Bacterial Succession in Microbial Biofilm as a Potential Indicator for Postmortem Submersion Interval Estimation.

Bacteria acts as the main decomposer during the process of biodegradation by microbial communities in the ecosystem. Numerous studies have revealed the bacterial succession patterns during carcass decomposition in the terrestrial setting. The machine learning algorithm-generated models based on such temporal succession patterns have been developed for the postmortem interval (PMI) estimation. However, the bacterial succession that occurs on decomposing carcasses in the aquatic environment is poorly understood. In the forensic practice, the postmortem submersion interval (PMSI), which approximately equals to the PMI in most of the common drowning cases, has long been problematic to determine. In the present study, bacterial successions in the epinecrotic biofilm samples collected from the decomposing swine cadavers submerged in water were analyzed by sequencing the variable region 4 (V4) of 16S rDNA. The succession patterns between the repeated experimental settings were repeatable. Using the machine learning algorithm for establishing random forest (RF) models, the microbial community succession patterns in the epinecrotic biofilm samples taken during the 56-day winter trial and 21-day summer trial were determined to be used as the PMSI predictors with the mean absolute error (MAE) of 17.87 ± 2.48 ADD (≈1.3 day) and 20.59 ± 4.89 ADD (≈0.7 day), respectively. Significant differences were observed between the seasons and between the substrates. The data presented in this research suggested that the influences of the environmental factors and the aquatic bacterioplankton on succession patterns of the biofilm bacteria were of great significance. The related mechanisms of such influence need to be further studied and clarified in depth to consider epinecrotic biofilm as a reliable predictor in the forensic investigations.

Production of Prebiotic Xylooligosaccharides via Dilute Maleic Acid-Mediated Xylan Hydrolysis Using an RSM-Model-Based Optimization Strategy.

Xylooligosaccharides (XOS) are functional feed additives that are attracting growing commercial interest owing to their excellent ability to modulate the composition of the gut microbiota. The acid hydrolysis-based processing of xylan-containing materials has been proposed to represent a cost-effective approach to XOS preparation, with organic acids being preferable in this context. As such, in the present study, maleic acid was selected as a mild, edible organic acid for use in the hydrolysis of xylan to produce XOS. A response surface methodology (RSM) approach with a central composite design was employed to optimize maleic acid-mediated XOS production, resulting in a yield of 50.3% following a 15 min treatment with 0.08% maleic acid at 168°C. Under these conditions, the desired XOS degree of polymerization (2-3) was successfully achieved, demonstrating the viability of this using a low acid dose and a high reaction temperature to expedite the production of desired functional products. Moreover, as maleic acid is a relatively stable carboxylic acid, it has the potential to be recycled. These results suggest that dilute maleic acid-based thermal treatment of corncob-derived xylan can achieve satisfactory XOS yields, highlighting a promising and cost-effective approach to XOS production.

LINC01535 Attenuates ccRCC Progression through Regulation of the miR-146b-5p/TRIM2 Axis and Inactivation of the PI3K/Akt Pathway.

lncRNAs, a group of eukaryotic cell genome-encoded transcripts, have been demonstrated to exert a notable impact on tumorigenesis. LINC01535, belonging to the lncRNA family, was reported to have an aberrant expression in certain types of cancers and thus affect cancer progression. Nevertheless, the expression pattern and potential roles of LINC01535 in clear cell renal cell carcinoma (ccRCC) remain to be elucidated. Here, LINC01535 expression was detected in ccRCC by RT-qPCR, cell proliferation by CCK-8 assays, and invasion by transwell assays. Besides, effects of LINC01535 on in vivo tumor growth were investigated by xenograft tumor models. The miR-146b-5p/LINC01535/TRIM2 interaction was evaluated via luciferase reporter assays. This study showed downregulation of LINC01535 in ccRCC. Moreover, LINC01535 upregulation attenuated in vitro ccRCC development and hindered in vivo tumor growth. Furthermore, LINC01535 sponged miR-146b-5p which had a negative correlation with LINC01535, and TRIM2 was a direct target of miR-146b-5p and mediated by LINC01535. Mechanically, LINC01535/miR-146b-5p/TRIM2 axis affected ccRCC progression by mediating the PI3K/Akt signaling. All in all, our observations suggest the LINC01535/miR-146b-5p/TRIM2 axis as a crucial role in ccRCC.

