Structural analysis and in vitro fermentation characteristics of an Avicennia marina fruit RG-I pectin as a potential prebiotic.

Avicennia marina (Forssk.) Vierh. is a highly salt-tolerant mangrove, and its fruit has been traditionally used for treating constipation and dysentery. In this study, a pectin (AMFPs-0-1) was extracted and isolated from this fruit for the first time, its structure was analyzed, and the effects on the human gut microbiota were investigated. The results indicated that AMFPs-0-1 with a molecular weight of 798 kDa had a backbone consisting of alternating →2)-α-L-Rhap-(1→ and →4)-α-D-GalpA-(1→ residues and side chains composed of →3-α-L-Araf-(1→-linked arabinan with a terminal ß-L-Araf, →5-α-L-Araf-(1→-linked arabinan, and →4)-ß-D-Galp-(1→-linked galactan that linked to the C-4 positions of all α-L-Rhap residues in the backbone. It belongs to a type I rhamnogalacturonan (RG-I) pectin but has no arabinogalactosyl chains. AMFPs-0-1 could be consumed by human gut microbiota and increase the abundance of some beneficial bacteria, such as Bifidobacterium, Mitsuokella, and Megasphaera, which could help fight digestive disorders. These findings provide a structural basis for the potential application of A. marina fruit RG-I pectic polysaccharides in improving human intestinal health.

A preliminary study characterizing temporal changes in soil bacterial communities after dismembered bones were buried.

Determining the burial time of skeletal remains is one of the most important issues of forensic medicine. We speculated that the microbiome of gravesoil may be a promising method to infer burial time by virtue of time-dependent. As we know, forensic scientists have established various models to predict the postmortem interval of a decedent based on the changes in body and soil microbiome communities. However, limited data are available on the burial time prediction for bones, especially dismembered bones. In this exploratory study, we initially conducted 16S rRNA amplicon high-throughput sequencing on the burial soil of 10 porcine femurs within a 120-day period and analyzed the changes in soil microbial communities. Compared with the control soil, a higher Shannon index in the microbial diversity of burial soil containing bones was observed. Correlation analysis identified 61 time-related bacterial families and the best subset selection method obtained best subset, containing Thermomonosporaceae, Clostridiaceae, 0319-A21, and Oxalobacteraceae, which were used to construct a simplified multiple linear regression model with a mean absolute error (MAE) of 56.69 accumulated degree day (ADD). An additional random forest model was established based on indicators for the minimum cross-validation error of Thermomonosporaceae, Clostridiaceae, 0319-A21, Oxalobacteraceae, and Syntrophobacteraceae, with an MAE of 55.65 ADD. The produced empirical data in this pilot study provided the evidence of feasibility that the microbial successional changes of burial soil will predict the burial time of dismembered bones and may also expand the current knowledge of the effects of bone burial on soil bacterial communities.

A human identification system for hair shaft using RNA polymorphism.

Hair is one of the common pieces of evidence at crime scenes, with abundant mitochondrial DNA but limited nuclear DNA in its shaft. It also helps to narrow the investigation scope to maternal lineage but fails to provide unique individual information. We assumed that RNA in hair shafts would be an alternative resource used to perform human identification based on the facts that (1) RNA retains the polymorphic information; (2) the multi-copy of RNA in a cell resists degradation as compared to the one-copy of nuclear DNA. In this study, we explored the potential of RNA polymorphism in hair shafts for forensic individual identification. A SNaPshot typing system was constructed using 18 SNPs located on 11 genes (ABCA13, AHNAK, EXPH5, KMT2D, KRT35, PPP1R15A, RBM33, S100A5, TBC1D4, TMC5, TRPV2). The RNA typing system was evaluated for sensitivity, species specificity, and feasibility for aged hair samples. Hair samples from a Shanxi population in China were used for the population study of the system. The detection limit of the assay was 2 ng RNA. The CDP of these 11 genes was 0.999969 in the Shanxi population. We also identified the concordance of the RNA and DNA typing results. In summary, we developed an RNA typing method to perform human identification from hair shafts, which performed as accurately as nuclear DNA typing. Our method provides a potential basis for solving the human identification problem from hair shafts, as well as other biological materials that lack nuclear DNA.

