanomotors are investigated in detail. Correspondingly, a new chemotherapy mode called recognition‐penetration‐reversal‐elimination is proposed, whose effectiveness is verified by in vitro cellular experiments and in vivo animal tumor model, which can not only provide effective solutions to these challenges encountered in cancer chemotherapy, but also apply to other therapy methods for the special deep‐tissue penetration ability of a therapeutic agent.Poor permeation of therapeutic agents and multidrug resistance (MDR) in solid tumors are the two major challenges that lead to the failure of the current chemotherapy methods. Herein, a zero‐waste doxorubicin‐loaded heparin/folic acid/ Deep drug penetration and effective reversal of multidrug resistance in cancer chemotherapy can be efficiently realized by motion ability and sustained release of nitric oxide (NO) by NO‐driven nanomotor. Among them, the existence of the ECM mainly comprised of collagen has been regarded as the key barrier that should be conquered. Besides, MDR of cancer cells may also appear simultaneously in the process of chemotherapy, making the treatment situation even more complicated. So far, it has been reported that more than 90% of cancer deaths are affected by MDR, which is primarily caused by the overexpression of certain proteins such as P‐glycoprotein (P‐gp) on cell membranes that excrete antineoplastic drugs. Therefore, solving the above problems to improve cancer cure rate becomes the research focus of the biomedical science field.One of the major bottlenecks of cancer chemotherapy methods which lead to treatment failure is that most of the therapeutic agents cannot reach the inner tumor tissues due to their limited penetration ability,3, 4 However, the former may face the challenge that the different tumor types have different internal stimuli, which need to be studied and dealt with separately. And the weak tissue penetration of external stimuli also limits the systemic effect of the latter strategy. Most recently, viruses, pathogens, and lipoproteins have been utilized as natural nanocarriers owing to their high‐density lipoproteins, which are endogenous proteins that can be used to deliver different drugs because they can promote drug permeability in tumors. Yet, their clinical applications are still disputed, which may be attributed to the possibly occurred endogenous protein instability or immune disorders. In particular, most of the above strategies are to solve the problem of the cell membrane or intercellular permeability, and only a few studies involve strategies for the degradation of ECM outside the cancer cells. As for the reverse of MDR of cancer cells, based on the fact that the phenomenon of MDR is due to the increased expression of certain protein in cell membrane, which has the function of pumping out drugs, leading to drugs being blocked or discharged before reaching the intracellular target. The traditional way to solve this problem is to inhibit the MDR pumping effect or use nanocarriers to load a large number of drugs, which often brings unnecessary toxic substances into the organism. Besides, the problem of poor drug permeability also limits the effect of reversing MDR.Currently, many researchers have used different strategies including internal  and external stimuli  to improve the tumor penetration of therapeutic agents.14 How In particular, previous studies have shown that NO can react with superoxide anion in the tumor environment to form oxidant peroxynitrite (ONOO−), so as to stimulate the production of active matrix metalloproteinases (MMPs) in the tumor stroma which can degrade almost all collagen components in ECM, improving the penetration of the therapeutic agent in the tumor site. In addition, NO can also be used as an effective reverser of the MDR of the tumor by inhibiting P‐gp expression. Yet, the application of NO has been greatly limited by its short half‐time and high sensitivity to the biological environment.It is quite necessary to design a new therapy system that can simultaneously solve the above problems for cancer chemotherapy. The special physiological function of NO draws our attention to this field. As a signal molecule produced by organism body, NO has been selected as the molecule of the year in 1992 owing to its specific physiological and pathological function.21 In  Especially, nanomotors with self‐propelled motion ability display rapid cell internalization owing to their cell friendly surface chemistry and fast movement inside or across cells that are driven by their fuels. Besides, such strategies depending on physical motion behavior to promote intracellular access may overcome the heterogeneous property of different tumors and cause no immune disorders, making them among the most proper candidates to accomplish the above tasks. This prompted us to design a new kind of NO‐driven nanomotor to solve the existing problems of poor drug permeability, MDR, and unstable supply capacity of NO for cancer chemotherapy simultaneously, which has never been reported before.Our previous work reported a novel NO‐driven nanomotor, which can provide a continuous supply of NO to overcome its short half‐time and has the ability to move in cellular environment.24 Espl‐arginine and anticancer drug doxorubicin (DOX), forming HFLA‐DOX nanomotors  and reactive oxygen species (ROS) in a tumor environment than normal cells, HFLA nanomotors can produce NO to drive the nanomotors, facilitating their intracellular distribution and intercellular transportation process. In the meantime, the produced NO can form ONOO− to promote the degradation of collagen components in ECM, further promoting extracellular penetration of nanomotors in tumor tissues. Subsequently, the MDR of cancer cells can be reversed by NO and the released DOX can further efficiently complete the anticancer effect, thereby achieving better chemotherapeutic effect. Besides, anticancer drug HF and NO with a higher concentration can also assist DOX in killing cancer cells during this process.Specifically, we choose the heparin/folic acid (HF) nanoparticle (anticancer drug and cancer cell‐targeting agent) with a cage‐like structure as carrier to load s Figure 1A, which , 3D MTSs penetration, detailed mechanism of tumor tissue ECM degradation, and in vivo penetration. We fully demonstrate the role of NO and the effect of motion ability of HFLA‐DOX nanomotors during this process, which may provide a scientific strategy and method to study the effectiveness of nanomotors in cancer therapy.The important and complex effectiveness of NO in chemotherapy have been proposed by researchers for a long time.35 Tab2 The chemical structure of HF can be verified by 1H NMR spectrum  and the mass ratio of l‐arginine in HFLA nanomotor is about 10% . Transmission electron microscopy (TEM) results display that the average particle size of HF is about 30 nm .29 Them Figure . HFLA shm Figure ,D. The pd Figure  and veriR2 = 0.999), indicating that the motion of nanomotors can be regarded as a self‐propulsion process.Then, we tested the duration of NO release from nanomotors under a cellular environment. The results display that the HFLA nanomotors can release NO for at least 12 h Figure . Besidesl‐NMMA acetate) to treat the cancer cells. It was found that the amount of NO production was obviously decreased, and the movement of the nanomotor was also notably suppressed , indicating that the existence of NOS in cells play an important role for NO production and motion ability of the nanomotors ‐2‐amino‐5‐(2‐methylguanidino) pentanoic acid compound with acetic acid, s Figure . The low uptake of the nanomotors by macrophages was also confirmed by results in Figures S11 and S12, Supporting Information, in which the nanomotors display low cytotoxicity to macrophages and few nanomotors (blue color) can be engulfed by macrophages during the incubation process (1 h).The biocompatibility of the nanomotors was also investigated, which shows that the hemolysis rates of HF and HFLA are below 2% , indicating their good biocompatibility. In the meantime, it has been regarded that the electronegative property of these nanoparticles can protect them from being swallowed by macrophages.37 TheThe above results imply that the nanomotors proposed in this work may be ideal for the sustained delivery of NO and drugs into tumor., cells (MCF‐7/ADR cells) with or without (HUVECs) overexpressed folic acid receptors were used in this part. The results suggest that folic acid in HFLA nanomotors may facilitate them to target cancer cells .Based on the fact that HF of the nanomotors can target the cancer cells by reacting with the overexpressed folic acid receptors in their membranes, the targeting ability of the nanomotors to cancer cells were detected. The overexpressed folate receptor in cancer cells were assessed by western blots and immunofluorescence assay. As shown in Figures S13 and S14, Supporting Information, the folate receptor protein (MW = 38 kDa) was highly expressed in MCF‐7/ADR cells, whereas it was lowly expressed in normal cells such as human umbilical vein endothelial cells (HUVECs). The nanomotors with  and without  targeting groups folic acid24, ce . The results depict that very weak red signals representing DOX can be detected in the lower chamber in HF‐DOX+l‐arginine group, indicating little effect of NO on the penetration of drugs between cells 38 (Fiβ‐cyclodextrin (blocking both clathrin and caveolae‐mediated pathways), cytochalasin D (macropinocytosis inhibitor), and 4 °C (energy inhibitor) were chosen to pretreat cancer cells for 2 h before treatment with nanomotors. It can be seen clearly that very weak red fluorescence signals appears in cells after being treated with chlorpromazine, nystatin, and β‐cyclodextrin and the relative fluorescence signals decrease to about 0.5, 0.2, and 0.1, respectively  appears in the cells for the DOX group , and then use the movement of the nanomotors to promote their endocytosis and exocytosis by the next adjacent cell, effectively completing cell‐to‐cell transmission.As intracellular distribution is a key factor in the transport and effectiveness of nanomotors, thus, we studied the distribution of nanomotors in cells after they entered the cells. The co‐localization of DOX/nanomotors and lysosomes after being treated with for 5 min illustrates that almost all the internalized DOX is located in the lysosomes  and (d) in Figure It is worth noting that by comparing the HF‐DOX+l‐arginine group are much lower than that of the nanomotor group (about 120 µm and 3000 a.u.), indicating that the motion behavior of nanomotors can greatly promote the penetration process of drugs in 3D MTSs  in which their motion ability can effectively facilitate these processes  and fluorescence intensity across the 120th µm section of the 3D MTSs (about 1000–2000 a.u.) for HF‐DOX+s Figure 4A,B. In   in ECM. In order to fully study the role of NO and ONOO− during this process, HFLA+uric acid group with motion ability while ONOO− would be eliminated by uric acid was set for comparison . The NO release in tumor tissues was assessed first. It was found that neither the control group nor the HF‐DOX group can generate obvious NO in tumor tissues due to their lack of l‐arginine. The active ingredient capable of producing NO include HFLA, HFLA‐DOX, and HFLA‐DOX+uric acid group can produce a plentiful amount of NO with similar fluorescence intensities (the fluorescence intensity is about three times that of the control group)  in proteins (biomarker of ONOO− activity) to characterize the formation of ONOO−. An obvious brown color (representing 3‐NT) can be found in HFLA and HFLA‐DOX group instead of HFLA‐DOX+uric acid group, indicating that the ONOO− scavenger uric acid can significantly suppress the ONOO− formation  were chosen for analysis by using in situ zymography method. A significantly enhanced expression and activity of the MMP can be found in HFLA and HFLA‐DOX groups while a low activity of MMP is found in HFLA‐DOX+uric acid group  in ECM.6 In on Figure ,D. Next,− production ability. The HFLA‐DOX group shows superior penetration ability, almost all the inside of the tumor is filled with red fluorescent signal, and the signal intensity is about three times that of the HF‐DOX group. The group of HFLA‐DOX+uric acid shows a certain degree of reduced penetration intensity , but it is still stronger than the fluorescence intensity of the HF‐DOX group, implying that the motion behavior of the nanomotors can also promote the penetration process.The above results confirm the unique effectiveness of motion ability and NO production of the nanomotors on promotion of cellular uptake, intracellular distribution, intercellular transport, and tumor ECM degradation ability using cellular and in vivo model, respectively. In this part, the synergistic promotion effect of motion ability and NO of the nanomotors proposed in this work to the deep penetration in tumor tissue will be demonstrated by in vivo characterizations. As shown in Figure − production of HF‐DOX. The HFLA‐DOX group with both motion ability and ONOO− exhibits much better drug diffusion performance in both amount and depth (relative fluorescence intensity is larger than 3.5). The red fluorescence signals not only appear around the blood vessel, but also penetrate to a considerable distance from the blood vessel. The uric acid‐treated group displays better penetration ability than HF‐DOX group (relative fluorescence intensity is about 1.7 times that of the HF‐DOX group) but poorer than that of HFLA‐DOX group. These results confirm the synergistic effect of the nanomotor's motion ability and the biomedical functions of NO on the deep penetration of drugs in tumor tissues.Furthermore, immunofluorescence staining for the blood vessels inside the tumor to determine the penetration of the nanomotor in the tumor blood vessels was performed, and the fluorescence signal intensity of the circular area with the same distance from the blood vessels was quantified , which further decrease with the increasing concentration of HFLA‐DOX nanomotors . In the meantime, 3D MTSs were also used as a tumor model in vitro. For HFLA‐DOX nanomotors, the 3D MTSs dissociated and finally lost 3D structure, which can be eliminated within about 8 days, implying that the enhanced drug penetration into tumor spheroids by the help of the motion ability of nanomotors can promote their antitumor efficiency . That is to say, the specific components of nanomotor participate in tumor recognition (HF), facilitated penetration (l‐arginine and NO), reversal of MDR (NO), and elimination of tumor  process, respectively. This is also the biggest difference between the zero‐waste/biomimetic NO‐driven nanomotor proposed in this work and various types of existing nanomotors. Using the important physiological process of the human body to solve the difficulties in the treatment of diseases in vivo is the original intention of our design of a bionic zero‐waste nanomotor, and the effect of the reaction of human body itself and its raw materials can always bring us more surprises.Based on the above results, we tentatively propose a recognition‐penetration‐reversal‐elimination model for drug‐resistant tumors based on drug‐loaded NO‐driven nanomotor (detailed discussion is shown in Supporting Information). The nanomotor first uses folic acid to target tumor sites by recognizing folic acid receptors that were overexpressed in tumor cells. Then, the specific high concentration of NOS and ROS at the tumor site could effectively trigger the movement of the HFLA‐DOX nanomotors, thereby enhancing their penetration ability in tumor cells. The released NO can reverse the MDR of the tumors, and DOX loaded on the tumors finally completes the task of eliminating the tumors. In particular, anticancer drug HF and NO with a higher concentration can assist DOX in completing the combined killing of tumors in this process.48 It 3In summary, a strategy for cell‐and‐tissue deep penetration for MDR solid tumor relying on a bio‐inspired zero‐waste nanomotor (HFLA‐DOX) was constructed, and a chemotherapeutic model called recognition‐penetration‐reversal‐elimination for cancer therapy was proposed and verified in this work. These zero‐waste nanomotors had good motion ability in both aqueous condition and 2D/3D cellular environment, which can accumulate in the tumor site and be triggered by specific higher ROS and NOS concentration in tumor to produce NO as driving force for nanomotors and the reversal agent for MDR tumor. Thanks to the motion behavior of nanomotors, the versatility of NO can be fully utilized, including driving force of the nanomotors, promoting transvascular penetration, escaping lysosomes, accumulating in Golgi and nucleus, ECM degradation, reversal