Pneumococcal conjugate vaccines (PCVs) effectively prevent pneumococcal disease, but the global impact of pneumococcal vaccination is hampered by its cost. The evaluation of reduced dose schedules of PCV includes measurement of effects on immunogenicity and carriage acquisition compared to standard schedules. The relevance and feasibility of trials of reduced dose schedules is greatest in middle- and low-income countries, such as The Gambia, where the introduction of PCV resulted in good disease control but where transmission of vaccine-type pneumococci persists. We designed a large cluster-randomised field trial of an alternative reduced dose schedule of PCV compared to the standard schedule, the PVS trial. We will also conduct a sub-study to evaluate the individual-level effect of the two schedules on carriage acquisition, immunogenicity, and co-administration of PCV with yellow fever vaccine, the PVS-AcqImm trial.PVS-AcqImm is a prospective, cluster-randomised trial of one dose of PCV scheduled at age 6 weeks with a booster dose at age 9 months  compared to three primary doses scheduled at 6, 10, and 14 weeks of age (i.e. standard ‘3+0’ schedule). Sub-groups within the alternative schedule group will receive yellow fever vaccine separately or co-administered with PCV at 9 months of age. The primary endpoints are (a) rate of nasopharyngeal vaccine-type pneumococcal acquisition from 9 to 14 months of age, (b) geometric mean concentration of vaccine-type pneumococcal IgG at 18 months of age, and (c) proportions with yellow fever neutralising antibody titre ≥8 four weeks after administration of yellow fever vaccine. Participants and field staff will not be masked to group allocation while the measurement of laboratory endpoints will be masked. Approximately equal numbers of participants will be resident in each of 28 geographic clusters ; 784 enrolled for acquisition measurements and 336 for immunogenicity measurements.Analysis will account for potential non-independence of measurements by cluster and so interpretation of effects will be at the individual level . PVS-AcqImm will evaluate whether acquisition of vaccine-type pneumococci is reduced by the alternative compared to the standard schedule, which is required if the alternative schedule is to be effective. Likewise, evidence of superior immune response at 18 months of age and safety of PCV co-administration with yellow fever vaccine will support decision-making regarding the use of the alternative 1+1 schedule. Acquisition and immunogenicity outcomes will be essential for the interpretation of the results of the large field trial comparing the two schedules.72821613.International Standard Randomised Controlled Trial Number The online version contains supplementary material available at 10.1186/s13063-021-05949-4. HoweverEPI programmes in low- and middle-income countries are becoming more complicated and costly with the introduction of new vaccines. The addition of vaccines such as PCV, rotavirus vaccine, injectable polio vaccine, meningococcal group A conjugate, human papillomavirus vaccine, and typhoid conjugate challenges the implementation, acceptance, cold-chain capacity, and sustainability of EPIs. The difficulty of introducing such new vaccines has its biggest impact in low- and middle-income countries where the burden of disease is greatest but resources are scarce.The Medical Research Council Unit The Gambia at London School of Hygiene & Tropical Medicine (MRCG at LSHTM) has a long history investigating the burden of pneumococcal disease and pneumococcal vaccination. In 2000–2004, a trial of a 9-valent PCV (PCV9) was conducted in Central and Upper River Regions (CRR and URR) of The Gambia. Vaccine efficacy in children aged 3–29 months was 37% against radiological pneumonia, 77% against vaccine-type (VT) invasive pneumococcal disease (IPD), and 16% against all-cause mortality . In 2009CI, −28–91%) and radiological pneumonia by 27%  [It is now evident that following the introduction of PCV13, herd protection has developed in The Gambia. The annual count of VT IPD in older children in PSP was six to ten before the introduction of PCV13 in 2011. Following the introduction of PCV13, the annual case counts in 2015, 2016, and 2017 were four, one, and zero, respectively (author’s own data). In the 5–14-year age group, IPD incidence declined by 69%  carriage of PCV13 VT in the BHDSS area before the introduction of vaccine was 47% in the under-5-year age group. In 2015 and 2017, the prevalence of vaccine types was 17% and 15%, respectively (author’s own data). The downward trajectory of vaccine-type prevalence from 2009 to 2015 and 2017 suggests that the introduction of PCV13 has substantially reduced the prevalence of VT carriage. However, it is evident that transmission of VT pneumococci continues in the population.The Gambian EPI schedule currently includes birth doses of BCG, hepatitis B, and oral polio vaccine (OPV); visits at 2 and 3 months of age when OPV, pentavalent, rotavirus, and PCV13 vaccines are scheduled; a visit at 4 months of age when OPV, injectable polio (IPV), pentavalent, rotavirus, and PCV13 vaccines are scheduled; a visit at 9 months of age when measles-rubella and yellow fever vaccines are scheduled; conjugate meningococcal group A vaccine scheduled at 12 months of age was introduced in 2018; at 18 months of age, OPV and measles-rubella vaccines are scheduled. Human papilloma virus vaccine delivered to school-age girls was introduced in 2020. In the event that polio is eradicated, then OPV will be phased out and be replaced by IPV. The EPI is also considering the introduction of a conjugate typhoid vaccine. Thus, in recent years, the EPI schedule has introduced several additional injectable antigens and more may be added in the future.Several studies indicate that immunological priming for an optimal PCV booster dose-response may be more effective with fewer primary doses , 13. In This trial joins a global initiative to generate evidence about reduced dose schedules for PCV. WHO is engaged with this initiative having held a consultative meeting in February 2016. Studies investigating reduced dose schedules for PCV are underway in South Africa, Vietnam, India, and the UK. The UK introduced a 1+1 schedule nationwide in 2019. Our trial in The Gambia is critical to provide evidence from a typical African setting.In Fiji and The Gambia, one dose of PCV at 2 or 3 months of age significantly reduced carriage of VT pneumococci at 9 months of age , 15. A DData from a recent UK trial show that the immunogenicity of the PCV booster dose using a 1+1 schedule was equivalent to, or superior to, a 2+1 schedule for nine of the 13 serotypes in PCV13 . Of impoThe duration of protection of PCV is poorly defined, but the potential for greater antibody persistence following a booster dose compared to doses in early infancy , 19 suggDemonstrating a superior immune response at 18 months of age and reduced VT acquisition following the booster dose will provide supporting evidence to the PVS field trial assisting decision-makers in considerations of reduced dose schedules. Reducing the number of PCV doses in EPI schedules will impact on multiple elements of the challenges posed by this vaccine: reducing the costs to countries and GAVI, reducing the number of injections in schedules and providing greater flexibility for the inclusion of other vaccines, reducing staff time and cold-chain requirements, and ultimately making EPI programmes more acceptable and sustainable. If countries can safely transition to 1+1 schedules, the global uptake of PCV should accelerate with greater and more sustainable reductions in pneumococcal disease.The primary objectives will be achieved by testing the following hypotheses:• The serotype-specific IgG concentration for VT at 18 months of age will be greater in 1+1 compared to 3+0 schedule recipients.• The rate of NP VT acquisition from 9 to 14 months of age will be reduced in 1+1 compared to 3+0 schedule recipients.• The proportion of participants with protective titres of yellow fever (YF) neutralising antibodies will be non-inferior in those who receive co-administered compared to separately administered PCV and yellow fever vaccines.The secondary objectives are to compare in alternative versus standard schedule groups the:• Rate of NP acquisition of non-VT pneumococci between 9 and 14 months of age• Proportion of participants with NP VT colonisation at 6, 9, and 18 months of age• Proportion of participants with pneumococcal VT IgG concentration ≥0.35 μg/ml, 4 weeks after the primary series, 4 weeks after the booster dose at age 9 months, and at 18 months of age• Pneumococcal VT opsonophagocytic antibody (OPA) titres following a single dose at age 6 weeks, following three primary doses, following the booster dose at age 9 months, and at 18 months of age• Geometric mean concentrations (GMCs) of pneumococcal VT IgGs 4 weeks after administration of PCV13 at 9 months of age with and without co-administration with the YF vaccineThe PVS acquisition/immunogenicity (PVS-AcqImm) is a parallel group, unmasked, cluster-randomised trial of the individual-level effect  of two different schedules of PCV13. This trial is nested within the PVS field trial but designed for interpretation of effects at the individual level. This is a phase IV trial involving licenced products comparing alternative and standard schedules for PCV13 and separate versus co-administration with the YF vaccine. We will test the superiority of the 1+1 compared to the 3+0 schedule to reduce the rate of NP acquisition of VT pneumococci from 9 to 14 months of age and in terms of the immune response at 18 months of age. We will test the non-inferiority of the immune response following separate and co-administration of PCV and YF vaccines. Approximately equal numbers of participants will be enrolled in each cluster allocated to the two groups in a 1:1 ratio.The PVS-AcqImm trial is being conducted in Upper River Region (URR) in the area covered by the BHDSS Fig. . The triThe sampling frame for selection to receive the interventions will be all infants resident in the 28 selected clusters. Residency will be defined as:• Born to or cared for by a parent or guardian who is resident for greater than 4 months as confirmed by BHDSS records or a household visit with the report of the household or compound head. Provisional residency may be established by the verbal report of the parent or guardian.