Anti-Tumor Role of CAMK2B in Remodeling the Stromal Microenvironment and Inhibiting Proliferation in Papillary Renal Cell Carcinoma.

The tumor microenvironment (TME) is variable across tumor types and has diverse effects on malignant progression, based on the type and number of infiltrating stromal cells. In particular, TME effector genes and their competitive endogenous RNA (ceRNA) networks play a critical role in regulating malignant tumor progression. However, the core effector molecules involved in TME modulation of kidney renal papillary cell carcinoma (KIRP) are poorly understood. To address this question, a cohort containing 233 KIRP patients was derived from The Cancer Genome Atlas (TCGA) database, and the data were processed using the ESTIMATE algorithm. We further evaluated the relationship between immune scores (ISs) and stromal scores (SSs) and disease progression and found that high SSs were associated with a poor prognosis in KIRP. Differentially expressed genes (DEGs) were therefore screened based on SS scores, resulting in 2509 DEGs, including 1668 mRNAs, 783 long noncoding (lnc)RNAs, and 58 micro (mi)RNAs. DEGs were then filtered using the random variance and subjected to hierarchical clustering using EPCLUST. Weighted gene co-expression network analysis (WGCNA) was used to assess the prognostic capacity of these DEGs and identify target ceRNA networks, and lncRNA GUSBP11/miR-432-5p/CAMK2B in the turquoise module was selected as a promising ceRNA network. From this analysis CAMK2B was selected as the core gene predicted to be involved in stromal TMA regulation. We therefore explored the expression and function of CAMK2B in vitro and in vivo and provide evidence that this protein promotes stromal TME remodulation and inhibits proliferation in KIRP. Lastly, we show that vascular endothelial growth factor (VEGF), transforming growth factor (TGF)ß, and close homolog of L1 (CHL1) act as downstream effectors of CAMK2B in KIRP. Thus, in this study, we show that the TME determines prognosis of KIRP patients via the core effector molecule CAMK2B, which mediates both microenvironmental remodeling and tumor progression. Based on these findings, we propose that remodeling of the stromal microenvironment could represent an improved therapeutic approach relative to immunotherapy for KIRP.

Bilateral inguinal lymphadenectomy using simultaneous double laparoscopies for penile cancer: A retrospective study.

OBJECTIVE: To assess the feasibility, safety, and efficiency of bilateral inguinal lymphadenectomy using simultaneous double laparoscopies for penile cancer. MATERIALS AND METHODS: We reviewed retrospectively the records of 65 patients who underwent inguinal lymph nodes dissection (ILND) for penile cancer from January 2012 to May 2019. Treatments included open ILND (OILND, 19 patients), video-endoscopic inguinal lymphadenectomy (VEIL) using single laparoscopy (S-VEIL, 24 patients), and VEIL using double laparoscopies (D-VEIL, 22 patients). We evaluated the peri-operative and short-term oncological outcomes of the three groups. RESULTS: The mean operative time of D-VEIL (105.91 ± 10.87 minutes) was significantly shorter than the other two groups, OILND shorter than S-VEIL (160.47 ± 13.74 minutes, 191.67 ± 17.80 minutes, respectively) (P < 0.001). Intraoperative blood loss in the S-VEIL and D-VEIL groups were 53.54 ± 8.78 and 48.41 ± 13.22 ml, respectively; they were significantly lower than that of the OILND group (99.74 ± 9.64 ml; P < 0.001). The numbers of unilateral and total lymph nodes harvested were similar in all groups. The complication rates in the S-VEIL group (4.2%) and the D-VEIL group (4.5%) were significantly lower than that in the OILND group (63.2%; P < 0.001). Compared with open surgery (13.53 ± 1.74 days for hospitalization; 11.37 ± 1.92 days for the left side of drain, 11.95 ± 1.84 days for the right side), the two VEIL groups had significantly shorter drainage tube residence time (7.42 ± 2.02 and 7.32 ± 1.52 days, respectively for the left side; 7.63 ± 1.81 and 7.27 ± 1.58 days, respectively for the right side), shorter postoperative hospitalization (9.46 ± 1.64 and 9.00 ± 1.83 days, respectively) (P < 0.001). There were no statistically significant differences in rates of regional recurrence and short-term survival among the three groups. CONCLUSION: Bilateral inguinal lymphadenectomy using double laparoscopies simultaneously can provide adequate oncological outcomes safely and efficiently, and carry significantly lower morbidity than OILND, at a median follow-up of 33.5 months. It is a more time-saving surgical approach for penile cancer patients who need bilateral ILND.