of MDR, and anticancer effect. In vitro and in vivo results imply that the HFLA‐DOX nanomotors proposed in this work could overcome the drug‐resistant tumor and penetration obstacles existing in current tumor treatment modalities. This work provides the illustration of zero‐waste NO‐driven nanomotors to realize the simultaneously deep cell‐and‐tissue dual‐penetration and reversal of MDR of tumor, which is different from any existing nanomotors that use physical effects to improve the penetration ability of therapeutic agents in tumors, offering possible clues for the development of a new avenue for cancer therapy.4Methods and any associated references are available in the Supporting Information.All animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals published by the National Academy of Sciences, and all experiments procedures and protocols were approved by the Animal Research Committee of Drum Tower Hospital .The authors declare no conflict of interest.Supporting InformationClick here for additional data file.Supplemental Movie 1Click here for additional data file.Supplemental Movie 2Click here for additional data file.Supplemental Movie 3Click here for additional data file.Supplemental Movie 4Click here for additional data file.Supplemental Movie 5Click here for additional data file.Supplemental Movie 6Click here for additional data file.Supplemental Movie 7Click here for additional data file.Supplemental Movie 8Click here for additional data file.Supplemental Movie 9Click here for additional data file.


Saccharomyces cerevisiae found that Dox had no effect on genome‐wide gene expression as measured by microarray. In contrast, another study found that the use of Dox in common cell lines and several model organisms led to mitonuclear protein imbalance, suggesting an inhibitory role of Dox in eukaryotic mitochondria. Recently, a new Dox derivative, 4‐epidoxycycline (4‐ED) was developed that was shown to have less off‐target consequences on mitochondrial health. To determine the best tetracycline family chemical to use for gene expression control in S. cerevisiae, we performed RNA sequencing (RNA‐seq) on yeast grown on standard medium compared with growth on media supplemented with Tet, Dox or 4‐ED. We found each caused dozens of genes to change expression, with Dox eliciting the greatest expression responses, suggesting that the specific tetracycline used in experiments should be tailored to the specific gene(s) of interest when using the Tet‐On/Tet‐Off system to reduce the consequences of confounding off‐target responses.Tetracycline (Tet) and derivative chemicals  have gained widespread recognition for their antibiotic properties since their introduction in the late 1970s, but recent work with these chemicals in the lab has shifted to include multiple techniques in all genetic model systems for the precise control of gene expression. The most widely used Tet‐modulated methodology is the Tet‐On/Tet‐Off gene expression system. Tet is generally considered to have effects specific to bacteria; therefore, it should have few off‐target effects when used in eukaryotic systems, and a previous study in the yeast  Cells were then pelleted and used for RNA extraction. Cultures were grown in biological triplicate  for downstream transcriptional profiling  R1158 (URA3::CMV‐tTA MATa his3‐1 leu2‐0 met15‐0) which was derived from BY4741  with a modified protocol  under accession number GSE155989 (to be available at time of acceptance).33.1q < 0.05) with 15 upregulated and seven downregulated  whereas this was not observed for Tet treatment . When we compared the lists of differentially expressed gene (DEGs) for the three treatments, we found that there was very little overlap in identified genes that had annotations . Although only a few genes are found to overlap between identified gene sets, the amount observed is significantly greater than expected by chance in each pairwise comparison .To determine which common tetracycline was best suited for control of yeast gene expression, we compared yeast grown in control media to yeast grown in media containing 1.5 μg/ml of Tet, Dox or 4‐ED. When yeast were grown in media containing Tet, 22 genes were significantly differentially expressed , cell cycle processes and terms associated with the replication fork Table . Additio4S. cerevisiae, we performed RNA‐seq on yeast grown on standard medium compared with growth on media supplemented with Tet, Dox or 4‐ED to identify changes in gene expression caused by this exposure. We found that although the numbers of genes with response to Tet, Dox or 4‐ED are not large, there are dozens responding to each chemical. In fact, the results for Dox are in contrast with a prior report that found no genes responded to Dox using microarrays , suggesting an increase in cell cycle and mitosis when yeast are exposed to Dox, similar to that observed for DNA replication genes. This is in contrast to the slowed growth rate we observe for yeast exposed to Dox in their media, and the disconnect between these processes requires further studies. All three of the major GO terms enriched point to upregulation of processes associated with the S phase of the cell cycle suggesting some mechanistic link between these identified genes. It is also possible that all the genes identified have a common regulatory element(s) that are all responding to the presence of Dox directly or indirectly and there may not be a specific functional relationship among the regulated genes. Either way, our findings suggest that use of Dox may not be ideal for studies focused on DNA replication, DNA repair, cell cycle, chromosome cohesion or any genes associated with or downstream of these processes.When 4.2S. cerevisiae was grown on medium containing 4‐ED, various GO terms for arginine catabolism were enriched among the genes differentially expressed with both genes directly involved in arginine catabolism downregulated in the presence of 4‐ED (CAR1 and CAR2). Changes in the expression of these genes could alter processes including protein synthesis as well as nitrogen utilization. Genes with GO terms associated with glucose and fructose metabolism were also differentially expressed when yeast were grown on medium containing 4‐ED. Among the four genes involved in glucose and/or fructose metabolism, two were downregulated in the presence of 4‐ED (HXK2 and PFK27) and the other two were upregulated (PCK1 and FBP1). Reductions in expression for key enzymes in metabolic processes including glucose and/or fructose metabolism could explain previous reports of reduced mitochondrial function in widespread organisms including mammalian cells (human and mouse), nematodes, fruit flies and plants  for study of the genes identified herein as DEGs or genes involved in associated biological processes seems potentially problematic. That said, the majority of the genome does not respond to these chemicals suggesting they should still be available for widespread use in the testing of gene function as long as their limitations are first considered. Moving forward, we suggest that when using the yeast Tet‐On/Tet‐Off system for inferences about genome‐wide gene expression, controls similar to those performed here (comparing control to tetracycline exposure in background strains) should be included in experimental designs to ensure that off‐target effects are not the source of any observed effects on gene expression.Due to the flexibility, precision and specificity of the changes that can be achieved using the Tet‐On/Tet‐Off system, it has become a powerful tool in research aiming to control gene expression in many model systems. Despite these major benefits, caution when using these chemicals to study gene function and expression is recommended. Recent reports have already suggested a potential negative impact of these antibiotics on the environment and health (Moullan et al., The authors declare no conflicts of interest.Table S1: List of significantly differentally expressed genes upon exposure to 1.5 μL TetTable S2: List of significantly differentally expressed genes upon exposure to 1.5 μL DoxTable S3: List of significantly differentally expressed genes upon exposure to 1.5 μL 4‐EDTable S4: GO Term enrichment of significantly expressed genes when treated with DoxTable S5: GO Term enrichment of significantly expressed genes when treated with 4‐EDClick here for additional data file.


