Biochar enhanced the stability of toluene removal in extracted groundwater amended with nitrate under microaerobic conditions.

Groundwater pollution caused by the leakage of petroleum and various fuel oils is becoming a serious environmental problem. In this study, carbon-based materials including biochar and hydrochar were applied to investigate the effects of additives on the toluene removal in the extracted groundwater under microaerobic condition with the addition of nitrate. Biochar and hydrochar could adsorb toluene, and thus enhance the toluene removal in the system. The toluene removal efficiency was 8.2-8.9 mg/(g·h) at the beginning, and then decreased with time in the control and the hydrochar treatment, while it remained the stable values in the biochar treatment, owing to the fact that biochar could reduce the NO3--N loss by partial denitrification. Moreover, biochar could prompt the growth of toluene-degrading bacteria including Thauera, Rhodococcus, Ideonella and Denitratisoma, which had the capability of denitrification. However, hydrochar could stimulated the growth of denitrifiers without toluene-degrading capacity including Candidatus Competibacter and Ferrovibrio, which might play a key role in the partial denitrification of the system. The findings are helpful for developing remediation techniques of contaminated groundwater.

Experimental study on the effect and mechanism of adipose stem cell-derived exosomes combined with botulinum toxin A on skin trauma in rats.

BACKGROUND: Adipose stem cell-derived exosomes (ADSC-EXO) and botulinum toxin type A (BTX-A) individually showed a therapeutic effect on skin wound repair. AIMS: This study investigated their synergistic effect on promoting skin wound healing in vitro and in vivo and the underlying molecular events. METHODS: ADSCs were isolated from Sprague-Dawley (SD) rats to obtain ADSC-EXO by ultrafiltration and ultracentrifugation and were confirmed using nanoparticle tracking analysis and transmission electron microscopy. Human skin fibroblasts (HSF) were cultured and treated with or without ADSC-EXO, BTX-A, or their combination. Changes in cell phenotypes and protein expression were analyzed using different in vitro assays, and a rat skin wound model was used to assess their in vivo effects. RESULTS: The isolated ADSC-EXO from primarily cultured ADSCs had a circular vesicle shape with a 30-180 nm diameter. Treatment of HSF with ADSC-EXO and/or BTX-A significantly accelerated HSF migration in vitro and skin wound healing in a rat model. Moreover, ADSC-EXO plus BTX-A treatment dramatically induced VEGFA expression but reduced COL III and COL I levels in vivo. ADSC-EXO and/or BTX-A treatment significantly upregulated TGF-ß3 expression on Day 16 after surgery but downregulated TGF-ß1 expression, suggesting that ADSC-EXO plus BTX-A promoted skin wound healing and reduced inflammatory cell infiltration. CONCLUSIONS: The ADSC-EXO plus BTX-A treatment demonstrated a synergistic effect on skin wound healing through upregulation of VEGF expression and the TGF-ß3/TGF-ß1 and COL III/COL I ratio.

Exosomes combined with biosynthesized cellulose conduits improve peripheral nerve regeneration.

Peripheral nerve injury is one of the more common forms of peripheral nerve disorders, and the most severe type of peripheral nerve injury is a defect with a gap. Biosynthetic cellulose membrane (BCM) is a commonly used material for repair and ligation of nerve defects with gaps. Meanwhile, exosomes from mesenchymal stem cells can promote cell growth and proliferation. We envision combining exosomes with BCMs to leverage the advantages of both to promote repair of peripheral nerve injury. Prepared exosomes were added to BCMs to form exosome-loaded BCMs (EXO-BCM) that were used for nerve repair in a rat model of sciatic nerve defects with gaps. We evaluated the repair activity using a pawprint experiment, measurement and statistical analyses of sciatica function index and thermal latency of paw withdrawal, and quantitation of the number and diameter of regenerated nerve fibers. Results indicated that EXO-BCM produced comprehensive and durable repair of peripheral nerve defects that were similar to those for autologous nerve transplantation, the gold standard for nerve defect repair. EXO-BCM is not predicted to cause donor site morbidity to the patient, in contrast to autologous nerve transplantation. Together these results indicate that an approach using EXO-BCM represents a promising alternative to autologous nerve transplantation, and could have broad applications for repair of nerve defects.

