Generation of rat forebrain tissues in mice.

Interspecies blastocyst complementation (IBC) provides a unique platform to study development and holds the potential to overcome worldwide organ shortages. Despite recent successes, brain tissue has not been achieved through IBC. Here, we developed an optimized IBC strategy based on C-CRISPR, which facilitated rapid screening of candidate genes and identified that Hesx1 deficiency supported the generation of rat forebrain tissue in mice via IBC. Xenogeneic rat forebrain tissues in adult mice were structurally and functionally intact. Cross-species comparative analyses revealed that rat forebrain tissues developed at the same pace as the mouse host but maintained rat-like transcriptome profiles. The chimeric rate of rat cells gradually decreased as development progressed, suggesting xenogeneic barriers during mid-to-late pre-natal development. Interspecies forebrain complementation opens the door for studying evolutionarily conserved and divergent mechanisms underlying brain development and cognitive function. The C-CRISPR-based IBC strategy holds great potential to broaden the study and application of interspecies organogenesis.

Glia-to-Neuron Conversion by CRISPR-CasRx Alleviates Symptoms of Neurological Disease in Mice.

Conversion of glial cells into functional neurons represents a potential therapeutic approach for replenishing neuronal loss associated with neurodegenerative diseases and brain injury. Previous attempts in this area using expression of transcription factors were hindered by the low conversion efficiency and failure of generating desired neuronal types in vivo. Here, we report that downregulation of a single RNA-binding protein, polypyrimidine tract-binding protein 1 (Ptbp1), using in vivo viral delivery of a recently developed RNA-targeting CRISPR system CasRx, resulted in the conversion of Müller glia into retinal ganglion cells (RGCs) with a high efficiency, leading to the alleviation of disease symptoms associated with RGC loss. Furthermore, this approach also induced neurons with dopaminergic features in the striatum and alleviated motor defects in a Parkinson's disease mouse model. Thus, glia-to-neuron conversion by CasRx-mediated Ptbp1 knockdown represents a promising in vivo genetic approach for treating a variety of disorders due to neuronal loss.

One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering.

The type II bacterial CRISPR/Cas system is a novel genome-engineering technology with the ease of multiplexed gene targeting. Here, we created reporter and conditional mutant mice by coinjection of zygotes with Cas9 mRNA and different guide RNAs (sgRNAs) as well as DNA vectors of different sizes. Using this one-step procedure we generated mice carrying a tag or a fluorescent reporter construct in the Nanog, the Sox2, and the Oct4 gene as well as Mecp2 conditional mutant mice. In addition, using sgRNAs targeting two separate sites in the Mecp2 gene, we produced mice harboring the predicted deletions of about 700 bps. Finally, we analyzed potential off-targets of five sgRNAs in gene-modified mice and ESC lines and identified off-target mutations in only rare instances.

Generation of genetically modified mice by oocyte injection of androgenetic haploid embryonic stem cells.

Haploid cells are amenable for genetic analysis. Recent success in the derivation of mouse haploid embryonic stem cells (haESCs) via parthenogenesis has enabled genetic screening in mammalian cells. However, successful generation of live animals from these haESCs, which is needed to extend the genetic analysis to the organism level, has not been achieved. Here, we report the derivation of haESCs from androgenetic blastocysts. These cells, designated as AG-haESCs, partially maintain paternal imprints, express classical ESC pluripotency markers, and contribute to various tissues, including the germline, upon injection into diploid blastocysts. Strikingly, live mice can be obtained upon injection of AG-haESCs into MII oocytes, and these mice bear haESC-carried genetic traits and develop into fertile adults. Furthermore, gene targeting via homologous recombination is feasible in the AG-haESCs. Our results demonstrate that AG-haESCs can be used as a genetically tractable fertilization agent for the production of live animals via injection into oocytes.

Development of miniature base editors using engineered IscB nickase.

