Bovine tuberculosis (bTB) is globally significant due to its impacts on cattle production. A Brucellosis and Tuberculosis Eradication and Control (BTEC) program commenced in Fiji during the 1980's and has since been sustained by government funding and industry cooperation. A retrospective study of bTB data obtained during the Fiji BTEC program from 1999 to 2014 was undertaken at the University of Sydney with support from the Government of Fiji. It confirmed that bTB is well-established in dairy cattle farms in Naitasiri and Tailevu provinces of Central Division on the main island of Viti Levu, and suggested that the disease is present among cattle on farms in all or most provinces across three  of the four divisions in the country. It was evident that despite sustained efforts, disease reduction and containment was not being achieved. Reasons contributing to this situation included the appropriateness of the protocol for conduct of the single intradermal test (SID) in cattle, absence of regular quality assurance training of BTEC field staff, lack of standard procedures for bTB data collation and evaluation, unregulated cattle movements and the presence of stray cattle. The Fiji Ministry of Agriculture responded proactively to these findings by implementing revision to the use of the SID in cattle and refresher training for staff along with the Biosecurity Authority of Fiji who implemented cattle movement restriction. A subsequent apparent outbreak of bTB in some farms due to increased detection by the new test protocol raised concerns for the local dairy industry. To clarify the status and extent of bTB infection and the challenges faced by the industry, a stakeholder forum was held in May 2017, and a new BTEC strategy was formulated and endorsed by stakeholders. bTB remains a focus for cattle disease control by the government of Fiji. This case study highlights the challenges for bTB control in Fiji and underlines the importance of technical and social considerations to achieve success in disease control. Mycobacterium bovis, although other zoonotic members of the M. tuberculosis complex may be the cause, such as M. caprae, the common cause of bTB in central Europe  is a chronic bacterial disease of cattle caused mainly by l Europe . bTB resl Europe . Furtherast Asia .In Fiji, bTB leads to decreased production and opportunity for local trade due to sick animals and early culling of potentially productive stock. The bTB situation in Fiji is becoming an increasing concern for industry stakeholders as the culling of stock further aggravates the low milk production in the country. Data in 2014 from cattle sent to slaughter after a positive skin test on farm showed that one in three reactors  had generalized TB, and 85% had some form of gross TB lesion at post-mortem examination. Successful eradication of bTB is recognized by the Government of Fiji to be of benefit to individual cattle owners and to the country in relation to trade and potentially to human health. Hardest hit is the dairy sector which has suffered the greatest loss of cattle numbers  is administered at the caudal fold of the tail (CFT) with the result read 3 days after administration. Up to September 2014 a positive result was determined by the presence of a wheal not <4 mm in diameter. All log books and handwritten data collected from the field were filed by the Fiji Ministry of Agriculture in a government stock room. There was no written protocol for standard data management and analysis, and no systematic analysis of data to evaluate progress of bTB control over time. Quarterly and annual reports were prepared based on manual counts of records. Designated responsibility for the conduct of the bTB program was at the level of the division offices from 1999 to 2010 in an effort to increase surveillance coverage. This was centralized to the national office from 2011 to improve monitoring of the quality of testing.Cattle farms of all types  are included in the program and participation is mandatory although some farmers do not comply. There are no specific consequences for non-compliance other than ongoing transmission among cattle in non-compliant infected farms. All cattle aged 6 months and above are tested and have a metal tag with a unique number placed in the right ear to indicate the animal has been tested. The single intradermal test (SID) using purified protein derivative antigen from Abattoir monitoring consists of carcass inspection for tubercle lesions by government meat inspectors at the two main abattoirs of the Fiji Meat Industry Board (FMIB) located in Nasinu, Central Division and Vuda, Western Division, respectively . AffecteFarms with positive animals determined by on-farm testing or abattoir monitoring are classified as “Infected.” BTEC requires an infected farm to be free from bTB for 3 consecutive SID tests held at a minimum of 3-months intervals to obtain “Restricted,” “Provisionally clear,” and “Clear” statuses, respectively. It requires a minimum of 9 months from the time of detection for a farm to complete three consecutive clear tests and obtain “Clear” (bTB-free) status.The Fiji BTEC program, a long-term activity sustained by annual government funding and industry cooperation, demonstrates collective commitment to address bTB in the cattle population. To underpin a review of the BTEC program, a retrospective study of bTB surveillance data from 1999 to 2014 was conducted over 12 months during 2014–2015. The aim of the retrospective study was to document the progress of the BTEC program and to provide recommendations to strengthen it. The final results of the study were formally presented to Ministry of Agriculture in September 2016 and to the industry stakeholders during the BTEC Forum held in March 2017.This study was conducted using data collected by the BTEC program from 1999 to 2014. The Fiji Ministry of Agriculture granted approval for use of the Fiji BTEC data to conduct this study in March 2014. Hard copies of batch books, reactor books, field sheets, annual reports, memorandum and other documents related to bTB in Fiji were used to collate and cross-check data from 1999 to 2014. The dataset compiled by year included farm identification number, location, farm type, date of testing, total number of cattle tested, total number of cattle test positive and farm TB status. For 2011 to 2014, the dataset for each year listed tests conducted by individual animal tag number. The few bTB test results from species other than cattle  were excluded, as were farm record data on the number of cattle younger than 6 months. It was assumed that all TB test results were read 3 days after the date of tuberculin administration.Records of carcass inspection at slaughter from 2011 to 2014 were obtained for FMIB abattoirs at Nasinu and Vuda. Individual cattle records for slaughtered bTB positive animals were identified, including reactors identified during on-farm testing and subsequently sent for slaughter, and other animals identified at slaughter via detection of lesions during carcass inspection. The dataset compiled included farm identification number, animal identification number, date of slaughter and type of lesion detected. Complete records were only available for the Nasinu abattoir.Due to the absence of a formal national registration system for all cattle farms, no absolute total cattle number were available for use as a denominator to calculate the population coverage of testing or infection prevalence in this study. In place of this, cattle population estimates for 2011 to 2014 published in the World Animal Health Information Database (WAHIS Interface)  were useFarm ID spreadsheet included farm registration number, farm name, farm location , farm type , date of test, number of cattle on farm by age group, number of animals tested, number of animals tested positive, number of animals tested negative and TB status of farm. Animal ID spreadsheet included farm registration number, farm name, farm location , date of test, TB tag number, age-gender description , and TB test result.For each year from 1999 to 2014, data were transcribed from hard copy sources into a purpose-built spreadsheet in Microsoft Excel version 2003. Data transcription was performed by BTEC personnel from May 2014 to May 2015. Data sorting and validation conducted by the first author produced a comprehensive inventory of cattle farms and farmers from 1999 to 2014. This was verified for dairy farms by matching farm registration number and farm name to the MOA dairy farm registration list, and for beef farms based on familiarity of BTEC staff with farmers and farm operations as there is no formal registration system for beef farms. The list identified 2,141 cattle holding facilities  including subsistence or irregular cattle farm operations. When needed, missing values for farm location were entered based on recall. This list was sent to MOA Economic Planning and Statistics Division (EP&S) to validate location details recorded for each farm. To ensure that all testing data were for bTB SID tests, a cross-check against records for bovine brucellosis testing was performed.Farm ID data from 1999 to 2014 and Animal ID data from 2011 to 2014 were available for analysis. Descriptive statistics for the number of positive farms and animals were calculated, and the number of positive animals detected through on-farm surveillance and abattoir monitoring were tabulated separately by division and by province per year.Farm ID data were also analyzed to determine the number of tests conducted on infected farms each year and the status of each infected farm by year end. Within a calendar year, farms that had undergone one test with at least one reactor  were designated as “Infected.” Farms that had undergone one test round with at least one positive result and undergone one follow up test within the same calendar year with no positive result were designated as “Restricted.” Farms that had undergone two and three consecutive test rounds without any positive results were designated as “Provisionally clear” and “Cleared” farms, respectively.For 2011 to 2014, the status of Infected farms from one calendar year to the next was investigated to identify farms that had positive cattle detected over consecutive years and did not attain cleared status within a period of two or more consecutive calendar years.From 1999 to 2014 ~2,141 cattle holding facilities were included in the BTEC program across the 4 divisions of Fiji  reactors per year  in 6 years with positive cattle (termed reactors) detected in 1999 and 2004  in 9 years with three positive cattle from the two farms tested in Lomaiviti in 2005, and none in the other years  with positive cattle detected consistently Figure , particuFor the 16 years that testing was conducted in Naitaisiri province, reactors were detected each year except in 2010 and 2011 when lower numbers of cattle were tested . For the other years, higher numbers were tested with 7,046 animals (3 reactors) tested in 2009, 5,044 animals (4 reactors) tested in 2012, 4,466 animals (2 reactors) in 2013, and 7,831 animals (6 reactors) tested in 2014.Serua province had the second highest number of reactors (12 of 721 reactors) in 2014, compared to earlier years when Naitasiri commonly ranked next to Tailevu. Data showed that in 2014, reactors in Serua came from only 1 of 6 farms (17%) tested in the province.An average of 168 dairy cattle  were detected positive each year during the last 16 years compared to beef cattle . Ninety-nine percent  of positive dairy animals detected from 1999 to 2014 were from Central Division.For 1999 to 2014 in Central Division, a small proportion of dairy cattle tested positive every year (range 0.1–4.2%), from 0.1 to 1.5% of beef cattle tested positive in 8 of 16 years, and < 1% of cattle from