Adjuvant endocrine therapy (AET) reduces breast cancer recurrence and mortality in women with early-stage breast cancer. Unintentional nonadherence to AET is common . Forming habits surrounding medication taking could reduce reliance on memory and improve AET adherence. SMS text messaging interventions may offer a low-cost approach for promoting medication-taking habits. To optimize the likely effectiveness of such SMS text messages, the content should be developed using a transparent approach to ensure fidelity to relevant psychological theory and with user input to increase acceptability.This study aimed to develop a pool of brief SMS text messages promoting habit formation to support AET adherence, which are acceptable to women with breast cancer and show fidelity to theory-based behavior change techniques (BCTs).According to published literature, we selected 6 BCTs derived from the habit formation model: action planning, habit formation, restructuring the physical environment, adding objects to the environment, prompts/cues, and self-monitoring of behavior. In study 1, behavior change experts (n=10) created messages, each based on 1 of the 6 BCTs, in a web-based workshop and rated the fidelity of the messages to the intended BCT. In study 2, women with experience of taking AET discussed the acceptability of the messages in a focus group (n=5), and the messages were refined following this. In study 3, women with breast cancer rated the acceptability of each message in a web-based survey (n=60). In study 4, additional behavior change experts rated the fidelity of the remaining messages to the intended BCT in a web-based survey (n=12). Finally, a consultant pharmacist reviewed a selection of messages to ensure that they did not contradict general medical advice.In study 1, 189 messages were created targeting the 6 BCTs. In total, 92 messages were removed because they were repetitious, unsuitable, or >160 characters, and 3 were removed because of low fidelity . Following study 2, we removed 13 messages considered unacceptable to our target population. In study 3, all remaining messages scored above the midpoint on an acceptability scale (1-5); therefore, no messages were removed . Following study 4, we removed 13 messages owing to low fidelity . All the remaining messages showed fidelity to the intended BCTs . Following the pharmacist review, 2 messages were removed, and 3 were amended.We developed a pool of 66 brief SMS text messages targeting habit formation BCTs to support AET adherence. These showed acceptability to women with breast cancer and fidelity to the intended BCTs. The delivery of the messages will be further evaluated to assess their effect on medication adherence. Adjuvant endocrine therapy (AET) is routinely prescribed for 5 to 10 years to women with early-stage estrogen receptor–positive (ER+) breast cancer to reduce recurrence and mortality . It is aNonadherence to AET can be intentional or unintentional. Intentional reasons for nonadherence include difficulties with side effects, concerns about AET, and psychological distress -15. UninA Cochrane review of 182 randomized controlled trials on medication adherence interventions across multiple long-term conditions highlighted the need for more effective and novel interventions to support medication adherence . ExistinMaking medication-taking behaviors habitual could address unintentional nonadherence, as habits reduce reliance on memory. A habitual behavior is an action triggered by exposure to a contextual cue . HabituaAccording to habit theory, if habits for medication-taking behaviors are formed, taking medication could become more automatic, thereby reducing reliance on memory or the motivation to take AET . In a meSMS text messaging interventions are a low-cost method for supporting habit formation. They can serve as an initial reminder to take the medication and could lead to sustained behavior change through prompting medication taking in the same context repeatedly to support habit formation . Meta-anOutside of habit-based interventions, SMS text messaging interventions aimed at improving adherence to AET among women with breast cancer have shown mixed results -47. In tThe message development processes for existing SMS text messaging–based interventions targeting AET adherence generally lack detail and transparency, and there is rarely a theoretical justification for the message content. Intervention development guidance highlights the importance of having a theoretical basis for an intervention and advocates for detailed reporting of intervention development to improve transparency -52. TherUsing an established process ,54, we aIn preparing this manuscript, we followed the guidance for reporting intervention development studies in health research .Using the Target, Action, Context, Time framework for behavior specification , the tarhabit formation [also known as context-dependent repetition]), BCTs promoting the use of feasible environmental cues for medication taking  [self-monitoring of behavior [On the basis of evidence suggesting that habit interventions could be effective for improving medication adherence ,39 and tironment ,34,57), lanning) , and a Bbehavior ,57). TheWe followed an established process using a series of studies to develop the pool of SMS text messages ,54. ThisThe aim was to generate 12 to 15 messages for each of the 6 chosen BCTs targeting habit formation and to assess message fidelity to the intended BCTs.We emailed 25 UK experts in behavior change, identified through the research team’s networks and by searching university websites and Twitter profiles. We sent a reminder email to nonrespondents after 1 week. The participants were given a £100 honorarium for their time. A currency exchange rate of GBP £1=US $1.25 is applicable.We sent a web-based questionnaire asking for e-consent, demographic information, and expertise in behavior change. Participants who completed this questionnaire were sent the schedule for the 1-day web-based workshop. The schedule included the aims for the day, target behaviors for the messages, and the names and descriptions of the 6 target BCTs. The workshop was hosted on Zoom (Zoom video communications) and was split into 3 sections. For each section, participants were split into 2 groups (5 participants per group), facilitated by 2 researchers per group . Each group was introduced to a BCT with a short description and an example taken from the BCTTv1 . ParticiFollowing the workshop, 4 research team members  removed messages deemed unsuitable for the intervention . Then, each researcher rated each message based on how well it addressed the target behaviors on a scale of 1 to 10 (coherence to the behavior).Two working days after the workshop, the participants were sent a survey containing the remaining messages. Participants were asked to rate how relevant they felt the BCT was to support medication adherence within this population, how well they thought the aim of generating 12 to 15 good-quality messages had been achieved, and the fidelity of each message to the BCT it was intended to target. All 3 questions were assessed on a scale of 1 (not relevant or not very well) to 10 (very relevant or very well).We removed messages scoring <5.5 on the research team’s coherence rating, as this was the midpoint of the scale. Participant characteristics were summarized. We calculated the means and SDs of each SMS text message on fidelity, and an a priori threshold of <5.5 on the fidelity scale was used to remove messages, as this was the midpoint on the scale and has been used as a cutoff in a previous similar study . We summThe aim was to assess the acceptability of the SMS text messages generated in study 1 to women taking AET.Participants were recruited from a preexisting patient and public involvement group. Patient and public involvement group members were originally recruited via an advertisement circulated in a newsletter for a charity that supports people with cancer. All members had a diagnosis of ER+ breast cancer and were currently taking AET. All members were offered £37.50 compensation for their time, in line with the published guidelines .All participants completed demographic and clinical questions and were sent a copy of the SMS text messages 2 weeks before the meeting. The focus group was hosted on Microsoft Teams, lasted 90 minutes, and was facilitated by SMCG and ER. Each BCT was presented along with a short description taken from the BCTTv1 and the SMS text messages relating to that BCT. The participants were asked to discuss the acceptability of the messages and suggestions for rewording. A topic guide was used to structure the discussion .The focus group was recorded and transcribed verbatim. One author (SMCG) extracted all suggestions from the transcript and made amendments to the messages when there was no disagreement between participants. If there was disagreement among participants, these instances were discussed within the research team . Where the research team could not reach an agreement, the message was retained and included in study 3.The aim was to determine the acceptability of SMS text messages remaining after studies 1 and 2.In total, 60 women diagnosed with ER+ breast cancer were recruited via a recruitment company, Dynata, to participate in a web-based survey. Dynata provided the women with a small monetary incentive on completion of the survey.Dynata sent the web-based survey link to their panel members. The survey contained information on the study, an e-consent form, and demographic questions. Participants who were eligible and provided e-consent were asked to rate the SMS text messages based on their acceptability, with responses on a 5-point Likert scale (1=completely unacceptable and 5=very acceptable). The order in which the messages appeared was randomized.We summarized participant demographics, means, and SDs for the acceptability of each SMS text message. We calculated an acceptability score for each BCT by averaging the acceptability scores of the messages related to that BCT. On the acceptability scale, any messages scoring below an a priori threshold of 3 were removed, as this was the midpoint of the scale and has been used as a cutoff in a previous similar study .The aim was to assess the fidelity of the remaining SMS text messages to the intended BCTs.We emailed 41 UK experts in behavior change. This included 15 experts who had not responded to the study 1 invitation or could not attend the workshop. In total, 26 additional potential participants were identified from the research team’s networks and through further searching of university websites and Twitter profiles. Study 1 participants were ineligible and were not contacted. A £25 Amazon voucher was offered upon completion of the survey.We emailed a link to a web-based survey containing information about the study and an e-consent form. If the participants consented, they were asked to rate each message on fidelity to the intended BCT on a scale of 1 (not very well) to 10 (very well). A description and an example from the BCTTv1 were provided.We summarized participant demographics and the means and SDs for each text message and BCT. We removed messages scoring below an a priori threshold of 5.5 on fidelity, as this was the midpoint of the scale and has been used as a cutoff in a previous similar study .A selection of 20 messages were sent to a consultant clinical pharmacist with experience of AET to ensure advice in the messages was appropriate and not conflicting with general medical advice. The consultant pharmacist was a member of the trial management group for the larger program of research that this study is part of. The 20 messages to be reviewed included all messages in which there could be any risk of conflicting or dangerous advice. The review occurred at this stage to ensure that the final versions of the messages were checked.Ethics approval was granted by the University of Leeds School of Medicine Ethics Committee (MREC 20-038 July 2021). All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional research committee and with the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. Informed consent was obtained from all the participants included in this study. All data from the study were deidentified. Participants were compensated in the following ways: study 1: £100, study 2: £37.50, study 3: small incentive provided directly from the market research company, and study 4: £25 Amazon voucher.The flowchart of the 4 studies is shown in Of the 10 participants, 8 (80%) were research scientists and 2 (20%) were research scientists and health care professionals. Participants had been in paid research-related posts for between 10 and 25  years. Each participant had published between 3 and 71 papers related to behavior change, medication adherence, and breast cancer . Behavior change interventions were the research focus for most participants . Half of the participants  described medication adherence as central or somewhat central to their work, and 1 participant described breast cancer as central to their work.In total, 189 SMS text messages were created for the 6 BCTs during the expert workshop .In total, 92 messages were removed because they were considered unsuitable ; for exaWe removed 3 messages because they scored below the midpoint (5.5) on the 1 to 10 fidelity scale. These are related to “action planning” and “restructuring the physical environment” . MessageIn total, 5 women aged between 41 and 79 years participated in the focus group . All parAll suggestions from the focus group in which there was no disagreement between participants were