Cardiomyocytes contract keeping their sarcomere length (SL) close to optimal values for force generation. Transmural heterogeneity in SL across the ventricular wall coordinates the contractility of the whole-ventricle. SL heterogeneity (variability) exists not only at the tissue  level, but also presents at the level of a single cardiomyocyte . However, transmural differences in intracellular SL variability and its possible dependence on the state of contraction (e.g. end-diastole or end-systole) have not been previously reported. In the present study, we studied three aspects of sarcomere-to-sarcomere variability in intact cardiomyocytes isolated from the left ventricle of healthy guinea-pig: 1) transmural differences in SL distribution between subepi- (EPI) and subendocardial (ENDO) cardiomyocytes; 2) the dependence of intracellular variability in SL upon the state of contraction; 3) local differences in SL variability, comparing SL distributions between central and peripheral regions within the cardiomyocyte. To characterize the intracellular variability of SL, we used different normality tests for the assessment of SL distributions, as well as nonparametric coefficients to quantify the variability. We found that individual SL values in the end-systolic state of contraction followed a normal distribution to a lesser extent as compared to the end-diastolic state of contraction . The relative and absolute coefficients of sarcomere-to-sarcomere variability in end-systolic SL were significantly greater (∼1.3-fold) as compared to end-diastolic SL. This was independent of both the transmural region across the left ventricle and the intracellular region within the cardiomyocyte. We conclude that the intracellular variability in SL, which exists in normal intact guinea-pig cardiomyocytes, is affected by the contractile state of the myocyte. This phenomenon may play a role in inter-sarcomere communication in the beating heart. Sarcomeres form the basic contractile unit of cardiac muscle. The coordinated activation of hundreds of sarcomeres that are mechanically and structurally coupled to each other underlies the Frank-Starling law of the heart . CardiomCardiomyocytes from distinct myocardial layers of the ventricular wall, sub-endocardial (ENDO) and sub-epicardial (EPI), differ both in their SL properties as well as force generation . HeterogIn the present study, we compared sarcomere-to-sarcomere variability in the end-diastolic and end-systolic SL in mechanically unloaded isolated guinea-pig cardiomyocytes obtained from ENDO and EPI layers of the left ventricle (LV). To examine whether the variability in SL may differ within a single cardiomyocyte, we also compared individual SL distributions in two distinct regions  of single cardiomyocytes. We found that individual SL values in the end-systolic state of contraction follows normal distribution to a lesser extent as compared to the end-diastolic state of contraction . The relative and absolute coefficients of sarcomere-to-sarcomere variability in end-systolic SL were ∼1.3-fold higher compared to end-diastolic SL. This was independent of both the transmural region across the left ventricle and the intracellular region within the cardiomyocyte. We conclude that the intracellular variability in SL, which exists in normal intact guinea-pig cardiomyocytes, is affected by the contractile state of the myocyte and may play a role in the inter-sarcomere communication in the beating heart.Male and female guinea-pigs, weighing 350–500 g, were obtained from the institutional vivarium and maintained under standard conditions (12 h light/dark cycle with ad libitum access to water and food). The animals were injected intramuscularly with heparin sodium  to prevent blood clotting in coronary vessels, anesthetized 30 min later with Zoletil-100  and 2% Xylazine  and rapidly euthanized 15–20 min later by exsanguination following removal of the heart.via the aorta and perfused using a Langendorff apparatus with a sequence of three solutions equilibrated with 95% O2 + 5% CO2 at a rate of 5 ml/min at 36°C. The perfusion was started with heparinized Krebs-Henseleit bicarbonate buffer for 5 min after the heart has started normal beating (80–110 beat/min). Next, perfusion was switched to low Ca2+ Krebs-Henseleit buffer for 15 min (causing the heart to stop beating). Next, the heart was perfused with enzyme solution, containing 0.2 mg/ml collagenase  and 0.06 mg/ml protease XIV (∼3.5 U/mg) for 6 min. After the appearance of viscous drops at the heart apex, the heart was transferred to a Petri dish with enzyme solution. The LV was gently digested by the Langendorff-free injection technique  HEPES-buffered Tyrode solution  at 22–24°C and used within 4–6 h. For measurements of sarcomere dynamics, suspensions of ENDO and EPI cardiomyocytes were diluted in fresh HEPES-buffered Tyrode and transferred to the experimental chamber. Measurements were performed at 30°C and pacing rate of 1 Hz using an electronic pacing device . Unless otherwise noted, all chemicals and reagents were purchased from Sigma-Aldrich .The removed heart was immediately flushed with heparin-containing cold (10–15°C) Krebs-Henseleit bicarbonate buffer  was used to measure the sarcomere striation profile in a single isolated cardiomyocyte contracting in mechanically unloaded conditions. The optical settings were as follows: transmitted light mode (T-PMT channel); phase-contrast imaging mode with a 63x oil-immersion objective (Plan-Apochromat 63x/1.40 Oil DIC M27); excitation by 488 nm . The physical spatial resolution of the measured sarcomere striation profile using the given optical settings was ∼0.12 µm. The acquisition settings for time-series measurements of a sarcomere striation profile in electrically paced cardiomyocytes were set as follows: region-of-interest (ROI) mode, where the horizontal ROI size was adjusted optimally for the selected optical settings  along the long cell axis (ROI width) and the vertical ROI size was 2-3 pixels along the short cell axis . To measure individual SL we determined the positions at which the sarcomere striation profile curve either crossed its baseline  an averaged end-diastolic inclusion SL criterion was set to: 1.7 µm ≤ SL ≤ 1.9 µm, and 2) a fractional sarcomere shortening (sarcomere shortening divided by the end-diastolic SL) criterion was set to: 5% ≤ sarcomere shortening ≤ 20%.p-value of <0.05.Data analyses were carried out with GraphPad Prism 7.0  and custom-made software EqapAll6. We used one-sample Shapiro-Wilk, and one- and two-sample Anderson-Darling statistical tests to assess the normality of SL value distributions. For all tests, the hypothesis of normality was rejected at a p-value of <0.05 was considered to indicate a significant difference between the parameters.For each SL set we used non-parametric measures: median, median absolute deviation (MAD), and MAD divided by median (MADM). A two-way and three-way ANOVA with Sidak multiple comparison tests were used for statistical analyses between the parameters. The parameters of all analyzed sets were distributed normally  and had similar variance between groups (checked using Bartlett’s test). A On average for pooled data , the passage rates for Shapiro-Wilk and Anderson-Darling one-sample tests were, respectively, 72.9 ± 3.2 and 75.8 ± 4.8% for end-diastolic state of contraction, while the passage rates for end-systolic state were, respectively, 53.8 ± 4.9% and 56.1 ± 4.7%. If both Shapiro-Wilk and Anderson-Darling tests for the given cell were required to satisfy , the passage rate was 70.4 ± 3.6% and 51.3 ± 4.8% for end-diastolic and end-systolic state of contraction, respectively. According, the percentage of cells in which end-diastolic and end-systolic SL distributions did not pass both Shapiro-Wilk test and Anderson-Darling test was 21.7 ± 4.2% and 41.5 ± 4.8%, respectively . Moreover, if the passage of Shapiro-Wilk and Anderson-Darling one-sample tests in both central and peripheral intracellular regions was required, the passage rate further decreased to 50.4 ± 7.6% and 29.1 ± 4.9% for end-diastolic and end-systolic state of contraction, respectively. The Anderson-Darling two-sample test for two individual SL sets, which were measured in the central and peripheral intracellular regions of the same cell in the same state, showed a higher passage rate: 84.5 ± 4.3% and 80.9 ± 5.9% for end-diastolic and end-systolic state of contraction, respectively. On average, for all four types of normality tests , the number of cells in which end-systolic SL distributions pass normality tests was ∼1.3-fold lower in ENDO cells and ∼1.6-fold lower in EPI cells compared to end-diastolic SL distributions. The percentage of cells in which individual SL distributions passed Shapiro-Wilk test were close to that for Anderson-Darling test, while this percentage was lower when these tests were applied together.Thus, our analysis showed that individual SL values in a single cardiomyocyte can be distributed by a function other than normal. Therefore, in our further analysis we used non-parametric measures of absolute and relative sarcomere-to-sarcomere variability in SL .To reveal regional differences in end-diastolic and end-systolic SL between ENDO and EPI cardiomyocytes or between central and peripheral intracellular regions, we compared median SL values obtained from individual SL sets see . End-diaTo access regional differences in the variability of individual SL, we analyzed the measures of absolute variability  and relative variability  obtained from individual SL sets. No significant differences in the mean values of MADM or MAD of end-diastolic or end-systolic SL were found between ENDO and EPI cells or between central and peripheral intracellular regions see . Note thp < 0.001, vs. EPI, and bottom panels for centre vs. periphery. The significance of this difference was invariant to the intracellular region  or whether we used pooled data from both regions. For example, for pooled data from central and peripheral intracellular regions the mean value of MADM in ENDO myocytes was 4.28 ± 0.99% and 5.80 ± 1.69% for end-diastolic and end-systolic states of contraction, respectively (p < 0.0001). Likewise, for pooled data obtained in EPI myocytes, the mean value of MADM was 4.02 ± 1.02% and 5.40 ± 1.64% for end-diastolic and end-systolic states of contraction, respectively (p < 0.0001). Therefore, MADM was ∼1.35-fold greater (CV was ∼1.3-fold greater) for the end-systolic state compared to the end-diastolic state, indicating that sarcomere-to-sarcomere SL variability increases during contraction.Finally, we assessed the effect of contraction state on the SL distribution variability. For each cellular or sub-cellular region, we found that relative and absolute coefficients of variability in end-systolic SL were significantly greater compared to coefficients of variability in end-diastolic SL . A minority of cardiomyocytes (∼15% for pooled ENDO + EPI cells) showed the opposite effect during the contractile transition with, again, no transmural differences in this value. The separated SL sets for ENDO or EPI pools, either of central or peripheral intracellular regions, showed non-significant differences between each other in the slopes of the relationship between SL and MADM.To demonstrate the correlation between the contractile state of a cardiomyocyte and the extent of sarcomere-to-sarcomere SL variability in a particular cardiomyocyte, we plotted end-diastolic (for resting state) or end-systolic  mean SL values against MADM values for the corresponding individual SL values in each individual cell . Most ofvs. end-diastole, independent of either the transmural origin of a cell or intracellular region. The magnitude of SL variability was found to be non-different between pools of subepicardial (EPI) and subendocardial (ENDO) cardiomyocytes or between two intracellular regions within the same ENDO/EPI cardiomyocyte.In this study, we used mechanically unloaded membrane intact isolated guinea-pig cardiomyocytes from the subepi- and subendocardial layers of the left ventricle and measured individual sarcomere length (SL) in two intracellular regions  for each cell in its relaxed state (end-diastole) and at peak contraction (end-systole). The sarcomere-to-sarcomere variability in their lengths was evaluated by absolute and relative non-parametric measures, median absolute deviation (MAD) and MAD divided by median value (MADM), respectively. The main finding is that SL variability is higher at end-systole in vivo  during a complete contraction-relaxation cycle. The time-course of individual sarcomere(s) can be analyzed to determine to what extent they are distinct from each other and whether a single sarcomere is “synchronized” with the others during the contraction cycle . The timin vivo under a natural beat frequency  differences in the variability of individual sarcomere lengths.Likewise, we failed to find transmural differences in the extent of SL variability. It has been shown recently that the subtle inter-cellular  differences in the mechanical properties of living cardiomyocytes can only be revealed employing relatively large sample sizes, containing at least several hundreds of cardiomyocytes . For exaThe phenomenon of myocardial heterogeneity is manifested not only at the tissue or inter-cellular level, but also at the intracellular level. The extent of intracellular heterogeneity in sarcomere length correlated with the contractile state of the cardiomyocyte and was higher at peak systole compared to diastole. On the other hand, this phenomenon displayed no regional-specific differences, indicating a universal character (independent of cell origin) of SL heterogeneity. Sarcomere-to-sarcomere SL variability and its “sensitivity” to the activation state of a cardiomyocyte may be an important feature, allowing for optimization of the contractile response of the cardiomyocyte to naturally occurring transmural stress and strain gradients in the whole heart. However, elucidation of the functional role of intracellular variability in sarcomere length and other mechanisms of electro-mechanical coupling requires further studies focused on this issue.