As a miniature RNA-guided endonuclease, IscB is presumed to be the ancestor of Cas9 and to share similar functions. IscB is less than half the size of Cas9 and thus more suitable for in vivo delivery. However, the poor editing efficiency of IscB in eukaryotic cells limits its in vivo applications. Here we describe the engineering of OgeuIscB and its corresponding ωRNA to develop an IscB system that is highly efficient in mammalian systems, named enIscB. By fusing enIscB with T5 exonuclease (T5E), we found enIscB-T5E exhibited comparable targeting efficiency to SpG Cas9 while showing reduced chromosome translocation effects in human cells. Furthermore, by fusing cytosine or adenosine deaminase with enIscB nickase, we generated miniature IscB-derived base editors (miBEs), exhibiting robust editing efficiency (up to 92%) to induce DNA base conversions. Overall, our work establishes enIscB-T5E and miBEs as versatile tools for genome editing.

RNA base editing therapy cures hearing loss induced by OTOF gene mutation.

Otoferlin (OTOF) gene mutations represent the primary cause of hearing impairment and deafness in auditory neuropathy. The c.2485C>T (p. Q829X) mutation variant is responsible for approximately 3% of recessive prelingual deafness cases within the Spanish population. Previous studies have used two recombinant AAV vectors to overexpress OTOF, albeit with limited efficacy. In this study, we introduce an enhanced mini-dCas13X RNA base editor (emxABE) delivered via an AAV9 variant, achieving nearly 100% transfection efficiency in inner hair cells. This approach is aimed at treating OTOFQ829X, resulting in an approximately 80% adenosine-to-inosine conversion efficiency in humanized OtofQ829X/Q829X mice. Following a single scala media injection of emxABE targeting OTOFQ829X (emxABE-T) administered during the postnatal day 0-3 period in OtofQ829X/Q829X mice, we observed OTOF expression restoration in nearly 100% of inner hair cells. Moreover, auditory function was significantly improved, reaching similar levels as in wild-type mice. This enhancement persisted for at least 7 months. We also investigated P5-P7 and P30 OtofQ829X/Q829X mice, achieving auditory function restoration through round window injection of emxABE-T. These findings not only highlight an effective therapeutic strategy for potentially addressing OTOFQ829X-induced hearing loss but also underscore emxABE as a versatile toolkit for treating other monogenic diseases characterized by premature termination codons.

Ptbp1 knockdown failed to induce astrocytes to neurons in vivo.

The conversion of non-neuronal cells to neurons is a promising potential strategy for the treatment of neurodegenerative diseases. Recent studies have reported that shRNA-, CasRx-, or ASO-mediated Ptbp1 suppression could reprogram resident astrocytes to neurons. However, some groups have disputed the interpretation of the data underlying the reported neuron conversion events. These controversies surrounding neuron conversion may be due to differences in the astrocyte fate-mapping systems. Here, we suppressed Ptbp1 using Cas13X and labelled astrocytes with an HA tag fused to Cas13X (Cas13X-NLS-HA). We found no astrocyte-to-neuron conversion in the mouse striatum via the HA-tagged labelling system compared with the GFAP-driven tdTomato labelling system (AAV-GFAP::tdTomato-WPRE) used in previous studies. Our findings indicate that Cas13X-mediated Ptbp1 knockdown failed to induce neuron conversion in vivo.

Hepatocyte-specific TAK1 deficiency drives RIPK1 kinase-dependent inflammation to promote liver fibrosis and hepatocellular carcinoma.

