Worldwide incidence of type 2 diabetes (T2D) is rapidly increasing. Given the numerous negative health consequences associated with T2D, prevention of this disease has become a priority. Lifestyle changes, including regular exercise, can reduce the onset of T2D in those at elevated risk. However, long-term adherence to exercise is often poor in this population. Existing lifestyle interventions targeting exercise are labor intensive and costly for staff and participants. Evidence-informed counseling delivered in a manner that reduces dependence on staff and facilitates self-regulatory skills could alleviate time and financial barriers while promoting independent exercise.This protocol outlines the design, recruitment, and proposed analysis of a brief, 2-week evidence-informed exercise counseling intervention combined with either high-intensity interval training (HIIT) or traditional moderate-intensity continuous training (MICT).Small Steps for Big Changes is a 2-arm randomized controlled trial that will examine the effectiveness of combining brief exercise counseling with HIIT or MICT on adherence to moderate and vigorous exercise over 1 year. Cardiorespiratory fitness will be assessed at baseline, post intervention (2 weeks), and at 6- and 12-month follow-up. Physical activity behavior will be examined at baseline, post intervention, and 3-, 6-, 9-, and 12-month follow-up. The impact of the intervention on psychosocial outcomes pertinent to exercise adherence will be examined.Data collection was complete in March 2017. Data analysis is currently underway, and the first results are expected to be submitted for publication in 2019.The results of this brief intervention have the potential to inform future public health efforts designed to increase exercise in individuals at risk of T2D.ClinicalTrials.gov NCT02164474; https://clinicaltrials.gov/ct2/show/NCT02164474 (Archived by WebCite at http://www.webcitation.org/74Hx1ipj6)DERR1-10.2196/11226 GloballSmall Steps for Big Changes is a brief, evidence-informed lifestyle intervention that seeks to overcome the limitations of feasibility and sustainability in previous interventions with the aim of increasing physical activity adherence in individuals at elevated risk of T2D.Physical activity has been highlighted as beneficial for preventing the progression of prediabetes to T2D ,8. HowevTo increase the potential for long-term behavior change, it is important to target theoretically proposed mechanisms of change and incorporate evidence-based behavior change strategies to maximize potential intervention success. Hardeman et al  refer toself-regulatory efficacy beliefs). Self-regulatory efficacy is essential in promoting long-term engagement in behaviors such as physical activity [Social cognitive theory (SCT) provides a conceptual framework through which to design physical activity interventions. A key construct of SCT is the confidence in one’s ability to carry out and self-manage behavior  in relation to both social cognitions and thereafter physical activity behavior in individuals at risk of T2D -28. For Small Steps for Big Changes was developed to target the underlying theoretical- and evidence-informed mechanisms of behavior change within a brief intervention to promote long-term physical activity behavior in individuals at risk of T2D. Findings from a pilot feasibility study provided preliminary evidence that the Small Steps for Big Changes intervention protocol resulted in significant increases in self-regulatory efficacy and outcome expectations and a significant increase in purposeful moderate-to-vigorous physical activity (MVPA) 1 [(MVPA) 1  and 6 mo(MVPA) 1 .In addition to consideration of the psychological constructs to be targeted within a physical activity intervention, it is also important to consider the modality of exercise prescribed. Traditionally, individuals at risk of T2D embarking on a new exercise regime are prescribed moderate-intensity continuous training , with limited free-living adherence typically reported . Given tHIIT has received considerable attention as it elicits positive metabolic and cardiovascular adaptations that are similar, or even superior, to MICT in a variety of populations with lower time commitments ,34. Speclong term . Furthermore, trials with greater power are needed to explore whether psychological responses associated with these modalities of activity impact adherence rates.HIIT involves brief, repeated bursts of vigorous exercise separated by periods of recovery. In a cross-over study of inactive adults, comparable exercise enjoyment and confidence were found between HIIT and MICT, with a greater proportion of participants reporting a preference to engage in HIIT (62%) over MICT (20%) after a single bout of each exercise modality . SimilarThe objectives of this randomized controlled trial are to:Compare differences in cardiorespiratory fitness between HIIT and MICT at 6 and 12 months after the intervention.Examine accelerometry-measured purposeful moderate-to-vigorous physical activity in bouts of ≥10 minutes (MVPA10+) and total accelerometer counts throughout the follow-up year  after the HIIT and MICT intervention. Self-reported adherence to HIIT and MICT exercise prescriptions will be collected at 3-, 6-, 9-, and 12-month follow-up.Examine the following psychological outcomes to explore their potential relationship with free-living adherence: task self-efficacy, self-regulatory efficacy, concurrent self-regulatory efficacy, self-monitoring, outcome expectations, exercise enjoyment, and instrumental and affective attitudes. In addition, differences in acute affect, in-task enjoyment, and perceived exertion between HIIT and MICT will be compared.Examine differences in cardiometabolic health markers. Glucose control and insulin sensitivity will be assessed at baseline, post intervention, and at 6- and 12-month follow-up. Body composition will be assessed at baseline, post intervention, and at 12-month follow-up. Differences in average blood glucose levels between conditions will be examined immediately after the intervention in a subsample of participants.Small Steps for Big Changes is a 2-arm, parallel group randomized controlled trial comparing adherence to HIIT with MICT in individuals at risk of T2D. This single-center trial will be conducted at The University of British Columbia, Okanagan Campus. The design of the study and flow of participants is described in 2 and 40 kg/m2; and are cleared to engage in vigorous exercise ; have af needed ).Participants will be recruited using posters administered in the local community, social media, newspaper advertisements, and word of mouth. Advertisements will invite inactive adults aged between 30 years and 65 years to contact the researchers about potential participation in a lifestyle change program, specifically designed to assist individuals who wish to become regular exercisers. A contact phone number and email address will be provided for individuals to contact the research team. Individuals who contact the research team will be able to ask questions regarding the program, and if wishing to proceed, will be scheduled for a previsit telephone screening interview. Telephone screening will take approximately 30 minutes during which the research coordinator will outline the time commitment of the study; record the participants’ age, current medications, height, and weight ; and administer verbally and record responses to the Godin Leisure Time Physical Activity questionnaire  and the Baseline testing will last approximately 150 minutes. Participants will be provided with a detailed consent form and will be invited to ask any questions. After written consent is obtained, weight, height, and blood pressure will be taken and a 7-day physical activity recall interview  will be Eligible individuals will be stratified based on sex and then randomly assigned to either the HIIT or MICT exercise conditions. An external statistician, who has no other involvement in the trial, will generate random allocation sequences, which will be linked to an interactive Web page. Permuted blocks of random size will be used. Once an individual is identified as eligible, the research coordinator will access the password-protected Web page, and after entering requisite information on patient eligibility and sex, participants will be randomly allocated. Participants and researchers will be aware of group allocation. Following group assignment, participants will have a fasting blood sample taken, consume a 75 g glucose drink as part of a modified oral glucose tolerance test, complete several Web-based questionnaires, have a second blood sample taken 30 minutes following the glucose drink consumption, undergo a body composition scan, and complete a maximal cardiorespiratory fitness test to exhaustion on a cycle ergometer. At the end of the session, participants will be provided with an accelerometer to wear for 7 consecutive days, commencing the day after baseline testing. Participants will be shown how to wear the device and will be instructed to remove the device while sleeping or for water-based activities. In addition, instruction will be provided to remind participants how to correctly wear the accelerometer. Short message service (SMS) text messages or emails will be sent on days 1, 3, and 5 of monitoring to remind participants to wear the device (see A voluntary subsample of 8 to 10 participants per group will return to the laboratory 3 days after the preassessment for insertion of a continuous glucose monitor (CGM). The CGM will be worn for 2 consecutive days, and participants will be provided with a standardized diet. Participants will return after 2 days of wearing the CGM to have the CGM removed. These 2 additional visits to the laboratory will last approximately 30 minutes in total. No sample size calculation will be conducted as this aspect of the study represents an exploratory aim to determine if CGM can be used to detect changes in glucose control in this population.In this study, 10 days after completing baseline testing, eligible participants will begin the 2-week supervised exercise training. Participants in each condition will participate in 10 sessions of exercise performed over a 12-day period . Seven of these sessions will be supervised in the laboratory and will last approximately 30 to 60 minutes each, while 3 sessions will be conducted at home to foster independence and work through plausible barriers participants may encounter in the future. Each supervised exercise session will occur one-on-one with a counselor and will consist of an exercise component  and 10 minutes of counseling. Participants will be exposed to a variety of exercise formats within their given modality . For 4 of the 7 supervised sessions, participants will be able to self-select the exercise format to encourage autonomy. A total of 2 sessions will be performed on the cycle ergometer during which in-task affect and enjoyment will be assessed to more precisely control exercise intensity and control for the influence of exercise format on enjoyment. The exercise prescriptions for each condition