Begomovirus represents a group of multipartite viruses that significantly damage many agricultural crops, including papaya, and influence overall production. Papaya leaf curl disease (PaLCD) caused by the complex begomovirus species has several important implications and substantial losses in papaya production in many developing countries, including India. The increase in the number of begomovirus species poses a continuous threat to the overall production of papaya. Here, we attempted to map the genomic variation, mutation, evolution rate, and recombination to know the disease complexity and successful adaptation of PaLCD in India. For this, we retrieved 44 DNA-A and 26 betasatellite sequences from GenBank reported from India. An uneven distribution of evolutionary divergence has been observed using the maximum-likelihood algorithm across the branch length. Although there were phylogenetic differences, we found high rates of nucleotide substitution mutation in both viral and sub-viral genome datasets. We demonstrated frequent recombination of begomovirus species, with a maximum in intra-species recombinants. Furthermore, our results showed a high degree of genetic variability, demographic selection, and mean substitution rate acting on the population, supporting the emergence of a diverse and purifying selection of viruses and associated betasatellites. Moreover, variation in the genetic composition of all begomovirus datasets revealed a predominance of nucleotide diversity principally driven by mutation, which might further accelerate the advent of new strains and species and their adaption to various hosts with unique pathogenicity. Therefore, the finding of genetic variation and selection emphases on factors that contribute to the universal spread and evolution of Begomovirus and this unanticipated diversity may also provide guidelines toward future evolutionary trend analyses and the development of wide-ranging disease control strategies for begomoviruses associated with PaLCD.The genus  Geminiviridae) are the largest group of plant viruses that pose a significant yield loss to many economically important crops in tropical and sub-tropical regions of the world  encapsulated in a quasi-isometric non-enveloped twinned particle . Begomovwhitefly .Carica papaya), a major tropical, sweet, large, and herbaceous food crop, belongs to the order Brassicales (family: Caricaceae) and is cultivated throughout India (Papaya leaf curl virus (PaLCuV), Papaya leaf crumple virus (PaLCrV), Chilli leaf curl virus (ChiLCV), Chilli leaf curl India virus (ChiLCINV), Duranta leaf curl virus (DLCV), Papaya yellow leaf curl virus (PaYLCV), Papaya severe leaf curl virus (PaSLCV), Tomato leaf curl New Delhi virus (ToLCNDV), Tomato leaf curl virus (ToLCV), Cotton leaf curl Multan virus (CLCuMuV), Tomato leaf curl Gujarat virus (ToLCGV), Croton yellow vein mosaic virus (CYVMV), Pedilanthus leaf curl virus (PeLCV), Ageratum enation virus (AEV), and Cotton leaf curl Burewala virus (CLCuBuV).Papaya  tree based on CLUSTAL W pairwise alignment and the best fit nucleotide substitution model, i.e., (GTR + G) for DNA-A, its ORFs (AV1: TN93 + G + I); (AV2: HKY + G); (AC1 and AC3: GTR + G); (AC2: TN93 + G); (AC4: HKY + G), and (TN93 + G + I) for betasatellites, based on the corrected Bayesian Information Criterion (BIC) score of the MEGA X program . Moreovep and default detection thresholds, datasets were subjected. To reduce false-positive results, out of seven, at least three algorithms were considered appropriate to detect recombination events.The recombination analysis was done by using seven methods, i.e., RDP , BOOTSCA7 run lengths to provide a 95% highest probability density (HPD) interval for determining statistical uncertainty. For assessing temporal structure, which is important for the estimation of substitution rates, we repeated the BEAST analysis by reshuffling the sampling times for each dataset, which were randomised on the tips  in the sequence datasets of begomoviruses, their ORFs and betasatellite were determined by using the Bayesian Markov Chain Monte Carlo (MCMC) parameter of Bayesian Evolutionary Analysis Sampling Tree  software were used . The aliD, which signifies the difference between the two measures of genetic diversity, i.e., the number of segregating sites and the mean number of pairwise differences, Fu and Li’s D* for identifying the difference between the total number of mutations and the number of singletons, and Fu and Li’s F* for identifying the difference between the average number of nucleotide differences between paired sequences and the number of singletons, i.e., detecting the neutrality of mutations in DNA populations  software (ulations .Papaya leaf curl virus (PaLCV), 13 sequences of Papaya leaf crumple virus (PaLCrV), and 10 sequences of Chilli leaf curl virus (ChiLCV), infecting papaya plants in India , (PaLCrV II), (PaYLCuV III), and , (ToLCBDB II), (ChiLCB III), (CroYVMB IV), and  interval ranging from 4.126 × 10−5 to 6.647 × 10−3], which was found to be higher when compared with the range of nucleotide substitution rates of RNA viruses reported so far . However, a relaxed molecular clock is used to get the suitable value of the mean substitution rate, and the obtained high substitution value is most likely to have resulted from strong positive selection  for DNA-A datasets. Similarly, the analysis was performed for ORF datasets and we found the maximum number of polymorphic sites (s) and number of mutations (η) in AC1 (Rep) and AV1 (CP) genes having nucleotide diversity π = 0.1. Moreover, for betasatellite, we found the total number of polymorphic sites (s) 834, with a total 1,482 number of mutations (η) having a nucleotide diversity (π) of 0.30509 (π = 0.3) was again high. However, the resultant maximum π value explains the non-random distribution of nucleotides throughout viral and sub-viral genome regions, which significantly contributes to a high degree of genetic variability. Therefore, the estimation suggests diverse populations within and among populations.Demography structure analysis was used to estimate the degree of genetic variability (>0.08) within and between populations . HoweverFurther, genetic diversity within and among populations was also determined by the number of haplotypes (H) and haplotype diversity (Hd). Therefore, using DnaSP software v. 6.0; .begomoviruses and associated satellite molecules. For evaluation, Tajima’s D test was used, which statistically reflects the negative Tajima’s D value for DNA-A datasets at −0.77977, for ORFs range between −0.42227 and −1.45344, and for associated betasatellites at −1.06710. Predominantly, the statistically significant values were negative, which indicates that a large proportion of genetic segregation might be present within sequence datasets that are unique to individual sequences. Similarly, the other parameters, such as Fu & Li’s D and Fu & Li’s F tests of population statistics, were also evaluated, resulting in negative values for DNA-A, its ORFs, and betasatellites datasets, indicating reiterating of purifying selection and population expansion, which might be due to the inherent diversity. Nevertheless, the combination of Tajima’s D, Fu & Li’s D, and Fu & Li’s F negative values for DNA-A, its ORFs, and associated satellite population revealed the conserved nature of the gene. Such evidence of nucleotide diversity might be expected when a selective sweep succeeds in the expansion of the population and when most observable segregation functioning at the nucleotide level in a population is momentary and is eventually withdrawn by purifying selection  is found to infect papaya plants and cause PaLCD, which affects plant growth, fruit size, quality, and quantity, slowing its yield  for DNA-A, ORFs (AV1:TN93 + G + I); (AV2: HKY + G); (AC1& AC3: GTR + G); (AC2:TN93 + G); (AC4: HKY + G); and (TN93 + G + I) for betasatellites. The recurrent occurrence of recombination and nucleotide substitution alike in RNA viruses is mostly attributed to factors contributing to the high degree of genetic variability among begomovirus populations, which may significantly step up their evolution by expanding the combinations of pre-existing nucleotide segregation created by mutation (To explore the evolution of begomoviruses across India, a full-length sequence of reported begomoviruses infecting mutation , 2009. AThe phylogenetic results revealed homological diversification and the mean branch length observed for DNA-A and betasatellites datasets shows the possibility of a recombinational event in the studied population of begomovirus . Thus, tbegomovirus populations that use a conserved feature, i.e., a rolling circle mechanism for replicating their genomes and making them mechanistically recombination-prone, thus generating recombination breakpoints in a non-random location (The phylogeny-based partitioning method was qualitatively estimated for eight datasets, resulting in mean branch length, and was found useful in quantifying the effect of recombination events. Previous studies have revealed that recombination happens at high frequencies in location . As recolocation , showinglocation . Howeverlocation .Significant aspects of population genetics are possibly accompanied by mutation, along with recombination, neutral selection, genetic drift, and gene flow, which encourage shaping the genetic structure of populations . We calcbegomovirus genomes. In this context, the combination of various factors is responsible for affecting genetic variability and acts by distributing polymorphisms in a non-random manner in the genomic regions of begomoviruses (Additionally, it is important to address the key issue that refers to the uneven distribution of variation across oviruses . Howeveroviruses . FurtherD, and Fu & Li’s F negative values for DNA-A, its ORFs, and associated satellite population revealed the conserved nature of the gene. Such evidence of nucleotide diversity might be expected when most observable segregation functioning at the nucleotide level in a population is momentary and is ultimately withdrawn by purifying selection. Our results indicate that begomoviruses infecting Carica papaya are not restricted to any solitary geographical region of India.Moreover, to understand the population genetics and selection forces acting on the begomovirus population and satellite molecules, neutrality tests were evaluated, resulted in a negative value, suggesting that populations might be influenced by purifying selection or have experienced recent expansion  rather tAlthough the number of sequence data for DNA-A, betasatellites, and particularly alphasatellites is insufficient, the current study’s findings may provide meaningful basic information that contributed significantly to the diversification of begomoviruses, particularly those causing PaLCDs, and acknowledge the evolutionary potential, particularly in the context of recombination, adaptation, genetic diversity, emergence, and evolution.The present study, based on bioinformatics approaches, provides the current understanding of the genetic diversification of the begomovirus population associated with PaLCD. The coexistence of the begomovirus population and its satellite molecules, as explained by geographical distribution, indicates their interdependence in accelerating disease severity and spread among economically important crops such as papaya across India. A wide begomovirus population having a common host might be attributed to an uncontrolled evolutionary variation in its DNA genome, which is mainly driven by a high frequency