Antimicrobial resistance (AMR) is one of the biggest threats to global public health. Selection of resistant bacteria is driven by inappropriate use of antibiotics, amongst other factors. COVID-19 may have exacerbated AMR due to unnecessary antibiotic prescribing. Country-level knowledge is needed to understand options for action.To review the current situation with respect to AMR in India and initiatives addressing it. Identifying areas where more information is required will provide a call to action to minimize further rises in AMR and to improve patient outcomes.Streptococcus pneumoniae and Haemophilus influenzae) were identified. National and international antibiotic prescribing guidelines for specific CA-RTIs  commonly used locally were also reviewed, plus local antibiotic availability. Insights from a local clinician and clinical microbiologist were sought to contextualize this information.National AMR initiatives, antibiotic use and prescribing in India, and availability of susceptibility data, in particular for the key community-acquired respiratory tract infection (CA-RTI) pathogens  and Antimicrobial Testing Leadership and Surveillance (ATLAS). For common RTIs, clinicians use a range of international and national guidelines. However, a more standardized inclusive approach to developing local guidelines, using up-to-date local surveillance data from community-acquired infections, could make guidelines more locally relevant. This would encourage more appropriate antibiotic prescribing and improve adherence. This would, in turn, potentially limit AMR development and improve patient outcomes. Even if new medicines are developed, without behaviour change, antibiotic resistance will remain a major threat’.3 The first paper in this Supplement included details about the multiple factors which can drive a rise in AMR, along with the global initiatives that are in place to address this threat.2 Each country and/or region must also play their part through local initiatives.Antimicrobial resistance (AMR) is one of the biggest threats to public health throughout the worldStreptococcus pneumoniae and Haemophilus influenzae. National and international antibiotic prescribing guidelines for CA-RTIs  commonly used by healthcare professionals were also reviewed, along with how these link to local antibiotic availability. Insights from a clinician and from a clinical microbiologist in India were sought to contextualize this information. In addition, we aimed to identify areas where more information is required and present a call to action to improve clinical outcomes for patients and to minimize further rises in AMR within India.In order to identify how AMR can be addressed in India in the future, it is necessary to review what is happening now. In this paper, we present the current situation in India, determined by using published information  to ascertain any national initiatives to address AMR, antibiotic use and prescribing, and availability of susceptibility data, in particular for the key community-acquired respiratory tract infection (CA-RTI) pathogens 4 Organizations such as the Indian Academy of Paediatrics, the Global Antibiotic Resistance Partnership and the Indian Council of Medical Research (ICMR) have worked to build awareness concerning rising AMR amongst professional bodies, the media, and the general public. A key milestone was the Chennai Declaration of 2012, the output from the first ever meeting of medical societies in India which aimed to address AMR by formulating a 5 year strategy to develop a road map for combating the global challenge of AMR from the Indian perspective.4In recent years, addressing AMR has been recognized in India as a national priority and a range of strategies has been introduced, including educational and awareness initiatives, infection control guidelines, audit and feedback, and antimicrobial stewardship (AMS).4In 2016, the ‘Medicines with the Red Line’ campaign was launched as a way of addressing the increasing misuse of antibiotics by educating and informing patients. All prescription-only antibiotics were marked with a red line on the packaging and patients were informed that any antibiotic marked in this way should only be taken following prescription and advice from their doctor and the complete course should be taken. The aim was to discourage over-the-counter (OTC) purchase of antibiotics and unnecessary antibiotic prescribing through raised awareness.5 many countries began to develop their own National Action Plan (NAP) on AMR. In April 2017, in response to this WHO initiative,5 the Indian Ministry of Health and Family Welfare published a 5 year NAP (2017–21) on AMR outlining the priorities and implementation strategies for curbing AMR in India.6 The first five strategic priorities of NAP-AMR are firmly aligned with the GAP on AMR and an additional sixth strategic priority highlights India’s aspirational aim to contain AMR at an international level with other countries and organizations, national disease control programmes and at a sub-national/state level through development of state action plans on AMR.6Following the formulation by the World Health Assembly in 2015 of a Global Action Plan (GAP) for AMR7 is considered to be amongst the highest in the world and includes infections due to multiresistant pathogens.6 In 2010, India was the world’s largest consumer of antibiotics in humans.4 Antibiotic consumption in India is high due to: easy access to OTC medicines, self-medication based on word of mouth advice or on internet searching, non-availability or non-utilization of clinical microbiology laboratory services for cultures and antibiotic susceptibility testing (AST), variation in the approach of clinicians to antibiotic prescribing, lack of antibiotic de-escalation, regulatory issues and commercial antibiotic promotion.6 Underuse of antibiotics due to lack of access to good quality and affordable antibiotics is also common in India, leading to significant mortality (particularly in paediatrics). In addition to human healthcare, antibiotics are also used in animal husbandry, fisheries (for treatment and growth promotion), as well as in agricultural sectors. The lack of knowledge among the public is also a matter of concern regarding appropriate use of antibiotics along with poor compliance and self-medication.6 Another factor contributing to the crisis of AMR is environmental pollution due to pharmaceutical, livestock and hospital waste.8The burden of infectious disease in India 8 There are also a small number of studies from individual units or hospitals which have provided information on local AMR. Whilst these local data are extremely useful within a specific hospital or city, they cannot provide a full picture nationally. Therefore, in response and acting on the aims of the NAP, the Indian Council of Medical Research (ICMR) established an AMR surveillance network (ICMR-AMRSN) in tertiary care hospitals across India.9 Alongside this network, ICMR has developed a fully customizable tool for the collection, management, analysis and reporting of the AMR data. It is a software package and tool for management and analysis of AST and antimicrobial consumption data collected through multiple laboratories across India and has the additional aim of enforcing quality antimicrobial susceptibility testing in laboratories. The National Centre for Disease Control (NCDC) is the focal point for implementation of the programme and has the overall aim of understanding the extent and pattern of AMR and making use of this information when formulating strategies designed to prevent AMR spread.6 The Indian NAP also recognizes that, since microbiologists and clinical microbiology laboratories are the cornerstones for surveillance, it is vital to improve the quality of laboratories by ensuring increased availability of trained personnel and adequate infrastructure, reinforced quality controls, and hands-on training support from tertiary care institutions.4In India, several systematic national surveillance studies generating local data have been described.S. pneumoniae and H. influenzae, the most commonly isolated bacterial respiratory pathogens in the community.10 Key features of the SOAR study are that the focus is on only these pathogens, and that identification and susceptibility testing are performed in an independent centralized laboratory using standardized methodology (CLSI) allowing for comparisons to be made between countries or regions and for the identification of trends over time.10 New SOAR data is analysed based on three different breakpoints: CLSI, EUCAST dose specific, and PK/PD breakpoints.Several global surveillance studies provide antibiotic susceptibility data in India. Survey of Antibiotic Resistance (SOAR) is a multinational antibiotic surveillance study, ongoing in an expanding range of countries since 2002. The study aims to collect and make available in published, peer-reviewed papers, antibiotic susceptibility data, specifically for 10 Two main international organizations define breakpoint values: CLSI and EUCAST. Due to variation in criteria for their definition, there are some differences between CLSI and EUCAST in the clinical breakpoint values for certain bacteria for some antibiotics and this can impact susceptibility interpretation of clinical isolates.11 EUCAST breakpoints are dose-specific and use the EMA-approved doses that are included in the Summary of Product Characteristics of an antibiotic. This means that by application of breakpoints for higher doses, the effect of using a raised dose on the clinical efficacy of a particular antibiotic can be predicted. Currently, most clinical microbiology laboratories in India use CLSI breakpoints, however the international application of the EUCAST breakpoints is expanding,12 so it is possible that dose-specific breakpoints could at some time also be applied in India. The EUCAST dose-specific breakpoints can also be used retrospectively to calculate the susceptibility of previously collected isolates, to show the susceptibility levels that would have been achieved at higher doses.Clinical breakpoints are cut-off MIC values used to classify microorganisms into the clinical categories susceptible (S), intermediate (I) and resistant (R) to assist in prediction of the clinical success or failure of a specific antibiotic.S. pneumoniae, the most isolated respiratory pathogen14 from infections such as CAP, AOM and ABRS, has over time become less susceptible to amoxicillin/clavulanic acid in some countries15 since the MICs of some isolates have increased. When treating infections, it is important to be able to eradicate bacterial pathogens with raised MICs to optimize clinical outcome while at the same time minimizing the risk of selecting variants with even higher MICs. This is possible because β-lactams, unlike many other antibiotics, have time-dependent killing properties. Their efficacy depends on the amount of time the required drug concentration is present at the site of action. Although increasing the concentration at the infection site above a particular value will not have any effect on the efficacy, the use of higher doses and/or more frequent dosing allows for successful eradication of infections caused by pathogens with higher MICs because the time above the MIC at the site of infection is increased.16The use of the EUCAST dose-specific breakpoints shows the effect of increasing the antibiotic dose on the susceptibility of a pathogen, providing additional information so the prescriber can decide if a higher dose would be of benefit. For example, S. pneumoniae (n = 219) isolates were susceptible to amoxicillin/clavulanic acid, but this fell to 66.3% and 54.8% for the macrolides azithromycin and clarithromycin, respectively. For the fluoroquinolone, levofloxacin, S. pneumoniae susceptibility was 85.8%10  from India, a high susceptibility of 97% to amoxicillin/clavulanic acid was seen, applying CLSI criteria.  