Resolving the dynamics of chrysolaminarin regulation in a marine diatom: A physiological and transcriptomic study.

Diatom containing different active biological macromolecules are thought to be an excellent microbial cell factory. Phaeodactylum tricornutum, a model diatom, is a superb chassis organism accumulating chrysolaminarin with important bioactivities. However, the characteristic of chrysolaminarin accumulation and molecular mechanism of the fluctuated chrysolaminarin in diatom are still unknown. In this study, physiological data and transcriptomic analysis were carried out to clarify the mechanism involved in chrysolaminarin fluctuation. The results showed that chrysolaminarin content fluctuated, from 7.41 % dry weight (DW) to 40.01 % DW during one light/dark cycle, increase by day and decrease by night. The similar fluctuated characteristic was also observed in neutral lipid content. Genes related to the biosynthesis of chrysolaminarin and neutral lipid were up-regulated at the beginning of light-phase, explaining the accumulation of these biological macromolecules. Furthermore, genes involved in carbohydrate degradation, cell cycle, DNA replication and mitochondria-localized ß-oxidation were up-regulated at the end of light phase and at the beginning of dark phase hinting an energy transition of carbohydrate to cell division during the dark period. Totally, our findings provide important information for the regulatory mechanism in the diurnal fluctuation of chrysolaminarin. It would also be of great help for the mass production of economical chrysolaminarin in marine diatom.

Chondroitin Sulfate/Dermatan Sulfate Hybrid Chains from Swim Bladder: Isolation, Structural Analysis, and Anticoagulant Activity.

Glycosaminoglycans (GAGs) with unique structures from marine animals show intriguing pharmacological activities and negligible biological risks, providing more options for us to explore safer agents. The swim bladder is a tonic food and folk medicine, and its GAGs show good anticoagulant activity. In this study, two GAGs, CMG-1.0 and GMG-1.0, were extracted and isolated from the swim bladder of Cynoscion microlepidotus and Gadus morhua. The physicochemical properties, precise structural characteristics, and anticoagulant activities of these GAGs were determined for the first time. The analysis results of the CMG-1.0 and GMG-1.0 showed that they were chondroitin sulfate (CS)/dermatan sulfate (DS) hybrid chains with molecular weights of 109.3 kDa and 123.1 kDa, respectively. They were mainly composed of the repeating disaccharide unit of -{IdoA-α1,3-GalNAc4S-ß1,4-}- (DS-A). The DS-B disaccharide unit of -{IdoA2S-α1,3-GalNAc4S-ß1,4-}- also existed in both CMG-1.0 and GMG-1.0. CMG-1.0 had a higher proportion of CS-O disaccharide unit -{-GlcA-ß1,3-GalNAc-ß1,4-}- but a lower proportion of CS-E disaccharide unit -{-GlcA-ß1,3-GalNAc4S6S-ß1,4-}- than GMG-1.0. The disaccharide compositions of the GAGs varied in a species-specific manner. Anticoagulant activity assay revealed that both CMG-1.0 and GMG-1.0 had potent anticoagulant activity, which can significantly prolong activated partial thromboplastin time. GMG-1.0 also can prolong the thrombin time. CMG-1.0 showed no intrinsic tenase inhibition activity, while GMG-1.0 can obviously inhibit intrinsic tenase with EC50 of 58 nM. Their significantly different anticoagulant activities may be due to their different disaccharide structural units and proportions. These findings suggested that swim bladder by-products of fish processing of these two marine organisms may be used as a source of anticoagulants.

Endoplasmic reticulum-quality control pathway and endoplasmic reticulum-associated degradation mechanism regulate the N-glycoproteins and N-glycan structures in the diatom Phaeodactylum tricornutum.