• Born to or cared for by a parent or guardian who intends to be resident for greater than 4 months with verification at a household visit and report of the household or compound head. Provisional residency may be established by the verbal report of the parent or guardian.The sampling frame will be developed as part of the BHDSS in the trial area. Pregnancies and births in all households are registered from 4-monthly BHDSS enumerations of each household, village reporter records, and registration at EPI clinics. These data are electronically recorded in the field and synchronised centrally on a weekly basis. A verified and updated sampling frame is available for use each week. The sampling frame lists the mother’s name, infant’s name, date of birth, father’s name, name of household head, village name, compound number, and individual ID number.All resident infants are eligible for enrolment in the large PVS field trial. Approximately 28 infants in each of the 28 clusters will be enrolled in the PVS-AcqImm trial with 784 participants for the study of pneumococcal carriage and 336 for measurements of immunogenicity .Consent for the collection and use of participant data and biological specimens is specified in the trial information sheet and consent form. The consent form includes specific confirmation, marked on the form and entered in the database, confirming consent for the collection of specific numbers and types of specimens, future research using the specimens, shipping of specimens overseas, and use of unidentified data via MRCG authorised data repositories.Our choice to compare the 1+1 (doses of PCV scheduled at 6 weeks and 9 months of age) versus 3+0  schedules for PCV vaccination is based on the hypotheses that the 1+1 schedule will provide sufficient, but inferior, direct protection between 2 and 9 months of age, during which time the risk of VT disease in our setting is very low, and superior herd protection, and similar overall effectiveness compared to the 3+0 schedule. We also hypothesise that the 1+1 schedule will provide superior direct protection between 9 and 18 months of age compared to the 3+0 schedule. Finally, we chose to compare co-administration and separate administration of PCV and YF vaccines at 9 months of age as the YF vaccine is scheduled at 9 months of age in most African countries.The experimental intervention in this trial is the scheduling of PCV13 for infants resident in geographic clusters of villages to receive the first dose due at 6 weeks of age and a booster dose scheduled at 9 months of age. The standard intervention is the scheduling of PCV13 for infants with doses due at 6, 10, and 14 weeks of age. A further intervention in the alternative schedule group is the scheduling in one group of the YF vaccine to be administered at 10 months of age and PCV13 at 9 months of age and in another group co-administration of PCV13 and YF vaccines at 9 months of age. Following informed consent, infants are registered on the trial and a trial identification sticker is fixed to their infant welfare card. At each visit, trial staff identify the infant and the group allocation, based on the trial sticker and residential location. Trial staff record whether infants are migrating, or intend to migrate, within or out of the trial area.PCV13 (Prevnar 13®) vaccine, manufactured by Pfizer Ltd, is licenced in many countries and has been approved for use in The Gambia since 2011. Stabilised YF vaccine is manufactured by Institut Pasteur in Dakar, with WHO prequalification in 2001. The EPI procures these vaccines through global systems coordinated by UNICEF. This trial delivers PCV13 and YF vaccines in collaboration with, and through the structures of the Gambian EPI, and under the operational conditions of the public health system. The EPI receives PCV13 into a central cold storage facility. The vaccine is transported to the regional centres either by a specially designed EPI ‘cold van’ or by cold storage units carried by Regional Health Directorate (RHD) vehicles. The RHD in URR is based in Basse. The URR RHD stores vaccine in solar refrigerators in Basse. From Basse, small volumes of vaccines are distributed on a monthly basis to five ‘base clinics’ in Basse, Gambisara, Demba Kunda, Fatoto, and Sabi. These five base clinics administer vaccines in the 28 different geographic locations involved in PVS-AcqImm. Solar vaccine refrigerators are used for storage at the base clinics. The trial uses the existing EPI procedures to monitor and record the vaccine accountability and cold-chain documentation with daily logs.Immunisation is undertaken at four of the fixed health centres in the study area on 1 or 2 days each week. Mobile clinics in the other 24 sites are held once or twice per month. Given the infrequency of EPI clinics, there is generally some delay in the time that vaccines are actually received. In 2016, coverage in the BHDSS of three doses of PCV at 12 months of age was 92% and coverage of one dose of measles vaccine was 82%.Trial participants are discontinued from participation in the study if:• Any clinically significant adverse event, intercurrent illness, or other medical condition or situation occurs such that continued participation in the study would not be in the best interest of the participant• The parent or guardian so desiresParticipants who attend an EPI clinic outside their cluster and within the study area, but continue to reside in their original cluster, receive the trial schedule originally indicated on their infant welfare card and on the trial sticker. If participants migrate within the study area before completing their PCV schedule, they continue to receive the trial schedule allocated in the cluster of their new residence. Participants who migrate after completing their PCV schedule do not receive any further doses of PCV in the cluster of their new residence. Parents in the 1+1 group are advised that if they migrate permanently out of the trial area they should attend the next available EPI clinic to complete the routine schedule for PCV. If an infant allocated to the 1+1 group visits an EPI clinic outside the study area, the parent is instructed to request that their child receive the trial schedule indicated on the infant welfare card and study sticker. The study sticker includes free-call telephone numbers so that parents, or EPI staff outside the study area, may call for guidance. Infants resident in the alternative schedule clusters who decline consent are assigned to the national standard schedule.Adherence to the trial vaccination schedules is facilitated by exclusion criteria including an intention to migrate out of the study area in the next 18 months. Also, lists of infants allocated to the 3+0 group who have not completed three doses by the age of 5 months, and lists of infants allocated to the 1+ 1 group who have not received the booster dose by the age of 11 months, are generated every month to guide defaulter tracing at home visits throughout the study area.PCV13 has been co-administered with measles-mumps-rubella  and measParticipants admitted to the hospital with an acute medical problem will have a blood culture taken, a plasma aliquot stored, a rapid malaria test done, and haemoglobin concentration measured. Samples will not be collected from children admitted electively or those with surgical problems, trauma, acute burns, or non-infectious neonatal problems. Participants admitted with suspected sepsis defined according to standardised criteria will have a blood culture done and those with suspected meningitis will have a blood culture done and a lumbar puncture performed. For those admitted with clinical pneumonia, a danger sign, or focal chest signs, a blood culture and chest X-ray will be performed and pleural fluid or lung aspirate obtained as clinically indicated. Other investigations will be done according to the clinical judgement of the attending clinician.The trial may be stopped early if there is evidence that the risk of pneumococcal disease is greater in one compared to the other trial group. If the Data Monitoring Committee (DMC) recommends that the trial be stopped early, a joint meeting of the DMC, Trial Steering Committee (TSC), and Central Stakeholder Committee will make a recommendation to the Sponsor regarding post-trial procedures, including whether a dose of PCV be administered to children in a group found to be inferior. LSHTM carries clinical trial/non-negligent harm insurance and medical malpractice insurance applicable to this trial.The primary outcome of the immunogenicity study is serotype-specific IgG for VT at 18 months of age, analysed as the ratio of the geometric mean concentrations (GMC) in 1+1 compared to 3+0 participants. If serotype-specific IgG concentrations at 18 months of age are greater in the 1+1 compared to the 3+0 age group, we will infer that the schedule including the PCV booster dose provides a superior immune response to the standard schedule at 18 months of age. This superior immune response may be associated with reduced acquisition of VT colonisation and reduced risk of VT disease in the second year of life.The primary outcome of the acquisition study is the rate of NP VT acquisition from 9 to 14 months of age in 1+1 compared to 3+0 schedule participants. The rate of acquisition will be the number of VT pneumococcal carriage acquisitions detected in six monthly NP specimens collected between the ages of 9 and 14 months. If the rate of VT acquisition is reduced in 1+1 compared to 3+0 participants at this age, we will conclude that the PCV booster dose provides superior protection from acquisition at this age with the implication of reduced transmission and potentially greater herd protection.The primary outcome of the investigation of co-administration of PCV13 and YF vaccines is the proportion of participants with YF neutralising antibody titres of ≥1:8 comparing those who receive the vaccines separately or co-administered. If the difference in proportions in the two groups is non-inferior, we will conclude that there is no immune interference when the vaccines are co-administered. This finding would support a policy of co-administration of PCV13 and YF vaccines.Pneumococcal VT opsonophagocytic activity (OPA) following a single dose at age 6 weeks, following three primary doses, following the booster dose at age 9 months, and at 18 months of ageThe proportion of participants with GMC of pneumococcal VT IgGs ≥0.35 μg/ml, 4 weeks after the primary series, 4 weeks after the booster dose at age 9 months, and at 18 months of ageGeometric mean concentrations of pneumococcal VT IgGs 4 weeks after administration of PCV13 at 9 months of age, with and without co-administration with the YF vaccineSecondary immunogenicity outcomes include comparison in alternative versus standard schedule groups, of:These secondary immunogenicity outcomes will demonstrate the functionality of anti-pneumococcal IgG antibodies throughout the different schedules, which is required when investigating PCVs . In addiSecondary acquisition outcomes include the:• Rate of NP acquisition of non-VT pneumococci between 9 and 14 months of age• The proportion or participants with NP VT colonisation at 6, 9, and 18 months of ageThese secondary outcomes will provide information on the potential for the PCV13 booster dose to increase the rate of non-VT pneumococcal acquisition and the comparative effects of the two schedules on VT prevalence at different age points.Participants in the trial are enrolled in one of three different groups with administration of intervention PCV13 and YF vaccine schedules as shown in Table For measurement of the effect of the PCV booster dose on the rate of acquisition of VT pneumococci, the smallest clinically important hazard ratio to detect is 0.75. A Dutch study of VT colonisation following a PCV booster dose at 12 months of age compared to no booster showed a relative risk reduction of 0.64 . Thus, t5; that is, the expected proportion of participants who acquire VT pneumococci is 0.67. Using a survival analysis logrank test