other farm types tested positive in 10 of 16 years  and other farm types (10 or 16 years) suggests that bTB infection is established throughout the cattle population.Further the on-farm testing results and abattoir detections provide evidence that bTB is present among cattle farms in the other three divisions of the country, and in all 4 provinces of Western Division and in 3 of 4 provinces of Northern Division. Given the substantially lower numbers of dairy cattle in these other 3 divisions, this suggests that bTB is established at least among some beef cattle farms in Western Division and Northern Division.From 1999 to 2014, the consistent positive status of a small number of farms and the fact that no farms were cleared of bTB infection within a calendar year (whilst acknowledging that a minimum of 9 months is required to progress from infected to clear status) is clear evidence that the test and cull plus quarantine procedures as applied for infected farms were inadequate to clear infection from a farm. The example of nine dairy farms located along one road in Tailevu province that were consistently positive for 2011–2014 exemplifies the situation with persisting infection.The descriptive analysis of the BTEC data from 1999 to 2014 provided disturbing evidence that despite sustained efforts in on-farm testing and carcass inspection at abattoirs, BTB disease reduction and containment was not being achieved. This situation is well-illustrated although limitations of the 1999–2014 BTEC data, such as considerable variation in number of farms and animals tested between years and the positive SID designation based on wheal ≥4 mm, restricted the retrospective study to descriptive analyses.Factors contributing to this situation and recommendations to strengthen the BTEC program are presented in Table The Fiji Ministry of Agriculture responded proactively to the findings of the retrospective study along with the Biosecurity Authority of Fiji (BAF) and the Fiji Cooperative Dairy Company Limited (FCDCL). The earliest responses commenced in late 2014 initiated following a preliminary analysis of the bTB records for 2011–2013. The response actions taken from 2014 to 2018 are described in detail below.n = 301), and 26, 40, 34% in 2015 (n = 1101), respectively   . During To clarify the status and extent of bTB infection and the challenges faced by the industry and to promote communication and collaboration in delivery of the BTEC program, a stakeholder forum was held in May 2017 with government MOA, Biosecurity Authority of Fiji (BAF) and Ministry of Health (MOH) representatives, Fiji industry stakeholders and relevant experts from Australia and New Zealand. Presentations highlighted the needs for a clear policy and strategy for bTB eradication and rehabilitation, action to address overlapping and unclear legislative and stakeholder responsibilities (particularly between MOA and BAF), immediate removal of infected cattle from farms, auditing and capacity building programmes, and a data recording system for monitoring, evaluation and learning . StakehoThe Ministry of Agriculture further refined and finalized this strategy in early 2018. Input to this process included review and recommendations on meat hygiene, bTB control strategies and diagnostic test selection by a technical team under the Government of Chile funded project “Strengthening the institutions responsible for the inspection and certification of agricultural products, and the coordination of the national system of food safety in Fiji” .The goal of the Fiji BTEC Strategy is total eradication of bovine tuberculosis and brucellosis by 2037. The documented strategy lays out the direction for future implementation of the Fiji BTEC Program to attain the long term goal of official recognition by OIE of Fiji as free from both bovine brucellosis and bovine tuberculosis and maintaining this disease-free status . The strAlong with improvements to field testing and cattle movement control, opportunities for simultaneously improving the laboratory diagnostic capacity for bTB early detection and confirmatory diagnosis have been sought. The BTEC veterinarians and managers have established laboratory network links with Australia, India, New Zealand and Thailand to support diagnostic capacity building in Fiji. Under discussion with the World Organization for Animal Health (OIE) is funding for a Laboratory Twinning Program between FVPL and Animal and Plant Quarantine (QIA) Korea for proficiency testing and laboratory management training.Concerned about the potential contribution of zoonotic TB to the human TB burden in Fiji, the MOA in collaboration with the Fiji Ministry of Health and Medical Services and the University of Sydney funded by the Marie Bashir Institute will undertake geospatial analysis of human tuberculosis cases and bTB-infected cattle farms, pilot TB surveillance of households in identified high risk areas for bTB exposure, and send samples from human extra-pulmonary cases and cattle cases for species determination. This investigation arises from concern about levels of extra-pulmonary tuberculosis (EPTB). During 2016 among the 312 notified human TB cases, 29% were classified as extra-pulmonary tuberculosis, nearly double the 15% of extra-pulmonary tuberculosis cases among the global total of human TB notifications in 2016 . The conWhen the preliminary analysis of 2011–2013 bTB records indicated wide spread endemic infection, the Fijian government acted swiftly in September 2014 to revise SOP for SID testing. This was followed up by actions from 2014 to 2018 that have enhanced identification of infected cattle farms and removal of infected cattle, strengthened implementation of restrictions on cattle movements, and led to the endorsement of a new Fiji Brucellosis and Tuberculosis Eradication Strategy. These are critical steps on the journey to reduce bTB in the national cattle herd, and then subsequently to progress to bTB eradication. This staged process of bTB reduction and containment followed by eradication can be guided by the lessons learnt by other countries on the road to bTB control and eradication, such as Australia, Ireland and New Zealand. The generic components, first of bTB control and containment while ensuring continuity of the industry, and second of bTB eradication and proof of freedom must be contextualized to the bTB situation in Fiji. A policy based on contemporary scientific evidence and international best practice in bTB control needs to be accompanied by specific research in Fiji, given its particular geoclimatic and cultural features. It is crucial for the Fijian government and the dairy and beef industries to be aware that the current policy will need to be modified over time and the commitment to implementation maintained when the BTEC program transitions to the final eradication stage. Industry concern about an increasing proportion of SID test positive cattle with no visible lesions at slaughter is expected with continuation of current SOPs. This provides an example of a situation where technical expertise is required to inform future decisions on test protocol, and where specific research would be beneficial to determine if false positive cases are present and to understand the basis and the extent of these. The international community also needs to consider its role in supporting the Fijian government and industry to attain bTB freedom for the benefit of animal and human health in the Pacific. As Fiji serves as a regional hub, providing live animal stocks and animal products to the neighboring island countries, addressing bTB in Fiji supports the long-term goals of sustainable livelihood and food security in the Pacific island region.The case study of bTB control in Fiji offers lessons within a Pacific context about the importance of the following technical and social aspects to achieve success in animal disease control.Objective, ongoing assessment of bTB distribution using agreed performance measures  is internationally accepted as essential for critical assessment of progress toward control and eradication . This reRobust and accurate diagnostics able to minimize false farm or animal designation given bTB prevalence level at the relevant stage of the control and eradication process must be applied. Selection of the most appropriate diagnostic test/s given the stage of control program and the field conditions for animal testing requires expertise in test protocols. Understanding is needed of the costs of false designation to bTB maintenance and spread  and to unnecessary loss of productive animals and prohibition to trade . The Fiji MOA recognizes that early identification of infected farms and infected animals is critical. To date the SID PPD-B in the caudal fold is the single diagnostic applied in the Fiji BTEC program due principally to its low cost and practical suitability to on-farm conditions. While the combination of SID test in the caudal fold (assuming use of potent tuberculin) and carcass inspection at slaughter is reasonable for detection of infected farms, a more sensitive testing regime is needed to support eradication from known infected farms. Thus, the MOA is considering application of other diagnostics, such as the interferon-γ test as a confirmatory test for SID positive animals in known infected farms, and increased use of culture to confirm status of lesions identified at abattoir carcass inspection. A cost-benefit analysis on the use of single intradermal comparative tuberculin test (SICTT) and interferon-γ [with sensitivity when applied in parallel approaching 93% ] in placQuality control (QC), the managerial process to compare actual and desired performance of a service or product, will act to ensure an animal disease control program is meeting its objective at the best possible return for the funds invested , 25. WheFarmer cooperation with control and surveillance activities is vital for the success of animal disease programs. Active participation requires farmer knowledge of bTB risk and impact on cattle production and health, and farmer confidence that BTEC requirements are feasible and effective. Strengthening incentives, such as compensation for culling of positive animals will encourage more farmer cooperation. Effective communication about bTB via farmer targeted and general community campaigns is required to generate farmer action and community support. Clear messaging is proving challenging for bTB due to confusion about tuberculin skin test performance, the involvement of wildlife reservoirs in some countries, and local cultures and beliefs, particularly in countries where despite sustained control programs bTB remains endemic, such as Spain and the United Kingdom. Recent qualitative research involving farmers and veterinarians in Spain articulated the link between farmer non-participation in on-farm testing and distrust of official veterinary services and with farmer perception of little benefit to be gained from bTB freedom .The Government of Fiji has demonstrated sustained commitment to reduce bTB in the cattle population. The determination to succeed in a resource limited setting with challenging field conditions is to be commended. The history of bTB control elsewhere shows that the use of tuberculin tests (SID PPD-B and/or SICTT) needs to be relevant to the context  and the EB collated and analyzed the data for the retrospective study. RP assisted EB with collation and entry of the bTB data 1999–2014. LB, RP, and TT assisted with interpretation of the findings of the retrospective study. All authors contributed to the content and the preparation of this manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer MG declared a shared affiliation, with no collaboration, with one of the authors, EB, to the handling editor at time of review.


