Genetic identification of human remains from the Korean War.

The genetic identification of skeletal remains from Chinese People's Volunteers (CPVs) of the Korean War has been challenging because of the degraded DNA samples and the lack of living close relatives. This study established a workflow for identifying CPVs by combining Y-chromosome short tandem repeats (Y-STRs), mitochondrial DNA (mtDNA) hypervariable regions I and II, autosomal STRs (aSTRs), and identity-informative SNPs (iiSNPs). A total of 20 skeletal remains of CPVs and 46 samples from their alleged relatives were collected. The success rate of DNA extraction from human remains was 100%. Based on Y-STRs, six remains shared the same male lineages with their alleged relatives. Meanwhile, mtDNA genotyping supports two remains sharing the same maternal lineages with their alleged relatives. Likelihood ratios (LRs) were further obtained from 27 aSTRs and 94 iiSNPs or 1936 iiSNPs to confirm their relationship. All joint pedigree LRs were >100. Finally, six remains were successfully identified. This pilot study for the systematic genetic identification of CPVs from the Korean War can be applied for the large-scale identification of CPVs in the future.

A bioresponsive diselenide-functionalized hydrogel with cascade catalytic activities for enhanced local starvation- and hypoxia-activated melanoma therapy.

Glutathione (GSH) consumption-enhanced cancer therapies represent important potential cancer treatment strategies. Herein, we developed a new multifunctional diselenide-crosslinked hydrogel with glutathione peroxidase (GPx)-like catalytic activity for GSH depletion-enhanced glucose oxidase (GOx)-mediated tumor starvation and hypoxia-activated chemotherapy. By increasing acid and H2O2 during GOx-induced tumor starvation, the degradation of the multiresponsive scaffold could be promoted, which led to accelerated release of the loaded drugs. Meanwhile, the overproduced H2O2 led to accelerated intracellular GSH consumption under the cascade catalysis of small molecular selenides released from the degraded hydrogel, further enhancing the curative effect of in situ H2O2 and subsequent multimodal cancer treatment. Following the GOx-induced amplification of hypoxia, tirapazamine (TPZ) was transformed into the highly toxic benzotriazinyl radical (BTZ·), exhibiting enhanced antitumor activity. This GSH depletion-augmented cancer treatment strategy effectively boosted GOx-mediated tumor starvation and activated the hypoxia drug, leading to significantly enhanced local anticancer efficacy. STATEMENT OF SIGNIFICANCE: There has been a growing interest in depleting intracellular GSH as a potential strategy for improving ROS-based cancer therapy. Herein, a bioresponsive diselenide-functionalized dextran-based hydrogel with GPx-like catalytic activity was developed for GSH consumption-enhanced local starvation- and hypoxia-activated melanoma therapy. Results showed that the overproduced H2O2 led to accelerated intracellular GSH consumption under the cascade catalysis of small molecular selenides released from the degraded hydrogel, further enhancing the curative effect of in situ H2O2 and subsequent multimodal cancer treatment.

The compensation effect between safety and efficiency in xylem and role in photosynthesis of gymnosperms.

The classical theory of safety-efficiency trade-off is a common theme in plant sciences. Despite safety and efficiency partly compensating for each other physiologically (namely, there is a compensation effect, CE, among traits from the "whole" organism perspective), they are always mathematically described as a trade-off against one another. However, the compensation effect has never been defined and quantified, let alone its role in the xylem water transport and subsequently photosynthesis. Here, we developed an alternative theory to define the CE as a positive relationship between safety and efficiency, and further define a new trade-off index, SETO, that is expressed as CE multiplied by a trade-off factor (differing from the classical average trade-off value). Then, we tested SETO- and CE-photosynthetic rate relationships across different levels based on a common garden experiment using nine conifers and published data for gymnosperms. The results demonstrated that the compensation effect in xylem functions was the dominant force in facilitating photosynthetic rates from species- to phylum-scale. By integrating the compensation effect into the xylem hydraulic functional strategy, our study clearly indicated that the compensation effect is the evolutionary basis for the coordination of xylem hydraulic and photosynthesis physiology.