of mutational changes. Furthermore, the variable frequency of mutation demonstrated the random allocation of the recombination breakpoints, leading to a diversified distribution of recombinational patterns. Moreover, this phenomenon uses different mechanisms to overwhelm selection pressure and to effectively adapt to environments with new hosts. Thus, that geographically separates begomovirus and its associated satellites. The continuous evolution of new recombinants and their satellites might lead to efficient vector transmission, expansion of host range, and breaking of resistance, which poses a threat to crop production at an alarming rate, thus affecting the agroeconomic value and disease management. However, for expanding knowledge in an area, the depth study of molecular fundamentals together with vector-mediated and host-dependent dispersal might provide an understanding of the expanding begomovirus disease complex.The original contributions presented in the study are included in the article/AS and VP performed experiments and wrote the manuscript. AKS helped in data collection. RG and DY guided the design of the whole test scheme, and AA-S and MS critically reviewed the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Spirobolus bungii plays a key role in soil ecolsystems, but there are few studies on this species, especially regarding genetics. In this study, 166 S. bungii individuals were collected from two cities in China (Nanjing and Tianjin), and COX2 and Cytb gene sequences of mitochondrial DNA and 18S rRNA gene fragments were sequenced. We conducted a population genetic analysis based on mtDNA sequences, revealing the population genetic diversity and genetic structure of S.bungii in the two cities, and confirming the hindering effect of geographical barriers on the gene flow of the populations.Spirobolus bungii, are an important component of ecosystems. However, systematic studies of the genetic diversity, population genetic structure, and the potential factors affecting the genetic differentiation of S. bungii are lacking. We performed a population genetic study of 166 individuals from the mountains to the south of the Yangtze River, north of the Yangtze River in Nanjing city, and near Tianjin city, in order to investigate the correlations between geographical distance and genetic diversity. A total of 1182 bp of COX2 and Cytb gene sequences of mitochondrial DNA, and 700 bp of the 18S rRNA gene sequence were analyzed. There were two haplotypes and one variable site in the 18S rRNA gene, and 28 haplotypes and 78 variable sites in the COX2 and Cytb genes. In this study, the 18S rRNA gene was used for species identification, and mtDNA (concatenated sequences with Cytb and COX2) was used for population genetic analysis. Structure cluster analysis indicated that the genetic structures of the different populations of S. bungii tended to be consistent at small geographical scales. Phylogenetic trees revealed that the haplotypes were clearly divided into three branches: the area south of the Yangtze River, the area to the north of the Yangtze River in Nanjing, and the area in Tianjin. Large geographical barriers and long geographical distance significantly blocked gene flow between populations of S. bungii. Our results provide a basic theoretical basis for subsequent studies of millipede taxonomy and population genetic evolution.Soil macrofauna, such as  Soil macrofauna is an important part of biodiversity and include diverse macroinvertebrates living in the soil. They play a key role in regulating the physical, chemical, and microbiological properties of soils . Soil maIn addition to annelids, platyhelminths, mollusks, and arthropods are important soil macrofauna. However, most genetic studies on soil macrofauna have been restricted to earthworms of the annelids. Furthermore, mitochondrial DNA is the most common molecular marker used in these studies ,6. In a COI and 16S rRNA) and morphology to define four new Apeuthes species from Southeast Asia [Spirobolus bungii is a common millipede species in the mountain forests of China. In this study, mitochondrial DNA was used as a marker to study the phylogeny and genetic diversity of S. bungii, to reveal the characteristics of the genetic diversity of millipedes. The 18S rRNA gene was used for species identification, to ensure that all the individuals studied were S. bungii.As common soil macro-invertebrates, millipedes of the arthropods are one of the oldest terrestrial organisms and are widely distributed, with the earliest millipede fossils dating back to the late Silurian . Millipeast Asia . SpiroboS. bungii in eastern and northern China and to reveal the genetic structure and diversity of millipedes and their influencing factors. To study the genetic diversity of S. bungii, we needed to sample from two cities. First, considering the possibility of mass sampling, we selected Nanjing, East China. Second, S. bungii was found in the Yan Mountains of China [S. bungii distributed in different regions could be divided into three groups: the southern bank of the Yangtze River (Group A), the northern bank of the Yangtze River (Group B), and in Tianjin (Group C).This experimental study aimed to determine the population differentiation of of China . Pan Mouof China ; therefoof China ,21. Nanjof China ,23. The S. bungii individuals were collected from seven sites: Zijin Mountain , Qixia Mountain , Tang Mountain , Fang Mountain , Lao Mountain , Longwang Mountain  in Nanjing, and Pan Mountain  in Tianjin , a set of PCR primers were separately designed for 18S rRNA, COX2, and Cytb: Spbu-18S-F (5′-GGA GAG CAA GAA TTA AGA-3′) and Spbu-18S-R (5′-TCG GTC AAT TCA CTC TAA-3′), Spbu-COX2-F (5′-TTA GTT AGA TGC TGT CAC-3′) and Spbu-COX2-R (5′-CAC AAA CAC TTC TCT TCC-3′), and Spbu-Cytb-F (5′-AAG TAC AGG ACG GTT AGA AC-3′) and Spbu-Cytb-R (5′-CTC CAT CTA ACC TGA ACG TC-3′). Each 40-μL reaction mixture contained 2 μL of template DNA (25–50 ng/μL), 20 μL of 2× Rapid Tap Master Mix , 1.5 μL of each primer (10 μM), and 15 μL H2O. The PCR procedure consisted of pre-denaturation at 95 °C for 5 min, denaturation at 95 °C for 30 s, annealing at 50–55 °C for 30 s, extension at 72 °C for 40 s, 35 cycles, and a final extension at 72 °C for 5 min. Following agarose gel electrophoresis, the PCR products were sent to the Tsingke Biotechnology Co., Ltd.  for sequencing in one direction.The genome DNA were extracted from the tissue samples using DNAiso Reagent . According to the nuclear and mitochondrial sequences of −1 NaHCO3) were analyzed using the standard Kjeldahl and Olsen methods, respectively. The available potassium (AK) extracted with 1 mol L−1 NH4OAc was determined using an atomic absorption spectrophotometer . Nitrate was extracted using 1 mol L−1 KCl via ultraviolet spectrophotometry  [The soil pH and electrical conductivity (EC) were measured in a 1:5 soil:water solution using an electronic pH meter  and a conductivity meter, respectively. The soil moisture content was measured after oven-drying at 105 °C for 24 h. The soil organic matter (SOM) was determined using the Walkley–Black potassium dichromate oxidation method [, China) .COX2 and Cytb genes of each individual millipede were concatenated using Editseq and Seqman. DnaSP5.0 [Hd), nucleotide diversity (pi), and the mean number of pairwise differences (K). The pairwise fixation index (STF), gene flow  values, and analyses of molecular variance (AMOVA) using Arlequin version 3.5 [S. bungii population, which was then analyzed using Structure 2.3.4 software [The DnaSP5.0  was utilsion 3.5  were empsoftware . The setS. bungii were estimated from an unrooted statistical parsimony network using Popart v1.7 [The results were uploaded to Structure Harvester , to obtaart v1.7 . TBtoolsart v1.7 .COX2 (534 bp) and Cytb (648 bp) gene sequences of mtDNA and 700 bp of the 18S rRNA gene sequence from 166 individuals were analyzed (GenBank accessions: OL449413–OL449447). Gene sequence analyses revealed that there were two haplotypes and one variable site in the 18S rRNA gene, and 28 haplotypes and 78 variable sites in the COX2 and Cytb genes of the mtDNA , all population data indicated that Cytb had a higher genetic diversity than COX2. In the concatenated sequence, the number of haplotypes ranged from two to nine per sampling site, with ZJ having the fewest (n = 2) and PS having the most (n =9); 12 haplotypes were found in the southern bank of the Yangtze River, seven haplotypes were found in the northern bank of the Yangtze River, and nine haplotypes were found in Tianjin, and the haplotypes in each group were unique haplotypes. Haplotype diversity (Hd) ranged from 0.153 (ZJ) to 0.653 (PS), and the nucleotide diversity (pi) ranged from 0.00013 (ZJ) to 0.0085 (PS).Although the 18S rRNA gene has often been used for phylogenetic analysis among different species ,35,36, iS. bungii and its influencing factors  to 0.99368 (ZJ and LS populations) for the mtDNA, Cytb, and COX2 genes. The results showed that on the south bank of the Yangtze River, the level of gene flow among populations was relatively high (1.04729–8.74928), and genetic differentiation was not obvious (0.02778–0.19271).Gene flow occurs because of contact between different populations of a species. We studied the gene flow between each population, to explore the genetic diversity and population structure of  factors , Table 2S. bungii. The results showed that K = 4  was the most likely number for the S. bungii genetic clusters  identified only 32.14% of the variation as being present between groups, whereas 66.40% was among populations within groups and 1.46% was within populations  indicated that most of the genetic variation was between groups (98.74%), whereas little genetic variation was within populations (1.02%) or among populations within groups (0.24%) . These rulations .S. bungii populations into three groups according to their geographical distribution was supported by the results of the phylogenetic tree.The ML tree a showed The TCS haplotype network c showed S. bungii populations distributed along the Yangtze River in the Nanjing areas) were more distant than those distributed in the Tianjin area. The influence of the Yangtze River on the genetics of S. bungii was more profound than that of the Yellow River.A dominant role of intrapopulation variations in intragroup variations was found in the analyses of these two geographical regions. This result indicated that two populations within the same geographical region . The corresponding physical and chemical soil properties represented the average value for this group. Hap6, Hap7, Hap8, and Hap9 were distributed only on Lao Mountain (LS), where the levels of organic soil carbon and soil moisture were lower than the average for Group B. Hap10 and Hap11 were distributed only on Longwang Mountain (LW), where the levels of organic soil carbon and soil moisture were above average and EC was at a significantly lower level. Hap12–20 were all found in Group C (PS), and the results showed that the physical and chemical soil properties on the Pan Mountain of Tianjin (PS) were different from those of other sample areas in Nanjing.Dispersal is an important ecological process, which is widely believed to play a role in determining the community structure of species, together with local factors ,39. TherS. bungii shows lower levels of population genetic differentiation than some soil animals proven to have limited dispersal ability (such as earthworms) [S. bungii in Nanjing is relatively strong and that the habitat patches connecting habitats serve as bridges between