Background: Heat shock factor 1 (HSF1) is now considered to have the potential to be used as a prognostic biomarker in cancers. However, its clinical significance and potential function in acute myeloid leukemia (AML) remain underexplored.Methods: In this study, the expression pattern and clinical significance of HSF1 in AML were examined by integrating data from databases including The Cancer Genome Atlas (TCGA), The Genotype–Tissue Expression (GTEx), Vizome, Cancer Cell Line Encyclopedia (CCLE) and Gene Expression Omnibus (GEO). Linkedomics was applied to collect HSF1–related genes in AML. GeneMANIA was applied to outline HSF1–related functional networks. CancerSEA analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and Gene Set Enrichment Analysis (GSEA) were performed to mine the potential mechanism of HSF1 in leukemogenesis. Single–sample Gene Set Enrichment Analysis (ssGSEA) was applied to explore the correlation between HSF1 and infiltrating immune cells in AML.Results: HSF1 expression was elevated in AML compared to healthy controls and indicate a poor overall survival. HSF1 expression was significantly correlated with patients age, associated with patient survival in subgroup of bone marrow blasts (%) >20. Functional analyses indicated that HSF1 plays a role in the metastatic status of AML, and is involved in inflammation–related pathways and biological processes. HSF1 expression was significantly correlated with the immune infiltration of nature killer cells and T cell population.Conclusion: HSF1 plays a vital role in the molecular network of AML pathogenesis, and has the potential to be a biomarker for prognosis prediction. Acute myeloid leukemia (AML) is a highly malignant disease with a poor survival rate despite continuous advances in treatment . AdvanceHeat shock factors (HSFs) are a class of evolutionarily conserved transcription factors, that regulate the heat shock response (HSR) by mediating the expression pattern of heat shock proteins (HSPs) , 5. HSPsPrevious research on HSP expression from both the stress response and cancer fields involves HSF1. A study confirmed that HSF1 is elevated in AML cell lines including HL–60 and OCI–AML3, acting as a promoter of cell proliferation . AdditioIn this study, the expression level, diagnostic efficiency, and prognostic significance of HSF1 in AML, as well as the correlations between HSF1 and clinical parameters, were investigated and validated based on The Cancer Genome Atlas (TCGA), Genotype–Tissue Expression (GTEx), Gene Expression Omnibus (GEO), Cancer Cell Line Encyclopedia (CCLE), and Beat AML data viewer (Vizome). Then, the potential mechanism of HSF1 in AML was explored based on annotation of transcriptome and single–cell sequencing data. The molecular regulatory association between HSF1 and its related genes was explored by constructing a protein–protein interaction network. Additionally, the correlation between HSF1 expression and immune cell subset abundance was further explored. This study aimed to elucidate the role of HSF1 in AML at a systems level in order to explore the clinical implications of HSF1 and the relevant molecular mechanisms in AML pathogenesis.The HSF family in humans includes HSF1, HSF2, HSF4 and HSF5. According to data from AML based on TCGA and healthy controls derived from GTEx, HSF1 and HSF5 were elevated, while HSF2 and HSF3 were decreased in AML compared to healthy controls  and 1G. A logistic model was constructed to explore the association between HSF1 expression and clinical parameters of AML patients, and the results showed that HSF1 is significantly associated with patient age and peripheral blood (PB) blast proportion based on data from TCGA. Evidence from the Vizome indicated that HSF1 is significantly associated with patient age . In bothThe top 50 negatively and positively correlated genes related to HSF1 expression in AML were obtained from linkedomics . CorrelaBased on single–sample Gene Set Enrichment Analysis (ssGSEA), correlation between HSF1 expression and immune cell infiltration was analyzed. HSF1 expression was significantly related to the abundance of infiltrating immune cells in AML including the T cell population, nature killer (NK) cells, B cells, and immature dendritic cells (iDCs) . There wHSF–induced upregulation of HSPs is a key part of HSR. Among members of the HSF family, HSF1 is reported to be the major regulator of increased HSPs in cancer, and switches on during the development of cancer . GeneralC. elegans [HSF family members expressed in humans include HSF1, HSF2, HSF4 and HSF5, of which expression pattern, diagnostic efficiency and prognostic significance were investigated in this study. HSF1 and HSF5 were highly expressed, while HSF2 and HSF4 were decreased in AML compared to healthy controls, and elevated expression of HSF1 has prognostic significance, indicating a worse survival probability, which has the potential to be a prognosis–predicted biomarker. Further logistic analysis showed that HSF1 expression is correlated to patient age. Previous studies have suggested that elevated HSF1 increases both stress tolerance and lifespan of  elegans , 24. HSF elegans . Althougin vitro and in vivo [in vitro and in vivo.It is known that HSF1 exerts its pro–oncogenic effects by mediating HSR–related biological processes in the oncogenic network. To further explore the underlying role of HSF1 in AML pathogenesis, multi-omics analyses were integrated in this study. In the results from single–cell sequencing data form CancerSEA, HSF1 was significantly related to various aspects of leukemogenesis, and a remarkable association was observed for HSF1 and leukemia stem cell metastasis in AML. The correlation between HSF1 expression and metastatic potential has been reported in breast, colon, lung, prostate, hepatocellular tumors and malignant melanoma, indicating that HSF1 supports anchorage–independent growth . In AML, in vivo , 34. HSFIn the present study, we integrate data from several sources of publicly available databases and aimed to systematically investigate the expression and functional roles of HSF1 in AML as well as its clinical implications. The results analyzed based on TCGA were validated using independent datasets sourced from GEO and Vizome. The credibility of the prediction was improved, but some inconsistency exists between different cohorts. Validated results provide an important perspective for understanding the potential role of HSF1 in AML, and contradictory or conflicting results represent open questions for future studies. Additionally, datasets are limited in diversity and sample sizes, large–scale prospective cohort studies should be implemented to warrant the screening value of HSF1 for clinical purposes.In summary, HSF1 was screened as a potential diagnostic and prognostic biomarker for AML patients. The leukemogenesis mechanisms of HSF1 are a multifactor regulatory process that is involved in inflammatory states, leukemia stem cell metastasis and related signaling pathways. This study comprehensively elucidated the clinical significance and potential functions of HSF1, which provides a perspective to understand the complex molecular network of leukemogenesis.https://xenabrowser.net/datapages/), which were sourced from the TCGA and GETx databases and processed using the Toil pipeline to minimize batch effects [https://www.broadinstitute.org/ccle) [http://vizome.org/) [In this study, mRNA sequencing data for AML and healthy controls were downloaded from UCSC XENA  , and Vizme.org/) .t test was used for independent samples, and the Mann–Whitney test was used if the assumptions of normality for the t tests were not fulfilled. The expression levels of HSF1, HSF2, HSF4, and HSF5 in AML cell lines was further examined in CCLE. The ROC curve was then generated to evaluate the diagnostic value of HSF1 in AML based on data from TCGA. An AUC closer to 1 means better classification of the samples, and indicates a test of perfect diagnostic value, whereas an AUC of 0.5 indicated no diagnostic value.The HSF family includes HSF1, HSF2, HSF4 and HSF5. The expression level, diagnostic efficacy and prognostic significance of HSFs in AML were detected based on the data sourced from TCGA and GTEx. Expression levels were analyzed for normality using the Kolmogorov–Smirnov test. An unpaired High (top 50%) and low (bottom 50%) HSF1 expression groups were divided according to the median expression of HSF1. OS of the high expression group and the low expression group was assessed using the Kaplan-Meier method by log-rank test based on data sourced from TCGA. Subsequently, AML patients were divided into different subgroups based on clinical variables, and the prognostic value of HSF1 among different subgroups was assessed to further elucidate the prognostic significance of HSF1 in AML patient subgroups.P-value < 0.05 was considered to be statistically significant.HSF1 expression level and diagnostic efficacy were validated using GSE9476, and the prognostic significance was validated using GSE12417 and Vizome. Statistical analyses and visualization were performed using GraphPad Prism , and a P-value < 0.05 were identified as independent clinical factors associated with HSF1 expression.The association of HSF1 expression with clinical parameters including patient gender, age, BM blast proportion, PB blast proportion, white blood cell (WBC) counts, cytogenetic risks, NPM1 mutation and FLT3 mutation was estimated using logistic regression models based on TCGA data, which was validated using data sourced from the Vizome database. This analysis was conducted using IBM SPSS Statistics  by taking HSF1 as an independent variable, and relevant clinical parameters were set as dependent variables. Clinical parameters with a threshold http://biocc.hrbmu.edu.cn/CancerSEA/) is an integrative database depicts functional states of cancer cells at a single–cell resolution, covering 14 functional phenotypes of 41,900 cancer single cells from 25 cancer types [CancerSEA (er types . In ordehttp://www.linkedomics.org/login.php), a database containing multiomics data and clinical data for 32 cancer types and a total of 11,158 patients from TCGA project [LinkedOmics ( project . Genes r project .http://www.genemania.org) is a tool for generating hypotheses about gene function, analyzing gene lists and prioritizing genes for functional assays [GeneMANIA (l assays . Positivhttp://kobas.cbi.pku.edu.cn/) [P. adjust value <0.05 were considered to be significantly enriched.To understand the functional implications of HSF1 in AML. KEGG analyses of positively and negatively correlated genes of HSF1 were performed based on KOBAS–i (edu.cn/) . DiffereP-value < 0.05 was considered to be a significant association.HSF1 expression with immune cell infiltration levels in AML was further explored. The gene markers of tumor–infiltrating immune cell markers are referenced in prior studies . The infhttps://osf.io/nw23m/. The data supporting the conclusions of this article are available upon request to the corresponding author.All R code used for the pre-processing of the data is available at Open Science Framework (OSF),


