Lay community health workers and facility-based health care providers in Benin were trained to administer DMPA-SC safely and effectively in 10 health zones. Community-based DMPA-SC was popular, particularly among new users of contraception, and could help the country achieve its family planning goals. Lay community health workers and facility-based health care providers in Benin were trained to administer DMPA-SC safely and effectively in 10 health zones. Community-based DMPA-SC was popular, particularly among new users of contraception, and could help the country achieve its family planning goals. relais communautaires (RCs). DMPA-SC is an easy-to-administer, discreet injectable contraceptive that provides 3 months of protection from pregnancy. Beginning in May 2017, the government introduced DMPA-SC through a phased approach in 10 health zones, which encompassed 149 health centers and 614 villages. Between June 2017 and June 2018, the MOH and APC trained 278 facility-based providers and 917 RCs to provide DMPA-SC, and nearly 11,000 doses were subsequently administered to 7,997 women at facilities and in communities. This article presents findings from an assessment of community-level and health facility service data collected during the first 13 months of DMPA-SC introduction in Benin. Because of this intervention, nearly 35,000 women received family planning counseling and 7,997 women chose DMPA-SC. At the community level, 3,111 DMPA-SC users were first-time users of modern contraception. The initial success of the DMPA-SC rollout in Benin shows promise for helping the country meet its reproductive health commitments.The Republic of Benin faces high maternal, newborn, and child mortality; low modern contraceptive use; and a critical shortage of health workers. In 2013, the Government of Benin made 3 reproductive health commitments to improve national health indicators, including expanding provision of family planning services at the community level through task sharing. Since 2016, the Advancing Partners & Communities (APC) project has been helping the Benin Ministry of Health (MOH) provide subcutaneous depot medroxyprogesterone acetate  through facility-based health care providers and community health workers known as  Desire for large families and myths and misconceptions about family planning contribute to a low modern contraceptive prevalence rate of 12% and a high total fertility rate of 5.7.Expanding access to contraception—especially in communities that have an unmet need and demand for it—is essential to reach the country's family planning goals. At the 2013 International Conference on Family Planning in Addis Ababa, the Government of Benin (GOB) made 3 commitments to improve reproductive health indicators.With an expanding population of 11 million, nearly half of whom reside in rural areas with limited access to health care services, Benin continues to face high maternal and child mortality. This problem is compounded by the country's critical shortage of health workers. Other countries with similar shortages have introduced task-sharing approaches, as recommended by the World Health Organization (WHO),aides-soignantes to administer the 2-month intramuscular injectable Noristerat in Adja-Ouèrè commune in southeastern Benin. Aides-soignantes are a cadre of paraprofessional nurses' aides who are stationed at health centers and provide immunization and assist in health promotion campaigns in communities. Previously, contraceptives were available exclusively at health facilities, which limited rural women's access to family planning. During the 9-month pilot, aides-soignantes counseled 1,809 women on a range of voluntary family planning methods, and 449 women adopted Noristerat. The aides-soignantes referred 249 women to health centers for other methods including pills, intrauterine devices, and implants.In 2014, the Benin Ministry of Health's (MOH's) Maternal and Child Health Directorate approved a pilot project to allow relais communautaires (RCs), the lowest-level CHW cadre, to provide high-quality community-level primary health services including family planning in 10 USAID priority health zones  project, funded by the United States Agency for International Development (USAID), has worked in more than 40 countries to advance and support programs that improve the overall health of communities and other health-related indicators, especially related to family planning. In Benin, between 2015 and 2018, the APC project built the technical and organizational capacity of 3 local NGOs—Dedras, Bupdos, and Sia N'son—to train th zones .BOX 1Relais communautaires (RCs) constitute a key component of the national community health strategy. They typically receive supervision from staff at public health facilities and training and small monthly stipends from local NGOs. RCs are volunteers chosen by residents of their villages to provide a package of high-impact health services (paquet d'interventions à haut impact) related to water, sanitation, and hygiene; maternal and child health; nutrition; malaria; and family planning . RCs are required to be proficient in French. An estimated 15,000 are deployed nationally and 2,080 work in the United States Agency for International Development's 10 priority health zones. The Ministry of Health offers RC refresher trainings every 2–3 years.Benin has 2 types of RCs: those who provide curative services and those who provide preventive services only. Preventive RCs conduct health education sessions in their communities, while curative RCs also provide basic health care interventions and refer community members who need complex care to a local health facility. According to the national community health strategy, 1 curative RC should serve between 30 and 50 households that are 5 kilometers or more from the nearest public health facility. In practice, however, there are not enough trained RCs to fulfill this ideal.The APC project built the capacity of local NGOs to train community health workers to provide high-quality community-level primary health services in Benin.In 2016, the MOH authorized the introduction of DMPA-SC in health facilities and via RCs. Midwives administered DMPA-SC at health facilities, adding to their routine family planning service provision. RCs selected for training were linked to NGOs that received APC technical assistance.–,DMPA-SC has been described as a “game-changing” injectable contraceptive because it is discreet and requires minimal training to administer.After authorization for DMPA-SC introduction was granted, Beninese stakeholders, including the MOH and implementing partner staff, visited DMPA-SC pilot sites in Burkina Faso and Uganda to learn from their introduction processes . These vBOX 2Introduce DMPA-SC in health zones where DMPA-IM is already being offered—because providers and clients are already familiar with DMPA—including eligibility criteria, reinjection timeline, and side effect profiles.Develop targeted, regional communication materials in accordance with local requirements and manufacturer guidelines.Develop specific monitoring and evaluation tools for DMPA-SC.Establish an appropriate pharmacovigilance strategy.Engage national and local political and administrative authorities early on to garner buy-in.Abbreviations: DMPA-IM, intramuscular depot medroxyprogesterone acetate; DMPA-SC, subcutaneous depot medroxyprogesterone acetate.A national technical steering committee provided overall guidance and technical leadership for the introduction and implementation of DMPA-SC. The committee comprised technical and financial partners including APC, the United Nations Population Fund, USAID, the Benin Association for Social Marketing and Communication for Health (ABMS), the Beninese Association for Family Planning, and University Research Company's Advancing Newborn, Child, and Reproductive Health project. The committee adapted the training curriculum, job aids, and monitoring and evaluation tools, all in French, that PATH developed for DMPA-SC introduction in Senegal.The 3-day RC training curriculum included a review of all available family planning methods ; how to conduct family planning counseling and referrals; and how to complete data collection tools. The process of adapting and formatting the tools for Benin took 9 months and several committee meetings to solicit input and ensure that content adhered to national health policies, strategies, and norms. For example, the committee decided that DMPA-SC would be injected only in the back of a client's upper arm, rather than in the thigh or abdomen because of concerns for client privacy with male providers.  curriculum . Beginning in May 2017, introduction proceeded in a phased rollout in 10 USAID priority health zones where RCs had been trained on family planning methods and counseling through the MOH's rriculum . DMPA-SCDMPA-SC introduction began in May 2017 in USAID priority health zones with RCs trained on family planning methods and counseling.Health worker training was cascaded  to fosteAfter certification to provide community-based DMPA-SC, RCs received several forms of support. PIHI NGOs and RC supervisors at local health facilities held monthly group supervision sessions at which RCs discussed challenges, successes, and lessons with their peers and were reminded to follow up with clients for reinjection after 3 months. Health center in-charges also conducted quarterly on-site supervision visits with RCs. Lastly, supervision teams of staff from APC, the MOH, ABMS, and the district health zone and health center in-charges visited RCs on site 4 to 6 weeks post training to provide support and resolve challenges.DMPA-SC is delivered to health centers through the national supply chain, which operates a “pull” system, whereby each level orders commodities; products flow from the central medical stores to district warehouses to health centers. Upon certification, health centers give RCs 5 free doses of DMPA-SC. RCs obtain product resupply at health centers at the nationally set cost of 200 CFA (∼US$0.35) per dose and resell it to clients for the same price. RCs are provided with safety boxes to dispose used DMPA-SC devices, which are discarded per medical waste disposal guidelines at the health center during routine monthly group supervision sessions.ABMS and APC led community sensitization and national advocacy sessions to raise awareness of family planning, specifically DMPA-SC. Sensitization sessions in each introduction health zone included local elected officials and traditional and religious leaders, and featured short films on the demographic dividend, maternal mortality, and community-based injectables programming, followed by discussions. These workshops prepared traditional and political leaders to support RC DMPA-SC administration. A total of 536 local officials and 578 traditional and religious leaders participated in family planning sensitization and advocacy sessions. At the national level, ABMS and APC led briefings to inform parliamentarians and journalists of the benefits of family planning and the GOB's commitment to make DMPA-SC widely available through RCs.The APC project assessed community-level and health facility service data collected by RCs and midwives between June 2017 and June 2018, including number of DMPA-SC doses administered, DMPA-SC uptake, and number of women receiving family planning counseling. Results and lessons from the first 13 months of DMPA-SC introduction are described below.DMPA-SC was introduced in 10 health zones in Benin through a phased approach that allowed course corrections during rollout to 149 health centers and 614 villages. The 278 trainers  and 917 RCs who were trained administered nearly 11,000 doses of DMPA-SC over the 13-month period. RCs administered 1,309 doses of DMPA-SC to women as reinjections, and facility staff administered 1,403 doses, for a total of 2,712 reinjections. Few clients reported