A fluorescence background is one of the common interference factors of the Raman spectroscopic analysis in the biology field. Shifted‐excitation Raman difference spectroscopy (SERDS), in which a slow  modulation to excitation wavelength is coupled with a sequential acquisition of alternating shifted‐excitation spectra, has been used to separate Raman scattering from excitation‐shift insensitive background. This sequential method is susceptible to spectral change and thus is limited only to stable samples. We incorporated a fast laser modulation (200 Hz) and a mechanical streak camera into SERDS to effectively parallelize the SERDS measurement in a single exposure. The developed system expands the scope of SERDS to include temporary varying system. The proof of concept is demonstrated using highly fluorescent samples, including living algae. Quantitative performance in fluorescence rejection and the robustness of the method to the dynamic spectral change during the measurement are manifested. We present parallelized shifted excitation Raman difference spectroscopy by the synchronized operation of a mechanical streak camera with a wavelength tunable light source. The parallel detection scheme facilitates the effective and accurate fluorescence rejection from a temporary varying system having faster dynamics than the spectral acquisition rate. Quantitative performance in fluorescence rejection and the robustness of the method to the dynamic spectral change during the measurement are manifested. CCDcharge‐coupled deviceCWcontinuous wavedeform.deformationMCR‐ALSmultivariate curve resolution‐alternate least squaresPCAprincipal component analysisP‐SERDSparallelized shifted‐excitation Raman difference spectroscopystr.stretch1In recent decades, the application of Raman spectroscopy to biological and medical studies has been expanded vastly, especially in the field of a living cell and tissue characterization Currently, the most frequently used branch of fluorescence eliminating techniques is an ad hoc baseline subtraction by polynomial curve fitting To overcome above issues, numbers of fluorescence rejection methods based on solid physical principles, such as temporal In this study, we took a further step to expand the scope of the SERDS technique to include unstable, dynamic samples by effectively parallelizing the measurement. Herein, we first outline the principle of the developed parallelized SERDS (P‐SERDS). Then, proof of concept studies using stationary fluorescing artificial dye solution and dynamic living algae cells are reported. High‐quantitative performance in fluorescence rejection, as well as the robustness of the method to the dynamic spectral change during a signal accumulation, is manifested. Finally, we demonstrate the superior compatibility of the proposed method to multivariate chemometrics in comparison with the conventional fluorescence removal method using the polynomial baseline fitting.2Similar to other SERDS instruments, the P‐SERDS utilizes a wavelength tunable laser for fast wavelength switching. Parallelization of the measurement is realized by synchronization of fast laser tuning with a modulation to a mechanical streak camera. A mechanical streak camera is composed of a galvano mirror and a spectrograph, in which the galvano unit is used to control the height of signal light entering the spectrometer. Without invoking a detector readout, the device can designate and modulate the vertical position on the two‐dimensional detector pixels on which signal light is exposed. By driving the streak device to switch detection pixels repeatedly in conjunction with the laser tuning, it is possible to parallelly record multiple spectra excited by the different laser wavelengths in a single exposure.The advantage of the present parallel configuration is the independence of the spectral acquisition rate and the modulation frequency. Long (seconds to minutes) exposures for weak Raman scatterers can be performed, while employing orders of magnitude faster wavelength switching frequency (up to 200 Hz). Owing to this fast wavelength switching, a dynamically changing sample may be considered virtually unchanged during a single set of shifted‐excitation in 5 ms. Slower changes in the spectrum during the total exposure will be recorded equally in all the parallelly accumulated spectra, therefore will not affect the subsequent fluorescence separation.We note that, recently, the concept of parallelization of SERDS has also been demonstrated by Sowoidnich et al. 33.13.1.1Figure 3.1.2The output wavelength of the laser system and the vertical detection position of the streak unit were both controlled by external voltage signals. The laser tuning and the streak scanning were synchronized by the application of concerted voltage waveforms produced by a PC‐controlled function generator , shown in Figure Waveforms were designed so that each spectrum excited at different wavelengths was separately recorded at each designated vertical position of the CCD pixels.−1) range with the help of a built‐in feed‐forward adjustment circuit, we deliberately operated the laser without the feed‐forward function to achieve repeated mode‐hopping between two discrete frequencies with wider wavelength difference. By optimizing the center voltage and the modulation amplitude of the input damped square waveform, we manage to generate mode‐hopping cycle between two wavelengths of ca. 0.1 nm (5 cm−1) difference in 5 ms period (200 Hz), which was appropriate for P‐SERDS experiments.Here, we note that although the DL Pro laser system was capable of mode‐hop free continuous frequency tuning in 60 GHz  of the CCD readout, and the short duration intermediate level is used to discard the emission during the laser wavelength transition to outside of ROI. The typical spatial distribution of charges on the detector after a single P‐SERDS exposure is represented in Figure 3.1.3−1). The average laser power at the sample and the exposure duration were 1.0 mW and 60 seconds, respectively. For the time‐lapse P‐SERDS measurements of algae cells, the excitation wavelength shift and the laser power were set to 0.13 nm (5.3 cm−1) and 2.7 mW, respectively. Other measurement parameters were the same as those for the dye samples.The P‐SERDS measurements of artificially prepared dye samples were performed under the synchronized modulation frequency of 200 Hz, with the excitation wavelength shift of 0.07 nm , was performed using laboratory‐developed analysis programs on Igor Pro 7 or 8 . MCR‐ALS with the non‐negativity constraint was performed using Sci‐kit learn package  3.2.1−1). Resampling was necessary because the raw spectra from different ROIs were sampled at slightly different wavelength points.Prior to SERDS analysis of the acquired spectra, the obtained spectra were subject for spectral preprocessing for the correction and calibration. Spectral abscissa of each ROI is individually calibrated to wavelength by reference to atomic emission lines of a neon lamp, and Raman shift by Raman peaks of indene. The spectra were, then, individually processed for dark count subtractions, pixel sensitivity corrections by white light, and then resampled by interpolation at common wavelength series of a constant wavenumber interval  to yield a differential Raman spectrum. A reconstructed Raman spectrum was calculated by a simple one‐dimensional numerical integration of the differential spectra; wherein, the integral constant was chosen so that the minimum value of the output spectrum became zero. Note that this choice of integration constant assumes that the observed Raman spectra contain the region of no Raman band in the observation window. This assumption may be generally valid when the “silent” region is included. However, to avoid ambiguity in the discussion, the reconstructed spectra are mainly used for the qualitative evaluation of