will be progressive in nature see  and matcsprints, uphill treadmill walking, uphill outdoor walking, and elliptical machine) at approximately 80% to 90% peak oxygen uptake (VO2 peak), interspersed with 1-minute rest periods at approximately 40% VO2 peak. Results from the pilot study preceding this randomized controlled trial demonstrated that individuals with prediabetes respond well to this protocol [HIIT involves sessions of 4 to 10 ×1-minute high-intensity intervals (cycling protocol .2 peak. This prescription of MICT matches the progression of HIIT (percent increase over time) and modeled the exercise program that has been shown to prevent the progression of prediabetes to T2D  at approximately 45% to 55% VOwalking; ). An int walking , has bee walking ,48 and i walking  for inacThe 2-week supervised exercise training program will incorporate 3 unsupervised sessions to enable participants to practice engaging in exercise independently. Following completion of the 2-week program, participants will be asked to engage in their randomized exercise protocol  a minimum of 3 times per week for the next 12 months. Specifically, participants in HIIT will be prescribed a 5-minute warm-up, 10 ×1-minute high-intensity intervals, and a 5-minute cooldown. Participants in MICT will be prescribed 50 minutes of moderate-intensity exercise.To assist participants with the transition to independent exercise and to promote long-term exercise adherence, participants in both conditions will receive a brief behavioral exercise counseling intervention built into the 2-week supervised training program. The counseling involves 10 minutes of one-on-one structured discussion at the end of each supervised exercise session, totaling 70 minutes over the 2 weeks. Participants will see the same counselor each session to bolster rapport. A detailed description of the BCTs used in the behavioral intervention and the social cognitive mechanisms they are hypothesized to target is reported elsewhere . Participants will receive a worksheet for each of the 3 home-based sessions, each reviewing specific behavior change constructs.In summary, the key aims of the behavioral exercise counseling are to:Enhance confidence to perform the exercise : providing instruction on how to perform the behavior, opportunities to practice engaging in exercise, helping participants identify physiological cues associated with the assigned exercise intensity, sharing the success stories of similar individuals, and providing positive support.Increase confidence to self-manage exercise : providing opportunities to practice self-monitoring behavior, preplan exercise behavior, work through exercise barriers, and practice independent exercise.Increase awareness of the psychological and physiological outcomes associated with exercise engagement : through education and drawing on the experiences of similar individuals and of the participant throughout the 2-week intervention.The counseling will follow a semistructured format, targeting specific psychological and behavioral constructs each session, while allowing the participants to share their questions and concerns. The content of the program, tools, and materials to be used; session description; and the underlying theoretical constructs are presented in check-ins) regardless of whether purposeful exercise was planned or completed that day. Participants will be permitted to record 4 nonexercise days within the app per week, coinciding with the prescribed 3 bouts of planned exercise for each week. Once all 4 rest days had been used, participants will record a missed session on days on which no exercise is completed.To promote exercise adherence after the 2-week intervention, participants will be provided with a self-monitoring mobile application . The coParticipants will be reminded to record their exercise behavior each day via an automated SMS text message sent from the app if they have not checked in by 9:00 pm. Participants will be informed that the counselor is able to view their exercise behavior through the counselor portal of the app. In the event of 3 consecutive missed check-ins, the participant will be contacted by the counselor via the app messaging function. During the 12-month follow-up period, the participant will be messaged monthly using the app messaging system on months with no scheduled follow-up appointments . These SMS text messages will target verbal persuasion, performance accomplishment, vicarious experience, awareness of physiological and affective cues, social support, self-monitoring, relapse prevention, and action planning.A timeline for when each measure was assessed is provided in 2 peak at 6- and 12 months postrandomization. VO2 peak will be assessed by a continuous incremental ramp maximal exercise test on an electronically braked cycle ergometer . Expired gas will be collected continuously by a metabolic cart  that is calibrated with gases of known concentration and a 3.0 L syringe before every test. The test begins with a 4-minute warmup at 30 Watts, after which Watts increase by 1 every 4 seconds (15 Watts per minute). Verbal encouragement will be provided to the participant throughout the test. The test will be terminated upon volitional exhaustion or when revolutions per minute fall below 50. VO2 peak is defined as the highest 30-second average for VO2 (in L/min and mL/kg/min). Criteria for achieving VO2 peak are (1) respiratory exchange ratio >1.15; (2) plateau in VO2; (3) reaching age-predicted heart rate (HR) peak (220-age); and/or (4) volitional exhaustion. A Polar chest strap, which is integrated with the metabolic cart and cycle ergometer software (Lode Exercise Manager), will capture HR. HR peak and peak power output will be recorded as the highest values attained in the test. Cardiorespiratory fitness will be assessed at baseline and 6- and 12-month follow-up.The primary outcome is cardiorespiratory fitness determined by measuring VOObjectively measured purposeful MVPA adherence  will be assessed by triaxial accelerometry  at baseline and 3-, 6-, 9-, and 12-month follow-up. MVPA10+ is appropriate for measuring purposeful exercise  and provSelf-reported physical activity adherence will be assessed through an exercise log completed through the mobile application. Participants will be asked to record (1) whether exercise was complete, (2) type of exercise completed, (3) the duration of the exercise, (4) the number of intervals conducted (for the HIIT condition exclusively), and (5) how hard the session was . The peTask self-efficacy, self-regulatory efficacy, concurrent self-regulatory efficacy, outcome expectations, and self-monitoring will be assessed at baseline, post intervention, and at 6- and 12-month follow-up , waist circumference (measured at the level of the umbilicus), and blood pressure  will be measured in the morning after an overnight fast at baseline, post intervention, and at 6- and 12-month follow-up using standard procedures. Information on ethnicity, household income, marital status, education level, medication history, and current smoking status will be collected at baseline.Dual-energy x-ray absorptiometry (Hologic Discovery A) scans will be used to assess percentage body fat mass at baseline, post intervention, and at 12-month follow-up.A qualified and experienced phlebotomist will obtain a blood sample in the fasted state (≥8 hours overnight fast) and exactly 30 minutes following consumption of the 75 g glucose drink  from an antecubital vein. Samples will be placed on ice and processed within 30 minutes to obtain plasma via centrifugation for 15 minutes at 1550 g at 4℃.r=.88) with the hyperinsulinemic-euglycemic clamp [Samples will be batch-analyzed in duplicate after storage at −80℃. Plasma glucose will be assessed via the enzymatic hexokinase method using a commercial assay (Pointe Scientific) on a clinical chemistry analyzer . Insulin will be measured by enzyme-linked immunosorbent assay , as described previously . Glucoseic clamp  and is sic clamp ,67,68. OCGM will take place using the iPro 2 Professional CGM and Enlite sensor  in a subsample of participants. The first 20 participants randomized in the trial will be invited to participate in the CGM assessment. The CGM records glucose values in a blinded fashion using a small microneedle inserted into the subcutaneous abdominal adipose tissue that is connected to a recorder that quantifies interstitial glucose values every 5 minutes. Finger-stick blood glucose samples are taken 4 times per day and are analyzed by a software program (CareLink Pro) upon device removal and downloaded to create 24-hour blood glucose curves. CGM data will be analyzed for (1) average blood glucose, (2) 2-hour postprandial area under the glucose curve, (3) postmeal spikes, and (4) glycemic variability (mean amplitude of glycemic excursions and SD). Participants who wear a CGM will be provided a standardized diet  to consume during the day before and on the day of each 24-hour monitoring period. CGM data will be collected for 24 hours at baseline and beginning approximately 24 hours after the last day of supervised training. To accurately interpret CGM data, each participant will consume the same meals at the same times of the day during each assessment period.The primary safety concerns for participants in this trial will be associated with completing the cardiorespiratory fitness test and increased physical activity in daily life. These concerns include cardiovascular and musculoskeletal events. All participants will be cleared for exercise using the PAR-Q+ , and if reviewed and discussed barriers sheet completed by participant. The 2-day training workshop covers theory and BCTs targeted in the intervention and is mandatory for all counselors. Each counselor will participate in role-play activities during the workshop that will be observed by the primary investigator, and feedback will be provided. Counselors will record intervention attendance, missed sessions, session time changes, or dropout for each participant throughout the intervention and follow-up.To promote the fidelity of intervention delivery and to ensure the underlying BCTs are administered as intended, a comprehensive 2-day counselor training workshop will be delivered by the principal investigator (an expert in BCTs). To augment the workshop, a standard operating procedure manual will guide intervention delivery for each day of the 2-week training program and for all follow-up face-to-face contact points . The manual consists of detailed scripts and protocols for each exercise session, phone call, or app interaction. Counselors, who will be health psychology graduate students, will complete a checklist for each training day and follow-up session to ensure all key intervention components are administered. Checklists will align with information contained within the intervention scripts, for example, During the follow-up phase, counselors will be provided with standardized monthly messages to send to each participant either using the app messaging system or via email in the months when no face-to-face contact was scheduled. Counselors will