side effects.The trainers and lay community health workers administered nearly 11,000 doses of DMPA-SC over 13 months.Between June 2017 and June 2018, 9,296 women at the health facility level and 24,947 women at the community level received family planning counseling. A total of 7,997 women—including those who were adopting a modern family planning method for the first time and those switching from other family planning methods—chose DMPA-SC. DMPA-SC uptake at the community level climbed steadily during the first year of introduction and peaked in June 2018. At the facility level, DMPA-SC uptake increased from September 2017 onward, peaking at nearly 600 new users in June 2018.At the community level, 3,111 women were first-time users of modern contraception, and 769 women switched to DMPA-SC from another contraceptive method . FurtherDuring the same time frame, Noristerat uptake at the health facility level was high, with 13,698 women adopting the method, followed by DMPA-SC  and DMPA-IM (972 women) . NoristeAlthough stock-outs were not measured systematically, anecdotal reports suggest they occurred at both the facility and community levels during the first months of DMPA-SC introduction. In facilities, stock-outs likely occurred because staff had to adapt to their new and additional responsibility for reordering DMPA-SC from district warehouses. In communities, either RCs did not realize that they should seek DMPA-SC resupply from the health center or when they did seek it, health centers were out of the product. It is important to note that RCs must contend with frequent stock-outs of products related to other public sector health interventions because Benin still struggles to maintain its national health commodity supply chain.DMPA-SC was introduced through the public sector at health centers and in communities to expand family planning access to and method choice for women. RCs administered 49% of the total DMPA-SC doses to women who chose the method. Furthermore, 80% of DMPA-SC doses at the community level were administered to first-time family planning users, suggesting that community-based distribution of DMPA-SC by RCs is an effective way to increase use of family planning. Although there were challenges throughout the process, the phased introduction allowed course corrections along the way, providing useful lessons for future DMPA-SC programming.80% of DMPA-SC doses at the community level were administered to first-time family planning users.–Community-based distribution of injectables via CHWs is a promising way to expand access to family planning services and commodities, particularly for women who live in remote areas. Our results are in line with findings from DMPA-SC introduction in Burkina Faso, Niger, Senegal, and Uganda, which demonstrated that community-based distribution of DMPA-SC has the potential to reach new acceptors of family planning as well as youth and adolescents.The introduction of DMPA-SC corresponded with an increase in the number of women receiving family planning counseling at both the community and facility levels. As this experience shows, adding a contraceptive option to the method mix often prompts programs to provide refresher trainings for providers to strengthen skills, thus enhancing the overall quality of the family planning program. Many RCs reported being happy with their new DMPA-SC-related responsibilities because they were providing a useful service to community members and were eager to continue.The lessons learned during DMPA-SC expansion in Benin were used to improve subsequent trainings and strengthen implementation and can provide guidance to countries introducing DMPA-SC .Master trainers realized that many RC trainers had little experience facilitating sessions, which resulted in a low-quality training experience for RCs. The MOH and APC added a module on how to conduct effective trainings to the RC trainer curriculum. This half-day training increased RC trainer competency and led to higher-quality RC training sessions.During the post training supervision phase, many RCs displayed weak family planning counseling skills. To remedy this problem, the APC project developed a job aid that gave concise guidance on how to counsel on all family planning methods and ensure informed choice and voluntarism. This job aid was given to trained RCs and incorporated into the training curriculum. A half-day counseling refresher session was added to subsequent trainings to supplement the family planning information that RCs received under the government's PIHI curriculum. The refresher training included time to review the job aid and practice its contents through role play. Lastly, a day of on-site counseling capacity building for RCs who continued to exhibit poor counseling skills post training and certification was added to MOH, APC, and ABMS routine monitoring and supervision visit schedule.RC certification took longer than expected, sometimes exceeding the 4-week validation period initially established. Of the 917 RCs trained, approximately half were certified to administer DMPA-SC by June 2018. Those who did not become certified within 2 months of training are preventive RCs, conducting family planning and DMPA-SC awareness-raising activities in their communities. One cause of delayed certification was supervisors' lack of adequate follow-up with RCs. In response, APC invited health zone coordinating doctors, who oversee health center staff, to attend the supportive supervision visits so they could note challenges, develop monitoring and action plans, and encourage supervisors to be available to RCs.Another cause of delayed RC certification was low client uptake of DMPA-SC, which prevented RCs from administering the 5 voluntary client injections to demonstrate competence. Low client uptake of an unfamiliar  family planning option is common. To rectify this, RCs strengthened their mobilization efforts to challenge social norms that hinder family planning uptake. Client demand was also low, likely due to the delayed launch of a mass media DMPA-SC educational campaign. It is critical to launch corresponding communication campaigns  and product introduction simultaneously to create momentum and attract clients. The campaign should include information on the benefits and risks of family planning in general and the new method in particular, as well as where to access it.It is critical to launch communication campaigns and product introduction simultaneously.National health worker strikes, which took place during RC training, also delayed RC certification. Facility-level health care providers serve as RC supervisors, so when they were unavailable during the strike, RCs were unable to complete the requisite number of client injections for certification. Many trained RCs were unable to provide community-based services during this period, either because they were not yet properly certified to do so, were not being directly supervised, or were not able to resupply stock.Product stock-outs were initially problematic in the first health zone because health center staff were unaware of their responsibility for reordering commodities. The reason for this is likely twofold: (1) health center staff assumed that because APC was leading the DMPA-SC trainings, the project was also responsible for product resupply; and (2) the national commodities resupply form was not updated to include DMPA-SC until June 2017. Stock-outs at the facility level trickled down and affected community-level provision because RCs sought DMPA-SC resupply at health centers.A continuing challenge is the collection of high-quality data. RCs record community-level data in family planning registers, which are compiled at the facility level. During the early phases of implementation, some RCs did not have the registers because of delayed printing by the affiliated NGOs and therefore did not collect or record data in a standardized manner. Once those RCs received their registers, APC and MOH staff provided technical support during monitoring visits to ensure data were collected properly. Subsequent trainings emphasized the importance of monitoring, evaluation, and data collection and allowed time for RCs to practice completing the data registers. Data quality also remains a problem at the health facility level because reporting is primarily paper based, which often results in poor record keeping. APC and MOH staff provide technical assistance to facility-level health care providers during routine supervision visits to ensure that family planning data are captured and reported accurately. High-quality data are critical for measuring the impact of DMPA-SC introduction in the health zones.Collection of high-quality data is a continuing challenge, and more data on side effects experienced by clients are needed.Another ongoing challenge is limited data on client side effects. Women rarely reported side effects to RCs or facility-level health care providers, and although it is possible that this reflects a low rate of experienced side effects, it is also possible that clients fail to report side effects that they consider “minor” or health workers are not collecting or reporting this information accurately. We suggest establishing a more robust system to monitor and capture potential minor and major side effects.Our final lesson is the importance of involving communities and their gatekeepers. We found that inviting local stakeholders—including religious and traditional leaders, local and national politicians, and communities at large—to orientation sessions during the DMPA-SC introduction process was critical to gaining their support, without which the program would not have succeeded.A limitation of this analysis is the lack of unique client identifiers within the data. Consequently, it is impossible to track individual women over time to determine if they elected to be reinjected with DMPA-SC and if so how many times; therefore, calculating continuation and discontinuation rates is also impossible. The indicators simply captured the total number of reinjections in communities and at health facilities during the specified period. Moreover, this type of routine service statistics data does not measure the number of contraceptive discontinuers, only users, so it is difficult to assess if and how much the program increased contraceptive use and contributed to national reproductive health goals. Further, because this analysis is restricted to service statistics data, client motivations for using family planning for the first time or choosing to switch to DMPA-SC from another method cannot be determined.Additionally, given the MOH's current facility-level indicators, it is impossible to determine if women adopting DMPA-SC were new users to family planning or had switched from another method. Having this disaggregated information in the future will help determine user profiles for women who choose DMPA-SC. The data show that in health centers where DMPA-SC and DMPA-IM were both offered, DMPA-SC was the more popular method, surpassing the number of DMPA-IM users during each of the 13 assessed months. It would be helpful to monitor from which methods users were switching to determine if DMPA-SC introduction decreases use of other methods, including DMPA-IM and Noristerat. Further, age-disaggregated data are not collected at either the facility or community level because the indicator has yet to be added to national data collection forms. This information is critical for improving youth-focused programming.Having disaggregated information would help monitor family planning method preferences and improve youth-focused programming.Results from the first 13 months of DMPA-SC introduction in Benin demonstrate the demand for community-based family planning provision, as uptake of the injectable contraceptive increased steadily at the community level. Between June 2017 and June 2018, 7,997 women chose to adopt DMPA-SC as their preferred method of family planning, from either a community- or facility-based health worker. Results suggest that service provision through RCs is feasible and acceptable among clients, as nearly 4,000 women—nearly 80% of whom were first-time users of modern contraception—chose to receive this method at the community level.As the popularity of DMPA-SC grows in Benin, service provision through additional channels, including self-injection, should be considered to make the method available to all who would chose it. Recent findings from a DMPA-SC self-injection trial in Malawi show a user discontinuation rate of 56% when CHWs administer injections. However, when women self-inject, the discontinuation rate drops by almost half, to 30%.As a midwife, I approve this initiative because it solves the problem of geographical accessibility because the health centers often serve remote villages. The relais communautaires were chosen on the basis of certain well-defined criteria and they have been trained to offer Sayana Press, so for me they have the competence. [Health care provider]I am happy to have been chosen and trained to offer this service because now women in my community no longer need to pretend to take their children for care at the health center to receive a contraceptive method, [they] now can access a method close to home. [Relais communautaire]