Modelling the equilibrium partitioning of low concentrations of airborne volatile organic compounds in human IVF laboratories.

RESEARCH QUESTION: Can volatile organic compounds (VOC) be modelled in an IVF clinical setting? DESIGN: The study performed equilibrium modelling of low concentrations of airborne VOC partitioning from the air phase into the oil cover layer into the water-based culture media and into/onto the embryo (air-oil-water-embryo). The air-phase VOC were modelled based on reported VOC concentrations found in modern assisted reproductive technology (ART) suites, older IVF clinics, and hospitals, as well as at 10 parts per billion (ppb) and 100 ppb for all compounds. The modelling was performed with 23 documented healthcare-specific VOC. RESULTS: Based on the partitioning model, seven compounds (acrolein, formaldehyde, phenol, toluene, acetaldehyde, ethanol and isopropanol) should be of great concern to the embryologist and clinician. Acrolein, formaldehyde, phenol, toluene and acetaldehyde are the VOC with the most potent cytotoxic factor and the highest toxic VOC concentration in media. In addition, ethanol and isopropanol are routinely found in the greatest air-phase concentrations and modelled to have the highest water-based culture concentrations. CONCLUSIONS: The results of the equilibrium partitioning modelling of VOC provides a fundamental understanding of how airborne VOC partition from the air phase and negatively influence human IVF outcomes. The results presented here are based on the theoretical model and the values presented have not yet been measured in a laboratory or clinical setting. High air-phase concentrations and toxic concentrations of VOC in culture media are likely indicators of poor clinical outcomes. Based on this model, improved air quality in IVF laboratories reduces the chemical burden imparted on embryos, which supports findings of improved IVF outcomes with reduced air-phase VOC concentrations.

Construction of Exosomes that Overexpress CD47 and Evaluation of Their Immune Escape.

Our general purpose was to provide a theoretical and practical foundation for the use of exosomes (EXOs) that have high levels of CD47 as stable and efficient drug carriers. Thus, we prepared EXOs from adipose tissue-derived mesenchymal stromal cells (ADMSCs) that had high levels of CD47 (EXOsCD47) and control EXOs (without CD47), and then compared their immune escape in vivo and their resistance to phagocytosis in vitro. Nanoflow cytometry was used to determine the CD47 level in these EXOs, and the amount of EXOsCD47 that remained in rat plasma at 3 h after intraperitoneal injection. Phagocytosis of the EXOs was also determined using in vitro rat macrophage bone marrow (RMA-BM) experiments. Our in vitro results showed that macrophages ingested significantly more control EXOs than EXOsCD47 (p < 0.01), with confirmation by ultra-high-definition laser confocal microscopy. Consistently, our in vivo results showed that rats had 1.377-fold better retention of EXOsCD47 than control EXOs (p < 0.01). These results confirmed that these engineered EXOsCD47 had improved immune escape. Our results therefore verified that EXOsCD47 had increased immune evasion relative to control EXOs, and have potential for use as drug carriers.

The de novo assembly of a European wild boar genome revealed unique patterns of chromosomal structural variations and segmental duplications.