and trimethoprim/sulfamethoxazole (23.0% susceptible).SOAR results for India, collected from outpatients with confirmed CA-RTIs at four sites in 2012–14, show that when applying CLSI breakpoints, 91.8% of 17 ATLAS data is analysed based on CLSI and EUCAST breakpoints. Susceptibility data for India is currently available for only a small number of S. pneumoniae isolates (n = 11) from 2019 and H. influenzae isolates from 2016 (n = 23) and 2019 (n = 37)  as seen in the SOAR data. In the ATLAS data, H. influenzae isolates remained fully susceptible to amoxicillin/clavulanic acid and ceftriaxone in 2016 and 2019.17The Antimicrobial Testing Leadership and Surveillance (ATLAS) database is a global AMR surveillance programme which is fully searchable. It is available for general access and covers susceptibilities of a range of bacterial and fungal pathogens to various antimicrobials.016 n = 2 and 2019 Figures . S. pneu19 During the first 4 years, 91 countries or territories enrolled in GLASS and data for over two million patients from 66 countries are included.19 Pathogens currently included in GLASS-AMR are: Acinetobacter spp., Escherichia coli, Klebsiella pneumoniae, Neisseria gonorrhoeae, Salmonella spp., Shigella spp., Staphylococcus aureus, and S. pneumoniae and a new and important component is the inclusion of antimicrobial consumption (AMC) surveillance at the national level.20 GLASS data is analysed based on CLSI and EUCAST breakpoints. India is participating in the GLASS initiative but, according to the early implementation report for 2020, whilst S. pneumoniae isolates from blood samples will be included, no results are currently available within the system.19In 2015, WHO launched the Global Antimicrobial Resistance and Use Surveillance System (GLASS). GLASS is the first global system to collect national AMR data for selected bacterial pathogens that cause common infections. The aim is to monitor the prevalence of AMR among major pathogens in clinical settings to provide the supporting data required to ensure that countries can design cost-effective, evidence-based AMR response strategies.For management of the common RTIs, CAP, AOM and ABRS, in India clinicians make use of a range of national/international antibiotic prescribing guidelines, examples of which are shown in Table 27 the IDSA36 and from the UK’s National Institute for Health and Care Excellence (NICE).35 For example, a first-line antibiotic treatment recommendation for CAP management from the IDSA 2019 guideline for treating adults with no comorbidities or risk factors for MRSA or Pseudomonas aeruginosa is amoxicillin or doxycycline or a macrolide  but if the patient has comorbidities, the recommendation is combination therapy with amoxicillin/clavulanic acid or a cephalosporin plus a macrolide or doxycycline, or monotherapy with a respiratory fluoroquinolone. The amoxicillin/clavulanic acid dose would be 875 mg/125 mg twice daily or 500 mg/125 mg three times daily or amoxicillin/clavulanic acid 2000 mg/125 mg twice daily plus either a macrolide or doxycycline36 and the NICE 2019 guidelines recommend amoxicillin/clavulanic acid for young people over 1 month and under 18 years and for adults as first-line treatment for severe symptoms.35 For the management of AOM, the international guidelines referred to in India include those from the American Academy of Pediatrics30 and NICE guidance.34 For the management of ABRS in adults and paediatrics, international guidelines used include those from the IDSA29 and NICE.33 For ABRS in children , the IDSA recommend initial empirical treatment as first-line, amoxicillin/clavulanic acid 45 mg/kg/day twice daily and in adults  first-line amoxicillin/clavulanic acid 500 mg/125 mg three times daily or 875 mg/125 mg twice daily.29For the management of CAP in adults and paediatrics, the international guidelines referred to by clinicians in India include those from the British Thoracic Society (BTS)22 Treatment Guidelines for Antimicrobial Use in Common Syndromes 2019,23 and Joint ICS/NCCP(I) 2012 Guidelines for Diagnosis and Management of Community and Hospital-acquired Pneumonia in Adults.21 For example, the NTG 2016 National Treatment Guidelines for Antimicrobial Use in Infectious Diseases recommends amoxicillin/clavulanic acid for the treatment of ABRS in adults at a dose of 1 g/day twice daily and for severe ABRS in children, 45 mg/kg/day twice daily.22Concerning local Indian guidelines for management of CAP, AOM and ABRS, currently the commonly referred to documents are National Treatment Guidelines (NTG) for Antimicrobial Use in Infectious Diseases 2016,38Access to antibiotics may be an issue for patients in low- and middle-income countries. Drug supply chains may also contribute to the problem. Limited access to the most appropriate antibiotic to treat a specific infection may result in increased mortality from treatable bacterial infections and, the use of suboptimal amounts of antibiotic facilitates resistance development and allows resistant strains to persist.25 for outpatients with comorbidities; IDSA 2011 guidelines for the treatment of paediatric CAP in outpatients;28 IDSA 2012 treatment guidelines for sinusitis29 as initial treatment for children and in adults with risk of treatment failure; AAP 2013 guidelines as initial or delayed treatment of AOM in children30 or after failure of initial treatment; and by the IDSA 2019 guidelines for adults CAP patients with comorbidities.36In India, taking amoxicillin/clavulanic acid as an example, several currently available formulations are mentioned as first-line or second-line recommendations by the RTI management guidelines. Examples include combination therapy of amoxicillin/clavulanic acid 500 mg/125 mg three times daily with a macrolide, which is recommended in the NICE 2019 guidelines for the treatment of CAP in adults and children with severe CAP. In the ICMR 2019 guidelines for adult CAP patients with no comorbidities, amoxicillin/clavulanic acid 1 g twice daily or 625 mg three times daily is recommended. It was not until April 2021 that amoxicillin/clavulanic acid 90 mg/kg or 4 g/0.25 g per day (high-dose) was available in India. High-dose regimens are recommended by: IDSA 2007 CAP treatment guidelines39 since poor-quality antibiotics are unlikely to contain the full dose needed to eliminate all of the infecting pathogens, which will encourage resistance to develop and allow resistant strains to survive and be transmitted.40 The quality of medicines, specifically antibiotics, is an important consideration for countries worldwide. The WHO launched a Global Surveillance and Monitoring System (GSMS) for substandard and falsified products.40 The GSMS aims to work with WHO member states to improve the quality of reporting of substandard and falsified medical products, and, importantly, to ensure the data collected are analysed and used to influence policy, procedure, and processes to protect public health, at the national, regional and the global level. Use of substandard or falsified antibiotics not only compromises clinical outcome but also risks increased AMR. The most recent summary (2013–17) reported substandard and falsified medicines in 46 member states (including India) and antibiotics represent 16.9% of all products reported, second only to malaria drugs (19.6%).40Substandard, poor-quality or falsified antibiotics promote AMR and the spread of drug-resistant infections,S. pneumoniae in India which reveal a range in penicillin resistance from 3.8% to 30%.41 In such a large country as India, it is important to capture susceptibility variation in order to optimize antibiotic use.From a clinical microbiologist’s perspective, AST, performed for clinically relevant bacterial isolates based on the site of infection is vital, not only for guiding therapy for a specific patient but also for understanding epidemiological trends of different pathogens and for the choice of antimicrobials used for empirical or targeted therapy. Every healthcare facility should maintain its local antibiogram for important pathogens due to variation between areas, as illustrated by studies of 42 Further risk factors for AMR include counterfeit medications and sharing antibiotics between family members. Balancing access and excess therefore remains key to minimizing antibiotic abuse and AMR. Primary care and outpatient settings need to adopt AMS, diagnostic stewardship (making diagnostics readily available) and infection prevention and control practices to use antibiotics effectively. Capacity building of community health workers, pharmacists and doctors towards appropriate antibiotic use and improving community involvement through education and awareness will minimize antibiotic abuse in the long term. The success of any of these interventions requires strong political will and leadership support.Antibiograms, along with clinical outcome data, contribute to the preparation of local, national and international guidelines for empirical antibiotic prescribing. Without local or national data, one can still refer to international guidelines especially for community-acquired pathogens to define empirical choices. Antibiotic prescribing guidelines allow for standardization of therapy with respect to primary and secondary choices, route, dose, frequency and optimum duration for treatment. In the absence of such guidelines, antibiotic abuse prevails which further drives AMR. Another factor driving antibiotic use is access. In India, access to antibiotics is a major cause of concern because of the extensive availability of OTC antibiotics. It has been suggested that in some situations, when there is limited access to a healthcare professional (HCP) or in resource-limited settings, OTC purchase of antibiotics may be needed, but self-medication risks antibiotic misuse and can result in increasing resistance.Common challenges faced by clinical microbiology laboratories when undertaking and reporting the results of AST include inappropriate specimen provision (e.g. sending swabs instead of fluids or tissue samples), AST reports being requested: (i) for colonizers; (ii) when there is ‘no growth’; (iii) for antibiotics