implemented, resulting in the removal of 13 messages . AmendmeIn total, 60 women with ER+ breast cancer completed the web-based survey . The aveAll individual messages and BCTs scored above the midpoint on the acceptability scale (3); therefore, none were removed . The meaIn total, 12 experts in behavior change participated in the survey: 11 were research scientists and 1 was a research scientist and health care professional. All participants described behavior change interventions as central or somewhat central to their work. A total of 5 participants described medication adherence as central or somewhat central to their work. In addition, 3 participants described breast cancer as central or somewhat central to their work. The participants had been in paid research-related posts for between 5 and 16  years and had published between 5 and 25 papers related to medication adherence, behavior change, and breast cancer (median 10.5).We removed 13 messages because they scored below the midpoint (5.5) of fidelity to the intended BCT , but these effects were not sustained at 12 months (P=.62) [This study builds on previous attempts to use SMS text messaging interventions in women taking AET by developing explicitly theory-based messages. Previous SMS text messaging interventions aimed at improving AET adherence have produced equivocal findings -47. One  (P=.62) . This nu (P=.62) -33. Form (P=.62) -33.Our iterative text message development process using 4 interlinked studies enabled us to continually optimize the pool of messages throughout the development process. We used the key steps recommended by the intervention development guidelines . For exaThe final pool of text messages demonstrated prospective acceptability in women with breast cancer. This was based on a single assessment of acceptability. Our approach was effective in ensuring that no messages considered unacceptable to women with breast cancer were included in the final pool of text messages. Previous studies involving SMS text messages to support adherence to diabetes medications found prospective and experienced acceptability to be correlated . In the Despite our rigorous intervention development process, some key uncertainties remain. First, there are uncertainties surrounding how these text messages should be used in an intervention; which messages to use, the frequency with which messages should be sent, and the duration for which they need to be sent to support habit formation . Such unWe adapted an established approach previously used to develop SMS text messages to support diabetes self-management and demonstrated how the process can be conducted remotely . Our appIn conclusion, we conducted a series of 4 linked studies using mixed methods to develop a pool of 66 brief messages to support medication adherence to AET in women with breast cancer. The messages were based on 6 BCTs theorized to support habit formation. The messages were rated as acceptable to women with breast cancer and showed fidelity to the intended BCTs. Further evaluation of these messages is needed to establish whether they can support medication adherence behaviors.


Following amputation, patients with lower limb amputations (LLA) are classified into different functional mobility levels (K-levels) ranging from K0 (lowest) to K4 (highest). However, K-level classification is often based on subjective criteria. Objective measures that are able to differentiate between K-levels can help to enhance the objectivity of K-level classification.The goal of this preliminary cross-sectional study was to investigate whether differences in hip muscle strength and balance parameters exist among patients with transfemoral amputations (TFA) assigned to different K-levels.Twenty-two participants with unilateral TFA were recruited for this study, with four participants assigned to K1 or K2, six assigned to K3 and twelve assigned to K4. Maximum isometric hip strength of the residual limb was assessed in hip flexion, abduction, extension, and adduction using a custom-made diagnostic device. Static balance was investigated in the bipedal stance on a force plate in eyes open (EO) and eyes closed (EC) conditions. Kruskal-Wallis tests were used to evaluate differences between K-level groups.Statistical analyses revealed no significant differences in the parameters between the three K-level groups (p>0.05). Descriptive analysis showed that all hip strength parameters differed among K-level groups showing an increase in maximum hip torque from K1/2-classified participants to those classified as K4. Group differences were also present in all balance parameters. Increased sway was observed in the K1/2 group compared to the K4 group, especially for the EC condition.Although not statistically significant, the magnitude of the differences indicates a distinction between K-level groups. These results suggest that residual limb strength and balance parameters may have the potential to be used as objective measures to assist K-level assignment for patients with TFA. This potential needs to be confirmed in future studies with a larger number of participants. The irreversible loss of an extremity affects physical integrity and leads to social and psychological burdens.4 To identify functional mobility, several classification systems exist to assign patients with lower limb amputations (LLA) to different mobility levels.5 In the United States, the Medicare's Functional Classification Level (MFCL) distinguishes patients with LLA into five functional levels ranging from K0 (lowest) to K4 (highest). This classification, which is intended to reflect the individual's abilities to ambulate with the prosthesis, strongly influences the selection and assignment of the different prosthetic components. Patients with LLA classified as K2 will not have the possibility to receive high functioning prosthesis components as patients classified at K4. In Germany, a similar classification system is used with the same categories as the MFCL system. The assignment into the different levels is based on the so-called profile survey sheet, in which doctors or orthopedic technicians subjectively evaluate abilities concerning functional mobility.6 As objective parameters, only the range of motion (ROM) of joints of the lower extremities are documented. Further objective evaluation criteria are missing. Due to the relevance of K-level assignment for the patients with LLA, the lack of objectivity has been recognized and 75% of orthopedic technicians would support additional objective measures to improve the subjectivity of K-level classification.7 In a recent study, Sions et al. emphasized the necessity of reliable and valid objective measures to differentiate between K-level classifications.8The prescription of the type of prosthesis and its specified components as well as the financial coverage by health insurance are based on the expected functional mobility of the patient.4 to objectively assess the patient's functional abilities. The AMP consists of 21 ambulation and balance tasks with and without prostheses, which are individually rated by an examiner using a point system. The AMP was shown to have the potential to distinguish between K-levels.9 In two recent papers, physical performance tests were performed and tested if they are suitable measures to improve the objectivity of K-level assignment.10 Differences between patients with LLA classified as K3 and K4 were seen in the Timed Up and Go test and the 6-minute walk test.8 In the study of Beisheim et al., functional strength and dynamic balance tests were performed with patients with LLA. K4-classified patients showed higher functional strength and better dynamic balance when compared to participants classified as K3.10 These studies show that walking tests, as well as functional tests, may help to assign patients with LLA to the different K-levels.Addressing the subjectivity of the existing K-level classification, Gailey et al. were the first to develop a clinical tool, the Amputee Mobility Predictor (AMP),12 The muscles surrounding the hip are important to stabilize the pelvis during standing and locomotion. Weak hip abductors are one cause of the compensatory trunk shifting over the prosthetic side13 and poor balance performance.14 In a recent review, Hewson et al. concluded that muscle strength deficits exist in lower limb prosthesis users and contribute to balance and mobility impairments.12 In patients with TFA, these strength deficits are particularly pronounced in the hip of the residual limb.12Performance in functional tests and walking tests are often associated with lower limb strength. Several studies have demonstrated that patients with transfemoral amputation (TFA) have significantly reduced strength in the residual limb compared to the sound leg as well as to controls.However, no study has included standardized hip strength tests of the residual limb as possible measures for assisting to objectify K-level classification. Objective evaluation methods must be tested for their suitability in assisting in K-level assignment, particularly methods that evaluate lower limb strength and balance. Therefore, the goal of this preliminary study was to investigate whether there are differences in hip muscle strength of the residual limb as well as differences in static balance parameters among patients with TFA assigned to different K-levels. The authors hypothesized that participants classified at higher K-levels would demonstrate higher performance on the strength and balance tests than participants that were assigned to lower K-levels.Participants were recruited from January 2018 to September 2019 through calls and articles in official journals of amputee organizations as well as in a local newspaper. Inclusion criteria were a unilateral transfemoral amputation with a post-amputation time of at least one year, an age ≥18 years and the current use of the prosthesis. Due to the measurement setup, one further inclusion criterion was a minimum residual limb length of 15 cm. Participants were excluded if the amputation was caused by diabetes mellitus, or if they had open wounds, edema, or acute pain in the residual limb. All participants gave written consent to participate in this study after being informed about the procedure and its purpose. The study was approved by the local ethics committee of the Otto von Guericke University Magdeburg and carried out in line with the Declaration of Helsinki .For this cross-sectional study, the participants attended a single testing session, in which all measurements were conducted. Measurements were performed in two institutions, University of Magdeburg and University of Applied Sciences Brandenburg, which were equipped with the same measurement systems. Prior to physical performance tests, demographic and anthropometric data were collected and participants were asked to answer amputation related questions . The K-level assignment was obtained from medical records in collaboration with the respective orthopedic technician. The physical performance tests included isometric strength tests of the hip muscles of the residual limb as well as examinations of static balance. The strength assessment of the hip muscles was performed without the prosthesis whereas the static balance tests were performed with the prosthesis.Figure 1). This diagnostic device was built specifically for patients with LLA. An individually adjustable pelvic support provides stability and safety during the measurements. The 270° rotatable base plate enables hip muscle strength diagnostics in different directions  while participants do not need to change position within the device. An additional resting chair, which can be slid under the participants, is integrated into the device to provide relief of the standing leg between examinations.The measurement of the maximum isometric strength of the hip muscles of the residual limb was performed in a custom-made diagnostic device . This brace served as an attachment possibility for the cuff of the hauling rope. A force transducer  integrated into the hauling rope was used to measure the isometric strength at a sampling rate of 1000 Hz for hip flexion, extension, abduction, and adduction in the neutral hip position . Before the measurements, participants were asked to familiarize themselves with the setup. For each motion direction, one submaximal test (pretest) and three maximum tests (main tests) were performed with one minute of rest between trials. Participants were instructed to successively build up strength and pull maximally without an abrupt push. They could follow their current measured strength values live on the screen during the measurement. The maximum achieved strength value from the pretest was visualized on the screen as a threshold value and participants were verbally encouraged to exceed this in the main tests. The threshold value was readjusted after exceeding the previous threshold value to ensure that the maximum possible force value was reached within the three main tests. The distance between the greater trochanter and the point of applied force (center of the cuff) served approximately as the lever arm .For the strength measurement, participants were standing in an upright position supported by the pelvis support without the prosthesis. A neoprene brace was placed around the residual limb  and filtered with a 4th order Butterworth low-pass filter (5 Hz). Torques for each motion direction were calculated from the force and the lever arm and normalized to the body mass of the participants. Out of the three main trials for each motion direction, the trial with the highest torque was used for