populations, making gene exchange possible  . Therefopossible . On an eS. bungii in Nanjing has a certain dispersal ability, which is, however, only one of the factors influencing the genetic diversity and structure of the population in the area. Local factors  may also play a role [We believe that y a role . Our curS. bungii population analyses revealed a significant relationship between phylogeny and geography, demonstrating the presence of a phylogeographic structure. S. bungii exhibited a high level of genetic differentiation, with many unique haplotypes and without shared haplotypes, which showed that the S. bungii populations were differentiated from each other. The AMOVA results  ,48,49. IS. bungii might be intimately and directly related to the formation of the Yangtze and Yellow rivers. Their formation could have carved large populations into smaller ones, leading to neutral evolutionary processes such as genetic drift. Prior to the Miocene, there may have been no obvious genetic differentiation in the eastern coastal areas of China. Following the formation of the Yangtze River, the populations to the north (the predecessor species of Groups B and C) could not contact the populations to the south (the predecessor species of Group A). Over thousands of years of evolution, the genetic distance between the two populations increased, and genetic differentiation may have become evident. The formation of the Yellow River in the early Pleistocene might have hindered the gene exchange of S. bungii once again, and genetic differentiation may have appeared between the populations distributed to the north of the Yangtze River and in the Tianjin region. Due to the later divergence time, the genetic distance between Groups B and C was smaller than that between Groups A and B.Therefore, we considered that the obvious differences in the haplotypes of S. bungii. Soil is the main living space that supports the activities of soil-dwelling animals [S. bungii and the soil environment.In the present study, we investigated the relationships between the environmental factors represented by the basic physical and chemical properties of the soil and the genetic diversity of  animals . Biologi animals ,59, and  animals ,61. ThusS. bungii genotypes. The results (We speculated that several physical and chemical soil properties may influence the  results  revealedS. bungii. Mitochondria have the advantages of a simple structure, low molecular weight, and maternal inheritance [S. bungii to study their genetic diversity. Our results suggested that the relationship between soil properties and millipede mitogenomes is not clear; however, the effects of other habitat conditions on genetic diversity cannot be neglected. Therefore, in subsequent experiments, we aim to use further methods, such as simple sequence repeats, and more genes, such as nuclear genes, to assess the potential impacts of habitat conditions on the genetic diversity of S. bungii.Although there was no evident correlation between haplotype distribution and soil properties, habitat conditions had an undeniable influence on the genetic diversity of eritance ; therefoS. bungii in Nanjing and Tianjin. The results revealed that the gene flow between populations of S. bungii increased the consistency of the genetic structure. However, large geographical barriers, such as between the Yangtze and Yellow rivers, significantly blocked the gene flow between S. bungii populations, leading to high levels of genetic differentiation. Moreover, the correlation between the soil physical and chemical properties and haplotype distribution of S. bungii is not obvious. Our study fills research gaps in the genetic diversity of S. bungii at different geographical scales and provides a theoretical basis for subsequent studies of millipede phylogenetic analyses, community composition, and genetic diversity.This study focused on the genetic diversity and population structure of


Studies conducted in healthy subjects have clearly shown that different hypnotic susceptibility, which is measured by scales, is associated with different functional equivalence between imagery and perception/action (FE), cortical excitability, and information processing. Of note, physiological differences among individuals with high (highs), medium (mediums), and low hypnotizability scores (lows) have been observed in the ordinary state of consciousness, thus independently from the induction of the hypnotic state, and in the absence of specific suggestions. The potential role of hypnotic assessment and its relevance to neurological diseases have not been fully explored. While current knowledge and therapies allow a better survival rate, there is a constant need to optimize rehabilitation treatments and quality of life. The aim of this paper is to provide an overview of hypnotizability-related features and, specifically, to discuss the hypothesis that the stronger FE, the different mode of information processing, and the greater proneness to control pain and the activity of the immune system observed in individuals with medium-to-high hypnotizability scores have potential applications to neurology. Current evidence of the outcome of treatments based on hypnotic induction and suggestions administration is not consistent, mainly owing to the small sample size in clinical trials and inadequate control groups. We propose that hypnotic assessment may be feasible in clinical routine and give additional cues into the treatment and rehabilitation of neurological diseases. Hypnotizability is a psychophysiological trait predicting the proneness to modify perception, memory, and behavior according to specific suggestions, and to enter the hypnotic state. It is measured by scales , 2, is sIn this scoping review, we propose that hypnotic assessment can assist in the prognosis and treatment of a few neurological diseases. With respect to lows and medium hypnotizables (mediums), in fact, highs display stronger functional equivalence between imagery and perception/action (FE) and different modes of cortical information processing , 12, greThe term “functional equivalence” (FE) refers to the similar brain activation occurring during actual and imagined perception/action , 26. As 28. Topological analyses revealed that during sensori-motor imagery lows display significant task-related changes in specific areas, whereas highs exhibit largely distributed, very small changes in their brain activity with respect to resting conditions [Highs and lows differ also in the mode of information processing. Spectral analysis of EEG signals recorded during motor imagery failed to detect significant differences between basal and imagery conditions in highs, in contrast to lows nditions , 28. In nditions , thus thnditions  and to tnditions .Overall, experimental findings obtained in healthy participants suggest that being highly hypnotizable may be a favorable prognostic factor for imagery training aimed at neurorehabilitation. Current literature supports this hypothesis and indicates that imagery training with and without hypnosis is a useful complementary intervention in several movement disorders not only of functional origin . SeveralIn patients with multiple sclerosis (MS) guided imagery increases walking speed and distance, reduces fatigue, improves the quality of life, and positively influences dynamic balance and perceived walking ability , 36. ImaIn Parkinsonian (PD) patients, motor imagery of gait in gait freezers improved gait velocity, stride length, stance time, swing time, single support time, and total double support time . DynamicAlso in amyotrophic lateral sclerosis (ALS) a 4-week hypnosis-based treatment consisting of a hypnosis induction followed by a core phase of guided visual imagery with patient-tailored suggestions and a conclusive phase aiming at self-hypnosis, not only improved wellbeing and quality of life and reduced depression , but was even associated with a slower functionality loss at a 6-month follow-up, in a study with fifteen patients compared to fifteen one-to-one matched controls .A meta-analysis including 51 studies showed that hypnosis is effective in the control of autoimmune diseases . DerangeAnother chronic neurological autoimmune disorder is myasthenia gravis (MG), in which, in the usual forms, autoantibodies are directed against acetylcholine receptors (AChR) or muscle-specific kinase receptors (MuSK) impairing the proper formation of end-plate potential in the muscle fibers and, thus, muscle contraction. In a recent study, patients affected with MG and a higher level of depression or stress had also a higher level of relapse rate, suggesting that emotional factors may influence the course of MG . Thus, a2 and their ability to further increase it after hypnotic induction [Hypnotic treatments may influence the immune system by modulation of the autonomic activity. The highs’ higher parasympathetic tone during relaxation, with respect to lows nduction  could ennduction . After induction , 16, 20.nduction  showed tnduction  togethernduction . More renduction . In gerinduction .In brief, a multidisciplinary approach including suggestions for relaxation, pleasant experiences, and/or motor imagery may improve the treatment outcome, which could be predicted by hypnotic assessment.Suggestions for analgesia and/or pleasant imagery reduce pain whichever the pain origin and duration –56. TheySuggestions for analgesia and self-hypnosis have been efficaciously used to reduce pain in post-stroke patients, multiple sclerosis, amyotrophic lateral sclerosis Parkinson’s disease, Guillain-Barré syndrome, HIV, post-polio syndrome, diabetes, and cancer . MoreoveIn the general population, both physical stimulation and motor imagery (MI) are used to control pain, in line with the concept of functional equivalence between imagery and perceptions see the “” sectionA specific motor imagery protocol—graded motor imagery (GMI) consisting of three sequential phases —has been used in several patients. A non-randomized controlled trial on twenty-eight patients with first-ever stroke  treated In tetraplegic patients, a modified GMI protocol  induced In brief, suggestions for analgesia and pleasant imagery as well as motor imagery are useful tools for pain control.Finally, we must underline that the pharmacological treatments of acute/chronic pain can be oriented by hypnotic assessment. Highs, in fact, exhibit the µ1 polymorphism  associated with low sensitivity to opiates , 22. SucImagery abilities such as the strength of the functional equivalence between imagery and perception/action, the proneness to control the immune system through relaxation by enhancing the parasympathetic tone, to reduce pain through cognitive strategies, and to take advantage from certain pharmacological pain, can be predicted by hypnotic assessment. Suggestions with and without induction of hypnosis also allow an improvement of the patients’ quality of life including physical symptoms and disease progression. Thus, we propose hypnotic assessment as a routine evaluation of neurological patients.A limitation of the role of hypnotic assessment could be the fact that highs and lows represent 15% of the population each, while mediums represent its largest part . NonetheIn conclusion, our opinion is that novel approaches to neurological patients suffering from sensory, motor, and cognitive symptoms of various origins should consider also hypnotizability among the classical psychophysiological traits potentially influencing symptoms and recovery and usually investigated in the context of the body-mind interaction.


