Application of a newly constructed NGS panel with 45 X-linked microhaplotypes demonstrates the unique value of X-MH for kinship testing and mixture analysis.

X-linked microhaplotypes (X-MHs) have the potential to be a valuable supplementary tool in complex kinship identification or the resolution of DNA mixtures, because they bring together the distinctive genetic pattern of X chromosomal markers and the benefits of microhaplotypes (MHs). In this study, we used the 1000 Genome database to screen and select 63 X-MHs; 18 MHs were filtered out though a batch sequencing assessment of the DNA samples collected from 112 unrelated Chinese Han individuals. The resulting 45-plex panel performed well in comprehensive assessments including repeatability, sensitivity, species specificity, resistance to PCR inhibitors or degradation, mutation rate, and accuracy in detecting DNA mixture samples. The minimum amount of DNA template that can be tested with this panel is 0.5 ng. Additionally, the alleles of the minor contributor can be accurately detected when the mixture rate is larger than 1:9 in female-male mixture or 1:19 in male-male mixture. Then, we calculated population parameters on each MH based on the allele frequency data obtained from the sequence results of the aforementioned 112 unrelated samples. Combining these parameters on each MH, it can be calculated that TDPm, TDPf, CPET, CPEDFM, CPEDFF and CNCEP3 of the 45-plex system were 1-8.99×10-13, 1-1.62×10-19, 0.9999999995, 0.9999981, 0.9955, 0.9999971 and 0.99940, respectively, indicating that the panel is capable in personal identification and parentage testing. To reveal the unique advantage of X-MHs in the analyses of complex kinship and male DNA mixture, further assessments were made. For complex kinship identification, 22 types of individual pairs with different second-degree kinship were simulated and different types of likelihood ratios (LR) were calculated for each. The results revealed that the panel can achieve accuracy of approximately 70 %∼80 % when dividing each of the three types of second-degree kinships into three or four groups. Theoretically, such sub-division cannot be done by using independent autosomal markers. For male DNA mixture analysis without suspects, the maximum likelihood ratio strategy was derived and employed in the estimation of the number of male contributors (NOMC). Simulations were conducted to verify the efficacy of the 45-plex panel in the field and to compare it with autosomal markers by assuming the 45 MHs as autosomal ones. The results showed that X-MHs can achieve higher accuracy in the estimation of NOMC than autosomal ones when the mixed males were unrelated. The results highlighted the unique value of X-linked MHs in complex kinship and male mixture analyses.

The association between DNA methylation and human height and a prospective model of DNA methylation-based height prediction.

As a vital anthropometric characteristic, human height information not only helps to understand overall developmental status and genetic risk factors, but is also important for forensic DNA phenotyping. We utilized linear regression analysis to test the association between each CpG probe and the height phenotype. Next, we designed a methylation sequencing panel targeting 959 CpGs and subsequent height inference models were constructed for the Chinese population. A total of 11,730 height-associated sites were identified. By employing KPCA and deep neural networks, a prediction model was developed, of which the cross-validation RMSE, MAE and R2 were 5.62 cm, 4.45 cm and 0.64, respectively. Genetic factors could explain 39.4% of the methylation level variance of sites used in the height inference models. Collectively, we demonstrated an association between height and DNA methylation status through an EWAS analysis. Targeted methylation sequencing of only 959 CpGs combined with deep learning techniques could provide a model to estimate human height with higher accuracy than SNP-based prediction models.

Differences of circular RNA expression profiles between monozygotic twins' blood, with the forensic application in bloodstain and saliva.