further analyses.15 These results suggest that the custom-made diagnostic device provides an environment to reliably quantify maximum isometric hip strength.The reliability of the isometric hip strength measurement of the diagnostic device had been investigated in a test-retest design before the examinations. The calculated intraclass correlation coefficients (ICCs) showed values ranging from 0.85 to 0.95 for the isometric hip strength measurement . According to Koo and Li, these ICCs indicate good to excellent reliability.16 as poor socket fit might influence static balance parameters. Socket score has been deemed a valid and reliable outcome measurement.16 The participants were asked to rate the comfort of their socket on a scale from 0 to 10 with 0 being the most uncomfortable and 10 being the most comfortable socket imaginable. Mean comfort scores were between 7 and 8 points for all K-level groups.Static balance was assessed in two different conditions: bipedal stance with eyes open (EO) and bipedal stance with eyes closed (EC). Before generating balance data, the prosthetic socket comfort was determined with the Prosthetic Socket Fit Comfort Score17For the bipedal stance, subjects were instructed to stand hip-width apart on a 45x45 cm force plate  with the arms hanging down at the sides and to remain as still as possible. For the EO conditions, participants were asked to focus on a fixed point at eye-level on the wall in front of them whereas for the EC condition the participants closed their eyes. Prior to collecting data, participants practiced both poses for a few seconds. For safety reasons, an examiner stood near the participants during the entire familiarization and measurement period. Once the familiarization period was over, two trials with a duration of twenty seconds were recorded for both conditions with a sampling frequency of 250 Hz. Balance data were further processed using Matlab and filtered applying a 4th order Butterworth low-pass filter with a 10 Hz cut-off frequency. The total length of the center of pressure (COP) during the two standing conditions was computed as well as the maximum and mean deviations in mediolateral (ML) and anteroposterior (AP) directions. These COP-based measures have been used in most studies examining static balance in participants with LLA.Statistical analyses were performed using IBM SPSS Statistics 26 . Based on the K-level assignment, participants were divided into groups. For each K-level group, descriptive statistics were determined for all anthropometric, demographic and measurement variables. Due to the small sample size of participants and the unequal distribution of participants across K-levels, variables were described using the median and interquartile range . To detect differences between K-level groups, Kruskal-Wallis tests were performed for each variable. Pairwise posthoc comparisons with Bonferroni correction for multiple testing followed where appropriate. The significance level was set at p< 0.05.Table 1, participants' anthropometric and demographic data are represented according to the K-level assignment. Due to the small numbers of patients classified as K1 or K2, they were combined as one group. The Kruskal-Wallis test showed that age differed significantly between K-level groups. Posthoc tests revealed that participants of the K4 group were significantly younger than the ones of the K1/2 group (p=0.03). The K4 group was not only younger on average, but also had a longer residual limb length and the amputation had not occurred as long ago as for K1/2 and K3-classified participants.Twenty-two participants fulfilled the inclusion criteria and were considered for the study. All participants were able to complete all tests and were included in the data analysis. In Table 2. A significant difference across K-level groups was only detected for hip flexion (p=0.04). The posthoc tests did not reveal significant differences between the individual groups (p>0.05). If the medians of the parameters are considered, an increase in maximum hip torque can be observed from participants classified as K1/2 to those classified as K4. Differences were especially visible between the K3 and K4 groups with a mean difference of 0.97 Nm/kg for hip flexion, 0.51 Nm/kg for hip abduction, 0.45 Nm/kg for hip extension and 0.44 Nm/kg for hip adduction. A graphical representation of the data in form of boxplots is shown in Figure 3.The results of the hip muscle strength test of the residual limb are shown in Table 3. For neither the EO nor the EC condition, the Kruskal-Wallis tests revealed significant differences in the parameters across the three K-level groups (p>0.05). The descriptive analysis showed that differences in the medians were especially seen between K1/2 classified participants and the ones assigned to K4. For the EO condition, the mean and maximum deviation in ML and AP directions decreased from K1/2 to the K4 group. For the maximum deviation in ML and AP directions, a mean difference of 8 mm and 6 mm was determined between K1/2 and K4. For the EC condition, differences between groups became more evident. For the COP length, the mean difference between the K1/2 group and K4 group was 340 mm, and the mean differences of the maximum excursions in ML and AP directions were 23 mm and 20 mm, respectively. Figure 4 shows an example of sway paths from one participant of the K1/2 group and one K4-classified participant in both EO and EC conditions. An increase in sway from the EO condition to the EC condition is visible for both K-groups, although the increase is considerably more pronounced for the K1/2-participant than for that of the participant of the K4 group.The parameters from the examination of the static balance are presented in In this study, examinations of hip muscle strength of the residual limb and examinations of static balance were performed in patients with TFA. The goal was to investigate whether these objective measures could differentiate between patients who were classified at different K-levels. As hypothesized, participants classified at higher K-levels performed better on the strength and balance tests than participants assigned to lower K-levels. However, statistical analyses revealed no significant differences in the parameters between the three K-level groups.While previous studies showed that participants with TFA suffer from a strength deficit of the residual limb, this is the first study that included an isometric muscle strength assessment of the hip muscles on the affected side for potential K-level distinction. Strength differences were particularly visible between the K3 and the K4 group as well as between the K1/2 and the K4 group. For maximum torque, mean group differences ranged from 0.4 Nm/kg to 0.9 Nm/kg for hip flexion, hip abduction, hip extension and hip adduction. Although not statistically significant, the magnitude of these values implies a distinction between K-level groups.11 Reasons for the lack of statistical significance in our study may include the small number of participants in the K1/2 and K3 groups, the uneven distribution of participants among K-level groups and the individuality of each participant. Beisheim et al. examined lower extremity strength differences between K3 and K4 classified participants with TFA applying the functional 5-Times Sit-to-Stand Test and found a significant difference.10 Functional strength tests were shown to have the potential to differentiate between K-levels. However, they could not explain the reasons why participants of the K3 group performed worse than those assigned to K4. Muscle strength tests of isolated muscle groups of the residual limb, as performed in this study, have the advantage to identify specific muscle weaknesses. Knowing the individual strength deficits are especially important for patients with LLA as hip abductor strength is associated with gait deviations18 and hip extension strength has been reported as the greatest predictor of performance on the 6-minute walk test.19Heitzmann et al. investigated maximum hip torque in a similar measurement setup and the hip torque differences between participants with TFA  and healthy participants were in the same numerical range (0.5-0.7 Nm/kg) and statistically significant.17 This may be explained by the missing ankle plantar and dorsiflexor muscles on the amputated leg which are relevant for stability control in the AP direction.20As lower extremity strength of lower limb prosthesis users is linked to postural control, parameters of static balance were also investigated to find potential outcome measures that may be able to distinguish between K-levels. The statistical analyses, however, did not reveal significant differences in the parameters across the three K-level groups. Concerning the medians of the parameters, participants classified as K1/2 showed greater COP length as well as greater mean and maximum sway deviations in ML and AP directions in the bipedal stance than the participants of the K3 and K4 groups. For all groups greater sway was observed in the AP than in the ML direction, which has been observed in previous studies.17 The eyes-closed condition has a great effect on patients with LLA as vision is especially relevant to compensate for balance impairments due to missing somatosensory feedback from the prosthetic leg.21 Although not proven in this study, using a closed-eye condition in quiet standing might be a sensitive method for distinguishing between different K-levels as balance control mechanisms differ in relation to functional abilities. Measures of static balance have been criticized that they cannot reflect postural demands in daily life.22 However, they may be helpful to identify weaknesses in postural control differentiated in AP or ML directions, which can be relevant for patients with LLA. Static balance tests could be performed in addition to dynamic balance tests, which have been shown to be suitable for K-level distinction among participants with TFA.10In the eyes-closed condition, COP parameters increased and the differences in COP between K-level groups became larger. Group differences of maximum excursions in AP and ML directions were observed up to 14 mm as well as a mean difference in COP length of 240 mm. Increased COP sway due to the absence of visual input is in line with previous studies investigating patients with LLA during quiet standing.Several limitations have to be addressed. The major limitation is the small number of participants in each K-level group as well as the heterogeneity between groups, which may be the reason that no significant differences were detected. The groups differed in age, residual limb length and in post-amputation time, which may have affected the results of physical performance tests. Therefore, generalization of the data is not possible and studies with larger and more homogeneous samples need to confirm the presented results. Further, the cause of amputation was not recorded in this study except that patients who experienced LLA due to diabetes mellitus were excluded. The amputation etiology may impact physical performance and should be recorded in future studies. In balance examinations, prosthetic alignment and different types of prosthetic components  may also affect performance and should be controlled in future studies. However, this is the first study that included participants with TFA classified as K1/K2 to find objective measures for supporting K-level classification. Future studies should not only focus on differentiating between participants with LLA classified as K3 and K4 but should also include participants classified as K2.This study was the first one to perform hip strength tests of the residual limb and static balance tests with participants with TFA classified at different K-levels to find parameters that may be suitable to enhance objective K-level classification. Statistical analyses could not reveal any significant group differences but the value of the magnitude of the group differences detected may be relevant to differentiate between K-level groups. The results of the study suggest that residual limb strength and balance parameters may have the potential to serve as objective measures to support K-level classification but this potential needs to be confirmed by future studies with a larger number of participants.The authors declare that they have no competing interests.Stefanie John: Contributed to the study concept and design, participated in data gathering, analyzed, and interpreted data, contributed to the drafting of the manuscript, read, and approved the final manuscript.Katja Orlowski: Contributed to the study concept and design, participated in data gathering, contributed to the drafting of the manuscript, read, and approved the final manuscript.Kai-Uwe Mrkor: Participated in data gathering, contributed to the drafting of the manuscript, read, and approved the final manuscript.Jürgen Edelmann-Nusser: Contributed to the study concept and design, contributed to the drafting of the manuscript, read, and approved the final manuscript.Kerstin Witte: Contributed to the study concept and design, analyzed, and interpreted data, contributed to the drafting of the manuscript, read and approved the final manuscript.German Central Innovation Program for small and medium-sized enterprises for the project ‘Multifunctional diagnostic device for patients of lower limb amputations’ (ZF4096303TS6).The study was approved by the local ethics committee of the Otto von Guericke University Magdeburg and carried out in line with the Declaration of Helsinki . Signed informed consent was obtained from all participants.


