Glioma is the most common malignant primary brain tumour in adults. Despite improvements in neurosurgery and radiotherapy, the prognosis of glioma patients remains poor. One of the main limitations is that there are no proper clinical therapeutic targets for glioma. Therefore, it is crucial to find one or more effective targets. Signal transducer and activator of transcription 3 (STAT3) is a member of the STAT family of genes. Abnormal expression of STAT3 is involved in the process of cell proliferation, migration, invasion, immunosuppression, angiogenesis, dryness maintenance, and resistance to radiotherapy and chemotherapy in glioma. Therefore, STAT3 has been considered an ideal therapeutic target in glioma. Noncoding RNAs (ncRNAs) are a group of genes with limited or no protein-coding capacity that can regulate gene expression at the epigenetic, transcriptional and posttranscriptional level. In this review, we summarized the ncRNAs that are correlated with the ectopic expression of STAT3 in glioma. Glioma is the most common primary intracranial malignant tumour. Despite the great progress that has been made in neurosurgical technology, radiotherapy and chemotherapy, the prognosis of glioma patients is still poor . ParticuNoncoding RNAs (ncRNAs) are a group of genes with limited or no protein-coding capacity . Based oSignal transducer and activator of transcription 3 (STAT3) is a member of the signal transducer and activator of transcription (STAT) protein family, which can be activated by a variety of cytokines or growth factors . STAT prThe expression and activation of STAT3 are precisely regulated, including its proteasomal degradation and the activation-inactivation mechanism that regulates the transient activation of STAT3 . It is cIn glioma, ncRNAs can play an important role in upstream signals and mechanisms to regulate the expression and activation of STAT3. In addition, STAT3, acting as a transcription factor, can directly target and then regulate the expression of ncRNAs. To further understand the occurrence of glioma and provide new ideas for the development of STAT3-targeted tumour therapy, this article mainly summarizes the ncRNAs involved in the STAT3-mediated signalling pathway in glioma.MiRNAs are endogenous, noncoding small RNAs with an approximate length of 20–22 nucleotides that mainly participate in regulating gene transcription, translation, or epigenetic processes. They are widely found in eukaryotes, with a high degree of conservation, cell and tissue specificity and timing . The proMiRNAs play important regulatory roles in eukaryotes by binding to corresponding mRNA transcripts and then leading to their degradation and/or translation inhibition at the mRNA level . StudiesAn increasing number of studies have shown that miRNAs are involved in the occurrence and development of gliomas by acting alone or in the form of gene clusters. These miRNAs may regulate the expression of nearly one-third of the genes in the genome. Different miRNAs are involved in distinct pathophysiological processes, including the proliferation, migration, invasion, apoptosis, radiotherapy, and chemotherapy resistance of glioma cells. The occurrence of glioma is closely related to the increase in the expression of tumour-promoting miRNAs and the decrease in the expression of tumour-suppressing miRNAs . In addiIn GBM, on the one hand, miRNAs can directly target STAT3 to regulate the expression of related proteins in the STAT3 pathway, thereby affecting tumour cell function . In termOn the other hand, miRNAs can directly target STAT3 and function as therapeutic tools for GBM treatment. In terms of chemotherapy resistance, temozolomide (TMZ) is a standard chemotherapy drug for GBM multiforme. However, the occurrence of TMZ resistance is increasing rapidly in clinical treatment. In recent years, miRNAs have been confirmed to act as important regulators in the process of drug resistance . OverexpIt has been reported that p-STAT3 is key for the function of STAT3. P-STAT3 forms homodimers or heterodimers (STAT1/STAT3) through mutual phosphotyrosine-SH2 domain interactions, enters the nucleus, and regulates the transcription of target genes. Regarding the negative regulation of STAT3 activation, the SOCS and PIAS protein families play important roles. Suppressor of cytokine signalling 3 (SOCS3) belongs to the SOCS family and harbours an SH2 domain, which can bind to the active region of JAKs through the SH2 domain, preventing it from phosphorylating STAT3 . RT-qPCRIn addition to SOCS3 and PIAS3, miRNAs can regulate growth factor/cytokine receptors, which are common ways to activate STAT3. Connective tissue growth factor (CTGF) is related to the occurrence and development of tumours; participates in cell proliferation, development, adhesion, migration, and angiogenesis; and predicts prognosis . MiRNA-1MiRNAs also regulate other proteins upstream of STAT3 signalling pathways. Kinesin family member 20A (KIF20A), a member of kinesin superfamily 6, plays a significant role in the occurrence and development of tumours . FunctioExosomes, containing mRNAs, microRNAs, and long noncoding RNAs, deliver their contents to recipient cells and function as regulators of intercellular communication . Based oAs a member of the STAT protein family, STAT3 acts as an important transcription factor, which can initiate the transcription of downstream target genes. For example, chromatin immunoprecipitation (ChIP) analysis has shown that STAT3 binds to the miR-21 promoter region to regulate the expression of miR-21; thus, IFN-β can downregulate miR-21 transcription . There iIn addition to the above three miRNAs, other miRNAs can also affect the expression of STAT3, but the specific mechanism has not yet been elucidated. A study has shown that overexpression of miR-184 promotes cell proliferation and invasion through the JAK2/STAT3 signalling pathway . MoreoveIn summary, some miRNAs serve as target genes and are transcriptionally regulated by STAT3. Meanwhile, to regulate the expression and activation of STAT3 in glioma, the main mechanism underlying miRNA function is through direct binding of the 3′-UTR of the target mRNA. In addition to the traditional mechanism, miRNAs can be present in exosomes to induce M2 macrophage polarization and maintain the GBM phenotype. However, some miRNAs need more in-depth study to uncover their specific mechanism. In addition, studies should focus on investigating other mechanisms of miRNA function. MiRNAs can bind to the 5′-UTR of the target gene and promote its transcription. For example, a study showed that miR-27a-3p could act as an oncogene by directly interacting with the 5'-UTR of cyclin D1 to enhance its expression in bladder cancer .Long noncoding RNAs, which are more than 200 nucleotides in length, generally have no protein coding ability or can only code a few short peptides. LncRNAs are widespread in human organisms and are essential for human gene expression regulation and physiological and pathological processes. Most types of lncRNAs are transcribed by RNA polymerase II and are then capped at the 5' end, polyadenylated at the 3' end and edited through a series of splicing processes that occur in the nucleus. In addition, there are other mechanisms involved in the lncRNA maturation process. According to the position and directional relationship with the gene encoding protein, lncRNAs can be divided into five categories: sense lncRNAs, antisense lncRNAs, intronic lncRNAs, bidirectional lncRNAs, and intergenic lncRNAs . In termNumerous studies have proven that there are a large number of abnormally expressed lncRNAs in gliomas, and they play a vital role in the occurrence and development of gliomas. The abnormal expression and role of these lncRNAs in gliomas makes them promising targets for the early diagnosis and treatment of gliomas. LncRNAs regulate the expression and activation of various signalling pathways and target genes in gliomas . Among tTheir ability to act as competing endogenous RNAs (ceRNAs) or miRNA sponges is the most common function of lncRNAs. The lncRNAs located in the cytoplasm likely regulate the expression of downstream genes through this ceRNA mechanism. The expression of lncRNA CASC9 (cancer susceptibility candidate 9) is significantly increased in glioma tissue and is positively related to advanced pathological grade of glioma. Knocking down lncRNA CASC9 inhibits glioma cell invasion and proliferation. In terms of the mechanism responsible