record the details of all contact with participants during this time.2 peak as compared with MICT (approximately 10%) in participants with lifestyle-induced metabolic disease. We, therefore, used a 9% difference between HIIT and MICT as a meaningful exercise-induced increase in our population of interest, corresponding to a Cohen d effect size of 0.48 (or Cohen f of 0.24). Using these data, with a two-tailed alpha of .05 and 80% power, assuming a medium correlation among repeated measures of r=.5, it is calculated that 15 participants are required per group for a significant within-between interaction .The a priori sample size was derived in relation to the primary outcome for this trial, cardiorespiratory fitness assessed at 6 and 12 months. To calculate the sample size, a pooled mean of 20.8 mL/kg/min and a SD of 4.0 mL/kg/min were used based on our previous pilot study . At the age=47 years, and female=54.53% (1150/2109). Using these data, an increase in 10 minutes of MVPA per day is associated with a 15% reduction in cardiometabolic risk [P<.05, 41 participants per group are required. A conservative loss to follow-up of approximately 20% is anticipated, and therefore, the trial aimed to recruit 50 participants per group .However, given that the secondary outcome of exercise adherence typically produces greater measurement variability, the final sample size is determined to be powered for detecting a clinically relevant within-group increase in average daily MVPA assessed by an accelerometer. To calculate sample size, data from the Framingham Heart Study  and the lic risk . An incrlic risk . Thus, wlic risk  or Natiolic risk . To detePreliminary analyses will be conducted to test for univariate and multivariate outliers and to test for normality. Data transformations will be conducted if required. Variance between the conditions in baseline outcome variables will be examined. Data will be analyzed on an intention-to-treat basis, with individuals retained within their randomized groups regardless of participation. All significance tests will be assessed at a 5% level of significance and effect sizes will be reported. Sensitivity analysis will be conducted to examine the impact of missing data. We will account for missing data by performing multiple imputation by chained equations to determine whether the results are impacted .Mixed-effects regression with baseline as a covariate and 6- and 12-month outcomes as dependent variables will be used to examine differences in cardiorespiratory fitness between HIIT and MICT. Physical activity behavior assessed by accelerometer will be analyzed similarly but will include 3-, 6-, 9-, and 12-month time points. Mixed-effects regression using baseline and all other time points will be used to determine whether the treatments result in a significant change from baseline.The mediating effects of task self-efficacy, self-regulatory efficacy, concurrent self-regulatory efficacy, outcome expectations, and self-monitoring will be examined using the INDIRECT macro developed by Preacher and Hayes . The macA series of mixed-effects regressions will be conducted to examine changes over time between groups in anthropometrics, body composition, biochemical markers, CGM, and additional psychological constructs.The administrative database  and questionnaire data will be managed in-house. Random checks will be performed on the entered survey data against paper records. All errors will be logged and corrected. All physiological, anthropometric, and biochemical measures will be conducted in 1 laboratory with established quality assurance systems. All data will be stored in a locked laboratory on password-protected and encrypted computers. Paper records will be stored in a locked filing cabinet.Maximizing retention is an important issue to fully understand exercise adherence rates over the 12-month follow-up. At recruitment, all laboratory visits and community visits will be outlined for the participants and the importance of follow-up testing emphasized regardless of exercise behavior. During the 2-week supervised intervention, participants will book the same appointment time for each day and will be sent all the dates and times by the counselor before day 1 of the intervention. Moreover, 2 of the follow-up appointments will be conducted within the local community at a location and time that suits the participant. It is anticipated that this will help reduce some of the burden on participants in attending a laboratory visit.Ethical approval was provided by the University of British Columbia Clinical Research Ethics committee (H12-02268-A008), with informed consent obtained from all participants in the study.The project was funded in 2013 and enrollment began in July 2014. Data collection was complete in March 2017. Data analysis is currently underway, and the first results are expected to be submitted for publication in 2019.Small Steps for Big Changes program will be the first randomized trial to examine the efficacy of low-volume HIIT compared with traditionally prescribed MICT as a means of increasing long-term physical activity behavior in individuals at elevated risk of T2D. The impact of HIIT on cardiorespiratory fitness, physical activity adherence, and psychosocial outcomes will be directly compared with current exercise recommendations for this population .The The authors acknowledge potential limitations in the proposed methodology. Specifically, exercise studies suffer from potential selection bias, which could impact the generalizability of the results. Furthermore, the considerable number of outcome measures may limit an individual’s willingness to participate. However, the number of outcome measures included in this study is similar to other exercise randomized controlled trials ,77. GeneHowever, the findings from this study have the potential to make substantive contributions to understanding how different types of exercise protocol are tolerated and adhered to and the comparative health outcomes associated with these protocols. These findings could have significant implications for exercise recommendations provided for individuals at risk of diabetes.In addition, this trial examines the effectiveness of a brief behavioral intervention designed to support the promotion of independent physical activity behavior through facilitation of self-regulatory strategies and efficacy beliefs. This intervention brings together theory and evidence in a carefully constructed intervention that will have the opportunity to examine the causal pathways from cognitions to exercise adherence. Together, the results of this trial have the potential to impact future public health campaigns designed to increase physical activity with individuals at risk of T2D.


The expression of miR-302 over the period of early embryogenesis could possibly regulate the maternal transcript clearance. Zygotic transcription activation is mostly related to maternal messages degradation.In this study, the effects of in-vitro maturation technique (IVM) on the expression of miR-302 in human embryo produced from immature and mature human oocytes  obtained from females under gonadotrophin therapy were evaluated for assisted reproduction.Immature oocytes were cultured in vitro. The injection of oocytes-producing polar bodies was given using fresh sperm. Then, the embryo quality score was assessed in the IVM group compared with the control group. In both the groups, embryos with normal morphology were included in the molecular study. Only one blastomere was removed from three-day embryos and then the embryos were frozen. The expression of miR-302 in embryos was measured through quantitative real-time polymerase chain reaction.Our data showed a significant reduction of miR-302 expression in the IVM group vs. the control group (p = 0.02). The embryo quality score showed a significant difference between the two groups (p = 0.01).The present study demonstrated that the IVM process had a negative effect on the expression level of miR-302 in human pre-implantation embryos. Considering the major role of expression miR-302, a reduced potential in miR-302 expression could be related to a decrease in the early embryonic development. The complete maturation of oocytes is very substantial in supply and accumulation of maternal transcription factors (TFs) particularly at the final step of the oocyte growth phase  progresses to the metaphase II stage) (1). The recovery of immature oocytes, and subsequent in-vitro maturation (IVM), of such oocytes could be established as a procedure to cure women with infertility or to obtain more oocytes or more chances for infertile couples. No aspect of this therapy technique was known so far (2). There are many uncertainties over IVM culture, complete and comprehensive of oocyte maturation; such as nuclear, membrane, and cytoplasmic maturation. For the time of early stages of embryogenesis, development is primarily controlled by maternal parameters contributed by the egg cytoplasm, but the zygotic nuclear genome is inactive. Consequently, the activation of this genome occurs, products of the embryonic gene are moved, and the clearance of maternal parameters occurs. This developmental control transfer is known as maternal-to-zygotic transition (MZT) and is coupled with arising embryonic genome activation (3).During MZT and over cellular reprogramming, the history of the cell is deleted to make possible the formation of new cellular states via particular transcription parameters (zygotic state or pluripotency). Despite the variety of mechanisms recognized among diverse species to regulate the expression of a gene, uniting themes, such as the roles of central TFs and microRNAs (miRNAs) to direct the MZT emerge, miRNAs were indicated to control temporal gene expression by downregulating mRNAs transcribed over earlier developmental steps (4-7). However, it is important to note that such miRNAs might play significant roles in the maternal transcript clearance in mammals. There are numerous lines of evidence suggesting that such miRNAs not only contribute to pluripotency but also have key roles in cellular reprogramming. Importantly, miR-430 (one of the earliest and most highly transcribed genes originated from the zygotic genome (8-11)) and miR-427 have abundant expression during MZT in zebrafish  and Xenopus , respectively. miR-302 and miR-372 are its orthologous in humans (16-19).According to the previous studies, maternally prepared parameters have the ability to activate zygotic gene transcription through the function of TFs involving the early embryonic genome. In addition, zygotic genes, such as miRNAs, play an important role in maternal RNA clearance. The failure in the activation of the zygotic genome through the inhibition of maternal TF activity leads to stabilized maternal program and arrested development. miR-302 and miR-372 expression occurs over early development stages in humans and primates (20).Based on the aforementioned information, the present study was designed to assess the effects of oocyte culture on miR-302 expression in the human embryo. The aim of this study was to