Tissue-Specific Distribution and Maternal Transfer of Persistent Organic Halogenated Pollutants in Frogs.

Persistent organic pollutants pose a great threat to amphibian populations, but information on the bioaccumulation of contaminants in amphibians remains scarce. To examine the tissue distribution and maternal transfer of organic halogenated pollutants (OHPs) in frogs, seven types of tissues from black-spotted frog (muscle, liver, kidney, stomach, intestine, heart, and egg) were collected from an e-waste-polluted area in South China. Among the seven frog tissues, median total OHP concentrations of 2.3 to 9.7 µg/g lipid weight were found (in 31 polychlorinated biphenyl [PCB] individuals and 15 polybrominated diphenyl ether [PBDE], dechlorane plus [syn-DP and anti-DP], bexabromobenzene [HBB], polybrominated biphenyl] PBB153 and -209], and decabromodiphenyl ethane [DBDPE] individuals). Sex-specific differences in contaminant concentration and compound compositions were observed among the frog tissues, and eggs had a significantly higher contaminant burden on the whole body of female frogs. In addition, a significant sex difference in the concentration ratios of other tissues to the liver was observed in most tissues except for muscle. These results suggest that egg production may involve the mobilization of other maternal tissues besides muscle, which resulted in the sex-specific distribution. Different parental tissues had similar maternal transfer mechanisms; factors other than lipophilicity (e.g., molecular size and proteinophilic characteristics) could influence the maternal transfer of OHPs in frogs. Environ Toxicol Chem 2024;00:1-12. © 2024 SETAC.

Targeting OXCT1-mediated ketone metabolism reprograms macrophages to promote antitumor immunity via CD8+ T cells in hepatocellular carcinoma.

BACKGROUND & AIMS: The liver is the main organ of ketogenesis, while ketones are mainly metabolized in peripheral tissues via the critical enzyme OXCT1. We previously found that ketolysis is reactivated in hepatocellular carcinoma (HCC) cells through OXCT1 expression to promote tumor progression; however, whether OXCT1 regulates antitumor immunity remains unclear. METHODS: To investigate the expression pattern of OXCT1 in hepatocellular carcinoma in vivo, we conducted multiplex immunohistochemistry (mIHC) experiments on human HCC specimens. To explore the role of OXCT1 in mouse hepatocellular carcinoma tumor-associated macrophages (TAMs), we generated LysMcreOXCT1f/f (OXCT1 conditional knockout in macrophages) mice. RESULTS: Here, we found that inhibiting OXCT1 expression in tumor-associated macrophages reduced CD8+ T-cell exhaustion through the succinate-H3K4me3-Arg1 axis. Initially, we found that OXCT1 was highly expressed in liver macrophages under steady state and that OXCT expression was further increased in TAMs. OXCT1 deficiency in macrophages suppressed tumor growth by reprogramming TAMs toward an antitumor phenotype, reducing CD8+ T-cell exhaustion and increasing CD8+ T-cell cytotoxicity. Mechanistically, high OXCT1 expression induced the accumulation of succinate, a byproduct of ketolysis, in TAMs, which promoted Arg1 transcription by increasing the H3K4 trimethylation (H3K4me3) level in the Arg1 promoter. In addition, Pimozide, an inhibitor of OXCT1, suppressed Arg1 expression as well as TAM polarization toward the protumor phenotype, leading to decreasing CD8+ T-cell exhaustion and deceleration of tumor growth. Finally, high expression of OXCT1 in macrophages was positively associated with poor survival in HCC patients. CONCLUSIONS: In conclusion, our results demonstrate that OXCT1 epigenetically suppresses antitumor immunity, suggesting that suppressing OXCT1 activity in TAMs is an effective approach for treating liver cancer. IMPACT AND IMPLICATIONS: The intricate metabolism of liver macrophages plays a critical role in shaping HCC progression and immune modulation. Targeting macrophage metabolism to counteract immune suppression presents a promising avenue for HCC. Here, we found that ketogenesis gene OXCT1 was highly expressed in tumor-associated macrophages and promoted tumor growth by reprogramming TAMs toward a protumor phenotype. And the strategic pharmacological intervention or genetic downregulation of OXCT1 in TAMs enhances the antitumor immunity and decelerated tumor growth. Our results suggest that suppressing OXCT1 activity in TAMs is an effective approach for treating liver cancer.

