This cross-sectional study assesses trends in the percentage of applicants and matriculants to US surgical specialties who identified as part of a racial/ethnic group underrepresented in medicine from the 2010-2011 to 2018-2019 academic years From 2010 to 2018, did the percentage of applicants and matriculants who identified as part of a racial/ethnic group that is underrepresented in medicine change among US surgical specialties?In this cross-sectional study evaluating 737 034 applicants and 265 365 matriculants to US residency programs, no statistically significant change was seen in the percentage of applicants or matriculants to most US surgical programs among individuals who identified as part of a racial/ethnic group underrepresented in medicine.The findings suggest that persons belonging to racial/ethnic minority groups continue to be largely underrepresented in surgical fields despite continued efforts to diversify the US surgical workforce. Surgical programs across the US continue to promote and invest in initiatives aimed at improving racial/ethnic diversity, but whether this translates to changes in the percentage of applicants or matriculants from racial/ethnic minority groups remains unclear.To examine trends in the percentage of applicants and matriculants to US surgical specialties who identified as part of a racial/ethnic group underrepresented in medicine from the 2010-2011 to 2018-2019 academic years.This cross-sectional study examined trends in self-reported racial/ethnic identity among applicants and matriculants to US residency programs to evaluate demographic changes among surgical programs from 2010 to 2018. Data were obtained from the Association of American Medical Colleges.P = .63). Thoracic surgery was the only surgical specialty in which there was a statistically significant change in the percentage of applicants  or matriculants  underrepresented in medicine based on race/ethnicity. Obstetrics and gynecology had the highest mean percentage of applicants  and matriculants  underrepresented in medicine among surgical specialties. Thoracic surgery had the lowest mean percentage of applicants  and otolaryngology the lowest mean percentage of matriculants  underrepresented in medicine.The study population consisted of a total of 737 034 applicants and 265 365 matriculants to US residency programs, including 134 158 applicants and 41 347 matriculants to surgical programs. A total of 21 369 applicants (15.9%) and 5704 matriculants (13.8%) to surgical specialties identified as underrepresented in medicine. There was no statistically significant difference in the percentage of applicants underrepresented in medicine based on race/ethnicity for all surgical specialties combined in 2010 vs 2018 (15.3%  vs 17.5% ; In this cross-sectional study, overall US surgical programs had no change in the percentage of applicants or matriculants who self-identified as underrepresented in medicine based on race/ethnicity, but the proportion remained higher than in nonsurgical specialties. Reevaluation of current strategies aimed at increasing racial/ethnic representation appear to be necessary to help close the existing gap in medicine and recruit a more racially/ethnically diverse surgical workforce. This study was determined to be exempt from review by the University of Washington institutional review board because it did not involve human participants. Data were deidentified. This study followed the Strengthening the Reporting of Observational Studies in Epidemiology , neurological surgery, obstetrics and gynecology, orthopedic surgery, otolaryngology, plastic surgery (integrated), thoracic surgery (integrated), urology, and vascular surgery (integrated). Medical specialties considered nonsurgical included anesthesiology, dermatology, emergency medicine, family medicine, internal medicine, neurology, pathology , pediatrics, psychiatry, and radiology (diagnostic). The URM designation followed the AAMC definition and represents individuals who identify as American Indian or Alaska Native; Black or African American; Hispanic, Latino, or of Spanish origin; and Native Hawaiian or other Pacific Islander. Race/ethnicity data were only available for US citizens and permanent residents and include individuals who may have completed their medical education outside the US. White, Asian, other race/ethnicity, unknown race/ethnicity, and non-US citizen or nonpermanent US resident categories were considered non-URM.Self-reported race/ethnicity  for applicants and matriculants to surgical and nonsurgical specialties from 2010-2011 to 2018-2019 academic years was obtained and analyzed in April and July 2020. Academic years have been abbreviated to the year that the individual submitted their application to residency . Medical specialties were categorized as surgical or nonsurgical based on the definition provided by the American College of Surgeons.2 test for trend was performed to assess changes in proportions across all years between 2010 and 2018 for URM groups and individual races/ethnicities, and the t test was performed to evaluate a difference between the mean proportions of individuals who identified as URM in surgical vs nonsurgical specialties using Prism, version 8.4.2 (GraphPad Software). Results were considered statistically significant at a 2-tailed P < .05.The χA total of 737 034 and 265 365 matriculants during the 9 years of the study period were included in the sample . Of thesApplicants who identified as URM accounted for 21 369 of the 134 158 surgical applicants (15.9%) from 2010 to 2018. Of the 134 158 surgical residents, 828 (0.6%) identified as American Indian or Alaska Native; 24 481 (18.2%) as Asian; 10 391 (7.7%) as Black or African American; 9955 (7.4%) as Hispanic, Latino, or of Spanish origin; 195 (0.1%) as Native Hawaiian or other Pacific Islander; and 58 584 (43.7%) as White.2 test for trend analyses for the period 2010 to 2018. There was no significant difference in the percentage of applicants who identified as URM in all surgical specialties combined . The P = .02). There was no significant difference in the percentage of applicants who identified as URM to nonsurgical specialties combined over the study period . From 2010 to 2018, obstetrics and gynecology had the highest mean percentage of applicants who identified as URM  among surgical specialties, and thoracic surgery had the lowest .All individual surgical specialties had no significant change in the proportion of applicants who identified as URM from 2010 to 2018 with the exception of thoracic surgery, which nearly doubled from 14 applicants  in 2010 to 27 applicants  in 2018 (P = .04). There were no statistically significant differences in the percentage of applicants of each race/ethnicity for nonsurgical specialties combined. From 2010 to 2018, surgical specialties had a significantly higher mean percentage of applicants who identified as URM than did nonsurgical specialties .Analysis for each race/ethnicity was consistent with the findings for applicants who identified as URM to surgical specialties combined. No race/ethnicity had a statistically significant change within the applicant pool from 2010 to 2018. Only Hispanic and Latino applicants to thoracic surgery showed a significant increase, from 1.2%  in 2010 to 5.9%  in 2018  identified as URM. Compared with the proportion of the applicant pool, Black and African American matriculants (2423 of 41 347 [5.9%]) were the most underrepresented group, and White matriculants (27 352 [66.2%]) were overrepresented by 20.1%. Among the other surgical matriculants, 276 (0.7%) identified as American Indian or Native American; 6996 (16.9%) as Asian; 2925 (7.1%) were Hispanic, Latino, or of Spanish origin; and 80 (0.2%) as Native Hawaiian or other Pacific Islander. P = .99). As with applicants, thoracic surgery was the only surgical specialty with an increase in matriculants who identified as URM, with 0%  in 2010 and 10.0%  in 2018 (P = .01).The percentage of matriculants who identified as URM did not significantly change for all surgical specialties combined ; Hispanic and Latino matriculants from 0%  in 2010 to 7.9%  in 2018 (P = .008); Native Hawaiian and other Pacific Islander matriculants from 0%  in 2010 to 5.9%  in 2011 and 0%  by 2018 (P = .004); and White matriculants from 62.4%  in 2010 to 65.0%  in 2018 (P = .004). One thoracic surgery matriculant in 2011 self-identified as Native Hawaiian or other Pacific Islander. For matriculants, obstetrics and gynecology had the highest mean percentage of individuals identifying as URM  among surgical specialties, and otolaryngology had the lowest .The proportion of surgical matriculants in all racial/ethnic groups did not change significantly from 2010 to 2018 for almost all specialties. The only surgical specialty with a statistically significant change in the percentage of matriculants by race/ethnicity was thoracic surgery. The percentages of Black and African American matriculants increased from 0%  in 2010 to 3.0%  in 2018 (P = .99). Analysis by individual race/ethnicity also did not show a significant change in the percentage of matriculants to all nonsurgical specialties combined. During the study period, surgical specialties had a significantly higher mean percentage of matriculants who identified as URM compared with nonsurgical specialties .For nonsurgical specialties, 86 482 of 602 876 applicants (14.3%) and 27 840 of 224 018 matriculants (12.4%) identified as URM . There wIn a comparison of the difference between the mean percentages of matriculants and applicants who identified as URM in all surgical specialties combined, there were 2.1% fewer surgical matriculants than applicants  who identified as URM . In nonsThe differences in URM representation increased over time, from −1.3% in 2010 to −2.7% in 2018 for all surgical specialties combined and from −0.8% to −2.8% during the same period for nonsurgical specialties combined. General surgery had a representation difference that increased each year after 2010. The mean difference from 2010 to 2018 between matriculants and applicants who did not identify as URM was 21.1% for surgical specialties  and 17.0% for nonsurgical specialties , with an overall decrease in differences in both surgical and