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.

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.

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.

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.

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.

Programmable deaminase-free base editors for G-to-Y conversion by engineered glycosylase.

Current DNA base editors contain nuclease and DNA deaminase that enables deamination of cytosine (C) or adenine (A), but no method for guanine (G) or thymine (T) editing is available at present. Here we developed a deaminase-free glycosylase-based guanine base editor (gGBE) with G editing ability, by fusing Cas9 nickase with engineered N-methylpurine DNA glycosylase protein (MPG). By several rounds of MPG mutagenesis via unbiased and rational screening using an intron-split EGFP reporter, we demonstrated that gGBE with engineered MPG could increase G editing efficiency by more than 1500 fold. Furthermore, this gGBE exhibited high base editing efficiency (up to 81.2%) and high G-to-T or G-to-C (i.e. G-to-Y) conversion ratio (up to 0.95) in both cultured human cells and mouse embryos. Thus, we have provided a proof-of-concept of a new base editing approach by endowing the engineered DNA glycosylase the capability to selectively excise a new type of substrate.

Prolonged hypoxia alleviates prolyl hydroxylation-mediated suppression of RIPK1 to promote necroptosis and inflammation.

The prolyl hydroxylation of hypoxia-inducible factor 1α (HIF-1α) mediated by the EGLN-pVHL pathway represents a classic signalling mechanism that mediates cellular adaptation under hypoxia. Here we identify RIPK1, a known regulator of cell death mediated by tumour necrosis factor receptor 1 (TNFR1), as a target of EGLN1-pVHL. Prolyl hydroxylation of RIPK1 mediated by EGLN1 promotes the binding of RIPK1 with pVHL to suppress its activation under normoxic conditions. Prolonged hypoxia promotes the activation of RIPK1 kinase by modulating its proline hydroxylation, independent of the TNFα-TNFR1 pathway. As such, inhibiting proline hydroxylation of RIPK1 promotes RIPK1 activation to trigger cell death and inflammation. Hepatocyte-specific Vhl deficiency promoted RIPK1-dependent apoptosis to mediate liver pathology. Our findings illustrate a key role of the EGLN-pVHL pathway in suppressing RIPK1 activation under normoxic conditions to promote cell survival and a model by which hypoxia promotes RIPK1 activation through modulating its proline hydroxylation to mediate cell death and inflammation in human diseases, independent of TNFR1.

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.

Engineered CRISPR-OsCas12f1 and RhCas12f1 with robust activities and expanded target range for genome editing.

The type V-F CRISPR-Cas12f system is a strong candidate for therapeutic applications due to the compact size of the Cas12f proteins. In this work, we identify six uncharacterized Cas12f1 proteins with nuclease activity in mammalian cells from assembled bacterial genomes. Among them, OsCas12f1 (433 aa) from Oscillibacter sp. and RhCas12f1 (415 aa) from Ruminiclostridium herbifermentans, which respectively target 5' T-rich Protospacer Adjacent Motifs (PAMs) and 5' C-rich PAMs, show the highest editing activity. Through protein and sgRNA engineering, we generate enhanced OsCas12f1 (enOsCas12f1) and enRhCas12f1 variants, with 5'-TTN and 5'-CCD (D = not C) PAMs respectively, exhibiting much higher editing efficiency and broader PAMs, compared with the engineered variant Un1Cas12f1 (Un1Cas12f1_ge4.1). Furthermore, by fusing the destabilized domain with enOsCas12f1, we generate inducible-enOsCas12f1 and demonstate its activity in vivo by single adeno-associated virus delivery. Finally, dead enOsCas12f1-based epigenetic editing and gene activation can also be achieved in mammalian cells. This study thus provides compact gene editing tools for basic research with remarkable promise for therapeutic applications.

Develop a Compact RNA Base Editor by Fusing ADAR with Engineered EcCas6e.