The current subjective histopathological assessment of cutaneous melanoma is challenging. The application of image analysis algorithms to histological images may facilitate improvements in workflow and prognostication. To date, several individual algorithms applied to melanoma histological images have been reported with variations in approach and reported accuracies. Histological digital images can be created using a camera mounted on a light microscope, or through whole slide image (WSI) generation using a whole slide scanner. Before any such tool could be integrated into clinical workflow, the accuracy of the technology should be carefully evaluated and summarised. Therefore, the objective of this review was to evaluate the accuracy of existing image analysis algorithms applied to digital histological images of cutaneous melanoma.Database searching of PubMed and Embase from inception to 11th March 2022 was conducted alongside citation checking and examining reports from organisations. All studies reporting accuracy of any image analysis applied to histological images of cutaneous melanoma, were included. The reference standard was any histological assessment of haematoxylin and eosin-stained slides and/or immunohistochemical staining. Citations were independently deduplicated and screened by two review authors and disagreements were resolved through discussion. The data was extracted concerning study demographics; type of image analysis; type of reference standard; conditions included and test statistics to construct 2 × 2 tables. Data was extracted in accordance with our protocol and the Preferred Reporting Items for Systematic Reviews and Meta-Analyses-Diagnostic Test Accuracy (PRISMA-DTA) Statement. A bivariate random-effects meta-analysis was used to estimate summary sensitivities and specificities with 95% confidence intervals (CI). Assessment of methodological quality was conducted using a tailored version of the Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2) tool. The primary outcome was the pooled sensitivity and specificity of image analysis applied to cutaneous melanoma histological images. Sixteen studies were included in the systematic review, representing 4,888 specimens. Six studies were included in the meta-analysis. The mean sensitivity and specificity of automated image analysis algorithms applied to melanoma histological images was 90%  and 92% , respectively. Based on limited and heterogeneous data, image analysis appears to offer high accuracy when applied to histological images of cutaneous melanoma. However, given the early exploratory nature of these studies, further development work is necessary to improve their performance. Moreover, the incidence of melanoma is predicted to rise by 7% from 2014 to 20352. Diagnosis of melanoma depends upon a histopathologist’s interpretation of the tissue at a cellular level, with subjective thresholds for morphological features. Histopathological interpretation can be challenging, resulting in high levels of interobserver variation3, which may in part be due to the wide range of histological appearances  remaining the most important predictor of survival for over 50 years5. Other prognostic biomarkers contained within the AJCC staging include ulceration, mitoses, lymph node involvement and metastatic disease detected in viscera. However, the staging system explains an insufficient proportion of the variance in survival6 with some thin tumours unexpectedly causing metastatic disease.The current prognostic biomarkers based on histological features are contained within the current staging system 7, there is a clear need for tools that aid pathologists and improve workflow. It is important that any new prognostic biomarkers not demand additional work of histopathologists.Given that there is currently a staffing shortage in pathology services globally, with only 3% of pathology departments reporting being fully staffed in the United Kingdom (UK)12.Digital microscopy has become an essential tool in pathological research over the past few decades. Initially, cameras mounted on microscopes enabled the generation of standard digital images, but the invention of the whole slide scanner over 20 years ago, glass slides can now be scanned to create a whole slide image (WSI) enabling the tissue to be viewed at multiple magnifications  and the Preferred Reporting Items for Systematic Reviews and Meta-Analyses-Diagnostic Test Accuracy (PRISMA-DTA) StatementWe included studies of any design that reported accuracy outcomes of IA applied to histological images of cutaneous melanoma.The target condition was cutaneous melanoma.The index test was any form of automated IA. This includes more conventional IA techniques as well as neural networks. Manual annotation of histological images was not included.The reference standard was any form of histopathological assessment of the haematoxylin and eosin (H&E) histological image and/or immunohistochemical staining.PubMed and Embase were searched from inception to 11th March 2022, restricted to English language (for full search strategy see Appendix 1). Citation checking was also conducted.All citations were independently deduplicated and screened by ELC and RGW. Where possible, the full texts of potentially eligible articles were obtained and independently assessed by the same two individuals with disagreements resolved by discussion. We included abstracts as well as full texts.Data were extracted concerning study demographics; type of IA; type of reference standard; conditions included and test statistics to construct 2 × 2 tables of the number of true-positives (TP), false-positives (FP), false-negatives (FN) and true-negatives (TN).14) and used to appraise the risk of bias and applicability of the included studies (Appendix 2).The QUADAS-AI tool was in development at the time of carrying out this work and therefore a tailored version of the Quality Assessment of Diagnostic Accuracy Studies QUADAS-2 was created  test set and whether all the cases were included in the analysis. Studies that do not use a separate test set are also at risk of overestimating a model’s performance. Bias of the reference standard included whether the reference standard results were interpreted without knowledge of the index test, alongside the ability of the reference standard to correctly identify melanoma. If the reference standard is interpreted with knowledge of the index test, then this may bias the reference standard to mirror the index test results, again overestimating performance. Finally, studies including cases with a time interval of more than 10 years between the diagnosis of the reference standard and the digital image creation indicated a high risk of bias for flow and timing, since diagnostic criteria and terminology changes with time and glass slides fade introducing risk that they may not clearly depict the pathology and underestimate the model’s performance.Applicability assessment involved whether the case selection, index test or reference standard matched the review question.15.This data was summarised using the Risk-of-bias VISualisation (robvis) tool17 was used to generate summary sensitivities, specificities, forest plots and summary receiver operating characteristic (SROC) plots using a bivariate random-effects model. A sensitivity analysis was performed including only those studies generating IA models concerned with melanoma tumour detection. A flow-diagram was generated using the PRISMA2020 tool18. Publication bias was not assessed because the determinants are not well understood for diagnostic accuracy reviews19 and the Deeks test has low power in the presence of substantial heterogeneity20. The significance level was set at 5%. Confidence intervals were generated to the 95% level.The MetaDTA: Diagnostic Test Accuracy Meta-Analysis v2.01 shinnyappUltimately, sixteen studies were included Fig. .Figure 321, Germany22, France23, Italy12, Sweden24, USA10, Canada28, Japan29 and China33(p202) and were performed between 2012 and 2021. Studies varied in size with a median sample size of 100 specimens or slides . Overall, 4,888 specimens were included, of which at least 2,715 were melanoma specimens. The diagnostic entities within the datasets varied between studies, with some only containing melanoma deposits33 and others containing more than one pathology32.Study characteristics are presented in Supplementary Table 32 and others performing diagnostic classifications including melanomas versus naevi31 and primary melanoma versus metastatic melanoma33. Five studies addressed more complex classifications into three or more diagnostics entities30. One study24 did not focus on a classification task, but instead studied automation of the proliferation index in melanoma.There was between-study variation in terms of intended use of the IA. Most studies focused on a binary classification task, with some focussing on detection and localisation of melanoma deposits in WSIs containing melanoma (melanoma versus not melanoma)33. Studies using CNNs were more recently conducted. Two of the earliest studies employed the use of a support vector machine (SVM)27. Two studies used a combination of a CNN and SVM as their index test32. A further study used more basic image processing and adaptive thresholding method29.There was some degree of variation in the IT used. Most employed the use of a convolutional neural networks (CNN), with the architecture differing considerably31, categorised specimens by histological features23, carried out manual annotation30 or interpreted immunohistochemical staining26. Some studies used a combination of these approaches32. Two studies did not detail the reference standard used33.There was heterogeneity in the reference standard. In most studies pathologists provided diagnostic labels26 or cell-level outcomes28, whereas others focussed on patch-level32 or slide-level classifications32. Three studies did not report on their unit for analysis33, whilst one appeared to be at the WSI-level23.There was variation in the reported units for performance analysis. Some studies reported pixel-based outcomes33, of which, four studies were at high risk since more than one histological image was included per patient and were spread across the training/ test sets33. The remaining studies were at unclear risk of patient selection bias33. Thirteen studies were at risk of bias from the index test33; seven studies were at high risk either due to the index test not being tested on an external test set 30, or not reporting results from a separate test set28, or the test set being derived from the same histological slide as the training and validation sets26. Six studies were at unclear risk of bias from the index test33. Eleven studies were at unclear risk of bias from the reference standard33 because it was not clear if the reference standard results were interpreted without knowledge of the IA or if the reference standard was likely to correctly classify the target condition. No studies were at high risk. Fifteen studies were at risk of bias due to the flow and timing33; one study was at high risk of bias since the reference standard was determined over 10 years prior to the index test being conducted24. The remaining fourteen studies were at unclear risk of bias since the timings for the determination of the reference standard and index test were not reported33.The risk of bias and applicability assessment are summarised in Fig. 33, due to it not being stated if the cases were purposively selected. There were applicability concerns for seven studies regarding the index test; three studies were of high concern28 