In a collaborative study involving six laboratories in the USA, Europe, and India the molecular weight distributions of a panel of heparin sodium samples were determined, in order to compare heparin sodium of bovine intestinal origin with that of bovine lung and porcine intestinal origin. Porcine samples met the current criteria as laid out in the USP Heparin Sodium monograph. Bovine lung heparin samples had consistently lower average molecular weights. Bovine intestinal heparin was variable in molecular weight; some samples fell below the USP limits, some fell within these limits and others fell above the upper limits. These data will inform the establishment of pharmacopeial acceptance criteria for heparin sodium derived from bovine intestinal mucosa. The method for MW determination as described in the USP monograph uses a single, broad standard calibrant to characterize the chromatographic profile of heparin sodium on high-resolution silica-based GPC columns. These columns may be short-lived in some laboratories. Using the panel of samples described above, methods based on the use of robust polymer-based columns have been developed. In addition to the use of the USP’s broad standard calibrant for heparin sodium with these columns, a set of conditions have been devised that allow light-scattering detected molecular weight characterization of heparin sodium, giving results that agree well with the monograph method. These findings may facilitate the validation of variant chromatographic methods with some practical advantages over the USP monograph method. Heparin preparations for medical use are polydisperse polymers derived from mast cell containing tissues, with molecular weights determined by both the tissue of origin and the processes involved in heparin manufacture . MeasureOnly heparin from porcine intestinal mucosa  is currently approved for use in the USA, but the US Food and Drugs Administration (FDA) is investigating the possibility of introduction of heparin sodium from bovine intestinal mucosa  into the US market . Bovine A multi-laboratory collaborative study has therefore been organized by the USP to obtain consensus molecular weight data using the USP Heparin Sodium monograph method ,5 for a Phase 1: Molecular weight parameters as described in the USP Heparin Sodium monograph  were meaPhase 2: Using the same heparin samples as Phase 1, a single laboratory assessed results from 12 distinct chromatographic methods, varying in column type, mobile phase, and calibration method. The aim of this phase of the study was to identify alternative chromatographic methods giving similar molecular weight values to those obtained using the USP Heparin Sodium monograph method, avoiding some practical disadvantages of the USP method.Results for 10 heparin samples were obtained from six participating laboratories, and for a further 14 samples from five laboratories. The sample codes are listed in wM , 24,000M (no more than 20%) and the ratio 8000–16,000M/16,000–24,000M (no less than 1.0) [An earlier study of heparin samples from porcine intestinal mucosa, currently the only acceptable source for heparin sodium in the USA, was used to derive appropriate acceptance criteria for molecular weight distribution by setting limits to the weight-average molecular weight wM), 24,000M) and 8000–16,000/M16,000–24,000M)   and summwM near 16,000 Da, a typical value for heparin sodium as previously determined [24,000M of about 9%, and the ratio 8000–16,000M/16,000–24,000M is about 1.5. Two bovine lung heparins H-2 and K-2 have lower wM, outside the acceptable range for heparin sodium at about 13,500 Da. This is consistent with values determined for bovine lung heparins dating from the 1950s to the 1990s [The two porcine mucosal heparins A-3 and D-1 have termined  and closhe 1990s . wM higher than the top limit of 19,000 Da, and the same three samples have more than the limit of 24,000M (20%). One of these samples also has a low value for the ratio 8000–16,000M/16,000–24,000M, though the mean value from five laboratories rounds up to 1.0, the lower limit for this value. For the 20 bovine mucosal heparins, variability between individual samples is similar to that for porcine heparin before introduction of molecular weight acceptance criteria, as determined previously . Three swM. Though there is considerable overlap between the two samples, it is clear that sample E-2 contains a major amount of low molecular weight material, with longer retention time, compared with sample R-2. At higher molecular weights (short retention time), sample R-2 has about 30% material over 24,000 Da (well outside the USP’s acceptable range for porcine heparin sodium) whereas sample E-2 only has about 8% , samples were analyzed with polymer column set C (Methods from 3 to 7) and finally with polymer column set D (Methods from 8 to 12). The chromatographic profiles overlay of the USP Heparin Sodium Identification RS is reported in wM range calculated for the USP Heparin Sodium Identification RS, as measured to assess system suitability (see Methods section), is between 15,200 and 17,700 Da, with an average value of 16,200 Da. Acceptance criteria for the system suitability test indicate that wM for this sample should lie within +/−500 Da of the established value of 16,000 Da (see Methods for the full set of system suitability requirements). Results for Methods 2, 4, 5 and 7, respectively 17,700, 15,200, 16,700 and 17,000 Da are out of the USP acceptance criteria, but considering all 12 methods wM results, the RSD% calculated is 4.17%, an acceptable value taking into account the instrument sensitivity and the different chromatographic conditions tested. One of the most critical points in the analysis with light-scattering detector is the use of the correct dn/dc value, a parameter that is a function not only of the sample but also of the chromatographic conditions, and this is the base of the differences observed in molecular weight distribution results [The weight-average molecular weight 24,000M in comparison with Method 1 ; instead Methods 3, 4 and 6, acquired at 30 °C with the same columns but different calibration, gave lower molecular weight in comparison to 5 and 7 . So, it is clear that column set C does not completely have the acceptable chromatographic properties required. The main reason of these differences could be the different particle size between columns sets C and A . Regarding to the columns set B, both molecular weight and ratio 8000–16,000M/16,000–24,000M are out of the acceptance criteria; again, the main problem could be the particle size of the columns set (5 µm). As a conclusion for this first part, it seems that with a chromatographic system in which the particle size is the same for each column used in series, the correct resolution required is not reached, with the only exception of Method 3 chromatographic conditions, very similar to the USP official method. Looking at Methods 8 to 12, each of these chromatographic conditions results are within the acceptance criteria; the fact that column set D particle sizes are different (respectively 10 µm for the TSKG4000SWXL and 7 μm TSKG3000PWXL) could be a confirmation that having a single particle size in a column set cannot reach the right resolution required. Particularly remarkable is that the use of a light-scattering detector allows us to reach results comparable with the official USP method for the analysis of heparin sodium.More in detail: Methods 5 and 7 were acquired at 40 °C with same columns and both gave high molecular weight and of course higher wM . This calibrant was characterized for use with the L59 silica columns specified in the monograph. The calibrant information for a broad standard is a slice table of mass fraction vs. molecular weight  of the duplicate injections of system suitability solution do not differ by more than 5% of the upper value.System suitability criteria were as follows, taken from the USP Heparin Sodium monograph: in the chromatogram of the system suitability sample (the USP Heparin Sodium Identification RS), there is a baseline resolution between the heparin and salt peaks. The linear regression coefficient of the calibration curve fitted to the broad standard table values must be not less than 0.990 in magnitude, using a third order polynomial equation. The mean of the calculated In Phase 2 of the study 12 different GPC methods were compared in a single laboratory. Details of the chromatographic conditions used in each of the 12 methods are given in the text of the wM, the percentage of heparin with molecular weight in the ranges 8000 to 16,000, 8000–16,000M, and 16,000 to 24,000, 16,000–24,000M, and the percentage of heparin with molecular weight greater than 24,000, 24,000M were evaluated.Two methods for derivation of molecular weights from chromatograms were employed. For methods 1 (the USP monograph method), 5, 6, 7, 10, 11 and 12, in which only the refractive index detector is involved , a broad standard calibration was performed using a chromatogram of the USP Heparin Sodium Molecular Weight Calibrant RS, as described for Phase 1. Methods 2, 3, 8 and 9 used both RI and Right Angle Laser Light Scattering (RALLS) detection, for which no calibrant