that do not concentrate at the infection site; (iv) for antibiotics that are not active against the pathogen; and (v) for antibiotics that are not included in CLSI/EUCAST or local guidelines. In addition, physicians may compare and prefer non-standard reporting patterns (+/−) with standard reporting (S/I/R) as per CLSI or EUCAST. There is also a lack of understanding among clinicians on how to use MICs, especially for drug-resistant pathogens. Lack of patient histories may result in physicians requesting AST reports for whatever has grown, which overestimates infection rates, especially at non-sterile sites, leading to unnecessary antibiotic prescribing. Poor communication between clinical microbiologists and physicians may result from lack of insight into each other’s field of expertise.Frequent informal and formal discussion between both experts on challenging cases or samples that need to be sent for appropriate diagnosis, antibiotics to be tested, mechanisms of resistance, and escalation or de-escalation strategies would greatly help. Good IT back up is needed for quick and sustained communication of AST results and hospital-based laboratories should ensure continuous availability of microbiology services along with written guidance to the clinical team on specimen collection, storage and transport, in order to optimize recovery of pathogens. Regular clinicopathological co-relation meetings/journal clubs to discuss cases from the clinical and microbiology perspectives would promote communication and understanding along with participation of microbiologists in clinical rounds to open channels for bedside case discussions and exchange of information. Inclusion of advisory notes on AST reports regarding preferred antibiotic choice, mechanism of drug resistance, additional tests that may support clinical diagnosis or management of the infectious disease would also improve the situation. There is also an urgent need to introduce compulsory refresher training programmes for all HCPs prescribing antimicrobial agents at intervals to understand and adopt good antimicrobial prescribing practices. Finally, annual, or more frequent, discussions between different specialities regarding antibiotic patterns of important pathogens and empirical choices of therapy to formulate the antibiotic policy would be beneficial.From a clinician’s perspective in India, recent local susceptibility data would assist in the selection of empirical antibiotics for community-acquired infection management and to support rational choices when treating these bacterial infections. In addition, HCPs should be regularly alerted to rational antibiotic use. A community-acquired pathogen susceptibility registry that is India-specific is lacking; only sporadic data are available in published papers. There are many good initiatives underway from the Indian government, such as the ICMR, but the data published here are mainly related to Gram-negative hospital-acquired infections, which is not helpful to the physician managing CA-RTIs. In addition, antibiograms produced by major hospitals do not reflect the situation within the community, however if they do exist for a specific hospital, they should be updated on an annual basis.In clinical practice in India, it is advisable to check any available local antibiotic surveillance data that exists ahead of prescribing antibiotics, along with local management guidelines including those produced by the ICMR, Indian Academy of Pediatrics (IAP) and NTG, and to check pathogen prevalence data, management guidelines and local hospital susceptibility data, if available. Patient education is also vital, covering the misuse of antibiotics, such as not purchasing OTC antibiotics and also the need to complete the full course as prescribed by the physician. This is where campaigns, such as ‘Medicines with the Red Line’, which educate the public and help enforce pharmacy laws can be effective but need to be regularly revisited and refreshed. AMS programmes are also important, and they should be implemented at all levels, including in the pharmacy. Most patients do not complete the full course of antibiotics as prescribed by HCPs. Parents should also be educated about the need for, and the importance of, completing the course of antibiotics as prescribed. There should be regular continuing medical education (CME) and awareness programmes for HCPs about rational selection and appropriate use of antibiotics.In an era of rising AMR throughout the world, this paper aims to define areas where action is required to address AMR by analysing and understanding the current situation within India. Information is presented concerning antibiotic use and prescribing, approach to AMR, availability of local susceptibility data, use of international and/or local management guidelines and how these link to antibiotic availability. To our knowledge, this is the first time this information has been reviewed and presented in detail by country.Antibiotic use in India is extremely high; in 2010, India was the world’s largest consumer of antibiotics for human health and the burden of infectious diseases in India is amongst the highest in the world. In terms of surveillance, there are a small number of studies from individual units or hospitals that have provided information on local AMR and the ICMR have established an AMR surveillance network (ICMR-AMRSN) in tertiary care hospitals across India. Several global surveillance studies also include data for India including SOAR and ATLAS. The WHO GLASS study will also provide useful data in the future, but complete and robust surveillance data is required for bacterial pathogens responsible for community-acquired infections.S. pneumoniae and H. influenzae remains high, supporting these recommendations in India.For management of the common RTIs, CAP, AOM and ABRS in India, clinicians make use of several country-specific local antibiotic prescribing guidelines plus a wide range of international antibiotic prescribing guidelines. For the management of CA-RTIs, amoxicillin/clavulanic acid is an antibiotic commonly recommended by both international and local prescribing guidelines. Susceptibility studies (ATLAS and SOAR) confirm that susceptibility of the common RTI pathogens, 2 This would pave the way for improved adherence and a higher level of appropriate antibiotic prescribing in CA-RTIs which could, in turn, potentially limit AMR development and improve clinical outcomes for patients.The clinical microbiologist and clinician are united in their wish for local up-to-date susceptibility data to support empirical antibiotic prescribing. An increase in local surveillance studies would allow local management guidelines to be updated regularly which, if applied along with guidelines relating to the management of viral diseases such as COVID-19 would improve clinical outcomes and minimize further rises in AMR. While a range of international guidelines is utilized by clinicians in India, a more standardized inclusive approach is needed to develop local country-specific guidelines. These guidelines would be based on up-to-date surveillance data of isolates from community-acquired infections, which would make them more locally relevant for clinicians, reiterating the Consensus Principles as described in the introductory paper to this Supplement.


The usefulness of neoadjuvant chemotherapy for patients with duodenal adenocarcinoma remains unclear. We report the case of a successfully resected duodenal adenocarcinoma managed by neoadjuvant chemotherapy using gemcitabine and S-1.A 72-year-old female presented with a one-week history of abdominal bloating and vomiting after meals. Esophagogastroduodenoscopy revealed a circumferential epithelial lesion in the second portion of the duodenum. Abdominal computed tomography scan revealed thickened walls and narrowing of the duodenum. Further, an adenocarcinoma was noted on biopsy. Though she was diagnosed with duodenal adenocarcinoma, pancreatic cancer could not be completely ruled out. Therefore, she underwent neoadjuvant chemotherapy using gemcitabine and S-1 after bypass surgery. After six chemotherapy cycles, the tumor significantly reduced in size. Further, lymph nodes and distant metastases were not noted on abdominal computed tomography. The patient underwent pancreaticoduodenectomy. Pathological examination revealed a 0.5-mm lesion and surrounding fibrosis at the duodenum, distal from the ampulla of Vater and the pancreas. Her postoperative course was almost uneventful, and she was discharged on the 31st postoperative day. The patient was followed up and had no tumor recurrence at 24 months after surgery.Neoadjuvant chemotherapy with gemcitabine and S-1 was useful in reducing the size of a duodenal adenocarcinoma. This finding would aid physicians in managing patients that present with a similar presentation. Duodenal carcinoma (DC) is an uncommon malignancy, accounting for only 0.4% of gastrointestinal cancers . BecauseThe usefulness of neoadjuvant chemotherapy has been reported in various types of cancers such as pancreatic and rectal cancers , 4. We r2) on the first and eighth days. S-1 was administered orally twice daily (80 mg/day) for 2 weeks. The size of the tumor gradually decreased without any lymph nodes or distant metastases (Fig. A 72-year-old female presented with a one-week history of abdominal bloating and vomiting after meals. She was previously diagnosed with diabetes mellitus. Esophagogastroduodenoscopy revealed a circumferential epithelial lesion in the second portion of the duodenum Fig. . Abdominses Fig. .Fig. 1EsExcept for the development of a postoperative grade B pancreatic fistula, her postoperative course was uneventful. She was discharged from the hospital on postoperative day 31 . At a foTo the best of our knowledge, this is the first study reporting the use of neoadjuvant GS in patients with duodenal adenocarcinoma. Interestingly, it was markedly effective in a 72-year-old Japanese woman.It was sometimes challenging for surgeons to differentiate duodenal adenocarcinoma from ampullary and pancreatic head cancers. Generally, it is pathologically impossible to differentiate these three cancers using biopsy alone. The only key feature differentiating the three would be the tumor location. Creating a preoperative diagnosis is essential because treatment plans for these tumors are different. In fact, we could not rule out pancreatic cancer preoperatively. Therefore, neoadjuvant GS treatment after bypass surgery was used in the case to manage pancreatic cancer, which had a worse prognosis .Studies have shown that neoadjuvant chemotherapy or chemoradiotherapy has better clinical efficacy in patients with DC . 5-FU, FPrevious reports on the use of neoadjuvant chemotherapy with S-1 for patients with duodenal adenocarcinoma are listed in Table Neoadjuvant chemotherapy with gemcitabine and S-1 was useful in reducing the size of the duodenal adenocarcinoma. This finding would aid physicians in managing patients who present with a similar presentation.