Application of nano-radiosensitizers in non-small cell lung cancer.

Radiotherapy stands as a cornerstone in the treatment of numerous malignant tumors, including non-small cell lung cancer. However, the critical challenge of amplifying the tumoricidal effectiveness of radiotherapy while minimizing collateral damage to healthy tissues remains an area of significant research interest. Radiosensitizers, by methods such as amplifying DNA damage and fostering the creation of free radicals, play a pivotal role in enhancing the destructive impact of radiotherapy on tumors. Over recent decades, nano-dimensional radiosensitizers have emerged as a notable advancement. Their mechanisms include cell cycle arrest in the G2/M phase, combating tumor hypoxia, and others, thereby enhancing the efficacy of radiotherapy. This review delves into the evolving landscape of nanomaterials used for radiosensitization in non-small cell lung cancer. It provides insights into the current research progress and critically examines the challenges and future prospects within this burgeoning field.

Determination of the water vapor transmission rate of cellulose-based papers by multiple headspace extraction analysis.

This paper presents a multiple headspace extraction (MHE) analysis technique to determine the water vapor transmission rate of cellulose-based papers. The water vapor passing through the sample in a closed headspace vial is determined by MHE-gas chromatography. The results show that the employed method offers good precision (the relative standard deviation < 3.49 %) and good accuracy. The method is rapid and accurate, and is promising for the determination of the water vapor transmission rate of cellulose-based papers in future studies.

Sex-Specific Bioaccumulation, Maternal Transfer, and Tissue Distribution of Legacy and Emerging Per- and Polyfluoroalkyl Substances in Snakes (Enhydris chinensis) and the Impact of Pregnancy.

The effects of sex and pregnancy on the bioaccumulation and tissue distribution of legacy and emerging per- and polyfluoroalkyl substances (PFASs) in Chinese water snakes were investigated. The bioaccumulation factor of PFASs showed a positive correlation with their protein-water partition coefficients (log KPW), and steric hindrance effects were observed when the molecular volume was > 357 Å3. PFAS levels in females were significantly lower than those in males. The chemical composition of pregnant females was significantly different from that of non-pregnant females and males. The maternal transfer efficiencies of perfluorooctane sulfonic acid were higher than those of other PFASs, and a positive correlation between the maternal transfer potential and log KPW was observed for other PFASs. Tissues with high phospholipid content exhibited higher concentrations of ∑PFASs. Numerous physiological changes occurred in maternal organ systems during pregnancy, leading to the re-distribution of chemicals among different tissues. The change in tissue distribution of PFASs that are easily and not-so-easily maternally transferred was in the opposite direction. The extent of compound transfer from the liver to the egg determined tissue re-distribution during pregnancy.

Inorganic Solid-State Nonlinear Optical Switch with a Linearly Tunable Tc Spanning a Wide Temperature Range.

Nonlinear optical (NLO) switch materials that turn on/off second-harmonic generation (SHG) at a phase transition temperature (Tc ) are promising for applications in the fields of photoswitching and optical computing. However, precise control of Tc remains challenging, mainly because a linearly tunable Tc has not been reported to date. Herein, we report a unique selenate, tetragonal P 4 ‾ ${\bar{4}}$ 21 c [Ag(NH3 )2 ]2 SeO4 with a=b=8.5569(2) Šand c=6.5208(2) Šthat exhibits a strong SHG intensity (1.3×KDP) and a large birefringence (Δnobv. =0.08). This compound forms a series of isostructural solid-solution crystals [Ag(NH3 )2 ]2 Sx Se1-x O4 (x=0-1.00) that exhibit excellent NLO switching performance and an unprecedented linearly tunable T c , x , e x p . = T 0 - k x ${{T}_{\left(c,{\rm \ }x\right),{\rm \ }\left({\rm e}{\rm x}{\rm p}.\right)}{\rm \ }={T}_{0}-kx}$ spanning 430 to 356 K. The breaking of localized hydrogen bonds between SeO4 2- and the cation triggers a phase transition accompanied by hydrogen bond length changes with increasing x and a linear change in the enthalpy Δ H x = Δ U 1 - Δ U 2 x + Δ U 2 ${{{\rm { \Delta{}}}H}_{x}=\left({\rm { \Delta{}}}{U}_{1}-{\rm { \Delta{}}}{U}_{2}\right)x+{\rm { \Delta{}}}{U}_{2}}$ .