and four studies33 were of unclear concern that the index test33 or its conduct or interpretation differed from the review question. Eight studies33 had unclear concerns for applicability of the reference standard because it wasn’t clear if the individual determining the reference standard was suitably qualified or there were unclear criteria for diagnosis.Twelve studies were of unclear concern regarding the applicability of patient selection33, six studies had data that could be meta-analysed33. The extracted data from five of these studies were from published work33 and additional data from one study was provided by the authors11. Over these 5 studies, 1,935 specimens were included, of which at least 1,088 were melanoma specimens. The true-positive, false-positive, false-negative and true-negative rates can be seen in Supplementary Table Of the sixteen studies included in this systematic reviewFigure 33. In total there were 1,853 specimens, of which at least 1,088 were melanoma specimens. The mean sensitivity of IA for cutaneous melanoma tumour detection was 88%  and a mean specificity of 90% .A sensitivity analysis was performed using the 5 studies concerned with tumour detectionFor all tasks, IA applied to cutaneous melanoma histological images has a high sensitivity and specificity Fig. . When in33 of the six meta-analysed studies reported very high sensitivities and specificities, whereas the other three32 were more modest. These three studies32 applied the IA to a reasonably sized separate test dataset containing more than one diagnostic entity. By contrast, the other three studies33 contained less varied datasets containing only melanoma specimens, which may explain the more accurate results.As shown in Fig. 33, there was no clear association between the type of index test or reference standard and the reported performance. Surprisingly, an increase in the number of data sources did not appear to temper performance; two studies12 contained data from three sources but reported highly accurate results.Across the 16 studies included in this reviewGiven the exploratory nature of this work, there is currently no consensus regarding whether a more sensitive or specific test is preferable. A more sensitive test would result in fewer false negatives, which in the context of melanoma detection is likely to be of greater utility as missing a melanoma would not be acceptable, particularly as these tests are likely to be used as a screening or triage tool prior to pathologist assessment.31, dice co-efficient26, area under the curve (AUC)30, F-score31, precision24, recall24, percentage correctly classified23, positive prediction rate28, under-segmentation rate28. The unit of analysis was also variable for the same reasons and included classifications at a pixel-level26, cell-level28, patch-level32 and slide-level32. This variety in reported unit of analysis and performance metrics presents challenges for interpretation and data amalgamation, but it is expected given the wide range of model tasks included in this review. It is essential that the unit of analysis is appropriate for the task to prevent inaccurate performance estimation, as detailed in a seminal paper on the subject34. However, regardless of the unit of analysis or performance metrics presented, we urge authors to report their raw data in a confusion matrix  for classification-based tasks as per existing guidelines35.The studies that could not be included in the meta-analysis due to the lack of raw data or data appropriate for back calculation, reported alternative performance metrics including accuracy33. However, many studies which detected melanoma at a cell, pixel or patch-level did not address the clinical utility of their models29, when these models are suited to prognostic biomarker generation. This may be due to difficulties acquiring the necessary data, but we would recommend that future studies detecting melanoma at a cell, pixel or patch-level, focus on how these models could be applied to predict patient outcome.The clinical utility of the studies presenting results at the slide-level was clear; to assist with specimen triage33. There was concern for risk of bias and applicability in all included studies, although reporting standards and methodological rigor did appear to improve with time. This variation in methodological rigor and reporting standards is likely due to a lack of reporting guidelines, although these are currently under development14. Additionally, our risk of bias and applicability assessments may be suboptimal since the QUADAS-AI tool was still in development at the time of completion of this work. Future reviewers should deploy this AI-specific tool.Our review had limitations. While 16 studies were included in the review, data extraction was only possible for six of the studies owing to deficient reporting14 once they are published.Based on limited and heterogenous data, IA offers high accuracy when applied to melanoma histological images. The focus of work to date has been on developing the technology in this field, which has accelerated over the past decade. Going forwards, future work should address the clinical application of such models and evaluate their use as a screening/ triage tool or for prognostic/ predictive biomarker generation. The quality of existing studies is variable but is improving with time—it is important that authors report their data according to AI-specific guidelinesSupplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.Supplementary Information 6.


This article presents a 3D-printed cylindrical dielectric resonator antenna operating at 5.8 GHz that achieves circular polarization by integrating a fully dielectric parasitic helix with a higher permittivity than the cylindrical resonator. The antenna polarization can be right-handed or left-handed depending on the turning sense of the helix. An extensive parametric study was done for the helix design to evaluate the effects of the dimensions and dielectric constant of the helix over the matching and axial ratio of the antenna. The manufacturing is made using low-loss dielectric filaments and a low-cost 3D printer. Simulation and measurement results show that both antennas are well-matched and operate with the corresponding circular polarization, with an axial ratio bandwidth compatible with UAV applications. For example, when using CP the delay spread can be reduced, ensuring higher levels of received power2, and this polarization has a higher resistance to multipath interference4. Different methods can be found in the literature to obtain circular polarized antennas, which are mainly based on modifying the radiating structure or the antenna feed network6.Circular polarization (CP) has been used in a wide variety of wireless communication applications, such as satellite communications and unmanned vehicles7. One technology that can overcome this issue is their implementation using additive manufacturing.In the perspective of implementing CP antennas, circular polarized dielectric resonator antennas (DRA) can also be found, which can be interesting candidates for the mentioned applications, due to their versatility in terms of shapes, radiation patterns, and the possibilities of implementation. Nevertheless, DRAs can be limited when designing complex shapes if traditional dielectric manufacturing techniques are used, resulting in higher costs8, including high-frequency topologies9 due to the availability of low-cost and low-loss dielectric filaments and high-precision 3D-printers. This made it possible to implement topologies with shapes that were either too expensive or impossible to implement without this technology10. Some examples of implementing DRAs using 3D printing can be found in the literature, such as high-gain structures11, multi-ring structures12, and the conference paper containing the preliminary work on the design presented here13.Additive manufacturing or 3D-printing is suitable for many applications in engineeringThe antenna presented in this article consists of the design, parametric study, modal analysis, implementation, and measurement of a cylindrical dielectric resonator antenna (DRA) operating at 5.8 GHz, which uses a parasitic dielectric helix with high permittivity to achieve circular polarization. The turning sense of the helix determines the sense of the polarization to be right-handed (RHCP) or left-handed circular polarization (LHCP).15, using a slot coupling feed7 defined by Eq. 16 and presented for each axis in Eqs.  and andx, y, in Eqs. :2\documeg is done by fixing the helix width at 3 mm. From the results shown in Fig. In Fig. The proposed antenna following the model presented Fig. mentclass2pt{minim18, resonating at 5.7 GHz and 6.1 GHz. For the axial ratios, as expected, both antennas have the same axial ratio, independently of the polarization, reaching values below 3 dB at the operational frequency.In Fig. In Fig. 19. Based on this analysis, we expect to obtain a low axial ratio at the design frequency of It is important to identify the modes generated on the CDRA with the dielectric helix and check their orthogonality. The E-field vector distribution obtained with full-wave simulation is shown in Fig. 20. For the helix, we use the ABS1500 filament, which has a 21 whose characteristics are summarized in Table Once the design is set, we proceed to manufacture the antenna using fused filament fabrication (FFF) 3D printing. The materials used for printing are the low-loss premix dielectric filaments from AVIENT23 in the band of interest. It is known that there can be differences between the nominal value of the relative permittivity of the filament and the actual printed value depending on the printing parameters24, and therefore the characterization of samples is relevant for these implementations. The samples were characterized in the 4.9 GHz to 7.0 GHz band, using a WR159 standard waveguide. The resulting measured permittivities were The 3D-printing parameters used for the deposition of the filaments were set using a nozzle temperature of 260The reflection coefficient of the antenna was measured using a Vector Network Analyzer Anritsu MS46122B, while the gain radiation pattern and axial ratio were measured in an anechoic chamber. It should be noted that as the RHCP and LHCP implementations are identical in structure, we have only implemented the RHCP version for validation. Fig. The simulated and measured gain radiation pattern in two cut planes of the DRA with the new dimensions are shown in Fig. The article presents a 3D-printed circular polarized cylindrical dielectric resonator antenna obtained by adding a parasitic dielectric helix to the structure. The design of this antenna is based on a simple CDRA, with a helix that gives the desired circular polarization depending on its turning sense, while the main characteristics of the antenna, such as maximum gain at the original resonance frequency and the matching at this same frequency remain close to the ones of the CDRA without the helix. In addition, the obtained axial ratio bandwidth is compatible with UAV applications. On the other hand, the possibility of fully implementing the structure using low-cost 3D printing makes it interesting for applications where cost, volume, and weight can be an issue, such as for unmanned vehicles. In addition, it is demonstrated how essential it is to characterize the printed filaments, as their final dielectric properties may vary depending on the printing parameters and printer characteristics. Finally, the versatility of this design makes it a good candidate topology to be tested using other types of DRA.