Tunicamycin inhibits the first step of protein N-glycosylation modification. However, the physiological, transcriptomic, and N-glycomic effects of tunicamycin on important marine diatom Phaeodactylum tricornutum are still unknown. In this study, comprehensive approaches were used to study the effects of tunicamycin stress. The results showed that cell growth and photosynthesis were significantly inhibited in P. tricornutum under the tunicamycin stress. The soluble protein content was significantly decreased, while the soluble sugar and neutral lipid were dramatically increased to orchestrate the balance of carbon and nitrogen metabolisms. The stress of 0.3 µg ml-1 tunicamycin resulted in the differential expression of ERQC and ERAD related genes. The upregulation of genes involved in ERQC pathway, the activation of anti-oxidases and the differential expression of genes related with ERAD mechanism might be important for maintaining homeostasis in cell. The identification of N-glycans, especially complex-type N-glycan structures enriched the N-glycan database of diatom P. tricornutum and provided important information for studying the function of N-glycosylation modification on proteins. As a whole, our study proposed working models of ERQC and ERAD will provide a solid foundation for further in-depth study of the related mechanism and the diatom expression system.

Estimating the time since deposition (TsD) in saliva stains using temporal changes in microbial markers.

Determining the time since deposition (TsD) of traces could be helpful in the investigation of criminal offenses. However, there are no reliable markers and models available for the inference of short-term TsD. The goal of this study was to investigate the potential of the succession pattern of human salivary microbial communities to serve as an efficiency TsD prediction tool in the resolution of the forensic cases. Saliva stains exposed to indoor conditions up to 20 days were collected and analyzed by 16S rRNA profiling using high-throughput sequencing technique. Noticeable differences in microbial composition were observed between different time points, and the indoor exposure time of saliva stains were inversely correlated with alpha diversity estimates across the measured time period. The sequencing results were used to identify TsD-dependent bacterial indicators to regress a generalized random forest model, resulting in a mean absolute deviation (MAD) of 1.41 days. Furthermore, a simplified TsD predictive model was also developed utilizing Enhydrobacter, Paenisporosarcina, and Janthinobacterium by quantitative PCR (qPCR) with a MAD of 1.32 days, and then forensic practice assessment were also performed by using mock samples with a MAD of 3.53 days. In conclusion, this study revealed significant changes in salivary microbial abundance as the prolongation of TsD. It demonstrated that the microbial biomarkers could be invoked as a "clock" for TsD estimation in human dried saliva stains.

Sperm-derived RNAs improve the efficiency of somatic cell nuclear transfer (SCNT) through promoting R-loop formation.

Mammalian sperm and oocytes are haploid cells that carry parental genetic and epigenetic information for their progeny. The cytoplasm of oocytes is also capable of reprograming somatic cells to establish totipotency through somatic cell nuclear transfer (SCNT). However, epigenetic barriers seriously counteract SCNT reprogramming. Here, we found that sperm-derived RNAs elevated chromatin accessibility of nuclear donor cells concurrent with the appearance of increased RNA amount and decreased cell proliferation, instead of activating DNA damage response. Additionally, tri-methylation of lysine 9 on histone H3 (H3K9me3) and the H3K9 methyltransferase SUV39H2 were significantly downregulated by the sperm-derived RNA treatment. Our findings thus raise a fascinating possibility that sperm RNA-induced R-loops may activate gene expression and chromatin structure, thereby promoting SCNT reprogramming.

Single-cell RNA-seq analysis of testicular somatic cell development in pigs.

Spermatogenesis is the process by which diploid male germ cells propagate and differentiate into haploid flagellated spermatozoa. This highly complicated process is dependent on testicular somatic cells maturation. While the role of these somatic cells in spermatogenesis is relatively well established, knowledge about their development and maturation, particularly at the molecular level, is limited. In this study, we profiled the testicular single-cell transcriptomes of Guanzhong black pigs at the age of 7, 30, 60, 90, and 150 days. Five types of Sertoli cells, five types of Leydig cells, and four types of peritubular myoid cells were identified. Histological analysis revealed the changes in proliferation levels and marker gene expressions, and the prion-like protein gene (PRND) was identified as a novel marker for Sertoli cells. Additionally, integrated analyses of porcine and human datasets revealed similarities between human and pig testicular somatic cells. Overall, the data obtained in this study contribute to the understanding of testicular development in pigs as a model species.