[Influence of bronchopulmonary dysplasia on cerebral blood flow in preterm infants: a prospective study based on arterial spin labeling]. / åŸºäºŽåŠ¨è„‰è‡ªæ—‹æ ‡è®°æˆåƒæŠ€æœ¯æŽ¢è®¨æ”¯æ°”ç®¡è‚ºå‘è‚²ä¸è‰¯å¯¹æ—©äº§å„¿è„‘è¡€æµé‡å½±å“çš„å‰çž»æ€§ç ”ç©¶.

OBJECTIVES: To investigate local cerebral blood perfusion in preterm infants with bronchopulmonary dysplasia (BPD) based on cerebral blood flow (CBF) values of arterial spin labeling (ASL). METHODS: A prospective study was conducted on 90 preterm infants with a gestational age of <32 weeks and a birth weight of <1 500 g who were born in the Department of Obstetrics and admitted to the Department of Neonatology in the Third Affiliated Hospital of Zhengzhou University from August 2021 to June 2022. All of the infants underwent cranial MRI and ASL at the corrected gestational age of 35-40 weeks. According to the presence or absence of BPD, they were divided into a BPD group with 45 infants and a non-BPD group with 45 infants. The two groups were compared in terms of the CBF values of the same regions of interest (frontal lobe, temporal lobe, parietal lobe, occipital lobe, thalamus, and basal ganglia) on ASL image. RESULTS: Compared with the non-BPD group, the BPD group had a significantly lower 1-minute Apgar score, a significantly longer duration of assisted ventilation, and a significantly higher incidence rate of fetal distress (P<0.05). After control for the confounding factors such as corrected age and age at the time of cranial MRI by multiple linear regression analysis, compared with the non-BPD group, the BPD group still had higher CBF values of the frontal lobe, temporal lobe, parietal lobe, occipital lobe, basal ganglia, and thalamus at both sides (P<0.05). CONCLUSIONS: BPD can increase cerebral blood perfusion in preterm infants, which might be associated with hypoxia and a long duration of assisted ventilation in the early stage.

Mesenchymal Stem Cell-Derived Extracellular Vesicles Therapy for Pulmonary Hypertension: A Comprehensive Review of Preclinical Studies.

Pulmonary hypertension (PH) is a type of clinical pathophysiological syndrome characterized by a progressive increase in pulmonary vascular resistance and subsequent progressive failure of the right heart function, and is a common complication of many diseases. Mesenchymal stem cells (MSCs) autonomously home to sites damaged by disease, repair damaged tissues, and participate in the regulation of systemic inflammation and immune responses, which have good clinical application prospects. Extracellular vesicles (EVs), such as exosomes and microvesicles, participate in various biological activities by regulating intercellular communication. Exosomes secreted into the extracellular environment also affect the host immune system. MSC-derived extracellular vesicles (MSC-EVs), as a mediator in the paracrine processes of MSCs, carry biologically active substances such as proteins, lipids, mRNA, and micro-RNA. MSC-EVs therapies, safer than cell-based treatments, have been shown to be effective in modulating macrophages to support anti-inflammatory phenotypes, which are strongly related to histological and functional benefits in preclinical models of pulmonary hypertension. The main effects of active substances and their potential medical value have attracted wide attention from researchers. This article reviews the role and relevant mechanisms of MSC-EVs in the treatment of pulmonary hypertension in recent studies and provides a basis for their future clinical applications.

Discoidin domain receptor 2 expression increases phagocytotic capacity in sertoli cells of sertoli cell-only syndrome testes.