TENM3 pathogenic variants cause isolated or syndromic microphthalmia. Syndromic microphthalmia 15 (MCOPS15) is characterized by microphthalmia, coloboma, and developmental delay. Currently, only four cases of MCOPS15 have been reported and the clinical features varied among the patients indicating potential broad phenotypic spectrum.Biallelic The present case was a 6‐month‐old male at diagnosis. The patient exhibited long philtrum, large ears, bilateral ptosis, and nystagmus. Ophthalmic tests showed that he had microcornea, iris and choroidal coloboma. The patient presented with global developmental delay (GDD). Trio‐whole exome sequencing and genome copy number sequencing were conducted to explore the disease‐causing mutations.TENM3, which were inherited from the mother and father, respectively. Sanger sequencing was conducted to verify association of the mutations with the disease in the present family.Exome sequencing and genome copy number sequencing showed the presence of L1471F and E661G compound mutations in TENM3 variants were identified in a patient with Syndromic microphthalmia 15 in the present study. However, further studies should be conducted to explore the pathogenicity of the variants.Two  To date, only four cases of MCOPS15 were reported and the clinical features of the patients were variable, which indicate potential broad phenotypic spectrum. In this study, using trio‐whole exome sequencing, we identified biallelic variants (L1471F and E661G) of TENM3, in a 6 months Chinese boy. Our findings expand the mutational spectrum of the TENM3 gene, which might help increases understanding of the heterogeneity of clinical manifestations of diseases. Currently, only four cases of MCOPS15 have been previously reported and the clinical features of the patients varied, indicating potential broad phenotypic spectrum  is an extremely rare disease caused by homozygous or compound pathogenic variants of the 22.1The patient in the present study was a 1‐year‐old male who was previously presented in our hospital at 6 months of age due to acute respiratory infection. The patient was the second child born to a non‐consanguineous couple without family history of congenital diseases. Perinatal and prenatal examinations were normal, and there was no history of exposure to toxic substances. The height and weight of the patient were within the normal range for children of the same age. He underwent normal breast feeding and received infant feeding. The patient had no history of previously unexpected or abnormal treatment. The patient presented with long philtrum, large ears, bilateral ptosis, and nystagmus  and mild hypertonia of lower limbs. The patient could not crawl or sit without assistance at 1 year of age. In addition, his cognitive and emotional responses were severely impaired, indicating possible severe global developmental delay (GDD). The patient underwent the full program of developmental assessment because he was not cooperating during the tests. Ophthalmic tests showed that he had microcornea, iris and choroidal coloboma Figure . Further9/L, elevated NE (neutrophilic granulocyte) ratio of 47.2%, platelet count of 384 × 109/L, and CRP (C‐reactive protein) level of 12.79 mg/L. Liver and kidney function, enzyme, and metabolic tests of the patient were normal. His urine and stool tests were normal.Blood and biochemical tests at the time of admission showed WBC (white blood cell) count of 7.13 × 10Doppler ultrasound showed that the patient had congenital heart defects (CHDs) including: atrial septal defect, patent foramen ovale, patent ductus arteriosus, mildly dilated right atrium and ventricle, mild right ventricular hypertrophy, mild tricuspid valve regurgitation, and pulmonary arterial hypertension Figure . EEG andDifferential diagnosis was conducted to review diseases of syndromic ocular coloboma with CHDs. The reviewing criteria were retrieved from the Online Mendelian Inheritance in Man (OMIM) database and previous research articles , an online protein structural annotation tool hosted by EMBL's European Bioinformatics Institute (EMBL‐EBI). The three‐dimensional protein structure of TENM3 has not been experimentally solved, therefore, the homolog structure of notch1 (pdb id: 5uk5), a Rattus norvegicus protein was used, and E661 was matched to E352 in the PDB structure.Genomic DNA was extracted from peripheral blood of the triad and whole exome sequencing (WES) and copy number variation sequencing (CNV‐seq) were performed  and c.1982A > G  in the patient and the variants were further verified through Sanger sequencing  diagnosis. However, microphthalmia was not observed. Stephen et al. gene and is expressed in interconnected areas of the developing nervous system and nonneural tissues  indicating potentially higher risk of TENM3 disease in China. Currently, Chinese ophthalmologists lack the skills for genetic tests required for accurate diagnosis of the disease.Lack of information on the pathogenicity of 5TENM3 L1471F and E661G variants were identified in a Chinese patient with MCOPS15. The findings of the current study showed a relatively high MAFs of these variants in East‐Asian population and the potential absence of neurological disorder, implying that TENM3 disease may be underreported in China and other East‐Asian countries.The The authors declare that the research was conducted in the absence of any commercial and financial relationships that could be construed as a potential conflict of interest.Youfeng Zhou was involved in writing—original draft, investigation, and resources. Ke Xu was involved in formal analysis and visualization. Weiyue Gu was involved in formal analysis, visualization, and writing—review and editing. Yan Huang was involved in conceptualization, resources, methodology, writing—review and editing, funding acquisition, and project administration.The study was reviewed and approved by the Ethics Committee of Fujian Provincial Maternity and Children's Hospital. Written informed consent to participate in this study was provided by the participants’ legal guardian.


Pval, κ, κ′, Plm) obtained for differentatom types were greatly influenced by neighboring atom types, hybridization,geometrical strain in the ring system, and charges on the molecule.Contrary to previous findings, the atoms showing variable oxidationstates and ions deviate from the linear dependence of monopole-derivedcharges on the expansion–contraction κ parameter.A fast and accurate operational model of electron densityis crucialin many scientific disciplines including crystallography, molecularbiology, pharmaceutical, and structural chemistry. In quantum crystallography,the aspherical refinement of crystal structures is becoming increasinglypopular because of its accurate description in terms of physicallymeaningful properties. The transferable aspherical atom model (TAAM)is quick and precise, though it requires a robust algorithm for atomtyping and coverage of the most popular atom types present in smallorganic molecules. Thus, the University at Buffalo Databank (UBDB)has been renamed to the Multipolar Atom Types from Theory and Statisticalclustering (MATTS) data bank, broadened, restructured, and implementedinto the software DiSCaMB with 651 atom types obtained from 2316 small-moleculecrystal structures containing C, H, N, O, P, S, F, Cl, and Br atoms.MATTS2021 data bank now covers most of the small molecules, peptides,RNA, DNA, and some frequently occurring cations and anions in biological,pharmaceutical, and organic materials, including the majority of knowncrystal structures composed of the above elements. The multipole modelparameters ( In reality, molecules have electronic density shared between bonded and interacting atoms.Atomic contributions to molecular electron densities are not spherical,and atoms in molecules are not neutral. This is not accounted forusing spherical atom models.ensities 1 composecore. The second component accounts for the sphericalexpansion and contraction (determined by the parameter κ) ofthe valence shell ρval as well as the populationof electrons in the valence shell . The third component describes the aspherical deformation of thevalence electron density through mathematical functions, in termsof real spherical harmonics (dlm) together with the radial terms (Rl). The radial terms are modified by the expansionand contraction parameter κ′. The populations of sphericalharmonics are identified by the parameters Plm. Pseudoatom functions are described interms of polar coordinates defined on a local coordinate system centered on each atom. Furtheranalysis of the multipole model, for example, by Bader’s approachof Quantum Theory of Atoms In Molecule (QTAIM), results in electron-density-basedbonding properties.5 These results thusobtained are useful in understanding interaction-based phenomena inthe solid state materials and macromolecules.7 However,growing a good-quality diffracting crystal and obtaining good-quality,high-resolution charge density data is a very time-consuming and tediousprocess. The multipolar modeling often is not reliable due to thepoor quality of data compromised with experimental errors, model limitations,and phase problems.The first component accounts for thefrozen core electron densityρ8 Contrary to themultipole model, the IAM-IAS model does not replace the IAM. Instead,it treats the delocalized density as spherical Gaussian scattererslocated at the centroid of the delocalized density and keeps the conventionalspherical atoms unchanged.9 Thus, the informationobtained from the IAM-IAS model is more detailed than IAM but simplerthan the multipole model. The model was applied to both small-moleculeand macromolecular systems.A more runtime-efficient and simpler approachis the use of dummybond electron or interatomic scatterers (IAS) along with the IAM,named the IAM-IAS model.Pval, κ, κ′, Plm) were found to be transferable;i.e., theparameters were found to be equivalent in similar chemical environments.10 It is possible to construct a multipole modelfor a new molecule with some previously known pseudoatom electrondensities. The early works were based on the transfer of experimentallyobtained pseudoatom parameters from one compound to construct theaspherical structural model of another compound, which was difficultto model using conventional experimental multipolar modeling.12 This research led to the advent of several pseudoatom data banks.Three such established data banks are the Experimental Library ofMultipolar Atom Models (ELMAM/ELMAM2),13 the Invariomdata bank,17 and the University at Buffalo