Integrated bioinformatics analysis of the NEDD4 family reveals a prognostic value of NEDD4L in clear-cell renal cell cancer.

The members of the Nedd4-like E3 family participate in various biological processes. However, their role in clear cell renal cell carcinoma (ccRCC) is not clear. This study systematically analyzed the Nedd4-like E3 family members in ccRCC data sets from multiple publicly available databases. NEDD4L was identified as the only NEDD4 family member differentially expressed in ccRCC compared with normal samples. Bioinformatics tools were used to characterize the function of NEDD4L in ccRCC. It indicated that NEDD4L might regulate cellular energy metabolism by co-expression analysis, and subsequent gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. A prognostic model developed by the LASSO Cox regression method showed a relatively good predictive value in training and testing data sets. The result revealed that NEDD4L was associated with biosynthesis and metabolism of ccRCC. Since NEDD4L is downregulated and dysregulation of metabolism is involved in tumor progression, NEDD4L might be a potential therapeutic target in ccRCC.

LncRNA NR2F2-AS1 positively regulates CDK4 to promote cancer cell proliferation in prostate carcinoma.

We in this study investigated the role of lncRNA NR2F2-AS1 in prostate carcinoma (PC). We showed that NR2F2-AS1 was upregulated in PC and positively correlated with CDK4. In PC cells, NR2F2-AS1 overexpression led to upregulated, while NR2F2-AS1 siRNA silencing led to downregulated CDK4. Follow-up study revealed that high levels of NR2F2-AS1 and CDK4 in PC tissues were closely correlated with the poor survival of PC patients. Cell proliferation data showed that NR2F2-AS1 overexpression led to increased, while NR2F2-AS1 siRNA silencing led to proliferation rate of PC cells. Moreover, NR2F2-AS1 also showed positive effects on cell cycle progression. In addition, CDK4 overexpression reduced the effects of NR2F2-AS1 siRNA silencing. Therefore, NR2F2-AS1 positively regulates CDK4 to promote cancer cell proliferation in PC.

Fungal succession during mammalian cadaver decomposition and potential forensic implications.

The necrobiome is the postmortem community that includes bacteria, fungi, arthropods, and other cadaver-associated organisms. It has been suggested as biological evidence for forensic investigation. Fungi form distinctive mildew spots in colonizing decomposing bodies, converting them into moldy cadavers. However, the postmortem fungal community consists of more than these visible species. Characterizing the succession pattern of the fungal community during decomposition is valuable not only for understanding the ecosystem composition of the cadaver decomposition islands but also for contributing to forensic investigations. In the present study, the fungal composition of pig cadavers and succession patterns during decomposition were investigated with high-throughput sequencing. The succession patterns were easier to discern in outdoor cadavers, compared with those that were placed indoors. The metabarcoding approach revealed trends linking particular fungal taxa with specific postmortem intervals (PMIs). Dominant species increased notably in cadavers and soil. Furthermore, the succession of the soil community was driven by the cadaver decomposition. Significant mycoflora differences were observed between environmental and cadaveric soil. The results obtained suggested that postputrefaction mycoflora have considerable potential for PMI estimation, particularly in cases that involve heavily decomposed bodies. In addition, the diversity of fungal communities revealed by the metabarcoding approach allowed us to discriminate the sites of cadaver decomposition, implying that postputrefaction mycoflora may be helpful in identifying the environment in which a cadaver has been placed, or the original location from which a cadaver has been moved. Our results provide an important step towards developing fungal evidence for use in forensic science and add to the growing body of work on postmortem microbial communities.