Catalytically inactive CRISPR-Cas13 (dCas13)-based base editors can achieve the conversion of adenine-to-inosine (A-to-I) or cytidine-to-uridine (C-to-U) at the RNA level, however, the large size of dCas13 protein limits its in vivo applications. Here, a compact and efficient RNA base editor (ceRBE) is reported with high in vivo editing efficiency. The larger dCas13 protein is replaced with a 199-amino acid EcCas6e protein, derived from the Class 1 CRISPR family involved in pre-crRNA processing, and conducted optimization for toxicity and editing efficiency. The ceRBE efficiently achieves both A-to-I and C-to-U base editing with low transcriptome off-target in HEK293T cells. The efficient repair of the DMD Q1392X mutation (68.3±10.1%) is also demonstrated in a humanized mouse model of Duchenne muscular dystrophy (DMD) after AAV delivery, achieving restoration of expression for gene products. The study supports that the compact and efficient ceRBE has great potential for treating genetic diseases.

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.

The Effects of Drug Addiction and Detoxification on the Human Oral Microbiota.

Drug addiction can powerfully and chronically damage human health. Detoxification contributes to health recovery of the body. It is well established that drug abuse is associated with poor oral health in terms of dental caries and periodontal diseases. We supposed that drug addiction and detoxification might have significant effects on the oral microbiota. To test the hypothesis, we assessed the effects of drug (heroin and methylamphetamine) addiction/detoxification on the oral microbiota based on 16S rRNA gene sequencing by an observational investigation, including 495 saliva samples from participants. The oral microbial compositions differed between non-users, current and former drug users. Lower alpha diversities were observed in current drug users, with no significant differences between non-users and former drug users. Heroin and METH addiction can cause consistent variations in several specific phyla, such as the enrichment of Acidobacteria and depletion of Proteobacteria and Tenericutes. Current drug users had significantly lower relative abundances of Neisseria subflava and Haemophilus parainfluenzae compared to non-users and former drug users. The result of random forest prediction model suggested that the oral microbiota has a powerful classification potential for distinguishing current drug users from non-users and former drug users. A cooccurrence network analysis showed that current drug users had more complex oral microbial networks and lower functional modularity. Overall, our study suggested that drug addiction may damage the balance of the oral microbiota. These results may have benefits for further understanding the effects of addiction-related oral microbiota on the health of drug users and promoting the microbiota to serve as a potential tool for accurate forensic identification. IMPORTANCE Drug addiction has serious negative consequences for human health and public security. The evidence indicates that drug abuse can cause poor oral health. In the current study, we observed that drug addiction caused oral microbial dysbiosis. Detoxication have positive effects on the recovery of oral microbial community structures to some extent. Understanding the effects of drug addiction and detoxification on oral microbial communities will promote a more rational approach for recovering the oral function and health of drug users. Furthermore, specific microbial species might be considered biomarkers that could provide information regarding drug abuse status for saliva left at crime scenes. To the best of our knowledge, this is the first report on the role of the oral microbiota in drug addiction and detoxification. Our findings give new clues to understand the association between drug addiction and oral health.

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.

Mini-dCas13X-mediated RNA editing restores dystrophin expression in a humanized mouse model of Duchenne muscular dystrophy.

Approximately 10% of monogenic diseases are caused by nonsense point mutations that generate premature termination codons (PTCs), resulting in a truncated protein and nonsense-mediated decay of the mutant mRNAs. Here, we demonstrate a mini-dCas13X-mediated RNA adenine base editing (mxABE) strategy to treat nonsense mutation-related monogenic diseases via A-to-G editing in a genetically humanized mouse model of Duchenne muscular dystrophy (DMD). Initially, we identified a nonsense point mutation (c.4174C>T, p.Gln1392*) in the DMD gene of a patient and validated its pathogenicity in humanized mice. In this model, mxABE packaged in a single adeno-associated virus (AAV) reached A-to-G editing rates up to 84% in vivo, at least 20-fold greater than rates reported in previous studies using other RNA editing modalities. Furthermore, mxABE restored robust expression of dystrophin protein to over 50% of WT levels by enabling PTC read-through in multiple muscle tissues. Importantly, systemic delivery of mxABE by AAV also rescued dystrophin expression to averages of 37%, 6%, and 54% of WT levels in the diaphragm, tibialis anterior, and heart muscle, respectively, as well as rescued muscle function. Our data strongly suggest that mxABE-based strategies may be a viable new treatment modality for DMD and other monogenic diseases.