Discoidin domain receptor 2 (DDR2) belongs to the receptor tyrosine kinase (RTK) family, other RTKs have been reported to regulate phagocytic function of Sertoli cells (SCs), yet little is known about the function of DDR2 in Sertoli cells. In the present study, we aim to explore the function and mechanism of ectopic discoidin domain receptor 2 (DDR2) expression in Sertoli cells of Sertoli cell-only syndrome (SCOS) testes. We found that discoidin domain receptor 2 (DDR2) was absent in Sertoli cells of normal testis but was expressed in Sertoli cells of SCOS testes. This Sertoli cell DDR2 expression was induced by impaired androgen receptor (AR) signaling, but was inhibited by increased AR signaling from testosterone administration. The Sertoli cell DDR2 expression led to an increase in phagocytosis through up-regulation of Scavenger receptor class B member 1 (SR-BI) levels. However, loss of DDR2 by knock-out or knock-down weakened the phagocytotic capacity of Sertoli cells. Furthermore, the expression of DDR2 in Sertoli cells activated matrix metallopeptidase 9 (MMP-9) to consume abnormal collagen increase in seminiferous tubules which was responsible for the block of testosterone transportation and AR loss and to compensate for the impaired blood-testis-barrier (BTB). Our data suggest that the AR/DDR2 cascade may serve as a negative feedback mechanism to help compensate for the homeostasis of seminiferous epithelium in SCOS testis.

Association of neutropenia at disease onset with severe surgical necrotizing enterocolitis and higher mortality: A retrospective study.

Background: Neutrophils are among the earliest immune cells recruited to the site of an intestinal injury, but their predictive role in the progression of necrotizing enterocolitis (NEC) has not been fully elucidated. This study aimed to evaluate if a reduction in neutrophils at the onset of NEC is associated with severe surgical NEC and/or NEC-associated deaths. Methods: This is a retrospective cohort study in which neonates underwent surgery due to NEC during 2015-2020. The data on absolute neutrophil count (ANC), before and at the onset of NEC, were collected from the complete blood count results. The primary exposure was the difference in absolute neutrophil count (ΔANC) at NEC onset. The primary outcome was severe surgical NEC, defined as the residual small bowel length after intestinal resection of <30 cm. Results: A total of 157 neonates were included in this study, of which 53 were diagnosed with severe surgical NEC. A decrease in ANC at the onset of NEC was associated with an increased probability of severe surgical NEC (crude odds ratio [OR] 1.248, 95% CI 1.107-1.407; P = 0.000). ΔANC (area under the curve [AUC] 0.729, 95% CI 0.653-0.797; P < 0.001] was a good predictor for severe surgical NEC. The addition of platelets to ΔANC at NEC onset (AUC 0.738, 95% CI 0.662-0.808; P < 0.001) resulted in a higher AUC and specificity for severe surgical NEC prediction than ΔANC alone. A reduction in the neutrophil count at NEC onset (ΔANC > 0) was associated with adverse outcomes (hazard ratio [HR] 3.48, 95% CI 1.64-7.36) and a lower survival probability (χ2 10.63; P < 0.001). Conclusion: A reduction in the ANC at the onset of NEC was associated with severe surgical NEC and higher mortality. The addition of platelets to ΔANC at NEC onset resulted in a higher predictive value of severe surgical NEC. This study may provide a new insight into the bedside evaluation of NEC by analyzing data from the day of NEC onset.

Spatial region-resolved proteome map reveals mechanism of COVID-19-associated heart injury.

Direct myocardial and vascular injuries due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection-driven inflammation is the leading cause of acute cardiac injury associated with coronavirus disease 2019 (COVID-19). However, in-depth knowledge of the injury characteristics of the heart affected by inflammation is lacking. In this study, using a quantitative spatial proteomics strategy that combines comparative anatomy, laser-capture microdissection, and histological examination, we establish a region-resolved proteome map of the myocardia and microvessels with obvious inflammatory cells from hearts of patients with COVID-19. A series of molecular dysfunctions of myocardia and microvessels is observed in different cardiac regions. The myocardia and microvessels of the left atrial are the most susceptible to virus infection and inflammatory storm, suggesting more attention should be paid to the lesion and treatment of these two parts. These results can guide in improving clinical treatments for cardiovascular diseases associated with COVID-19.

Construction of early risk prediction models for bronchopulmonary dysplasia in preterm infants. / 早产儿支气管肺发育不良早期风险预测模型的构建.