Potential use of high-throughput sequencing of bacterial communities for postmortem submersion interval estimation.

Microorganisms play vital roles in the natural decomposition of carcasses in aquatic systems. Using high-throughput sequencing techniques, we evaluated the composition and succession of microbial communities throughout the decomposition of rat carcasses in freshwater. A total of 4,428,781 high-quality 16S rRNA gene sequences and 2144 operational taxonomic units were obtained. Further analysis revealed that the microbial composition differed significantly between the epinecrotic (rat skins) and the epilithic (rocks) samples. During the carcass decomposition process, Proteobacteria became the dominant phylum in the epinecrotic, epilithic, and environmental (water) samples, followed by Firmicutes in the epinecrotic samples and Bacteroidetes in the epilithic and water samples. Microbial communities were influenced by numerous environmental factors, such as dissolved oxygen content and conductivity. Our study provides new insight about postmortem submersion interval (PMSI) estimation in aquatic environments.

Capn4 expression is modulated by microRNA-520b and exerts an oncogenic role in prostate cancer cells by promoting Wnt/ß-catenin signaling.

Accumulating evidence reports that Capn4 plays an important role in the development and progression of various malignant cancers. However, whether Capn4 is involved in prostate cancer remains unclear. Therefore, the aim of this study was to investigate the expression, biological function and regulatory mechanism of Capn4 in prostate cancer. Herein, we found that Capn4 was highly expressed in prostate cancer cell lines compared with normal prostate cells. Capn4 gene silencing markedly suppressed the growth, invasion and Wnt/ß-catenin signaling of prostate cancer cells, whereas Capn4 overexpression showed an oncogenic effect. Moreover, silencing of ß-catenin significantly blocked the oncogenic effect of Capn4 overexpression. Bioinformatics analysis predicted that Capn4 was a potential target gene of microRNA-520b (miR-520b), which has been reported as a tumor suppressive miRNA in various cancers. The dual-luciferase reporter assay confirmed that miR-520b directly bound to the 3'-untranslated region of Capn4. Real-time quantitative PCR and Western blot analysis showed that miR-520b negatively regulated Capn4 expression in prostate cancer cells in vitro. Furthermore, we found that miR-520b was significantly downregulated in prostate cancer cell lines and tissues. In addition, miR-520b expression was inversely correlated with Capn4 expression in prostate cancer clinical specimens. Overexpression of miR-520b mimicked the tumor suppressive effect of Capn4 siRNA, whereas inhibition of miR-520b had an oncogenic effect. Restoration of Capn4 significantly blocked the antitumor effect of miR-520b in prostate cancer cells. Overall, our findings demonstrate an oncogenic role of Capn4 in prostate cancer and show that its expression is epigenetically regulated by miR-520b. Our results reveal that suppression of Capn4 by miR-520b inhibits the growth and invasion of prostate cancer cells associated with downregulated Wnt/ß-catenin signaling, indicating an important role of the miR-520b/Capn4/Wnt/ß-catenin regulation axis in the molecular pathogenesis of prostate cancer. Our study suggests that miR-520b and Capn4 may represent potential and novel therapeutic targets for prostate cancer.

pH low insertion peptide mediated cell division cycle-associated protein 1 -siRNA transportation for prostatic cancer therapy targeted to the tumor microenvironment.