nonsurgical specialties from 2010 to 2018.10 reported no significant difference in applicants from 2008 to 2018 by race/ethnicity except a significant decrease in Asian applicants to otolaryngology, neurological, vascular, thoracic, orthopedic, and general surgery. The data set used started in 2008, at which time there were more applicants that identified as Asian than in 2010, which may account for the observed trend.Despite efforts to diversify the workforce, our study found no significant change in the percentage of individuals who identified as URM applying or matriculating into the collective 9 American College of Surgeons–recognized surgical specialties from 2010 to 2018. Those who identified as URM accounted for approximately 15.9% of applicants and 13.8% of matriculants to surgical programs in the US from 2010 to 2018. Choinski et al21 Future analysis should consider what happens to applicants who identified as URM who do not matriculate into a surgical program  and the implication of these findings.The only surgical specialty with a statistically significant change in our study was thoracic surgery, with a change in both applicants and matriculants, particularly an increase in Hispanic and Latino applicants and in Black and African American, Hispanic and Latino, and Native Hawaiian and other Pacific Islander matriculants. Thoracic surgery had the lowest proportion of applicants (8.1%) and matriculants (0%) who identified as URM in 2010 accompanied with a low total number of individuals who identified as URM; therefore, a small increase in the total number likely indicated a significant increase in the percentage of applicants (14.6%) and matriculants (10.0%) who identified as URM by 2018. Our findings are consistent with previous studies of individual surgical specialties that found no statistically significant increase in the number of individuals who identified as URM in plastic and orthopedic surgery and among otolaryngology applicants.7 In all specialties combined, a higher proportion of individuals who identified as URM applied and matriculated into surgical rather than nonsurgical specialties every year between 2010 and 2018 .25 This finding differs from the increases observed in surgery among individuals wo identify as URM and women in the 1990s and early 2000s.26 Of importance, the stagnant percentage of individuals who identify as URMs in surgery programs suggests that current efforts are falling short in increasing the relative proportions of medical school graduates with varying races/ethnicities and, in particular, those who ultimately decide to pursue surgical programs.Although individuals who identify as URM remain underrepresented as applicants and matriculants overall, these proportions are higher than the proportion of those in this group who graduate from medical school .and 2018 . Prior s30 The American College of Surgeons has underscored the importance of moving beyond superficial efforts toward diversity by urging inclusivity, the concept of creating a welcoming space for all health care participants from faculty to patients as individuals and members of the group.31 Framing recruitment and exposure initiatives in terms of value added over rote diversity metrics will be a necessity to overcoming the inertia of structural bias in creating sustainable growth in the proportion of surgical subspecialty training matriculants from URM backgrounds.32Several national surgical societies have committed to promote diversity through the development of diversity committees and societies for the advancement of specific groups that are URM to increase racial/ethnic representation.19 The difference in representation affects the entire training pathway: 14.9% of medical school applicants, 13.6% of medical school matriculants, 12.4% of 2019 graduates, 17.0% of surgical residency applicants, 14.3% of surgery residency matriculants, and 12.8% of practicing surgeons in the US identified as URM during the 2018-2019 academic year.36The difference in representation of matriculants compared with applicants who identify as URM in surgical specialties increased from 2010 to 2018 . Individ38 Diversity within a program has been shown to play some role in program selection by applicants, among multiple other factors, as has been shown in studies evaluating residency program choices by women and individuals who identify as URM.40 Some strategies that have been successful in increasing the representation of women and, to a certain extent, groups who identify as URM include meaningful mentorship experiences, changes in the culture of surgical departments, and shifts in the focus of residency interviews.41Although large disparities in gender among surgical trainees remain, the strategies used to increase representation from 15% in 2000 to 25% by 2013 may provide some insight for improving racial/ethnic diversity in the surgical workforce.42 Early exposure to surgical specialties through strategic support initiatives, such as summer internship programs, may help to describe these fields and establish mentorship relationships, which have been associated with significant increases in applicants who identify as URM.14 Cochran et al43 described a grounded theory model of successful mentorship in academic surgery that emphasizes the need for multiple mentors to engage with a diverse group of mentees over time, covering unique strategic domains as opposed to overreliance on fixed mentorship dyads.Directed exposure and longitudinal relationship-building efforts, such as the Diverse Surgeon’s Initiative and The Harold Amos Medical Faculty Development Program through the Robert Wood Johnson Foundation have been shown to be successful frameworks for providing skills and resources for applicants and trainees who identify as URM to advance in all stages of career development in academic surgical fields.44 Receiving an interview offer has been recognized as an important step of gatekeeping the number of applicants who identified as URM.20 A study assessing general surgery programs found that having more residents and faculty who identify as URM was not associated with an increased likelihood of applicants who identified as URM being offered an interview.45 Creative and new strategies should be developed to encourage more applicants, make the interview process more equitable, and decrease the overall attrition of individuals who identify as URM who matriculate.48 Continued active evaluation in a reiterative way will be needed to identify strategies that are or are not helping to diversify the workforce.There are also steps during the application process that should be studied further and potentially restructured. Websites are an underused way of communicating a program’s commitment to diversity and could be enhanced to further increase diversity among applicants.50 Studies about women identified that the environment of training affects why students choose to pursue surgery.52 Recent reports detail the discrimination and harassment that individuals who identify as URM and women experience in surgical training, which has been disavowed by the American Surgical Assocation.54 Further change in hierarchical and social dynamics is likely necessary to increase the number of individuals who identify as URM who ultimately pursue a surgical field.Addressing the work environment experienced by residents who identify as URM may improve the resident experience as well as influence the encounters of medical students who identify as URM during their clinical rotations and their perception of surgery.56 Because our focus was on surgical specialties, we did not investigate the trends for individual nonsurgical specialties; perhaps there is insight that can be gained from that analysis in future studies.This study has limitations. We used self-report data, which relies on participants’ willingness to accurately share personal information. Race is a complex social category with evolving meaning, shown by how AAMC permitted students to select more than 1 race/ethnicity after 2013. This change means that data after 2013 with race/ethnicity alone and in combination counts multiracial individuals in more than 1 category, potentially overestimating the percentage of students who identify as URM. We did not have access to identify which applicants who identified as URM attended osteopathic or non-US medical schools, which may be a confounder because these students tend to have lower matriculation rates into US residency programs, including surgical ones.In this cross-sectional study, there was no change in the proportion of individuals who identified as URM applying and matriculating to surgical and nonsurgical specialties overall from 2010 to 2018. These findings support the claim that underrepresentation of racial/ethnic minorities is a broader issue that extends beyond surgery, starting from entry into medical school and beyond residency. Therefore, to reach the goal of accurately representing the demographics of the US and improving patient care in surgery, further investment in innovative programs focused on increasing racial/ethnic diversity from the medical school to the surgical residency level appears to be needed.


Molecular machines and molecular robots are a highly interdisciplinary research field including material science, chemistry, biotechnology, biophysics, soft matter physics, micro-electromechanical systems (MEMS), and computer science ,2,3. MolIn this Special Issue, six research papers and two review articles related to molecular machine engineering and molecular robotics from a wide range of research fields have been presented. Three theoretical research papers, on reaction–diffusion simulation accelerated the finite-element method by Sellami et al. , on a stBy overviewing the recent advances in this field, we would like to ferment the seeds of future applications, such as medical microrobots, intelligent drug delivery systems, artificial cells/organelles, environmental nano/microsensor robots, agricultural nano/microrobots, and unconventional brain-like computers. We would like to thank all of the contributors to publish this Special Issue and we are happy if this issue helps to understand the recent advances in molecular machines and molecular robotics.