Raman responses in this study. Alternative reconstruction algorithm based on a deconvolution calculation 3.2.3Principal component analysis (PCA) mathematically reconstructs the input data based on its covariance to a set of principal components (PCs). Since the noise in the data has little covariance to physical signals, the process can effectively separate the signals from the noise. The number of significant (non‐noise) PCs was estimated based on a statistical test for the noise characteristics of both loading  and score  vectors 3.2.4Fluorescence rejection by polynomial baseline fitting was performed using the procedure outlined in the literature 3.3Chlamydomonas sp. JSC4, isolated from the coast of Southern Taiwan, were obtained through the courtesy of Dr. Shih‐Hsin Ho and Dr. Liang‐da Chiu. The cells were cultured in modified Bold 6 N medium Fluorescein  and ethanol (Guaranteed Reagent grade) were both purchased from Wako Pure Chemical Corporation and used as received. The algae cells, 4−3). The spectra are plotted against wavelength (bottom axis) and, for the reference, the corresponding Raman shift for one of the excitation wavelengths is displayed on the top axis. Inset displays the expanded view of the graph for the 2000 nmol dm−3 sample, in which paired spectra with shifted vibrational bands are made visible. As is expected, each pair of spectra almost perfectly overlaps one another except for the small Raman peak shifts due to excitation wavelength shift. Every spectrum exhibits characteristic vibrational bands of ethanol at 432, 882, 1051, 1094, 1274, 1452 and 1480 cm−3. On top of these bands, broad fluorescence background emerges to rise concertedly with dye concentrations.We examined the fluorescence rejection capability of our proposed method using artificially prepared samples that exhibit stationary fluorescence background. Figure −3) was identical to the original observed Raman spectrum. Hence, we conclude that no Raman vibrational information was lost by P‐SERDS analysis. In the case of fluorescent samples, slight band shape distortions with a fluctuating baseline become more apparent in the reconstructed spectra as the dye concentration increases. Nevertheless, all the reconstructed spectra present the characteristic peak pattern of ethanol and are proven to be useful for the qualitative analysis, such as general bands assignments and spectral matching. The possible origins of the band shape distortion are discussed in the latter paragraph.Figure −1 (estimated by the minimum to maximum intensity difference of the dispersive curve) against the background‐to‐signal ratio (B/S) of the original spectra. The result is shown in Figure In order to validate the quantitative Raman response in the differential spectra, we plotted the ethanol peak intensity at 882 cmOn the other hand, the polynomial subtraction methods show highly dependent results (open symbols) on the choice of polynomial order. When polynomial order is low , the estimated Raman intensity follows linear dependence to the B/S ratio. This behavior is due to the presence of the residual background component in their Raman spectra after the background subtraction. Although the use of higher polynomial order  seems to resolve the problem, the result demonstrates one of the inherent drawbacks of the method, in which the analysis is susceptible to the choices of the analytical parameter.Next, we applied our P‐SERDS method to living cells in order to demonstrate its feasibility to temporally unstable samples. We chose algae cells for this purpose. Microalgae are a large group of unicellular species possessing a photosynthesis system. They are considered as a prospective source of biofuel production, and much research has been devoted to engineering novel or enhanced synthesis pathways of energy‐rich metabolites, lipids and carbohydrates in algae −1, CH3 deformation [deform.] at 1449 cm−1, amide III at 1253 cm−1, phenylalanine ring at 1001 cm−1); lipids  and glycogens  Time‐lapse spectra series Figure  of a sinNext, to assess the efficacy of fluorescence rejection in multivariate analysis, we constructed a P‐SERDS time‐lapse spectral data set, and it was subjected to PCA. We also performed the polynomial baseline subtraction to the same time‐lapse series. This polynomial subtracted data set and the original fluorescence unremoved time‐lapse series were also processed by PCA for comparison. Table PCA analysis revealed that the original observed time‐lapse data set of a single alga has a little complicated nature than expected, carrying three independently varying components Figure B. In genIn contrast to the fluorescence unremoved result, the PCA result of the P‐SERDS data set exhibited benefits of prior fluorescence rejection by much simplified composition as it yielded only one significant component Figure A. This sUnlike the P‐SERDS result, the polynomial fitting approach Figure C did notNext, we further extended our study to demonstrate the P‐SERDS application to assess cell‐to‐cell individuality of algae by multivariate resolution techniques. A time‐lapse Raman spectra series of six individual algae cells were concatenated to form a large matrix. This total data set was then processed for PCA analysis. As a result, we found two significant components Figure . The pre−1, as well as triplets at 1128, 1099 and 1061 cm−1, which are the signature of all‐trans C—C chain conformation of saturated fats in solid phase The decomposed Raman spectra obtained from MCR‐ALS analysis, presented in Figure Next, we remark the applicability of the P‐SERDS method to various Raman measurements. The method can also be used to reject non‐fluorescent background, for example, spontaneous emissions from the sample and ambient light from the environment, as long as they are independent of the excitation wavelength shift. The essential devices, the tunable laser and the galvano mirror unit, can be easily incorporated into a working single‐focus Raman spectroscopic system equipped with a two‐dimensional detector, with the least modification to the optical layout. Therefore, the method is not limited to a biological investigation and applies to a wide variety of research subjects whenever the interference of background is inevitable.Finally, we discuss some of the limitations of the present P‐SERDS method. Firstly, as is partly seen in Figure 5In this study, we have developed P‐SERDS by the synchronized operation of a mechanical streak camera with a wavelength tunable light source. The parallel detection scheme proposed by the present method facilitates the effective and accurate fluorescence rejection from the temporary varying system with much faster dynamics than the spectral acquisition rate, thus expanded the scope of SERDS technique to include broader and more practical biological application. The highly accurate background rejection performance of P‐SERDS for such dynamic samples is demonstrated to be beneficial, especially for the multivariate analysis of Raman data set by reduction of the number of retained compositions. This simplification of the analytical result not only helps in further quantitative and qualitative assessments but also promotes the development of clearer biochemical insight into the target system. We believe that the present method has enormous potential in boosting the reliability and applicability of Raman spectroscopy in biological research, and thus empowers related discrimination, diagnosis and quantification applications in the near future.The authors declare no financial or commercial conflict of interest.Figure S1. Time‐lapse Raman spectra series of a single alga by parallelized shifted‐excitation Raman difference spectroscopy (P‐SERDS).Figure S2. First five loading spectra of principal component analysis for the concatenated time‐lapse series of six algae cells.Click here for additional data file.