Prostate cancer (PCa) is a common malignancy in male urinary system. Cell division cycle-associated protein 1 (CDCA1) is expressed highly in many cancer cells. Yet, whether CDCA1 play an important role in PCa progression is uncertain. pH low insertion peptide (pHLIP), a PH-induced transmembrane structure, can pass through the cell membrane into intracellular in an acidic environment. In this study, we try to confirm the expression status of CDCA1 in the PCa patients' tissues and PCa cell line. In addition, to make the CDCA1-siRNA efficiently targeting the PCa cells, pHLIP and CDCA1-siRNA were combined with disulfide bond to become effector molecules. By the characteristics of the pHLIP allosteric occurring in cancer tissue acidic microenvironment, CDCA1-siRNA may be transported specificity into prostatic cancer cells and released in the cytoplasm. The interference effect of the effector molecules on the CDCA1 was detected in vitro and in vivo. The results showed that CDCA1 was highly expressed in PCa cell line and human PCa clinical samples. Knock down CDCA1 significantly inhibit the growth and promote the apoptosis of prostatic cancer cells. In the intracellular translocation experiment, CDCA1-siRNA could be delivered into cytoplasma at pH 6.2, but not at pH 7.4. In the in vivo test, the tumor size was reduced obviously in the NOD/SCID mice treated with pHLIP-CDCA1-siRNA compared to the CDCA1-siRNA and the bioluminescent signal of Cy5-pHLIP-CDCA1-siRNA was focused detected in the tumor site. Our findings indicated that CDCA1 might be a very key molecule regulating survival and proliferation of PCa. pHLIP-CDCA1-siRNA might be a promising targeting therapy for PCa.

Developing a new nonbinary SNP fluorescent multiplex detection system for forensic application in China.

Nonbinary single-nucleotide polymorphisms (SNPs) are potential forensic genetic markers because their discrimination power is greater than that of normal binary SNPs, and that they can detect highly degraded samples. We previously developed a nonbinary SNP multiplex typing assay. In this study, we selected additional 20 nonbinary SNPs from the NCBI SNP database and verified them through pyrosequencing. These 20 nonbinary SNPs were analyzed using the fluorescent-labeled SNaPshot multiplex SNP typing method. The allele frequencies and genetic parameters of these 20 nonbinary SNPs were determined among 314 unrelated individuals from Han populations from China. The total power of discrimination was 0.9999999999994, and the cumulative probability of exclusion was 0.9986. Moreover, the result of the combination of this 20 nonbinary SNP assay with the 20 nonbinary SNP assay we previously developed demonstrated that the cumulative probability of exclusion of the 40 nonbinary SNPs was 0.999991 and that no significant linkage disequilibrium was observed in all 40 nonbinary SNPs. Thus, we concluded that this new system consisting of new 20 nonbinary SNPs could provide highly informative polymorphic data which would be further used in forensic application and would serve as a potentially valuable supplement to forensic DNA analysis.

Population genetics of 26 Y-STR loci for the Han ethnic in Hunan province, China.

To study the population data of Y-chromosome STRs (Y-STRs) of Han population resided in Hunan province, we analyzed haplotypes of 26 Y-STRs (DYS19, DYS385a/b, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS481, DYS533, DYS549, DYS570, DYS576, DYS635, DYS643, DYS388, DYS449, DYS460, and YGATAH4) in 310 unrelated male individuals using a commercially available Goldeneye® DNA ID 26Y system. The calculated average gene diversity values ranged from 0.4211 to 0.9590 for DYS438 and DYS385a/b loci, respectively. The discriminatory capacity was 96.77 % with 300 observed haplotypes. Population relationships between Hunan Han and eight other populations available from Y-chromosome haplotype reference database (YHRD) were compared. The results showed that the Han population resided in the Hunan district is significantly different from other populations. Our results also indicated that these 26 Y-STR loci were highly genetically polymorphic in the Hunan Han population and of great value in forensic application.

MicroRNA-382 inhibits prostate cancer cell proliferation and metastasis through targeting COUP-TFII.