Significance: Deep-tissue penetration by x-rays to induce optical responses of specific molecular reporters is a new way to sense and image features of tissue function in vivo. Advances in this field are emerging, as biocompatible probes are invented along with innovations in how to optimally utilize x-ray sources.Aim: A comprehensive review is provided of the many tools and techniques developed for x-ray-induced optical molecular sensing, covering topics ranging from foundations of x-ray fluorescence imaging and x-ray tomography to the adaptation of these methods for sensing and imaging in vivo.Approach: The ways in which x-rays can interact with molecules and lead to their optical luminescence are reviewed, including temporal methods based on gated acquisition and multipoint scanning for improved lateral or axial resolution.Results: While some known probes can generate light upon x-ray scintillation, there has been an emergent recognition that excitation of molecular probes by x-ray-induced Cherenkov light is also possible. Emission of Cherenkov radiation requires a threshold energy of x-rays in the high kV or MV range, but has the advantage of being able to excite a broad range of optical molecular probes. In comparison, most scintillating agents are more readily activated by lower keV x-ray energies but are composed of crystalline inorganic constituents, although some organic biocompatible agents have been designed as well. Methods to create high-resolution structured x-ray-optical images are now available, based upon unique scanning approaches and/or a priori knowledge of the scanned x-ray beam geometry. Further improvements in spatial resolution can be achieved by careful system design and algorithm optimization. Current applications of these hybrid x-ray-optical approaches include imaging of tissue oxygenation and pH as well as of certain fluorescent proteins.Conclusions: Discovery of x-ray-excited reporters combined with optimized x-ray scan sequences can improve imaging resolution and sensitivity. The biological utility and goals of x-ray-based sensing to better understand tissue physiology and pathophysiology are the driving motivations that underpin technical advances in this area. The major benefits of x-rays as a probe excitation source are (i) the high penetrance and (ii) the wide availability and acceptance of x-ray sources in biomedical imaging. When compared to other methods of molecular sensing in tissue, the strengths of x-ray-based molecular sensing may be less clear, because this methodology only began emerging during the last decade.Perhaps the most scientifically enticing part of this methodology is the concept of utilizing one radiation source  as the excitation probe, combined with another radiation type as a signal. This conceptual framework for developing a hybrid imaging modality is illustrated in i.unique molecular probe, able to bind with high affinity to biological targets;ii.high contrast or specificity via high signal-to-background ratio;iii.an emission signal type that can be detected with high signal-to-noise ratio; andiv.an excitation radiation with high penetrance for imaging through tissue , are also a commercially available paradigm.In the case of optical emission, the single largest attraction is that optical molecular probes comprise arguably the most developed and diverse group of sensors for biological imaging. There are thousands of optical probes, and a number of them are commercially available for use in preclinical imaging. Consequently, preclinical optical imaging systems are the most widely used for whole body animal imaging,The choices of x-ray sources, type, and mode of detectors are also quite diverse. The energy range of the x-rays needed depends upon the type of probe and its physical interaction mechanisms, as outlined in 2––in vivo molecular imaging tools. The limits of detection are near in vivo use.,,The basic principles of using x-rays to sample tissue comes from the successful origins of XRF sensing, where the concentration of trace metals such as Fe, Zn, Cu, Hg, and Se are quantified within tissue by detection of the secondary characteristic x-rays emitted when irradiating the sample with an excitation of incident x-rays or gamma rays.From these origins in XRF over a few decades, an exciting direction of development was to utilize other excitation modes, or other emitted signals, that might provide molecular-specific signals. Additionally, these developments might provide a wider range of available signals,–A natural transition then is to utilize a more sensitive detection mode that can reach down to the single photon level, such as either isotopic gamma emission in nuclear medicine or optical luminescence methods such as fluorescence or phosphorescence, to increase the signal level and/or suppress the background and noise levels.The excitation mode of an optically active contrast agent depends upon the mechanism of interaction with the radiation, as illustrated in 1.Direct or indirect electronic excitation of a scintillator through the upper singlet states of pi electrons, resulting in eventual radiative decay. The complexity of processes depends upon the nature of both medium and scintillator, which determines their interactions. The end products of water hydrolysis  or beam depth of scanning, as illustrated in –If the depth of penetration is to be restricted, it is feasible to use electron irradiation which has limited depth of penetration, and even apply a variable bolus or buildup region to the beam before the tissue, illustrated in 4.4,–,,,,Excitation with x-rays implicitly leads to a broadband signal that can excite more than one molecular probe. Both scintillation and Cherenkov-excited signals can occur, and it is possible to detect multiple emission signals, based either on temporal gating or wavelength separation of intensity. The excitation of multiple molecular dyes by Cherenkov has been shown, with optimal spectral windows being in the red,55.1,X-ray photons experience much less scattering than optical radiation in tissue, and therefore x-ray-induced radioluminescence largely originates within or very near the volume that is directly in the pathway of the scanning beam. The limits on resolution though can be defined by the x-ray Compton scattering, which can happen both inside and outside the tissue. For keV sources, the scattering is usually not significant because it largely produces soft x-rays that have very short penetration depth in tissue. This is true both outside the tissue and within the tissue. In the MeV approach, the Linac MLC  provides5.2,––,,The radiative transfer of photon diffusion through turbid media can be solved either numerically through Monte Carlo simulation or approximated analytically by the diffusion approximation. Monte Carlo solutions generally maintain high accuracy and wide applicability,Cherenkov light is generally modeled in a comprehensive manner that involves distinct behavior of a broadband wavelength spectrum of photons peaked in the ultraviolet and decaying in intensity with an inverse square dependence upon the wavelength.5.3,,,,–Currently, image reconstructions could be coarsely classified into three categories: model-basedBy taking advantage of a careful measurement geometry and utilization of coded illumination techniques, a straightforward direct deconvolution has been used for reconstruction, e.g., Cherenkov-excited luminescence scanned imaging (CELSI). CELSI uses the collimating system  of a Lin,,–in-vivo imaging is similar to CT imaging in that the image is always better with more radiation, which can lead to excessive radiation dose, and so care must be taken to design the scan sequence. To address this issue, a lot of effort has focused on sparse-view XLCT by taking advantage of improved measurement geometry and algorithms.,,Backprojection or filtered backprojection (FBP) reconstruction algorithm originally developed for CT has been used, known as x-ray luminescence CT (XLCT).6The range of x-ray choices, as described already, also affects the radioluminescent reporter agent that is optimally used, as the sources in the keV range excite scintillators exclusively, whereas the sources in the MeV range can excite all of fluorophores, phosphors, and scintillators.ex vivo use.in vivo optical imaging with ultrahigh spatial resolution and negligible tissue autofluorescence.,––––,,in vitro or in vivo is presented in Development of scintillating nanoparticles that can convert x-ray radiation into UV–vis–NIR light is a very active area of research. While there is incredible promise, all known scintillating reporters are in preclinical molecular imaging research or ,,,MeV x-ray-induced Cherenkov emission can be used as a controllable indirect light source that can scan the imaging objects to excite optical molecular probes, without the need for scintillation.77.1–,,,Tumor oxygenation significantly affects the outcome of radiotherapy, and hypoxic tissues such as tumors  makes it feasible to integrate diagnosis and tumor therapy for tumor theranostic applications. A theranostic mesoporous silica nanoparticle encapsulated a photosensitizer, 2,3-naphthalocyanine and a X-ray deposited chemotherapies have been examined in several delivery moieties. Delivery via vitamin B12 uptake via the transcobalamin receptor was shown for delivery of agents and photorelease via the alkylcobalamin scaffold that is light sensitive.While this last section has focused on therapeutic effects from x-rays mediated by optical signals, there is a linkage to diagnostic scanning that could be very important, both scientifically and practically. As these therapeutic studies advance, it seems inevitable that the diagnostic potential for their use will become more apparent as well.8Optical molecular sensing from x-ray excitation describes a range of technologies and research studies where an incident x-ray beam is used for deep tissue sensing. The common theme is that through excitation by x-rays and active scanning or active delivery of molecular probes fundamentally new biological information could be sampled from tissue deeper than before and with higher spatial resolution. The field involves the intersection of (i) molecular probes that have high potential for radioluminescence or interaction with radioluminescence, (ii) x-ray technologies that provide specific energy, lateral or axial control, and scanning, and (iii) biomedical needs where there is not good potential for diagnostic information already. The strengths are in the widespread application and acceptance of x-rays as a diagnostic tool and the diversity of systems, energies, and controls that are well understood and developed. The challenges remain in the understanding and refinement of molecular probes that intake excitation energy from x-ray origins and maximize the output of optical signals in a way that retains meaningful molecular information by the location, intensity, or lifetime. While this field is not well defined as a single entity, it is inevitable that research will continue to define what is possible and that niche uses will become adopted, such as oxygen sensing, pH sensing, receptor uptake of nanomaterials, or X-PDT applications.