Pseudoatom Data Bank (UBDB).21 With the availability of these data banks, it is possible to constructthe transferable aspherical atom model (TAAM) and replace the conventionalIAM in the crystal structure refinement. TAAM refinement is able toprecisely and accurately determine atomic positions , describe electron density deformations due to lonepairs and bonding, and deconvolute thermal motion from static electrondensity more accurately than IAM.25The pseudoatom (multipole model)parameters  are assignedto chemically unique atoms based on the nearest-neighbor approachhaving the same element and bond order.14 The next nearest neighbors are most often replaced by hydrogen atoms.The electron densities of Invarioms were generated from quantum chemicalapproximation while their transferability was found to be very accurate.29 The Invariom data bank has been generalizedto most of the common elements such as C, H, N, O, S, F, Cl, P, andSi and has been utilized in various studies on small molecules, aminoacids, and peptides.31 In the caseof UBDB, the theoretical electron densities are calculated on theexperimental geometry of a set of model molecules obtained from theCambridge Structural Database (CSD)32 whilechemically similar atoms are grouped into families with closely relatedpseudoatom populations and expansion–contraction parameters.UBDB is also based on the nearest-neighbor approach while in specificatom types the second and third neighbors are also defined. UnlikeInvariom, the next nearest neighbors are kept as they are in the modelmolecule. The electron density reconstructed with the help of pseudoatomdatabanks is accurate enough not only to see benefits of replacing IAMby TAAM in structure refinements on modern diffraction data but alsoto model in a fast and quantitative manner, various electrostaticproperties of molecules including biological macromolecular systemsor a large set of small molecules. Electrostatic intermolecular interactionenergies (Ee’s), for example, computed from UBDB-based electrondensities are by far more accurate than the ones from point-chargeapproximations commonly used in molecular mechanics and approach theaccuracy of quantum chemical calculations with much shorter computationaltime.33 This is because asphericity ofcharge distribution and charge penetration is taken into account.34 With Ee’s well balanced, much richerinformation on a drug molecule interactions with its protein partnercan be obtained.36 Molecular electrostatic potentialsreconstructed with UBDB are chemically accurate not only at the vander Waals region but also in the entire volume of a molecule.21 Therefore, the data bank may help in the nearfuture to better model the experimental potentials measured with single-particlecryo-EM techniques.Each pseudoatom data bank is constructed slightly differently.The ELMAM/ELMAM2 data bank was derived from experimental charge densityscattering factors obtained from high-resolution experimental datacollected on small peptides and amino acids.38 and their exemplaryapplications to crystal structure refinementand molecular electrostatic properties estimations were shown.Databanks based on interatomic scatterermodeling were also proposed,39 and the software had limitedcapabilities to integrate with software from outside of the originalquantum crystallography field. To overcome the limitations, we designeda new structure and a new file format for a pseudoatom data bank asa part of a new software library called DiSCaMB.40 The UBDB201821 was translatedto the new structure, and the data bank was renamed the MultipolarAtom Types from Theory and Statistical clustering (MATTS) data bank.The newly created MATTS data bank has already been used for determiningthe accurate and precise atomic positions of nonhydrogen and hydrogenatoms from standard X-ray diffraction experiments,25 and its usability in TAAM refinements of 3D electron diffraction data was explored.41Our focus now was to extend the UBDB, restructure it, include anew software implementation, allow for the assignment of more diverseatom types with a more reliable algorithm, and hence improve the reachof modeling toward more diverse chemical structures and fields ofapplication. The early developments of UBDB were mainly focused onmodeling proteins, nucleic acids, peptides, and druglike molecules,In the presented work, the newly created MATTS data bank was widelyextended to cover most of the atom types present in crystal structuresdeposited in the Cambridge Structural Database (CSD) and consistingof C, H, N, O, P, S, F, Cl, and Br atoms. The MATTS2021 data banknow allows a full model of electron densities of most known organicsmall molecules and determined crystal structures, including aminoacids, peptides, nucleic acids, and proteins. Here, we present a detaileddescription of the new data bank structure, atom type algorithm, andthe new entry parametrization. We describe how we optimized the procedureof defining the missing atom types to minimize the percentage of unrecognizedatoms (atoms for which no atom type is present in the data bank).We also perform a basic analysis of the content of the new data bankby presenting various statistics based on selected multipole modelparameters. A deeper analysis, with the use of statistical clusteringmethods, is presented in ref .22.1Pval, κ, κ′, Plm), with mean values resulting from averagingover model atoms fulfilling the atom type definition, constitute theprime information stored in the pseudoatom data bank. The atom typedefinition in the MATTS2021 data bank is based on a set of connectivityrules that defines the environment of the central atom, enriched byinformation about element type, group planarity, ring size, and ringplanarity. Unlike the UBDB and the LSDB software implementation associatedwith it, the connectivity list is now explicitly defined for eachatom type and directly stored in the data bank. Information on theorientation of the local coordinate system with respect to neighboringatoms, along with the local symmetry associated with multipole functions,is stored in the data bank directly as well, in contrast to the previousimplementation. The format of the MATTS2021 data bank is shown in SI.The multipole model parameters , provided thatthe local coordinate system is oriented in a way that is optimal forthat symmetry. The local (pseudo)symmetry of an atom type is predeterminedby the character of its close neighbors and the character of chemicalbonds. However, it is the local (pseudo)symmetry of an atom type electrondensity, not atom type topology, that is evaluated and then assignedto that atom type. Only with a proper orientation of local coordinatesystems can the local (pseudo)symmetry of an atom type be revealedfrom the values of multipole model parameters.Assignment of a local coordinate system to anatom type is an essential requirement of the pseudoatom data bank.The multipole model parameters of equivalent pseudoatoms should berepresented in an analogous local frame to achieve consistency inexpression of spherical harmonics functions centered at these atoms.Only then, the averaging of 42 using a procedure that was independent from atom type definition.In fact, LSDB is a multipurpose program, includes a fully automaticindependent procedure for the definition of unique local coordinatesystems based on the coordination environment of each atom, and thusis an essential tool in experimental charge density investigationsbased on multipolar refinement with high-resolution diffraction data.Information about assigned local coordinate systems was not directlystored in the UBDB file. In most cases, this behaved properly, butin the case of more exotic atom types, the errors appeared to be relatedto the variable orientation of the axes with respect to neighborswhile transferring the parameters from model molecules to the databank for averaging, or while transferring them from the data bankto an investigated molecule at the data bank application stage.Previously, ateach stage of data bank creation and application, the local coordinatesystem was assigned by the program LSDBSI.Within the new MATTS2021 data bank format, the definition of localcoordinate systems is a part of the atom type definition stored inthe data bank. The local coordinate systems are defined manually whilethe new atom type entry is created. The local coordinate system definedin the atom type definition may differ from the local coordinate systemsused during the refinement of individual atoms of model molecules.While atom type is parametrized from model molecules, the softwarefrom the DiSCaMB library recognizes how the local coordinate systemwas oriented in the model molecule and, if needed, reorients it tothe one defined in the data bank entry. Reorientation of local coordinatesystems is done before averaging multipole parameters. In this way,the creator of a new entry has an easy tool to check which coordinatesystem gives the highest transferability of multipole parameters . Local symmetry cannow also be set at the stage of atom type entry creation and mightbe independent from local symmetry constrains used during the refinementof model molecules. However, care must be taken to ensure that thelocal symmetry used during the refinement is of the same point group,or of lower symmetry (subgroup), as compared to the one set in theatom type definition. Further details about the local coordinate systemassignment can be found in the m, mm2, 3m, 4̅3m, etc.) or by writing “no”(meaning the 1 symmetry point group). Currently, the symmetry is assignedmanually on the basis of similarities among topologies of the firstneighbors, and care is taken in defining a new atom type so that itis consistent with values of multipole model parameters.Local symmetry is set by providing the symbol of one of the possiblepoint groups  correspondingto components of “colors” of the vertices.Each atom type 2.nth neighbor, where n is the smallest number whichis sufficient for correct assignment of atom types. A connectivityof atoms in the crystal is represented as a periodic graph. For eachatom in an asymmetric unit, a set of its neighbors up to the nth one is calculated with a breadth-first search algorithm,and a union of the sets forms the molecule M.An algorithm for atom type assignmentworks on a molecule  hereinafter referredto as M. Therefore, it is necessary to extract a subset of atoms froma crystal structure for further processing. This set of atoms consistsof atoms of an asymmetric unit and their neighbors up to 3.GM.Similarly as for atom types, atomsand bonds of M are represented with graph 4.a ofM that belongs to an asymmetric unit, a subgraph Ga of GM consisting of the atom a and its neighborsup to nth one is formed.Foreach atom 5.a is of type t, then Gt mustbe a subgraph of Ga.This is tested by searching for graph–subgraph isomorphismusing the VF2 algorithm. If