present the first expression library of miRNAs within the human embryos derived from the IVM technique to provide a necessary starting point for future investigations to improve the IVM.In the present research, two different experiments were studied. The control group included 16 couples who underwent intracytoplasmic sperm injection (ICSI) cycles and then pre-implantation genetic diagnosis (PGD) was done for them. In the control group, after embryo transfer, the excess of normal biopsied embryos that were donated for scientific research. The IVM group included 36 couples who underwent Repeated Implantation Failure (RIF) treatment and immature oocytes used at the GV stage in the present study. The exclusion criteria were any infertility with female factor as well as male factor. The age of the female and male patients were less than 35 and 45 yr old, respectively.For controlled ovarian stimulation, all the patients received the standard long protocol of pituitary suppression with gonadotrophin-releasing hormone agonist. Hypophyseal suppression of patients was started by administering 0.1 mg of Diphereline  or Decapeptyl  in the mid luteal phase of the previous cycle. Subsequently, we decreased gonadotropin-releasing hormone analog dose to 0.05 mg until the administration day of human chorionic gonadotropin (HCG). The patient's ovaries were stimulated with recombinant follicle-stimulating hormone (FSH) . The initial doses were determined and administrated based on the patient's age as well as basal serum FSH and estradiol levels. The patients received transvaginal ultrasound. HCG  was administrated. After 36-38 hr of the HCG injection, follicles larger than 11 mm were punctured, using transvaginal ultrasonography.GV oocytes were collected. The oocytes were denuded with 1% hyaluronidase enzyme , gently aspirated using a hand-drawn glass pipette, and incubated under mineral oil  for a maximum of 1-hr in 6% COOocytes showing liberated polar bodies were injected by sperm, based on the World Health Organization criteria . The injected oocytes were evaluated following 16-18 hr for the appearance of pronuclei according to our routine screening procedure. Zygotes were maintained in the culture media for further 24-30 hr. Three days after ICSI, the embryos were evaluated and scored for their cleavage and quality. The morphological features of embryos were determined according to a previously published study (23).Three days after ICSI, the embryos moved into microdrops of biopsy media  that were under mineral oil. Zona pellucida drilling was performed mechanically and one blastomere was biopsied for PGD analysis. The embryo was transferred into a drop for embryo vitrification. The embryo was maintained into an equilibration solution  for 15 min at room temperature (20-27°C), after an initial shrinkage and recovery. Subsequently, the embryo was aspirated and placed in the vitrification solution  at room temperature for a period of 1 min and then placed on a cryotop with a minimum volume of VS solution (24). Cryotop quickly plunged them into liquid nitrogen.The samples were applied to isolate RNA and synthesize cDNA, followed by quantitative real-time (qRT)-polymerase chain reaction (PCR) analysis. Extraction of RNA, synthesis complementary of DNA (cDNA) and analysis were done using the protocol explained in the previous studies. (25-27). In brief, the samples were pipetted into Eppendorf tubes consisting of 1.5 µL lysis buffers. cDNA was synthesized by the addition of 2 µL poly N, 2 µL mir-302 primer, and 5 µL nuclease-free water to each of 2 µL embryo samples. The samples were then placed in a Bio-Rad thermocycler for 5 min at 75°C until the reaction was complete. Subsequently, the tubes were placed on ice, and 5Table I indicates the primer sequences used in this study for qRT-PCR to study the frequency of the level of mir-302 via Rotor-Gene Q instrument . We performed RT-PCR reactions and reaction conditions  based on the protocol described in the previous study (27). A single gene-specific peak of different amplification reactions was verified by melting curve analysis. One reference gene, Snord, was used for the normalization of the RT-PCR as an endogenous internal gene. Additionally, we performed three replications and normalized fold-change in the mRNA level for each sample to that of endogenous internal mRNA levels .This study was approved by the Ethics Committee of the Shahid Beheshti University of Medical Sciences, Tehran, Iran (SBMU.REC.1393.78). In current research, written and verbal informed consent was achieved from the couples who had undergone ICSI/PGD according to standard protocols.All statistical analyses were carried out using the Statistical Package for the Social Sciences software, version 22 . The embryo quality score was compared using the non-parametric analysis test (Mann–Whitney U test). The results are expressed as means % Germinal vesicle oocytes were collected from 36 couples undergoing treatment. The frequency of harvested GV oocytes ranged from 3 to 6. We used the results of 16 couples undergoing ICSI-PGD cycles (Table II). After the development of the embryos at the 8-cell stage, the embryos having normal morphological features were chosen for molecular studies. Embryo quality score reduced significantly in the IVM group when compared with the control (p = 0.01) (Table II).Real-time PCR was performed to assess the quantitative expression of the studied gene. The miR-302 levels were measured by qRT-PCR in 8-cell stage embryos derived from ICSI using freshly prepared sperm. According to the criteria explained by the World Health Organization, sperm parameters  were determined (21). The morphological features of sperms were also determined using the Kruger strict criteria (22). The comparison of the relative transcript abundance miR-302 mRNA in embryos derived from ICSI indicated a significant difference at the levels of this micoRNA, during human early developmental stages, between the IVM group and the control group (p = 0.02). Figure 1 indicates the relative levels.The purpose of the present study was to investigate the effects of the IVM technique on the expression of miR-302 in human embryos grades A and B, produced from immature and mature oocytes  and obtained from females under gonadotrophin therapy for assisted reproduction. The main aspect of the present study was to assess one of the major miRNAs over early human embryonic development in the Assisted Reproductive Technique (ART). Our findings demonstrated that miR-302 expression diminished significantly in the IVM group when compared with the control group (p = 0.02).Previously published studies in this field demonstrated the high expression of miR-302 in the development of an early human embryo. The results obtained from those studies, indicating increased expression over the early period of the embryo, document a putative role for such miRNAs already identified in embryonic stem cells (ESC), demonstrating their major roles in maintaining pluripotency (20). miRNAs, small non-coding RNA molecules, regulate genes in numerous biological systems, including the oocyte and embryo (28). Previous studies indicated that the stem-cell factors, such as Oct4, link miR-302 as a promoter and stimulate its expression in ESCs (6). Epigenetic reprogramming events have the ability to stimulate the expression of ESC specific genes, particularly Oct4, Sox2, and Nanog, in turn further induce the expression of miR-302 for the production of a positive feedback loop cycle having a key role in maintaining somatic cell reprogramming (SCR) (29). It has been reported that several possible miR-302 targets regulate the cell cycle. Therefore, the regulation of cell-cycle development mediated by miR-302 may be ascribable to directing multiple mRNAs. This suggested that miR-302a might be coordinately directing regulators of multiple G1 phases toward cyclin D1 and Cdk4 for serving as a negative regulator of G1 (6). Particularly, this emphasize the main role of miR-302a in the proliferation of EC cells. Interestingly, miR-294 in mice and miR-302/372 in humans are plentifully stated in ESCs and embryonic tissues . Collectively, miR-302 functions in a single-cell range, such as contributing to erase the history of cells to make easy the formation of new cellular states by specific TFs (zygotic state) and pluripotency, have important roles in cell reprogramming and cell cycle regulators .Reviewing how mammalian oocytes evolve, we can find that they obtain a sequence of abilities over follicular progress (growth of oocyte and maturation of cytoplasm) having important effects on fertilization and following early embryonic progress . The final preparation of oocytes for fertilization is carried out during oocyte maturation (from the GV to metaphase II stage) (2). These factors are applied to maintain the early phase of embryonic progresses before the beginning of embryo DNA transcription . During the cytoplasmic maturation synthesis, essential maternal materials including maternal TFs are synthesized that involve the early embryonic genome and contain miR-302 in human early embryonic development. miR-302 is one of the human embryonic genes whose expression is govern by a wide variety of factors, containing various functions in embryonic and stem cells (2). Therefore, based on our findings, a decrease in the expression of miR-302 (considering its main role) in the embryo derived from in-vitro matured oocyte can be related to a decrease in early embryonic development so that this reduction cannot be overlooked.Generally, the routine assessment of oocyte morphology using the phase-contrast microscopy is a key predictive marker used for oocyte quality, therefore mature oocytes, with full nuclear maturation followed by the first polar body extrusion, are currently utilized to measure the success or failure of a given ART program (34). Initial studies emphasize to “nuclear maturation could occur suddenly subsequent to the culture in vitro of animal and human immature oocytes” (2). Nevertheless, the developmental competence after fertilization in the later stages of these oocytes is doubtful. Culture conditions considerably affect the maturation of in-vitro oocytes. Not only normal morphology and nuclear maturation of oocyte but also many factors are involved and active in the early progress of the embryo development, which is neglected in the IVM method. Therefore, due to the lack of the probable clinical methods, cytoplasmic maturation is ignored in ART programs, and the embryo development sequences in the next phases remain unknown and more studies are required in this area.The present study indicated that IVM process had a negative effect at the expression level of miR-302 in human pre-implantation embryos . Therefore, these results could change several events involved in establishing pluripotency and in improving the activation of zygotic transcription in the development of the embryo, requiring further and advanced studies at the molecular level and improvement of culture media for in-vitro maturation.Authors declare that they have no conflict of interests.