During their life span, concrete structures interact with many kinds of external mechanical loads. Most of these loads are considered in advance and result in reversible deformations. Nevertheless, some of the loads cause irreversible, sometimes unnoticed changes below the macroscopic scale depending on the type and dimension of the impact. As the functionality of concrete structures is often relevant to safety and society, their condition must be known and, therefore, assessed on a regular basis. Out of the spectrum of non-destructive monitoring methods, Coda Wave Interferometry using embedded ultrasonic sensors is one particularly sensitive technique to evaluate changes to heterogeneous media. However, there are various influences on Coda waves in concrete, and the interpretation of their superimposed effect is ambiguous. In this study, we quantify the relations of uniaxial compression and uniaxial tension on Coda waves propagating in normal concrete. We found that both the signal correlation of ultrasonic signals as well as their velocity variation directly reflect the stress change in concrete structures in a laboratory environment. For the linear elastic range up to 30% of the strength, we calculated a velocity variation of −0.97‰/MPa for compression and 0.33%/MPa for tension using linear regression. In addition, these parameters revealed even weak irreversible changes after removal of the load. Furthermore, we show the time-dependent effects of shrinkage and creep on Coda waves by providing the development of the signal parameters over time during half a year together with creep recovery. Our observations showed that time-dependent material changes must be taken into account for any comparison of ultrasonic signals that are far apart in time. The study’s results demonstrate how Coda Wave Interferometry is capable of monitoring stress changes and detecting even small-size microstructural changes. By indicating the stated relations and their separation from further impacts, e.g., temperature and moisture, we anticipate our study to contribute to the qualification of Coda Wave Interferometry for its application as an early-warning system for concrete structures. Reinforced concrete is used for a large part of our built environment today , especiaRegular inspections, in most cases based on visual examination or tap tests, ensure structural integrity and operability. On the downside, they are only able to represent the structure’s state at accessible areas at one point in time, whereas monitoring methods, usually from the field of non-destructive testing (NDT), complement the spectrum of available tools for safety assurance. Reinforced concrete, however, poses a special challenge for NDT techniques due to the material’s heterogeneity, which complicates the separation between naturally present scatterers and undesired material changes, or damage. To this end, the evaluation of ultrasonic signals by means of Coda Wave Interferometry (CWI) is a promising and exceptionally sensitive method to detect even weak changes in the material and has been subject to previous studies on both the laboratory ,3,4,5,6,With this study, we quantify the effects of uniaxial compression, uniaxial tension, shrinkage and creep of a normal concrete on signal changes in the Coda that we describe by means of correlation coefficients and velocity variations of collected ultrasonic signals.Ultrasonic (US) waves in inhomogeneous media are scattered, depending on the wave properties such as frequency, and the size, shape, and type of the inhomogeneities. The effect of such scattering on ultrasonic measurements is shown in Generally, CWI methods take advantage of the accumulation of weak changes over a great number of wave paths between a sender-receiver pair that are caused by the heterogeneities. Multiple methods for CWI analysis have been proposed in recent decades ,14,15, aWith this method, two parameters,  changes . Both thConcrete is a complex heterogeneous material consisting of coarse aggregates and a system of air pores embedded into a cementitious mortar matrix. Depending on the design case and objective, concrete can be investigated on different scales, whereas most structural and material design models consider the material on the macro level as a homogeneous material. However, evaluation of crack formation starts with pre-existing defects on the micro level, followed by a multilayered process of damage evolution .From the monitoring perspective, it is most ideal to detect weak changes in the material already on the microstructural level and use the collected information as a precursor for material failure on the above-lying scales. Besides the impact of environmental conditions such as temperature or moisture, changes are in many cases caused by mechanical loads, i.e., compression, tension, or multidimensional stress states. We show the crack formation process for a normal concrete for the uniaxial compression and tension cases in Pre-existent micro-cracks evolve primarily in tensile sections along the comparably weaker boundary between the cement matrix and single aggregates, the Interfacial Transition Zone . For theBesides direct impacts such as short-term loading, time-dependent phenomena such as shrinkage and creep are characteristic for concrete. Shrinkage causes a reduction in the material’s volume and is affected by the ambient humidity, the component geometry, the drying process, and the concrete mix. It is a superimposed process of drying shrinkage due to moisture loss to the environment and autogenous shrinkage due to the internal reaction of water during the hydration from fresh to hardened concrete . AlthougThe immediate strain Equation  23] sum sumεci(tn (εc(t)  for a loWe used an identical concrete mix for all experiments. For the compression test, we used three cuboid specimens with an edge length of 400 mm and side lengths of 100 mm with a transducer distance of 300 mm symmetrical to the center axis . We decoHilti HIT-RE 500 V3 having cut the end faces in a chessboard pattern to improve the strength of the joint. The tensile strength was determined to be 3.0 MPA beforehand from three identical samples. The subsequent loading rate was 5 N/s while recording every 10 s up to maximum loads of 100%, 60%, and 30% of the tensile strength For the centric tension test, we used three cylindrical bone-shaped specimens with a length of 300 mm and a decreasing cross section  to definThe specimens for the shrinkage and creep tests were cuboids, geometrically equal to the ones from the compression test. The edge length was 400 mm and side lengths were 100 mm with a transducer distance of 300 mm symmetrical to the specimen’s center axis . The sawThe normal strain of every specimen was individually tracked by a system of strain gauges in a temperature-compensated circuit of two active and two passive strain gauges each. All specimens were equipped with embedded piezoelectric transducers (described in ) to collFor each experiment, we evaluated the collected ultrasonic signals using Coda Wave Interferometry methods. By comparing every signal to a fixed, non-perturbed reference signal at an unloaded state, we present the velocity variations Equation  during trefer to  for the The compressive loading scenario is presented in As the experiment covers not only weak changes in the stress state, using one fixed reference for the entire experiment may cause discontinuities of velocity perturbations due to great signal variations in comparison to an unloaded state. We considered this method-specific phenomenon by establishing an additional evaluation technique picking a new reference signal each time the overall signal correlation coefficient drops below a threshold, which was defined as 0.7 for this analysis. Therefore, we ensure keeping the signal changes within the thus generated windows small enough to satisfy the principles of Coda Wave Interferometry regarding the sensitive evaluation of weak changes to the material. Up to a compressive stress of 6.4 MPa, equaling Analysis of the velocity variation yielded similar development over time, or equally load. We observed a steady signal compression for increasing loads up to approximately 25.6 MPa, equaling 60% slope in . The coeAnalysis of the signal correlation coefficient development also yielded similar results for all specimens up to 12.8 MPa, equaling 30% Both parameters were translated into the compressive stress domain during the loading process. We show the results in The velocity variation is plotted in accordance with both methods for one fixed reference (top) and switching reference signals for windows with Considering building codes , the elaThe centric tensile loading scenario is presented in In comparison to the compressive loading experiment, the stress range is smaller due to the characteristic behavior of concrete regarding tension. For this concrete mix, the ratio between the tensile strength Analysis of the velocity variation yielded similar forms for all specimens over time, or equally load. Signals were steadily stretched for increasing tensile loads up to 100% Signal correlation coefficients steadily decreased during the experiment time, or load up to We translated both CWI parameters into the tensile stress domain during the loading process and show the results for the three specimens in Both processes, shrinkage and creep, are concrete-specific phenomena. In contrast to external load variations, which have a direct impact on the ultrasonic signal, these phenomena cause a characteristic material reaction over time. In this experiment, we show the CWI parameters, together with the measured strain, for pure shrinkage and superimposed creep at different constant loads of Raspberry Pi-based measuring system [The specimens were loaded for the duration of half a year and unloaded after that period. The spike after 7 days results from a restart of the load-applying hydraulic system, where further specimens were added to the experiment. Therefore, the experiment duration for g system . CWI parThe CWI analysis by means of a fixed reference signal, immediately after load application, showed a signal velocity variation corresponding to the characteristic strain evolution during concrete shrinkage and creep. For pure shrinkage, we observed −0.4% Signal correlation coefficients The propagation of Coda waves in concrete is affected by various external impacts that may cause microstructural changes to the material. To interpret the total signal changes, the single effects must be separable, particularly regarding reversible and irreversible changes, or damage. By means of our experiments, we can specify the relations between externally applied mechanical loads, i.e., uniaxial compression and tension, and immediate changes to the ultrasonic signals, i.e., signal correlation and velocity variation, using Coda Wave Interferometry methods. We indicate these relations as calibration curves for a normal concrete, whereas we assess a full range of such calibration curves as key to separate reversible from irreversible changes. This range must be extended by further impacts, i.e., temperature and moisture for the identical material in the future.As we produced a standard concrete using Ordinary Portland Cement, quartzitic aggregates, and a w/c-ratio of 0.45, we evaluate the relations as references valid for a broad range of concrete mixes. However, we expect deviations for concrete mixes with varying cement stone and aggregate stiffness due to the modified crack formation process. During the compression experiment, we consider CWI evaluation techniques with a single fixed reference not able to cover the entire stress range between the unloaded state and material failure due to major material changes, especially while irreversible damage advance. The evolution of Furthermore, we stated a calibration curve for the impact of uniaxial tension on the same concrete mix using one fixed reference signal at the unloaded state. In this experiment, we found nearly linear relations of In addition to immediate responses of the CWI parameters, we extended our experiments by investigation of concrete-characteristic shrinkage and creep behavior. These phenomena were monitored in the time domain and are, in contrast to calibration curves, considered to be long-term features. We showed that shrinkage and creep behavior is, in addition to conventional techniques such as strain measurement, representable by evaluation of the velocity variation Coda Wave Interferometry is a superiorly sensitive method to detect weak changes to heterogeneous materials such as concrete. To establish this technology as an early-warning system for real structures, we analyzed the relations between the correlations and velocity variations of the ultrasonic signals using the stretching technique and externally applied mechanical loads, which we call calibration curves. These curves could serve as the basis for translation of multidimensional fields of velocity variations into stress changes and damage, even for more-than-one-dimensional stress states and on larger specimen scales beyond our investigations. We calculated the relation between velocity variation of the full-length ultrasonic signals for both uniaxial compression and uniaxial tension using a linear regression for elastic material behavior up to 30% of the respective stress which agrees with previous studies ,6,27. Fu