The initial high molecular fraction of Fn with Mw ~320–340 kDa is not completely hydrolyzed even after three days of control. The presence of low molecular fractions with Mw ~17 and Mw ~10 kDa in the solution slightly increases within an hour and noticeably increases for three days. The destruction of macromolecules of high molecular collagen to hydrolysis products appears almost completely within the first minute mainly to the polymer with Mw ~10 kDa, and enzymatic hydrolysis of fibrinogen proceeds slower than that of collagen, but also mainly to the polymer with Mw ~10 kDa. Comparative photos of the surfaces of native collagen, fibrinogen and the scaffold based on them were obtained.Enzymatic hydrolysis of native collagen and fibrinogen was carried out under comparable conditions at room temperature. The molecular weight parameters of proteins before and after hydrolysis by thrombin were monitored by gel-penetrating chromatography (GPC). An analysis of the experiment results shows that the molecular weight parameters of the initial fibrinogen (Fn) and cod collagen (CC) are very similar. High molecular CC decays within the first minute, forming two low molecular fractions. The main part (~80%) falls on the fraction with a value of M Scaffold technology is the most dynamically developing direction in tissue engineering. Fibrinogen/fibrin and collagen are among the most popular natural polymers for the formation of hydrogel scaffolds ,2. TheseComparatively, not long ago, marine collagen isolated from fish scales, skin, and bones was used in scaffold technology. A structural analysis of the protein confirmed that fish have collagen type I similar to collagen type I of mammals and birds ,13. MariA number of methods of scaffold formation based on these biopolymers have been carried out in the presence of the proteolytic enzyme—thrombin, hydrolyzing peptide bonds ,18. NewlIt is not entirely clear how collagen interacts with fibrin during the formation of composite scaffolds under enzymatic hydrolysis conditions, however it is well known that thrombin hydrolyzes the bonds formed by arginine and lysine  24]..24].It was found earlier that the hydrolysis of native high molecular collagen of type I, isolated from the skin of different animals, under standard conditions in the presence of the enzyme—pancreatin proceeds at a sufficiently high rate .Due to the fact that both proteins-collagen and fibrinogen/fibrin- are structure-forming proteins in fibrin-collagen scaffolds, the purpose of this work is to analyze the hydrolysis rate of high molecular cod collagen (CC) and fibrinogen (Fn) isolated from blood plasma in the presence of the enzyme—thrombin  and the scaffold based on them are presented in It can be seen that fibrinogen does not have a fibrillar structure a,b, whicw according to the literature data [These conclusions suggest a scaffold formation scheme based on fibrinogen and collagen. First, we should refer to the literature data, from which it is known that fibrin-polymer is formed from fibrinogen in three stages . Two pepure data  is abouture data .w ~10 kDa are observed. Fibrinogen is destroyed more slowly; its destruction occurs according to the scheme described in the literature [Most likely, the scaffold formation during thrombin hydrolysis of fibrinogen and collagen mixture occurs in the following way. Hydrolysis of two proteins at room temperature is quite fast, especially collagen—almost a minute later, only oligomeric peptides with mainly Mterature . At the distilled water;solutions of acetic acid with a concentration of 3% and 4%, which were prepared from glacial acetic acid by diluting with distilled water;acetic acid solution of cod collagen with a concentration of 3%;fibrinogen (Fn)—commercial product extracted from human blood plasma ;enzyme—thrombin, proteolytic activity in 1 mL—50 IU ;1 M of sodium hydroxide solution, which was prepared from solid caustic soda by dissolving in a certain volume of distilled water;fibrin-collagen cell-free scaffold .The following was used during the study:Collagen was extracted according to the author’s (copyright) method . The cov3: 1.Enzymatic hydrolysis was carried out at a temperature of 25 °C in an aqueous solution at pH ~7.0. Thrombin was used as an enzyme. The substrate–enzyme ratio in solution protein: thrombin 10The solution for hydrolysis of high molecular collagen was prepared by diluting the initial CC in acetic acid. Then 1 mol/L NaOH was added to the CC solution. In the case of Fn hydrolysis, its aqueous solution was prepared. Further, samples (1 mL) were taken in certain intervals after the addition of the enzyme: 1, 2, 3, 5, 8, 10, 20, 30 min, 1 h, 2 h, 3 days. To interrupt the hydrolysis, 1 mL of 4% acetic acid solution was added to the samples.w) of 1000–410,000 Da (Fluca) were used for calibration.Molecular weight characteristics of hydrolysate of collagen and fibrinogen were determined by gel-penetrating chromatography (GPC) using a high-performance liquid chromatograph manufactured by Shimadzu CTO20A/20AC  with the LC-Solutions-GPC software module. Separation was performed using columns Tosoh Bioscience TSKgel G3000SWxl  with a pore diameter of 5 microns. The low temperature light scattering detector ELSD-LT II  was used as a detector. The 0.5 M acetic acid solution was used as eluent. The flow rate was 0.8 mL/min. Narrowly dispersed dextran samples with a molecular weight range  and the initial proteins—high molecular weight cod collagen and fibrinogen, was carried out on the scanning electron microscope JSM-IT300 . The scaffold sample was previously dissolved in a 3% solution of acetic acid and dried lyophilically. Samples of dehydrated proteins and scaffold were visualized. Dehydration of the samples took place directly in the chamber JSM-IT300 under the low vacuum impact.w ~17–18 kDa and 10 kDa.Low molecular fractions of collagen and fibrinogen hydrolysates have similar molecular weight values: Mw ~10 kDa.The destruction of macromolecules of high molecular collagen to hydrolysis products takes place almost completely within the first minute mainly to the polymer fraction with Mw ~10 kDa.Enzymatic hydrolysis of fibrinogen goes slower than collagen, but it also occurs mainly to the polymer fraction with MThe presented data allow three important conclusions to be drawn:


The capacity to increase one’s gait speed is critical for maintaining safe community ambulation. There is limited work on the longitudinal changes in this capacity and its predictors. Because lower dopamine is associated with lower task adaptation and motivation, we hypothesized that lower dopamine would predict more decline in rapid gait speed. Catechol-O-methyltransferase (COMT) polymorphism and at least 3 repeated rapid and usual pace gait speed assessments were obtained over 10 years in 1,261 older adults . Linear mixed models computed person-specific rapid and usual pace gait speed trajectories. Regression models adjusted for usual gait trajectory tested whether COMT predicted rapid gait trajectory; covariates included, demographic, psychological, cognitive, and physical factors. Val/Val carriers (lower dopamine) declined more in rapid gait compared to Met/Met carriers . Modifying dopamine may positively influence the ability to maintain rapid gait over time.


A cross-sectional survey was conducted in selected districts of Bangladesh to estimate the prevalence of bovine tuberculosis (bTB), and to identify the risk factors for bTB. We included 1865 farmed cattle from 79 herds randomly selected from five districts. Herd and animal level data were collected using semi-structured interviews with cattle herd owners. The single intradermal comparative tuberculin test (SICTT) was used to estimate the prevalence of bTB. The risk factors were identified using mixed-effect multiple logistic regression analyses. The overall herd and animal level prevalences of bTB were estimated to be 45.6%  and 11.3 (95% CI = 9.9–12.8%), respectively, using the OIE recommended >4 mm cut-off. The true animal level prevalence of bTB was estimated to be 11.8 . At the herd level, farm size, bTB history of the farm and type of husbandry were significantly associated with bTB status in univariable analysis. Similarly, age group, sex, pregnancy status and parity were significantly associated with bTB at cattle level. However, in multivariable analysis only herd size at the herd level and age group and pregnancy status at the cattle level were significant. Compared to a herd size of 1–10, the odds of bTB were 22.8 (95% CI: 5.2–100.9) and 45.6 times (95% CI: 5.0–417.7) greater in herd sizes of >20–50 and >50, respectively. The odds of bTB were 2.2 (95% CI: 1.0–4.5) and 2.5 times (95% CI: 1.1–5.4) higher in cattle aged >3–6 years and > 6 years, compared to cattle aged ≤1 year. Pregnancy increased the odds of bTB by 1.7 times (95% CI: 1.2–2.4) compared to non-pregnant cattle. Taken together, the results suggest high herd and animal level prevalence of bTB in these 5 districts, with the greatest risk of bTB in older and pregnant cattle within large herds (>20), and highlight an urgent need for continued surveillance and implementation of bTB control programs in Bangladesh. Mycobacterium bovis, a member of M. tuberculosis complex. [M. orygis has been reported to be the main causative agent of bTB in Bangladesh [M. bovis, but other captive wild mammals  as well as humans can also be infected. The disease can occur as subacute or chronic forms, with a variable degree of progression. A small number of animals may show clinical signs within a few months of infection while others may require several years to develop clinical signs. In low and middle income countries (LMICs), bTB is still common and it induces severe economic losses that can occur from livestock deaths, chronic disease, and reduced production. For example, in Ethiopia the economic cost of bovine TB was US$75.2 million and US$358 million in 2005 and 2011, respectively [Bovine tuberculosis (bTB) is a bacterial disease of cattle mainly caused by complex. . Howeverngladesh , 3. Cattectively . MoreoveM. bovis occurred in 2018. Bangladesh is located in the WHO’s South East Asian region where the total regional burden of zTB is 44,800  and an estimated number of deaths of 2,090  [M. bovis accounts for 3.1% of all human TB cases, 2.1% of all pulmonary and 9.4% of all extra-pulmonary TB (EPTB) cases [Globally, it was estimated that 143,000  cases and 12,300  deaths due to zoonotic tuberculosis (zTB) caused by M. tuberculosis complex (MTC) to the OIE. However, the actual bTB burden is most likely to be underestimated due to a lack of active surveillance in Bangladesh. Several studies have been conducted in the past to estimate the prevalence of bTB in different geographical locations of Bangladesh. Pharo et al. (1981) reported 6.0% tuberculin skin test (TST) positive cattle in Sirajgonj district [Bangladesh is routinely notifying