Klebsiella pneumoniae strain (ST14) under different phage pressures. The population yield remained stable after 30 days. Surprisingly, the initially sensitive strain remained in all populations and its frequency was highest when phage pressure was strongest. Resistance to phages in these populations emerged initially through mutations preventing capsule biosynthesis. Protection through lysogeny was rarely observed because the lysogens have increased death rates due to prophage induction. Unexpectedly, the adaptation process changed at longer time scales: the frequency of capsulated cells in BJ1 populations increased again because the production of the capsule was fine-tuned, reducing the ability of phage to absorb. Contrary to the lysogens, these capsulated-resistant clones are pan-resistant to a large panel of phages. Intriguingly, some clones exhibited transient non-genetic resistance to phages, suggesting an important role of phenotypic resistance in coevolving populations. Our results show that interactions between lysogens and sensitive strains are shaped by antagonistic co-evolution between phages and bacteria. These processes may involve key physiological traits, such as the capsule, and depend on the time frame of the evolutionary process. At short time scales, simple and costly inactivating mutations are adaptive, but in the long term, changes drawing more favorable trade-offs between resistance to phages and cell fitness become prevalent.Many bacterial genomes carry prophages whose induction can eliminate competitors. In response, bacteria may become resistant by modifying surface receptors, by lysogenization, or by other poorly known processes. All these mechanisms affect bacterial fitness and population dynamics. To understand the evolution of phage resistance, we co-cultivated a phage-sensitive strain (BJ1) and a polylysogenic  Parasites shape the life history and fitness of their hosts. They also impact community structure via predation and competition, and thereby affect numerous ecological and evolutionary processes . BacteriK. pneumoniae, an ubiquitous species, of which at least 75% of the species’ genomes are polylysogenic (K. pneumoniae strain (ST14) isolated from a urinary tract nosocomial infection. ST14 produces multiple infectious virions for which the BJ1 is known to be sensitive  theensitive . Based oensitive . We hypoensitive . To testKlebsiella, after 24 hr there is still a large amount of citrate remaining, that is sufficient to inhibit infection  LB, (ii) LB supplemented with 0.2% citrate to inhibit phage infection due to calcium chelation , and ii, and desnfection . We alsonfection .i(j), which measures the effect of mixing two strains i and j on the viable population size of strain i, relative to pure culture controls. This measure accounts for the absolute performance of each competitor in mixed groups (see Methods). Negative Ci(j) values indicate that strain i have a lower population yield during growth in the presence of j than in pure culture, and positive values indicate the opposite. For the phage producer, strain ST14, the competition had no positive or negative effect on total population yield, most likely because the increased release of viruses resulting in ST14 death is outweighed by an exacerbated death of phage-sensitive BJ1 (i(j) was significantly lower than zero, indicating a negative effect on the growth of strain BJ1. This effect was dependent on the amount of phage released into the environment, as an increased production of phages by ST14 due to MMC leads to an even lower Ci(j) for BJ1  at an initial ratio of 1:1 for 24 hr. First, we tested if mixing the two strains affected the total growth or the population yield. We observed no increased cell death due to the competition in the different growth conditions . Then, wtive BJ1 . In the We also tested whether ST14 could sense the presence of competition, for instance by quorum sensing mechanisms, and induce prophages and the production of viral particles . Our resca 103 CFU/mL. Taken together, in 30 days of coevolution, ST14 did not completely displace BJ1 from the populations, even in conditions where the production of the phage by strain ST14 is exacerbated. Indeed, across all conditions, and throughout all the experimental evolution, infectious virions were actively produced and released . To follow the evolution of each strain, we plated the populations every day on selective media and counted CFU. As expected, no significant changes in the group yield were observed . This isreleased , represeKlebsiella , and in the absence of induction (87 clones out of 94)  .Interestingly, we observed that when new lysogens are grown in LB, in the absence of induction, there is a detectable amount of cell death, and growth delay at the end of the exponential phase in at least in 29 out of 94 tested clones . This coTo study the impact of prophage acquisition on the long-term stability of lysogens in a population, we used eVIVALDI, an individual-based model for microbial interactions and evolution . We usedWe observed two main patterns. When prophages have low spontaneous induction rates (1 to 5%), they generate stable, non-costly lysogens. Consequently, phages spread slowly in the bacterial population, which gives time for the phage-resistant mutants to emerge and increase to high frequencies. However, because these mutations are costly, lysogens slowly displace these mutants. This results in a sigmoidal-like temporal frequency of lysogens, where at the end of the simulations most of the resistant population is composed of lysogens . These dP=0.03)  . Taken tTo test the competitive fitness of the evolved clones having different phage-resistance mechanisms, we compared the area under growth curves of all BJ1 evolved clones isolated in the presence or in the absence of phage . In the AUC analyses also revealed that lysogens are less fit than the resistant capsulated clones, in the absence of phage. However, this is not so, in the presence of phage at short evolutionary time scales. At longer time scales (after ten days of evolution), resistant capsulated clones are fitter than lysogens, in the presence of phage, supporting our previous observations that lysogeny incurs a high fitness cost. This suggests that resistant mechanisms that emerge more frequently or that are accessible in evolutionary terms, such as capsule inactivation and lysogeny could be initially selected for, but become less advantageous at longer timescales where other less costly resistance mechanisms seem to provide higher fitness. Overall, our results show a hierarchy in the competitive advantages of phage resistance mechanisms, that varies across time scales and phage pressure.rcsB mutant, with reduced capsule expression, and a non-capsulated ∆wcaJ mutant , as well as an ∆J mutant . AdditioJ mutant .orf13, and potentially leading to a change in the capsule’s biochemical composition  and in wcaJ (9H3) and an 11 base-pair deletion in the capsule regulator wzi (9H7). These clones have reduced capsule expression comparable to mutations 3E1 and 9G11 in orf13, reduced phage adsorption, and an increased resistance to phage in liquid media  had frameshift mutations in a gene coding for an acyltransferase, position , Table 1 surface . The thrid media . SurprisInterestingly, the remaining seven clones that were identified in our initial screens to be resistant seem to be susceptible to phage lysate in all subsequent tests. Despite their marginally lower capsule production, we could not detect mutations in their genome relative to their ancestors (except for one clone with an intergenic mutation). To discard the possibility that this could be due to a problem in our initial screen for resistant clones, we returned to the original glycerol stocks and retested these clones for their resistance to phage during growth in liquid (i.e. the same conditions as the screen) . To avoiWe sought to test whether resistance to phages from ST14 could result in resistance to phages produced by other strains. To test potential cross-resistance between lysates, we produced phage lysates from strains 03–9138, ST17, and T69, all of which were previously shown to successfully infect BJ1 . We firsWe then tested whether the lysogenized BJ1 clones, resistant to ST14 lysate, were also resistant to other lysates. To do so, we assessed plaque formation on lysogen lawns rather than growth inhibition, as these new lysogens already exhibited significant cell death due to phage outbursts . DespiteK. pneumoniae strains under different degrees of parasite pressure. Based on theoretical works  whose changes are difficult to detect with the current genome assemblers and variant calling software. SSR are often mutational hotspots allowing rapid evolution of traits by changes that are easily reversible  that incur in lower fitness costs relative to other constitutive changes such as permanent loss of receptors  and ST14 (ENA: SAMN22024794) are two phylogenetically-distant K. pneumoniae, both bearing the K2 capsule serotype. Bacteria were grown at 37° in Luria-Bertani (LB) agar plates or in 4 mL of liquid broth under vigorous shaking (250 rpm). Chloramphenicol (30 μg/ml) and trimethoprim (100 μg/ml) were used to select for strain BJ1 and ST14, respectively.BJ1  were performed as reported in i(ji) Unidirectional mixing-effect parameter C). The effect of mixing two strains i and j on the population yield during the growth of focal strain i was quantified by the one-way mixing effect parameter i(jC). To calculate this parameter, the expected log10-transformed yield of strain i based on pure-culture performance (corrected for the frequency at which strain i was added) was subtracted from its actual log-transformed yield during competition with strain j.Calculations of i(jC) values indicate that strain i grew to a higher population size in the experiments in the presence of strain j than in pure culture, whereas a negative value indicates that mixing with j negatively affected the growth yield of ij. Biji. (ii) Bidirectional mixing effect parameter B is the difference between the actual total (log10-transformed) group cell count in a mix of strains i and j and the value expected from pure culture performance of the same strains .Positive ijB values indicate that total productivity is higher or lower, respectively, than expected from pure culture performance.Positive and negative Three clones from each strain were used to inoculate overnight cultures, which were then diluted at 1:100 and used to initiate the three independent mixed populations in a ratio 1:1, in a final volume of 4 mL. Each of the three mixed populations evolved in three different environments: (i) LB, (ii) LB supplemented with 0.2% citrate, and (iii) LB with mitomycin C . Cultures were allowed to grow for 24 hr at 37 °C and diluted again to 1:100 in fresh media. This was repeated for 30 days. Each day, each independently evolving population was plated and serially diluted. CFUs were counted (three plates per sample) and the emergence of non-capsulated mutants was recorded. Non-capsulated mutants are easily visualized by the naked eye as mutants produce smaller, rough, and translucent colonies.i) Growth curves: 200 µL of diluted overnight cultures of Klebsiella spp. (1:100 in fresh LB) were distributed in a 96-well plate. Cultures were allowed to reach OD = 0.2 and either mitomycin C to 1 µg/mL or 20 µl of PEG-precipitated induced and filtered supernatants at 2 × 108 PFU/mL were added. Growth was then monitored until the late stationary phase. (ii) PEG-precipitation of phages. Overnight cultures were diluted 1:500 in fresh LB and allowed to grow until OD = 0.2. Mitomycin C was added to the final 5 µg/mL. After 4 hr at 37 °C, cultures were centrifuged at 4000 rpm and the supernatant was filtered through 0.22 µm. Filtered supernatants were mixed with chilled PEG-NaCl 5 X (PEG 8000 20% and 2.5 M of NaCl) and mixed through inversion. Phages were allowed to precipitate for 15 min and pelleted by centrifugation for 10 min at 13,000 rpm at 4 °C. The pellets were dissolved in TBS . (iii) Calculating plaque forming units (PFU). Overnight cultures of susceptible or tested strains were diluted 1:100 and allowed to grow until OD = 0.8. 