Prostate cancer (PCa) continues to be one of the most common cancers in men worldwide. Prostate specific antigen (PSA) measured in blood has been used for decades as an aid for physicians to detect the presence of prostate cancer. However, the PSA test has limited sensitivity and specificity, leading to unnecessary biopsies, overdiagnosis and overtreatment of patients. For these reasons, there is an urgent need for more accurate PCa biomarkers that can detect PCa with high sensitivity and specificity. Urine is a unique source of potential protein biomarkers that can be measured in a non-invasive way. This review comprehensively summarizes state of the art approaches used in the discovery and validation of urinary biomarkers for PCa. Numerous strategies are currently being used in the discovery of urinary biomarkers for prostate cancer including gel-based separation techniques, mass spectrometry, activity-based proteomic assays and software approaches. Antibody-based approaches remain preferred method for validation of candidate biomarkers with rapidly advancing multiplex immunoassays and MS-based targeted approaches. In the last decade, there has been a dramatic acceleration in the development of new techniques and approaches in the discovery of protein biomarkers for prostate cancer including computational, statistical and data mining methods. Many urinary-based protein biomarkers have been identified and have shown significant promise in initial studies. Examples of these potential biomarkers and the methods utilized in their discovery are also discussed in this review. Prostate cancer (PCa) is the most prevalent cancer in males and it is estimated that approximately 116, 000 men living in USA were diagnosed with prostate cancer in 2017 . The mos-1 and PSA has only a 25-40% positive predictive value to detect PCa [-1 thereby leaving certain cancers undetected [In 1986, to guide clinical decision making, the US Food and Drug Administration approved the PSA test as a diagnostic, screening, and monitoring tool for the early detection of prostate cancer . Prostattect PCa , 19. Up detected . To addrdetected . The potdetected  lead thedetected  criteriaUrine is a biofluid enriched with proteins that reflect the physiological or pathological state of major urological tissues, including the prostate . Urine aProteomics is the large-scale study of proteins  which ofP< 0.001 for each) [In mass spectrometry-based proteomics there are two distinct approaches in proteomics analysis: the less used, less mature “top-down” proteomics, and the almost universally used “bottom-up” proteomics . Proteinor each) .The label-free protein quantification approach is based either on the comparison of extracted peptide MS peak intensities from different biological samples (intensity-based quantitation) or on the total number of MS/MS acquired for the same peptide  to represent the relative abundance of this peptide in the mixture . The samTo accurately and quantitatively analyze data in MS-based approaches, computational methods and common search engines are used to help facilitate quick data analysis . The mosIn general, there are two commonly used proteomic separation techniques, gel-based and gel-free methods Figure .P < 0.001) and MMP-9/NGAL (P = 0.003) by multivariable regression as independently predictive in differentiating prostate cancer patients and controls. In addition, MMP-9 and MMP-9 dimer were identified as multivariable predictors for differentiating prostate from bladder cancer (P < 0.001) [The two most common gel-utilizing methods to fragment and set apart proteins in a gel-based substance are 1-dimensional (1D) and 2-dimensional (2D) gel electrophoresis. After gel electrophoresis, proteins are cut out from the gel, processed with a protease such as endopeptidase trypsin, and the resulting peptide fragments are examined using MS , 74. In < 0.001) . In a re< 0.001) . Due to < 0.001) .Development of new technologies and tools in the last decade significantly accelerated progress in proteomics approaches and biomarker discovery. However, an open question remaining is the determination of which specific proteomic approach is the most appropriate to use in biomarker discovery and validation when using clinical samples . SeveralOvertreatment and overdiagnosis of PCa patients significantly raises the cost of health care. Therefore, a novel economical, non-invasive approach, which would limit the number of biopsies, could also lead to the reduced cost. For example, analysis of urine from PCa patients performed by capillary electrophoresis mass spectrometry (CE/MS) outperformed the biopsy approach as well as the PSA test. Moreover, a majority of cost savings were related to the significant reduction in the number of necessary biopsies by 49% . Cost-efThere is a limited literature regarding reproducibility of the urinary proteomic profile  and therWhile advances such as new instruments, techniques and software have led to the evolution of novel proteomic approaches for identification and validation of protein biomarkers, there also exist a variety of challenges and limitations associated with this approach. For example, gel-based techniques are frequently used in discovery but are not applicable in clinical settings due their time consuming nature, lack of high-throughput capabilities, requirement for high antibody quality and need for continuous optimization. MS-based methods are very sensitive, specific and generate significant amounts of information and, as such, are now gradually being incorporated into clinical settings. However, there are also some drawbacks associated with MS-based methods such as the need for expensive hardware, specific software, expensive equipment maintenance fees, high cost of certain chemicals, low-throughput and the requirement of highly skilled personal. Functional assays are highly specific and provide activity readouts, but their use can be limited due the availability and cost of commercial antibodies or reporter tags. ELISA-based methods are simple, fast, have high-throughput, can be performed by trained technician, and can be highly sensitive and specific. This technique is frequently used in validation of proteomic discoveries and in clinical settings as well. However, detection of only a single antigen at a time is an important limitation which is being addressed by the development of a number of multiplex immunoassays.P <0.001) in patients with metastatic disease at presentation than in patients with local-regional disease or normal controls (P <0.001). The MMP levels were also significantly associated with 1-year progression-free survival as was the individual MMP-2 trend  [Proteases play a key role in normal physiological functions in the body. However, dysfunction in the biological control mechanisms of proteases may contribute to various diseases, including cancer and cancer metastasis . Therefoctively) . These rctively) . In summctively) . Recentlctively) .in vivo imaging [Activity-based protein profiling (ABPP) has recently developed as an unconventional approach that is complementary to gene expression analysis and represents a tool to assist in decoding the overflow of genomic information . Selecti imaging . ABPs ha imaging . Recentl imaging . Moreove imaging . Lastly, imaging . TherefoBioinformatics plays a key role in the analysis of MS data  used to 2 (Beckman Coulter), FAST Quant (Whatman Schleicher & Schuell BioScience), and FlowCytomix (Bender MedSystems) among others [The biomarker validation stage is a necessary requirement indispensable for successful biomarker implementation in clinical practice . Only thg others . In partg others  and cling others . The MSDg others . Recentlg others . The PLAg others . Quantifg others . Anotherg others . Promisig others . Selecteg others . Howeverg others , 55. In g others , 55. In Recent advances in proteomic technologies, molecular biology and better understanding of carcinogenesis with respect to PCa has led to the discovery of new protein biomarkers  and to iUrine is an ideal body fluid and liquid biopsy resource for the identification and measurement of protein biomarkers  for pros


Transfer RNA (tRNA) is a dynamic molecule used by all forms of life as a key component of the translation apparatus. Each tRNA is highly processed, structured, and modified, to accurately deliver amino acids to the ribosome for protein synthesis. The tRNA molecule is a critical component in synthetic biology methods for the synthesis of proteins designed to contain non-canonical amino acids (ncAAs). The multiple interactions and maturation requirements of a tRNA pose engineering challenges, but also offer tunable features. Major advances in the field of genetic code expansion have repeatedly demonstrated the central importance of suppressor tRNAs for efficient incorporation of ncAAs. Here we review the current status of two fundamentally different translation systems (TSs), selenocysteine (Sec)- and pyrrolysine (Pyl)-TSs. Idiosyncratic requirements of each of these TSs mandate how their tRNAs are adapted and dictate the techniques used to select or identify the best synthetic variants. Ka or redox potential [Genetic code expansion (GCE) involves the engineering of protein synthesis machinery to site-specifically incorporate non-canonical amino acids (ncAAs) into a desired protein ,2. This otential .Pyl, a natural UAG suppressor. In archaea, PylRS is a single polypeptide chain; however, bacteria harbor a split protein where the C-terminal catalytic domain is only active in the presence of the N-terminal domain [Pyl pairs are used to engineer proteins with unique properties and functions in bacteria, viruses, insects, yeast, and animals [The most versatile aaRS for incorporating ncAAs is pyrrolysyl-tRNA synthetase (PylRS). Naturally, PylRS attaches pyrrolysine (Pyl), the 22nd genetically encoded amino acid, to its cognate tRNAl domain ,5. PylRSl domain . Moreove animals ,9,10,11.Another valuable building block for protein engineering is the 21st amino acid, selenocysteine (Sec). Sec is a naturally occurring amino acid that resembles cysteine but has a selenol group instead of the thiol. Sec is found in the active site of redox enzymes of species that span all three domains of life, providing enhanced nucleophilic and reducing properties . The sitPyl, there is no aaRS to form Sec-tRNASec. Rather, Sec is biosynthesized in a tRNA-dependent manner  for conversion to Sec-tRNASec (Sec is phosphorylated to form O-phosphoseryl-tRNASec (Sep-tRNASec) by Sep-tRNA