the presence of bTB in cattle as district . Samad adistrict . The predistrict  and 7.78district . Howeverdistrict  and a fedistrict , 14. Eigdistrict . Indigendistrict . These pdistrict . MoreoveThe present study is part of the research protocol No. PR-17121, which was approved by the Research Review Committee (RRC) and Ethical Review Committee (ERC) of the International Center for Diarrhoeal Disease and Research , Bangladesh and by the Animal Welfare and Experimentation Ethical Committee (AWEEC) of Bangladesh Agricultural University (AWEEC/BAU/2019/24). Both written and oral consent were taken from the owner/manager of the cattle farm before conducting tuberculin skin test and data collection.The study was conducted in five dairy intensive districts of Bangladesh viz: Dhaka, Munshiganj, Mymensingh, Gazipur and Jamalpur  from JunAmong the five districts, Dhaka , Gazipur  and Munshiganj  districts are located in the central part of the country under Dhaka division, whereas Mymensingh  and Jamalpur  districts are located in north-eastern part of the country under Mymensingh division . There aThe list of dairy farms were obtained from sub-district (Upazila) livestock offices posted in respective district. The list of farms (sampling frame) from five dairy intensive districts of Bangladesh were entered into a spreadsheet (Microsoft Excel 2010). Each farm was assigned with an Excel generated random number using “rand” function. Then the herds were randomly selected from the sampling frame. These districts were: Dhaka, Munshignaj, Mymensingh, Gazipur, and Jamalpur . The farZ = Z—score at 95% confidence interval = 1.96, Se = average sensitivity of the SICTT = 0.88, Sp = Specificity of SICTT = 0.84 [Pexp = expected prevalence = 5% = 0.05 and d = level of precision = 5% = 0.05. These assumptions produced a sample size of 468. As cluster sampling was used, the design effect (D) of the study was calculated using the formula given in b the average is number of samples per cluster (15) and ρ is the intra-cluster correlation coefficient. The intra-cluster correlation coefficient for bTB is considered to be 0.2 [D = 3.8; when multiplied by the calculated sample size, the minimum sample size then becomes 1778. We assumed average herd size of 15 and hence 119 farms to be tested to reach target sample size. As the herd size varied we needed to visit only 79 herds to reach a sample size of 1865.The sample size was calculated using the formula given in T = 0.84 , Pexp = o be 0.2 . The desThe intradermal test (SICTT) was performed as per standard procedure , 24. In Information on herd level risk factors was collected from farm owners/managers using a semi-structured and pre-tested questionnaire. The questionnaire contained open-ended and closed-questions and was completed at the time of tuberculin testing. The questions in the questionnaire were translated into the local dialect so that the respondents could easily understand them. The objectives of the survey were explained to each farm owner/farm manager and they could withdraw from the study at any time.Herd level data on farm type, age of farm, farm size, husbandry type, manure use, feeding of silage, introduction of new animals within last two years, veterinary healthcare provider, and biosecurity status and animal level data on breed, age, sex, parity, weight, milk production, pregnancy status, and body condition score were collected using semi-structured face to face interviews with cattle herders and herd owners at the day 0 of PPDs inoculation . The dataset was coded, checked for integrity and exported to STATA 13  and R 3.6.0  for analWe summarized the data using descriptive statistics for demographic characteristics and factors. We calculated the mean and standard deviation (SD) for continuous variables and calculated proportions and frequency distributions for categorical variables. All continuous predictor variables  were categorized prior to logistic regression analysis. Based on the average sensitivity and specificity of the SICTT  the animInitially, univariable mixed effects logistic regression analyses were performed by including herd and district as random intercepts for animal and herd level, respectively  value. Then the remaining variables were added in turn, based on AIC. The final model selected also had the lowest AIC. Confounding was checked by observing the change in the estimated coefficients of the variables that remained in the final model by adding a non-selected variable to the model. If the inclusion of this non-significant variable led to a change of more than 25% of any parameter estimate, that variable was considered to be a confounder and retained in the model . The twoThe 95% confidence interval of the ICC was bootstrapped using the “bootMer” function of the R package “lme4” . All of SICTT was performed on 1865 cattle from 5 districts. The study included 79 randomly selected dairy farms with a median  herd size of 11 (6–36). Most farms were in Dhaka (67.0%), followed by Mymensingh (16.5%), Jamalpur (14.0%), Munshiganj (1.3%) and Gazipur districts (1.2%) . Nearly,The distribution of key demographics of the cattle tested are shown in Of 1865 cattle tested, 16.3% (n = 303) demonstrated a measurable increase in skin thickness after 72 h of the bovine and avian PPD inoculation both at bovine and the avian sites respectively which reflects cross reaction of bTB with paratuberculosis and or environmental mycobacterium. Of these 303 cattle, 22.8% (n = 69), 15.2% (n = 46) and 62% (n = 188) were interpreted as positive, inconclusive and negative animals respectively .As a whole, herd level prevalence of bTB was 45.6% (95% CI: 34.3–57.2%) . The witThe herd size, husbandry type and history of bTB in the herd were significantly (P < 0.05) associated in univariable logistic regression analyses . At the Compared with a herd size of 1–10, the odds of bTB were 22.8 (95% CI: 5.2–100.9) and 45.6 times (95% CI: 5.0–417.7) greater in herd sizes of >20–50 and > 50, respectively. The odds of bTB were 2.2 (95% CI: 1.0–4.5) and 2.5 times (95% CI: 1.1–5.4) higher in cattle aged >3–6 years and >6 years compared to cattle aged ≤1 year. Pregnancy increased the odds of bTB infection by 1.7 times (95% CI: 1.2–2.4) compared to non-pregnant cattle . No confIn this study, we estimated herd and cattle level bTB prevalence in five dairy-intensive districts in Bangladesh and identified risk factors for bTB in cattle. A substantial proportion of cattle and herds tested positive, with herd size, age of individuals, and pregnancy status significantly associated with bTB reactor status in cattle in these selected districts in Bangladesh. The study further suggests that frequent screening of bTB of larger herds and especially targeting older and pregnant cattle could reduce within herd transmission and minimize the risk of zoonotic transmission of tuberculosis in Bangladesh context.As a whole, herd level prevalence was found to be 45.6% 95% CI = 34.3–57.2%). No previous published report on herd level bTB in Bangladesh is available to compare to this estimate. In addition, 31% 11/36) of the positive herds had a within-herd bTB prevalence of ≥25%. Of note was the observation that of the 11 high-prevalence herds, 7 were located in Dhaka city. With an average density of 44,500 people per square kilometer, Dhaka ranks amongst the most densely populated cities in the world. While in general, most people within urban environments in Dhaka boil milk prior to consumption, individuals living in close proximity to bTB positive animals may be at increased risk for acquiring zoonotic TB infection via aerosol or direct transmission .3–57.2%. and 52.2 of the pWe identified herd size as a potential risk factor for likelihood of bTB test-positivity at farm level. Size of herds has been reported as a risk factor for bTB –49, howeAs a whole, animal level prevalence was found to be 11.3% (95% CI = 9.9–12.8%). This is substantially higher than reported previously studies that suggested between 2–7.8% reactor animals from different geographical locations and in different cattle breeds of Bangladesh , 50. HowAt animal level, age and pregnancy were found to be potential risk factors for bTB in Bangladesh. Two age group of cattle >3–6 and > 6 years were found to be at higher risk of bTB. Our findings are consistent with previous studies , 52, 53.Pregnancy status was found to be an important risk factor for bTB which corroborates findings of other authors , 56. PreMycobacterium avium subsp. paratuberculosis (MAP) or environmental mycobacterium infection, of which only 22.8% (n = 69) were found to be bTB positive  positivity status.(DOCX)Click here for additional data file.S2 TableThe weighting was completed in relation to bovine tuberculosis (bTB) positivity status of individual animal level.(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S1 Questionnaire(DOCX)Click here for additional data file.S2 Questionnaire(PDF)Click here for additional data file.S3 Questionnaire(DOCX)Click here for additional data file.S4 Questionnaire(PDF)Click here for additional data file.S1 File(TXT)Click here for additional data file.


Antimicrobial resistance (AMR) is one of the most challenging worldwide health threats facing modern medicine (Centers for Disease Control, 1The prognosis of AMR is increased morbidity and mortality from uncontrolled growth of a microorganism that is resistant to one, or even all, available antimicrobial therapies (Centers for Disease Control, In the long‐term, the threat from a postantimicrobial therapy world is that today's relatively minor infections—including practically unavoidable exposures to microbes associated with labour and delivery—may once again contribute to increased morbidity and mortality in both developed and in developing countries (Centers for Disease Control, Currently, nurse practitioners are unable to easily and quickly screen and diagnose many types of AMR because specimens must be sent to a centralized diagnostic laboratory for evaluation of antimicrobial susceptibility that often takes several days of testing (Mitsakakis, Kaman, Elshout, Specht, & Hays, 2AMR needs to be viewed by nurse practitioners not just as a primary care issue but also an ethical issue (Basu & Garg, An important distinctive feature of AMR is the collective and reciprocal vulnerability of all people both as the vectors and victims of AMR organisms (Basu & Garg, Unfortunately, the combination of monetary incentives from some hospitals a few different countries and limited knowledge about prescription guidelines for antimicrobial therapies can contribute to unethical prescribing practices (Basu & Garg, 3AMR brings to the forefront ethical issue that requires a new way of thinking for nurse practitioners, not just about the nature of the ethical issues AMR raises, but about how to best address these moral dilemmas (Johnstone, All authors contributed equally to this article.