and assuming a 5% level for statistical significance and 80% power, the study requires 784 participants with 28 participants in 14 clusters in each group to detect a hazard ratio of 0.75. The sample size was inflated from 392 to 532 with the intra-cluster correlation factor and then to 784 when taking into account rates of death, loss-to-follow-up, withdrawal, cross-over, and censoring due to no detection of vaccine-type colonisation.The current prevalence of NP VT pneumococcal colonisation in those aged less than 2 years in the study area is 17% (author’s own data). This measurement used selection of one or more morphologically different colonies, while the latex sweep serotyping method used in this study is more sensitive to multiple serotype colonisation so we assume a slightly higher prevalence. The assumptions included in the sample size calculations are that the baseline risk of VT colonisation at any one point in time is 20%, participant withdrawal of 2%, death rate of 2%, loss to follow-up of 3%, a 4% rate of cross-over between clusters, and 35% censoring of participants who do not experience VT colonisation. We have used local baseline data on the variability of cluster-specific pneumococcal carriage to calculate an intra-cluster correlation of 0.02. The probability that VT pneumococci will be detected in the five NP swabs collected between 10 and 14 months of age in the 3+0 group is 1–0.80α = 0.025 and β = 0.9. For the first primary endpoint, our interest is in the relative magnitude of the IgG response in the two groups. Thus, we will test the alternative hypothesis of a ≥1.8-fold difference in GMCs between the 1+1 and 3+0 groups for ≥10/13 serotypes at 18 months of age. Baseline GMCs and their standard deviations for each serotype in the 3+0 group were taken from findings in the USA [m − 1) × ICC = 1 + (8 − 1) × 0.01, the effective sample size in each group is n/VIF = 106/(1 + (8 − 1) × 0.01) = 99. Based on local cluster-wise variation in the mean prevalence of VT colonisation, the intra-cluster correlation = 0.01. Thus, simulations assumed 99 participants per group. Data were simulated so that there was a 1.8-fold difference in the IgG levels in the two groups, and differences in serotype-specific GMCs were tested using a t-test. Within-subject correlation between the multiple serotype-specific endpoints was captured by a subject-level variation term with standard deviation 0.4 in a random-effects linear regression model. Superiority was declared for the overall conclusion if at least 10 out of 13 tests rejected the null hypothesis in 5000 simulations. Table The immunogenicity study includes two primary endpoints and the sample size calculation for each used  the USA  and Fiji the USA . Non-indICC = 0.01, and seven or eight participants per cluster (seven to account for loss to follow-up). Power to detect a 10% absolute difference in proportions with seven participants per cluster was 86% and 90% with eight participants per cluster. With the inclusion of 28 clusters and seven or eight participants per cluster, this leads to a sample size of 196 or 224 participants.For the second primary endpoint, power was calculated using a formula for cluster-randomised non-inferiority study using Nquery + nTerim software. Calculations assumed that 95% of participants had YF neutralising antibody titres ≥ 1:8,  ICC = 0.All resident infants will be eligible for enrolment in the PVS field trial, which enrols approximately 150 participants per week. PVS participants may also be enrolled in PVS-AcqImm; 28 infants in each of the 28 clusters will be enrolled with a total of 784 participants for repeated measures of pneumococcal colonisation. Of the 392 participants resident in the 1+ 1 clusters, a sub-sample of 224 will be enrolled for measurement of immunogenicity endpoints, 16 in each of the 14 clusters. Of the 16 infants targeted in each 1+1 cluster, a target of eight will be allocated to 1+1 with PCV13–YF vaccine co-administration and a target of eight allocated to separate administration, i.e. 112 infants will be allocated to each of the co-administration and separate administration groups . In the 1+1 clusters, infants will be allocated to PCV13–YF vaccine co-administration or separate administration groups according to a prepared random allocation list including 14 clusters with a target of 16 infants (less or more than 16 may be enrolled per cluster) in each cluster. In each of the 3+0 clusters, a target of eight infants will be enrolled in each cluster for measure measurement of immunogenicity endpoints, i.e. a total of 112 participants. If insufficient numbers of participants are enrolled in a cluster in 1 month, then additional participants may be enrolled in that cluster in the following month. If a participant withdraws or is lost to follow-up, additional participants may be enrolled in both groups to ensure sufficient enrolment and the use of identical procedures in the two groups.Sixty-eight PVS trial clusters were randomised using a blocked scheme to ensure similar numbers of clusters were assigned to each group. Randomisation was stratified by a binary variable correlated with ‘high’ or ‘low’ cluster-level incidence of clinical pneumonia. Randomisation was carried out in permutations using the above stratification until selections achieved balance in terms of the presence of a health centre and balance on population size between the two groups. In order to select the stratifying variable of cluster-level incidence of clinical pneumonia, the cluster-level prevalence of ‘high’ or ‘low’ VT carriage in children with clinical pneumonia in the BHDSS was correlated with cluster-wise population density, rates of hospitalisation, clinical pneumonia, radiological pneumonia, IPD, and mortality. Of the five listed outcomes, clinical pneumonia incidence had the closest correlation with VT carriage prevalence and thus was chosen as the stratifying variable.Individuals selected for participation in PVS-AcqImm will be selected from the 28 clusters closest to Basse Fig. . Of thesPre-prepared random assignment lists, by cluster, are used at the time of enrolment to determine participant assignment to different schedules of blood collection. In the 1+1 clusters, the pre-prepared random assignment lists specify assignment to the PCV–YF vaccine co-administration or separate administration groups.A public event was held to announce group allocations of each cluster of villages in the PVS trial area. Representatives of each cluster were present at the public event. Selection of the randomisation list at the public event involved random selection of one of 100 valid randomisation lists. Thus, the investigators and cluster representatives had no knowledge of the allocation sequence at the time of group allocation.An independent statistician prepared the cluster randomisation lists. Trial staff enrol the participants. The trial data manager produced the lists for random assignment of participants to different schedules of blood collection and co-administration or separation administration of PCV–YF vaccines.Vaccinators and parents will be aware of the schedules used. Laboratory staff will be blinded as specimens will be labelled with a unique identification number that does not identify the study group. Blinding of laboratory staff will avoid bias given the laboratory-based objectives of the study. Statisticians will analyse date in a pseudo-blinded fashion with the two groups identified by an indicator label rather than the identity of each group.Given that participants are not blinded to their group allocation, a procedure for unblinding is not needed.Trials, TRLS-D-21-01145).Participants are under passive surveillance for clinical events as per the procedures of the PVS field trial. If participants present to a health facility in the trial area, staff will provide standardised evaluation, investigation, and recording of clinical safety events in an electronic medical record . Details of the surveillance for clinical safety events are provided in an accompanying protocol paper for the PVS field trial  is done offline using encrypted devices which is then synchronised with a central server on a weekly basis. Trial staff attend all EPI clinics, confirming the identity of all infants and record immunisation data in real time. Trial staff generate source data on electronic devices at regular visits as per the trial timeline.Data entered into eCRFs will be monitored for completeness and consistency against the relevant source documents. Independent trial monitors undertake 100% verification of source data for the primary endpoints and informed consent. Anomalies identified are reconciled with the source.Front-end data quality checks are programmed into the data capture application. Backend edit checks and validation checks are built-in to monitor the validity of data . Data queries are generated weekly by the data manager for resolution by trial staff. Reports of data quality are generated periodically.The trial database is housed on a secure network server with restricted access to the backend. The backend comprises a MSSQL database which will be regularly backed up as part of the organisation’s disaster recovery plan. An in-house Web-based application is used for the database structure, using the PHP\ASP.Net platform connecting to the MSSQL backend database. A recovery point objective is set so that systems and data will be restored back to their prior status 24 h prior to a failure. Daily and monthly backups of all media servers are done to achieve this objective.The e-CRF is used as the specification for the design of the database. An annotated CRF indicates the relationship between the variable names in the database and the fields in the CRF. A data dictionary uses a standard template and includes a list of database variable names, variable descriptions, data types, and sources, valid values, and in-built edit checks. Standard data coding will be used (e.g. latest MedDRA version accessed through the Internet will be used for adverse event reporting).The long-term storage of research records is done in accordance with MRCG policies and procedures for archiving. All paper records will be held for at least 10 years in the Unit’s archiving facility. Data in electronic format will be held indefinitely on our Electronic Data Repository. The trial is run in compliance with the MRC Corporate Information Security Policy  and the No subject identifiable information  is entered into the database. Identifiable information is stored in the electronic medical record, vaccination and BHDSS databases, encrypted and password-protected, and accessible to only a limited number of staff involved in the care of patients. Trial monitors only access pre-specified, non-identifiable data. Informed consent documents are stored in locked, fire-resistant filing cabinets to which only the Principal Investigator and a limited number of delegated clinical trials personnel have access. Data are backed up at the end of each day in the field on encrypted flash drives to prevent any data loss during transit. All computers within MRCG are access controlled with strong password policies that prevent unauthorised access to networked user machines. Users to whom network access has