Introduction: Asymmetrical sensorimotor function after stroke creates unique challenges for bipedal tasks such as walking or perturbation-induced reactive stepping. Preference for initiating steps with the less-involved (preferred) leg after a perturbation has been reported with limited information on the stepping response of the more-involved (non-preferred) leg. Understanding the capacity of both legs to respond to a perturbation would enhance the design of future treatment approaches. This pilot study investigated the difference in perturbation-induced stepping between legs in stroke participant and non-impaired controls. We hypothesized that stepping performance will be different between groups as well as between legs for post-stroke participants.Methods: Thirty-six participants  were given an anterior perturbation from three stance positions: symmetrical (SS), preferred asymmetrical (PAS−70% body weight on the preferred leg), and non-preferred asymmetrical (N-PAS−70% body weight on the non-preferred leg). Kinematic and kinetic data were collected to measure anticipatory postural adjustment (APA), characteristics of the first step , number of steps, and velocity of the body at heel strike. Group differences were tested using the Mann-Whitney U-test and differences between legs tested using the Wilcoxon signed-rank test with an alpha level of 0.05.Results: Stepping with the more-involved leg increased from 11.5% of trials in SS and N-PAS up to 46% in PAS stance position for participants post-stroke. Post-stroke participants had an earlier APA and always took more steps than controls to regain balance. However, differences between post-stroke and control participants were mainly found when stance position was modified. Compare to controls, steps with the preferred leg (N-PAS) were earlier and shorter (in time and length), whereas steps with the non-preferred leg (PAS) were also shorter but took longer. For post-stroke participants, step duration was longer and utilized more steps when stepping with the more-involved leg compared to the less-involved leg.Conclusions: Stepping with the more-involved leg can be facilitated by unweighting the leg. The differences between groups, and legs in post-stroke participants illustrate the simultaneous bipedal role (support and stepping) both legs have in reactive stepping and should be considered for reactive balance training. Sensorimotor dysfunction is a common outcome after a stroke that contributes to deficits in gait and voluntary stepping as well as impaired postural control –6. StrokSeveral studies on perturbation stepping in persons post-stroke report a predilection for initiating steps with the less-involved leg, even with lateral perturbations –22. Two The asymmetry in stepping preference and sensorimotor function creates challenges in investigating the bipedal nature of induced reactive stepping. Comparing perturbation induced reactive stepping responses between individuals' post-stroke and controls would provide some insight into the alterations in inter-limb control of reactive stepping. To our knowledge, only one study has compared persons post-stroke and controls and only during a posterior perturbation. In that study, post-stroke participants initiated fewer backward steps, took more steps when they did initiate stepping, and were less stable with shorter step length and delayed onset compared to controls . HoweverDifferences between the inter-limb control of stepping for different directions of imbalance in unimpaired controls and persons post-stroke are important to identify given the bipedal nature of the induced stepping task and the direction-dependent neuromotor requirements for recovering balance . IncomplTo further address the foregoing issues, the purpose of this pilot study was to investigate potential differences in reactive perturbation-induced stepping characteristics between: (1) post-stroke survivors and healthy control participants, and (2) between legs in the post-stroke participants. Using a simple method of modifying the initial stance symmetry, we hypothesized that for perturbation induced reactive steps in the anterior direction: (1) stance asymmetry will alter the selection of the stepping leg, (2) stepping performance would be different between post-stroke participants and age-matched controls for both legs, and (3) the more-involved leg would be less efficient in reactive step performance than the less-involved leg.Data sharing is fully appreciated. The raw data supporting the conclusions of this manuscript will be made available by the corresponding author, without undue reservation, to any qualified researcher upon reasonable request.Thirty-six individuals (20 persons post-stroke and 16 controls) participated in this study. The post-stroke participants were recruited from the Clinical Neuroscience Research Registry. They all met the following criteria: history of a single unilateral non-cerebellar stroke at least 1 year prior to the study, ambulated independently in the community and did not use a wheelchair or a long leg brace. Additional selection criteria included: no history of other neurological and orthopedic diseases or surgery to the lower extremities, and not currently receiving occupational or physical therapy. In addition, post-stroke participants were physically screened for the ability to stand and walk independently 10 feet without an assistive device or orthosis, unilateral paresis, ability to give consent. They were excluded if they scored below a five on the Functional Ambulation Categories , 27. ConThe clinical tests were selected to give a fuller picture of the participants' characteristics . The ActSensorimotor measures included sensation on the plantar surface of feel and extension control in standing. Deep pressure sensation in the feet were measured using Semmes-Weinstein aesthesiometer , 36. TheParticipants stood with feet shoulder-width apart, each foot on a separate force platform . They weThree different body weight distributions were used to encourage stepping with both legs . In the After three practice trials of increasing weight bearing to the predetermined level, 10 trials at each of the three weight distribution conditions and five catch trials  were presented in the same predetermined standard randomized order. Participants were given a seated rest after 18 trials or as needed. A research personnel was always standing next to the participants for safety.Two force platforms  recorded the forces under each foot and allowed the calculation of the total center of pressure (CoP) position. Reflective markers were attached bilaterally over key landmarks of the leg and trunk  to record foot, shank, and trunk movements using an eight-camera motion analysis system . Perturbation, kinetic, and kinematic data was collected in Qualisys at 100 Hz for 15 s, beginning at least 1 s prior to perturbation. Kinematic data was filtered using a second order Butterworth filter with 1 bidirectional pass resulting in a fourth order filer. The cutoff frequency was set at 10 Hz , 43.Following the perturbation, pre-step postural activity, referred to as anticipatory postural adjustment (APA), is often characterized by an initial displacement of the CoP toward the stepping leg that initiates weight transfer toward the upcoming single stance leg . Its chaFor each trial, the execution of the step was evaluated by determining the number of steps, first step onset timing, first step length, width, height, and duration. These characteristics were identified from the ankle markers of the stepping side. The beginning and end of the step were identified from the vertical velocity of the marker in order to determine the step duration and the mediolateral and anteroposterior displacement of the foot . The step clearance was defined as the maximal vertical excursion of the step ankle marker above the ground. The onset time of stepping was calculated relative to the onset of the perturbation.To quantify the termination of the whole-body movement which influences the maintenance of balance and the necessity of a second step, the velocity of the body at the end of the first step (first heel strike) was calculated using the midpoint of the bilateral acromion markers. The velocity was multiplied by the subjects' mass to calculate the body momentum as the difference in momentum between first and second heel strike.Descriptive statistics were used to describe the data for both groups and are reported as median (Mdn) and interquartile range (25 and 75th percentile). The Shapiro-Wilk and Levene tests indicated that the reactive step measures were not normally distributed, and that the variance was different between groups. In addition, an unequal number of stepping responses between groups and legs were observed , thus reBased on the number of participants who took steps with each leg , the difAcross all trials, one control participant resisted the pull and did not step on the first trial, otherwise all participants stepped in all trials. Two participants  needed external assistance to regain their balance during one trial and those two trials were dropped from the analyses.The control group stepped more often with their non-preferred stance leg (68.5%) in the SS condition (5 persons used that leg only); whereas in the asymmetrical conditions, they stepped more often with the leg initially supporting less weight, which was the preferred leg in the N-PAS condition (79.1% - 6 persons used that leg only) and the non-preferred leg in the PAS condition (92.4% - 12 participants used that leg only) . The posp = 0.009) and N-PAS condition . Post-stroke participants took more steps  than controls  to regain balance. They also took significantly  more steps (Mdn = 2.78 [2 3]) than controls (Mdn = 2.00 [1.27 2]) when stepping with the non-preferred/more-involved leg in the PAS conditions.A significant difference was observed between groups for the number of steps taken when stepping with the preferred leg in both the SS  was observed in most trials for both groups of participants (96% for controls and 94% for post-stroke participants). APA was absent in 54 trials: 17 trials (3.5%) involving 7 controls and 37 trials (6.2%) involving 15 post-stroke participants.p = 0.538) or amplitude  in SS condition or in N-PAS condition . This indicates that, when post-stroke participants stepped with their less-involved leg, the step preparation had the same characteristics as controls. However, APA onset occurred significantly earlier in post-stroke participants compared to controls in both the SS conditions  and N-PAS .p = 0.363) or amplitude . However, APA onset occurred earlier post-stroke compared to controls .When stepping with the non-preferred leg in the PAS condition, there was also no significant difference in APA duration . However, the step length  and step width  were different between groups showing that post-stroke participants took shorter and slightly more lateral (Mn = 1.52 cm [−0.05 2.96]) steps than controls (Mn = −0.05 cm [−1.51 1.25]). In the N-PAS condition, step onset was earlier , step length  and step duration  were shorter for the post-stroke participants compared to controls. No significant difference was found for step height  or width .p = 0.018) and step length was significantly shorter  for post-stroke participants compared to controls. No differences were found for other step characteristics in this condition .When stepping with the non-preferred leg in the PAS condition, step duration was significantly longer  or in the change in momentum between the first and the second step .p = 0.008) and the change of momentum between first and second steps  tended to be smaller for post-stroke participants.Asymmetrical conditions uncovered differences between groups. When stepping with the preferred leg in the N-PAS condition, the velocity at the end of the step was smaller for post-stroke participants compared to controls  and there was a smaller change of momentum  between first and second steps for post-stroke participants compared to controls indicating that post-stroke participants were less efficient arresting the body momentum.When stepping with the non-preferred leg in PAS condition, the velocity at the end of the step was smaller , for stepping measures (p > 0.53) or the landing characteristics (p > 0.15). The only almost significant difference concerned the step width . Controls tended to place their foot slightly more medially when stepping with the preferred leg (Mdn = 1.51 cm [−0.6 2.96]) than when stepping with the non-preferred leg (Mdn = −0.20 cm [−1.29 2.03]).No significant difference between legs was found for controls in postural activity preceding the step (p = 0.65) and