Genomic sequence data from worldwide human populations have provided a range of novel insights into our shared ancestry and the historical migrations that have shaped our global genetic diversity. However, a comprehensive understanding of these fundamental questions has been impeded by the lack of inclusion of many Indigenous populations in genomic surveys, including those from the Wallacean archipelago  and the former continent of Sahul . Notably, these regions have been important areas of human evolution throughout the Late Pleistocene, as documented by diverse fossil and archaeological records which attest to the regional presence of multiple hominin species prior to the arrival of anatomically modern human (AMH) migrants. In this review, we collate and discuss key findings from the past decade of population genetic and phylogeographic literature focussed on the hominin history in Wallacea and Sahul. Specifically, we examine the evidence for the timing and direction of the ancient AMH migratory movements and subsequent hominin mixing events, emphasising several novel but consistent results that have important implications for addressing these questions. Finally, we suggest potentially lucrative directions for future genetic research in this key region of human evolution. Homo erectus in the island of Java [H. erectus likely reached Java while it was still contiguous with mainland Asia due to lower sea levels during the mid-Pleistocene, further east lies the maritime barrier known as Wallace’s Line, which has separated mainland Asia from the Wallacean and Philippine archipelagos and Sahul throughout hominin evolutionary history. This persistent barrier was responsible for the diversification of marsupial and placental mammals around 50 Mya, though notably, it did not prevent multiple hominin groups from venturing further east , including the oldest known hominin remains outside of Eurasia; i.e.,  of Java . While Hher east . Indeed,f Flores ,4,5, whihipelago . The thrhipelago  and northipelago ,10,11,12Crucially, efforts to obtain robust estimates of the timing and direction of the historical migratory movements that eventually brought AMH and other hominin groups into Sahul have been complicated by the sparse archaeological record in Wallacea. Indeed, the current oldest evidence for AMH occupation in Wallacea recorded in Timor ,13 and SWith the current limited availability of archaeological sites across much of Wallacea, researchers have recently turned to genetic sequence data from local modern populations and ancient remains as an alternative empirical tool to help resolve fundamental questions around AMH arrival and hominin mixing events in the region. In the following sections, we review key findings from the small but growing number of population genetic and phylogeographic studies examining the human history in Wallacea and Sahul, with a particular focus on studies of Wallacean populations from the past decade. These studies have begun to illuminate a complex population history marked by multiple migrations and population mixing events, which may have erased much of the ancestry descending from the original AMH founders. We discuss the implications of these findings for our understanding of the broader human and hominin regional history and conclude by highlighting potentially fruitful areas of future research.While human genetic research has accumulated steadily for populations in most parts of the world, comparatively few population genomic and phylogeographic studies leveraging complete mitogenome  sequences or Y-chromosomes currently exist for present-day Indigenous peoples from the former continent of Sahul ,25,26,27These genomic results are consistent with phylogeographic studies of mitogenomes and Y chromosomes ,36,37,38To further investigate AMH migratory pathways into Sahul, Brucato and colleagues  comparedThe findings from these recent phylogeographical studies strongly suggest that the movement of Papuan lineages may have reconfigured the genetic profile of AMH populations in Wallacea, with impacts likely extending into surrounding regions . ImportaThe handful of prior population genomic studies that have investigated historical Wallacean demography have revealed a highly dynamic history, with multiple genetic ancestries being introduced into the region around the arrival of Austronesian-speaking seafarers during the late Holocene. One of the first genomic studies attempting to reconstruct the history of ISEA by Lipson and colleagues  used a vHighly consistent genetic patterns were also reported in a more recent study by Hudjashov and colleagues  which utThese studies indicate that the genetic profile of modern Wallacea was largely determined by movements and mixing events in the late Holocene, possibly through the arrival of admixed Austronesian-speaking seafarers that carried multiple ancestry sources. Crucially, these interpretations implicitly assume that the Papuan-related ancestry observed in Wallaceans most likely reflects the AMH founder ancestry endemic to each island. However, the strict partitioning of Negrito and Papuan-related genomic ancestries reported for populations situated either west or east of Wallace’s Line, respectively, may instead signal the effective replacement of an ancient AMH genetic profile across Wallacea by incoming migrants bearing Papuan and potentially additional ‘Asian’ related ancestries. Indeed, it remains possible that the underlying mixing events may not have been local to each Wallacean island and could even have occurred outside of Wallacea. The extent to which these mixing events have decoupled Wallacean genetic profiles from their original island context remains an outstanding empirical question, and its resolution will be crucial for future genetic reconstructions of the deep human history of the region.The complex population history of Wallacea evident in genetic studies suggests that ancient DNA (aDNA) research may provide the key to recovering key historical details. By situating genetic sequences within specific historical, geographical, and archaeological contexts, aDNA can illuminate aspects of human population history that may no longer be evident in modern genetic data. The field of ancient aDNA has developed rapidly since researchers first retrieved fragments of DNA from the desiccated muscle of an extinct subspecies of an Equus quagga museum specimen in 1984 . In the f-statistic analyses showed that the Leang Panninge specimen has two distinct genetic ancestries, with one component that is genetically equidistant to both modern Indigenous Australians and Papuans, and another that is genetically closer to ancient East Asian and modern Andamanese (Onge) populations. Admixture graph models suggest that the Australo-Papuan-like component may reflect the genetic profile of the initial AMH migrants to ISEA, with subsequent mixing with an unknown Asian lineage resulting in the Leang Panninge specimen’s distinctive dual ancestry [In 2021, the first Wallacean paleogenome was published from an ~8000-year-old female from Leang Panninge in southern Sulawesi , which nancestry . InteresA more recent study of 16 paleogenomes from multiple sites in Sulawesi, Moluccas, and Lesser Sundas, which range from several hundred to ~2300 years old, also found no evidence for genetic continuity between these 16 individuals and the Leang Panninge specimen , indicatThese paleogenomic studies provide the first direct support for the erosion of genetic profiles associated with founder AMH migrants across Wallacea through the introduction of external genetic ancestries. While the timing and ordering of the underlying mixing events appear to have varied across Wallacea, estimates of admixture times from ancient and modern genomes consistently point to initial mixing events occurring around 1–2.5 kya ,45, withThe landmark sequencing of hominin genomes from two distant AMH relatives, namely Neanderthals and Denisovans, led to the momentous finding that these now-extinct hominin species had interbred with AMH as they dispersed from Africa across the rest of the planet ,34,55,56Ongoing investigations of the geographic patterning of hominin admixture in populations from ISEA and Sahul attest to a particularly complex set of historical interactions in this region, suggestive of multiple distinct mixing events occurring in situ across different islands ,61,62,63H. erectus, H. floresiensis, and H. luzonensis—were present in the region prior to the arrival of AMH in the region by 50–70 kya [The high levels of Denisovan-related ancestry found across ISEA and Sahul is particularly surprising given that all currently identified Denisovan fossils come from mainland Asia . However0–70 kya ,4,5,6,650–70 kya . To addrWallacea and Sahul are key regions of hominin evolution whose history remains only partially explored, partly due to conditions that have made fossils and archaeological records challenging to retrieve and preserve. In the past two decades, a small but growing number of genetic and phylogeographic studies from Wallacea and Sahul’s populations have started to explore the deep human history of this region, attempting to unravel the timing and location of the original human migrations and mixing events with now-extinct hominins. Notably, these studies have revealed a complex demographic history marked by multiple migrations that appear to have largely erased the deep genetic signals needed to properly resolve these fundamental questions. Moving forward, further genomic studies are sorely needed to determine the timing and origin of the migrations that brought Papuan-related ancestry into Wallacea and beyond, and to ascertain the extent to which modern Wallacean populations retain genetic information from the original AMH migrants. Notably, even if this genetic information has been retained, the complexity and extent of historical migrations impacting Wallacea may have sufficiently decoupled these genetic signals from their initial geographical context, to make the resolution of the migration routes impossible from modern genomes alone. Accordingly, the path ahead may ultimately require the generation of paleogenomes from AMH specimens across Wallacea and ISEA in the period preceding these mixing events. Similarly, determining the identity of hominin ancestry in modern populations east of Wallace’s Line may require ancient DNA or proteomic data from regional hominin fossils. Encouragingly, aDNA recovery from tropical regions has improved rapidly in recent times, suggesting that at least some of the paleogenomic data necessary to address these important questions may yet emerge in the foreseeable future.