for this effect, the 3′-UTR of CASC9 can interact with miR‐519d, which acts as a tumour suppressor by directly targeting the 3′‐UTR of STAT3 mRNA, to increase the expression of STAT3. Moreover, ChIP and luciferase reporter assays demonstrated that STAT3 can increase the expression of CASC9 by binding to the promoter of CASC9. Taken together, in glioma, lncRNA CASC9 increases STAT3 expression by sponging miR‐519d, while STAT3 interacts with the CASC9 promoter to accelerate CASC9 expression .The activation of STAT3 is precisely regulated. In normal cells, the activation of STAT3 is short-lived, while in glioma cells, STAT3 exhibits constitutively high activation and participates in the occurrence and development of tumours due to the lack of upstream regulatory signals. Similar to miRNAs, lncRNAs can also increase the expression and activity level of STAT3 by regulating its upstream regulatory signals. Coimmunoprecipitation and immunoprecipitation experiments have shown that piwi-like RNA-mediated gene silencing 4 (PIWIL4), belonging to the PIWI subfamily, could induce activation of STAT3. The expression of colorectal neoplasia differentially expressed (CRNDE) is high in glioma. Knockdown of CRNDE induced glioma cell apoptosis, while overexpression of CRNDE promoted U87 and U251 cell proliferation, migration and invasion. RT-PCR assays showed that the expression of miR-384 was decreased in glioma tissues and glioma cell lines and negatively correlated with advanced glioma pathological grade. Luciferase assays showed that CRNDE could bind to miR-384, while miR-384 could target PIWIL4 to function as a tumour suppressor. LncRNA CRNDE increased PIWIL4 expression to induce p-STAT3 through a ceRNA mechanism . MoreoveSimilar to miRNAs, lncRNAs can also serve as target genes of STAT3 in gliomas and undergo transcriptional regulation. Previous studies have shown that STAT3 interacts with the promoter region of CASC9, while CASC9 competitively binds with miR‐519d to increase the expression of STAT3. Similarly, p-STAT3 directly binds to the miR155HG promoter region. MiR155HG inhibits miR-185-5p expression through a ceRNA mechanism and then promotes ANXA2 expression to accelerate the activation of STAT3.In addition, some lncRNAs can affect the expression of STAT3 without a clear and specific mechanism. For example, knockdown of lncRNA PEG10 inhibited U251 cell proliferation, migration, and invasion and inactivated the Raf/MEK/ERK and JAK1/STAT3 signalling pathways by increasing miR-506 expression . In addiIn summary, although lncRNA PEG10 and GHET1 require future studies to reveal the specific mechanism, it is clear that the ceRNA mechanism plays an important role in the regulation of the STAT3 pathway by lncRNAs. Moreover, studies established 2 positive feedback loops: the miR155HG/miR-185-5p/ANXA2 loop and the CASC9/miR‐519d/STAT3 loop. Regarding transcription factor research in glioma formation and progression, future studies can also try to verify the existence of such a positive feedback loop. For instance, Lin and colleagues, found that the positive feedback loop HOXC-AS2/miR-876-5p/ZEB1 could regulate epithelial–mesenchymal transition (EMT) in GBM . In addiFollowing the investigation of miRNAs and lncRNAs, circRNAs have become a new research focus. Unlike linear RNA, circRNAs have a covalent single-stranded closed loop structure, lacking a 5' cap and 3' tail, and the structure of circRNA is highly stable compared with linear RNA. According to the various positions and formation mechanisms of circRNAs, they can be divided into three subclasses: exonic circRNAs, intronic circRNAs and EIciRNAs . It has The expression of circRNAs is tissue specific and developmental stage dependent. Many studies have shown that abnormally expressed circRNAs are involved in the pathophysiological process of glioma. For example, the circRNA HIPK3  regulateThere is only one study on the role of circRNA in regulating the STAT3 pathway. Based on qRT-PCR assays, the expression of the circular RNA HIPK3 was significantly higher in the U251 and U87 cell lines than in the normal cell line (HEB), while miR-124-3p was expressed at a low level in the U251 and U87 cell lines. Functional experiments confirmed that circ-HIPK3 knockdown by targeted siRNAs inhibited cell proliferation, migration, and invasion and induced cell apoptosis. Mechanistic experiments have shown that circ-HIPK3 directly binds to miR-124-3p and that miR-124-3p can directly target the 3'-UTR of STAT3 mRNA. In summary, circ-HIPK3 suppresses the mRNA expression of miR-124-3p, an inhibitor of STAT3, through a ceRNA mechanism. Due to the low expression of miR-124-3p, STAT3 expression is increased .In summary, studies should investigate more circRNAs involved in the glioma STAT3 signalling pathway. Mechanistically, similar to lncRNAs, the ceRNA mechanism is the most common functional mechanism of circRNas, while few studies have found that circRNAs can induce STAT3 nuclear translocation. In colorectal cancer, circSPARC not only regulates the expression of JAK2 through the ceRNA mechanism to affect the activation of STAT3 but also enhances the nuclear translocation of p-STAT3 through the recruitment of FUS RNA binding protein .In summary, an increasing number of studies have shown that noncoding RNAs play an important role in a variety of malignant tumours. The role and mechanism of noncoding RNAs in glioma has been a research hotspot in recent years, and they are expected to become diagnostic and prognostic markers and therapeutic targets for glioma. Moreover, the continuous high expression and activation of STAT3 is an important factor in the occurrence and development of glioma. STAT3 is considered a potential therapeutic target for glioma, but there is no effective targeted STAT3 clinical treatment method for glioma. In this review, we analysed the noncoding RNAs that regulates the expression of STAT3 and that are regulated by STAT3 in gliomas Fig. .Fig. 4NcIn terms of STAT3, as a transcription factor, there is no doubt that STAT3 can play an important role in regulating the expression of ncRNAs. LncRNAs CASC9 and miR155HG promote STAT3 through the ceRNA mechanism, and STAT3, in turn, acts as a transcription factor in the nucleus of glioma cells to promote the expression of CASC9 and miR155HG. These molecules form a positive feedback loop to promote their expression. For miRNAs, activated STAT3 can promote the expression of many oncogenic miRNAs, such as miR-182-5p, miR-21 and miR-30b-3p. Similarly, STAT3 can also inhibit the expression of miR-218, a tumour suppressor. In terms of miRNAs, the most common mechanism of miRNA function is binding to the 3′-UTR of the target mRNA. In the STAT3 signalling pathway of glioma, miRNAs can directly target STAT3 mRNA, genes closely related to STAT3 activation , and upstream genes of STAT3. In addition, miRNAs, such as miR-1246, can also play a role by being packaged inside exosomes. However, some miRNAs can regulate the expression and activation of STAT3, but the specific mechanism is still not clear; thus, it is necessary to explore these specific mechanisms. In terms of lncRNAs and circRNAs, these RNAs can regulate the expression of target genes at the epigenetic, transcriptional and posttranscriptional level. However, studies have mainly focused on lncRNAs and circRNAs that regulate the transcription level of STAT3 through the ceRNA mechanism. In addition to the ceRNA mechanism, other mechanisms of lncRNAs and circRNAs have been reported. For example, in nasopharyngeal carcinoma, lncRNA differentiation antagonizing nonprotein coding RNA (DANCR) can interact with STAT3 to enhance JAK1 binding to STAT3 . LncRNA In recent years, research on tumour immunity has gained more attention. Interferon, as an immune-related cytokine, can play an important role in the antitumour process, and interferons play important roles in STAT3 activation. Of course, some ncRNAs are also involved in this process. In glioma, IFN-β regulates miR-21 through STAT3, which is an interesting finding. IFN-β can induce STAT3 activation, but the role of STAT3 is distinct. The p-STAT3 induced by IFN-β can directly bind to the promoter of miR-21 and then inhibit the transcription of this carcinogenic miRNA, but STAT3 promotes the transcription of miR-21 without IFN-β . DiffereTherefore, further exploration of noncoding RNAs related to STAT3 expression and activation in glioma may provide an avenue for STAT3-targeted glioma treatment.