a match is found, i.e., a mapping fromvertices of Gt to verticesof Ga is found, thenan additional check has to be performed. Atom type specificationsallow a definition of whether some atoms in the atom type definitionbelong to the same or different rings and also verification if thematch fulfils this aspect of the atom type definition or requiresan additional test.If atom 6.For each atom of M that belongs toan asymmetric unit, an attempt to match each atom type in the databank is undertaken until the match is found or until all of the atomtypes are examined.7.Essentially, some atom types can bea more general version of other atom types. In such a situation, themore general atom type should be specified in the data bank file afterthe more detailed one, since then, the more detailed one will be testedfirst.The main idea behind the atom type assignment  algorithm can be summarized with the following points:2.42.4.1To find the most popular atom types that frequentlyexist in small-molecule crystals but are still missing in the MATTSdatabank, we focused our attention on the largest available database oforganic and organometallic crystal structures, the Cambridge StructuralDatabase (CSD). We retrieved from the CSD a subset of structures containingonly those chemical elements for which we aimed to have all possibleatom types in the MATTS2021 data bank: C, H, N, O, P, S, F, Cl, andBr. We processed the entire subset in order to identify atoms to whichnone of the already existing atom types was assigned. We categorizedunassigned atoms into groups of atoms having similar topological featuresand sorted them according to the size of the group, thus identifyingthe most popular groups. Groups with the largest number of unassignedatoms were starting points for introducing new atom types to the MATTS2021data bank. The details of entire procedure were as follows.44 and was limited to structures which containeda desired set of chemical elements. All possible combinations of Cand one or more of H, N, O, P, S, F, Cl, and Br elements were considered.As search criteria, queries determining the following features wereused: (a) a list of elements that must be present in same structure,(b) a list of excluded elements, and (c) the C–H distance toensure the presence of coordinates for at least one hydrogen atomfor structures allowed to have hydrogen. The filters available inConQuest such as 3D coordinates determined, R-factorlower than 5%, nondisordered structures, no unresolved errors, onlysingle crystal structures, and only organics were applied to avoidthe selection of crystal structures with the wrong geometry or missingatoms. The resulting subset still contained structures with missingor disordered hydrogen atoms. Custom-made filters were applied toremove most of the structures with problematic hydrogen atoms.The CSD (Nov. 2019) search was performed with the ConQuest program40 typeQuest read atomcoordinates from .cif or SHELX.res format files, characterized topologicalfeatures of each atom, and, if possible, assigned atom types fromthe provided MATTS2021 data bank file to the analyzed atoms. Unassignedatoms were further arranged by the program into groups of atoms havingsimilar topology, i.e., having the same atomic number, the same typeand number of chemical elements among first neighbors, the same typeand number of chemical elements among second neighbors, and the samevalues of the remaining central atom descriptors such as group planarityor belongingness to various rings. In addition, typeQuest providedappropriate statistics documenting (a) how many atoms were assignedto which atom type, (b) the total number and fraction of all assignedatoms in relation to all analyzed atoms, (c) the total number andfraction of structures with all assigned atoms in relation to allanalyzed structures, and (d) how many unassigned atoms belong to eachidentified group. The statistics along with detailed information aboutassigned and unassigned atoms were printed to various log files.The selected subset of the CSD was next analyzed by a new utilityprogram, typeQuest, created from the developer version of the DiSCaMBsoftware library.On the basis of topological features of highly populated groupsof unassigned atoms, new entries to the MATTS2021 data bank were manuallydefined. While setting the definitions, decisions about the levelof details of the first and the second neighbors to be included inthe definition had to be arbitrarily made. Decisions were made onthe basis of knowledge gained while building UBDB. The number andelement type of the first neighbors were preserved. The total numberof second neighbors was usually preserved as well, but element typesof second neighbors were often ignored. Next, electron density pseudoatomparameters for each atom type entry were computed following the proceduresgiven in 2.4.221 A set of good-quality crystal structures with moleculesrepresenting the missing atom types was retrieved from the CSD.32 The hydrogen bond distances  in model molecules were extended to neutronbond distances using the LSDB program.42 Single-point calculations were performed on selected molecules usingthe GAUSSIAN03 program45 to obtain molecularwave functions. Density functional theory (DFT) with the standardsplit-valence double-exponential 6-31G** basis set with polarizationfunctions46 and the B3LYP functional48 was used for the calculations. The theoretical valence-only structurefactors were calculated in the range 0 < sin θ/λ < 1.1 Å–1 for reciprocal-lattice pointscorresponding to a cubic cell with 30 Å edges and the P1 space group. Subsequently, the obtained structure factorswere fitted with the Hansen–Coppens multipole model4 using the method of least-squares in the XD programsuite.49 Structure factor phases were constrainedto the one obtained from the structure factors calculations. For thedeformation functions of multipoles, default values of Slater radialfunction coefficients nl were used except for sulfur and phosphorus atoms. nl =  and nl =  were applied for sulfurand phosphorus atoms, respectively, following the previously establishedprocedure.50 The multipole expansion wastruncated at the hexadecapolar level (lmax = 4) for the nonhydrogen atoms and at the quadrupolar level (lmax = 2) for hydrogen atoms, for which onlybond-directed functions of l,m =1,0 and 2,0 were refined. Both radial screening factors (κ andκ′) were refined independently for each atom, with theexception of the chemically equivalent hydrogen atoms that sharedthe same κ and κ′ parameters. Local symmetry constrains,set by the LSDB program, were applied except for atoms having 6̅2m (Dh3), 4̅3m (Td), and m3m (Oh) local symmetry. Atoms with 6̅2m symmetrywere refined with the 3m constraints, and atoms withthe 4̅3m and m3m symmetries were refined with no constraints.The MATTS2021 data bank was constructedfollowing the procedure previously established for the older versionsof UBDB.Pval, κ, κ′, Plm) were averaged over a family of chemically equivalent atomsusing the bankMaker utility program from the DiSCaMB library and atomtype definitions provided with the external data bank file. Wheneverthe local coordinate system of an atom used during the refinementwas different from the one defined in the atom type definition, beforeaveraging, the program rotated the multipole functions52 to the coordinate system defined for the atom type. Mean valuesof multipole model parameters and their sample standard deviationswere computed for all of the parameters and were printed to the databank file following the selection rules given in the SI.The values ofmultipole model parameters thus obtained , or atomtypes that were extremely rare in the analyzed subset of the CSD.Finally, definitions of all atom types, includingthose translatedfrom the UBDB2018, were reexamined with the focus on large samplestandard deviations for any of the multipole model parameters obtainedfrom averaging over the whole set of model molecules. Care was takenso that, for most of the multipole model parameters, their samplestandard deviations did not exceed their desired values: 0.1 e for 33.121 were translatedto the new format, modified to benefit from the new design, splitif necessary, and reparametrized by reaveraging over atoms collectedfrom the newly added model molecules along with the previous molecules.This resulted in 476 atom types imported to the MATTS2021 data bankfrom the UBDB2018. 175 new atom types were added following the specificneeds of users and the here-described procedure of identifying themost popular missing types among crystal structures deposited in theCSD and containing exclusively one or more elements like C, H, N,O, P, S, F, Cl, and Br.The oldatom types from UBDB2018SI. Each atom type resulted from averaging multipolemodel parameters of many individual atoms. We targeted to have ca.five representative atoms per atom type. Some atom types were verypopular among model molecules and thus were parametrized on the basisof a much larger number of individual atoms. More than 50% of thetypes resulted from averaging 15 individual atoms or more. One thirdof the types are based on more than 30 atoms, whereas the record holdersare the H104, H101, and C330 types, which are built from more than10 000 atoms each. The achieved transferability errors, measuredby sample standard deviations of multipole model parameters, wererelatively low. More than 90% of the Pval parameters have their ssds in the range 0.01–0.1 e. For 90%of the κ parameters, ssd is in the range 0.001–0.01.In the case of κ′ parameters, more than 80% have theirssds in the range 0.01–0.1. Finally, only 11% of atom typeshave at least one Plm with its ssd exceeding 0.05 e; usually, ssds are only slightly abovethe limit.The MATTS2021 version of the extendeddata bank now contains 651 atom types, among which there are 29 hydrogen,378 carbon, 104 nitrogen, 82 oxygen, 35 sulfur, 12 phosphorus, 6 chlorine,3 fluorine, and 2 bromine atom types. A total of 2516 model moleculeswere used, and these were taken from 2316 crystals structures. TheCSD refcodes of all of the structures are given in the 3.2Pval, κ, and κ′parameters. A global analysis of Plm values was not possible here and will be given in ref .55 It was also reported that aspherical TAAM refinement increased thebond lengths of hydrogen atoms toward more accurate values. However,it is very necessary to have all of the proper atom types with theirneighboring atom types well-defined to achieve an accuracy approachingthe reference neutron bond distances using TAAM. The MATTS2021 databank contains 29 hydrogen atom types with a hydrogen atom attachedto aliphatic and aromatic carbons and nitrogen, aliphatic and aromaticalcohol, carboxylic acids, esters, etc. The MATTS2021 data bank alsocontains hydrogen atom types attached to sulfur and phosphorus. Additionally,hydrogen atom types attached to hydronium and