While significant effort has been devoted to understand the role of intraurban characteristics on sustainability and growth, much remains to be understood about the effect of interurban interactions and the role cities have in determining each other’s urban welfare. Here we consider a global mobility network of population flows between cities as a proxy for the communication between these regions, and analyze how it correlates with socioeconomic indicators. We use several measures of centrality to rank cities according to their importance in the mobility network, finding PageRank to be the most effective measure for reflecting these prosperity indicators. Our analysis reveals that the characterization of the welfare of cities based on mobility information hinges on their corresponding development stage. Namely, while network-based predictions of welfare correlate well with economic indicators in mature cities, for developing urban areas additional information about the prosperity of their mobility neighborhood is needed. We develop a simple generative model for the allocation of population flows out of a city that balances the costs and benefits of interaction with other cities that are successful, finding that it provides a strong fit to the flows observed in the global mobility network and highlights the differences in flow patterns between developed and developing urban regions. Our results hint towards the importance of leveraging interurban connections in service of urban development and welfare. Significance StatementDetermining the factors behind the economics success of urban areas is a complex endeavor involving many factors. Nevertheless, the flow of intellectual capital and resources between them likely plays a role. Here we analyze the global mobility network of cities, finding that their position in the network, combined with their topological properties are good predictors of their success. Developing cities leverage their mobility neighborhood, connecting to developed cities to rise up the success ladder.science of cities aimed at uncovering the physical and structural features driving their growth and function  to aggregate flows over time (https://policies.google.com/technologies/anonymization). No individual user data were ever manually inspected, only heavily aggregated flows of large populations were handled.To produce this dataset, machine learning methods are applied to logs data to automatically segment it into semantic trips . To provn unique users traveled from location a to location b within week w, the corresponding cell  in the mobility tensor would have a value of n ± η, where η is noise drawn from a Laplace distribution with mean 0 and scale 1/0.66. All metrics are removed for which the noisy number of users is lower than 100. This automated Laplace mechanism yields a -differential privacy guarantee of ϵ = 0.66 and δ =2.1 × 10−29 per metric. The parameter ϵ controls the noise intensity in terms of its variance, while δ represents the deviation from pure ϵ-privacy. The closer each value is to zero, the stronger the privacy guarantees. All data processing is done before being made available to researchers, and the analysis in this paper is done on the resulting heavily aggregated and differentially private data.All anonymized trips are processed in aggregate to extract their origin and destination location and time. For example, if T whose elements ijT represent the flows from location i to j and whose diagonal elements correspond to flows within cells on a weekly basis. For the purposes of our analysis, we aggregate the flows to the full year, that is ijT corresponds to the total flow between locations i, j over the year 2019. We note that the dataset excludes mobility information from China.The mobility flows are encoded in an origin–destination matrix https://seoulsolution.kr/sites/default/files/gettoknowus/jll-global300-2015.pdf).To quantify the economic success of cities, we collected data of city-level socioeconomic variables from the company Jones Lang LaSalle IP, Inc. (JLL) for the year 2018 . The repEarly Growth, Developing, Transitional, and Mature, which are assigned following information concerning the city’s real estate liquidity, the depth of its corporate occupier base, and the quality and range of its commercial stock. The geographical sub-regions indicated are North America, LATAM , and Sub-Saharan Africa. For our analysis, we consider 268 metropolitan areas, excluding China, for which we do not have mobility data.  are http://www.oecd.org/cfe/regional-policy/functionalurbanareasbycountry.html). Using a gridded population dataset, urban cores are defined as clusters of adjoining grid cells with a population density above a certain threshold—1,500 inhabitants per km2 for all regions except Mexico and the United States, where due to lower density the threshold is 1,000 inhabitants per km2. Many Asian and African cities are not part of the dataset. For these, we use data from the Atlas of Urban Expansion , which provides a definition of city boundaries based on the extension of the built-up area.The Organization for Economic Cooperation and Development (OECD) provides boundaries for functional urban areas in member countries  and E = 39,868 directed edges. To incorporate, in a tunable way, the contribution of long-distance travel to economic activity, we define the edge weights of the mobility network asβ ≥ 0. In this expression, ijT is the total flow from city i to j and the ijd is the geodesic distance between them. Incorporating this allows for including the fact that long-range connections indicate greater levels of implied connectivity between the cities, given the associated cost of travel , whereas in panel (b) we show an illustrative example for β > 0. As the figure shows, incorporating the effect of distance makes long-range flows more prominent where colors indicate magnitude of flow, increasing from yellow to red, and node sizes correspond to PageRank [maps generated using the Shapely (https://pypi.org/project/Shapely/) and GeoPandas (https://geopandas.org/) packages in Python].While we have metadata for 268 cities, for the purposes of our analysis, we construct the static mobility network of aggregated intercity flows. The network consists of f travel , 57. Thei is computed as \docuα = 0.85 ). In addα = 0.85 ,64, diseα = 0.85 , roads oα = 0.85 , ecologiα = 0.85 , and higα = 0.85  among ots that measures the strength and direction of association between two ranked variables. In Fig. β = 0 finding a monotonic relationship with ρs = 0.53. Including distance in the edge-weights reveals an increase in the association between the two variables for any non-zero β. In s as well as Pearson’s correlation coefficient ρp between log (BHI) and log (PR) as a function of β, finding a peak value for both correlation coefficients at β = 2.19 (ρs) and β = 1.88 (ρp). In Fig. β = 2.19, indicating a much stronger correlation (ρs = 0.77). In ijT, the distance ijd as well as Eq.  through Transitional and Developing cities  and finally a weak correlation in Early Growth cities (sρ = 0.38). Indeed, the differences become rather stark when looking at the other socioeconomic indicators as seen in Fig. Next, we disaggregate the cities based on their level of development and geographical region to check whether the results are consistent across these categories. In Fig. s = 0.86, 0.93, and 0.76, respectively), in areas with mixed levels of prosperity such as Latin America, Asia, and the Middle East, the correlations are slightly weaker  with the weakest correlations being in Sub-Saharan Africa (ρs = 0.38).Differences also exist when grouping cities based on their geographical location as shown in We note, the possible confounding factor of agglomeration effects. Indeed, urban agglomeration is a major facilitator of socioeconomic development  by provik-core decomposition, that identifies strongly connected core-nodes and sparsely connected peripheral nodes, revealing meso-scale network structure (s between PageRank and BHI as a function of the layer-number k (numbered in increasing order from the outer to inner layer) as shown in To check whether these observed differences based on maturity and location are consequences of higher-order topological features of the mobility network, we next employ the tructure ,72. Applr is a richness parameter. Two flavors of the parameter that we consider are the out-degree kout and the out-strength sout. Given this, ϕ(k) is a fraction that measures the number of edges between nodes of degree ≥kout compared to the edges they would share if all these nodes were connected to each other. The quantity ϕnull(kout) is the corresponding measure under out degree-preserving reshuffling of links. For sout, ϕnull(sout) is computed through both edge and weight reshuffling. Values of ρ(r) > 1 indicate a set of nodes that are connected to more cities, with higher exchange of populations, than one would expect merely as a consequence of the distribution of links and edge-weights. In Fig. kout) and in Fig. 4(b)ρ(sout), finding that nodes with high connectivity and strength of connections are between two to three times more connected to each other than would be expected by random chance. The results indicate that Mature and Transitional cities form a rich-club dominating mobility flows in the network.The existence of large core clusters concentrated in Western Europe and North America  suggest that they form a densely interconnected backbone of the mobility network and thus control most of human capital and resource flow. This can be quantified by studying the weighted rich-club organization , which ik-core decomposition allows for assigning nodes to layers, there is no clear way to define a boundary between the core and periphery. To do so, we use a method introduced in  that are successful in terms of socioeconomic indicators. This success can be reasonably well predicted by their centrality, or just their topological features in relation to the mobility network. For cities in the periphery, and those that are not so tightly connected, their level of development cannot be simply captured by topological aspects. In particular PageRank is a poor measure to capture such cities’ welfare, given that it is noisy for nodes with low connectivity and edge-weights .jS denotes the success/welfare of a given city j (measured through the socioeconomic indicators) and WI contains only international flows from j to i. The quantity 〈S〉i can then be interpreted as the weighted average of the success of the international visitors to city i. For instance, if we take BHI as the measure of success, then this number will be high if most visitors are from Mature cities, and low if they are from Early Growth cities. Based on this, we define an estimator for a city i’s success.iPR is its PageRank and γ is a tunable parameter that measures the extent to which the level of development of a city’s neighbors plays any role in predicting its own development. For γ ≃ 0, the node’s network topological properties are the best indicator of its welfare, whereas for γ ≥ 0 incorporating information about the development levels of its neighbors provides a better estimate.Furthermore, it does not capture an important feature, that is the maturity level of the cities from which travelers come to a given city. For instance, it stands to reason that for two Developing cities with similar connectivity profiles, if one of them has visitors from Mature cities, and the other only from Developing cities, then the former may benefit more from the flow of intellectual and human capital from more developed regions. To account for this effect, we define the quantityγ (see s(γ), between the estimator iS as quantified by the BHI which captures the city’s maturity level. We note that the analysis excludes six cities in the USA  and two from Mexico (Puebla and Querétaro). For these cities there is no BHI data on any of their international connections. The role of the average success of neighbors 〈 S〉 is the most prominent for Early Growth cities, and practically non-existent for Mature cities  = 0.47) unlike for the other regions (ρs(γ) ≥ 0.76).Table γ see , maximizentclass1pt{minimai has a certain amount of total outflow ic which is incident on different cities j within some set of target cities ∂i—its mobility network neighborhood. We assume that city i’s outgoing flow ic to neighbors j ∈ ∂i is partitioned based on the success jS of city j, as well as the distance ijd, so that μ and ν are free parameters, and i to j from the model (whose dependence on μ and ν has been made explicit). More precisely, we havejS makes travel to city j more attractive to travelers from city i, then one would expect μ ≥ 0. On the other hand, if the cities are far apart in distance ijd, then it would make it more costly to travel from i to j, and in that case ν ≤ 0. The ratio