been given are granted necessary privileges to the trial database based on their trial roles.The trial collects blood and NP specimens but procedures do not include genetic or molecular analyses of human material.2. Identification of pneumococcal colonies follows recommended methods [Flocked nylon swabs are placed in media and transported to the MRCG laboratory in Basse within 6 h or placed in a dry shipper for later transport to Basse. NP specimens are processed in the Basse laboratory. A 10-μl loop of vortexed NP specimen is inoculated onto blood agar with 5% gentamicin and incubated in CO methods . Specime methods . The BasBlood specimens collected in serum separation tubes are transported in a cool container to the Basse laboratory within 6 h, centrifuged, and aliquoted. Aliquots are stored in Basse at −70 °C prior to shipment to MCRI for pneumococcal serology and to the Bernard Nocht Institute for Tropical Medicine (BNITM), Hamburg, for YF serology.Serotype-specific anti-pneumococcal IgG and OPA will be measured in the Pneumococcal Laboratory at MCRI. A standardised ELISA will be used. Microtitre wells are coated with pneumococcal polysaccharide, depending on the serotype. To neutralise cell wall polysaccharide antibodies, diluted serum samples are incubated with cell wall polysaccharide and polysaccharide of serotype 22F, before further dilutions. A reference serum  is used and incubated with cell wall polysaccharide and 22F polysaccharide. Horseradish peroxidase-conjugated anti-human IgG and the TMB Peroxidase Substrate system is used for detection. Three control sera are used to assess inter-assay variation. A standardised and validated OPA  will meaNeutralising antibodies against the YF virus will be assayed at BNITM. Neutralisation titres will be expressed as the serum dilution yielding ≥50% neutralisation . If a complete infection is observed at all serum dilutions, the neutralisation titre is recorded as < 1:4. Seroprotection is defined as a neutralisation titre ≥1:8.The primary analysis of the effect of the PCV13 booster dose on pneumococcal acquisition will use data from the six NP specimens collected between 10 and 14 months of age. Detection of homologous serotypes within an individual on multiple occasions will not be counted as multiple acquisitions. That is, the maximum number of acquisitions for one participant will be five. Analysis will employ a Cox proportional hazards regression model comparing the hazard ratio for VT acquisition in the 1+1 compared to the 3+0 group including variables to adjust for previous serotype-specific acquisition and trial cluster. The interpretation of the models will be at the individual level.Secondary analyses of the endpoint of non-VT acquisition will use the same approach as described above. For the endpoint of VT prevalence at 6, 9, and 18 months of age, we will compare proportions while also taking into account the trial cluster.Analyses of immunogenicity data will be per-protocol for non-inferiority endpoints and otherwise by modified intention-to-treat with all individuals included by randomised group if they have a laboratory result. For the primary endpoint, we will compare the VT serotype-specific GMCs in the two groups using linear regression including co-variates of baseline IgG, cluster, and season of PCV13 booster if associated with GMC, using separate models for each serotype. For calculation of GMCs, a logarithmic transformation will be applied to the IgG concentration value prior to analysis. The 1+1 schedule response will be deemed superior if the ratio of GMCs is ≥1.8 for ≥10/13 serotypes at the 5% level of significance.Proportions of participants with YF neutralising antibody titre ≥1:8 will be calculated with exact binomial 95% confidence intervals (CI). The CI for the difference in the proportions will be computed using the Miettinen-Nurminen method (or another appropriate method), including cluster and/or season as needed. If the lower limit of the CI for the difference in proportions is greater than −10%, response in the co-administration group will be deemed non-inferior.There are no planned interim analyses.An exploratory analysis will include repeated acquisitions of homologous serotypes, that is counting repeated acquisitions separately, testing whether the rate of individual VT acquisitions, including repeated acquisitions, is reduced in the 1+1 compared to the 3+0 group.The protocol specifies enrolment of additional participants if a participant is lost to follow-up. Analysis will handle events of non-adherence by restricting the data included based on certain conditions. Receipt of the third dose of PCV13 in the 3+0 group at greater than 23 weeks of age will be considered outside the eligible window for per-protocol analysis. Receipt of the booster dose of PCV13 in the 1+1 group at greater than 11.5 months of age will be considered outside the eligible window for per-protocol analysis. Receipt of the YF vaccine in the 1+1 YF co-administration group at greater than 11.5 months of age will be considered outside the eligible window for per-protocol analysis. Receipt of the YF vaccine at greater than 12.5 months of age by infants allocated to separate administration of PCV13 and YF vaccines will be considered outside the eligible window for per-protocol analysis. Inclusion of the age 18 months blood specimen in the per-protocol analysis will require collection no later than 20 months of age. Analyses will be conducted with the available data.For practical reasons, this time period for trial-related publications may be indicative and might need to be revised if delays occur. Different periods may be applied to different datasets, e.g. to take account the complexity of cleaning and documentation.Timing will depend on the trial’s data collection patterns.In relation to timing, the terms could, for instance, be expressed as follows: ‘6-months after the end of the current grant period’, ‘12-months after new data collection to allow for data cleaning and documentation’, or ‘3-months following the first publication of findings based on the data’.The protocol is available on request. The data generated will be suitable for sharing in an anonymised format. The data will be in CDISC ODM format that is an internationally recognised standard. Data will not be deposited into a central repository but held securely by MRCG. The trial is registered with the International Standard Clinical Trial Registry Network (ISCTRN) to maximise its visibility to other interested parties. Summary data will be provided through ISCTRN at the end of the study. Data sharing will follow MRCG policy . ApplicaStatistical code will be available on request.The trial management group meets every week and includes the Principal Investigator, Trial Epidemiologist, Trial Coordinator, Project Manager, Data Manager, and Higher Laboratory Scientific Officer. The Trial Steering Committee (TSC) is composed of a Chairperson, an expert clinician, an expert trialist, an expert laboratory scientist, the national EPI Programme Manager, members of the URR and CRR RHDs, two community representatives, and a Sponsor representative.The DMC is independent of the Sponsor and composed of a Chairperson, expert clinician, expert statistician, and an independent statistician. The role of the DMC is described in its Charter see  and is tAdverse events are defined according to the ICH Harmonised Guideline for GCP E6(R2). Due to the established safety record of PCV13 and YF vaccines, the trial does not record solicited events of reactogenicity. The trial uses an electronic vaccine record system to record unsolicited events of reactogenicity when reported by caregivers within 7 days of a dose of PCV13. Unsolicited events of reactogenicity following a dose of the YF vaccine are recorded up to 1 month post-administration. Serious adverse events (SAEs) are tabulated in reports every 3 months. Severe adverse drug reactions (SADRs) and serious, severe unexpected serious adverse reactions (SUSARs) are reported to the Sponsor within 24 h. The Sponsor reports SADRs and SUSARs to the Gambia Medicines Control Agency (GMCA), while PI reports these events to the Gambia Government/MRCG Joint Ethics Committee, LSHTM Ethics Committee, TSC, and DMC.Follow-up and resolution of SAEs are recorded in electronic reports. The Sponsor, TSC, and DMC are informed of SAEs at each meeting. The MRCG Clinical Trials Department Coordinator reports SAEs to the GMCA. The PI reports SAEs to the TSC and DMC. SADRs and SUSARs will be reported to the GG/MRC JEC within 5 working days. Deaths unrelated to the intervention are reported to the GG/MRC JEC at the next meeting. Information on unanticipated changes that may increase the risk to participants or may affect adversely the safety of the participants or the conduct of the trial or that could alter the EC’s approval to continue the trial will be reported to the Sponsor, GG/MRC JEC, LSHTM EC, GMCA, TSC, and DMC in writing within 2 working days.The GG/MRCG JEC may audit the conduct of the trial at any time, independent of the investigators and Sponsor. The trial management group meets weekly to review progress in recruitment, clinical endpoint surveillance, and data quality. The trial management group meets monthly to review standardised indicators of quality assurance of all study procedures and complaints. The TSC meets 1 to 2 months after every DMC meeting, which occur approximately every 4–6 months. The TSC sets targets for recruitment, data collection, and protocol compliance. All trial-related complaints are reviewed by the TSC. The trial statistical analysis plan will be submitted for approval by the TSC. The TSC considers new information relevant to the trial, including reports from the DMC and the results of other studies that may have a direct bearing on the future conduct of the trial. Annual reports are submitted to the GG/MRCG JEC and LSHTM EC which document progress in recruitment, SAEs, and protocol deviations and violations.Protocol amendments are reported to the Sponsor, trial monitors, SCC, GG/MRCG JEC, LSHTM EC, GMCA, DMC, TSC, and clinical trial registry (ISRCTN). Deviations from the protocol are fully documented using a non-adherence/compliance report form.The investigators intend to publish the results of the trial in peer-reviewed scientific journals. All trial publications will follow MRC guidelines for Open Access publishing. The results of the study will be disseminated to the parents/guardians of each participant and the communities in the study area. The findings of the trial will be presented to the Central Stakeholders Committee that includes representatives of the Ministry of Health EPI and other regional and central health authorities. The results of the trial will be presented to the WHO EPI department and its Strategic Advisory Group of Experts on Immunisation.The start of recruitment in the trial was delayed in 2020 due to restrictions related to the COVID-19 pandemic. Since the beginning of recruitment, we have not experienced any significant interruptions to trial implementation.The current protocol version is 5.0, dated 24 May 2021. Recruitment began on 14 September 2020. Recruitment was completed on 28 October 2021.Additional file 1:. Model Informed Consent form.


