Bce). Our results indicate the presence of high steppe ancestry in the Somogyvár–Vinkovci culture. They were then replaced by the Kisapostag group, who exhibit an outstandingly high (up to ∼47%) Mesolithic hunter–gatherer ancestry, despite this component being thought to be highly diluted by the time of the Early Bronze Age. The Kisapostag population contributed the genetic basis for the succeeding community of the Encrusted Pottery culture. We also found an elevated hunter–gatherer component in a local Baden culture–associated individual, but no connections were proven to the Bronze Age individuals. The hunter–gatherer ancestry in Kisapostag is likely derived from two main sources, one from a Funnelbeaker or Globular Amphora culture–related population and one from a previously unrecognized source in Eastern Europe. We show that this ancestry not only appeared in various groups in Bronze Age Central Europe but also made contributions to Baltic populations. The social structure of Kisapostag and Encrusted Pottery cultures is patrilocal, similarly to most contemporaneous groups. Furthermore, we developed new methods and method standards for computational analyses of ancient DNA, implemented to our newly developed and freely available bioinformatic package. By analyzing clinical traits, we found carriers of aneuploidy and inheritable genetic diseases. Finally, based on genetic and anthropological data, we present here the first female facial reconstruction from the Bronze Age Carpathian Basin.In this study, we report 21 ancient shotgun genomes from present-day Western Hungary, from previously understudied Late Copper Age Baden, and Bronze Age Somogyvár–Vinkovci, Kisapostag, and Encrusted Pottery archeological cultures (3,530–1,620 cal  Bce) and Bronze  Ages. A number of cultural transformations occurred in the Carpathian Basin, often attributed to population changes and genetic influxes. The observed shifts in ancestry compositions sparsely covered intensive or direct European HG introgressions into EEF or steppe ancestry groups  , their ay groups   or human leukocyte antigen (HLA) genes and pigmentation markers, are beginning to thrive , Kisapostag culture , and the Encrusted Pottery culture  that are referred to as Bk-I, II, and III phases in this study, respectively  and clinical variant analyses. Moreover, we present a series of bioinformatic tools for biological relatedness, ploidy, and variant analyses implemented in a new bioinformatic package for ancient DNA analysis. We analyzed archeological finds from the Balatonkeresztúr-Réti-dűlő site in Western Hungary (Transdanubia), where—among others—Bronze Age features and human remains were found during roadwork in 2003. Three Bronze Age archeological horizons were distinguished based on ectively  online. ectively . Howeverectively . AdditioBce), excavated ∼30 km away from Balatonkeresztúr, was added to the data set. Our data highlight not only detailed population events in a microregion but also reveal hidden processes that formed the genetic landscape of East-Central Europe at the beginning of the Bronze Age.In order to provide an additional data point to our analysis of population ancestry of the region, a Late Copper Age individual from the Baden culture  and whole mitochondrial DNAs (mtDNAs) of all individuals. The shotgun- and the capture-sequenced samples ultimately resulted in an average ∼144k SNPs/individual using the 1240k SNP panel for genotype calling  see Mat online. mobility .Bce). A majority of the samples came from the site of Balatonkeresztúr-Réti-dűlő. We sampled and sequenced 20 individuals, labeled S1–S45, skipping numbers that did not belong to the Bronze Age horizon or were not suitable for genetic testing. One male individual (S9) that was associated with Bk-I by 14C data has a very long (ultradolichocran) skull type, which differentiates him from most individuals in Bk-II and Bk-III, who have very short (brachycranic) skull types ; however, with Bayesian analysis using the OxCal software  were possibly slightly earlier (Bce) with skeletal remains of eight people of various ages. This is an unusual burial treatment as most bodies were cremated or buried as single inhumations in that culture. For detailed descriptions of the sites and burials, see We included only one Infans II (late childhood) individual (BAD002) from the site of Balatonlelle belonging to the early phase of the Baden culture  lineages provides rough estimates of genetic composition and is essential to assess biological relatedness and social structure for the studied population. Additionally, we performed phylogenetic analysis using MrBayes software  on mtDNAMPMR; fig. 1b) of Bk-II approximately follows the distribution of individuals in A–B grave groups (fig. 1a), which were likely established along family relationships and chronology. Individuals buried in the Bk-III mass grave only show a few blood relations (fig. 1c): a half-brother or uncle–nephew, a father–son, and a dizygotic twin; to our knowledge, the latter is the oldest detection of such relatedness. None of the distant inhumations  show biological relationship to any other individuals up to second degree. For further details, see Contrary to the diverse mtDNA makeup, most male individuals in Bk-II and Bk-III belong to the Y chromosome haplogroup I2a-L1229, except for two individuals that belong to haplogroup R1b-Z2103 . SimilarInvestigating genetic disorders in archeological data sets can be useful to improve our knowledge of the history of health and medicine and also highlights the overall genetic health of past populations. Genetic disorders, if accompanied with severe phenotypic anomalies, could also explain unusual burial practices, for example, as it was described in cases of dwarfism in the Byzantine era  or in thZ-score–Adjusted Coverage (ZAC) for ploidy estimates by using a set of reference genomes. This can estimate ploidy for samples as low as 0.01x average genomic coverage, enabling genetic sex determination and aneuploidy assessment for all new samples . After running the panel, we excluded low-coverage transitional variants from the final evaluation due to the possible presence of DNA damage. We only made exceptions when skeletal features supported the presence of the low-quality variant or when more than one sample possessed the same allele. Nevertheless, we listed all alternate variant hits in We also examined the nuclear genomes to find regions with clinical significance. Since a complete panel for determining disease susceptibility only exists in commercial DNA kits with nonavailable descriptions similar to the 1240k panel, we created a new SNP calling panel focused on various conditions including amyotrophic lateral sclerosis, Alzheimer disease, autism, Crohn's disease, diabetes, lactose intolerance, mental disorders, Parkinson disease, schizophrenia, and ulcerative colitis. For this study, we used a ∼5k set of clinically significant SNPs, which were marked as “pathogenic” or “likely pathogenic” in the ClinVar database , by ignoLig4 syndrome is a transitional mutation (rs104894421) induced disease with skeletal abnormalities , for whic), although some variants for lighter pigmentation exist within this group too. Members of Bk-III on the other hand show a wide range from dark to light pigmentation tones and even the presence of variants for red hair , Bk-I is clearly separated from Bk-II and Bk-III, where Bk-II has a strong shift toward European HGs overlapping with only a fraction of known ancient samples  for assessing genetic components revealed ∼17% HG, ∼40% EEF, and ∼43% steppe ancestry for Bk-I, similar to average Bronze Age Europeans –related population and Ukraine_EBA with almost equal contributions , whereas “dilution” of Bk-II to Bk-III is mostly driven by contact with various local populations, genetically best represented by later Transdanubian Hungary_LBA or Serbia_Mokrin_EBA_Maros (Maros culture) groups  with software  based onsoftware  and ADMIsoftware  analysessoftware . Accordisoftware a, Bk-I i samples  and Bk-Ianalyses b for assuropeans   groups  online.qpAdm analysis of the basic composition resulted in EEF ∼40 ± 2%, Eastern HG (EHG) ∼39 ± 3%, Western HG (WHG) ∼13 ± 2.7%, and Caucasus HG ∼8 ± 2%, P = 0.0917, in par with Admixture analysis, pointing toward a rather EHG characteristic composition. Next, we performed an f4 test in the form of f4 , but only marginal relations with the EHG (Lithuania_Mesolithic) populations , or Scandinavia, although we can see some weak connections to older (down to sixth millennium Bce) Lithuanian HGs. These results may reflect the population turnover in the sixth millennium Bce in the Baltics (qpAdm provides negative weights for this component when we model Bk-II as a combination of WHG (Loschbour_WHG), EHG (Lithuania_Mesolithic), EEF (Turkey_N), and Yamnaya (Russia_EBA_Samara_Yamnaya), suggesting that Lithuania_Mesolithic is not a good proxy for the actual EHG component.We aimed to investigate further the exact composition of HG ancestry in Bk-II. buti.DG) , to see buti.DG) . Contrar Baltics , suggestDATES  on all of the groups  moved northward from the region of today's Eastern Romania, Moldavia, or Western Ukraine, subsequently mixed with FBC- or Globular Amphora culture (GAC)–related populations and then split into two groups: one taking a route to Transdanubia and the other moving further North. These results are highly in par with 87Sr/86Sr isotopes to evaluate whether individuals were born in the area of their burial. The results  excluded the contribution of BAD002 to Bk-II  shows affinity to the Iberian Bell Beaker culture–associated individuals  online. to Bk-II  online.PAPline (Performing Archaeogenetic Pipeline), written in linux bash, R, and python v3.8.10 programming languages. One can use this package primarily to analyze next-generation sequencing data of archeogenomic samples, supplemented by tools, including ploidy test, MPMR relatedness analysis, and clinical variant test. The standalone tools and the core workflow of PAPline are available at https://github.com/ArchGenIn/papline. In the future, PAPline is aimed to be compared with the EAGER  and genetic continuity for hundreds of years at the studied site. Observable dilution of HG ancestry in Bk-III compared with Bk-II can be connected to continuous female-biased admixture with nearby communities according to our and previous genetic  and arch87Sr/86Sr isotope data show local values for both sexes, which along with similar genomic makeup of females and males suggest exogamy most probably between villages of the same population. The overlap between outlier parallels of Bk-II/III and archeological contact regions is also noteworthy, as it suggests smaller scale migrations of Kisapostag/Encrusted Pottery individuals or groups along trading networks, such as mobility possibly connected to wandering merchants.In both periods, the homogeneity of paternal lineages suggests a patrilocal residence system, similarly to previously described social organizations . HoweverBce Maros culture individuals from Mokrin, Serbia  of the uniparental lineages are the same at the haplogroup level with the individuals from the Croatian Encrusted Pottery culture Jagodnjak site, despite high similarities in cultural traits, social structure, and genomic composition of the communities . This su, Serbia , that shThe relatively limited presence of female and children burials in both Bk-II and Bk-III periods may suggest distinctive treatment or another (here undiscovered) burial group for women and children at the same site. However, in other cemeteries of the culture, for example, Ordacsehi and Bonyhád in Hungary, males, females, and children were buried close to each other, suggesting high variance in burial practices .Although low genomic coverages did not allow fine SNP recovery, we did find evidence for malignant variants within all of the tested groups and undoubtedly showed the presence of LHON and Jacob's syndrome within Bk-II. Whereas it only remains a possibility, the presence of autism risk factor in the CNTNAP2 gene, signs for severe bruxism, and the distinctive burial treatment of individual S45 suggest the actual onset of symptoms. Additionally, the disease panel we created and made freely available can be extended and used in future studies, providing insight into past population health qualities.87Sr/86Sr isotope analyses on the first and third molars of the individuals in the BK-III mass grave indicate that subadult males—including a severely disabled individual (S15) with hip dysplasia—left their community for a while and then returned to their birthplace prior to their death, raising further questions for future studies on prehistoric lifeways and social organization.Considering the unstructured age and biological relatedness distribution in the mass grave of Bk-III compared with Bk-II, the coetaneous death of eight people at least, the absence of traumatic or ritual events on bones, and the noncremated nature of the burial all signal a sudden tragic event in the Middle Bronze Age period (Encrusted Pottery population), most likely an epidemic, as first suggested based on the anthropological analyses . Careful87Sr/86Sr isotope measurements were performed in the ICER Centre, Institute for Nuclear Research Debrecen, Hungary, and at Quinnipiac and Yale University, Connecticut, USA  with AmpFLSTR Yfiler PCR Amplification Kit (Applied Biosystems), having one blank and one positive control in each reaction. The workflow followed the recommended protocol except the PCR cycles were increased from 30 to 34, and reactions were halved in volume. Two repeats were done where at least four markers yielded results. Data analyses were carried out in GeneMapper ID Software v3.2.1 (Applied Biosystems), and results are summarized in PAPline (https://github.com/ArchGenIn/papline). We used the GRCH37.p13 reference sequence to call the pseudohaploid genomes. For relatedness inferences, we applied the READ software (MPMR; see MrBayes v3.2.6 (BEAST v1.10.4 (Popgenome (R package (see Yleaf v1 (Network v10.1.0.0 and Network publisher v2.1.2.5 (ADMIXTURE v1.3.0 software (f statistics and qpAdm were performed using the admixr v0.9.1 (admixtools v2.0.0 (R packages. The timing of the admixture events was inferred by using the DATES software (Illumina sequencing paired-end reads were processed by the software  and a cus v3.2.6  and the  v1.10.4  softwareopgenome  R packagkage see  online. Yleaf v1  softwarev2.1.2.5  programsv2.1.2.5  online. v2.1.2.5  using thv2.1.2.5 ; for othv2.1.2.5  was usedv2.1.2.5  online. software . f statir v0.9.1  and the s v2.0.0  R packagsoftware .msad182_Supplementary_DataClick here for additional data file.