The rapid progress of sequencing technology has greatly facilitated the de novo genome assembly of pig breeds. However, the assembly of the wild boar genome is still lacking, hampering our understanding of chromosomal and genomic evolution during domestication from wild boars into domestic pigs. Here, we sequenced and de novo assembled a European wild boar genome (ASM2165605v1) using the long-range information provided by 10× Linked-Reads sequencing. We achieved a high-quality assembly with contig N50 of 26.09 Mb. Additionally, 1.64% of the contigs (222) with lengths from 107.65 kb to 75.36 Mb covered 90.3% of the total genome size of ASM2165605v1 (~2.5 Gb). Mapping analysis revealed that the contigs can fill 24.73% (93/376) of the gaps present in the orthologous regions of the updated pig reference genome (Sscrofa11.1). We further improved the contigs into chromosome level with a reference-assistant scaffolding method. Using the 'assembly-to-assembly' approach, we identified intra-chromosomal large structural variations (SVs, length >1 kb) between ASM2165605v1 and Sscrofa11.1 assemblies. Interestingly, we found that the number of SV events on the X chromosome deviated significantly from the linear models fitting autosomes (R2  > 0.64, p < 0.001). Specifically, deletions and insertions were deficient on the X chromosome by 66.14 and 58.41% respectively, whereas duplications and inversions were excessive on the X chromosome by 71.96 and 107.61% respectively. We further used the large segmental duplications (SDs, >1 kb) events as a proxy to understand the large-scale inter-chromosomal evolution, by resolving parental-derived relationships for SD pairs. We revealed a significant excess of SD movements from the X chromosome to autosomes (p < 0.001), consistent with the expectation of meiotic sex chromosome inactivation. Enrichment analyses indicated that the genes within derived SD copies on autosomes were significantly related to biological processes involving nervous system, lipid biosynthesis and sperm motility (p < 0.01). Together, our analyses of the de novo assembly of ASM2165605v1 provides insight into the SVs between European wild boar and domestic pig, in addition to the ongoing process of meiotic sex chromosome inactivation in driving inter-chromosomal interaction between the sex chromosome and autosomes.

Electrocatalysis degradation of coal tar wastewater using a novel hydrophobic benzalacetone modified lead dioxide electrode.

Coal tar wastewater is hard to degrade by traditional methods because of its toxic pollutant constituents and high concentration of aromatic hydrocarbons, especially phenolic substances. A new type of hydrophobic benzacetone modified PbO2 anode (BA-PbO2 electrodes) was used for the electrocatalytic treatment of coal tar wastewater in a continuous cycle reactor. The surface morphology, structure, valences of elements, hydrophobicity, hydroxyl radical (·OH) produced capacity, electrochemical properties and stability of BA-PbO2 electrodes were characterized by SEM (scanning electron microscopy), XRD (X-ray diffraction), XPS (X-ray photoelectron spectroscopy), contact angle, a fluorescence probe test, an electrochemical workstation and accelerated life test, respectively. The BA-PbO2 electrodes exhibited a compact structure and finely dispersed crystallize size of 4.6 nm. The optimum degradation conditions of coal tar wastewater were as follows: current density of 90 mA cm-2, electrode gap of 1 cm and temperature at 25 °C with flow velocity of 80 L h-1. The chemical oxygen demand (COD) removal efficiency reached 92.39% after 240 min of degradation under the optimized conditions and the after-treatment COD value was 379.51 mg L-1 which was lower than the centralized emission standard (less than 400 mg L-1). These findings demonstrated the feasibility and efficiency of electrocatalytically degrading coal tar wastewater by BA-PbO2 electrodes. The possible mechanism and pathway for phenol a specific pollutant in coal tar wastewater were investigated by quantum chemistry calculations (Multiwfn) and gas chromatography-mass spectrometry (GC-MS). The toxicity of each intermediate was predicted by the ECOSAR program.

Analysis of whole-genome re-sequencing data of ducks reveals a diverse demographic history and extensive gene flow between Southeast/South Asian and Chinese populations.

BACKGROUND: The most prolific duck genetic resource in the world is located in Southeast/South Asia but little is known about the domestication and complex histories of these duck populations. RESULTS: Based on whole-genome resequencing data of 78 ducks (Anas platyrhynchos) and 31 published whole-genome duck sequences, we detected three geographic distinct genetic groups, including local Chinese, wild, and local Southeast/South Asian populations. We inferred the demographic history of these duck populations with different geographical distributions and found that the Chinese and Southeast/South Asian ducks shared similar demographic features. The Chinese domestic ducks experienced the strongest population bottleneck caused by domestication and the last glacial maximum (LGM) period, whereas the Chinese wild ducks experienced a relatively weak bottleneck caused by domestication only. Furthermore, the bottleneck was more severe in the local Southeast/South Asian populations than in the local Chinese populations, which resulted in a smaller effective population size for the former (7100-11,900). We show that extensive gene flow has occurred between the Southeast/South Asian and Chinese populations, and between the Southeast Asian and South Asian populations. Prolonged gene flow was detected between the Guangxi population from China and its neighboring Southeast/South Asian populations. In addition, based on multiple statistical approaches, we identified a genomic region that included three genes (PNPLA8, THAP5, and DNAJB9) on duck chromosome 1 with a high probability of gene flow between the Guangxi and Southeast/South Asian populations. Finally, we detected strong signatures of selection in genes that are involved in signaling pathways of the nervous system development (e.g., ADCYAP1R1 and PDC) and in genes that are associated with morphological traits such as cell growth (e.g., IGF1R). CONCLUSIONS: Our findings provide valuable information for a better understanding of the domestication and demographic history of the duck, and of the gene flow between local duck populations from Southeast/South Asia and China.