reference standard is needed. The relationship between refractive index increment and concentration of the analyte, known as the dn/dc parameter, changed as a function of the mobile phase used, as described in wM value determined for USP Heparin Sodium Identification RS (the system suitability sample) has to be within 500 Da of the assigned value of 16,000 Da [Acceptance criteria were as for Phase 1: The chromatographic system is suitable if the chromatographic profile of samples does not overlap the mobile phase peak; secondly, the 6,000 Da .A panel of 20 lots of bovine mucosal heparin had average molecular weights similar to those of porcine mucosal heparin samples, but with a wider variation from sample to sample, probably reflecting differences in manufacturing methods for heparin from a single species and tissue source. Some molecular weight values fall outside current USP acceptance criteria for heparin sodium.Bovine lung heparin samples were lower in average molecular weight than mucosal heparin, as has been reported in the past. Alternative SEC methods for molecular weight analysis of heparin sodium give varying degrees of comparability with the USP monograph method. Use of the USP Heparin Sodium Molecular Weight Calibrant RS with long-lived polymer based columns gave comparable results with the USP monograph method for samples within the current acceptable range of heparin sodium samples; some methods using polymer-based columns with light scattering detection gave good agreement throughout the full range of heparin samples investigated in this study.


Mycobacterium bovis (or M. caprae) infection. Moreover, since 2012, a PCR method with similar sensitivity and specificity values of histopathology and bacteriology respectively is additionally employed in France, partially compensating for the weaknesses of classical diagnostic methods. We analysed a collection of bTB-like lesions from cattle presenting positive histological results albeit with negative PCR results. We present here the results of these samples, recovered from 292 animals culled between 2013 and 2016, analysed with a second line molecular diagnosis approach that consists in a combination of PCRs targeting the M. tuberculosis-M. avium complexes as well as the Mycobacterium genus and sequencing of hsp65 gene. These molecular analyses disclosed to identify the presence of non-tuberculous bacteria which could be responsible for most of these non-specific TB lesions: non tuberculous mycobacteria (24%) or Actinomycetales (56%) such as Rhodococcus equi (53%); 24% of the samples were negative. M. bovis -or any other MTBC members- was neither detected by molecular methods nor isolated in any of them at the end of the 3 months of culture. In conclusion, these results highlight the lack of specificity of histopathology and the usefulness of a first line PCR with a second line molecular diagnostic test to circumvent it. This diagnostic strategy makes it possible to reduce the number of suspect bTB cases raised at the abattoir or shortening their lock-up periods. By simplifying diagnostic schemes, the use of this tool could improve bTB surveillance and make eradication programs more efficient in the future.Surveillance of bovine tuberculosis (bTB) is partly based on the sanitary inspection of carcasses at the abattoir to detect bTB-like lesions which, in compliance with EU recommendations, are analysed by bacteriology and histopathology to disclose  Mycobacterium bovis, is an important re-emergent zoonotic disease in Europe [Bovine tuberculosis (bTB), mainly due to n Europe . Althougn Europe , 3. The n Europe . In thisn Europe , 6. Stiln Europe .Mycobacterium tuberculosis complex (MTBC) mycobacteria [BTB diagnosis by bacteriology can take up to three months due to the slow growth of the bacteria . Alternabacteria . Actuallbacteria . This PCA significant and increasing number of cattle samples were analysed at the NRL, after discordant histopathological positive–first line PCR negative results. In this study we summarise the results of 4 years analyses on such samples with our second line molecular diagnosis scheme, which made it possible to identify non-tuberculous bacterial agents giving rise to nonspecific bTB-like lesions, to avoid cumbersome -albeit official- diagnostic alternatives such as culture for confirming the bTB-free status of the herd, thus gaining diagnostic specificity and confidence for bTB status confirmation.BTB is a notifiable disease for which there are control and surveillance campaigns in France. Official methods for diagnosis of this disease are culture, PCR and histopathology. Therefore, all the samples included in this study are issued from animals analysed within an official context. No purpose killing of animals was performed for this study. All samplings were in complete agreement with national and European regulations. No ethical approval was necessary.https://info.agriculture.gouv.fr/gedei/site/bo-agri/instruction-N2013-8123) follow the regulation(EC) No 854/2004 . Typically, lesions due to M. bovis have a centre of caseous necrosis, sometimes associated with calcification, surrounded by epithelioid cells, lymphocytes and neutrophils [M. bovis. Two to 5 g of sampled tissues were crushed with a 4% sulfuric acid solution to decontaminate the tissue. After 10 min, the acid was neutralized by adding a 6% sodium hydroxide solution. After decontamination, the supernatant was seeded on two different solid media: Löwenstein-Jensen and Coletsos. All seeded media were incubated at 37°C +/- 3°C for three months and exanimated every two weeks. Any isolated mycobacterial strain is submitted to the NRL for further characterisation. DNA from each sample was extracted by using the QIAamp DNA mini kit  or by Magvet MV384  with a King Fisher KF96 automate, following the manufacturer’s instructions and analysed with the LSI VetMAX Mycobacterium tuberculosis Complex Real-Time PCR Kit . Samples submitted to the NRL for further molecular analyses were those that (i) presented a histopathological result suggesting tuberculosis, and (ii) showed a negative result with the first-line PCR.Samples included in our study presented macroscopic bTB-like lesions at routine abattoir inspection between 2013 and 2016. Inspection procedures for bovine carcasses implemented in France  identification, and the 65 kDa heat shock protein gene (hsp65) for Mycobacterium sp. detection  [rpoB) [Identification of non-tuberculous mycobacteria species was done by sequencing using primers targeting the 65 kDa heat shock protein gene (hsp65)  or the β) [rpoB) . The obtThis retrospective analysis includes all cattle cases sent to the NRL which had a negative result for MTBC PCR but with a bTB suggestive histopathology. Between 2013 and 2016, we analysed samples of 292 cattle from 278 herds . The majMycobacterium sp. (n = 1), R. equi (n = 1) and a Nocardia sp. (n = 1).The histopathological profiles of these animals were almost the same, i.e. encapsulated granulomas with necrosis areas and the presence of Langhan’s giant cells. For some samples, other types of cells, like lymphocytes or macrophages, were observed as well as a partial to complete mineralisation. The identification of acid-alcohol resistant bacillus by Ziehl Neelsen staining was positive for a few samples (15/292), latter identified as MAC (n = 11) or hsp65 sequencing: 13 were MAC, 15 were other non-tuberculous mycobacteria (NTM)  and four Rhodococcus equi. The identification by bacteriology was congruent with the one on tissue for 22 samples (Only 31 out of the 292 samples were bacteriology positive. Identification of these isolates was performed by  samples . For theM. avium avium and M. nonchromogenicum) or between a NTM  and an Actinomycetale (Rhodococcus equi). Identification of the bacteria species by sequencing highlighted that 95% of the Actinomycetales were R. equi. Among the NTM, MAC represents 26% of the cases, while the other half were various mycobacteria species  and 8 actinobacteria from various genus: Corynebacterium sp., Streptomyces sp., Arthobacter sp. and Nakamurella sp. These results confirm that bacteria identified in nonspecific bTB-like lesions are not ubiquitous and accordingly that they are the real causative agents of them.Of the 240 LN from control animals without bTB-like lesions and a negative first-line PCR results, 96% remained completely negative to the second line diagnosis . Only twNocardia species [Nocardia, Rhodococcus or Corynebacterium) shared the same tinctorial properties and thus are identified as acid resistant bacilli by Ziehl Neelsen staining [We studied bTB suspect cases resulting from abattoir inspection which presented nonspecific histopathology bTB-like lesions and a negative first line MTBC PCR and focused on the identification of the bacteria responsible for them. The number of this type of nonspecific suspicions increased since 2013 as a result of awareness campaigns organised for abattoir agents, one of a series of measures introduced to reinforce the national bTB control campaign in 2011 . None of species . Moreovestaining .M. nonchromogenicum, few MAC, few M. kansasii and only one R. equi [R. equi, have been recognised in lymph node infection of domestic and wild