Abundant citizen science data on species occurrences are becoming increasingly available and enable identifying composition of communities occurring at multiple sites with high temporal resolution. However, for species displaying temporary patterns of local occurrences that are transient to some sites, biodiversity measures are clearly dependent on the criteria used to include species into local species lists. Using abundant opportunistic citizen science data from frequently visited wetlands, we investigated the sensitivity of α‐ and β‐diversity estimates to the use raw versus detection‐corrected data and to the use of inclusion criteria for species presence reflecting alternative site use. We tested seven inclusion criteria (with varying number of days required to be present) on time series of daily occurrence status during a breeding season of 90 days for 77 wetland bird species. We show that even when opportunistic presence‐only observation data are abundant, raw data may not produce reliable local species richness estimates and rank sites very differently in terms of species richness. Furthermore, occupancy model based α‐ and β‐diversity estimates were sensitive to the inclusion criteria used. Total species lists  may therefore mask diversity differences among sites in local communities of species, by including vagrant species on potentially breeding communities and change the relative rank order of sites in terms of species richness. Very high sampling effort does not necessarily free opportunistic data from its inherent bias and can produce a pattern in which many species are observed at least once almost everywhere, thus leading to a possible paradox: The large amount of biological information may hinder its usefulness. Therefore, when prioritizing among sites to manage or preserve species diversity estimates need to be carefully related to relevant inclusion criteria depending on the diversity estimate in focus. Very high sampling effort can produce a pattern in which many species are observed at least once almost everywhere, thus leading to a possible paradox: The large amount of biological information may hinder its usefulness. The distinction between site use based on ecologically informed species inclusion criteria for determining composition of species communities is particularly relevant when prioritizing among sites to manage or preserve. These data are largely composed of citizen science presence‐only records  at a site i during day d and year t, following Kéry et al. . For computational reasons, we restricted the maximum number of visits to 40 per day and site, prioritizing visits with the longest species lists, thus reducing the number of single observations in our data to c. 31%. In order to construct data on pseudo‐nondetections, any species not reported during a visit j was considered not detected in that visit. A pseudo‐nondetection then corresponds to a focal species not being observed or reported by an observer reporting at least one other species at the wetland on that day.We obtained data from Artportalen  occurrence status of the species in day d, year t, and site i, and j,d,t,ip is the detection probability of the species in visit j, given that the species is present. To control for variation in effort, we modeled detection probability as an increasing function of a visit's SLL. The steepness of the increase in detection probability with SLL was further allowed to vary among sites, on whether the visit was done during the first or second half of the season, and with the annual proportion of long species lists . In other words, the parameter in the detectability saturation function will vary with each year's general observation behavior (PLL), the species behavior according to whether it is early or late during the season, and independently for each site. See more details on the modeling approach in Supplementary Information In order to estimate daily site‐ and species‐specific occupancy probabilities, we employed a site‐use model  and random effects for site and year. We modeled the effect of the doy as a quadratic function to allow the colonization and persistence parameters to increase, decrease, or both within the season. In this way, the model may be suitable for a wider range of species with different phenologies.The occurrence status The models were fitted separately to data for each species in the Bayesian framework using JAGS Plummer, . For detWe fitted the site‐use model to data over all 10 years from 2005 to 2014. Using multiple years as input to the model allows us to better estimate detection probability parameters (via species list length and yearly proportion of long species lists) and the colonization–extinction process by sampling more independent annual colonization and extinction events. However, to simplify the presentation of results we only estimated local species richness and derived bird diversity measures for year 2014.2.3iSday) obtained by summing the posterior mean of daily occurrence probabilities for each species and site. As shown by other authors, probability‐based richness is not prone to be biased by the amount of suitable habitat occupied by a species  to the estimated daily local species richness . For example, the observed richness under the 10‐day criterion at a wetland is the number of species that were observed on at least 10 different days.We also estimated local species richness based on the estimated daily occurrence status that was corrected for detection and reporting probability. Given we estimated the daily occurrence dynamics per site, the criteria for inclusion  were considered either in any sequence spread‐out over the season (nonconsecutive) or strictly on consecutive days within the season (consecutive). The occupancy model for each species was used to compute the posterior probabilities that the species were present for at least the number of days required by the threshold at each wetland (see Supplementary Information 2.5βSOR), turnover of species , and dissimilarity by reduction in number of species , using the beta.pair function from the R package "betapart"  in opportunistic observations of birds typically varied from day to day and decreased at the end of the season inducing lower numbers of observed species  into turnover (Simpson index) and nestedness components Baselga,  showed t4In theory, very high sampling effort can produce a pattern in which almost every species is observed almost everywhere, especially for organisms with high dispersal abilities . If the aim is to investigate the stable part of local community, for example, species reproducing at a site, then a large fraction of transient occurring species will also be included when data are abundant and spread over time. Therefore, as the amount of data provided by citizen science is increasing rapidly and can be huge at some frequently visited sites  within 10, 20, and even 30 km radius around a city were shown to give erratic estimates of annual measures of biodiversity, which was suggested to be due to underreporting of mainly common species  and occupancy (any occurrence without consideration of the use of the site) for determining composition of species communities is particularly relevant when prioritizing among sites to manage or preserve species. For instance, complete species lists can mask diversity patterns regarding only species that use the site for reproduction  and for species groups with, generally, somewhat smaller number of observations available ; data curation ; formal analysis ; investigation ; methodology ; validation ; visualization ; writing – original draft ; writing – review and editing . Debora Arlt: Conceptualization ; funding acquisition ; investigation (supporting); validation ; writing – review and editing . Åke Berg: Data curation ; investigation ; resources ; writing – original draft ; writing – review and editing . Jonas Knape: Conceptualization ; formal analysis ; funding acquisition ; methodology ; writing – original draft ; writing – review and editing . Michał Żmihorski: Conceptualization (supporting); methodology (supporting); writing – original draft (supporting); writing – review and editing (supporting). Tomas Pärt: Conceptualization ; funding acquisition ; investigation ; methodology ; project administration ; supervision ; writing – original draft ; writing – review and editing .Appendix S1Click here for additional data file.Appendix S2Click here for additional data file.