A. carmichaelii Debx., is a typical traditional herbal medicine with both poisonousness and effectiveness, and often used in the treatment of heart failure and other heart diseases. In this review, we searched domestic and foreign literature to sort out the molecular mechanisms of cardiotonic and cardiotoxicity of Fuzi, also including its components. The major bioactive components of Fuzi for cardiotonic are total alkaloids, polysaccharide and the water-soluble alkaloids, with specific mechanisms manifested in the inhibition of myocardial fibrosis, apoptosis and autophagy, and improvement of mitochondrial energy metabolism, which involves RAAS system, PI3K/AKT, JAK/STAT, AMPK/mTOR signaling pathway, etc. Diester-diterpenoid alkaloids in Fuzi can produce cardiotoxic effects by over-activating Na+ and Ca2+ ion channels, over-activating NLRP3/ASC/caspase-3 inflammatory pathway and mitochondria mediated apoptosis pathway. And three clinically used preparations containing Fuzi are also used as representatives to summarize their cardiac-strengthening molecular mechanisms. To sum up, Fuzi has shown valuable cardiotonic effects due to extensive basic and clinical studies, but its cardiotonic mechanisms have not been systematically sorted out. Therefore, it is a need for deeper investigation in the mechanisms of water-soluble alkaloids with low content but obvious therapeutic effect, as well as polysaccharide.Fuzi, the lateral root of  The pathogenesis of cardiovascular diseases is related to pathological links and processes such as hypertension and arrhythmias, which have become the number one cause of death worldwide due to their high incidence and incurability. Nevertheless, heart failure, the end stage of many heart diseases, mainly manifests as a series of clinical syndromes such as dyspnea and edema, and nowadays has become an issue of concern of people all over the world. According to the data published by the GBD , there were an estimated 64.3 million people with heart failure all over the world between 1990–2017 , with 6,The treatment of heart failure is usually based on chemical drugs, including diuretics and angiotensin-converting enzyme inhibitors, etc. Although these drugs are more significant in relieving some symptoms, patients may experience serious side effects after long-term use, such as bradycardia and hypotension, which bring poor quality of life to patients . TraditiAconiti Lateralis Radix Praeparata, named Fuzi in Chinese, is derived from the lateral roots of A. carmichaelii Debx. Compendium of Materia Medica , including alkaloids, flavonoids, saponins, sugars and other components. Among these components, mainly alkaloids, including more than 80 kinds of C19-diterpenoid alkaloids, followed by C20-diterpenoid alkaloids, more than 20 kinds, and more than 10 other alkaloids such as amides, quaternary ammonium salts and aporphines, in addition, nearly 20 kinds of non-alkaloid components  Table 1Table 1.C19-diterpenoid alkaloids are the most isolated components from Fuzi and the most toxic components, mainly composed of aconitine skeleton . If diffFuzi is known as the best botanical drug of restoring Yang (阳) for resuscitation, and its effect of “restore Yang (阳) and rescue patient from collapse and tonify fire to help Yang (阳)” is explained by modern medicine as “strengthening the heart.” So that in addition to analgesia, it is often used in the treatment of cardiac arrhythmia, myocardial infarction, heart failure and other heart diseases. As for the bioactive components of Fuzi in treating heart failure, there are evidences that total alkaloids , water-sThe total alkaloids in Fuzi include diester-diterpenoid alkaloids, monoester-diterpenoid alkaloids and alcohol amine alkaloids. After compatibility and decoction, the diester type alkaloids of toxic components can be converted to the monoester form, and it can be further converted into alcohol amine type alkaloids after absorption by human stomach and intestinal mucosa, thus reducing the toxicity , Kuang .In order to explain which components of Fuzi have cardiotonic effect, It is worth mentioning that the total alkaloids of Fuzi contain many chemical components. At low doses, it has positive effects on cardiac function and isolated hearts, but with the increase of concentration, it shows cardiac toxicity . It can Fuzi polysaccharide (FPS), a water-soluble polysaccharide with hydroxyl group of reduced hemiacetals, has antioxidant, apoptosis inhibiting and autophagic activity enhancing effects.In vitro myocardial ischemia-reperfusion injury cell model, post-treatment with FPS inhibited apoptosis by enhancing the expression of manganese superoxide dismutase (Mn SOD) activity as well as gene expression, promoting the expression of the anti-apoptotic gene Bcl-2, and scavenging excess oxygen radicals produced by mitochondria  Qi” method, believes that the use of Fuzi must be used in large doses in order to be sufficiently effective in saving patients with acute illnesses. Therefore, modern masters of traditional Chinese medicine evaluated Fuzi as “the most useful and most difficult medicine” . A searcvia the p38-MAPK pathway, and stimulates p53 expression and activates caspase-3, all of which together promote apoptosis in ventricular myocytes  in the inner mitochondrial membrane, finally, making the outflow of contents such as cytochrome C and thus causing apoptosis, and the elevated ROS also reduced the oxidative phosphorylation function and decreased the production of ATP; in addition, it were found to inhibit the mRNA and protein expression of PGC-1α, an important factor of mitochondrial biosynthesis, suggesting that PGC-1α may be one of the important factors of myocardial injury triggered by water extract of Fuzi . The alcThe cardiotoxicity of Fuzi is objective, but the specific mechanism of toxicity has not been elucidated in detail, and the current studies have focused on ion channels, oxidative damage and mitochondrial dysfunction and other related pathways. In order to more comprehensively evaluate the toxic and potent effects of Fuzi, it is necessary to scientifically interpret the contents of the botanical drug from six aspects: variety, quality, processing, property, efficacy, application . And thaAconitum (Ranunculaceae) in the world about 400 kinds of plants, mainly in the northern hemisphere temperate regions, and in China there are about 211 species, which are mainly divided into three subgenera  (The genus conitum) . Historiconitum) .According to the historical records, the first time that the Fuzi was recorded as a great poison in the “Shennong’s Classic of Materia Medica” (Shennong Bencao Jing), However, in the “Treatise on Febrile Diseases” (Shang Han Lun) compiled by Zhang Zhongjing in the Han Dynasty, the prescriptions using Fuzi accounted for 16.8% of the total . Some ofShenfu Injection (SFI) is derived from the “Shenfu Decoction,” which was originally published in “Zheng Ti Lei Yao” by Xue Ji in the Ming Dynasty , containIn vitro and in vivo studies revealed that SFI inhibits apoptosis not only by upregulating the anti-apoptotic protein Bcl-2 and inhibiting the activation of caspase-3, but also by regulating the two major apoptotic pathways of Fas/Fas-L and Bcl-2/Bax, thereby altering the balance of microRNA to inhibit apoptosis  is a proprietary Chinese medicine made of 11 botanical drugs, including  in 2004   is one of the catecholamine adrenergic receptor agonists. Studies showed that isoprenaline can lead to cardiac pathological changes such as cardiac hypertrophy and fibroblast proliferation, and that catecholamines levels are elevated in the progression of heart failure  deficiency, and processed Fuzi should be used for mild patients . In the


Background and Objectives: Eosinophilic angiocentric fibrosis (EAF) is an indolent but sometimes locally destructive lesion with a predilection for the sinonasal tract. Although it was first described in 1983, its etiology remains unknown. Some authors initially attributed EAF to trauma, hypersensitivity, and/or surgical manipulation, while it has been recently suggested to include EAF within the spectrum of IgG4-related systemic diseases. Materials and Methods: We report an uncommon case of idiopathic EAF in a 76-year-old male who developed two bilateral tumefactive masses in the nasal cavities. Results: As the histological examination showed a subepithelial proliferation of fibroblasts along with sclero-hyaline fibrosis around small-sized vessels (an “onion skin-like” pattern) and an eosinophils-rich inflammatory infiltrate, a diagnosis of EAF was rendered. The differential diagnosis included granuloma faciale, Wegener’s granulomatosis, and Churg–Strauss syndrome. Conclusions: Pathologists should be aware of the possibility that this lesion can be part of the wide spectrum of IgG4-related systemic diseases by performing IgG4 investigations to assess adherence to IgG4-related systemic disease criteria. Eosinophilic angiocentric fibrosis (EAF) is a benign, rare lesion of unknown etiology. It was first described in 1983 by Holmes and Panje as an “intranasal granuloma faciale” .Females are affected more often than males. EAF clinically presents as a tumefactive lesion, more often occurring in the upper respiratory tract, the sinonasal region, and the orbit, frequently causing progressive and prolonged airway obstruction ,5,6,7,8.Depending on its stage, EAF can histologically exhibit multiple morphological features. In the early stages, eosinophilic vasculitis of the submucosal small-sized vessels, as well as eosinophilic and lymphoplasmacytic infiltrates, are the predominant features. As the disease progresses, fibrosis arises. The late-stage morphology consists of subepithelial thickening due to dense fibrosis and loss of plasma cells and lymphocytes. The deposition of collagen in a concentric lamellar way around the vessels gives the characteristic “onion skin-like” appearance, while eosinophils are prominent. Although immunohistochemistry is not mandatory to diagnose EAF, it is fundamental to relate it to IgG4-RSD .+ plasma cells in the perivascular infiltrate; the possible etiologies and differential diagnoses are also discussed.We report a case of a male patient affected by idiopathic EAF, who lacked both elevated IgG4 serum levels and tissue IgG4A 76-year-old male patient without previous history of nasal obstruction developed two tumefactive masses in both nasal cavities A. No preeosinophilic angiocentric fibrosis” was rendered. Accordingly, the lesion was immunohistochemically tested with an anti-IgG4 antibody, but no IgG4-positive cells were seen. In addition, IgG4 serum levels were also normal (92 mg/dL). Serologic diagnostic work-up for the patient included anti-proteinase 3, c-ANCA, p-ANCA, and anti-myeloperoxidase, but no positivity was found. The erythrocyte sedimentation rate (ESR) was normal. Full body-computed tomography did not show other localizations of disease. The patient is now healthy without local recurrence of disease after 11 months of follow-up.Histological examination of both lesions showed a subepithelial proliferation of bland-looking fibroblasts with an expansive growth pattern and well-circumscribed borders A. As a cEAF is an indolent lesion, sometimes locally destructive and with a predilection for the sinonasal tract. Its etiology is still unclear; it was initially attributed to trauma, hypersensitivity, and/or surgical manipulation, while some authors recently suggested including EAF within the spectrum of IgG4-RSD ,14,15.Conventional histopathological features of EAF include perivascular concentric fibrosis (“onion skin-like”) and eosinophils-rich inflammatory infiltrate. However, the histopathology tends to differ depending on the stage of the disease; while in the early stage the eosinophilic vasculitis is patchy, as the disease progresses, fibrosis arises, until it is the only prominent feature in the late stage . Differe+ plasma cells in four out of five biopsies [+ plasma cells (more than 50 IgG4+ per high power field), and an IgG4+:IgG+ ratio greater than 40% [+ plasma cells in the perivascular infiltrate and high IgG4 serum levels [In the last few decades, EAF was suggested to be part of IgG4-RSD, due to a casual discovery of high IgG4 serum levels and IgG4biopsies . Hallmarbiopsies . Histolothan 40% ,23. Cructhan 40% ,11,22,24m levels ,10. Tabl+ plasma cells were negative, it was crucial to rely on the conventional EAF histopathological features in order to avoid misdiagnoses and mistreatments. The evaluation of the presence of IgG4+ plasma cells is helpful to determine whether the lesion is responsive to glucocorticoid treatment and whether surgery can be avoided, since IgG4-RDs are extremely responsive to medical treatment, but it should not rule out EAF diagnosis when negative.In the present case, in which the immunohistochemical tests for IgG4Recently, an attempt to evaluate the relationship between EAF and IgG4-RSD has been made by reviewing literature and using a validated set of criteria . The resThe present report emphasizes that EAF must be assessed by identifying the classical histopathological findings; however, we would like to emphasize that pathologists should be aware of the possibility that this lesion can be part of the wide spectrum of IgG4-RSD and that they should perform IgG4 investigations to assess the adherence to IgG4-RSD criteria to guarantee the best treatment and outcome for the patient.