Understanding urban travel behaviour is crucial for planning healthy and sustainable cities. Africa is urbanising at one of the fastest rates in the world and urgently needs this knowledge. However, the data and literature on urban travel behaviour, their correlates, and their variation across African cities are limited. We aimed to describe and compare travel behaviour characteristics and correlates of two Kenyan cities (Nairobi and Kisumu). We analysed data from 16,793 participants  in a 2013 Japan International Cooperation Agency (JICA) household travel survey in Nairobi and 5790 participants (2760 households) in a 2016 Institute for Transportation and Development Policy (ITDP) household travel survey in Kisumu. We used the Heckman selection model to explore correlations of travel duration by trip mode. The proportion of individuals reporting no trips was far higher in Kisumu (47% vs 5%). For participants with trips, the mean number [lower - upper quartiles] of daily trips was similar (Kisumu (2.2 [2–2] versus 2.4 [2–2] trips), but total daily travel durations were lower in Kisumu (65 [30–80] versus 116 [60–150] minutes). Walking was the most common trip mode in both cities (61% in Kisumu and 42% in Nairobi), followed by motorcycles (17%), matatus (minibuses) (11%), and cars (5%) in Kisumu; and matatus (28%), cars (12%) and buses (12%) in Nairobi. In both cities, females were less likely to make trips, and when they did, they travelled for shorter durations; people living in households with higher incomes were more likely to travel and did so for longer durations. Gender, income, occupation, and household vehicle ownership were associated differently with trip making, use of transport modes and daily travel times in cities. These findings illustrate marked differences in reported travel behaviour characteristics and correlates within the same country, indicating setting-dependent influences on travel behaviour. More sub-national data collection and harmonisation are needed to build a more nuanced understanding of patterns and drivers of travel behaviour in African cities. It helps us estimate the population risk of road traffic injuries, the physical activity resulting from active travel, and transport-related air pollution and greenhouse gas emissions . MeasuriKnowledge of urban travel behaviour is limited in low- and middle-income countries (LMICs), especially in Africa . It is oThe gaps in the literature on African urban travel behaviour stem primarily from the lack of routine surveys and the inadequate exploitation of the few available data. The scant literature provides insights into transport challenges in some thematic areas, such as gender and socioeconomic inequities in mobility, but these are generally not based on representative survey data. Moreover, the available literature  mainly rComparing travel behaviours among cities provides not only valuable information on the risk of road traffic injuries, air pollution, and transport-related physical activity but also yields transferable lessons which cities at different growth stages can use to tackle urban transport challenges. For example, if the factors favouring active travel in one city are modifiable and applicable in another. This information is also crucial for national governments, which need to design policies on urban transport that account for the different needs of cities at different growth stages. Given that Africa is urbanising at one of the fastest rates in the world  and in aThis study compares travel behaviour characteristics and correlates between two Kenyan cities: Nairobi  and Kisumu (the third-largest city in Kenya). Travel behaviour characteristics include trip number, mode, duration, and purpose . The stu22.1We used two cross-sectional travel surveys from two cities in Kenya  household travel survey in Nairobi  and the 2.2The study is based in two Kenyan cities: Nairobi and Kisumu a and b. 2 in 2013. As Kenya's largest city, it attracts many people from other urban and rural areas in search of better economic and livelihood opportunities. This has led to rapid and uncontrolled urban growth, congestion of streets and residential areas, and proliferation of slums, with 60%–70% of Nairobi's population currently living in slums , was used to compare key indicators in the JICA and ITDP data.JICA surveyed a total of 10,000 households and 16,580 individuals in Nairobi in 2013 . The JICITDP surveyed 2760 households and 5970 individuals in Kisumu in 2016 . The teaBoth surveys included household questions on household composition, income, and vehicle ownership; individual questions on demographics, income and occupation; and 24-h trip diaries covering the number of trips taken, trip stages, modes, purposes and costs. Additional questions in Kisumu covered perceptions of the travel environment and opinions to improve travel in the city.To account for differences that potentially stemmed from comparing datasets collected by two different institutions at different locations and times, we compared commute characteristics from the two analysis datasets with those from the 2013 World Bank survey in Kenya. The World Bank survey collected baseline data on the state of the cities in Kenya, including data on usual commutes to school and work.2.5Predictor variables included household and individual characteristics. Household characteristics included household size , household monthly income, and household vehicle ownership. Individual characteristics included age, age group , sex, monthly income, and occupation . Individual monthly income was grouped into three categories to reflect the minimum monthly wage in Kenya, which was <10,000 KES (Kenya Shilling) between 2013 and 2016  Trading . One KenOutcome variables included individual trip characteristics. A trip was defined as one journey undertaken by a participant from origin to destination for a single purpose during the reference day. ‘No trip’ was defined as not making a trip on the reference survey day. A trip could have multiple stages, denoting the different stops to change transport modes during the trip. A trip mode was defined as the largest transport mode used during the trip, while the stage mode was the transport mode used in each trip stage. Transport modes included walking, cycling, motorcycle, tricycle, car, taxi, matatu (minibus), bus, truck, and train. Matatu refers to privately owned small-capacity (14–30) minibuses used as shared taxis, while a bus refers to a publicly owned larger-capacity vehicle running on fixed routes and schedules. Transport modes were grouped into non-motorised transport (NMT)  and motorised transport (MT) . Motorised transport was further grouped into private MT  and public MT ; and public MT was also grouped into formal public MT (bus) and informal public MT . The trip duration was the sum of stage durations within one trip, and the daily travel duration was the sum of all trip durations for trips travelled by an individual. Trip purposes included travel for work, school, recreation, shopping, personal business, return home and others.2.6For our exploratory analysis, we used a combination of summary statistics and plots to check for missingness, normality, zero inflation, and near-zero variance in data variables. With 3% and 5% of observations with missing data points in the Nairobi and Kisumu travel datasets, respectively, we simply excluded missing records from each relevant analysis. Our variables of interest did not show near-zero variance . Total daily travel duration and daily travel duration by mode were right-skewed; these were log-transformed.We summarised and compared household and participant characteristics for each site. We also summarised and compared travel behaviour characteristics  travel times by mode and trip purposes) by age group and sex for each study site. We used the mean (lower - upper quartile) to summarise variables with higher proportions of zeros, which could otherwise be all zeros if the median (interquartile range) was used. Our descriptive statistics were weighted using postratification weights derived from sex and age distribution. These demographic distributions were obtained from the 2014 population demographic data, which was a projection from the 2009 national population census.We used a Toby type 2 Heckman selection modelling framework to evaluate correlates of trip making, use of different transport modes , daily travel duration and travel durations in different transport modes. Our model choice was informed by the high frequencies of unobserved zeros in the data, especially for Kisumu. The total zeros were far more than the expected zeros in the case of a corner solution, suggesting unobserved zeros, which could result from soft refusal (participants reporting travel to avoid answering long survey questions). The model framework comprised two equations. The first equation was a probit model that evaluated the likelihood of making a trip and using a transport mode. The probit model also generated the inverse mills ratios (imr) which were used to parameterise the second equation. The second equation was an ordinary least squares (OLS) model that evaluated the correlates of the log-transformed daily travel durations. In addition, we used a Poisson regression to predict correlates of trip frequencies. All analyses were performed in the R software, notably using the “sampleSelection” package (version 1.2–12) for our modelling.We reported the outputs of the probit model as average marginal effects (AME), indicating the change in probability for any unit change in predictor variables. The outputs of the Poisson regression were reported as rate ratios. We reported the outputs of the OLS as per cent change in the daily travel time for a unit change in the predictors since the dependent variable was log-transformed. We reported the point estimates and their 95% confidence intervals for each dependent variable.33.1p < 0.001), while ownership of all forms of motorised vehicles was lower in Kisumu. Participants from Kisumu were younger and comprised more females, students, and people with no employment.We analysed data from 10,000 households and 16,794 participants in Nairobi and 2760 households with 5790 participants in Kisumu. Household and participant characteristics are summarised in 3.2p < 0.001), but when participants with no trips were excluded, the mean number of trips per participant with trips was similar in both cities . The mean duration per trip, mean total travel time per survey participant (including participants with no trips) and total travel time per survey participant (excluding participants with no trips) in Kisumu were half of those in Nairobi.The trip characteristics in each study site are summarised in Walking was the most common trip mode in both cities for both sexes, with most walking trips undertaken by females . CompareWalking was the transport mode for >50% of trips and more often for females than males in both cities . The meaWhen we grouped transport modes, NMT and informal public MT were the predominant modes, with similar transport mode shares in both cities . Formal With regard to trip purposes, Kisumu had lower proportions and shorter durations of work commute trips compared to Nairobi. Main trip purposes included work, school, personal business and return home. All other trip purposes followed similar patterns in both cities .3.2.1The proportion of participants with a walking stage during any trip in Kisumu was half of that in Nairobi (40% vs 83%) . Among tThe proportion of participants who cycled any trip in Kisumu was double that of Nairobi (4% vs 2.1%). Where trips were cycled in both cities, they were predominantly (90%) cycled all the way. In Nairobi, cycling was sometimes combined with other modes, mainly buses, matatus, and motorcycles.3.33.3.1For those with at least one trip on the reference day in Nairobi, females , younger participants, and homemakers and those with no work (compared to those employed) had shorter total travel times. Higher-income earners and students travelled for longer durations. In Kisumu, females and homemakers travelled for shorter durations, while older people were more likely to travel for longer durations.3.3.23.3.33.3.43.4The Nairobi (2013) and Kisumu (2016) travel surveys, and the World Bank (2013)  baselineIn terms of commute time by mode, the World Bank data showed that apart from cars, commute times for all modes were higher in Kisumu than in Nairobi. On the contrary, when using the Nairobi (2013) data and the Kisumu (2016) data, commute times were higher for all modes in Nairobi than in Kisumu. Although commute times were generally lower in the World Bank data, the time difference was more pronounced with the Nairobi (2013) data than with the Kisumu (2016) data.Overall the Kisumu travel survey closely matches the World Bank estimates in terms of commute duration and mode shares for NMT but not other modes. Similarly, the Nairobi travel survey closely matches the World Bank estimates in terms of NMT shares but significantly deviates when it comes to commute durations.44.1This is one of the very few studies addressing the literature gap on travel behaviour in African cities. It describes and compares travel behaviour characteristics and correlates between Nairobi, the capital city of Kenya, and Kisumu, the third-largest city in Kenya. We found a higher rate of no reported trips in Kisumu compared with Nairobi (47% vs 5%), with double the average number of trips per survey participant in Nairobi . The mean trip duration and daily travel time in Nairobi were double those in Kisumu. Walking was the most common transport mode, followed by matatus in both cities. Most walking (53%) was undertaken in combination with other modes in Nairobi, whereas walking was the sole transport mode in most walking trips in Kisumu (77%).Gender, income, occupation, and household vehicle ownership were variedly associated with individual trip making; use of NMT, informal PT, and private MT; and individual daily travel times in different transport modes. For example, in Nairobi, females, compared to males, were less likely to make trips , and those who made trips travelled for shorter times . In Kisumu, gender was not associated with trip making; for those who travelled, females travelled for shorter times than males . Living in households earning >10,000 KES ($100) per month was associated with a higher probability of trip-making and longer daily travel times in both cities. Females and people living in households with higher monthly incomes travelled for shorter durations in NMT modes and had higher chances of using informal PT in both cities.Other relevant studies in the literature focused on testing advanced travel survey designs , analysi4.2Despite filling an important gap in the literature, our study does have limitations, most of which are related to the data sources. Firstly, because the datasets originated from different organisations, collected at different times and for slightly different purposes, with possible contextual adaptations, there are likely inherent differences between the data sources. To account for differences resulting from data types, we explored our two main datasets and compared them with other available data. In Nairobi, four other representative surveys with travel-related questions have been conducted since 2004. One of these surveys was conducted by the Japan International Cooperation Agency (JICA) in 2004; the second survey by the Kenya Institute for Public Policy Research and Analysis (KIPPRA) in 2004, the third survey in 2010 by the African Centre of Excellence for Studies in Public and Non-Motorised Transport (ACET), and the fourth survey conducted by the World Bank in 2013. Many parameters have been found to substantially differ among these surveys, with JICA surveys reporting higher car mode share, fewer walkers and longer travel durations compared to all the other surveys . There i4.34.3.1The two cities have very contrasting rates of reporting no trips on the survey day (5% Nairobi vs 47% Kisumu). These values deviate substantially from the admitted 8 to 12% of the population that will be captured as immobile at some point , even ifHowever, the large observed differences strongly suggest considerable differences in participation and travel times between two major cities in the same country.Our findings that females were less likely to make trips, and when they travelled, they travelled for shorter times than males support recent claims on gender inequity in travel in Africa . In addi4.3.2The high use of NMT modes is expected in most African cities, and cities with higher NMT use such as Kisumu have taken steps to preserve NMT in their mobility plans with actions including education and advocacy for NMT, the introduction of car-free days, and improving private car parking facilities to create space for public transport and NMT use . ParticiOur regression analysis also showed some unexpected results. The positive coefficients between daily trips and no work and the negative correlation between ownership of motorised vehicles and the likelihood of trips are intriguing results that warrant further exploration. One possible explanation for the positive correlation between daily trips and no work could be that individuals who are not employed have more flexible schedules and are, therefore, more likely to engage in non-work-related trips and job searches. The negative correlation between ownership of motorised vehicles and the likelihood of trips could be explained by the fact that individuals may own cars for luxury and not for essential travel. The results also showed that wealthier people are more likely to use informal public transport, probably because this study's wealthy bracket (≥10000KSH) still includes most people who would typically use informal PT. However, it is important to note that these explanations are speculative and further research is needed to understand the underlying factors that fully contribute to these findings.An ad-hoc analysis of the perceptions of the environment and opinions on improving travel modes in Kisumu shed more light on our findings. Most participants report acceptable neighbourhood public transport, low road traffic congestion, high crime rates, inadequate footpaths, and poor street lighting. The observed low mobility means that the high crime rates, inadequate footpaths and poor lighting could be strong barriers to mobility. The perception of a good transport environment in terms of access and low traffic does not seem to imply high mobility. Page et al.  reported4.4The high proportion of participants with no trips in Kisumu needs further exploration, especially using improved data, but highlights the importance of consistent study designs with attempts to overcome soft refusals in travel surveys. From the lenses of income, gender, adolescence, and the elderly, it was more inclusive and equitable when people were mobile. The low proportions of people with trips raise concerns about potential immobility in smaller towns and cities that make up most of Africa's urban areas. Most African cities are slightly smaller than Kisumu in size and stage of urban development, with over 97% of African urban areas having fewer than 300,000 inhabitants . Much ofThe observed growth in motorcycles equally concerns from two perspectives. Firstly, motorcycle trips seem to pull from NMT and other public transport modes such as matatus. This reduces opportunities for active travel and obscures the demand for further development of walking, cycling and public transport infrastructure. Secondly, motorcycle riders and passengers typically face high injury risks and pose a high risk to pedestrians.Kisumu's relatively higher cycling uptake indicates the possible success of promoting cycling use in similar settings. To encourage and consolidate high cycling levels, there is a need for investments to reduce danger and increase the safety of the mode, as reflected by the participants' opinions on improving the safety of the different transport modes. Efforts to increase cycling must also consider the competition induced by the rapid increase in motorcycling.The data challenges encountered in this study, including limited datasets, gross disparities in estimates, and omission of key variables, highlight known problems with data that inform travel behaviour. Travel data from cities worldwide have often posed challenges that impede the comparison and monitoring of travel patterns. These challenges include the frequent unclear definition of trips, inconsistent inclusion of short trips, poor selection of travel days to adequately capture weekly and seasonal variation, the granularity of travel time estimates and the designation of trip modes. Efforts to harmonise and triangulate travel data are necessary to paint more complete pictures of travel behaviour and the evolution of travel patterns.5Our findings show that the proportion of individuals travelling is very high in Nairobi and very low in Kisumu; also, the travel durations are longer in Nairobi and shorter in Kisumu. Higher household income is associated with more travel in both cities, while the female gender is associated with less travel in Nairobi and shorter travel times in both cities. The safety of different transport modes is considered a key strategy for improving mode usage in Kisumu. The marked differences in travel behaviour characteristics and correlates between cities in the same country indicate setting-dependent correlates of travel behaviour. This suggests the need for more sub-national data collection to build a more nuanced understanding of travel behaviour profiles, patterns, and drivers in African cities.10.13039/501100000272National Institute for Health Research (NIHR) (GHR: 16/137/34) using UK aid from the UK Government to support Global Health Research. The views expressed in this publication are those of the authors and not necessarily those of the NIHR or the UK Department of Health and Social Care. LT and JW were supported with funding from the 10.13039/501100000781European Research Council (ERC) under the Horizon 2020 Research and Innovation Programme (grant agreement No 817754).This research was funded by the