Monozygotic twins (MZTs) possess identical genomic DNA sequences and are usually indistinguishable through routine forensic DNA typing methods, which can be relevant in criminal and paternity cases. Recently, novel epigenetic methods involving DNA methylation and microRNA analysis have been introduced to differentiate MZTs. In this study, we explore the potential of using epigenetic markers, specifically circular RNAs (circRNAs), a type of non-coding RNA (ncRNA), to identify MZTs, and investigate the unique expression patterns of circRNAs within pairs of MZTs, enabling effective differentiation. Epigenetics regulates gene expression at the post-transcriptional level and plays a crucial role in cell growth and aging. CircRNAs, a recently characterized subclass of ncRNA, have a distinct covalent loop structure without the typical 5' cap or 3' tail. They have been reported to modulate various cellular processes and play roles in embryogenesis and eukaryotic development. To achieve this, we conducted a comprehensive circRNA sequencing analysis (circRNA-seq) using total RNA extracted from the blood samples of five pairs of MZTs. We identified a total of 15,257 circRNAs in all MZTs using circRNA-seq. Among them, 3, 21, 338, and 2967 differentially expressed circRNAs (DEcircRNAs) were shared among five, four, three, and two pairs of MZTs, respectively. Subsequently, we validated twelve selected DEcircRNAs using real-time quantitative polymerase chain reaction (RT-qPCR) assays, which included hsa_circ_0004724, hsa_circ_0054196, hsa_circ_004964, hsa_circ_0000591, hsa_circ_0005077, hsa_circ_0054853, hsa_circ_0054716, hsa_circ_0002302, hsa_circ_0004482, hsa_circ_0001103, novel_circ_0030288 and novel_circ_0056831. Among them, hsa_circ_0005077 and hsa_circ_0004482 exhibited the best performance, showing differences in 7 out of 10 pairs of MZTs. These twelve differentially expressed circRNAs also demonstrated strong discriminative power when tested on saliva samples from 10 pairs of MZTs. Notably, hsa_circ_0004724 displayed differential expression in 8 out of 10 pairs of MZTs in their saliva. Additionally, we evaluated the detection sensitivity, longitudinal temporal stability, and suitability for aged bloodstains of these twelve DEcircRNAs in forensic scenarios. Our findings highlight the potential of circRNAs as molecular markers for distinguishing MZTs, emphasizing their suitability for forensic application.

Prediction of BMI traits in the Chinese population based on the gut metagenome.

BACKGROUND: Identifying individual characteristics based on trace evidence left at a crime scene is crucial in forensic identification. Microbial communities found in fecal traces have high individual specificity and could serve as potential markers for forensic characterization. Previous research has established that predicting body type based on the relative abundance of the gut microbiome is relatively accurate. However, the long-term stability and high individual specificity of the gut microbiome are closely linked to changes at the genome level of the microbiome. No studies have been conducted to deduce body shape from genetic traits. Therefore, in this study, the vital role of gut bacterial community characteristics and genetic traits in predicting body mass index (BMI) was investigated using gut metagenomic data from a healthy Chinese population. RESULTS: Regarding the gut microbial community, the underweight group displayed increased α-diversity in comparison to the other BMI groups. There were significant differences in the relative abundances of 19 species among these three BMI groups. The BMI prediction model, based on the 31 most significant species, showed a goodness of fit (R2) of 0.56 and a mean absolute error (MAE) of 2.09 kg/m2. The overweight group exhibited significantly higher α-diversity than the other BMI groups at the level of gut microbial genes. Furthermore, there were significant variations observed in the single-nucleotide polymorphism (SNP) density of 732 contigs between these three BMI groups. The BMI prediction model, reliant on the 62 most contributing contigs, exhibited a model R2 of 0.72 and an MAE of 1.56 kg/m2. The model predicting body type from 44 contigs correctly identified the body type of 93.55% of the study participants. CONCLUSION: Based on metagenomic data from a healthy Chinese population, we demonstrated the potential of genetic traits of gut bacteria to predict an individual's BMI. The findings of this study suggest the effectiveness of a novel method for determining the body type of suspects in forensic applications using the genetic traits of the gut microbiome and holds great promise for forensic individual identification.

Feature selection with a genetic algorithm can help improve the distinguishing power of microbiota information in monozygotic twins' identification.

Introduction: Personal identification of monozygotic twins (MZT) has been challenging in forensic genetics. Previous research has demonstrated that microbial markers have potential value due to their specificity and long-term stability. However, those studies would use the complete information of detected microbial communities, and low-value species would limit the performance of previous models. Methods: To address this issue, we collected 80 saliva samples from 10 pairs of MZTs at four different time points and used 16s rRNA V3-V4 region sequencing to obtain microbiota information. The data formed 280 inner-individual (Self) or MZT sample pairs, divided into four groups based on the individual relationship and time interval, and then randomly divided into training and testing sets with an 8:2 ratio. We built 12 identification models based on the time interval ( ≤ 1 year or ≥ 2 months), data basis (Amplicon sequence variants, ASVs or Operational taxonomic unit, OTUs), and distance parameter selection (Jaccard distance, Bray-Curist distance, or Hellinger distance) and then improved their identification power through genetic algorithm processes. The best combination of databases with distance parameters was selected as the final model for the two types of time intervals. Bayes theory was introduced to provide a numerical indicator of the evidence's effectiveness in practical cases. Results: From the 80 saliva samples, 369 OTUs and 1130 ASVs were detected. After the feature selection process, ASV-Jaccard distance models were selected as the final models for the two types of time intervals. For short interval samples, the final model can completely distinguish MZT pairs from Self ones in both training and test sets. Discussion: Our findings support the microbiota solution to the challenging MZT identification problem and highlight the importance of feature selection in improving model performance.