kinase (PSTK) [Sec delivery to the ribosome is aided by a selenocysteine-specific elongation factor (SelB in bacteria or EFSec in eukaryotes) [Sec to the ribosome for the recoding of a UGA stop codon [While PylRS directly ligates an ncAA onto tRNAiewed in ). In bac-tRNASec . In eukae (PSTK) , to whice (PSTK) ,18,19. Saryotes) ,21. Furtop codon ,23  components for GCE, it is ideal to produce a high amount of the ncAA-tRNA while retaining orthogonality and limiting the effects on cellular fitness. Heterologous aaRS•tRNA pairs for the OTS of a particular host organism are often imported from a different domain of life, since tRNA identity elements and substrate recognition are dissimilar enough to function orthogonally . Moreovesequence ,29,30,31Sec is naturally inefficient compared to canonical aminoacyl-tRNA formation; SerRS serylates tRNASec 100-fold less efficiently than tRNASer [Sec to ensure limited amounts of Ser misincorporation during selenoprotein expression [Pyl formation is inefficient compared to other aaRSs and PylRS has a moderate level of catalytic activity [The production of Sec-tRNA tRNASer . It is lpression . Similaractivity ,33. It hactivity ,35.Pyl pairs and components of the Sec pathway must be manipulated to out-compete host tRNAs or release factors for the targeted codon, while maintaining cellular fitness. Furthermore, altering the stoichiometry of the Sec-OTS components is important for the efficiency and homogeneity of selenoprotein production [As a result of increasing ncAA-tRNA concentrations, the cellular levels of the PylRS•tRNAoduction ,29. ThusDue to their interactions with various parts of the translation machinery, tRNAs are central to achieving highly efficient ncAA incorporation, and both Sec- and Pyl-OTSs can be significantly improved through tRNA engineering. This is often accomplished through rational design, structure-guided mutagenesis, and random mutagenesis. Current molecular biology techniques facilitate the construction of large libraries of mutants, while combining positive and negative selection has been a successful approach to finding better variants. Here, we discuss the aspects of tRNA biology that should be carefully considered prior to OTS engineering and review the recent developments of Sec- and Pyl-OTSs with a main focus on tRNA design.1G37 modification of tRNAAsp in yeast is required to inhibit erroneous charging by ArgRS [Asp [The identity elements of tRNAs are nucleotides and their modifications, which function as substrate recognition determinants. These determinants are found throughout the tRNA molecule and are essential for interaction with enzymes for aminoacyl-tRNA formation, as well as elongation factors. Moreover, tRNA recognition involves anti-determinant nucleotides and modifications to prevent the binding and charging of non-cognate tRNAs. In some cases, a single nucleotide mutation can change the tRNA identity and allow aminoacylation by a non-cognate aaRS . SimilargRS [Asp ), maintagRS [Asp . For thiGenetic code expansion designates a particular stop codon, or an “open” codon in genetically recoded organisms, for the insertion of an ncAA. Nonsense suppression is the most common way to insert ncAAs, since recoding is less detrimental to the proteome, given the low occurrence of stop codons. In this regard, the tRNAs of interest for GCE are typically those without identity elements in the anticodon, as their anticodons can be mutated to decode a stop codon of interest, while retaining aminoacylation capabilities. Conversely, if the active site of an aaRS is suitable for engineering ncAA substrate specificity, the anticodon binding domain can be evolved to recognize a nonsense suppressor tRNA ,41,42,43Sec and tRNAPyl. Thus, ncAA insertion can be easily directed towards a codon of interest using tRNASec and tRNAPyl, within the limitations of the host organism fitness and proteome perturbation.The genetic code naturally expanded to include Sec and Pyl, through the recoding of UGA and UAG, respectively. However, Sec can be efficiently inserted at sense codons ,45 and iE. coli, orthogonal tRNAs can be transcribed from “standard” constitutive and inducible promoters . To mimic the coding sequences of bacterial tRNAs, the naturally absent terminal CCA sequence is added to the 3′-end of the archaeal tRNA gene. In contrast, to ensure proper processing in eukaryotes, the 3′-CCA sequence of bacterial orthogonal tRNA genes is typically removed.Various factors influence the available pools of the aa-tRNA that can be used for peptide synthesis in the cell. These include amino acid and nutrient availability, tRNA expression and maturation , aaRS levels, and tRNA stability and degradation . For GCEPyl), bacterial tRNAs are utilized for GCE in eukaryotic hosts [E. coli tRNAs [Arg (used as a part of a dicistronic construct) fused upstream of the target tRNA [While archaeal tRNAs in principle are not orthogonal to eukaryotic aaRSs . Furthermore, tRNA processing is quite complex, sometimes involving intron splicing, trafficking to several subcellular locations [Between 6.5% and 16.5% of tRNA nucleosides are post-transcriptionally modified, depending on the organisms , and oveocations , and eveocations . While tocations , structuocations .iMet lacking m1A58 is polyadenylated by Trf4, which then triggers nuclease degradation by Rrp6 and the nuclear exosome [7G and m5C also play a role in tRNA stability. The rapid tRNA decay pathway (RTD) in yeast, involving 5′–3′ exonucleases Rat1 and Xrn1, targets mature tRNA that lack the m7G and m5C modifications [To ensure that an aberrant tRNA is not used for protein synthesis, tRNAs lacking certain modifications are targeted by nucleases for degradation. The nuclear surveillance turnover pathway ensures that a tRNA is properly modified during biosynthesis. For example, yeast pre-tRNA exosome ,58. The ications ,60. Thesications .E. coli suppressors depend on the isopentenylation of adenosine 37 for full activity [E. coli or yeast strain collections containing deletions and/or the overexpression cassettes of metabolic genes. Recently it was shown that the yield and specificity of O-phosphoserine incorporation is significantly improved by the deletion of cysteine desulfurase and the overexpression of E. coli dimethylallyltransferase (MiaA) and pseudouridine synthase (TruB) [SUP4) and other non-suppressor tRNAs that lack modifications [The modification of tRNA nucleotides also affects codon–anticodon interactions, binding at the ribosomal A site , and ultactivity ,64. A gee (TruB) . Furtherications . Thus, wSec and its delivery to the ribosome is complex compared to the canonical amino acid pathway and involves several interactions with different portions of tRNASec. The major challenge in engineering tRNASec for the more efficient incorporation of Sec is to improve serylation, while also having complete conversion of Ser-tRNASec to Sec-tRNASec. In addition to this, the requirement of a SECIS sequence directly after UGA necessitates an EF-Tu-mediated Sec insertion pathway for the design and expression of selenoproteins in bacteria.The biosynthesis of Sec-tRNASec with Ser by SerRS  that is recognized by EF-Tu/eEF1a, tRNASec has a longer 13-base-pair acceptor branch (8/5 or 9/4). The deletion of a base pair from the acceptor stem of E. coli tRNASec to resemble that of canonical tRNASer abolishes UGA read-through with Sec [Sec with SelA and SelB [Ser, which is the basis of SelA-tRNASec interaction [Identity elements of the tRNAwith Sec , likely and SelB . In addieraction .Sec complex obtained from single-particle cryo-electron microscopy depict how the linker region between D3 and D4 binds and distorts the variable arm of tRNASec, while an extended loop of D3 interacts with the acceptor and T stems [Sec compared to tRNASer, D3 and the linker between D3 and D4 of SelB provide Sec-tRNASec specificity.Comparisons of SelB and EF-Tu complex structures show similarities of acceptor stem binding, but also unique domains and motifs that provide tRNA specificity. The N-terminal half of SelB consists of three domains, named D1, D2, and D3, that are analogous to those of EF-Tu . D1 make T stems . In conjSec posed a challenge for engineering the EF-Tu-mediated Sec insertion. Although the binding specificity of tRNASec can be switched from SelB to EF-Tu by shortening the acceptor stem [Sec has different bases. Moreover, the last base pair of the acceptor stem and the first two base pairs of the T stem of tRNASec are anti-determinants of EF-Tu complexed with GTP [The acceptor stem of the tRNAtor stem , the eigtor stem . In the with GTP .Sec for EF-Tu recognition, named tRNAUTu (U for Sec and Tu for EF-Tu), was designed using E. coli tRNASer as a scaffold with the first seven base pairs of the E. coli tRNASec acceptor stem [UTu acceptor stem was transplanted from tRNASer to eliminate the EF-Tu anti-determinant position. Serylation of tRNAUTu was as efficient as canonical tRNASer, however, the Ser to Sec conversion was hampered, which led to ~30% Ser misincorporation. Nonetheless, tRNAUTu was successfully used to site-specifically incorporate Sec into selenoproteins of bacterial and human origin in a SECIS-independent manner.The first generation tRNAtor stem . The lasUTu to Sec-tRNAUTu. E. coli tRNASec was used as a scaffold for the random mutagenesis of the EF-Tu anti-determinant base pairs C7:G66, G49:U65, and C50:G64. A Sec-specific NMC-A β-lactamase reporter was selected as an efficient tRNASec suppressor containing G7:C66, U49:G65, and C50:U64, which was named tRNASecUX [SecUX dosage was decreased, and PSTK was co-expressed to form a Sep-tRNASecUX intermediate, which would remain a substrate for SelA but not for EF-Tu prior to Sec conversion.Complementary approaches were taken to address the incomplete conversion of Ser-tRNARNASecUX . In ordeUTu to improve Ser to Sec conversion. Using the structure of Aquifex aeolicus SelA in a complex with Thermus tengcongensis tRNASec, twenty-nine different tRNAUTu variants were rationally designed to include tRNASec features that interact with SelA, while maintaining those that are required for EF-Tu binding. E. coli FDHH was used as a Sec insertion reporter in a sensitive colorimetric assay to identify the best variant, named tRNAUTuX, which differed from tRNAUTu at 11 positions [UTuX was comparable to tRNAUTu and tRNASec. Ser-to-Sec conversion was increased to 90%, reaching a similar conversion rate as E. coli tRNASec. Furthermore, Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry analysis confirmed Sec insertion by tRNAUTuX into the selenoprotein, Grx1, but did not detect a peak corresponding to Ser insertion. More recently, tRNAUTu was used as a template for the generation of chimeric molecules to improve Sec incorporation and selenoprotein yields. It was found that a single base change of A59C in tRNAUTu, generating a molecule named tRNAUTu6, resulted in the highest expression levels of human GPx1 and nearly 90% Sec incorporation [Other studies have built on tRNAositions . Kineticporation .Sec molecules, a group of tRNAs with unusual cloverleaf structures were identified, named allo-tRNA [Ser identities and functioned as efficient amber suppressors with Ser [Sec molecules. SelA from Aeromonas salmonicida subsp. pectinolytica 34mel (As) was coupled with allo-tRNA for selenoprotein expression, since its cognate tRNASec also possesses a 12-base-pair acceptor branch [As tRNASec identities. In addition, the stoichiometry of allo-tRNA to As SelA was altered to ensure the complete sequestration of the tRNA for Ser-to-Sec conversion while also maintaining non-toxic levels of As SelA. Further optimizations and metabolic engineering efforts created a Sec-OTS consisting of allo-tRNAUTu2D, As SelD, As SelA, and Treponema denticola Trx1. Along with the high selenoprotein yields obtained with a Sec incorporation efficiency estimated at >90%, the stand-alone capabilities of this