The RHEA with 15% Al2O3 exhibits a high compressive yield strength (1837 MPa) and a low density (7.75 g/cm3) with an adequate ductility retention. The yield strength and density are 32% higher and 15% lower, respectively, compared to the RHEA without Al2O3 addition. The specific yield strength (237 MPa cm3/g) of the RHEAs is much higher than that of other reported RHEAs, and is mainly ascribed to the introduction of high volume fraction of Al2O3 additives, resulting in solid solution strengthening and precipitation strengthening. Meanwhile, the ductile matrix is responsible for the good compressive plasticity.A series of novel lightweight TaNbVTi-based refractory high entropy alloys (RHEA) were fabricated through ball-milling and spark plasma sintering (SPS). The reinforced phase of TiO precipitates were in-situ formed due to the introduction of Al HEAs 0.4Hf0.6NbTaTiZr RHEA by adding Al into the HfNbTaTiZr RHEA. The density of the alloy decreased from 9.94 to 9.05 g/cm3, and the specific yield strength increased from 93.56 to 203.43 MPa∙cm3/g [0.25 RHEA with Al addition still have outstanding compressive plasticity greater than 50% [3/g, which has the potential for further improvement. In this work, a series of lightweight TaNbVTi-based RHEAs were fabricated by mechanical alloying (MA) followed by spark plasma sintering (SPS). The microstructural evolution and its effects on mechanical properties were analyzed.Adding lightweight elements into RHEAs can effectively reduce the density and improve the specific strength ,13,14,15Pa∙cm3/g . Howeverthan 50% . Neverth2O3 powders as the raw materials, and then blended with a planetary ball-millers for 6 h at 120 rpm under argon atmosphere at a temperature of 25 °C. The average particle size of Ta, Nb, V, Ti and Al2O3 powders is 24.5 μm, 26.1 μm, 22.6 μm, 29.1 μm and 0.3 μm, respectively. The content of interstitial impurities of these powders are listed in 2O3 powders  were added to in-situ generate particles reinforced RHEAs, namely, TaNbVTi-0, TaNbVTi-1 and TaNbVTi-2, respectively. 2O3 powders. Finally, the as-milled powders were consolidated via SPS machine  at 1700 °C with a heating rate of 100 °C/min. The sintering process was held for 10 min with a constant pressure of 30 MPa. The dimension of the sintered bulks is 10 mm in height and 40 mm in diameter.The TaNbVTi-based RHEAs were prepared by powder metallurgy (P/M) method. High purity (>99.5 wt.%) Ta, Nb, V, Ti elemental powders in equiatomic composition were mixed with Al−3 s−1. All the specimens were compressed until fracture. Tensile tests were carried out on an MTS Landmark test machine at a strain rate of 1 × 10−3 s−1 by using “I” sharp samples with a gauge length of 10 mm and a rectangular cross-section of 2 mm × 2 mm.The oxygen content was determined by the fusion method on a Leco O/N analyzer. The composition was analyzed by chemical methods. The particle size distribution was investigated by a laser particle size analyzer (MICRO-PLUS). The phase analysis was performed by an X-ray diffraction analyses  with Cu Ka radiation. The XRD analyses were conducted at a 2θ from 10–80° with a scan rate of 5°/min. The microstructure was characterized by a scanning electron microscope  equipped with a backscatter electron (BSE) detector and an energy dispersive X-ray spectroscopy (EDX) device. The elemental distribution was analyzed by an electron probe microanalysis . The density of the specimens was measured by Archimedes method. Cubic specimens with a dimension of 8 mm × 8 mm × 8 mm for density test were obtained from the center of the sintered bulks. Three samples were tested for the same composition, and the average value was adopted. Cylindrical specimens (Φ 6 mm × 9 mm) for compressive test were cut from the sintered bulks by using an electrical discharge machining. Before testing, the surface of test specimens was polished by a 2000-grit SiC paper to eliminate scratches. Room temperature compressive test was carried out on a universal testing machine  at a strain rate of 1 × 102O3, some extra diffraction peaks are detected in the TaNbVTi-1 RHEA, and the intensity increased as the increase of Al2O3 addition, indicating more second phase forms in the TaNbVTi-2 RHEA. According to the JCPDS cards (No. 72-0020), the second phase can be identified as TiO phase, implying the formation of TiO phase in the sintered bulks, which is consistent with the results reported by Xin et al. [n et al. .3 of TaNbVTi-0 RHEA to 7.75 g/cm3 of TaNbVTi-2 RHEA with the incremental Al2O3 addition. The embedded images show the microstructural characteristic of the TaNbVTi-0, TaNbVTi-1 and TaNbVTi-2 RHEAs. After sintering, the bulks can be consolidated to nearly full dense, and there are few residual pores or shrinkage defects in the microstructure. Slight component segregation can be observed in the TaNbVTi-0 RHEA, and the microstructure is composed of two distinguishable regions, bright regions and dark regions, indicating an incomplete alloying process. 2O3, a large number of particles can be clearly found in the TaNbVTi-1 RHEA . The compressive curves of the TaNbVTi-0, TaNbVTi-1 and TaNbVTi-2 RHEAs are shown in (1)2O3 ceramic particles. The introduction of Al2O3 can promotes the formation of TiO precipitates in the BCC matrix. Meanwhile, Al atoms decomposed from Al2O3 could diffuse and dissolve into the matrix.Novel lightweight TaNbVTi-based RHEAs has been successfully developed through the addition of Al(2)3 with a high compressive yield strength of 1837 MPa, which are 32% higher and 15% lower, respectively, compared to the RHEA without Al2O3 addition. The specific yield strength of TaNbVTi-2 RHEA is better than most reported RHEAs. The newly developed RHEAs are promising for applying in aerospace field  due to the high specific strength.The TaNbVTi-2 RHEA has a relative low density of 7.75 g/cm(3)2O3 additives, resulting in solid solution strengthening and precipitation strengthening. Moreover, the ductile matrix is responsible for the good compressive plasticity.The improved strength is mainly ascribed to the introduction high volume fraction of Al


Defining transcriptional profiles of substantia nigra pars compacta (SNc) and ventral tegmental area (VTA) dopamine neurons is critical to understanding their differential vulnerability in Parkinson’s Disease (PD). Here, we determine transcriptomes of human SNc and VTA dopamine neurons using LCM-seq on a large sample cohort. We apply a bootstrapping strategy as sample input to DESeq2 and identify 33 stably differentially expressed genes (DEGs) between these two subpopulations. We also compute a minimal sample size for identification of stable DEGs, which highlights why previous reported profiles from small sample sizes display extensive variability. Network analysis reveal gene interactions unique to each subpopulation and highlight differences in regulation of mitochondrial stability, apoptosis, neuronal survival, cytoskeleton regulation, extracellular matrix modulation as well as synapse integrity, which could explain the relative resilience of VTA dopamine neurons. Analysis of PD tissues showed that while identified stable DEGs can distinguish the subpopulations also in disease, the SNc markers SLIT1 and ATP2A3 were down-regulated and thus appears to be biomarkers of disease. In summary, our study identifies human SNc and VTA marker profiles, which will be instrumental for studies aiming to modulate dopamine neuron resilience and to validate cell identity of stem cell-derived dopamine neurons. Aldh1a7, Sox6, Cbln1, Vav3, Atp2a3, and VTA e.g., Calb1, Otx2, Crym, Cadm1, and Marcks  and the ventral tegmental area (VTA) . SNc dopd Marcks . Transcrd Marcks  except fd Marcks . These ain vitro derivation of SNc dopamine neurons, which represent the ideal cell type for transplantation in PD . The slides with sections were kept at −20°C during the sectioning and subsequently stored at −80°C until further processed. The laser capture procedure followed by sequencing library preparation (LCM-seq) was carried out as described (Sample preparation prior LCM-seq was carried out as follows. Frozen midbrain tissues (controls and PD), of rostral to intermediate midbrain level , obtaineescribed , 2018. Described . To limiescribed , we trieescribed .Samples were sequenced using an Illumina HiSeq2000, HiSeq2500, or NovaSeq platforms (reads of 43 or 50 bp in length). The uniquely mapped reads were obtained by mapping to the human reference genome hg38/GRCh38 using STAR with default settings. The reads per kilobase of transcript per million mapped reads (RPKM) were estimated using “rpkmforgenes”  to 10.88P-value of 0.05. Identified DEGs  are shown in Differentially expressed genes were identified using the R package “DESeq2” (version: 1.16.1)  where thTo counteract the variability among human subjects and identify the most reliable DEGs between SNc and VTA neurons across datasets we developed a bootstrapping approach coupled with DESeq2 . The staIn detail:(1)Define ¨N¨ and ¨M¨ as the number of samples in Groups 1 and 2, respectively. Choose ¨I¨ as a reference representing a given number of samples from ¨N¨ and ¨M¨.(2)Define ¨i ¨ as the number of randomly selected samples from Groups 1 and 2, where i ∈{3, 4, 5, …, ¨I-1¨}. In the human dataset, as we have 12 paired samples, the I ∈{3, 4, 5, 6, 7, 8, 9, 10, 11}.(3)Pool ¨i¨ samples (temporary considered a ¨new data set¨) and calculate DEGs with DESeq2.(4)Repeat steps (2) and (3) for ¨j¨ times .(5)For every round of random selection and DESeq2, save the full list of DEGs, compute and rank their frequency.(6)Set a threshold (30% ratio in this study) to consider DEGs with higher frequency as stable genes.Reliable genes appear when frequencies are above: Total times of bootstrapping × ratio (300 in this study). A stringent, but fair ratio can be defined by comparing the percentage of identified stable genes overlapping with the top (most significant) 10%, 20%, 30%, …, DEGs identified by DESeq2 alone.To reliable identify DEGs between human SNc and VTA samples, while minimizing subject variability, we selected 12 control individuals  where both neuronal populations were available and sequenced. Hence, the number of randomly selected samples  was from three to 11 and the algorithm repeated 1000 times .N = 73 cells) and VTA . To compensate the unbalance in cell number and adjust dataset representation compared to the human analysis (66%), we first randomly collected a subset of 73 VTA cells, pairing both SNc and VTA. Similarly, the number of randomly selected samples was 20, 25, 30, …, 70 and the algorithm repeated again 1,000 times.For this adult mouse dataset  we definBased on the DE genes between SNc and VTA , two STRData visualization was achieved using Principal Component Analysis (PCA) and Hierarchical Clustering (H-cluster). PCA was calculated with the function “prcomp” in R with default parameters. Then samples are projected onto the first two dimensions, PC1 and PC2. For H-cluster we used the R function “pheatmap” (version 1.0.12) with the clustering method of “ward.D2.”SEZ6) and one VTA-preferential gene (CDH13) based on the sequencing data. In brief, midbrain sections of human fresh frozen tissue (dapB-C1) and a positive control probe against tyrosine hydroxylase  (SEZ6 (Cat. 411351-C1) or CDH13 probes (Cat. 470011-C1). Slides were counterstained with fresh 50% Gill Solution  for 2 min, washed in water and dried for 15 min at 60°C before mounting with Pertex . For