Identification Strategy of Biological Half Sibling Relationship.

OBJECTIVES: To compare the application value of the likelihood ratio (LR) method and identity by state (IBS) method in the identification involving half sibling relationships, and to provide a reference for the setting of relevant standards for identification of half sibling relationship. METHODS: (1) Based on the same genetic marker combinations, the reliability of computer simulation method was verified by comparing the distributions of cumulated identity by state score (CIBS) and combined full sibling index in actual cases with the distributions in simulated cases. (2) In different numbers of three genetic marker combinations, the simulation of full sibling, half sibling and unrelated individual pairs, each 1 million pairs, was obtained; the CIBS, as well as the corresponding types of cumulative LR parameters, were calculated. (3) The application value of LR method was compared with that of IBS method, by comparing the best system efficiency provided by LR method and IBS method when genetic markers in different amounts and of different types and accuracy were applied to distinguish the above three relational individual pairs. (4) According to the existing simulation data, the minimum number of genetic markers required to distinguish half siblings from the other two relationships using different types of genetic markers was estimated by curve fitting. RESULTS: (1) After the rank sum test, under the premise that the real relationship and the genetic marker combination tested were the same, there was no significant difference between the simulation method and the results obtained in the actual case. (2) In most cases, under the same conditions, the system effectiveness obtained by LR method was greater than that by IBS method. (3) According to the existing data, the number of genetic markers required for full-half siblings and half sibling identification could be obtained by curve fitting when the system effectiveness reached 0.95 or 0.99. CONCLUSIONS: When distinguishing half sibling from full sibling pairs or unrelated pairs, it is recommended to give preference to the LR method, and estimate the required number of markers according to the identification types and the population data, to ensure the identification effect.

Development and evaluation of a novel panel containing 188 microhaplotypes for 2nd-degree kinship testing in the Hebei Han population.

Distant kinship identification is one of the critical problems in forensic genetics. As a new type of genetic marker defined and discussed in the last decade, the microhaplotype (MH) has drawn much attention in such identification owing to its specific advantages to traditional short tandem repeat (STR) or single nucleotide polymorphism (SNP) markers. In this study, MH markers were screened step by step from the 1000 Genomes Project database, and a novel multiplex panel containing 188 MHs (in which 181 are reported the first time, while 1 was reported in a previous study and the other 6 have partial overlaps with known markers) was constructed for application in 2nd- and 3rd-degree kinship identification. Along with the construction, a novel MH nomenclature was proposed, in which the SNP position information they contained was taken into account to eliminate the possibility that the same locus was named differently interlaboratory. After a series of evaluations, the panel was shown to have good sequencing accuracy, high sensitivity, species specificity, and resistance to anti-PCR inhibitors or degradation. Population data of the 188 MHs were calculated based on the genetic information of 221 unrelated Hebei Han individuals, and the effective number of alleles (Ae) ranged from 2.0925 to 8.2634 (with an average of 2.9267). For the whole system, the cumulative matching probability (CMP), the cumulative power of exclusion in paternity testing of duos (CPEduo) and that of trios (CPEtrio) reached 2.8422 × 10-137, 1-1.3109 × 10-21, and 1-2.8975 × 10-39, respectively, indicating that this panel was satisfactory for individual identification and paternity testing. Then, the efficiency of the 188 MHs in 2nd- and 3rd-degree kinship testing was studied based on 30 extended families consisting of 179 2nd-degree and 121 3rd-degree relatives, as well as simulations of 0.5 million pairs of those two kinships. The results showed that clear opinions would be given in 83.36% of 2nd-degree identifications with a false rate less than 10-5, when the confirming and excluding thresholds of cumulative likelihood ratio (CLR) were set as 104 and 10-4, respectively. This panel is still not sufficient to solve the problem of 3rd-degree kinship identification alone, and approximately 300 or 870 MH loci would be needed in 2nd- or 3rd-degree kinship identification, respectively, to achieve a system efficiency not less than 0.99 with such a threshold set; such necessary numbers would be used only as a reference in further research.