OBJECTIVES: To construct risk prediction models for bronchopulmonary dysplasia (BPD) in preterm infants on postnatal days 3, 7, and 14. METHODS: A retrospective analysis was performed on the medical data of 414 preterm infants, with a gestational age of <32 weeks and a birth weight (BW) of <1 500 g, who were admitted to the neonatal intensive care unit from July 2019 to April 2021. According to the diagnostic criteria for BPD revised in 2018, they were divided into a BPD group with 98 infants and a non-BPD group with 316 infants. The two groups were compared in terms of general status, laboratory examination results, treatment, and complications. The logistic regression model was used to identify the variables associated with BPD. The receiver operating characteristic (ROC) curve was used to evaluate the predictive value of models. RESULTS: The logistic regression analysis showed that BW, asphyxia, grade III-IV respiratory distress syndrome (RDS), acute chorioamnionitis, interstitial pneumonia, fraction of inspired oxygen (FiO2), and respiratory support mode were the main risk factors for BPD (P<0.05). The prediction models on postnatal days 7 and 14 were established as logit (P7) =-2.049-0.004×BW (g) +0.686×asphyxia (no=0, yes=1) +1.842×grade III-IV RDS (no=0, yes=1) +0.906×acute chorioamnionitis (no=0, yes=1) +0.506×interstitial pneumonia (no=0, yes=1) +0.116×FiO2 (%) +0.816×respiratory support mode (no=0, nasal tube=1, nasal continuous positive airway pressure=2, conventional mechanical ventilation=3, high-frequency mechanical ventilation=4) and logit (P14) =-1.200-0.004×BW (g) +0.723×asphyxia+2.081×grade III-IV RDS+0.799×acute chorioamnionitis+0.601×interstitial pneumonia+0.074×FiO2 (%) +0.800×respiratory support mode, with an area under the ROC curve (AUC) of 0.876 and 0.880, respectively, which was significantly larger than the AUC of the prediction model on postnatal day 3 (P<0.05). CONCLUSIONS: BW, asphyxia, grade III-IV RDS, acute chorioamnionitis, interstitial pneumonia, FiO2, and respiratory support mode are the main risk factors for BPD and can be used to construct risk prediction models. The prediction models on postnatal days 7 and 14 can effectively predict BPD.

G protein pathway suppressor 2 suppresses gastric cancer by destabilizing epidermal growth factor receptor.

G protein pathway suppressor 2 (GPS2) is expressed in most human tissues, including the stomach. However, the biological functions of GPS2 in cancer, as well as the underlying molecular mechanisms, remain poorly understood. Here, we report that GPS2 expression was aberrantly downregulated in gastric cancer (GC) tissues compared with control tissues. Clinicopathologic analysis showed that low GPS2 expression was significantly correlated with pathological grade, lymph node stage, and invasive depth. Kaplan-Meier analysis indicated that patients with low GPS2 expression showed poorer overall survival rates than those with high GPS2 expression. Moreover, GPS2 overexpression decreased GC cell proliferation, colony formation, tumorigenesis, and invasion. Overexpression of GPS2 reduced the protein expression of epidermal growth factor receptor (EGFR) and inhibited its downstream signaling in GC cells. Interestingly, GPS2 decreased EGFR protein expression, which was reversed by a lysosome inhibitor. Furthermore, GPS2 reduced EGFR protein stability by enhancing the binding of EGFR and an E3 ligase, c-Cbl, which promoted the ubiquitination of EGFR, ultimately leading to its degradation through the lysosomal pathway. Further analysis indicated that GPS2 activated autophagy and promoted the autophagic flux by destabilizing EGFR. Taken together, these results suggest that low GPS2 expression is associated with GC progression and provide insights into the applicability of the GPS2-EGFR axis as a potential therapeutic target in GC.

Developing a Peptide That Inhibits DNA Repair by Blocking the Binding of Artemis and DNA Ligase IV to Enhance Tumor Radiosensitivity.