Previous estimates of nucleotide substitution rates are routinely applied as secondary or “universal” molecular clock calibrations for estimating evolutionary timescales in groups that lack independent timing information. A major limitation of this approach is that rates can vary considerably among taxonomic groups, but the assumption of rate constancy is rarely evaluated prior to using secondary rate calibrations. Here I evaluate whether an insect mitochondrial DNA clock is appropriate for estimating timescales in Collembola—a group of insect-like arthropods characterized by high levels of cryptic diversity. Relative rates of substitution in cytochrome oxidase subunit 1 (COI) were inferred via Bayesian analysis across a topologically constrained Hexapod phylogeny using a relaxed molecular clock model. Rates for Collembola did not differ significantly from the average rate or from the rates estimated for most other groups (25 of 30), suggesting that (1) their apparent cryptic diversity cannot be explained by accelerated rates of molecular evolution and (2) clocks calibrated using “universal” insect rates may be appropriate for estimating evolutionary timescales in this group. However, of the 31 groups investigated, 10 had rates that deviated significantly from the average , underscoring the need for caution and careful consideration when applying secondary insect rate calibrations. Lastly, this study exemplifies a relatively simple approach for evaluating rate constancy within a taxonomic group to determine whether the use of secondary rates are appropriate for molecular clock calibrations. The concept of the molecular clock has revolutionized the field of evolutionary biology by providing a foundation for evaluating the tempo of biological processes and mechanisms shaping patterns of biodiversity . MoleculCollembola (or springtails) present an interesting, yet challenging, case of exploring evolutionary hypotheses for taxa without available node calibrations or taxon-specific molecular clocks. These minute insect-like arthropods are among the most abundant organisms in the soil mesofauna, occupy nearly all terrestrial ecosystems, and serve vital roles in detrital decomposition, food webs, and soil structure . DespiteDrosophila flies [Patterns of morphological stasis or ‘cryptic speciation’ can be driven by directional selection on non-morphological traits such as sexual recognition, physiology, or behavior , and/or la flies . Wessel la flies  also attCollembola share these peculiar population dynamics: they are non-vagile, with high degrees of ecological specificity, and are seemingly robust to climatic oscillations and ecological change . These cDespite the circumstantial plausibility for accelerated molecular rates in Collembola, mitochondrial DNA clocks calibrated using rates estimated for insects, such as Papadopoulou et al.’s  estimateNevertheless, estimating molecular rates in relative, rather than absolute, time can offer an alternative way to test evolutionary hypotheses concerning the rate of genetic change or molecular divergence for groups that lack robust calibrations. This comparative approach has been used to estimate relative divergence times for hydrobiid snails  and membAlthough not within the primary scope of their study, Ciccondardi et al.  estimateTo expand upon the work of Cicconardi et al.  I use thComplete COI sequences for a total of 204 taxa were acquired from NCBI GenBank  Table S: 188 taxR2) was computed independently for each codon position. To determine if the inclusion of codon 3 would impair phylogenetic estimation, R2 for codons 1 and 2 combined was compared with the 2R for all codon positions combined. If the relationship between uncorrected and model-corrected p-distances is approximately linear, then there is no saturation. If saturation is present, the relationship will deviate and begin to plateau because uncorrected p-distances will be underestimating the number of substitutions between taxa due to a loss of information after multiple substitutions at single sites [Substitution saturation is a major concern when estimating phylogenetic parameters across deep evolutionary time , especiale sites . At thesle sites , and thele sites . All uncle sites . p < 0.5). The mean proportion of invariant sites used for this test (mean = 0.21) was determined via Bayesian phylogenetic analysis.Substitution saturation was also assessed for COI  using the substitution saturation test developed by Xia et al.  with DAMTo estimate relative rates of COI nucleotide substitution across Hexapoda I performed a Bayesian phylogenetic analysis using a fixed tree topology  and an uThe Bayesian phylogenetic analysis was performed using BEAST2 v. 2.4.8  with theR2 = 0.666). Plots for codon 1, codon 2, and codons 1 and 2 combined, indicate strong linear correlations between uncorrected and model-corrected genetic distances . When codon 3 was combined with codons 1 and 2, the coefficient of determination remained high (R2 = 0.998), supporting the inclusion of codon 3 for estimating evolutionary rates.Simple linear regression plots of genetic distances (uncorrected vs. model corrected)  identifip < 0.01)  , indicatThe Bayesian phylogenetic analysis of 204 arthropod COI sequences revealed significant variation in relative rates of nucleotide substitution among hexapod groups . Visual Saturation tests indicated that the phylogenetic analysis would not be significantly impacted by substitution saturation . This waThe comparative rate analysis  revealedBecause springtail substitution rates were similar to most other hexapods, their apparent morphological-molecular disparity cannot be explained by accelerated rates of molecular evolution and is instead more consistent with the hypothesis of long-term morphological stasis. However, given the limited taxonomic sampling (average rates of clades may be biased) and low precision in the rate estimates, these findings should be considered with caution, especially for hypotheses concerning mechanisms driving cryptic diversity within Collembola. Nevertheless, this study serves as a useful starting point towards understanding the rate of molecular evolution in springtails with respect to patterns of cryptic diversity and provides limited validation of previous studies that have used molecular clocks calibrated with secondary rates estimated for Coleoptera  to infer


Prokaryotes represent the largest living biomass reservoir in aquatic environments and play a crucial role in the global ocean. However, the factors that shape the abundance and potential growth rate of the ecologically distinct prokaryotic subgroups  along varying trophic conditions in the ocean remain poorly understood. This study conducted a series of modified dilution experiments to investigate how the abundance and potential growth rate of HNA and LNA prokaryotes and their regulating factors  change along a cross-shore nutrient gradient in the northern South China Sea. The results showed that the abundance of both HNA and LNA cells was significantly positively correlated with the abundance of heterotrophic nanoflagellates and viruses, whereas only HNA abundance exhibited a significant positive correlation with nutrient level. With a decreasing nutrient concentration, the potential growth rate of the HNA subgroup declined significantly, while that of the LNA subgroup was significantly enhanced, leading to an elevated relative potential growth rate of the LNA to HNA subgroup under decreasing nutrient levels. Furthermore, our data revealed different regulatory roles of protozoan grazing and viral lysis on the HNA and LNA subgroups, with HNA suffering higher mortality pressure from grazing than from lysis in contrast to LNA, which experienced equivalent pressures. As the nutrient levels declined, the relative contribution of lysis to the mortality of the HNA subgroup increased significantly, in contrast to the insignificant change in that of the LNA subgroup. Our results indicated the elevated role of LNA cells in the prokaryotic community and the enhanced viral lysis pressure on the total prokaryotes under oligotrophic conditions. This implies a weakened efficiency of carbon cycling within the microbial loop and enhanced viral lysis to shunt more carbon and energy flow in the future ocean, in which oligotrophication will be strengthened due to global warming. Representing the largest living biomass reservoir  and critThe flow cytometry technique has revealed that prokaryotes cluster into high nucleic acid (HNA) cells and low nucleic acid (LNA) cells, and the two subgroups may be physiologically and ecologically distinct . InitialIt is widely recognized that prokaryotic abundance and potential growth rate are dynamically controlled by both bottom-up   and top-Previous studies demonstrated that the relative importance of protozoan grazing and viral lysis on prokaryotes varies by trophic status  and wateThis study aims to investigate how the abundance and potential growth rate of two distinct prokaryotic subgroups  and their regulating factors, including protozoan grazing and viral lysis, change under different trophic conditions in the natural environment. A series of modified dilution experiments were conducted along a transect spanning the nutrient-rich coastal zone to an area of nutrient-depleted open ocean in the northern South China Sea (SCS) in November 2016. Being the largest marginal sea in the northern Pacific, the SCS is an ideal testbed because it harbors both a river-dominated shelf region  and a vast area of an oligotrophic deep basin, facilitating the investigation of the differing regulatory impacts of protozoan grazing and viral lysis on the HNA and LNA subgroups under various nutrient conditions in the ocean.Six sampling sites along a transect spanning the shelf water neighboring the Pearl River Estuary (PRE) to the deep basin of the northern SCS  were visSamples for determining abundance of prokaryotes and virus-like particles (VLP) were prepared via the following steps: 2-mL samples were fixed with glutaraldehyde at a final concentration of 0.5%, held at room temperature for 15 min in the dark, shock-frozen in liquid nitrogen, and stored at −80°C until analysis. Prokaryotic abundance was determined using flow cytometry  with a laser emitting light at 488 nm. Prior to analysis, the samples were thawed to room temperature, and 990-μL subsamples were stained in the dark with SYBR green I solution  and then held at room temperature for 15 min . ProkaryThe high and low nucleic acid content (HNA and LNA) prokaryotic subgroups were discriminated based on their respective signature in the cytogram plot of SSC versus green fluorescence . All flo–1 (Heterotrophic nanoflagellate (HNF) abundance was measured by the following steps: 50-mL subsamples were fixed with glutaraldehyde at a final concentration of 1%, filtered through 0.45-μm polycarbonate black filters, and stained with DAPI  at a final concentration of 10 μg mL–1 . HNFs onA modified parallel dilution technique was utilized to estimate prokaryotic mortality mediated by protozoan grazing and viral lysis following 0 dilution series . From each of these t0 bottles, triplicate 50-mL polycarbonate bottles were rinsed twice with diluents and then gently filled with diluents by siphoning to minimize physical damage to the grazers, viruses, and prokaryotes. Immediately after completing the above preparation steps, the 50-ml polycarbonate bottles  were incubated in an on-deck incubator for 24 h with in situ light simulated by covering with a neutral density plastic sheet and under the same temperature as the seawater at the time they were sampled by running seawater at that temperature. Triplicate 2-mL samples were collected at the start and the end of incubation to measure the prokaryotic abundance.Briefly, seawater was first passed through a 20-μm mesh and then filtered through a tangential flow filtration system with a 0.2-μm and 30-kDa pore size polyvinylidene difluoride cartridges  to generate the grazer-free and virus-free diluents, respectively . The pol–1) was calculated for each sample based on the prokaryotic abundance at the start and the