Evaluation of the performance of Illumina's ForenSeq™ system on serially degraded samples.

The use of next generation sequencing is increasing in the field of forensic genomics. This study assesses the performance of Illumina's MiSeq FGx System in the recovery of forensic genomic sequencing information from degraded and low-template DNA. The analysis involved a sensitivity study where a range of 1 ng to 5 pg of 2800M DNA was utilized. DNA was artificially and systematically degraded by incubating 2800 M DNA at 98°C for 10, 20, 30, 40, 50, 60, 70, 120 and 180 min (resulting in degradation index values ranging from 0.837 to 232.247). The results revealed the detected allele call frequencies were greater than 80% when the DNA input was > = 50 pg or the degradation index was lower than 72.28. The allele balance was lower than 0.6 when the samples were heated for more than 120 min or the input quantity was less than 25 pg. Our data also indicated that the stutter type and ratio depend on the specific locus, while the sequencing run was the most significant factor in noise occurrence. The results of this study will help laboratories to develop workflows for the analysis of challenging samples using the ForenSeq FGx system.

A Simple and Efficient Method of Extracting DNA from Aged Bones and Teeth.

DNA is often difficult to extract from old bones and teeth due to low levels of DNA and high levels of degradation. This study established a simple yet efficient method for extracting DNA from 20 aged bones and teeth (approximately 60 years old). Based on the concentration and STR typing results, the new method of DNA extraction (OM) developed in this study was compared with the PrepFiler™ BTA Forensic DNA Extraction Kit (BM). The total amount of DNA extracted using the OM method was not significantly different from that extracted using the commercial kit (p > 0.05). However, the number of STR loci detected was significantly higher in the samples processed using the OM method than using the BM method (p < 0.05). This study aimed to establish a DNA extraction method for aged bones and teeth to improve the detection rate of STR typing and reduce costs compared to the BM technique.

Antisense Oligonucleotides Targeting Raf-1 Block Japanese Encephalitis Virus In Vitro and In Vivo.

Japanese encephalitis virus (JEV) infections represent a major health concern in Southeast Asia since no effective treatments are available. Recently, several reports have demonstrated that inhibition of certain host cell proteins prevents viral infection. Raf-1 kinase is a central component of many signaling pathways involved in normal cell growth and oncogenic transformation, and Ras/Raf/ERK signaling activation has been observed during viral infections (including JEV infection). In this study, Raf-1 was confirmed to be upregulated by JEV infection, which suggested that Raf-1 might be important for JEV infection and might be a target for novel anti-JEV drugs. To determine the role of Raf-1 during the JEV infection process, antisense oligonucleotides (ASODNs) were used to downregulate Raf-1 expression in JEV-infected baby hamster kidney (BHK-21) cells and African green monkey kidney (Vero) cells. From five ASODNs candidates tested, Raf-1-1 (Raf-1 antisense) significantly downregulated Raf-1 protein expression levels, significantly inhibited cytopathic effect (CPE) in cultured cells, and reduced JEV RNA levels in cell medium without affecting cell viability. Furthermore, it also demonstrated that ASODN Raf-1-1 possessed therapeutic effects by using a lethal JEV infection mouse model. In conclusion, data presented in this report demonstrated that ASODN Raf-1-1 could suppress Raf-1 protein and that Raf-1 inhibition suppressed JEV replication in vitro and in vivo. These data provided evidence for targeting Raf-1 in the development of novel anti-JEV therapies. In addition, Raf-1-1 represents potential drugs that can be adapted for treating JEV infections.

Influenza virus infection induces translocation of apoptosis-inducing factor (AIF) in A549 cells: role of AIF in apoptosis and viral propagation.