PURPOSE: Artemis and DNA Ligase IV are 2 critical elements in the nonhomologous end joining pathway of DNA repair, acting as the nuclease and DNA ligase, respectively. Enhanced cellular radiosensitivity by inhibition of either protein contributes to a promising approach to develop molecular targeted radiosensitizers. The interaction between Artemis and DNA Ligase IV is required for the activation of Artemis as nuclease at 3'overhang DNA; thus, we aim to generate an inhibitory peptide targeting the interaction between Artemis and DNA Ligase IV for novel radiosensitizer development. METHODS AND MATERIALS: We synthesized the peptide BAL, which consists of the interaction residues of Artemis to DNA Ligase IV. The radiosensitization effect of BAL was evaluated by colony formation assay. The effects of BAL on radiation-induced DNA repair were evaluated with Western blotting and immunofluorescence. The effects of BAL on cell proliferation, cell cycle arrest, and cell apoptosis were assessed via CCK-8 and flow cytometry assays. The potential synergistic effects of BAL and irradiation in vivo were investigated in a xenograft mouse model. RESULTS: The generated peptide BAL blocking the interaction between Artemis and DNA Ligase IV significantly enhanced the radiosensitivity of GBC-SD and HeLa cell lines. BAL prolonged DNA repair after irradiation; BAL and irradiation showed synergistic effects on cell proliferation, cell cycle, and cell apoptosis, and these functions are all DNA Ligase IV-related. Finally, we confirmed the endogenous radiosensitization effect of BAL in a xenograft mouse model. CONCLUSIONS: The inhibitory peptide BAL targeting the binding of Artemis and DNA Ligase IV successfully functions as a novel radiosensitizer that delays DNA repair and synergizes with irradiation to inhibit cell proliferation, induce cell cycle arrest, and promote cell apoptosis.

Recent Progress in Saikosaponin Biosynthesis in Bupleurum.

BACKGROUND: Chaihu is a popular traditional Chinese medicine that has been used for centuries. It is traditionally used to treat cold fever and liver-related diseases. Saikosaponins (SSs) are one of the main active components of chaihu, in addition to essential oils, flavonoids, and polysaccharides. Considerable effort is needed to reveal the biosynthesis and regulation of SSs on the basis of current progress. OBJECTIVE: The aim of this study is to provide a reference for further studies and arouse attention by summarizing the recent achievements of SS biosynthesis. METHODS: All the data compiled and presented here were obtained from various online resources, such as PubMed Scopus and Baidu Scholar in Chinese, up to October 2019. RESULTS: A few genes of the enzymes of SSs participating in the biosynthesis of SSs were isolated. Among these genes, only the P450 gene was verified to catalyze the SS skeleton ß-amyrin synthase. Several UDP-glycosyltransferase genes were predicted to be involved in the biosynthesis of SSs. SSs could be largely biosynthesized in the phloem and then transported from the protoplasm, which is the biosynthetic site, to the vacuoles to avoid self-poisoning. As for the other secondary metabolites, the biosynthesis of SSs was strongly affected by environmental factors and the different species belonging to the genus of Bupleurum. Transcriptional regulation was studied at the molecular level. CONCLUSION: Profound discoveries in SSs may elucidate the mechanism of diverse the monomer formation of SSs and provide a reference for maintaining the stability of SS content in Radix Bupleuri.

The Combination of Icotinib Hydrochloride and Fluzoparib Enhances the Radiosensitivity of Biliary Tract Cancer Cells.

BACKGROUND: Radiotherapy and chemotherapy are the main clinical treatments for biliary tract cancers (BTCs). Patients with advanced disease have a very poor prognosis, yet no molecular targets have been proven effective for the adjuvant therapy of BTCs. In this study, we aimed to explore the effect of combination treatment with icotinib hydrochloride (IH) and fluzoparib (FZ) on radiosensitivity and clarify its underlying mechanism in the HCCC-9810 and GBC-SD human BTC cell lines. METHODS: Cell proliferation was measured by Cell Counting Kit-8 (CCK-8) assay. The cell cycle distribution and apoptosis were analyzed by flow cytometry. The phosphorylation of EGFR and its downstream signaling molecules and the expression of RAD51 were measured by Western blot analysis. γ-H2AX foci in the cellular nuclei were visualized using immunofluorescence staining. A colony formation assay was performed to demonstrate cell radiosensitivity with IH and FZ combination treatment. RESULTS: In the HCCC-9810 and GBC-SD human BTC cell lines, combined treatment with IH and FZ with synergetic radiation significantly inhibited cell proliferation, redistributed the cell cycle, enhanced apoptosis and delayed DNA damage repair by suppressing activation of the EGFR signaling pathway and attenuating expression of the homologous recombination (HR) protein RAD51. CONCLUSION: This study demonstrates that combined treatment with IH and FZ may be an applicable therapy to enhance the radiosensitivity of BTCs and that RAD51 may serve as a biomarker for this combination treatment.