BRCA1 Reversion Mutation Confers Resistance to Olaparib and Camrelizumab in a Patient with Breast Cancer Liver Metastasis / 한국유방암학회지

Reversion mutations are associated with clinical resistance to poly(ADP-ribose) polymerase inhibitors (PARPi). Here, we describe the detection of a BRCA1 reversion mutation in a 39-year-old woman with metastatic breast cancer harboring a heterozygous germline BRCA1 exons 7–8 deletion who received PARPi olaparib combined with immune checkpoint inhibitor camrelizumab as third-line therapy. During progression from the olaparib and camrelizumab combination therapy, we identified via genomic sequencing a novel 7-base pair somatic deletion in BRCA1 (c.617_623delACAAATC). Sequence analyses indicated that this mutation realigned the reading frame of BRCA1, which potentially led to the reversal of its normal function and conferred resistance to PARPi.

Synthesis and appraisal of a hydroxyapatite/pectin hybrid material for zinc removal from water.

A simple method to modify hydroxyapatite and pectin into an efficient zinc sorbent was investigated. Process and formulation modifications enabled the formation of a flower-like hydroxyapatite/pectin hybrid material. The hybrid material was characterized with scanning electron microscopy, elemental analysis, and zeta potential tests. Sorption data were analyzed with different kinetic and isotherm models. The results showed that the pseudo-second order kinetic model and two-staged isotherm curves with Langmuir at the first stage and a Freundlich model the at second stage could best describe the zinc sorption on the hybrid. The maximum experimental sorption capacity was 330.4 mg Zn2+ per gram of sorbent, which was obtained with an initial concentration of 260 mg L-1 Zn2+ at pH 5.0. pH monitoring and Zeta potential tests suggested surface complexation and electrostatic attraction were fundamental in the zinc sorption process.

Selective constraints in cold-region wild boars may defuse the effects of small effective population size on molecular evolution of mitogenomes.

Spatial range expansion during population colonization is characterized by demographic events that may have significant effects on the efficiency of natural selection. Population genetics suggests that genetic drift brought by small effective population size (Ne) may undermine the efficiency of selection, leading to a faster accumulation of nonsynonymous mutations. However, it is still unknown whether this effect might be balanced or even reversed by strong selective constraints. Here, we used wild boars and local domestic pigs from tropical (Vietnam) and subarctic region (Siberia) as animal model to evaluate the effects of functional constraints and genetic drift on shaping molecular evolution. The likelihood-ratio test revealed that Siberian clade evolved significantly different from Vietnamese clades. Different datasets consistently showed that Siberian wild boars had lower Ka/Ks ratios than Vietnamese samples. The potential role of positive selection for branches with higher Ka/Ks was evaluated using branch-site model comparison. No signal of positive selection was found for the higher Ka/Ks in Vietnamese clades, suggesting the interclade difference was mainly due to the reduction in Ka/Ks for Siberian samples. This conclusion was further confirmed by the result from a larger sample size, among which wild boars from northern Asia (subarctic and nearby region) had lower Ka/Ks than those from southern Asia (temperate and tropical region). The lower Ka/Ks might be due to either stronger functional constraints, which prevent nonsynonymous mutations from accumulating in subarctic wild boars, or larger Ne in Siberian wild boars, which can boost the efficacy of purifying selection to remove functional mutations. The latter possibility was further ruled out by the Bayesian skyline plot analysis, which revealed that historical Ne of Siberian wild boars was smaller than that of Vietnamese wild boars. Altogether, these results suggest stronger functional constraints acting on mitogenomes of subarctic wild boars, which may provide new insights into their local adaptation of cold resistance.