Transforming growth factor ß-activated kinase1 (TAK1) encoded by the gene MAP3K7 regulates multiple important downstream effectors involved in immune response, cell death, and carcinogenesis. Hepatocyte-specific deletion of TAK1 in Tak1ΔHEP mice promotes liver fibrosis and hepatocellular carcinoma (HCC) formation. Here, we report that genetic inactivation of RIPK1 kinase using a kinase dead knockin D138N mutation in Tak1ΔHEP mice inhibits the expression of liver tumor biomarkers, liver fibrosis, and HCC formation. Inhibition of RIPK1, however, has no or minimum effect on hepatocyte loss and compensatory proliferation, which are the recognized factors important for liver fibrosis and HCC development. Using single-cell RNA sequencing, we discovered that inhibition of RIPK1 strongly suppresses inflammation induced by hepatocyte-specific loss of TAK1. Activation of RIPK1 promotes the transcription of key proinflammatory cytokines, such as CCL2, and CCR2+ macrophage infiltration. Our study demonstrates the role and mechanism of RIPK1 kinase in promoting inflammation, both cell-autonomously and cell-nonautonomously, in the development of liver fibrosis and HCC, independent of cell death, and compensatory proliferation. We suggest the possibility of inhibiting RIPK1 kinase as a therapeutic strategy for reducing liver fibrosis and HCC development by inhibiting inflammation.

Actual mutational research of 19 autosomal STRs based on restricted mutation model and big data.

Short tandem repeat (STR) markers have been widely used in forensic paternity testing and individual identification, but the STR mutation might impact on the forensic result interpretation. Importantly, the STR mutation rate was underestimated due to ignoring the "hidden" mutation phenomenon in most similar studies. Considering this, we use Slooten and Ricciardi's restricted mutation model based on big data to obtain more accurate mutation rates for each marker. In this paper, the mutations of 20 autosomal STRs loci (D3S1358, D1S1656, D13S317, Penta E, D16S539, D18S51, D2S1338, CSF1PO, Penta D, TH01, vWA, D21S11, D6S1043, D7S820, D5S818, TPOX, D8S1179, D12S391, D19S433, and FGA; The restricted model does not include the correction factor of D6S1043, this paper calculates remaining 19 STR loci mutation rates) were investigated in 28,313 (Total: 78,739 individuals) confirmed parentage-testing cases in Chinese Han population. As a result, total 1665 mutations were found in all loci, including 1614 one-steps, 34 two-steps, 8 three-steps, and 9 nonintegral mutations. The loci-specific average mutation rates ranged from 0.00007700 (TPOX) to 0.00459050 (FGA) in trio's and 0.00000000 (TPOX) to 0.00344850 (FGA) in duo's. We analyzed the relationship between mutation rates of the apparent and actual, the trio's and duo's, the paternal and maternal, respectively. The results demonstrated that the actual mutation rates are more than the apparent mostly, and the values of µ1"/µ2"(apparent) are also greater than µ1/µ2 (actual) commonly (µ1", µ1; µ2", µ2 are the mutation rates of one-step and two-step). Therefore, the "hidden" mutations are identified. In addition, the mutations rates of trio's and duo's, the paternal and maternal, exhibit significant difference. Next, those mutation data are used to do a comparison with the studies of other Han populations in China, which present the temporal and regional disparities. Due to the large sample size, some rare mutation events, such as monozygotic (MZ) mutation and "fake four-step mutation", are also reported in this study. In conclusion, the estimation values of actual mutations are obtained based on big data, they can not only provide basic data for the Chinese forensic DNA and population genetics databases, but also have important significance for the development of forensic individual identification, paternity testing and genetics research.

DNA typing from skeletal remains: a comparison between capillary electrophoresis and massively parallel sequencing platforms.