ammonium ions and fusedwater molecules were added to the MATTS2021 data bank. Thus, the TAAMrefinement using the current version of the MATTS2021 data bank resultedin a precise and accurate determination of hydrogen atom positions,approaching reference neutron bond lengths with an accuracy similarto that of HAR.25 The valence populations for hydrogen types vary in therange from 0.58(1) e to 1.10(9) e  and 1.09(4) e . In general, the values of Pval and expansion and contraction κ andκ′ parameters for hydrogen atom types depend on the typeof neighboring atoms attached to the hydrogen atom gen atom 1. The Pv3.2.23 hybridized,93 for sp2 hybridized, and 4 for sp hybridized carbon atoms.There are 149 atom types for aromatic carbon atoms, out of which 16are on the border of two or three fused rings of various sizes . Pval for carbonatom types was found to be in the range of 4.47(5) e  and 3.50(8) e . The expansion and contraction κ and κ′parameters with maximum values were found for C410 [1.027(5)] andC783 , respectively. Minimum κ and κ′ parametervalues were found for C314c and C201 [0.79(2)], respectively. Depending upon the hybridization,the values of the Pval parameters decreasein the order C sp > C sp2 > C sp3, whilethevalues of κ parameters do not change significantly  and the highest mean value of κ′(1.073). The atom types describing the sp3 hybridized nitrogenatoms with four neighbors have the highest mean value of Pval (5.195 e) and lowest mean value of κ′(0.796). The mean values of Pval and κ′parameters for atom types of the sp3 hybridized aromaticnitrogen atoms with three neighbors are higher and lower, respectively,compared to atom types of analogous nitrogen atoms but from aliphaticgroups. In the case of atom types describing sp2 hybridizednitrogen atoms with two neighbors, belongingness to aromatic or aliphaticgroups does not influence the mean values of Pval and κ′.The nitrogen atom typescan be divided into seven categories, based on hybridization, numberof neighbors, and belongingness to aromatic systems. The variationin values of 3.2.42 hybridized oxygen atomsattached to one neighbor (O sp2) and sp3 hybridizedoxygen atoms attached to two neighbors (O sp3). There wasno significant difference found in the mean values of Pval, κ, and κ′ parameters computedfor all O sp2 atom types when compared to all O sp3 atom types. A further analysis on the type of neighbors attachedto O sp3 atom types reveals that there is no significantdifference in the mean values of parameters  are higher than for sp3 hybridizedsulfur atom types , while for κ′parameters, the reverse is true  > S sp3 (3) >S sp3 (4). There is no significant difference between themeanvalues of κ and κ′ parameters across various Ssp3 sulfur atom types.The MATTS2021 data bankcontains sulfur atom types for sp is true 6. All of3.2.6Pval, κ, and κ′ values; the Pval values vary from 5.20(6) e to 5.42(2) e,κ parameters from that 0.932(3) to 0.948(3), and κ′parameters from 1.021(8) to 1.094(4). In the case of PF6–, the phosphorus atom type (P601) has the lowest Pval (4.76(6) e) and κ′ (0.962(6))values, whereas the κ (0.940(1)) parameter has a value similarto other those for phosphorus atom types.Out of the 12 phosphorusatom types added to the MATTS2021 data bank, 11 of them have similar 3.2.7Pval values for all ofthe halogen atom types are consistent [range between 7.17(3) e and7.3(1) e] except the ClO4 atom type for the ClO4– ion which has the lowest Pval [6.127(3) e]. The κ parameters for all of the halogenatom types were found to be consistent and to vary from 0.985(2) to0.9946(2). The κ′ parameters for fluorine atom typeshave the highest values, followed by those for bromine atom types,and the lowest values are observed for chlorine atom types. The κ′parameter for the ClO4 atom type is the lowest [0.878(2)].The mean values of Pval parameters forhalogen types increase in the order F < Cl < Br; the mean valuesof κ parameters remain unchanged, while the mean values of κ′parameters follow the order Cl < Br < F (56 which has tabulated wave function of atoms upto krypton (Kr) and does not include iodine.Halogen types includethree fluorine atom types, six chlorine atom types, and two bromineatom types. The < Br < F 7. Iodine3.358 Linear dependences of κ parameters on monopole-derived charges q were reported with a high correlation coefficient19 computed on the basis of 104 atom types presentin an earlier version of UBDB2006. However, in the MATTS2021 databank, which has more diverse atom types including charged ions andatoms showing different oxidation states, the correlation betweenκ parameters and monopole-derived charges q was found to be very weak , methyl , andmethylene  groups 9. These 6– and ClO4– anions .Interestingly, the largest populations of atomtypes which weremissing in the UBDB2018 and are now present in the MATTS2021 are aromaticcarbons existing in heavily distorted aromatic rings  and observed in such molecules like fullerenes,buckybowls, strained pyrenophanes, and others. Among the most commonmissing atom types, there were also fluorine and oxygen types fromsimple inorganic PFOneof the main reasons that some atom types are still lackingis the limitation of the filtering procedure applied to remove improperstructures. Using ConQuest filters while searching the CSD still resultedin many structures with clutter. The usage of ConQuest filters duringthe CSD search resulted in many structures with wrong geometries anddisorders or missing hydrogen atoms. Some structures were furtherremoved by custom-made additional filters, but not all. This meansthat the level of atom recognition with the MATTS2021 data bank iseven better than that reported above.60 have shown that theymay have charge and expansion–contraction coefficients farfrom the formal one. To be able to do so, the atom-typing algorithmwould need to take into account not only covalent neighbors but alsoall other neighbors associated with the investigated atom by weakinteractions. This is a cumbersome task at the moment, as it wouldrequire modeling hundreds of possible configurations and robust definitionsof possible weak interactions . The halfway solution is to add Cl– andBr– atom types to the MATTS2021 data bank with Pval values following their formal charge (q = −1 e) and atomic scattering functions computedfor isolated anions. A noticeable improvement in atom recognitioncan be achieved in such a way , each level with atom typesbeing more and more general at the expense of a lower and lower levelof multipole model parameter transferability. More general types willcover those types that are very rare, and it is not profitable toadd them with atom type definitions at the same level of detail asothers. Generalization of atom types will be based on some advancedstatistical analysis like multivariate data clustering.425The MATTS2021 was formedby restructuring and modifying the previousversions of UBDB which allows for a more flexible methodology in definingnew atom types. The current version of MATTS2021 contains 651 atomtypes, among which there are 29 hydrogen, 378 carbon, 104 nitrogen,82 oxygen, 35 sulfur, 12 phosphorus, 6 chlorine, 3 fluorine, and 2bromine atom types. The atom types include the most general and popularatom type in the CSD along with specific atom types to cover the majorityof organic small molecules, including different amino acids, peptides,nucleic acids, and most macromolecular proteins. Application of MATTS2021on a set of 80 crystal structures including amino acids and peptidesresulted in accurate and precise hydrogen atom positioning and bonddistance descriptions.4–, SO42–, PF6−, NH4+, H3O+, and the Zundel cation werealso added to MATTS2021.Apart fromthe aromatic five- and six-membered planar rings, three-and four-membered ring atom types were also defined which have distinguishedgeometry and multipole populations. Atom types describing aromaticcarbons existing in heavily distorted aromatic rings were also addedinto MATTS2021 along with some missing two or three fused ring atomtypes. The atom types with most common and frequently appearing ionicsystems such as ClOAveraging multipole model parameterswas performed using the DiSCaMBlibrary which allows for a manual and more flexible definition ofcoordinate systems, compared to LSDB used in previous versions, whichadhere to the symmetry rules and can be directly stored in the databank.Pval and expansion–contraction parameters κ andκ′reveals that the neighboring atom types, hybridization, geometricalstrain in the ring system, and charges on the molecule impose significantimpact on these parameters, especially Pval and κ′, across all of the atom types belonging to differentelements in MATTS2021. The influence of charges on molecules in theionic system were more prominent in all of the cases, and their Pval and κ′ parameters differ mostin a particular subgroup of atom types. The κ parameter wasfound to be more stable with the changes in neighboring atom types,hybridization, geometrical strain in the ring system, and chargeson the molecule. Some other factors must influence the variabilityof the κ parameter.Analysis of valence population Contrary to what was seen before,the monopole-derived chargescorrelate to κ parameters only in those systems showing loweroxidation states. The third row element belonging to groups 5 and6 such as phosphorus and sulfur which can have higher oxidation statesof +5 and +6, respectively, deviates significantly. Also, the ionicsystems were majorly found to be outliers. There were no correlationsfound between monopole-derived charges and κ′ parametersexcept in the case of carbon.The recognition of the atoms,which now covers 98.17%, has beenimproved by 1.61% and that of the whole crystal structures by 18.45%which now covers 66.76% of all of the structures consisting of C,N, O, H, P, S, F, Cl, and/or Br atoms, compared to UBDB2018. Structureshaving a very strong intra-/intermolecular hydrogen bond with veryshort distances, which do not follow the criteria set for noncovalentbond lengths, cannot be included in the present data bank.63 With the additionof the diverse yet most common atom types in MATTS2021, it will bepossible to evaluate the electrostatic interactions in the screeningof organic small molecules and macromolecular structures accuratelyand quickly.33 MATTS2021 can be appliedin drug discovery and docking studies as it enhances the physicalmeaning of molecular electrostatic potential descriptors used to constructpredictive quantitative structure–activity relationship/quantitativestructure–property relationship (QSAR/QSPR) models.21Furtherapplication of MATTS2021 will be explored for refinementof electron diffraction data which is possible with the earlier versionof UBDB.61More statistics on the clustering of multipolesand their variationsacross different atom types will be presented in a future article.