While the mechanisms by which chemical signals control cell fate have been well studied, the impact of mechanical inputs on cell fate decisions is not well understood. Here, using the well-defined system of keratinocyte differentiation in the skin, we examine whether and how direct force transmission to the nucleus regulates epidermal cell fate. Using a molecular biosensor, we find that tension on the nucleus through linker of nucleoskeleton and cytoskeleton (LINC) complexes requires integrin engagement in undifferentiated epidermal stem cells and is released during differentiation concomitant with decreased tension on A-type lamins. LINC complex ablation in mice reveals that LINC complexes are required to repress epidermal differentiation in vivo and in vitro and influence accessibility of epidermal differentiation genes, suggesting that force transduction from engaged integrins to the nucleus plays a role in maintaining keratinocyte progenitors. This work reveals a direct mechanotransduction pathway capable of relaying adhesion-specific signals to regulate cell fate. Physical forces and the architecture of the extracellular environment are an emerging area of cell fate regulation . Cells sThe interfollicular epidermis is an excellent model to explore how mechanical inputs regulate cell fate. Epidermal stem cells adhere to the underlying basal lamina through β1 integrin-based adhesions and β4 integrin-based hemidesmosomes , which mHere, we explore whether mechanical cues can regulate keratinocyte cell fate via tension on the nucleus via the linker of nucleoskeleton and cytoskeleton (LINC) complex. The LINC complex, composed of tail-anchored Nesprins integrated into the outer nuclear membrane and SUN proteins integrated into the inner nuclear membrane, spans the nuclear envelope to mechanically integrate the cytoplasmic cytoskeleton and the nuclear interior – specifically the nuclear lamina and its associated chromatin . While tSeveral lines of evidence suggest that epidermal differentiation is regulated by the nuclear lamina, yet the mechanisms remain unknown. First, during basal stem cell differentiation, a large chromosomal region termed the epidermal differentiation complex (EDC), which consists of 60 consecutive genes necessary for epidermal stratification and the production of the cornified envelope, relocates away from the nuclear lamina toward the nuclear interior to be transcriptionally activated . FurtherHere, we provide evidence that the LINC complex regulates epidermal differentiation in vitro and in vivo. Using a novel molecular biosensor to measure forces exerted on LINC complex molecules, we find that tension is high on the LINC complex and A-type lamins in epidermal stem cells in an integrin-dependent manner and is reduced upon differentiation. In mouse keratinocytes (MKCs) lacking LINC complexes, we observe precocious differentiation in vitro and expansion of the differentiated, suprabasal layers of the skin in vivo. This work suggests that tension from integrins is communicated through the LINC complex to the nuclear lamina to maintain the progenitor state of basal keratinocytes, providing a potential mechanism by which cell fate is regulated directly by mechanical cues.In order to visualize tension on the LINC complex in living cells, we generated a molecular biosensor to measure force on individual Nesprin proteins. To this end, we inserted a tension sensor module composed of the fluorescence resonance energy transfer (FRET) pair mTFP and Venus connected by an elastic flagelliform linker  into theWe transfected primary undifferentiated MKCs cultured on fibronectin-coated glass bottom dishes with either the N2G-JM-TSMod or NoT_TSMod construct; both were targeted efficiently to the nuclear envelope when expressed at moderate levels . Indeed,β1 integrin (Itgb1) null MKCs plated on glass coverslips coated with FN had higher N2G-JM-TSMod FRET ratios (lower tension) than WT controls  substrates coated with fibronectin (FN) . We obsecontrols , K. As Mntegrins , these oPrimary MKCs can be induced to differentiate in vitro by elevating extracellular calcium levels, leading to the formation of cohesive colonies that engage E-cadherin-based cell-cell adhesions and subsequently differentiate as assessed by upregulation of markers expressed in epidermal suprabasal layers in vivo . In our Given that the LINC complex has been shown to transmit tension from the cell substrate to the lamin network at the nuclear periphery , we examSprr1b mRNA, which is upregulated in differentiated MKCs and is found in the EDC. We find that MKCs at the colony interior express more Sprr1b and Ivl (involucrin) mRNA 24 and 48 hr after induction of differentiation compared to cells at the colony periphery  for eriphery . These d-/-/Sun2-/-Sun1 (hereafter Sun dKO) mice die after birth .Histological analysis of E15.5 skin tissue revealed a thickening of the epidermis in the Sun dKO mouse compared to WT littermates . ImmunosTo further examine whether the thickened epidermis of Sun dKO mice resulted from changes in differentiation and/or proliferation, we performed an EdU pulse chase experiment. We labeled proliferating cells in embryonic WT and Sun dKO mice with EdU and examined the skin after 24 hr . ImmunosSun1/2+/-) and Sun dKO mice at P0.5. The basal layer-basal lamina interface in Sun1/2+/- and Sun dKO skin appeared indistinguishable, displaying tight association in mice from both genotypes , and S100a14 mRNA levels in β1 integrin null MKCs. β1 integrin null MKCs displayed precocious expression of all three of these epidermal differentiation genes  relative to WT MKCs (19 peaks) . SpecifiSprr2a3 . We nextSprr2a3 . We furtSprr2a3 . Thus, tMyod1) and the HOX  gene families is in a closed chromatin state in both WT and Sun dKO MKCs (Olig2 and Neurog2) also remained in a closed state while the others (Olig3 and Neurog2) revealed a novel peak in Sun dKO cells  or WT pups as previously described (2). After 2–4 passages, keratinocytes were plated on plastic dishes without feeder cells and maintained in media containing 0.05 mM CaCl2 . Cells at low passage were stored under liquid nitrogen, and thawed cells were only used to passage 16. Integrin β1 null keratinocytes and corresponding WT controls . MKCs were tested and found to be mycoplasma-free. Sun dKO MKC cell lines were validated by western blotting (see below).MKCs were isolated from skin from E18.5 embryos or newborn whole-body escribed . Under scontrols  (kind giThe N2G-JM-TSMod construct was derived from pEGFP-C1 harboring mini-Nesprin2  kind gi kind gi. AcceptoThe soft 3 kPa PDMS gels were prepared by mixing a 1:1 ratio (w/w) of CY 52-276A and CY 52-276B (Dow Corning). Components were mixed thoroughly and immediately degassed for 15 min in a desiccator. The surface of 35 mm glass-bottomed culture dishes (MatTek Corporation) were covered with 200 µL of the mixed gel. Each dish was then spin-coated at 1000 rpm for 60 s using a spin coater instrument . Gels were cured at room temperature overnight. The next morning gels were sterilized for 30 min under UV light and then washed once with sterile PBS. Rheometry analysis (ARES-LS1) confirmed that the Young’s modulus of the PDMS was ~3 kPa.involucrin (Thermo Fisher Cat# VB1-3030396-VC) and Sprr1b (Thermo Fisher Cat# VB4-3117172-VC) according to manufacturer’s instructions. Coverslips were mounted onto glass slides with Prolong Gold with DAPI (Invitrogen P36935) and sealed with clear nail polish. Images were acquired with the Zeiss Imager M1 using Zen software. Images for the same marker were acquired at the same exposure, pixel range, and gamma values. Acquired images were equally brightened, contrasted, and cropped using ImageJ/Fiji (version 1.50e) software  onto glass coverslips coated with 50 μg/mL fibronectin (Sigma-Aldrich), MKCs were cultured in E-low calcium media overnight before switching to 1.2 mM calcium media to induce differentiation for 24 or 48 hr. RNA FISH was performed using the ViewRNA ISH Cell Assay kit (Thermo Fisher Cat# QVC0001) with probes for software . For quaThis study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (IACUC protocol number 2018-11248). All animal care and experimental procedures were conducted in accord with requirements approved by the Institutional Animal Care and Use Committee of Yale University. At harvest, embryos were submerged in O.C.T. compound (Tissue-Tek) and sectioned using a cryostat . Sections were cut in a specific and consistent orientation relative to embryo morphology and stained with hematoxylin and eosin for routine histopathology or incubated with primary antibodies and Alexa Fluor-conjugated secondary antibodies for indirect immunofluorescence as previously described . PrimarySun2-/-, and Sun dKO MKCs were prepped as previously described  and stored at −80°C until sectioning using a cryostat . After sectioning, the Click-iTEdU Cell Proliferation Kit for Imaging, Alexa Fluor 488 dye (Invitrogen C10337), was used according to manufacturer’s instructions to evaluate EdU incorporation. Tissue sections were then co-stained for K10 . Before imaging, coverglass was mounted onto slides using Prolong Gold with DAPI (Invitrogen P36935) and sealed with clear nail polish. Images were acquired with the Zeiss Imager M1 using Zen software. Total EdU-positive cells were counted and normalized to the total number of epidermal cells (determined by DAPI staining). Location of proliferating cells was determined by K10 staining  and then normalized to total EdU-positive cells. Statistical analysis was performed using Prism 8 software.Sun 1+/-/Sun2-/- and Sun dKO pups were isolated at age P0.5; three mice were examined for each genotype. Tissue blocks were fixed in 2.5% glutaraldehyde/2% paraformaldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, for 30 min at RT and 1.5 hr at 4°C. The samples were rinsed in sodium cacodylate buffer and were postfixed in 1% osmium tetroxide for 1 hr. The samples were rinsed and en bloc stained in aqueous 2% uranyl acetate for 1 hr followed by rinsing, dehydrating in an ethanol series to 100%, rinsing in 100% propylene oxide, infiltrating with EMbed 812 (Electron Microscopy Sciences) resin, and baking overnight at 60°C. Hardened blocks were cut using an ultramicrotome . Ultrathin 60 nm sections were collected and stained using 2% uranyl acetate and lead citrate for transmission microscopy. Carbon-coated grids were viewed on a transmission electron microscope  at 80 kV. Images were taken using a CCD camera  and iTEM (Olympus) software. The length and number of hemidesmosomes was quantitated from sections from three mice of each genotype.Transmission electron microscopy was performed in the Yale School of Medicine Center for Cellular and Molecular Imaging Electron Microscopy core facility. Back skin sections from WT and Sun dKO keratinocytes were plated at low density  onto glass coverslips coated with 50 μg/mL fibronectin (Sigma-Aldrich), MKCs were cultured in E-low calcium media overnight before switching to 1.2 mM calcium media to induce differentiation for 48 hr. Cells were fixed with methanol at −20°C for 5 min and washed with PBS. Cells were permeabilized using 0.5% Triton X-100 in PBS at RT for 20 min and blocked using 10% goat serum, 5% bovine serum albumin (BSA), and 0.5% Tween 20 in PBS for 1 hr. Cells were incubated in primary antibody, conformationally sensitive lamin A/C , diluted in blocking buffer at 4°C overnight. Coverslips were washed with PBS for three 5 min intervals and incubated with Alexa Fluor 488-conjugated secondary antibody  diluted in blocking buffer at RT for 1 hr. Coverslips were then costained with Hoechst 33342  and Alexa Fluor 594-conjugated Wheat Germ Agglutinin  diluted in PBS for 5 min at RT. Coverslips were washed with PBS, mounted using Fluoromount-G, and sealed with clear nail polish.2. Images were acquired using Zen software. FRET imaging was performed in a similar manner as previously described  as described  was imaged using a 488 nm laser line (ex) and PMT detector set for AF488 emission; and the WGA (AF594) using a 594 nm laser line (ex) and PMT detector set for AF594 emission. PMT gain was adjusted for each sample to avoid over-/under-exposure. Acquisition settings were standardized and maintained during experiments. Single-plane images to determine the location of each cell within a colony were acquired using a 40× 1.25 NA air objective, imaging the AF488 and AF594 channels sequentially. Next, Z-stacks of each images were acquired using a 63×, 1.4 NA oil objective in the AF488 channel. Image zoom and size was adjusted such that voxel size was 55–65 nm in the xy-plane and 130 nm in the z-plane. Image stacks were deconvolved using Huygens Professional software  or were switched to high calcium media to induce adhesion formation and differentiation for 48 hr. Total RNA was isolated using the RNeasy Plus kit (QIAGEN) according to the manufacturer’s instructions for three biological replicates for each condition. cDNA was synthesized using reagents from the TruSeq RNA sample preparation kit (Illumina) according to the manufacturer's instructions. cDNA libraries were sequenced (paired end 75 nts) at the Yale Stem Cell Center Genomics and Bioinformatics Core on the HiSeq4000 platform. Reads were mapped using BowTie/TopHat2  to the mAGGTCGGTGTGAACGGATTTG and reverse: TGTAGACCATGTAGTTGAGGTCA; Sprr1b forward: GATCCCAGCGACCACAC and reverse: GCTGATGTGAACTCATGCTTC; Sprr2d forward: GTGGGCACACAGGTGGAG and reverse: GCCGAGACTACTTTGGAGAAC; involucrin forward: GCAGGAGAAGTAGATAGAG and reverse: TTAAGGAAGTGTGGATGG; S100a14 forward: GGCAGGCTATAGGACA and reverse: CCTCAGCTCCGAGTAA. PCR product levels were normalized to GAPDH mRNA levels.WT and Sun dKO MKCs were plated in a fibronectin-coated  6-well dish such that they were 70–80% confluent. They were cultured in E-low calcium media overnight before switching to 1.2 mM calcium media to induce differentiation for 48 hr. Total RNA was isolated using the RNeasy Plus kit (QIAGEN) according to the manufacturer’s instructions. The iScript cDNA Synthesis Kit (Bio-Rad) was used to generate cDNA from equal amounts of total RNA (1 mg) according to the manufacturer’s instructions. Quantitative real-time PCR was performed with a Bio-Rad CFX96 using iTaq Universal SYBR Green Supermix (Bio-Rad) for 40 cycles. Primers used include GAPDH forward: ATAC-seq libraries were generated as previously described . Briefly In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:This work will be of broad interest, because it provides new insights into how cells sense tension and transmit it to the cell nucleus. With ever increasing excitement as to how cells sense tension and transmit it to the nucleus, your new work will now be an important addition to this arena.Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Anna Akhmanova as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "The LINC complex transmits integrin-dependent tension to the nuclear lamina and represses epidermal differentiation" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife". Please let us know if you would like to pursue this option. As the editors have judged that your manuscript is of interest, but as described below that additional experiments are required before it is published, we would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv  along with this decision letter and a formal designation that the manuscript is "in revision at Summary:The reviews of your manuscript are now in. As you can see, the consensus is that your manuscript strives to address a fundamental and largely understudied question, and your tools and strategy have the potential to significantly advance our understanding of how connections between cell-matrix adhesions and the nucleus have direct consequences on cell fate. That said, all your reviewers encourage you to provide better support for the central claims of your manuscript. There are five pressing concerns summarized below.Essential revisions:1. Their central hypothesis is that a relay of tension between integrins and the LINC complex maintains nuclear organization such that EDC gene expression is repressed. As tension on basally localized integrins is released, either by delamination or by redistribution of forces to cell-cell junctions (as happens in the middle of keratinocyte colonies), the chromatin state changes such that transcription of the differentiation program may be de-repressed. This attractive hypothesis needs to be tested more rigorously using their current models and existing experimental tools. For example, DNA-FISH approaches  to test whether the positioning of the EDC chromosomal region within the nucleus is differentially located the interior of colonies (where tension is low) compared to the outside of colonies (where tension is high), or spatially correlates with LINC tension at the single-cell level (if their tension sensor is suitable for fixation and DNA-FISH conditions). The authors should also test whether nuclear positioning of the EDC cluster and/or genes associated with epidermal differentiation is misregulated in the Sun1/2-dKO epidermis. The inclusion of such data would significantly strengthen the central claim and broader impact of the manuscript.2. There is a paucity of validation for the FRET sensor construct. To make conclusions about tension, a number of experiments can be conducted. A version of the construct lacking the actin-binding or KASH domains of Nesprin2 is important; similar controls were vital to the conclusions drawn in the original Schwartz paper  and in other contexts where the TS-module has been used . Having the TS "free floating" on the N-terminus would also be valuable. These controls are also very important as e.g. treatment with latruculin or other inhibitors can affect the FRET efficiency in similar directions independent of tension. Other manipulations of cell tension (besides poly-lysine coating and Latrunculin treatment), such as coating with varying concentrations of fibronectin, culturing cells on substrates of different stiffness, or exogenous force application to culture substrates, would significantly improve confidence that their construct indeed reports on mechanical tension in the nuclear envelope. Standard for validation of FRET experiments is to include an acceptor photobleaching experiment. Also, although the raw data for Venus/mTFP channels and bleedthrough controls with single colour constructs should be shown.3. The data with the force-sensitive epitope LaminA/C antibody is not of sufficient quality. In the original paper where this tool was developed , the signal of the LaminA/C was normalized against signal from a force-insensitive LaminB antibody. Otherwise, how do we know we are looking at the nuclear envelope? Or that there aren't overall differences in nuclear envelope morphology/composition that contribute to the differences in A/C signal that we see, rather than an effect that can be specifically attributed to tensional differences?4. Some baseline description of the LINC complex in the epidermis would provide insights that may lend support for (or refute) their hypothesis. For example, where in the epidermis is Sun1/2 expressed? Is there any developmental regulation of LINC complex organization? One might predict reduced expression or altered localization of LINC complex components (such as SUN1/2) in the differentiated layers of the epidermis. Can the force-sensitive Lamin-A/C antibody be used in vivo to see if there are differences between staining patterns in the basal layer where cells have integrin-mediated contacts compared to the suprabasal, differentiating layers where they do not? Are there differences in nuclear morphology in the presence or absence of sun1/2 dKO epidermis that might support their model? Does loss of Sun1/Sun2 alter nuclear stiffness? If the authors can show that the regulation of molecular tension across the Linc complex directly correlates to changes in nuclear tension, this will strengthen the paper.5. The molecular links how integrins control tension on the Linc complex, or how altered tension at the Linc complex controls differentiation are not at all addressed, and the paper should at least provide some insight into either upstream or downstream control of how integrin dependent regulation of Sun1/2 regulates differentiation; e.g. explore the molecular link between integrin and the linc complex , alterations in epigenetic marks, chromatin organization of e.g. relocalization of the EDC locus etc.The conditional b1 and KO of integrin a3 integrins do not result in altered/precocious differentiation in the skin . Presumably the link between integrins and the LINC complex under these conditions would be altered as well? The idea that LINC complex may control differentiation is possible, however the role of integrin based adhesions in this process has not been parsed out. At the very least the authors could have disrupted integrin signaling using KO cells or blocking antibodies. So it is possible that there may be alternative mechanisms? This should be considered.eLife. Your article has now been re-reviewed by a Reviewing Editor and by Anna Akhmanova as the Senior Editor.Thank you for submitting your revised article "The LINC complex transmits integrin-dependent tension to the nuclear lamina and represses epidermal differentiation" for consideration by eLife. That said, there still several items remaining, that we feel need to be addressed prior to publication.We agree that your manuscript is much improved and has an important message for Three issues remain. First, changes in nuclear structure and likely mechanics in keratinocytes induce rather global changes in chromatin and transcription , and hence it would be helpful if you can present the ATAC data more generally and not just pick a few loci to analyze. Are your data specific for the EDC locus or are other differentiation genes also affected? One would also predict changes in the so-called stem cell genes-can you comment on whether this is observed? These minor adjustments will provide even a better global vision on how the nuclear envelope tension regulates gene expression and differentiation.Second, the b1 KO cells have to be cultured in high calcium media and have very high levels of Rho activity, strong focal adhesions (nucleated through integrin b6) and massive stress fibers . Given the role of the actin cytoskeletal network in transmitting the forces via the LINC complex to the nucleus, it seems surprising that the tension sensor shows such high FRET (low tension) compared to the WT cells. It would be helpful if you could add a bit of discussion as to how you interpret these data and reconcile this point. Toning down some of the conclusions made regarding this point might be warranted.Finally, it would seem that you should add a brief discussion and referencing of the recent Nava et al. Cell paper, that shows heterochromatin driven changes in nuclear mechanics in keratinocytes. Essential revisions:1. Their central hypothesis is that a relay of tension between integrins and the LINC complex maintains nuclear organization such that EDC gene expression is repressed. As tension on basally localized integrins is released, either by delamination or by redistribution of forces to cell-cell junctions (as happens in the middle of keratinocyte colonies), the chromatin state changes such that transcription of the differentiation program may be de-repressed. This attractive hypothesis needs to be tested more rigorously using their current models and existing experimental tools. For example, DNA-FISH approaches  to test whether the positioning of the EDC chromosomal region within the nucleus is differentially located the interior of colonies (where tension is low) compared to the outside of colonies (where tension is high), or spatially correlates with LINC tension at the single-cell level (if their tension sensor is suitable for fixation and DNA-FISH conditions). The authors should also test whether nuclear