Skeletal remains encountered frequently in forensic applications are a challenging specimen, since their DNA is usually degraded due to harsh conditions, limiting the utilization of skeletal DNA. Forensic scientists have tried various methods to extract DNA from skeletal remains of low quantity and poor quality or improve detecting technology for more information from compromised DNA. Compared with traditional capillary electrophoresis (CE), massively parallel sequencing (MPS) is more sensitive to shorter fragments, able to detect allele sequences for variations from core motif or flanking regions, and able to detect more markers with a higher discrimination power. In this study, short tandem repeats (STR) and single nucleotide polymorphisms (SNP) from 35 human skeletons were genotyped by MPS platform, and CE method was also used to perform STR genotyping. The results indicated that the detection rates reached 100.00% in 16 of 35 samples with MPS method, while the same 100.00% was reached in only 9 samples with CE. The success rates of MPS were also higher than that of CE method in shared 21 loci (excluding Y-indel, DYS391, and SE33), especially in loci detected by MPS method only. Besides, all SNPs (124 and 90 SNPs in males and females) were detected in 18 samples of 35 samples by MPS method. Some intra-allelic sequence variants were observed in eight loci (D21S11, D8S1179, D5S2800, D3S1358, vWA, D2S1338, D1S1656, D12S391) using MPS technology. Interestingly, there is a sample showing genotyping disagreement in FGA locus. The clone sequencing verified that a "T" deletion discovered in flanking sequence of FGA led to wrong genotyping on Ampliseq Converge. Our results indicated that MPS could be adopted in qualified labs as a supplementary when the DNA of skeletal remains are hard to identify.

Correction to: DNA typing from skeletal remains: a comparison between capillary electrophoresis and massively parallel sequencing platforms.

The name of an author was misspelled. The correct spelling is "Linlin Gao" instead of Linin Gao.

Multi-omic Analyses Reveal Minimal Impact of the CRISPR-Cas9 Nuclease on Cultured Human Cells.

The CRISPR-Cas9 system is a genomic editing tool widely used in basic research and under investigation for potential applications in gene therapies for human diseases. To accomplish genomic editing, the system requires the expression of a prokaryotic DNA endonuclease enzyme, Cas9, in host cells. Previous studies have mainly focused on the specificity of Cas9 on the host genome, and thus it is unclear whether this bacterium-derived enzyme affects the protein homeostasis of host cells. Here we applied multi-omic analyses, including transcriptome, proteome, phosphoproteome, Cas9-associated protein interactome, protein synthesis, and histone epigenetic modification, to investigate the cellular response of human cells upon the expression of Cas9. We demonstrate that Cas9 has minimal impact on host cells. Our assessment of intracellular effects of Cas9 paves a path for its broad applications in biological studies and potential clinical translations.

A Single Intrathecal or Intraperitoneal Injection of CB2 Receptor Agonist Attenuates Bone Cancer Pain and Induces a Time-Dependent Modification of GRK2.

The objective of this study was to explore the potential role of G-protein-coupled receptor kinase 2 (GRK2) in the progression of cannabinoid 2 receptor (CB2) agonist-induced analgesic effects of bone cancer pain. Female Sprague-Dawley rats, weighing 160-180 g, were utilized to establish a model of bone cancer pain induced by intra-tibia inoculation of Walker 256 mammary gland carcinoma cells. JWH-015, a selective CB2 agonist, was injected intrathecally or intraperitoneally on postoperative day 10. Bone cancer-induced pain behaviors-mechanical allodynia and ambulatory pain-were assessed on postoperative days -1 (baseline), 4, 7, and 10 and at post-treatment hours 2, 6, 24, 48, and 72. The expressions of spinal CB2 and GRK2 protein were detected by Western Blotting on postoperative days -1 (baseline), 4, 7, and 10 and at post-treatment hours 6, 24, and 72. The procedure produced prolonged mechanical allodynia, ambulatory pain, and different changes in spinal CB2 and GRK2 expression levels. Intrathecal or intraperitoneal administration of JWH-015 alleviated the induced mechanical allodynia and ambulatory pain, and inhibited the downregulation of spinal GRK2 expression. These effects were in a time-dependent manner and reversed by pretreatment of CB2 selective antagonist AM630. The results affirmed CB2 receptor agonists might serve as new treatment targets for bone cancer pain. Moreover, spinal GRK2 was an important regulator of CB2 receptor agonist-analgesia pathway.

The role of Tet3 DNA dioxygenase in epigenetic reprogramming by oocytes.