250 µL of bacterial cultures were mixed with 3 mL of top agar (0.7% agar) and poured into prewarmed LB plates. Plates were allowed to dry before spotting serial dilutions of induced PEG-precipitated phages. Plates were left overnight at room temperature and phage plaques were counted. (iv) Phage adsorption. Adsorption of phage particles to the cell surface was performed as previously described (ca. 5*106 phage particles) was added. The mix was allowed to sit for 2 min at room temperature prior to incubation at 37 °C for 15 min with shaking at 140 rpm. Phage adsorption was measured by quantifying the free phage remaining in the solution, after centrifugation for 10 min at 10,000 rpm, to get rid of bacterial cells. The supernatant was serially diluted and the non-adsorbed phage was quantified by spot titer on a bacterial lawn of strain BJ1. Finally, to quantify how many phage was adsorbed, the non-adsorbed phage was subtracted from the initial amount of phage added to the culture.(escribed . Brieflyi) Genomes of phage-resistant clones. Single clones were allowed to grow overday in LB supplemented with 0.7 mM EDTA, to limit capsule production. We performed DNA extraction with the guanidium thiocyanate method, with few modifications  wcaJ gene. PCR of wcaJ was performed using the primers that hybridized 150 base pairs upstream and downstream of the wcaJ gene; K2.wcaJ.150–5 (5’- GGCGTTCCAGCAAGGGTTATC –3’) and K2.wcaJ.150–3 (5’-ACGTTCGCGCTTAAATGTG-3’), respectively. To allow full coverage of the gene, PCR products were also sequenced with primer K2.wcaJ.inseq-5 (5’-CTGGGTCTTTACAGAGGAATC-3’). PCR products were sequenced by Sanger and analyzed using APe.. The uronic acid concentration in each sample was determined from a standard curve of glucuronic acid.The bacterial capsule was extracted as described in 24 hr cultures of BJ1, ST14, their coculture, or blank tubes with citrate, were centrifuged for 10 min at 4000 rpm and the supernatant was sterilized with 0.22 μm filter, prior to deproteination by centrifugation in an Amicon tube (10 kDa). Citrate concentration was measured using a Citrate assay kit (Sigma-Aldrich MAK333).Simulations were performed based on the model described in Phage similarity was calculated as described in i and Bi is the pair i of homologous proteins present in A and B (containing respectively #A and #B proteins), id is the percent sequence identity of their alignment, and min is the total number of proteins of the smallest prophage, i.e., the one encoding the smallest number of proteins (A or B). wGRR varies between zero and one. It amounts to zero if there are no orthologs between the elements, and one if all genes of the smaller phage have an ortholog 100% identical in the other phage. Hence, the wGRR accounts for both frequencies of homology and the degree of similarity among homologs.where A The overarching question of the manuscript is important and the findings inform the patterns and mechanisms of phage-mediated bacterial competition, with implications for microbial evolution and antimicrobial resistance. The strength of the evidence in the manuscript is compelling, with a huge amount of data and very interesting observations. The conclusions are well supported by the data. This manuscript provides a new co-evolutionary perspective on competition between lysogenic and phage-susceptible bacteria, that will inform new studies and sharpen our understanding of phage-mediated bacterial co-evolution. public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 2 peer reviewers, including Samuel L Díaz-Muñoz as the Reviewing Editors and Reviewer #2, and the evaluation has been overseen by Christian Landry as the Senior Editor.Thank you for submitting your article "Competition between lysogenic and sensitive bacteria is determined by the fitness costs of the different emerging phage-resistance strategies" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:1) The manuscript concludes that selection favors more subtle capsule mutations because they are less costly than capsule-loss mutants (lines 497-500). However, there are no data to support this conclusion, as fitness costs of the various resistance phenotypes analyzed were not measured.The revised manuscript should either include such data if available or, without such data, substantial changes in the focus of the paper would have to be made, as the manuscript places considerable emphasis on the importance of fitness costs in resistance evolution, notably in the title.2) The manuscript attempts to directly relate the emergence of the various resistance mechanisms to phage infection pressure during the coevolution experiment. However, while bacterial population dynamics data are monitored meticulously, the reviewers were not able to find the phage dynamics data. It seems likely to the reviewers that these data exist and these data are needed to make conclusions that link resistance mechanisms to phage pressure.In the absence of these data, a major change in the narrative would be needed to bring the conclusions of the paper in line with the evidence (i.e. conclusions would be related to competition with the lysogenic strain and avoid a mention of phage pressure).3) We note that if there is no further evidence to support both conclusions noted in 1) and 2) above, then the conclusions made by the manuscript would be substantially altered and the significance of the contribution would also substantially change.Reviewer #1 (Recommendations for the authors):The authors have gathered a huge amount of data and made some very interesting observations but unfortunately, a clear narrative is missing and the data do not support the main conclusions. One relatively simple thing would be to measure phage titers alongside bacterial densities over time – this would allow the authors to directly say something about phage infection pressure. To understand whether resistance mechanisms confer different fitness costs in the presence/absence of phage (and high/low phage pressure) should in my view be tested separately and directly in short-term experiments by directly competing clones with different resistance mechanisms isolated from the experiment. If the latter is not possible  the authors could measure bacterial growth curves instead to make inferences about their fitness.Reviewer #2 (Recommendations for the authors):I think this is a very well-done and very interesting study! Please see below my recommendations:1) Rearrange the discussion to provide the salient overall, general points. Make it. I think those major take-home messages are that 1) lysogenization might not be as prevalent a phage resistance mechanism as we thought previously (or at least in as wide a set of conditions as we thought), particularly in the long-term and 2) that receptors continue to be the primary mechanism, importantly through loss at first and then through changes in expression/composition. Secondarily, the multiple mechanisms and the transience can be discussed, but as written these aspects detract from the overall message.2) Please acknowledge previous work on context dependence (i.e. agar/liquid) from multiple groups, but Britt Koskella and Ellie Harrison come to mind immediately. Essential revisions:1) The manuscript concludes that selection favors more subtle capsule mutations because they are less costly than capsule-loss mutants (lines 497-500). However, there are no data to support this conclusion, as fitness costs of the various resistance phenotypes analyzed were not measured.The revised manuscript should either include such data if available or, without such data, substantial changes in the focus of the paper would have to be made, as the manuscript places considerable emphasis on the importance of fitness costs in resistance evolution, notably in the title.We now provide a novel figure in the main text, Figure 4, comparing the growth of all isolated clones with different resistance mechanisms, in the presence and absence of phages, and at different periods during the evolution experiment. Our data further support our conclusion that new lysogens are less fit than other resistant clones.2) The manuscript attempts to directly relate the emergence of the various resistance mechanisms to phage infection pressure during the coevolution experiment. However, while bacterial population dynamics data are monitored meticulously, the reviewers were not able to find the phage dynamics data. It seems likely to the reviewers that these data exist and these data are needed to make conclusions that link resistance mechanisms to phage pressure.In the absence of these data, a major change in the narrative would be needed to bring the conclusions of the paper in line with the evidence (i.e. conclusions would be related to competition with the lysogenic strain and avoid a mention of phage pressure).We agree with the reviewer that including phage titers is important.In light of these comments, we provide a new supplementary figure  showing that phages are being produced in large numbers in our coevolution setting until the end of the experiment. This strongly suggests that evolutionary outcomes are still affected by the presence of phage and not solely caused by clonal competition.3) We note that if there is no further evidence to support both conclusions noted in 1) and 2) above, then the conclusions made by the manuscript would be substantially altered and the significance of the contribution would also substantially change.We hope that the modifications we have made to address points (1) and (2) prove that our conclusions are fully supported by our data.Reviewer #1 (Recommendations for the authors):The authors have gathered a huge amount of data and made some very interesting observations but unfortunately, a clear narrative is missing and the data do not support the main conclusions. One relatively simple thing would be to measure phage titers