every sample (n = 5), we imaged 5–6 random fields within the SNc and VTA regions. On average 194.25 ± 43.02 cells were imaged per region and staining. Pictures were made at 40X magnification using the bright-field of a Leica microscope (DM6000/CTR6500 and DFC310 FX camera). After randomization and coding of all the images, the number of dots within melanised cells (dopamine neurons) were counted using ImageJ (version 1.48) and later the average number of dots per cells determined for each region. Cells were classified as having either 0, 1, 2, 3, 4, 5 dots/cell. If cells had >5 dots they were classified within the 5 dots/cell category.RNAscope  was usedn tissue  were quin tissue , we firs, TH-C1) . Once weInvestigators performing the quantification were blinded to the sample, target region (SNc and VTA) and probe staining.t-test  was used to compare the mean average dots per cell (for SEZ6 or CDH13 staining) between the SNc and VTA. Where applicable, individual statistical tests are detailed in the figure legends where significance is marked by P < 0.05. The number of subjects/cells used for each experiment is listed in the figure or figure legends. Results are expressed as mean ± SD or SEM as specified in the figure legend.For this study, statistical analyses were performed using ¨R¨. For the RNAscope analysis a paired 1 (GEO) under accession number GSE114918. Human samples re-analyzed from the Nichterwitz study , sample handling and preservation. Therefore, prior to conducting differential gene expression analysis we performed extensive quality control analysis to rule out undesired influences from sample processing (P = 0.515) , and the general neuronal marker NEFH, while they lack glial markers MFGE8, CX3CR1, or GPR17. This clearly demonstrates the selective enrichment of dopamine neurons using the LCM-seq methodology (KCNJ6 (GIRK2) and CALB1, two genes often used to distinguish between SNc or VTA dopamine neurons, were also expressed  . However= 0.515)  and thushodology . KCNJ6  (N = 18), even though the same experimental method was used. In fact, only seven SNc and 21 VTA DEGs overlapped across the current and the previous cohorts (N = 3)  . However cohorts . Notably (N = 3) , failed  (N = 3) . This suP < 0.05. This random sampling of three subjects, followed by DESeq2 analysis, was performed a total of 1,000 times, and the DE frequency over these 1,000 comparisons was recorded for this iteration (i = 3 subjects). Subsequently, this process was repeated using subsets of four subjects, then five, up to a maximum of 11 of the 12 subjects. For each subset size (i3–i11)the DEG frequency was calculated by the 1,000x comparisons of that iteration  and 16 of the VTA stable genes  were among the aforementioned seven and 21 overlapping DEGs, that we identified across the two studies.To evaluate how sample size may affect DEG detection, we used a bootstrapping algorithm in combination with DESeq2. To reduce the biological variability, we considered only those subjects for which both SNc and VTA samples were available . To begin with this approach, a subset of three subjects were randomly chosen from the pool of total 12 subjects. Differential expression analysis was then performed between the SNc and VTA samples of these subjects (DESeq2), and DEGs were selected with adj teration . By consation , separated genes into SNc or VTA enriched lists, and ranked the lists from most to least frequently DEs   as the output of the overall bootstrapping approach showed genes with the 30% threshold  represent SNc and VTA stable genes  . To idenle genes . We alsole genes . All eigP < 0.05  or a mor01, SEZ6  and the 6 , and a strict selection-criteria  we identify 33 of the most stable DEGs. Among these, 25 of the genes define VTA identity, while eight define SNc identity, which together accurately classify LCM-seq samples from our previous , and current  subject cohorts. We confirm the utility of our bootstrapping approach on a larger published mouse single cell data set and show that identified DEGs there could correctly classify SNc and VTA dopamine neurons.Using an iterative bootstrapping without replacement coupled with DESeq2  is a serine protease inhibitor which can promote neurite extension by inhibiting thrombin, and which appears down-regulated in Alzheimer’s disease . Interestingly, mice lacking Rgs6, a related family member of the human SNc stable gene RGS16, develop specific degeneration and cell loss of SNc dopamine neurons at the age of 12 months  can be found in the article/EH, QD, and JA: conceptualization and funding acquisition. JA, MC, SC, MW, NK, QD, and EH: methodology, investigation, writing—review, and editing. SC, JA, MW, and NK: software, formal analysis, and visualization. JA, SC, QD, and EH: writing-original draft. EH and QD: supervision and project administration. 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.


The built environment of cities is complex and influences social and environmental determinants of health. In this study we, 1) identified city profiles based on the built landscape and street design characteristics of cities in Latin America and 2) evaluated the associations of city profiles with social determinants of health and air pollution. Landscape and street design profiles of 370 cities were identified using finite mixture modeling. For landscape, we measured fragmentation, isolation, and shape. For street design, we measured street connectivity, street length, and directness. We fitted a two-level linear mixed model to assess the association of social and environmental determinants of health with the profiles. We identified four profiles for landscape and four for the street design domain. The most common landscape profile was the “proximate stones” characterized by moderate fragmentation, isolation and patch size, and irregular shape. The most common street design profile was the “semi-hyperbolic grid” characterized by moderate connectivity, street length, and directness. The “semi-hyperbolic grid”, “spiderweb” and “hyperbolic grid” profiles were positively associated with higher access to piped water and less overcrowding. The “semi-hyperbolic grid” and “spiderweb” profiles were associated with higher air pollution. The “proximate stones” and “proximate inkblots” profiles were associated with higher congestion. In conclusion, there is substantial heterogeneity in the urban landscape and street design profiles of Latin American cities. While we did not find a specific built environment profile that was consistently associated with lower air pollution and better social conditions, the different configurations of the built environments of cities should be considered when planning healthy and sustainable cities in Latin America. Cities are highly complex systems in which dynamic networks between people and the built environment give rise to patterns in behaviors and health , 2. SpecCertain features of Latin American cities make an examination of the built environment of these cities of special interest. Latin America is dense and highly urbanized , 12 wherIn this context, understanding factors that explain the configuration of the built environment in Latin American cities may provide useful insights for urban policies. Factors that may impact the configuration of cities include historical legacies related to when the area was settled, how it was settled, and urban form indicators . SpecifiIn this context, it is crucial to better understand the heterogeneity of the built environment of Latin American cities . The fewSeveral studies have been conducted to assess the associations of the built environment with social determinants of health and air pollution . Social One empirical approach to managing the complexity of describing widely varying cities, and the interconnectedness of built environment attributes that characterize them, is to develop multi-dimensional typologies or profiles. Profiles are ways of describing units according to an ensemble of characteristics and allow for further studies into the mechanisms that connect the built environment to health. Profiles serve both as descriptions of current conditions and as triggers for policy action, either to address concerns or to transform the built environment in pursuit of specific planning or policy goals.There are several existing examples of profiles based on indicators involving the built environment. These profiles include 1) transit-oriented development profiles , 2) bus This study is innovative and makes unique contributions to the current literature evaluating relationships of built environment with social and environmental determinants of health. First, this study is part of the SALURBAL project  with theThe aims of our study were to: 1) empirically identify profiles of the urban landscape and street morphology of 370 cities of 100,000 residents of more across the region and 2) to evaluate the associations of these urban landscapes and street design profiles with social determinants of health  and air pollution. Characterizing the built environment profiles of cities is critical to understanding the impact of built environment features on health and environmental outcomes and to the development of urban policies that promote health and environmental sustainability. These types of analyses may inform global and local policies linked to the New Urban Agenda and the Sustainable Development Goals (SDGs), particularly SDG 11– Make cities and human settlements inclusive, safe, resilient and sustainable and its interaction with SDG 3-of health and wellbeing , ‘moderate’ when the 50th percentile of the indicator of interest in profile i is between the 25th and the 75th percentile for the overall profiles indicators [P25≤ Pi50 ≤P75], and ‘high’ when the 50th percentile of the indicator in profile i is higher than the 75th percentile for the overall profiles indicators [Pi50>P75]. The FMM models were fitted in R ) showed a large variability. The country with the highest street length average was Argentina (164.9 m), and the country with the shortest streets was Guatemala (113.1 m). The city with the highest average street length (510.1 m) was Barreiras (Brazil) and the city with the shortest average street length (82.8 m) was Santiago (Chile).Within the subdomain of directness, the circuity average (1.0646 [1.0470 1.0891]) showed a large variability. The country with the highest average circuity, indicating less direct streets was Costa Rica (1.12), and the country with the most direct streets was Perú (1.05). The city with the least direct streets (1.37) was Huaraz (Peru) and the city with the most direct streets (1.01) was Santa Rosa (Argentina). Histograms of each Street design metric can be found in We found that the largest positive Pearson correlation coefficient among metrics of urban landscape and street design domains was between street density and patch density 0.75, , indicat25; P75], ), moderate area-weighted mean patch size , irregular shape  and moderate isolation . We labeled this profile proximate stones. Cities with a high conditional probability of belonging to this profile included Pocos de Caldas, Araras, Birigui and Parnaiba (Brazil) and San Nicolas de los Arroyos (Argentina). The second profile was characterized by cities with lower patch density , lower area-weighted mean patch size (<1082.4), a compact shape (<4.33) and higher isolation . This profile was labeled scattered pixels and included 91 cities (24.6%). Cities