system make it ideal for use in other organisms [In a bioinformatic search for novel tRNAllo-tRNA ,83. Certwith Ser . Allo-tRr branch . Allo-tRrganisms .Pyl, and the AA-binding pocket can be separately adapted to accept some bulkier ncAAs [Pyl engineering include those aiming to improve its compatibility with the cellular machinery of the host. In E. coli, tRNAPyl was evolved by targeting the EF-Tu-binding regions [PylOPT may be more suitable for the delivery of one particular ncAA and less for the other  [Pyl was evolved. Mutations in the D-stem, D-loop, T-loop and the anticodon-stem U29aC proved to be indispensable for high activity [Pyl, the use of this variant in HEK293 cells improved the incorporation of two ncAAs, Nε-carbobenzyloxy-lysine (Z-lysine) and Nε-(tert-butoxycarbonyl)-lysine (Boc-lysine). Interestingly, a chimera between mitochondrial (mt) tRNASer and Methanosarcina mazei tRNAPyl improved the insertion of Boc-lysine selectively (C15) [Ser in E. coli failed, due to the lack of orthogonality [Compared to the Sec system, the use of Pyl-OTS is comparatively less challenging, as its tRNA is orthogonal in the majority of model organisms used for GCE ; the enzer ncAAs . Attempt regions , althougalanine) . The neealanine) ,89, the alanine) . For effalanine) ,92. By iactivity ; comparegonality . The impgonality .Pyl, which together with the T-loop forms a dipped surface [M. mazei PylRS it is evident that this minimalistic variable loop is a prerequisite for effective binding, as a larger variable loop would sterically clash with the N-domain [M. mazei, Desulfitobacterium hafniense Pyl-OTS was employed in E. coli, either with its original N-terminal domain, or as a fusion with the recombinant (chimeric) N-domain of the archaeal system [Pyl with extremely high affinity [One of the distinct features of the Pyl system is the minimal variable loop of tRNA surface ,95  are highly active in E. coli [Pyl features  but also diverge in the nucleotide composition of the acceptor stem and in the probable structure of the anticodon stem  [Pyl anticodon to CCG this almost universally orthogonal tRNA becomes a substrate for endogenous ArgRS. In conclusion, while the anticodon-blind recognition of PylRS allows the anticodon of tRNAPyl to be mutated into any nucleotide triplet, synonymous anticodons (such as CCU and CCG) can be recognized by host aaRSs with very different affinity, causing one tRNAPyl variant to lose its initial orthogonality.The creation of multiple, mutually orthogonal OTSs is inherently related to the number of liberated codons that can be targeted for ncAA incorporation. In addition to UAG-directed incorporation, Pyl-OTS was also employed for ncAA incorporation in response to rare arginine (AGG) codons in i, alone  or in ta Tyr-OTS . A simil-to-Pyl) . HoweverImprovements to OTSs have been emerging rapidly in recent years and are valuable for the accurate and efficient production of proteins containing ncAAs. The increasing amount of sequence data and bioinformatic/structural analyses reveal new molecules and novel mechanisms that help enhance each system. Moreover, advanced molecular cloning and directed evolution techniques help further shape the molecules that nature has provided into molecules that are better suited for the incorporation of ncAAs. tRNAs interact with each component of an OTS in the process of bringing the ncAA to the ribosome to insert a particular ncAA during peptide synthesis. For this reason, finding the best tRNA variant is critical for OTS developments. Our expanding knowledge of tRNA processing, maturation, and interaction mechanisms has guided tRNA engineering towards this goal. As we continue to learn more from nature and as technologies advance, it is conceivable that peptides with unique properties will be produced with significant industrial and medical implications.


Identifying single nucleotide polymorphism (SNP) interactions is considered as a popular and crucial way for explaining the missing heritability of complex diseases in genome-wide association studies (GWAS). Many approaches have been proposed to detect SNP interactions. However, existing approaches generally suffer from the high computational complexity resulting from the explosion of candidate high-order interactions. In this paper, we propose a two-stage approach  to detect high-order genome-wide SNP interactions based on significant pairwise SNP combinations. In the screening stage, to alleviate the huge computational burden, ClusterMI firstly applies a clustering algorithm combined with mutual information to divide SNPs into different clusters. Then, ClusterMI utilizes conditional mutual information to screen significant pairwise SNP combinations in each cluster. In this way, there is a higher probability of identifying significant two-locus combinations in each group, and the computational load for the follow-up search can be greatly reduced. In the search stage, two different search strategies (exhaustive search and improved ant colony optimization search) are provided to detect high-order SNP interactions based on the cardinality of significant two-locus combinations. Extensive simulation experiments show that ClusterMI has better performance than other related and competitive approaches. Experiments on two real case-control datasets from Wellcome Trust Case Control Consortium (WTCCC) also demonstrate that ClusterMI is more capable of identifying high-order SNP interactions from genome-wide data. Genome-wide association study (GWAS) has become a popular and powerful tool for studying human complex diseases . Many GWThere are two challenges in SNP interactions’ detection. The first is the intensive computational burden caused by the exponential growth of the search space. For moderate genome-wide data, even only considering pairwise SNP combinations, more than ten billion combinations need to be evaluated. The second is developing statistical tests that can effectively detect SNP interactions. To attack the first challenge, some computationally-efficient approaches have been proposed ,7,8,9,10Currently, there are four categories of SNP interactions’ detection approaches: exhaustive search, stochastic search, machine learning search and stepwise search. The exhaustive search-based approaches enumerate all SNP combinations and performs the chi-square test (or logistic regression) for each combination. Ritchie et al.  proposedStochastic search-based approaches apply random sampling procedures to search the space of SNP interactions. BEAM   firstly screen an SNP subset based on some low-order statistic tests, then conduct multi-locus detection based on the selected SNP subsets. EDCF   to detect high-order SNP interactions based on two-locus combinations. In the screening stage, considering there is a higher probability of identifying significant two-locus combinations in a high-associativity SNP group, ClusterMI firstly utilizes clustering to divide SNPs into several clusters and applies the mutual information as the association measurement. Then, ClusterMI applies conditional mutual information to screen significant pairwise SNP combinations in each cluster. This strategy avoids exhaustively analyzing all two-locus combinations and greatly reduces the search space and computation load. In the search stage, to be adaptive to different data sizes and to obtain high detection accuracy, ClusterMI provides two alternative strategies to search high-order SNP interactions. For a small candidate set, ClusterMI employs the exhaustive search. For a large candidate set, ClusterMI employs an improved ant colony optimization (ACO) algorithm . ExtensiD is the number of all datasets. Power can be seen as a measurement of accuracy in simulation experiments to compare the performance of different approaches. Since there was no training set and test set throughout the detection process, cross-validation was not suitable for ClusterMI. In actual fact, almost all SNP interaction detection approaches adopt power as the measurement. We firstly analyze the parameter sensitivity of ClusterMI in To evaluate the performance of ClusterMI quantitatively, the power and runtime were used as the evaluation metrics. We adopt the same measurement of power suggested by Wan et al. :(1)Powerk) and cMI  threshold λ=pAag et al.  to simulg et al. , SNPHarvg et al.  and HiSeg et al. . Particui et al. . For ModSince ClusterMI grouped high-associativity SNPs into clusters and screened two-locus combinations from these clusters, ClusterMI(E) had a higher power than other approaches on the five models (Model 1–Model 5), except a few cases. In such cases, ClusterMI(E) had lower power than DCHE and EDCF. That was because DCHE and EDCF measured the significance via the chi-square test with a lower degree of freedom than ClusterMI(E), and they could report more interactions in such cases. For all models, when ClusterMI(E) had better performance than DECMDR, HiSeeker(A) and ClusterMI(A), since ClusterMI(E) employed exhaustive search to find optimal solutions without considering the time complexity, while DECMDR, HiSeeker(A) and ClusterMI(A) applied heuristic search. ClusterMI(A), HiSeeker(A) and DECMDR performed poorly when The high-dimensional simulation dataset with 8000 samples (4000 cases and 4000 controls) and 3000 SNPs for Model 5 was further used to test ClusterMI and other comparison approaches. The parameter settings including MAF and LD were the same as above described. We also used the same simulation program as Zhang et al.  to simulp-value (Hardy–Weinberg equilibrium) <0.0001 in controls or a sample with a call rate of <98%. Then, SNPs with MAF <0.1 were further excluded. This process produced a BC dataset containing 1045 cases and 3893 controls with 5607 SNPs.It has been reported that breast cancer is caused by a combination of genetic and environmental risk factors . A real p-value was below the chi-square threshold. In this way, 3812 two-locus combinations were retained for the next stage. Many retained two-locus combinations were significant, and several representative ones are reported in ClusterMI took 10 min to analyze the BC dataset. In the screening stage, ClusterMI employed conditional mutual information combined with the chi-square test following igenesis . This gerognosis . rs37851t cancer .B, which belongs to HLA class I heavy chain paralogues. Antigen expression of HLA class I is associated with the aggressiveness and prognosis of breast cancer [In the search stage, ClusterMI also identified some significant three-locus combinations, and a representative one is reported in the last row of t cancer . These sFurthermore, we also ran HiSeeker, EDCF and DCHE on the BC dataset. HiSeeker took 15 min to analyze this dataset, and selected 2576 two-locus combinations in the first stage. SNP interactions identified by HiSeeker were  on chromosome 3,  on chromosome 16 and  on chromosome 6. rs1108842 in gene GNL3, rs3785181 in gene GAS11, rs879882 in gene POU5F1 and rs2523608 in gene HLA-B are reported to have a high probable association with tumor or breast cancer. All of these SNPs can be identified by ClusterMI. In addition, ClusterMI also identified another SNP interaction  on chromosome 16. rs17822931 is in gene ABCC11, which is reported to be associated with breast cancer. EDCF and DCHE heavily suffered from the marginal effects, and they did not identify interactions associated with breast cancer in the BC dataset.Celiac disease (CD) is a common heritable chronic inflammatory condition of the small intestine induced by dietary wheat, rye and barley in susceptible individuals . After tClusterMI took 15 min to analyze the CD dataset. In the screening stage, ClusterMI also employed conditional mutual information combined with the chi-square test to screen two-locus combinations. For the threshold of cMI, since the values of conditional mutual information between two SNPs for CD dataset were generally