Wildlife corridors play a pivotal role to support free ranging wildlife in rapid changing landscapes which remains vulnerable due to growing human population, together with unplanned urbanization, industrialization, developmental activities and the inverse impacts of climate change. This triggers for undertaking urgent actions to not only ensure their long-term survival, but also securing sustainable socio-economic and environmental development. Using landscape genetics approach, we in the present study examined the movement and dispersal patterns of a wild mountain ungulate, Himalayan ibex which is considered an ecological engineer and also imparts a major prey base to the large carnivores in Indian trans-Himalayan Region. We identified the Lahaul–Zanskar–Sham valley (L–Z–SV) corridor supports the bidirectional movement of Himalayan ibex, and suggest science driven management interventions for the conservation of Himalayan ibex and other sympatric ungulates in the trans-Himalayan region. Capra sibirica has widespread distribution, and its southern peripheral population is distributed in the Indian trans-Himalayan region (ITR). In the present study, we studied the spatial distribution and genetic make-up of Himalayan ibex from the ITR following the landscape genetics approach. We obtained 16 haplotypes at the mitochondrial d-loop region and found a stable demography in the past with a recent decline. With 10 nuclear microsatellites, we ascertained 111 unique individuals assigned into two clusters following Bayesian and non-Bayesian clustering analysis with several admixed individuals. We also recorded 25 first-generation migrants that reflected relatively high dispersal and gene-flow across the range. We identified a 19,835 sq.km suitable area with 13,311 sq.km in Ladakh and 6524 sq.km in Lahaul-Spiti. We identified a novel movement corridor for Himalayan ibex across the Lahaul–Zanskar–Sham valley (L–Z–SV) that displayed a fairly good conductance with low genetic divergence among the samples collected on the L–Z–SV corridor. We propose declaring a protected area in the Lahaul and Kargil districts to prioritize dedicated conservation efforts for the Himalayan ibex and other sympatric ungulates that impart a major role in the diet of large carnivore and balancing ecosystem services in the trans-Himalayan region.Wildlife corridors that connect mosaic habitats in heterogeneous mountainous landscapes can be of high significance as they facilitate the genetic and demographic stability of free-ranging populations. Peripheral populations of widespread species are usually ignored in conservation planning. However, these populations retain locally common alleles and are genetic reservoir under the changing climatic conditions.  Biological corridors connect large landscapes and facilitate the movement of wildlife for various purposes ,2,3. TheRecently, the Convention on Biological Diversity (CBD) proposes a post-2020 global biodiversity framework where monitoring and maintaining genetic diversity within all species (domestic and wild) is prioritized under the Goals and Action Targets . In light of this, we believe that the landscape genetics approach bridges the ecological information and population genetic estimates to spatially represent genetic diversity patterns, as well as the functionality of the biological corridors ,8,9,10. Capra sibirica) is the largest species of ibex in the genus “Capra” which occupies mountains, deserts and plains of many Asian countries  ,,29,28,29http://www.worldclim.org/bioclim accessed on 24 April 2022) (https://sedac.ciesin.columbia.edu accessed on 25 April 2022). Each variable raster was re-sampled at 1 km resolution and converted into ascii format in ArcMap 10.8 software (ESRI 2019) to bring uniformity in the data. Based on the Pearson correlation coefficients (r) threshold of 0.8, highly correlated variables were omitted from the analysis (ENM tool-R package). Finally, we selected 17 variables for species distribution modelling of ibex  . Topogra of ibex . A bias −7 substitutions/site/year following Tarekegn et al. (2018) [All generated sequences were aligned on MEGA X  using Cl. (2018)  for d-lo. (2018) . A media. (2018)  and the ID) and siblings (PID sibs) was calculated using the identity analysis module in GenAlEx version 6.51 [We repeated genotyping at least three times with a large fraction of samples and heterozygotes were ascertained with two independent runs. Then, we calculated genotype errors such as allele drop out (ADO) and false allele (FA) using PEDANT v 1.0 involving 10,000 search steps for enumeration of per-allele error rates using the repeat genotypes. The PIC value, an indicator of marker’s informativeness and null allele frequencies, was calculated using CERVUS version 3.0 . Since iion 6.51 .a) and effective alleles (Ne), as well as observed (Ho) and expected (He) heterozygosity using GenAlEx version 6.51 [IS was calculated according to Weir and Cockerham (1984) [Furthermore, the population genetic analysis was attempted using ten microsatellite markers with identified unique genotypes having > 70% success rate. The per-locus diversity was quantified by estimating the numbers of observed (Nion 6.51 . For theion 6.51  using thion 6.51 . The unbm (1984)  using FSm (1984) . Linkagem (1984) .K value was determined by calculating ad hoc quantity (ΔK) following Evanno et al.’s 2005 work [q), i.e., q ≥ 0.80; otherwise, an individual was determined as admixed if the q value was less than 0.80.The population’s genetic structure was attempted using three spatially and non-spatially explicit Bayesian and non-Bayesian clustering methods. In the Bayesian clustering method, we run STRUCTURE software v.2.3.4 with 500,000 MCMC and 50,000 burn-in with 20 runs at each K. The appropriate 005 work . Followi005 work ,48,49,50The second spatial Bayesian clustering was performed using GENELAND, which takes in account both the genotype data and geo-referenced information. We allowed K to vary from 1 to 10 with both correlated and uncorrelated allele frequency models. We conducted 20 independent runs with 1,000,000 iterations and a thinning of 1000 while other parameters were kept as default. The top run with the highest posterior probability algorithm was selected and post-processing was performed using 100 × 100 pixels in spatial domain with a 100 burn-in period.6 MCMC iterations [6 were discarded as burn-in and sampling frequency was conducted every 2000 intervals. Runs were carried out at a migration rate prior to 0.05 while other parameters were kept at default. We used GENECLASS2 [h/Lmax with algorithm as described by Paetkau et al. (2004) [p < 0.01.In the non-Bayesian clustering method, we used DAPC in the “adegenet” package  of R   and the . (2004)  was contIn order to evaluate the relationship between the genetic and geographic distances, isolation by distance (IBD) analysis was conducted using Allele in Space (AIS) , followiWe evaluated the current connectivity among the habitats of the Himalayan ibex in the landscape based on the circuit theory as implemented in the Circuitscape software V.4.0  followinWe found a 19,835 sq.km area suitable from the 106,014 sq.km area for the Himalayan ibex, which comprises 13,311 sq.km in Ladakh and 6524 sq.km in Lahaul-Spiti . The AUCOf the 337 pellet samples, 148 samples were identified as ibex, of which 126 samples were amplified with 10 microsatellite markers. We obtained 16 haplotypes characterized by 34 substitutions with an average of 6.977 nucleotide differences in the control region  with thrID of 3.2 × 10−8 and cumulative PID sibs of 1.4 × 10−3). Locus-wise PID and PID sibs ranged from 0.05 to 0.1 and from 0.35 to 0.46, respectively  with a select panel of seven microsatellite loci  value (p > 0.01)  . The Man > 0.01) .In the landscape connectivity model, we obtained high intensity current flow from the Spiti valley to Lahaul and from Lahaul to Zanskar to Sham valley in a vertical manner. This indicated that the study area represents a good physical connectivity A, and weCapra nubiana), Korean goral (Naemorhedus caudatus), Hangul (Cervus hanglu hanglu) and blue sheep (Pseudois nayaur) [The genetic diversity estimates of the Himalayan ibex were in the range of other similar ungulates such as the Nubian ibex ( nayaur) ,61,62,63https://hplahaulspiti.nic.in/climate/ accessed on 15 December 2022) is higher than in Leh (~200 mm) [The population assignment showed two distinct clusters with 48 admixed individuals that reflected that the ibex in LS and LA is well-connected by means of the L–Z–SV corridor A. The ib~200 mm) ,69,70,71~200 mm)  which me~200 mm) , while t~200 mm) . The hig~200 mm) ,75,76. TThrough the present study, we suggested the management and protection of the predicted novel corridor, ‘‘L-Z-SV’’, from urbanization and fragmentation in order to facilitate gene flow. This is important because a decline in the connected population will result in a survival of the species in small and isolated patches, which in turn may result in the loss of genetic diversity and upraise in genetic inbreeding. The population at the periphery should also be given equal priority in conservation to maintain and protect the genetic diversity of the population from loss of genetic diversity. We recommend the long-term monitoring of the ibex population and other species’ diversity to prevent inbreeding and local extinction. It is imperative to develop a management policy between both states for community conservation of the predicted corridor and to declare a protected area in these regions.