positioning of the EDC cluster and/or genes associated with epidermal differentiation is misregulated in the Sun1/2-dKO epidermis. The inclusion of such data would significantly strengthen the central claim and broader impact of the manuscript.We have carried out a variety of additional experiments to provide more direct evidence for (1) changes at the level of the chromatin at the EDC upon loss of the LINC complex and (2) the ability of disrupting β1-integrin to release tension on the LINC complex and recapitulate loss of the LINC complex with respect to regulation of epidermal differentiation genes.Specifically, in this revision we added:Sun dKO MKCs .1) Additional analysis of individual EDC genes in WT and Sun dKO MKCs .2) Direct assessment of MKCs lacking β1-integrin to demonstrate that β1-integrin is essential to 1) drive the high tension state on the LINC complex  and 2) repress epidermal differentiation markers, thereby recapitulating our observations in Sun dKO mouse keratinocytes (MKCs). We observe a clear increase in chromatin accessibility as revealed by gains in ATAC peaks in the EDC of Sun dKO MKCs across the EDC in the low calcium condition when these genes are repressed in WT MKCs, indicative of a more open chromatin state and consistent with our model of precocious differentiation.3) We believe the most compelling addition are our results from Assay for Transposase Accessible Chromatin followed by sequencing (ATAC-seq) in WT and Sun dKO MKCs, we were not able to robustly detect signal in WT MKCs for comparison. We believe this could reflect our inability to sufficiently denature the probe targets when the region is heterochromatized, as we expect it to be in WT MKCs cultured in low calcium media. Despite our inability to complete this experiment, the new data described above provide additional support for the role of the LINC complex in regulating chromatin accessibility at epidermal differentiation genes and their expression.We tried extensively to analyze the EDC position by FISH as suggested. Although we were able to detect the EDC at a central nuclear position in 2. There is a paucity of validation for the FRET sensor construct. To make conclusions about tension, a number of experiments can be conducted. A version of the construct lacking the actin-binding or KASH domains of Nesprin2 is important; similar controls were vital to the conclusions drawn in the original Schwartz paper  and in other contexts where the TS-module has been used . Having the TS "free floating" on the N-terminus would also be valuable. These controls are also very important as e.g. treatment with latruculin or other inhibitors can affect the FRET efficiency in similar directions independent of tension. Other manipulations of cell tension (besides poly-lysine coating and Latrunculin treatment), such as coating with varying concentrations of fibronectin, culturing cells on substrates of different stiffness, or exogenous force application to culture substrates, would significantly improve confidence that their construct indeed reports on mechanical tension in the nuclear envelope. Standard for validation of FRET experiments is to include an acceptor photobleaching experiment. Also, although the raw data for Venus/mTFP channels and bleedthrough controls with single colour constructs should be shown.We thank the reviewers for their feedback and suggestions. We have provided additional controls to ensure that changes in the FRET indexes of the mini-Nesprin2 TSMod (N2G-JM-TSMod) indeed reflect tension on the LINC complex. To this end, we made a tension insensitive construct (no-tension construct) by inserting the TSMod at the N-terminus of the mini-Nesprin2  where it cannot bear any load. Comparing FRET index measurements at the nuclear envelope for MKCs transfected with the TSMod inserted at the juxtamembrane region (N2G-JM-TSMod) and the NoT_TSMod control  indeed revealed that the N2G-JM-TSMod is under tension in WT MKCs. We also compared the response of the two TSMods to treatment with latrunculin A to depolymerize actin. While the N2G-JM-TSMod was under higher tension that was relaxed upon treatment of latrunculin A , the tension insensitive NoT_TSMod control showed no change in tension at the nuclear envelope . Last, we provide new evidence that tension on the N2G-JM-TSMod is sensitive to the mechanics of the substrate . Bolstered by these additional data we conclude that (1) the TSMod is sensitive to actin-dependent tension at the nuclear envelope; and (2) the changes in FRET efficiency reported by the N2G-JM-TSMod reflects intramolecular tension exerted on the juxtamembrane region, as the no tension control is insensitive to load. As suggested by the reviewers, we also now show additional experimental controls including “dark” donor only and acceptor only constructs used for bleed through correction .3. The data with the force-sensitive epitope LaminA/C antibody is not of sufficient quality. In the original paper where this tool was developed , the signal of the LaminA/C was normalized against signal from a force-insensitive LaminB antibody. Otherwise, how do we know we are looking at the nuclear envelope? Or that there aren't overall differences in nuclear envelope morphology/composition that contribute to the differences in A/C signal that we see, rather than an effect that can be specifically attributed to tensional differences?We thank the reviewers for this critical feedback. We agree that ideally we would co-stain nuclei with a force-insensitive lamin A/C or lamin B antibody to address the concerns raised by the reviewers. However, we have tested numerous commercially available tension insensitive lamin antibodies, including those used in the 2015 Nature Materials paper, as well as antibodies to other nuclear envelope and nuclear pore complex proteins. Ultimately, we were unable to identify an antibody that stained the nuclear envelope of the mouse keratinocytes under conditions compatible with the tension-sensitive lamin A/C antibody with the performance required for this experiment . Since we found that we could successfully co-stain human epidermal keratinocytes, we further attempted to validate our system in this model. However, the nuclei of these cells are flatter than the mouse keratinocyte nuclei, and we were unable to achieve sufficient resolution with our confocal imaging and deconvolution set-up to resolve the apical and basal surfaces of the nucleus, even with manipulation of substrate conditions. This said, we developed our approach specifically because it allows us to robustly resolve the nuclear and basal surfaces. We have softened the language we use in the revised manuscript to acknowledge that while we observe decreased staining with this conformationally-sensitive antibody, this could reflect multiple changes in the state of the basal lamina, including a higher tension state.The revised manuscript also includes abundant further characterization of the our N2G-JM-TSMod including its sensitivity to the presence of β1 integrin  and the stiffness of the substrate on which the MKCs are plated . As our observations using the tension-sensitive lamin A/C antibody mirror the results obtained from our now thoroughly-characterized N2G-JM-TSMod, we continue to favor the model that LINC complexes are under more tension in cells at the colony periphery versus the interior.4. Some baseline description of the LINC complex in the epidermis would provide insights that may lend support for (or refute) their hypothesis. For example, where in the epidermis is Sun1/2 expressed? Is there any developmental regulation of LINC complex organization? One might predict reduced expression or altered localization of LINC complex components (such as SUN1/2) in the differentiated layers of the epidermis. Can the force-sensitive Lamin-A/C antibody be used in vivo to see if there are differences between staining patterns in the basal layer where cells have integrin-mediated contacts compared to the suprabasal, differentiating layers where they do not? Are there differences in nuclear morphology in the presence or absence of sun1/2 dKO epidermis that might support their model? Does loss of Sun1/Sun2 alter nuclear stiffness? If the authors can show that the regulation of molecular tension across the Linc complex directly correlates to changes in nuclear tension, this will strengthen the paper.Sun dKO epidermis, but would argue that our lack of understanding of the tension network in keratinocytes in vivo does not rule out there are nonetheless changes in tension exerted on the nucleus. Indeed, we have noted in our prior work that changes in nuclear shape can reflect both LINC complex-dependent and -independent factors .We performed immunostaining for SUN1 and SUN2 in E15.5 mouse skin. As described for post-natal skin in our prior manuscript , we find that both SUN1 and SUN2 are expressed throughout the basal and suprabasal layers of the epidermis . We have been unable to perform staining for the force sensitive lamin-A/C antibody in skin sections due to poor antibody performance in IHC. Further, measuring nuclear stiffness directly, a technique with which we have experience, requires a substantial additional commitment and we have not attempted this approach here. We note, however, that the literature suggests that while LINC complexes are essential for force transduction to the nucleus, they have little effect on the force response . We do not observe any gross changes in the nuclear morphology in 5. The molecular links how integrins control tension on the Linc complex, or how altered tension at the Linc complex controls differentiation are not at all addressed, and the paper should at least provide some insight into either upstream or downstream control of how integrin dependent regulation of Sun1/2 regulates differentiation; e.g. explore the molecular link between integrin and the linc complex , alterations in epigenetic marks, chromatin organization of e.g. relocalization of the EDC locus etc.The conditional b1 and KO of integrin a3 integrins do not result in altered/precocious differentiation in the skin . Presumably the link between integrins and the LINC complex under these conditions would be altered as well? The idea that LINC complex may control differentiation is possible, however the role of integrin based adhesions in this process has not been parsed out. At the very least the authors could have disrupted integrin signaling using KO cells or blocking antibodies. So it is possible that there may be alternative mechanisms? This should be considered.We appreciate the reviewer’s feedback and agree that further investigation into the role of integrins in the regulation of LINC complex tension and keratinocyte differentiation is warranted. In this revision, we include new experiments to directly address the role that integrins play in regulating tension on the LINC complex by examining the tension on the N2G-JM-TSMod in β1 integrin KO MKCs. Our FRET analysis demonstrates that the FRET index of the N2G-TSMod was significantly higher in β1 integrin KO MKCs compared to WT MKCs  indicating that β1 integrin is required for the high LINC complex tension state. Our additional data demonstrating that the N2G-TSMod is also sensitive to substrate stiffness  further reinforces this relationship.Sprr1b, Involucrin (Ivl), and S100a14 revealed a significantly elevated expression of these mRNAs in β1 integrin KO MKCs cultured in low calcium conditions . These data mirror our observations in Sun dKO MKCs and support a role for β1 integrin in mediating tension on the LINC complex and its role in suppressing epidermal differentiation.We also examined the expression of differentiation genes in β1 integrin KO MKCs. RT-qPCR analysis of We agree that your manuscript is much improved and has an important message for eLife. That said, there still several items remaining, that we feel need to be addressed prior to publication.Three issues remain. First, changes in nuclear structure