end of the incubation experiment (Nt0 and Nt), assuming exponential growth , whereas the slope of the regression for the virus-free dilution series reflects the combined impact of protozoan grazing and viral lysis. Therefore, the viral lysis-mediated prokaryotic mortality (VMM) can be obtained from the difference between the slopes of the regression lines from the two-dilution series . PotentiF-test to assess whether a significant difference between the two sources of mortality existed, and a P-value less than 0.05 was considered significant. Shapiro–Wilk W tests were used to examine the data normality before analysis, and logarithm transformation was performed if necessary. Significant differences between samples were determined using paired t-tests. The relationships between the prokaryotic parameters and abiotic or biotic variables were examined using Spearman’s rank correlation analysis with a significance level (α) of 0.05.GraphPad Prism 7  software was used to perform all statistical analyses. Briefly, a least-square regression analysis was conducted to analyze the relationship between the net growth rate and fractions of the grazer-free and virus-free dilution series. The PMM and VMM rates were analyzed using an The temperature of the upper 70 m water generally increased from nearshore station A to offshore station SEATS . Vertica–1), whereas distinctly lower surface (0.11–0.35 μmol L–1) but higher subsurface concentrations (0.55–3.96 μmol L–1) were observed at the other offshore stations that were minimally affected by the plume  was significantly higher than the surface average (1.59 ± 0.68 d–1) , where the maximum subsurface PPG-H (at A) was nearly fourfold greater than the minimum surface PPG-H (at SEATS).The potential growth rate of the HNA group (PPG-H) at the surface generally decreased offshore , while n–1) was significantly  higher than the subsurface average (0.55 ± 0.17 d–1).The potential growth rate of the LNA subgroup (PPG-L) at the surface generally increased offshore from A to E but slightly declined to SEATS , while PR2 = 0.65, P < 0.005)  reflects the relative activity of the HNA cells to the LNA cells. The maximum ratio was recorded at the subsurface layer of A (8.54), and the minimum was at the surface layer of SEATS (0.87) . Linear < 0.005) .t-test, P < 0.05)  while there is no significant correlation between VMM-H and DIN  was higher at nearshore station A than at the offshore stations and was significantly higher in the subsurface than surface layers (paired  < 0.05) . No clea < 0.05) . Linear  and DIN . The sum and DIN .R2 = 0.41, P < 0.05) (The ratio of PMM-H to VMM-H (PMM-H/VMM-H) represents the relative contribution of protozoan grazing over viral lysis to the mortality of the HNA subgroup, where a ratio value higher than 1 indicates greater contribution by grazing than lysis and vice versa. The ratio of PMM-H/VMM-H was distinctly higher than 1 for all surface and subsurface samples . At the  < 0.05) .t-test, P < 0.05), with the surface value of each station generally higher than its subsurface counterpart   did not show a clear trend along the transect, although a significant difference was found between layers  . The sum < 0.05) .No clear spatial trend was found for the ratio of protozoan grazing to viral lysis of the LNA subgroup (PMM-L/VMM-L) in the surface  or subsur = 0.44, P < 0.05), HNF abundance , VLP abundance , and protozoan grazing on the HNA subgroup  and was negatively related to temperature . Conversely, LNA abundance was not related to any nutrient but was strongly, positively related to HNF abundance  and VLP abundance . HNA% exhibited similar correlation analysis results as the HNA abundance except that HNA% was also significantly positively related to DIN  but not VLP abundance, despite the positive correlation between the abundance of VLP and each subgroup.Spearman’s rank correlation analysis was used to reveal the potential drivers of prokaryote abundance and potential growth rate . HNA abur = 0.80 and 0.82, P < 0.01), HNF abundance , and protozoan grazing and viral lysis of the HNA subgroup . Specifically, the correlation between PPG-H and grazing was more significant than that between PPG-H and lysis. In contrast to PPG-H, PPG-L showed negative correlations with DIN and silicate concentrations  and had comparable significant, positive correlations with protozoan grazing and viral lysis on the LNA subgroup .The potential growth rate of the HNA group was positively related to DIN and silicate concentrations  respond to various nutrient conditions. The transect survey was carried out in the early winter season when the relatively strong northeast wind prevailed, and the Pearl River discharge was less than the annual mean, resulting in a narrower offshore plume extension . This faThe abundance of the HNA and LNA subgroups responded differently to the abiotic and biotic variables. Specifically, the spatial distribution of HNA abundance exhibited an environmental gradient along the transect that was similar to the physical and chemical characteristics, i.e., decreasing from coastal to open ocean, and was generally higher at the subsurface than the surface waters of the open ocean. In contrast, the spatial distribution of LNA abundance showed a weaker gradient along the transect. The different responses of the abundance of the two subgroups to environmental factors were further reflected by the fact that HNA abundance was significantly correlated with both silicate concentration and temperature, while LNA abundance was not related to any abiotic factor. It follows that the HNA subgroup is more responsive to environmental variations than the LNA subgroup. A similar phenomenon was observed in the Atlantic Ocean where HNA abundance changed more than twofold between seasons, acting similar to an r-strategist or opportunist, while the LNA subgroup demonstrated more equilibrium dynamics over the same period, likely following the k-strategy . This teSimilar to HNA abundance, the spatial distribution of HNA prokaryotic potential growth rate (PPG-H) showed a clear environmental gradient, largely mirroring the distribution pattern of DIN and silicate concentration, i.e., declining offshore, and was significantly higher at the nutrient-rich subsurface of the open ocean than the nutrient-poor surface of the open ocean. The spatial distribution of LNA prokaryotic potential growth rate (PPG-L) showed the opposite pattern to PPG-H and nutrient distributions, which generally increased from coastal to open ocean and was higher at the surface than subsurface waters of the open ocean. This reverse response of HNA and LNA cells to nutrient levels is also reflected by the significant positive correlations between PPG-H and DIN and silicate concentration in contrast to the significant negative correlations between the PPG-L and DIN and silicate concentration. As a result, a significant positive linear relation was found between the PPG-H/PPG-L ratio and DIN concentration, suggesting that the relative activity of the HNA cells decreased while that of the LNA cells increased under oligotrophication. This finding is consistent with other studies that found that open ocean oligotrophic communities, compared to coastal or shelf communities, have a higher proportion of heterotrophic activity ascribed to the LNA subgroup , 2006. TThe increasing fraction of LNA abundance in total prokaryotic abundance (indicated by declining HNA%) and increasing relative activity of LNA cells (indicated by declining PPG-H/PPG-L ratio) with decreasing nutrients revealed in our data may suggest a reduced capacity of the total prokaryotic community to degrade and utilize DOC under the more oligotrophic conditions. This is associated with that LNA cells, compared to the HNA cells, have lower metabolic rate and weaker capability to degrade and utilize DOC due to their small streamlined genomes, limited genetic repertoires, and simpler extracellular hydrolase system . A similProtozoan grazing and viral lysis are well recognized as the major agents of the top-down control of prokaryotes . PreviouImportantly, our results indicated the contrasting regulating impacts of protozoan grazing and viral lysis on the abundance and potential growth rates of the HNA and LNA subgroups. The protozoan grazing profoundly impacted HNA abundance but not the LNA abundance, as suggested by the correlation analysis. On the contrary, VLP abundance was found to be significantly, positively correlated with both HNA and LNA abundance and the correlation between VLP abundance and LNA subgroup was stronger and more significant than that of HNA. The contrasting regulatory role of protozoan grazing and viral lysis on the HNA and LNA subgroups is also reflected in the prokaryotic potential growth rates. The HNA subgroup exhibited a stronger and more significant correlation with protozoan grazing than viral lysis, whereas the correlations between the LNA subgroup and grazing and lysis were more or less equivalent. In other words, the HNA cells were subjected to greater top-down control from grazing than lysis, while the LNA cells experienced equivalent pressures from the two regulators. This is well evidenced by the higher grazing to virus lysis ratio for HNA (ranging from 1.45 to 3.58) than for the LNA subgroup (0.47 to 1.54). We attribute the greater protozoan grazing pressure on the HNA than LNA cells to the selective grazing strategy of protozoa. Namely, the protozoa preferentially graze HNA cells that belong to the actively growing portion of the prokaryotic assemblage , whereasR2 = 0.32, P < 0.05; Another interesting finding is that the relative role of protozoan grazing to viral lysis (indicated by the ratio of grazing to lysis) in regard to the HNA and LNA subgroups responded differently to varying nutrient levels. Specifically, the relative importance of grazing on the HNA subgroup declined with decreasing nutrient concentration, which may be explained by the reduced food quality or availability under lower nutrients, because food quality  and availability are known to influence protozoan grazers and grazing rates . The dec2.Our data revealed that as the nutrient concentrations declined, the proportion of LNA abundance to total prokaryotes, the relative prokaryotic potential growth rate of the LNA to HNA cells, and the relative impact of viral lysis to protozoan grazing on total prokaryotes all increased. Such responses of prokaryotic abundance and potential growth rate and their top-down regulating factors to the decreasing nutrient levels have important implications for carbon cycling and sequestration in the future ocean . The antThis study revealed that the potential growth rate of the HNA and LNA subgroups responded reversely along the nutrient gradient. Namely, as the nutrient concentrations decreased, the HNA potential growth rate significantly decreased, while that of the LNA markedly increased, thus yielding an increasing relative potential growth rate of the LNA subgroup with declining nutrient levels. Furthermore, there existed contrasting regulatory impacts of protozoan grazing and viral lysis on the HNA and LNA subgroups, where the HNA subgroup experienced higher pressure from grazing than lysis while the LNA subgroup was subjected to an equivalent impact from grazing and lysis. The relative role of lysis to grazing on the HNA subgroup increased with decreasing nutrients. Taken together, our results revealed an elevated contribution of LNA cells to the prokaryotic community and a relatively greater virus-mediated mortality pressure on total prokaryotes under more oligotrophic conditions. This implies a weakened efficiency of carbon cycling within the microbial loop and enhanced viral lysis to shunt more carbon and energy flow in the anticipated future ocean oligotrophication driven by global warming.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.CH designed and performed the experiments under the supervision of DX and NJ. CH carried out the formal analysis with assistance and inputs from XC and LY. CH wrote the manuscript with contributions from all co-authors. 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.