The National Agrobiodiversity Center under the Rural Development Administration (RDA) in Jeonju, Republic of Korea stands as the foremost national genebank in the country. Over the years, the National Agrobiodiversity Center has remained committed to enriching its collection with foreign genetic resources, elevating its status to a world-class plant genetic resources (PGR)- holding genebank. Currently, several steps are being undertaken to improve the accessibility of the collection to national as well as international researchers, improve the data available on the resources, and amend the passport information for the accessions. With the implementation of the Nagoya Protocol, the origin of genetic resources is being highlighted as an important input in the passport information. The RDA-Genebank actively responds to the Nagoya Protocol by supplementing passport data for resources lacking information on their origin. In addition, a large number of conserved resources are continuously multiplied, and agronomic traits are investigated concurrently. With the traditional methods of characterization of the germplasm requiring a significant amount of time and effort, we have initiated high-throughput phenotyping using digital techniques to improve our germplasm data. Primarily, we have started adding seed phenotype information followed by measuring root phenotypes which are stored under agronomic traits. This may be the initial step toward using largescale high-throughput techniques for a germplasm. In this study, we aim to provide an introduction to the RDA-Genebank, to adopted international standards, and to the establishment of high-throughput phenotyping techniques for the improvement of passport information. The National Agrobiodiversity Center in Jeonju, Republic of Korea, under the administration of the Rural Development Administration (RDA), plays a pivotal role in the collection, evaluation, conservation, and distribution of diverse plant genetic resources, including both seeds and vegetative germplasms. With the establishment of a low-temperature seed storage facility in 1975 and a seed management office in 1976, around 3300 germplasms were managed at local agricultural institutions. Later, in 1980, official documentation of 12,865 rice germplasms conserved by local agricultural institutions and universities was carried out, followed by their addition to the low-temperature seed storage collection. While seed germplasms were conserved at the genebank, external management institutions managed vegetative germplasms. In 1993, the RDA-Genebank began international cooperation starting with the N.I. Vavilov All-Russian Institute of Plant Genetic Resources (VIR) (1993–2019). At the RDA-Genebank, researchers aim to secure germplasm diversity by continuously collaborating with international joint research institutes including the Uzbek Research Institute of Plant Industry (UzRIPI) in Uzbekistan (1996–2004), the Department of Agriculture (DOA) Genebank in Thailand (1998), the Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) (1998–1999) in Germany, the International Rice Research Institute (IRRI) in the Philippines, the Plant Science and Agricultural Research Institute (PASRI) in Mongolia, the National Bureau of Plant Genetic Resources (NBPGR) in India, and the Georgian Academy of Agricultural Sciences (GAAS) in Georgia. Adoption of international policies such as the Convention on Biological Diversity (CBD) at the United Nations conference on environment and development (UNCED) in 1992 and the Nagoya Protocol on Access to Genetic Resources and the Fair and Equitable Sharing of Benefits Arising from Their Utilization in 2010 has further reinforced germplasm conservation efforts. Despite the limitations on data disclosure and international distribution of germplasms, Korea remains one of the significant countries conserving plant biodiversity. The Nagoya Protocol is an international agreement that aims to promote the fair and equitable sharing of benefits arising from the use of genetic resources, including those held in genebanks. The protocol was adopted in 2010 under the United Nations Convention on Biological Diversity (CBD) and entered into force in 2014 . The hisOverall, the Nagoya Protocol represents a significant milestone in the international effort to promote the conservation and sustainable use of genetic resources, including those held in genebanks, and to ensure that the benefits of these resources are shared fairly and equitably. In accordance with the Nagoya Protocol, RDA-Genebank continuously updates passport data on the origins of germplasms to actively respond to the utilization of genetic resources and the sharing of benefits accordingly. As a method of supplementing origin information, the passport data disclosed on the homepage of the relevant genebank was reflected on the basis of the original international genebank resource number.http://genebank.rda.go.kr/plantMain.do, accessed on 23 Mar 2023). Whereas the Korean version provides access to essential passport data, including conserved genetic resources of plants for distribution, the English version falls short in divulging the current status of resource conservation and the pertinent passport data. Consequently, this omission poses challenges for foreign researchers seeking to obtain and investigate our germplasms.The website of our institution presently offers visitors the option to select either the Korean or the English version  were domestically distributed in the Republic of Korea, while only a small proportion (0.73%) was circulated internationally . The treIn 2018, the RDA-Genebank initiated a collaboration research program, the Technology Development Project for Customized Agricultural, Forestry, and Fishery Products and Services, partnering with agricultural organizations such as seed companies and private research institutions. The objectives of this program were to broaden and improve the characterization and evaluation information of the conserved germplasms, augment their diversity, propagate them, and concurrently conduct characterization research. Through chemical composition profiling or disease resistance evaluation, commendable genetic resources have been shortlisted from the outcomes of these research endeavors, to be recommended for breeding and further scientific investigations. During 2010–2013, a total of 1343 accessions were distributed for local propagation of rice to the IRRI, the Philippines. For the purpose of germplasm exchange with international genebanks, the RDA-Genebank has introduced 568 accessions in the ARS (2012) in the US, 995 accessions in the UzRIPI in Uzbekistan (2011–2014), 10 in the VIR in Russia (2014), 235 in the AVRDC in Taiwan (2013–2016), and 232 in the GAAS in Georgia (2015–2016) as a part of its germplasm exchange policy. We have also supplied to private entities, including the distribution of 62 sesame accessions to the Tainan District Agricultural Research and Extension Station in Taiwan. The distribution overseas can be categorized into two types: those sent through Korea Project to the International Agriculture (KOPIA) center as part of Official Development Assistance (ODA) projects, which provide country-specific farming techniques, and those distributed through the 3-FACI. The 3-FACI initiative includes the Asian Food & Agriculture Cooperation initiative (AFACI), the Korea–Africa Food & Agriculture Cooperation initiative (KAFACI), and the Korea–Latin America Food & Agriculture Cooperation initiative (KoLFACI). These programs aim to facilitate multi-continental research and collaboration to address common farming problems. Despite RDA-Genebank’s possession of world-class germplasms, limited overseas distribution has occurred due to the unavailability of individual resource information on online platforms such as Genesys, managed by the Global Crop Diversity Trust. Exceptions to this pattern have occurred only when researchers with Korean affiliations have utilized publicly available history information to apply for resource distribution. Our institution is diligently making preparations, including the dissemination of passport information, to ensure the continuous availability and accessibility of our plant genetic resources for international distribution.In order to improve the utilization of the conserved germplasms, systematic passport data management is indispensable . GermplaGlycine max), the morphological characteristics of 650 landraces were investigated, about 40 of them were selected as a core collection, and a molecular genetic profile was promoted [Capsicum spp.), the analysis of capsaicin content, the cultivation of anthracnose-resistant intermediate seedlings, the evaluation of