above 2.1, we selected a larger value 2.19 as the cMI threshold. For the threshold of the chi-square, we used the same value as the BC dataset  on chromosome 6, in which rs542441 in gene N and number of SNPs M. The recorded runtimes of these approaches are shown in Computational efficiency is another key performance index that needs to be considered in high-order SNP interactions’ detection. The runtime of ClusterMI was compared to those of the other four approaches under varying sample sizes N individuals , heterozygous genotype (Aa) and homozygous variant genotype (aa). Generally, they are coded as {1, 2, 3}, respectively. The whole framework of ClusterMI is illustrated in Given a genotype dataset consisting of W. The following two subsections elaborate on these two stages.ClusterMI is a two-stage approach. In the first stage, it screens candidate two-locus combinations using clustering and mutual information. In the second stage, it detects high-order SNP interactions based on two alternative search strategies (exhaustive search and improved ACO search) for different sizes of candidate set We assume that SNPs with high associativity usually have a higher probability of being identified as significant SNP interactions. Given this, ClusterMI clusters SNPs into several groups and then selects two-locus combinations from each group. Since only two-locus combinations in each group need to be analyzed, clustering SNPs can greatly reduce the search space and computation complexity. For a GWAS dataset containing 1 million SNPs, the number of two-locus combinations that need to be analyzed is D with N samples and M SNPs, the clustering process is described as follows:(a)k SNPs are randomly selected from M SNPs as initial centroids of k clusters k is the preset number of SNP groups.Initialization: (b)u (or v) = {1,2,3} denotes the three genotypes of m-th Clustering: Mutual information can measure the dependency or associativity between two variables ,41. Give(c)k clusters. Suppose m-th cluster; ClusterMI measures the sum of mutual information of centroid Update centroids: In each iteration, ClusterMI updates each centroid after each SNP has been divided into one of the Then, for each SNP If Given a dataset k clusters no longer change or the maximum number of repetitions is reached. In this way, ClusterMI obtains k SNP groups. Each cluster has a disjoint subset of all the SNPs, and two-locus interactions with high-associativity SNPs are more likely to be significant and placed into the same subset. As such, ClusterMI can efficiently screen significant pairwise SNP combinations from a reduced number of SNP combinations, instead from all SNP combinations.Steps (b) and (c) are repeated until the centroids of k clusters, ClusterMI then applies conditional mutual information to screen two-locus combinations in each cluster as follows:(a)m-th cluster, the association between a two-locus combination and the disease can be measured by conditional mutual information [y = 1) can be calculated as:For the ormation ,41. The (b)ClusterMI takes a two-locus combination After dividing all SNPs into W.For all two-locus combinations in each cluster, ClusterMI applies the same two-step procedure (a) and (b) described above to screen significant SNP combinations. Next, it places all the significant two-locus combinations of each cluster into the candidate set W.In the search stage, ClusterMI provides two alternative strategies (exhaustive search and improved ACO search) to search high-order SNP interactions based on the size of W is small, and it has a larger chance to detect high-order SNP interactions than heuristic search. To exhaustively search K-SNP  algorithm [W obtained in the first stage. Here, we take K-SNP (i)W is initialized as Initialization: The pheromone value of all two-locus combinations in (ii)i-th two-locus combination t can be defined as [q is a randomly-generated number with a uniform distribution in  and R and T can be described as:i-th two-locus combination t and a at iteration t, and a randomly selects a two-locus combination from Combinations selection: ACO introduces fined as :(8)Pai((iii)u-th joint genotype for the three-locus combination and the v-th disease status, u-th joint genotype under the case i-th two-locus combination i-th two-locus combination t, a at iteration t and Pheromone update: In each iteration, each ant selects two two-locus combinations, the corresponding pheromone of each combination is updated as:When the cardinal of lgorithm  to searclgorithm ,42,43,44K-SNP combinations of all these reported combinations. It finally takes K-SNP subsets, the p-values of which are below the Bonferroni-corrected significance level K-SNP interactions.The improved ACO algorithm introduces a memory strategy to save the optimal solutions in each iteration and speed up the convergence . The aboIn this paper, we developed a two-stage approach called ClusterMI to detect high-order SNP interactions from genome-wide case-control data. Considering SNP groups with high associativity usually have a higher probability to encompass high-order SNP interactions and the high computational complexity with the exponential growth of high-order SNP combinations, ClusterMI employs a clustering algorithm that utilizes mutual information as the association measurement, to divide all SNPs into several clusters. Next, it applies conditional mutual information to screen significant two-locus combinations in each group. As such, ClusterMI avoids exhaustively analyzing all two-locus combinations and greatly reduces the computational complexity. In the search stage, ClusterMI provides two alternative search strategies (exhaustive search and improved ACO search) to detect high-order SNP interactions based on the size of candidate two-locus combinations. Exhaustive search is used for a small candidate set, and improved ant colony optimization (ACO) is used for a large candidate set. Extensive simulation experiments compared to representative approaches show that ClusterMI has a better performance. Experiments on two real case-control datasets also demonstrate that ClusterMI can identify high-order SNP interactions from genome-wide data.The clustering algorithm utilized in the screening stage of ClusterMI can place SNPs with high-associativity into a cluster, in which the true SNP interactions can be more easily identified. In addition, it greatly reduces the computational complex by avoiding analyzing the whole set of two-locus combinations.The conditional mutual information-based evaluation strategy in each high-associativity cluster can effectively screen two-locus combinations and reduce the search space of ACO; it also can improve the power of SNP interactions’ detection and make high-order SNP interactions’ detection on genome-wide data more efficient.ClusterMI can be an effective methods for detecting high-order interactions, and its main contributions are:Although ClusterMI shows good performance on both simulated and real datasets, it still has some limitations and can be improved. In the future work, we will investigate more efficient and effective clustering algorithms ,46,47, d


Drosophila visual system is an excellent model for understanding the developmental control of the nervous system. By performing a systematic transgenic RNAi screen, we investigated the requirements of secreted proteins and cell-surface receptors for the development of photoreceptor neurons (R cells) and wrapping glia (WG) in the Drosophila visual system. From the screen, we identified seven genes whose knockdown disrupted the development of R cells and/or WG, including amalgam (ama), domeless (dome), epidermal growth factor receptor (EGFR), kuzbanian (kuz), N-Cadherin (CadN), neuroglian (nrg), and shotgun (shg). Cell-type-specific analysis revealed that ama is required in the developing eye disc for promoting cell proliferation and differentiation, which is essential for the migration of glia in the optic stalk. Our results also suggest that nrg functions in both eye disc and WG for coordinating R-cell and WG development.The establishment of the functional nervous system requires coordinated development of neurons and glia in the embryo. Our understanding of underlying molecular and cellular mechanisms, however, remains limited. The developing  Building brain architecture requires the coordinated development of neurons and glia. In mammals, it is shown that neuronal-derived signals promote the proliferation and differentiation of glia such as astrocytes and oligodendrocytes , 2. AccuDrosophila visual system is an excellent model for understanding the control of coordinated neuron and glia development. Photoreceptor neurons (R cells) and wrapping glia (WG) originate from different tissue compartments. R cells are born in the eye-imaginal disc, an epithelial monolayer covered by the peripodial membrane, at the third-instar larval stage [The al stage . In the al stage .Recent studies identify several neuron-derived factors that coordinate the development of R cells and WG , 9. It iamalgam (ama), domeless (dome), epidermal growth factor receptor (EGFR), kuzbanian (kuz), N-Cadherin (CadN), neuroglian (nrg), and shotgun (shg). Cell-type-specific knockdown experiments show that while nrg acts in both eye disc and WG, the remaining six genes are only required in the developing eye disc for R-cell and WG development.To identify additional cell-surface players that are involved in regulating the coordinated development of R cells in the eye disc and WG in the sub-retinal region, we set out to perform a transgenic RNAi screen targeting 177 secreted proteins and cell-surface receptors   whose expression affected the projections of R-cell axons without causing obvious defects in WG development  and Thrombospondin (Tsp), respectively. In wild type , kuz, myospheroid (mys), nrg, shg, and SP2353.From the screen, we identified ten RNAi lines that disrupted the development of both R cells and WG Table , Fig. 4.mys (i.e. BDSC#33642) did not affect the proliferation and differentiation of R cells and WG , the organization of R-cell cluster in the eye disc was severely disrupted  proliferate in the optic stalk and subsequently migrate from the optic stalk into the sub-retinal space of the eye disc, where they differentiate into WG [To further understand the cause of severe WG phenotype in some knockdown animals, we analyzed the effects of  into WG .ama knockdown animals . Ho. HoDrosoCadN and shg encode for the fly orthologs of mammalian N-Cadherin and E-Cadherin, respectively, both of which belong to the classical Cadherin family of cell adhesion molecules. Consistent with previous loss-of-function studies [CadN disrupted the organization of R-cell axonal terminals in the optic lobe, and also caused abnormal axonal terminal morphology. However, the fact that WG projected normally in CadN knockdown animals suggests that unlike that of Tutl and Bdl [ studies , we show and Bdl , 10, theCadN knockdown that only affected R-cell axonal projection pattern in the optic lobe, shg knockdown severely disrupted the organization of R cells in the developing eye disc. Like its mammalian homolog E-Cadherin, Shg is well known for its role in regulating cell-cell adhesion during epithelial morphogenesis [shg knockdown animals. There are two possible explanations for the WG phenotype. First, since WG processes follow R-cell axons along the path of migration, the disorganization of R-cell axonal bundles in shg knockdown eye disc may present physical barriers for WG extension. And second, Shg, like Tutl, may mediate specific recognition between R-cell axons and WG, thus facilitate the migration of WG processes along R-cell axons.Unlike ogenesis . For insogenesis ). Thus, ama, dome, EGFR and kuz function in the developing eye disc, but not in WG, for the proper development of R cells and WG. The severity of defects in WG development appeared to correlate with the