jS and ijd, the relative importance of these measures can be compared directly using this ratio regardless of units.The enhanced ability to predict the true socioeconomic indicator of a city in the less developed categories by incorporating the maturity level of its neighborhood suggests that perhaps success plays a role in how flows are allocated. To check for this, we construct a simple model for the nature of outgoing flows of a city among its destinations that accounts for both the success of the target destinations, as well as the distance between these targets and the source city. Suppose a city ic can be inferred from the raw (unweighted) origin–destination matrix i is restricted to the subset of cities j that i connects to that also have BHI metadata jS. We then fit Eq.  significantly improves our predictive model of outflows over a baseline model, where only the distance between cities is considered. To do this, we identify the exponent ν0 that optimizes the correlation between the true and predicted flows while ignoring the success parameter (μ = 0). Since the model with μ has one more free parameter than the model with μ = 0, the fit between the true and predicted flows will always be better according to the Pearson correlation ρp. Thus for a fair comparison, the more complex model needs to be penalized using some model selection criteria. Here we opt for both the Akaike Information Criterion (AIC) and the Bayesian Information Criterion (BIC)  , which ar2 for each city subcategory. As discussed, the ratio jS and distance ijd with the outflows ic from city i. According to the model fits, in general outflows from Developing cities are more strongly associated with success than outflows from more developed cities, as indicated by the shaded red box. More specifically, outflows from cities in the Developing, Early Growth, and Transitional subgroup have a higher relative magnitude of association with the success of the cities they flow to, as compared to outflows from cities in the Mature subgroup. We also see a geographic dependence with outflows from cities in North America and (Western and Eastern) Europe having lower relative levels of association with BHI and having weaker model fits in general. This suggests that other factors are more important in determining the corresponding flows (perhaps consistent with these regions having many developed cities). As insets, we illustrate the variables used in the flow model . Their level of development is partially connected to their network centrality in some cases, and very weakly so in others, in particular in Africa.For those regions, whose success is weakly connected to their centrality, we find that adding information on their mobility neighborhood (i.e. the average development level of the cities they are connected to) leads to a marked increase in correlations with their socioeconomic indicators. This effect is less pronounced for more developed cities, whose socioeconomic indicators are connected primarily with their network centrality. Once again Sub-Saharan Africa is an outlier, its development being weakly connected to network properties and the socioeconomics of their mobility neighborhood. To check whether the connection of cities to other cities is indeed influenced by their development-level, we propose a simple model that disentangles the relevant aspects influencing the success/welfare of urban areas. The model assumes that the outflow of cities is distributed among destinations in proportion to their relative benefit (maturity) and cost (distance). The predicted outflows are compared to the empirical outflows to determine the contribution of each individual component of the model. We find that all cities incorporate the development level of the cities they connect to, and are less inclined to connect to cities at great distance. However, the former is much more pronounced in developing regions as compared to developed regions.Taken together, our analysis indicates that the welfare of cities and the interurban mobility network are strongly correlated, but that this correlation depends on their level of maturity. The global urban ecosystem appears to be a combination of a well-established set of core urban areas whose interconnections describe their performance according to network science principles, and a subset of Developing cities whose welfare is influenced by the extent of connections to the core. The core set of cities also exhibit a rich-club phenomenon, whereby the density of internal connections within the core is higher than expected, which might be related to the existence of global cities as proposed in sociological studies .While we do not have data on individual mobility-flows the results are akin to that seen in , where aThese results should be interpreted in light of several important limitations. First, the Google mobility data are limited to smartphone users who have opted in to Google Location History feature, which is off by default. These data may not be representative of the population as whole, and may vary by location. Importantly, these limited data are only viewed through the lens of differential privacy algorithms, specifically designed to protect user anonymity and obscure fine detail. Moreover, comparisons across, rather than within locations are only descriptive since these regions can differ in substantial ways. For instance, privacy choices and income thresholds may vary from country to country. Nevertheless, it has been shown that the dataset provides equal coverage across populations irrespective of their income profiles .Another limitation is the fact that the dataset does not provide information about the composition of population flows. Namely, the fraction of capital, knowledge, and trade flows cannot be distinguished in the mobility network, given that it is aggregated across all types of flow. This precludes one from making more granular analysis on the differences between trade and population flows, for instance. Furthermore, due to the static nature of the network, temporary and permanent migration patterns of human labor cannot be identified. This introduces an issue of endogeneity, whereby it is difficult to ascertain whether migration creates better economic conditions, or is it that migrants tend to move to locations that already are economically attractive. Likewise, our analysis overlooks other socioeconomic factors beyond mobility which might also play a crucial role to shape urban welfare. Availability of longitudinal data and the ability to distinguish between the types of flow will prove invaluable for more detailed investigations. Finally, the exclusion of China from the analysis will necessarily affect the set of cities identified to be part of the well-developed urban core.pgac178_Supplemental_FileClick here for additional data file.


Ropalidia plebeiana constructs extremely dense colony aggregations consisting of hundreds of colonies within a few square meters; however, little is known about the aggregation structures. We genetically analyzed the colony and population structure of R. plebeiana, and concomitant variations in colony sex ratios. In spring, the foundress (candidate queen) group started their colonies on a single old comb from the previous season, subsequently dividing these old combs via relatedness-based comb-cutting. Female philopatry, a prerequisite condition of Local Resource Competition (LRC), was confirmed. The colony sex ratio of reproductive individuals  became slightly male-biased in larger colonies, as predicted under LRC. However, the number of foundresses was positively associated with the number of reproductive individuals, suggesting that Local Resource Enhancement (LRE) also operates. Although the population structure appears to meet the prerequisites of LRC, the sex ratio appears to be modulated by factors other than LRC. Rather, through LRE, the availability of female helpers at the founding stage is likely to mitigate the sex ratios predicted under LRC.Social insects are highly diverse in their social structures, aside from the consistent presence of reproductive castes. Among social insects, the Australian paper wasp  Colony size also varies, in some species consisting of a queen and a few workers, and others consisting of hundreds of queens and tens of thousands of workers2.Few groups of animals exhibit more extremes in lifestyle than social insects. Common characteristics of social Hymenoptera include the presence of one or more breeding queen(s) and sterile helpers who assist with reproduction, but beyond these traits, they exhibit remarkable variation. Although they are thought to have originally been monogamousRopalidia plebeiana falls at one extreme of population structure owing to its exceedingly dense colony aggregations consisting of a group of colonies . Richards5 in 1978 described R. plebeiana colony aggregations in Cabbage Tree Creek in New South Wales; these aggregations were still extant as of 2018.The Australian paper wasp ies Fig. . Each co6; Fig. 2. In R. plebeiana, overwintered foundress groups start their colonies (founding stage) in spring (September). Comb-cutting is frequently observed from the beginning of the colony cycle from spring (October) to the first worker emergence  in which queens and workers co-exist. Upon comb-cutting, each old colony, consisting of a single old comb, is divided into several new colonies, each of which comprises a single new comb  the following season, as old combs are divided into several new combs  under haplodiploidy. We removed these data from further analyses of population structure.Using KinshipFollowing sequential Bonferroni correction, no significant departures from Hardy–Weinberg equilibrium were detected across the five loci assessed for the remaining 148 individuals. Similarly, no significant linkage disequilibrium or null alleles were detected in the five loci.K) was largest at K = 1 or K = 2, and Evanno’s method8 estimated nine populations, our STRUCTURE analysis did not detect any genetic structure among the 14 aggregations at K = 2  in the microsatellite dataset was 0.007 (P = 0.077), whereas the inbreeding coefficient (FIS) was 0.038 (P = 0.042), and the overall fixation index (FIT) was 0.045 (P = 0.018), implying no apparent subdivision in the population, but indicating a slight trend toward inbreeding.Although the mean likelihood (ФST fixation index of this dataset was 0.126 (P = 0.001), indicating apparent population subdivision. In the absence of sex-biased gene flow, the expected relationship between microsatellites (represented by FST) and COI (ФST) is as follows: ΦST = 4FST/(1 + 3FST)9. Both FST and ФST are known to be influenced by expected heterozygosity (Hs)10; we, therefore, calculated G’ST fixation index following Hedrick11 and inserted it into the equation. The calculated value of G’ST was 0.027 , indicating female philopatry.We found 18 haplotypes of the cytochrome oxidase subunit I (COI) (accession nos. LC533797–LC533806 and LC554438–LC554447). The global rs = 0.425, P = 0.009) but not in the COI data . These results indicate that gene flow and drift are balanced in microsatellites, whereas drift is more prominent than gene flow in mitochondrial DNA (mtDNA). We can thus conclude that the population structure of females is more subdivided than that of males.IBD (Isolation By Distance) relationships , which is an expected contagious distribution.Prior to the onset of comb-cutting in spring 2002, we re-sighted 59 foundresses that were marked during the previous season. We counted the number of combs between the natal comb and the comb on which each foundress was re-sighted . The frequency distribution of the relatedness within combs and between combs  and that at post-comb-cutting was 7.297 ± 0.924 (n = 37), respectively  or among new foundresses in the following spring  following sequential Bonferroni correction.Relatedness was significantly higher among new gynes (0.589 ± 0.053) than among foundresses   overwinter. Although their wintering sites are unknown, most foundresses return to the same colony aggregation sites the following spring4.The paper wasp Ф’ST for mtDNA was higher than the standardized G’ST for microsatellites, even when values were corrected based on differences in the effective population size between the two markers. In contrast, we observed a weaker population structure in the microsatellite data  of foundresses start their colonies with their nestmates from the previous year. This value corresponds closely to the frequency of foundresses observed to settle on their natal combs . Therefore, this might indicate the presence of a relatedness-based threshold for comb-cutting, i.e., the comb is cut when a foundress group comprises < 60% natal nestmates.The estimated global standardized R. plebeiana reuse old combs from the previous season, and those combs are often gnawed into several separate