Sperm and eggs carry distinctive epigenetic modifications that are adjusted by reprogramming after fertilization. The paternal genome in a zygote undergoes active DNA demethylation before the first mitosis. The biological significance and mechanisms of this paternal epigenome remodelling have remained unclear. Here we report that, within mouse zygotes, oxidation of 5-methylcytosine (5mC) occurs on the paternal genome, changing 5mC into 5-hydroxymethylcytosine (5hmC). Furthermore, we demonstrate that the dioxygenase Tet3 (ref. 5) is enriched specifically in the male pronucleus. In Tet3-deficient zygotes from conditional knockout mice, paternal-genome conversion of 5mC into 5hmC fails to occur and the level of 5mC remains constant. Deficiency of Tet3 also impedes the demethylation process of the paternal Oct4 and Nanog genes and delays the subsequent activation of a paternally derived Oct4 transgene in early embryos. Female mice depleted of Tet3 in the germ line show severely reduced fecundity and their heterozygous mutant offspring lacking maternal Tet3 suffer an increased incidence of developmental failure. Oocytes lacking Tet3 also seem to have a reduced ability to reprogram the injected nuclei from somatic cells. Therefore, Tet3-mediated DNA hydroxylation is involved in epigenetic reprogramming of the zygotic paternal DNA following natural fertilization and may also contribute to somatic cell nuclear reprogramming during animal cloning.

[Changes of Mu-opioid receptor and neuron-restrictive silencer factor in periaquductal gray in mouse models of remifentanil-induced postoperative hyperalgesia].

OBJECTIVE: To determine the changes of Mu-opioid receptor (Mor) and neuron-restrictive silencer factor (NRSF) in periaquductal gray (PAG) in mouse models of remifentanil-induced postoperative hyperalgesia. METHODS: Thirty-two Kun-Ming mice were randomly divided into 4 groups (8 mice in each group): Group C (mice underwent a sham procedure and saline was infused subcutaneously over a period of 30 min), Group I (mice underwent a surgical incision and the same volume of saline), Group R (mice underwent a sham procedure and remifentanil was infused subcutaneously at the moment of surgical incision over a period of 30 min), and group IR (mice underwent a surgical incision and remifentanil). Paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) tests were performed 24 h before the operation and 2, 6, 24, and 48 h after the operation. The specimens were collected after behavioral testings at 48 h. The expressions of Mor and NRSF in mice's PAG neurons were determined by Western blot. RESULTS: Mechanical allodynia and thermal hyperalgesia developed in Group I, R and IR (P<0.01). Intraoperative infusion of remifentanil enhanced mechanical allodynia and thermal hyperalgesia in mice with planta incision (P<0.01). In Group R and Group IR, the expression of Mor was significantly lower (P<0.01) and NRSF was significantly higher (P<0.01) when compared with Group C and Group I. CONCLUSION: Intraoperative infusion of remifentanil induces postoperative hyperalgesia in mouse models, accompanied with decreased expressions of Mor and increased of NRSF level in PAG neurons, which may be involved in remifentanil induced hyperalgesia.

Body fluids should be identified before estimating the time since deposition (TsD) in microbiome-based stain analyses for forensics.