amplitude  before the step were the same between the legs but APA duration tended to be longer  when stepping with the more-involved leg compared with the less-involved leg (p = 0.363), step width , step height  or step length . However, a significant difference was found in number of steps  and for step duration . Post-stroke participants took more steps (Mdn = 2.78 [2 3]) when stepping with the more-involved leg than when stepping with the less-involved leg (Mdn = 2.3 [2 2.8]) and step duration was longer with the more-involved leg compared to the less-involved one (p = 0.388), the change in momentum was significantly smaller  when stepping with the more-involved leg compared to the less-involved side in participants post-stroke. Thus, although the body was moving at the same speed at the end of the first step, post-stroke participants had difficulties in reducing the body momentum  leg was the strategy observed most often among the post-stroke group as previously reported , 19, parIn the asymmetrical stance condition, a shorter step was observed in the post-stroke group compared to controls when they stepped with the less-involved leg and more weight was on the more-involved leg. Standing with greater weight on the more-involved leg is often challenging for people post-stroke and can be observed clinically. The shorter step length in this condition may reflect not only a desire to return to bipedal stance as soon as possible, but possibly lateral instability on the more-involved stance side during the step. In this case, the generation of hip abduction torque is an important contributor to the regulation of mediolateral stability during the single limb stance phase of stepping as well as for ongoing gait . DeficitAlthough we anticipated that stepping with the less-involved leg in post-stroke participants would be similar to that of controls, it was not the case. Despite a smaller velocity of the body at the end of the first step and similar change in momentum, post-stroke participants took more than two steps to stop their forward progression when using the less-involved leg whereas the controls stopped generally with <2 steps. Thus, the need for additional steps might be related to instability arising at the end of the second step when the more-involved leg contacts the ground, creating the necessity for additional steps.Stepping with the more-involved leg was achieved by changing the initial weight distribution and initially reducing the amount of weight on that leg. The longer step duration and shorter step length of the more-involved leg in post-stroke participants compared to controls is not surprising given the greater sensorimotor impairments of hemiparetic side. The lack of differences between groups and legs for the other step characteristics when stepping with the more involved leg was unexpected. Given that the perturbation was standardized and the velocity at landing was not significantly different between legs, the problem for stepping with the more-involved leg was likely not directly related to the execution phase of the reactive step. Instead, the smaller change in momentum between heel strikes for the post-stroke participants suggested that they were less able to slow the momentum of the body between the first step with the more-involved leg and the second step, and thus that the control of the landing phase of the first step was problematic. This could be influenced by decreased extension strength or control in the more involved leg , 52 thatWhen using the more-involved leg, no group differences in the APA duration and amplitude were observed. However, the comparison was made in the asymmetrical condition when the stepping leg supported only 30% of the body weight and thus when the need for weight transfer postural requirements were minimized. Despite the lack of differences between the groups, a deficit in pre-step mediolateral postural control when stepping with the more-involved leg cannot be ruled out. Specifically, when comparing the legs in post-stroke participants for the same weight distribution condition, the pre-step postural adjustment tended to be longer for steps with the more-involved leg compared to steps with the less-involved leg. Even though the legs were partially unweighted, additional time might be needed to complete the postural adjustment before a step with the more-involved leg thus illustrating deficits in postural control. This may result from the larger variability in the amplitude of the APA. In contrast with controls or less-involved leg steps, several of the post-stroke participants displayed multiple peaks in their APA performance before reaching their maximum amplitude prior to releasing a step with the more-involved leg. This observation resembled that of previous studies showing differences in postural adjustments during gait initiation between post-stroke and control participants  and betwPost-stroke participants took more steps when stepping with the more-involved leg than with the less-involved one. The need for more than two reactive steps when stepping with the more-involved leg may be less about the step execution and more about the postural challenge before the step and/or at landing. The smaller change in momentum see  observedThe increased use of the more-involved leg for stepping when initially bearing 30% of body weight support illustrates the difficulty of releasing a step when more weight is on the leg and may reflect an underlying issue with the postural demands prior to stepping. It is thus important to implement situations facilitating the use of the more-involved leg by varying (reducing or increasing) postural demands prior to the step. In a prior study using diagonal anterior waist-pull perturbations, a pull toward the less-involved leg appeared to force increased stepping with the more-involved leg . This diAs a therapeutic approach, induced step training has been used in other populations and has shown improvement in step initiation timing in healthy older and younger adults for both reactive and voluntary stepping , and incThis pilot study results are limited to community dwelling ambulatory chronic post-stroke survivor's reaction to sudden anterior perturbations of balance in controlled conditions. Due to the unrestrained responses of the participants, differences between legs for all stance symmetry posture could not be analyzed. Although the laboratory setting tries to mimic real life perturbation situations, only the onset of the pull was unknown whereas the perturbation direction was predictable. Lastly, specific neuromuscular measures were not taken so we can only speculate on the precise underlying neuromotor factors that may contribute to these findings. The findings however illustrate several issues with reactive stepping that involve both legs post-stroke.These results highlight an altered stepping strategy involving both legs in persons post-stroke and illustrate the complexity of perturbation-induced stepping with either leg. Stepping with the more-involved leg can be facilitated by unweighting the leg which constitutes a simple intervention to encourage stroke survivors to use their weaker limb. The predilection for initiating a step with the less-involved leg may be, at least in part, a learned behavior as stepping with either leg after a perturbation appears to be challenging for ambulatory stroke survivors. Therefore, consideration of the simultaneous roles (support and stepping) of both legs during reactive stepping is important for reactive balance training and should be included when designing rehabilitation approaches to enhance balance function.KM designed and collected the data, conducted the data analysis, and drafted the initial manuscript. M-LM assisted with design, data analysis, and manuscript revisions. MR, MB, and MC assisted with results interpretation and gave critical comments for the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Glycans and glycan binding proteins (GBPs or lectins) are essential components in almost every aspect of immunology. Investigations of the interactions between glycans and GBPs have greatly advanced our understanding of the molecular basis of these fundamental immunological processes. In order to better study the glycan-GBP interactions, microscope glass slide-based glycan microarrays were conceived and proved to be an incredibly useful and successful tool. A variety of methods have been developed to better present the glycans so that they mimic natural presentations. Breakthroughs in chemical biology approaches have also made available glycans with sophisticated structures that were considered practically impossible just a few decade ago. Glycan microarrays provide a wealth of valuable information in immunological studies. They allow for discovery of detailed glycan binding preferences or novel binding epitopes of known endogenous immune receptors, which can potentially lead to the discovery of natural ligands that carry the glycans. Glycan microarrays also serve as a platform to discover new GBPs that are vital to the process of infection and invasion by microorganisms. This review summarizes the construction strategies and the immunological applications of glycan microarrays, particularly focused on those with the most comprehensive sets of glycan structures. We also review new methods and technologies that have evolved. We believe that glycan microarrays will continue to benefit the growing research community with various interests in the field of immunology. Immunology as a field is continuously evolving and expanding. With thousands of publications every year, it fundamentally impacts many aspects of biomedical sciences and has led to fascinating breakthroughs in a large number of biomedical areas, including primary immunodeficiencies, immunometabolism, neuroimmunology, mucosal immunology, cancer immunotherapy, and vaccine development. It is becoming increasingly clear that major components of our immune system include glycoproteins and recognition of glycans by glycan-binding proteins (GBPs) or lectins  facilitated the analyses of many GBPs in various aspects of immunology. With the technology developments at the National Center for Functional Glycomics (NCFG), these key immunological players have been re-examined with a larger variety of glycan sequences. This review introduces glycan microarray technologies from a historical point of view, summarizes the recent developments in the chemical and chemoenzymatic methods and new glycan microarrays with the focus on the CFG and NCFG approaches. We highlight the biological applications of glycan microarrays in immunology studies, and discuss current challenges and future perspectives of this technology.Identifying the interactions between glycans and GBPs is, therefore, key to understanding the molecular mechanisms of these immunological events. There is a need for facile methods to study such interactions in a miniaturized and high throughput manner. A major breakthrough in this effort was the development of printed glycan microarrays  epitopes by Morgan and Watkins, and Kabat and colleagues between the 1950's and 1970's Watkins, . They haSolid phase direct binding assays were developed thereafter, in which GBPs were directly radio-labeled and the binding assays were performed on thin-layer chromatography (TLC). Breakthrough discoveries in this stage include the identification of GM1 as the ligand for cholera toxin  ester- or epoxy-derivatized glass slides, much effort has been devoted to the development of amine-containing bi-functional linkers, which can be coupled to the reducing end of the glycan and contains a reactive amine group to covalently attach to solid supports.The strategy taken by the CFG and NCFG to generate glycan microarrays is covalent attachment, which requires glycans to be covalently derivatized with a specific bi-functional chemical group that can subsequently be used to covalently react with a functionalized solid surface. Although glycans produced by Initially, the commercially available 2,6-diaminopyridine (DAP) -benzamide AEAB,  Song et. AEAB seIt should be noted that another approach using 2-aminobenzamide (2-AB) and 2-aminobenzoic acid (2-AA) has also been developed, whereby glycans reductively labeled with 2-AB can be isolated by chromatography and then directly covalently coupled to epoxy-activated glass slides via secondary amine chemistry to generate 2-AB or 2-AA-glycan microarrays  to regenerate free reducing glycans, which particularly facilitates glycomics and functional glycomics. This “catch and release” approach provided by F-MAPA enables selective enrichment of free reducing glycans from complex biological samples, which can be