In Myanmar, men who have sex with men (MSM) and transgender women (TW) are disproportionately affected by HIV, despite national HIV program scale‐up. However, limited HIV surveillance capacity prevents monitoring of epidemic trends and program impact. This study aimed to estimate HIV prevalence and incidence and explore associated sexual risk behaviours among MSM and TW clients attending HIV testing clinics in Myanmar.An electronic data management system was implemented in two community‐based, MSM and TW ‐tailored HIV testing clinics in Myanmar in August 2016. Unique client identifiers enabled prospective monitoring of service engagement, testing frequency and outcomes. We estimated HIV incidence and rate of HIV diagnosis at baseline testing visit among clients over a 15 month period. Correlates of HIV diagnoses were identified using multivariable logistic regression.2794 MSM and TW were tested for HIV. At their baseline test, 38% of clients reported any previous testing and 93% reported being sexually active over the previous three months, with 74% reporting sex with casual male partners and 28% reporting consistent condom use with casual partners. 291 clients tested positive for HIV for the first time at baseline . Twelve incident cases were detected among 279 clients receiving ≥2 tests . HIV diagnosis at baseline was significantly associated with being a transgender woman or a non‐openly disclosing man who has sex with men, age 26 to 39 years, and reporting no testing history.High HIV incidence and new diagnoses being associated with reporting no testing history points to undiagnosed HIV driving transmissions in Myanmar. Repeat testing was uncommon. HIV programs in Myanmar must focus on promoting frequent HIV testing alongside adequate coverage of education and primary prevention interventions among MSM and TW. Homosexual sex remains criminalised under Myanmar's penal code, and while enforcement of this law appears rare, it acts to legitimise the intimidation or harassment of sexual minorities, contributing to stigma and discrimination as key barriers to HIV testing, treatment and care for MSM and TW HIV epidemiological data in Myanmar is limited largely to infrequent integrated bio‐behavioural surveys In 2016, we developed and implemented an electronic data management system (eDMS) at two community‐based HIV testing clinics targeting MSM and TW in Yangon and Mandalay with the aim of strengthening data collection and reporting practices, and enabling the monitoring of HIV trends in a service delivery context. Using this data, we describe socio‐demographic characteristics, sexual behaviours and HIV testing behaviours and outcomes among clients; identify factors associated with HIV positivity, and; calculate HIV incidence among clients returning to test at the services.22.1The eDMS was implemented within two MSM and TW‐ focused community‐based drop‐in centres (DICs) in Yangon and Mandalay, the two largest urban areas in Myanmar with large, documented MSM and TW populations, relative to other areas 2.2apone ; apwint ; and, thange  The eDMS was designed to replace previous paper‐based reporting systems used to record client visits and testing outcomes at the DICs and was piloted in Yangon and Mandalay over a 15‐month period (August 2016 to November 2017). At their first presentation to the DIC during this period, clients were registered into the system using a unique numeric identifier (UID) to enable prospective data linkage of testing episodes. Client contact information  and basic demographic details  were captured. Consistent with indigenous Myanmar labels of sexuality and gender expression among MSM and TW, clients were categorised through self‐identification as either: At first presentation and every subsequent visit during the 15 month pilot period, peer educators collected the following behavioural information during pre‐test counselling using handheld tablets: reason for HIV testing ; source of referral (self/referred by peer educator/referred by other organization); history of HIV testing (yes/no); time since last HIV test (<6 months/6 to 12 months/1 to 2 years/>2 years); and any contact with peer outreach worker in the past three months (yes/no). Clients who reported being sexually active in the past three months were asked about: sex with casual male partners (yes/no); sex with regular male partners (yes/no); sex with female partners (yes/no); condom use by partner type ; HIV status of regular male partner (HIV positive/HIV negative/unknown/prefer not to answer); experience of sexually transmissible infection (STI) symptoms in the past three months ; paid money or gifts in exchange for sex (yes/no) or received money or gifts in exchange for sex in past three months (yes/no); and means of meeting casual male partners during past three months . A paper‐based version of all eDMS fields was developed in Myanmar language and available for use by DIC staff who did not wish to use the English‐version tablets, or when the tablets were unavailable. Information from these paper‐based forms was subsequently entered into the eDMS by a senior DIC staff member.Following pre‐test counselling, a trained nurse authorised to conduct HIV testing used a two‐test algorithm – Alere Determine third generation HIV 1/2 , followed by HIV 1/2 STAT‐PAK Assay  – to test clients for HIV. Final results were recorded as HIV positive/HIV negative/indeterminate in the eDMS, and delivered to the client with post‐test counselling. Three‐monthly testing was recommended to all HIV negative clients. Clients returning HIV positive or indeterminate results were referred to a local NGO for confirmatory HIV testing as part of initiation of free ART.2.3Service‐level permission was sought and granted from MBCA prior to the implementation of the eDMS to access to de‐identified client records. At the first visit during the pilot period, clients were informed of the use of their de‐identified data for research purposes and provided with a plain language participant informed consent form by peer educators. After describing the aims and duration of the research, permission was sought from clients to include their de‐identified clinic records in future data extractions during the pilot period; clients were informed of their ability to opt‐out and withdraw consent at any stage during this period. A specific field was built into the eDMS which, when checked, meant that records of clients who did not consent would be automatically excluded during data extraction.2.4All data entered into the eDMS between 01 August 2016 and 30 November 2017 were extracted for analysis. To maintain data continuity and fidelity during the eDMS pilot period and for ongoing reporting to program donors, DIC staff continued to use a data entry spreadsheet for recording HIV testing outcome, date of visit and basic demographic information at each testing episode. The eDMS and spreadsheet used the same UID, and the latter was used to validate testing episodes recorded in the eDMS and enhance data completeness. When consolidating the data sources, duplicate records matched by UID and occurring within 28 days of one another were consolidated into a single testing event; this cut off accounted for the reported low likelihood of repeat testing within one month and allowed for delays in data entry into either data source.2.5Data collected during the first HIV test received at the DIC during the pilot period (‘baseline test') was used to describe socio‐demographic characteristics, reported HIV testing history and sexual behaviours, and HIV testing outcomes of MSM and TW. We describe the proportion of clients HIV positive at baseline; defined as the number of individual clients who tested positive at their first test, divided by the total number of individual clients with a valid test result during the pilot period. We examined factors associated with HIV positivity at baseline test through logistic regression and included all variables in both unadjusted and adjusted models. Collinear variables or variables with insufficient observations were excluded from the adjusted model.Repeat testing behaviour was identified among clients receiving more than one HIV test at the service during the pilot period and median follow up time between first and last recorded test was calculated. HIV incidence was calculated among repeat testers, defined as the total number of incident