reduction in the number of differentiating R cells in the eye disc. The R-cell developmental defects suggest a role for these genes in promoting cell proliferation and differentiation in the eye disc. Indeed, dome, EGFR and kuz have been shown previously to function in key signaling pathways for regulating cell growth and differentiation [dome encodes for a transmembrane receptor for the cytokine-like ligand Upd, and functions in activating the intracellular JAK/STAT pathway [kuzbanian (kuz) encodes for a member of membrane-anchored metalloprotease-disintegrins (ADAMs) [kuz is required for Notch activation [kuz mutation severely disrupted the regular array of ommatidia in the adult eye [dome, EGFR and kuz, ama has not been previously implicated in promoting cell proliferation and differentiation. ama encodes for a secreted Ig-like protein [ama may also function as a growth factor to promote eye morphogenesis.Our results indicate that ntiation . For ins pathway . Upd/Jak pathway . The EGF pathway . And kuz (ADAMs) . It is rtivation . Previoudult eye , 27. Unl protein  and is r protein , 30. Ourama and nrg knockdown animals, suggest that a failure of glial migration due to defective eye development may also contribute to the defects in WG development.One likely explanation for the correlation between WG defects and the decrease in the number of R cells in the eye disc is the requirements of R-cell-derived signals for WG differentiation and projection. It is shown that R-cell-derived FGF8-like ligand Thisbe is required for the differentiation of WG . Thus, anrg encodes for the fly ortholog of the mammalian neural cell adhesion molecule L1, which is a member of the Ig superfamily [Drosophila, it has been shown that Nrg is involved in regulating axonal pathfinding and synaptic development (e.g. [nrg was knocked down in the eye only or in WG only. One possible explanation is that when Nrg on one cell type (e.g. eye-disc cell) was removed, opposing cell type (e.g. WG) still has Nrg on its surface, which allows Nrg to mediate eye-WG interactions by binding to another cell-surface receptor on opposing cell surface. The Nrg-mediated cell-cell recognition between WG and eye-disc cells may directly promote cell proliferation and differentiation for coordinating the development of R cells and WG. Alternatively, Nrg may function indirectly by antagonizing the effects of pro-apoptotic factors. Future studies will be needed to distinguish between these possibilities.The results from Cell-type-specific knockdown suggest that Nrg may mediate specific interactions between WG and certain cell types (R cells and/or other cell types) in the eye disc for coordinating the development of R cells and WG. erfamily . In Drosnt e.g. –34). Sin. Sinnrg nt  were either obtained from BDSC or generated in our previous studies [All the fly stocks are maintained at 25 °C with 50% humidity and 12/12 h light-dark cycle. The RNAi stocks were obtained from Bloomington Drosophila Stock Center (BDSC) and Vienna Drosophila Resource Center (VDRC) , rabbit polyclonal anti-GFP , rabbit polyclonal anti-Bdl (1:1000), Alexa 647-conjugated goat anti-HRP . Secondary antibodies including Alexa Fluor 488 goat anti-Mouse IgG  and Alexa Fluor 647 goat anti-Rabbit IgG , were used at 1:500 dilution.Epifluorescent images were analyzed by confocal microscopy (Olympus FV1000). Photo stacks are performed using Z-stack projection by FluoView software.Additional file 1: Table S1. A list of transgenic RNAi lines targeting 177 genes encoding for secreted proteins and cell-surface receptors. The positive genes are highlighted.


Findings from participants who attended the second post-Ebola SORT-IT course are reported in five short papers on the “legacy of Ebola on health Systems in Sierra-Leone”. After a decline of health service provision and utilisation during the Ebola outbreak, present findings show recovery over time in the post-Ebola period. However, important challenges remain, including an important health workforce gap. Findings from participants who attended the second post-Ebola SORT-IT course are reported here in five short papers on the “legacy of Ebola on health Systems in Sierra-Leone”. Consideration of these findings is an important step in building resilience to future challenges.Operational research studies, using the Structured Operational Research Initiative (SORT-IT) model3, present findings show recovery over time in the post-Ebola period. There are some encouraging signs that some health indicators may even be better than before the outbreak. Following investments in staff and community involvement, a steep increase of utilization of malaria diagnostic testing was reported post-Ebola3. In addition, post-Ebola childhood mortality is reported to be lower compared to pre-Ebola4. Encouragingly, the Community Health Worker programme, with a focus on the diagnosis and treatment of malaria, pneumonia and diarrhea, performed consistently throughout the pre-, intra-, and post-Ebola period5.After a decline of health service provision and utilisation during the outbreak6. The recovering health system was unable to cope with the increased demand for anti-malaria drugs, which resulted in stock outs3. In addition, a 12-fold increase of measles cases post-Ebola is an alarming indicator of reduced herd immunity and requires urgent action4. Finally, care for non-communicable diseases (NCD) has not yet returned to pre-Ebola levels7.However, some important challenges still remain. Although service utilization has increased, staff shortages remain unaddressed. Medical staff and non-medical staff deficits hover around 60% and 90%, respectively, and did not substantially change post-Ebola9. From the 2014–15 Ebola outbreak operational research initiatives we learnt that capacity building in programme data collection, analysis and reporting and timely access to information are absolutely needed to ensure learning-by-doing and evidence-informed adaptations to practice and policies during outbreak response10. Open access publication of operational research manuscripts even before the peer review process is completed, for instance through the F1000 portal, is a mean to achieve this. Moreover, in the post-Ebola era, more operational research such as that reported here will be key to inform programmes about progress beyond the pre-Ebola health status, and to monitor the journey to the Sustainable Development Goal “ensure healthy lives and promote wellbeing for all at all ages”.To fully recover from the Ebola outbreak, the health systems, including decentralized health care provision, needs to be strengthened further. Training of health care workers, robust information systems and reliable drug supply are needed to further increase and maintain access to health care provision. Moreover, community involvement is needed to re-establish trust, promote long-term utilization of health services and support disease surveillance and eventual future outbreak responseNo data is associated with this article


Although the amygdala’s role in shaping social behavior is especially important during early post-natal development, very little is known of amygdala functional development before childhood. To address this gap, this study uses resting-state fMRI to examine early amygdalar functional network development in a cross-sectional sample of 80 children from 3-months to 5-years of age. Whole brain functional connectivity with the amygdala, and its laterobasal and superficial sub-regions, were largely similar to those seen in older children and adults. Functional distinctions between sub-region networks were already established. These patterns suggest many amygdala functional circuits are intact from infancy, especially those that are part of motor, visual, auditory and subcortical networks. Developmental changes in connectivity were observed between the laterobasal nucleus and bilateral ventral temporal and motor cortex as well as between the superficial nuclei and medial thalamus, occipital cortex and a different region of motor cortex. These results show amygdala-subcortical and sensory-cortex connectivity begins refinement prior to childhood, though connectivity changes with associative and frontal cortical areas, seen after early childhood, were not evident in this age range. These findings represent early steps in understanding amygdala network dynamics across infancy through early childhood, an important period of emotional and cognitive development. Structurally, the amygdala shows precocious development in utero. The structure of the amygdala is discernable in 8 week-old embryos, and cytoarchitectonic differentiation begins by 12 weeks post-conception . The amyThe amygdala also demonstrates early functionality in humans. Studies have revealed a robust functional responsiveness to emotional stimuli by childhood (4–10 years) . In the An emerging focus in the study of the developing human amygdala is on understanding the early functional organization of the amygdala’s anatomical sub-regions. A large animal literature has established how these sub-regions serve different behavioral roles in maturity. Specifically, the laterobasal nuclei are central to aversive and appetitive valuation through cortical and subcortical connections . In contTherefore, the current study assessed amygdala functional networks in a cross-sectional sample from infancy through early childhood using resting-state functional magnetic resonance imaging (RS-fMRI). Specifically, we investigated early network connectivity of the whole amygdala as well as two of the amygdala’s major subdivisions, the laterobasal and superficial sub-regions, across infancy and early childhood. RS-fMRI provides a robust measure of functional network composition indexing the maintenance and stability of functional connections . Additio22.1The participants from this study were enrolled as part of a larger longitudinal study of typical brain development . InformeA total of 159 datasets were initially collected from 133 individual children between 3 and 54 months of age. Of the initial 159 datasets, 50 datasets were excluded based on an initial screening of motion characteristics of the functional data . A further 8 were excluded due to low quality of the structural scan. Finally, for children who were scanned on more than one occasion, only their later dataset was included, leading to the exclusion of a further 21 datasets. Unfortunately, the small number of longitudinal scans at present precluded robust mixed effects modeling of the data, so these additional scans were discarded for the present cross-sectional analyses . The rem2.21 weighted SPoiled GRadient echo (SPGR) structural image used here was acquired as part of the mcDESPOT sequence . This also means that the acquisition parameters  varied with age .ge group  and expe age see . These city e.g. .Table 1A1-weighted image was first corrected for intensity non-uniformities using the N4BiasFieldCorrection tool : repetition time = 2.4 s, echo time = 34 ms, flip angle = 80°, 32 interleaved 3.6 mm slices acquired at an orientation parallel to the anterior-posterior commissure line, in-place resolution of 2.97 mm, and a GRAPPA acceleration of 2. A total of 132 volumes were initially collected but the first 4 were discarded to allow magnetization to reach equilibrium leading to just over 5 min of data (307.2 s) per subject. Each subject contributed 307.2 s of data to analysis. That is, included scan length did not vary by age (due to the artifact correction procedures employed that do not censor entire volumes). Each resulting time-series was corrected for inter-scan motion using MCFLIRT (FMRIB Software Library (FSL), Oxford, United Kingdom  and had https://github.com/rhr-pruim/ICA-AROMA). AROMA is an independent component analysis (ICA) based method that automatically classifies and removes components identified as noise according to a combination the correlation