and likely mechanics in keratinocytes induce rather global changes in chromatin and transcription , and hence it would be helpful if you can present the ATAC data more generally and not just pick a few loci to analyze. Are your data specific for the EDC locus or are other differentiation genes also affected? One would also predict changes in the so-called stem cell genes-can you comment on whether this is observed? These minor adjustments will provide even a better global vision on how the nuclear envelope tension regulates gene expression and differentiation.We have now added a supplemental panel  and accompanying Supplementary File 4 to provide genome-wide analysis of the ATAC-seq data. Across the genome, most ATAC-seq peaks are found at the same genes for WT and Sun dKO MKCs. Sun dKO MKCs also show both gains and losses of ATAC-seq peaks associated with genes compared to WT MKCs, with a bias towards gains. Further analysis revealed that additional ATAC-seq peaks are found at genes encoding keratins and cell adhesion genes in Sun dKO MKCs, suggesting that epidermal differentiation genes outside of the EDC also show increased chromatin accessibility (Supplementary File 5). However, we do not see premature loss of ATAC-seq peaks at proliferation genes or “stem cell genes” (Supplementary File 5), arguing that precocious expression of epidermal differentiation genes is the primary alteration upon loss of the LINC complex. Of note, these data differ from the results of Le et al., 2016, who found global down-regulation of genes upon critical stretch of keratinocyte monolayers. We conclude, as outlined in the Discussion, that stretch achieved with the device employed by Le et al. and constitutive tension on the LINC complex dictated by integrin engagement represent two distinct contexts.Second, the b1 KO cells have to be cultured in high calcium media and have very high levels of Rho activity, strong focal adhesions (nucleated through integrin b6) and massive stress fibers . Given the role of the actin cytoskeletal network in transmitting the forces via the LINC complex to the nucleus, it seems surprising that the tension sensor shows such high FRET (low tension) compared to the WT cells. It would be helpful if you could add a bit of discussion as to how you interpret these data and reconcile this point. Toning down some of the conclusions made regarding this point might be warranted.We appreciate this perspective. However, our data from this manuscript and other published work clearly indicate that high Rho activity and actin contractility are not sufficient to drive tension on the LINC complex – indeed this represents the critical point that lies at the heart of our model. For instance, cells at the interior of cohesive MKC colonies also display extensive stress fibers integrated at cell-cell adhesions  and show low tension on the LINC complex and the nuclear lamina, as demonstrated in Figure 2 A-D. Thus, our data indicate that actomyosin contractility is not sufficient, in and of itself, to drive a high-tension state on the LINC complex. While we agree that these findings may be, on the surface, surprising, this model provides an explanation for how the LINC complex could specifically contribute to repressing epidermal differentiation genes in basal but not suprabasal keratinocytes. As for the specificity for integrin b1 and not integrin b6, further studies will be required to define why only engagement of the former is sufficient to exert high tension on the LINC complex. We now include a more detailed discussion on these points in the discussion of the revised manuscript.Finally, it would seem that you should add a brief discussion and referencing of the recent Nava et al. Cell paper, that shows heterochromatin driven changes in nuclear mechanics in keratinocytes.We now cite and discuss this paper, as suggested. However, we would point out that stretched cells and constitutive tension on LINC complexes with engaged b1-integrins likely represent distinct regimes as we do not observe the global changes observed by Le et al. and. Nava et al.


Klebsiella pneumonia (CRKP) blood stream infection is still largely unknown. The aim of this study was to explore the relationship between postoperative NLR and mortality in those patients.The neutrophil to lymphocyte count ratio (NLR) has been recognized as a useful marker of inflammation. But, the prognostic function of NLR in patients with Carbapenem-resistant We performed a retrospective study based on the database from Computerized Patient Record System in Sir Run Run Shaw Hospital from 1/1/2017 to 31/10/2020. Logistic analysis was performed to assess the associations between NLR and 28-day mortality. Multivariate analyses were used to control for confounders.th day , NLR on the 4th day  were significant risk factors for the 28-day mortality of CRKP BSI patientsA total of 134 CRKP blood stream infection inpatients were included in this study, including 54 fatal cases and 80 survival cases on the 28-day after the onset of CRKP BSI, the overall 28-day mortality rate of patients with a CRKP BSI episode was 40.3% (54/134). We conducted a multivariate analysis on these 134 patients and found that APACHE II score on the 4th day after first positive culture.NLR is promising to be a readily available and independent prognostic biomarker for patients with CRKP blood stream infection.Elevated NLR was significantly associated with increased 28-day mortality as well as APACHE II score on the 4All Authors: No reported disclosures


Several reports have compared spinal cord tumor removal techniques but none have clearly described the appropriate site and level of indication for laminectomy or laminoplasty. The approach method for tumor removal depends on the type and localization of the tumor and the surgeon's skill. Therefore, a system that can suggest various surgical techniques is useful for spinal cord tumor surgery. The mixed reality system introduced in this paper is an excellent system that can suggest various surgical procedures. Using this system for spinal cord tumor removal, we made the surgery less invasive; therefore, we introduced this system and demonstrated its usefulness. Stereoscopic data of the patients with spinal cord tumors were obtained from preoperative myelogram-CT data. Stereoscopic laminectomy models including tumors were created using Blender, a free three-dimensional (3D) image editing software. We observed these data as 3D object images using a head-mounted display (HMD). This HMD is commercially available and relatively inexpensive. The surgical procedure is determined by considering those 3D images, radiological diagnosis, and the skill of surgeons. Intraoperative confirmation of the laminectomy site could be performed using the HMD. The 3D visualization of pathological conditions resulted in correct preoperative surgical planning and less invasive surgery in all five cases. Stereoscopic images using HMDs allow us a more intuitive understanding of the positional relationship between the tumor and spinal structure. These 3D object images can bring us more accurate preoperative planning and proper determination of surgical methods. Spinal cord tumors are often removed by laminectomy or laminoplasty -10. To pPlain images such as CT, myelogram-CT, and MRI have been widely used to determine the site and extent of bone resection or laminoplasty. Recent advances in image editing technology have made it easy to display stereoscopic images of bones and nerves on a flat display. However, if a stereoscopic image appears on a conventional flat display, this image is only a two-dimensional (2D) image. It takes time for the evaluators to convert a 2D image to a three-dimensional (3D) one in their brain. On the other hand, advances in mixed reality (MR) technology using head-mounted displays (HMDs) have made it possible to observe stereoscopic images in 3D projected with HMDs -14. TherApplied casesCase 1This is a case of a 63-year-old man with L3 schwannoma Preoperative MRI showed a neoplastic lesion including a cyst posterior to the L3 vertebral body. The tumor was removed by L3 laminectomy with the longitudinal split of the L3 spinous process Figure . PostopeCase 2This is a case of a 66-year-old man with C2-C3 schwannoma. Preoperative images showed a spinal cord tumor extending from C2 to C3  or Brilliance 64-CT scanner  to create 3D images for the spine and spinal cord tumors. These images were created using a workstation . When identifying the tumor was difficult, the myelogram-CT and MRI images were checked individually and fine-tuned to achieve the best position of the spinal cord tumor. The STL data of both bony elements and the spinal cord tumor were then output by a workstation. A free 3D image editing software  was applied to the STL data to create the laminectomy model. The planned decompression area of the bone element was deleted from the original spinal data using the Blender software  or virtual reality device , using a dedicated application . In the Holoeyes MD software, the bone and tumor can be observed in three dimensions Figure , case 2.With MR technology, this stereoscopic image is displayed as 3D as it is in the real body and can be fixed in place. Therefore, by displaying the stereoscopic data as true 3D using an HMD, the stereoscopic data can be intuitively grasped in the brain of surgeons. Holoeyes MD can arbitrarily display and hide each uploaded 3D image: bony elements, spinal cord tumor, planned laminectomy model, and other images including nerves and vessels. In addition, Holoeyes MD can adjust the transparency of each image Figure  and magnWe can observe these images from any angle at 360°. Spinal cord tumors can be easily seen transparently when the spine is viewed in wireframe mode Figures . The wirResultsCase 1: Lumbar CaseWe were unsure whether to perform a partial L2 caudal laminectomy from the preoperative images Figure . After oCase 2: Cervical CaseDue to the large size of the tumor based on the preoperative images, we considered a double door laminoplasty of C2 and laminectomy of C3. HMD showed that the tumor was localized to the left side, and hemilaminectomy seemed sufficient to remove the tumor at the C3 level. In addition, the extension of the tumor to the cephalad was limited to the cephalic margin of the C2 lamina, and it seemed possible to remove the tumor if it could be approached through half of the lamina of C2 and C3 Figures , 7. TherVarious methods have been attempted for the removal of spinal cord tumors -10. TherUsing the Holoeyes MD software, it is possible to change the bone transparency and observe the tumor from any angle at 360°, which is very useful for planning spinal cord tumors removal. We could see the full-scale spine and tumor in front of us as if we were performing the spine surgery. Therefore, this system could intuitively suggest various surgical plans. In addition, the system can magnify the spine and tumor up to 10 times during the planning process, which is useful for observing fine details when we assess the margins of the tumor.In our four cases of laminectomy, we had intuitively understood the extent of bone resection in the cephalocaudal and lateral directions preoperatively. In the case of laminoplasty, this system supported the decision to perform a less invasive surgery and visualized the tumor removal by laminoplasty with a preoperative prediction of the adequate location of the bony gutter.The MR system resulted in an intuitive determination of the surgical approach and spinal cord tumor removal area. In addition, by creating a laminectomy model using the 3D editing software Blender, we can predict and confirm the details of the tumor removal procedure before surgery. This planning allowed us to perform less invasive surgery with confidence. Furthermore, we could use it to identify the laminectomy site during surgery. This novel technique of the MR system can be useful to determine the surgical procedure for spinal cord tumors.