Chinese cabbage (Brassica rapa ssp. penkinensis) landraces, the cultivation of superior groups, and the cultivation of intermediate seedlings resistant to wart disease of cabbage [In response to consumer preferences, there is a notable increase in the incorporation of evaluation data concerning chemical composition profiles, such as glucosinolate levels in Brassica ssp. and disease resistance. This highlights the importance of considering these factors in the evaluation process. Since the 1990s, the RDA-Genebank has systematically investigated its conserved genetic resources for their qualitative and quantitative traits to promote the use of germplasms by consumers of the end product. During the early stages, the characterization centered on seed viability and primary characteristics of germplasms such as seed coat color, width, and length. From 1995, the organization was subdivided into several divisions for cereal crops, horticultural crops, and special crops, respectively, and their evaluation data was augmented. In addition, chemical composition profiles such as the oil and fatty acid content of sesame seeds; the protein content, amylose and amylopectin content, and starch characteristics of rice landraces; and the DNA polymorphism analysis of rice and barley were initiated . In the promoted . Among t cabbage . The gerB. rapa subsp. pekinensis), our studies focused on analyzing the individual glucosinolates of the mustard leaf (B. juncea) and the Choy Sum (B. rapa subsp. chinensis var. parachinensis) [Starting in 2019 with Chinese cabbage (inensis) ,14. The With further work carried out on expanding the information available in the evaluation data for germplasms conserved at the RDA-Genebank, substantial consideration has been given to using high-throughput techniques to study the germplasms. One such aspect is using digital phenotyping for largescale analysis and screening of the germplasms.r2) between image-based and actual height and the width measurements of soybean seeds  [Glycine max), and diversity analysis was performed using origin  and status  [Glycine max) and adzyki bean (Vigina angularis) germplasms for the first time across international genebanks [Glycine max cv. Enrei Japanese varieties, which are known for their excellent nodulation characteristics. Additionally, these digital phenotyping techniques can be applied to various other crops conserved at the RDA-Genebank. For instance, in wheat, RGB images obtained from a digital camera can be utilized to measure agronomic traits such as tiller number per plant, canopy (including leaf number and length), phenotypic characteristics of wheat spike, and tomato internode length. In the future, RGB images captured using unmanned aerial vehicles can also be used to measure plant heights in crops such as rice, wheat, barley, and corn [The significance of seed traits is widely acknowledged, and seeds are selected on the basis of their phenotypic characteristics to improve crop yield and quality, making this one of the oldest phenotyping techniques . Phenotyctively) . Therefond wild) . Currentnd wild) . In geneenebanks ,23. Specenebanks . We confand corn .Hyperspectral sensors have been utilized to measure photosynthesis and respiration, which are directly related to crop growth and are currently being studied in tobacco, corn, and wheat . In addiThe augmentation of phenotype data with existing characterization information, as well as the integration of genomic, phenomic, and other molecular data, can significantly aid researchers in selecting optimal accessions for breeding and elucidating various evolutionary characteristics . In the Given the accelerating loss in biodiversity worldwide due to changing climatic conditions and anthropogenic activities, the importance of agricultural genetic resources is becoming more prominent. At plant genebanks throughout the world, continuous efforts are being taken towards improving the quality and quantity of germplasm collection along with ease of access. The germplasms we conserve at the National Agrobiodiversity Center are continuously investigated for agricultural traits and building an updated database. Recently, we have also concentrated on methods for investigating agricultural traits by applying digital techniques. We expect information on the germplasms conserved in the RDA-Genebank to be made accessible at an early juncture to researchers all over the world. Ensuring timely access to vital genetic resources will foster international collaboration and contribute to the advancement of research and agricultural development worldwide. By promoting transparency and facilitating the exchange of germplasms, plant biologists can collectively work towards enhancing global food security, biodiversity conservation, and sustainable agriculture. As custodians of invaluable genetic resources, the RDA is committed to promoting their widespread availability and fostering fruitful partnerships with researchers and institutions across the globe.


It is well-established that most Hepatocellular carcinoma (HCC) patients die of metastasis, yet the potential mechanisms orchestrating metastasis remain poorly understood. Current evidence suggests that the dysregulation of METTL3-mediated m6A methylation modification is closely associated with cancer progression. STAT3 is an oncogenic transcription factor that reportedly plays a central role in the occurrence and development of HCC. However, the relationship between METTL3 and STAT3 in HCC metastasis remains unclear.The relationship between METTL3 expression and the survival of HCC patients was assessed by online tools GEPIA and Kaplan–Meier Plotter. Western blotting, Tissue microarray (TMA), and immunohistochemistry (IHC) staining were used to evaluate the expression levels of METTL3 and STAT3 in HCC cell lines and metastatic and non-metastatic tissues. Methylated RNA immunoprecipitation (MeRIP), MeRIP sequencing (MeRIP-seq), qRT-PCR, RNA immunoprecipitation (RIP), Western blotting and luciferase reporter gene assay were utilized to clarify the mechanism of METTL3 regulating STAT3 expression. Immunofluorescence staining, Western blotting, qRT-PCR, Co-immunoprecipitation (Co-IP), IHC staining, TMA and Chromatin immunoprecipitation (ChIP) assay were performed to explore the mechanism of STAT3 modulating METTL3 localization. Cell viability, wound healing and transwell assay, and orthotopic xenograft model were used to evaluate the role of METTL3-STAT3 feedback loop in the promotion of HCC metastasis in vitro and in vivo.METTL3 and STAT3 are both abundantly expressed in high-metastatic HCC cells and tissues. Moreover, a positive correlation was found between the expression of STAT3 and METTL3 in HCC tissues. Mechanistically, METTL3 could induce the m6A modification of STAT3 mRNA, and then promote the translation of m6A-contained STAT3 mRNA by interacting with the translation initiation machinery. In contrast, STAT3 promoted nuclear localization of METTL3 via transcriptionally upregulating WTAP, a vital member of the methyltransferase complex, and facilitated the methyltransferase function of METTL3. METTL3 and STAT3 form a positive feedback loop to accelerate HCC metastasis in vitro and in vivo.Our findings reveal a novel mechanism of HCC metastasis and uncover the METTL3-STAT3 feedback signaling as a potential target for the anti-metastatic treatment of HCC.Video AbstractThe online version contains supplementary material available at 10.1186/s12964-023-01148-7. Hepatocellular carcinoma (HCC) is widely acknowledged as a common malignant tumor and a severe threat to human health . NotwithN6-methyladenosine (m6A) is a dynamic and reversible chemical modification in eukaryotes, which affects the fate of many mRNAs and non-coding RNAs by modulating RNA processing, transport, translation, and degradation , 5. The It is widely acknowledged that signal transducer and activator of transcription (STAT) family proteins can be activated by different cytokine receptors and participate in cell signal transmission and gene expression regulation . Among tIn the present study, we discovered a positive correlation between STAT3 expression and METTL3 in HCC. METTL3 induced m6A modification and enhanced the translation of STAT3 mRNA, upregulating STAT3 protein levels. Moreover, STAT3 promoted nuclear localization of METTL3 by upregulating WTAP expression, potentiating the methyltransferase function of METTL3. Overall, METTL3 and STAT3 formed a positive feedback loop to accelerate HCC metastasis in vitro and in vivo. These findings provide a foothold for further studies on identifying novel therapeutic targets for HCC.The MHCC97H, MHCC97L, HCCLM3, HepG2, L-O2, SMCC-7721 and HEK293T cell lines were purchased