with a high conditional probability of belonging to this profile included Barreiras (Brazil), Quibdó (Colombia), and Chilpancingo, Fresnillo and Tecoman (Mexico). The third profile was characterized by cities with a moderate patch density , larger area-weighted mean patch size (>3928.5), complex shape (>6.27) and moderate isolation . This profile was labeled as proximate inkblots and included 90 cities (24.3%). Cities with a high conditional probability of belonging to this profile included Cartagena (Colombia), and Chihuahua, Hermosillo, Mexicali and Torreon (Mexico). The fourth profile was characterized by cities with a higher patch density (>0.56), higher area-weighted mean patch size (>3928.5), complex shape (>6.27) and lower isolation (<72.2). This profile was labeled as contiguous large inkblots and included 21 cities (5.7%). Cities with a high conditional probability of belonging to this profile included Buenos Aires (Argentina), and Belo Horizonte, Curitiba, Porto Alegre y Recife (Brazil). For specific information of profiles by country and cities see visualization tool  and street density , streets with moderate length  and moderately direct streets . This profile was labeled semi-hyperbolic grid and included 130 cities (35.1%). Cities with a high conditional probability of belonging to this profile included Sao Carlos, Araras, Londrina, Marilia and Arapongas (Brazil). The second profile was characterized by cities with low street connectivity due to low intersection density  and street density (<529.3), streets with moderate length  and streets with moderate directness . This profile was labeled labyrinthine and included 110 cities (29.7%). Cities with a high conditional probability of belonging to this profile included Rio Cuarto, Rio Gallegos and Villa Mercedes (Argentina), and Uruguaiana and Barreiras (Brazil). The third profile was characterized by cities with higher street connectivity due to high intersection density  and street density (>1956.9), shorter streets (<118.6) and streets moderately directed . This profile was labeled as the spiderweb and included 80 cities (21.6%). Cities with a high conditional probability of belonging to this profile included Buenos Aires (Argentina), Quetzaltenango , and Recife, Rio de Janeiro and Sao Paulo (Brazil). The fourth profile was characterized by cities with moderate street connectivity due to moderate intersection  and street density , long streets (>163.7) and streets with low directness (>1.0891). This profile was labeled hyperbolic grid and included 50 cities (13.5%). Cities with a high conditional probability of belonging to this profile included Petropolis, Teresopolis and Nova Friburgo (Brazil), Huaraz (Peru) and Manizales (Colombia). For specific information of profiles by country and cities see visualization tool (The FMM yielded four profiles according to the BIC criteria . The ention tool .Labyrinthine and Scattered pixels (53.44% of cities), characterizing cities with isolated patches and a less well-connected street design network. A substantive overlap between Semi-hyperbolic grid and the Proximate stones (39.26% of cities) profiles was also identified. The cities belonging to these two profiles were characterized by moderate connectivity and moderate fragmentation with irregular shape  were positively associated with more piped water access and negatively associated with overcrowding. Cities with moderate patch density, moderate to larger patch size, irregular to complex shape and moderate isolation (proximate stones and proximate inkblots) were positively associated with higher congestion. The semi-hyperbolic grid and spiderweb profiles were positively associated with higher annual mean concentration of PM2.5  presents an interactive map illustrating the geographical distribution of the 370 cities. It includes a dashboard where users can select the profiles for urban landscape or street design, and then explore the distribution of cities within them.The visualization tool . Similarly, four profiles emerged for the street design domain. The most common street design profile was the semi-hyperbolic grid, and home to 19.4% of the population living in 130 cities, characterizing cities with moderate connectivity, moderate street length, and moderate directness. The least common profile was the hyperbolic grid with 5.1% of the population living in 50 cities. This profile is characterized by a less directed street network.Among the four profiles emerging in the urban landscape domain, the most common one was the semi-hyperbolic grid, spiderweb, and hyperbolic grid profiles were associated with higher access to piped water and less overcrowding, but not population education. The cities in these profiles exhibit higher socioeconomic conditions compared to the profile with less connectivity and moderate directness (labyrinthine). Likewise, the semi-hyperbolic grid and spiderweb profiles were associated with higher air pollution. Urban landscape profiles were associated with congestion. Specifically, the proximate stones and proximate inkblots profiles were associated with higher congestion.The built environment profiles were associated with social determinants of health and air pollution. Street design profiles were associated with socioeconomic conditions. The contiguous large inkblots characterized by the largest and highest number of patches is similar to what has been previously described in other multi-country studies that included only cities like Bogotá, Buenos Aires, Brasilia, Córdoba, Lima, and Santiago . More information about the project can be found at http://journals.plos.org/plosone/s/submit-now.  Please also include the statement “There was no additional external funding received for this study.” in your updated Funding Statement.Please provide an amended statement that declares *all* the funding or sources of support  received during this study, as detailed online in our guide for authors at Please include your amended Funding Statement within your cover letter. We will change the online submission form on your behalf.[Note: HTML markup is below. Please do not edit.]Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The authors identify city profiles based on the built landscape and street design characteristics of cities in Latin America and evaluate the associations of city profiles with social determinants of health and air pollution. The manuscript is well-structured and contributes to studies related to morphological classifications of cities/urban areas and their associations with social aspects of health determinants. In addition, the manuscript also contributes to the literature on morphological classification of cities in Latin American context.Some specific comments and suggestions are listed below:1. A literature review of related past studies is missing and it is important to justify the methods used, to identify the gaps and to highlight the originality and contribution of this study to literature. More particularly, past studies using different pattern analysis to identify various profiles of cities should be highlighted. One such study is: Southworth, M., & Owens, P. M. (1993). The evolving metropolis: Studies of community, neighborhood, and street form at the urban edge. Journal of the American Planning Association, 59(3), 271-287. A more recent study is Peponis, J., Allen, D., Haynie, D., Scoppa, M., & Zhang, Z. (2007). Measuring the configuration of street networks: the spatial profiles of 118 urban areas in the 12 most populated metropolitan regions in the US. These are based on the classification of street connectivity of urban areas. What are the key metrics used previously? More regarding different measures should be cited. In addition, studies investigating the relationship between these patterns and health outcomes need to be cited too. Many previous studies are cited in the Discussion, but these should have been introduced in the Lit Rev.2. In Table 1, why are no definitions provided for certain metrics used: i.e. directness? These already have been defined in the literature and indeed the authors define them later in the text.3. Line 198: why is the threshold set to 25?4. Table 2 shows the correlations of individual measures with social aspects of health determinants. I believe these need some discussion/explanation. For example, to me it is interesting and unexpected why intersection density is negatively correlated with air pollution whereas street density is positively correlated with the same measure.5. Line 331: should be edited as “95% confidence interval” to be consistent with the note on Table 6.6. The manuscript can be stronger if, in the Discussion, the authors discuss briefly the policy/planning implications of their findings, particularly the associations of urban landscape and street design with health aspects. Can any suggestions for urban planners be drawn based on the findings?7. There are a couple sentences that need to be revised grammatically.line 467: “of the fragmentation an density of…” and?line 509: “Latin American…” should be Latin AmericaReviewer #2: This is an interesting study describing landscape and street patterns across Latin American cities. I believe it requires some revision prior to be acceptable for publication. In particular, it needs a clearer motivation and takeaway message, as well as more clarity in some of the methods.Throughout: change "OpenStreetMaps" to "OpenStreetMap"Introduction: I'd like to see a clearer motivation here for why these research questions are important. What is the value of generating this new knowledge? Who will use it and how will they do things differently after reading your paper than they would have beforehand?Where do the study site boundary geometries come from? Is that the AUE specifically? Worth making that explicit.Pages 5-9: For both the urban landscape domain and street design domain, you mention that you selected a few metrics to measure different aspects. But how did you select these specific metrics? Why these? I'd like to see more theoretical development here or in the background to make a case for why these specific dimensions capture the most important physical aspects of these cities for your study. More motivation for their selection.Page 10: you mention that the socioeconomic variables come from the nations' census bureaus. Do these variables have the same meaning, coverage, interpretation, etc across the various nations? They are directly comparable with one another?Page 10: regarding the particulate matter concentration raster, it strikes me that it may be better to population-weight the mean so that you measure exposure. For example, if half the pollution is concentrated in an industrial area where very little of the population is exposed to it, an unweighted mean may not give an accurate snapshot.Page 11: you briefly introduce what FMM is capable of, but it may be useful to briefly explain what it does and how it does it for the general readership.Pages 11-12: please provide mathematical equations for your models to make their specification clearerResults section: rather than just presenting who was lowest/highest on different indicators, it would help to briefly explain/interpret why they were lowest or highest. What are the unique characteristics or histories of these places to explain their extreme values? Also, consider presenting a table to summarize some of these values, or perhaps a figure showing box plots to demonstrate their distributions.The visualization tool on your web site is a nice feature.Discussion section: I feel like the paper concludes without a takeaway message right now. This is a correlational/descriptive study, but I'd like to see more motivation for why these descriptions matter. How can policymakers use these findings? What new theoretical knowledge do they generate? Why is that new knowledge urgently needed to understand and improve urban living?The last paragraph uses causal language  that does not appear to be warranted as the study did not employ a causal research design such as a quasi-experimental framework.