alongside bacterial densities over time – this would allow the authors to directly say something about phage infection pressure.See answer to Editor’s comment #2.The new supplementary figure S4 shows phage titers at three different time points of the experiment  indicating that phages are being produced in large numbers in our coevolution setting until the end of the experiment. This strongly suggests that evolutionary outcomes are, at least partially, influenced by the presence of phage and not solely due to clonal competition.To understand whether resistance mechanisms confer different fitness costs in the presence/absence of phage (and high/low phage pressure) should in my view be tested separately and directly in short-term experiments by directly competing clones with different resistance mechanisms isolated from the experiment. If the latter is not possible  the authors could measure bacterial growth curves instead to make inferences about their fitness.See answer to Editor’s comment #1.i.e. those for which we already know the genetic basis of the resistance-. As expected, our results show that different mechanisms of resistance have different fitness costs, but these may change with time. This is now addressed in a new section of the results.We now provide an extended analysis deriving from the growth curves from all clones isolated during the experiment, in the presence and absence of phage. The use of genetic markers or performing qPCRs would have reduced the analysis to only a few resistant clones -Reviewer #2 (Recommendations for the authors):I think this is a very well-done and very interesting study! Please see below my recommendations:1) Rearrange the discussion to provide the salient overall, general points. Make it. I think those major take-home messages are that 1) lysogenization might not be as prevalent a phage resistance mechanism as we thought previously (or at least in as wide a set of conditions as we thought), particularly in the long-term and 2) that receptors continue to be the primary mechanism, importantly through loss at first and then through changes in expression/composition. Secondarily, the multiple mechanisms and the transience can be discussed, but as written these aspects detract from the overall message.We have rewritten our discussion to better emphasize our key findings and streamline our thoughts, in light of the reviewer’s comment. Specifically, we better highlight that lysogenization is not as prevalent as previously thought in longer time scales. As suggested, we also discuss the loss of receptor as a defence mechanisms, but we chose to also focus on the emergence of transient changes (phenotypic resistance) as we believe (and provide large evidence from recent publications) that it is a wide-spread resistance mechanism, and very often neglected.2) Please acknowledge previous work on context dependence (i.e. agar/liquid) from multiple groups, but Britt Koskella and Ellie Harrison come to mind immediately.We have expanded our discussion to cite other previous works more including references by the mentioned scientists.


Lift-off processing is a common method of pattern transfer for different nanofabrication applications. With the emergence of chemically amplified and semi-amplified resist systems, the possibilities for pattern definition via electron beam lithography has been widened. We report a reliable and simple lift-off process for dense nanostructured pattern in CSAR62. The pattern is defined in a single layer CSAR62 resist mask for gold nanostructures on silicon. The process offers a slimmed down pathway for pattern definition of dense nanostructures with varied feature size and an up to 10 nm thick gold layer. The resulting patterns from this process have been successfully used in metal assisted chemical etching applications. Lift-off methods are an important step for pattern transfer or etch mask definition in nanofabrication ,2. TheseA common method of pattern definition is electron beam lithography (EBL), which is suitable for defining very fine structured patterns or patterns with a high demand on precision and small feature size. For EBL systems, single lift-off processes have previously been made with the resists Poly(methyl methacrylate) (PMMA) ,5 or HydHere we describe in detail a lift-off process using the chemically semi-amplified resist CSAR62  for the fabrication of narrow as well as thin gold zone plate nanostructures. Zone plates make for ideal test patterns, as they show a wide range of feature sizes. Both micron sized structures and sub 50 nm structures are printed and evaluated in the same experiment, and the lift-off process has to be versatile enough to facilitate processing for both. The patterns are dense in nature and have a smallest feature size of 30 nm in the outermost zone. Through the use of a single layer resist mask combined with a 50 kV EBL system, it is possible to obtain very fine structures in the desired metal on a silicon wafer . A possiFor this study, Fresnel zone plates were used as an example of dense nanostructured patterns. The widths of the zones ranged from micron-sized in the centre towards 30 nm at the outermost zone. Each zone is split in sections as the final gold structures are supposed to be used as catalyst in metal assisted chemical etching . Thus, tEach zone plate pattern is 150 µm in diameter and nine zone plates are printed on each chip together with supporting structures for identification and future dicing. The designed structures’ parameters were defined in MATLAB code generating the input file for the pattern generator in the electron beam lithography system.2 pieces before they were cleaned by sonication in isopropanol (IPA) and blown dry under N2 gas flow. A 5 min reactive ion-etch step  followed in preparation for resist application. All chips were then spin coated in CSAR-6200.9 , 7000 RPM for 1 min before baked on a hotplate at 150 °C for 1 min. The resulting resist layer measured approximately 90 nm in thickness.Silicon chips were diced to 0.5–1 cm2. The printed structures were developed in amyl acetate  for 1 min before the development process was stopped through a 10 s IPA soak. After a 20 s subsequent soak in penthane, the developed patterns were allowed to air dry.Structures were then printed in a Voyager  50 kV system at 120 µC/cmThe samples were coated with a 3 nm Ti adhesion layer and a 10 nm Au layer to facilitate future etching experiments. This was done through a physical vapour deposition process (PVD) utilising UHV E-gun  deposition in-house built instrument.The lift-off process was adapted from a previously described method utilized for similar applications . The resAn initial soak in AR 600-71  was conducted as soon as the Ti and Au layers had been deposited. Each sample was left in 20 mL of the AR 600-71 remover solution for 2 days at room temperature. Samples were rinsed in the soak remover solution and manually agitated vigorously to remove any larger resist residues. Samples were then transferred to individual crystallisation dishes for Cycle 1 of the lift-off process. Each step was conducted in a sonication bath at 80 kHz at a slightly elevated temperature of 30 °C. The different cycles are described in Samples were then removed from the DI-water and gently blown dry under nitrogen gas flow.Samples were imaged utilising scanning electron microscopy  with 10 kV at 5.4 mm working distance.Successful and failed lift-off results are shown in The process itself has been adapted from previously used methods . With thFirst and foremost, the remover solution used was changed from dimethylsuccinate to remover AR600-71. The main component of this remover solution is dioxolane, which offers a higher solubility for the CSAR62 resist, even when the film has been baked at relatively high temperatures. To further facilitate the solubility of the resist, the baking temperature before exposure was lowered from 180 °C to 150 °C. The over-night soak was extended from one to two days, as this showed significant improvement in loosening the resist from the silicon. Also, the suggested lift-off process now consists of several cycles to further increase the amount of exposure the resist receives from the remover solution.Two different sonication frequencies were tested. The starting point from Akan et al.  suggesteComparing the process described by Akan el al.  to the cThe biggest challenge is to complete the lift-off process for the smallest structures, as these seem to be more prone to failure than the larger features towards the middle. After two nights of soaking, cycle 1 and 2 will be efficient in removing the bulk of the resist, but not all. Thus, cycle 3 and 4 were added so to ensure any remaining flakes and areas in the more densely structured parts of the pattern were cleared. Cycle 5 and 6 are the only steps that remains relatively unchanged in comparison to the previously used method, since they do not add to any efficiency of the lift-off process itself, only acting as a cleaning step for following processing.In The resist applied measured approximately 90 nm in thickness. With 10 nm gold deposited at both the bottom of the narrow trenches and the trench wall tops, a narrow window of 80 nm is all that is left exposed to the solvents on the sidewalls of the trenches. A lift-off process with a 15 nm gold layer was attempted, but failed. This could be due to the metal-free resist openings being too small, or due to the (thin) metal layer deposited on resist sidewalls. Maintaining an acceptable ratio of exposed resist seems critical for the lift-off process to work, as no residual resist can be tolerated for the subsequent processing steps. A thicker layer of resist would be ideal, as this would enable a thicker layer of gold to be deposited. Such layers are expected to further facilitate the MACE process . This caThe main advantage of CSAR62 is it’s high sensitivity compared to resists such as ZEP or PMMA ,9, offerIt can be discussed whether adding a lift-off resist (LOR) layer could facilitate the process. This could potentially increase the yield of samples with a successful pattern transfer. Presently, no LOR-layer matrices for CSAR62 are offered by the supplier. Further studies on both single layer and bi-layer lift-off processes with CSAR62 could offer insight on how to make an even more reliable method.We report a successful single layer lift-off process for dense nanostructured patterns in CSAR-62. The process has been used numerous times in preparation of samples with gold nanostructures for metal assisted chemical etching demanding clean patterns with good pattern definition. The resist layer is stripped by a cyclic solvent exchange procedure that combines repeated dissolution of the resist helped by sonication at high frequencies. The deposited gold is used to define the pattern on the silicon in a satisfying, repeatable and reliable fashion. The process is well suited for patterns and processes demanding small feature size structures, but can also be used for larger feature sizes in the micrometer regime.