cases divided by total follow‐up time accrued during the pilot period. Incident cases were defined as clients who returned a HIV positive test result during the pilot period following a negative result at their first valid (i.e. determinate positive or negative) test result. Follow‐up time was measured in person‐years (PY) and defined as the time between the first negative result and either a HIV positive result or last recorded negative result.p ≤ 0.05.Analyses were conducted using Stata . Statistical significance in all analysis was set at 2.6Ethics approval was granted by the Alfred Hospital Ethics Committee, Australia and the Department of Medical Research, Myanmar.3apone (44.7%) and 7.7% identified as apwint or TW. Most clients were aged 15 to 25 years (77.1%) and 63.5% were educated to a secondary or tertiary level. Approximately one third of clients reported a lifetime history of HIV testing (38.2%), with over half (55.4%) reporting a previous test at the DIC. Nearly all MSM and TW were sexually active during the past three months (93.0%). Among these clients, most reported sex with casual male partners (74.8%) and approximately one third reported sex with a regular male (31.9%), or a female partner (34.3%). Among those reporting sex with casual partners, 28.7% reported consistent use of condoms. The most commonly reported way of meeting casual male sex partners was via cruising sites (55.0%) and internet/phone‐based applications or websites (28.7%). Approximately one in five clients (18.9%) reported selling sex and 11.8% reported buying sex in exchange for money/gifts during the past three months. Ten per cent of clients reported experiencing STI symptoms in the past three months  and Yangon . Among reporting clients, most identified as 3.1apone (70.8%), 41 were apwint (14.1%), 42 were thange (14.4%) and 2 were identity unknown . Unadjusted and adjusted correlates of testing HIV positive are presented in Table apone  or apwint , be aged 26 to 39 years , and were significantly less likely to report a lifetime history of HIV testing  , of whom 206 were 3.2The 291 clients who tested positive at their first test were excluded from incidence analysis. Among the remaining 2503 clients, 2224 had a single test during the pilot period (88.9%) and 279 were repeat testers . There were no significant differences in sexual risk behaviours among repeat and single‐testing clients (data not reported). Among repeat testers, 12 incident HIV infections were detected across 344 post‐baseline visit testing events and 118.8 PY. Median follow‐up time was 147 days. All incident infections occurred among clients with male gender identity. HIV incidence was 10.1 per 100 PY (95% CI: 5.73 to 17.8). Limited number of incident infections and missing variable data prevented analysis of potential correlates.4To our knowledge, this is the first study to report on correlates of HIV positivity and the first to generate an estimate of HIV incidence among MSM and TW attending a HIV testing service in Myanmar. Approximately 10% of MSM and TW tested positive at their baseline test, lower than the 14% HIV positivity among previously undiagnosed participants in the 2015 IBBS This study used service‐level data to estimate HIV burden and factors associated with HIV positivity at baseline among MSM and TW presenting for testing. As we elaborate below, service level data can be associated with substantial biases. However, with limited epidemiological data on MSM and TW in Myanmar and known challenges of achieving population‐based estimates for hard‐to‐reach and marginalised groups HIV incidence among MSM and TW documented in this study – 10.1 cases per 100 person years – is the first assessment of HIV incidence for any group in Myanmar, and demonstrates a higher rate of HIV transmission among MSM and TW compared to other estimates in Asia Despite participating services recommending three‐monthly repeat testing, only about one in 10 clients repeat tested during the 15‐month pilot period. Moreover, clients who did not report a testing history at baseline were 70% less likely to repeat test during the pilot period , with no significant difference in risk behaviours between those who did and did not repeat test (data not reported). While MSM and TW in Myanmar self‐report high levels of regular HIV testing Encouraging frequent HIV testing and improving retention in HIV prevention programs must be a key focus of local HIV prevention strategies for MSM and TW in Myanmar apwint clients were more than eight times more likely to test positive at baseline than thange or behaviourally bisexual men. A range of documented risk factors may contribute to the disproportionate HIV burden among TW relative to other sexual and gender identities in Myanmar, including being more likely to report receptive anal sex positioning, higher numbers of sexual partners, lower levels of condom use, earlier sexual debut, and experiences of forced sex apone clients – men who are sexually oriented towards other men but typically do not openly identify as gay – had seven‐times higher risk of HIV positivity at baseline than thange. Across Asia, non‐disclosure of sexual identity has been associated with higher levels of condomless sex apone clients in this study. Awareness of sexual orientation is an important precursor to provider‐initiated HIV testing Sexual identity was significantly correlated with baseline HIV positivity. Transgender identity was identified as a significant predictor of prevalent HIV; apwint which prevented a reliable calculation HIV prevalence and incidence by gender identity. Nonetheless, we believe that given the absence of data to date on HIV epidemiology among MSM and TW in Myanmar, the novelty of the data collection system in the local context and the implicit need for more robust data collection systems to better monitor HIV infection and to inform programming priorities in Myanmar, the findings presented in this paper represent an important starting point for further research into factors associated with HIV infection among MSM and TW in Myanmar.Our findings should be considered with the following limitations in mind. First, as mentioned above, the self‐selection bias inherent in service‐level data may limit the generalizability of these findings to the wider MSM and TW population in Myanmar. Engagement in HIV prevention and testing may be motivated by perceptions of HIV risk which may overestimate our estimates of HIV burden. Conversely, the risk of HIV may be lessened among clients actively engaging in HIV prevention services. Second, our incidence estimates may be underestimated due to the limited number of clients engaging in repeat testing behaviour – potentially an artefact of the short period of observation. Third, this data was collected from MSM and TW residing in the two largest cities in Myanmar with the highest prevalence of HIV 5Despite the implementation challenges noted above, the use of electronic health data in low‐income countries remains limited and the data presented here highlights both the utility and potential of novel electronic data collection system to capture data that can inform HIV prevention priorities. This pilot project demonstrated high HIV incidence among MSM and TW clients, relative to estimates from the region, combined with high levels of risk behaviours and infrequent engagement with HIV testing services. These factors strongly support the scale‐up of HIV prevention activities, the prioritisation of regular HIV testing and enhanced retention among MSM and TW in Myanmar, as well as consideration of more contemporary testing and prevention approaches such as peer‐delivered and self‐HIV testing, and PrEP into the national HIV response in Myanmar. Such strategies must be cognisant of socio‐demographic and other factors that may influence engagement with HIV testing services. This study supports the utilisation of community‐based HIV testing services as an important source of ongoing epidemiological data in Myanmar, as well as in other low‐resource settings in the region.None to declare.VV, MS and CH designed the study and developed the data collection tools. VV, ZMO, TTT, NNO and HM managed in‐country data collection efforts. Data cleaning and analysis was led by VV, MT and MS. AP provided input into data analysis and manuscript preparation. All authors have read and approved the final manuscript.