Preliminary exploratory research on the application value of oral and intestinal meta-genomics in predicting subjects' occupations-A case study of the distinction between students and migrant workers.

Background: In the field of forensic science, accurately determining occupation of an individual can greatly assist in resolving cases such as criminal investigations or disaster victim identifications. However, estimating occupation can be challenging due to the intricate relationship between occupation and various factors, including gender, age, living environment, health status, medication use, and lifestyle habits such as alcohol consumption and smoking. All of these factors can impact the composition of oral or gut microbial community of an individual. Methods and results: In this study, we collected saliva and feces samples from individuals representing different occupational sectors, specifically students and manual laborers. We then performed metagenomic sequencing on the DNA extracted from these samples to obtain data that could be analyzed for taxonomic and functional annotations in five different databases. The correlation between occupation with microbial information was assisted from the perspective of α and ß diversity, showing that individuals belonging to the two occupations hold significantly different oral and gut microbial communities, and that this correlation is basically not affected by gender, drinking, and smoking in our datasets. Finally, random forest (RF) models were built with recursive feature elimination (RFE) processes. Models with 100% accuracy in both training and testing sets were constructed based on three species in saliva samples or on a single pathway annotated by the KEGG database in fecal samples, namely, "ko04145" or Phagosome. Conclusion: Although this study may have limited representativeness due to its small sample size, it provides preliminary evidence of the potential of using microbiome information for occupational inference.

Forensic age estimation from human blood using age-related microRNAs and circular RNAs markers.

Aging is a complicated process characterized by progressive and extensive changes in physiological homeostasis at the organismal, tissue, and cellular levels. In modern society, age estimation is essential in a large variety of legal rights and duties. Accumulating evidence suggests roles for microRNAs (miRNAs) and circular RNAs (circRNAs) in regulating numerous processes during aging. Here, we performed circRNA sequencing in two age groups and analyzed microarray data of 171 healthy subjects (17-104 years old) downloaded from Gene Expression Omnibus (GEO) and ArrayExpress databases with integrated bioinformatics methods. A total of 1,403 circular RNAs were differentially expressed between young and old groups, and 141 circular RNAs were expressed exclusively in elderly samples while 10 circular RNAs were expressed only in young subjects. Based on their expression pattern in these two groups, the circular RNAs were categorized into three classes: age-related expression between young and old, age-limited expression-young only, and age-limited expression-old only. Top five expressed circular RNAs among three classes and a total of 18 differentially expressed microRNAs screened from online databases were selected to validate using RT-qPCR tests. An independent set of 200 blood samples (20-80 years old) was used to develop age prediction models based on 15 age-related noncoding RNAs (11 microRNAs and 4 circular RNAs). Different machine learning algorithms for age prediction were applied, including regression tree, bagging, support vector regression (SVR), random forest regression (RFR), and XGBoost. Among them, random forest regression model performed best in both training set (mean absolute error = 3.68 years, r = 0.96) and testing set (MAE = 6.840 years, r = 0.77). Models using one single type of predictors, circular RNAs-only or microRNAs-only, result in bigger errors. Smaller prediction errors were shown in males than females when constructing models according to different-sex separately. Putative microRNA targets (430 genes) were enriched in the cellular senescence pathway and cell homeostasis and cell differentiation regulation, indirectly indicating that the microRNAs screened in our study were correlated with development and aging. This study demonstrates that the noncoding RNA aging clock has potential in predicting chronological age and will be an available biological marker in routine forensic investigation to predict the age of biological samples.

Antibiotics enhancing drug-induced liver injury assessed for causality using Roussel Uclaf Causality Assessment Method: Emerging role of gut microbiota dysbiosis.

Drug-induced liver injury (DILI) is a disease that remains difficult to predict and prevent from a clinical perspective, as its occurrence is hard to fully explain by the traditional mechanisms. In recent years, the risk of the DILI for microbiota dysbiosis has been recognized as a multifactorial process. Amoxicillin-clavulanate is the most commonly implicated drug in DILI worldwide with high causality gradings based on the use of RUCAM in different populations. Antibiotics directly affect the structure and diversity of gut microbiota (GM) and changes in metabolites. The depletion of probiotics after antibiotics interference can reduce the efficacy of hepatoprotective agents, also manifesting as liver injury. Follow-up with liver function examination is essential during the administration of drugs that affect intestinal microorganisms and their metabolic activities, such as antibiotics, especially in patients on a high-fat diet. In the meantime, altering the GM to reconstruct the hepatotoxicity of drugs by exhausting harmful bacteria and supplementing with probiotics/prebiotics are potential therapeutic approaches. This review will provide an overview of the current evidence between gut microbiota and DILI events, and discuss the potential mechanisms of gut microbiota-mediated drug interactions. Finally, this review also provides insights into the "double-edged sword" effect of antibiotics treatment against DILI and the potential prevention and therapeutic strategies.