It is recognized that influenza virus induces caspase-dependent apoptosis by activating caspase-3. Apoptosis-inducing factor (AIF) is a caspase-independent cell death effector, and its mitochondrial-nuclear translocation plays an important role in apoptosis. It is demonstrated in this study how influenza virus infection can induce caspase-independent apoptosis in the human alveolar epithelial cell line A549. AIF is translocated from the mitochondria to the nucleus in a caspase-independent manner in response to infection with influenza virus. Knockdown of AIF expression by small interfering RNA (siRNA) led to a reduction in virus-infection-induced apoptosis and virus yield. These results indicate that AIF translocation has a role in influenza-virus-induced apoptosis.

Proteomic analysis at the subcellular level for host targets against influenza A virus (H1N1).

Influenza viruses (IVs) trigger a series of intracellular signaling events and induce complex cellular responses from the infected host cell. Accumulating evidence suggests that host cell proteins play an essential role in viral propagation and represent novel antiviral therapeutic targets. Subcellular proteomic technology provides a method for understanding regional differences at the protein level. The present study, which utilized subcellular proteomic technology, aimed to identify host cell proteins involved in influenza virus (HIN1) infection. Two-dimensional gel electrophoresis (2-DE) combined with mass spectrum (MS) was performed on protein extracts from the nuclei, cytoplasm, and mitochondria of infected and control human lung epithelial cells (A549). In total, 112 differentially expressed protein molecules were identified; 80 protein spots were successfully validated using MS. The differential expression of ISG15, MIF, PDCD5, and UCHL1 was confirmed by western blot. Furthermore, antisense oligodeoxyribonucleotide (ODN) targeting ISG15, MIF, PDCD5, and UCHL1 significantly mitigated HIN1 propagation, cytopathic effects, vRNA by RT-qPCR, and rescued cell viability in A549 cells. Taken together, the differentially expressed proteins identified in this study might provide novel targets for anti-influenza drug development.

MicroRNA-141 represses HBV replication by targeting PPARA.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression primarily at the post-transcriptional level and play critical roles in a variety of physiological and pathological processes. In this report, miR-141 was identified to repress HBV expression by screening a small miRNA expressing library and synthetic miR-141 mimics could also significantly suppress HBV expression and replication in HepG2 cells. Bioinformatic analysis and experiment assays indicate that peroxisome proliferator-activated receptor alpha (PPARA) was the target of hsa-miR-141 during this process. Furthermore, knockdown of PPARA by small interfering RNA (siRNA) inhibited HBV replication similar to levels observed for miR-141. Promoter functional analysis indicated that repression of HBV replication by miR-141 mimics or siRNA was mediated by interfering with the HBV promoter functions, consistent with previous studies demonstrating that PPARA regulated HBV gene expression through interactions with HBV promoter regulatory elements. Our results suggest that miR-141 suppressed HBV replication by reducing HBV promoter activities by down-regulating PPARA. This study provides new insights into the molecular mechanisms associated with HBV-host interactions. Furthermore, this information may facilitate the development of novel anti-HBV therapeutic strategies.

Cellular microRNA let-7c inhibits M1 protein expression of the H1N1 influenza A virus in infected human lung epithelial cells.

The influenza virus (IV) triggers a series of signalling events inside host cells and induces complex cellular responses. Studies have suggested that host factors play an essential role in IV replication. MicroRNAs (miRNAs) represent a class of small non-coding RNAs that target mRNAs, triggering either translation repression or RNA degradation. Emerging research suggests that host-derived cellular miRNAs are involved in mediating the host-IV interaction. Using miRNA microarrays, we identified several miRNAs aberrantly expressed in IV-infected human lung epithelial cells (A549). Specifically, miR-let-7c was highly up-regulated in IV-infected A549 cells. PITA and miRanda database screening indicated that the let-7c seed sequence is a perfect complementary sequence match to the 3' untranslated region (UTR) of viral gene M1 (+) cRNA, but not to PB2 and PA. As detected by a luciferase reporter system, let-7c directly targeted the 3'-UTR of M1 (+) cRNA, but not PB2 and PA. To experimentally identify the function of cellular let-7c, precursor let-7c was transfected into A549 cells. Let-7c down-regulated IV M1 expression at both the (+) cRNA and protein levels. Furthermore, transfection with a let-7c inhibitor enhanced the expression of M1. Therefore, let-7c may reduce IV replication by degrading M1 (+) cRNA. This is the first report indicating that cellular miRNA regulates IV replication through the degradation of viral gene (+) cRNA by matching the 3'-UTR of the viral cRNA. These findings suggest that let-7c plays a role in protecting host cells from the virus in addition to its known cellular functions.