animals (swine and wild boar (Sus scrofa), roe deer (Capreolus capreolus) or red deer (Cervus elaphus)), which could lead to a possible misdiagnosis of M. bovis [Many environmental mycobacteria may interfere with the bTB surveillance program at post-mortem inspections at the abattoir . NTM hav R. equi . NTM as M. bovis –22. IndeM. bovis , i.e. thR. equi and the presence of nonspecific bTB-like lesion as this species was not identified in the LN of cattle without any bTB-like lesions. Rhodococcus equi and non-tuberculous mycobacteria  are facultative intracellular pathogens surviving inside macrophages and inducing granulomatous inflammation [Rhodococcus equi (formerly Corynebacterium equi) is a coccobacillus bacterium commonly found in soil which is pathogenic for domesticated animals such as horses, pigs and cattle [R. equi in the monitoring of bTB was already acknowledged 35 years ago [R. equi are most frequently observed in retropharyngeal, bronchial and mediastinal lymph nodes [M. bovis granulomas, even if the presence of a heavy infiltration of neutrophils and/or extensive sheets of macrophages could presumably allow distinction [Mycobacterium nonchromogenicum which is known to interfere with ante-mortem diagnosis of bTB [Mycobacterium bourgelatii, closely related to M. intermedium and described for the first time in 2013, was isolated from cattle lymph nodes [M. intermedium is classified as a ‘pathogen’, together with M. gordonae and M. kansasii, in a recent phylogenetic analysis [Mycobacterium aichiense closely related to M. gilvum [M. petroleophilum closely related to M. aurum [M. pyrenivorans and M. thermoresistibile are all rapidly growing mycobacteria found in the environment, potentially opportunistic in humans and included in the same phylogenetic group [Mycobacterium genavense, slow growing mycobacteria, is responsible for infection both in birds and humans and has also been isolated from the environment [Mycobacterium shimoidei, slow growing mycobacteria, is an opportunistic pathogen of humans but few pulmonary cases have been reported worldwide [Our results strongly suggest the link between ammation , 23. Rhod cattle , 25. Eved cattle . The intears ago . This baears ago , 25. Gratinction . Non-tubtinction , 22. Inftinction  and Mycos of bTB , 28, 29.ph nodes . M. inteanalysis . Thus, tanalysis . Mycobac. gilvum , M. petrM. aurum , M. pyreic group . Mycobacironment . MycobacM. bovis antigen immuno-localisation [M. bovis culture. In conclusion, second line molecular tests used at the NRL could confidently be added as an official test in order to accelerate the diagnosis process and improve bTB surveillance in Europe and to render control and eradication programs more efficient in the future.In our study, the majority of the samples presented a histological profile with an encapsulation of the granuloma and the presence of giant Langhans cells, sometimes in association with others cells . The presence of epithelioid macrophages and Langhans cells is not pathognomonic as these cells are seen in immunologic granulomas , especialisation . In a prlisation . HistopaS1 Table(DOCX)Click here for additional data file.


The physico-chemical processes supporting life’s purposeful movement remain essentially unknown. Self-propelling chiral droplets offer a minimalistic model of swimming cells and, in surfactant-rich water, droplets of chiral nematic liquid crystals follow the threads of a screw. We demonstrate that the geometry of their trajectory is determined by both the number of turns in, and the handedness of, their spiral organization. Using molecular motors as photo-invertible chiral dopants allows converting between right-handed and left-handed trajectories dynamically, and droplets subjected to such an inversion reorient in a direction that is also encoded by the number of spiral turns. This motile behavior stems from dynamic transmission of chirality, from the artificial molecular motors to the liquid crystal in confinement and eventually to the helical trajectory, in analogy with the chirality-operated motion and reorientation of swimming cells and unicellular organisms. Converting chemical energy into movement is essential to all forms of life, but the molecular processes are yet to be uncovered. Here the authors show a light-responsive reorientation behavior in liquid crystal droplets which stems from dynamic inversion of chirality from molecules to liquid crystals in confinement. However, these motile systems have yet to demonstrate the sophisticated and purposeful behavior of their living counterparts12, and, in particular, encoding directionality from the molecular level upwards has remained elusive. The frequently used catalytic decomposition of hydrogen peroxide to achieve propulsion, typically leads to erratic motion, with a notable lack of control over rotational degree of freedom13. In contrast, swimming cells and aquatic microorganisms are steered by biomolecular machines and can thus orient their swim. For example, it is the flagellar motor of the Escherichia coli bacterium that steers its motion and encodes its tumbling, with chirality a salient feature in this operation15.In the search for creating molecular systems with life-like qualities, droplets, proto-cellular constructs, and other small-scale motile entities have been designed and synthesized18 and in recent years, the asymmetry and chirality of dynamic and responsive molecules has been harnessed to drive complex macroscopic motion19, e.g., the twisting of springs20, the rotation of patterns21, and the emergence of pulsating vortices22. Even so, in all these examples motion sees the center of mass of the object fixed, or in other words the molecular motion fails to convert into motile behavior.Chemists have succeeded in designing and synthesizing a large variety of artificial molecular machines24. Movement at small scales requires a shape transformation of the body that breaks a time-reversal invariance25, and this is arguably why swimming microorganisms must be able to change the shape of their chiral bodies. For example, E. coli uses a rotating helical flagellum to swim14, whereas helix-shaped body of Helicobacter pylori and Campylobacter jejuni mediates their efficient swimming in challenging media26. Besides often relying on chiral body shape transformations, the vast majority of aquatic microorganisms and cells actually swim along helical trajectories, including zooplankton, ciliates, and bacteria27. Although the evolutionary advantages of helical motion remain unclear, it appears that the directionality of helical motion is less sensitive to extracellular and intracellular random variations, compared with rectilinear motion28. Significantly, swimming in a screw-like fashion offers opportunity for efficient reorientation behaviors set to follow chemical and physical gradients30. Besides the well-known ‘run and tumble’ behavior of E. coli, these chirality-derived reorientation mechanisms often feature deterministic directional changes, such as in the ‘run, reverse, and flick’ of Vibrio alginolyticus, a marine bacterium that also moves along helical pathways, and for which the flick angle is pre-programmed, i.e., the re-direction does not occur randomly32.Chirality is key to impart sophisticated motile behavior36. Recent work has shown that such chiral droplets follow helical trajectories in surfactant-rich water37, based on the observation that instabilities can form when micelles solubilize oils from droplets in water40.Our general strategy is inspired by the chirality-derived reorientation mechanisms of living systems, where molecular machines drive a change in chiral structures to initiate a reorientation from the molecular level upwards. We have incorporated molecular motors in an  nematic liquid crystal. The chirality of the motors induces a twist in the liquid crystal and leads to the formation of a light-responsive cholesteric liquid crystal, which is characterized by a helical organization of the molecules Fig. . This liHere, we show that photo-inverting the handedness of the molecular motors initiates a cascade of bottom-up events. This molecular-scale event leads to inversion of the liquid crystal helix, further into inversion of the spiral organization of the director field in the droplet, and eventually leading to modification of the flow of fluid matter that steers the helical propulsion. Droplets subjected to such an inversion of handedness reorient their swimming in a direction that is encoded by the number of spiral turns in the droplet.m and Ph-m, Fig. 41. These large photo-induced changes in pitch are combined with the possibility to invert helical handedness43.Overcrowded alkenes were used as light-driven molecular rotary motors , Me-m concentrates in the droplet during the motion and translates into an increase of spiral turns. This observation is consistent with previous report on chiral dopants structurally more complex than CB15, which have been proven to enrich in shrinking droplets38.Liquid crystals doped with (10.00 ± 0.01) wt% CB15 have an initial pitch of ca. 1.5 μm, with initial values of ion Fig. . During m offers complementary data that point at a threshold value of N over which helical motion occurs. The