A study of strong nucleosomes in the human genome.

Micrococcal nuclease (MNase) is widely used to map nucleosomes. However, nucleosomes are highly dynamic and susceptible to experimental conditions, resulting in extreme variability across nucleosome maps, which complicates the generation of accurate nucleosome organization data. We mapped nucleosomes from different individuals using improved MNase-seq. The improvements included setting different digestion levels (low, medium, high) and naked DNA correction to remove the noise caused by experimental manipulation and comparing maps to obtain the accurate position and occupancy of strong nucleosomes (SNs) in the whole genome. In addition, the characteristics of SNs were further excavated. SNs were enriched in Alu elements and near the centromere of Chr12. SNs contain some specific sequences, and the GC content of SNs is different from that of dynamic nucleosomes. The findings suggest that nucleosome location in the genome and the DNA sequence may affect nucleosome stability.

Characterization of a new L-carnosine synthase mined from deep-sea sediment metagenome.

L-Carnosine is a natural biologically active dipeptide with critical physiological functions, such as antioxidant, antiglycation, and cytoplasmic buffering properties. Direct enzymatic synthesis is a promising way for L-carnosine production. In this study, a new aminopeptidase (gene_236976) with synthetic activity toward L-carnosine was identified by a metagenome mining approach from deep-sea sediment and functionally expressed in Escherichia coli. The enzyme shared a low identity of 14.3% with reported L-carnosine dipeptidase (SmPepD) from Serratia marcescens. ß-Alanine methyl ester was proven to be the best substrate for the synthesis, and no ATP was needed for the enzymatic reaction. The enzyme activity was increased by structure-guided rational design. Only the mutant of G310 site gave positive results, and G310A mutant showed the best performance among the site-direct saturation mutagenesis, indicating that the additional CH3 group of mutant G310A was the main factor affecting the enzymatic activity. The engineered enzyme produced about 10 mM L-carnosine was produced from substrates of 50 mM ß-alanine methyl ester and 50 mM L-histidine, under a tentatively optimized condition. This study enriched the enzyme resources for developing the microbial synthesis process of L-carnosine production.

Genome and transcriptome of Papaver somniferum Chinese landrace CHM indicates that massive genome expansion contributes to high benzylisoquinoline alkaloid biosynthesis.

Opium poppy (Papaver somniferum) is a source of morphine, codeine, and semisynthetic derivatives, including oxycodone and naltrexone. Here, we report the de novo assembly and genomic analysis of P. somniferum traditional landrace 'Chinese Herbal Medicine'. Variations between the 2.62 Gb CHM genome and that of the previously sequenced high noscapine 1 (HN1) variety were also explored. Among 79,668 protein-coding genes, we functionally annotated 88.9%, compared to 68.8% reported in the HN1 genome. Gene family and 4DTv comparative analyses with three other Papaveraceae species revealed that opium poppy underwent two whole-genome duplication (WGD) events. The first of these, in ancestral Ranunculales, expanded gene families related to characteristic secondary metabolite production and disease resistance. The more recent species-specific WGD mediated by transposable elements resulted in massive genome expansion. Genes carrying structural variations and large-effect variants associated with agronomically different phenotypes between CHM and HN1 that were identified through our transcriptomic comparison of multiple organs and developmental stages can enable the development of new varieties. These genomic and transcriptomic analyses will provide a valuable resource that informs future basic and agricultural studies of the opium poppy.

Highly accurate mtGenome haplotypes from long-read SMRT sequencing can distinguish between monozygotic twins.