Objective: Lingual lymph node (LLN) metastasis is regarded as an indicator of unfavorable prognosis and a crucial sign of the high degree of primary tumor aggressiveness. However, detecting LLN metastasis is an important but frequently overlooked aspect of diagnosis and surgical treatment planning. The study aims to identify LLNs by intraoperative near-infrared (NIR) fluorescence imaging with indocyanine green absorbed into human serum albumin (ICG: HSA) and describe the presence of lymphatic drainage channels from the floor of the mouth in patients with tongue carcinoma.Materials and Methods: 21 patients diagnosed with cT1-T4 squamous cell carcinoma (SCC) of the tongue margin and scheduled to undergo tumor resection and unilateral neck dissection were enrolled. After exposing the neck, the patients were injected with 0.3 ml of ICG: HSA (500 μM) in three quadrants around the tumor, excluding the mucous membrane of the basal region cavity. Employing a near-infrared fluorescence imaging system, the fluorescence of levels I, II, III, and IV was measured during neck dissection.Results: LLNs were detected in four patients and were identified as metastatic LLNs in all 21 patients. The near-infrared fluorescence imaging system showed the existence of lymphatic drainage channels in the floor of the mouth. In patients receiving peritumoral injection of ICG: HSA, the mean fluorescence intensity (MFI)of metastatic lymph nodes (LNs)  was higher than that in non-metastatic LNs  (p < 0.05).Conclusion: NIR fluorescence imaging with ICG: HSA could be used for intraoperative identification of LLNs and assist in the determination of metastatic lymph nodes for tongue carcinoma patients. Additionally, this finding demonstrates the feasibility of near-infrared fluorescence imaging in defining lymphatic drainage channels in the head and neck. The oral cavity is the most common subregion for head and neck cancer. The most common type is squamous cell carcinoma (SCC), which makes up approximately 90% of the cancers found in the oral cavity. The oral tongue has the highest incidence of SCC involvement (35.3%) . CervicaThe LLNs are defined as in-transit lymph nodes that appear infrequently in lymphatic drainage channels. The surgical classifications of LLNs were divided into four groups, including median LLNs, anterolateral LLNs, posterolateral LLNs, and LLNs positioned near the horn of the hyoid bone . LLN metin vivo and real-time fluorescent pictures of the lymphatic system and collateral drainage routes have been obtained using this method . From September 2021 to March 2022, this research included 21 patients with cT1-T4 SCC of the mobile tongue margin who were scheduled for tumor excision and unilateral neck dissection. Neck dissections included resecting levels I, II, and III, as well as level IV in some cases. The primary tumor was removed according to conventional surgical techniques. Exclusion criteria were recurrent oral tongue SCC; oral mucosal disease; earlier head and neck radiotherapy; earlier history of allergy to ICG and iodine; women who were pregnant, lactating, or trying to become pregnant; severe liver or kidney problems ; and serious cardiac problems (cardiac function class III or under). A written informed consent was obtained from each patient.A total of 25 mg of ICG  was dissolved in 5 ml of distilled water to obtain a concentration of 5 mg/ml ICG. Then, 7.8 ml ICG was transferred to 50 ml Cealb  solution) to obtain a final concentration of 500 μM ICG: HSA.According to our previous studies , a NIR fFollowing neck exposure, 0.3 ml ICG: HSA (500 μM) was injected into three quadrants around the tumor, excluding mucous membranes in the basilar region cavity. We observed intraoperative fluorescence of levels I, II, III, and IV using the NIR fluorescence imaging system with the shadowless lamp in the operating room turned off. Measurements were taken at 5, 10, 15, 20, 25, 30, 45, and 60 min after injection to monitor the lymphatic outflow from the tongue cancer. The images, including white light, fluorescence, and merged images, were transmitted to a digital video processor to be displayed on a monitor in real time. The first NIR fluorescence hotspot was documented, and the LNs that fluoresced were marked. During neck dissection, we searched for fluorescent lymph nodes along the central and lateral directions of the sublingual area, the larger cornu of the hyoid bone, and the lingual vascular system. Intraoperative attentive palpation was also employed to detect LLNs.LN dissection was performed in accordance with the preoperative plans. Following surgery, all LNs were separated for regular pathological evaluation, and a handheld NIR fluorescence spectrometer was used to evaluate the fluorescence intensities. All LNs were dispatched to a pathology department for a routine pathological investigation that included hematoxylin and eosin (HE) staining and immunohistochemistry (IHC). To determine whether the lymph nodes were colonized by tumor cells, two senior pathologists examined the pathological slides. They were blinded to fluorescence. We collected statistics on the location, number, fluorescence intensity, and histological status of all LNs.p < 0.05, differences were considered meaningful.GraphPad Prism Software  was used to analyze the data and generate graphs. Data for comparing the fluorescence intensity between nonmetastatic LNs and metastatic LNs are displayed as the mean ± SD (range). Differences were compared using a two-sided Student’s t test. The receiver operating characteristic (ROC) curve was employed to determine the sensitivity and specificity of peritumoral injection of ICG: HSA for recognizing metastatic LNs. When The study included 21 patients diagnosed with cT1-T4 SCC of the mobile tongue margin and radiological and clinical stage N0. The characteristics of all 21 patients receiving peritumoral injection of ICG: HSA were shown in All first draining NIR fluorescent LNs were considered SLNs. Of the 21 subjects, SLNs were observed in 11 patients within 5 min after injection, in four patients at 10 and 15 min after injection, and in one patient each at 30 and 45 min after injection. No adverse responses or complications associated with ICG: HSA injections occurred during the study.LLNs were detected in four patients and were identified as metastatic LLNs . Among tDue to the different locations of primary lesions, the first draining fluorescent LN and metastatic LNs were found in different regions of the neck. For lesions located in the anterior 1/3 and middle 1/3 of the tongue, the first draining fluorescent LN was most often found in level I (both 4/6). Seven of nine of the first draining fluorescent LNs were found in level II for lesions located in the posterior 1/3 of the tongue . For lesp < 0.05)  . The rel < 0.05) .Ozeki et al.  recognizAs noted in the literature review, anatomical presence and frequency of metastatic LLN involvement may not be deemed low . Medial As shown in Based on our pre-experimental findings, ICG mixed with HSA outperforms ICG alone or methylene blue. As shown in To determine the optimal dose of ICG: HSA, we consulted the literature. Previous studies have shown that peritumoral injection with ICG mixed with HSA at a concentration of 500 μM could provide enough contrast to visualize lymphatics, prolong the observation time and achieve more retention in SLNs . SurprisIn a study conducted by Bredell et al. , the timOur study concluded that the use of near-infrared fluorescence imaging combined with ICG: HSA for intraoperative LLN detection in patients with tongue cancer is effective. In addition, we confirmed the presence of lymphatic drainage channels in the floor of the mouth, which has important implications for delineating lymphatic drainage channels in the head and neck. Resection of connective tissue on the floor of the mouth at the same time as tongue lesions in patients with tongue SCC is necessary to improve local control. Furthermore, further clinical research on the diagnosis and management of LLN metastases is required before these nodes may be included in the TNM staging scheme. Future investigations should concentrate on optimal dosage, lymphatic tracer enhancement, multimodal hybrid tracers, and surgical procedures.


BRCA variants in twenty-two different dogs. The amplicon mean coverage was 5499× and uniformity was higher than 98% in all samples. The results of germline single nucleotide variants (SNVs) and insertions/deletions (INDELs) were fully concordant regardless of the types of samples considered . Moreover, despite the high DNA degradation observed in older FFPE blocks (>5 years), the assay allowed full coverage of all amplicons for downstream analyses. We consider the NGS panel developed in this study as a useful tool for expanding information on BRCA genes in the veterinary field and for human health from a comparative oncology perspective.Mammary carcinomas are the most common neoplasms observed in women and in female dogs. Canine mammary tumors show epidemiological, clinical, genetic, and prognostic characteristics comparable to human breast cancers. The recent introduction of next generation sequencing (NGS) technologies has greatly improved research and diagnostics for humans, while these new tools still need to be implemented in animal models. In this study we developed and validated an AmpliSeq Panel assay for the identification of  BRCA1 and BRCA2 are tumor suppressor genes that have been associated with increased risk, mainly for breast and ovarian human cancers, when carrying specific mutations. These genes are also involved in canine mammary tumors (CMTs), which are the most common neoplasms in female animals, representing a major health threat also for dogs [BRCA genes [Similarities in the biological, epidemiological, and genetic characteristics of canine and human mammary tumors suggested that dogs may provide a useful model for human breast cancer research. Humans and dogs share over 80% of the aminoacidic residues of CA genes . MoreoveCA genes ,4. BRCA due to its high throughput and cost-effectiveness [The recent introduction of next-generation sequencing (NGS) is changing the genetic analysis and research of human tiveness ,6,7. TheThe most common technique for downstream diagnosis, classification, and storage of cancer tissues in many veterinary pathology laboratories is formalin-fixed, paraffin-embedded (FFPE). The use of this technique has also intensified to enlarge the biobanks. All this has led to a significant increase in FFPE samples that could be available for further analysis, such as retrospective investigations. FFPE blocks also provide useful information on histotypes, in addition to the presence of precancerous lesions. Such data, combined with other techniques such as immunohistochemistry and NGS, can complete the scenario of specific lesions. BRCA1 and BRCA2 has recently been published for cats [BRCA research and diagnostics in dogs. To date, studies on dog BRCA mutations and the possible association with mammary cancer are very limited. Unfortunately, most investigations have focused on highly conserved regions only, which often represent sites of interaction with the RAD51 protein [In human medicine, many studies have shown the effectiveness of using the NGS technique applied to FFPE tissues ,9; howevfor cats , while t protein ,13,14.BRCA Panel assay on an Illumina platform for the identification of variants across BRCA1 and BRCA2 genes in dogs. Rather than the differentiation between somatic or germline variations introduced in neoplastic tissues and those inherited, this study focuses instead on the analysis of FFPE blocks. It is also worth considering that this method may open a new window for the retrospective analysis of historical samples. Aiming to fill in this gap, we present here the development and validation of a new AmpliSeq Finally, we compared variants found in dogs with those already described in humans to evaluate the possible clinical effects and a new strategy for classification in canine samples. Twenty-two FFPE tissue samples from pathological (n. 20) and normal (n. 2) canine