Inhibition of Hepatitis B virus replication by phospholipid scramblase 1 in vitro and in vivo.

Human Phospholipid scramblase 1 (PLSCR1) is an α/ß interferon-inducible protein that mediates antiviral activity against RNA viruses including vesicular stomatitis virus (VSV) and encephalomyocarditis virus (EMCV). In the present study, we investigated the antiviral activity of PLSCR1 protein against HBV (Hepatitis B virus). Firstly, PLSCR1 mRNA and protein expression was found to be downregulated in HepG2 cells after HBV infection. Then by performing co-transient-transfection experiments in cells and hydrodynamics-based transfection experiments in mice using a HBV expression plasmid and a PLSCR1 expression plasmid, we found that PLSCR1 inhibited HBV replication in vitro and in vivo through a significant reduction in the synthesis of viral proteins, DNA replicative intermediates and HBV RNAs. We also demonstrated that the antiviral action of PLSCR1 against HBV occurs, partly at least, by activating the Jak/Stat pathway. In conclusion, our results suggest that the expression of PLSCR1 is involved in HBV replication and that PLSCR1 has antiviral activity against HBV.

Antisense oligonucleotides targeting abhydrolase domain containing 2 block human hepatitis B virus propagation.

Hepatitis B virus (HBV) infection is a major health concern worldwide and only a minority of treated patients develop a sustained protective response following a short course of therapy, and most patients require prolonged treatment to suppress viral replication. However, several recent reports showed that inhibition of certain host cell proteins prevented viral infection, specifically the human abhydrolase domain containing 2 (ABHD2) has been confirmed by our previous study to be upregulated in HepG2.2.15 cells but downregulated by lamivudine. These observations suggested that ABHD2 was important for HBV propagation and could be a target of novel anti-HBV drugs. To assess the importance of ABHD2 to the HBV infection process, antisense oligonucleotides (ASODNs) were used to downregulate ABHD2 expression in HepG2.2.15 cells. From 5 ASODNS candidates tested, AB3 significantly downregulated ABHD2 mRNA and protein expression levels. Further, AB3 significantly reduced HBV DNA, hepatitis B surface antigen, and hepatitis B "e" antigen protein expression levels in cell medium without affecting cell viability. These results suggest that downregulation of ABHD2 using ASODNs blocked HBV replication and expression without affecting host cell physiology. Further, data demonstrated an essential role of ABHD2 in HBV propagation, suggesting it can serve as a novel target for anti-HBV drug development.

Fibronectin and asialoglyprotein receptor mediate hepatitis B surface antigen binding to the cell surface.

Both fibronectin and the asialoglycoprotein receptor (ASGPR) have been identified by some investigators as partners for hepatitis B virus (HBV) envelope proteins. Because fibronectin is a natural ligand for ASGPR, we speculated that HBV might attach to ASGPR expressed on the hepatocyte surface via fibronectin. To test this hypothesis, we first confirmed by co-immunoprecipitation that ASGPR, fibronectin and HBsAg bind to each other in HepG2.2.15 cells, and possible binding domains were identified by GST pull-down. In addition, by measuring binding of HBsAg to cells, we found that ASGPR and fibronectin enhanced the binding capability of HBsAg to HepG2 cells, and even to 293T and CHO cells, which normally do not bind HBV. In conclusion, our findings suggest that both fibronectin and ASGPR mediate HBsAg binding to the cell surface, which provides further evidence for the potential roles of these two proteins in mediating HBV binding to liver cells.