Detecting the Population Structure and Scanning for Signatures of Selection in Horses (Equus caballus) From Whole-Genome Sequencing Data.

Animal domestication gives rise to gradual changes at the genomic level through selection in populations. Selective sweeps have been traced in the genomes of many animal species, including humans, cattle, and dogs. However, little is known regarding positional candidate genes and genomic regions that exhibit signatures of selection in domestic horses. In addition, an understanding of the genetic processes underlying horse domestication, especially the origin of Chinese native populations, is still lacking. In our study, we generated whole genome sequences from 4 Chinese native horses and combined them with 48 publicly available full genome sequences, from which 15 341 213 high-quality unique single-nucleotide polymorphism variants were identified. Kazakh and Lichuan horses are 2 typical Asian native breeds that were formed in Kazakh or Northwest China and South China, respectively. We detected 1390 loss-of-function (LoF) variants in protein-coding genes, and gene ontology (GO) enrichment analysis revealed that some LoF-affected genes were overrepresented in GO terms related to the immune response. Bayesian clustering, distance analysis, and principal component analysis demonstrated that the population structure of these breeds largely reflected weak geographic patterns. Kazakh and Lichuan horses were assigned to the same lineage with other Asian native breeds, in agreement with previous studies on the genetic origin of Chinese domestic horses. We applied the composite likelihood ratio method to scan for genomic regions showing signals of recent selection in the horse genome. A total of 1052 genomic windows of 10 kB, corresponding to 933 distinct core regions, significantly exceeded neutral simulations. The GO enrichment analysis revealed that the genes under selective sweeps were overrepresented with GO terms, including "negative regulation of canonical Wnt signaling pathway," "muscle contraction," and "axon guidance." Frequent exercise training in domestic horses may have resulted in changes in the expression of genes related to metabolism, muscle structure, and the nervous system.

De novo assembly of mitochondrial genomes provides insights into genetic diversity and molecular evolution in wild boars and domestic pigs.

Up to date, the scarcity of publicly available complete mitochondrial sequences for European wild pigs hampers deeper understanding about the genetic changes following domestication. Here, we have assembled 26 de novo mtDNA sequences of European wild boars from next generation sequencing (NGS) data and downloaded 174 complete mtDNA sequences to assess the genetic relationship, nucleotide diversity, and selection. The Bayesian consensus tree reveals the clear divergence between the European and Asian clade and a very small portion (10 out of 200 samples) of maternal introgression. The overall nucleotides diversities of the mtDNA sequences have been reduced following domestication. Interestingly, the selection efficiencies in both European and Asian domestic pigs are reduced, probably caused by changes in both selection constraints and maternal population size following domestication. This study suggests that de novo assembled mitogenomes can be a great boon to uncover the genetic turnover following domestication. Further investigation is warranted to include more samples from the ever-increasing amounts of NGS data to help us to better understand the process of domestication.

Population size may shape the accumulation of functional mutations following domestication.

BACKGROUND: Population genetics theory predicts an important role of differences in the effective population size (N e ) among species on shaping the accumulation of functional mutations by regulating the selection efficiency. However, this correlation has never been tested in domesticated animals. RESULTS: Here, we synthesized 62 whole genome data in eight domesticated species (cat, dog, pig, goat, sheep, chicken, cattle and horse) and compared domesticates with their wild (or ancient) relatives. Genes with significantly different selection pressures (revealed by nonsynonymous/synonymous substitution rate ratios, Ka/Ks or ω) between domesticated (Dω) and wild animals (Wω) were determined by likelihood-ratio tests. Species-level effective population sizes (N e ) were evaluated by the pairwise sequentially Markovian coalescent (PSMC) model, and Dω/Wω were calculated for each species to evaluate the changes in accumulation of functional mutations after domestication relative to pre-domestication period. Correlation analysis revealed that the most recent (~ 10.000 years ago) N e (s) are positively correlated with Dω/Wω. This result is consistent with the corollary of the nearly neutral theory, that higher N e could boost the efficiency of positive selection, which might facilitate the overall accumulation of functional mutations. In addition, we also evaluated the accumulation of radical and conservative mutations during the domestication transition as: Dradical/Wradical and Dconservative/Wconservative, respectively. Surprisingly, only Dradical/Wradical ratio exhibited a positive correlation with N e (p < 0.05), suggesting that domestication process might magnify the accumulation of radical mutations in species with larger N e . CONCLUSIONS: Our results confirm the classical population genetics theory prediction and highlight the important role of species' N e in shaping the patterns of accumulation of functional mutations, especially radical mutations, in domesticated animals. The results aid our understanding of the mechanisms underlying the accumulation of functional mutations after domestication, which is critical for understanding the phenotypic diversification associated with this process.