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool, AttachmentPONE-D-20-18493__review.docSubmitted filename: Click here for additional data file. 27 Jul 2021Dear reviewers,We greatly appreciate your feedback and constructive comments. We addressed each comment by thoroughly revising our manuscript. The actions taken to respond to each comment are detailed in the following text. All the changes are also highlighted in blue within the word text of the manuscript and we provided page and line numbers where you can find those changes. We included the word file with the track changes and without track changesReviewer 1Summary of work “The authors identify city profiles based on the built landscape and street design characteristics of cities in Latin America and evaluate the associations of city profiles with social determinants of health and air pollution. The manuscript is well-structured and contributes to studies related to morphological classifications of cities/urban areas and their associations with social aspects of health determinants. In addition, the manuscript also contributes to the literature on morphological classification of cities in Latin American context”.Answer:We greatly appreciate the encouraging comments of the reviewer.Remarks “A literature review of related past studies is missing, and it is important to justify the methods used, to identify the gaps and to highlight the originality and contribution of this study to literature. More particularly, past studies using different pattern analysis to identify various profiles of cities should be highlighted. One such study is: Southworth, M., & Owens, P. M. (1993). The evolving metropolis: Studies of community, neighborhood, and street form at the urban edge. Journal of the American Planning Association, 59(3), 271-287. A more recent study is Peponis, J., Allen, D., Haynie, D., Scoppa, M., & Zhang, Z. (2007). Measuring the configuration of street networks: the spatial profiles of 118 urban areas in the 12 most populated metropolitan regions in the US. These are based on the classification of street connectivity of urban areas. What are the key metrics used previously? More regarding different measures should be cited. In addition, studies investigating the relationship between these patterns and health outcomes need to be cited too. Many previous studies are cited in the Discussion, but these should have been introduced in the Lit Rev”.Answer:We appreciated the recommendation of the reviewer. We included the following changes in the manuscript: 1) we expanded the introduction with a literature review including the literature on the built environment (urban landscape and street design metrics) in previous studies in Latin America; 2) These metrics were compared with studies in other countries; 3) we included the literature review of the association of built environment and social determinants of health with emphasis on those conducted in Latin America, 3) we included a literature review of urban profiles, 4) we included a paragraph on the innovation of the study and the potential use of this results for inform global and local policies, 5) lastly, we added your suggested references regarding profiles and typologies. Changes can be found on: 1) Introduction section: pages: 3-7; lines 77-1772) Discussion section: pages: 33-39; lines 605-774 “In Table 1, why are no definitions provided for certain metrics used: i.e. directness? These already have been defined in the literature and indeed the authors define them later in the text”.Answer:We completed table 1 with the missing definitions for the number of urban patches, patch density, and circuity average. Changes can be found on: Page 9: Line: 223 “Line 198: why is the threshold set to 25?”Answer:The threshold was set according to the metrics in the SALURBAL project. This variable has been used for characterizing variability and predictors of infant mortality in urban settings. Specifically, 25 years is the minimum age standard for measuring adult educational attainment defined by UNESCO and OECD. This age is used because it assumes that most adults will have already completed basic, high school, and college education in most countries. To clarify this cut point we included the following reference:Ortigoza AF, Tapia Granados JA, Miranda JJ, et al. Characterising variability and predictors of infant mortality in urban settings: findings from 286 Latin American cities. J Epidemiol Community Health 2021;75:264-270. Changes can be found on: Page: 14 ; Lines: 310-315 “Table 2 shows the correlations of individual measures with social aspects of health determinants. I believe these need some discussion/explanation. For example, to me it is interesting and unexpected why intersection density is negatively correlated with air pollution whereas street density is positively correlated with the same measure”.Answer:We appreciate the comment we want to clarify that the table had an error. When we made the table, one column was shifted and that was the reason why some of the correlations seem odd. We fixed this error, and table 2 now is updated. In the discussion section, we also expanded the hypothesis regarding the associations of air pollution with urban landscape profiles and metrics and street design profiles and metrics. Changes can be found on: Page: 22; Lines: 471-482Page: 23 for table 2 “Line 331: should be edited as “95% confidence interval” to be consistent with the note on Table 6”.Answer:According to the recommendation of the reviewer, we changed the confidence interval to 95% confidence interval.The corrected table footnote is: The 95% confidence intervals are shown in square brackets. Change can be found on: Page: 30; line: 580 “The manuscript can be stronger if, in the Discussion, the authors discuss briefly the policy/planning implications of their findings, particularly the associations of urban landscape and street design with health aspects. Can any suggestions for urban planners be drawn based on the findings?”Answer: According to the reviewers, suggestions we included the policy recommendations in the discussion section. “There are a couple sentences that need to be revised grammatically.line 467: “of the fragmentation an density of…” and?line 509: “Latin American…” should be Latin America”Answer:We changed the sentences to be grammatically correct.  Reviewer 2Summary of work “This is an interesting study describing landscape and street patterns across Latin American cities. I believe it requires some revision prior to be acceptable for publication. In particular, it needs a clearer motivation and takeaway message, as well as more clarity in some of the methods”.Answer:We included the following changes in the manuscript: 1) we expanded the introduction with a literature review including the literature on the built environment (urban landscape and street design metrics) in previous studies in Latin America; 2) These metrics were compared with studies in other countries; 3) in the methodology we expanded the justification of the use of our metrics; 4) we included the literature review of the association of built environment and social determinants of health with emphasis on those conducted in Latin America, 5) we included a literature review of urban profiles, and 6) we included a paragraph on the innovation of the study and the potential use of this results for inform global and local policies. Changes can be found on the following sections: 1) Introduction section: pages: 3-7; lines 77-1772) Discussion section: pages: 33-39; lines 605-774Remarks “Throughout: change "OpenStreetMaps" to "OpenStreetMap"”.Answer:According to the recommendation of the reviewer, we corrected the word OpenStreetMap.Changes can be found on lines 160, 285 and 286  “Introduction: I'd like to see a clearer motivation here for why these research questions are important. What is the value of generating this new knowledge? Who will use it and how will they do things differently after reading your paper than they would have beforehand?”Answer:We included the following changes in the manuscript: 1) we expanded the introduction with a literature review including the literature on the built environment (urban landscape and street design metrics) in previous studies in Latin America; 2) These metrics were compared with studies in other countries; 3) we included the literature review of the association of built environment and social determinants of health with emphasis on those conducted in Latin America, 4) we included a literature review of urban profiles, and 5) we included a paragraph on the innovation of the study and the potential use of this results for inform global and local policies. Changes can be found on the following sections: 1) Introduction section: pages: 3-7; lines 77-1772) Discussion section: pages: 33-39; lines 605-774 “Where do the study site boundary geometries come from? Is that the AUE specifically? Worth making that explicit”.Answer:Cities were included in SALURBAL based on city size >=100,000 as determined by the AUE and official census data and boundaries were based on official administrative boundaries of subcity units in each country that encompassed the visual urban extent of each city. We obtained administrative boundaries from official country sources and overlayed those into satellite imagery of each city to determine the urban extent.Changes can be found on the methods section: page 8: Lines: 193-197  “Pages 5-9: For both the urban landscape domain and street design domain, you mention that you selected a few metrics to measure different aspects. But how did you select these specific metrics? Why these? I'd like to see more theoretical development here or in the background to make a case for why these specific dimensions capture the most important physical aspects of these cities for your study. More motivation for their selection”.Answer:The three landscape subdomains, together with area and population, encompass attributes that can be used to characterize urban development. Previous studies developed various strategies to measure urban form in particular, which may differ among application objectives. Furthermore, a number of spatial metrics have been proposed to assess landscape configuration based on geometry and spatial relationships among discrete units, or patches. To more effectively identify typologies of urban morphology landscape structure for SALURBAL cities in this study, we chose a parsimonious set of landscape metrics based the principles of strength, universality and consistency - i.e., metrics that represent different components of landscape structure such as size, shape, texture, and contiguity, and that, by design, would not be expected to strongly correlate with each other.We expanded the definition and rationale for using these metrics. Changes can be found on the methods