Discriminating between monozygotic twins (MZ) remains a challenge in the field of forensics globally. It is very difficult to find sequence variants within MZ twins, despite using ultra-deep next generation sequencing (NGS) for nuclear DNA. However, mitochondrial DNA might be a potential marker owing to its higher mutation rate and easier sequencing via NGS. Here, we aimed to introduce a long-read single molecule real-time sequencing (SMRT) strategy, with better continuity and fewer alignment errors, to obtain more accurate mitochondrial genome (mtGenome) sequence on the Sequel platform. Compared to Ion Torrent Personal Genome Machine (PGM), the long-read SMRT sequencing strategy generated highly accurate and mapped circular consensus sequence (CCS) reads and exhibited robust performance in terms of reliable repeatability, consistent coverage pattern, and balanced strands in mtGenome recovery. Moreover, the long-read SMRT strategy exhibited superior ability to not only identify accurate haplotypes but also discover a total of 785 low-level variants within 16 MZ twin pairs with threshold of 2% and 20 CCS reads with Q30 quality. Taken together, our findings suggested the long-read SMRT technology as an appreciable strategy for obtaining accurate mitotypes and providing a promising solution for distinguishing between MZ twin pairs in forensic genetics.

Development of an NGS panel containing 42 autosomal STR loci and the evaluation focusing on secondary kinship analysis.

High-throughput next-generation sequencing (NGS) is a feasible technique to detect considerably more markers and simultaneously obtain length and sequence information in a single reaction. In this study, we developed an NGS panel including 42 commonly used autosomal short tandem repeats (STRs) and amelogenin on the Illumina MiSeq FGx™. Sequencing accuracy was validated by the consistency of 2800M Control DNA detected using the ForenSeq™ DNA Signature Prep Kit and Sanger sequencing. Nomenclature incompatibility was found between NGS-STR and CE-STR typing at 9 loci (D3S3045, D6S477, D7S3048, D9S925, D14S608, D17S1290, D18S535, D21S1270, GATA198B05), despite the correct sequence. The difference was caused by the two different methods of identifying motif sequence and a one-to-one correspondence can be found. We evaluated the panel by investigating consistency, sequencing sensitivity and the effectiveness of the 2nd-degree relationship identification. Herein, we present sequencing results from 58 unrelated individuals of the Hebei Han population. The total discrimination power (TDP) and cumulative probability of exclusion for trio paternity testing (CPEtrio) of the 42 NGS-STR panels reached 1-2.84 × 10-57 and 1-9.87 × 10-21, respectively. By family simulation and likelihood ratio (LR) calculation, this panel was shown to have effectiveness for the 2nd-degree kinship identification similar to the ForenSeq™ DNA Signature Prep Kit and certain advantages compared with it due to the relatively small number of loci. As expected, it provides new data for the development of NGS-STR typing technology.

The glucose-lowering effects of α-glucosidase inhibitor require a bile acid signal in mice.

AIMS/HYPOTHESIS: Bile-acid (BA) signalling is crucial in metabolism homeostasis and has recently been found to mediate the therapeutic effects of glucose-lowering treatments, including α-glucosidase inhibitor (AGI). However, the underlying mechanisms are yet to be clarified. We hypothesised that BA signalling may be required for the glucose-lowering effects and metabolic benefits of AGI. METHODS: Leptin receptor (Lepr)-knockout (KO) db/db mice and high-fat high-sucrose (HFHS)-fed Fxr (also known as Nr1h4)-KO mice were treated with AGI. Metabolic phenotypes and BA signalling in different compartments, including the liver, gut and endocrine pancreas, were evaluated. BA pool profiles were analysed by mass spectrometry. The islet transcription profile was assayed by RNA sequencing. The gut microbiome were assayed by 16S ribosomal RNA gene sequencing. RESULTS: AGI lowered microbial BA levels in BA pools of different compartments in the body, and increased gut BA reabsorption in both db/db and HFHS-fed mouse models via altering the gut microbiome. The AGI-induced changes in BA signalling (including increased activation of farnesoid X receptor [FXR] in the liver and inhibition of FXR in the ileum) echoed the alterations in BA pool size and composition in different organs. In Fxr-KO mice, the glucose- and lipid-lowering effects of AGI were partially abrogated, possibly due to the Fxr-dependent effects of AGI on decelerating beta cell replication, alleviating insulin hypersecretion and improving hepatic lipid and glucose metabolism. CONCLUSIONS/INTERPRETATION: By regulating microbial BA metabolism, AGI elicited diverse changes in BA pool composition in different host compartments to orchestrate BA signalling in the whole body. The AGI-induced changes in BA signalling may be partly required for its glucose-lowering effects. Our study, hence, sheds light on the promising potential of regulating microbial BA and host FXR signalling for the treatment of type 2 diabetes. DATA AVAILABILITY: Sequencing data are available from the BioProject Database (accession no. PRJNA600345; www.ncbi.nlm.nih.gov/bioproject/600345).