Exploring the Genetic Resistance to Gastrointestinal Nematodes Infection in Goat Using RNA-Sequencing.

Gastrointestinal nematodes (GINs) are one of the most economically important parasites of small ruminants and a major animal health concern in many regions of the world. However, the molecular mechanisms of the host response to GIN infections in goat are still little known. In this study, two genetically distinct goat populations, one relatively resistant and the other susceptible to GIN infections, were identified in Yichang goat and then four individuals in each group were chosen to compare mRNA expression profiles using RNA-seq. Field experiment showed lower worm burden, delayed and reduced egg production in the relatively resistant group than the susceptible group. The analysis of RNA-seq showed that 2369 genes, 1407 of which were up-regulated and 962 down-regulated, were significantly (p < 0.001) differentially expressed between these two groups. Functional annotation of the 298 genes more highly expressed in the resistant group yielded a total of 46 significant (p < 0.05) functional annotation clusters including 31 genes (9 in innate immunity, 13 in immunity, and 9 in innate immune response) related to immune biosynthetic process as well as transforming growth factor (TGF)-ß, mitogen-activated protein kinase (MAPK), and cell adhesion molecules (CAMs) pathways. Our findings provide insights that are immediately relevant for the improvement of host resistance to GIN infections and which will make it possible to know the mechanisms underlying the resistance of goats to GIN infections.

MiR-99a and MiR-491 Regulate Cisplatin Resistance in Human Gastric Cancer Cells by Targeting CAPNS1.

Cisplatin is the first-line agent utilized for the clinical treatment of a wide variety of solid tumors including gastric cancer. However, the intrinsic or acquired cisplatin resistance is often occurred in patients with gastric cancer and resulted in failure of cisplatin therapy. In order to investigate if miRNA involves in cisplatin resistance of human gastric cancer, we first screened and compared the expression of miRNAs between cisplatin resistant gastric cancer cell lines SGC-7901/DDP and BGC-823/DDP and their sensitive parental cells by miRNAs microarray and followed by analysis of 2D-GE/MS to identify their target proteins. We found both miR-99a and miR-491 were upregulated while their target gene calpain small subunit 1 (CAPNS1) was downregulated in resistant gastric cancer cells. Dual-luciferase- reporter assays with wild-type and mutated CAPNS1 3'-UTR confirmed their specificity of targeting. Inhibition of miR-99a and miR-491, or overexpress CAPNS1 can enhance cisplatin sensitivity of the resistant cells while transfection of two miRNAs' mimics or si-CAPNS1 in the sensitive cells can induce their resistance. Moreover, our results demonstrated CAPNS1 positively regulated calpain1 and calpain2, the catalytic subunits of CAPNS1, and cleaved caspase3 which further cleaved PARP1 and directly induced apoptosis. Therefore, miR-99a and miR-491 might be work as novel molecules regulate cisplatin resistance by directly targeting CAPNS1 associated pathway in human gastric cancer cells.

The complete mitochondrial genome of Mong Cai pig (Sus scrofa) in Vietnam.