Identification of the vaginal secretion donor in mixture stains using polymorphic cSNPs on mRNA biomarkers.

In recent years, RNA profiling has become an important technique in identifying the origin of human body fluids/tissues. Both perpetrators and victims can be identified from stains involving vaginal secretions (VS), such as sperm/VS mixtures left on condoms, bed sheets, or papers, etc. Body fluid specific RNA typing could link the source of body fluids/tissues and the identity of the donor. In this study, we aimed to trace the donor of VS in mixture stains using body fluid-specific mRNA markers and construct a coding single nucleotide polymorphism (cSNP) typing system for VS. We screened 8 VS-specific mRNA biomarkers (MUC4, SFTA2, CYP2A6, MYOZ1, FUT6, ESR1, SPINK5, and SERPINB13) encompassing 18 cSNPs. The RNA obtained from various body fluid/tissue samples was treated with reverse transcription polymerase chain reaction (RT-PCR) and then followed by a multiplex PCR and SNaPshot mini-sequencing assay. The detection limit of the assay was 0.08 ng RNA. For single-source body fluid, the positive cSNP typing was only shown in VS and void in non-VS body fluids/tissues. For laboratory-generated VS-containing mixtures, the minor VS contributor could be successfully detected at a ratio of 1:10-1:500. We also confirmed the concordance of DNA typing and mRNA typing for the cSNPs in this system. In summary, we established an 18-cSNP typing system for VS with high sensitivity and specificity, which could identify both the donor and the tissue origin simultaneously. This was shown to be a powerful tool for identifying the VS donor in those VS-containing mixture stains.

A new set of 20 Multi-InDel markers for forensic application.

Insertion/deletion markers (InDels) become an important marker for forensic medicine because of their compatible typing techniques with STRs and lower mutation rates. Recent years, a new kind of DNA marker named Multi-InDel was reported as characterized by two or more tightly linked InDel loci within a short length of physical position, usually 200-300 nucleotides. Many pieces of research showed that Multi-InDels had excellent application values in ancestry inference and forensic medicine. Since the identical number of insertion/deletion nucleotides of the InDel markers that composing the Multi-InDel marker, the genotypes of most reported Multi-InDels could not be directly typed by capillary electrophoresis (CE) due to the lack of length discrepancy among the composing InDel sequence. In this study, we applied a typing system of 20 Multi-InDels including 41 InDels, whose genotypes could be deduced by CE and assessed their potential applications in forensic medicine. A total of 200 unrelated Chinese Han individuals and five mother-child-father trios with proven paternity with one STR locus transmission incompatibilities from Shanxi province were genotyped by the multiplex system. The results showed that a total of 70 specific alleles were observed, more than three alleles were observed in 19 loci and seven alleles were observed in one locus. The combined probability of exclusion and the combined power of discrimination were 0.992 and 0.99999999993, respectively. This study demonstrates their potential usefulness for individual identification and paternity tests. The development of Multi-InDels provided another genetic tool inherent in higher polymorphic and lower mutation rates.

SETDB1 Regulates Porcine Spermatogonial Adhesion and Proliferation through Modulating MMP3/10 Transcription.

The transition from gonocytes into spermatogonia takes place during the homing process. A subpopulation of undifferentiated spermatogonia in niche then shifts to spermatogonial stem cells (SSCs), accompanied by the self-renewal ability to maintain life-long fertility in males. Enormous changes in cell morphology, gene expression, and epigenetic features have been reported during spermatogenesis. However, little is known about the difference of these features in SSCs during aging. Here, we examined the dynamics of SET domain bifurcated 1 (SETDB1) expression in porcine testes. SETDB1 was expressed in postnatal undifferentiated spermatogonia, while gradually disappeared after being packed within the basal compartment of seminiferous tubules. In addition, the cell-adhesion ability, proliferative activity, and trimethylation of the histone H3 lysine 9 (H3K9me3) level were significantly altered in SETDB1-deficient porcine SSCs. Moreover, the matrix metalloproteinases 3/10 (MMP3/10) was upregulated at both mRNA and protein levels. These results illustrate the significance of SETDB1 in modulating early male germ cell development.