mammary tissues were retrieved from the archives of the Diagnostic Department of Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise . Seven o® RSC DNA FFPE Kit (Promega) with a slight modification to the protocol at point 7 where the samples were incubated on a heat block at 65 °C overnight instead of 80 °C for 4 h. Cartridges provided by the three different kits were loaded on the Maxwell 16 Instrument (Promega). Quality control was performed on DNA by the Agilent 4200 TapeStation System  using the Genomic DNA ScreenTape and Genomic DNA reagents (Agilent Technologies). DNA was also quantified by the Qubit 2.0 Fluorometer  and then diluted to 4 ng/µL using Low TE buffer (10 mM Tris-HCl (pH 8.0) + 0.1 mM EDTA) for library preparation.Genomic DNA was extracted from blood, fresh tissues, and FFPE specimens. More precisely, DNA from blood samples (200 µL) and fresh tissues (25 mg) were extracted by means of the Maxwell 16 Blood DNA Purification Kit  and the Maxwell 16 Tissue DNA Purification Kit (Promega), respectively. FFPE samples were extracted using the MaxwellBRCA1 and BRCA2 exonic regions of Canis familiaris (UCSC canFam3). The amplicon average length was 162 bp, with a maximum length of 175 bp. Libraries were prepared by means of the AmpliSeq Library PLUS  and AmpliSeq UD Indexes for Illumina , following the protocol described in the AmpliSeq for Illumina On-demand, Custom, and Community Panels Reference Guide (Document # 1000000036408 v09). Quality control of the individual libraries (size and concentration) was fulfilled by the Agilent 4200 TapeStation System (Agilent Technologies), using the High Sensitivity D5000 ScreenTape and reagents (Agilent Technologies), and Qubit 2.0 Fluorometer (Thermo Fisher Scientific). Manually normalized libraries were then pooled, diluted to 1.2 pM and loaded in the MiniSeq Mid Output reagent cartridge (Illumina). Sequencing was performed by the Illumina MiniSeq Sequencer setting 300 cycles (150 bp paired-end reads), followed by primary analysis performed by the on-instrument software SCS/RTA (Illumina). Secondary analysis was accomplished by the DNA Amplicon workflow available on Local Run Manager Version 2.4.1 , using the manifest file (IAA19398_179_manifest.txt) (https://genome.ucsc.edu/ (accessed on 27 May 2022), on CanFam3/CanFam3.1 genome, and Alamut Plus software v.1.0 , in order to gain biological significance for BRCA1/BRCA2 variants from dog versus human comparisons. Statistical analysis was completed by the Kruskal–Wallis non-parametric test. A total of 197 custom amplicons  were useest.txt) . AnalyseResults from Qubit quantification showed a DNA concentration higher than the input required by the protocol (4 ng/µL) in all the samples considered in this study. DNA integrity (DIN) evidenced different grades of fragmentation according to the time of storage of FFPE samples and to the different matrices considered . As expected, the highest level of degradation was observed for FFPE samples and, within them, for those stored for a larger period .p > 0.05). The amplicon mean (deep) coverage was 5499×, and a mean of 1.2 M passing filter reads (Q score > 30) were obtained from the samples. Uniformity of coverage was higher than 98% in all samples . Reprodup-values 0.00819 and 0.00174 for total reads and coverage, respectively. Conversely, FFPE samples stored for different numbers of years showed progressively better results from the oldest to the recent ones in terms of total reads number and amplicon mean coverage . These rBRCA1, while 23 exonic and 3 intronic variants were identified in BRCA2 , and not referenced (n. 14), while mutations in BRCA2 were classified as likely benign (n. 3), variants of unknown significance (n. 5), not referenced (n. 15), and not found in humans (n. 3).A total of 42 different genetic variants were identified in 22 dogs. In particular, 12 exonic and 4 intronic variants were found in in BRCA2 . AccordiBRCA1, whereas in BRCA2, 17 variants were coding SNPs, 4 synonymous SNPs, 1 deletion and 1 insertion both of 3 bp  and 2 synonymous SNPs in  of 3 bp . BRCA1 and BRCA2 variants in dogs (Canis familiaris). The Amplicon panel has been designed to cover the whole BRCA1/BRCA2 exome and the proximal intronic regions (~30–50 bp), in both directions. The short-length PCR panel has been properly designed for fragmented DNA . Indeed, despite the high degradation of DNA observed in FFPE samples, particularly true for DNA obtained from older FFPE blocks (>5 years), the assay was able to produce good quality results for all the samples. In the first sequencing attempt, we pooled together all the samples without previous individual quantification and normalization. The pool was then quantified and diluted as recommended but the resulting coverage was unbalanced between samples and sometimes unsuitable for downstream analysis. The poorer the DNA quality the lower the total reads number and amplicon mean coverage occurred. The same libraries were thus quantified and normalized at the equal molar concentration before pooling and sequencing. Following this procedure, we obtained more standardized reads number and amplicon mean coverage among the samples. Despite the variation observed in FFPE samples, depending on the time of storage, all the samples were successfully analyzed. Individual normalization is thus a critical point to obtain reliable quality results for strong fragmented DNA samples. Amplicon sequencing (AmpliSeq) is a targeted approach which enables researchers to analyze genetic variation in specific genomic regions. The ultra-deep sequencing of PCR products allows efficient variant identification and characterization in a wide range of fields ,18,19. AVariant analysis of the seven dogs for which all three matrices were available in this study, confirmed the high reproducibility of the whole workflow, from DNA extraction to variant calling. In such samples, we could establish with certainty the germinal nature of the variants found, although this was not among our goals.In addition to the method validation, this study also attempted the interpretation of the variants found in dogs. In humans, family information is generally integrated to evaluate the probability that sequence variants are associated with genetic predisposition . In contIn this study, the dog versus human BRCA1/BRCA2 comparison was established by using Alamut Visual Plus, while the interpretation of the dog variants found in both humans and dogs were carried out using ClinVar, a software that archives and aggregates information about relationships among variation and health in humans. Only two BRCA1 variants were referenced in humans and classified as likely benign, while eight variants of BRCA2 were also reported in humans; five were classified as of uncertain significance (UV) and three as likely benign. Among UVs, the human BRCA2 variants p.(Lys1440Arg), p.(Arg1997Lys) and p.(Trp395Cys), showed the same amino acid substitution described here in dogs, while at the position of human BRCA2 p.(His150Arg), we found p.(His143Pro). BRCA2 and previously described in the literature as implicated in carcinogenesis of dogs [Four out of 22 different dogs  showed c.4314A>G—p.(Lys1435Arg) together with c.4283A>C—p.(Thr1425Pro), both in exon 11 of  of dogs ,14,22. BIn healthy dogs (n = 2), we did not find c.4314A>G nor c.4283A>C in FFPE samples, nor in fresh tissues. These variations are located in the BRC3 repeat domain, which plays a fundamental role in the interaction with the protein RAD51, and affect well conserved positions ,24 in avBRCA2. Finally, it is worth considering the c.10204_10206insAAA variation that is positioned in the nuclear localization signal 2 (NLS2), which plays a key role in the mislocation of BRCA2 in humans . Recent BRCA1 and BRCA2 will help to generate reproducible and robust results. The data obtained here are part of a database of variants that will be further increased by new findings from pathologic and healthy tissues of both historical and new samples. All these data will certainly lead to a better understanding of the disease and a more careful management of health care in both humans and dogs.Canine and human tumors share numerous traits: genetic and histopathological characteristics, biological behavior, molecular targets and response to standard antineoplastic treatments. For this reason, dogs offer an extraordinary opportunity to improve our knowledge of tumor biology in both species. The AmpliSeq NGS assay developed in this study for dog


The use of black soldier fly (BSF) larvae to recycle various organic materials while producing biomass for use as feed is well established. Variety selection is important from the perspective of application. In the current study, morphometric and life-history traits of a Wuhan-domesticated BSF colony (Wuhan strain) were compared to those of a ‘selectively inbred’ population . In terms of morphological characteristics, the results showed that both strains had dichoptic compound eyes, club-shaped antennae, blue halters, and blue-green metallic luster wings with a hexagon discal cell. In both strains, the body and wing length of female adults were slightly larger than those of male adults. The first four larval stages of the BSF occurred rapidly (1–12 days) with transitions across stages resulting in doubling of size for both populations. Selective inbreeding did not alter the life-history traits of the larval exuviate stage in terms of age, size, weight, and feed reduction rate. Overall egg production for the inbred strain was significantly higher (1.5 times greater) than the Wuhan strain. This is explained by increased adult emergence and individual oviposition performance. It was speculated that inbreeding improved the reproductive success of inbred adult female offspring and selection process steadied it. The findings indicate that selective inbreeding could enhance overall oviposition performance and provide a strategy to selectively breed BSF with high egg production for future applications. Hermetia illucens; Diptera: Stratiomyidae) is a widely distributed insect worldwide. Larvae of the BSF (BSFL) feed on decaying organic matter and are often collected from manure in livestock facilities [Musca domestica L. (Diptera: Muscidae) [The black soldier fly  ; reductiuscidae) ,5, odorsuscidae) , and antuscidae) ; and conuscidae) . Furtheruscidae) ,10.The impact of BSF inbreeding has not previously been examined. Zhou et al.  suggesteThe Wuhan BSF strain (Wuhan strain) was established from eggs collected at a poultry facility in Wuhan, China in 2011. The ‘inbred’ strain came from one cluster of eggs from a single female BSF of the Wuhan strain, whose egg weight, larval weight, and larval survival rate were not significantly different from those of Wuhan strains in the same generation. These eggs were selectively inbred for 10 generations. Each generation of the inbred strain was propagated from a single selected egg clutch (with the most hatched larvae) from the previous generation  of the 10th generation inbred BSF, the following generation was propagated from all clusters of the previous generation. This was carried out for 20 more generations to eliminate the inbreeding depression and increase the inbred population.w:w, final water content: 60–70%), on the basis of the standard scheme established by Sheppard et al. [The Wuhan and inbred BSF colonies were located in a greenhouse, where the temperature was controlled to be within the range of 27 °C to 30 °C, with a relative humidity of 60–70%. Larvae from both strains were fed wheat bran and wheat flour at a ratio of 6:4  with a sour odor to attract adults for spawning. The collected eggs were incubated at 28 °C and 60–70% relative humidity in a dark place to avoid sunlight and water evaporation. The whole colony process lasted between 38–45 days for one generation , which was lit from 7:00 to 17:00 every day following pupa eclosion. In the mating cage, the adults were mating and spawning. The eggs were collected from corrugated paperboards, which were positioned above the fermented artificial feed  and fed with artificial feed . Ten