Novel nucleic acid detection strategies based on CRISPR-Cas systems: From construction to application.

Beyond their widespread application as genome-editing and regulatory tools, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems also play a critical role in nucleic acid detection due to their high sensitivity and specificity. Recently developed Cas family effectors have opened the door to the development of new strategies for detecting different types of nucleic acids for a variety of purposes. Precise and efficient nucleic acid detection using CRISPR-Cas systems has the potential to advance both basic and applied biological research. In this review, we summarize the CRISPR-Cas systems used for the recognition and detection of specific nucleic acids for different purposes, including the detection of genomic DNA, nongenomic DNA, RNA, and pathogenic microbe genomes. Current challenges and further applications of CRISPR-based detection methods will be discussed according to the most recent developments.

Carboxyl terminal activating region 3 of latent membrane protein 1 encoded by the Epstein­Barr virus regulates cell proliferation and protein expression in NP69 cells.

In the present study, the mechanism by which carboxyl terminal activating region 3 (CTAR3) of latent membrane protein 1 (LMP1), encoded by the Epstein­Barr virus, regulated cell proliferation and protein expression was investigated in the nasopharyngeal epithelial cell line NP69. The deletion mutant LMP1 (LMP1Δ232­351; amino acid residues including 232­351 codons in CTAR3 deleted) was generated by polymerase chain reaction. An NP69­LMP1Δ232­351 cell line was established by retroviral infection. Finally, cell proliferation and protein expression of NP69 cells expressing LMP1Δ232­351 were examined using a cell growth curve and western blot analysis. The results demonstrated: i) The proliferation of NP69­LMP1Δ232­351 cells was significantly decreased compared with cells expressing wild type LMP1 (LMP1WT; n=3; P<0.05); ii) 17 proteins exhibited differential protein expression (>2­fold change) in NP69­LMP1Δ232­351 cells compared with NP69­LMP1WT cells; and iii) LMP1WT was involved in activating the Janus kinase 3 (JAK3) promoter and regulating the expression of JAK3 protein, while LMP1Δ232­351 was almost defective in ability to activate the JAK promoter. These results suggested that LMP1­CTAR3 may be an important functional domain for regulating cell proliferation and protein expression in nasopharyngeal epithelial cells.

[Genome survey analysis and SSR loci mining of Bupleurum falcatum].

Buplewrum falcatum is a traditional Chinese medicine,which is mainly used for the treatment of cold and liver protection. B. falcatum is dominantly cultivated in Japan as well as planted in China,Korea and other countries and regions. In order to determine the appropriate sequencing strategy,the genome survey before large-scale genome sequencing is needed. This survey can provide information about the size and complexity of the whole genome of the target species. In the present study,the next generation sequencing technology( Illumina Hiseq 2000) was used to analyze the genome size and complexity of B. falcatum. In addition,SSR loci were analyzed from the sequenced data. Primer 3 was used to design specific primers and 33 pairs of primers were randomly selected for PCR with template DNA of B. falcatum,and the PCR system and optimal annealing temperature were screened. A total of 288. 64 G genome sequence data was obtained,and the estimated genome size of B. falcatum was 2 119. 58 Mb. The measured genome data depth was138×; the rate of heterozygosity was 1. 84%; and the ratio of repeat sequence was 83. 89%. It is speculated that the genome of B. falcatum is complex. The preliminary assembly was performed with K-mer = 41,and the contig N50 was 224 bp,the total length 896. 97 Mb,the scaffold N50 313 bp,and the total length was 922. 67 Mb. A total of 91 377 SSR sequences were detected in the sequenced genome data which were distributed in 70 809 unigenes.The main type is dinucleotide repeats,with 49 680 sequences,accounting for70. 16%. Among the 33 pairs of primers randomly synthesized according to the obtained SSR sequences,21 pairs were successfully amplifying the target sequences. The results will be helpful for later large scale genome sequencing and SSR molecular markers development for germplasm identification and trait mapping.