helical twisting power of Ph-m is larger than that of Me-m in the same liquid crystal, and was used as a dopant to form droplets of initial pitch ca. 1.6 μm . These chiral droplets doped by Ph-m are characterized by a confinement ratio that is similar to that of the CB15-doped drops, and, similarly, over time, the chiral confinement increases in these droplets, until the high concentration of motor disrupts the liquid crystal order and the droplet turns isotropic , the droplets reorient significantly in three-dimensional space and the handedness of their trajectories inverts also Fig. –10. We nsee Fig. , we estiθ that defines a cone of probability for the reorientation . In such experimental conditions, the liquid crystal is allowed to transition in the compensated nematic state, where chirality is not expressed because the chiral contributions of the motor isomers compensate each other. As the number of spiral turns decreases in the droplet, the radius of the helical motion decreases and eventually the droplet swims along a straight trajectory that merges with the axis of the helical trajectory, as shown in Fig. 25 such as bacteria and the malaria parasite57, as well as chiral neuronal motility58. In a context where accumulating evidence suggests that cells are intrinsically chiral and that the cytoskeleton and motor proteins steer their chiral motility59, our findings show how asymmetric molecular operations are able to effectively regulate motility in fully artificial molecular systems.The helical motion of spiral droplets in surfactant-rich water is determined by their handedness  and by the confinement of the liquid crystal helix in the droplets (which defines the pitch and radius of the trajectories). The system features transmission of chirality across length scales: the chirality of the molecular motors is transmitted to the spiral organization of the liquid crystal in the droplets, which is in turn transmitted into a chiral trajectory. Our experiments demonstrate that when the chiral dopants used to make the droplets are light-driven molecular motors, the light-induced inversion of handedness operated by the motor is associated with a deterministic reorientation of the droplets. Key to the emergence of directionality in response to light is the possibility of a dynamic conversion between right-handed and left-handed trajectories. The chirality-derived and deterministic reorientation of the spiral droplets shows similarities with the motile behavior of unicellular organismsm and Me-m were synthesized by following a procedure on which we reported earlier22.The nematic liquid crystal 5CB and the chiral dopant CB15 were purchased from Merck and used as received. TTAB (tetradecyl-trimethylammonium bromide) was purchased from Wako. The light-responsive chiral dopants Ph-m, or Ph-m) were weighed in glass vials and then the desired volume of nematic liquid crystal 5CB was added. The contents of the vial were heated above the isotropization temperature, where the opaque material becomes transparent. The isotropic mixture was stirred at 40°C and was cooled down to room temperature.The chiral dopants , until no change in texture was observed anymore  and its pitch was measured using the Grandjean Cano method, with a polarized optical microscope (Olympus BX51). The pitch of the liquid crystal helices doped with Me-2O) surfactant solution . The chamber containing the aqueous solution was prepared by applying an adhesive spacer (≈ 1 mm in thickness) onto a microscope slide. After injection, the chamber was closed with a glass coverslip. The size of the droplets was tuned by changing the injection pressure and injection time of the Femtojet.Motor-doped liquid crystals were injected with a microinjector , using commercially available needles  in an aqueous . The time-lapse images were acquired using a CMOS camera . Tracking was performed by adjusting the focus on the droplet manually. Experiments in which the droplets were out of focus were not included in the statistical analysis. The plane coordinates of the stage  and the Z coordinate of the motorized objective lens  were recorded using Labview. The spiral droplets were allowed to move over at least twice the pitch before irradiation started. Confocal fluorescence imaging was performed with a using a Nikon confocal microscope and laser sources with −2) or low intensity (50 mW cm−2) non-polarized collimated UV light (λ = 365 nm). Light intensity was measured using a PM-100D power meter (Thorlabs).The droplets were irradiated using high intensity (300 mW cm38.The movies were analyzed using a custom-written program in pythonSupplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4


Systems chemistry has been a key component of origin of life research, invoking models of life’s inception based on evolving molecular networks. One such model is the graded autocatalysis replication domain (GARD) formalism embodied in a lipid world scenario, which offers rigorous computer simulation based on defined chemical kinetics equations. GARD suggests that the first pre-RNA life-like entities could have been homeostatically-growing assemblies of amphiphiles, undergoing compositional replication and mutations, as well as rudimentary selection and evolution. Recent progress in molecular dynamics has provided an experimental tool to study complex biological phenomena such as protein folding, ligand-receptor interactions, and micellar formation, growth, and fission. The detailed molecular definition of GARD and its inter-molecular catalytic interactions make it highly compatible with molecular dynamics analyses. We present a roadmap for simulating GARD’s kinetic and thermodynamic behavior using various molecular dynamics methodologies. We review different approaches for testing the validity of the GARD model by following micellar accretion and fission events and examining compositional changes over time. Near-future computational advances could provide empirical delineation for further system complexification, from simple compositional non-covalent assemblies towards more life-like protocellular entities with covalent chemistry that underlies metabolism and genetic encoding. In his last paper, Alexander Oparin, life’s origin pioneer, wrote: “The process of evolution of organic compounds that led to the emergence of life can be divided into two major stages: chemical and prebiological. Chemical evolution developed at the molecular level, obeying chemical laws to reach abiogenic synthesis of polymers that resulted in a spontaneous assembly of phase-separated thermodynamically open systems, or probionts. The appearance of probionts was accompanied by the development of a new law they obeyed, i.e., natural Selection” . TranslaWhat Oparin called probiont, also termed protobiont or protocell, is defined by the Oxford dictionary as “An entity consisting of a small drop of aqueous solution surrounded by a membrane and containing complex organic molecules, hypothesized as ancestral to living cells”. Evidently, Oparin alludes to the idea that the transition from abiotic entities to such capable of natural selection was not centered on a single molecule, but rather constituted a multicomponent system. In this respect, Oparin may be legitimately considered as a pioneer of systems chemistry and its relationship to the origin of life. This distinction between what single molecules can do and what necessitates a molecular ensemble is indeed reflected in the general definition of systems chemistry. This field seeks insight into complex networks of interacting molecules deriving systems-level characteristics that emerge through collective behavior .Notably, from its inception, systems chemistry has been tightly linked to studies on the origin of life, and is sometimes thought of as focusing on the origin of replication at the molecular level . Some ofAn appreciable sector of life’s origins models adheres to the credo of systems chemistry, asserting that life likely began as a multi-molecular system capable of reproduction, selection, and evolution. Along these lines, early abiogenesis was followed by systems protobiology , a stageMolecular Dynamics simulation was first developed in the late 1970s ,18, awarOver the past four decades, molecular dynamics has advanced from simulating several hundreds of atoms to systems having 50,000–500,000 atoms . This enMolecular dynamics simulations have thus evolved into a mature technique that can help effectively understand molecular and macromolecular structure and dynamics, as reviewed ,22. ThisIn the past, experimentation and theory of molecular interactions and dynamics were distinct entities. Theoretical models were usually depicted in a set of thermodynamic and kinetic equations leading to overall numerical predictions. The validation of such models depended on their ability to predict the experimental results . A severThe recent massive improvement of high-speed computing led to the introduction of computer experiments, a new mediator between experiment and theory, substantially modifying the traditional chasm between theory and experiment. Computing power now allows more realistic systems to be modeled, leading to a better understanding of real-life experiments . FurtherSystems chemistry is already leveraging the above-mentioned revolution. In one example , moleculThe graded autocatalysis replication domain (GARD) model belongs to the mutually catalytic networks approach to life’s origin, specifically embodied in a lipid world scenario ,11,39. GA known deficiency of GARD is the paucity of experimental verification of many of its predictions. Such experimental evidence should address the question of whether lipid assemblies, e.g., micelles, are capable of homeostatic growth and even rudimentary transfer of compositional information to fission-generated progeny. These experiments would require complex setups and accurate compositional monitoring of individual microscopic amphiphile assemblies, currently mostly outside the realm of experimental scrutiny. Some promising leads do however exist in the recent experimental exploration of multi-component lipid vesicles ,43,44,45The prospect of full-fledged molecular dynamics simulations of the GARD model could provide an important supplement to the expected laboratory experimental evidence. This would specifically generate a path to analyzing the dynamic behavior of complex molecular mixtures, e.g., in heterogeneous lipid micelles, utilizing specific models for molecularly realistic amphiphiles. Lipid micelles are particularly attractive as a platform for GARD molecular dynamics analyses, because their smaller size (a few hundred molecules) is more compatible with the emergence of homeostatic growth , when coAmphiphilic molecules are endowed with the capacity to self-assemble and generate supramolecular structures. The possibility to obtain relevant information on the emerging structures, compositions, and energy parameters, as well as the capacity to model and predict the self-organizing mechanisms of such molecules, was augmented by the advent of computational chemistry. This prominently includes molecular dynamics analyses, initially coarse-grained and more recently increasingly accurate scrutiny, which allows for better understanding of amphiphile dispersion, self-assembly, and segregation.The history of using molecular dynamics for understanding micelles of lipids and surfactants dates back >30 years ,48,49, wOne study  implemenAnother study  aimed toMixed micelles, colloidal particles, and nanoparticles have attracted considerable attention, including in the realm of drug delivery. Recently, this field has been boosted by the use of molecular dynamics, in conjunction with other computational methods , leadingOther studies probe the synergistic effects of components within mixed amphiphiles, with direct relevance to the potential prebiotic micellar behavior. For example,  studied The foregoing micellar renderings address only non-covalent transformations, such as lipid entry and exit, as well as micelle nucleation growth and fission. These are directly relevant to the inner works of the basic GARD model, which invokes mutually catalyzed lipid accretion. Such relevance is underlined by molecular dynamics studies of life’s origin models that invoke lipid involvement. One study reported the suitability of lipid phases for heterogeneous catalysis, i.e. the increase of reaction rates at the interface . AnotherIn parallel, for several decades, research has been done on the capacity of lipid micelles and vesicles to also exert covalent catalysis in enhancing the rate of amphiphile production from chemical precursors ,78. SuchPerforming GARD model simulations with molecular dynamics requires crucial proficiencies. Thus, the simulation must be able to accurately recognize micellar assemblies and ascribe each molecule either to a micelle or to the surrounding simulated environment as a free monomer. This could be achieved by calculating local centers of mass  or, moreRegarding system size, if a simulation can contain about 500,000 atoms , it coulThe GARD model is opportunistic, affording the participation of diverse amphiphile types , and thuApplying molecular dynamics to GARD allows one to take advantage of its existing molecular definition and detailed kinetic equations ,41,84,85We envisage two major approaches to the provision of molecular dynamics evidence for the validity of the GARD model. The first approach to be explored involves full atomistic simulations of individual micelles, as described in , focusinThe second approach to be explored involves a relatively recent development in the field, employing Markov state modeling (MSM) methods ,90. In tAn advantage of the MSM method is related to the fact that all atom molecular dynamics simulations for long time-scales may be limited by computational resources. Such a problem may be alleviated by clever MSM sampling techniques, whereby many independent simulations can be easily parallelized. This can be done by using the molecular dynamics simulation software for large systems (MODYLAS), a highly parallelized general-purpose program with scalable fine-grained algorithms ,94. SuchWith the environment being equimolar in the different lipid types , we willWe will examine whether compositional fluctuations are amplified by the anticipated mutually catalytic effects, portraying attractor phenomenology . Such atWe will examine the net incoming time-dependent amphiphile fluxes for a correlation of their compositional direction to that of the initial premicelles. Such correlation will provide evidence of compositional preservation, the hallmark of homeostatic growth, and an equivalent to compositional replication/reproduction .We will seek simulated fission events affecting the biasedly grown micelles , showingThe molecular dynamics simulations will follow the kinetics of micellar growth, e.g., from premicelles to micelle, seeking evidence for the following two predicted phenomena:In addition to the aforementioned molecular dynamics simulation plans for the standard GARD model, whereby all catalysis event are non-covalent, we should lay the foundations for analyzing the behavior of the new metabolic GARD model ,97. For The GARD model for life’s origin is based on the notion that the first rudiments of life were not individual molecular replicators, but rather self-reproducing multi-molecular systems, a concept pioneered by Oparin . As reprAmong the strengths of the GARD model are its realistic depiction of the emergence of life-like entities from a prebiotic mixture of random chemicals , and itsThere are severe limitations in providing laboratory evidence for GARD. This is because a mutually catalytic micellar lipid network is expected to initially involve weak, relatively low-specificity interactions, and hence would likely portray low fidelity compositional reproduction. Further, any process in which the fidelity might gradually improve would require long-term evolutionary chemistry, suggesting the necessity for very long-term laboratory experiments. In this respect, molecular computerized experiments could be the only realistic path for the validation of GARD.Given the riches of the presently available molecular dynamics capabilities, it is likely that such proposed initial steps will serve as roots for a much more detailed scrutiny. The concrete predictions for the increased capacities of the methodology over the next two decades , potenti


In the electrospinning process, a modified parallel electrode method (MPEM), conducted by placing a positively charged ring between the needle and the parallel electrode collector, was used to fabricate highly aligned carbon nanotubes/polyacrylonitrile (CNTs/PAN) composite nanofibers. Characterizations of the samples—such as morphology, the degree of alignment, and mechanical and conductive properties—were investigated by a combination of scanning electron microscopy (SEM), transmission electron microscopy (TEM), universal testing machine, high-resistance meter, and other methods. The results showed the MPEM could improve the alignment and uniformity of electrospun CNTs/PAN composite nanofibers, and enhance their mechanical and conductive properties. This meant the successful preparation of highly aligned CNT-reinforced PAN nanofibers with enhanced physical properties, suggesting their potential application in appliances and communication areas. Polymer nanocomposites with nanoparticles show amazing mechanical, electrical, and thermal properties due to the interaction between the polymer matrixes and nanoparticles ,2,3. CarElectrospinning (ES) is an efficient technique for the preparation of CNTs/polymer composite nanofibers. The mechanical properties of the electrospun nanofibers are expected to be enhanced through stretching and making composites. There are some reports concerning the preparation of electrospun CNTs/polymer nanofibers ,11,12,13Some techniques have been proposed to obtain aligned nanofibers ,21,22,23N-vinylpyrrolidone (PVP) [Recently, using the ES technique, many functional nanomaterials have been developed using various polymers, such as poly(vinylidene fluoride) (PVDF) , poly(mene (PVP) ,30. Polyne (PVP) .Based on the above considerations, highly aligned PAN composite nanofibers containing single-walled carbon nanotubes (SWCNTs) with concentrations ranging from 0 to 2 wt % were prepared directly by the MPEM technology without any additional postprocessing. Characterizations of the SWCNTs/PAN composite nanofibers—such as morphology, the degree of alignment, and mechanical and conductive properties—were studied by means of scanning electron microscopy (SEM), transmission electron microscopy (TEM), universal testing machine, high-resistance meter, and other methods. The results showed the MPEM could improve the uniformity of diameter distribution and the degree of alignment of electrospun SWCNTs/PAN composite nanofibers. In addition, mechanical and conductive properties of the composite nanofibers were enhanced. As a result, for the CNT-reinforced PAN nanofibers, strong interfacial adhesion, uniform dispersion, and high alignment were more crucial factors for improving the physical properties of the composite nanofibers.N,N-dimethylformamide (DMF)  was purchased from Shanghai Chemical Reagent Co. Ltd. . The SWCNTs were purchased from Shanghai Aladdin Biochemical Technology Co. Ltd.  . All materials were used without any further purification.PAN powder, the biodegradable polymer used in this study, with a molecular weight of 150,000 g/mol, was supplied by Beijing Lark Branch Co. Ltd. . w/w). Initially, different weights of SWCNTs were dispersed in the DMF by using an ultrasonic cleaner  for 4 h at 25 ± 2 °C (room temperature). Then, the electrospinning solution was prepared by dissolving 8 wt % of PAN in SWCNTs/DMF solution under magnetic stirring for 6 h at room temperature (25 ± 2 °C) until it became homogeneous. The calculated SWCNTs’ concentrations for each component of the various samples were 0, 0.5, 1, 1.5, and 2 wt % (mass ratios to PAN).All concentration measurements were done in weight per weight  at a relative humidity of 50%. The prepared SWCNTs/PAN/DMF solution was dropped into a 10 mL syringe. The flow rate was 0.5 mL/h. The applied spinning voltage was 15 kV and the applied ring voltage was +5 kV. The distance from the tip of the needle to the parallel auxiliary electrodes was 18 cm, the distance from the ring to the parallel electrode collector was 5 cm, and the gap between two parallel auxiliary electrodes was 4 cm.The morphologies of highly aligned SWCNTs/PAN composite nanofibers were examined by a scanning electron microscopy  at an acceleration voltage of 3 kV. All samples were dried at room temperature, and then sputter-coated with gold by an IB-3  for 10 min. The matrix morphology and fibrous diameter characterization were carried out using ImageJ software .The distribution of SWCNTs in the composite nanofibers was characterized by a transmission electron microscopy  operating at 200 kV. The samples were prepared by placing the carbon-coated copper grids on the collector and directly depositing a thin layer of electrospun nanofibers onto the copper grids. TEM images were subsequently obtained by passing a beam of electrons through the copper grids containing the composite nanofibers at a high voltage of 200 kV, a dark current of 10.57 μA, and an emission current of 64 μA.−1 and a resolution of 4 cm−1. Raman spectra for the pure SWCNTs and the SWCNTs/PAN composite nanofibers were obtained on a Micro-Raman Spectrometer  at 532 nm laser excitation (KIT-532-25) with edge filter and laser filter. The laser power density was kept as 25 mW. Energy range of Raman spectrum was from 200 to 4000 cm−1, all the integration time of the Raman tests was 5 s, and the accumulation was 10 times.The PAN structure and its interactions with the SWCNTs were investigated through FTIR spectroscopy . For each measurement, each spectrum was obtained by the performance of 32 scans with the wavenumber ranging from 400 to 4000 cmθ > 15°) and silver behenate (2θ < 10°). Cu-Kα radiation was used with diffraction angle 2θ range of 5°–60° at 40 kV and 40 mA, and the diffraction patterns were collected at a scanning rate of 2°/min.X-ray diffraction (XRD) analyses were performed to elucidate the crystalline structure of powdered SWCNTs, PAN, and SWCNTs/PAN composites using Philips X’Pert-Pro MPD  with a 3 kW ceramic tube as the X-ray source (Cu-Kα) and an X’Celerator detector. The TTK sample stage was set horizontally. The reflection peak positions were calibrated with silicon powder  at room temperature and ambient condition. The tensile strength and elongation-at-break values of PAN and SWCNTs/PAN composite nanofiber membranes were investigated by a universal electromechanical test machine Instron-3365 . The electromechanical test machine is well known for its ability to perform a wide range of quasi-static tension and compression tests at test speeds up to 40 ipm (1000 mm/min). All samples were 50 mm × 10 mm rectangle membranes. All measurements were carried out at room temperature. The measurement was repeated three times.σ) are high due to estimation of diameters of fibers based on observed sample data. Therefore, a confidence interval gave an estimated range of values, which was likely to include unknown diameters of fibers. The estimated range was calculated from a given set of sample data [The morphologies of SWCNTs/PAN composite nanofibers were carried out by SEM. To determine the diameter distribution of nanofibers, 50 SEM images were chosen for diameter distribution analysis using ImageJ software. ple data .θ) between the long axis of the nanofibers and its expected direction  was used as the parameter to quantify the alignment. The degree of nanofiber alignment was defined as the ratio of the number of nanofibers, whose θ is between −6° and 6°, to the total number of nanofibers. To determine the degree of nanofiber alignment, several SEM images were captured from each sample. Finally, 50 images were chosen for alignment analysis. With the concentration of SWCNTs increased from 0 to 2 wt %, the average diameters of composite nanofibers were 536, 539, 542, 634, and 737 nm and the degrees of their alignment were 70%, 60.9%, 78.1%, 78.1%, and 85.2%. In this study, the viscosity and electrical conductivity of the spinning solution increased with the increase of SWCNTs concentration from 0 to 2 wt %. With the increase of the viscosity, the polymer chains resist electric field stretching, and the bending instability of jets can be suppressed. As a result, both the diameter and degree of alignment of composite nanofibers increased [ncreased . This woncreased . As a reThe distribution of SWCNTs in the highly aligned SWCNTs/PAN composite nanofibers were researched by TEM. −1 region, indicating that the π electrons present in SWCNTs interact with the hydrogen (free and bonded) attached to the nitrogen in the urethane bond, thus changing the shape of the band. Moreover, the spectra showed sharp peaks at 1616 cm−1 , D-band (1341 cm−1), and G’-band (2648 cm−1) for the samples, indicating their effective carbonization. Moreover, in the region from 350 to 120 cm−1, SWCNTs yielded a series of bands that had been assigned to the radius breathing mode (RBM). From the spectra  crystal planes of PAN [θ = 26.5° indicated the typical signal of CNTs or graphite structures. This peak was associated with the (002) diffraction of the hexagonal graphite structure in the carbon materials. θ = 17° and a less intense peak at 2θ = 21.5°. The principal peak of graphene structure at 26.5° did not appear, probably because of the high ratio of PAN composite with respect to SWCNTs [In order to elucidate the crystalline structure of SMWNTs, PAN, and SMWNT/PAN composites, their XRD patterns with distinctive crystalline peaks are shown in s of PAN . The cryo SWCNTs . The XRDIn addition, these XRD patterns contained both sharp as well as defused bands. Sharp bands corresponded to crystalline orderly regions and defused bands corresponded to amorphous regions. The crystallinity was calculated by separating intensities due to amorphous and crystalline phase on diffraction phase. Computer-aided curve-resolving technique was used to separate crystalline and amorphous phases of the diffracted graph. After separation, the total area of the diffracted pattern was divided into crystalline , carried out by placing a positively charged ring between the needle and the parallel electrode collector, was successfully used to fabricate highly aligned SWCNTs/PAN composite nanofibers with the different SWCNT concentrations. The properties—such as morphology, the degree of alignment, crystallinity, mechanical properties, and electrical conductivity—of highly aligned SWCNTs/PAN composite nanofibers with SWCNT concentrations, which ranged from 0 to 2 wt %, were investigated. The results showed the properties of PAN nanofibers were improved by adding SWCNTs.SEM and TEM photographs showed that with the increase of SWCNT contents, the diameters of the composite nanofibers increased and their surface became rough moreover, the SWCNTs were embedded in the polymer matrix and almost aligned along the composite nanofiber axis. In addition, the measurement of mechanical properties and surface resistances revealed that the tensile strength and electrical conductivity of aligned composite nanofibers were obviously improved over that of random composite nanofibers. The results showed that the MPEM could improve the alignment of electrospun CNTs/PAN composite nanofibers, and enhance their mechanical and conductive properties.