Mesenchymal–epithelial transition (MET) exon 14 skipping mutation (METex14) is a low-frequency driver mutation in metastatic non-small cell lung cancer (NSCLC) (3%–4%) and is associated with a poor prognosis. With the advent of selective MET inhibitors such as capmatinib, tepotinib, and savolitinib, the outcome for these patients was significantly improved. Here, we report a 76-year-old male patient with marginally resectable stage IIIB lung adenocarcinoma harboring METex14 who was successfully treated with savolitinib for neoadjuvant therapy. An 82% shrinkage of the primary tumor was observed, and only 5% of the tumor was viable by pathology in the following radical surgery. A dozen of studies tested the efficiency of neoadjuvant immunotherapy or immunochemotherapy, but for NSCLC with driver mutations, neoadjuvant targeted therapy might be more appropriate. We advocated the neoadjuvant MET TKI treatment for NSCLC. The mesenchymal–epithelial transition (MET) factor, as a receptor for the hepatocyte growth factor (HGF), is encoded by the MET gene and plays a vital role in cancer progression. The major MET alterations were MET amplification and overexpression and MET exon 14 skipping mutation (METex14) –5. In ChTo improve the prognosis of lung cancer, neoadjuvant strategies for NSCLC have aroused great interest. The significance of a molecular targeted agent as a preoperative treatment is currently unknown, whereas immunotherapy (IO) has shown promising results in phase 2 or 3 studies such as the LCMC3 , CheckMaA 76-year-old male patient with dyspnea, slight cough, weight loss (3 kg) in 2 months, and a history of smoking, without fever, chills, or syncope, visited our hospital on 7 September 2021. He had an Eastern Cooperative Oncology Group (ECOG) performance status of 2. A routine chest computed tomography (CT) scan showed a mass in the right lung, highly likely to be a tumor. A percutaneous pulmonary biopsy established the diagnosis of a poorly differentiated carcinoma  was significantly longer in the erlotinib group. Also, the overall survival  was numerically longer , 24. In It should be noted that most, if not all, studies were performed on patients with resectable diseases. For those with marginally or potentially resectable lesions, preoperative therapy followed by surgery might also be possible. In an elegant pilot study, patients with initiative “unresectable” locally advanced lung cancer were successfully transformed into “resectable” disease by preoperative immunochemotherapy . This stMETex14 is a low-frequency driver mutation in metastatic NSCLC 3%–4%) and is associated with a poor prognosis . Patient%–4% and Considering the high response rate of savolitinib, the neoadjuvant transformation strategy was explored in our patient. This treatment led to objective tumor regression, confirmed by the pathological response in the following radical surgery. In our case, we advocated the neoadjuvant MET TKI treatment for NSCLC. In support of our proposal, a phase 2 trial (NCT04926831) of perioperative capmatinib in NSCLC with METex14 or MET amplification is being conducted. The results of this trial are warranted.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.All authors conceived, directed, and supervised this paper. JT and ZL were responsible for data collection and manuscript writing. ZD revised the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Concrete presents different internal micro-structure and damage characteristics because of the different content of steel fibers and the randomness of its distribution. Therefore, the failure process of steel-fiber-reinforced concrete (SFRC) should be divided into different stages and the damage types should be classified to further clarify the strengthening mechanism of steel fibers. The role of volume fractions of steel fibers in the splitting-tensile strength of concrete was investigated by split tensile tests for concrete with four different volume fractions of steel fibers . The acoustic emission energy and horizontal displacement of concrete in the splitting-tensile process were monitored by combing digital image correlation (DIC) and acoustic emission (AE) techniques, and the microscopic failure mechanism of SFRC was analyzed emphatically. The results showed that the addition of steel fibers improved the splitting-tensile strength of concrete. With the increase of the volume fraction of steel fibers, the splitting-tensile strength of concrete increased first and then decreased, and reached the maximum value of 5.294 MPa when the content was 1.5%. It was observed that the overall failure mechanism could be divided into four stages: slow accumulation of elastic energy (I); rapid accumulation of elastic energy (II); rapid accumulation of dissipated energy (III); a slow decrease of elastic energy and a slow increase of dissipated energy (IV). Tensile failure dominated the failure process of concrete splitting-tensile resistance, while there was a part of shear failure. In recent years, SFRC has been widely used in some fields including highway bridges, hydraulic dams, ports, and marine engineering. Due to the significant brittleness and low tensile strength of ordinary concrete, adding an appropriate amount of steel fibers can effectively improve its tensile and flexural strength ,2, crackThe DIC technique has been widely used in material strain measurement and crack propagation because of its simple operation, lack of contact, and high precision . Li et aIn summary, in previous studies, DIC and AE technologies were mainly used to study the development of FPZ in the process of concrete fracture damage and to analyze the damage degree of specimens. However, the development law of cracks in concrete in the process of splitting-tensile resistance is still unclear. Moreover, due to the disordered and random distribution of steel fibers, concrete exhibits different microstructure and damage characteristics, so it is necessary to further clarify the crack resistance and strengthening mechanism of steel fibers in the process of splitting-tensile resistance. To provide the relationship between the overall splitting-tensile failure process of SFRC and the local energy dissipation and crack propagation, the splitting-tensile failure process of concrete with four different volume fractions of steel fibers  was monitored by combining the AE and DIC techniques in this study. The full-field horizontal displacement and the parameters such as AE energy were obtained, and the influence mechanism of the volume fraction of steel fibers on the splitting and tensile properties of concrete was studied.Conch brand P.O 42.5 ordinary Portland cement produced by Liquan Conch Cement Factory in Xianyang, China was used in this test. The physical and chemical properties of the cement are shown in fμ = 3.7~1.6. Coarse aggregate used in this test is 5 mm~20 mm continuously graded gravel, which meet the standard requirements of “Specification of Pebble and Crushed Stone for Building” (GB/T14685-2011).The particle size distribution graph of the aggregate as shown in According to the Chinese standard “Fly Ash Used for Cement and Concrete” (GB 1596-91), fly ash is Grade I, with an average particle size of 12.71 μm and is produced by Shaanxi Weihe Power Plant. Its chemical properties are listed in The water reducer was produced by Shaanxi Qinfen Building Materials Co., Ltd. . Its physical properties are listed in The air-entraining agent used was produced by Jiangsu Sobute New Material Co., Ltd. , and the content was 0.01%.The milled steel fiber used was 2.0~2.6 mm in width, 0.4~0.8 mm in thickness, and about 38 mm in length, and was produced by Hengshui Junye Road and Bridge Maintenance Engineering Co., Ltd. , as shown in Four different contents of steel fibers  were used in the test. The mix proportions are listed in According to the “Standard for Test Method of Performance on Ordinary Fresh Concrete” (GB/T 50080-2002), the slump and air content of the fresh concrete were measured. The test results are shown in (1)Firstly, the premixed mortar with a water-binder ratio of 0.4 was used to paint the HJS-60 double-horizontal axis concrete mixer manufactured by Cangzhou Yixuan Test Instrument Co., Ltd.  to avoid water absorption.(2)Cement, fly ash, sand, coarse aggregates, and water reducer agent were added into the mixer and mixed at a low speed of 62 rpm for 30 s. After that, steel fibers were dispersed manually into the mixer and blended at a high speed of 125 rpm for 60 s. Finally, the air-entraining agent was dissolved into water and then the liquid was added into the mixer and mixed for 120 s.(3)The fresh concrete mixture was poured into moulds. The molds were then placed on a vibrating table and vibrated for 30 s to avoid segregation of fresh concrete. The specimens were demolded 24 h after casting and then cured for 28 days in a curing box with a temperature of 20 ± 2 °C and a relative humidity of 95%.(4)After 28 days’ curing, the specimens were taken out and painted by white-black speckles. The typical specimens prepared are shown in Specimens with dimensions of 150 mm × 150 mm × 150 mm (three specimens for each mix group) were prepared for testing the splitting-tensile strength of SFRC. The SFRC specimens were prepared in the following steps:The splitting-tensile test, with reference to GB/T 50081-2019 , was carThe splitting-tensile test was performed on a MTS2000 kN universal testing machine produced by Shanghai Qingbo Test Equipment Co., Ltd.  by applying a load at a rate of 0.05 MPa/s and plotting the load-vertical displacement curve by computer, as shown in The DIC technique was to monitor the splitting-tensile process with the 2018 version of MatchID-2D non-contact, full-field strain measurement and simulation optimization analysis system developed by Match ID company. Two cameras with a resolution of 2048 × 2048 were fixed on a tripod and placed vertically 2 m away from the surface of the specimen being photographed. The crack was expected to occur in the center of the specimen, so the camera’s horizon was focused there . The camWhile starting the tester, the DIC system and AE system were triggered at the same time to dynamically track the crack opening and propagation of the specimen during the splitting and tensile process; the loading was terminated when the specimen suffered macroscopic damage, and the DIC system and the AE system were stopped at the same time.The splitting-tensile strength of concrete with four different contents of steel fibers at 28 days of age is shown in The effect of the volume fraction of steel fibers on the The effect of volume fraction of steel fibers on the W is the work done by an external force on the system, U is the initial strain energy, T is the strain energy released after cracking, and D is the increased surface energy after cracking. When all of D was used to form a new crack area At, Equation (2) was satisfied:Griffith ,34,35 deE is the elastic modulus.The energy release rate of crack propagation  and high AF values (ring count/duration), while shear cracks have high RA values and low AF values, and tension-shear cracks have low RA values and low AF values ,44. The The percentage of cracks at different stages of the whole splitting-tensile process for concrete with different volume fractions of steel fibers is shown in The full-field horizontal displacements of SFRC at different stages during the splitting-tensile process are shown in (1)The addition of steel fibers significantly improved the splitting-tensile strength of concrete. With the increase of the volume fraction of steel fibers, the splitting-tensile strength of concrete increased first and then decreased, and reached the maximum value of 5.294 MPa when the content was 1.5%.