Transcriptome-wide Dynamics of m6A mRNA Methylation During Porcine Spermatogenesis.

Spermatogenesis is a continual process that occurs in the testes, in which diploid spermatogonial stem cells (SSCs) differentiate and generate haploid spermatozoa. This highly efficient and intricate process is orchestrated at multiple levels. N6-methyladenosine (m6A), an epigenetic modification prevalent in mRNAs, is implicated in transcriptional regulation during spermatogenesis. However, the dynamics of m6A modification in non-rodent mammalian species remains unclear. Here we systematically investigated the profile and role of m6A during spermatogenesis in pigs. By analyzing the transcriptomic distribution of m6A in spermatogonia, spermatocytes, and round spermatids, we identified a globally conserved m6A pattern between porcine and murine genes with spermatogenic function. We found that m6A was enriched in a group of genes that specifically encode the metabolic enzymes and regulators. In addition, transcriptomes in porcine male germ cells could be subjected to the m6A modification. Our data showed that m6A played the regulatory roles during spermatogenesis in pigs, which is similar to that in mice. Illustrations of this point were three genes (SETDB1, FOXO1, and FOXO3) that were crucial to the determination of the fate of SSCs. To the best of our knowledge, this study has for the first time uncovered the expression profile and role of m6A during spermatogenesis in large animals and contributes to insights into the intricate transcriptional regulation underlying the lifelong male fertility in non-rodent mammalian species.

Predicting human age by detecting DNA methylation status in hair.

Age prediction is of great importance for criminal investigation and judicial expertise. DNA methylation status is considered a promising method to infer tissue age by virtue of age-dependent changes on methylation sites. In recent years, forensic scientists have established various models to predict the chronological age of blood, saliva, and semen based on DNA methylation status. However, hair-inferred age has not been studied in the field of forensic science. In this study, we measured the methylation statuses of potential age-related CpG sites by using the multiplex methylation SNaPshot method. A total of 10 CpG sites from the LAG3, SCGN, ELOVL2, KLF14, C1orf132, SLC12A5, GRIA2, and PDE4C genes were found to be tightly associated with age in hair follicles. A correlation coefficient above 0.7 was found for four CpG sites (cg24724428 and Chr6:11044628 in ELOVL2, cg25148589 in GRIA2, and cg07547549 in SLC12A5). Among four age-prediction models, the multiple linear regression model consisting of 10 CpG sites provided the best-fitting results, with a median absolute deviation of 3.68 years. It is feasible to obtain both human identification and age information from a single scalp hair follicle. No significant differences in methylation degree were found between different sexes, hair types, or hair colors. In conclusion, we established a method to evaluate chronological age by assessing DNA methylation status in hair follicles.

Identification of coding region SNPs from specific and sensitive mRNA biomarkers for the deconvolution of the semen donor in a body fluid mixture.

mRNA markers provide a very promising method for the identification of human body fluids or tissues in the context of forensic investigations. Previous studies have shown that different body fluids can be distinguished from each other according to their specific mRNA biomarkers. In this study, we evaluated eight semen-specific mRNA markers (KLK3, NKX3-1, CKB, KLK2, PRAC1, SEMG1, TGM4, and SORD) that encompass 12 coding single nucleotide polymorphisms (cSNPs) to identify the semen contributor in a mixed stain. Five highly specific and sensitive mRNA markers for blood, menstrual blood, saliva, vaginal secretions, and skin were also incorporated into the PCR system as body fluid-positive controls. Reverse transcription polymerase chain reaction (RT-PCR), multiplex PCR and SNaPshot mini-sequencing assays were established for the identification of semen-specific mRNA. The amplicon size ranged from 133 to 337 bp. The semen-specific system was examined against blood, menstrual blood, saliva, vaginal secretions, and skin swabs. The eight mRNA biomarkers were semen-specific and could be successfully typed in laboratory-generated mixtures composed of different body fluids supplemented with 1 ng of semen cDNA. This system possessed a high sensitivity that ranged from 1:10-1:100 for detecting trace amounts of semen in semen-containing body fluid mixtures. Additionally, our results demonstrated that the cSNPs polymorphisms included in the mRNA markers were concordant with genomic DNA (gDNA). Despite the presence of other body fluids, the system exhibited high sensitivity and specificity to the semen in the mixture. In future studies, we will add other cSNPs from the semen-specific genes using massively parallel sequencing to further improve our system.