from American Type Culture Collection (ATCC). L-O2 cells were cultured in RPMI Medium 1640  supplemented with 10% FBS (Gibco). The other cell lines were cultured in high-glucose Dulbecco’s Modified Eagle’s Medium  with 10% FBS. The HCCLM3-KD-STAT3 (stably knockdown STAT3) and HCCLM3-control cell lines were gifts from Wenzhou medical university. The MHCC97H-control and MHCC97H-KD-STAT3 (stably knockdown STAT3) cell lines were stably transfected with pSilencer 4.1 CMV vector containing the nonsense RNAi fragment or STAT3 RNAi fragment. The related plasmid and RNA interference lentivirus were packed and purchased from GeneChem  to generate the stable cell lines used in Figs. The association of METTL3 and STAT3 was assessed by IHC staining in HCC tissue microarray . The association between WTAP and STAT3 was assessed in HCC tissue microarray . The clinical characteristics of patients are presented in supplementary tables Sn = 10) and Fig. Sn = 4) were collected from the patients at First Affiliated Hospital of Wenzhou Medical University. This study was evaluated and reviewed by the Ethics Committee of Wenzhou Medical University. Written consent was obtained from all patients prior to the study. The clinical characteristics of patients are presented in supplementary table SThe paired HCC tissues and non-tumor tissues used in Figs. Total RNA was extracted from corresponding tissues or cells using Trizol reagent  according to the manufacturer's instructions. Total RNA concentration was determined by NanoDrop 2000 . According to the standard protocol of m6A RNA Methylation Assay Kit , the RNA was incubated with m6A capture antibody solution in each well for 1 h. After incubation, the m6A capture antibody solution was removed and the well was washed by wash buffer for three times. Similarly, the detection antibody was added for incubation (30 min) and then removed. After washing by wash buffer, the diluted enhancer solution was added and incubated for 30 min. Removed the diluted enhancer solution and washed. Finally, the absorbance was read at 450 nm after the addition of developer solution and stop solution. According to the manufacturer's requirements, the m6A content was calculated based on the standard curve.The methylated RNA immunoprecipitation (MeRIP) was performed according to a previously described protocol . In brie4 cells were seeded into the upper chamber with 150 µl medium without FBS (two replicates per sample). The chambers were then placed in the medium with 18% FBS for 48 h to drive cell invasion. Cells were removed from the upper chamber with a cotton swab, and invasive cells in the lower chamber were fixed in 4% paraformaldehyde and stained with 0.5% crystal violet. For the migration assay, the experiment was performed with the same method but without supplementation of matrigel in the upper chamber. Chambers were photographed, and total invasive (/migrated) cells were quantified by Image J software. For wound healing assays, cells cultured in 6-well plates were scratched by the tip of a small 200 µl pipette to create a "wound" and monitored for "healing" at indicated time points. The closure area was quantified by Image J software.For transwell assay, a polycarbonate transwell chamber  with an 8-μm pore size was used to evaluate the migration and invasion of indicated cells. For the invasion assay, the matrigel  was spread on the upper chamber. 5 × 106) with luciferase signal were prepared and resuspended in 150 µl PBS/Matrigel . The mice were randomly divided into 3 groups (n = 5 per group). The cells were injected into the mice's hepatic lobe. After 2 weeks, the mice were anesthetized with isoflurane and intraperitoneally injected with 150 mg/kg D-Luciferin once a week to evaluate tumor metastasis. D-Luciferin was obtained from Keyuandi Biotech Co. Ltd . The IVIS system was used to analyze the bioluminescence of mice. For direct assessment of lung metastasis, the lower body of mice was covered with black cardboard. After 7 weeks of injection, the nude mice were sacrificed by cervical dislocation. The weight of the tumors was recorded. After paraffin embedding, tumor samples were used for IHC and HE staining. In this experiment, researchers who conducted measurements were blinded to avoid biases. All animal experiments were approved by the Ethics Committee of Xinxiang Medical University for medical laboratory animal sciences.Six-week-old male BALB/C athymic nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd. The mice were housed according to the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The corresponding cells  and Kaplan–Meier Plotter . The correlation of gene expression in HCC tissue microarray was analyzed by Spearman correlation analysis. The statistics of IHC staining and nuclear fluorescence were assessed by image J.Statistical significance was assessed using GraphPad Prism 8. The values in the graphs represent the mean of three biologically independent experiments. Error bars represent mean ± SD. The significance was set as *r = 0.656, p < 0.001). Notably, strong staining of METTL3 and STAT3 was observed in metastatic tissues compared with non-metastatic tissues  . The m6A levels in HCC tissues were upregulated compared to non-tumor tissues  were used to analyze the binding site of STAT3 on WTAP promoters. Five putative binding sites of STAT3 were identified on the promoter of WTAP. The ChIP assay was performed, and the specific primers were used to amplify the five regions containing the corresponding binding sites. The data showed that STAT3 could occupy the promoter of WTAP at region 3  showed that WTAP mRNA levels in HCC tissues were significantly elevated than in non-tumor tissues (Fig. SFurthermore, tissue microarray analysis uncovered a positive relationship between the expression levels of STAT3 and WTAP in cancer tissues and adjacent non-cancerous tissues (Fig. STo further verify the relationship among STAT3, WTAP, and METTL3, we performed Co-IP assays in MHCC97H and HCCLM3 cells. Consistent with the previous study , we founThe above data revealed the comprehensive interplay between METTL3 and STAT3. They formed a positive feedback loop in HCC. Next, we intended to investigate the role of the feedback loop in HCC metastasis. Based on MHCC97H cells, we constructed different cell lines with luciferase signal, including stably depleted-METTL3, stably depleted-METTL3-overexpressed-STAT3, and corresponding control cells. The efficiencies of knockdown and overexpression were verified, as shown in Fig. In this study, we analyzed the expressions of METTL3 and STAT3 in tissue microarray. The close relationship between METTL3 and STAT3 in primary and metastatic specimens suggested their potential role in promoting HCC metastasis. To clarify the regulatory mechanism between them, we first evaluated the modulatory role of METTL3 on STAT3 expression. A recent study showed that METTL3 enhanced JAK1 translation via a m6A-YTHDF1 axis, and subsequently promoted the activation of STAT3 . Herein,Gregory's team have reported that METTL3 could interact with translation initiation machinery to facilitate m6A-modified mRNA translation independently of m6A readers , 30. MorNuclear speckle localization is required for the interaction of METTL3 with its target mRNA , 32. HerIn this study, we clarified the interplay between METTL3 and STAT3, which formed a positive feedback loop in HCC. To evaluate the role of this feedback loop in cell metastasis, in vitro and in vivo experiments were performed by knocking down METTL3 with or without STAT3 overexpression. STAT3 could alleviate the METTL3 depletion-induced inhibitory effect on cell metastasis, indicating that METTL3 could promote metastasis by acting on STAT3 in HCC. Notably, STAT3 could not totally reverse the METTL3 knockdown-induced inhibition on lung metastasis, which may be attributed to the fact that other target genes of METTL3 also play a role in cell metastasis. According to current reports, SOCS2, Snail, RDM1, and non-coding RNA LINC00958, etc. are all target genes of METTL3 in HCC; METTL3 could affect proliferation, migration, invasion and lipogenesis to facilitate HCC progression by regulating these target genes , 37–39. In addition, we found that WTAP was closely associated with STAT3 expression in HCC tissues. Since STAT3 and METTL3 exhibited a pro-metastatic role in HCC, as a part of the feedback loop, we explored whether WTAP yielded a similar effect. As expected, WTAP overexpression enhanced the migration and invasive capacities of HCC cells Fig. S. HoweverIn summary, we documented a new mechanism of HCC metastasis Fig.  whereby Additional file 1.Additional file 2.Additional file 3.Additional file 4.Additional file 5.Additional file 6.