section: page 8: Lines: 219-228Within the street design domain, we selected five metrics that represent the subdomains of street connectivity, street length, and directness. We selected these metrics that capture the cities’ general street network structure and have been used in multiple studies allowing comparabilityWe expanded the definition and rationale for using these metrics. Changes can be found on the methods section: page 13: Lines: 291-294 “Page 10: you mention that the socioeconomic variables come from the nations' census bureaus. Do these variables have the same meaning, coverage, interpretation, etc across the various nations? They are directly comparable with one another?”Answer:These metrics were standardized among countries according to common thresholds. As such they are comparable. We clarified this in the manuscript and provided a reference.Ortigoza AF, Tapia Granados JA, Miranda JJ, et al. Characterising variability and predictors of infant mortality in urban settings: findings from 286 Latin American cities. J Epidemiol Community Health 2021;75:264-270.Changes can be found on the methods section: page 14: Lines: 321-324 “Page 10: regarding the particulate matter concentration raster, it strikes me that it may be better to population-weight the mean so that you measure exposure. For example, if half the pollution is concentrated in an industrial area where very little of the population is exposed to it, an unweighted mean may not give an accurate snapshot”.Answer:We thank the reviewer for this insightful comment. While we agree there are many situations where a population-weighted air pollution metric would be ideal, given the emphasis of the paper on urban landscape profiles of cities as a whole, we feel that it is appropriate to use an unweighted air pollution metric that represents mean characteristics within the same geographic area as the urban landscape variables in the analysis. In examining city profiles, we do not distinguish between built environment characteristics in residential, industrial, or green spaces within each city because the explicit purpose is to look at each city as a whole. The urban characteristics  used to create the city profiles in the analysis represent the entire geographic area within the political boundaries of the city. Given the specific urban characteristics we used to develop profiles of urban landscape and street design, we expect that associations between these built environment profiles and air pollution are largely driven by city-level differences related to road networks and vehicular traffic profiles. Within each city, traffic-related pollution may or may not have a strong spatial correlation with residential areas, and these residential areas would, of course, be given more weight with a population-weighted air pollution metric. We believe that for our aim of determining associations between different built environment city profiles and differences in city-level air pollution concentrations, the most accurate approach is to use an unweighted air pollution metric that spatially matches the urban landscape and street design characteristics.Clarifications can be found on the methods section: page:15 Lines: 344-347 “Page 11: you briefly introduce what FMM is capable of, but it may be useful to briefly explain what it does and how it does it for the general readership”.Answer:We have expanded the description of the FMM including the following sentences: “FMM uses a mixture of distributions to model the heterogeneity of group structures. This methodology is defined in terms of the measurements and their probability density functions. Let Y_1,…,Y_n the realizations of the p measurements in the sample Y [28]. The probability density function f(y_j ) can be described as:f(y_j )=∑_(i=1)^g▒〖π_i*f_i (y_j)〗where f_i (y_j) denotes the component densities of the mixture, g is the number of components of the finite mixture distribution, π_i are called the weights or conditional probabilities to belong to each component. These weights are nonnegative quantities that sum one:■)@∑_(i=1)^g▒π_i =1)The value of g and the conditional probabilities π_i are unknown and must be inferred from the data [28]. The selection of the number of components was conducted using as criteria the Bayesian Information Criteria (BIC) and the entropy. The BIC was used to evaluate the goodness of fit of the model and the entropy was used to evaluate a good classification of the observations within latent constructs. The observations were assigned to the latent construct with the highest conditional probability”. Changes can be found on the methods section: pages 15-16: Lines: 355-366 “Pages 11-12: please provide mathematical equations for your models to make their specification clearer”.Answer:We provided the mathematical equations The first level can be described as:■(■((1)&Y_ij=b_0j+b_1j X_ij+ε_ij )&,ε_ij~N )where Y_ij is the outcome for the i^th individual in the j^th group, b_0j is the intercept for the j^th group, b_1j is the coefficient for the matrix of variables for the j^th group, X_ij is the variables matrix for the i^th individual in the j^th group and ε_ij is the i^th individual error in the j^th group. The outcome Y_ij correspond to the social determinants of health and air pollution. The variables used to define the X_ij matrix were the urban landscape and street design profiles.The second level can be described as:■((2)&■)@■)))where γ_00 is the common intercept across groups, γ_10 is the common slope associated with X_ij across groups, γ_01 is the change in the intercept per unit change C_j, γ_11 is the change in the slope per unit change C_j, U_0j is the unique deviation of the intercept of each group from the overall intercept, U_1j is the unique deviation of the slope within each group from the overall slope, τ_00 is the variance of the group specific intercept and τ_11 is the variance of the group specific slope.”Changes can be found on the methods section: pages 17-18: Lines: 394-410 Results section: rather than just presenting who was lowest/highest on different indicators, it would help to briefly explain/interpret why they were lowest or highest. What are the unique characteristics or histories of these places to explain their extreme values? Also, consider presenting a table to summarize some of these values, or perhaps a figure showing box plots to demonstrate their distributions.Answer:According to your recommendations, we included examples of the cities within each profile underscoring those cities with the highest conditional probability. We included a supplementary table including indicators of type colony, population density and area that could explain in part the configuration of the city. Future studies should take into account our results in the context of the history of each city, transport and urban planning policies, and economic development. We also included two figures (figure 1 and figure 2) with histograms showing the scale and dispersion of each metric for all studied cities. We did not include box plots due to different scales and the large dispersion among the metrics, but included the histograms for helping the visualization of the results. In addition, we want to point out that these suggested tables can be complemented with the information of the dashboard.Changes can be found on the results section: pages 20 and 21: Lines: 454 and 479In addition we included a Table of supplementary material including indicators of type colony, population density and area “The visualization tool on your web site is a nice feature”.Answer:Thank you for the comment. “Discussion section: I feel like the paper concludes without a takeaway message right now. This is a correlational/descriptive study, but I'd like to see more motivation for why these descriptions matter. How can policymakers use these findings? What new theoretical knowledge do they generate? Why is that new knowledge urgently needed to understand and improve urban living?”Answer:According to the reviewers comment we expanded the policy and research implications of our study.Our study characterizes the urban landscape and street design of 370 cities in Latin America and their association with social and environment determinants of health We identified four profiles for the urban landscape and four for street design. The characteristics and composition of the profiles highlight the heterogeneity and complexity of cities in the region. When examining associations with social and environmental determinants of health, we did not find a best “potentially healthier” profile. Profiles of cities including higher street connectivity with moderate directed streets exhibit better socioeconomic conditions, but more air pollution; while profiles including more fragmentation, less isolation and more complex shape were associated with more traffic congestion.Prior applications of profiles have emphasized their descriptive nature and their use as triggers of policy change, either to address concerns such as fragmentation and isolation, or as an aspirational vision to be achieved. Yet, the profiles in this study were not consistently and unequivocally associated with positive environmental and social outcomes. They underscore the tradeoffs often present in areas of rapid growth and economic need: economic development and productivity improving housing and social development but beget air pollution and congestion. Together, these profiles provide a novel conceptual perspective to understanding urban areas. Especially for Latin America, the inclusion of cities with different sizes sheds new light on the organization of cities and can serve as indicators to guide decisions about urban planning, transport and health. Additionally, the profiles can be used as indicators when evaluating interventions that can alter the trajectory of cities towards being healthier, less unequal, and more sustainable. At the global level, our results could also be useful for The New Urban Agenda and the SDG 11 of Sustainable Cities & Communities and the incorporation of the ‘Health in All Policies’ approach.Changes can be found on the discussion section: pages 33-34: Lines: 620-640 “The last paragraph uses causal language  that does not appear to be warranted as the study did not employ a causal research design such as a quasi-experimental framework”.Answer:Following the reviewer’s suggestion, we adjusted the language for avoiding confusion about causality conclusions of cities towards more healthy, less unequal, and sustainable outcomes.AttachmentResponse to Reviewers.docxSubmitted filename: Click here for additional data file. 7 Sep 2021Built Environment Profiles for Latin American Urban Settings: The SALURBAL studyPONE-D-20-18493R1Dear Dr. Sarmiento,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. As disclosure, note that I was one of the reviewers of the initial submission.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Geoff BoeingGuest EditorPLOS ONE 14 Oct 2021PONE-D-20-18493R1 Built Environment Profiles for Latin American Urban Settings: The SALURBAL study Dear Dr. Sarmiento:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staffon behalf ofDr. Geoff Boeing Guest EditorPLOS ONE