A new strategy to confirm the identity of tumour tissues using single-nucleotide polymorphisms and next-generation sequencing.

With growing cancer morbidity, forensics cases in which archived tumour tissues can be used as biological samples are increasing, and an effective method to identify the body source of tumour tissues is needed. Single nucleotide polymorphisms (SNPs) may be a promising biomarker to identify the source of tumour tissues because of their low mutation rate and small amplicon size. Next-generation sequencing techniques offers the ability to detect hundreds of SNPs in a single run. The Precision ID Identity Panel (Thermo Fisher Scientific, Waltham, MA, USA) detects 90 autosomal SNPs for individual identification and 34 lineage-informative SNPs on Y chromosome using the Ion PGM system (Thermo Fisher Scientific). In this study, we evaluated performance of the panel for individual identification of tumour tissues. One hundred and fifty pairs of tumour tissues and corresponding normal tissues were analysed. Loss of heterozygosity was detected only in tumour tissues. The identity-by-state (IBS) scoring system was adopted to identify the body source of tumour tissues. The IBS score, as well as the number of loci with 2 alleles (A2), 1 allele (A1) and 0 alleles (A0) shared, were analysed within each tumour-normal pair, unrelated individual pairs, parent-offspring pairs and full-sibling pairs. According to the probability distribution, threshold of A2 in the range of 69 to 89 could achieve accuracy > 99% in identifying the source of tumour tissues. Thus, we developed a new strategy (process and criteria) to identify the source of tumour tissues that could be used in practice.

Extracellular production of active-form Streptomyces mobaraensis transglutaminase in Bacillus subtilis.

Transglutaminase (TG) from Streptomyces mobaraensis has been widely used in the food industry. It is secreted naturally as an inactive zymogen, which is then activated by the removal of the N-terminal pro-peptide. In this study, the mtg gene from S. mobaraensis was expressed in a food-grade strain of bacterium, Bacillus subtilis. When its native signal peptide was replaced by signal peptide SacB (SPsacB) and the pro-peptide was replaced by that derived from S. hygroscopicus, an extracellular activity of 16.1 U/mg was observed. A modified Saccharomyces cerevisiae vacuolar ATPase subunit (VMA) intein was introduced into the zymogen to simplify its activation process by controlling temperature. When the cleavage site in the C-terminal of VMA was placed between the pro-peptide and core domain, the activation process was carried out at 18 °C. Promoter replacement further increased the enzymatic activity. Finally, the extracellular enzymatic activity reached 2.6 U/mg under the control of the constitutive promoter PyvyD. This is the first report on the extracellular production of active-form Streptomyces TG in B. subtilis without splicing with the cleavage enzyme.

Atorvastatin Targets the Islet Mevalonate Pathway to Dysregulate mTOR Signaling and Reduce ß-Cell Functional Mass.

Statins are cholesterol-lowering agents that increase the incidence of diabetes and impair glucose tolerance via their detrimental effects on nonhepatic tissues, such as pancreatic islets, but the underlying mechanism has not been determined. In atorvastatin (ator)-treated high-fat diet-fed mice, we found reduced pancreatic ß-cell size and ß-cell mass, fewer mature insulin granules, and reduced insulin secretion and glucose tolerance. Transcriptome profiling of primary pancreatic islets showed that ator inhibited the expression of pancreatic transcription factor, mechanistic target of rapamycin (mTOR) signaling, and small G protein (sGP) genes. Supplementation of the mevalonate pathway intermediate geranylgeranyl pyrophosphate (GGPP), which is produced by 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, significantly restored the attenuated mTOR activity, v-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MafA) expression, and ß-cell function after ator, lovastatin, rosuvastatin, and fluvastatin treatment; this effect was potentially mediated by sGP prenylation. Rab5a, the sGP in pancreatic islets most affected by ator treatment, was found to positively regulate mTOR signaling and ß-cell function. Rab5a knockdown mimicked the effect of ator treatment on ß-cells. Thus, ator impairs ß-cell function by regulating sGPs, for example, Rab5a, which subsequently attenuates islet mTOR signaling and reduces functional ß-cell mass. GGPP supplementation could constitute a new approach for preventing statin-induced hyperglycemia.