MicroRNAs (miRNAs) have emerged as important regulators in cancer that are implicated in regulation of various cellular processes. miR-382 has been proposed as a tumor suppressor by several recent studies. However, the function of miR-382 in prostate cancer remains unknown. In this study, we aimed to investigate the potential function of miR-382 in prostate cancer. We found that miR-382 was significantly decreased in prostate cancer specimens and cancer cell lines. The overexpression of miR-382 in prostate cancer cells markedly inhibited cell proliferation, migration, and invasion. In contrast, miR-382 suppression exhibited an opposite effect. Target analysis predicted that chicken ovalbumin upstream promoter transcription factor II (COUP­TFII) was a direct target of miR-382. This prediction was experimentally confirmed by dual-luciferase reporter assay, real-time quantitative polymerase chain reaction, and western blot analysis. Our results further demonstrated that miR-382 inhibited the downstream genes of COUP­TFII, including Snail and matrix metalloproteinase 2 (MMP2). Moreover, the restoration of COUP­TFII expression significantly blocked the inhibitory effect of miR-382 on cell proliferation, migration, and invasion, and Snail expression. Taken together, this study suggests that miR-382 inhibits prostate cancer cell proliferation and metastasis through inhibiting COUP­TFII, representing an important new mechanism for understanding prostate cancer pathogenesis and providing a novel therapeutic candidate target for prostate cancer therapy.

Genetic polymorphisms of 26 Y-STR loci in the Mongolian minority from Horqin district, China.

To study the population data of Y chromosome STR (Y-STRs) of the Mongolian minority population residing in the Horqin district, we analyzed haplotypes of 26 Y-STRs (DYS19, DYS385a/b, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS481, DYS533, DYS549, DYS570, DYS576, DYS635, DYS643, DYS388, DYS449, DYS460, and YGATAH4) in 298 unrelated Chinese Mongolian individuals using the commercially available Goldeneye® DNA ID 26Y system. We also investigated blood stains, saliva spots, semen spots, hair follicles, fingernails, and sweat latent fingerprints from ten healthy males for testing the efficiency of direct amplification of this new Y-STRs system. The calculated average gene diversity values of the Mongolian population ranged from 0.3024 to 0.9510 for the DYS389I and DYS385a/b loci, respectively. The discriminatory capacity was 92.95 % with 277 observed haplotypes using 23 Y-STR loci (DYS19, DYS385a/b, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS481, DYS533, DYS549, DYS570, DYS576, DYS635, DYS643, and YGATAH4). By adding three more Y-STRs (DYS388, DYS449, and DYS460) to the 26Y system, the discriminatory capacity was increased to 94.63 % with a total of 282 observed haplotypes. Population relationships were calculated and compared with seven populations available from the Y chromosome haplotype reference database and data from ten Asian populations published previously. The Mongolian minority population residing in Horqin district is significantly different from other populations. Our results indicated that these 26 Y-STRs were highly genetically polymorphic in the Mongolian group and this contributes greatly to existing Chinese ethnic genetic information. As a result of direct amplification, we have obtained full profile from all blood stains, saliva spots, hair follicles, and fingernails; six semen spots; and one sweat latent fingerprint. It revealed that the 26 Y-STR system was a valuable tool for male sex analysis in forensic field and the kit was highly adaptive to direct amplification of various samples including blood stain, saliva spot, hair follicle, and fingernail.

Potential use of bacterial community succession for estimating post-mortem interval as revealed by high-throughput sequencing.

Decomposition is a complex process involving the interaction of both biotic and abiotic factors. Microbes play a critical role in the process of carrion decomposition. In this study, we analysed bacterial communities from live rats and rat remains decomposed under natural conditions, or excluding sarcosaphagous insect interference, in China using Illumina MiSeq sequencing of 16S rRNA gene amplicons. A total of 1,394,842 high-quality sequences and 1,938 singleton operational taxonomic units were obtained. Bacterial communities showed notable variation in relative abundance and became more similar to each other across body sites during the decomposition process. As decomposition progressed, Proteobacteria (mostly Gammaproteobacteria) became the predominant phylum in both the buccal cavity and rectum, while Firmicutes and Bacteroidetes in the mouth and rectum, respectively, gradually decreased. In particular, the arrival and oviposition of sarcosaphagous insects had no obvious influence on bacterial taxa composition, but accelerated the loss of biomass. In contrast to the rectum, the microbial community structure in the buccal cavity of live rats differed considerably from that of rats immediately after death. Although this research indicates that bacterial communities can be used as a "microbial clock" for the estimation of post-mortem interval, further work is required to better understand this concept.