pieces by them5. These observations led to hypothesize that each comb is nepotistically divided into smaller new combs and that foundress groups are divided into smaller groups based on kin relationships. Indeed, we found that genetic relatedness among foundresses is higher within combs than between combs , but some subordinate foundresses may be allowed to reproduce in some colonies.The genetic relatedness of workers was 0.235, and the effective number of mothers for workers was 3.94 based on the following equation if queen’s monandry is a rule:18. However, the opportunity for subordinates to inherit colonies might be quite limited in R. plebeiana, as we did not observe any queen turnover at our study site. Another possibility is that the fate of the foundress is determined in a pre-imaginal fashion, in which the future queen is determined prior to the death of the existing queen. Her nestmates recognize her as the future queen, and the pheromonal control by the queen over the colony is more important than physical aggression20. In R. plebeiana, future queens among foundresses are docile during the founding stage and never forage , implying that future queens may also signal their presence via pheromones. Such possibilities should be examined in future chemical studies.The frequency distribution of the relatedness among foundresses within combs indicated that several combs exhibit zero relatedness . The sex ratio became significantly male-biased in larger colonies Fig. . The con25. We found a cooperative breeding advantage in R. plebeiana: the number of foundresses in spring had a positive effect on total productivity , which comprises positive interactions among cooperative breeding females31, such that protogyny is associated with a moderately female-biased sex ratio. The caste plasticity hypothesis proposes that females are produced before males (protogynous production) whenever there is uncertainty regarding the optimal time to begin producing reproductive individuals. Thus, we might expect that the sex ratio will be female-biased because females may become either workers or new gynes (future queens) until late in their development and respond more flexibly than males30. In this study, the reproductive timing of R. plebeiana was protogynous , and this might have also counteracted the male-biased sex ratios favored under LRC.Another factor that may mitigate LRC is caste plasticity32. The relatively short annual colony cycle in such wasps may constrain worker control of the sex ratio; colony maintenance is a stronger driver than reproduction, mainly because, unlike perennial species, they have only a single opportunity33. Such seasonal constraints may not allow for the evolution of the adaptive traits predicted by theory.We found that colony fission via comb-cutting is kinship-based. Furthermore, we detected female philopatry in both the mark–release–recapture and genetic studies. We thus conclude that the kinship structure of this species is such that the decline in kinship during the founding stage, assuming that foundresses form groups randomly, is mitigated by female philopatry and kin recognition and subsequently increases due to reproductive skewness among foundresses. This cycle is repeated each season. Our results imply that overwintering foundresses may retain some natal nest-related information during the overwintering period. This ability to discriminate relatives among foundresses is likely to prevent the degree of relatedness within a colony from declining significantly. In addition, we found that sex ratios become more male-biased as colony size increases. However, LRC, which leads to a male-biased sex ratio, may counteract LRE, which leads to a female-biased sex ratio, thus evening out the population sex ratio. In general, no worker control of the sex ratio has been confirmed among primitively eusocial paper waspsWe censused 26 colonies from an aggregation at Dinner Creek in New South Wales, Australia , and stored them in 99% EtOH. After each round of collection, we visually inspected whether the old combs had been cut. We successfully monitored this process for 16 combs with 16 foundress groups (n = 269 foundresses). Each of the 16 combs was divided into two or three new combs during the founding stage, and, in total, 37 combs were constructed by 37 foundress groups.We used insect nets to collect 14–40 adults from each of 14 aggregations (n = 309) from March to September 2004, including the Dinner Creek aggregation  as described by Walsh et al.2), and 6.85 µL of distilled water, yielding a total volume of 10 µL. PCR was performed using a thermal cycler . After denaturation for 4 min. at 94 °C, the samples were subjected to 30–35 cycles of 1 min. at 94 °C, 1 min. at annealing temperatures appropriate for each primer pair, and 45 s. at 72 °C, with a final extension for 7 min. at 72 °C. We used five primer pairs (Supplementary Table 35.Polymerase chain reaction (PCR) was performed using 1 µL genomic DNA (approximately 1 ng) diluted in a mixture consisting of 1 µL of primer mix (2.5 µM), 0.1 µL of 10 mM dNTP mix, 0.05 µL of Taq polymerase , 1 µL of 10 × buffer  and a Big-Dye Terminator Cycle Sequence Kit (Applied Biosystems). PCR fragments were sequenced in both directions to ensure accuracy.36 based on the genotype data for the 309 adults. Departures from Hardy–Weinberg assumptions were calculated using GenAlEx ver. 637. Linkage disequilibrium was tested using Genepop ver. 1.238. The 14 aggregations were sampled using arbitrary hand netting. Using Kinship7, we identified full-sib relationships and removed them from our population data.For the microsatellite dataset, we checked for null alleles across loci using MICRO-CHECKER39. This method assesses mtDNA haplotype differentiation based on an analog of the F statistic known as the Ф statistic40. FST (for microsatellites) and ФST (for mtDNA) indicate the overall genetic variation among populations and imply sex-biased dispersal patterns. We evaluated genetic differentiation among aggregations using pairwise FST with 1000 permutations in GenoDive. Significant correlations between genetic differentiation (based on FST/(1 – FST) values for both markers) and the corresponding geographic straight-line distance (in km) were assessed using a Mantel test with 1000 permutations.For both the microsatellite and mtDNA datasets, we quantified genetic variation between hierarchical levels in each aggregation and calculated their fixation indices using analysis of molecular variance in Genodive ver. 3.041 as an alternative measure of the degree of genetic differentiation, using the microsatellite data to delineate the number of genetically identified clusters (K) and assign individuals to clusters without using prior information about their locality of origin. We calculated an ad hoc criterion of ΔK8 to determine optimal K values between 1 (panmixia) and 10 and performed 10 runs for each K value using 1,000,000 Markov chain Monte Carlo iterations with a burn-in period of 100,000 iterations. These calculations were conducted using Structure Harvester42. The assignment index for each individual in each aggregation was calculated using CLUMPAK43.We applied Bayesian clustering in STRUCTURE ver. 2.044. We calculated the relatedness among foundresses within combs and between combs after comb-cutting and compared the values with a paired test45.We genotyped four classes of females, namely, foundresses, workers, new gynes, and new foundresses from the following season. We did not genotype the subordinate foundresses, which later became workers after comb-cutting. The genotypes of new foundresses were assessed based on females collected on September 15–16, 2002. We calculated coefficients of genetic relatedness and inbreeding using Relatedness ver. 4.2cUnless otherwise specified, all statistical analyses were performed in R ver. 3.4.1 (R Core Development Team 2020).Supplementary Information.


The combined use of a vital dye and radioactive colloid reportedly performs better in detecting sentinel lymph nodes (SLNs) for cancers than the use of either of them alone. However, especially for gastric cancer, two endoscopic procedures are required to administer these two tracers, which burdens the patients and practitioners. Here we propose the use of stannous colloid (SnC) mixed with indocyanine green (ICG) as a new mixed tracer (SnC–ICG); its characteristics were investigated in vivo and in vitro to estimate its usefulness for SLN navigation. The tracers were administered to rats and the accumulation of radioactivity and/or near-infrared fluorescence were evaluated in the regional lymph nodes (LNs) using single positron emission computed tomography and near-infrared fluorescence imaging, respectively. SnC–ICG showed significantly better clearance from the injection site and better migration to primary LNs than the single administration of SnC or ICG aqueous solution. SnC–ICG demonstrated a wide particle size variability, stabilized to 1200-nm upon the addition of albumin in vitro; These properties could contribute to its behavior in vivo. The use of SnC–ICG could contribute better performance to detect SLNs for gastric cancer with less burden on both patients and medical practitioners. SLNs are defined as the first one or first few lymph nodes (LNs) which receive lymphatic flow from the primary tumor, and the absence of metastasis in the SLNs indicates no metastasis in the other regional LNs4. Therefore, the extent of the lymphatic dissection could reasonably be reduced based on the metastatic status of the nodes revealed intraoperatively. Survival rates after surgeries without regional LN resection in patients without SLN metastasis are comparable with those after conventional radical surgeries5. This SLN navigation surgery was first applied to the treatments for skin melanoma4 and breast cancer2, and subsequently to gynecologic and other solid malignancies6. Radioactive colloid or vital dye is used to detect SLN8. Although radioactive colloids require radiation-controlled handling area and specific visualizing equipment such as a gamma camera or single positron emission computed tomography (SPECT), they show long retention in the LNs, enabling a quantitative evaluation from outside the body with high sensitivity9. In contrast, vital dyes, such as isosulfan blue, patent blue violet, and indocyanine green (ICG), are easy to handle, inexpensive, and can help visualize every lymphatic route to the SLNs without the need for a detection equipment. The near-infrared (NIR) fluorescence properties of ICG have recently attracted the attention of surgeons as they allow the penetration of biological tissues up to a depth of approximately 1 cm; ICG can be detected with better sensitivity than other dyes using NIR fluorescence video systems12. However, vital dyes must be administered intraoperatively because they tend to diffuse quickly and reach secondary or downstream LNs almost immediately.In recent years, sentinel lymph node (SLN) navigation has gained much attention as a tool that can reasonably minimize surgical invasiveness for the treatment of various malignant diseases15. Therefore, a multicenter clinical trial designed to evaluate the usefulness of SLN navigation in gastric cancer surgery in Japan16 recommended the combined use of technetium-99m (99mTc)–stannous colloid (SnC) and a vital dye such as ICG or indigocarmine. SLN navigation for gastric cancer surgery could enable function-preserving and less invasive procedures by reducing the required degree of gastric and regional LN resection17.Single use of these tracers has achieved clinically practical identification rates and accuracy levels, while their combined use has shown an improved performance18. This multimodal tracer was constructed from human serum albumin aggregates and ICG, and it has already been adopted into the clinical management routine of various cancers23 in several hospitals in European countries. However, this ICG-99mTc-nanocolloid is not available in our country and its utility in gastrointestinal surgeries remains unclear.Nevertheless, differences in the pharmacokinetics of the two types of tracers were noted, and a multimodal tracer that could be detected with both NIR and radioactive methods was proposed to overcome this problem24 reported that, in an animal model, the addition of phytate colloid to ICG significantly prolonged the retention of fluorescence in primary LNs and retarded its migration to downstream nodes. However, they did not analyze the mixture’s particle profile; The use of phytate colloid for visceral organs is tricky because it reportedly accumulates heavily in the liver24 and behaves differently in rodents and primates25. Therefore, we tested the utility of the SnC and ICG mixture (SnC–ICG), both approved by the Pharmaceuticals and Medical Devices Agency in Japan for the detection of SLN.In this context, Araki et