of each components timeseries with motion, its spatial overlap with a mask at the edge of the brain, a mask of cerebrospinal fluid, and finally the proportion of high-frequency signal content. ICA-based motion correction approaches, including AROMA, are robust approaches for removing motion artifact in infant and child data .Amygdala sub-regions, laterobasal and superficial, were derived from a population atlas of architectonic profiles transformed to a standard population MRI atlas  provided3 unilaterally in the adult, larger than the partial volumes deduced from manual tracing with structural MRIs or extracted from standard atlases   . Studieslly e.g. , and stu greater . That is greater . The amytion see . Moreovetion see .All sub-regions of interest were bilateral, given both the high degree of similarity between left and right amygdala functional connectivity maps previously reported in adults  as well 2.5http://www.uniklinik-ulm.de/struktur/kliniken/psychiatrie-und-psychotherapie/klinik-fuer-psychiatrie-und-psychotherapie-iii-ulm/home/forschung/clinical-neuroimaging/digital-atlas.html. See Supplementary For the whole amygdala seed and for each amygdala sub-region individually, we calculated the mean time-series for every subject. Due to the proximity of the amygdala to areas of high vascular density, we also created a mask of regions associated with vascularization using a freely downloadable population atlas  availablWe tested whole-brain functional connectivity with the amygdala in a single-subject general linear model (GLM). In a separate single-subject GLM, we also tested the two amygdala sub-regions together. The inclusion of both LB and SF subregion timeseries in a single GLM model facilitated the identification of average connectivity and age-related connectivity patterns unique to each subregion (i.e. unrelated to the other subregion’s connectivity). These models also included the timeseries of the vascular region as a confound regressor. For each subject, the GLMs were tested using FSL’s Feat .A cross-sectional GLM was designed to test the average connectivity per contrast, including age, sex, average absolute scan-to-scan motion and average relative scan-to-scan motion as further covariates. Our contrasts of interest were average connectivity  and relationships with age and sex. The resulting parameter estimates for the whole amygdala and the amygdala sub-region contrasts were then input to a higher-level group analysis using this GLM and tested by permutation using FSL’s Randomise. Multiple comparison correction for each contrast was performed using the cluster extent option in Randomise (thresholding based on the null distribution of cluster size), with an initial cluster forming threshold of t>3.1.In addition, to explore non-linear age-related changes during this developmental period, two additional sets of GLMs were run as exploratory analyses, where the first set included a quadratic age term, and the second set included a natural log of age term. Both covariates were orthogonalised with respect to the linear age term. Results were corrected for multiple comparisons as before, using the cluster extent option in Randomise, with an initial cluster forming threshold of t>3.1. There were no statistically significant functional connectivity patterns associated with the quadratic age term that survived correction, so the following results are from set of analyses examining only the linear age-related and non-linear natural log(age) effects.33.1The whole amygdala had average positive connectivity with ventral cortical and subcortical structures. Significant positive connectivity with cortical structures was limited to the temporal cortex (middle and inferior gyri), insula, and posterior orbitofrontal cortex (OFC), while connectivity with subcortical structures included the basal ganglia, striatum , and thalamus a. The whThe LB sub-region had average positive connectivity with frontal and ventral cortical and subcortical regions in the paediatric sample a. The coThe SF sub-region had predominantly average negative connectivity in the paediatric sample. However, positive connectivity was observed with subcortical structures including bilateral amygdala, thalamus, and striatum  a. The SFNo significant sex effects were found in any analyses of whole amygdala, LB, or SF functional connectivity during this developmental period.3.2For the whole amygdala and amygdala sub-regions, associations between connectivity and increasing age were localized to sensory and motor-related regions b and c. The LB sub-region had positive linear age-related changes in connectivity across sensory and motor cortical regions, reflecting both reductions in negative connectivity and increases in positive connectivity. The LB sub-region showed a linear reduction in negative connectivity with the bilateral postcentral gyrus (somatosensory cortex), shifting from negative connectivity at age 3 months to no significant connectivity by age 5 years b and c. In contrast, the SF sub-region showed negative age-related changes in connectivity across subcortical and cortical sensory and motor regions. The SF sub-region connectivity with the thalamus shifted linearly from positive connectivity at age 3 months to negative connectivity by age 5 years b and c. 3.3Additional conjunction analyses directly compare the amygdala connectivity profiles in the pediatric sample to those of a publically available dataset of adult resting-state data  are included . Average functional connectivity maps for the pediatric datasets as well as their slopes with age are also included as both raw t-statistic images, and the t-statistic images after correcting for multiple comparisons (masked by the significant clusters). All images are in nifti format.4Despite the amygdala’s critical and basic role in shaping emotional and social behavior early in life, we currently have little knowledge regarding the human amygdala’s functional development during this period. Here, we characterized the functional (resting-state) networks of the amygdala and its LB and SF sub-regions. We also demonstrated their age-related changes in a large cohort of typically-developing children from 3-months to 5-years of age. We showed that the spatial pattern of functional connectivity pattern seen in later life is already largely present by early childhood , with the interesting exception of medial prefrontal cortex. Similarly, we distinguished functional connectivity patterns for each amygdala sub-region, suggesting network distinctions between sub-regions are evident and observable in early life. We have also shown age-related maturational changes in the whole amygdala and amygdala sub-regions’ resting-state connectivity limited to sensory and motor-related regions, a pattern unique to this early post-natal developmental period.Distinct patterns of average connectivity and age-related changes in connectivity separated each of the amygdala sub-regions in infancy and early childhood, and were consistent with connectivity observed later in development. The observed pattern of both positive LB connectivity with temporal and ventral cortical regions and negative LB connectivity with dorsal and posterior cortical regions is highly consistent with stable patterns observed later in development and in maturity . MoreoveAge-related changes in LB connectivity were also observed during infancy and early childhood with sensori-motor and occipito-temporal regions. LB-occipital connectivity increased with age during infancy and early childhood, in parallel with reports of extensive changes in the face-processing capacities subserved in part by amygdala-occipito-temporal connections . Future SF patterns of functional connectivity and age-related changes in connectivity were also highly consistent with those identified in later development. In particular, the positive functional connections with ventral caudate and anterior putamen regions and negative connectivity with broad posterior-cingulate and superior-frontal regions that were concordant between the paediatric and adult samples have all been observed as stable patterns in older populations. The SF’s positive subcortical connectivity is also in line with preliminary studies in adult humans implicating the SF in olfactory and social incentive processing . AlthougThe age-related changes in SF connectivity were also consistent with changes that continue through adolescence in older samples, suggesting contiguous, gradual refinement occurs in the SF-network. Specifically, while there was robust positive SF connectivity with much of the thalamus, consistent with both mature resting-state and stimulus-elicited SF-networks , there wAlthough functional networks continue segregating through adolescence, these spatial patterns of connectivity suggest core network components differentiating the sub-regions are in place early. This set of findings is consistent with evidence that amygdala sub-regions segregate structurally early in development . MoreoveSeveral patterns emerged across the sub-regions’ connectivity in infancy and early childhood distinguishing this period from amygdala networks in later developmental periods. First, the period of infancy is unique in that amygdala reactivity is absent to stimuli during this time that elicit responsiveness in childhood, but we and others have observed extensive functional connectivity during infancy . Thus, fIn particular, positive connectivity of the whole amygdala or either sub-region was not observed during infancy and early childhood with the peri-genual medial anterior cingulate. This finding is consistent with other reports in infancy . These rSeveral caveats should be noted in considering these results. First, large changes in brain size occur during this early developmental period . To addrIn summary, these findings illustrate that the amygdala’s LB and SF sub-regions have functional circuits in place by infancy, and progressive shaping of motor and sensory circuit components occurs during this period. Sub-regions can be distinguished from each other by their distinctive connectivity patterns as well as their age-related changes in connectivity. Indeed, these results suggest that the sub-regions may have disparate trajectories of network construction across development, requiring future longitudinal study. The robust amygdala connectivity observed in infancy seems to temporally precede observations of reactivity. These findings present the first information about typical age-related changes in human amygdala functional network development in early childhood and, cumulatively, these results represent an important initial step in understanding the early development of amygdala networks and their dynamics, central to sculpting emotional and social behavior.This work was supported by the National Institutes of Mental Health (RO1 MH087510) and The Bill & Melinda Gates Foundation (0PP1120016) to SD. SD is also supported by the Eunice Kennedy Shriver National Institute of Child Health & Human Development under the National Institutes of Health (NICHD UG3OD023313); LJG-D is supported by the Graduate Research Fellowship from the National Science Foundation (DGE-11-44155). J.O.M. is supported by a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (Grant Number 206675/Z/17/Z). This work was supported by the Wellcome EPSRC Centre for Medical Engineering at King's College London (WT 203148/Z/16/Z), the Department of Health through an NIHR Comprehensive Biomedical Research Centre Award  Foundation Trust in partnership with King’s College London and King’s College Hospital NHS Foundation Trust), DCD is supported by a T32 Postdoctoral Training Fellowship awarded by the Eunice Kennedy Shriver National Institute of Child Health & Human Development under the National Institutes of Health (T32HD007489 and U54HD090256) and a National Institute of Mental Health K99 award (K99MH11059).None.