Aedes koreicus is a mosquito species characterized by marked anthropophilic behavior, and a potential vector of nematodes and viruses. It is native to East Asia, but its presence has recently been reported in many regions of Europe. In Italy, these mosquitoes had been detected in the northeast since 2011 and are now spreading towards the southwest of the country.COI, ND4, ITS2 and D2).In 2020, during a surveillance program for invasive mosquito species in the district of Bergamo , about 6000 mosquito larvae were collected. Emerged adults were assigned to mosquito species according to morphological analyses, followed by amplification and sequencing of genetic markers (Ae. koreicus.According to the morphological and genetic data, about 50 individuals belonged to the species Ae. koreicus in the district of Bergamo, which confirms the spread of this species in the north of Italy and raises concerns about its possible role as a vector of diseases in the Alpine area.We report the presence of The online version contains supplementary material available at 10.1186/s13071-021-05031-7. Aedes albopictus, another species of this genus, Ae. koreicus, is spreading in Europe [In recent decades, the spread of alien mosquito species, as potential vectors of pathogens of public health importance, has raised serious concern in Europe , 2. In an Europe . This spn Europe  and was n Europe . Subsequn Europe –10, and n Europe , in 2013n Europe , in 2015n Europe , in 2016n Europe .Ae. koreicus in Italy, in the Bergamo district (in the Lombardy region). This novel record highlights that this species is likely expanding in Lombardy, especially in mountain areas. Identification of the collected mosquitoes was achieved by morphological observations and molecular analysis of three genetic markers, which were then also used to reconstruct phylogenetic trees. Our record further emphasizes the urgent need for coordinated efforts for the monitoring of invasive mosquitoes in Italy, to prevent the spread of vectors of emerging pathogens.Here we report the detection of a novel population of Culex, based on the recognition of the peculiar traits of this genus at the larval instar. Some of the remaining larvae were observed and morphologically characterized under a stereomicroscope before pupation; most of them emerged as adults and were observed for further identification. Based on the morphological identification keys described in Tanaka et al. [Ae. koreicus. The remaining mosquitoes were identified as belonging to the genus Culiseta.In a surveillance program for invasive mosquito species in the Bergamo district   lining the frontal clipeal edge and about 18 evenly spaced teeth on siphonal pecten. Moreover, adult mosquitoes of Ae. koreicus show a pale basal band at the hind tarsomeres IV and the typical scutal pattern. The base of the posterior femur is completely pale, and a subspiracular patch of pale scales is usually present. These features distinguish Ae. koreicus from the closely related invasive mosquito Ae. japonicus japonicus, which instead usually presents dark hind tarsomeres IV and a dark subbasal band on the posterior femur. Moreover, Ae. j. japonicus mosquitoes lack the subspiracular patch [Ae. koreicus have been identified as invasive mosquitoes in Europe: the Korean mainland population and the South Korean volcanic island Jeju population [Ae. koreicus mosquitoes collected and maintained in our insectarium. Thus, we could attribute the individuals of Ae. koreicus collected in Lombardy to the Jeju morphological variant, which was previously reported in Belgium [As reported in Fig. ar patch . In recepulation , 5. The pulation . On the  Belgium .Fig. 1MoAe. koreicus can be misleading because of peculiar features partially overlapping with those of Ae. japonicus subspecies. Moreover, samples can be damaged: from our experience, some morphological characters, such as the scales forming the design of the mesonotum, can be lost if mosquitoes are stored for a long time, making the identification difficult. In this study, following polymerase chain reaction (PCR)-based identification as described in Cameron et al. [COI, D2, ND4 and ITS) allowed us to further confirm the identification of the collected samples and to reconstruct the phylogenetic relationship between Ae. koreicus and other mosquito species. DNA was extracted from whole bodies of individual mosquitoes . A first molecular analysis was performed following Cameron et al. [ND4) gene of Ae. koreicus has enabled the development of a PCR tool for rapid identification of this species. This PCR protocol uses the Ae. koreicus-specific primer ND4korF (5′-CCCCATTTAACCCCCAATAT-3′), together with the primers N4J8502D(F) and N4N-8944D(R), to amplify a single band of 465 bp in the control species Ae. albopictus and an additional band of 283 bp for Ae. koreicus  , are usen et al. , the seqn et al. : the con010 , areCOI) and ND4, and two nuclear  loci, the internal transcribed spacer 2 (ITS2) and the gene portion coding for the 28S ribosomal subunit 2 (D2). To amplify these DNA fragments, specific primers and protocols, already published, were followed, as reported in the Additional file Four other molecular markers were amplified and sequenced: two mitochondrial loci, cytochrome oxidase I  were used to build phylogenetic trees including sequences from closely related species, retrieved using the nucleotide Basic Local Alignment Search Tool (BLAST) . AlignmeCOI gene. However, we obtained a sequence for Ae. koreicus that showed 98–100% identity with those in the databases. This sequence and the other three were all deposited in GenBank .Because of the lack of enough sequences of related species in GenBank, no tree was constructed for the ND4 region  are 100% identical to the Ae. koreicus sequences from Germany (GenBank: KT945239.1) and Italy , while only a single nucleotide in the sequence MW365451 was different. As reported in Fig. ND4 phylogenetic tree highlighted that our sequences clustered with a Korean ND4 sequence  with 90.7% bootstrap support. As expected, the Ae. koreicus monophylum clustered with Ae. japonicus sequences with 91.6% bootstrap support. In fact, according to the phylogenetic analysis of Cameron et al. [Ae. koreicus forms a monophyletic group with the four Ae. japonicus subspecies.The sequences obtained from the amplification of the n et al. , Ae. korD2 nuclear region sequences : our sequences clustered with an Ae. formosensis sequence (GenBank: KF687496.1) and an Ae. japonicus sequence (GenBank: MG242580.1) with 98.3% bootstrap support. Indeed, the three species are recognized members of the Hulecoeteomyia subgenus [Figure ITS2 region  were identical to Ae. koreicus sequences from Russia, Belgium and Hungary .All the four sequences of the Ae. koreicus, Ae. japonicus and the other species belonging to the Hulecoeteomyia subgenus [Our results confirm the high genetic similarity between subgenus .Ae. koreicus in the study area. This species is an Asian mosquito species native to Korea, Japan, China and Eastern Russia, which has become increasingly diffused in Europe [Ae. koreicus tolerates low temperatures and prefers mountain regions (23–28 °C) [Ae. koreicus have greater persistence during the late summer and autumn seasons [All these analyses confirm the presence of n Europe , 12, 16.3–28 °C) ; further seasons .Ae. koreicus has colonized an area of more than 3000 km2 in the hills and pre-Alpine zones (from Liguria to Trentino and Veneto regions). The spread of Ae. koreicus mosquitoes is rapidly increasing towards southern and western Italy, from the original infested area in the district of Belluno, probably due to the presence of dense road connections and suitable habitats in other areas [Ae. koreicus mosquitoes to avoid competition for breeding sites occupied by species that have demonstrated a considerable advantage in terms of number and speed of replication, such as Ae. albopictus [Ae. albopictus were found at the collection site of Ae. koreicus near Bergamo. Larval competition between Ae. albopictus and Ae. koreicus under natural conditions needs to be thoroughly studied and should be integrated with investigations on the interspecific interactions with other species such as Culex spp. and Culiseta spp., found at the same breeding site of Ae. koreicus.In Italy, er areas . The abibopictus . In thisAe. koreicus in the pre-Alpine area in the Bergamo district, particularly at the end of the summer season, is a further confirmation of the adaption of these mosquitoes to specific climate and environmental conditions; our georeferenced record will thus contribute to the prediction of future expansion patterns for this species. The distribution area of Ae. koreicus is indeed widening, under the influence of global warming and the heavier international trade and travels in Europe [Ae. koreicus is a potential vector of pathogens, such as the nematodes Dirofilaria immitis and Brugia malayi [Ae. koreicus during a sampling that was not focused on the collection of Aedes mosquitoes highlights the importance of the monitoring activity and the need for wider knowledge of the introduction routes and the ecological niches occupied by this invasive mosquito. The clarification of some aspects of the biology of the Korean mosquito, such as biting habits, together with a deeper investigation of its vector status, will be fundamental to evaluate the risk of the spread of Ae. koreicus for public health in Europe.The finding of n Europe . Additioa malayi  and virua malayi  and Japaa malayi . The ideAe. koreicus mosquitoes caught in the Bergamo district are comparable to those typical of the Jeju Island population, from which the majority of European populations seem to derive. This observation raises questions about the introduction and expansion of this alien species in Italy and, more broadly, in Europe. One plausible way of introduction in the Alpine area of Lombardy is the exchange of goods and people occurring in the international airport of Orio al Serio, in the Bergamo district. The presence of an international airport was also suggested as the possible importation route to the Liguria region [Ae. koreicus could have been introduced in the Bergamo area from other infested areas in Italy. It must be noted that the road transport in this region is very intense, and previous surveys have reported the presence of the species in the nearby districts of Vicenza and Verona, in the Veneto region [Ae. koreicus in the next decade due to the presence of trade roads [The morphological features of the population of a region . Alternao region . Dispersde roads . Since cAe. koreicus in the district of Bergamo, which confirms the rapid spread of this species in this area and raises concerns about its possible role as a vector of diseases in the Alpine area.In conclusion, we report the presence of Additional file 1: Figure S1. Localization of the collection site of Aedes koreicus mosquitoes in the Bergamo district  on Google Earth.Additional file 2: Figure S2. Agarose gel electrophoresis showing results of Aedes koreicus assay with N4J8502D(F), N4N-8944D(R) and ND4korF primers. Ae. albopictus specimen displayed a single fragment of 465 bp (code number 1). Ae. koreicus specimens produced two fragments, one common fragment of 465 bp and the species-specific fragment of 283 bp (code number 3–7). M molecular weight standard .Additional file 3: Table S1. Primer sequences and references used for Aedes koreicus identification. PCRs were performed for two mitochondrial loci, cytochrome oxidase I (COI) and nicotinamide adenine dinucleotide dehydrogenase subunit 4 (ND4), and for two nuclear  loci, the internal transcribed spacer 2 (ITS2) and the 28S ribosomal subunit 2 (D2).