Application of Intravenous Anesthesia in Laparoscopic Hiatal Hernia Repair of Children.

In order to solve the stress problem in laparoscopic hiatal hernia repair of children, improve surgical safety, and reduce surgical risk, this study compared the perioperative changes of epinephrine, norepinephrine, IL-6, IL-10, and hemodynamics in children undergoing laparoscopic surgery under intravenous general anesthesia and general anesthesia combined with an epidural block. In this study, 40 children aged 1-3 years who planned to undergo laparoscopic ortopexy and those who planned to undergo laparoscopic high ligation of hernia sac, aged 23.84 1.6 months and weighed 14.9 1.1 kg, were randomly divided into general anesthesia combined with the epidural block group (group A) and a total intravenous anesthesia group (group B), with 20 subjects in each group. The results are as follows: There were no differences in age, gender, body weight, anesthesia time, pneumoperitoneum duration, and functional time between the two groups. Cytokines: Compared with T0, the levels of IL-6 in T2, T3, T4, and T5 groups were significantly increased (P < 0.01). IL-10 levels: T2, T3, T4, and T5 groups were further increased, and the difference was statistically significant compared with T0 (P < 0.01). There was no difference between groups (P > 0.05). The recovery time in group B was shorter than that in group A (P < 0.01), and the total amount of propofol and fentanyl in group B was less than that in group A (P < 0.01). Through research on intravenous anesthesia treatment, it has been proved that total intravenous anesthesia can relieve perioperative pressure, reduce intravenous injection, and reduce the recovery time of children. However, its effect on cytokines is not obvious, so intravenous anesthesia is the most appropriate anesthesia mode in laparoscopic hiatal hernia repair surgery, which has practical significance.

Does Cyberostracism Reduce Prosocial Behaviors? The Protective Role of Psychological Resilience.

To reduce the negative consequences of cyberostracism on prosocial behaviors, we developed a coping strategy based on psychological resilience, and revealed its effectiveness in combating the adverse effects of cyberostracism on prosocial behavior through two studies. Study 1 demonstrated that psychological resilience could mitigate the negative impact of cyberostracism on prosocial behaviors through experimental manipulation. By targeting continuously ostracized people with low resilience for an online self-help resilience intervention program, Study 2 confirmed that psychological resilience was effective in alleviating the detrimental effects of cyberostracism. These studies not only help us to recognize the negative effects of cyberostracism, but also extend Williams' temporal need-threat model of ostracism in the context of online ostracism. As emerging technologies represent a promising new approach to intervention delivery, the most valuable contribution of this study is that we developed an online self-help psychological resilience intervention program that showed encouraging therapeutic effects and advantages for assisting in caring for a larger population of people who are at elevated risk for being cyberostracized.

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.

Predicting the postmortem interval of burial cadavers based on microbial community succession.

Previous studies have demonstrated that microbial community succession during the decomposition of cadavers could be used to estimate the postmortem interval (PMI). However, the vast majority of the existing studies focused on exposed cadavers. In fact, burial cadavers are common scenarios for forensic investigations. In this study, the microbial communities from gravesoil, rectum and skin of burial SD rat cadavers during decomposition were characterized using 16S rRNA gene high-throughput sequencing. We predicted PMI based on the microbial community succession. Obvious differences in microbial community structures were observed between different stages of decomposition. Later decay stages had a lower alpha diversity compared to earlier decay stages. Significant linear relationships between similarities of the microbial communities and postmortem intervals were observed, manifesting regular succession over the course of decomposition. Furthermore, we combined random forest models with postmortem microbial features to predict PMI. The model explained 86.83%, 84.55% and 81.67% of the variation in the microbial community, with a mean absolute error of 1.82, 2.06 and 2.13 days within 60 days of decomposition for gravesoil, rectum and skin of burial cadavers, respectively. Overall, our results suggested that postmortem microbial community data could serve as a potential forensic tool to estimate accurate PMI of burial cadavers.