Human sperm tsRNA as potential biomarker and therapy target for male fertility.

Infertility caused by male factors is routinely diagnosed by assessing traditional semen parameters. Growing evidence has indicated that the tsRNAs carried in sperm act as epigenetic factors and potential biomarkers for the assessment of sperm quality. We recently demonstrated that tRNAGln-TTG derived small RNAs played notable roles in the first cleavage of a porcine embryo. However, the function of human sperm tRNAGln-TTG derived small RNAs as a diagnostic biomarker and its role in early embryo development remains unclear. In this study, we found that human sperm tRNAGln-TTG derived small RNAs were highly associated with sperm quality. By microinjecting the antisense sequence into human tripronuclear (3PN) zygotes followed by single-cell RNA-sequencing, we found that human sperm tRNAGln-TTG derived small RNAs participated in the development of a human embryo. Furthermore, Gln-TTGs might influence embryonic genome activation by modulating noncoding RNA processing. These findings demonstrated that human sperm tRNAGln-TTG derived small RNAs could be potential diagnostic biomarkers and could be used as a clinical target for male infertility.

DIP-microhaplotypes: new markers for detection of unbalanced DNA mixtures.

The identification of a suspect in a degraded and unbalanced DNA mixture has been a challenge for the standard short tandem repeat polymorphisms (STR) typing. Several methods have been introduced to solve this problem, such as DIP-STR, DIP-SNP, and SNP-STR markers. In this study, we proposed DIP-microhaplotype (deletion/insertion linked a chain of SNPs) as a kind of new genetic marker to type the unbalanced and degraded DNA mixture. We established the detection method with ten DIP-microhaplotype markers including 26 SNPs using allele-specific multiplex PCR followed by SNaPshot assay. This novel compound marker allows us to detect the minor DNA with a sensitivity of 1:100 to 1:1000 in a DNA mixture of any gender. Most of the DIP-microhaplotype markers had a relatively high probability of informative alleles with an average informative value (I value) of 0.308. In all, we proposed DIP-microhaplotype as a novel type of DNA marker for the detection of minor contributor from unbalanced DNA mixtures. Due to their inherent shorter length, higher polymorphism, and sensitivity, DIP-microhaplotypes are promising markers for the examination of the degraded and unbalanced mixtures in forensic stains or clinical chimeras.

A method of identifying the blood contributor in mixture stains through detecting blood-specific mRNA polymorphism.

In the past decades, messenger RNA (mRNA) biomarkers have been employed to identify the origin of body fluids in forensic medicine. We hypothesized that the polymorphism of mRNA could be applied to identify individuals in mixture samples composed of two body fluids. In this study, we selected five blood-specific mRNA biomarkers of venous blood (SPTB, CD3G, AMICA1, ANK1, and GYPA) that encompass 16 SNPs to identify the mixture contributor(s). Five specific gene markers for menstrual blood, semen, skin, saliva, and vaginal secretions were amplified and typed as body-fluid positive controls. We established the system of multiplex PCR and single base extension (SBE) reaction followed by CE. The amplicon size was between 90bp and 294bp. The peripheral blood specificity was examined against other human body fluids, including saliva, semen, skin, menstrual blood, and vaginal secretion. The 16 SNPs were peripheral blood specific and could be successfully typed in homemade mixtures which are composed of different body fluids with 1 ng peripheral blood mRNA added. This system showed a supersensitivity (1:100) in detecting the trace amount of peripheral blood mixed in other body fluids and a combined discrimination power (CDP) of 0.99929 in Chinese population. It was the first time to establish a method for identifying the blood donors and deconvoluting mixtures through detecting mRNA polymorphism with SNaPshot assay. This peripheral blood specific SNP typing system showed high sensitivity to the typing of blood source specific markers regardless of other body fluids in the mixture.