Nuclear medicine has evolved over the last half-century from a functional imaging modality using a handful of radiopharmaceuticals, many of unknown structure and mechanism of action, into a modern speciality that can properly be described as molecular imaging, with a very large number of specific radioactive probes of known structure that image specific molecular processes. The advances of cancer treatment in recent decades towards targeted and immune therapies, combined with recognition of heterogeneity of cancer cell phenotype among patients, within patients and even within tumours, has created a growing need for personalised molecular imaging to support treatment decision. This article describes the evolution of the present vast range of radioactive probes – radiopharmaceuticals – leveraging a wide variety of chemical disciplines, over the last half century. These radiochemical innovations have been inspired by the need to support personalised medicine and also by the parallel development in development of new radionuclide imaging technologies – from gamma scintigraphy, through single photon emission tomography (SPECT), through the rise of clinical positron emission tomography (PET) and PET-CT, and perhaps in the future, by the advent of total body PET. Thus, in the interdisciplinary world of nuclear medicine and molecular imaging, as quickly as radiochemistry solutions are developed to meet new needs in cancer imaging, new challenges emerge as developments in one contributing technology drive innovations in the others. Advances in gene sequencing and single cell transcriptomics have revolutionised our understanding of clonal heterogeneity and cancer evolution . These nsystems imaging.On the other hand, molecular imaging techniques, such as positron emission tomography (PET) and single photon emission computed tomography (SPECT), present the opportunity to spatiotemporally monitor, in the living patient, the expression and activity of key biomarkers associated with the cancer phenotype. Direct conjugation of positron or gamma-emitting radionuclides to small molecule substrates, inhibitors, and larger biomolecules such as peptides, antibodies, and even whole cells, has enabled visualisation, repeatedly and across the whole body, of a wide variety of biological processes in living subjects. Moreover, the substitution of an imaging radionuclide for a therapeutic radioisotope  has been effectively used for molecular-targeted cancer treatment. Nuclear medicine imaging techniques, however, have been traditionally difficult to multiplex and hence cannot probe large numbers of different processes/genes, restricting the longitudinal monitoring of disease progression often to a single biomarker. In this review, we explore how recent developments in radiochemistry, alongside scanner development, have evolved from early functional imaging radiopharmaceuticals to provide the current tool-kit to image a wide range of molecular processes and gene expressions - “molecular imaging” – and how further development may overcome this limitation to herald a new era of molecular Immunotherapy has led to durable, and in some cases, curative responses in patients with non-solid tumours . The primolecular imaging to the use of radioiodines in this context.Eighty years on from the first administration of radioiodine to a human subject,  the huma99mTc]pertechnetate is the chemical form of technetium-99 m eluted from the generator, hence no further chemistry development was required to apply this in the clinic.However, the accumulation of iodine in thyroid tissue is not solely dependent on the activity of hNIS; hNIS mediates the initial entry of iodide ions into the thyrocyte, but its retention in and efflux from thyroid tissue is mediated by other processes including transport into the thyroid “colloid,” oxidation by peroxidase, incorporation into thyroid hormones, and later release of these hormones . Thus, r99mTc]pertechnetate is therefore more suitable for this reporter gene application, [18F]tetrafluoroborate, [18F]fluorosulfate [18F]hexafluorophosphate [18F]tetrafluoroborate is in the vanguard of clinical and preclinical use and has been evaluated in healthy human volunteers, perunteers,  thyroid unteers,  and precunteers, . These nunteers,  and cell18F]-2-fluoro-2-deoxyglucose (FDG) dihydroxyphenylalanine, a dopamine precursor analogue used in neuroendocrine and brain tumours) [18F]fluoromisonidazole, a nitroimidazole hypoxia imaging agent) and related nitroimidazole derivatives [18F]DCFPyL (a prostate-specific membrane antigen (PSMA) ligand used for prostate cancer imaging) and other 18F-labelled PSMA ligands [18F]fluoroethylcholine [18F]fluoroethyltyrosine), [18F]fluoropropyl)-L-glutamic acid  [18F]fluciclovine DC ligands ; [18F]fllcholine  and relarosine),  FSPG (3-sporter)  and fluoride is extremely challenging fluoride into a pre-formed aluminium-chelate-biomolecule conjugate as the final synthetic step  emitter lending itself to both PET and therapeutic application. A “theranostic” pairing, in which 64Cu PET imaging provides quantitative and accurate biodistribution data to underpin dosimetry for therapeutic use of 64Cu and 67Cu, can be envisaged. The half-life of 64Cu is 12.7 h, reasonably compatible with antibodies, peptides and small molecules and sufficiently long that rapid labelling is less important than in vivo stability. Because Cu2+ is intrinsically more kinetically labile than other radiometals discussed above, no acyclic chelators have found use for 67Cu and 64Cu. Against this background, macrocyclic bifunctional copper chelators  the priorities are reversed: fast labelling is paramount while prolonged in vivo stability is of little consequence.As clinical PET developed, interest turned to the copper-64, which can be produced using a typical biomedical cyclotron. ors Fig.  have devors Fig.  as the p89Zr a suitable bifunctional chelator emerged quickly and fortuitously without the long years of trial and optimisation of dozens of ligand systems that characterised the development of copper bifunctional chelators. Desferrioxamine-B , has not enjoyed such widespread use so far.A core demand of imaging in the age of targeted and immune therapy is tracking the fate of antibodies, whether used therapeutically, diagnostically or theranostically. Radiolabelling antibodies has been a focus of radiochemists since the 1980s, as highlighted above, but the demand to extend imaging with antibodies from gamma scintigraphy to PET has highlighted the need for accessible long half-life positron-emitting radionuclides. Zirconium-89   and iodi in bone . Despite52Mn-immunoconugates, labelled without undue difficulty by means of the DOTA , may provide the solution. The first Among the armoury of emerging immunotherapeutics are cell-based therapies such as mesenchymal stem cells and tumour-targeted CAR-T cells. The risks of adverse effects of these therapies, and the need to understand reasons for their success or failure, have highlighted the need for clinical trials to incorporate methodology to track the migration, survival and proliferation of administered therapeutic cells  and to e18F]fluoro-3-(hydroxymethyl)butyl]guanine , by PET with nucleoside analogue 9-[4-[18F]FHBG .4] -compatible kit-based method of producing the 89Zr-oxine complex has been reported recently, [89Zr(oxinate)4], and analogous [64Cu(oxinate)2] and [52Mn(oxinate)2], have also been used to label liposomal drug formulations for PET imaging of their biodistribution fluoride under mild, aqueous conditions remains sought-after but elusive.Similarly, the wide availability of fluorine-18 has made possible clinical use of a variety of small organic molecular tracers for PET, but the complexity of their synthesis and the associated need for high-capital value facilities and expert radiochemistry staff has limited widespread use of all but a few, and made them very cumbersome to use with proteins. Consequently The many radiochemical innovations described above have been driven not only by the wide range of molecular targets associated with targeted therapy and immunotherapy in cancer, but also by developments in imaging physics which change the expectations of what can be achieved. Most obviously, the availability of PET-CT (PET-computed tomography) scanners and cyclotrons, in turn driven by the clinical importance of FDG, has catalysed production of new radionuclides and development of new radiochemistry to exploit the advantages of PET over scintigraphy and SPECT. The advent of PET-MR (PET-magnetic resonance) has also contributed to new combined modality tracer design .Despite the plethora of molecular targets of interest to imagers to help characterise disease heterogeneity and treatment response, radionuclide imaging of more than one of the molecular characteristics in an individual patient, as outlined in the introduction, is fraught with difficulties including exceeding acceptable radiation doses, the inconvenience of multiple visits for scanning, and extended use of scanner time. Now a new technology is emerging which promises to be a game-changer in this respect, perhaps as important as the advent of clinical PET itself – Total Body PET . The abiIn the interdisciplinary world of nuclear medicine and molecular imaging, as quickly as radiochemistry solutions are developed to meet new needs in molecular imaging, new challenges emerge, driven both by the advances in therapy they support and by parallel advances in scanning technology, as developments in one contributing technology drive innovations in the others.


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