The Mong Cai pig is an indigenous breed and popularly raised as maternal line in northern Vietnam. In this study, the complete mitochondrial genome sequence of the Mong Cai pig is reported. The total length of this mitochondrial genome is 16 632 bp, including 1 non-coding control region (D-loop region), two ribosomal RNA genes, 13 protein-coding genes and 22 transfer RNA genes. The phylogenetic tree of 162 pig complete mitogenomes reveals a very close relationship between Mong Cai pig in Vietnam and Bama miniature pig in southern China. This complete mitochondrial genome sequence of Mong Cai pig is useful to further genetic studies on adaptation and performance.

[sgRNA design for the CRISPR/Cas9 system and evaluation of its off-target effects].

The third generation of CRISPR/Cas9-mediated genome editing technology has been successfully applied to genome modification of various species including animals, plants and microorganisms. How to improve the efficiency of CRISPR/Cas9 genome editing and reduce its off-target effects has been extensively explored in this field. Using sgRNA (Small guide RNA) with high efficiency and specificity is one of the critical factors for successful genome editing. Several software have been developed for sgRNA design and/or off-target evaluation, which have advantages and disadvantages respectively. In this review, we summarize characters of 16 kinds online and standalone software for sgRNA design and/or off-target evaluation and conduct a comparative analysis of these different kinds of software through developing 38 evaluation indexes. We also summarize 11 experimental approaches for testing genome editing efficiency and off-target effects as well as how to screen highly efficient and specific sgRNA.

Development of a graphical user interface for sgRNAcas9 and its application.

The CRISPR/Cas9 genome editing technique is a powerful tool for researchers. However, off-target effects of the Cas9 nuclease activity is a recurrent concern of the CRISPR system. Thus, designing sgRNA (single guide RNA) with minimal off-target effects is very important. sgRNAcas9 is a software package, which can be used to design sgRNA and to evaluate potential off-target cleavage sites. In this study, a graphical user interface for sgRNAcas9 was developed using the Java programming language. In addition, off-target effect for sgRNAs was evaluated according to mismatched number and "seed sequence" specification. Moreover, sgRNAcas9 software was used to design 34 124 sgRNAs, which can target 4691 microRNA (miRNA) precursors from human, mouse, rat, pig, and chicken. In particular, the off-target effect of a sgRNA targeting to human miR-206 precursor was analyzed, and the on/off-target activity of this sgRNA was validated by T7E1 assay in vitro. Taken together, these data showed that the interface can simplify the usage of the sgRNAcas9 program, which can be used to design sgRNAs for the majority of miRNA precursors. We also found that the GC% of those sgRNAs ranged from 40% to 60%. In summary, the sgRNAcas9 software can be easily used to design sgRNA with minimal off-target effects for any species. The software can be downloaded from BiooTools website (http://www.biootools.com/).

TXNL1 induces apoptosis in cisplatin resistant human gastric cancer cell lines.

Cisplatin is one of the most commonly used drugs in the treatment of gastric cancer. However, drug resistance is a major obstacle for effective treatment and originates in multiple mechanisms such as enhanced DNA repair and anti-apoptosis. Our previous results demonstrated that XRCC1 was a key regulator of cisplatin induced DNA damage and apoptosis. TXNL1, a member of the thioredoxin family, negatively regulated the expression of XRCC1 via the ubiquitin-proteasome pathway. Here, we investigated the role of TXNL1 in the apoptosis induced by cisplatin. Our data showed that the expression of TXNL1 in the cisplatin resistant gastric cancer cell lines BGC823/DDP and SGC7901/DDP cells was significantly lower compared with the cisplatin sensitive cell lines BGC823 and SGC7901. Inhibition of the expression of TXNL1 in BGC823 and SGC7901 cells led to increased resistance to cisplatin induced apoptosis and cell death detected by Tunel and clonogenic assay, respectively. In contrast, over expression of TXNL1 in BGC823/DDP and SGC7901/DDP cells lead to higher cisplatin induced apoptosis and cell death. Moreover, our results demonstrated that the mechanism of TXNL1 regulating cisplatin-induced apoptosis was closely associated with Bcl-2 mediated mitochondria apoptosis pathway. In conclusion, these findings suggest that TXNL1 was a feasible modulator and potential chemotherapeutic target for the cisplatin resistant phenotype of human gastric cancer cells.