Identification and the time since deposition (TsD) estimation of body fluid stains from a crime scene could provide valuable information for solving the cases and are always difficult for forensics. Microbial characteristics were considered as a promising biomarker to address the issues. However, changes in the microbiota may damage the specific characteristics of body fluids. Correspondingly, incorrect body fluid identification may result in inaccurate TsD estimation. The mutual influence is not well understood and limited the codetection. In the current study, saliva, semen, vaginal secretion, and menstrual blood samples were exposed to indoor conditions and collected at eight time points (from fresh to 30 days). High-throughput sequencing based on the 16S rRNA gene was performed to characterize the microbial communities. The results showed that a longer TsD could decrease the discrimination of different body fluid stains. However, the accuracies of identification still reached a quite high value even without knowing the TsD. Correspondingly, the mean absolute error (MAE) of TsD estimation significantly increased without distinguishing the types of body fluids. The predictive TsD of menstrual blood reached a quite low MAE (1.54 ± 0.39 d). In comparison, those of saliva (6.57 ± 1.17 d), semen (6.48 ± 1.33 d), and vaginal secretion (5.35 ± 1.11 d) needed to be further improved. The great effect of individual differences on these stains limited the TsD estimation accuracy. Overall, microbial characteristics allow for codetection of body fluid identification and TsD estimation, and body fluids should be identified before estimating TsD in microbiome-based stain analyses.IMPORTANCEEmerged evidences suggest microbial characteristics could be considered a promising tool for identification and time since deposition (TsD) estimation of body fluid stains. However, the two issues should be studied together due to a potential mutual influence. The current study provides the first evidence to understand the mutual influence and determines an optimal process for codetection of identification and TsD estimation for unknown stains for forensics. In addition, we involved aged stains into our study for identification of body fluid stains, rather than only using fresh stains like previous studies. This increased the predictive accuracy. We have preliminary verified that individual differences in microbiotas limited the predictive accuracy of TsD estimation for saliva, semen, and vaginal secretion. Microbial characteristics could provide an accurate TsD estimation for menstrual blood. Our study benefits the comprehensive understanding of microbiome-based stain analyses as an essential addition to previous studies.

Enthesitis and Dactylitis Resolution with Risankizumab for Active Psoriatic Arthritis: Integrated Analysis of the Randomized KEEPsAKE 1 and 2 Trials.

INTRODUCTION: The presence (vs absence) of enthesitis/dactylitis is associated with greater psoriatic arthritis (PsA) activity and reduced health-related quality of life. Risankizumab, an interleukin 23 antagonist, demonstrated superior treatment efficacy over placebo in patients with PsA, including enthesitis/dactylitis. Herein, we report the efficacy of risankizumab on complete resolution of enthesitis and/or dactylitis and improvements in patient-reported outcomes in patients with PsA. METHODS: This integrated post hoc analysis of data from KEEPsAKE 1 and KEEPsAKE 2 included patients with baseline enthesitis (Leeds Enthesitis Index > 0) and/or dactylitis (Leeds Dactylitis Index > 0). Efficacy outcomes at weeks 24 and 52 included proportion of patients achieving enthesitis and/or dactylitis resolution and minimal clinically important differences (MCID) in pain, Health Assessment Questionnaire-Disability Index, and Functional Assessment of Chronic Illness Therapy-Fatigue. RESULTS: Of 1407 patients, approximately 63%, 28%, and 20% had baseline enthesitis, dactylitis, and both enthesitis/dactylitis, respectively. At week 24, higher response rates were observed for risankizumab vs placebo for resolution of enthesitis, dactylitis, and both enthesitis/dactylitis (differences of 13.9%, 16.9%, and 13.3%, respectively; p < 0.05). By week 52, risankizumab treatment resulted in complete resolution of enthesitis, dactylitis, and both enthesitis and dactylitis in 55.0%, 76.1%, and 52.3% of patients; similar resolution rates occurred among patients who switched from placebo to risankizumab. Among risankizumab-treated patients who achieved resolution of enthesitis and/or dactylitis, MCIDs were also attained in patient-reported pain, disability, and fatigue at week 24 (all p < 0.05; except fatigue in patients with resolution of both enthesitis/dactylitis); responses were sustained through week 52. CONCLUSIONS: Higher proportions of risankizumab-treated (vs placebo-treated) patients achieved enthesitis and/or dactylitis resolution and meaningful improvements in patient-reported outcomes at week 24 and generally sustained responses at week 52. Thus, risankizumab may result in sustained alleviation of PsA-related pathognomonic musculoskeletal lesions of enthesitis/dactylitis. GOV IDENTIFIERS: NCT03675308, and NCT03671148.

Adenine base editing-mediated exon skipping restores dystrophin in humanized Duchenne mouse model.