larvae were randomly collected every two days, and their size (body length and width) was measured after cleaning. Then, the larvae were returned into the container.The larvae in the prepupal stage were separated from the residue when 50% of them had turned black. The water content of the substrate (10 g) was determined by collecting and drying to constant weight at 60 °C. The feed reduction rate was calculated using Equation (1). All experiments were carried out in triplicate.n = 3). The volume of the cage was approximately 0.025 m3 (0.05 m2 × 50 cm). The eggs were collected from honeycomb cardboard every day (placed on the fermented feed at 8:00 a.m. and collected at 4:00 p.m.) and weighed to check the hatch percentage of each strain.In the greenhouse previously described, , 250 pupae  of the Wuhan and inbred strains were placed into separate adult-mating cages (n = 9). The eclosion rates, female–male ratios, and weights of eggs laid by one female adult were calculated using Equations (2)–(4), respectively.The above mating experiment was repeated four times due to the significant difference in the spawning characteristics between the two strains ; α = 0.05 was used as the benchmark for statistical significance.The extent of differences between the two strains was gauged using Student’s p > 0.05). For both strains, the body and wing lengths of female adults are slightly greater than those of male adults for both strains.Morphological characteristics of adult BSF from the two strains under the same breeding conditions are summarized in The external morphological structures of female and male adult BSF are shown in The thorax consists of the prothorax, mesothorax, and metathorax. The mesothorax C is wellThe adults have six legs, composed of coxa, trochanter, femur, tibia, and tarsus for each leg. The tarsus is white, and the others are black with short setae. The abdomen is long and slender, covered with dense short hairs, and has five visible segments D. They mMale genitalia, which consists of aedeagus (center) and parameres (sides), are short and exposed to the environment G. The feH. illucens L., according to the taxonomic key list [These morphological characteristics of adults conform with those of key list , for StrObserving the exuviate by using a stereomicroscope reveals that the BSFL pass through six instars with five exuviate stages. The exuviate and growth characteristics of BSFL are shown in The first exuviate of larvae occurs 1–2 days after hatching, in which the body length of the larva is only 1.5–2.0 mm. The shedding skin is semipermeable A. NormalThe 4–5-day old larvae entered into the second exuviate period, and the body length was twice that of the 1st instar. When the exuviate started, many bubbles separated the old skin and the body B. The laThe larva was macroscopic from the 3rd exuviate, when larval length reaches about 8 mm, growing for 6–7 days D. The 4tThe growth parameters of the two BSFL strains are shown in t-test was used to evaluate significant differences for each individual trial and the paired-samples t-test for the all data  in this study was longer than those where BSF were fed by meat meal (8.06–9.79 mm) and similar to those fed by hen feed (15.47–16.22 mm) . The larThe body and wing lengths of female adults are slightly larger than those of male adults for both strains, and these results are consistent with those of a previous study . This maH. illucens (Diptera: Stratiomyidae) [The details of external morphological structure of female and male adult BSF for both strains are characterized and show no statistically significant difference. All external morphological structures conform to the characteristics of omyidae) ,14.The BSFL grow through six instars with five exuviate stages, and this phenomenon is consistent with previous research . ResultsThe larval size and weight reach their maximum  when they grow to the 5th instar stage for both strains. By contrast, the BSFL from a private company  achieved maximum length and weight of approximately 18 mm and 18 g/100 larvae, respectively, for vegetable and fruit waste rearing  and emptIn terms of feed reduction, both strains can reduce 56.4 ± 0.9%–55.5 ± 3.2% of dry feed mass. In previous studies, BSFL of the Wuhan strain have been fed with dairy manure and soybean curd residue, mushroom root waste, and chicken manure with 26–72% , and 35.Hermetia Baruth GmbH , the total egg weight was 64.8–68.9 mg in cages containing 80 adults  under LED light (202–215 mg per 250 adults) [A significant difference in gained eggs is observed between the two strains, and the inbred strain displays better oviposition performance than the Wuhan strain. The final gained egg weights of the Wuhan and inbred strains are 411 ± 213 mg 31,647 ± 16,401 eggs) and 637 ± 227 mg , respectively, in the systems containing 250 pupae. Light sources are reportedly a key factor for fertilizing eggs ,401 eggs. In thisThis beneficial oviposition performance of the inbred strain is due to a high eclosion rate (96.8 ± 0.9%) and egg weight laid per female adult  of the inbred strain. The egg weights per female of the BSF from Baruth, Germany and Tsukuba, Japan are 1.6–1.7 mg and 289.0 ± 27.0 eggs, respectively ,31. Our The mechanism of the reproductive advantage for the inbred strain is still unclear. Inbreeding causes inbreeding depression in survival, mating, reproduction, and fitness and leads to an increase in genome-wide homozygosity and increased expression of deleterious recessives ,33. The Drosophila and flour moths (Ephestia kühniclla Z.) [Forficula auricularia) parents and reveals that inbreeding dramatically depresses the reproductive success of inbred adult male offspring but only has little effect on the reproductive success of inbred adult female offspring. The current results suggest that the inbreeding of BSF may also have little effect on the reproductive success of inbred adult female offspring but improves the egg production, which may be a self-strategy to prevent population inbreeding depression at the macro level. Moreover, the selection process may steady the traits of reproductive advantage—more egg production.Studies have revealed that inbreeding does not influence fertility or productivity in clla Z.) ,35. Furtclla Z.)  demonstrFor the genetic mechanisms, the inbreeding depression is because part of the genetic load of populations, known as inbreeding load, is only expressed in homozygotes . The inbH. illucens (Diptera: Stratiomyidae), are observed. The larval growth characteristics show that both strains have size and weight advantages. Furthermore, the inbred strain displays a higher reproductive advantage than the Wuhan strain and other strains in previous studies. This may reflect inbreeding selection, which could improve the egg production of inbred BSF adult female offspring. The results therefore provide a basis for one BSF inbreeding selection strategy which could be used in future applications.In conclusion, the external morphological structure characteristics of the Wuhan and inbred strain, which are identical and conform to the characteristics of


Skin malformations in dermatology are mostly evaluated subjectively, based on a doctor’s experience and visual perception; an option for objective quantitative skin assessment is camera-based spectrally selective diagnostics. Multispectral imaging is a technique capable to provide information about concentrations of the absorbing chromophores and their distribution over the malformation in a noncontact way. Conversion of spectral images into distribution maps of chromophores can be performed by means of the modified Beer–Lambert law. However, such distribution maps represent only single specific cases, therefore, some extensive method for data comparison is needed. This study aims to develop a more informative approach for identification and characterization of skin malformations using three-dimensional (3D) representation of triple spectral line imaging data. The 3D-representation method is experimentally tested on eight different skin pathology types, including both benign and malignant pathologies; an imaging device ensuring uniform three laser line  illumination is used. Three spectral line images are extracted from a single snapshot RGB image data, with subsequent calculation of attenuation coefficients for each working wavelength at every image pixel and represented as 3D graphs. Skin chromophore content variations in malformations are represented in a similar way.Clinical measurement results for 99 skin pathologies, including basal cell carcinomas, melanoma, dermal nevi, combined nevi, junctional nevi, blue nevi, seborrheic keratosis, and hemangiomas. They are presented as 3D spectral attenuation maps exhibiting specific individual features for each group of pathologies. Along with intensity attenuation maps, 3D maps for content variations of three main skin chromophores , calculated in frame of a model based on modified Beer–Lambert law, are also presented. Advantages and disadvantages of the proposed data representation method are discussed.The described 3D-representation method of triple spectral line imaging data shows promising potential for objective quantitative noncontact diagnosis of skin pathologies. One possible approach is multispectral imaging of skin diffuse reflectance where the specular reflection of skin is minimized by using two orthogonally oriented polarizers – one in front of the image sensor (camera) and the second in front of the light source.–To ensure reliable and accurate diagnostics of skin malformations, direct The potential of the snapshot triple spectral line imaging method for noncontact assessment of various skin malformations has been successfully demonstrated in Refs. 2Skin can be modeled as a structure comprising a number of homogeneous layers. In simple models only two layers are considered: epidermis and dermis,2.1A modified BLL model for diffusely reflected light was tested experimentally in this work. Reflected intensity from the surrounding healthy skin was always taken as a reference in analysis of the malformation-reflected light intensity;The corresponding distribution of the backscattered photon path lengths in skin was found as ,It was assumed that only skin melanin, oxyhemoglobin, and deoxyhemoglobin are absorbing the incident light in this model. Scattering was excluded because diffusely reflected light intensities from the skin pathology and the healthy skin were compared, assuming that scattering properties in the healthy skin and the pathology region are very similar. Reduced scattering coefficient 2.2Our previously created device2.3Image processing scheme is illustrated in λ)/I0(λ) , where k2.4in-vivo measurements were taken under permission of the local ethics committee having written consent of all 77 volunteers with skin photo-types I or II (Fitzpatrick classification), aged between 20 and 68, in cooperation with certified dermatologist Anna Berzina . In total, 99 skin pathologies were examined in this study: three basal cell carcinomas, 27 dermal nevi, 12 hemangiomas, 16 combined nevi, one melanoma, 17 junctional nevi, 22 seborrheic keratoses, and one blue nevus. Data for dermal, combined, and junctional nevi, as well as those for hemangiomas and seborrheic keratoses, may be considered as sufficient for primary conclusions while the few cases of skin cancers , and blue nevus can serve only for general illustration.Clinical 33.1The measured attenuation coefficients To summarize, the graphs in 3.2All possible values of chromophore concentration variations for melanin, oxyhemoglobin, and deoxyhemoglobin in moles  by means of the triple spectral line snapshot imaging method were represented as 3D plots of spectral attenuations, so demonstrating specific shape/volume features of each pathology group. Concentration variations of three main skin chromophores in the examined malformations were calculated in frame of a modified Beer–Lambert model and 3D-represented, as well. Further development of this technique may contribute to improved noncontact objective diagnostics of skin pathologies, including skin cancers.