al.The combined SnC–ICG administration revealed a significantly better flow from injection sites and retention in primary LNs than the administration of SnC or ICG alone. We also investigated the physical and chemical characteristics of this mixed tracer and the results revealed that the interaction between SnC and ICG contributes to its behavior in vivo.The NIR fluorescence camera revealed an accumulation of fluorescence in the popliteal LNs of all the evaluated rats at 18 h after the administration of ICG aqueous solution or SnC–ICG  and relatively stronger fluorescence in the iliac nodes (p = 0.091) of the rats administered with SnC–ICG than those administered ICG aqueous solution alone Fig. .Figure 2A histological analysis of the popliteal LN showed disseminated fluorescence in almost all LNs in rats administered ICGaq or SnC–ICG; however, the fluorescence in the marginal sinuses was stronger in the rats administered SnC–ICG versus ICG aqueous solution Fig. .Figure 399mTc-SnC–ICG  visualized hot spots in the tracer injection sites of all rats; however, those within the popliteal and iliac node areas were revealed only in rats administered ICG Fig. .Figure 499mTc-SnC–ICG, and the fluorescence intensity appeared stronger in the popliteal versus iliac nodes  radioactivity counts in the foot (injection site) and significantly higher radioactivity counts (p = 0.004) in the popliteal LNs than those administered 99mTc-SnC.After the rats were euthanized, the part of the injected foot and the regional LNs  were retrieved, and their radioactive counts were measured ex vivo Table . Mean %I26. SnC–ICG administration showed slightly smaller median particle sizes (334 nm) but a much higher size variability  than SnC . The median particle size of SnC or SnC–ICG increased significantly when albumin was added , and the median SnC size was approximately 500 nm larger than the mean SnC–ICG size (p = 0.003). Interestingly, the particle size variability of SnC–ICG was considerably smaller  after the addition of albumin, making it comparable with that of albumin-added SnC .To investigate the background of the differences in the biodistribution analyses, particle size distributions and zeta potentials of the tracers were examined. Albumin-added samples were also measured to estimate their behaviors in vivo Fig. A. The meThe zeta potentials of these samples were estimated as well Fig. B. While In this study, we proposed the combination of SnC and ICG as a new tracer (SnC–ICG) for SN navigation, which can be detected with both NIR fluorescence and radioactivity since both are widely used for the same purpose in our country. At 18 h after administration, SnC–ICG showed a better clearance from the administration site and LN retention compared with SnC alone in our rat model. Fluorescence levels of the same LN at the same time point were considerably low but detectable; those with SnC–ICG were significantly higher than those with ICG alone. Two LNs, the popliteal and iliac LN, visualized using an NIR fluorescence camera or SPECT/CT, were thought to be primary LNs from a foot pad in this rat model, since our preliminary study showed two lymphatic routes from a foot pad . These data suggested that SnC–ICG was expected to perform better than SnC alone in the radiocolloid-guided method with an acceptable level of performance in the NIR fluorescence-guided method for SN navigation surgery.30. SnC–ICG prepared without albumin showed a relatively lower size  and a much wider size variation  than SnC and this could have accounted for the better clearance of SnC–ICG from the injection site. SnC–ICG was observed to increase in size  and converge to a smaller dispersion  after the addition of albumin, which could have contributed to a better retention in primary LNs.The differences in biodistribution characteristics between SnC–ICG and SnC could be accounted for by the differences in the particle size Fig. A and sur31. Similarly, the separate administration of immunogenically modified liposome and pentameric immunoglobulin M in a rat model resulted in their aggregation and retention in LN for prolonged periods of time32. Araki et al. also mentioned that small phytate colloidal particles (100–200 nm in diameter) migrate immediately after their injection into LN, where they increase in size upon reacting with ionized calcium and are retained for much longer periods without passing to the downstream nodes24. However, they did not investigate the detailed particle profile of phytate colloid mixed with ICG. Our in vivo and in vitro data indicated that SnC and SnC–ICG increase in size in association with serum albumin in vivo.Particle size change in vivo is one of the essential concepts for SN tracers for better clearance from the injection site and longer retention by LNs. Philips et al. showed that a subcutaneous injection of avidin after the administration of biotin-coated liposome greatly enhanced its LN retention in a rabbit model33. Our zeta potential analysis indicated a high positivity for SnC, but a neutral to negative charge for SnC–ICG 22+, etc.), which are formed by the hydrolysis of stannous (II) ions in a solution containing chloride ions36. Meanwhile, ICG has two negatively charged sulfate residues and a much higher molecular weight than chloride ions; this factor may influence the SnC during the process of nucleation, growth, and cohesion, generating turbulence in the electrostatic repulsion of the colloid particles.The wide variability of SnC–ICG particle sizes and zeta potential distribution suggested an instability of the colloidal product; however, its mechanism requires further elucidation. The zeta potential of SnC was largely positive because its formation occurs via bonding with cationic hydroxide salts . However, it was difficult to explore the actual degree of unity between these two components in 99mTc-SnC–ICG since the particle size of SnC and quantum yield of ICG fluorescence changes with the dilution of the suspension. Nevertheless, the addition of ICG caused considerable changes in SnC in terms of physical conditions and biodistributions, indicating certain interaction between the two tracers.Furthermore, the mechanism of SnC–ICG responsible for its significantly higher fluorescence in primary LNs compared with that of ICG aqueous solution Figs.  and 2 re99mTc. Although the concentration of 99mTc was extremely low, we believe these properties would be altered to some extent in its presence. However, Higashi et al. reported that the colloidal size was the same in the preparations with radiolabeling and those without37. Second, we used a normal rat model for the biodistribution analyses of the tracers. However, lymphatic flow of the regional area for tumors is modified in tumor-bearing animals. Third, the pharmacokinetics of SnC–ICG were not investigated because we set a single time point to assess the biodistribution of this tracer with reference to our routine procedure for SN navigation of gastric cancer in a clinical setting. Assessment of different time points would have been more informative but we prioritized the minimization of the number of test subjects for ethical animal welfare reasons. The lymphatic system is much smaller in rats than in humans; thus, the assessment interval would have been too long to estimate the biodistribution in humans. Therefore, the in vivo behaviors of tracers in humans require further verification in clinical settings.Our study has some limitations. First, the physical properties of the colloidal samples were measured without the addition of In conclusion, here we proposed a new mixed tracer (SnC and ICG) for SLN detection that can be detected through both radioactivity and near-infrared fluorescence. A biodistribution study in an animal model and physical property analyses indicated that the single administration of SnC–ICG achieved better clearance from the injection site and showed longer retention in primary LNs than the administration of either alone. Although further investigations are required, this new tracer could improve the SLN detection and reduce patient and physician burden in SLN navigation for intra-abdominal and intrathoracic tumors.99mTc-SnC was dispensed using a SnC Tc-99m kit . Isotonic sodium chloride solution  supplied by Otsuka Pharmaceutical Co. Ltd.  was used as the diluting solution, while Albuminar® 25% purchased from CSL Behring K.K.  was used to prepare the human serum albumin. The 99mTc was eluted from a 1.1 GBq 99Mo-99mTc generator . The isoflurane used for general anesthesia in the rats was purchased from Pfizer Japan Inc. . All reagents were used without further purification.The ICG aqueous solution was prepared using DIAGNOGREEN® purchased form Daiichi-Sankyo Co., Ltd. . The 99mTc-labeled samples (99mTc-SnC–ICG and 99mTc-SnC) were prepared following the same procedure; however, the saline was replaced with the 99mTc extract with approximately 740 MBq/mL radioactivity. For the preparation of the albumin-added samples, 0.15 mL of Albuminar® 25%  was added at the final preparation step .The concentration of ICG was adjusted to 5 mg/mL using 5 mL of distilled water and then diluted tenfold with saline . Next, stannous chloride solution, saline, and tenfold diluted ICG aqueous solution at a volume ratio of 5:4:1 .Ten-week-old female Sprague–Dawley rats (190–230 g) were purchased from Japan SLC, Inc.  and housed in plastic cages in an animal housing facility at Chiba University, with a time routine of 12:12 h light/dark cycle, under controlled temperature and humidity, and water and food ad libitum. All animal experiments were performed under general anesthesia. Inhalation of isoflurane was started at a concentration of 4 v/v% and maintained at 2–3 v/v%. All animals were euthanized with an overdose inhalation of isoflurane (5 v/v%) for 20 min before retrieving regional LNs and administration sites. All animal experimental protocols in this study were reviewed  and approved by the Institutional Animal Care and Use Committee of Animal Experimentation of Chiba University. All methods were carried out in accordance with the relevant guidelines and regulations in Japan. The reporting in the manuscript follows the recommendations in the ARRIVE guidelines . The data acquisition was performed at 240 s/projection with a stepwise rotation of 16 projections over 360˚. The SPECT Triumph-RECON software  was used to obtain the SPECT images, while the data were reconstructed using a three dimensionally ordered subset expectation maximization algorithm using two subsets and eight iterations.NIR fluorescence images were obtained by an NIR fluorescence camera . The system enables the real-time speed monitoring of both bright-field and NIR fluorescence video images through a high-contrast charge-coupled device (CCD) camera. Bright-field and NIR fluorescence microscope images of the frozen sections were obtained using a microscope  equipped with an electron-multiplying CCD camera  and an NIR fluorescence detection filter set . The in vivo distribution of With each rat under general anesthesia, 10 Tissue sections . The mean brightness of the fluorescent area determined by a five-time repeated discriminant analysis in the CIE Lab color space on an apparent LN image was used as the representative fluorescence intensity.99mTc-SnC–ICG  or 99mTc-SnC was administered to the right foot pad of four animals each and the injection site was massaged a few times. The radioactivity of the samples at the time of administration was 120–220 MBq as measured with a Curiemeter . SPECT/CT imaging studies were conducted 18 h later under general anesthesia. The rats were then sacrificed and the fluorescent nodes were identified using the NIR fluorescence camera. Regional LNs from the injection site and foot were removed to determine their radioactivity counts with an automatic gamma counter .With each rat under general anesthesia, 10 Particle size distribution and zeta potential were measured using a zeta potential/particle size analyzer  and a data processing software . Particle size distribution was estimated based on the dynamic light scattering method of this equipment. The zeta potential was estimated according to the laser Doppler method. Each sample was measured three times at 25 °C.Mean values of the two groups in the in vivo experiments were compared using Welch’s t-test. Multiple comparisons of mean values from the in vitro experiments were performed using Tukey’s honest significant differences test. All statistical analyses were conducted using the R software version 3.6.3 , and p values < 0.05 were considered statistically significant.The authors declare no competing interests.Supplementary Video 1.Supplementary Video 2.Supplementary Video 3.Supplementary Video 4.Supplementary Video 5.Supplementary Video 6.Supplementary Video 7.Supplementary Video 8.Supplementary Video 9.Supplementary Video 10.Supplementary Information 1.