Duchenne muscular dystrophy (DMD) affecting 1 in 3500-5000 live male newborns is the frequently fatal genetic disease resulted from various mutations in DMD gene encoding dystrophin protein. About 70% of DMD-causing mutations are exon deletion leading to frameshift of open reading frame and dystrophin deficiency. To facilitate translating human DMD-targeting CRISPR therapeutics into patients, we herein establish a genetically humanized mouse model of DMD by replacing exon 50 and 51 of mouse Dmd gene with human exon 50 sequence. This humanized mouse model recapitulats patient's DMD phenotypes of dystrophin deficiency and muscle dysfunction. Furthermore, we target splicing sites in human exon 50 with adenine base editor to induce exon skipping and robustly restored dystrophin expression in heart, tibialis anterior and diaphragm muscles. Importantly, systemic delivery of base editor via adeno-associated virus in the humanized male mouse model improves the muscle function of DMD mice to the similar level of wildtype ones, indicating the therapeutic efficacy of base editing strategy in treating most of DMD types with exon deletion or point mutations via exon-skipping induction.

Development of deaminase-free T-to-S base editor and C-to-G base editor by engineered human uracil DNA glycosylase.

DNA base editors enable direct editing of adenine (A), cytosine (C), or guanine (G), but there is no base editor for direct thymine (T) editing currently. Here we develop two deaminase-free glycosylase-based base editors for direct T editing (gTBE) and C editing (gCBE) by fusing Cas9 nickase (nCas9) with engineered human uracil DNA glycosylase (UNG) variants. By several rounds of structure-informed rational mutagenesis on UNG in cultured human cells, we obtain gTBE and gCBE with high activity of T-to-S (i.e., T-to-C or T-to-G) and C-to-G conversions, respectively. Furthermore, we conduct parallel comparison of gTBE/gCBE with those recently developed using other protein engineering strategies, and find gTBE/gCBE show the outperformance. Thus, we provide several base editors, gTBEs and gCBEs, with corresponding engineered UNG variants, broadening the targeting scope of base editors.

Tracing recent outdoor geolocation by analyzing microbiota from shoe soles and shoeprints even after indoor walking.

The microbial communities on shoe soles and shoeprints could carry microbial information about where someone walked. This is possible evidence to link a suspect in a crime case to a geographic location. A previous study had shown that the microbiota found on shoe soles depend on the microbiota of the soil on which people walk. However, there is a turnover of microbial communities on shoe soles during walking. The impact of microbial community turnover on tracing recent geolocation from shoe soles has not been adequately studied. In addition, it is still unclear whether the microbiota of shoeprints can be used to determine recent geolocation. In this preliminary study, we investigated whether the microbial characteristics of shoe soles and shoeprints can be used to trace geolocation and whether this information can be destroyed by walking on indoor floors. In this study, participants were asked to walk outdoors on exposed soil, then walk indoors on a hard wood floor. High-throughput sequencing of the 16S rRNA gene was performed to characterize the microbial communities of shoe soles, shoeprints, indoor dust, and outdoor soil. Samples of shoe soles and shoeprints were collected at steps 5, 20, and 50 while walking indoors. The PCoA result showed that the samples were clustered by geographic origin. The shoeprint showed a more rapid turnover of microbial community than the shoe sole during indoor walking. The result of FEAST showed that the microbial communities of shoe sole and shoeprint were mainly (shoe sole, 86.21∼92.34 %; shoeprint, 61.66∼90.41 %) from the soil of the outdoor ground where the individual recently walked, and a small portion (shoe sole, 0.68∼3.33 %; shoeprint, 1.43∼27.14 %) from the indoor dust. Based on the matching of microbial communities between geolocation and shoe sole or shoeprint, we were able to infer the recent geolocation of the individual with relatively high accuracy using the random forest prediction model (shoe sole: 100.00 %, shoeprint: 93.33∼100.00 %). Overall, we are able to accurately infer the geolocation of an individual's most recent outdoor walk based on the microbiota of shoe sole and shoeprint, even though these microbiotas show a turnover when walking indoor floor. The pilot study was expected to provide a potential method for tracing recent geolocation of suspects.