followed by mass spectrometry (MS)-based structural analysis upon release of the linker, and parallel microarray binding analysis upon release of Fmoc. Moreover, the successful recycling of the reaction system and synthesis of neoglycoprotein indicate that F-MAPA has great potential for industrial application in the synthesis of neoglycoprotein therapeutics and vaccines. The facile and scalable synthesis, high conjugation efficiency and operational simplicity of the derivatization reaction make the linker highly accessible to general research laboratories, thereby enabling general accessibility of glycan microarrays to the research community.F-MAPA is more versatile than ordinary bi-functional linkers. It can be facilely cleaved under mild conditions, such as treatment by de novo chemical synthesis or isolation from natural sources had been intimidating for N-glycan production. Although chemoenzymatic synthesis has provided an alternative route, many of the methods rely on sophisticated chemical synthesis to produce the core structures to start with. These suffered from low yield and efficiency due to the usage of non-mammalian glycosyltransferases and poor substrates in which the aglycone potentially interferes with the reactivity. Moreover, evidence has shown that the linkers also play a role in microarray binding experiments  guarantees the beta-configuration of the N-glycans, thus at least partially retains the natural presentation of N-glycans within context of N-glycopeptides. Asn-linked biantennary N-glycan core is abundant in chicken egg yolk. Fmoc can be selectively installed on the primary amine of the asparagine residue. Similar to F-MAPA, it renders strong fluorescence, adequate hydrophobicity and can be easily removed. The Asn-linked N-glycans have also been shown to be great substrates for enzymatic reactions. They are often quantitatively converted to products with the presence of mammalian glycosyltransferases.Using this approach, we successfully synthesized a library of 32 multiantennary Asn-linked N-glycans and prepared the Asn-linked N-glycan array (designated N-glycan array). All of them are naturally occurring complex N-glycan structures found in human and other mammals. We will discuss the application of this glycan microarray in detail in the next section.The Asn-linked chemoenzymatic approach holds great promise in expansion to more asymmetric complex N-glycan structures. In combination with specifically designed sugar donors, it is now possible to generate asymmetric multiantennary N-glycans without specially synthesized core structures in the initial steps, and thus can significantly reduce the difficulty in total chemical synthesis of N-glycans , as well as self-associated molecular patterns (SAMPs) and many of these interactions involve binding to carbohydrates. Due to their myriad of functions in anti-microbial defense and immune homeostasis, understanding of the fine binding specificities of these receptors is critical in our understanding of how the innate immune system responds selectively in the context of infections, autoimmunity and cancer.Siglecs are a family of sialic acid-binding lectins that are mostly located on the surface of hematopoietic cells. They are involved in cell signaling and adhesion, and are particularly important in immune cell regulation GlcNAc- and Neu5Gcα2-6Galβ1-4GlcNAc-, We also observed novel specificities that have not been reported previously. Siglec-3 bound strongly to Neu5Acα2-3(6S)Galβ1-4GlcNAc- , which iNotably, none of the N-glycans on the CFG glycan microarray were among the highest binders. This may be due to multiple factors. First, most of the multi-sialylated highly branched N-glycans  on the CFG array contain a bisecting GlcNAc , a key sGalectins are another key family of GBPs implicated in virtually all aspects of the immune system  to all of the LacNAc -containing glycans, with or without core fucose . Not surMoreover, our result identified the branching preference of galectin-1 and -3: the 2,2,4-form triantennary structures were always more strongly bound than the 2,2,6-isomeric triantennary counterparts; in galectin-3, the binding to 2,2,6-form triantennary structures was completely diminished . This isIt has been known that the human milk glycans (HMGs) are not significantly digested in the infant GI tract  , however these individuals never progress to active disease or result in a positive Mtb test. These resistant individuals were profiled for their anti-glycan antibody response, and compared to individuals with active and latent Mtb infections. The results of the NCFGv1 antibody profile from these distinct cohorts were indistinguishable, meaning that the resisters did not possess an anti-glycan antibody response that differed from active and latent patients. However, the data in its entirety has suggested that individuals resistant to Mtb infection have a unique and distinctive adaptive immune profile against Mtb specifically, although their deficiency in antibody response to carbohydrate antigens was eliminated by our results from the NCFGv1 array  bound the biantennary and 2,2,6- triantennary forms but not to the 2,2,4-counterparts or tetra-antennary N-glycans, which suggests that these lectins prefer branching; among the N-glycan backbone-binding lectins, only LCA, Pisum sativum agglutinin (PSA), and Concanavalin A (ConA) can accommodate both the α2,3- and α2,6-linked sialic acid. Other lectins, such as WGA, Phaseolus vulgaris erythroagglutinin (E-PHA), Phaseolus vulgaris leukoagglutinin (L-PHA), and Datura stramonium agglutinin (DSA) can only accommodate the α2,3-sialic acid . In contrast to the gnathostomes which produce antibodies made of immunoglobulin domains, the agnathans or jawless vertebrates, utilize a class of proteins constructed with leucine rich repeat motifs called Variable Lymphocyte Receptors (VLRs) as antigen receptors  platform  in the form of neoglycoproteins  of samples in parallel. As a screening method, the glass slide-printed microarrays do not allow for analyses of large numbers (hundreds) of samples in a high-throughput manner. Recently, She et al. developed a multiplex glycan bead array platform based on the Luminex technology  (Chen et al., A cell-based platform has been reported in characterization of the structure of glycan ligands (Nonaka et al., As a robust high-throughput and high-content screening platform, glycan microarray represents a unique tool to define protein-glycan interactions in immunological studies. Glycan microarrays require minute amount of glycans that can be assayed with low background and high specificity. They enable functional screening and comparison in binding specificity of GBPs including antibodies, proteins, and viruses. Glycan microarrays provide information on both positively and negatively bound glycan structures which is particularly informative when placed in the relevant biological context.Under current parameters, glycan microarray analyses cannot provide solutions to all scientific demands. For instance, this platform is not suitable for quantitative studies such as determining the binding constants. Several technical details are also yet to be addressed, and array robustness is related to the number and diversity of the printed compounds. The printing efficiency is generally low due to the efficiency in the underlying chemical reaction. The density and the presentation have also been shown to have a variable impact on the glycan interactions, and there may be cases where in-solution assays are more appropriate. Nonetheless, we and other groups have made many biologically important observations and advancements through glycan microarray interaction studies.hypothesis-generating discovery tool that can be effectively used to screen many types of immune proteins against hundreds of glycans simultaneously and provide leads to further experiments into these interactions. Sequence-defined and Shotgun microarrays are well complemented from distinct aspects of biological research: shotgun glycan arrays capture natural glycans that cannot be synthesized for defined arrays, sequence-defined glycan arrays corroborate the discovery made by shotgun glycan arrays. Therefore, they serve as a first-line screening that can guide further experimentation. An enormous amount of information can be gained from the array binding experiments themselves, especially if multiple types of arrays are utilized in a comparative fashion. They can also be coupled with creative steps such as enzyme treatments of the glycans, hapten inhibition, titration curves, and mutant proteins to obtain next-level binding characteristics. With increased sensitivity and throughput and the development of new formats including beads-based, cell-based and DNA-coded, glycan microarrays are now playing more and more important roles in discovering new GBPs and defining their essential functions in the host and microbiome interactome and in the immune system.The glycan microarrays represent a CG, MW, TM, AM, JH-M, and RC contributed to the writing and editing of the final review.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Himalayan-Tibetan orogeny has considered as a natural black box in the context of geodynamic evolution and tectonic complexity. The eastward extrusion model of Tibetan crust contradicts with the oblique convergence model in the NE-Himalaya , where the overall convergence rate accommodated in the Himalaya is about 20–25% less than that in the neighbouring central Himalaya and Eastern Himalayan syntaxis (EHS). We propose that instead of partitioning in the backarc, the NE-Himalaya has developed an active sliver along the Assam-Brahmaputra valley in the outer deformation front, in order to accommodate the deficiency in long-term plate convergence between Himalaya and southern Tibet. We argue that the strong eastward extrusion of Tibetan crust along NE-Himalaya is the main driving force for the unusual development of the Assam-Brahmaputra sliver. This new hypothesis can explain active convergence along EHS, low convergence and subdued topography in Bhutan and Arunachal Himalaya, kinematic and space-problem of Indo-Burmese wedge, and finally solves the contradiction between Tibetan extrusion and oblique convergence model of the HimalayanTibetan orogeny. The spectacular topographic features and tectonic history of Himalayan-Tibetan orogen has received global attention in terms of continental collision and related geodynamic processes, seismic hazards, and Asian climatic patterns, among other themes4. Among the geodynamic implications, two popular models have been in focus during the past decades: (a) ductile “glacier-like flow” of the Tibetan lower or upper crust and associated distribution in deformation7; and (b) interference of rigid lithospheric blocks and localised deformation along active faults9.The dynamic topography of the Earth’s surface, particularly those along continental margins, has been linked to mantle processes in recent studies8, and the lateral extrusion model which mainly considers deformation of Tibet8, appear to be mutually contradictory in view of the tectonics and distributed deformation (or slip partitioning) in the NE-Himalaya8. It has been proposed that in the NW-Himalaya, the motion between India and southern Tibet is oblique with respect to the structural trend, which is partitioned between right-lateral strike-slip motion on the Karakorum fault system in the back arc, and slightly oblique motion in the Kashmir Himalaya in the frontal arc, leading to the formation of the NW-Himalayan sliver10. However, in the adjoining Garhwal-Kumaun and Nepal Himalaya, there is no partitioning, and the entire convergence of 18–20 mm/yr occurs in arc-normal manner12.The oblique convergence model which describes the deformation between Himalaya and southern Tibet along the frontal-arc13.In the NE-Himalaya , the motion between India and southern Tibet is again oblique with respect to structural trend of the arc Fig. . In orde33, is mainly responsible for such unusual development of a sliver in the outer deformation front of the NE-Himalaya. Based on the GPS observations, we address the oblique convergence and outer deformation-front strain partitioning process along the Assam-Brahmaputra valley adjacent to NE-Himalaya.This raises question on applicability of the oblique convergence model in the NE-Himalaya. Here we address this issue and propose that instead of partitioning in the back arc, an active sliver in NE Himalaya has developed along the Assam-Brahmaputra valley in the outer deformation front which is accommodating the oblique motion through distributed deformation. The strain partitioning in the outer deformation front of Himalayan