Epiregulin as a key molecule to suppress hepatitis B virus propagation in vitro.

Hepatitis B virus (HBV) infection is a major health concern around the world, and only a minority of patients receiving current treatments achieve a sustained response following a short course of therapy. The majority of patients require prolonged therapy to suppress viral replication. However, several recent reports show that inhibiting host-cell proteins can prevent viral infection. Human epiregulin (EREG) was confirmed by our previous study to be up-regulated in HepG2.2.15 cells while down-regulated by lamivudine. Therefore, the question was asked whether inhibiting EREG could prevent viral infection. To address this question, antisense oligonucleiotides (ASODNs) were used to down-regulate EREG expression in HepG2.2.15 cells. We were able to show that HBV propagation in HepG2.2.15 cell culture was reduced in a dose-dependent manner by EREG inhibition. In addition, we found that treatment with ASODNs did not affect HepG2.2.15 cell viability. To probe the role of EREG in HBV replication, the interaction between EREG and HBsAg was also verified using co-immunoprecipitation and GST pull-down assays. We observed that EREG can contribute to HBsAg binding to HepG2 cells. In summary, this study demonstrates that EREG is essential for HBV replication and also provides some evidence for the potential role of EREG in HBsAg binding.

Protocatechuic aldehyde inhibits hepatitis B virus replication both in vitro and in vivo.

Natural compounds provide a large reservoir of potentially active anti-hepatitis B virus (HBV) agents. We examined the direct effects of protocatechuic aldehyde (PA; derived from the Chinese herb, Salvia miltiorrhiza) on HBV replication in HepG2 2.2.15 cell line and duck hepatitis B virus (DHBV) replication in ducklings in vivo. The extracellular HBV DNA, hepatitis B e antigen (HBeAg) and hepatitis B surface antigen (HBsAg) concentrations in cell culture medium were determined by quantitative real-time PCR and ELISA, respectively. DHBV in duck serum was analyzed by dot blot. PA appeared to downregulate the secretion of HBsAg and HBeAg as well as the release of HBV DNA from HepG2 2.2.15 in a dose- and time-dependent manner at concentrations between 24 and 48 microg/mL. PA (25, 50, or 100 mg/kg, intraperitoneally, twice daily) also reduced viremia in DHBV-infected ducks. We provide the first evidence that PA, a novel anti-HBV substance derived from traditional Chinese herb S. miltiorrhiza, can efficiently inhibits HBV replication in HepG2 2.2.15 cell line in vitro and inhibit DHBV replication in ducks in vivo. PA therefore warrants further investigation as a potential therapeutic agent for HBV infections.

Fibronectin is essential for hepatitis B virus propagation in vitro: may be a potential cellular target?

Previous studies in our laboratory strongly suggested that fibronectin was upregulated by hepatitis B virus (HBV) in HepG2.2.15 cells. Report by Budkowska A also indicated that human liver fibronectin could bind HBV in a species-restricted manner. Therefore, it is reasonable to ask whether inhibiting fibronectin expression might have anti-HBV activity and whether fibronectin might be developed as a new potential cellular target for anti-HBV drugs. By using fibronectin antisense oligonucleotide (ASODN), fibronectin antibody, and Protocatechuic aldehyde (PA), we were able to show that HBV productions in HepG2.2.15 cell culture were reduced in a dose-dependent manner by fibronectin inhibition. In addition, we found that treatment with ASODNs, fibronectin antibody, and PA did not affect HepG2.2.15 cell viability. Furthermore, we observed that fibronectin inhibition sensitized HBV to anti-HBV drugs. In summary, this study demonstrates that fibronectin is essential for HBV propagation and also provides some evidences for the potential of fibronectin as a new cellular target for HBV infection therapy.