(2)AE energy can reflect the four stages of concrete fracture characteristics; the critical state of instability can be judged effectively by the sudden increase of accumulated AE energy. The splitting-tensile process of SFRC mainly included the following four stages: Stage I (0 < (3)Tensile cracks dominated the whole process of splitting and tensile failure of SFRC. The percentage of shear cracks in SFRC with volume fractions of steel fibers of 0.0% was high in stage I, 1.0%, and 1.5% was high in stage I and stage IV. However, the proportion of shear cracks in concrete with volume fractions of steel fibers of 2.0% is between 30–40% at every stage.(4)DIC is possible to measure and accurately determine parameters of crack, such as its actual length, shape, and trajectory of propagation. The combination of AE and DIC is an effective experimental research method, which has great development potential in the field of non-destructive monitoring of SFRC structures.In this study, the splitting-tensile strength of concrete with four different volume fractions of steel fibers  was tested, and the splitting-tensile failure process of concrete was monitored by combining AE and DIC techniques. The evolution law of AE energy and the variation law of full-field displacement of concrete were obtained, and the microscopic failure mechanism of SFRC under macroscopic failure and the crack resistance mechanism of steel fiber were studied. The main conclusions are as follows:


Antibiotic susceptibility testing is vital to tackle the emergence and spread of antimicrobial resistance. Inexpensive digital CMOS cameras can be converted into portable digital microscopes using 3D printed x-y-z stages. Microscopic examination of bacterial motility can rapidly detect the response of microbes to antibiotics to determine susceptibility. Here, we present a new simple microdevice-miniature microscope cell measurement system for multiplexed antibiotic susceptibility testing. The microdevice is made using melt-extruded plastic film strips containing ten parallel 0.2 mm diameter microcapillaries. Two different antibiotics, ceftazidime and gentamicin, were prepared in Mueller-Hinton agar (0.4%) to produce an antibiotic-loaded microdevice for simple sample addition. This combination was selected to closely match current standard methods for both antibiotic susceptibility testing and motility testing. Use of low agar concentration permits observation of motile bacteria responding to antibiotic exposure as they enter capillaries. This device fits onto the OpenFlexure 3D-printed digital microscope using a Raspberry Pi computer and v2 camera, avoiding need for expensive laboratory microscopes. This inexpensive and portable digital microscope platform had sufficient magnification to detect motile bacteria, yet wide enough field of view to monitor bacteria behavior as they entered antibiotic-loaded microcapillaries. The image quality was sufficient to detect how bacterial motility was inhibited by different concentrations of antibiotic. We conclude that a 3D-printed Raspberry Pi-based microscope combined with disposable microfluidic test strips permit rapid, easy-to-use bacterial motility detection, with potential for aiding detection of antibiotic resistance. The analysis of cellular behavior by cytometry can arguably provide insight into bacterial behavior more rapidly than most other methods. This can be useful for making quick phenotypic observations, including antibiotic susceptibility. Rapid and portable measurements of antibiotic susceptibility are needed to combat the increasing spread of antibiotic resistance. Bacterial motility can provide some information on bacterial identification, but can also be used to distinguish between live and dead cells and can be used as a measure of antibiotic susceptibility .Vibrio species; active motility by Salmonella; sluggish motility by Bacillus and Clostridia genus; and thumbing motility by Listeria. Initial developments of bacterial motility tests included the use of culture medium and naked-eye observation, such as in the Craigie, J. (1993) tube method, which was then advanced by the inclusion of microscopic examination  . Bacterial visible stab line and cloudiness were recorded as positive motile, and is considered resistant if antibiotic is present. In contrast, the visible stab line with clear agar was recorded as negative and thus non–motile, which was considered susceptible.The novel microfluidic method was compared to a conventional multiwall stab method. To prepare semi-solid agar, Mueller-Hinton agar . Motility and single-cell imaging provides one of the fastest measurements of phenotypic AST, providing results in as little as 20 min. However, these often require complex and bulky imaging or cytometry systems, such as microscopes or flow cytometers that require a laboratory environment to be operated. Previously, we have demonstrated the use of microcapillary film as a scalable and accurate AST device suited for point-of-care (PoC) applications, using a colour-based indicator to determine growth after several hours . HoweverHere, we miniaturised a soft agar motility assay into microcapillaries . SamplesThis test device was combined with a 3D-printed holder to fit the stage of the 3D-printed OpenFlexure microscope to record videos and thereby determine motility. In this way, a small and compact bacterial cytometer can be produced for under $200 . The 3D-printed OpenFlexure microscope is much smaller and lighter than a conventional laboratory microscope. The system is controlled by a Raspberry Pi, and the sample is lit by a single LED, allowing the system to be powered by 5V USB or even by battery rather than requiring mains power, making it ideal for field use or low-resource settings.There is a trade-off between cost of instrument, field of view and resolution. Biological samples often use phase contrast microscopy or differential interference contrast (DIC) microscopy, allowing unstained samples and cellular structures to be more visible. The OpenFlexure lack these modifications, and the sample is lit simply from above using a single white LED. This provides limited contrast for cellular samples, yet the OpenFlexure microscope was originally developed for water quality testing .P. aeruginosa was loaded into the devices and imaged on a conventional laboratory microscope equipped with a digital camera, and these were compared with the OpenFlexure system  containing TTC and doubling dilutions of ceftazidime and gentamicin were poured into 96-well plate format microtitre stripwells. Cultures of E. coli and P. aeruginosa were stabbed into each well, and movement of bacteria away from the stab site was monitored during overnight incubation at 37 °C using Piramid, a time-lapse Raspberry Pi imaging box recently developed by our group . The MIC can be recorded as the lowest concentration of antibiotic that fully inhibits growth  methods, such as disc diffusion, broth microdilution or agar dilution, are used to measure antibiotic susceptibility . We thers growth . The stalocation B. The moThe feasibility of motility-based AST was then assessed in the low-cost, portable combined microfluidic cytometry system, with bacterial movement recorded directly at the inlet of microcapillaries using the OpenFlexure digital microscope. Results were recorded for two bacteria and two antibiotics at concentrations where motility was known to be inhibited in the soft agar-filled capillaries. Videos of 6 min duration were recorded using a frame-rate of 15 frames per second, which gave the best compromise between image resolution and stable video capture by the Raspberry Pi computer, resulting in movies that run at double speed of real-time microbial . The vidThe results of the microcapillary motility follow the same trends as the stab culture, although as is commonly a challenge for AST, the exact MIC is less clear cut and can be challenging to define. One aspect of this is due to timing. Motility was assessed after just 3 min, compared to 16 h under standard AST conditions. Furthermore, the test involved the bacteria moving into the soft agar capillaries to be exposed to the antibiotics; while motile bacteria can clearly migrate into soft agar, this process is likely to be slower than motility in liquid due to increased viscosity. One adjustment that could be made to this system would be to use liquid media cultures in capillaries containing dried antibiotics; this would expose the bacteria much faster to the antibiotic of interest. The disadvantage of such a liquid system is the risk of rapid mixing or flow of antibiotic solution out of the microcapillary, leading to less confidence in the antibiotic concentration. Another disadvantage of the agar-loaded antibiotic test strips is the limited stability of antibiotic in solution. Our test strips are unlikely to have long shelf-life for many antibiotics. However, the results presented here with this soft agar method clearly demonstrate the concept of low-cost compact 3D-printed microscope combined with melt-extruded microfluidic deviceTo measure motility in the 200 µm microcapillary film, individual bacteria need to be identifiable, as proven to be possible using the OpenFlexure microscope configured with an approximately 400–500 micron field of view. However, this is not possible with lower magnification that allows monitoring all ten capillaries at once, as the resolution is no longer enough to identify individual bacteria, so capillaries have to be monitored individually, and the stage moved to monitor the next capillary. This makes the system more involved to use, but this compromise between resolution and field of view is also true of conventional microscopes.The OpenFlexure microscope provides a low-cost, portable microscopy that combined with increasingly more affordable portable devices, such as microfluidic systems, can allow field use cytometry, for example as near-patient diagnostics, in this example illustrated by rapid antibiotic resistance detection. The microscope has sufficient resolution to identify individual bacteria under simple single-LED bright field illumination, and video recording can readily capture the motility of unstained bacteria in the microcapillaries used in this study. The increase in availability of microelectronics such as low-cost but high-quality optoelectronics combined with microfluidics, will drive the development of new portable diagnostic devices, improving healthcare needs in especially in rural or low-resource settings.