region has not been well evaluated in previous studies. Here we take into account the Assam-Brahmaputra valley sliver in the outer deformation front of NE Himalaya, in order to explain the complex geodynamic process. We also propose that the strong eastward flow of Tibetan crust around the EHS, investigated using different methods in different time scale37. The estimate varies from ~13 mm/yr in Kashmir35 to 18–20 mm/yr in the central Himalaya34 to 15–16 mm/yr in Bhutan Himalaya37 and 19–20 mm/yr in the Eastern Himalayan syntaxis38. To accommodate the oblique motion between India and southern Tibet in the NW-Himalaya, the convergence is partitioned into right-lateral strike-slip motion on the Karakorum fault system in the back arc and oblique motion in the Kashmir Himalaya along frontal arc, forming NW-Himalayan sliver10. On the other hand, it has argued that in the NE-Himalaya, the Brahmaputra valley has broken apart from the Indian plate and has rotated in a clockwise direction, resulting in the lowering of relative plate convergence rate across the NE-Himalaya37.The GPS derived convergence rates along the Himalayan arc between the Indian plate and southern Tibet have been reported by several authorsIn view of relative contradiction in oblique convergence and eastward extrusion model of Tibet, we propose here a new hypothesis for the NE Himalaya that is reasonably more compatible in the context of regional tectonics and deformation. We argue that the uncertainties in the GPS site velocities (~3.5 mm/yr) and poor spatial distribution of the data points from the Arunachal and Assam-Brahmaputra valley region appear to be inconsistent for demarcation of micro-block’s boundaries and associated deformation patterns. Also, the differential rotation is difficult to constrain (or distinguish) the variation in slip-rate along the strike of the fault system.39. However, lacking of enough common sites among some of the available velocity fields, we estimated an unique velocity field for Bhutan, Arunachal, Assam, Tibet, Bangladesh, Shillong and Indo-Burmese wedge44, by transforming all available velocity field into the ITRF200845 using the National Geodetic Survey’s horizontal time dependent positioning tool (https://www.ngs.noaa.gov/TOOLS/Htdp/Htdp.shtml). Finally, all the measurements were transformed into two relative reference frames, with India-fixed34 and the Assam block fixed37 respectively for two transects B-B’ and A-A’  absorbs about ~16 mm/yr arc normal convergence, although uncertainties in fault parallel velocity limit further interpretations  act50. It has argued that rapid uplift of Shillong Plateau and westward encroachment of the outer Indo-Burmese wedge are closely related49. Maurin and Rangin49, suggested that during the time range from Late Eocene to Early Oligocene, the Shillong Plateau was significantly uplifted, that facilitated the rapid westward propagation of the outer wedge. Further, it has argued that the systematic westward overprinting from thin-skinned tectonics to thick-skinned tectonics was a manifestation of rapid onset of westward wedge propagation. Recently, using seismic, sedimentological, stratigraphic constrains from the Surma Basin and palaeodrainage analyses of the Brahmaputra river, Najman et al50., upholds the westward wedge propagation hypothesis.Geodynamic evolution of the Indo-Burmese arc associated fold-and-thrust belt structure is significantly influenced by the interaction with the Shillong Plateau and Assam-Brahmaputra valley formation processFurther, existence of Shillong Plateau/Assam-Brahmaputra valley in north of the Indo-Burmese arc, a hyper-oblique to strike-slip plate boundary region may acts as a structural buttress for the outer wedge. Therefore, as a consequence westward wedge propagation and systematic change in fold-and-thrust belt spacing are expected in order to resolve space problem in the region. However, fold-and-thrust belt spacing of the outer wedge does not show any systematic variation with respect to increasing distance further south from the Shillong Plateau Fig. . Moreove52. The space and kinematic problems can be resolved by lengthening the merged faults (zipping) or splitting it (unzipping), named as “Zipper junctions”52. We proposed an opening “Zipper junctions” analogy to explain Shillong Plateau/Assam-Brahmaputra valley sliver and Indo-Burmese wedge interaction process , which forms in the backarc domain are the result of the obliquity of relative plate convergence55  are essential conditions for the outer trench (or deformation front) slip partitioning. Similarly, the outer deformation front of MFT in the NE-Himalaya, the Assam-Brahmaputra valley region, also satisfies the essential condition for the slip partitioning  is rare and ing Fig. .Clark and Royden30 has explored the rheological property , by quantifying geometry of the topographic margins  of the material in the channel can be expressed by integrating the velocity of the material over the channel height (h),Again, it can be related to changes in crustal thickness (c) over time:Further, considering c is the average crustal density (2800 kg/m3), g is the gravitational acceleration, and T(x) is the topographic elevation. By considering topographic wavelengths that are long compared to the flexural wavelength of the crust (according to concept of Airy isostatic equilibrium58), changes in topographic relief can be linearly related to variations in crustal thickness. Therefore, the change in topographic elevations over time as a result of flux of crustal material in the lower crust can be expressed by:m = mantle density = 3300 kg/m3.It has been assumed that the lateral pressure gradient in the channel is a function of topography, where ρhttp://www.cgiar-csi.org). We assumed a spatially uniform lower crustal viscosity in the 15 km lower crustal channel. Flux rate was considered to allow the plateau margin of average 5 km elevation to be produced over 20–25 Ma30. Model topographic profile in the Tarim, Sichuan and Nepal Himalaya fault margins, yields an excellent fit with the topography margins for a higher crustal viscosity of about 1021 to 1022 Pa s, however in the Bhutan and Arunachal Himalaya segments a good fit is obtained with lower viscosity of about 1020 Pa s (Fig. Using the above concept, we generated swath topographic profiles across Himalaya and surrounding margins of Tibet Fig.  using toa s Fig. . Using sa s Fig. . FurtherSupplementary Information


As photonic circuits grow in complexity, full quantum tomography becomes impractical, and therefore an efficient method for their characterization8 is essential. Here we propose and demonstrate a fast, reliable method for reconstructing the two-photon state produced by an arbitrary quadratically nonlinear optical circuit. By establishing a rigorous correspondence between the generated quantum state and classical sum-frequency generation measurements from laser light, we overcome the limitations of previous approaches for lossy multi-mode devices10. We applied this protocol to a multi-channel nonlinear waveguide network and measured a 99.28±0.31% fidelity between classical and quantum characterization. This technique enables fast and precise evaluation of nonlinear quantum photonic networks, a crucial step towards complex, large-scale, device production.Integrated photonics is a leading platform for quantum technologies including nonclassical state generation Spontaneous parametric down-conversion (SPDC) and spontaneous four-wave mixing are the two most common processes used for photon generation on chip with the former being the most efficient by far, needing only a few microwatts of pump power for generation rates exceeding several MHz15. Monolithic integration of SPDC sources with multi-port optical circuits has been achieved in several contexts, with applications in quantum communication16, quantum metrology1, spatial multiplexing of heralded single-photon sources17, quantum state generation in nonlinear waveguide arrays2 and small-scale reconfigurable quantum photonic circuits18.Practical applications of quantum photonic technologies19, requiring the collection of statistics from coincidence counts and a quadratically increasing number of measurements with system size. Here we propose and demonstrate a practical method for the characterization of the two-photon wavefunction generated by an arbitrary device with quadratic nonlinearity that uses only laser probes and power measurements. This technique fully reconstructs the spectral and spatial properties of the generated photon pairs from the measurements of bright optical beams and, with optimized hardware, it performs the same number of measurements at least four orders of magnitude faster than the corresponding quantum characterization. Our protocol is of both fundamental and practical importance for the development of integrated quantum photonics technologies including characterization of large-scale wafer production.The near future of quantum photonics will involve an expansion in scale and applications of integrated circuits. However, the characterization of the two-photon state generated by a nonlinear waveguide network is a cumbersome experimental task9 for predicting the two-photon wavefunction produced by a nonlinear device using the analogy between spontaneous nonlinear processes and their classical stimulated counterparts, that is, difference-frequency generation or stimulated four-wave mixing. This technique was demonstrated for spectral characterization of two-photon states23, and fast reconstruction of the density matrix of entangled-photon sources25.A method based on stimulated emission tomography (SET) was proposed26. A possible workaround is to inject the seed beam into each single channel individually then perform a transformation through supermode decomposition to obtain quantum predictions. Regardless, complete knowledge of the linear light dynamics inside the whole structure is required, making SET a multi-step procedure prone to errors and not applicable to ‘black-box’ circuits. Additionally, SET is strictly valid only in the limit of zero propagation losses10, posing a fundamental limitation for the characterization of real optical circuits.However, SET has never been realized on multimode optical networks since it requires injection of the seed beam into the individual supermodes supported by the structure10, posing a stringent restriction for the characterization of more complex devices. In this work, we uncover a fundamentally important equivalence between the biphoton wavefunction and the classical sum-frequency field generated in the reverse direction of SPDC for any multimode non-linear device, overcoming the limitations of previous approaches. Our theoretical analysis is based on the rigorous use of the Green-function method27 (χ2 -nonlinear process (Characterization via sum-frequency generation (SFG), the reverse process of SPDC, gives exact results in the presence of any type of losses. This approach was previously formulated only for single, homogeneous waveguidesmethod27 , and hol process .ωp is injected into waveguide np at the input of the device it produces, by SPDC, the biphoton state  is the index for signal(idler) output waveguide number, n with the frequency ω, and 20. In the classical SFG process shown in ωs and idler frequency ωi are injected into waveguides ns and ni from the SPDC output directions. The generated sum-frequency electric field np.where PSFG, and predict the absolute photon-pair generation rates for SPDC through the relation:We reveal that the sum-frequency field in the undepleted pump regime is directly proportional to the two-photon wavefunction  . From thPp is the pump beam power, dNpair/dωsdt is the photon-pair generation rate per unit signal frequency, and Here, 29 and heated to T=84 °C to obtain phase matching at λ=1550 nm. The waveguides have an inhomogeneous and asymmetric poling pattern along the propagation direction in order to test the generality of the method where laser light propagates in the opposite direction of the SPDC process  contributions were first measured by inputting signal and idler beams into each channel individually. SHG powers were then subtracted from SFG-power measurements. The procedure was repeated and automated with Labview. For SFG-phase measurements, SFG and SHG contributions were separated at the output of the array with the aid of a diffraction grating.ANP, JT, ASS and AAS developed the theory for the SFG-SPDC analogy. FL, AB, SK and BH fabricated the nonlinear device. FL, PF, AB and MV performed the experimental measurements. AM, ASS, AAS and ML supervised the project. FL wrote the manuscript with contributions from all authors.